Maillard products and coffee roasting products activate NF ... · 1.1.1 The coffee bean Each coffee...

Maillard products and coffee roasting products

activate NF-κB and Nrf2

in cell culture and intact human gut tissue ex vivo

Maillard Produkte und Röstprodukte aktivieren NF-κB und Nrf2

in verschiedenen Zelltypen und humanem Darmgewebe ex vivo

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg

zur Erlangung des Doktorgrades Dr. rer. nat.

vorgelegt von

Tanja Sauer

aus Bad Dürkheim

Als Dissertation genehmigt von der

Naturwissenschaftlichen Fakultät der

Friedrich-Alexander-Universität Erlangen-Nürnberg

Tag der mündlichen Prüfung: 20.09.2011

Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatterin: Prof. Dr. Monika Pischetsrieder

Zweitberichterstatter: Prof. Dr. Gerald Muench

Parts of this thesis have already been published:

Publications

Tanja Sauer, Martin Raithel, Jürgen Kressel, Gerald Münch and Monika Pischetsrieder

Nuclear translocation of NF-κB in intact human gut tissue upon stimulation with coffee and

roasting products

Accepted in August 2011 in Food & Function

Tanja Sauer, Martin Raithel, Jürgen Kressel, Gerald Münch and Monika Pischetsrieder

Activation of the transcription factor Nrf2 in macrophages, Caco-2 cells and intact human gut

tissue by Maillard reaction products and coffee

Submitted in April 2011

Talk

Tanja Sauer

Coffee-induced regulation of redox-sensitive transcriptions factors - Protective mechanism

against oxidative stress associated diseases

Neuroscience Colloquium 2010, Kioloa, Australia

Poster

Tanja Sauer and Gerald Muench

Coffee roasting products as a trigger for the cellular antioxidant defense system in oxidative

stress associated diseases

Annual meeting of the Society for Free Radical Biology and Medicine (SFRBM) 2010,

Orlando, Florida (USA)

Tanja Sauer and Gerald Muench

Coffee roasting products as a trigger for the cellular antioxidant defense system in oxidative

stress associated diseases

Student Conference & Career Day 2010 – School of Medicine, University of Western

Sydney, Australia

Tanja Sauer and Monika Pischetsrieder

Einfluss von Kaffee auf zelluläre antioxidative Mechanismen in Makrophagen

39. Deutscher Lebensmittelchemikertag 2010, Stuttgart, Germany

Tanja Sauer, Gerald Muench and Monika Pischetsrieder

Coffee, coffee-related Maillard products and Alzheimer’s Disease

International Conferences for Alzheimer’s Disease (ICAD) 2010, Honolulu, Hawaii (USA)

Tanja Sauer, Gerald Muench and Monika Pischetsrieder

Biphasic effects of food-derived AGEs

Alzheimer’s and Parkinson’s Disease Symposium on Pathomechanisms in Neuro-

degeneration 2010, Sydney, Australia


Die H2O2-vermittelte, zelluläre Wirkung von Maillard-Produkten auf redox-sensitive

Transkriptionsfaktoren im Modell-System und in Kaffee

38. Deutscher Lebensmittelchemikertag 2009, Berlin, Germany


Isolierung H2O2-produzierender Komponenten aus einer Maillard-Mischung

59. Arbeitstagung LChG-Regionalverband Bayern der GDCh 2008, Munich, Germany

TABLE OF CONTENTS

TABLE OF CONTENTS

1 LITERATURE OVERVIEW ...................................................................................................1

1.1 Coffee .............................................................................................................................1

1.1.1 The coffee bean .......................................................................................................1

1.1.2 Processing of raw coffee beans ...............................................................................2

1.1.2.1 Coffee roasting..............................................................................................2

1.1.3 The chemical composition of roasted coffee beans .................................................3

1.1.4 Brewing methods .....................................................................................................6

1.1.5 Coffee as bio-active beverage .................................................................................8

1.1.5.1 Physiological effects of coffee brew..............................................................8

1.1.5.2 Bio-active components in coffee ...................................................................8

1.2 The Maillard reaction ......................................................................................................9

1.2.1 The Maillard reaction scheme according to Hodge................................................10

1.2.2 Primary effects of food-derived Maillard products..................................................14

1.2.3 Secondary effects of food-derived Maillard products and advanced glycation endproducts (AGE) in vivo ......................................................................................18

1.3 Reactive oxygen species (ROS)...................................................................................23

1.3.1 Endogenous generation of ROS ............................................................................24

1.3.2 Detoxification of cellular ROS ................................................................................24

1.3.3 Impact of ROS on cell components and signalling pathways ................................26

1.3.4 Maillard-dependent generation of ROS in a cell free system.................................27

1.4 The human gastrointestinal tract ..................................................................................29

1.4.1 The histology of the intestine .................................................................................29

1.4.2 Pathology of gut associated diseases....................................................................32

1.4.3 Macrophages - immune associated cells ...............................................................34

1.5 Aims of this study..........................................................................................................36

TABLE OF CONTENTS

2 CHARACTERIZATION OF H2O2 GENERATION IN MAILLARD TEST SYSTEMS...........37

2.1 Introduction ...................................................................................................................37

2.2 Results..........................................................................................................................38

2.2.1 Generation of H2O2 by Maillard products ...............................................................38

2.2.1.1 The role of temperature in the generation of H2O2......................................42

2.2.1.2 The role of pH in the generation of H2O2.....................................................43

2.2.2 De novo generation of H2O2 by Maillard products ..................................................44

2.2.3 Activity-guided fractionation of Maillard products...................................................48

2.2.3.1 Size exclusion chromatography (SEC) .......................................................48

2.2.3.2 Ultra-filtration...............................................................................................51

2.2.4 De novo generation of H2O2 by the active fraction of Maillard products ................52

2.2.5 Generation of H2O2 by coffee extract .....................................................................54

2.3 Discussion ....................................................................................................................56

3 NUCLEAR TRANSLOCATION OF NF-κB .........................................................................63

3.1 Introduction ...................................................................................................................63

3.2 Results..........................................................................................................................65

3.2.1 Cell growth and cell viability of macrophages ........................................................66

3.2.2 NF-κB activation by Maillard products....................................................................68

3.2.2.1 Stimulation of different cell types with Maillard products.............................68

3.2.3 NF-κB activation by coffee extract .........................................................................69

3.2.3.1 Stimulation of different cell types with coffee extract ..................................69

3.2.3.2 Stimulation of macrophages with raw coffee extract...................................72

3.2.4 Ex vivo stimulation of intact human gut tissue .......................................................74

3.2.4.1 Establishing a method for ex vivo stimulation of intact human gut tissue ...74

3.2.4.2 Stimulation of intact human gut tissue with Maillard products and coffee extract .........................................................................................................77

3.3 Discussion ....................................................................................................................78

TABLE OF CONTENTS

4 NUCLEAR TRANSLOCATION OF NRF2 ..........................................................................82

4.1 Introduction ...................................................................................................................82

4.2 Results..........................................................................................................................85

4.2.1 Detection of early, intermediate & late stage Maillard products in Maillard mixtures .................................................................................................85

4.2.2 Nrf2 activation by Maillard products .......................................................................86

4.2.2.1 Stimulation of different cell types and tissue with Maillard products during short-term incubation .......................................................................87

4.2.2.2 Stimulation of macrophages with Maillard products during long-term incubation ........................................................................89

4.2.3 Nrf2 activation by coffee extract.............................................................................93

4.2.3.1 Stimulation of different cell types and tissue with coffee extract during short-term incubation .......................................................................94

4.2.3.2 Stimulation of macrophages with coffee extract during long-term incubation ........................................................................94

4.2.4 Involvement of Maillard-dependent H2O2 in Nrf2 activation ...................................96

4.2.4.1 The role of extracellular H2O2 in Nrf2 activation by Maillard products.........96

4.2.4.2 The role of extracellular H2O2 in Nrf2 activation by coffee extract ............101

4.2.5 The effect of a pure H2O2 solution on Nrf2 translocation .....................................102

4.3 Discussion ..................................................................................................................103

5 EXTRA- AND INTRACELLULAR ROS DURING STIMULATION ...................................109

5.1 Introduction .................................................................................................................109

5.2 Results........................................................................................................................109

5.2.1 Extracellular H2O2 concentration during stimulation of macrophages and human gut tissue.....................................................................109

5.2.1.1 Stimulation of macrophages with Maillard products..................................109

5.2.1.2 Stimulation of macrophages with coffee extract........................................110

5.2.1.3 Stimulation of intact human gut tissue with Maillard products and coffee extract .........................................................112

5.2.2 Intracellular oxidative stress level ........................................................................114

TABLE OF CONTENTS

5.3 Discussion ..................................................................................................................118

6 SUMMARY........................................................................................................................121

7 DEUTSCHE ZUSAMMENFASSUNG ...............................................................................131

8 MATERIALS AND METHODS..........................................................................................142

8.1 Materials .....................................................................................................................142

8.1.1 Instrumentation ....................................................................................................142

8.1.2 Laboratory equipment ..........................................................................................143

8.1.3 Chemicals and reagents ......................................................................................143

8.1.4 Buffers and solutions............................................................................................146

8.1.5 Cell lines and primary cells ..................................................................................150

8.1.6 Primary antibodies ...............................................................................................150

8.1.7 Secondary antibodies...........................................................................................150

8.2 Methods ......................................................................................................................151

8.2.1 Preparation of Maillard reaction mixtures and control solutions...........................151

8.2.2 Preparation of roasted and raw coffee extract .....................................................151

8.2.3 Cell culture ...........................................................................................................152

8.2.4 Tissue culture.......................................................................................................152

8.2.5 Detection of hydrogen peroxide (H2O2) ................................................................153

8.2.6 De novo generation of hydrogen peroxide (H2O2)................................................155

8.2.7 Fractionation of Maillard products via size exclusion chromatography ................155

8.2.8 Fractionation of Maillard products via ultra-filtration.............................................156

8.2.9 Detection of lysine: Ninhydrin assay ....................................................................157

8.2.10 Detection of ribose: Orcinol assay .....................................................................157

8.2.11 Nuclear translocation of Nrf2 and NF-κB ...........................................................158

8.2.12 Nuclear protein content in cells: Dc Protein assay.............................................162

8.2.13 Total protein content in tissue: Bicinchoninic acid (BC) assay...........................163

8.2.14 Oxidative stress level: 2’,7’-Dichlorofluorescein (DCF) assay............................163

TABLE OF CONTENTS

8.2.15 Cytotoxicity of the Maillard reaction mixture and coffee extract .........................164

8.2.16 Cell viability of intact human gut tissue during mucosa oxygenation .................166

8.2.17 Statistical analysis..............................................................................................167

BIBLIOGRAPHY..................................................................................................................168

LIST OF ABBREVIATIONS.................................................................................................187

LIST OF FIGURES...............................................................................................................190

LIST OF TABLES ................................................................................................................192

1 LITERATURE OVERVIEW 1

1 LITERATURE OVERVIEW

1.1 Coffee

Coffee is a popular and worldwide consumed beverage. In 2009, the per capita consumption

in Germany averaged 150 L coffee [1]. The coffee plant (Figure 1.1A), which is grown mainly

in Brazil and Columbia, belongs to the Rubiaceae family. There are about 80 species which

slightly differ in their composition of ingredients and thus their taste and aroma. Coffea

arabica and coffea canephora variant robusta are of most commercial importance with about

75 % and 25 % of world production respectively [2].

1.1.1 The coffee bean

Each coffee cherry (Figure 1.1B) contains two separate coffee seeds (beans). The seed is

surrounded by a spermoderm tissue, the silverskin, and is embedded into the coffee hull, the

endocarp. A layer of mucilage encloses the hull, followed by the pulp, a thick layer of spongy

cells. Reaching maturity the coffee cherry shows an intense dark red colour [2].

A

Figure 1.1: The coffee plant. (A) Rubiaceae – Coffea arabica [3]. (B) A longitudinal cut through the coffee cherry

(modified [2]).

pulp

spermoderm(silver skin)

endocarp

coffee seed

B

pulp


endocarp

coffee seedpulp


endocarp

coffee seed

B


1.1.2 Processing of raw coffee beans

The ripe coffee bean can be processed via two independent approaches, (i) wet or (ii) dry

preparation. (i) During the wet processing, the cherries are pulped mechanically. The so

called pulper squeezes the soft pulp away from the beans. In a following fermentation and

washing step, the slippery mucilage will be removed and the beans will be dried thereafter.

(ii) Alternatively, the coffee cherries can be dried until the pulp can be easily removed by

peeling. Independent of the process, remnant layers will be separated in subsequent

treatments to gain the beans [2].

1.1.2.1 Coffee roasting

In the final step of processing, the raw coffee beans are roasted giving the coffee beans its

characteristic flavour, aroma and colour. During this step, the volume of the coffee beans

increase about 50 - 80 % and the weight decreases about 13 - 20 %. The roasting process

can be subdivided into four stages: (i) dehydration, (ii) development, (iii) disruption and (iv)

full roasting. (i) During the drying step at 50°C, the proteins of the beans coagulate. (ii) With

increasing temperature up to 150°C, the beans swell due to the decomposition of organic

compounds leading to the concomitant formation of gases such as carbon dioxide and water

vapor. Simultaneously, a toasty aroma arises. (iii) The first crack of the beans starts at

temperatures between 180 - 200°C. The beans get a light brown colour and the specific

coffee flavour develops. (iv) The last step is mainly marked by further caramelisation of the

sugars until the second crack arises between 225 - 230°C. [2], [4]

The browning and the fragrance of roasted coffee beans are attributed to (i) the caramel-

isation of sugars, (ii) the Maillard reaction, which occurs between reactive carbonyl species

and amino groups, and (iii) the degradation of compounds present in the raw coffee bean

such as chlorogenic acid. Thereby, coloured polymeric melanoidins and characteristic coffee

odorants such as 2-furfurylthiol, methional, furanones and alkylpyrazines are formed [4].


1.1.3 The chemical composition of roasted coffee beans

The changes in the composition of coffee bean ingredients, which occur during the roasting,

are illustrated in Table 1.1.

Table 1.1: The composition of green and roasted coffee beans (Coffea arabica) in % dry weight. (Data from [5]).

0.1tracesVolatile aromatic compounds

00.5Amino acids

25.4-Caramelization products

1.31.2Caffeine

1.0*1.0Trigonelline

0.80.4Quinic acid

2.56.5Chlorogenic acid

1716.2Lipids

08.0Sucrose

0.31.1Other sugars

38.049.8Polysaccharides

1.61.1Aliphatic acids

4.54.2Minerals

7.59.8Proteins

Roasted coffee beans[%] dry weight

Green coffee beans [%] dry weightConstituent

0.1tracesVolatile aromatic compounds

00.5Amino acids

25.4-Caramelization products

1.31.2Caffeine

1.0*1.0Trigonelline

0.80.4Quinic acid

2.56.5Chlorogenic acid

1716.2Lipids

08.0Sucrose

0.31.1Other sugars

38.049.8Polysaccharides

1.61.1Aliphatic acids

4.54.2Minerals

7.59.8Proteins

Roasted coffee beans[%] dry weight

Green coffee beans [%] dry weightConstituent

* Inclusive decomposition products from roasting

As shown in Table 1.1, the total amounts of amino acids, which were determined as acid

hydrolyzates, and carbohydrates are decreased in roasted coffee beans compared to the

amount in raw coffee beans. This drop of both substance classes is traced back amongst

others to the Maillard reaction which takes place between carbohydrates and amino acids

causing a decrease in their concentration and the formation of Maillard products. The loss of

carbohydrates is secondary due to caramelisation and fragmentation to low molecular weight

products such as aliphatic acids. [4] The residual carbohydrates in roasted coffee beans are

mainly insoluble polysaccharides of mannose, galactose and arabinose or soluble fragments


of those polysaccharides. Whereas the concentration of proteins and carbohydrates

decrease significantly during roasting, the amount of lipids and caffeine remains mostly

constant. However, the diterpenoid lipids cafestol and kahweol partly degrade [2]. Trigonellin

(1) is another alkaloid in coffee beans besides caffeine which on the contrary to caffeine,

largely disappears [4]. Trigonellin (1) degrades into N-methylpyridinium (NMP) (2) or nicotinic

acid (3) (Figure 1.2) and its esters depending on whether a decarboxylation or demethylation

of trigonellin occurs [6].

N+

CH3

O

OH

N+

CH3 N

O

OH

Trigonellin (1)

Decarboxylation Demethylation

N-Methylpyridinium (NMP) (2)

Nicotinic acid (3)

Figure 1.2: Proposed thermal degradation of trigonellin (1) into N-methylpyridinium (NMP) (2) by decarboxylation

or into nicotinic acid (3) by demethylation [6].

The last parameter which varies crucially between green and roasted coffee beans is the

concentration of the chlorogenic acids.


OH

OH O

OOH

COOH

OHOH

OH

OH OHOH

COOH

OH

O

OHOH

OHOH

OH

OHOH

OH

OHOH

OHOH

OHOH

5-O-Caffeoylquinic acid (Chlorogenic acid) (4)

Quinic acid (5)

Caffeic acid (6)

Pyrogallol (7)

Hydroxyhydroquinone (8)

Catechol (9)

4-Ethylcatechol (10)

4-Methylcatechol (11)

Figure 1.3: Proposed thermal degradation of 5-O-caffeoylquinic acid (chlorogenic acid) (4) into quinic acid (5) and

caffeic acid (6) which further degenerate into pyrogallol (7), hydroxyhydroquinone (8), catechol (9), 4-

ethylcatechol (10) and 4-methylcatechol (11) (modified [7]).

Chlorogenic acids are summarized as the esters between trans-cinnamic acids and quinic

acid [9]. 5-O-caffeoylquinic acid is the quantitatively predominating chlorogenic acid


composed of caffeic acid and quinic acid. Due to thermal degradation of 5-O-caffeoylquinic

(4), the precursors quinic acid (5) and caffeic acid (6) are formed which further degrade into

several roasting products amongst others pyrogallol (7), hydroxyhydroquinone (8), catechol

(9), 4-ethylcatechol (10) and 4-methylcatechol (11) (Figure 1.3) [7]. The amount of chloro-

genic acid decreased about 60 % after mild and almost 100 % after severe roasting [8].

1.1.4 Brewing methods

The roasting process and the brewing method crucially influence the composition of

compounds in the coffee beverage. Different brewing methods exist which can be

categorized into decoction, filtration or pressure methods.

The decoction preparation describes brewing methods in which water is added to the coffee

powder at a given temperature for an appropriate time. (i) For a standard boiled coffee, water

is added to the coffee powder and heated up to the boiling point. The insoluble coffee

powder is separated by decantation. (ii) Another alternative is the use of a Percolator, a

continuous refluxing machine. Water is heated up until it boils. The steam impels water up-

wards through a filter with ground coffee powder from where it trickles back into the base

vessel. This process is repeated over and over again. (iii) In case of a turkish coffee, coffee

beans are grinded to a very fine powder and boiled three times in water. Finally, the coffee

extract is poured gently into a cup while avoiding the insoluble coffee fraction to enter.

The filtration (infusion) methods, on the contrary, allow the contact of water and coffee

powder just for a short time. (i) During filtered coffee preparation, for example, coffee powder

is put into a filter and boiled water is then poured into the filter and allowed to seep through.

(ii) Napoletana coffee is similar to the filtered coffee. The only difference is that the coffee

powder is immobilised in the napoletana version between two filtering plates. Thus, the

coffee granules can not move and do not swim in the water as in the case of filtered coffee.

In the last category, the pressure preparation, boiling water is added to the coffee powder. (i)


In the French press, the insoluble coffee powder is separated after a couple of minutes from

the coffee beverages on the bottom of a beaker by pushing down a mesh filter. (ii) Espresso

is also gained by the pressure method; hot but not boiling water is pressured through the

coffee powder. As a consequence of the pressure, the total amount of extracted solids is

crucially higher in espresso than other coffee preparations. [4] (iii) Likewise, Moka is

prepared by pressing water through the coffee powder but with lower pressure as in the case

of espresso. Table 1.2 summarizes the total solids in coffee beverages according to their

brewing method.

Table 1.2: The amount of total solids in coffee brews prepared by different brewing methods [4].

13.0Filter

26.9Napoletana

14.2French press

41.1Moka

52.5Espresso

10.9Percolator

13.0Boiled

Total solids in brew [g/L]Brewing method

13.0Filter

26.9Napoletana

14.2French press

41.1Moka

52.5Espresso

10.9Percolator

13.0Boiled

Total solids in brew [g/L]Brewing method

The discrepancy in dry weight in different coffee preparations is associated with the varying

extraction time, water temperature and the use of a filter. The concentration of acids, sugars,

caffeine and lipids are restrained by a filter. [4] Unfiltered coffee contains between 0.2 and

18 mg/100 mL diterpenoid lipids, cafestol and kahweol, whereas in filtered coffee less than

0.1 mg/100 mL diterpenes were observed [10], [11]. Likewise, the amount of Maillard

products varies within the different coffee preparations used worldwide. In detail, coffee may

contain between 1.60 and 13.60 kU/100 mL Maillard products (measured as Nε-

(carboxymethyl)lysine (CML), a marker for the Maillard reaction) depending on the coffee


processing [12].

Brewed coffee is a complex mixture of hundreds of phytochemicals differing in their yield and

type amongst others due to the plant specie, processing and brewing method. Indeed, the

composition of the coffee brew is of certain interest since those phytochemicals trigger many

physiological effects in the human body after coffee consumption.

1.1.5 Coffee as bio-active beverage

1.1.5.1 Physiological effects of coffee brew

Besides the known short-term effects of coffee such as its stimulating activity,

epidemiological studies also show a long-term effect of coffee on human health. The intake

of coffee was associated with a reduced risk of Alzheimer’s disease [13], atherosclerosis

[14], Parkinson’s disease [15], type 2 diabetes mellitus [16] and cancer amongst others in the

brain, liver and colon [17-19]; diseases which are associated with inflammation and oxidative

stress [20-23]. To date, the underlying mechanisms are still discussed and mostly can not be

related to a specific compound or group of structures in coffee.

Indeed, in vitro and animal model investigations of coffee brew demonstrated that coffee

influences the nuclear factor-κB (NF-κB) [24-26] and the nuclear factor-erythroid-2-related

factor 2 (Nrf2) [24], [27], [28] pathway. The redox-sensitive transcription factor NF-κB is a cell

signalling biomarker for inflammation [29], [30] and regulates the expression of immuno-

logically relevant proteins such as cytokines [31]. On the other hand, Nrf2 regulates the

transcription of proteins with detoxification and antioxidant capacities which counteract the

unbalanced redox homeostasis during oxidative stress for example in age-related diseases.

1.1.5.2 Bio-active components in coffee

Indeed, numerous bioactive compounds have been identified in coffee. Caffeine is a natural

alkaloid which is probably the most investigated compound in coffee. Its well-known


stimulating effect on the central nervous system is traced back to the mobilization of

intracellular calcium, inhibition of specific phosphodiesterases and most important to the

antagonism to the adenosine receptor [32]. Besides this short-term effect, caffeine is recently

associated with a neuroprotective effect in a mouse model of Parkinson’s disease [33] as

well as a reduced risk of stroke and Alzheimer’s disease after long-term exposure [34]. The

diterpenoid lipids cafestol and kahweol, on the other hand, have been shown to enhance the

risk of cardiovascular diseases by increasing the serum cholesterol level [35]. This fact is not

of relevance for filtered coffee in which the amount of the diterpenes is insignificant.

Moreover, coffee contains bio-active polyphenols such as chlorogenic acid, an ester of

caffeic acid with quinic acid. Both, chlorogenic acid and caffeic acid possess antioxidative

activity [4], [36]. During coffee roasting, the amount of chlorogenic acid and caffeic acid

decreases due to destruction and deformation, but the antioxidative activity of coffee does

not attenuate with the roasting degree as measured by the oxygen consumption, chain

breaking activity and radical scavenging activity. [37] It was shown that melanoidins, the

brown Maillard products which are formed during the coffee roasting, also exert antioxidative

activity. [38] Thus, the antioxidative activity of coffee was attributed to both, phenolic

compounds and Maillard products. Compared to other polyphenol-rich beverages such as

tea, the antioxidative activity of coffee is significantly higher [39]; coffee was ranked sixth in

the amount of total antioxidants among 1113 foods in the US. [40]

1.2 The Maillard reaction

The roasting of raw coffee beans is an important step in the processing of coffee beans in

which the coffee-specific aroma, flavour and colour develop. In this context, the Maillard

reaction plays an important role. Besides aroma and colour, Maillard products are also

physiologically active showing beneficial or/and adverse health effects such as antioxidant

and mutagenic properties [41], [42].


1.2.1 The Maillard reaction scheme according to Hodge

Maillard products are formed during heat-treatment such as the roasting by non-enzymatic

reactions between amino groups and carbohydrates. Besides carbohydrates, phenolic

compounds might contribute to the formation of Maillard products as shown in coffee [43].

The Maillard reaction was named after the French scientist Louis Camille Maillard who

described a browning in a heated solution of glycine and glucose. It’s not a single but a

complex cascade of reactions forming low to high molecular weight products. The Maillard

products are classified into early, intermediate and late stage Maillard products according to

the three stages of the development pursuant to Hodge (Figure 1.4):

(i) Initial/Early stage (colourless)

(ii) Intermediate stage (colourless or yellow)

(iii) Late/Advanced stage (highly coloured)

carbonyl + amino

Amadori product Initial stage (i)

furfuralreductone-structure

dicarbonyls aldehydes Intermediate stage (ii)

Late stage (iii)Melanoidins(brown nitrogenous polymers)

carbonyl + amino

Amadori product Initial stage (i)

furfuralreductone-structure

dicarbonyls aldehydes Intermediate stage (ii)

Late stage (iii)Melanoidins(brown nitrogenous polymers)

Figure 1.4: The Maillard reaction scheme according to Hodge subdivided in initial, intermediate & final stage [44].


(i) Initial stage: • Carbonyl-amine condensation

• Amadori rearrangement

The initial (early) stage describes a condensation between reactive carbonyls and amines.

The amino group of amino acids, peptides or proteins, for example, react in a nucleophilic

attack with the carbonyl group of e.g. reducing sugars. A Schiff base (14) is formed under the

abstraction of water (Figure 1.5). The Schiff base is relatively unstable and rearranges

spontaneously to the Amadori product (15). [44]

NH

O

NH2

proteinprotein

O

OH

OHOH

OH

OH

OHOH

OH

Nprotein

OH

OOH

OH

Nprotein

L-Lysine-protein (12)

D-Ribose (13)

Schiff base (14)

Amadori product (15)

Amadori rearrangement

- H2O

Figure 1.5: Initial stage of the Maillard reaction according to Hodge. The free amino group of the protein (L-Lysine-

protein) (12) reacts non-enzymatically with the carbonyl group of the reducing sugar (D-Ribose) (13) forming an

unstable Schiff base (14) under abstraction of water. The Schiff base rearranges spontaneously to the Amadori

product (15) (modified [44]).


(ii) Intermediate stage • Dehydration of sugar moiety

• Fragmentation of sugar moiety

• Strecker degradation of amino acid moiety

In the intermediate stage, the Amadori product undergoes further dehydration of the sugar

moiety forming furfurals, reductones and aminoreductones. It was already shown that

reductones are a source of browning. Moreover, a retro-aldol reaction of the sugar moiety of

the Amadori product can occur to form triose and other fragmentation products such as α-

dicarbonyls. On the other hand, also the α-amino acid moiety can fragment through the

strecker degradation. As a result, aldehydes and carbon dioxide are formed. Both, fission

products of the sugars and the amino acid moiety are a source of browning. [44] Figure 1.6

illustrates Maillard products with dicarbonyl structures (3-deoxy-D-erythro-hexos-2-ulose (3-

DG) (16)) or aminoreductone / reductone (3-hydroxy-4-(morpholino)-3-buten-2-on (C4-AR)

(17) / 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-on (DDMP) (18)) as examples of

intermediate Maillard products.

OH

O OH

OH

O

O

OHO

OH

CH3

ON

OH

CH3

O

3-Deoxy-D-erythro-hexos-2-ulose (3-DG) (16)

2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-on (DDMP) (18)

3-Hydroxy-4-(morpholino)-3-buten-2-on (C4-AR) (17)

Figure 1.6: The chemical structures of three intermediate Maillard products are illustrated with their characteristic


group highlighted. 3-Deoxy-D-erythro-hexos-2-ulose (3-DG) (16) with dicarbonyl structure, 3-hydroxy-4-

(morpholino)-3-buten-2-on (C4-AR) (17) with aminoreductone structure and 2,3-dihydro-3,5-dihydroxy-6-methyl-

4H-pyran-4-on (DDMP) (18) with reductone structure.

(iii) Final stage • Aldol condensation

• Aldehyde-amine polymerization

The final (advanced) stage summarizes the formation of unsaturated, fluorescent and brown-

coloured polymers of the intermediates called melanoidins. One main reaction is the aldol

condensation between aldehydes which are generated in the former stages. Thus,

depending on the aldehydes, nitrogen-free and nitrogen containing melanoidins are formed.

However, nitrogen-free aldols might further react with amines forming nitrogenous

melanoidins. Besides the aldol condensation, it is suggested that aldehydes react with

amines and aldimines generating unsaturated conjugated aldimines and dihydropyridines.

Furthermore, pyrroles, imidazoles and other nitrogenous heterocyclic compounds are found

in model browning mixtures. Even though the structures of the melanoidins are rather

heterogeneous, melanoidins are usually negatively charged. [44]

It is not easy to summarize the products generated by the Maillard reaction since a broad

spectrum of heterogeneous Maillard products exists whose composition is crucially

influenced by the reaction conditions. The course of the Maillard reaction is directed by

various parameters showing the highest reaction rate with increasing temperature, time, pH

value and decreasing water activity. [45], [46] Also, the type and concentration of the

reactants regulate the extent and course of the Maillard reaction. Moreover, it was reported

that depending on the particular food matrix structures others than sugars and amines can

be incorporated into melanoidins. In coffee for instance, polysaccharides, chlorogenic acid

and its subunits quinic acid and caffeic acid are incorporated into melanoidins [43], [47].

Besides the Maillard reaction, two further types of browning reactions exist: the


caramelisation and oxidation of sugars. However, the browning reaction is highly accelerated

in the presence of amino acids emphasizing the importance of the Maillard reaction in

browning of food. [44]

1.2.2 Primary effects of food-derived Maillard products

The Maillard reaction does not only take place during roasting but also during baking, frying,

drying, pasteurization and sterilization of food; processes in which enhanced temperatures

are applied to prepare food. Thus, Maillard products are found in various foods such as

coffee, bakery products (cookies, bread, cakes etc), roasted malt, soy bean, beer, potato

fries, potato chips, popcorn and meat. The amount of Maillard products, measured as CML,

in raw and processed foods respectively is illustrated in Table 1.3.

Table 1.3: Summary of literature reports of CML content of various foods [48].

9.3 Raw milk

40.5Wholemeal bread crump

329Wholemeal bread crust

6 - 8Cornflakes

343Sterilized milk

0 - 1015Evaporated milk

5 - 35Cookies

29 - 259UHT milk

16Pasteurised milk

CML [mg/kg protein]Food

9.3 Raw milk

40.5Wholemeal bread crump

329Wholemeal bread crust

6 - 8Cornflakes

343Sterilized milk

0 - 1015Evaporated milk

5 - 35Cookies

29 - 259UHT milk

16Pasteurised milk

CML [mg/kg protein]Food

A regular meal contains 1570 ± 7.1 mg CML/kg protein. However, the total amount of

Maillard products in a meal can be reduced by low-heat processing reaching a concentration


of 530 ± 5.7 mg CML/kg protein. Since Maillard products are largely consumed with heat-

treated foods and beverages, several studies focused on the impact of Maillard products on

the food itself and on human health. To date, Maillard products can be characterized by

various functional properties:

(i) Fragrance, taste & colour

(ii) Antimicrobial activity

(iii) Mutagenic/carcinogenic & antimutagenic potential

(iv) Antioxidant & prooxidant ability

(v) Others

(i) Fragrance, taste & colour

On the contrary to flavonoids and carotenoids, which are a class of plant secondary meta-

bolites and responsible for the colour in fruits and vegetables [2], Maillard products are rather

known for giving particularly heated food a specific colour. Besides colour, Maillard products

are responsible for fragrance and taste in heat treated food. The melanoidins, for example,

are heterogeneous polymers giving processed food its brown colour [44]. Depending on the

food, colour- and aroma-specific Maillard products may or may not be desired. In coffee, for

example, the Maillard product 2-furfurylthiol is one of the key odorants giving coffee its

specific roasty aroma [49]. On the other hand, off-flavours might emerge due to the Maillard

reaction such as 2-methylpropanal and 3-methylbutanal in ultra heat-treated milk [50].

(ii) Antimicrobial activity

Einarsson et al reported that sugar-amino acid mixtures possess an antimicrobial activity

against Bacillus subtilis, Staphylococcus aureus and Escherichia coli; bacteria which cause

food spoilage and are pathogenic for humans. The antibacterial activity depends on the


sugar-amino acid combination and the type of bacteria. In the study of Einarsson et al,

incubation with especially the high molecular weight fraction of the mixtures inhibited

bacterial growth. [51] To date, Maillard products with aminoreductone structure are identified

to be antimicrobial active against Helicobacter pylori [52]. The hypothesis of Maillard

products as antimicrobial components was further underlined by studies with Maillard-rich

foods such as coffee and biscuits. Both foods had an antibacterial effect on several

microorganisms such as Staphylococcus aureus and Escherichia coli [53], [54]. Moreover,

the antibacterial activity increased further with the degree of coffee roasting emphasizing the

Maillard products as potent key players [53]. The mechanisms by which Maillard products

might be antibacterial active are not fully understood. It was demonstrated that coffee

melanoidins chelate essential metals such as iron and Mg2+. [55] Moreover, the cell

membrane is disrupted after incubation of Escherichia coli with coffee and biscuit

melanoidins which is possibly linked to the Mg2+ chelating [56].

(iii) Mutagenic/carcinogenic & antimutagenic potential

Depending on the reactants, Maillard products are also mutagenic in various bacterial

strains. Arginine- and lysine-glucose mixtures showed a strong mutagenic activity on

Salmonella typhimurium [57] whereas mixtures with glycine, proline and cysteine had no

detectable mutagenicity [57], [58]. The Maillard structures 2-amino-3,8- dimethylimidazo [4,5-

f]quinoxaline [59] and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine [60], which are

formed during cooking in meat, have already been identified as mutagens. One of the most

studied mutagenic Maillard products is acrylamide, which is classified as ‘probably

carcinogenic to humans’ by the International Agency for Research on Cancer (IARC

Monographs on the Evaluation of Carcinogenic Risks to Humans; Vol. 60). Increased

acrylamide levels were found in various heat-treated foods such as coffee, potato chips and

potato fries [61]. Moreover, it was demonstrated that the intake of heat treated food


increased the in vivo level of acrylamide, as measured as haemoglobin adduct in blood of

diet fed rats [62].

On the contrary, dichlormethane extracts from various Maillard mixtures were rather

antimutagenic. It was suggested that the Maillard mixtures are antimutagenic by inhibiting

cytochrome P-450 isozymes [63]. In a study of Wagner et al, Maillard mixtures possessed

mutagenic and/or antimutagenic activity in dependence on the concentration and the

experimental conditions. A glucose-cysteine mixture, for example, was antimutagenic active

against H2O2 induced mutations. The antimutagenic activity was associated with an

antioxidant potential to scavenge reactive oxygen species such as hydrogen peroxide (H2O2).

[64]

(iv) Antioxidant & prooxidant ability

The antioxidant properties of Maillard products were extensively studied in various sugar-

amino acid mixtures. It was observed that Maillard products have antioxidative activity by

chelating metals and scavenging oxygen/peroxyl radicals [65], [66]. The affinity to scavenge

free radicals varied with the reactant showing the highest effect in a ribose-lysine mixture. It

was further demonstrated that most notably the Maillard products with high molecular weight

(HMW; ethanol insoluble) are antioxidative active in in vitro scavenging assays [67]. As

active components, Maillard products with (amino)reductone or furanone structure were

identified [65], [68], [69]. The Maillard-dependent antioxidative activity was not only studied in

sugar-amino acid model systems but also in Maillard-rich foods. Coffee [37], [38], [70], bread

crust [68] and soy bean sauce [71] were antioxidative active as measured by their radical

scavenging activity. The antioxidative potential was traced back to the Maillard products

since the dark brown pigments possessed the highest activity [37], [38], [68], [70], [71].

It is well known that dietary antioxidants inhibit lipid oxidation in food and thus prevent food

spoilage and prolong their shelf life. Moreover after consumption, dietary antioxidants


prevent the oxidation of low density lipoproteins (LDL) in the human body which is associated

with a reduced risk of cardiovascular diseases [72]. Even though the involved mechanism is

not fully clarified, it was hypothesized that the Maillard products in antioxidative-rich diets

account at least in part to the reduced level of oxidized LDL. Antioxidative Maillard products

further protect cells from cytotoxic radicals and H2O2 by their scavenging ability [67].

On the contrary, Maillard products do not only scavenge but also generate H2O2 [25], [73].

This pro-oxidative effect was demonstrated for Maillard mixtures of glucose-, lactose- and

ribose-lysine solutions [73]. Furthermore, the formation of H2O2 was determined in coffee but

not in raw coffee [25], [73]. As H2O2 generating Maillard structures enediols [74], enaminols

[74], which might tautomerize from Amadori products and dicarbonyls, and

(amino)reductones [69] were already postulated.

(v) Others

Besides the formation of new compounds, the modification of the initial compounds due to

the Maillard reaction needs to be considered as well. Amino acids, for example, can be

crosslinked by the Maillard reaction causing a reduced digestibility. Thus, the bioavailability

of essential amino acids is diminished impairing the nutritional value of the foods. This side-

effect is of special importance for the heat-treatment of milk during which the amount of

bioavailable lysine gets reduced. [75]

1.2.3 Secondary effects of food-derived Maillard products and advanced

glycation endproducts (AGE) in vivo

In the 1950s, it was already demonstrated in sugar-amino-model systems that the Maillard

reaction does not only take place at enhanced temperatures but also at 37°C, raising the

question of its relevance in vivo [44]. To date, it is well known that the Maillard reaction,

indeed, occurs in the human body. Intra- and extracellular reactive carbonyl groups react


with amino groups forming the so-called advanced glycation endproducts (AGEs). Thus in

the general terminology, AGEs are generated by an endogenous source in vivo whereas

Maillard products are formed exogenously particularly in foods and tobacco smoke.

The formation and accumulation of AGEs inside the human body is favoured in various

diseases. Indeed, endogenous AGE levels are elevated in patients suffering from diabetes

mellitus type 2 [76], [77], uraemia [78], liver cirrhosis [79] as well as coronary heart disease

[77] and neurodegenerative diseases such as Alzheimer’s disease [80]. Moreover, increased

levels of AGEs were found in elderly persons [81] due to the normal process of aging. The

enhanced formation and accumulation of AGEs in diseased and/or elderly people can be

traced back to hyperglycemia and thus increased levels of AGE precursors (diabetes mellitus

type 2) [77], impaired elimination of AGEs and AGE precursors (uraemia, liver cirrhosis) [79],

and prolonged reaction time (aging) [81]. These enhanced levels of AGEs are associated

with the pathogenese of coronary heart disease [77] and Alzheimer’s disease [80]. Indeed,

AGEs can affect the pathogenese of those diseases in two different ways.

Firstly, endogenous proteins can be structurally modified due to AGE formation. The

structural modifications of the proteins can alter the proteins’ functionality which may trigger a

hindered etiopathology of those diseases [76]. In this context, long-living proteins such as

collagen can be glycated and therewith irreversible cross-links are formed [82]. The

formation of collagenous cross-links increases the stiffness of the collagen network which

may trigger pathophysiological complications such as osteoarthritis [83]. Besides collagen,

other proteins such as albumin and hemoglobin are found to be glycated [84]. Not only

proteins, but also amino groups of cellular lipids and nucleic acids can be modified by

reactive carbonyls. Phosphatidylethanolamine, for instance, is a membranous lipid which can

be glycated by D-glucose [85]. The glycation causes structural changes of the membranous

lipids and proteins respectively which as a result may affect the membranes’ functionality by

e.g. inactivating membrane receptors/enzymes [86]. In the case of deoxyribonucleic acid


(DNA) nucleosides, deoxyguanosine, deoxyadenosine and deoxycytidine were significantly

glycated by glyceraldehyde [87]. Glycated DNA can lead to depurination [88] and other DNA

modifications such as single-stranded regions and destabilized hydrogen bonds [89].

Secondly, AGEs can activate cells by specific triggering signalling pathways inside the cell.

These responses are thought to be mediated via interaction with specific receptors including

RAGE, OST-48 (AGE-R1), 80K-H (AGE-R2) and galectin (AGE-R3). Moreover, AGEs are

actively absorbed via endocytosis by macrophage scavenger receptors class A and B.

(reviewed in [90], [91]) Indeed, it was demonstrated that the specific AGE structure CML

(19) (Figure 1.7), which is one of the best characterized AGE, binds RAGE [92]. RAGE

ligation by CML was found in human umbilical vein endothelial cells, vascular smooth muscle

cells and mononuclear phagocytes [92] but not in lung epithelial cells [93].

NH

O

OHlysine

Nε-(Carboxymethyl)lysine (19)

Figure 1.7: The chemical structure of Nε-(carboxymethyl)lysine (CML) (19).

Interactions of AGEs with the abovementioned receptors can provoke several intracellular

responses involving the activation of the following mitogen-activated protein kinases

(MAPKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38 kinases, c-jun NH2-

terminal kinases (JNK) [94] and p21ras [95]. The downstream cascade can follow various

pathways. In fibroblasts, for instance, the nuclear factor-κB (NF-κB) pathway was activated

by AGEs [94]. Depending on the signalling pathway, the expression of different cytokines

and growth factors is enhanced. Exemplarily, an increased expression of the vascular cell


adhesion molecule-1 (VCAM-1) was found in vitro in human umbilical vein endothelial cells

[92] and adventitial fibroblasts [96], and in vivo in mice [92]. Furthermore, the expression of

tissue factor on monocytes and monocyte-like cells was enhanced after stimulation with

AGEs [97]. In this context, it was also demonstrated that after exposure to AGEs, monocytes

highly migrate across an intact endothelial cell monolayer and also trigger the expression of

platelet-derived growth factors [98]. Moreover, the expression of key mediators in the

inflammatory response such as interleukin-1/6/8 (IL-1/IL-6/IL-8), tumour necrosis factor α

(TNF-α) and monocyte chemotactic protein-1 (MCP-1) is activated in adventitial fibroblasts

[96], in monocytic leukemia cells [99], in macrophages [100] and in osteoarthritis

chrondocytes [101]. However in mast cells, cytokine expression remained unchanged after

AGE stimulation compared to untreated mast cells. At the same time, intracellular production

of reactive oxygen species (ROS) was enhanced. [102] Increased production of ROS was

also detected in endothelial progenitor cells after AGE stimulation [103]. This ROS

generation might be at least in part due to an up-regulation of the nicotinamide adenine

dinucleotide phosphate (NADPH) oxidase as shown for glycated albumin [104]. Not only

reactive oxygen species but also reactive nitrogen species (RNS) such as nitrite oxide are

generated via the inducible nitrite oxide synthase (iNOS) after stimulation with AGEs as

shown in glomerular mesangial cells [105]. Interestingly, AGE-RAGE interactions also up-

regulate the expression of RAGE itself which initiate a positive feedback loop [103], [106].

Taken together, endogenous AGEs can provoke several signalling pathways through the

interaction with various cell surface receptors. Thus, depending on the AGEs’ structure and

the cell type, AGEs individually modulate the expression of cytokines, growth factors and

adhesions molecules and the production of ROS/RNS. Unfortunately, a prognosis of the

cellular signalling, triggered by specific endogenous AGE, is impeded due to the

heterogeneous nature of the AGEs.

Maillard products, which are food-derived AGE analogues, possess a complex and unstable


nature likewise. Thus, no prediction can be made about the impact of the daily influx of

dietary Maillard products on cellular activation and thus human health. On the top of this

comes the fact that only little is known about the intestinal digestion, uptake and metabolism

of the broad range of food-derived Maillard products since the chemical structure of most

food-derived Maillard products still remains unknown. In a human study using an ovalbumin-

fructose rich Maillard diet, increased serum and urine levels of Maillard products were

determined compared to a diet low in Maillard products. [107] Faist and Erbersdobler

summarized the results of various studies in rats and humans on the absorption and

excretion of Maillard products, differentiated in early and advanced Maillard products. Free

and protein-bound Amadori compounds (early Maillard products) were excreted to about

60 – 80 % and 3 –10 % in the urine. In the case of advanced Maillard products, the excretion

in urine was 16 – 30 % for the low molecular weight (LMW) melanoidins and 1 – 5 % for high

molecular weight (HMW) melanoidins. [108] These results indicate that food-derived Maillard

products are at least partially absorbed by the intestine with an enhanced absorption for low

molecular weight products compared to high molecular weight products. In another study, the

bioavailability of CML, a specific Maillard product, was analysed in rats fed CML containing

diets. The results indicated that the urinary level of CML correlated with the CML amount of

the diets. [109] This result was underlined by a human study using a standard western diet

(rich in Maillard products) compared to a steam diet (low in Maillard products). The CML

levels in urine and blood were higher in the standard western diet after four weeks [110].

Moreover, accumulation of CML and Nε-(carboxyethyl)lysine (CEL) were determined in the

liver of rats after intravenous injection into a rat tail [111]. Besides CML, the biodistribution of

other Maillard products such as pyrraline and free pentosidine were analysed in healthy

volunteers who received test meals of pretzel sticks, brewed coffee or custard. After 24 h, 50

and 60 % of pyrraline and free pentosidine respectively were determined in the urine [112].

In conclusion, it can be summarized that dietary Maillard products are absorbed into blood


and to some extent excreted via the urinary system. In patients with renal failure, the amount

of Maillard products in the serum increases with the severity of the kidney disease [107]

indicating major role of the kidney in the elimination of circulating Maillard products. Since

dietary Maillard products demonstrably increased the in vivo pool of AGEs, the question was

raised whether or not dietary Maillard products may provoke similar cellular signalling as

endogenous AGEs. Indeed, human studies in diabetes patients indicated that Maillard rich

diets increase levels of inflammatory serum markers such as VCAM-1, mononuclear cell

TNF-α and oxidative stress. [113-115] Recently, a study was published highlighting the

impact of Maillard rich diets not on diabetic but on healthy humans [110]. Besides the effect

determined in blood serum and tissue, dietary Maillard products also activated endothelial

cells which form the inner layer of blood vessels which are found e.g. in the intestine [116]. In

this context, it was already demonstrated that human microvascular endothelial cells and

intestinal epithelial cells interact via cytokine signalling [117]. Thus, it can be assumed that

dietary Maillard products trigger not only a systemic effect in the blood serum and the tissue

but might also induce a local effect in the intestine.

1.3 Reactive oxygen species (ROS)

In general, the term ‘reactive oxygen species (ROS)’ encompasses several radical and non-

radical oxygen derivates such as superoxide anion radical (O2• -), hydrogen peroxide (H2O2),

hydroxyl radical (OH•), perhydroxyl radical (HOO•) and lipid peroxides. Nitric oxide (NO) and

peroxynitrite (ONOO-) can also be classified as reactive nitrogen species (RNS) (reviewed in

[118]).

These oxygen derivates are crucially reactive due to their electron configuration [119]. The

reactivity of the individual species slightly differs showing the shortest half-life of 10-9 sec and

thus the highest reactivity for OH• [120]. However, certain attention needs to be paid to all

species of reactive oxygen since ROS can inter-convert (Figure 1.8). Briefly, O2• -, which is


generated as a byproduct in the mitochondrial respiratory chain due to oxygen consumption,

can dismutate spontaneously or enzymatically via superoxide dismutase (SOD) into H2O2.

H2O2 in turn can be degenerated in the presence of iron and/or other metals into the highly

active OH• (Fenton reaction (1.2)). [119]

O2 (triplet)

↑ ↑

e-O2

• -

↓↑ ↑

e-/2H+H2O2

↑↓ ↑↓

Fe3+ + OH• + OH-

2pπ*

H2O2 + Fe2+

(1.1)spontaneous/SOD

(1.2)

Figure 1.8: (1.1) Activation of molecular oxygen (O2) to superoxide anion radical (O2• -) and dismutation to

hydrogen peroxide (H2O2). (1.2) H2O2 degenerates in the presence of ferrous ions via Fenton to hydroxyl radicals

(OH•).

1.3.1 Endogenous generation of ROS

Several metabolic reactions such as the mitochondrial respiratory chain proceed in aerobic

conditions under the participation of molecular oxygen. Thereby, ROS are formed, mostly as

undesirable byproducts. Besides the respiratory chain, other biological processes involving,

for instance, the xanthine oxidase, cytochrome P450 dependent monooxygenase and

NADPH oxidase systems are considered as endogenous sources of ROS. In addition,

soluble cellular compounds such as flavin and catecholamines activate oxygen during

autoxidation processes. [119]

1.3.2 Detoxification of cellular ROS

In order to maintain a steady state formation of oxidants, the cell exerts an antioxidative

defence mechanism counteracting an enhanced level of intracellular ROS. Enzymes such as

SOD, catalase and glutathione peroxidase scavenge different kinds of ROS (Figure 1.9). In


the cytoplasm and the mitochondria, SOD degrades O2• - into H2O2. H2O2 is a small molecule

which can easily diffuse through cell membranes and thus may migrate out of the

mitochondria into different compartments of the cell. H2O2 can be detoxified to water and

molecular oxygen by catalase and glutathione peroxidase. Glutathione peroxidase is located

in the mitochondria whereas catalase is for the most parts in the peroxisomes. Thus,

depending on the location of H2O2 different enzymes perform. The catalytic activity of

glutathione peroxidase is, unlike SOD and catalase, limited by the cellular supply of

glutathione (GSH) as reducing co-factor. The oxidized co-factor gets recycled by glutathione

reductase thereafter. [119] Besides its function as a co-factor, glutathione may also

scavenge radicals directly. In addition to the scavenging enzymes, further non-enzymatic

antioxidants such as α-tocopherol, ascorbic acid and uric acid are involved in the

detoxification of ROS. [119]

2O2• - + 2H+ H2O2SOD Cat

O2

1/2 O2 + H2O

GPx

2 H2O

2 GSH

GSSG

NADPH + H+

NADP+

GR

2O2• - + 2H+ H2O2SOD Cat

O2

1/2 O2 + H2O

GPx

2 H2O

2 GSH

GSSG

NADPH + H+

NADP+

GR

Figure 1.9: Detoxification mechanism of ROS by superoxide dismutase (SOD), catalase (Cat) and glutathione

peroxidase (GPx) (modified [119]). GSH = Glutathione; GSSG = Glutathioneoxidized; GR = Glutathione reductase


1.3.3 Impact of ROS on cell components and signalling pathways

In the case of an overproduction of ROS or attenuated antioxidative defence, the balance

between the generation and elimination of ROS destabilizes and the cell enters a state of

oxidative stress. [119] An excess of ROS is cytotoxic by irreversibly modifying DNA, cellular

lipids and proteins. In detail, in the mitochondria, where ROS are formed within the

mitochondrial respiratory chain as byproducts, the level of oxidized DNA bases is crucially

increased compared to the nuclear DNA [121]. Furthermore, ROS can modify membrane

lipids causing irreversible damages, disruption of the cell membrane and cell death. Similarly,

proteins, preferentially consistent of unsaturated and sulphur containing amino acids, can be

oxidized by ROS. Oxidative modifications may provoke a conformational change which

probably deactivates the proteins’ functionality such as inactivation of receptors or enzymes.

[119]

Contrary to the former assumption that ROS exclusively cause irreversible damage on

cellular structures, recent publications highlight the role of physiological concentrations of

ROS as intra- and extracellular messenger in cell signalling. Exemplarily, the transcription

factor NF-κB is activated in human T cells by a H2O2 dependent mechanism [122]. Likewise,

the activity of the protein kinase C (PKC) in COS-7 cells [123] and of MAPK kinase in HeLa

cells [124] was enhanced in response to H2O2. On the other hand, reactive oxidants such as

H2O2 can also inhibit enzyme activity as in the case of protein tyrosine phosphatase [125]. It

can be summarized from these studies that depending on the cell type, concentration and

type of ROS, various cell signalling pathways can be triggered which are involved in

physiological processes such as cellular metabolism, gene induction, cell replication,

differentiation, migration and also apoptosis [118], [126].

Moreover, various extracellular stimuli may induce the generation of intracellular ROS and

thus provoke intracellular pathways. Amongst others, exposure of human fibroblasts to IL-1

and TNF-α triggered the release of ROS consisting mostly of O2• - [127]. Stimulation of


human epidermoid carcinoma cells with the epidermal growth factor (EGF) increased the

intracellular ROS level predominantly by cellular generation of H2O2 [128]. Likewise, other

growth factors such as transforming growth factor-β1 and platelet-derived growth factor

enhanced the generation of H2O2 in a mouse osteoblastic cell line [129] and in pre-

adipocytes [130]. Interestingly, also a mixture of AGEs, Maillard similar structures, stimulated

the production of ROS in mast cells [131] and hepatic stellate cells [132]. CML, a specific

AGE, also increased the intracellular ROS level in human umbilical vein endothelial cells and

human pulmonary microvascular endothelial cells [133]. The increase in intracellular ROS

after cell stimulation with AGEs was attributed to the interaction of AGEs with AGE-receptors

such as RAGE, the major membrane-bound receptor for AGEs [131-133]. Moreover, co-

treatment with diphenyleneiodonium chloride (DPI), a NADPH oxidase inhibitor, attenuated

the AGE dependent increase in ROS. This result indicates that AGE may induce the

generation of ROS via an interaction with NADPH oxidase [132]. Likewise, IL-1/TNF-α

activated human fibroblasts by a NADPH oxidase dependent pathway [127]. During

inflammation, macrophages, cells of the innate immune system, take advantage of the

NADPH oxidase dependent ROS generation. In this case the macrophages’ oxidative burst

acts as a defence mechanism against pathogens and foreign material in phagocytosis [134].

1.3.4 Maillard-dependent generation of ROS in a cell free system

Besides the NADPH oxidase-mediated ROS generation in cells stimulated with AGEs, it was

postulated that specific substructures of Maillard products generate ROS in a cell free

system. A definite reaction mechanism is not clarified until now but a couple of mechanisms

are proposed illustrating the generation of ROS by Maillard products. Among the suggested

mechanisms, autoxidation of α-hydroxyaldehydes [135], [136] and generation of ROS by

Amadori products [135], [137], [138], dicarbonyls [137], [139] reductones and

aminoreductones respectively [140] are described. The mechanisms for ROS formation by α-


hydroxyaldehydes, Amadori products and dicarbonyls such as methylglyoxal show a

common intermediate with enediol substructure which in the presence of metals produce

O2• - (Figure 1.10).

OH OH

RR

OO

R R

Mn+ M(n-1)+O2 O2. -

- 2H+

Figure 1.10: Proposed mechanism for the generation of superoxide radical anion (O2• -) by enediol substructures

according to [135]. M = metal.

Moreover, a mechanism is proposed for the generation of ROS by reductones and

aminoreductones respectively in the presence of metals [140] as illustrated exemplary for

aminoreductones in Figure 1.11.

OOH

NHR

R

R

O

NHR

R

R

OO O+ Mn+ + O2 Mn+ ROS

Figure 1.11: Proposed mechanism for the generation of ROS by aminoreductone substructure according to [140].

M = metal.

Based on these mechanisms, Maillard products can be described as a natural source of

ROS which are consumed with heat-treated foods such as coffee.


1.4 The human gastrointestinal tract

The gastrointestinal tract is responsible for the ingestion and the digestion of food, and thus

the resorption of vital nutrients, electrolytes and water. [141]

The human gastrointestinal tract can be divided into the upper and the lower tract. The upper

tract consists of the oesophagus, stomach and parts of the duodenum and the lower tract of

the small and large intestine. The small intestine includes parts of the duodenum, jejunum

and ileum and the large intestine the caecum, vermiform appendix, colon (ascending colon,

transverse colon, descending colon and sigmoid flexure) and rectum (Figure 1.12). [141]

Oesophagus

Stomach

Duodenum

AscendingColon

CaecumRectum

AnusSigmoid Colon

Small Intestine

Descending Colon

Transverse Colon

Oesophagus

Stomach

Duodenum

AscendingColon

CaecumRectum

AnusSigmoid Colon

Small Intestine

Descending Colon

Transverse Colon

Figure 1.12: The upper and lower gastrointestinal tract [142].

1.4.1 The histology of the intestine

The microscopic anatomy of the different sections of the gastrointestinal tract is to a great

extent uniform with small variations for functional specifications [141]. The tissue between

the gut lumen and the abdominopelvic lumen are split into the following layers illustrated in

Figure 1.13 [141]: Tunica mucosa, tela submucosa, tunica muscularis propria, tunica serosa

(tela subserosa).


Figure 1.13: General diagram of a longitudinal section of the wall of the gastrointestinal tract (modified) [143].

In this study, endoscopic biopsies of the terminal ileum and the ascending colon were

analysed. Since endoscopic biopsies are limited by experience to the tunica mucosa, this

epithelial layer is of certain interest in this study.

The tunica mucosa can be subdivided into an (i) epithelial layer (lamina epithelialis

mucosae), (ii) a layer of connective tissue (lamina propria mucosae) and (iii) a thin layer of

smooth muscle cells (lamina muscularis mucosae).

(i) The lamina epithelialis mucosae is a single-layer of columnar, absorptive enterocytes.

Depending on the section of the gastrointestinal tract, various secreting cells exist in addition

in the lamina epithelialis mucosae (Table 1.4) [141].

Table 1.4: Secreting cells in the ileum und colon of the gastrointestinal tract (data from [141]).

++Enterochromaffin cells (endocrine)

-+Paneth cells (exocrine)

++++Goblet cells (exocrine)

ColonIleum

++Enterochromaffin cells (endocrine)

-+Paneth cells (exocrine)

++++Goblet cells (exocrine)

ColonIleum

- = non existing, + = existing; +++ = many


(ii) The lamina propria mucosae is a connective tissue involving vascular and neural cells.

Gut sections in the small intestine also show sporadically smooth muscle cells. During

inflammation, immune associated cells such as macrophages, plasma cells, granulocytes,

mast cells and lymphocytes are highly infiltrated into the tissue. The lymphocytes can form

lymphoid follicles as can be seen in the ileum as peyer’s patches. The peyer’s patches and

the immune cells in the lamina propria mucosae belong to the so-called gut associated

lymphoid tissue (GALT) which plays an important role in the intestinal immune response.

(iii) The lamina muscularis mucosae is the final zone in the tunica mucosa consisting of a thin

layer of smooth muscle cells.

In order to accomplish an enhanced resorption of nutrients, the mucosal surface is enlarged

by mucosal folds, intestinal villis and intestinal crypts. Mucosal folds are excrescences of the

tunica mucosa and tela submucosa on the top of which the intestinal villis arise. Between the

intestinal villis, the intestinal crypts form tubular emarginations. Along the gastrointestinal

tract, the height and number of folds and villis decrease from the duodenum to the colon

(Figure 1.14). The crypts exist along the whole lower gastrointestinal tract with increasing

depth in the colon. [141]

Ileum (A)

(1) tunica mucosa, (2) tunica submucosa, (3) tunica

muscularis propria, (4) tunica serosa, (5) villi, (6) epithelium

of the mucosa (covers villi), (7) connective tissue of the

lamina propria of the mucosa, (8) glands (crypts) in the

lamina propria of the mucosa


Colon (B)

(1) tunica mucosa, (2)

tunica submucosa, (3)

tunica muscularis propria,

(4) tunica serosa, (5)

lymphoid follicle in the

lamina propria of the

mucosa

Figure 1.14: Microscopic histological images of the ileum (A) and the colon (B) [144].

1.4.2 Pathology of gut associated diseases

A healthy gut is regulated by the ratio of pathogenic to apathogenic bacteria, an intact

intestinal epithelium and a regulated intestinal immune response. A dysfunction in one of

these three components leads to one of the many diseases of the gut such as celiac disease,

food allergy, inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative

colitis, and cancer. In both Crohn’s disease and ulcerative colitis ulcers are formed due to

chronic inflammation (Figure 1.15). Ulcers are defined as lesions for example of the mucous

membrane which are produced by sloughing of necrotic inflammatory tissue (ulceration).

Healthy Crohn’s disease Ulcerative colitis

crypts UlcerationSwelling of the gut wall

A





A



B

Figure 1.15: (A) Histology of Crohn’s disease and ulcerative colitis. Ulceration in Crohn’s disease spreads across

the entire intestine wall but in ulcerative colitis only affects the tunica mucosa of the intestine wall. In both, the gut

wall is swollen compared to a healthy gut wall (modified [145]). (B) Endoscopic diagnostic of Crohn’s disease and

ulcerative colitis. In the healthy gut, sharp blood vessels are easily observable. In Crohn’s disease the diagnostic

findings show a red bordered ulcer. In the image illustrating the gut of a patient with ulcerative colitis obliterated

vessels and redness is visualized (modified [146]).

In Crohn’s disease, the entire intestine but mostly the ileum and the colon can be affected

whereas in ulcerative colitis the disease begins at the rectum and spreads at the most to the

colon. [147] Moreover, Crohn’s disease spreads across the entire intestine wall but ulcerative

colitis only affects the tunica mucosa and tunica submucosa of the intestine wall [145]. The

underlying pathological processes in IBD are not fully identified yet. However, genetic

predisposition, environmental factors, changing intestinal flora and dysregulation of the

intestinal immune response are considered to contribute to the development of the gut

disorders [146], [148].

To date, the therapeutic treatment is based on a suppression of the inflammatory process

since its dysregulation seems to be the primary issue in IBD [149]. In fact, the immunological

balance is shifted towards the pro-inflammatory side as can be seen by high levels of pro-

inflammatory cytokines like TNF-α, IL-6 and interferon-gamma (IFN-γ). These immune

mediators are produced particularly by macrophages, lymphocytes and epithelial cells with


macrophages playing the major role. Interestingly, the transcription factor NF-κB, which

regulates the expression of inflammatory and immunoregulatory cytokines, is highly

expressed and activated in macrophages of IBD patients. Thus, it was concluded that the

activation of NF-κB in macrophages is one key aspect in the aetiopathology of IBD. [150]

1.4.3 Macrophages - immune associated cells

Macrophages belong to the immune associated cells like T- and B-lymphocytes.

Macrophages mature out of monocytes which grow inside the bone marrow. The monocytes

migrate from the bone marrow into the peripheral blood and spread through the body passing

different tissues and organs such as the gut. Due to infection, monocytes differentiate into

macrophages which highly infiltrate into the affected tissue. Especially in IBD, macrophages

are extensively infiltrated in the tunica mucosa and become strongly activated.

Depending on their functionality, immune cells respond to infections either through

innate/non-specific or adaptive/specific defence mechanisms. Unlike lymphocytes,

macrophages primarily initiate the innate, non-specific immune response. In this context,

macrophages function primarily as phagocytes digesting invading microorganisms and

damaged cells. Thereby, macrophages induce an oxidative burst of O2• - which together with

lysozyme, a degradation enzyme, destroys foreign material. Parts of the phagocytised cells

are incorporated into the membrane of macrophages activating cells of the adaptive immune

defence such as T-lymphocytes and thus provoke the specific immune response. [141]

Moreover, macrophages can secret pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6

but also anti-inflammatory cytokines such as IL-1 receptor antagonist and IL-10 [151]. The

secretion of cytokines stimulates immune precursor cells to proliferate, differentiate and

produce again cytokines [141]. Furthermore, activated macrophages release chemokines

such as the monocyte chemotactic protein-1 to recruit other immune cells from the blood

circulation [151].


IFN-γ, Lipopolysaccharides (LPS) and TNF-α activate macrophages via a NF-κB dependent

pathway causing the release of cytokines [152]. Moreover, activated macrophages produce

O2• - and NO to kill and degrade microorganisms. Recent publications highlight other

functions of activated macrophages besides phagocytic actions in the absence of

abovementioned stimuli. In this context, the function of activated macrophages was focused

on tissue repair and immunosuppression rather than microbial killing emphasizing the multi-

functional role of macrophages in the human body (reviewed in [153]).


1.5 Aims of this study

Coffee is one of the most consumed beverages in the world. Besides its characteristic

flavour, the beverage gained popularity due to its stimulating effect by caffeine. To date,

coffee consumption is further associated with an inverse risk of Alzheimer’s disease,

Parkinson’s disease, cancer (liver, brain and colon) and type 2 diabetes mellitus; diseases

which are associated with inflammation and oxidative stress. However, the active

compounds of coffee, which counteract the risk of those diseases, have not been identified

yet. Indeed, coffee contains many phytochemicals besides caffeine such as polyphenols,

diterpenes and so-called Maillard products which are considered to be biologically active.

This study focused on the Maillard products which are formed in the Maillard reaction

between carbonyls and amines during the roasting. Maillard products are not only formed

during food processing but also at 37°C in the human body (AGEs). It has been shown

before that these AGEs trigger various signalling pathways in the human body. Since dietary

Maillard products are demonstrably absorbed during digestion, the question was raised

whether or not dietary Maillard products also provoke cellular signalling similar to AGEs. In

this context, the impact of Maillard products on the activation of the transcription factors NF-

κB and Nrf2, which are involved in the regulation of inflammation and oxidative stress, was

analysed in vitro in cells present in the intestine and ex vivo in human gut tissue. Thereby,

the involvement of H2O2, which is permanently generated in the extracellular space by

Maillard products, was investigated since H2O2 is proved to function as second messenger in

cell signalling. Thus, not only the extracellular concentration of H2O2 but also the intracellular

level of ROS during stimulation was monitored. Hence, this study was structured as follows:

Characterization of H2O2 generation in Maillard test systems (Chapter 2)

Activation of NF-κB by Maillard products and coffee extract (Chapter 3)

Activation of Nrf2 by Maillard products and coffee extract (Chapter 4)

Extracellular and intracellular ROS during stimulation (Chapter 5)

2 CHARACTERIZATION OF H2O2 GENERATION IN MAILLARD TEST SYSTEMS 37

2 CHARACTERIZATION OF H2O2 GENERATION IN MAILLARD TEST SYSTEMS

2.1 Introduction

Since the discovery of H2O2 in the early 1800’s, its strong oxidizing properties have been

applied in versatile fields as a bleaching and antimicrobial agent, propellant or disinfectant of

surfaces. Due to its ability to act as a strong oxidant which can easily cross cell membranes,

H2O2 is widely thought to be excessively cytotoxic in vivo. High concentrations of H2O2 might

cause irreversible modifications of proteins, lipids and DNA and thus cellular damage to a

wide range of animal, plant and bacterial cells. [154] However, at physiological concentration,

H2O2 can act as an intra- and intercellular signalling messenger. In this context, H2O2

functions as a second messenger in the activation of a multitude of signalling pathways

including NF-κB, activating protein-1, MAPK and phosphatidyl inositol-2 kinase (PI3K). [134]

Interestingly, it was postulated that specific substructures of Maillard products, which occur

naturally in heat-treated food such as coffee, generate ROS including H2O2 [135], [155-158].

Given that Maillard products exist naturally in processed foods, it was suggested that these

compounds might generate H2O2 in heat-treated foods. In order to exploit these Maillard

products as H2O2 generators for technological purposes for example as antimicrobial or

bleaching agent and to evaluate the potential physiological and toxicological consequences,

it is important to study the conditions favouring or attenuating the generation of H2O2. In this

context, the formation of H2O2 by Maillard products was investigated in two different Maillard

systems. Firstly, a heated mixture of ribose (rib) and lysine (lys) was tested. This mixture

represents an optimized Maillard model due to (i) the high level of acyclic ribose [159] and (ii)

the use of lysine with two amino groups in α- and ε-position [160] as highly active

components for the Maillard reaction. Moreover, the reaction was conducted in (iii) a buffered

system which quenches the interfering acids formed during the Maillard reaction [161].


Secondly, the generation of H2O2 was analysed in coffee, a beverage rich in Maillard

products [162]. Particular interest was directed towards the reaction conditions such as pH

and temperature that favour the generation and the de novo generation of H2O2. Moreover,

active fractions of the Maillard reaction mixture were isolated and their ability to generate

H2O2 was tested likewise.

2.2 Results

2.2.1 Generation of H2O2 by Maillard products

In order to study the formation of H2O2 by Maillard products, Maillard mixtures were heated

for 30 min at temperatures between 37°C and 130°C and H2O2 concentration was measured

via ferrous oxidation xylenol orange with perchloric acid (FOXPCA) assay. The content of H2O2

increased as a function of temperature levelling off at 120°C with 120 µM H2O2 (Table 2.1).

Table 2.1: H2O2 was quantified in Maillard reaction mixtures which were heated between 37°C and 130°C for

30 min. H2O2 concentration was detected in all mixtures (10 mM) via FOXPCA assay. Data is expressed as

mean ± SD (n = 3).

Temperature 37°C 60°C 80°C 100°C 120°C 130°C

H2O2 [µM] 3.68 ± 10.31 13.54 ± 4.62 45.42 ± 1.96 111.60 ± 17.06 123.61 ± 20.00 102.92 ± 26.69

Not only the H2O2 concentration of the Maillard reaction mixtures was measured but also the

absorbance was read at 280 nm, 350 nm and 420 nm detecting early, intermediate and late

stage Maillard products (Figure 2.1). The mixtures heated at 37°C and 60°C showed low

absorbance not exceeding 1 Absorbance Unit (AU) at 420 nm whereas at temperatures

higher than 60°C the mixtures significantly absorbed at 280 nm, 350 nm and 420 nm in a

temperature-dependent manner plateauing at 120°C.


0

100

200

300

400

500280 nm350 nm420 nm

20 40 60 80 100 120 140Temperature °C

Abso

rban

ce U

nit

Figure 2.1: Maillard reaction mixtures were heated at various temperatures between 37°C and 130°C for 30 min.

The absorbance was read at 280 nm, 350 nm and 420 nm indicating the presence of early, intermediate and late

stage Maillard products. Therefore, mixtures were diluted not to exceed the Absorbance Unit (AU) of 1. The

absorbance was extrapolated to undiluted concentration. Data is expressed as mean ± SD (n = 3).

As the Maillard reaction proceeds in response to temperature treatment, the pH values

decrease [163]. However, buffering has also an impact on the pH values. To evaluate the

influence of the heating temperature on the pH value of the buffered Maillard reaction

mixtures, the pH value was measured depending on the temperature treatment. The pH

value in Maillard mixtures heated at 37°C and 60°C did not decrease during heat treatment.

At temperatures higher than 60°C, the pH value declined in correlation to the temperature

(Table 2.2).

Table 2.2: Maillard reaction mixtures were heated for 30 min at temperatures between 37°C and 130°C. The

mixtures were cooled down to room temperature and the pH value was measured immediately. Data is mean ±

SD (n = 3).

Temperature 37°C 60°C 80°C 100°C 120°C 130°C

pH value 9.50 ± 0.14 9.41 ± 0.12 9.19 ± 0.04 8.52 ± 0.16 7.75 ± 0.04 7.16 ± 0.18


The Maillard mixture which was heated at 120°C for 30 min (Mrmh) was further analysed. In

detail, a dilution series of Mrmh was prepared and H2O2 content was measured in a

concentration dependant manner in Mrmh and in various control solutions by the FOXPCA

assay (Figure 2.2).

0 200 400 6000

200

400

600MrmhMrmu

RibhRibuLyshLysu

Concentration [mM]

H 2O

2 [µ

M]

Figure 2.2: H2O2 was quantified in the heated Maillard reaction mixture (Mrmh) and in various control solutions

which were prepared as described in the methods section. H2O2 concentration was detected in a dilution series of

each solution via FOXPCA assay. The concentrations used in the figure refer to the initial concentration of the

reactants D-(-)-ribose and L-lysine prior to heating (0.5 M). Mrm = Maillard reaction mixture; Rib = Ribose;

Lys = Lysine; u = unheated; h = heated. Data is expressed as mean ± SD (n ≥ 2).

Within a concentration range from 10 mM to 500 mM, H2O2 was detected neither in an

unheated ribose solution (ribu), an unheated lysine solution (lysu) nor in a heated lysine

solution (lysh). However, H2O2 could be quantified in Mrmh and unheated Maillard reaction

mixture (Mrmu) as well as in a heated ribose solution (ribh). In both, ribh and Mrmu, the

amount of H2O2 was raising concentration-dependently reaching 85 µM and 440 µM H2O2 at

500 mM. In the case of Mrmh, H2O2 concentration increased up to an absolute maximum of

180 µM in a 22.5 mM Mrmh and afterwards decreased. Focusing on the lower concentration

range, Mrmh generated significantly higher amounts of H2O2 than Mrmu and ribh.

To rule out any interference between the FOX reagent and the Maillard products of Mrmh in


high concentrations, H2O2 was measured alternatively via oxygen electrodes. Therefore,

Mrmh was treated with catalase which decomposes H2O2 into water and molecular oxygen

(O2). O2 was detected electrochemically with both the Clark- and the Luminescence

Dissolved Oxygen (LDO)-electrodes. In both electrochemical approaches, the H2O2 yield was

increasing concentration-dependently up to a maximum beyond which the H2O2

concentration declined (Figure 2.3) resembling the response curve monitored by the FOXPCA

assay. Moreover, the generation of H2O2 by the unheated Mrmu was determined with the

LDO-electrode. Similar to the results obtained by the FOXPCA assay, the amount of H2O2 in

Mrmu increased in a concentration dependent manner up to 270 µM at 500 mM Mrmu.

0 20 40 60 80 1000

50

100

150LDO electrodeClark electrode

Mrmh [mM]

H 2O

2 [µ

M]

Figure 2.3: The concentration of H2O2 was measured in the heated Maillard reaction mixture (Mrmh) using the

Clark- and the Luminescence Dissolved Oxygen (LDO)-electrode. The Mrmh was diluted in a range from 0.1 mM

to 0.1 M referring to the initial concentration of the reactants D-(-)-ribose and L-lysine prior to heating (0.5 M).

Then, Mrmh was treated with catalase decomposing H2O2 into H2O and O2 which could be detected by the

electrodes. Data is expressed as mean ± SD (n ≥ 3).

Since H2O2 generation in Mrmh showed an unexpected course progression in high

concentrations, solutions of 5 - 500 mM rib-lys were prepared individually and heated at

120°C to rule out any dilution-caused error. Likewise, a concentration-dependent H2O2

generation was detected which however raised concentration-dependently up to 100 mM


Mrmh reaching 350 µM H2O2. In concentrations higher than 100 mM no significant amount of

H2O2 was detected (data not shown). Similarly, various concentrations of the unheated Mrmu

were prepared by dilution and individually. The H2O2-generating ability was tested in each

mixture. However, no significant difference in H2O2 generation was detected between both

approaches.

2.2.1.1 The role of temperature in the generation of H2O2

As illustrated above, the temperature during heating of Maillard mixtures had a crucial impact

on the Maillard-dependent H2O2 generation. Next, the influence of post-heating exposure of

Mrmh to 37°C on the H2O2 concentration was investigated. In this context, two different

experimental approaches were used; dilution before and after prolonged incubation at 37°C

for 96 h. The H2O2 concentration differed significantly between both assay approaches

(Figure 2.4). When diluted after the incubation at 37°C, H2O2 generation was completely

unaffected by the temperature exposure. Thus, the concentration course of H2O2 remained

constant for 96 h.

0 200 400 6000

100

200

300

400

5000 h

2 h10 h23 h96 h

0 h2 h10 h23 h

96 h

pre-dilution

post-dilution

30 100 300 500Mrmh [mM]

H 2O

2 [µ

M]

Figure 2.4: The heated Maillard reaction mixture (Mrmh) was incubated at 37°C for up to 96 h and the H2O2


concentration was measured after various incubation periods using FOXPCA assay. Two different approaches

were used for this experiment. In the first procedure, 0.5 M Mrmh was incubated at 37°C and the dilution series

was prepared after incubation at 37°C shortly before H2O2 detection (post-dilution). In the second approach, the

dilution series was carried out prior to incubation which means that the individual dilutions were incubated at 37°C

and the H2O2 content was measured afterwards (pre-dilution). Data is expressed as mean ± SD (n = 3).

In contrast, when diluted before the 37°C exposure, the H2O2-generating activity was highly

influenced by the treatment at 37°C. More precisely, H2O2 concentration increased with

incubation time at 37°C reaching levels of H2O2 about 370 µM after 96 h. Irrespective of the

incubation time, the general curve progression of H2O2 generation remained similar but on

different H2O2 level. Moreover, with increasing incubation time at 37°C, the peak of maximal

H2O2 content was shifted towards higher sample concentrations.

2.2.1.2 The role of pH in the generation of H2O2

Besides temperature, changes in pH value intensively alter the course of Maillard reaction. In

order to figure out the influence of pH on H2O2 generating Maillard products, the pH of Mrmh

was adjusted prior to heating to values between 1 and 14. After heating, the mixtures were

treated as aforementioned and the H2O2 content was measured using the FOXPCA assay. As

depicted in Figure 2.5, pH influenced the generation of H2O2 in Mrmh to a large extent.

0 100 200 300 4000

200

400

600pH 1

pH 4pH 7

pH 10

pH 14

Mrmh [mM]

H 2O

2 [µ

M]

Figure 2.5: H2O2 concentration was quantified via FOXPCA assay in heated Maillard reaction mixtures which were


pH adjusted between 1 and 14 before heating. The concentrations used in the figure belong to the initial

concentrations of the reactants D-(-)-ribose and L-lysine prior to heating (0.5 M). Data is mean ± SD (n = 3).

Within a pH range from 1 to 7, H2O2 concentrations increased with both, raising sample

concentration and basicity, reaching concentrations between 200 µM (pH 1) and 370 µM

(pH 7). In the case of the alkaline mixtures, the H2O2 content was enhancing in a con-

centration dependant manner. Thereby, an absolute maximum of 170 µM H2O2 (pH 14) was

detected. Regarding high sample concentrations, the H2O2 yield declined as reported before.

Not only pH adjustment prior to heating but also after heating influenced the H2O2 generation

by Maillard products forming less H2O2 at pH 5.5 than at pH 8 (Table 2.3). To rule out any

effect of pH on the FOXPCA assay itself, the H2O2 standard curve was prepared at both pH 5.5

and pH 8 showing no difference in their H2O2 amount detected by the FOXPCA assay (data

not shown).

Table 2.3: In order to evaluate the influence of the pH value of the Maillard reaction mixtures on the H2O2

generation after heat treatment, the concentration of H2O2 was measured via FOXPCA assay in Mrmh which were

adjusted to pH 5.5 and pH 8. Data is mean ± SD (n ≥ 2).

H2O2 [µM] Mrmh

Mrmh pH 5.5 Mrmh pH 8

5 mM 83.44 ± 5.37 82.51 ± 4.15

15 mM 129.83 ± 2.31 153.38 ± 12.83

25 mM 123.91 ± 4.17 161.28 ± 4.72

2.2.2 De novo generation of H2O2 by Maillard products

The fact that the H2O2 concentration of Mrmh increased during storage at 37°C (Figure 2.4)

indicates that H2O2 is not only generated during heating at 120°C but also formed at 37°C


during storage. To test this hypothesis, H2O2 was completely removed in Mrmh by catalase-

treatment (Table 2.4). Catalase was then heat-inactivated and H2O2 neoformation was deter-

mined dependent on temperature, incubation time and concentration (Figure 2.6). It was con-

firmed that catalase heat-inactivation did not affect H2O2 generation by Maillard products

(Table 2.4).

Table 2.4: Concentration of H2O2 was measured via FOXPCA assay in the Mrmh ± catalase or heat-treatment. For

catalase treatment, Mrmh was incubated with catalase (150U/mL) for 30 min. Heat-treatment was at 95°C for

5 min. Data is expressed as mean ± SD (n ≥ 3).

H2O2 [µM] Mrmh Mrmh Mrmh + catalase Mrmh + 95°C/5 min

10 mM 127.82 ± 8.86 1.71 ± 0.83 140.75 ± 19.21

20 mM 165.60 ± 10.65 0 ± 0.38 176.63 ± 32.06

30 mM 188.22 ± 17.42 0 ± 1.29 195.96 ± 13.21

After 1 h incubation at 4°C, 25°C and 37°C, H2O2 could be detected in all reaction mixtures

between 65 µM and 110 µM according to sample concentration and temperature. Whereas

the H2O2 content slightly decreased during prolonged storage for 24 h at 4°C, the H2O2

concentration stayed nearly constant up to 4 h at 25°C. The situation after 24 h at 25°C was

highly dependent on the concentration of Mrmh; whereas H2O2 content did not exceed 10 µM

at 5 mM Mrmh, it increased up to 140 µM at 25 mM Mrmh. At 37°C, the neoformation of H2O2

was most pronounced and increased with incubation time reaching more than 210 µM H2O2

at 25 mM Mrmh after 24 h. Interestingly, H2O2 concentration of 5 mM Mrmh was decreased

after 24 h at 37°C as in the case of 4°C and 25°C.


4°C

0

50

100

1505 mM15 mM25 mM

A

1 2 4 24Incubation time [h]

H 2O

2 [µ

M]

25°C

0

50

100

150

2005 mM15 mM25 mM

B


H 2O

2 [µ

M]


37°C

0

50

100

150

200

2505 mM15 mM25 mM

C


H 2O

2 [µ

M]

Figure 2.6: De novo H2O2 generation was investigated in the heated Maillard reaction mixture (Mrmh) depending

on incubation temperature after various time intervals. After abolishing pre-existing H2O2 by catalase treatment,

Mrmh was incubated at 4°C (A), 25°C (B) and 37°C (C) for up to 24 h and H2O2 concentration was measured

using the FOXPCA assay. Data are expressed as mean ± range (n = 2).

The assay was also carried out with a heated Maillard reaction mixture which was adjusted

to pH 5.5 (data not shown). These data were in agreement with the previous results

exhibiting the same tendency in H2O2 neoformation due to incubation time, temperature and

concentration but on lower H2O2 level compared to pH 8.

Due to the fact that H2O2 concentration substantially altered within 24 h, the experiment was

carried out with an extended incubation time for 96 h (Figure 2.7). The lower Mrmh

concentrations at 4°C and 37°C as well as all samples stored at 25°C showed severely

reduced H2O2 levels during prolonged storage for 96 h. Interestingly, stationary or even

increasing H2O2 contents could be detected in the case of higher concentrated Mrmh at 4°C

and 37°C but not at 25°C.

In order to exclude that the procedure of the FOXPCA assay itself at room temperature did

influence the H2O2 generation, the FOXPCA assay was conducted with identical samples at


room temperature as well as at 4°C. The results of the FOXPCA assay did not differ in their

H2O2 generation (data not shown).

In further experiments, it could be observed that an increased air volume above the solution

had only a minor effect on the efficiency of the Maillard products to produce H2O2 (data not

shown).

0

50

100

150

200

250

5 mM 15 mM 25 mM 5 mM 15 mM 25 mM 5 mM 15 mM 25 mM

4°C 25°C 37°C

H2O

2 [µ

M]

1 h24 h48 h72 h96 h

Figure 2.7: H2O2 generation was analysed in the heated Maillard reaction mixture (Mrmh) depending on incubation

temperature after various time intervals. After abolishing pre-existing H2O2 by treating Mrmh with catalase, Mrmh

was incubated at 4°C, 25°C and 37°C for up to 96 h and H2O2 concentration was measured using the FOXPCA

assay. Data is expressed as mean ± range (n = 2).

2.2.3 Activity-guided fractionation of Maillard products

2.2.3.1 Size exclusion chromatography (SEC)

Next, H2O2-generating Maillard products were isolated from the crude reaction mixture Mrmh

via SEC. First, the SEC column was calibrated using bovine serum albumin (BSA) (66 kDa),

vitamin B12 (1.4 kDa) and phenylalanine (165 Da) as molecular weight markers (Figure 2.8).

20 fractions (F), each 0.5 mL, were collected. The yields of recovery of the standards were

94.75 %, 113.7 % and 98.7 % respectively across the total of 20 fractions (F).


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 200

20

40

60

80Phenylalanine 165 DaVitamin B12 1.4 kDaBSA 66 kDa

Fractions F

Rec

over

y [%

]

Figure 2.8: A D-SaltTM polyacrylamide desalting column (cut off 1.8 kDa) was calibrated with 0.5 mL bovine serum

albumin (BSA) (10 mg/mL) as high molecular weight standard, vitamin B12 (0.1 g/mL) as intermediate molecular

weight standard and D-phenylalanine (0.1 M) as low molecular weight standard. 20 fractions (F) of 0.5 mL were

collected with water as eluent. The recovery was investigated by photometric means at 278 nm, 360 nm and

257 nm respectively. Data is expressed as mean ± SD (n = 2 - 6).

Mrmh was fractionated by a 3-step fractionation scheme (Figure 8.1) according to the column

calibration (Figure 2.8) into a high (HMW; F1 - F7) and a low molecular weight fraction

(LMW; F8 - F20). Thereafter, the H2O2 content was measured in each fraction (Figure 2.9).

0 50 100 1500

20

40

60HMW

LMW

Concentration of the fraction [mM]

H 2O

2 [µ

M]

Figure 2.9: Mrmh was fractionated via SEC into a high (HMW) and a low molecular weight fraction (LMW). The

lyophilized fractions were each reconstituted to the original volume yielding 500 mM HMW/LMW. H2O2

concentration in each fraction was measured via FOXPCA assay. Data is expressed as mean ± SD (n = 2 - 8).


Depending on the concentration of the fraction, HMW generated significantly more H2O2 than

LMW. In order to exclude any synergistic effects between HMW and LMW, the concentration

of H2O2 was measured in each fraction separately as well as in a recombined mixture of

HMW and LMW. The total concentration of H2O2 of the separate fractions compared with the

H2O2 concentration of the recombined mixture of HMW and LMW did not differ significantly

(data not shown). Thus, any synergistic effects between HMW and LMW can be excluded. It

was further shown that this tendency, HMW generating more H2O2 than LMW, was already

found after the first fractionation step (data not shown). Due to this observation and the fact

that H2O2 was detected in both HMW and LMW, an optimized method was used to collect an

active, H2O2-generating fraction from the Mrmh. In detail, Mrmh was not separated into HMW

and LWM but rather into 20 separate fractions of 0.5 mL each. H2O2 content was measured

in each fraction with the FOXPCA assay (Figure 2.10).

1

10

100

1000

10000

100000

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fractions F

Mol

ecul

ar w

eigh

t [D

a]

0

20

40

60

80

100

120

140

160

H2O

2 [µ

M]

BSAvitamin B12phenylalaninelysine

riboseMrmh

Figure 2.10: The Maillard reaction mixture (Mrmh) and the precursor lysine/ribose were fractionated into 20

fractions F via size exclusion chromatography on a polyacrylamide column (cut off 1.8 kDa). H2O2 concentration

was measured in each Mrmh fraction via FOXPCA assay (dots). As reference substances bovine serum albumin

(BSA), vitamin B12 and phenylalanine were used for column calibration. The figure shows the main elution

fraction for the standards, ribose and lysine (bars). Each experiment was carried out at least 3 times

independently.


F5 - F9 were highly active in generating H2O2 contributing 63 % of the dry weight of the crude

Mrmh. Fractionation of the Mrmh reactants, ribose (150 Da) and lysine (146 Da), confirmed

that very low molecular weight substances inclusive of ribose were removed from the active

fractions of Mrmh. After fractionation of a pure H2O2 solution (102 mM), only 0.45 % H2O2 of

the starting concentration was recovered in the fractions F10 - 13 (data not shown).

Based on the fact that H2O2 could be detected in relatively high concentrations in F5 - F9 of

the Mrmh, these fractions were combined into one fraction representing the active Maillard

products. Residual fractions were unified to combine all the inactive Maillard products. H2O2

content of both fractions was detected in a concentration dependent manner (Table 2.5). The

specific activity for H2O2 generation (H2O2 concentration/dry weight) was notably higher in the

active fraction than in the inactive fraction but not significantly exceeding the specific activity

of the crude Mrmh.

Table 2.5: The concentration of H2O2 was measured using the FOXPCA assay in the crude Mrmh as well as in the

active and inactive fraction. H2O2 concentrations are given as specific activity based on the dry weight of each

sample. Data is expressed as mean ± SD (n = 3).

Specific Activity [H2O2 µM/dry weight] Sample crude Mrmh active fraction inactive fraction

5 mM 0.6458 ± 0.0325 0.4036 ± 0.0614 0.000 ± 0.000

15 mM 1.2004 ± 0.1004 0.9825 ± 0.1432 0.0525 ± 0.0147

25 mM 1.2622 ± 0.0369 1.2651 ± 0.1666 0.1324 ± 0.0130

2.2.3.2 Ultra-filtration

For up-scaling the active components of the Mrmh were also isolated by ultra-filtration instead

of SEC since higher sample volume can be used. Thereby, four fractions depending on their

molecular masses were isolated (x > 30 kDa, 30 kDa > x > 3 kDa, 3 kDa > x > 1 kDa and


x < 1 kDa). H2O2 concentration was detected in each fraction. The formation of H2O2 was

most pronounced in the fraction < 1 kDa (Figure 2.11).

0 200 400 6000

100

200

300x > 30 kDa3 kDa < x < 30 kDa1 kDa < x < 3 kDax < 1 kDa

Concentration [mM]

H 2O

2 [µ

M]

Figure 2.11: The Maillard reaction mixture (Mrmh) was fractionated by ultrafiltration into four fractions (x > 30 kDa,

30 kDa > x > 3 kDa, 3 kDa > x > 1 kDa and x < 1 kDa). H2O2 concentration was measured in a dilution series of

each Mrmh fraction via FOXPCA assay. Data is expressed as mean ± SD (n = 3).

Thereby, it was assured that H2O2 did not resist fractionation but was rather generated by the

fraction < 1 kDa (data not shown). The membranes sufficiency were verified by ultra-filtrating

BSA (66 kDa), lysozyme (14.3 kDa) and vitamin B12 (1.4 kDa) as standards reaching a

recovery of more than 94 % each in the respective fraction (data not shown).

2.2.4 De novo generation of H2O2 by the active fraction of Maillard products

H2O2 can be formed permanently within the crude Mrmh at various conditions. Since the

active Maillard products (F5 - F9) have been successfully isolated out of the crude Mrmh via

SEC, the H2O2 generating properties were analysed analogously in the active fraction.

Briefly, after catalase-treatment, the isolated fraction of active Maillard products (pH 5.5) was

incubated at 4°C, 25°C and 37°C for up to 96 h and their H2O2 content was measured time-

dependently via FOXPCA assay (Figure 2.12). After 2 h incubation, H2O2 could be detected in


all samples between 50 µM and 115 µM according to sample concentration and temperature.

Whereas the concentration of H2O2 remained mostly constant for 96 h at 4°C, H2O2 levels at

37°C noticeably increased after 24 h, reaching afterwards a steady state up to 96 h.

Prolonged storage at 25°C for 96 h led to a severe decrease of H2O2 content at low sample

concentrations and to an increase to 180 µM H2O2 at high sample concentrations.

4°C

0

50

100

1505 mM15 mM25 mM

A

2 24 48 72 96Incubation time [h]

H 2O

2 [µ

M]

25°C

0

100

200

3005 mM15 mM25 mM

B


H 2O

2 [µ

M]


37°C

0

100

200

3005 mM15 mM25 mM

C


H 2O

2 [µ

M]

Figure 2.12: H2O2 generation was investigated in the active fraction (F5 - F9) of Maillard reaction mixture (Mrmh)

depending on incubation temperature after various time intervals. After abolishing pre-existing H2O2 by treating

the active fraction of Mrmh (pH 5.5) with catalase, the fraction was incubated at 4°C (A), 25°C (B) and 37°C (C) for

up to 96 h and H2O2 concentration was measured using the FOXPCA assay. Data is mean ± range (n = 2).

2.2.5 Generation of H2O2 by coffee extract

Coffee roasting products contain Maillard structures. Thus, it was investigated whether coffee

extract shows similar ability to generate H2O2.

Coffee extract was prepared as described in the methods section and H2O2 was measured

via FOXPCA assay. Coffee extract produced a significant amount of H2O2. H2O2 content

increased constantly with coffee concentration reaching a plateau of 440 µM H2O2 when

diluted 1:1.25 (Concentration factor of coffee extract = 0.8) (Figure 2.13). Besides roasted

coffee, the H2O2 concentration was also determined in raw coffee extract (Figure 2.13).

Interestingly, H2O2 was not only detected in roasted coffee extract but also in raw coffee

extract but to a significantly lower extent. In undiluted raw coffee extract, 205 µM H2O2 was

measured via FOXPCA assay.


0.0 0.2 0.4 0.6 0.8 1.00

200

400

600Roasted coffee extract

Raw coffee extract

Concentration factor of coffee extract

H 2O

2 [µ

M]

Figure 2.13: The concentration of H2O2 was measured in roasted and raw coffee extract in various dilutions via

FOXPCA assay. Data is expressed as mean ± SD (n = 3).

Alternatively, the concentration of H2O2 was also measured electrochemically in roasted and

raw coffee extract by a Luminescence Dissolved Oxygen (LDO) electrode. Roasted but not

raw coffee extract generated H2O2 in a concentration dependent manner reaching up to

300 µM H2O2 (Figure 2.14). This data was confirmed with the Clark electrode, an

independent method for H2O2 measurement (data not shown).

0.0 0.2 0.4 0.6 0.8 1.00

100

200

300

400Roasted coffee extractRaw coffee extract

Concentration factor of coffee extract

H 2O

2 [µ

M]

Figure 2.14: The concentration of H2O2 was analysed in roasted and raw coffee extract using the Luminescence

Dissolved Oxygen (LDO) electrode. The coffee extracts were diluted in water and treated with catalase

decomposing H2O2 into H2O and O2 which was detected by the oxygen electrode. The release of O2 was linearly

correlated to the initial concentration of H2O2 by an external H2O2 standard curve. Data is mean ± range (n = 2).


2.3 Discussion

Maillard products, which exist naturally in heat-processed foods, were shown to function as a

natural source of H2O2 [73]. In this study the formation of H2O2 in two independent Maillard

systems, a sugar-amino acid model and coffee extract, a Maillard-rich beverage [162], was

monitored under various conditions.

Up to 180 µM H2O2 was detected in Mrmh heated at 120°C with the FOXPCA assay. However

H2O2 was also generated in ribh and Mrmu. As expected the concentration of H2O2 was

significantly higher (at low sample concentration) in Mrmh than in ribh and Mrmu highlighting

the Maillard products as active generators of H2O2. Iida et al showed that Maillard mixtures

emit photons which could be inhibited by co-incubation with catalase implying that the

chemoluminescence during the Maillard reaction was deeply involved with free radical

development. Thus, it was postulated that the electron-rich Maillard products transfer

electrons to oxygen with simultaneous emission of light which leads to the formation of ROS.

This hypothesis was underlined by the fact that H2O2 was previously identified as one form of

ROS via oxygenometer [164], a peroxidase-phenol red system [165] as well as luminol-

chemiluminescence-high performance liquid chromatography [166] in Maillard systems.

Besides the Maillard reaction, autoxidation of sugars during caramelisation contributes to the

formation of H2O2 [135], [167] which leads to a detectable amount of H2O2 in the ribh solution.

Since neither ribu nor lysu generated H2O2, the amount of H2O2 measured in Mrmu had to be

due to a reaction between lysine and ribose. Indeed, it was shown before that the Maillard

reaction can take place at low temperatures at 22°C but with decelerated reaction rate [168].

The rate-limiting early steps of the Maillard reaction can be however favoured by high initial

reactant concentrations as used in this study [169]. With increasing temperature, the reaction

rate accelerates and thus the nature of Maillard products might vary which leads to the

formation of brown products as in Mrmh with more unsaturated character at high

temperatures [168].


Taking into consideration that H2O2 can be generated in various conditions in different

Maillard systems, Maillard-related substructures rather than defined Maillard products seem

to induce the formation of H2O2. In this context, protein enediols and enaminols which

tautomerize from Amadori products [135], [155], [156] and dicarbonyls [156], [170] have been

identified as H2O2-generating Maillard structures (Figure 1.10). Furthermore, a mechanism

was postulated by which reductones specifically aminoreductones were able to form H2O2

[157] (Figure 1.11). This hypothesis is further clarified by the fact that aminoreductone-

dependent NF-κB activation can be inhibited by co-incubation with catalase, implying that

aminoreductone-related H2O2 acts as a signalling molecule [171]. In conclusion, it can be

emphasized that Maillard products of especially the early and the intermediate stage were

identified as H2O2-forming structures [172].

Unexpectedly, it was monitored with the FOXPCA assay that the formation of H2O2 in Mrmh

quickly reached its maximum with increasing sample concentrations and decreased

thereafter. In order to verify this observation, the H2O2 content was also measured with an

alternative method. When detected by oxygen electrodes, the H2O2 levels showed a similar

curve progression with increasing sample concentration but at a lower level. Thus, the

FOXPCA assay itself can be ruled out as error source. Both assays were used previously for

detecting H2O2 in human urine. In agreement with our data, the H2O2 content in urine was

slightly lower when measured by the electrodes than by the FOXPCA assay. [173] However,

the existence of ferrous ions in the FOX reagent was unlikely to influence the generation of

H2O2 by Maillard products. In this context, it was demonstrated via Chelex-treatment that

excessive metal ions do not significantly affect Maillard-dependent H2O2 generation [174].

Since the detection method for H2O2 was excluded as error source, the pronounced

decrease in H2O2 levels in high concentrations of Mrmh was attributed to the ROS scavenging

activity of Maillard products [175]. Indeed, the antioxidative effect of Maillard products was

previously studied in multiple Maillard mixtures [176]. In this context, it was published before


that reductones especially aminoreductones act not only as prooxidants but also as

antioxidants depending on the concentration and reaction conditions. Ascorbic acid

exemplifies an analogous reductone which has been described extensively in the literature

for its dual behaviour [157]. Indeed, various antioxidants do not become active until they

pass a certain threshold [177] explaining the antioxidative action in high but not in low Mrmh

concentrations. This effect was also found in individually prepared concentrations of Mrmh

excluding dilution dependent side-effects as major error source. According to literature, the

antioxidant activity correlates to high molecular weight [176] and brown products [178]. This

observation does not only explain the H2O2-scavenging action in the Mrmh but also the fact

that this effect of antioxidant activity can be observed in the Mrmh but not in the Mrmu in

which no extensive browning occurred.

It is well established that both the pH value and the temperature crucially influence the

Maillard reaction [161], [168], [179]. Considering the low concentration range of Mrmh

between 7 mM and 50 mM, the H2O2 level increased between pH 1 and 10 and decreased at

pH values > 10. These findings were in agreement with a study in which a pH dependent rise

in radical development in an amino-sugar systems was measured via ESR spectrometry

which dropped at a pH above 10 [155]. The promoted H2O2 generation under basic

conditions might be due to the higher Maillard reaction rate which is caused by (i) the

predominant acyclic form of the sugar, (ii) the increased nucleophilic potential of the amino

acid [180] and (iii) the preferred Schiff base formation under alkaline conditions [177]. At a

pH higher than 10, the colour development dropped which implies a deceleration of the

Maillard reaction as shown in various sugar-amino acid models with pH values from pH 6 to

12 [181]. The slow Maillard reaction rate [160] as well as the radical instability in extreme

alkaline conditions [155] might explain the decrease in radicals at a pH above 10. Not only

pH adjustment during the preparation of Maillard reaction mixtures at high temperatures but

also pH changes during storage were found to be a distinct influence on H2O2 generation in


Maillard systems.

As expected, not only did the pH value but also the temperature influence H2O2 formation.

The H2O2 generation was provoked at increasing temperature between 37°C and 130°C

which was postulated to be due to a higher Maillard reaction rate. This hypothesis was

underlined by the fact that the pH value significantly decreased after high temperature-

treatment indicating an enhanced Maillard reaction rate [161]. Moreover with increasing

temperature, an enhanced absorbance at 280 nm and 350 nm for early and intermediate

stage Maillard products respectively was detected which significantly increased at

temperatures > 80°C. At the same time, a low absorbance at 420 nm was measured implying

the presence of brown, late stage Maillard products at high temperature-treatment above

80°C. As discussed above, especially the early and intermediate stage Maillard products are

associated as H2O2-generating compounds. The late stage Maillard products, on the other

hand, are rather related with antioxidative activity. Indeed, it was shown before that with

increasing temperature, the amount of carbons in Maillard structures was amplified while the

hydrogen content declined implying less saturation ergo enhanced antioxidant activity in

brown Maillard products [168].

Not only pre-adjustment of high temperatures during preparation of the Maillard mixtures

influenced the generation of H2O2 in Mrmh, but also prolonged storage of Mrmh at 37°C had a

crucial impact on the H2O2 formation. During storage at 37°C, H2O2 was crucially generated

depending on the concentration of Mrmh. This formation of H2O2 can be attributed to H2O2-

generating Maillard products which were formed during the preparation of Mrmh at high

temperatures as well as to the ongoing Maillard reaction at low temperature during storage

[168]. Interestingly, only low concentrated Mrmh (≤ 300 mM) were affected in their H2O2-

generating ability during storage at 37°C; the H2O2 level of highly concentrated Mrmh

(> 300 mM) remained low. It was suggested that in highly concentrated Mrmh, late stage

Maillard products were antioxidative active and thus rather scavenge than generate ROS.


These data substantially demonstrated that the generation of H2O2 by Maillard products is

linked to the sample concentration, pH value and heating temperature. Moreover, it was

suggested that H2O2 was not only formed during high heat-treatment but also neo formed

permanently at 37°C under storage conditions. Taking these findings into account, the de

novo generation of H2O2 was monitored at 4°C, 25°C and 37°C. After 1 h, between 50 µM

and 100 µM H2O2 were generated under all tested conditions. During prolonged storage for

96 h, the amount of H2O2 increased at 37°C to 200 µM, stayed almost constant at 4°C and

surprisingly decreased at 25°C in the highest concentrated sample. It was postulated that

many processes such as the generation of H2O2 by Maillard products, the scavenging of

H2O2 by Maillard products due to antioxidative activity and also the decomposition of instable

H2O2 contributed to the final concentration of H2O2. There is growing evidence that not only

the Maillard-dependent formation of H2O2 but also its scavenging by Maillard products is

correlated with temperature. It was demonstrated before that during prolonged storage of a

Maillard mixture at 25°C, the antioxidative activity remained constant for 180 days [172]. On

the other hand, it was also shown that the H2O2 level in coffee extract, which is rich in

Maillard products, equilibrated at room temperature but increased at 37°C as in the Maillard

mixture [73]. In order to obtain a Maillard mixture with higher H2O2-generating activity, the

H2O2-generating Maillard products were systematically isolated from the Mrmh by an

optimized SEC method and unified into one active Maillard fraction. The dry weight of the

active fraction was about 63 % of the dry weight of the crude Mrmh. To compare the capacity

of the Maillard products in the Mrmh and the active fraction respectively to generate H2O2, the

specific activity in H2O2 generation was calculated by relating the concentration of H2O2 to

the respective dry weight of the sample. The specific activity in generating H2O2 was only

slightly lower in the active fraction compared to the crude Mrmh. Subsequently, the de novo

generation of H2O2 was tested in the active fraction similar to the crude Mrmh. The active

fraction generated H2O2 permanently under all tested conditions between 50 µM and 100 µM


after 2 h. On the contrary to the crude Mrmh, the level of H2O2 stayed constant or even

increased at all tested temperatures reaching up to 260 µM H2O2 during prolonged storage

for 96 h. Thus, it can be confirmed that the active fraction of Mrmh exhibited an optimized

behaviour in H2O2 generation compared to the crude Mrmh. The fractionation method was

up-scaled by using ultra-filtration instead of SEC with which a higher sample volume can be

fractionated. The results obtained were in agreement with those of the SEC fractionation

confirming the low molecular weight Maillard products as active compounds.

Since up to 25 % of the dry matter of coffee beverages consists of Maillard products [162],

coffee is considered to be a good example for a Maillard-rich natural product. Indeed, coffee

extract did also generate H2O2 reaching levels up to 440 µM. However, H2O2 was also

generated in raw coffee extract but to a lower extent. Maillard products as well as

polyphenols have been described previously in literature as potent generators of H2O2 [174],

[182]. Since the polyphenols highly degrade during roasting [4], the generation of H2O2 in

roasted coffee extract was attributed to degradation products and the Maillard products

which are formed during roasting. In earlier studies, caffeic acid, pyrogallol and

hydroxyhydroquinone (1,2,4-Benzenetriol), the degradation products of chlorogenic acid,

were already identified as H2O2-generating structures in roasted coffee [182]. However, the

overall concentration of H2O2 formed by these degradation products [182] is significantly

lower compared to the amount of H2O2 found in coffee extract in the present study. Thus, the

formation of H2O2 was not only attributed to degradation products but also to Maillard

products. However, a specific Maillard product has not been identified yet as H2O2 generator

but rather Maillard substructures are associated with the formation of H2O2 as discussed

above. Even though H2O2 was detected in both, the Maillard model as well as coffee as a

Maillard-rich beverage, the concentration of H2O2 varied strongly. This might be due to the

fact that Mrmh mimics a model system for Maillard products, in which the yield of Maillard

products is concentrated and the type of Maillard products can differ from those of coffee.


Moreover, coffee specific compounds like polyphenols, which are missing in the Mrmh, might

influence the generation of H2O2 since polyphenols do not only possess H2O2-generating

activity but also antioxidative activity [182].

The Maillard mixture formed permanently H2O2 under all tested conditions underlining the

hypothesis of their use as bleaching agent and antimicrobial agent respectively with a wide

application range. Moreover, an active fraction, which showed improved behaviour in H2O2

neoformation, was successfully isolated of the Maillard mixture. Furthermore, this protocol for

the isolation of the active fraction was effectively up-scaled for industrial processing.

Moreover, it was shown in this study that not only Maillard test systems but also coffee, a

Maillard-rich beverage, generated crucial amounts of H2O2. Since H2O2 was not only formed

at high temperatures but also at 37°C, the question was raised if coffee might induce

physiological and/or toxicological effects in the human body after consumption due to H2O2

generation.

3 NUCLEAR TRANSLOCATION OF NF-κB 63

3 NUCLEAR TRANSLOCATION OF NF-κB

3.1 Introduction

Coffee contains numerous non-nutritional constituents, which possess biological activities

other than flavour, colour and taste but with potentially beneficial and/or adverse impact on

human health. High coffee drinking increases cardiovascular disease risk factors such as

plasma cholesterol levels in human studies, whereas moderate consumption seems to have

some cardiovascular benefits [10]. Moreover, epidemiological studies associated regular

coffee consumption with a reduced risk of Alzheimer’s disease [13], Parkinson’s disease [15],

type 2 diabetes mellitus [16] and cancer amongst others in the brain, liver and colon [17-19];

diseases which are associated with inflammation and oxidative stress [20-23].

To date, the underlying mechanisms are still unclear. It was shown that coffee and coffee

components considerably influence the activation of NF-κB, a transcription factor which

regulates the expression of immune response-related proteins. In unstimulated

macrophages, coffee induces a strong activation of NF-κB. This effect was attributed to

H2O2-generating Maillard products, which are formed during the roasting process [25]. In

LPS-stimulated monocytes as well as in the liver and the kidney of transgenic reporter mice

on the other hand, high concentrations of coffee rather inhibit LPS-induced activation of NF-

κB. This observation is traced back to the diterpenoid lipid kahweol existing in coffee [183].

NF-κB is an inducible transcription factor which is ubiquitously expressed in tissue. NF-κB is

a dimeric complex of the Rel/NF-κB family of polypeptides consisting of p105 and p100 or

their mature forms p50 and p52, and of Rel (c-Rel), v-Rel, Rel A (p65) and RelB proteins.

Among these various combinations, p50/p65 is the classical formation present essentially in

all cells. Without stimulation, the NF-κB dimer is localized in the cytoplasm bound to the

inhibitory κB (IκB) which prevents the translocation of the NF-κB:IκB complex into the


nucleus. Upon NF-κB activation, IκB kinase is induced causing the phosphorylation and

degradation of IκB which enables NF-κB to translocate into the nucleus. Potent NF-κB

activators are cytokines (TNF-α, IL-1/2), bacterial products (LPS), viral products (Human T

cell leukaemia virus-1) and also physical stress (UV light) and oxidative stress (H2O2).

Activated NF-κB accumulates in the nucleus where it binds to specific κB sites in the DNA.

Thereby, NF-κB regulates the expression of genes, which control diverse cellular processes

including immune response, inflammation and stress response, coding for cytokines (IL-1β

and TNF-α), growth factors (transforming growth factor-β2), immune receptors (tissue factor-

1) and adhesion molecules (vascular cell adhesion molecule-1 (VCAM-1)). Moreover, the

NF-κB pathway is self-regulating by inducing the transcription of Rel, p105 but also inhibitor

IκB. [184], [185] Figure 3.1 depicts a basic overview of the activation pathway of NF-κB.

cytoplasm

nucleus

p65p50

IκB

IκB kinase

p65p50

IκB

PP

IκB

PP

p65p50

Ubiquitinationdegradation

NF-κB target genetranscription

Stimulus

cytoplasm

nucleus

p65p50p50

IκBIκB

IκB kinase

p65p50p50

IκBIκB

PP

IκBIκB

PP

p65p50p50

Ubiquitinationdegradation

NF-κB target genetranscription

Stimulus

Figure 3.1: Simplified illustration of the activation of the NF-κB pathway


After coffee consumption, the gut encounters coffee and coffee components. In case of low

molecular weight components up to 30 % of dietary Maillard products are absorbed in the gut

[186], [187]. In the gut, NF-κB is a key regulator of the intestinal immune system.

Perpetuated activation of NF-κB is associated amongst others with inflammatory bowel

diseases (IBD) such as Crohn’s disease and ulcerative colitis [188]. Thus, the activation of

NF-κB by Maillard products and coffee was investigated in intestinal cells as well as

macrophages which are increasingly infiltrated in the mucosa of the intestine during IBD such

as Crohn’s disease [188]. Therefore, time, concentration and cell-type-dependent NF-κB

activation upon cell stimulation with coffee and Maillard products were studied. In detail,

NR8383 macrophages, cells of the immune system, and Caco-2 cells, adenocarcinoma cells

which are derived from the epithelium of the human colon, were investigated. Moreover,

primary cell culture of the endothelium of the human intestine (HIMEC) was analysed.

Furthermore, a method was established to investigate the influence of food components on

intact human gut mucosa tissue ex vivo. The model was then applied to analyse the effects

of coffee and Maillard products on NF-κB activation.

3.2 Results

In this study, various cell lines, primary cells and gut tissue were exposed to Maillard

mixtures, which contain a broad range of Maillard products, roasted coffee extract and raw

coffee extract (Figure 3.2). NF-κB activation was determined due to its nuclear translocation.

Moreover, the role of H2O2 in NF-κB activation by Maillard products was investigated by co-

treatment with catalase.


Roasted coffee extract± catalase

Raw coffee extract

Macrophages Caco-2

Maillard products± catalase

HIMEC Human gut tissue

Roasted coffee extract± catalase

Raw coffee extract

Macrophages Caco-2

Maillard products± catalase

HIMEC Human gut tissue

Figure 3.2: Experimental design for NF-κB analysis after stimulation with coffee extracts or Maillard products.

3.2.1 Cell growth and cell viability of macrophages

Cell growth of NR8383 macrophages was determined under cell culture conditions to

evaluate the cell growth characteristics. Therefore, 3 x 106 or 4.5 x 106 cells were seeded in

cell culture flasks and incubated without any stimulant for 96 h at 37°C.

0 20 40 60 80 1000

5

10

15

203 x 106

4.5 x 106

Growing time [h]

Cel

l num

bers

x 1

06

Figure 3.3: The cell growth of NR8383 macrophages was determined. Cells (3 x 106 or 4.5 x 106) were seeded in

cell culture flasks for 96 h at 37°C and cell numbers were counted every 24 h for 4 days with a counting chamber.

Data is mean ± range (n = 2).


As illustrated in Figure 3.3, with a starting cell number of 3 x 106, cell growth increased over

the entire culture period. A starting cell number of 4.5 x 106, on the other hand, cells reached

a plateau phase after 72 h after which cell growth stagnated. The doubling time for a starting

cell number of 3 x 106 was 43 h and for 4.5 x 106 was 47 h. For further experiments, a

starting cell number of 3 x 106 was chosen to assure optimal cell growth conditions without

exceeding the cell saturation density for at least 96 h.

In order to investigate cell viability, NR8383 macrophages were incubated with Mrmh (10 -

100 mM) for 2 h in phosphate buffered saline (PBS buffer). According to the MTT assay, cell

viability was significantly reduced reaching values between 49 ± 11 % and 33 ± 6 % cell

viability depending on the Mrmh concentration. Co-treatment with catalase increased the cell

viability between 16 % and 26 %. In another experiment, NR8383 macrophages were

incubated in supplement-free cell culture medium with Mrmh (10 - 100 mM) for 24 h. Cell

viability of NR8383 macrophages was not negatively impaired but cell growth was rather

enhanced after incubation with low concentrations of Mrmh. After exposure to 25 mM Mrmh,

cell viability was about 173 ± 23 %. The supplementation of catalase did not show any

significant impact on cell viability of Mrmh-treated NR8383 macrophages. However, 50 mM

and 100 mM Mrmh possessed cytotoxic potency (59 ± 21 and 23 ± 7 % cell viability) which

was reduced by co-treatment with catalase (121 ± 15 and 42 ± 5 % cell viability). In order to

test the influence of the incubation medium itself on the cell viability, NR8383 macrophages

were incubated without any stimulants in supplement free medium, PBS, PBS/catalase or

supplemented cell culture medium for 2 h. The lack of foetal calf serum (FCS) (supplement

free medium) reduced the cell viability to 62 ± 24 % compared to the cell viability in

supplemented medium. After incubation in PBS buffer, less than 50 % of the NR8383

macrophages (44 ± 7 %) were viable compared to the incubation in supplemented cell

culture medium. Exposure to catalase in PBS did not significantly influence cell viability

compared to the cell viability in PBS buffer alone.


Thus, short-term experiments for 2 h were conducted in PBS to fully exclude any effects of

the cell culture medium. However, long-term experiments for up to 24 h were carried out in

supplement-free medium to assure optimal cell viability with minimal potential effects of the

cell culture medium.

3.2.2 NF-κB activation by Maillard products

3.2.2.1 Stimulation of different cell types with Maillard products

NR8383 macrophages and Caco-2 cells were exposed to Mrmh (25 mM) for 2 h. Indeed, NF-

κB was significantly activated in both macrophages and Caco-2 cells (Figure 3.4). However,

the level of NF-κB activation was considerably higher in macrophages (5-fold) compared to

Caco-2 cells (2-fold). Moreover, it was shown that NF-κB activation in Caco-2 cells increased

with raising Mrmh-concentration. Next, NF-κB activation was determined in primary cell

culture of human intestinal endothelial cells (HIMEC) after exposure to Mrmh. Mrmh up-

regulated NF-κB 2-fold after 2 h compared to PBS treated control cells (Figure 3.4).

Control 25 25 25 25 50 100 100 25 250

1

2

3

4

5

6

- - + - + - - + - + Catalase

Mrmh [mM]

****

**

**

* *

Macrophages (B) Caco-2 (C) HIMEC (D)

***

A

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol


B

Control

-

25 mM

-

25 mM

+

p65

β-actin

[Mrm]

Catalase

D

Control 25 mM

p65

β-actin

[Mrm]

Control

-

25

-

50

-

100

-

p65

β-actin

25

+

100

+

C

Mrmh [mM]

Catalase

B

Control

-

25 mM

-

25 mM

+

p65

β-actin

[Mrm]

Catalase

B

Control

-

25 mM

-

25 mM

+

p65

β-actin

[Mrm]

Catalase

D

Control 25 mM

p65

β-actin

[Mrm]

D

Control 25 mM

p65

β-actin

[Mrm]

Control

-

25

-

50

-

100

-

p65

β-actin

25

+

100

+

C

Mrmh [mM]

Catalase

Control

-

25

-

50

-

100

-

p65

β-actin

25

+

100

+

C

Mrmh [mM]

Catalase

Figure 3.4: NF-κB activation was investigated in various cell types. NR8383 macrophages, Caco-2 cells and

HIMEC were stimulated with different concentrations of Mrmh for 2 h. In the case of catalase co-treatment,

catalase (150 U/mL) was added 10 min prior to stimulation. (A) The intensity of the p65 signal (NF-κB subunit)

was related to the loading control β-actin and expressed as n-fold increase compared to PBS treated control cells.

Data is mean ± SD (n = 2 - 8). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blots of p65 and

β-actin in NR8383 macrophages (B), in Caco-2 cells (C) and HIMEC (D).

3.2.3 NF-κB activation by coffee extract

3.2.3.1 Stimulation of different cell types with coffee extract

NR8383 macrophages were incubated with 2 mg/mL coffee extract between 30 min and 6 h.

Coffee extract up-regulated nuclear NF-κB amounts in NR8383 macrophages in a time

dependent manner reaching a maximum of a 3-fold up-regulation after 2 h (Figure 3.5A).

Thereafter, the activation declined slightly, but nuclear NF-κB concentration was still

significantly up-regulated after 6 h. A cell viability higher than 90 % during the entire


incubation period was determined by the trypan blue dye exclusion test. NF-κB activation

was not only time but also concentration dependent (Figure 3.5C). In fact, 1 mg/mL coffee

extract did not activate NF-κB in macrophages after 2 h, while 4 mg/mL coffee extract

caused a 4-fold up-regulation. Cell viability, which was measured by the trypan blue dye

exclusion test, exceeded 90 % of vital cells for all tested concentrations. Given that coffee

extract activated NF-κB in macrophages, its impact on the NF-κB translocation in Caco-2

cells was investigated likewise. However, coffee extract did not induce NF-κB translocation in

Caco-2 cells after an incubation period of 2 h, which was shown to be the incubation period

causing maximum NF-κB activation in macrophages (Figure 3.5E).

Control 0.5 1 2 4 60

2

4

6

8

0

50

100

***

***

***

***

A

1

Incubation time [h]

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

Cell viability [%

]

Control 1 h

p65

β-actin

Proteinmarker

Incubation time

4 h2 h 6 h0.5 h

B

81.8 kDa

68.5 kDa

55.0 kDa

Control 1 h

p65

β-actin

Proteinmarker

Incubation time

4 h2 h 6 h0.5 h

B

81.8 kDa

68.5 kDa

55.0 kDa


Control 1 mg/mL 2 mg/mL 4 mg/mL0

2

4

6

8

10

0

50

100

*** ***

C

Coffee extract

1

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

Cell viability [%

]

Control 4 mg/mL2 mg/mL1 mg/mL

p65

β-actin

Proteinmarker

Coffee extract

D81.8 kDa

68.5 kDa

55.0 kDa


p65

β-actin

Proteinmarker

Coffee extract

D81.8 kDa

68.5 kDa

55.0 kDa


Control 1 mg/mL 2 mg/mL 4 mg/mL 100 ng/mL0

2

4

6

8

TNFαCoffee extract

E

1Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

p65

β-actin


Coffee extract TNFα

100 ng/mL

F

p65

β-actin



100 ng/mL

p65

β-actin



100 ng/mL

F

Figure 3.5: NF-κB activation was investigated in (A - D) NR8383 macrophages and (E) Caco-2 cells. Cells were

stimulated with coffee extract (1 - 4 mg/mL) or TNF-α as positive control for 0.5 - 6 h. (A/C/E) The intensity of the

p65 signal (NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared

to water treated control cells (bars). Cell viability was assured by trypan blue dye exclusion test (dots). Data is

mean ± SD/range (A: n = 3 - 5; B: n = 3; C: n = 2). * p < 0.05, ** p < 0.01, *** p < 0.001. Representative Western

blots of p65 and β-actin in (B/D) NR8383 macrophages and Caco-2 cells (F). The p65 signal was identified via

p65-anti blocking peptide.

3.2.3.2 Stimulation of macrophages with raw coffee extract

NR8383 macrophages were incubated with 2 mg/mL roasted or raw coffee extract for 2 h.

Roasted but not raw coffee extract significantly activated NF-κB (Figure 3.6). After


stimulation of macrophages for 2 h with roasted coffee extract, the nuclear NF-κB amount

was increased 3-fold.

0

1

2

3

4

5

Control Coffee extract Raw coffeeextract

Coffee extract+

catalase

Coffee extract+

heat-inactivatedcatalase

***

***

A

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

Con

trol

p65

β-actin

Prot

ein

mar

ker

Cof

fee

extra

ct

Cof

fee

extra

ct +

cat

alas

e

Cof

fee

extra

ct +

in

activ

ated

cat

alas

e

Raw

cof

fee

extra

ct

B

81.8 kDa

68.5 kDa

55.0 kDa

Con

trol

p65

β-actin

Prot

ein

mar

ker

Cof

fee

extra

ct

Cof

fee

extra

ct +

cat

alas

e

Cof

fee

extra

ct +

in

activ

ated

cat

alas

e

Raw

cof

fee

extra

ct

B

81.8 kDa

68.5 kDa

55.0 kDa

Figure 3.6: NF-κB activation was investigated in NR8383 macrophages. Cells were stimulated with coffee extract

(2 mg/mL) or raw coffee extract (2 mg/mL) for 2 h. In the case of catalase co-treatment (150 U/mL), catalase or

heat-inactivated catalase (5 min, 95°C) was added 10 min prior to stimulation. (A) The intensity of the p65 signal

(NF-κB subunit) was related to the loading control β-actin and expressed as n-fold increase compared to water


treated control cells. Data is mean ± SD (n = 4 - 23). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative

Western blot of p65 and β-actin in macrophages after stimulation with coffee extract ± (heat-inactivated) catalase.

The p65 signal was identified via p65-anti blocking peptide.

3.2.4 Ex vivo stimulation of intact human gut tissue

3.2.4.1 Establishing a method for ex vivo stimulation of intact human gut tissue

Since the induction of nuclear translocation of NF-κB by coffee and Maillard products proved

to be strongly dependent on the cell type, stimulation should be repeated using intact human

gut tissue. Thereby, the actual cellular composition of human gut as well as the complex

cellular interaction can be investigated. Since ex vivo stimulation of human tissue is not an

established model for nutritional studies, uniformity of the tissue samples as well as cell

viability during ex vivo stimulation was studied. The protein content was used as a criterion to

prove the uniformity of the tissue samples. Thus, the total and the nuclear protein

concentrations of the human gut tissue samples were measured. A correlation between the

protein concentration and the wet weight of human gut tissue samples was calculated. Both

the total and the nuclear protein concentration increased with the wet weight of the tissue

samples (Figure 3.7). The correlation coefficient r according to Pearson was 0.7089 for the

total protein concentration and 0.8185 for the nuclear protein concentration.


0 2 4 6 8 100.0

0.2

0.4

0.6

0.8

A

Biopsy wet weight [mg]

Tota

l pro

tein

con

cent

ratio

n[m

g/m

L]

5 10 15 20 25-0.5

0.0

0.5

1.0

1.5

2.0

2.5

B

Biopsy wet weight [mg]

Nucl

ear p

rote

in c

once

ntra

tion

[mg/

mL]

Figure 3.7: The (A) total and the (B) nuclear protein concentrations were detected in each human gut tissue

sample and correlated to its wet weight. Data is expressed as xy pairs with n = 32 for total and n = 19 for nuclear

protein determinations. The correlation coefficient r was calculated according to Pearson with r = 0.7089 for the

total protein concentration and r = 0.8185 for the nuclear protein concentration.

Cell viability of the tissue cells during mucosa oxygenation ex vivo was investigated with the

lactate dehydrogenase assay (LDH) assay. The release of LDH into the supernatant was


associated with cell membrane damage and thus cell death and cell viability respectively.

Therefore, intact human gut tissue samples were kept in modified PBS or Hank’s balanced

salt solution (Hanks) for mucosa oxygenation between 0.5 and 24 h. The amount of secreted

LDH into the supernatant compared to the overall LDH amount raised time-dependently in

both media indicating a decrease in cell viability over time (Figure 3.8). After 4.5 h, cell

viability was higher than 75 % in both media. However, after 6 and 24 h cell viability was

about 50 % and nearly 0 % respectively, independent of the cell culture medium. The

difference in cell viability between the both media was not significant. A similar LDH release

was obtained when one single human gut tissue sample was monitored between 0.5 and

24 h (data not shown). According to these results, which again prove that the individuality of

the biopsies can be neglected, a stimulation time of 2 h was chosen for further experiments

avoiding notable cell death during the ex vivo incubation.

0

20

40

60

80

100 PBSHanks

1.5 3 4.5 6 24Incubation time [h]

Cel

l via

bilit

y [%

]

Figure 3.8: Time-dependent cell viability of human gut tissue samples was measured during mucosa oxygenation

ex vivo. The secretion of lactate dehydrogenase (LDH) into the supernatant was used as an indicator of cell

death/cell viability. Briefly, for each time point (up to 24 h) a single human mucosal gut specimen was incubated in

modified PBS or Hank’s balanced salt solution (Hanks). LDH concentration was measured in the supernatant and

the tissue. The cell viability was calculated via cell death values which are expressed as the ratio between LDH

concentrations in the supernatant and the overall LDH concentration. Data is expressed as mean ± SD (n = 2 - 3).


3.2.4.2 Stimulation of intact human gut tissue with Maillard products and coffee extract

Human gut tissue samples were stimulated with Maillard products from Mrmh for 2 h during

mucosa oxygenation ex vivo. Mrmh significantly activated NF-κB compared to the control

specimen in a concentration dependant manner (Figure 3.9). A 2.8-fold increase in NF-κB

translocation was detected with the highest Mrmh (100 mM) concentration.

Control 10 25 25 50 100 1000

1

2

3

4

**

- - - + - - + Catalase

Mrmh [mM]

A

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

Control

-

25

-

50

-

100

-

p65

β-actin

100

+

10-

B

Mrmh [mM]

Catalase

25

+

Control

-

25

-

50

-

100

-

p65

β-actin

100

+

10-

B

Mrmh [mM]

Catalase

25

+

Figure 3.9: NF-κB activation was investigated in human gut tissue ex vivo. The specimens were stimulated with

Mrmh (10 - 100 mM) in modified PBS for 2 h. In the case of catalase co-treatment, catalase (150 U/mL) was

added 10 min prior to stimulation. (A) The intensity of the p65 signal (NF-κB subunit) was related to the loading

control β-actin and expressed as n-fold increase compared to PBS treated control. Data is mean ± SD (n = 4 - 7).

* p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blots of p65 and β-actin in mucosal human

tissue samples of the gut. The p65 signal was identified by p65 anti-blocking peptide.


The experiment was repeated with coffee extract as stimulant. Coffee extract induced a 2-

fold higher NF-κB translocation compared to the control. The difference, however, was not

statistically significant (Figure 3.10).

Control 1 2 40

1

2

3

4

Coffee extract [mg/mL]

Nucl

ear N

F-kB

/ [β

-act

in]

fold

incr

ease

com

pare

d to

con

trol

Figure 3.10: NF-κB activation was investigated in human gut tissue ex vivo. The specimens were stimulated with

coffee extract (1 - 4 mg/mL) in modified PBS for 2 h. The intensity of the p65 signal (NF-κB subunit) was related

to the loading control β-actin and expressed as n-fold increase compared to water treated control cells. Data is

mean ± SD (n = 3 - 5).

3.3 Discussion

NF-κB is a multifunctional transcription factor which is involved in diverse cellular reactions

such as inflammation, immune function or cell survival [30]. In the healthy gut, NF-κB is a

critical factor of the intestinal immune system. Inflammatory bowel diseases (IBD), such as

Crohn’s disease or ulcerative colitis, are associated with a chronic activation of NF-κB [188].

Therefore, a balanced regulation of NF-κB is a crucial factor to maintain or attain gut health.

Among food products, coffee seems to have a major influence on NF-κB regulation. In LPS-

activated cell lines, coffee extract as well as coffee diterpene kahweol alone reversed NF-κB

activation [183], [189]. A similar effect of coffee was also observed in transgenic NF-κB-

luciferase mice [183]. In the absence of co-stimulants, coffee extract rather activated NF-κB


in macrophages [25]. Both effects, NF-κB down regulation of LPS-activated cells and NF-κB

activation in unstimulated macrophages seem to be very dependent on Maillard products

[25], [183]. These results are supported by a study on bread crust, which is another example

for food rich in Maillard products, triggering up-regulation of NF-κB in cardiac fibroblasts

[190].

Intestinal digestion and metabolism of foods are characterized by diverse interaction of

various cell types. Therefore, the present study focused on the influence of coffee and

Maillard products on NF-κB regulation in several cell types present in the intestine.

Furthermore, intact human gut tissue is proposed as a mean to study the effect of coffee

stimulation on complex cell structures ex vivo. Since mixtures of Maillard products as well as

coffee generate H2O2, the role of H2O2 in the NF-κB activation was investigated likewise.

Roasted coffee extract, but not raw coffee extract, as well as Maillard products produced in

Mrmh significantly up-regulated NF-κB in macrophages in a concentration and time

dependent manner. In Caco-2 cells, which are colorectal epithelial cells, stimulation with

different concentrations of coffee extract in contrast did not lead to an enhanced nuclear

translocation of NF-κB whereas Mrmh significantly increased nuclear amounts of NF-κB. The

response in Caco-2 cells after Mrmh stimulation was considerably lower compared to the

effect in macrophages. Additionally HIMEC, human intestinal microvascular endothelial cells,

which are primary human intestinal cells, were treated with Mrmh, since it has to be expected

that the metabolisms of immortalized cell lines differ from those of in vivo. As a matter of

fact, NF-κB responded not only in multiple cell lines to Maillard products but also in primary

intestinal cells which are a more representative model for cellular reaction in vivo. In earlier

studies it was shown that Maillard products induce NF-κB translocation in kidney cells [25].

Besides, individual Maillard products such as aminoreductones [171] and glycated ovalbumin

[191] were already identified as active triggers of NF-κB in macrophages and dendritic cells

respectively. Thus, it can be summarized that Maillard products can induce nuclear


translocation of NF-κB in various cell types. The degree of NF-κB activation however, varied

depending on the cell type showing the maximum effect in macrophages. NF-κB activation

by coffee was lower under the applied conditions and only visible in macrophages.

The in vitro screening raised the question of how NF-κB responds in human intestinal tissue

which consists of a wide range of cell types. Moreover, the human intestine, as part of the

alimetary organ, represents a primary target for food-induced inter- and intracellular reactions

of the human body. The mucosa layer of the gut consists of a variety of cell types including

immune cells such as macrophages which were found to show the highest NF-κB response.

Particularly during IBD such as Crohn’s disease, macrophages are increasingly infiltrated in

the mucosa of the intestine. Not only IBD but also food allergies are triggered by a specific

reaction of the immune system of the gut after food intake. Therefore, the immunomodulatory

effect of coffee extract and Maillard products was studied in the mucosa of the gut, as the

largest immunological organ of the body.

First, a method was established which allows the stimulation of human gut tissue ex vivo

[192], [193]. It was guaranteed that (i) the tissue samples per se were uniform and

comparable and (ii) the cell viability of the tissue samples was not harmfully affected by the

mucosa oxygenation ex vivo. Whereas Maillard products formed in Mrmh elevated nuclear

NF-κB levels in gut tissue significantly after 2 h, coffee extract showed only a trend towards

activation. In agreement with the in vitro studies, both coffee and Maillard products showed

similar effects on nuclear NF-κB translocation in human gut tissue, but with differences in the

level of activation. The discrepancy between Mrmh and coffee may be attributed to different

concentrations and compositions of active Maillard products in coffee and Mrmh, but also to

coffee ingredients which may counteract NF-κB activation such as kahweol and 3-methyl-

1,2-cyclopentanedione [189].

Furthermore, mechanisms were investigated which may explain how NF-κB activation in

different cell types and complex human gut tissue can be affected by coffee and Maillard


products. In this context, H2O2 seems to be a major factor of cell signalling induced by coffee

and Maillard products. Indeed, H2O2 has been recognized as an important second

messenger in redox signalling [194]. In fact, co-treatment with catalase blocked NF-κB

activation for both Mrmh and coffee extract incubations in macrophages [25]. Similar

suppression was achieved in the present study for macrophages, Caco-2 cells and HIMEC

for Mrmh and coffee extract respectively. Heat-inactivated catalase on the other hand, did not

show any impact on coffee-induced NF-κB activation in macrophages. In human gut tissue,

co-treatment with catalase reduced but not completely reversed NF-κB activation induced by

Mrmh and coffee extract respectively. Since extracellular catalase can not penetrate cellular

membranes, its action is restricted to the decomposition of extracellular H2O2 [195]. It is well

documented that coffee and Maillard products are able to generate H2O2 in a cell free system

[25], [73], [196], [197]. Extracellular H2O2 would then be able to penetrate the cell, activating

cell signalling including NF-κB. Indeed, supplements of pure H2O2 were shown to trigger the

activation of NF-κB [198]. The susceptibility of cells and tissue samples to nuclear NF-κB

translocation is then dependent on the activity of the stimulant to generate H2O2 and on the

ability of H2O2 to diffuse through cell membranes which is mediated by aquaporins [199].

Besides Maillard-dependent H2O2 generation, alternative sources of H2O2 such as the

NADPH oxidase might also influence H2O2-triggered cellular reactions [200]. Cells can

detoxify ROS such as H2O2 by antioxidative enzymes such as glutathione peroxidase,

peroxiredoxins or catalase and numerous non-enzymatic antioxidants such as glutathione.

Thus, also the cell-specific antioxidative capacity will have a major impact on the level of

H2O2 and thus on the level of NF-κB activation.

4 NUCLEAR TRANSLOCATION OF NRF2 82

4 NUCLEAR TRANSLOCATION OF NRF2

4.1 Introduction

The health risks and benefits of coffee drinking were intensively studied in several

epidemiological studies worldwide within the last decades. However, an unambiguous

assessment of the health effect of coffee is impeded by the fact that the compositions of

coffee compounds vary due to different species of coffee beans, brewing methods and also

polymorphism in metabolism and lifestyles of coffee drinkers. Several cohort- and case-

control studies were published between 2000 and 2004. The majority of the epidemiological

studies suggested an inverse risk of developing several chronic diseases such as type 2

diabetes mellitus, Parkinson’s disease, colorectal and liver cancer for coffee drinkers [201].

Likewise, a decreased risk was found in dementia with a specific focus on Alzheimer’s

disease [202]. Moreover, in vivo studies in models of Alzheimer’s disease and Parkinson’s

disease in drosophila [203] and in rats [204] with either coffee brew or coffee components

such as caffeine profoundly underline the protective effect of coffee.

The above mentioned diseases most commonly occur with age. The underlying mechanisms

are not fully understood but are considered to be associated with inflammation and oxidative

stress [205]. The altered redox homeostasis during oxidative stress can be due to either a

weakened antioxidant defence or increased ROS production. Indeed, brewed coffee was

ranked sixth in the amount of total antioxidants among 1113 foods in the US, promising

counteractive capability against oxidative stress. [40] Since antioxidative phytochemicals

such as chlorogenic acid degrade during roasting (Figure 1.3) [206], the antioxidant capacity

of coffee brew was attributed only to a minor extent to residual chlorogenic acid but primarily

to products which were formed during the roasting process such as the Maillard products

[207]. The Maillard products are considered to be a major part of the physiological active


compounds of coffee. Indeed within the last decades, various Maillard products with

antioxidative activity were identified. Several studies focused on the antioxidant capacity of

coffee and especially of Maillard products emphasizing an oxygen scavenging and a chain

breaking activity [162], [207], [208].

Interestingly, the antioxidant activity was not only detected in the beverage but also in human

plasma after coffee consumption [209]. In this context, it has been previously reported that

Maillard products are bioavailable with an absorption rate of up to 30 %. Thus, the

antioxidant capacity after coffee consumption might be attributed to absorbed Maillard

products. However, the antioxidant activity of Maillard products is unlikely to explain all the

beneficial effects, especially since metabolism of these compounds in vivo may lead to

altered antioxidant properties.

It has been suggested that the favourable action of coffee also might be due to the enhanced

transcription of cytoprotective proteins. Indeed, the concentration of glutathione (GSH), an

endogenous antioxidant, was increased in plasma after coffee consumption [210]. Moreover,

pre-incubation of HepG2 cells with coffee-specific Maillard products increased the

concentration of intracellular GSH. [211] GSH is synthesized enzymatically by γ-glutamyl-

cysteine- and glutathione-synthetase; enzymes which are both regulated by Nrf2. The redox-

sensitive transcription factor Nrf2 is ubiquitously expressed in the tissue with extensively high

levels in the intestine, lung and kidney [212], [213]. In non-activated cells, Nrf2 is bound to

the repressor Kelch-like ECH-associated protein 1 (Keap1) causing ubiquitination and de-

gradation of Nrf2. Since Keap1 itself is anchored to the actin cytoskeleton, the inactive

complex is restricted to the cytoplasm. However during activation, Nrf2 separates from

Keap1. To date, two Nrf2 activation mechanisms (stabilization and phosphorylation) are

proposed, each responsible for inhibiting Keap1-dependent Nrf2 degradation. Firstly, ROS

and electrophiles might stabilize cytoplasmic Nrf2 by modifying cysteine thiols of Keap1

(stabilization). Secondly, phosphorylation of serine and threonine residues of Nrf2 via protein


kinases is also suggested to cause Nrf2 release from Keap1 (phosphorylation) (Figure 4.1).

cytoplasm

Stimulus

nucleus

Nrf2

Nrf2 target genetranscription

Keap1

Nrf2 OH

Cys-SHHS-Cys

Keap1

Nrf2 OH

Cys**Cys

Keap1

Nrf2 P

Cys-SHHS-Cys

Nrf2 OH

Nrf2 P

ARE/EpRE

Nrf2 stabilization Nrf2 phosphorylation

cytoplasm

Stimulus

nucleus

Nrf2


Keap1

Nrf2 OH

Cys-SHHS-Cys

Keap1

Nrf2 OH

Cys**Cys

Keap1

Nrf2 P

Cys-SHHS-Cys

Nrf2 OH

Nrf2 P

ARE/EpRE


cytoplasm

Stimulus

nucleus

Nrf2


Keap1

Nrf2 OH

Cys-SHHS-Cys

Keap1

Nrf2

Keap1

Nrf2 OH

Cys-SHHS-Cys

Keap1

Nrf2 OH

Cys**Cys

Keap1

Nrf2

Keap1

Nrf2 OH

Cys**Cys

Keap1

Nrf2 P

Cys-SHHS-Cys

Keap1

Nrf2

Keap1

Nrf2 P

Cys-SHHS-Cys

Nrf2 OHNrf2 OH

Nrf2 PNrf2 P

ARE/EpRE


Figure 4.1: Simplified illustration of the activation of the Nrf2 pathway (modified [214]). Nrf2 = Nuclear factor-

erythroid-2-related factor 2, Keap 1 = Kelch-like ECH-associated protein 1, Cys = Cysteine, Cys* = modified

cysteine, ARE = antioxidant response element, EpRE = electrophile response element

After detachment, Nrf2 translocates into the nucleus and binds to the antioxidant response

element (ARE)/the electrophile response element (EpRE). Genes which possess these

elements in their promoter regions are thus regulated by Nrf2. Besides γ-glutamylcysteine

synthetase and glutathione synthetase, phase II target genes such as nicotinamide adenine

dinucleotide (phosphate) (NAD(P)H):quinone oxidoreductase 1, catalase and thioredoxin are

up-regulated by Nrf2. [214] These proteins are responsible for detoxification and antioxidant

capacities of the cell which might counteract the unbalanced redox homeostasis during


oxidative stress. Thus, dietary activation of Nrf2 may reverse biochemical dysfunctions which

result from diseases related to oxidative stress [215].

Activation of Nrf2 in the gut appeared to have several beneficial effects since inflammatory

bowel diseases (IBD) are characterized by an imbalance between reactive oxygen species

and mucosal antioxidant response [216]. Indeed, as shown in a mouse model, activators of

Nrf2 such as prohibitin can improve colitis [217]. Enhanced intestinal Nrf2 signalling was

further associated with a beneficial effect on gut barrier dysfunction [218], intestinal mucosal

injury [219] and intestinal inflammation other than colitis [218]. Moreover, there is growing

evidence that Nrf2 is also a promising target for the prevention of colorectal cancer [220],

[221].

In the present study, the impact of coffee extract and Maillard products on Nrf2 translocation

was screened in vitro in intestinal Caco-2 cells and macrophages which are important

intestinal tissue components. Furthermore, the Nrf2 activation was investigated ex vivo in

human gut tissue samples.

4.2 Results

The purpose of this study was to investigate the activity and the mechanisms of Maillard

products and coffee to induce nuclear translocation of Nrf2 in various cell lines and intact

human gut tissue. Therefore, Maillard products were generated from ribose and lysine under

conditions which have been previously applied to investigate physiological effects of Maillard

products [73], [174].

4.2.1 Detection of early, intermediate and late stage Maillard products in

Maillard mixtures

According to Hodge (1953), early, intermediate and late stage Maillard products are formed

during the Maillard reaction. The nature of Maillard products as well as the reaction rate is


crucially influenced by the reaction temperature showing a decelerated reaction rate at low

temperatures such as room temperature [44]. A Maillard reaction mixture heated at 120°C

(Mrmh) and an unheated Maillard reaction mixture (Mrmu) were prepared as previously

described. The content of early, intermediate and late stage Maillard products was detected

photometrically in both mixtures (Figure 4.2). As expected, Mrmh contained early to late

stage Maillard products whereas Mrmu consisted mainly of early and intermediate stage

Maillard products and only to a minor extent of late stage Maillard products.

Mrmu Mrmh

0.1

1

10

100

1000

280 nm

350 nm420 nm

Abso

rban

ce U

nit

Figure 4.2: The Absorbance Unit of the heated (Mrmh) and the unheated (Mrmu) Maillard reaction mixture was

measured at 280 nm, 350 nm and 420 nm to detect early, intermediate and late stage Maillard products

respectively. Data is mean ± SD (n = 3)

4.2.2 Nrf2 activation by Maillard products

Both the heated and the unheated Maillard reaction mixtures were tested for their Nrf2

activating ability in different cells and tissue during short-term (2 h) and long-term incubation

(2 - 24 h) (Figure 4.3). The activation of Nrf2 was defined as the translocation of

cytoplasmatic Nrf2 into the nucleus. Nuclear Nrf2 was detected by Western blot.


Stimulation

0 h

4.2.2.1 Short-term incubation (macrophages, Caco-2 cells, human gut tissue)

2 h 6 h 12 h 24 h

4.2.2.2 Long-term incubation (macrophages)

analysis analysis analysis analysis

4.2.2.2 Short-term + stimulant free post-incubation (macrophages)

Stimulation

0 h


2 h 6 h 12 h 24 h



4.2.2.2 Short-term + stimulant free post-incubation (macrophages)

Figure 4.3: Experimental design for cellular stimulation with Maillard mixtures to investigate Nrf2 translocation

4.2.2.1 Stimulation of different cell types and tissue with Maillard products during

short-term incubation

In order to investigate whether Maillard products activate Nrf2 in vitro, NR8383 macrophages

and Caco-2 cells were incubated for 2 h with a heated Maillard reaction mixture (Mrmh). Mrmh

significantly induced Nrf2 activation concentration dependently in macrophages and Caco-2

cells (Figure 4.4). The lowest Nrf2 activation was found in Caco-2 cells and the highest in

macrophages reaching a 2-fold and 5-fold up-regulation at 100 mM Mrmh compared to PBS

treated control cells. Moreover, Nrf2 activation in human mucosal gut tissue samples of the

lower gastrointestinal tract of five patients was analysed ex vivo. In the gut tissue samples,

the amount of nuclear Nrf2 was 2-fold higher at 100 mM Mrmh compared to PBS treated

control tissue.


Macrophages Caco-2 Human gut tissue0

2

4

6

8Control10 mM Mrmh

25 mM Mrmh

50 mM Mrmh

100 mM Mrmh

*** ******

***

* *

n.d.1

ANu

clea

r Nrf2

fold

incr

ease

com

pare

d to

con

trol

Figure 4.4: Nrf2 activation by a heated Maillard reaction mixture (Mrmh) was investigated in NR8383

macrophages, Caco-2 cells and human gut tissue samples during short-term incubation. Cells and tissue were

incubated with Mrmh (10 mM - 100 mM) for 2 h. (A) The intensity of nuclear Nrf2 is expressed as n-fold increase

compared to PBS treated control cells/tissue. Data is mean ± SD (macrophages: n = 2 - 5; Caco-2 cells: n = 5 - 6;

tissue: n = 5 - 6). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blot of Nrf2 in NR8383

macrophages, Caco-2 cells and human gut tissue after incubation with Mrmh (10 - 100 mM) for 2 h. N.d. = not

determined.

Macrophages

Caco- 2 cells

Human gut tissue

Control 10 mM 25 mM 50 mM 100 mM

n.d.

B

Caco- 2 cells

Human gut tissue

Control 10 mM 25 mM 50 mM 100 mM

n.

B


4.2.2.2 Stimulation of macrophages with Maillard products during long-term

incubation

It was investigated if a long-term incubation with Mrmh leads to a more pronounced activation

of Nrf2. From the previous experiment, it was known that the NR8383 macrophages were the

most active cells; therefore, the influence of Maillard products on Nrf2 translocation during

prolonged stimulation for up to 24 h was monitored in NR8383 macrophages (Figure 4.5A).

Nuclear Nrf2 level increased consistently in a time and concentration dependent manner. In

the case of 10 mM Mrmh, no influence on Nrf2 translocation was measured over an

incubation period of 24 h. However, exposure of macrophages to ≥ 25 mM Mrmh significantly

enhanced Nrf2 translocation, reaching 50-fold higher Nrf2 levels after stimulation for 24 h

with 50 mM Mrmh compared to PBS treated control cells. Furthermore, Nrf2 activation by

caramelization products, which are formed in sugar-rich foods besides Maillard products

during heat-treatment [161], was investigated. For this purpose, macrophages were

incubated with a heated ribose solution (100 mM). Nrf2 was not activated after stimulation

with the heated ribose solution for 24 h (data not shown). Thus, the observed Nrf2 activation

triggered by Mrmh was attributed to Maillard products and not to caramelization products. It

was confirmed by the MTT assay that the cell viability after 24 h was not attenuated but

rather enhanced by the incubation with low concentrations of Mrmh; only 50 mM Mrmh led to

a cell viability of 60 ± 21 % after 24 h.

In order to study how Maillard products induce the nuclear translocation of Nrf2, it was

investigated whether long-term activation of Nrf2 depends on the continuous presence of the

Maillard products as stimulant. Thus, macrophages were incubated for 2 h with 25 mM Mrmh

but instead of analysing the nuclear Nrf2 amount directly after stimulation, macrophages

were post-incubated without any stimulant for another 22 h at 37°C and Nrf2 was assayed

thereafter. Indeed, the nuclear Nrf2 levels continued to increase even in the absence of the

stimulus to about 8-fold higher levels compared to the experiment without post-incubation


(Figure 4.6). After continuous stimulation for 24 h with 25 mM Mrmh, the Nrf2 levels were 26-

fold increased. Taking these results into consideration, it can be suggested that Nrf2

translocation is triggered by Mrmh via a signal transduction chain which is still activated

during stimulant-free post-incubation. It was demonstrated that the cell viability was not

reduced but increased after incubation with Mrmh (25 mM) for 24 h reaching a cell viability of

173 ± 23 % compared to PBS treated control cells.

To verify whether nuclear translocation of Nrf2 was indeed induced by Maillard products and

not by a high concentration of amino acids and sugars, an unheated mixture of ribose and

lysine (Mrmu) was tested in the same way as Mrmh. The unheated Mrmu consisted of low

amounts of early and intermediate stage Maillard products, whereas the heated Mrmh

contained higher amounts of early and intermediate stage Maillard products plus advanced

Maillard products. Indeed, not only the Mrmh but also the Mrmu activated Nrf2 in

macrophages (Figure 4.5C). Mrmu up-regulated Nrf2 in a concentration and time dependent

manner causing nearly 20-fold higher levels of nuclear Nrf2 after stimulation with 50 mM

Mrmu for 24 h compared to PBS treated control cells. However, the nuclear Nrf2 level was

significantly lower after stimulation with Mrmu than after exposure with the equivalent

concentration of Mrmh.


0

20

40

60

80Control

10 mM Mrmh

25 mM Mrmh

50 mM Mrmh

2 6 12 24

***

**

****

****

A

Incubation time [h]

Nucl

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rf2fo

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crea

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ompa

red

to c

ontro

l

2 h

6 h

12 h

24 h

Control 10 mM

B

25 mM 50 mM

Mrmh

2 h

6 h

12 h

24 h

Control 10 mM

B

25 mM 50 mM

Mrmh


0

20

40

60

80 Control25 mM Mrmu

50 Mrmu

C

2 6 12 24 Incubation time [h]

Nucl

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crea

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to c

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2 h

6 h

12 h

24 h

Control 50 mMMrmu

25 mMMrmu

D

Control

2 h

6 h

12 h

24 h

Control 50 mMMrmu

25 mMMrmu

D

Control

Figure 4.5: Nrf2 activation by (A) a heated Maillard reaction mixture (Mrmh) and (C) an unheated Maillard reaction

mixture (Mrmu) was investigated in NR8383 macrophages during long-term incubation. Cells were stimulated with

Mrmh (10 mM - 50 mM) or Mrmu (25 mM - 100 mM) for 2 h - 24 h. (A/C) The intensity of Nrf2 is expressed as n-

fold increase compared to PBS treated control cells. Data is mean ± SD (Mrmh: n = 3 - 5; Mrmu: n = 2 - 3). * p <

0.05, ** p < 0.01, *** p < 0.001. (B/D) Representative Western blots of Nrf2 in NR8383 macrophages after

incubation with (B) Mrmh and (D) Mrmu.


Control 2 h Stimulation 2 h Stimulation 24 h Stimulation0

10

20

30

40

+ 22 h stimulant-free

post-incubation

**

***

*

Nucl

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rf2fo

ld in

crea

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ompa

red

to c

ontro

l

Figure 4.6: Nrf2 activation by Mrmh was investigated in NR8383 macrophages. Cells were stimulated with the

heated Maillard mixture (25 mM) for 2 h - 24 h. Nuclear Nrf2 levels were analysed directly after stimulation or after

a following post-incubation at 37°C without stimulant. The cell viability was confirmed by the MTT assay. The

intensity of Nrf2 is expressed as n-fold increase compared to PBS treated control cells. Data is mean ± SD

(n = 2 - 5). * p < 0.05, ** p < 0.01, *** p < 0.001.

4.2.3 Nrf2 activation by coffee extract

In order to investigate whether also natural Maillard products such as the roasting products in

coffee possess the ability to induce Nrf2 activation, different cells and tissue were stimulated

with roasted and raw coffee extract in short-term (2 h) and long-term (2 - 24 h) experiments

(Figure 4.7).

Stimulation

0 h


2 h 6 h 12 h 24 h


analysis analysis analysis analysisStimulation

0 h


2 h 6 h 12 h 24 h



Figure 4.7 Experimental design for cellular stimulation with coffee extract to investigate Nrf2 translocation.


4.2.3.1 Stimulation of different cell types and tissue with coffee extract during short-

term incubation

Nrf2 translocation was determined in various cell lines and tissue after short-term incubation

with coffee extract (Figure 4.8). Stimulation with coffee extract for 2 h showed a slight trend

to activate Nrf2 in macrophages but not in Caco-2 cells and human gut tissue samples.

Macrophages Caco-2 Human gut tissue0

2

4

6Control1 mg/mL Coffee extract2 mg/mL Coffee extract4 mg/mL Coffee extract

1

Nucl

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ld in

crea

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ompa

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to c

ontro

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Figure 4.8: Nrf2 activation by coffee extract was investigated in NR8383 macrophages, Caco-2 cells and human

gut tissue. Cells and tissue were incubated with coffee extract (1 - 4 mg/mL) for 2 h. The intensity of nuclear Nrf2

is expressed as n-fold increase compared to water treated control cells. Data is mean ± SD (macrophages: n = 4 -

8; Caco-2 cells: n = 3; human gut tissue: n = 2 - 5).

4.2.3.2 Stimulation of macrophages with coffee extract during long-term incubation

Nrf2 translocation was monitored during prolonged stimulation of macrophages with coffee

extract for up to 24 h. The maximal concentration of coffee extract tested (4 mg/mL)

corresponds to a regular coffee brew which was diluted 1:3.35. Coffee extract significantly

activated Nrf2 in macrophages during prolonged stimulation (Figure 4.9). The activation of

Nrf2 increased gradually with stimulant concentration and incubation time. Whereas 1 mg/mL

of coffee extract slightly elevated nuclear Nrf2 amounts 2-fold compared to water treated

control cells after incubation for 24 h, 4 mg/mL of coffee extract up-regulated nuclear Nrf2


levels nearly 20-fold. On the contrary, stimulation with raw coffee extract (4 mg/mL), which

does not contain roasting products, did not provoke any convincing Nrf2 activation in

macrophages after 24 h exposure. A cell viability higher than 90 % was assured by the

trypan blue dye exclusion test for coffee extract incubations (1 - 4 mg/mL) for up to 6 h.

0

5

10

15

20

25

2 h

6 h12 h24 h

Control 1 mg/mL 2 mg/mL 4 mg/mL 4 mg/mL

Roasted coffee Raw coffee

* *

* *

*****

***

***

**

A

1

Nucl

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crea

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2 h

6 h

12 h

24 h

Control 1 mg/mL

B

2 mg/mL 4 mg/mL 4 mg/mL


2 h

6 h

12 h

24 h

Control 1 mg/mL

B

2 mg/mL 4 mg/mL 4 mg/mL


Figure 4.9: Nrf2 activation by roasted and raw coffee extract was investigated in NR8383 macrophages. Cells

were stimulated with coffee extract (1 - 4 mg/mL) or raw coffee extract (4 mg/mL) for 2 h - 24 h. A cell viability

higher than 90 % was assured by the trypan blue dye exclusion test for coffee extract incubations (1 - 4 mg/mL)

for up to 6 h. (A) The intensity of Nrf2 is expressed as n-fold increase compared to water treated control cells.

Data is mean ± SD (n = 3 - 8). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western blots of Nrf2 in

NR8383 macrophages after incubation with roasted or raw coffee extract for 2 - 24 h.

4.2.4 Involvement of Maillard-dependent H2O2 in Nrf2 activation

The mechanisms of Nrf2 activation by Maillard products from model mixtures or coffee

remain unknown until now. However, it was previously shown that Maillard products activate

other redox-sensitive transcription factors such as NF-κB via a H2O2-mediated mechanism

[174]. Since Nrf2 is also a redox-sensitive transcription factor with similar activation

mechanism to NF-κB, the involvement of H2O2 in the Nrf2 activation was most likely. Thus,

the role of H2O2 in Nrf2 activation mechanism was investigated.

4.2.4.1 The role of extracellular H2O2 in Nrf2 activation by Maillard products

According to the results listed above (Figure 4.5), both Mrmh and Mrmu significantly activated

Nrf2 in macrophages after long-term incubation. Furthermore, it was previously shown that


both Maillard mixtures generate H2O2 (Chapter 2). Thus, the involvement of H2O2 in the

activation mechanisms of Nrf2 by Mrmh and Mrmu was investigated.

NR8383 macrophages were simultaneously incubated with Mrmh and catalase or heat-

inactivated catalase and Nrf2 translocation was analysed after several time points. As

illustrated in Figure 4.10, co-treatment with catalase but not heat-inactivated catalase

significantly blocked the Mrmh-induced translocation of Nrf2 in macrophages. It was

demonstrated that the cell viability was not reduced but increased after incubation with Mrmh

(25 mM) for 24 h reaching a cell viability of 173 ± 23 % compared to PBS treated control

cells. Co-treatment with catalase increased the cell viability exceeding 200 % cell viability

compared to PBS treated control cells after incubation for 24 h.

Control 25 25 250

20

40

602 h

6 h

12 h24 h

Catalase

Heat-inactivatedcatalase

++

- -

--

* ***

A

--

Mrmh [mM]

Nucl

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crea

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2 h

6 h

12 h

24 h

Control 25 25

B

25 Mrmh [mM]

Catalase


- - + -

- - - +

2 h

6 h

12 h

24 h

Control 25 25

B

25 Mrmh [mM]

Catalase


- - + -

- - - +

Figure 4.10: Nrf2 regulation by Mrmh ± (heat-inactivated) catalase was investigated in NR8383 macrophages.

Cells were stimulated with the heated Maillard reaction mixture (25 mM) for 2 h - 24 h. In some experiments, cells

were co-treated with catalase (150 U/mL) or heat-inactivated catalase (150 U/mL). The cell viability was

confirmed via MTT assay. (A) The intensity of Nrf2 is expressed as n-fold increase compared to PBS treated

control cells. Data is mean ± SD (n = 3 - 5). * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Representative Western

blots of Nrf2 after incubation of NR8383 macrophages with Mrmh (25 mM) ± (heat-inactivated) catalase for 2 h,

6 h, 12 h and 24 h.

Similarly, supplementation of catalase significantly abolished Mrmh-induced Nrf2 activation in

Caco-2 cells (Figure 4.11A) and also showed an inhibitory trend in human gut tissue samples

ex vivo (Figure 4.11C). These data indicate a similar Nrf2 activation mechanism in all tested

cell types and tissue.


Control 25 25 100 1000.0

0.5

1.0

1.5

2.0

2.5

- - + - + Catalase

**

A

Mrmh [mM]

Nucl

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B

Control 25 25 100 100 Mrmh [mM]

Catalase- - + - +

B

Control 25 25 100 100 Mrmh [mM]

Catalase- - + - +

Control 25 25 100 1000

1

2

3

- - + - +

C

Mrmh [mM]

Catalase

Nucl

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D

Control 25 25 100 100

catalase

Mrmh [mM]

- - + - +

D

Control 25 25 100 100

catalase

Mrmh [mM]

- - + - +

Figure 4.11: Nrf2 activation by Mrmh ± catalase was investigated in (A) Caco-2 cells and (C) human gut tissue

samples. Cells and tissue were stimulated with Mrmh (25 mM/100 mM) for 2 h. In some experiments, cells and

tissue were co-treated with catalase (150 U/mL). (A/C) The intensity of Nrf2 is expressed as n-fold increase

compared to PBS treated control cells/tissue. Data is mean ± SD (Caco-2 cells: n = 2 - 6; human gut tissue:

n = 3 - 5). * p < 0.05, ** p < 0.01, *** p < 0.001. (B/D) Representative Western blots of Nrf2 after incubation of (B)

Caco-2 cells and (D) human gut tissue with Mrmh (25 mM/100 mM) ± catalase for 2 h.

Like Mrmh, Mrmu is able to generate H2O2 but to a lesser extent (Chapter 2). Thus, the

involvement of extracellular H2O2 on Nrf2 activation was also investigated after stimulation of

NR8383 macrophages with Mrmu. However, Mrmu-induced Nrf2 activation was not blocked

by co-treatment with catalase (Figure 4.12).

Control 50 500

10

20

302 h

6 h

12 h

24 h

A

Mrmu [mM]

- - + Catalase

Nucl

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2 h

6 h

12 h

24 h

Control 50 50

B

- - +

2 h

6 h

12 h

24 h

Control 50 50

B

- - +

Figure 4.12: Nrf2 regulation by an unheated Maillard mixture (Mrmu) ± catalase was investigated in NR8383

macrophages. Cells were stimulated with the unheated Maillard mixture (50 mM) for 2 h - 24 h. In some

experiments, cells were co-treated with catalase (150 U/mL). (A) The intensity of Nrf2 is expressed as n-fold

increase compared to PBS treated control cells. Data is mean ± SD (n = 2). (B) Representative Western blots of

Nrf2 after incubation with Mrmu (50 mM) for 2 h, 6 h, 12 h and 24 h.

Contrary to Mrmh, Mrmu triggered Nrf2 not via a H2O2-mediated mechanism. Even multiple

addition of catalase did not influence Nrf2 activation excluding that the enzymatic activity of

catalase was not sufficient (data not shown).

4.2.4.2 The role of extracellular H2O2 in Nrf2 activation by coffee extract

Similarly to Mrmh and Mrmu, the effect of co-treatment with coffee extract and catalase on

Nrf2 activation was investigated. NR8383 macrophages were co-treated with coffee extract

and catalase for 24 h. There was a trend showing reduced nuclear Nrf2 levels in the

presence of catalase which was, however, not significant (Figure 4.13).


Control 4 40

5

10

15

20

252 h6 h12 h24 h

- - + Catalase


Nucl

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crea

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lA

2 h

6 h

12 h

24 h

Control 4 4

B


Catalase- - +

2 h

6 h

12 h

24 h

Control 4 4

B


Catalase- - +

Figure 4.13: The Nrf2 regulation by coffee extract ± catalase was investigated in NR8383 macrophages. Cells

were stimulated with coffee extract (4 mg/mL) for 2 h - 24 h. In some experiments, cells were co-treated with

catalase (150 U/mL). (A) The intensity of Nrf2 is expressed as n-fold increase compared to water treated control

cells. Data is mean ± SD (n = 5 - 6). (B) Representative Western blots of Nrf2 after incubation with coffee extract

(4 mg/mL) for 2 h, 6 h, 12 h and 24 h.

4.2.5 The effect of a pure H2O2 solution on Nrf2 translocation

Since extracellular H2O2 played a crucial role in Mrmh-induced Nrf2 activation, the impact of a

pure H2O2 solution on Nrf2 activation was investigated in macrophages. Neither 100 µM nor

500 µM H2O2 did increase the nuclear Nrf2 level in macrophages after incubation for 24 h.


4.3 Discussion

The present study focused on the influence of coffee and Maillard products as active coffee

compounds on the nuclear translocation of Nrf2 in different cell types, present in the

intestine, as well as in intact human gut tissue. Moreover, the role of H2O2 was investigated

since ROS are considered to participate in the activation of Nrf2 [214].

Nrf2 is a transcription factor which is ubiquitously expressed in tissue [213]. The activation of

Nrf2 triggers a pathway regulating the expression of detoxifying and antioxidative active

proteins such as glutathione S-transferase, heme oxygenase 1 and NAD(P)H: quinone

oxidoreductase 1 [214], [222]. Indeed, Nrf2 is classified as therapeutic target in various

diseases such as Parkinson’s disease [223], cardiovascular diseases [224] and type 2

diabetes mellitus [225]. Moreover, the intake of dietary Nrf2 activators is of crucial relevance

in intestinal diseases. In detail, the induction of Nrf2 is considered to reduce the risk of

colorectal cancer [220], [221] and has a beneficial impact on gut barrier dysfunction [218],

intestinal mucosal injury [219] and intestinal inflammation [218] such as colitis [226].

First, the impact of Maillard products on Nrf2 activation was studied after short-term

incubation for 2 h and long-term incubation for up to 24 h. During short-term experiments, a

heated Maillard reaction mixture (Mrmh), which contained high amounts of early, intermediate

and late stage Maillard products, significantly activated Nrf2 in human epithelial colorectal

adenocarcinoma Caco-2 cells and in NR8383 macrophages. The Nrf2 response increased in

a concentration dependent manner showing the highest effect in the NR8383 macrophages.

Interestingly, short-term incubations (2 h) of NR8383 macrophages with Mrmh followed by a

stimulant free post-incubation (22 h) further enhanced Nrf2 activation compared to short-term

incubations alone. Thus, it was suggested that Mrmh exhibits not only direct effects on

individual cell types, but possibly also long-lasting effects by induction of chain reactions

involving Nrf2. To date, two Nrf2 activation mechanisms are proposed for the inhibition of

Keap1-dependent Nrf2 degradation. Firstly, ROS and electrophiles such as H2O2 and


sulforaphane might stabilize cytoplasmic Nrf2 by modifying specific cysteine thiols of Keap1.

Secondly, phosphorylation of serine and threonine residues of Nrf2 via protein kinases such

as protein kinase C (PKC) and c-Jun N-terminal kinases (JNK) is also suggested to cause

Nrf2 release from Keap1. [214] Continuing activation of Nrf2 after removal of the stimulant

points to Nrf2 activation via a signal transduction chain instead of direct interaction with the

Nrf2-Keap1 complex. During long-term incubations of NR8383 macrophages with Mrmh for

24 h, the Nrf2 response increased over time. Very recently, it was shown that a heated

glucose-BSA solution (AGE-BSA) increased nuclear Nrf2 amounts in bovine aortic

endothelial cells. Moreover, an enhanced protein expression of Nrf2-regulated proteins such

as heme oxygenase 1 and NADPH:quinone oxidoreductase 1 was detected [227].

The in vitro screening raised the question of how Nrf2 responds in human intestinal tissue

consisting of a wide range of cell types. The human intestine represents a primary target for

food-induced inter- and intracellular reactions of the human body. The outer mucosa layer of

the gut consists of a variety of cell types including immune cells such as macrophages which

were found to show the highest Nrf2 response. Therefore, the immunomodulatory effect of

Maillard products was studied in the mucosa of the gut, as the largest immunological organ

of the body. Indeed, Mrmh did not only significantly activate Nrf2 in various cell lines but also

in human gut tissue ex vivo indicating a biological relevance of the results obtained in cell

culture experiments.

After co-treatment with catalase, the Mrmh-induced Nrf2 activation was reduced or fully

suppressed in NR8383 macrophages, Caco-2 cells and human gut tissue samples. Since

extracellular catalase can not penetrate the cell, catalase is ineffective against intracellular

but not extracellular H2O2. This observation indicates that extracellular H2O2 was involved in

Mrmh-induced Nrf2 activation. Nrf2 activation by H2O2 has been previously reported in rat

pulmonary microvascular endothelial cells [228]. Moreover, it was shown that Maillard

products generate H2O2 in a cell free system (Chapter 2) [73], [174]. Therefore, it was


assumed that H2O2 formation is not an artificial effect of cell culture in vitro but occurs in

various settings such as food preparation or in the gastrointestinal tract. Also, it was

previously shown that Maillard products trigger NF-κB via a H2O2 mediated mechanism [174].

Both, NF-κB and Nrf2 are redox-sensitive transcription factors with a similar activation

mechanism [229]. Besides a Maillard-dependent H2O2 generation, the NADPH oxidase, a

membrane-bound enzyme, can also liberate ROS into the extracellular space [230]. It was

hypothesized that extracellular H2O2, irrespective of its origin, was not completely detoxified

by the antioxidant system of the cell. Instead, extracellular H2O2 might penetrate the cell. The

increasing intracellular oxidative stress level then may trigger cellular signalling such as the

Nrf2 pathway. Indeed, Surh et al proposed a mechanism for Nrf2 activation involving

intracellular ROS [214]. Interestingly, incubation of macrophages with a single bolus of H2O2

did not activate Nrf2 in macrophages. It was suggested that Nrf2 activation by Mrmh was

rather enhanced by a synergistic activation mechanism of both H2O2 and Maillard products. A

similar effect was described for the DNA breaking ability of Maillard products [231].

To verify whether nuclear translocation of Nrf2 was indeed induced by Maillard products and

not by a high concentration of amino acids and sugars, an unheated mixture of ribose and

lysine (Mrmu) was tested in the same way as Mrmh. Even though the unheated Mrmu also

induced Nrf2 activation in macrophages after long-term incubation, the level of Nrf2

activation was profoundly lower compared to Mrmh-induced Nrf2 translocation. It was

suggested that during the incubation of Mrmu for 24 h at cell culture conditions, the Maillard

reaction takes place forming Maillard products which might activate Nrf2. Interestingly, co-

treatment with catalase did not block Mrmu-induced Nrf2 activation excluding a H2O2-

dependent Nrf2 activation mechanism. Since the Nrf2 activation mechanisms clearly differ

between the heated and the unheated Mrm, it can be concluded that activation of Nrf2 by the

heated Mrmh was not due to high concentrations of amino acids and sugars but rather due to

advanced Maillard products. Moreover, the involvement of caramelization products, which


exist in Mrmh besides Maillard products, can be excluded since a heated ribose solution did

not have any impact on Nrf2 translocation in macrophages.

Since Maillard products clearly induced nuclear translocation of Nrf2, the effect of coffee, a

Maillard-rich beverage, was investigated in the same test system. During coffee roasting,

amine- and sugar-components of the green coffee bean react extensively to give advanced

Maillard products, the so called melanoidins, which comprise about 25 % of the dry matter of

roasted coffee [232]. Thus, it can be assumed that Maillard products in roasted coffee induce

the nuclear translocation of Nrf2 in a similar way to the Maillard mixtures.

After short-term incubation, coffee extract did influence the nuclear Nrf2 amounts neither in

Caco-2 cells nor in human gut tissue but showed a trend of increased Nrf2 activation in

NR8383 macrophages. After long-term incubation however, coffee extract significantly

activated Nrf2 in NR8383 macrophages in a concentration and time dependent manner.

Since Nrf2 translocation increased over time in macrophages, a significant activation of Nrf2

in Caco-2 cells and human gut tissue is very likely after long-term incubation. However, a

cell- and organ-specific discrepancy in the level of Nrf2 activation was also reported in coffee

fed mice [24], [233]. The activation of Nrf2 by coffee was in alignment with studies on human

colon carcinoma cells [28], HepG2 cells transfected with a EpRE-Luc construct [24], [233],

ARE-luciferase reporter cells [27] as well as Nrf2-luciferase mice [24], [233]. The incubation

periods of the in vitro assays were 3 h, 17 h and 24 h respectively. Interestingly, the effect in

human colon carcinoma cells did not show a clear relation to the coffee concentration; in

contrast, the effect of 1 pg/mL coffee extract was even more pronounced than that of 1 or

500 µg/mL. This result indicates that the concentrations present in the human gut may be

sufficient to induce an effect in vivo.

Contrary to roasted coffee extract, raw coffee extract did not up-regulate Nrf2 in NR8383

macrophages. It was shown before that the level of Nrf2 activation is related to the degree of

coffee roasting [24]. Thus, it was suggested that the roasting products, which include Maillard


products, are the active compounds in coffee triggering Nrf2. Besides the Maillard products,

several coffee compounds such as the diterpenes cafestol and kahweol, N-methylpyridinium

(NMP) (Figure 1.2), hydroxyhydroquinone (Figure 1.3) and chlorogenic acid (Figure 1.3)

were identified as potential Nrf2 activators [24], [28], [234]. However, the diterpenes can be

excluded as major activators since both filtered and unfiltered coffee, which differ in their

diterpene content, significantly activated Nrf2 [24]. Furthermore, chlorogenic acid seems to

be of minor relevance since (i) the level of chlorogenic acid content decreases during

roasting whereas Nrf2 activation increases [24] and (ii) raw coffee extract which contains

high amounts of chlorogenic acid did not stimulate Nrf2 translocation. In the case of

hydroxyhydroquinone, the effective concentration was far higher than the amount present in

coffee extract [28]. Thus, none of these compounds could fully explain the coffee-induced

Nrf2 activation emphasizing the role of Maillard products.

The level of Nrf2 activation after coffee exposure was significantly lower compared to Mrmh.

This might be due to (i) variations in the concentration and type of Maillard products present

in the Maillard mixture compared to those in coffee extract and to (ii) coffee characteristic

ingredients not present in the Maillard mixture which do not activate Nrf2 but rather

counteract Nrf2 activation such as trigonelline [28].

Co-treatment with catalase showed only a slight trend to block the coffee-induced Nrf2

translocation indicating that coffee-induced Nrf2 activation can not be fully explained by

Maillard product-generated extracellular H2O2. These findings are consistent with a study on

coffee-induced Nrf2 activation in human colon cells [28].

Irrespective of the mechanism, Nrf2 activation by coffee extract caused an increase in the

level of messenger ribonucleic acid (mRNA) and/or protein expression of NAD(P)H: quinone

oxidoreductase 1, glutathione S-transferase, glutamate cysteine ligase, glutathione and

heme oxygenase 1 in human intestinal cells, liver cells and in primary hepatocytes [24], [27],

[234], [235]. This was also shown in animal studies of coffee-fed mice and rats in whose liver


and/or small intestine increased mRNA levels of NAD(P)H: quinone oxidoreductase 1,

glutathione S-transferase and glutamate cysteine ligase were determined [27], [234]. To

date, only one study is published about the activation of Nrf2 by Maillard products. Enhanced

mRNA levels and protein expression of heme oxygenase 1 and NAD(P)H: quinone

oxidoreductase 1 in bovine aortic cells were determined after treatment with heated glucose-

BSA solution as a consequence of Nrf2 activation [227].

In the conclusion, coffee and especially Maillard products may counteract oxidative stress of

the cell by the transcription of antioxidant and detoxifying enzymes via the Nrf2 pathway.

Thus, coffee and Maillard-rich foods exert not only direct [207], [208] but also indirect

antioxidative activity by inducing the expression of cytoprotective proteins.

5 EXTRA- AND INTRACELLULAR ROS DURING STIMULATION 109

5 EXTRA- AND INTRACELLULAR ROS DURING STIMULATION

5.1 Introduction

Mrmh as well as coffee significantly induced the activation of NF-κB and Nrf2 in various cell

types and intact human gut tissue. Co-treatment with catalase significantly blocked the Mrmh-

induced NF-κB and Nrf2 activation and coffee-induced NF-κB activation (Chapter 3 and 4).

These results indicate that extracellular H2O2 plays a significant role in the NF-κB and Nrf2

activation. Both Mrmh and coffee extract generate H2O2 as shown in Chapter 2. Since recent

publications highlighted H2O2 at physiological concentrations as intra- and extracellular

messenger in cell signalling [118], the involvement of H2O2 in NF-κB and Nrf2 activation was

suggested. Therefore, extracellular and intracellular development of ROS especially of H2O2

was measured after exposure of cells and tissue to Maillard mixtures and coffee extract.

5.2 Results

5.2.1 Extracellular H2O2 concentration during stimulation of macrophages and

human gut tissue

5.2.1.1 Stimulation of macrophages with Maillard products

In order to analyse the potential role of extracellular H2O2 in Mrmh-induced NF-κB and Nrf2

activation, NR8383 macrophages were incubated with Mrmh (10 - 100 mM) for up to 24 h and

extracellular H2O2 concentration was measured over time (Figure 5.1A). In addition, H2O2

levels were analysed in Mrmh which was stored at identical cell culture conditions but in the

absence of NR8383 macrophages (Figure 5.1A). In the presence of NR8383 macrophages,

between 100 and 450 µM extracellular H2O2 were detected after 2 h depending on the Mrmh

concentration. After 12 h, H2O2 levels were slightly decreased but not completely scavenged.


In comparison, the overall H2O2 concentrations were significantly higher in the absence of

NR8383 macrophages than in the presence of cells. H2O2 concentrations in Mrmh without

cells reached levels between 170 µM and 565 µM H2O2 after 2 h dependent on the Mrmh

concentration. Unlike in the presence of cells, H2O2 generation was promoted with increasing

incubation time. Both experimental approaches were repeated in the presence of catalase

(150 U/mL). Extracellular H2O2 was not detected within the first 12 h (data not shown).

5.2.1.2 Stimulation of macrophages with coffee extract

Extracellular H2O2 plays also a major role in coffee-induced NF-κB activation. Thus,

extracellular H2O2 was measured during coffee incubation in the presence and absence of

macrophages. Extracellular H2O2 concentrations after coffee incubation were similar to those

measured for Mrmh stimulation; H2O2 generation depended on coffee concentration and

increased with incubation time in the absence of cells and decreased in the presence of cells

(Figure 5.1B). In the presence of NR8383 macrophages, H2O2 levels were significantly lower

than in the absence of cells especially in higher coffee extract concentrations. Without cells,

H2O2 concentrations reached levels up to 800 µM whereas in the presence of cells H2O2

levels did not exceed 230 µM. In the presence of catalase, extracellular H2O2 was not

detected during 24 h in both approaches with and without cells (data not shown). Cell viability

of NR8383 macrophages after coffee extract (2 mg/mL) incubation for up to 6 h was always

higher than 90 % of vital cells.


Control 10 25 50 1000

200

400

600

800

1000

2 hcells

6 h12 h24 h

*** *** *** ***

*** *** *** ***

****** *** ***

******

*****

A

Mrmh [mM]

Extra

cellu

lar H

2O2

[µM

]

Control 1 2 40

200

400

600

800

1000

2 hcells

6 h12 h24 h

* ***

****

***

****

B

Coffee extract[mg/mL]

Extra

cellu

lar H

2O2

[µM

]

Figure 5.1: NR8383 macrophages were incubated with (A) Mrmh (10 - 100 mM) or (B) coffee extract (1 - 4 mg/mL)

for 24 h and extracellular H2O2 concentrations were measured after 2, 6, 12 and 24 h. Simultaneously, (A) Mrmh

and (B) coffee extract respectively were stored under similar cellular conditions but in the absence of NR8383

macrophages and H2O2 concentrations were investigated likewise. Data is mean ± SD (A: n = 4; B: n = 3). * p <

0.05, ** p < 0.01, *** p < 0.001.

H2O2 generation by active phytochemicals under cell culture conditions was attributed in the

past more likely to the instability of the substances in cell culture medium. Thus, production


of H2O2 was considered as an artefact of cell culture condition which does not occur in vivo

[236]. Therefore, the H2O2 generation of coffee and Maillard products was compared in

different solvents. For this purpose, the amount of H2O2 in coffee extract and Mrmh was

analysed in cell culture medium, water or PBS (Table 5.1). Even though the concentration of

H2O2 was higher when diluted in cell culture medium than in water or PBS, profound amounts

of H2O2 were detected under all conditions. Thus, the generation of H2O2 as an artefact of

cell culture conditions can be excluded. Any influence of the medium on the FOXPCA assay

itself can be ruled out since the respective standard curves were prepared in each medium

(data not shown).

Table 5.1: Mrmh and coffee extract were diluted in PBS/water and cell-free cell culture medium respectively and

H2O2 was detected immediately via FOXPCA assay. Data are expressed as mean ± SD (n = 3).

Mrmh [mM] Control 10 25 50 100

H2O2 [µM] in PBS 0 133.63 ± 05.33 196.12 ± 11.66 135.07 ± 21.82 8.99 ± 4.34

in medium 0 273.87 ± 08.52 368.14 ± 6.53 292.03 ± 32.16 94.34 ± 14.57

Coffee extract [mg/mL] Control 1 2 4

H2O2 [µM] in water 0 121.84 ± 12.95 193.54 ± 17.47 304.39 ± 12.82

in medium 0 193.27 ± 08.32 292.86 ± 17.89 445.11 ± 16.27

5.2.1.3 Stimulation of intact human gut tissue with Maillard products and coffee extract

The extracellular H2O2 content was also quantified after stimulation of human gut tissue ex

vivo. As illustrated in Figure 5.2, extracellular H2O2 could be detected in both the Mrmh

(Figure 5.2A) and the coffee extract (Figure 5.2B) incubation processes. In both experiments,

H2O2 concentrations were significantly higher in the absence than in the presence of the

tissue. In the presence of catalase, no H2O2 was detected independent of the presence or


absence of tissue samples (data not shown). An additional experiment clarified that air

bubbling during mucosa oxygenation, which prevent ischaemia and support the diffusion of

the medium-dissolved stimuli into the tissue, did not have any impact on the H2O2 generation

by Maillard products (data not shown).

Control 10 25 50 1000

100

200

300

400

500+ Human gut tissue- Human gut tissue

A

** **

***

Mrmh [mM]

Ext

race

llula

r H2O

2[µ

M]

Control 1 2 40

50

100

150

200+ Human gut tissue- Human gut tissue

B

**

**

***


Extra

cellu

lar H

2O2

[µM

]

Figure 5.2: Human gut tissue samples were incubated with (A) Mrmh (10 - 100 mM) or (B) coffee extract (1 -

4 mg/mL) during mucosa oxygenation ex vivo. Extracellular H2O2 concentrations were measured after 2 h.

Moreover, Mrmh and coffee extract were cultivated under the same conditions as aforementioned but in the

absence of the tissue samples and H2O2 concentrations were investigated likewise. Data is mean ± SD (A: n = 3;

B: n = 2 - 5). * p < 0.05, ** p < 0.01, *** p < 0.001.


5.2.2 Intracellular oxidative stress level

Recent publications highlighted the role of ROS as intra- and extracellular messenger in cell

signalling. In this context, it was demonstrated in earlier studies that intracellular ROS

triggers signalling pathways including the NF-κB pathway [122]. Thus, the intracellular ROS

content of activated macrophages was analysed. Therefore, macrophages were incubated

with Mrmh and the intracellular ROS level was detected with the 2’,7’-dichlorofluorescein

(DCF) assay. The DCF assay determines intracellular ROS including H2O2, peroxyl radicals,

nitric oxide and peroxynitrite by fluorescence signal [237]. Thus, the intracellular DCF

fluorescence can be used as an index to quantify the overall oxidative stress in cells.

In detail, J774 macrophages were incubated with Mrmh for 24 h and intracellular ROS level

was measured over time. After stimulation with Mrmh, the intracellular ROS level was

increased implying an elevated oxidative stress level (Figure 5.3). Figure 5.3A shows the

concentration-dependent raise in oxidative stress after stimulation for 6 h, reaching a 4-fold

higher fluorescence signal in cells treated with 5 mM Mrmh compared to PBS treated control

cells. Irrespective of the Mrmh-concentration, the intracellular oxidative stress level

significantly increased after 30 min of stimulation time-dependently (Figure 5.3B). After 4 h,

the level plateaued and remained constant for up to 24 h at each concentration. In order to

rule out that the steady state of fluorescence signal was due to a lack of excessive DCF

reagent, further experiments were conducted using 50 and 100 µM instead of 10 µM DCF

reagent (data not shown). Irrespective of the DCF concentration, the same curve response

for oxidative stress was detected excluding a lack of DCF reagent at high Mrmh concentration

and long incubation time. Cell viability of macrophages after incubation with Mrmh for 24 h

was always higher than 80 %.

Whereas incubation of J774 macrophages with a Maillard mixture heated at 130°C instead of

120°C further increased intracellular oxidative stress level, Mrmu did not show any impact on

the oxidative stress level at all (data not shown). Likewise, a heated ribose solution and pure


H2O2 solutions (100 µM and 500 µM) did not up-regulate the intracellular oxidative stress

level (data not shown).

0 0.25 0.5 1 1.5 2 2.5 3 4 50

200

400

600

800

0

50

100

A

******

*** ****** *** *** *** ***

100

Mrmh [mM]

Rel

ativ

e flu

ores

cenc

e si

gnal

com

pare

d to

con

trol [

%]

Cell viability [%

]

0 1/20 1/2 1 2 4 6 240

100

200

300

400

500

B

******

****** *** ***

Incubation time [h]

Rel

ativ

e flu

ores

cenc

e si

gnal

com

pare

d to

con

trol [

%]

Figure 5.3: As an indicator for intracellular oxidative stress, the fluorescence signal (Ex485 nm/Em520 nm)

obtained by the DCF assay was measured in J774 macrophages after stimulation with a heated Maillard mixture

(Mrmh). (A) Cells were stimulated with Mrmh (0.25 mM - 5 mM) and the fluorescence signal was detected after

6 h. (B) Cells were stimulated with 5 mM Mrmh and fluorescence signal was monitored for 24 h. The fluorescence


signal was normalized and given as percent compared to PBS treated control cells (■). Data is mean ± SD

(n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. Cell viability of macrophages after incubation with Mrmh for 24 h was

always higher than 80 % (●).

It is well established that activated macrophages release ROS for example via NADPH

oxidase, a membrane-bound enzyme [238]. The potential contribution of this oxidative burst

to the increase in intracellular ROS and thus in oxidative stress level was analysed after

stimulation with Mrmh.

First, the kinetics of the NADPH oxidase activation by a mixture of LPS and IFN-γ, which are

known to induce NADPH oxidase, were investigated. Stimulation of macrophages with

LPS/IFN-γ significantly raised intracellular oxidative stress level after 6 h. Co-incubation of

cells with LPS/IFN-γ and diphenyleneiodonium chloride (DPI), a NADPH oxidase inhibitor,

kept the ROS level on baseline during the whole incubation period (Figure 5.4A). This data

revealed the capacity of the NADPH oxidase to increase the oxidative stress level in

macrophages by producing intracellular ROS after stimulation with LPS/IFN-γ. In the case of

Mrmh-stimulated macrophages, the oxidative stress level was enhanced after 30 min of

exposure and increased further with time. However, co-treatment with DPI did not

significantly block the increase of the oxidative stress level until 24 h of stimulation

(Figure 5.4B).


LPS/IFN-γ LPS/IFN-γ0

200

400

600

800 30 min1 h2 h4 h6 h24 h

*** ***

Control

DPI - + - +

A

Control

100

Rel

ativ

e flu

ores

cenc

e si

gnal

com

pare

d to

con

trol [

%]

Mrmh Mrmh 0

200

400

600 30 min1 h2 h4 h6 h24 h

Control

DPI - + - +

**

B

Control

100

Rel

ativ

e flu

ores

cenc

e si

gnal

com

pare

d to

con

trol [

%]

Figure 5.4: The contribution of the oxidative burst of the NADPH oxidase to the intracellular oxidative stress level

was measured in J774 macrophages after LPS/IFN-γ (A) or Mrmh (B) stimulation. Cells were stimulated with LPS

(50 µg/mL)/IFN-γ (100 U/mL) (A) or Mrmh (2.5 mM) (B) for 24 h. In some experiments, cells were co-treated with

the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) (10 µM). Intracellular oxidative stress level was

detected with the fluorescence based 2’,7’-dichlorofluorescein (DCF) assay (ex 485 nm/em 520 nm) between

30 min and 24 h. The fluorescence signal was normalized and given as percent compared to PBS treated control

cells. Data is mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.


5.3 Discussion

Maillard products and coffee extract activated NF-κB and Nrf2 in various cell types and intact

human gut tissue. However, co-treatment with catalase partly blocked the activation implying

the involvement of H2O2 (Chapter 3 & 4). Thus, extracellular and intracellular ROS were mea-

sured since ROS are considered to be involved in the activation mechanisms of NF-κB and

Nrf2.

First, the development of extracellular H2O2 in Mrmh and coffee extract at 37°C over time was

compared with each other in the presence and absence of macrophages. In the presence of

macrophages, H2O2 was clearly detectable in the extracellular compartment when exposed to

coffee and Maillard products, but the concentration was significantly lower than in the

absence of cells. Without cells, more than 650 µM H2O2 was detected in Mrmh after 24 h

whereas in the presence of cells, H2O2 concentrations did not exceed 400 µM. A similar trend

was detected for coffee extract reaching up to 800 µM H2O2 in the absence of cells and only

200 µM H2O2 with cells. The decline of extracellular H2O2 in the presence of cells can be

attributed to the cellular detoxification by catalase, glutathione peroxidase and numerous

non-enzymatic antioxidants which scavenge ROS. In C6 glioma cells, for instance, a bolus of

H2O2 (≤ 100 µM) was completely detoxified [239]. But on the contrary to a one-time bolus of

pure H2O2, coffee and Mrmh generate H2O2 permanently (Chapter 2) which might

overcompensate cellular detoxification as shown in a similar model with Jurkat T-cells [240].

Thus, the generation of H2O2 by Maillard products counteracts the cellular detoxification

leading to the residual amount of extracellular H2O2 which was detected in this study. This

residual extracellular H2O2 may diffuse across cell membranes triggering cell signalling

pathways such as the NF-κB and Nrf2 pathway. This hypothesis was underlined by the fact

that co-treatment with catalase completely abolished extracellular H2O2 in macrophages as

well as cellular reactions such as NF-κB and partly Nrf2 activation induced by Mrmh and

coffee respectively.


Next, the extracellular H2O2 concentration was analysed after incubation of intact human gut

tissue with Mrmh and coffee extract. Similar to macrophages, human gut tissue was able to

reduce significantly extracellular H2O2 produced by coffee or Maillard products. However, the

detoxification rate of the tissue was higher compared to the rate of macrophages. The higher

detoxification rate can be caused by the diversity of cells present in the tissue samples, but

also by a higher cell density. Thus, the minor amount of residual H2O2 in the tissue samples

may cause a lower NF-κB and Nrf2 response compared to macrophages.

Since it was hypothesized above that residual extracellular H2O2 may penetrate the cell, the

intracellular ROS level of macrophages after incubation with Mrmh was measured via DCF

assay. Thereby, the level of intracellular ROS including H2O2, peroxyl radicals, nitric oxide

and peroxynitrite was determined by a fluorescence signal [237]. The fluorescence signal

was used as an index to quantify the overall oxidative stress in cells. Indeed, exposure of

macrophages to Mrmh caused an increase in the intracellular oxidative stress level. Likewise,

a heated mixture of glucose-BSA raised the intracellular oxidative stress level in bovine aortic

endothelial cells [227]. Thus, it was concluded that Maillard products raise the oxidative

stress level in cells. However, the mechanism remains unknown.

Since the NADPH oxidase, a membrane-bound enzyme, is known to produce ROS during

the oxidative burst [238], the involvement of the NADPH oxidase in the increase of the

oxidative stress level by Maillard products was investigated. Therefore, macrophages were

incubated with Mrmh in the presence and absence of DPI, a NADPH oxidase inhibitor. Since,

the intracellular oxidative stress level also increased in the presence of DPI, a major impact

of the NADPH oxidase on the oxidative stress level after Mrmh-incubation of macrophages

was excluded.

In course of the incubation period, the oxidative stress level in macrophages significantly

increased after 30 min of stimulation and plateaued after 4 h showing 4-fold higher

fluorescence signals compared to control cells. It seems likely that intracellular antioxidants,


which might be expressed as a consequence of Nrf2 activation (Chapter 4), attenuated any

further increase of the oxidative stress level. Indeed, a decrease of the oxidative stress level

in tert-butylhydroperoxides stimulated colon cells after incubation with Maillard-rich foods

was previously reported [235].

Unlike Mrmh, Mrmu and ribh did not increase the intracellular oxidative stress level in

macrophages. Likewise, a single bolus of H2O2 (100 µM or 500 µM) did not increase the

intracellular oxidative stress level of macrophages after 24 h. This might be due to a

complete cellular detoxification of H2O2 applied as one single bolus. Indeed, it was shown

that moderate concentrations of H2O2 can easily be fully detoxified by brain cells [241] and

glioma cells [239].

6 SUMMARY 121

6 SUMMARY

Coffee is one of the most consumed beverages worldwide. The characteristic flavour and the

stimulating effect of coffee are responsible for its popularity. Recent epidemiological studies

also correlated a reduced risk of oxidative stress-associated diseases such as Alzheimer’s

[13] and Parkinson’s disease [15], brain, liver and colon cancer [17-19] and diabetes mellitus

[16] to coffee consumption. To date, the bioactive coffee compounds causing a beneficial

health effects in these oxidative stress-related diseases have not been identified.

In the present study, Maillard products, which are formed during the roasting process in

coffee by carbonyls and amines, were analysed [44]. In detail, the role of Maillard products

as potential triggers of cell signalling was investigated. Therefore, the ability of a Maillard

model system (a ribose-lysine mixture which was heated at 120°C for 30 min) and of coffee

(a Maillard-rich beverage) to activate NF-κB and Nrf2 was analysed. Both, NF-κB and Nrf2

are redox-sensitive transcription factors. NF-κB regulates the expression of genes coding

amongst others for cytokines which control diverse cellular processes including immune

response, inflammation and stress response [185]. Nrf2, on the other hand, plays a role in

the induction of detoxifying and antioxidant enzymes such as γ-glutamylcysteine synthetase,

glutathione synthetase and phase II NADPH:quinone oxidoreductase 1 [214] counteracting

the unbalanced redox homeostasis during oxidative stress. Since both NF-κB and Nrf2 are

involved in the development and balance of oxidative stress [242], the activation of NF-κB

and Nrf2 by Maillard products was analysed. It was shown before that physiological

concentrations of reactive oxygen species (ROS) especially H2O2 trigger various cell

signalling pathways involving NF-κB [122], PKC [123] and MAPK kinase [124]. Indeed,

Maillard products generate H2O2 [73], [174] Thus, the development of H2O2 by Maillard

products and the role of H2O2 in the activation of NF-κB and Nrf2 were investigated in the

present study.

6 SUMMARY 122

In the first part of the study, the generation of H2O2 by Maillard products was analysed by the

FOXPCA assay. In a Maillard reaction mixture (Mrmh), consisting of 0.5 M D-ribose and L-

lysine, which was heated at 120°C for 30 min, up to 180 µM H2O2 was detected. Even though

a clear mechanism for the generation of H2O2 by Maillard products is not published yet, some

substructures of Maillard products are suggested to be involved in the H2O2 formation.

Amongst others, Maillard products with enediol and enaminol substructures, which

tautomerize from Amadori products and dicarbonyls such as methylglyoxal, as well as

reductones and aminoreductones are associated with the production of ROS in the presence

of metals [135], [155-158], [167]. Moreover, a mechanism is proposed highlighting the

formation of H2O2 by autoxidation of α-hydroxyaldehydes [135], [167]. Thus, it is not

surprising that H2O2 was also formed during caramelization of a heated ribose solution (ribh)

but to a lower extent than in Mrmh. H2O2 was also detected in an unheated Maillard reaction

mixture (Mrmu). Since neither an unheated ribose solution (ribu) nor an unheated lysine

solution (lysu) generated H2O2, the formation of H2O2 in Mrmu was related to a reaction

between ribose and lysine which can take place to a minor extent even at room temperature.

Indeed, the H2O2 concentration was significantly lower in Mrmu than in Mrmh underlining the

enhanced generation of H2O2 by Maillard products at increased temperatures.

The amount of H2O2 in Mrmh increased with the sample concentration up to 25 mM Mrmh.

However in Mrmh > 25 mM, the concentration of H2O2 decreased. To rule out any

interference between the FOX reagent and the Maillard products of the Mrmh, the

concentration of H2O2 in Mrmh was not only measured by the FOXPCA assay but also by

oxygen electrodes. In the case of the FOXPCA assay, the oxidation of ferrous ions of the FOX

reagent to ferric ions by H2O2 was measured photometrically. For the electrochemical

detection of H2O2, the Clark and the Luminescent Dissolved Oxygen (LDO) electrodes were

used. For this purpose, H2O2 was decomposed by catalase into water and oxygen which was

detected by the oxygen electrodes. Similar Mrmh concentration response curves were

6 SUMMARY 123

observed by all detection methods used. Thus, the reduced amount of H2O2 in the presence

of high Mrmh concentrations can be attributed to the radical scavenging activity of

antioxidative active Maillard products [175]. In this context, Maillard products with reductone

structures were already identified as structures which -depending on their concentration-

exhibit pro- as well as antioxidative activity [157].

Next, the formation of H2O2 in Maillard reaction mixtures was monitored under various

reaction conditions such as pH and temperature. Enhanced generation of H2O2 was

measured directly after preparation of the Maillard mixtures at basic pH values up to pH 10

and also at high temperatures up to 130°C. Also during prolonged incubation of Maillard

mixtures at 37°C for 96 h, the H2O2 content increased up to 370 µM. To test the hypothesis

that H2O2 is not only generated at elevated temperatures during preparation of the Mrmh but

also permanently at lower temperatures during prolonged storage, de novo generation of

H2O2 was monitored at 4°C, 25°C and 37°C. Therefore, any H2O2 was removed in Mrmh by

catalase and the generation of H2O2 was monitored for 96 h in each sample. After 1 h, H2O2

was generated under all tested conditions reaching levels between 50 and 100 µM. During

prolonged incubation of a 25 mM Mrmh for 96 h, the amount of H2O2 stayed almost constant

at 4°C, decreased at 25°C and increased at 37°C up to 200 µM. The same trend but at a

lower H2O2 level was observed when Mrmh was adjusted to pH 5.5 instead of pH 8 after

heating.

In order to enrich H2O2 generating Maillard products, Mrmh was fractionated by size

exclusion chromatography on a D-SaltTM polyacrylamide desalting column (cut off 1.8 kDa).

Five H2O2-generating fractions (F5 - F9) out of 20 fractions were collected and unified into

one active fraction. Next, the de novo generation of the active fraction was tested likewise.

The active fraction also permanently produced H2O2 for up to 96 h reaching higher levels

than the crude Mrmh (100 and 260 µM H2O2). Moreover, the amount of H2O2 in the active

fraction incubated at 25°C did not decrease as shown for the crude Mrmh but rather stayed

6 SUMMARY 124

constant or increased indicating a successful enrichment of H2O2-generating Maillard

products in the active fraction.

Subsequently, it was investigated if Maillard products modulate cellular signalling by a H2O2

dependent mechanism. The intestine as part of the gastrointestinal tract represents a primary

target for food-induced inter- and intracellular reactions of the human body. Thus,

macrophages, Caco-2 cells (colorectal epithelial cells) and HIMEC (primary cells of the

human intestinal microvascular endothelium), cells which are all present in the intestine,

were incubated with Maillard mixtures. The activation of the redox-sensitive transcription

factors NF-κB and Nrf2 was measured by their nuclear translocation.

Firstly, macrophages and Caco-2 cells were stimulated with Mrmh for 2 h (short-term). Both,

NF-κB and Nrf2 were significantly activated in a concentration dependent manner in Caco-2

cells and in macrophages up to 5-fold compared to PBS treated control cells. In primary

HIMEC, NF-κB was activated 2-fold compared to PBS treated control cells after exposure to

Mrmh for 2 h. Even though Mrmh activated both transcription factors in various cell lines and

primary cells, the degree of activation varied strongly with the cell type showing the

maximum effect in macrophages. Thus, long-term experiments for up to 24 h were

conducted on macrophages. First, macrophages were incubated for 2 h with Mrmh and the

nuclear amount of Nrf2 was analysed after a subsequent 22 h post-incubation without

stimulant. During the stimulant-free post-incubation, the nuclear translocation of Nrf2

continuously raised up to 8-fold higher levels compared to experiments without post-

incubation. When macrophages were continuously exposed for 24 h to Mrmh (25 mM), the

nuclear Nrf2 level was 26-fold higher compared to PBS treated control cells which

corresponds a 3-fold higher activation compared to the experiment with stimulant-free post-

incubation. Thus, it was concluded that Mrmh might induce Nrf2 by chain reactions which are

still active after removing Mrmh.

During long-term experiments for up to 24 h, the activation of Nrf2 increased with the Mrmh

6 SUMMARY 125

concentration and the incubation time. Exposure of macrophages to ≥ 25 mM Mrmh

significantly enhanced nuclear Nrf2 translocation reaching 50-fold higher Nrf2 levels after

stimulation for 24 h compared to PBS treated control cells. To test if nuclear translocation of

Nrf2 was indeed induced by Maillard products or by a high concentration of amino acids and

sugars, an unheated mixture of ribose and lysine (Mrmu) was tested in the same way as

Mrmh. Mrmu, which was shown not to induce NF-κB in macrophages under applied conditions

[25], increased nuclear Nrf2 levels up to 20-fold compared to PBS treated control cells.

However, the nuclear Nrf2 level was profoundly lower after stimulation with Mrmu than after

exposure with the equivalent concentration of Mrmh indicating the Maillard products of the

Mrmh as key players in NF-κB and Nrf2 activation. Moreover, the contribution of

caramelization products can be excluded since a heated ribose solution (ribh) did neither

induce NF-κB [25] nor Nrf2 in macrophages.

This in vitro screening raised the question of how the transcription factors respond in human

intestinal tissue consisting of a wide range of cell types present in the intestine. Thus, a

method was successfully established which allowed the stimulation of human gut tissue ex

vivo [192], [193]. During gastrointestinal endoscopy, human gut tissue samples were taken

from two localisations in the lower gastrointestinal tract; one biopsy from the terminal ileum of

the small intestine and one biopsy from the ascending colon of the large intestine. Next, the

human gut tissue samples were stimulated ex vivo with Mrmh for 2 h during mucosa

oxygenation. It was demonstrated that (i) the tissue samples were uniform and comparable,

and (ii) cell viability of the tissue samples was not harmfully affected by the mucosa

oxygenation ex vivo. Mrmh induced a 2.8-fold and 2-fold activation of NF-κB and Nrf2 ex vivo

in intact human gut tissue compared to PBS treated control cells. Thus, Mrmh did not only

activate NF-κB and Nrf2 in various cell types but also in human gut tissue which is

considered a valuable model to study nutritional effects on complex tissue systems for in vivo

evaluations.

6 SUMMARY 126

To date, defined mechanisms for NF-κB and Nrf2 activation by Maillard products had not

been established. However, it was shown before that Maillard-induced NF-κB activation in

macrophages was inhibited by catalase. Thus, there is growing evidence that H2O2, which is

permanently generated by Maillard products, is involved in the activation mechanism of NF-

κB [25]. As a matter of fact, the activation of both NF-κB and Nrf2 by Mrmh was fully

abolished in macrophages, Caco-2 cells and HIMEC and reduced in human gut tissue by co-

treatment with catalase. Heat-inactivated catalase did not show any effect. These results

reveal the involvement of extracellular H2O2, which is most certainly generated by the

Maillard products, in the activation of NF-κB and Nrf2 in all cell types tested. Interestingly,

pure solutions of H2O2 with concentrations up to 500 µM activated NF-κB in macrophages up

to 13-fold after stimulation for 2 h [25] but did not induce any Nrf2 response within 24 h.

Thus, it was hypothesized that the activation of Nrf2 by Mrmh might be induced not only by

H2O2 but rather an interaction of H2O2 with Maillard products. Since the generation of H2O2

by Maillard products seems to play a key role in NF-κB and Nrf2 activation in various cells

and tissue, the amount of H2O2 in Mrmh was analysed in the presence and absence of cells

and tissue respectively. First, macrophages were incubated with Mrmh at 37°C for up to 24 h

and extracellular H2O2 content was measured. In the presence of cells, the concentration of

H2O2 was significantly lower compared to the H2O2 concentration in the absence of cells. The

same trend was found during incubation of Mrmh in the presence and absence of intact

human gut tissue ex vivo. Cells can detoxify extracellular and intracellular reactive oxygen

species (ROS) such as H2O2 [241]. However, Maillard products continuously generate H2O2

which leads to an incomplete detoxification. It was hypothesized that the residual

extracellular H2O2 might penetrate the cell and increases the intracellular ROS level which

might induce signalling pathways including NF-κB and Nrf2. In order to verify this

assumption, the intracellular ROS level of macrophages after stimulation with Mrmh was

analysed using 2’,7’-dichlorofluorescein (DCF). The DCF assay determines intracellular ROS

6 SUMMARY 127

including H2O2, peroxyl radicals, nitric oxide and peroxynitrite by fluorescence signal [237].

Thus, the intracellular DCF fluorescence can be used as an index to quantify the overall

oxidative stress in cells. Indeed, the oxidative stress level was elevated after exposure to

Mrmh in a concentration and time dependent manner. After 4 h, the oxidative stress level

plateaued and stayed constant for up to 24 h at 4-fold higher levels compared to PBS treated

control cells. It seems likely that intracellular antioxidants, which are highly expressed as a

consequence of Nrf2 activation [214], attenuate any further increase of the oxidative stress

level after 4 h. Since the NADPH oxidase, a membrane-bound enzyme, is known to produce

ROS during the oxidative burst [238], the involvement of the NADPH oxidase in the increase

of the oxidative stress level by Maillard products was investigated. Therefore, macrophages

were incubated with Mrmh in the presence and absence of diphenyleneiodonium chloride

(DPI), a NADPH oxidase inhibitor. Since, the intracellular oxidative stress level also

increased in the presence of DPI, a major impact of the NADPH oxidase on the oxidative

stress level after Mrmh incubation of macrophages was excluded. Thus, it was proposed that

Mrmh generate permanently H2O2, which can not be fully detoxified by cells and thus

penetrate the cells. As a consequence, intracellular ROS increases causing the induction of

intracellular, signalling pathways such as NF-κB and Nrf2.

Not only Mrmh but also Mrmu activated Nrf2 in macrophages. Since Mrmu also generated

H2O2 but to a lower extent than Mrmh, the role of H2O2 in Mrmu induced Nrf2 activation was

determined likewise. In contrast to Mrmh, co-treatment of macrophages with Mrmu and

catalase did not reverse Nrf2 activation within 24 h. Thus, it was concluded that, contrary to

Mrmh, Mrmu activate Nrf2 in macrophages H2O2-independently. Moreover, exposure of

macrophages to Mrmu did not increase the intracellular oxidative stress level. Likewise, the

oxidative stress level was not elevated after stimulation with ribh or a pure solution of H2O2 up

to 500 µM. The content of H2O2 in Mrmu, ribh as well as a pure solution of 500 µM H2O2 was

less than in Mrmh especially when incubated at 37°C. Thus, it was proposed that cells may

6 SUMMARY 128

fully detoxify the concentration of H2O2 present in Mrmu, ribh, a pure solution of 500 µM H2O2

but not the amount of H2O2 present in Mrmh. Consequently, the activation of NF-κB and Nrf2

by Mrmh but not Mrmu involved H2O2 which was permanently generated by Maillard products.

Since Maillard products clearly induce NF-κB and Nrf2, coffee was investigated as an

example for foods rich in Maillard products in the same test systems. Similar to the Maillard

reaction mixtures, the generation of H2O2 was monitored in coffee extracts. With both

methods, the FOXPCA assay and the oxygen electrodes, up to 440 µM H2O2 was detected in

roasted coffee extract. However, also up to 200 µM H2O2 was detected in raw coffee extract.

It was not surprising that H2O2 was also found in raw coffee extract as polyphenols such as

chlorogenic acid, which are major components of raw coffee beans, are also considered as

potent generators of H2O2 [182]. Since the polyphenols highly degrade during roasting, the

generation of H2O2 in roasted coffee extract was attributed to degradation products and the

Maillard products which are formed during roasting. In earlier studies, caffeic acid, pyrogallol

and hydroxyhydroquinone (1,2,4-Benzenetriol), the degradation products of chlorogenic acid,

were already identified as H2O2 generating structures in roasted coffee [182]. However, the

overall concentration of H2O2 formed by these degradation products [182] is significantly

lower compared to the amount of H2O2 found in coffee extract in the present study. Thus, the

formation of H2O2 was not only attributed to degradation products but also to Maillard

products.

Next, coffee extract was tested for its ability to activate NF-κB and Nrf2 in various cells and

tissue. First, Caco-2 cells and macrophages were exposed for 2 h to 1 - 4 mg/mL coffee

extract (short-term). In Caco-2 cells neither NF-κB nor Nrf2 were activated. In macrophages

coffee significantly activated NF-κB up to 4-fold. Moreover, a trend towards Nrf2 activation

was found in macrophages. Next, intact human gut tissue samples were stimulated with 1 -

4 mg/mL coffee extract for 2 h. In human gut tissue samples, coffee extract did not activate

Nrf2 but induced a trend towards NF-κB activation. Short-term incubation with coffee showed

6 SUMMARY 129

the highest NF-κB and Nrf2 activation in macrophages amongst the cells and tissue samples

tested. Thus, macrophages were incubated next with coffee extract between 30 min and 24 h

and the activation of NF-κB and Nrf2 was investigated time-dependently. Nrf2 was activated

by coffee extract in a concentration and time dependent manner reaching nearly 20-fold

higher Nrf2 levels after 24 h compared to water treated control cells. NF-κB was activated up

to 3-fold higher levels after 2 h but declined slightly thereafter. After 6 h, the nuclear NF-κB

concentration was still 1.7-fold higher compared to control cells. It was suggested that the

pronounced activation of Nrf2 might attenuate the nuclear translocation of NF-κB. This

crosstalk between Nrf2 and NF-κB has been recently discussed for stimuli such as

sulforaphane which regulate both NF-κB and Nrf2. It was reported that for example

sulforaphane activate Nrf2 and concomitantly repress the activation of NF-κB. These results

may explain the pronounced activation of Nrf2 but not NF-κB after long-term incubation of

macrophages with coffee extract.

On the contrary to roasted coffee extract, raw coffee extract did activate neither NF-κB nor

Nrf2 after long-term incubation of macrophages. Thus, it was concluded that the roasting

products of coffee including the Maillard products are the key players triggering the nuclear

translocation of NF-κB and Nrf2. Since it was shown before that H2O2 was involved in the

activation mechanisms of NF-κB and Nrf2 by Mrmh, the role of H2O2 in coffee-induced NF-κB

and Nrf2 activation was investigated likewise. Similar to the experiments with Mrmh, the

activation of NF-κB after stimulation of macrophages with coffee extract was abolished by co-

treatment with catalase. However, co-treatment with catalase showed only a slight trend to

block the coffee-induced Nrf2 translocation in macrophages. Thus, it was concluded that the

generation of H2O2 by coffee might play a major role in coffee-induced NF-κB but not in Nrf2

activation. Hence, the amount of extracellular H2O2 after coffee stimulation was measured in

the presence and absence of macrophages. After incubation of coffee extract (1 - 4 mg/mL)

for 24 h at 37°C, H2O2 levels were higher in the absence than in the presence of

6 SUMMARY 130

macrophages. In the absence of cells, up to 800 µM H2O2 was measured whereas in the

presence of cells H2O2 levels did not exceed 230 µM. A similar trend was observed after

incubation of coffee extract with and without human gut tissue for 2 h. These results indicate

that coffee-generated H2O2 was not fully detoxified by cells and tissue respectively leading to

the activation of NF-κB as discussed above for Mrmh.

Thus, it was proposed that coffee and especially Maillard products may rather counteract

oxidative stress by Nrf2 activation than induce an inflammatory response by NF-κB activation

after long-term incubation. Activated levels of Nrf2 lead to an enhanced transcription of

antioxidant and detoxifying enzymes. Hence, coffee and Maillard-rich foods may exert not

only direct [207], [208] but also indirect antioxidative activity by inducing the expression of

cytoprotective proteins especially in the gut which represents a primary target for food-

induced inter- and intracellular reactions of the human body. Indeed, as shown in a mouse

model, activators of Nrf2 can improve colitis [217]. Enhanced intestinal Nrf2 signalling was

further associated with a beneficial effect on gut barrier dysfunction [218], intestinal mucosal

injury [219] and intestinal inflammation other than colitis [218]. Moreover, there is growing

evidence that Nrf2 is also a promising target for the prevention of colorectal cancer [220],

[221]. Indeed, epidemiological studies suggested for coffee drinker with regular coffee

consumption a reduced risk of colorectal cancer as well as other of oxidative stress-

associated diseases such as Alzheimer’s disease and type 2 diabetes mellitus [201].

7 DEUTSCHE ZUSAMMENFASSUNG 131

7 DEUTSCHE ZUSAMMENFASSUNG

In den letzten Jahrzehnten rückte Kaffee nicht nur auf Grund seines charakteristischen

Geschmackes and Aromas in den Vordergrund, sondern wurde ebenfalls hinsichtlich seines

gesundheitlichen Aspektes diskutiert. In zahlreichen epidemiologischen Studien wurde bei

Kaffeetrinkern ein vermindertes Risiko an Krankheiten diagnostiziert, die mit oxidativem

Stress assoziiert sind, wie beispielsweise Alzheimer [13], Parkinson [15], Gehirn-, Leber- und

Dickdarmkrebs [18], [19], [244] und Diabetes mellitus [16]. Bisher konnten allerdings dafür

noch keine aktiven Kaffeekomponenten identifiziert werden.

Ziel dieser Arbeit war es, den Einfluss von Maillard Produkten, die während des

Röstprozesses von Kaffee bei der Maillard Reaktion aus reduzierenden Carbonyl-

verbindungen und Aminen entstehen, auf die Transkriptionsfaktoren Nrf2 und NF-κB in

verschiedenen Zellen und Gewebe zu untersuchen [44]. Dazu wurde sowohl eine Maillard

Model Mischung aus Lysin und Ribose, die bei 120°C für 30 min erhitzt wurde, als auch

Röstkaffee, welcher reich an Maillard Produkten ist, eingesetzt. NF-κB reguliert die

Expression verschiedener Protein z.B. Zytokine, die bei der Regulierung von Entzündungs-

prozessen bzw. der Immun- und Stressantwort beteiligt sind [185]. Nrf2 hingegen kontrolliert

die Expression detoxifizierender und antioxidativer Enzyme wie die der γ-Glutamat-

cysteinligase, Glutathionsynthase und Phase II NADPH: Quinon-Oxidoreduktase, um

oxidativem Stress der Zelle entgegenzuwirken [29]. Da sowohl NF-κB als auch Nrf2 bei der

Entwicklung und Bekämpfung von oxidativem Stress beteiligt sind, wurde in diesem Projekt

der Einfluss der Maillard Produkte auf deren Aktivierung untersucht. Es wurde bereits in

verschiedenen Studien gezeigt, dass H2O2 in physiologischen Konzentrationen als sekun-

därer Messenger aktiv ist und dabei verschiedene zelluläre Signalwege auslösen kann, die

unter anderem durch NF-κB [122], PKC [123] oder MAPK Kinase [124] reguliert werden. Da

bereits bekannt ist, dass Maillard Produkte H2O2 generieren können [25], [73], wurde in


dieser Arbeit der Fokus auf die Generierung von H2O2 durch Maillard Produkte im Maillard

Model und in Kaffee und dessen Rolle in der Aktivierung von NF-κB und Nrf2 gelegt.

Im ersten Teil der Arbeit wurde die Generierung von H2O2 durch Maillard Produkte

untersucht. Dafür wurde eine 0,5 M Maillard Mischung aus D-Ribose und L-Lysin für 30

Minuten bei 120°C erhitzt (Mrmh) und deren H2O2 Gehalt mittels FOXPCA Assay quantifiziert.

In der Maillard Mischung konnten bis zu 180 µM H2O2 detektiert werden. Bisher konnte kein

eindeutiger Mechanismus aufgeklärt werden, der die Generierung von H2O2 durch

spezifische Maillard Produkte beschreibt. Allerdings werden in der Literatur u.a. Endiol- oder

Enaminol-Strukturen, die während der Maillard Reaktion gebildet werden, als H2O2-Bildner

diskutiert. Weiterhin zählen Reduktone und speziell Aminoreduktone zu den Maillard

Produkten, die ROS in Anwesenheit von Metallen generieren können [157]. Während der

Autoxidation von α-Hydroxyaldhyden, die auch bei der Karamelisierung von Zuckern

stattfindet, werden ebenfalls ROS gebildet [135], [167]. So scheint es nicht überraschend,

dass H2O2 nicht nur in Mrmh, sondern ebenfalls in einer erhitzten Ribose-Lösung (ribh)

detektiert wurde. Der H2O2-Gehalt der ribh war jedoch signifikant niedriger als in Mrmh. H2O2

konnte ebenfalls in einer nicht erhitzten Maillard Mischung (Mrmu) detektiert werden. Der

Gehalt an H2O2 war vor allem in niedrig-konzentrierter Mrmu signifikant geringer als in Mrmh

gleicher Konzentration. Da weder in einer nicht erhitzten Ribose-Lösung (ribu) noch in einer

nicht erhitzten Lysin-Lösung (lysu) H2O2 detektiert werden konnte, wurde die Generierung

von H2O2 in Mrmu auf eine Reaktion zwischen Lysin und Ribose und somit auf die Maillard

Reaktion zurückgeführt, die auch bei niedrigen Temperaturen von 22°C in nicht erhitzten

Mischungen mit verringerter Reaktions-rate ablaufen kann [168]. Dieses Ergebnis deutet

darauf hin, dass speziell die Maillard Produkte für die Generierung von H2O2 in der Maillard

Mischung verantwortlich sind.

Der Gehalt an H2O2 in Mrmh nimmt mit der Probenkonzentration bis 25 mM Mrmh zu. Ab

einer Probenkonzentration > 25 mM Mrmh fällt der H2O2 Gehalt allerdings ab und geht


schließlich gegen 0. Um eine eventuelle Störung des FOX Reagenzes durch hoch-

konzentrierte Maillard Produkte ausschließen zu können, wurde alternativ zum FOXPCA

Assay der H2O2-Gehalt in der Maillard Mischung mit zwei voneinander unabhängigen

elektrochemischen Methoden gemessen. Während beim FOXPCA Assay der oxidative Effekt

von H2O2 photometrisch über einen Eisen-Komplex gemessen wird [245], wird bei den

elektrochemischen Methoden vorhandenes H2O2 durch Katalasebehandlung in Wasser und

molekularen Sauerstoff abgebaut, welcher mit Hilfe einer Clark- bzw. einer Luminscent

Dissolved Oxygen (LDO)-Sauerstoffelektrode detektiert wurde. Unabhängig von der

angewandten Bestimmungsmethode wurde ein Abfall des H2O2-Gehaltes in höheren Mrmh-

Konzentrationen gemessen. Der geringe Gehalt an H2O2 wurde auf eine antioxidative

Wirkung der Maillard Produkte ab einer Mrmh Konzentration > 25 mM zurückgeführt.

Im nächsten Schritt wurde die Bildung von H2O2 in der Maillard Mischung unter

verschiedenen Bedingungen untersucht. Dabei wurde speziell der Einfluss des pH-Wertes

und der Temperatur analysiert. Direkt nach der Herstellung der Mischung war die Bildung

von H2O2 vermehrt bei basischem pH bis 10 und bei erhöhten Temperaturen von bis zu

130°C; Reaktionsbedingungen, die die Maillard Reaktion begünstigen. Nicht nur die

Temperatur bei der Herstellung der Maillard Mischung, sondern ebenfalls die Temperatur,

bei der die Mischung gelagert wird, regulierten die H2O2-Bildung. So wurde während der

Inkubation von Mrmh bei 37°C für 96 h bis zu 370 µM H2O2 gebildet. Dieses Ergebnis deutet

darauf hin, dass H2O2 nicht nur während der Erhitzungsphase bei hohen Temperaturen

generiert wird, sondern ebenfalls während der Lagerung bei 37°C permanent nachgebildet

wird. Um diese Hypothese zu überprüfen, wurde die de novo Generierung von H2O2 in Mrmh

bei 4°C, 25°C und 37°C untersucht. Dazu wurde das in der Mischung vorhandene H2O2

mittels Katalase abgebaut und die H2O2-Neubildung über 96 h beobachtet. Nach 1 h konnte

in allen Maillard Mischungen je nach Probenkonzentration zwischen 50 und 100 µM H2O2

detektiert werden. Innerhalb der folgenden 95 h war der H2O2-Gehalt nicht nur von der


Probenkonzentration, sondern ebenfalls von den Inkubationsbedingungen abhängig. Bei

einer Probenkonzentration von 25 mM Mrmh stieg der H2O2-Gehalt bei 37°C bis auf 200 µM

an, blieb annähernd konstant bei 4°C und nahm bei 25°C signifikant ab. Eine ähnliche

Entwicklung, aber mit einem niedrigeren H2O2 Gehalt, wurde mit einer Maillard Mischung, die

auf pH 5,5 anstelle von pH 8 eingestellt wurde, detektiert.

Um H2O2-produzierende Maillard Produkte aus der Maillard Mischung anzureichern, wurde

Mrmh mittels Größenausschlusschromatographie an einer D-Salt™ Polyacrylamid Säule (cut

off 1,8 kDa) fraktioniert. Mit einem optimierten Protokoll wurden aus 20 Fraktionen fünf H2O2-

generierende Fraktionen (F5 - F9) isoliert und zu einer aktiven Fraktion vereint. An-

schließend wurde die de novo Generierung von H2O2 in der aktiven Fraktion unter gleichen

Bedingungen wie bei Mrmh untersucht. Nach Inkubation für 96 h bei 4°C, 25°C und 37°C

wurden zwischen 100 µM und 260 µM H2O2 in der aktiven Fraktion gemessen. Im Gegensatz

zur unfraktionierten Mrmh, wurde bei der aktiven Fraktion nach Inkubation bei 25°C kein

Rückgang der H2O2-Bildung, sondern je nach Probenkonzentration entweder ein Plateau

oder ein Anstieg des H2O2-Gehaltes detektiert.

Maillard Produkte werden je nach Lebensmittelzubereitung vermehrt in erhitzten Lebens-

mitteln gebildet. Nun stellt sich die Frage, welche physiologischen Wirkungen durch H2O2-

generierende Maillard Produkte zellulär im Körper induziert werden. Um dieser Fragestellung

nachzugehen, wurden Zellen des Gastrointestinaltraktes, die primär mit Maillard Produkten

aus erhitzten Lebensmitteln in Kontakt kommen, untersucht. Dafür wurden Makrophagen,

Caco-2 Zellen (Kolonrektale Epithelzellen) und HIMEC (primäre, intestinale Endothelzellen)

mit Mrmh inkubiert und die Aktivierung der redox-sensitiven Transkriptionsfaktoren NF-κB

und Nrf2 untersucht. Als Indikator für die Aktivierung von NF-κB und Nrf2 wurde der nukleare

Gehalt der Transkriptionsfaktoren quantifiziert, die sich im inaktiven Zustand im Zytoplasma

befinden und erst nach Aktivierung in den Zellkern wandern.

Nach zweistündiger Stimulierung mit Mrmh, konnte sowohl in Makrophagen als auch in Caco-


2 Zellen eine signifikante Aktivierung von NF-κB und Nrf2 detektiert werden. Je nach

Konzentration der Mrmh war der nukleäre Gehalt der Transkriptionsfaktoren bis zu 5-fach

erhöht im Vergleich zu PBS stimulieren Kontrollzellen. Nicht nur in immortalisierten Zelllinien,

sondern auch in primären HIMEC aktivierte Mrmh NF-κB. Nach zweistündiger Inkubation von

HIMEC mit Mrmh war der nukleäre NF-κB Gehalt 2-fach erhöht. Der Grad der Aktivierung von

NF-κB und Nrf2 durch Mrmh variierte zwischen den verschiedenen Zelllinien und der

Primärkultur und zeigte die höchste Aktivität in den Makrophagen. Anschließend wurden

Langzeitstimulationsversuche für bis zu 24 h mit Makrophagen durchgeführt. Zuerst wurden

Makrophagen für 2 h mit Mrmh inkubiert und der Zellkernextrakt der Makrophagen nach einer

anschließenden 22-stündigen Stimulation ohne Probe untersucht. Der Gehalt an nukleärem

Nrf2 erhöhte sich 8-fach nach der Stimulanz-freien Post-Inkubation im Vergleich zu den

Versuchen ohne Stimulanz-freie Post-Inkubation. Daraus kann man schließen, dass bei der

Nrf2 Aktivierung durch Mrmh Kettenreaktionen induziert werden, die noch in der Stimulanz-

freien Post-Inkubation aktiv sind. Werden die Makrophagen kontinuierlich für 24 h mit Mrmh

stimuliert, liegt der nukleäre Nrf2 Gehalt 26-fach erhöht gegenüber der Kontrolle vor. Dies

entspricht einem 3-fach erhöhten Gehalt im Vergleich zu den Versuchen mit 22-stündiger

Stimulanz-freier Post-Inkubation.

Generell war der Gehalt an nukleärem Nrf2 in Makrophagen von der Konzentration der Mrmh

und der Inkubationsdauer abhängig und erreichte nach 24 h einen 50-fach erhöhten Wert im

Vergleich zu PBS stimulierten Kontrollzellen. Um zu untersuchen, ob die beobachteten

Effekte auf Maillard Produkte in der Mrmh zurückzuführen sind oder ob auch nicht

umgesetzte Edukte einen Effekt haben, wurde eine nicht erhitzte Maillard Mischung (Mrmu)

mit einem hohen Anteil an nicht reagierten Edukten ebenfalls untersucht. In früheren Studien

wurde bereits gezeigt, dass Mrmu keinen Einfluss auf NF-κB in Makrophagen hatte [174]. Der

nukleäre Gehalt an Nrf2 hingegen war nach 24-stündiger Stimulierung von Makrophagen mit

Mrmu 20-fach erhöht im Vergleich zu PBS stimulierten Kontrollzellen. Der nukleäre Nrf2


Gehalt Mrmh-stimulierter Makrophagen war allerdings stets um ein Vielfaches höher als der

nukleäre Nrf2 Gehalt von Makrophagen, die mit Mrmu gleicher Konzentration stimuliert

wurden. So wurde die Aktivierung sowohl von NF-κB als auch von Nrf2 auf Maillard Produkte

zurückgeführt, die unter erhöhten Temperaturen in der Mrmh vermehrt gebildet werden. Man

kann davon ausgehen, dass unter den angewendeten Versuchsbedingungen auch in der

nicht erhitzten Mischung geringe Mengen an Maillard Produkten gebildet werden, die für die

beobachtete Aktivierung verantwortlich sind. Karamelisierungsprodukte, die sich neben den

Maillard Produkten in Mrmh bilden können, wurden als Aktivatoren ausgeschlossen, da eine

erhitzte Ribose-Lösung weder den nukleären Gehalt von NF-κB [174] noch von Nrf2 in

Makrophagen beeinflusste.

Da die in vitro Ergebnisse für NF-κB und Nrf2 abhängig vom Zelltyp variierten, stellte sich die

Frage, welchen Einfluss Maillard Produkte auf NF-κB und Nrf2 in Gewebe haben, welches

aus verschiedenen Zelltypen besteht. So wurde eine Methode entwickelt, um humanes

Darmgewebe ex vivo zu untersuchen. Zunächst wurden im Rahmen einer gastrointestinalen

Endoskopie Bioptate aus dem unteren Gastrointestinaltrakt (terminales Ileum, Colon

ascendens) entnommen. Anschließend wurde das humane Darmgewebe ex vivo mit Mrmh

für 2 h während mukosaler Oxygenierung stimuliert [192], [246]. Mrmh aktivierte NF-κB bis zu

2,8-fach und Nrf2 bis zu 2-fach in humanem Darmgewebe. Es konnte gezeigt werden, dass

die Individualität der Darmbioptate die Versuchsergebnisse nicht beeinflussten. Weiterhin

konnte ausgeschlossen werden, dass die Zellviabilität durch die mukosale Oxygenierung ex

vivo beeinträchtigt wurde. Anhand dieser Ergebnisse konnte gezeigt werden, dass Mrmh NF-

κB und Nrf2 nicht nur in verschiedenen Zelltypen, sondern ebenfalls in humanem

Darmgewebe aktivierte. Die in vitro erzielten Daten waren auf ex vivo Inkubationen von

humanem Darmgewebe übertragbar, was ihre Relevanz in vivo verdeutlicht.

Bisher konnten allerdings keine Maillard Produkte eindeutig identifiziert werden, welche die

Aktivierung von NF-κB bzw. Nrf2 auslösen. In der Literatur wurde beschrieben, dass die NF-


κB Aktivierung durch Maillard Produkte in Makrophagen in Anwesenheit von Katalase

blockiert wurde [25]. Als Konsequenz wurde H2O2, welches nachweislich von Maillard

Produkten generiert wird, als potentieller sekundärer Messenger bei der Aktivierung von NF-

κB diskutiert. Um nun die Rolle von H2O2 bei der Aktivierung von NF-κB und Nrf2 nach Mrmh

Stimulierung in verschiedenen Zelltypen zu untersuchen, wurden die Zellen bzw. das

Gewebe mit Katalase ko-stimuliert. In Anwesenheit von Katalase wurde die Mrmh induzierte

Aktivierung von NF-κB bzw. Nrf2 in Makrophagen, Caco-2 Zellen und HIMEC völlig

aufgehoben und im Darmgewebe reduziert. Hitze-inaktivierte Katalase hatte keinen Einfluss

auf die Aktivierung. Diese Ergebnisse deuten daraufhin, dass bei der Aktivierung der beiden

Transkriptionsfaktoren NF-κB und Nrf2 durch Mrmh in allen getesteten Zell- und

Gewebesystemen H2O2 beteiligt war. Eine 24-stündige Inkubation mit einer reinen bis zu

500 µM H2O2-Lösung induzierte keine Aktivierung von Nrf2 in Makrophagen. Jedoch konnte

an der gleichen Zelllinie bereits gezeigt werden, dass reines H2O2 (500 µM) NF-κB bis zu 13-

fach aktivierte [25]. Anhand dieser Resultate wurde die Schlussfolgerung gezogen, dass

zumindest die Nrf2 Aktivierung durch die Maillard Produkte nicht nur auf H2O2 sondern eher

auf eine Interaktion von Maillard Produkten mit H2O2 zurückgeführt werden kann.

Da H2O2 eine entscheidende Rolle bei der Aktivierung beider Transkriptionsfaktoren durch

Mrmh spielte, wurde der Gehalt an H2O2 in Mrmh in Anwesenheit und Abwesenheit von Zellen

bzw. Gewebe untersucht. So wurden Makrophagen mit Mrmh für 24 h bei 37°C inkubiert und

der extrazelluläre H2O2-Gehalt mittels FOXPCA Assay detektiert. Zum Vergleich wurden

Proben ohne Zellen entsprechend untersucht. Der H2O2-Gehalt war in Anwesenheit der

Makrophagen signifikant niedriger als in ihrer Abwesenheit. Der gleiche Effekt wurde bei der

Stimulation der humanen Darmbioptate ex vivo beobachtet. Es ist bereits bekannt, dass

Zellen reaktive Sauerstoffspezies (ROS) wie H2O2 detoxifizieren können [241]. Maillard

Produkte in Mrmh bilden allerdings permanent H2O2 nach, sodass das extrazelluläre H2O2

nicht komplett durch die Zellen detoxifiziert werden konnte. Restliches H2O2, welches mit


dem FOXPCA Assay detektiert werden konnte, kann somit durch die Zellmembran

diffundieren. Dadurch steigt der intrazelluläre ROS-Level an und kann innerhalb der Zelle

Signalwege über NF-κB und Nrf2 induzieren. Um diese Hypothese zu überprüfen, wurde der

intrazelluläre ROS-Gehalt von Makrophagen nach Inkubation mit Mrmh mittels 2’,7’-

Dichlorofluorescein (DCF) untersucht. Bei dem DCF Assay werden intrazelluläre ROS wie

beispielsweise H2O2, Peroxyradikale, Stickstoffmonoxid oder Peroxynitrit mittels

Fluoreszenzspektroskopie detektiert [237]. So wurde das intrazelluläre Fluoreszenzsignal im

DCF Assay als Indikator für oxidativen Stress in der Zelle verwendet. Der oxidative Stress-

Level stieg mit der Mrmh- Konzentration und der Inkubationsdauer an. Nach 4 h wurde ein 4-

fach erhöhter oxidativer Stress-Level im Vergleich zu PBS stimulierten Kontrollzellen

gemessen, der über die nächsten 20 h konstant blieb. Es wird angenommen, dass durch die

Aktivierung von Nrf2 antioxidative Abwehrmechanismen hochreguliert wurden [29], die einem

weiteren Anstieg des oxidativen Stresses nach 4-stündiger Inkubation mit Mrmh

entgegenwirkten. Da die NADPH-Oxidase nach Aktivierung beim sogenannten „Oxidative

Burst“ ebenfalls ROS generiert [238], wurde untersucht, ob dieses membran-gebundene

Enzym an der Generierung von intrazellulärem oxidativen Stress durch Mrmh beteiligt ist.

Dazu wurden Makrophagen mit Mrmh und Diphenyleneiodoniumchlorid (DPI), einem

NADPH-Oxidase Inhibitor, ko-stimuliert. Da in Anwesenheit von DPI der intrazelluläre

oxidative Stress-Level ebenfalls anstieg, wurde ein signifikanter Beitrag der NADPH-Oxidase

an der Entstehung von intrazellulärem oxidativen Stress durch Mrmh ausgeschlossen.

Deshalb wurde angenommen, dass H2O2, welches von den Maillard Produkten in Mrmh

generiert wird, nicht vollständig von der Zelle detoxifiziert werden kann und durch die

Zellmembran ins Zellinnere diffundiert. Dadurch wird der oxidative Stress-Level der Zelle

erhöht und zelluläre Signalwege können über NF-κB und Nrf2 induziert werden.

Wie bereits beschrieben, induzierte nicht nur Mrmh sondern ebenfalls Mrmu eine Nrf2

Aktivierung. Gleichzeitig konnte gezeigt werden, dass Mrmu H2O2 generiert. Aus diesem


Grund wurde die Rolle von H2O2 an der Nrf2 Aktivierung durch Mrmu untersucht. Im

Gegensatz zu Mrmh wurde die Nrf2 Aktivierung in Makrophagen nach Inkubation mit Mrmu

für 24 h durch Zugabe von Katalase nicht blockiert. So wurde eine Beteiligung von H2O2 an

der Nrf2 Aktivierung durch Mrmu ausgeschlossen. Übereinstimmend zeigte sich, dass Mrmu

den intrazellulären oxidativen Stress-Level in Makrophagen nicht erhöht. Der oxidative

Stress-Level in Makrophagen wurde weiterhin weder nach Stimulierung mit einer reinen

H2O2-Lösung (500 µM) noch mit einer erhitzten Ribose-Lösung (ribh) erhöht. Es wurde

postuliert, dass der H2O2-Gehalt der Mrmu, ribh bzw. der reinen 500 µM H2O2-Lösung durch

Zellen vollständig detoxifiziert werden kann. Die Ergebnisse deuten daraufhin, dass H2O2 bei

der NF-κB und Nrf2 Aktivierung durch Mrmh jedoch nicht Mrmu eine Rolle spielt.

Da gezeigt werden konnte, dass Maillard Produkte NF-κB und Nrf2 induzieren, wurde

anschließend Kaffee untersucht, der aufgrund des Röstprozesses reich an Maillard

Produkten ist.

In Röstkaffee konnten sowohl mit der photometrischen FOXPCA Methode als auch

elektrochemisch mit der Sauerstoffelektrode bis zu 440 µM H2O2 nachgewiesen werden. In

Rohkaffee konnten bis zu 200 µM H2O2 gemessen werden. Die Rohkaffeebohne ist reich an

Polyphenolen wie beispielsweise Chlorogensäure, denen ebenfalls eine H2O2-generierende

Aktivität zugesprochen wird [182]. Da Polyphenole während des Röstprozesses degradieren

[4], wurde die Generierung an H2O2 in Röstkaffee sowohl auf die Degradierungsprodukte als

auch auf die Maillard Produkte zurückgeführt, die während der Röstung gebildet werden.

Im nächsten Schritt wurde der Einfluss von Kaffee auf die Transkriptionsfaktoren NF-κB und

Nrf2 in vitro und ex vivo in humanem Darmgewebe untersucht. Caco-2 Zellen und

Makrophagen wurden mit 1 - 4 mg/mL Kaffee-Extrakt für 2 h (Kurzzeitversuch) inkubiert. In

Caco-2 Zellen konnte nach 2 h weder eine Aktivierung von NF-κB noch von Nrf2 detektiert

werden. In Makrophagen war NF-κB bis zu 4-fach erhöht; es konnte ebenfalls ein Trend zur

Nrf2 Aktivierung nach 2 h beobachtet werden. In humanem Darmgewebe entwickelte sich


unter diesen Bedingungen nach 2 h eine konzentrationsabhängige Aktivierung von NF-κB,

die allerdings statistisch nicht signifikant war. Nrf2 wurde nicht hochreguliert. Da im

Kurzzeitversuch die stärkste Zellantwort auf Kaffee in Makrophagen detektiert wurde, wurden

anschließend Makrophagen im Langzeitversuch mit Kaffee für 30 min bis 24 h stimuliert. Der

nukleäre Nrf2 Gehalt stieg mit der Konzentration und der Inkubationszeit kontinuierlich an

und erreichte nach 24 h einen 20-fachen nukleären Nrf2 Gehalt im Vergleich zu mit Wasser

stimulierten Kontrollzellen. Der nukleäre NF-κB Gehalt stieg innerhalb von 2 h auf das 3-

fache an und fiel, im Gegensatz zu Nrf2, danach wieder ab. Nach 6 h war der Gehalt an

nukleärem NF-κB 1,7-fach im Vergleich zu mit Wasser behandelten Kontrollzellen erhöht.

Im Gegensatz zu Röstkaffee induzierte Rohkaffee keine Aktivierung von NF-κB oder Nrf2 in

Makrophagen. Dies deutet daraufhin, dass die Röstprodukte, zu denen die Maillard Produkte

zählen, für die Aktivierung beider Transkriptionsfaktoren durch Kaffee verantwortlich waren.

Weiterhin wurde die Beteiligung von H2O2, das in Röstkaffee produziert wird, an der

Aktivierung beider Transkriptionsfaktoren durch Röstkaffee in Makrophagen untersucht.

Während die Zugabe von Katalase die NF-κB Aktivierung vollständig inhibierte, wurde die

Nrf2 Aktivierung nur leicht reduziert. Daraus wurde geschlossen, dass H2O2 zwar bei der NF-

κB aber nur in geringem Umfang bei der Nrf2 Aktivierung durch Röstkaffee beteiligt ist.

Anschließend wurde der extrazelluläre Gehalt an H2O2 während der Stimulierung von

Makrophagen mit Kaffee (1 – 4 mg/mL) und in den entsprechenden Inkubationslösungen in

Abwesenheit von Zellen für 24 h gemessen. In Abwesenheit der Zellen wurden bis zu

800 µM H2O2 detektiert, wohingegen bei Anwesenheit von Makrophagen nur bis zu 230 µM

H2O2 gemessen wurden. Ein ähnlicher Trend ergab sich bei der Stimulierung von humanen

Darmbioptaten mit Kaffee für 2 h. Diese Ergebnisse deuten daraufhin, dass extrazelluläres

H2O2, welches in Röstkaffee gebildet wurde, nur unvollständig von Makrophagen bzw.

humanem Darmgewebe detoxifiziert wurde. Überschüssiges H2O2 kann in die Zellen

eindringen und wie bereits beschrieben zelluläre Signalwege wie NF-κB aktivieren.


Anhand der Ergebnisse dieser Studie wird postuliert, dass bei Langzeitinkubationen mit

Kaffee vor allem die Aktivierung von Nrf2 und weniger die von NF-κB in den Vordergrund

rückt. Nrf2 reguliert die Expression antioxidativer und detoxifizierender Enzyme, die

oxidativem Stress entgegenwirken. So können Kaffee und weitere Maillard-reiche

Lebensmittel nicht nur direkt [207], [208], sondern zusätzlich indirekt antioxidativ durch

Induzierung zytoprotektiver Proteine wirken. Dieser Effekt ist vor allem im Darm von

Bedeutung, der im Zuge der Verdauung in direkten Kontakt mit Maillard-reichen

Lebensmitteln kommt. So wurde bereits in einem Mausmodel gezeigt, dass sich die

Aktivierung von Nrf2 positiv auf eine Colitis auswirkt [217]. Darüber hinaus wird die

Aktivierung von Nrf2 mit einer Linderung intestinaler Barrierestörungen [218], mukosaler

Verletzungen im Darm [219] und weiterer intestinaler Entzündungen neben der Colitis [218]

assoziiert. Ferner gilt Nrf2 als vielversprechendes Target bei der Prävention von

Dickdarmkrebs [220], [221]. In der Tat deuten epidemiologische Studien daraufhin, dass bei

regulärem Kaffeekonsum Kaffeetrinker ein geringeres Risiko aufweisen, an Dickdarmkrebs

bzw. an anderen oxidativen Stress-assoziierten Krankheiten wie Alzheimer und Diabetes

mellitus zu erkranken [201].

8 MATERIALS AND METHODS 142

8 MATERIALS AND METHODS

8.1 Materials

8.1.1 Instrumentation

Amicon stirred cell Model 8050 Millipore

Centrifuges (Universal 32 R and 16 R) Hettich

Clark electrode (Type Sension 156) Hach-Lange

Counting chamber (Neubauer, improved) Carl Roth GmbH + Co. KG

D-SaltTM polyacrylamide desalting column Perbio Pierce® (107 mm x 12 mm; cut off 1.8 kDa)

Gel chamber Bio-Rad

Hypercassette autoradiography cassettes Amersham Biosciences

Incubator (CB 150) Binder

Luminescence reader (Lumat LB 9501) Berthold

Luminescent Dissolved Oxygen (LDO) electrode Hach-Lange (Type QD 40 D multi)

Microcentrifuge neoLab

Microplate reader (µQuant) Bio-Rad

Mucosa oxygenator Intestino-Diagnostics

Semi dry electroblotting system (Standard Power Pack P25) Biometra

Tissue culture hood (LaminAir HB2448) Heraeus Instruments

UltracellTMmembranes Millipore

Ultraturrax homogeniser IKA®Werke GmbH / CO.KG

Universal grinder (Type A 10) IKA®Werke GmbH / CO KG

VersaDoc™ Imaging System (Model 4000) Bio-Rad


8.1.2 Laboratory equipment

Blotting paper (GB002) Schleicher&Schuell GmbH

Coffee filter (Filtessa: brown, unbleached, size 4) Norma

Hyperfilm™ ECL Amersham Biosciences

Microcentrifuge tubes Eppendorf

Nitrocellulose transfer membrane (pore size: 0.45 µm) Schleicher&Schuell GmbH

Plastic for cell culture (sterile) Biochrom AG

Syringe filters (0.22 µm) Carl Roth GmbH + Co. KG

Tubes (15 mL, 50 mL) Sarstedt

96-Well polystyrene microtiter plates (Maxisorp F96) Nunc

8.1.3 Chemicals and reagents

Albumin Fluka-Sigma-Aldrich

Ammonium ferrous sulfate Fluka-Sigma-Aldrich

Anti-p65 antibody blocking peptide (sc-109p) Santa Cruz Biotechnology

Ascorbic acid Carl Roth GmbH + Co. KG

Bicinchoninic Acid assay (BC assay) kit Uptima Interchchim

Bis-tris hydrochloride Fluka-Sigma-Aldrich

Bovine serum albumin (BSA) Fluka-Sigma-Aldrich

Bromphenolblue Merck KGaA

N-Butanol Carl Roth GmbH + Co. KG

Catalase (from bovine liver) Fluka-Sigma-Aldrich

ChemiblotTM molecular weight marker Chemicon

2’,7’-Dichlorofluorescein diacetate (DCFH-DA) Merck KGaA

Dc-Protein assay kit Bio-Rad

Dimethyl sulfoxide (DMSO) Acros Organics


3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide Merck KGaA (MTT)

Diphenyleneiodonium chloride (DPI) Sigma-Aldrich

Di-sodium hydrogen phosphate dehydrate Fluka-Sigma-Aldrich

Dithiothreitol (DTT) Fluka-Sigma-Aldrich

Dulbecco’s modified eagle medium (DMEM) Invitrogen

Endo-Paractol Temmler Pharma GmbH

Endothelial cell medium ScienCell

Endothelial cell growth ScienCell

ECL Western Blotting detection reagent Amersham Biosciences

Ethylenediaminetetraacetic acid (EDTA) Carl Roth GmbH + Co. KG

Ethylene glycol tetraacetic acid (EGTA) Fluka-Sigma-Aldrich

Fetal calf serum (FCS) (Lot: 0565L) Biochrom AG

Fibronectin (from bovine plasma) Fluka-Sigma-Aldrich

L-Glutamine Biochrom AG

Glycerol anhydrous Fluka-Sigma-Aldrich

Glycine Fluka-Sigma-Aldrich

HAM’S F12 medium (1176 g/L NaHCO3 stable glutamine) Biochrom AG

HAM’S F-10 without phenol red medium Biochrom AG

Hanks’ balanced salt solution (Hanks) Invitrogen

HEPES Merck KGaA

Hydrochloric acid (35 %, 9.7 M) Evonik Degussa GmbH

Hydrogen peroxide (H2O2) (30 %) Merck KGaA

Interferon-γ (recombinant murine IFN-γ) PeproTech Inc.

Isopropanol BASF

Lactatdehydrogenase (LDH) assay kit Beckman Coulter


Lipopolysaccharide (LPS) (from Escherichia coli 055:B5) Sigma-Aldrich

L-Lysine Sigma-Aldrich

Lysozyme (L6876) Sigma-Aldrich

Magnesiumchloride Sigma-Aldrich

Magnesiumchloride hexahydrate Merck KGaA

2-Methoxyethanol Acros Organics

Minimal essential medium (MEM) Invitrogen

Molecular weight marker for proteins (14 - 66 kDa) Fluka-Sigma-Aldrich

Ninhydrin Merck KGaA

Non-essential amino acids Invitrogen

Nonident P-40 substitute (NP-40) Fluka-Sigma-Aldrich

Orcinol Acros Organics

Penicillin (10000 U/mL)/streptomycin (10000 µg/mL) Biochrom AG

Penicillin/streptomycin/amphotericin B Invitrogen

Perchloric acid (PCA) Merck KGaA

D-Phenylalanine Sigma-Aldrich

Phenylmethylsulfonylfluoride (PMSF) Sigma-Aldrich

Phosphate Buffered Saline (sterile) (PBS) Biochrom AG

Ponceau red S Fluka-Sigma-Aldrich

Potassiumchloride Merck KGaA

Protease inhibitor tablet complete (PIS) Roche Diagnostics GmbH

Raw coffee beans (100 % Coffea arabica) Koenigmanns Kaffeeroesterei Erlangen

Resazurin Sigma-Aldrich

D-(-)-Ribose Sigma-Aldrich

Roasted coffee beans (100 % Coffea arabica) Koenigsmanns (Fazenda Lagoa, Brazil) Kaffeeroesterei Erlangen


Skim milk powder Fluka-Sigma-Aldrich

Sodium chloride Acros Organics

Sodium dihydrogen phosphate dehydrate Fluka-Sigma-Aldrich

Sodium dodecyl sulphate (SDS) Fluka-Sigma-Aldrich

Sodiumhydroxide (2 N) Evonik Degussa GmbH

Trichloroacetic acid Merck KGaA

Tris base Fluka-Sigma-Aldrich

Tris-HCl Fluka-Sigma-Aldrich

Trypan blue Fluka-Sigma-Aldrich

Trypsin/EDTA (0.05 %/0.02 %) Biochrom AG

Tumour necrosis factor-α recombinant, human (TNF-α) Biochrom AG

Tween-20 Fluka-Sigma-Aldrich

Urea Riedel-de Haën

Vitamin B12 Sigma-Aldrich

Xylenol orange Sigma-Aldrich

8.1.4 Buffers and solutions

Chemiblot™ molecular weight marker

(14.8; 28.2; 41.6; 55; 68.5 and 81.8 kDa)

6 µL Chemiblot™ molecular weight marker stock solution

200 µL 5 x Loading buffer

200 µL Glycerol

600 µL H2Oddd

Aliquots were stored at -20°C


2’,7’-Dichlorofluorescein diacetate (DCFH-DA) stock solution

20 mM DCFH-DA in methanol

Dithiothreitol (DTT) stock solution

0.5 M DTT in H2O


10 x Electrophoresis buffer

0.25 M Tris base

1.89 M Glycine

0.035 M SDS

FOXPCA reagent stock solution

5 M Xylenol orange

5 M Ammonium ferrous sulfate in 0.11 M Perchloric acid

The stock solution was diluted 1:10 in H2Oddd before use

High salt extraction buffer B

20 mM HEPES

1 % NP-40

400 mM NaCl

10 mM KCl

1 mM MgCl2 x 6H2O

20 % Glycerol

0.5 mM EDTA

0.1 mM EGTA

Hypotonic lysis buffer Acell

10 mM HEPES

10 mM KCl

1 mM MgCl2 x 6H2O


5 % glycerol

0.5 mM EDTA

0.1 mM EGTA

Hypotonic lysis buffer Atissue

10 mM HEPES

1.5 mM MgCl2 x 6H2O

10 mM KCl

5 x Loading buffer

0.3 M Tris-HCl

0.35 M SDS

5.2 M Urea

0.32 M DTT

35 % Glycerol

Bromphenolblue

Adjusted to pH 7.4 with 2 NaOH and stored in aliquots at -20°C

Molecular weight marker for proteins

(14.2; 20.1; 24; 29; 36; 45 and 66 kDa)

100 µL Molecular weight marker for proteins stock solution

150 µL H2Oddd

75 µL 5 x Loading buffer

75 µL Glycerol


Ninhydrin reagent

3 % w/v Ninhydrin

0.3 % w/v Ascorbic acid

in 10 mL 2-Methoxyethanol


Orcinol reagent

0.2 % w/v Orcinol

1.6 % w/v Ferric ammonium sulphate

35 % Hydrochloric acid

PBS (Phosphate buffered saline)

137 mM NaCl

8.1 mM Na2HPO4 x 2H2O

2.7 mM KCl

1.6 mM NaH2PO4 x 2H2O

PBS/Tween washing buffer

0.1 % (v/v) Tween 20 in PBS (1 x)

Phenylmethylsulfonylfluoride (PMSF) stock solution

250 mM PMSF in Ethanol


Ponceau red S solution

2.6 mM Ponceau red S

180 mM Trichloroacetic acid

100 x Protease Inhibitor Solution (PIS)

1 Protease Inhibitor tablet complete was dissolved in 0.5 mL PBS and aliquots were stored at

-20°C

Skim milk blocking buffer

5 % (w/v) Skim milk powder in PBS/Tween washing buffer

Tissue incubation medium

PBS with 3 % Albumin

2.4 % HEPES buffer

0.0025 % Endo-Paractol


Transfer buffer

25 mM Tris base

200 mM Glycine

20 % Methanol

8.1.5 Cell lines and primary cells

Caco-2 cells ATCC

Human intestinal microvascular endothelial cells (HIMEC) ScienCell

J774 Macrophages UWS

NR8383 Macrophages ATCC

8.1.6 Primary antibodies

Mouse monoclonal anti-β-actin antibody (A5541) Fluka-Sigma-Aldrich

Mouse monoclonal anti-p65 antibody (Sc-8008) Santa Cruz Biotechnology

Rabbit polyclonal anti-Nrf2 antibody (AP06252-PU-N) Acris Antibody GmbH

Rabbit polyclonal anti-p65 antibody (sc-109) Santa Cruz Biotechnology

8.1.7 Secondary antibodies

Horseradish peroxidise (HRP)-conjugated anti-rabbit antibody Fluka-Sigma-Aldrich (A6154)

Horseradish peroxidise (HRP)-conjugated anti-mouse antibody Fluka-Sigma-Aldrich (A6782)


8.2 Methods

8.2.1 Preparation of Maillard reaction mixtures and control solutions

The heated Maillard reaction mixture (Mrmh) was prepared as follows unless otherwise

stated. An equimolar solution of D-(-)-ribose (rib) and L-lysine (lys) (each 0.5 M) were

dissolved in PBS buffer and adjusted from pH 9.7 to pH 9.4 with HCl (9.7 M). Sterile aliquots

of 20 mL were heated in capped screw neck bottles for 30 min at 120°C in a drying oven.

After cooling in an ice-bath, the pH was adjusted from pH 8.6 to pH 8.0 with HCl (9.7 M). An

unheated mixture of rib and lys (Mrmu), heated (h) and unheated (u) solutions of either rib

(ribh; ribu) or lys (lysh; lysu) were prepared under the same conditions. All solutions were filter

sterilized (0.22 µm) and aliquots were stored at - 20°C. Since the concentrations of the

reaction products in the final mixtures are not known, the given concentrations reflect the

initial concentration of the reactants.

The absorbance of the mixtures was read at 280 nm for early stage, 350 nm for intermediate

and 420 nm for late stage Maillard products. Therefore, the mixtures were diluted not to

exceed the absorbance unit (AU) of 1.

8.2.2 Preparation of roasted and raw coffee extract

Coffee extract was prepared using a standardized method. 15 g of coffee beans (100 %

Coffea Arabica, Fazenda Lagoa (Brazil)) were ground for 30 sec in an universal grinder. Raw

coffee beans (100 % Coffea arabica) were subjected first to 3 freeze/thaw cycles followed by

grinding for 60 sec. 3.75 g ground coffee powder was filter-brewed with hot tap water (Ø

85°C) to a total volume of 48 mL (coffee-temperature: Ø 60°C). The coffee was allowed to

stand for 1 hour in an ice-bath and pH was adjusted with NaOH (2 N) from pH 5.6 (Ø) to

pH 7.4. Thereafter, the coffee brew was filled up with hot tap water to an overall volume of

50 mL followed by filter-sterilization (0.22 µm). In all experiments, coffee extract was used

exactly 1 hour after preparation. The coffee concentrations used in the experiments refer to


the dry weight which was analysed in advance in at least 3 independent preparations per

coffee lot.

8.2.3 Cell culture

Cells (Table 8.1) were cultured at 37°C in a humidified atmosphere and 5 % CO2 according

to the manufacturer’s specifications. Cell culture media were supplemented with penicillin

(100 U/mL) and streptomycin (100 µg/mL) to avoid bacteria contamination. Rat macrophages

(NR8383) were maintained in HAM’s F12 medium supplemented with 15 % (v/v) heat-

inactivated FCS. Mouse macrophages (J774) were cultured in DMEM with 10 % (v/v) FCS, L-

glutamine (2 mM) and amphotericin B (0.25 µg/mL). Human epithelial colorectal

adenocarcinoma cells (Caco-2 cells) were grown in MEM supplemented with 20 % (v/v) FCS

and non-essential amino acid solution (0.1 mM). Primary human intestinal microvascular

endothelial cells (HIMEC) were cultivated in fibronectin-coated cell culture flasks in

endothelial cell medium containing 5 % (v/v) FCS and 1 % (v/v) endothelial cell growth

supplement. NR8383 macrophages and J774 macrophages were harvested by scratching;

Caco-2 cells and HIMEC were detached by trypsin (0.25 %)/EDTA (0.02 %).

8.2.4 Tissue culture

The study was approved by the ethics committee of the University Medical Centre in

Erlangen (Germany). Patients gave their informed consent to this local study. Mucosal gut

biopsies (3 - 15 mg) were obtained from seventeen patients during gastrointestinal

endoscopy because of various gastrointestinal complaints and to check-up for manifestations

of inflammation, neoplasm or food allergy. The specimens were taken from 2 localisations in

the lower gastrointestinal tract, from the terminal ileum of the small intestine and from the

ascending colon of the large intestine (ø 6 - 7 biopsies per localisation per patient)

(Table 8.1). In all patients, macroscopic as well as microscopic investigations of the biopsies


did not show any chronic inflammatory gut diseases such as Crohn’s disease. After

endoscopy, the biopsies were placed immediately into hard plastic tubes containing PBS

transport medium which was kept at 37°C and was bubbled constantly with room air

providing an adequate oxygen supply at a pO2 of 85–95 mm Hg (mucosa oxygenation, [192])

to avoid ischemia. Prior to stimulation, wet weight was noted.

Table 8.1: List of in vitro and ex vivo culture

Designation Characteristic Organ/Tissue Species Culture

NR8383 macrophages lung rat cell line

Caco-2 cells epithel/colorectal gut human cell line

HIMEC endothel/intestinal gut human primary cell

human gut tissue

mucosa/lower gastrointestinal tract

gut human tissue

8.2.5 Detection of hydrogen peroxide (H2O2)

Clark and Luminescent Dissolved Oxygen (LDO) electrodes

Both electrochemical methods are based on the indirect measurement of H2O2 as dissolved

O2. In the case of the Clark electrode, O2 is measured electrochemically on a catalytic

platinum electrode [247] whereas with the LDO electrode O2 is detected via an oxygen

sensitive luminophore [248].

Both methods were conducted according to Miyamoto et al (1997) with slight modifications

[247]. In detail, the O2 yield was detected in the sample before and after catalase treatment

(150 U/mL) whilst stirring constantly. Catalase decomposes H2O2 into water and oxygen (O2).

Thus, the release of O2 was linearly correlated to the initial concentration of H2O2. Finally, the

H2O2 concentration was calculated by an external H2O2 standard curve.


Ferrous oxidation xylenol orange in perchloric acid assay (FOXPCA assay)

For the FOXPCA assay, a protocol was used according to Gay and Gebicki [245].

In the absence of cells, a dilution series was prepared using PBS or water as solvent.

According to the protocol, aliquots of 60 µl of each dilution were mixed with 20 µl of solvent

or catalase solution (120 U/mL PBS) as a blank. Catalase converts H2O2 into oxygen and

water and thus detects oxidative effects independent of H2O2. After 15 min at room

temperature, aliquots of 20 µl were incubated with 180 µl FOX reagent (1:10 FOX stock

solution). Thereby, oxidants react with ferrous ions (Fe2+) forming ferric ions (Fe3+). Ferric

ions aggregate with xylenol orange to a coloured complex. After shaking for 30 min, the

absorbance of the complex was read at 550 nm. All readings were corrected for any

interference from brown colour of the samples. In order to detect exclusively the H2O2

induced oxidation, the absorbance of the catalase-blank expressing the H2O2 independent

oxidation was subducted from the absorbance of the pure sample. The H2O2 concentration

was calculated via an external H2O2 standard curve (26 - 520 µM H2O2). The concentration of

the H2O2 stock solution was determined by UV-spectroscopy, using the molar extinction

coefficient (0.0394 cm2/μmol) at 240 nm [249].

In the presence of cells, extracellular but not intracellular H2O2 was measured with the

FOXPCA assay. Therefore, NR8383 macrophages (1 x 106 cells; 2 x 105 cells/mL) were grown

for 4 days at 37°C. Subsequently, cells were stimulated with coffee extract (1 - 4 mg/mL) or

Mrmh (10 - 100 mM) for up to 24 h at 37°C in serum-/phenol red-free medium. In some

experiments, cells were treated with catalase (150 U/mL) which was added to the cells

10 min prior to stimulation. Aliquots of 150 µL were taken after 2, 6, 12 and 24 h. Prior to the

FOXPCA assay, floating cells were removed by centrifugation in a microcentrifuge. Next, the

FOXPCA assay was carried out according to the protocol as described above.

Similarly, extracellular H2O2 was measured during stimulation of human gut tissue samples.


8.2.6 De novo generation of hydrogen peroxide (H2O2)

Coffee extract or Maillard mixtures were incubated with catalase (600 U/mL) in an eppendorf

reaction tube while shaking for 30 min to decompose existing hydrogen peroxide. Next,

catalase was inactivated by heating at 95°C for 5 min in a block thermostate. After cooling on

ice, the samples were diluted and aliquots of 0.5 mL were incubated in eppendorf reaction

tubes at 4°C, 25°C and 37°C in a block thermostate. Within defined incubation times, H2O2

was detected via FOXPCA assay.

8.2.7 Fractionation of Maillard products via size exclusion chromatography

Mrmh was fractionated regarding the H2O2 generating ability by two methods using size

exclusion chromatography.

Firstly, the D-SaltTM polyacrylamide desalting column (cut off 1.8 kDa) was equilibrated with

0.5 mL BSA (10 mg/mL, 66 kDa) as high molecular weight standard, vitamin B12 (0.1 g/mL,

1.4 kDa) as intermediate molecular weight standard and D-phenylalanine (0.1 M, 165 Da) as

low molecular weight standard. In detail, 20 fractions (F) of each 0.5 mL were collected with

water as eluent. The recovery of the standards was investigated by photometric means at

278 nm, 360 nm and 257 nm respectively and by the Ninhydrin assay. In respect to the

elution patterns of the standards, 0.5 mL of 0.25 mM Mrmh (1:2) was fractionated collecting a

high molecular weight fraction (HMW) (F1 - F7) and a low molecular weight fraction (LMW)

(F8 - F20). The fractionation was conducted 3 times in a row according to the fraction

scheme (Figure 8.1) to assure a clear separation between high and low molecular weight

compounds. The concentration of H2O2 was subsequently detected in the HMW and LMW in

a concentration-dependent manner with the FOXPCA assay.


Figure 8.1: Schematic illustration of the 3-step fractionation of the heated

Mrmh into low (LMW) and high molecular weight fractions (HMW) using

size exclusion fractionation.

In a second approach, 0.5 mL of Mrmh (0.25 M, 1:2) was added to the column and eluted

with bidistilled water in 0.5 mL steps into 20 fractions (F). The fractions were collected

separately every 0.5 mL. H2O2 concentration was measured in each fraction with the FOXPCA

assay. Besides, a pure H2O2 solution (102 mM) and the reactants of the Maillard reaction

mixtures, D-(-)-ribose (0.1 M) and L-lysine (0.1 M), were fractionated likewise. Within each

fraction, the concentration of lysine was measured photometrically at 240 nm and by the

Ninhydrin assay and the concentration of ribose was analysed by the Orcinol assay.

8.2.8 Fractionation of Maillard products via ultra-filtration

Mrmh was fractionated regarding the H2O2 generating ability by an ultra-filtration method

using a stirred cell. The crude Mrmh was stepwise ultra-filtered through a stirred cell by

applying a pressure of 3 bar. The stirred cell was equipped with Ultracel membranes with

Mrmh

LMW1HMW1

HMW2 LMW2

LMW3HMW3

LMWHMW

Mrmh

LMW1HMW1

HMW2 LMW2

LMW3HMW3

LMWHMW


molecular mass cut offs of 30 kDa, 3 kDa and 1 kDa respectively. The filtrate was lyophilized

and dissolved in PBS to the starting volume. Each filtrate was used for the next ultra-filtration

step. Finally, H2O2 concentration was measured with the FOXPCA assay in each fraction.

Similarly, BSA (2 mg/mL, 66 kDa), lysozym (0.6 mg/mL, 14.4 kDa) and vitamin B12

(0.2 g/mL, 1.4 kDa) were fractionated as standards. Their concentration within each fraction

was measured by photometric means at 278 nm, 280 nm and 550 nm respectively.

8.2.9 Detection of lysine: Ninhydrin assay

The Ninhydrin assay is qualified to detect amino acids and was therefore used for the

quantification of lysine and phenylalanine in the fractions [250]. Briefly, 50 µl were mixed with

100 µl ninhydrin reagent. The solution was heated for exactly 3 min in a boiling water bath.

Afterwards the solution was cooled down for 5 min in a room tempered water bath. Next,

0.5 mL n-butanol was added and the absorbance was read at 570 nm in duplicate after

allowing standing for 15 min at room temperature. The concentration was calculated by an

external standard curve and corrected by subtracting the absorbance readings of reagent

blank.

8.2.10 Detection of ribose: Orcinol assay

Ribose was detected in the fractions by the Orcinol assay [251]. Briefly, 200 µl were mixed

with 200 µl trichloroacetic acid (10 %) and heated in a hot water bath at 100 °C for 15 min.

After cooling rapidly at 25 °C, 1.2 mL orcinol reagent was added and the mixed solution was

heated up in a boiling water bath for another 20 min. The absorption was read at 660 nm in

triplicate after cooling down to room temperature. Ribose equivalents were calculated based

on an external ribose standard curve and were corrected by subtracting the absorbance

readings of reagent blank.


8.2.11 Nuclear translocation of Nrf2 and NF-κB

In vitro stimulation of various cell types and nuclear protein extraction

Cells were stimulated with Maillard reaction mixtures, coffee extracts (roasted and raw), a

heated ribose solution or a H2O2 solution. In some experiments, cells were co-treated with

catalase (150 U/mL) or heat-inactivated (5 min; 95°C) catalase (150 U/mL) which was added

to the cells 10 min prior to stimulation. As negative control, cells were incubated with

water/PBS instead of stimulant.

NR8383 macrophages (3 x 106 cells; 2 x 105 cells/mL) were grown for 4 days in cell

culture medium followed by stimulation. In the case of NF-κB, cells were stimulated in

PBS for up to 6 h; for Nrf2, cells were stimulated in supplement free medium for 2 h

(short-term experiments) and up to 24 h (long-term experiments) respectively. In

order to change the incubation medium, floating cells were collected by centrifugation

(1500 rpm, 2 min) and both floating cells and adherent cells were washed separately

with PBS or supplement free medium respectively and unified thereafter.

Caco-2 cells (1 x 106 cells; 6.7 x 104 cells/mL) were grown for 5 days. Prior to

stimulation, medium was removed and adherent cells were washed with PBS.

Thereafter, cells were stimulated for 2 h in PBS.

HIMEC (7.5 x 105 cells; 2.5 x 104 cells/mL) were grown for 5 days in cell culture

medium. Medium was refreshed on the 4th day. On the 5th day, the medium was

removed, adherent cells were washed with PBS and cells were stimulated in PBS for

2 h.

The nuclear cell extracts were prepared on ice directly after stimulation, if not stated

otherwise, according to Andrews and Faller with slight modifications [252]. Briefly, adherent

cells were detached by scraping. Both floating cells (NR8383) and adherent cells were

collected by centrifugation (1500 rpm, 4 min, 4°C). The cell pellet was washed 3 times with

5 mL ice-cold PBS. Next, cells were lysed with 1 mL ice-cold hypotonic buffer Acell which was


freshly supplemented with protease inhibitor solution (PIS; 1 %), phenylmethylsulfonylfluoride

(PMSF; 2 mM) and dithiothreitol (DTT; 0.5 mM). After incubation for 15 min on ice, 65 µL of

10 % NP-40 was added. Supplementary, cells were mechanically lysed by vortexing for 15 s.

Cell nuclei, which were collected by centrifugation, were washed with 0.5 mL buffer Acell to

completely remove cytoplasmic proteins. Finally, nuclear proteins were extracted for 1 h with

52 µL ice-cold high salt extraction buffer B which was freshly supplemented with PIS (1 %),

PMSF (2 mM) and DTT (0.5 mM). The suspension was vortexed every 20 min. After 1 h

cellular debris were removed by centrifugation with a microcentrifuge and the supernatant

(which contained the nuclear proteins) was collected. Protein concentration of the nuclear

extract was measured directly according to the Dc Protein assay (BioRad) using BSA

dissolved in supplemented buffer B as standard. Nuclear cell extracts were stored at -80°C

until used for Western blotting.

Ex vivo stimulation of intact human gut tissue and nuclear protein extraction

Briefly, 2 mucosal gut specimens (terminal ileum/ascending colon) were exposed to the

Maillard reaction mixture (Mrmh) or roasted coffee extract for 2 h in the tissue incubation

medium. The negative control specimens were exposed to the solvent without any stimulant.

In some experiments, catalase (150 U/mL) was added to the human gut tissue samples 10

min prior to stimulation. During stimulation at 37°C, human gut tissue samples were

continuously oxygenated (mucosa oxygenation) to prevent ischaemia and to support the

diffusion of the medium dissolved stimuli into the tissue.

After stimulation, the nuclear proteins were extracted according to a modified method of

Thiele et al [253]. In detail, human gut tissue samples were washed in a 50 mL tube with ice-

cold PBS for 2 min while shaking at 400 rpm. In order to lyse the cells, the human gut tissue

samples were resuspended in ice-cold hypotonic buffer Atissue which was freshly

supplemented with DTT (0.5 mM), PMSF (0.2 mM), PIS (1 %) and NP-40 (0.56 %) followed


by 3 freeze-thaw cycles in liquid nitrogen. Afterwards, the human gut tissue was

mechanically disrupted with an Ultraturrax homogeniser for 1 min on ice and allowed to stand

for 30 min before the cell nuclei were collected by centrifugation. The pellet of cell nuclei was

washed with hypotonic buffer Atissue to ensure a complete removal of cytoplasmic proteins.

Subsequently, the nuclear proteins were extracted from the nuclei with 25 µL high salt

extraction buffer B including supplements (see above).

NF-κB/Nrf2 analysis by Western blotting

After in vitro and ex vivo stimulation respectively, the nuclear NF-κB/Nrf2 content in the

isolated nuclear protein extracts was determined immunochemically via Western blotting

according to a modified method described by Muscat et al [174] for Western blot analysis of

NF-κB. Briefly, nuclear proteins were denatured and coated with sodium dodecyl sulphate

(SDS) in loading buffer for 7 min at 95°C. Equal amounts of denatured protein (10 µg for cell

experiments; 5 µg for tissue experiments) in a total volume of 10 µL were separated

electrophoretically via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-

PAGE). Due to the coating with the anionic SDS, the proteins migrate in an electric field

through the gel independent of their charge but according to their molecular weight. The

electrophoretic separation was conducted on a 12 % (NF-κB) and 10 % (Nrf2) SDS-

polyacrylamide gel for 20 min at 80 V (stacking gel) followed by 90 min at 120 V (separation

gel) in electrophoresis buffer. Afterwards the proteins were transferred by a transfer buffer

from the gel onto a nitrocellulose transfer membrane for 1 h at 150 mA (Western blotting). In

order to control the protein transfer, the membranes were stained for 1 min with ponceau red

S solution.

In the case of NF-κB determination, the membranes were cut into 2 segments according to a

molecular weight marker for proteins. In the upper segment p65 (NF-κB subunit; 65 kDa) and

in the lower segment β-actin (42 kDa; loading control) were detected.


For both NF-κB and Nrf2 detection, the staining was removed with PBS and the membranes’

non-specific binding sites were blocked with 12.5 mL skim milk blocking buffer (5 %) for 1 h.

After washing with 10 mL PBS/Tween for 5 min, the membranes were incubated with the

primary antibodies under constant rolling in a 50 mL tube overnight at 4°C.

Primary antibodies included:

For NF-κB (p65 subunit):

rabbit polyclonal anti-p65 antibody (diluted 1:500 in skim milk blocking buffer)

mouse monoclonal anti-p65 antibody (diluted 1:100 in skim milk blocking buffer

(Caco-2 cells))

For β-actin:

mouse anti-β-actin antibody (diluted 1:13333 in skim milk blocking buffer)

For Nrf2:

rabbit anti-Nrf2 antibody (NR8383 macrophages: diluted 1:2000 in skim milk blocking

buffer; Caco-2 cells and human gut tissue: diluted 1:1000 in skim milk blocking buffer)

The next day, primary antibody incubations were continued for 1 h at room temperature.

Thereafter, the membranes were washed 3 times with 12.5 mL PBS/Tween for 5 min each

and incubated with the horseradish peroxidise (HRP)-conjugated secondary antibody under

constant rolling in a 50 mL tube for 1 h at room temperature.

For NF-κB (p65 subunit):

anti-rabbit (diluted 1:1500 in skim milk blocking buffer)

anti-mouse (diluted 1:2000 in skim milk blocking buffer)

For β-actin:

anti-mouse (diluted 1:2000 in skim milk blocking buffer)

For Nrf2:

anti-rabbit (diluted 1:2000 in skim milk blocking buffer)


Finally, the membranes were washed 3 times with 15 mL PBS/Tween for 15 min each and

exposed in the dark for 1 min to enhanced chemiluminescence (ECL) detection reagents

(H2O2, luminol, phenol) to visualize the protein bands. The mix of HRP/H2O2 catalyses the

oxidation of luminol under enhanced conditions in the presence of phenol. Immediately after

oxidation, luminol is in an excited state which then decays to ground state resulting in the

emission of light. The light emission can be detected on a Hyperfilm ECL which was exposed

to the membranes for about 5 sec (Nrf2/β-actin) to 1 h (p65 NF-κB subunit) in hypercassette

autoradiography cassettes. The film was developed and the relative intensity of the protein

bands was determined by densitometric analysis using the VersaDoc™ Imaging System.

Since more than one band arised after incubation with the polyclonal anti-p65 antibody, the

p65 NF-κB subunit was identified by (i) a chemiblot™ molecular weight marker (14.8 -

81.8 kDa), (ii) anti-p65 antibody blocking peptide and (iii) comparison of the polyclonal with

the monoclonal detection. The intensity of the p65 signal was normalized to β-actin which

was used as loading control besides the adjusted protein amount. NF-κB values without β-

actin adjustment showed similar results. Thus, fluctuations in the amount of β-actin due to

stimulation can be excluded. Finally, NF-кB and Nrf2 translocation were expressed as n-fold

increase relative to control which was exposed to the solvent PBS/water alone.

8.2.12 Nuclear protein content in cells: Dc Protein assay

The nuclear protein amount in cells and human gut tissue sample was determined according

to a protocol for the Dc Protein assay by BioRad, a modification of the Lowry procedure

[254]. Preliminary, nuclear proteins were extracted from the cells/tissue samples as

described above. For quantification, 5 µL nuclear protein extract (diluted 1:2 in high salt

extraction buffer B) / standard was mixed with 25 µL reagent A, an alkaline copper tartrate

solution, and with 200 µL reagent B, a diluted Folin reagent. The method relies on the

reaction of (i) proteins with cupric ions under alkaline conditions and (ii) the reduction of Folin


by cuprous ions. The colour development was detected photometrically after 15 min at

750 nm. Protein concentrations were finally calculated from an external standard curve

(0.05 - 3.5 mg/mL) using BSA in high salt extraction buffer B as standard.

8.2.13 Total protein content in tissue: Bicinchoninic acid (BC) assay

The total protein amount in human gut tissue samples was determined with the Bicinchoninic

acid (BC) assay (Uptima, Interchim). Briefly, immediately after weighting, the tissue sample

was homogenized with an Ultraturrax in a defined volume of distilled water on ice for 2 times

20 sec. Aliquots of 25 µL of the homogenate or of the standard were mixed with 200 µL of

the working reagent of the BC assay protein quantitation kit including bicinchoninic acid and

Cu2+-ions. After incubation for 30 min at 37°C, the absorbance of the developing

bicinchoninic acid-Cu+-complex was read at 570 nm. The protein concentrations were

calculated from an external standard curve using albumin as standard.

8.2.14 Oxidative stress level: 2’,7’-Dichlorofluorescein (DCF) assay

J774 macrophages were incubated with Maillard reaction mixtures, a heated ribose solution,

H2O2 or a mixture of LPS/IFN-γ. The intracellular oxidative stress level was monitored by the

DCF assay according to Wang and Joseph [237]. Specifically, J774 macrophages (0.3 x 106

cells/well) were plated in 96-wells overnight to attach and grow at 37°C in the incubator.

Shortly before stimulation, medium was removed, cells were washed with Hanks’ Balance

Salt Solution (Hanks) and pre-incubated with 10 µM 2’,7’-Dichlorofluorescein diacetate

(DCFH-DA) for 1 h. DCFH-DA penetrates into the cell where it gets deacetylated by cellular

esterases into non-fluorescent 2’,7’-dichlorofluorescein (DCFH) which can not diffuse through

the cell membrane anymore. After residual extracellular DCFH-DA was removed, the cells

were washed and exposed to the stimulants for up to 24 h. To rule out any interference,

stimulations were carried out in Hanks instead of cell culture medium. Wells with the


stimulant but no cells served as blank to exclude any impact of the stimulant itself on the

fluorescence signal. In some experiments, cells were co-treated with the NADPH oxidase

inhibitor DPI 30 min prior to the stimulant exposure. During oxidative stress, reactive oxygen

species (ROS) such as hydroxyl and peroxyl radicals, and reactive nitrogen species (RNS)

such as nitric oxide and hyponitrite radicals rapidly oxidize intracellular DCFH into the highly

fluorescent DCF. The fluorescence signal (ex 485 nm/em 520 nm) was monitored between

5 min to 24 h. All treatments and fluorometric determinations were performed in the dark.

The fluorescence signal of the negative control cells was set 100 %. The signal of the

stimulated cells was expressed as relative fluorescence signal compared to the control.

8.2.15 Cytotoxicity of the Maillard reaction mixture and coffee extract

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay

The viability of NR8383 macrophages, which were stimulated with Mrmh, was investigated

according to a modified method of Mosmann [255]. The method is based on the ability of

active cells to metabolize the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT) into dark blue insoluble formazan crystals. Specifically, macrophages (1 x 106

cells; 2 x 105 cells/mL) were allowed to settle overnight at 37°C in a 12-well plate. Next, cells

were stimulated with Mrmh (10 - 100 mM) for 2 or 24 h at 37°C in PBS or supplement free

cell culture medium respectively. In some experiments, catalase (150 U/mL) was added to

the cells 10 min prior to stimulation. Negative control cells were incubated with PBS without

any stimulant. As positive control, cells were treated with 10 % DMSO instead of Mrmh. After

stimulation, cells were incubated with MTT (1 mg/mL in PBS) for 3 h at 37°C. Dark blue

formazan crystals were formed which were dissolved by isopropanol/1 N HCl (25:1) solution

during shaking. The absorbance was read after 10 min at 595 nm. The absorbance of control

cells was considered 100 % viable cells. The cell viability of stimulated cells was expressed

as percent cell viability of the control cells. For medium change during the assay, cell


suspension was centrifuged at 1500 rpm for 2 min at room temperature.

Trypan blue dye exclusion test

The cell viability of NR8383 macrophages after stimulation with coffee extract was

determined by the trypan blue dye exclusion test. Depending on cellular membrane integrity,

which is one main characteristic of vital cells, the anionic diazo dye trypan blue is absorbed

into the cell and appears blue. Briefly, NR8383 macrophages (1 x 106 cells; 2 x 105 cells/mL)

were grown for 4 days. Then, cells were exposed to coffee extract (1 - 4mg/mL) for up to 6 h

in PBS. Control cells were incubated in PBS for up to 6 h alone. After stimulation, cells were

treated with trypan blue dye solution (5 mg/mL PBS). Viable as well as dead (blue) cells were

counted under the microscope via a counting chamber. Cell viability was calculated as the

ratio between living cells and the total cell number. Values obtained for control cells were

considered 100 % cell viability. Cell viability of stimulated cells was expressed as the

percentage of the control cells.

AlamarBlue assay

Cell viability of J774 macrophages after incubation with Mrmh was investigated with the

AlamarBlue assay according to a modified protocol of Nakayama et al [256]. Resazurin

(alamarBlue) is a non fluorescent dye which can diffuse through the cell membrane into the

inside of a cell. Intracellularly, active cells reduce resazurin to resorufin. Resorufin which

produces red fluorescence can be detected via fluorescence using ex 544 nm/em 590 nm. In

this study, J774 macrophages (0.3 x 106 cells; 1.5 x 106 cells/mL) were grown in a 96-well

plate over night in the incubator at 37°C. The following day, cell culture medium was

removed carefully and cells were washed with Hanks buffer. Next, cells were exposed to

Mrmh (0.25 - 5 mM) for 6 h or 24 h in Hanks buffer. Wells with Mrmh but no cells served as

blank to exclude any impact of the stimulant itself on the fluorescence signal. The negative


control cells were incubated with Hanks buffer without any stimulant. As positive control, cells

were exposed to 50 % DMSO. After the incubation time, the stimulant was removed

carefully, cells were washed with Hanks buffer and incubated with 0.001 % alamarBlue

solution (0.001 % resazurin in PBS) for 1 h in the dark. Fluorescence was read at

ex 544 nm/em 590 nm. The fluorescence signal of the negative control cells was set 100 %

cell viability. Cell viability of stimulated cells was expressed in percent according to the

control.

8.2.16 Cell viability of intact human gut tissue during mucosa oxygenation

Lactate dehydrogenase (LDH) assay

A kinetic study of the cell viability of human gut tissue samples during mucosa oxygenation in

ex vivo cultivation was measured using lactate dehydrogenase (LDH) assay. LDH is a

cytosolic enzyme which is released into extracellular medium due to cell membrane damage

and therefore used as an indicator for cell death and thus cell viability.

Human gut tissue samples were kept in either of the incubation medium, modified Hanks

(3 g/L albumin, HEPES (1 M) 2.4 % (v/v), FCS 1 % (v/v)) or modified PBS (3 g/L albumin,

HEPES (1 M) 2.4 % (v/v)). LDH was measured after 0.5, 1.5, 3, 4.5, 6 and 24 h (i) in the

supernatant and (ii) in the tissue. For each time point a single biopsy was used. (i) Aliquots of

the supernatant were taken to determine the LDH release into the medium. (ii) In order to

analyse the intracellular LDH amount, the human gut tissue samples were homogenized with

an Ultraturrax for 30 sec in a defined volume of bis-tris buffer (20 mM; pH 7) on ice. The

concentration of LDH was quantified according to a protocol from Beckman Coulter. Briefly,

aliquots and the homogenate respectively were mixed with L-lactate and NAD+ solution.

Existing LDH catalyses the oxidation of lactate into pyruvate with NAD+ as hydrogen

acceptor. The resulting amount of NADH, which can be finally detected at 340 nm, is directly

proportional to the LDH amount. The cell death is defined by the LDH release which is


calculated as the percentages of extracellular LDH related to total LDH amount. The cell

death values were finally used to calculate the cell viability.

In order to rule out any discrepancy between the human gut tissue samples themselves, the

kinetic LDH release was analysed in one single biopsy over time for 24 h. Therefore, a single

biopsy was incubated in a modified Hanks buffer for 24 h. Aliquots of the supernatant were

taken after several time points and the extracellular LDH amount was determined as

described above.

8.2.17 Statistical analysis

Statistical analysis was performed using Graphpad prism 5. Numeric data is expressed as

mean ± standard deviation (SD) of n-independent experiments. In the case of n = 2, numeric

data is expressed as mean ± range. Statistical significance of the data was calculated using

unpaired, two-tailed Student’s t-test with significance levels * p < 0.05, ** p < 0.01 and

*** p < 0.001.

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LIST OF ABBREVIATIONS 187

LIST OF ABBREVIATIONS AGE Advanced glycation endproducts

ARE Antioxidant response element

AU Absorbance unit

BC assay Bicinchoninic assay

BSA Bovine serum albumin

CEL Nε-(carboxyethyl)lysine

CML Nε-(carboxymethyl)lysine

DCF 2’,7’-Dichlorofluorescein

DCFH 2’,7’-Dichlorofluorescein

DCFH-DA 2’,7’-Dichlorofluorescein diacetate

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DPI Diphenyleneiodonium chloride

DTT Dithiothreitol

ECL Enhanced Chemiluminescence

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol tetraacetic acid

EpRE Electrophile response element

F Fraction

FCS Foetal calf serum

FOX Ferrous oxidation xylenol orange

GSH Glutathione

H2O2 Hydrogen peroxide


Hanks Hanks’ Balance Salt Solution

HIMEC Human intestinal microvascular endothelial cells

HMW High molecular weight fraction

HRP Horseradish peroxidise

IFN-γ Interferon-γ (recombinant, murine)

IκB Inhibitory κB

IL-1 Interleukin-1

IL-2 Interleukin-2

IBD Inflammatory Bowel disease

JNK c-Jun N-terminal kinases

Keap1 Kelch-like ECH-associated protein 1

LDO Luminescent Dissolved Oxygen

LDH Lactate dehydrogenase

LMW Low molecular weight fraction

LPS Lipopolysaccharides

Lysh Heated lysine solution

Lysu Unheated lysine solution

MAPK Mitogen-activating protein kinase

MEM Minimum Essential Medium

mRNA Messenger ribonucleic acid

Mrmh Heated Maillard reaction mixture

Mrmu Unheated Maillard reaction mixture

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide

NADPH Nicotinamide adenine dinucleotide

NF-κB Nuclear factor-κB

NMP N-methylpyridinium


NP-40 Nonidet P-40 substitute

Nrf2 Nuclear factor-erythroid-2-related factor 2

PBS Phosphate buffered saline

PCA Perchloric acid

PI3K Phosphotidyl inositol-2 kinase

PIS Protease inhibitor tablet complete

PKC Protein kinase C

PMSF Phenylmethylsulfonylfluoride

ribh Heated ribose solution

ribu Unheated ribose solution

RNS Reactive nitrogen species

ROS Reactive oxygen species

SD Standard deviation

SDS Sodium dodecyl sulphate

SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis

SEC Size exclusion chromatography

TNF-α Tumour necrosis factor-alpha

LIST OF FIGURES 190

LIST OF FIGURES

Figure 1.1: Rubiaceae – Coffea Arabica ................................................................................... 1

Figure 1.2: Proposed thermal degradation of Trigonellin .......................................................... 4

Figure 1.3: Proposed thermal degradation of 5-O-caffeoylquinic acid (chlorogenic acid) ......... 5

Figure 1.4: The Maillard reaction scheme according to Hodge............................................... 10

Figure 1.5: Initial stage of the Maillard reaction according to Hodge ...................................... 11

Figure 1.6: The chemical structures of intermediate Maillard products................................... 12

Figure 1.7: The chemical structure of Nε-(carboxymethyl)lysine (CML) .................................. 20

Figure 1.8: Activation of molecular oxygen (O2) ...................................................................... 24

Figure 1.9: Detoxification mechanism of ROS ........................................................................ 25

Figure 1.10: Proposed mechanism for the generation of O2• - by enediols.............................. 28

Figure 1.11: Proposed mechanism for the generation of ROS by aminoreductones .............. 28

Figure 1.12: The upper and lower gastrointestinal tract .......................................................... 29

Figure 1.13: Longitudinal section of the wall of the gastrointestinal tract ................................ 30

Figure 1.14: Microscopic histology images of the ileum and the colon ................................... 32

Figure 1.15: Histology of Crohn’s disease and ulcerative colitis ............................................. 33

Figure 2.1: Absorbance of Maillard reaction mixtures at 280 nm, 350 nm and 420 nm .......... 39

Figure 2.2: Detection of H2O2 in Maillard reaction mixtures by FOXPCA assay ........................ 40

Figure 2.3: Detection of H2O2 in Maillard reaction mixtures by electrodes.............................. 41

Figure 2.4: Generation of H2O2 in Maillard reaction mixtures at 37°C..................................... 42

Figure 2.5: Generation of H2O2 in Maillard reaction mixtures at various pH values ................ 43

Figure 2.6: De novo generation of H2O2 in the Maillard reaction mixtures for 24 h ................. 47

Figure 2.7: De novo generation of H2O2 in the Maillard reaction mixtures for 96 h ................. 48

Figure 2.8: Calibration of D-SaltTM polyacrylamide desalting column (SEC) ........................... 49

Figure 2.9: Generation of H2O2 in HMW and LMW of Maillard reaction mixtures ................... 49

Figure 2.10: Generation of H2O2 in 20 fractions of the Maillard reaction mixture collected by

SEC......................................................................................................................................... 50

Figure 2.11: Generation of H2O2 in 4 fractions of the Maillard reaction mixture collected by

ultrafiltration............................................................................................................................. 52

Figure 2.12: De novo generation of H2O2 in the active fraction of the Maillard reaction

mixture..................................................................................................................................... 54

Figure 2.13: Detection of H2O2 in roasted and raw coffee extract by the FOXPCA assay ......... 55

Figure 2.14: Detection of H2O2 in coffee extracts by electrodes ............................................. 55

LIST OF FIGURES 191

Figure 3.1: Simplified illustration of the activation of the NF-κB pathway................................ 64

Figure 3.2: Experimental design to investigate NF-κB activation ............................................ 66

Figure 3.3: The cell growth of NR8383 macrophages............................................................. 66

Figure 3.4: NF-κB activation by Maillard mixture ± catalase in various cell types (short-term) 69

Figure 3.5: NF-κB activation by coffee extract in various cell types (short-term) .................... 72

Figure 3.6: NF-κB activation by roasted and raw coffee extract ± catalase in macrophages.. 73

Figure 3.7: The total and the nuclear protein concentrations in human gut tissue samples.... 75

Figure 3.8: Cell viability of human gut tissue samples during mucosa oxygenation ex vivo ... 76

Figure 3.9: NF-κB activation by Maillard mixture ± catalase in human gut tissue ex vivo ....... 77

Figure 3.10: NF-κB activation by coffee extract in human gut tissue ex vivo .......................... 78

Figure 4.1: Simplified illustration of the activation of the Nrf2 pathway ................................... 84

Figure 4.2: Absorbance of Maillard reaction mixtures at 280 nm, 350 nm and 420 nm .......... 86

Figure 4.3: Experimental design to investigate Nrf2 translocation by Maillard reaction

mixtures................................................................................................................................... 87

Figure 4.4: Nrf2 activation by Maillard reaction mixtures in various cell types and tissue

(short-term).............................................................................................................................. 88

Figure 4.5: Nrf2 activation by Maillard reaction mixtures in macrophages (long-term) ........... 92

Figure 4.6: Nrf2 activation by Maillard reaction mixture (short-term) ± post-incubation .......... 93

Figure 4.7 Experimental design to investigate Nrf2 translocation by coffee extract................ 93

Figure 4.8: Nrf2 activation by coffee extract in various cell types and tissue (short-term) ...... 94

Figure 4.9: Nrf2 activation by coffee extract in macrophages (long-term)............................... 96

Figure 4.10: Nrf2 activation by Maillard reaction mixture (heated) ± catalase in macrophages..

................................................................................................................................................ 98

Figure 4.11: Nrf2 activation by Maillard reaction mixtures ± catalase in Caco-2 cells and

tissue ..................................................................................................................................... 100

Figure 4.12: Nrf2 activation by Maillard reaction mixture (unheated) ± catalase in

macrophages......................................................................................................................... 101

Figure 4.13: Nrf2 activation by coffee extract ± catalase in macrophages............................ 102

Figure 5.1: Extracellular H2O2 after exposure of macrophages with stimulants .................... 111

Figure 5.2: Extracellular H2O2 after exposure of human gut tissue samples with stimulants 113

Figure 5.3: Oxidative stress level in macrophages after stimulation with Maillard reaction

mixture................................................................................................................................... 115

Figure 5.4: The oxidative burst of the NADPH oxidase in oxidative stress ........................... 117

Figure 8.1: Schematic illustration of a 3-step fractionation of the Maillard reaction mixture . 156

LIST OF TABLES 192

LIST OF TABLES Table 1.1: The composition of green and roasted coffee beans (Coffea Arabica) in % dry

weight ........................................................................................................................................ 3

Table 1.2: The amount of total solids in coffee brew prepared by different brewing methods .. 7

Table 1.3: Content of Maillard products in various foods ........................................................ 14

Table 1.4: Secreting cells in the ileum und colon of the gastrointestinal tract ......................... 30

Table 2.1: Detection of H2O2 in various Maillard reaction mixtures......................................... 38

Table 2.2: The pH value of various Maillard reaction mixtures ............................................... 39

Table 2.3: Detection of H2O2 in Maillard reaction mixtures at pH 5.5 and pH 8 ...................... 44

Table 2.4: Detection of H2O2 in Maillard reaction mixtures ± (heat treated) catalase.............. 45

Table 2.5: Detection of H2O2 Maillard reaction mixtures and fractions.................................... 50

Table 5.1: Generation of H2O2 in PBS/water and cell-free cell culture medium .................... 112

Table 8.1: List of in vitro and ex vivo culture ......................................................................... 153

Acknowledgement / Danksagung

Allen voran möchte ich mich ganz herzlich bei meiner Doktormutter Frau Prof. Dr. Monika

Pischetsrieder für die Überlassung des äußerst interessanten Themas und die gewährten

Freiheiten bei dessen Bearbeitung bedanken. Ganz besonders möchte ich meinen Dank für

ihr Vertrauen und Verständnis in unterschiedlichsten Lebenslagen und die Ermöglichung

meines Forschungsaufenthaltes in Sydney zum Ausdruck bringen.

Des Weiteren möchte ich mich bei Herrn Prof. Dr. Gerald Münch für die Übernahme des

Koreferates und die herzliche Aufnahme in seine Arbeitsgruppe während meines

Forschungsaufenthaltes in Sydney bedanken.

Mein Dank gilt weiterhin Herrn Prof. Dr. Martin Raithel für die Übernahme der Doktorprüfung

und die äußerst erfolgreiche Kooperation mit dem Universitätsklinikum Erlangen.

Ferner gilt mein Dank Herrn Prof. Dr. Geoffrey Lee für die Übernahme des

Prüfungsvorsitzes.

Ganz besonders möchte ich mich bei Christine Meißner für Ihre Hilfestellungen in

bürokratischen Dingen und Ihre Unterstützung in sämtlichen Lebenslagen bedanken.

Mein herzlicher Dank gilt außerdem…

…Kerstin Augner, Ulla Müller, Jürgen Kressel, Melanie Deckert, Carolin Hausner und Dr.

Rohtraud Pichner für die gute Zusammenarbeit in den jeweiligen Kooperationsprojekten.

…dem gesamten Arbeitskreis Pischetsrieder inklusive ehemaliger Kollegen/-innen für eine

stets gute Arbeitsatmosphäre und die ein oder andere Einführung in die fränkische Kultur.

…dem Arbeitskreis Eichler insbesondere Marek, Christina, Arne, Uwe und Kalle für Ihre

liebevolle Aufnahme in ihre Runde.

…dem Arbeitskreis Gmeiner u.a. Stefan, Markus und Conny für die Unterstützung jeglicher

Projekte.

Zu guter letzt möchte ich mich bei Jasmin Meltretter und Wolfgang Utz für die herzliche

Aufnahme im Büro im 3.Stock bedanken.

My special thanks go to the postgrads of UWS in particular Megan, Nicky, Paul, Bernie,

Elise, Sam, Elie, Mary, Anette, Anton and Kib for the warm welcome in their group, for their

support in lab and computer issues, and for making my stay an amazing time.

I want to thank Bernadette Westcott, Paul D’Agostino, Pia Rücker and Christian Douniama

for their tireless efforts in proof-reading and correcting my thesis.

*Finally, I want to thank every single person who puts a smile on my face*

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