Nutritional phospholipid development and applications

Dr. Thierry COSTE

What are phospholipids?

Phospholipids

phosphatidic acid PA

Phosphatidylcholine PC

Phosphatidylethanolamine PE

phosphatidylserine PS

phosphatidylglycerol PG

phosphatidylinositol PI

Polar head(hydrophilic)

Non polar tails(hydrophobic)

Amphiphilic properties of phospholipids

Lecithins (phospholipids) => emulsifiers

Structures in aqueous solution

From liposome to cell membrane

Why phospholipids can be so important as food

supplements?

Dietary intake of phospholipids

Dietary intake of phospholipids has diminished due to: - mad cow disease (decrease in brain and liver consumption), - replacement of lecithin in food by other emulsifiers.

Triglycerides between 80 and 120 g Phospholipids 2-3 g instead of 4-6 g

No RDI for phospholipids

It exists no Reference Dietary Intakes (RDI) for phospholipids because we do not know the organism requirements and human body can synthesize them.

Innis et al. 2003, J Pediatr 143: 351-6

=> But for example as you can see in this figure, an excess in synthesis of PC can have a metabolic price: an increase in homocysteine levels.

Phospholipid applications

Health applications

1- Hyperlipidemia

Classic use of soybean lecithins

Dose g

Duration days

Cholesterolemia Triglyceridemia Reference

1-2 244 No effect No effect 1

6-18 84 Decrease Decrease 2

18 10 No effect No effect 3

1) ter Welle et al. 1974, Acta Med Scand 195: 267-71 2) Brook et al. 1986, Biochem Med Metab Biol 35: 31-9 3) Greten et al. 1980, Atherosclerosis 36: 81-8

2- Baby food

Feeding infants with Mother‘s milk is the Best !

The Golden Standard

One of the reason is the presence of LC-PUFA: DHA & ARA

Garcia et al. 2011, J Pediatr Gastroenterol Nutr 53: 206-12

Lipid composition of human milk

3-4 days postpartum 6-10 days postpartum 30 days postpartum

Garcia et al. 2011, J Pediatr Gastroenterol Nutr 53: 206-12

Respective contribution of TG & PL

LC-PUFA ARA DHA Proportion in TG, wt% TFA 0.31 ± 0.18 (0.05-0.76) 0.30 ± 0.28 (0.00-0.95) Proportion in PL, wt% TFA 2.83 ± 1.30 (0.81-4.59) 2.34 ± 0.91 (0.93-4.34)

Contribution of TG, % 90.0 ± 6.5 78.0 ± 32.8 Contribution of PL, % 9.9 ± 6.5 21.9 ± 32.8

Adapted from Table 3: Respective contribution of human milk TG and PL to ARA and DHA supply.

Thus, the contribution of phospholipids can be more important as much than their fatty acids are usually not used for ββ-oxidation.

Feeding infants with Mother‘s milk is the Best !

But, in case this is not possible, you absolutely need to provide DHA and ARA in

form of phospholipids with your formula

For this purpose, only egg yolk lipids bring DHA and ARA in form of phospholipids

similarly to Mother‘s milk lipids

Infant formula

3- Liver

Phosphatidylcholine and alcoholic hepatic steatosis

Baboons

Lieber et al. 1994, Gastroenterology 106: 152-9

=> PC with the dose of 2.8 g/1000 kcal, prevents the development of fibrosis and cirrhosis in baboons following a diet with 50% of caloric ration in form of ethanol.

Baboons with PC

Chronic hepatitis

Gundermann et al. 2011, Pharmacol Rep 63: 643-59

=> This meta-analysis of 9 studies (409 patients) shows global improvement of 26% in the phosphatidylcholine supplemented groups.

4- Stress

PS & PA against mental stress

3 weeks treatment 80 patients 20-45 years old

Hellhammer J et al. Stress 2004, 7: 119-26.

=> The ACTH and cortisol increases, normally induced by stress, are significantly lower with a preventive treatment of 400 mg of phosphatidylserine + phosphatidic acid.

PS & PA against mental stress: results

Hellhammer J et al. Stress 2004, 7: 119-26.

PS & PA against mental stress: results

=> The salivary cortisol increase is also significantly lower with the preventive treatment of 400 mg of phosphatidylserine + phosphatidic acid.

Hellhammer J et al. Stress 2004, 7: 119-26.

5- Brain

Phosphatidylserine levels in tissues

Tissues (human) PS in % of total cell phospholipid

Brain myeline 21

Brain white matter 16

Brain grey matter 13

Retina 8-16

Red blood cells 14

Platelets 9

Lung, spleen, amniotic fluid 8

Liver, heart, skeletal muscle 3

Kidney 1

Plasma Traces

First source of phosphatidylserine

Firstly, phosphatidylserine was manufactured from cerebral cortex of beef.

PS and cognitive decline

Patients (n)

Age years

PS dose in mg

Time weeks

Beneficial effects Ref

149 50-75 300 12 Amelioration of 3/5 tests in patients and 4/5 among most

affected patients

1

425 65-93 300 26 Amelioration in behaviour and cognitive parameters

2

1) Crook et al. Neurology 1991, 41 : 644-9 2) Cenacchi et al. Aging 1993, 5 : 123-33

First source of phosphatidylserine

=> But the mad cow disease made disappear this source.

Phosphatidylserine manufacturing

- bacteria (not so stable during time)

PS and cognitive decline

Patients (n)

Age years

PS dose in mg

Time weeks

Beneficial effects Ref

149 50-75 300 12 Amelioration of 3/5 tests in patients and 4/5 among most

affected patients

1

425 65-93 300 26 Amelioration in behaviour and cognitive parameters

2

120 > 57 300 or 600

12 No significant amelioration 3

1) Crook et al. Neurology 1991, 41 : 644-9 2) Cenacchi et al. Aging 1993, 5 : 123-33 3) Jorissen et al. Nutr Neurosci 2001, 4 : 121-34

Phosphatidylserine manufacturing

- bacteria (more stable with less residual activity) - vegetable (cabbage)

PS and cognitive decline

Patients (n)

Age years

PS dose in mg

Time weeks

Beneficial effects Ref

149 50-75 300 12 Amelioration of 3/5 tests in patients and 4/5 among most

affected patients

1

425 65-93 300 26 Amelioration in behaviour and cognitive parameters

2

120 > 57 300 or 600

12 No significant amelioration 3

78 50-69 100 or 300

26 Amelioration of memory in patients with memory

complaints

4

1) Crook et al. Neurology 1991, 41 : 644-9 2) Cenacchi et al. Aging 1993, 5 : 123-33 3) Jorissen et al. Nutr Neurosci 2001, 4 : 121-34 4) Kato-Kataoka et al. J Clin Biochem Nutr 2010, 47 : 246-55

Sources to manufacture the PS

Generally, soy phospholipids (rich in phosphatidylcholine) are used as raw material. There is also a new PS available, made from sunflower phospholipids.

Moreover, marine or egg yolk sources can be used (because they are also rich in phosphatidylcholine) as raw material to obtain phosphatidylserine containing omega 3.

PS-DHA/EPA and cognitive decline

Patients (n)

Age years

Dose PS-DHA/EPA

in mg

Time weeks

Beneficial effects Ref

8 (pilot study)

≥ 60 300-37.5 6 Amelioration of memory among patients with memory

complaints

1

157 50-90 300-79 15 Amelioration of memory among patients with memory

complaints

2

1) Richter et al. Clin Interv Aging 2010, 5 : 313-6 2) Vakhapova et al. Dement Geriatr Cogn Disord 2010, 29 : 467-74

Vectors for omega 3 fatty acids

What are omega 3 fatty acids?

Alpha linolenic acid (ALA, C18:3 ω3)

Eicosapentaenoic acid (EPA, C20:5 ω3)

Docosahexaenoic acid (DHA, C22:6 ω3)

Main omega 3 fatty acids

40

Why omega 3 fatty acids are so important as food

supplements?

Polyunsaturated fatty acid metabolism

18:2 ωω6

18:3 ωω6

20:3 ωω6

20:4 ωω6

22:4 ωω6

22:5 ωω6

ΔΔ 12 DS

E

24:4 ωω6

24:5 ωω6

Mammals

- Plants - Invertebrates

E AA

LA

18:1 ωω9

E

ββ-ox

Endoplasmic reticulum

Peroxisome

DS = Desaturase E = Elongase ββ-ox = ββ-oxidation

Arachidonic acid

18:3 ωω3

18:4 ωω3

20:4 ωω3

20:5 ωω3

22:5 ωω3

22:6 ωω3

ΔΔ 15 DS

24:5 ωω3

24:6 ωω3

E

E EPA

DHA

ALA

E

ββ-ox

Eicosapentaenoic acid

Docosahexaenoic acid

ΔΔ 6 DS

ΔΔ 5 DS

ΔΔ 6 DS

Strong competition for ΔΔ6 desaturase

18:3 ωω3

18:4 ωω3

22:6 ωω3

18:2 ωω6

18:3 ωω6

22:5 ωω6

24:5 ωω3

24:6 ωω3 24:4 ωω6

24:5 ωω6

DHA

LA ALA

ββ-ox

Endoplasmic reticulum

Peroxisome DS = Desaturase ββ-ox = ββ-oxidation

ββ-ox

SA GLA

DPA

24:6 ωω3 24:5 ωω6

ΔΔ6 DS Conversion < 1%

Why focusing on DHA ?

Omega 3 levels in human tissues

DHA is the most abundant omega 3 fatty acid in human cell membranes because this fatty acid is essential for the cell functionality.

Arterburn et al. 2006, Am J Clin Nutr 83: 1467S-76S

DHA and erythrocyte deformability Animal supplementation study

=> Supplementation with DHA induces an accretion of DHA in erythrocyte membranes resulting in a better deformability, contrary to supplementation with EPA, which can counteract this effect.

Poschl JM et al. Thromb Res 81: 283-8, 1996

Phosphatidylcholine DHA (PC-DHA)

500 ps dynamics of PC (C18:0 and DHA), courtesy from Dr. Scott Feller

Why focusing on DHA in form of phospholipids ?

A similar bioavailability

DHA 8mg/kg 40 days in female rats

Plasma µµg/ml

Erythrocyte mg/g PL

Hepatic tissue mg/g PL

Adipose tissue

mg/g lipids Placebo (olive oil) 80 12.5 45 4.5

Phospholipids 150 (+90%) 27.5 (+120%) 90 (+100%) 9 (+100%)

Monoacylglycerides* 160 (+100%) 25 (+100%) 80 (+80%) 8.5 (+90%)

Triglycerides** 145 (+80%) 22.5 (+80%) 70 (+55%) 7 (+55%)

Ethyl esters*** 150 (+90%) 17.5 (+40%) 40 (-10%) 5 (+10%)

* Not available in the market ** Available as fish or micro-algae oils *** Available as medical drug (for example Omacor®)

Animal supplementation study

Valenzuela A et al. Ann Nutr Metab 49: 325-32, 2005

Cell membrane

Wikimedia commons, LadyofHats Mariana Ruiz

A better bioaccretion in tissue membranes

DHA 8mg/kg 40 days in female rats

Plasma µµg/ml

Erythrocyte mg/g PL

Hepatic tissue mg/g PL

Adipose tissue

mg/g lipids Placebo (olive oil) 80 12.5 45 4.5

Phospholipids 150 (+90%) 27.5 (+120%) 90 (+100%) 9 (+100%)

Monoacylglycerides* 160 (+100%) 25 (+100%) 80 (+80%) 8.5 (+90%)

Triglycerides** 145 (+80%) 22.5 (+80%) 70 (+55%) 7 (+55%)

Ethyl esters*** 150 (+90%) 17.5 (+40%) 40 (-10%) 5 (+10%)

* Not available in the market ** Available as fish or micro-algae oils *** Available as medical drug (for example Omacor®)

Animal supplementation study

Valenzuela A et al. Ann Nutr Metab 49: 325-32, 2005

A better bioaccretion into brain

=> Less than 1% of the ingested dose of DHA was found in brain and the phospholipids form permits a two-fold accretion in older rats when compared to triglyceride form.

Graf et al. 2010, Prostaglandins Leukot Essent Fatty Acids 83: 89-96

A better resistance to oxidation In vitro study

=> DHA in the form of phospholipids is more resistant to the oxidative degradation than DHA in the form of triglycerides or ethyl esters.

Song et al. 1997, Biosci Biotechnol Biochem 61: 2085-8

-97%

-64%

-10%

To resume

DHA form Triglycerides Ethyl esters Phospholipids History of human consumption

++ - ++

Bioaccretion + +/- ++ Oxidative stability -- - ++ Choline - - ++ Phosphorus - - ++

=> Phospholipids are the better form to bring DHA to human organism.

Marine phospholipid developments

Marine phospholipids: raw materials

Marine phospholipids

Krill oil Fish eggs: Caviar phospholipids

Health applications of Krill oil

Krill oil Premenstrual syndrome2

Hyperlipidemia1

Inflammation/arthritis3

1) Bunea R et al. Altern Med Rev 2004, 9: 420-8 2) Sampalis F et al. Altern Med Rev 2003, 8: 171-9 3) Deutsch L J Am Coll Nutr 2007, 26: 39-48

Marine phospholipids: composition

Phospholipid distribution Fatty acid distribution

Marine phospholipids: comparison

Raw materials Krill Caviar History of human consumption

- ++

Oxidation stability ++ ++ Taste impact - - Choline ++ ++ EPA ++ + DHA + ++ Astaxanthin ++ ++ Vitamin E + + Bioavailability + +

=> Caviar phospholipids extracted from fish eggs present higher DHA levels than phospholipids extracted from Krill.

Marine phospholipids & psoriasis

Dupont 2006, Phytothérapie 1: 15-22

6 months of treatment

with 400 mg daily

Caviar phospholipids & psoriasis

Formulating agent

Overview on typical phospholipid formulations

Fricker et al. 2010, Pharm Res 27: 1469-86

Aspirin-Phosphatidylcholine

Thank you for your attention