hwb - jan 2011

Tissue partnerships – muscle: after a meal – fates of branched chain AA’s (BCAA’s)

Only valine, leucine & isoleucine are NOT catabolized extensively in liver; instead they go to ….

(peripheral) muscle where they are 1st transaminated, and then

resulting branched chain α-ketoacids catabolized for energy production (with large energy yield).

TPP(thiamine)

Degradation of intracellular proteinsLysosomal – acidic - enzymes (cathepsins) degrade prot’s continually; in

addition … Chemically modified proteins and those tagged by calpains & ubiquitins -

preferentially degraded

Half-life of a protein marked by N-terminal AA residueEg’s: if serine – ½ life = ~ 20 hrs

if aspartate – ½ life = ~ 3 min’sAspartate’s significance? Readily catabolized, so a logical signal – why?

Little long-term energy investment

*** Proteins rich in proline (P), glutamate (E), serine (S) & threonine (T) - (PEST) – typically have short 1/2-lives

Familiar example? – HMG-CoA reductase (4 hrs)

hwb - jan 2011

The process of protein degradation

1st step – always - conversion to N-free productGlucogenic AA’s –

converted to TCA / glycolytic intermediates Ketogenic AA’s –

degraded to Acetyl-CoA (final product) 

2 are purely ketogenic: leucine & lysine (essential) 

4 are both glucogenic and ketogenic: tryptophan, phenylalanine, isoleucine, tyrosine

14 - Purely glucogenic – with the exception of alanine & valine, none of these possess a hydrophobic methyl side group (-CH3)

WIF – Y?

hwb - jan 2011

Glutamate dehydrogenase: can either release NH3 or consume it

Very wide-spread enzyme in all mammalian tissues; highest activity in liver

Reaction: freely reversible and can function in either synthesis or degradation of glutamate

 Note – either NADP+ or NAD+ can serve as co-substrate (coenzyme).

Glutamate – unique – our only AA to undergo oxidative deamination

hwb - jan 2011

Glutamate dehydrogenase: a tightly regulated enzyme oxidative deamination vs reductive aminationActivate oxidation: ADP, GDP; Inhibit oxidation: ATP, GTPFit this into the energy charge concept; Note: 2 different coenzymes for 2 opposing reactions; Glutamate: non-essential & glucogenic

hwb - jan 2011

Glutamate metabolism:red. (NADPH) amination & oxid. (NAD+) deamination

Glutamate

The key turn-around AA

to either glutamine (ATP- utilizing synth)

or

α-ketoglutarate (ATP-yielding TCA cycle)

OD-ing onGlutamate

hwb - jan 2011

Pyridoxal phosphate (PLP)

- a prosthetic group for

transaminases:

Lysine’s -NH2 group

binds to the carbon of PLP’s aldehyde group; bond is a

Schiff base

hwb - jan 2011

Urea (Krebs-Henseleit) Cycle the synthesis of urea

First step occurs in liver, with nitrogen that has been transported to the liver via glutamine

 One key intermediate – carbamoyl phosphate 

Made from NH4+, CO2 & ATP

By carbamoyl phosphate synthetase (CPS I)

This enzyme has an absolute requirement for an allosteric activator – N-acetyl-glutamate

hwb - jan 2011

The Urea (Krebs-Henseleit) cycle.

Note partnership

of two separate compartments(text follows)

A: Aspartate provides one of the Nitrogens that becomes

part of UREA.

The Urea (Krebs-Henseleit) cycle.

Note partnership

of two separate compartments(text follows)

A: Aspartate provides one of the Nitrogens that becomes

part of UREA.

hwb - jan 2011

Carbamoyl phosphate synthetase I (CPS I)

Cost: 2 ATP equivalents/urea formed; irreversible step

Eukaryotes – have 2 forms of CPS - I (urea cycle) and II (pyrimidine synthesis)

1. CPS I, mitochondrial, high activity, requires NH3, and

NH 3 + HCO3- + 2 ATP carbamoyl phosphate + 2 ADP + Pi

2. CPS II, cytosolic, takes -NH2 from glutamine;

this 2nd variant is inhibited by UTP – reflecting its

involvement in pyrimidine synthesis

hwb - jan 2011

In the urea cycle: 6 main points

1. Carbamoyl phosphate & ornithine make citrulline;2. Citrulline & aspartate react, making argininosuccinate; 3. Original C-skeleton of aspartate (4-C) is released as fumarate - indirectly a 4-C gluconeogenic or TCA cycle intermediate - very important – as urea cycle is active when gluconeogenesis is activated; NB: fumarate → malate → OAA → → → glucose (next slide)

4. The nitrogens stay behind, to make arginine5. In last reaction, arginine is broken up by arginase, This removes urea and re-establishes ornithine, thus completing the “cycle”6. No reaction in urea cycle is coenzyme-dependent

hwb - jan 2011

Other key features of urea cycle

Note: the syntheses of carbamoyl phosphate &

citrulline are mitochondrial;

all other reactions - cytosolic.

High cost (4 ATP’s) of synthesizing one molecule of urea (yet - consider the alternatives)

How calculate 4 ATP’s – while only 3 ATP’s are used directly ?

ATP AMP + PPi: equate this with 2 ATP’s 

Argininosuccinate synthase

hwb - jan 2011

Failure of the urea cycle causes hyperammonemia & CNS dysfunctions

deficiencies of urea cycle enzymes – rare, usually recessive

but X-linked OTC def most common world-wide

Features of condition:

Orotic acid build-up; hyperammonemia & encephalopathy; feeding

problems, vomiting, lethargy or irritability, poor CNS devel’t,

tendency for coma → death

Condition aggravated by dietary protein → N’ous waste overload

*** Males with non-conservative mutations rarely survive 1st 72 h. Half of survivors die in 1st month, and half of the

remaining by age 5.

When treatment and diet inadequate, liver transplant may become a treatment option.

A: X LINKED OTC – MOST COMMON UREA CYCLE DEFICINEY

hwb - jan 2011

Failure of the urea cycle, cont’dPatient often develops aversion to protein-rich foods

In addition to rising NH4+, glutamine is usually elevated,

from diversion of extra α-ketoglutarate & NH 4+ for

glutamine synthesis. Thought: this could limit availability of α-KG for TCA cycle function, simultaneously depleting ATP, more of which is now used for the glutamine synthetase reaction – creating a vicious cycle.

Overall – toxic levels of NH4+ seem to interfere with very

high levels of ATP production required for normal brain function.

hwb - jan 2011

Treating the patient with urea cycle failure

1. Replace essential AA’s with their α-keto-acids - costly

2. Use of alternative routes of nitrogen excretionBenzoic acid, phenylacetate and phenylbutyrate

- these conjugate with 2 N-rich AA’s - glycine and glutamine

 Conjugated soluble N-rich products can then be readily excreted in urine

(this principle is exploited by the organism in many applications; e.g.: excretion of products of heme breakdown, bile salts, etc.)

(following slide for elaboration)

Catabolism of the individual amino acids

Alanine, aspartate, glutamate, asparagine & glutamine –

All 5 - closely related to common MAJOR metabolites

 

Typical relationships:

Eg: interconversion of

pyruvate & alanine by

alanine aminotrasferase

(transaminase)

hwb - jan 2011 16i.e. ALT

Interconversion of serine and glycine; can yield pyruvate

Hydroxy methyl transferase also requires pyridoxal phosphate (permanently bound); Tetrahydrofolate, stemming from folic acid, is a carrier of the 1-C methylene group.

Deficiencies of tetrahydrofolate – lead to loss of methylene carrying capacity and loss of the step

hwb - jan 2011 17

*** Tetrahydrofolate (THF) – from Folic acid

This cofactor/coenzyme - also related to tetrahydrobiopterin (BH4) – see later Source: Vitamin B-9 – folic acid

THF’s synthesis from folate requires NADPH in two successive reductions by

dihydrofolate reductase

NADPH + H+ NADPH + H+

Folate dihydrofolate tetrahydrofolate NADP+ NADP+

hwb - jan 2011 18

Degradation of methioninecoenzyme roles:

pyridoxine (B6) cystathionine synthase,

cystathioninase (see error C&H)

folate (B9)methyl-THF

(from methylene THF);

cobalamin (B12) accepts

methyl group from methyl-THF

- transfers methyl to homocysteine, remaking

methioninehwb - jan 2011 19

hwb - jan 2011 20

MUST KNOW THIS!!!!!! SEE NOTES BELOWAt least two Qs

A carrier of single carbon groups:

Tetrahydrofolate (THF),from folic acid – Vit B9, its interconversions from

formyl-THF → methyl-THF

(know differences between formyl (N10) methenyl, methylene (both

N5-10) and methyl (N5) Note error: second reducer ►

should also be NADPH !

hwb - jan 2011 21

SAM cycle Through donation of its methyl group, SAM becomes S-adenosylhomocysteine (SAH);

this is re-converted to homocysteine and then finally back to methionineMethylation of homocysteine → methionine

most important -requires BOTH methyl tetrahydrofolate &

cobalamin (vitamin B12) as methylcobalamin

This reaction is a central feature of vitamin B12 deficiency and pernicious anemia (see this later in the vitamin story)

hwb - jan 2011 22

Degradation of methionine to cysteine, cont’d

The condensation of homocysteine and serine - makes cystathionine

Cystathionine lyase (cystathioninase), nowcleaves cystathionine, & creates cysteine and α-ketobutyrate (5-C compound). Downstream –

conversion of propionyl-CoA →succinyl-CoA requires: 1st biotin & 2nd cobalamin.

So - through these reactions, cysteine is made from the carbon skeleton of serine and the sulfur of methionine.

Our daily excretion of about 20-30 mmol of SO4 is largely derived from dietary cysteine and methionine

hwb - jan 2011 23

Aberrant metabolism of methionine & cysteine

Homocystinuria

Defect/deficiency - cystathionine synthasemetabolite build-up always upstream of block; thushomocysteine, homocystine & methionine accumulate Frequency ~ 1:20,000 humans

Diagnostic: “funnel chest” ►(Pectus excavatum)

superficially, resembles Marfan syndrome, (also tall, thin build) but while Marfan has “loose” joints, in homocystinuria they are “tight”

hwb - jan 2011 24

Valine, Leucine & Isoleucine – 1st transaminated,

2nd oxidatively decarboxylated, then 3rd FAD-linked oxidized

NB: These 3 AA’s - transaminated in muscle & other extrahepatic tissues, and then catabolized

BC-α-keto acid dehydrogenase

resembles pyr. dehydrogenase; defects in PDH can cause

maple syrup urine diseasehwb - jan 2011 25

Disorders in metabolism of the branched chain amino acids (BCAA’s)

Maple syrup urine disease – 1:200,000 incidencemuch higher in Amish & Mennonite communities

(founder effect)

Cause: branched chain α-ketoacid dehydrogenase (quite nonselective): decarboxylates all 3 branched ketoacids & produces NADH + H+

Rx resembles pyruvate dehydrogenase reactionSigns: Severe mental retardation, acidosis, sweet-smelling urine (resembles burnt caramel), early death is frequent

Treatment:Megadoses of thiamin (why this?) can at times be effectiveRestrict dietary valine, leucine and isoleucine

hwb - jan 2011 26

Degradation of tryptophan –

1st - oxidative cleavage of pyrrol forms N-formylkynurenine - by tryptophan pyrrolase (oxidase) – requires O2, NADPH + H+

complex pathway; portion of molecule outside the ring becomes alanine & indole ring yields many products, incl acetoacetyl-CoA

One product - nicotinic acid – also considered a vitamin small, insufficient, amounts of related vitamin niacin can be formed in man

A number of tryptophan-derived products such as kynurenate and xanthurenate cannot be degraded further but are excreted, giving urine its characteristic color

hwb - jan 2011 27

Phenylalanine is degraded to tyrosine: both are glucogenic and ketogenic

Aromatic ring cannot be synthesized in the humanYet - tyrosine is not “essential” as it is made during the metabolism of phenylalanine by - 1st -Phenylalanine hydroxylase (PAH)

Requires ½ O2, &Tetrahydrobiopterin [BH4] as reducer (to form –OH group)

In active state: tetramer – Requires phosphorylationat critical serine(s)

hwb - jan 2011 28

Tyrosinemia

Two causal Deficiencies: Types I & II

I: fumaryl-acetoacetate hydrolase.

II: tyrosine transaminase (tyr phenylpyruvate)

Also note – homogentisate oxidase ► deficiency → alkaptonuria

Type I - more common – patient: cabbage-like odor from accumulated organic acids built up through inhibition of early steps in degradation of tyrosine; impairment of tubular absorption & liver failure frequent → death

hwb - jan 2011 29

A (lack of

homogentisate oxidase leads to alkaptonuria, a KEY SIGN IS BLACK URINE)

AA Synthesis via Transamination (3):

• Pyruvate Alanine • Oxaloacetate Aspartate

• α-ketoglutarate Glutamate

Transaminase (req. PLP!!)

Transaminase (req. PLP!!)

Transaminase (req. PLP!!)

AA synthesis via REDUCTIVE AMINATION (1):

α-KetoGlutarate Glutamate + NADP+

+ NADPH!!! + NH3

Glutamate Dehydrogenase

Make note

Of the three amino acids formed via Transamination (alanine aspartate and glutamate), ONLY GLUTAMATE can be formed via “REDUCTIVE AMINATION” as well

AA Synthesis via AMIDATION (2):

GlutAMINE+ NH3 (in the form of free amonia!!!!)

GlutamateGlutamine SynthetaseReqs ATP!

Aspartate AsparagineAsparagine SynthetaseReqs ATP!

+ NH3 (donated by glutamine!!!!!)

Serine, Glycine, Cysteine

Note the “succession” in the formation of these three amino acids 1. Serine: Formed from 3-phosphoglycerate (3PG) of glycolysis. First - 3 phosphopyruvate is formed

This is then transaminated (PLP) to 3-phosphoserine Hydrolysis of the phosphate ester creates serine

(It can also be formed by the attachment of a hydroxymethyl group to glycine – carried by N5-N10 -methylene tetrahydrofolate) – methylene-THF** 2. Glycine: by removal of hydroxymethyl from serine –

in a reaction that requires tetrahydrofolate (FH4) and PLP(see also previous file)

hwb - sept '09We will discuss Cysteine synthesis in a few slides

Synthesis of Serine

3PG phosphopyruvate 3-Phosphoserine SERINE(from glycolysis)

Transamination via PLP

Glycine + N5-N10-methylene tetrahydrofolate Serine and Tetrahydrofolate

Requires PLP!

AND

Synthesis of Glycine: from Serine Exact same reaction (directly above) but, in reverse!

Interconversion of serine and glycine and the central role of tetrahydrofolate

Note the removal/attachment of a single-carbon group – key step in interconversions of some amino acids

hwb - sept '09

In the interconversion of Glycine to Serine (going from right to left on this figure): A hydroxymethyl group is being transferred from methylene-THF to Glycine to form Serine

Synthesis of cysteine from methionine &

serine

NB: at branch point, homocysteine; can be

either (a) methylated or

(b) condensed with ► serine to create

cystathionine & on to Cysteine***

***note linked roles of vitamins B6 & B12

hwb - sept '09

B9 B12

B6

B6

Synthesis of cysteine –from

Methionine (sulfur group of cysteine comes from

Methionine).

Three products of terminal reaction:

1. ammonia, 2. α-ketobutyrate (to succinyl CoA),

3. cysteine

hwb - sept '09

**Carbon skeleton of cysteine provided by serine!!!!**

Rxn requires Vit. B6

Rxn requires Vit. B6

Vascular disease and high [homocysteine]

Patients with high homocysteine or homocystine(dimer) – unusually high risk for coronary heart disease – and/or

arteriosclerosisMost common cause – mutation in gene for cystathionine synthase!! (homocysteine + serine cystathionine)

Homocysteine’s action? Poorly understood, but some damage to endothelial cells & elevation of vascular smooth muscle growth

Also – raises oxidative stress – not known how

hwb - jan '10

Homocysteine Methionine

Methyl-THF

Homocysteine methyltransferaseREQ B12

Cystathionine

Cystathionine synthaseREQ B6

Cystathionase REQ. B6

CYSTEINE

(B9)

SERINE

Tyrosine

Created by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH)

Requirements: molecular O2 and key coenzyme tetrahydrobiopterin (BH4)*****;

In this reaction one O atom becomes –OH of tyrosine; the other is reduced to water

During this reaction sequence, the tetrahydrobiopterin is oxidized and then recycled,

via a reaction using NADPH

*BH4 and THF use NADPH as reducing power

hwb - sept '09

Synthesis of Tyrosine from Phenylalanine

Phenylalanine Tyrosine

Phenylalanine Hydroxylase REQ. O2

Tetrahydrobiopterin (BH4)

Dihydrobiopterin (BH2)

NADPHNADP+

Dihydropteridine Reductase

Deficiency in Phenylalanine Hydroxylase= Phenylketonuria (PKU)

NADPH is providing the reducing power to fuel this reaction

Products flowing from essential amino acids

Now – similar to cysteine – - tyrosine is not an essential AA in the strict sense. -synthesized from an amino acid (phenylalanine) which itself is essential. - can be synthesized only when adequate supplies of phenylalanine are available Deficiency of phenylalanine hydroxylase – basis of the well-know disorder phenylketonuria (PKU) - also hyperphenylalaninemia (HPA) (described earlier).

hwb - sept '09

Tetrahydrobiopterin and its roles in metabolism of the 3 aromatic amino acids

*NO BH4* = hyperphenylalanemia, NO catecholamines, NO serotonin (from Tryptophan)

hwb - sept '09

Cystinosis (ONLY AA disorder related to membrane transport defect!!!)

Mechanism: *** Lysosomal transport defect!Cystine storage in most organs and cell types, esp. cornea of the eye & renal tubular epithelium where damage most evident

Cystine: 2 cysteines in –SS- bond; transport/egress mechanism tocytosol is defective. Very poorly soluble: pure cystine – 4-sided crystals; cystine-HCl – prismatic needles (frequent in cornea)

Clinical presentations:Photophobia, retinal blindness, tearing, general (kidney) tubulardysfunction (Fanconi syndrome), acidosis, polyuria, weight loss, fevers, dehydration, muscle weakness (hypokalemia); major urinary ion losses; also glucosuria, diabetes mellitus, etc.; glomerular damage progresses → dialysis or transplant essential at 6-12 yrs.

hwb - sept 2010

Does not involve metabolic breakdown; problem is in Transport***

Synthesis and Degradation of PorphyrinReturn to the point that heme is synthesized in both erythropoietic and non-erythropoietic tissues.

Although the sequences are identical in such different cell types, differences in their regulation underscore strong divergences in function of the molecule created. Eg: heme in Hb – O2 transport; heme in Cyt450 – donate O in creation of –OH groups (usually)

There are different points at which heme synthesis is contolled/regulated in the two main tissue types:

1. in liver – control is at the first step (via allosteric negative feedback by heme)

2. in bone marrow – control only at final step (indirectly via heme-induced inhibition of iron uptake – not

allosteric in an enzymic sense. Enough complete heme means the tissue has sufficient Fe)

 

HIGHYIELD AS@#$%!

Erythropoietin and intracellular Fe availability

hwb - may 2011

Biosynthesis of Porphyrins

Major sites, in the adult - as stated: 1. RBC-producing cells of bone marrow

2. liver – here cytochrome P-450 enzymes are synthesized; thus major destination for heme

Initial and the last 3 steps of the process occur in the mitochondrion, - middle 4 take place in the cytosol.

Student Problem: why can the RBC NOT make heme?

HIGHYIELD AS@#$%!

HEME SYNTHESIS SUMMARY

glycine

succinyl-CoA

ALA

porphobilinogenno light absorption

Uroporphyrinogen IIIcan absorb light (ring)

protoporphyrin IX HEME

ALA synthase(PLP)

ALA dehydratase or porphobilinogen

synthase(Zn2+)

HMB synthase

uroporph. III synthase

Ferrochelatase

ALA

3 porphobilinogen

high [heme] or [hematin]low [heme]Drugs

Pb poisoning

Pb poisoning

1st step in mitochondria (ALA synthase)Middle 4 steps in cytosolLast 3 steps in mitochondria (copro heme)

Drugs that affect ALA synthase (inc activity):• phenobarbitols• hydantoins• griseofulvin

AIP

Fe2+ inserted into ring

CEP - AR!

*regulatory step in LIVER

*regulatory step in BONE MARROW

Hydroxymethylbilane = Linear tetramer of porphobilinogen - absorbs light cyclized to uroporphyrinogen

Added

4 molecules condensed

Coproporphyrinogen IIIMITO

(Symptoms of the acute hepatic porphyrias are worsened by these drugs)

hwb - may 2011

Heme synthesis summary.

Common porphyrias and their

causes

HIGHYIELD AS@#$%!

What is common to all the porphyrias? Decreased Heme synthesis increased ALA synthase in liver build up of toxic intermediates

Synthesis: Step 1 – in LIVER: Formation of δ-aminolevulinic acid (ALA)

Two simple & universally available starting metabolites:

succinyl CoA and glycine condense in a reaction catalyzed by

ALA synthase (requires pyridoxal phosphate - PLP) regulates the pathway in in liver rate-controlling step in liver

Complex regulatory scheme: heme controls

ALA synthase activity - 2 mechanisms

1. lower [heme] – raises catalytic activity & synthesis

2. higher [heme] – blocks synthesis & translocation of ALA synthase from cytosol (site of synthesis) to mitochondrion (site of action)

Neg feedback by heme on ALA synthaseLIVER regulation is at first step

HIGHYIELD AS@#$%!

hwb - may 2011

Formation of porphobilinogen

2 δ-aminolevulinates (ALAs) condense to form this molecule via

ALA dehydratase or porphobilinogen synthase (know both)

requires Zn++ ; bivalent cations can competitively inhibit; very sensitive to Pb++ poisoning (Plumbism).

HIGHYIELD AS@#$%!

What would you see in a patient with lead poisoning? Anemia and elevated ALA

hwb - may 2011

Formation of uroporphyrinogen

Aggregation of 4 molecules of porphobilinogen causes formation of uroporphyrinogen (hydroxymethylbilane is an

intermediate-1st to absorb light)

Two different – linked - enzymes involved:1. Hydroxymethylbilane synthase

2. Uroporphyrinogen III synthase

1st Synthase – makes chain of 4 porphobilinogens (linear)

2nd Synthase – creates ring from this chain; isomerizes itFinal product: asymmetric uroporphyrinogen III

(see side group arrangement)

condensation of the 4 porphobilinogen molecules can ABSORB LIGHT

closes the porphyrin ring/isomerization

HIGHYIELD AS@#$%!

Overview:Pathway of porphyrin synthesis

No light absorption @ 440 nm

by singlet porphobilingen but

Hydroxymethylbilane, a

Tetramer of porphobilinogen

absorbs light @ 440 nm

▼

▼

◄ note: NA - Pb levels in blood ↓ 30% last 25 yrs

▼hwb - may 2011

HIGHYIELD AS@#$%!

1. Hydroxymethylbilane synthase & 2. uroporphyrinogen III synthase

to be considered later in detail –

if insufficiency of second enzyme (uroIII synthase), hydroxymethylbilane causes accumulation to levels that exceed the body’s capacity to excrete the molecule, cell now has a Phototoxic metabolite.

What does “phototoxic” mean?(patients with an enzyme defect that causes an accumulation of phototoxic metabolites will

have itchy and burning skin when exposed to visible light)

Result - Congenital Erythropoietic Porphyria (AR)

HIGHYIELD AS@#$%!

Formation of heme

Through a set of oxidations and decarboxylations,Uroporphyrinogen III is modified to become protoporphyrin IX. Final step: Fe++ inserted into ring center by ferrochelatase;

Fe – must be in ferrous (reduced – Fe++) state **Control step in bone marrow – NOT allosteric

Final product: Heme or Fe-protoporphyrin IX

Ferrochelatase – 2nd enzyme in this pathway sensitive to Pb inhibition / poisoning (Plumbism)

Aug 20, ’09: CNN Headline – China, leadmajor smelters closed – Pb poisoning in ~ 1000 children

 

HIGHYIELD AS@#$%!

hwb - may 2011

Heme synthesis summary.

Common porphyrias and their

causes

HIGHYIELD AS@#$%!

hwb - may 2011

The porphyrias (Gr. “purple pigment”) 1877: Felix Hoppe-Seyler (coins Biological Chemistry – “Biochemistry”)

1889: Stokvis (“Acute” porphyrias)

Usually inherited – may be acquired through environmental events

If inherited – generally autosomally dominant, except for Congenital Erythropoietic Porphyria (CEP) - an autosomal recessive disease

Classified as hepatic or erythropoietic –Generally – cause is a defect in heme synthesis;

Student Problem: what happens to the porphyrins & their precursors?

what accumulates & what exists at lower concentrations?

HIGHYIELD AS@#$%!

PORPHYRIAS SUMMARY

LEAD POISONING ferrochetalase & copro and ALA accumulateALA dehydratase

ACUTE INTERMITTENT HMB synthase porphobilinogen +ALA accumulate PORPHYRIA (AIP) patients NOT photosensitive

liver enyzme deficiency

CONGENITAL uroporphyrinogen III AUTOSOMAL RECESSIVEERYTHROPOIETIC synthase patients are photosensitivePORPHYRIA (CEP) BM enzyme deficiency(Gunther’s Disease)

PORPHYRIA uroporphyrinogen uroporphyrin accumulates CUTANIA TARDA decarboxylase most common porphyria

patents are photosensitiveliver and BM enzymes affected

VARIGATE protoporphyrinogen liver enzyme deficiencyPORPHYRIA oxidase patients are photosensitive

ERYTHROPOIETIC ferrochetalase protoporphyrin accumulatesPROTOPORPHYRIA BM enzyme deficiency

photosensitive, build up in RBC, BM & plasma

Porphyrias decrease HEME synthesis. In the liver, heme inhibits ALA synthase. Porphyrias causes an increase in the synthesis of ALA synthase forms porphyrin intermediates prior to defective enzyme accumulation of TOXIC intermediates

Extra HIGHYIELD AS@#$%!

DEGRADATION OF HEME SUMMARY

1. Degrade Heme biliverdin • Via heme oxygenase – cofactors are NADPH and O2

• methenyl bridge hydrolyzed by one of the O in O2 (other O forms CO)• Fe2+ oxidized to Fe3+

2. Biliverdin Bilirubin• Soluble insoluble compound, anti-oxidant (in babies)

3. Bilirubin liver• In blood non-covalent association with albumin• In liver binds to ligandin enters hepatic circulation• Ligandin: glutathione-S-transferase activity

4. Conjugation of 2 glucuronates to bilirubin• Via bilirubin glucuronyltransferase• donor molecule is UDP-glucuronic• now SOLUBLE and can be removed via active transport into bile• defect in transport of conjugated bilirubin = Dubin-Johnson Syndrome: black liver,

autosomal recessive, conjugated hyperbilirubinema

5. Intestine• Resident bacteria form urobilins: urobilinogen (colorless), • If goes to kideny urobilin (yellow), large colon stercobilin (brown)

Added

Degradation of Heme & formation of bilirubin

RBC’s life span of ~ 120 days means continual turnover;taken up & degraded by spleen’s reticulo-endothelial cells1st - the spleen – and 2nd liver. ~ 85% heme destined for degradation comes from RBC,

remainder from immature or badly formed RBC’s & cytochromes from other systems

Step 1: Creates biliverdin – water soluble & thus easily excretable (birds, reptiles, amphibians) catalyzed by Heme oxygenase requires: NADPH & molecular O2

Methenyl bridge betw 2 neighboring pyrrole rings is hydrolyzed by addition of one atom of O and two protons.

The other atom of oxygen becomes part of carbon monoxide (CO)Further - ferrous iron is oxidized to ferric iron (Fe+++)

hwb - may 2011

Step 3: Bilirubin passes to liverBilirubin’s low H2O solubility necessitates transport to liver via non-covalent association with albumin

In hepatic circulation, bilirubin leaves albumin, binds to ligandin, & enters hepatocyte. Here – Ligandin – hepatocyte transporter of organic anions, non-polar cpds; has glutathione-S-transferase activity

Note: some drugs (eg barbiturates, steroids, etc) & other conjugates can displace bilirubin from

albumin;

When [bilirubin] too high, unconjugated bilirubin accumulates in certain lipid-rich brain regions (basal nuclei, globus pallidus, lentiform nucleus, caudate nucleus: → → “kernicterus” – German & Greek: nuclear jaundice) → neural damage, esp in developing CNS … (see later)

Step 4: Heme’s degradation, cont’d

To create an easily excreted product, bilirubin is made more soluble

Now -

2 glucuronates added byBilirubin

glucuronyltransferase – donor: UDP-glucuronic

Bilirubin diglucuronide (soluble) – removed via active transport into bile canaliculi, & proceeds to bile (defect/deficiency in this TRANSPORT mechanism → Dubin-Johnson syndrome:

black liver, auto rec., hyperbilirubinemiaNB: unconjugated bilirubin cannot be

easily excreted

DEGRADATION OF HEME SUMMARY

1. Degrade RBC heme biliverdin • Via heme oxygenase – cofactors are NADPH and O2

• methenyl bridge hydrolyzed by one of the O in O2 (other O forms CO)• Fe2+ oxidized to Fe3+

2. Biliverdin Bilirubin• Soluble insoluble compound, anti-oxidant (in babies)

3. Bilirubin liver• In blood non-covalent association with albumin• In liver binds to ligandin enters hepatic circulation• Ligandin: glutathione-S-transferase activity

4. Conjugation of 2 glucuronates to bilirubin• Via bilirubin glucuronyltransferase• donor molecule is UDP-glucuronic• now SOLUBLE and can be removed via active transport into bile• defect in transport of conjugated bilirubin = Dubin-Johnson Syndrome: black liver,

autosomal recessive, conjugated hyperbilirubinema.

5. Intestine• Resident bacteria form urobilins: urobilinogen (colorless), • If goes to kideny urobilin (yellow), large colon stercobilin (brown)

Major Groupings in Jaundice:Hemolytic Jaundice

Hemolytic JaundiceLiver – normally capable of handling a heme load of over 10 times the daily rate of heme production, sogenerally, able to process all that comes But on occasion, as in massive lysis of RBC’s (eg: malaria or sickle cell anemia) the patient will produce bilirubin at a rate that exceeds the capacity of the liver to degrade it.

Now unconjugated bilirubin levels rise sharply in blood

no mutation, defect, or deficiency… a quantity problem – too much bilirubin

hwb - may 2011

Jaundice in the Newborn refer also to hemoglobin lecture

If this occurs - and it often does - it is due to low activity of the enzyme bilirubin glucuronyl transferase in liver

This enzyme reaches adult activity levels about two weeks after birth Note: elevated bilirubin can exceed the carrying capacity of serum albumin, diffuse into the basal ganglia of the CNS and cause encephalopathies (kernicterus). of varying severity

Neonatal jaundiceRecall from our previous discussion –

succession of Hb’s in human development:Fetal Hb is ~ 2x as concentrated as HbA(210 g/L in HbF vs ~120 g/L vs HbA)

Review the functional significance of this The “excess” Hb is rapidly dismantled shortly after birth,

giving rise to the observed neonatal jaundice

Thus, two main reasons for the neonatal jaundice: 1. Low activities of the transferase and 2. High levels of bilirubin

hwb - may 2011

Bilirubin is light sensitive – becomes polarupon light exposure

Practice:newborns with noticeably elevated bilirubin readings are placed under fluorescent light;

This converts the bilirubin to a water-soluble structure, in turn enabling the organisms to excrete it far more easily, without the normally necessary (and expensive) step of conjugation to glucuronate.

Clinical Interest: – normal light destroys riboflavin, so infants receiving such treatment routinely get riboflavin (vit B–2) fortification in their formula

JAUNDICE TYPES SUMMARY

hemolytic jaundice

• increased destruction of RBCs (and therefore heme also)• heme is broken down faster than liver can degrade it• unconjugated bilirubin reabsorbed

neonatal jaundice

• Either deficiency of bilirubin glucuronyltransferase or too much HbF breakdown (remember: baby born with more hemoglobin than present in adults – levels drop shortly after birth).

• Unconjugated bilirubin• Treat with fluorescent light & give Vit. B2 (riboflavin) b/c it’s destroyed by light.

obstructive jaundice

• obstruction of bile duct (ex: bile stones)• prevents passage of bilirubin into intestine for excretion so liver “regurgitates” conjugated

bilirubin into blood

hepatocellular jaundice

• damage to liver cells (ex: cirrhosis in alcoholics)• decreased conjugation and the conjugated bilirubin “leaks” into blood

hwb - jan 2011

Synthesis of Creatine

Universal starting materials: Glycine & guanidino group of arginine, & methyl group of S-adenosylmethionine (Ado Met / SAM).

Note integration of several sequences – via key compounds – directly no essential nutrients

* Synthesized in liver and pancreas in significant amounts

* But neither tissue contains creatine kinaseSo – separate sites for creatine synthesis & use of phosphocreatine Uptake & concentration in myocyte by specific

Na+-dependent transporter.

?what makes up Creatine?

hwb - jan 2011

Synthesis of the catecholamines from

tyrosinenote –

4 Rx’s in succession –

1st hydroxylation with BH4 & O2,

2nd PLP-dependent decarboxylation,

3rd ascorbate dependent hydroxylation and finally

4th SAM-dependent methylation by phenyl-ethanolamine-N-methyl

transferase (PNMT)

KNOW WHAT EACH RXN REQUIRES!

• Arginine + NADPH + O2 Nitric oxide• Histidine + pyridoxal phosphate-dependent

decarboxylation (PLP/non-oxidative decarboxylation) HISTAMINE

• Glutamate with PLP-dependent GABA• Tryptophan with BH4-dependent hydroxylation which

makes 5-Hydroxytryptophan, then decarboxylation with PLP Serotonin

• Serotonin with acetyl-CoA and SAM ( acetyltransferase and SAM/methylation) Melatonin

SYNTHESIS OF BIOGENIC AMINES

hwb - jan 2011

Melanin

The pigment that colors hair, hide & eyes.Synthesis from tyrosine, via tyrosinase – a Cu-containing enzyme

a 2-step process: uses DOPA as a cofactor and produces dopaquinone Following exposure to UV light, higher levels of tyrosinase and a protein called “tyrosinase related protein” are induced. Albinism – often caused by lack of tyrosinase

Know the main pointsActivatorsEnzymesThe difference between purine and pyrimidine and not the total reactionKnow regulatorsKnow how to salvage purines

hwb - jan 2011

Purine nucleotide synthesis – de novo

Synthesized as the relevant nucleotide on a required“scaffold” – Phosphoribosyl-pyrophosphate (PRPP)

Requirements:Precursors: 3 common AA’s:

aspartate, glutamine, glycine; CO2’

Folic acid derivatives (vitamin B-9) as tetrahydrofolate (TH4) to donate 1-C formyl groups

Ribose-5-P (via PRPP - of HMP origin)Energy from ATP: large amounts

hwb - jan 2011

Sources of atoms that comprise

the purine nucleus:

aspartate & glutamine donate

N only;all of glycine;

formyl-FH4 donates (1-C) formyl groups

hwb - jan 2011

Purine ring fabricated by series of reactions that add nitrogen and

carbons to already existing PRPP (pyro-phosphorylated - at C-1- of ribose-5-P, shunt sugar)

1st Step: PRPP made by:

Ribose phosphate pyrophosphokinase (or PRPP synthetase – which requires ATP)

Note: activator (Pi) and inhibitors (ie – IMP, AMP & GMP)

hwb - jan 2011

2nd step: synthesis of 5’-phospho-ribosylaminePyrophosphate is removed from C-1, and the amide group from

glutamine replaces it - leaving one Pi remaining, at C-5

This step is regulatory: glutamine:phospho-ribosyl-pyrophosphate amido-transferase

(second –NH2 group - “amido” as in Gln & Asn) inhibited by the mononucleotides AMP, GMP, IMP - the pathway’s end

products

Note: this enzyme has a Km for PRPP significantly higher than intracellular concentrations of PRPP – thus any small change in [PRPP] causes a proportional change in reaction rate. Significance? PRPP – a potent activator. Also, synthesis accurately reflects availability of platform

KNOW THIS

hwb - jan 2011

The 3rd “step”: ends with synthesis of inosine monophosphate (IMP) – the stem (parent) purine nucleotide

nine (yes – 9!) steps are involved hereA derivative of tetrahydrofolate – FH4 - (formyl-tetrahydrofolate) is required here in two key reactionsthat transfer 1-C groups. Inhibition here blocks purine & therefore nucleic acid synthesisFolate is essential in attachments of one-carbon groups – such as formate, methyl groups, and methylene (review serine/glycine metabolism).

Note: analogues of folic acid can stop the assembly of purines, explaining their use in treatment of cancers

hwb - jan 2011

Purine nucleotide synthesis;

ends in stem IMPPRPP – platform for assembly of groups.

Pyrimidine nucleotide synthesis? – NO such

platform.

The cell’s investment? PRPP, 4 ATP,

2 Gln, asp, glycine,2 formyl-THF,

CO2 – NO biotin needed!! Note: inhib’s & activ’s;

Antibiotics - 2 places in formyl transfers

• FOLATE TRAP No Vitamin B12 OR NO HOMOCYSTEINE METHYLTRANSFERASE

• remember back to the Methionine pathway that in order to remake methionine you need to have vitamin B12, WITHOUT THIS YOU NO LONGER CAN REMAKE METHIONINE.

• ALSO, THE POINT OF THIS SLIDE, YOU CAN’T GENERATE TETRAHYDROFOLATE(THF) FROM METHYL THF. SO YOU HAVE “TRAPPED” THE FOLATE WHERE IT IS ALL STUCK IN THE METHYL THF FORM

• NO PURINE SYNTHESIS

hwb - jan 2011

4th Step: Conversion of IMP to GMP and AMPnote point of action of Mycophenolate

2-steps; reciprocal energy sources (GTP for AMP; ATP for GMP); in each case 1st step is regulated

Similar to arginosuccinate synthetase back in the urea cycle =)

en.wikipedia.org

hwb - jan 2011

The 5th step: Conversion of nucleoside monophosphates to di- & triphosphates

Energetic exchange bank - base-specific. Base-specific nucleoside mono- di- phosphate kinases

hwb - jan 2011

Enzymes that salvage the free purine bases

1. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)2. Adenine phosphoribosyl transferase (APRT)

2 different enzymes, yet similar reactions: each requires PRPP HGPRT

1. Hypoxanthine + PRPP inosine monophosphate (IMP) HGPRT

1. Guanine + PRPP guanosine monophosphate (GMP) APRT

2. Adenine + PRPP adenosine monophosphate (AMP)

Hydrolysis of pyrophosphate to 2 Pi’s makes reaction irreversible

hwb - jan 2011

Gout – uric acid, cont’d

Refer to previous slide – see 2 consecutive reactions involving xanthine (1 as product and 2 as substrate):

1. Hypoxanthine + O2 xanthine + H2O2

2. Xanthine + O2 uric acid + H2O2

Both consecutive reactions: run by xanthine oxidase (XO), which produces the ROS H2O2

in each reaction -

Allopurinol – competitive inhibitor of XO, drug to combat gout

hwb - jan 2011

Hyperuricemia/gout, cont’d

Uric acid and its salt crystals precipitate in synovial fluid of joints arthritis & degeneration of joint

Often associated with “rich” (DNA-purine-rich) foods: liver, sweetbreads, anchovies, red wine, often historically associated with excessively high life, ie – abundance of good food, dietary protein.

BUT – gout can also arise from cancer chemotherapy – likely due to overload of purines caused by nucleic acid degradation after death of cancer cells.

Gout, Lesch-Nyhan, cont’d

Mutations of HGPRT account for ~33% of all cases of Lesch-Nyhan

*** Some evidence of Parkinson-like 70-90% reduction of dopamine synthesis in basal ganglia – BUT – Parkinson’s patients do not show the aggression or self-mutilation of the L-N patient

Further – hyper-uricemia - NOT an etiologic factor in neuropathy of L-N disease

Q: What enzyme is deficient in Lesch Nyhan Syndrome?!

hwb - jan 2011

Disorders of Purine metabolism, cont’d: deficiency of adenosine deaminase (ADA)

Deficiencies of ADA will cause deoxy-adenosine levels to rise; These inhibit ribonucleoside reductase

and in turnprevent sufficient production of DNA

In severe form, this will cause a lack of T and B lymphocytes, causing Severe Combined Immunodeficiency Disease (SCID)

hwb - jan 2011

The Pyrimidines Sources of carbons and nitrogens

Starters: glutamine, aspartate and – without biotin - CO2

Carbamoyl phosphate Synthesis:

Committed step via cytosolic Carbamoyl

phosphate synthetase II\ (CPS II)

no biotin required Inhibitor: UTP

Activators: ATP, PRPP

*C skeleton from aspartate*N from glutamine

No G here (Glycine required for purines)

hwb - jan 2011

Pyrimidine synthesis: note involvement of PRPP to create stem product

Multifunctional Enzyme3 domains of same polypeptide chain

CPS II CO2 + 2ATP + glutamine carbamoyl phosphate inhibited by UTP activated by ATP, PRPP

aspartate transcarbamoylase (ATCase) carbamoyl phosphate + aspartate carbamoyl aspartate in prokaryotic cells, this is the regulatory step and is inhibited by CTP

dihydroorotase carbamoyl aspartate + H2O dihydroorotate (1st ring formed)

THEN… dihydroorotate + NAD orotate (complete ring added) via Dihydroorotate DH

orotate + PRPP OMP via Orotate phosphoribosyl transferase

OMP – CO2 UMP via OMP decarboxylase

last 2 enzymes: transferase & decarboxylase are separate domains on a single polypeptide – UMP synthase

Extra*

Final step

CTP is synthesized by the amination of UTP

This is catalyzed by CTP synthetase

amido nitrogen donated byglutamine

Transfering this costly amido group from glutamine toanother compound hydrolysis of anhydrous phosphate (ATP, pyrophosphate, etc)

has to be UTP form!!!

hwb - jan 2011

All N-ous bases stem from only 2 compounds by the simple addition or removal of functional groups:

a unifying & very cost-effective concept

Purines IMP + -NH2 (at different points) AMP or GMP

Pyrimidines

OMP - CO2 UMP UTP + - NH2 CTP

UDP reduction → dUDP - Pi → dUMP dUMP + -CH3 dTMP; + 2 Pi’s → dTTP → DNA

Note: these very similar modifications are specific for the phosphorylation status of the acceptor molecule

web2.airmail.net

Conversion of ribonucleotides to deoxyribo nucleotides

Necessary for DNA

Ribo-nucleoside di-phosphates are reduced to their deoxy forms (dADP, dUDP, dGDP, dCDP)

donors of the hydrogen atoms are the pair of sulfhydryl groups on the enzyme itself

Enzyme – ribonucleotide reductase (NADPH maintains it in reduced state)

hwb - jan 2011

Reduction of ribonucleosides to produce deoxyribonucleosides

Note flow of reducing power: NADPH → thioredoxin → rNDP. Overall inhibitor: dATP. 2 Reductases: i: ribonucleotide reductase & ii: thioredoxin reductase - operate serially. Thioredoxin: has 2 –SH groups in –Cys-Gly-Pro-Cys- sequence

NADPH essential

hwb - jan 2011

Regulation of deoxyribonucleotide synthesis

An adequate supply of the deoxy ribonucleotides has to be present at all times, so regulation of their synthesis is criticalControl of this enzyme: complex yet logicalSeparate sites of regulation & for catalysis

The process: Binding of dATP (finished product) blocks Activity Site,

prevents reduction of any of the 4 nucleoside diphosphates Substrate specificity site –

binding of nucleoside triphosphate at an additional site identifies the diphosphate substrate to be reducedE.g.: ATP tells enzyme that ADP is to be reduced;

CTP informs enzyme that CDP will be reduced, etc

ribonucleotides (RNA) to deoxyribonucleotides (DNA) SUMMARYthe ribonucleosides MUST BE in di-form (2 phosphates attached)

Ribonucleotide reductase is the enzyme that catalyzes ribo deoxyribo inhibited by dATP thioredoxin is the molecule that reduces the ribonucleoside the reduction releases H2O

Thioredoxin reductase is the enzyme that reduces thioredoxin this allows thioredoxin to continue to reduce ribo deoxyribo NADPH is the molecule that reduces the thioredoxin (from S-S to 2SH)

regulation of Ribonucleotide reductase via 2 sites:

1.dATP binding to activity site inhibits (blocks) reduction of ANY of the 4 NDPs sort of like negative feedback… there is enough deoxyNT’s (dATP) so don’t need to create any more

2.binding of a NTP at an additional site regulates identity of substrate ATP identifies that ADP is to be reduced, CTP tells enzyme CDP needs to be reduced, and so on…

extra

Production of dTTP (to be used in DNA) SUMMARY…

UMP UDP dUDP dUMP dTMP dTTP

UMP created from “stem molecule” of pyrimidines – OMP

needs to be phosphorylated to UDP in order to undergo reduction to deoxy state

UDP is converted to dUDP via ribonucleotide reductase

dUDP needs to return back to mono-state, dUMP in order to be methylated

dUMP is methylated to dTMP via Thymidylate synthase (methyl from THF)

dTMP is converted to dTDP and then to dTTP via kinases and ATP

to put it plainly… needs to be in DI-form to become deoxy

needs to be in MONO-form to become methylated

hwb - jan 2011

Now – the conversion of dUMP and dTMP

catalyzed by

Thymidylate synthase (no ATP requirement)

This utilizes methylene tetrahydrofolate (THF or FH4) as the source of the methyl group.

Sidebar note: Methylene-THF: (-CH2) methylene held at N5 & N10 (2 bonds) – reduced to methyl-THF by NADPH

Methyl-THF: -CH3 methyl held at N5 (one bond)

hwb - jan 2011

dUMP yields dTMP

Note connection between methylene tetrahydrofolate and

NADPH;

linkage of two enzymes required:

dihydrofolate reductase & thymidylate

synthase

KNOW what inhibits each enzyme…

5-Fluorouracil inhibitsthymidylate synthase

methotrexate inhibits dihydrofolate reductase

either of these 2 inhibitors results in blocking synthesis of dTMP

aaronlogan.com

Tetrahydrofolate (H4-folate)

recycled from dihydrofolate by the action of dihydrofolate reductase, which requires NADPH Methylene H4-folate supplies the methyl group in the synthesis of

dTMP from dUMP. Recall - serine donates this 1- C group, controlling TH4’s role here.

Clin. Corr.: Functional H4-folate essential for ultimate production of dTTP & DNA); → considerable scope for cancer treatment;

eg: methotrexate (amethopterin) competitively & strongly (x100 than substrate) inhibits dihydrofolate reductase (model out LW-Burk plot for this!)

remember… competitive inhibition = same binding site, decreased affinity ( Km), same Vmax

hwb - jan 2011

• Purine nucleotides• Start with PRPP• 1st corner then addedPrecursors: 3 AA’s:

aspartate, glutamine, and glycine (skeleton) ;

CO2’

Derivatives of folic acid (vitamin B-9) as tetrahydrofolate

Very large ATP cost

Pyrimidine nucleotidesRing built firstThen added to PRPP

Precursors:Glutamine (gives NH2)

Aspartate (gives 3 C skeleton and NH2)

CO2

Modest energetic cost (2 ATP’s)

Main points of nucleotide metabolism: Summary

hwb - jan 2011

Purine nucleotidesIMP yields AMP, GMP

Indestructible ring structure

Salvage pathway very important

Yields uric acid as end product

PyrimidinesOMP yields UMP, and indirectly, CMP and TMPEasily catabolized for energy yield, yields NH3

No salvage pathway

Summary of main points of nucleotide metabolism, cont’d

All reductions to deoxy state require NDP, the reduced enzyme and, indirectly, a steady supply of NADPH

To create dUMP: convert dCDP to dCMP and then add NH3 to create dUMP or take UDP dUDP –

To create dTTP – add methyl group to dUMP (methylene-THF)

All single carbon groups donated by THF

All Pi additions require ATP - mainly of mitochondrial origin (in bacteria – oxphos is on plasma membrane)

Main points of nucleotide metabolism: Summary

CopperTotal body stores: app 80-110 mg; liver is centrall Dietary need: app 1-3 mg/day; Excretion: bile ~1.5-2 mg/day

~ 60% Cu bound to ceruloplasmin; rest bound loosely to albumin or in histidine-complex

Ceruloplasmin (cpm) – abundant; 95+% plasma Cu;Glycoprotein – single polypeptide with 6 Cu’s; α2-globulin; site of synthesis – hepatocyte; ½-life = 5.5 d.;

Normally – 10% cpm apoprotein, secreted w/o Cu, ½-life ~ 5 hrs; Changes in Cu or cpm synthesis easily effect Wilson’s disease

Functions: In Cu-containing metalloenzymes; Generally these use either molecular O2 or an oxygen derivative as a substrate.

Examples:

cytochrome oxidase, dopamine β-hydroxylase, tyrosinase, lysyl oxidase (compare this with lysyl hydroxylase - with its Fe++), and cytoplasmic superoxide dismutase (see Lou Gehrig’s disease)

*Cu necessary for SOD functionHWB - Sept '09

Some trace metals - a two-edged problem: Deficiency and Toxicity

E.g.: Copper1. Deficiency – Menke’s Disease (kinky hair syndrome)

Rare, X-linked recessive, untreated – fatal; Infants – with condition, exposed to normal amounts of dietary Cu, cannot absorb & retain enough for normal metabolism;→ growth retardation, mental deficiency, seizures, arterial aneurysms, bone demineralization, brittle hair.

Apparent cause: deficiency of ATP-dependent Cu transporter; Possibly also - improper binding to histidine & other AA’s

Treatment – absorbable copper-histidine complex2. Toxicity – Wilson’s Disease *affects liver/brain mainly (accumulation of Cu)

*Kayser-Fleischer rings!

HWB - Sept '09

Copper Need To Knows:

• Most of plasma Cu is bound to a copper carrying-protein: Ceruloplasmin*

• GI uptake with Tranferrin• Cu deficiency= Menke’s Disease “Kinky Hair

Syndrome” deficiency in ATP dependent Cu transporter

• Cu excess= Wilson’s disease**- deficiency of a different Cu transporter biliary copper excretion blocked Kayser-Fleischer rings!

• Example of an enzyme requiring Cu: Cytoplasmic Superoxide Dismutase (Rmbr: Lou Gehrig’s Disease)

Zinc

Key in many enzymes; Next to Fe – most abundant trace mineral; Total stores ~ 1.5 – 2.5 g; in adult male RDA ~15 mg/day

Many Zn metalloenzymes (300+ identified –2000+ proteins)Eg’s: carbonic anhydrase, cytoplasmic superoxide dismutase (contains both Cu & Zn), alcohol dehydrogenase, carboxypeptidases A & B, DNA & RNA polymerases

Sources: meat, nuts, beans, wheat germ – broad variety of foodsAvg daily diet contains ~ 10-20 mg

Absorption - upper reach of small intestine incomplete; depends largely upon food substances that could interfere

No specific binding blood protein: transport - serum albuminStorage – with metallothionein present - many tissues,

synthesis induced by heavy metals : zinc, cadmium, arsenicZn absorption – follows metallothionein levels in intestinal mucosa.*** (Q!!) Metallothionein(s) protect(s) cell from toxic effects of free, unbound metal ions.

HWB – S:9

Zinc:• Zinc is transported in the blood via Albumin• GI uptake via Transferrin• Zinc is stored by metallothionein-- a zinc-binding

protein present in many tissues• Metallothionein’s synthesis induced by heavy

metals!!!!!• Metallothionein’s main role is to protect the cell

from toxic effects of free, unbound metal ions.• Zinc deficiency- may see Dermatitis and rare

autosomal recessively inherited condition called Acrodermatitis Enteropathica

Trace metals and their specific functions Manganese (Mn)

Cofactor of many enzymes, can often be replaced by Mg Eg: mitochondrial superoxide dismutaseAdequate intake ~ 2-5 mg/day.

Excess Mn – toxic, → psychosis and Parkinsonism (“manganese madness”) resembling Parkinson’s disease

Molybdenum: Found in a few oxidase enzymes, including xanthine oxidase (competitively inhibited by Allopurinol for Tx Gout)

Selenium: as selenocysteine - in glutathione peroxidase Both deficiency and toxicity of Se described

Keshan disease (cardiomyopathy endemic in some parts of Asia) - caused by low Se content of locally grown foodstuffs. Se content of soils varies widely, worldwide; reflected in Se content of locally grown plants

HWB - Sept '09

Ferritin Fe storage

shell: 24 similar subunits (H – 178 AA’s & L 171 AA’s); ratios vary. H – heart, nucleated (nascent) blood cells ; L- liver, spleen

hollow core – up to 4,500 Fe+++ as ferric oxide hydroxide (FeOOH) crystals; usually under 3,000. Fe/protein not constant

Most abundant storage form when iron stores lowsmall amounts of ferritin, mostly apoferritin with little bound iron, is also present in blood; released during normal cell turnover

Hemosiderincontains FeOOH & same subunits of ferritin, soluble.Rich in Fe+++

Hemosiderin predominates when tissue stores of Fe high – or Fe is in excess.

HWB - Sept '09

Iron:

• Most Iron (2/3) is in hemoglobin; the rest is stored in Ferritin and Hemosiderin

• Can be very toxic in the free state– can form free radicals

• Ferritin = storage form when Fe stores are low• Hemosiderin = storage form when Fe stores are

high/in excess• Most common nutritional deficiency worldwide

Iron accumulation: Hemochromatosis (systemic)Fe overload - with progressive hemosiderosis and organ damage

often associated with homozygosity for a recessive geneAbout 1/400 white Americans are homozygous for it, and about 10% of the white American public is heterozygous.

****most common inherited metabolic disorder in Caucasian population in US. Avoid Fe- fortified foods – esp males

Potentially lethal:Liver damage, diabetes mellitus from pancreatic

involvement, cardiomyopathy, hyperpigmentation, joint pain

Elevated Fe levels often seen in liver biopsies of patients with alcoholic cirrhosis - (alcohol stimulates Fe absorption!)

*** In such cases, difficult to say if the liver disease or the alcoholism caused the condition

HWB - Sept '09

B-vitamins• B-1 (TPP)

• Oxidative decarboxylations, transketolases• B-2 (riboflavin)• B-3 (Niacin)• B-5 (pantothenic acid)• B-6 (pyroxidine – PLP)

• Transaminations and non-oxidative decarboxylation• B-7 (Biotin)

• Carboxylations• B-9 (Folic acid)

• Methionine metabolism, Purine/Pyrimidine synthesis• B-12 (Cobalamine)

B-1 Deficiencies – moderate

GI complaints, weakness, burning feet (?!), peripheral neuropathy, reduced mental acuity and ataxia will appear

Advanced deficiency –

Beriberi, cardiovascular & neuromuscular disorders, weakness, delirium, muscle wasting, paralysis of eye muscles, memory loss, high venous return

Deficiencies uncommon in industrialized countries, except in the alcoholic who has impaired intestinal absorption and thus a poor dietary

intake of most things

Acutely - Wernicke-Korsakoff syndrome (Wernicke encephalopathy) – very serious –

Metal derangements and amnesia – irreversible**Korsakoff syndrome is the most common amnesic syndrome in

the USQ) what do you give alcoholics? TPP supplements

Best sources of B-1: pork, whole grains, nutshwb - Sept '09

B1-TPP

• Coenzyme form= thiamine pyrophosphate – TPP• Participates in transfers of 2-carbon -keto groups to

phosphorylated aldehyde sugars: For ex. In transketolase rxns of HMP

• And participates in oxidative decarboxylations of pyruvate, and other α-ketoacids

• Advanced deficiency – Beriberi• Acutely - Wernicke-Korsakoff syndrome, seen often

in alcoholics

Riboflavin B-2

hwb - Sept '09

Structure - dimethylalloxasine ring bound to ribitol – a sugar alcohol related to ribose

Only biological function known – precursor to FAD and FMN, - electron acceptor; Yellow color - absorbs at 450 nm.

Uptake energy dependentDietary sources: Liver, yeast, eggs, meat, enriched bread, milkDeficiencies: Glossitis, sore throat, moist dermatitis of nose;****destroyed by natural light (UV); therapy routinely given in hyperbilirubinemia (neonatal jaundice) treated with phototherapy

B2- Riboflavin

• –precursor to FAD and FMN• Riboflavin is destroyed by UV light- which is

why it needs to be supplemented in patients being treated with phototherapy for hyperbilirubinemia/neonatal jaundice who are

Niacin (original term): Vitamin B-3 – nicotinic acidPrecursor of NAD+ & NADP+ - pyrimidine derivatives of nicotinic

acid & nicotinamideDietary forms - hydrolyzed in gut & direct absorption asas nicotinic acid & nicotinamideIn Humans - tryptophan → small amounts Niacin

very inefficient; *** pathway also requiresthiamin, riboflavin; In multiple deficiencies (quite common), impairment is strong

Niacin’s conversion to NAD+ and NADP+:

Synthesis of NADP+

hwb - jan '10

Only vitamin that can be made from one of our aa Tryptophan (W)

B3-Niacin

• Precursor of NAD+ & NADP+

• Only vitamin that can be made from one of our amino acids– Tryptophan

B5-Pantothenic Acid

• Required to synthesize Coenzyme A • Essential prosthetic group of several Acyl

Carrier Protein (ACP) of the fatty acid synthase complex

• Pantothenic acid in the form of CoA is required for acylation and acetylation rxns

B6

• Active form is pyridoxal phosphate= PLP• Key Prosthetic group of Aminotransferases

(Transamination and Deamination) and Non-Oxidative Decarboxylases

• It is also an essential cofactor for glycogen phosphorylase (glycogneloysis)

Folic Acid – Vitamin B-9 Supplied only by dietSynthesis - entirely by bacterial work

Structure: conjugate of p-aminobenzoic acid (PABA) & glutamate (“reasonably” inexpensive AA)

In the body –

it is usually reduced to form tetrahydrofolate (FH4) bydihydrofolate reductase, using NADPH

hwb - Sept '09

Partnership between methyltetrahydrofolate and cobalamin cobalamineHomocysteine + Methyl-THF methionine + THF

MeTHF donmates Me group to Homocysteine, & is reconstituted as THF

**** If - a deficit in Vit B12, Methyl THF cannot give up its Me group, the THF remains locked as 5’-Methyl-THF (folate trap)

Best sources: heat labile; extensive cooking can destroy itYeast, liver, kidney, fish, fruits, green leafy vegetables (Latin – folium = leaf) Low levels often seen in late pregnancyThus (pernicious anemia) megaloblastic anemia can be precipitated in the pregnant woman who is a on a diet that is marginally low in folate

Alcoholism, malabsorption diseases - can cause folate deficiency.

hwb - Sept '09

B9- Folic Acid

• Supplied only by diet• conjugate of p-aminobenzoic acid (PABA) & glutamate• usually reduced to form tetrahydrofolate (FH4) by

dihydrofolate reductase, using NADPH -- this step is blocked by Methotrexate***

• Rmbr: Methyl-tetrahydrofolate donates a methyl group to homocysteine to re-form methionine

• B9 Deficiency: DNA replication/cell division delayed can lead to Megaloblastic Anemia

• Severe deficiency in pregnant women: can impair embryonic development and lead to NEURAL TUBE DEFECTS like spina bifida and anencephaly

Vitamin B12

In mammals - only 2 reactions known to require cobalamin (15+ overall):

1. Methylation of homocysteine to methionine (homocysteine methyltransferase) and (methionine metab.) and methyl-THF2. Methylmalonyl CoA mutase reaction (see consecutive carboxylation of propionyl-CoA to methylmalonyl-CoA and – finally – mutation of methylmalonyl-CoA to succinyl-CoA) Odd chain FA oxidation

Deficiencies: As with folic acid, lack of this vitamin will cause

megaloblastic anemia (pernicious anemia) looking just like the folate deficiency – which also will cause incorporation of odd FA’s in nerve membranes – mechanisms? Insufficient methylations of RNA, DNA, etc via MeTHF as folate remains “trapped” in a sink from which it cannot escape

hwb - Sept '09

Vitamin B12

Carboxylation (biotin) of propionyl-CoA creates methylmalonyl-CoA,

subsequent role of vitamin B12 in mutase

reaction creating succinyl-CoA

hwb - Sept '09

B12- Cobalamin

• Only coenzyme that has Cobalt--in center of a corrin ring• Must come from animal products• INTRINSIC FACTOR absolutely required for absorption of

B12!!! (Thus, loss of intrinsic factor can cause B12 deficiency)• Only 2 reactions require Cobalamin: A) Methylation of

homocysteine to methionine via homocysteine methyltransferase and B) Conversion of methylmalonyl-CoA to succinyl-CoA via Methylmalonyl CoA mutase

• Deficiencies: can lead to megaloblastic anemia (pernicious anemia) looking just like the folate deficiency

AscorbateDeficiencies:

Rare; switch to Vitamin C-free diet, symptoms appear after 2-3 monthsDry mouth, eyes; peeling & decaying gums, small petechial hemorrhages, loose teeth, slow wound healing & scar formation; bleeding from old scars, weakness, sore legs, joint pain Scurvy ►►

Unstable athigh heat or under neutral pH

hwb - Sept '09

Vitamin C-Ascorbic acid

• Synthesis - via glucuronate pathway• Except man lacks the last step in this pathway,

because lack Gulonolactone oxidase (GULO) • Therefore: Ascorbic acid recycling occurs via

reduction of dehydro-ascorbate by reduced Glutathione (GSH)

• Deficiency: SCURVY

Biotin

Deficiencies uncommoninducible diet that binds biotin, as in large consumption of raw egg whites (12+/d!); contains Avidin - binds biotin & prevents uptake Cooking denatures avidin & destroys its ability to bind biotin.

Proteolysis of Biotin-containing enzymes (in gut and tissues) yields biocytin; Biotinidase – hydrolyzes biocytin & releases biotin for re-use

Biotinidase Deficiency non-dietary biotin deficiency hypotonia, seizures, optic atrophy dietary supplements curative

*** Biotinidase deficiency often included in newborn screening programs for treatable congenital diseases such as PKU, galactosemia, maple syrup urine disease, hypothyroidism

Best sources:Yeast, liver, eggs, peanuts, milk, chocolate, fish

hwb - Sept '09

Biotin• Required as a prosthetic group of ATP-dependent carboxylases• 3 major enzymes require Biotin: (1) Pyruvate carboxylase

(2) Acetyl-CoA carboxylase(3) Propionyl-CoA carboxylase

• Bound to the enzyme via the –NH group of a lysyl residue• Deficiency (dietary): though uncommon, can be caused by consumption of raw

eggs (bodybuilders; Raw eggs contain Avidin which binds biotin tightly and prevents uptake

• Deficiency (non dietary): can also be cause by Biotinidase deficiency– hydrolyzes biocytin & releases biotin for re-use

Summary B-12 (Cobalamin)

o “Folate Trap” Hyperhomocysteinemiao Intrinsic factor binds B-12 deficiency = pernicious anemia

Folic acid (B-9)o Depression and hyperhomocysteinemiao Neural tube defects (MTHFR gene)

B-6 (Pyroxidine – active form = PLP)o Hyperhomocysteinemiao Serotonin production depression

B-1 (TPP) – [CNS impairment]o Wernick’s/Korsakoff’s encephalopathy o Beriberi

B-2 (riboflavin)o FMN/FAD co-enzymes (metabolism/energy)o Oral-ocular-genital syndrome (FYI)

Vitamin Co Scurvy – important in collagen formation (hydroxylation)

Vitamin A

Active forms: retinal, retinol, retinoic acid (trans – straight form)NB: in foods of animal origin – most in retino(y)l ester between retinol & long-chain FA

  Esters – hydrolyzed by pancreatic enzymes, with aid of bile salts, Free retinol absorbed @ ~ 40 – 80% efficiency

In mucosal cell,most retinol - re-esterified with long-chain FA to retinyl esters; incorporated into chylomicrons

  then as cargo of chylomicron remnants – reach liver; esters temporarily stored in “stellate” cells.

hwb -jan 2011

Vitamin A:absorption,

transport and storage

RBP – retinol binding protein

Metabolism of Vitamin A: Important bio-forms: retinoic acid (at level of gene), retinal (vision)

Retinol - initially in cis form (bent) Small amount oxidized irreversibly to retinoic acidRetinoic acid’s main target cells:

epithelial cells - oxidize retinol, retinal to retinoic acidRetinal’s function: prosthetic group of rhodopsins – visual

pigment of rods & conesHow light is “sensed”

1. Its long fatty tail imbeds retinal in retina’s plasma membrane; 2. Light absorption changes cis- to trans- (straight) form

  3. configuration change “sensed” by opsin, which holds retinal at its center4. Change in shape triggers action potential, informs brain that the cell has just “seen” a photon of light.

Deficiency of vitamin A night blindness

Pathway from retinol to retinoic acid to the gene

Retinoic acid binds nuclear receptor proteins;Receptor-Retinoic Acid complex regulates gene expression after binding to  

Response elements on DNA;

Process resembles actions of steroid hormones in target tissues

 Retinoic acid receptors belong to same

superfamily of ligand-regulated transcription factors as do steroid hormone receptors.

hwb -jan 2011

Retinoic acid – functions, cont’d

Essential for maintenance of epithelia & membranes – not involved in vision

 Deficiencies:

Transformation of columnar epithelia into heavily keratinized squamous epithelia – (“gooseflesh” look)

In extreme cases: conjunctiva of eye loses mucus-secreting cells and becomes keratinized

Loss of glycoprotein content of tears – leading to xerophthalmia (“dry eyes”) – which may lead to infections,

and blindnessLeading cause of blindness in developing countries

hwb -jan 2011

VITAMIN A: Retinoic acid acts like steroid hormone & regulates of epithelial membrane maintenance

Retinal is used in the retina

Vitamin A deficiency = night blindness (rods not working).“Retinol” is Vitamin A.“RBP” = retinol binding protein(carries vitamin A)

Vitamin D and its synthesisNutritionally essential for those out of the sun (aka medical students)

 Under UV – 1st step occurs photochemically in skin

so no dietary source necessary

7-dehydrocholesterol → cholecalciferol (vitamin D3)

transport from skin via non-covalent binding to vitamin D-binding plasma protein.

In (1st) Liver & (2nd) kidney – transformed to active1, 25 – dihydroxycholecalciferol (calcitriol) by 2

successive hydroxylations 

Calcitriol – hormone-like action

hwb -jan 2011

KNOW WHERE RXNS OCCUR!

Synthesis of Vitamin D

1st step - 25 hydroxylation is NOT rate limiting or regulatory major circulating form - 25-hydroxy-D3.

 Last step – producing calcitriol - 1α-hydroxylase - is

regulatorytight control by parathyroid hormone (PTH) & the 2 linked states of hypocalcemia & hypophosphatemia.

Note –25-hydroxy-D3 & cholecalciferol have half-lives of ~ 30-40 days; on the other hand ---

mature calcitriol persists for only 2-4 hours (why so short?); single known action: up-regulate plasma [Ca++]

hwb -jan 2011

Vitamin D metabolism:

Cholecalciferol is produced in skin by UV

irradiation of 7-dehydrocholesterol;

in liver a -OH is added at C-25;

in kidney a second –OH is added at C-1 (in the

regulatory step), producing the mature

molecule 1,25-

dihydroxycholecalciferol(calcitriol – or vitamin

D3)hwb -jan 2011

Vitamin D deficiency and Rickets:Left: 2.5 year old boy with severe rickets; right: the same boy at 5 years

of age after 14 months of Vitamin D therapy

hwb -jan 2011

Genesis of Vitamin D3 (active):

•7-dehydrocholesterol cholecalciferol ( by sunlight)•Liver you have hydroxy reaction, attach in the 25’ position (LIVER = 25).•Final step occur in kidney, attach a hydroxy group in 1’ position ( regulated by PTH) (“KIDNEY #1!”)•1, 25 – dihydroxycholecalciferol = calcitriol = ACTIVE FORM!•Calcitriol (Vit. D3)’s job Help increase Ca+ levels in blood, also helps absorption in the intestine without the help of PTH.•Vitamin D deficiency in kids-> rickets!•More lactose= more Ca and Pi absorbed in the gut

Vitamin E – α-Tocopherol

Over 8 related substances, all with vit E activity, isolated from natural sourcesMost potent & prevalent : α-tocopherol – double methylated

RDA – 10 mg/day for men; 8 mg/day for womenBest sources: nuts, seeds, wheat germ & vegetable oils, leafy vegetables

Requirement rises as intake of polyunsaturated FA’s increases 

hwb -jan 2011

VITAMIN E (TOCOPHEROL) = ANTIOXIDANTKNOW IT!!!!

Vitamin E:

•Antioxidant- reduce amount of free radicals.

•Promote prostacyclin- vasodilate blood vessel and inhibit platelet aggregation- less thrombus occur

Vitamin K – the “koagulation vitamin”Only known role:

post-translational carboxylation of specific AA residues, esp in clotting factors, certain snake venoms (Australian)

Only major manifestation of deficiency: deranged clotting cascade

(eg: Romanov family; for fun & culture, consider historically disastrous effects of genetically borne metabolic disorders)

1938: U of Iowa Pathology Dept: 1st report of successful treatment of life-threatening hemorrhage in patient with prothrombin deficiency

1943: Nobel Prize – Dam & Doisy (St Louis Univ)

Phylloquinone – plant product – covers part of human’s needs

Parsley – especially potent source of Vit K

hwb -jan 2011

Vitamin K – production, availability

Menaquinone(K2) synthesis by bacteria in large intestine & absorbed there.Menadione - analogue after enzymic alkylation in body.Significantly – menadione absorbed in absence of bile salts; thus can offset problems in natural production of Vit K due to long-term antibacterial therapy Parsley – especially rich in Vit K; also meat eggs, dairy prod’sAs with vitamin E - no specific vit K binding protein identified

Tissue Distribution: via plasma lipoproteins & chylomicrons  Not stored to any extent, reserves low ~ 50 – 100 mg;

However – rapid & continual turnover

Vit K - first fat-soluble vitamin to be deficient in acute fat malabsorption.

hwb -jan 2011

Vitamin K deficienciesOnly one known disorder:

prolonged-clotting time (prothrombin time) – period over which prothrombin → thrombin; this converts prothrombin to make more of itself (multiplicative cascade)

Newborns - esp premature – very prone to Vit K deficiency; gut still sterile & maternal milk has insufficient Vit KSeen in ~ 1/400 live births: “hemorrhagic disease of the newborn” most common disease of the neonate

  Europe, most US states: neonatal Vit K prophylaxis – mandatory

One other avenue can cause deficiency of vit K (in adults):

Combination of: Vit K-deficient diet & prolonged antibiotic therapy that can kill off flora in GI tract.

In cases requiring extended antibiotic therapy, dietary fortification with Vit K is strongly advised.

  hwb -jan 2011

KNOW*: HEMORRHAGIC DISEASE OF THE NEWBORN AND WHAT VITAMIN TO USE VITAMIN K

Vitamin K = Koagulation!•post-translational carboxylation.•enzyme: Gamma-glutamyl carboxylase requires Vitamin K.•deranged clotting cascade.•Vit K - first fat-soluble vitamin to be deficient in acute fat malabsorption•Factos II VII IX X ( always factor C and S) are Vitamin K-dependent!!•Warfarin is vitamin K antagonist.•most common disease of the neonate- Vitamin K deficiencies

hwb - jan 2011

The respiratory quotient (RQ): CO2 produced/O2 consumed

RQ’s for different foodstuffsCH2O 1.0 (less O2 consumed, less energy needed)

Fat (TG) 0.7Protein 0.8

Why the differences? the more reduced the substrate, the more O2 is consumed to produce equal amounts of CO2. Very significant!

3 Problems: i. Two equal (in Calories) diets consumed: (a) 50 / 50 CH2O/fat(b) 75 / 25 CH2O/fatWhich elicits the higher RQ? B

ii. Three diets of equal caloric value. (a) 50% fat, 50% protein; (b) 33% fat, 33% protein, 34% CH2O; ( c) 75% CH2O, 12% fat, 13% protein.

Which yields the lowest RQ? Aiii. design a diet that yields All Fat 100%= lowest RQ & highest O2 uptake

RQ is a ratio: C02

O2

If RQ =Higher value (CH20) = Less energy required to break it down (less O2)

Lower value (fat & protein) = More energy to break it down (more O2 consumption)

***A diet with more CH20 than other ingredients Higher RQ!!!

HIGHYIELD AS@#$%!

hwb - jan 2011

Major factors in the liver’s response to CHO

Hepatic GLUT 2 – unaffected by insulin

Liver retains ~ 60% of glucose after a meal - via

a.  via glucokinase (high Km for glucose)

b.  accelerated glycogen synthesis

c.  increased HMP activity - from high use of NADPH in FA synthesis – accounts for ~ 5-10% of G-6-P use in liver

Glucose flux increases for these reasons:

a. huge standing #’s of GLUT - 2’s;

b. Thus entry is never rate-limiting;

c. glucose - rapid conversion Acetyl-CoA; in turn → citrate → FA synthesis

d. In sum: glycolysis, HMP, TCA & ETS fully active; all power FA & triacylglycerol synthesis

HIGHYIELD AS@#$%!

hwb - jan 2011

Logically, a concomitant drop in the rate of stored TAG hydrolysis

The high insulin/glucagon ratio favors the dephosphorylated (inactive) state of

hormone-sensitive lipase (inactive) –

also PFK-2 & PyK (ACTIVE)

Also favors de-phosphorylation of

HS-lipase & perilipin (inactive)

so - hydrolysis of triacylglycerol

in the recently

fed state is inhibited.

Summary:Insulin is an effective antagonist of lipolysis (& breakdown of energy stores in general);-High Insulin → TAG’s synthesized after a meal;

In contrast, TAGs are degraded during fasting/long-term work via glucagon

HIGHYIELD AS@#$%!

hwb - jan 2011

Skeletal muscle: Carbohydrate metabolismTemporary rise in glucose & insulin after a normal (carbohydrate)-rich

meal → accelerated glucose uptake via GLUT-4

Recall – muscle hexokinase has low Km for glucose, so phosphorylation to glucose-6-phosphate is favored.

In turn this stimulates: …. Glycogen synthesisif glycogen stores depleted by prior exercise, then glycogen synthesis; via glucose-1-phosphate, UDP-glucose - is

heavily favored.

Under these conditions, glycogen phosphorylase and glycogen synthase are dephosphorylated – the first is inactive and the synthase is active

 

HIGHYIELD AS@#$%!

hwb - jan 2011

Carbohydrate metabolism in brain

No significant glycogen stores in brain; uses ~ 140 grams of glucose over a 24-h period;

compare this to ~30 grams of glucose used by the RBC mass over the same time; why this huge difference?

Brain glucose - totally oxidized to CO2 & water

Fat metabolism in brainRestrictive blood-brain barrier: no FA’s pass, but ketones are

permitted, and as there are no stores of triacylglycerol ….. fat metabolism contributes very little to overall budget of brain.

*** However – the ability of virtually all other tissues to utilize fat makes it possible for brain & RBC mass to be predominant consumers of glucose

 

HIGHYIELD AS@#$%!

hwb - jan 2011

The well-fed state: glucose and insulin

In sum – with respect to glucose uptake

The organism may be divided into two discrete halves:

1. One whose glucose transporters are

insulin-independent

(liver, RBC and brain)

2. One whose glucose transporters are

insulin-dependent

(muscle and the adipocytes)

HIGHYIELD AS@#$%!

hwb - jan 2011

First – the insulin-INDEPENDENT portion: liver

glycolysis – stimulated (very broad G-6-P utilization)

gluconeogenesis - inhibited,

pentose shunt - activated to provide reducing power for lipogenesis,

chylomicron remnants – dismantled in liver

VLDL’s - assembled for distribution of triacylglycerol to peripheral tissues,

amino acids - taken up rapidly, proceed to protein synthesis

or pass into blood plasma

Brain aerobic glycolysis continues, at a constant rate

Hence the brain’s O2 demand – in relative terms – can vary widely, depending on total metabolic rate of the body

HIGHYIELD AS@#$%!

hwb - jan 2011

Second - the insulin-DEPENDENT portion:

Muscle

insulin-dependent glucose & FA transporters very active

Glycogen synthesis spurred,

triacylglycerols deposited &

amino acids imported for building blocks for protein synthesis.

Adipose tissue – especially insulin-sensitiveGlucose-6-phosphate: essential, as the source of glycerol backbone for triacylglycerol synthesis –

Of course

majority of FA’s synthesized de novo come from liver and then are stored in the adipocyte

HIGHYIELD AS@#$%!

hwb - jan 2011

Integrating the “well-fed state” into body weight regulation

Proposed: “Lipostatic” model for regulation of body weightinvolves 2 hormones: Leptin & Resistin (resistin renders

adipocyte insulin resistant) 

can partially explain long-term relative constancy of somatic energy stores, effects of their depletion on ingestion, energy expenditure, linear growth, and fertility.

Leptin - produced by adipocyte; promotes feeling of satiation & inhibits food intake

Resistin – produced by adipocyte; promotes resistance to insulin

Output of both – directly proportional to fat mass – in absence of other factors

HIGHYIELD AS@#$%!

Well-fed Stateadipocyte – GLUT-4Muscle – GLUT-4Liver- Glut 2Pancreas- Glut 2respiratory quotient (RQHighest= lots of carbs consume least O2Lowest= lots of fat’; consume most O2

Trypsin, chymotrypsin, elastase: Active only at neutral pH carboxypeptidases A & B – both are Zn++

metalloenzymes

Brain only uptake glucose ; with Glut-3, completely oxidize glucose to CO2 and waterAlso use ketones but in starvation mode

HIGHYIELD AS@#$%!

Hormones from the pancreas: Insulin, glucagon, somatostatin

Glucagon – α-cells; Insulin – β-cells; Somatostatin – δ-cells, stomach, intestine; also hypothalamus

All three - regulators of homeostasis: largely independent of direct CNS input; internal monitors

1. Insulin: X plasma [glucose] from rising too high2. Glucagon: X plasma [glucose] from falling too low3. Somatostatin: X excessive release of growth hormone

(somatotropin) - thereby preventing unnecessary investment of energy, nutrients, etc. Also counters release of several digestion-related hormones

hwb - jan 2011

Clin. Corr’n: Hyper-pro-insulin-emia

Insulin insufficiently processed before secretion Immature forms of insulin (prepro- & proinsulin) make up

bulk of immunoreactive insulin (all forms detectable with insulin-antibody)

More frequent in DM Type 2 (NIDDM)

Attributed to 1. defective β-cells or 2. (indirectly) to dysregulation under sustained high [glucose]’s

hwb - jan 2011

Main factors cause increased secretion of insulin (essentially – in the well-fed state)

Glucose – most powerful stimulator (after uptake)

Amino acids – especially arginine

Gastrointestinal hormones - secretin, especially right after the ingestion of food

Glucagon - this stimulates the secretion of insulin and inhibits further release of itself;

*** this feature is an especially significant finding in the Type I diabetic

hwb - jan 2011

GAGS! (mnemonic)GlucoseArginineGlucagonSecretin!!

Metabolic effects of insulin: well-fed state

Carbohydrate Metabolism3 major cell types primarily affected: liver, muscle, adipocyte Liver: gluconeogenesis inhibited, glycogenolysis sharply diminished,

Glucose uptake stimulated & glycogenesis stimulated (not a paradox – synthesis of glucose and glycogen must not be confused)

Peripheral tissues

Muscle: glucose uptake raised, glycogen synthesis elevated

Adipocyte: glucose uptake stimulated – to produce glycerol-3-P for triacylglycerol synthesis & storage

hwb - jan 2011

Revisit our glucose transporters (GLUT’s)

Variant tissue location commentGLUT1 heart muscle, plasma membrane In muscle-

brain, placenta, by hypoxia, erythrocyte

GLUT2 liver, kidney, “ independent pancreatic β-cells, of insulin

intestine

GLUT3 neuron, kidney, “ insulin-inde-placenta pendent

GLUT4 * muscle, adipocyte “ myo-, fat cell * heart activity

by insulinGLUT5 muscle, sarcolemmal . dietary

sperm vesicles fructosehwb - jan 2011

It’s BACK! Don’t miss it on this block!

What events in the pancreatic β-cell trigger it to release insulin?

1. Recall from glycolysis – pancreatic β-cell has GLUT-2, Glucokinase (high Kmglu); now glucose is in the cell ….

2. This sets off a rapid chain of events in the β-cell:accelerated catabolism → ATP (energy charge) levels rise → closing of ATP-dependent K+ channels → Ca++ entry → membrane depolarization → insulin secretion via exocytosis → enhanced glucose uptake & storage in peripheral tissues

(credit – Dr. M. Goodman, U. Mass)

*** See upper left corner of next figure

hwb - jan 2011

Glucose transporter 4 (GLUT 4) is activated

Specific to skeletal muscle and adipose tissueBUT

Some evidence that early in development – shortly after birth – this GLUT is also in heart muscle.

Conceivable function – take up glucose and possibly store some as glycogen - in conducting fibers - at a time the organism is establishing itself and needs to stockpile energy-ready, quick-response, carbon

hwb - jan 2011

Glucagon – the counter-regulatory hormone

Identified 1920 – named 1923From pancreatic α-cells 3,500 MW polypeptide, 29 AA’s

Both synthesis and release controlled by insulinPrimary targets – 1. Liver, 2. adipocytePrincipal effect: Liver: elevation of cAMP → promote

glycogenolysis and inhibit both glycogen synthesis and glycolysis; this activates gluconeogenesisAdipocyte: R cAMP; protein kinase A phosphorylates hormone-sensitive lipase to yield FFA’s and glycerol ( hepatic gluconeogenesis)

hwb - jan 2011

Glucagon, cont’d

From pancreatic α-cells (near outer edge of Islet)Primary structure:

note –

simple, small & no cysteine12 AA residues (41 %) are essential or synthesized (Tyr) from essential AA’s (40% of our 20 – essential)

NH2

-His-Ser-Gln-Gly-Thr-Phe- Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-Asp- Phe-Val-Gln-Trp-Leu- Met-Asn-Thr- COOH

hwb - jan 2011

Glucagon

Stimulators of its secretionFall in plasma [glucose]

High catecholamines for some timeIncreased [AA’s] – to prevent hypoglycemia after an all-protein meal (arginine key role)

AcetylcholineCholecystokinin/pancreazymin

Inhibitors of its secretionSomatostatin

Insulin

Control of output? Mechanism not yet understood: except that α -cells seem to respond directly to changes in [glucose]

hwb - jan 2011

Body Weight Homeostasis, Obesity and Leptin

Current “lipostatic” model for regulation of body weight:- Only partially explains:

1. long-term homeostasis of somatic energystores,

2. effects of fat depletion on ingestion, energy expenditure, linear growth, fertility.

Leptin Males, [leptin] are ¼ - ½ in premenopausal femalesOther: pregnancy – levels double; cannot cross placenta

hwb - sept '09

Note the differences!!!

LEPTIN - Promotes the feeling of being full and inhibits food intake- Produced by adipose tissue, like a negative feedback mechanism on eating

Leptin

Product of adipose tissue - especially white adipose tissue (WAT); also BAT & stomach

proportional to fat tissue mass167 AA’s – incl 21 AA signal sequence,

MW ~ 16,000.4 helices; one essential SS bond

member - cytokine family

Production - directly proportional to fat tissue mass good candidate for afferent signal from “central” energy

stores to a “peripheral” receptor

Name - from “leptos” (Greek for thin or lean)

hwb - sept '09

Leptin’s mechanism & meaning:

Food intake → leptin secretion from adipose tissuesMessage: “energy reserves sufficient - stop eating!” → release

of appetite-suppressing (anorexigenic) hormonesEffect: ↑sensitivity to insulin (liver, muscle, fat cell) &

inhibit insulin secretion (this indirectly inhibits excessive storage of food as lipid – its primary storage form)*********

hwb - sept '09

Additionally - complex neuronal and hormonal signals regulate food intake and conservation

**Ghrelin (stimulates hunger) – from stomach ; causes release of appetite-stimulating (orixogenic) hormones

PYY – from intestine & colon – appetite reducerAdiponectin – from adipocyte; ▲ β-oxidation,

▼ lipogenesis & gluconeogenesis

NB: “Redundant” overlay of gene suppression & expression for seamless & precisely-graded long-term regulation

hwb - sept '09

Adiponectin works through AMPK

Adiponectin

▼ AMPK (AMP activated protein kinase)

Muscle Liver↑ FA uptake ↓ FA synthesis↑ β-oxidation ↓ gluconeogenesis↑ glucose uptake ↑ glycolysis

***AMPK – inhibits virtually all biosynthetic processesHigh AMP’s message? Low energy charge !

hwb - sept '09

Adiponectin= low energy charge—STOP SYNTHESIS

Ghrelin and PYY’s roles in control of short-term eating behavior

Ghrelin (28 AA’s) & PYY3-36 34 AA’s) precede a meal

Insulin profile mirrors food intake

* Prader-Willi syndr:high ghrelin levels:extreme obesity →early death hwb - sept '09

AA Synthesis via Transamination (3):

• Pyruvate Alanine • Oxaloacetate Aspartate

• α-ketoglutarate Glutamate

Transaminase (req. PLP!!)

Transaminase (req. PLP!!)

Transaminase (req. PLP!!)

Metabolism of Starving State

• Dietary glucose 2-3 hrs• Liver glycogen 2-24 hrs• Gluconeogenesis 24 hrs ~ 3 days• Fat stores 3-5 days• After 32 hrs, all blood glucose is maintained

via gluconeogenesis

hwb - sept '09

Chronic “semi-starvation”: leads to Kwashiorkor or Marasmus

Kwashiorkor (!!) – more or less normal caloric intake and

diminished protein intake;Decreased serum albumin → edema (distended

abdomen)Combination of body fat and edema

creates impression of a well-nourished person

Marasmus (!!) - starved in all aspects of nutrition: fat, carbohydrate and protein are in normal proportions

hwb - sept '09

Kwashiorkor(↓ protein)

ObserveDeceptively plump belly – due to enlarged, often fatty liver and excessive edema

hwb - sept '09

Marasmus – (↓ calories & ↓ all aspects of nutrition)

Causes:inadequate intake of protein and calories in all food categories.

Some symptoms:stunted growth, pronounced weight loss including loss in muscle in shoulders and buttocks, hair loss, darkened skin and apathy.

hwb - jan 2011

DM: two dominant syndromes

1. Insulin-dependent diabetes mellitus (IDDM – Type I) 2. Non-insulin-dependent diabetes mellitus

(NIDDM – Type II). Patients with NIDDM can be further subdivided between the (a) obese (85%) and the (b) non-obese (15%).

**Persistent HYPERglycemia – in both DM I and II many 2o causes of diabetes mellitus, most of which arise from increased secretion of the counter-regulatory hormones

(epinephrine & esp. glucagon)Effects: microvascular & macrovascular

Overview: as not painful (at first) most can’t appreciate how serious it will be later – especially in DM II

hwb - jan 2011

hwb - jan 2011

Result: HIGH glucagon = no insulin

Protein: AA transport (liver); protein synthesis (in general); protein degradation; gluconeogenesis and ureagenesis from AA’s (urea cycle)

CHO: glycogenolysis; gluconeogenesis;¯ glycolysis & when [glucose] is low – glucose secretion into

plasma

Fats: hormone sens. Lipase; FA export from adipocyte; ketogenesis (liver) & ketogenic enzyme synthesis (liver); cytoplasmic carnitine acyl transferase (I); triglyceride release from liver

(Basically, all the effects of glucagon in the body!)

HIGHYIELD AS@#$%!

hwb - jan 2011

Diabetes mellitus 1 and starvation compared

1. Insulin is effectively absent in Type I diabetics;

it is low in the starving

2. All diabetics show hyperglycemia,

the starving individual shows near normal glucose levels in blood

3. Ketosis: in both diabetic and starveling, FA mobilization is very rapid, but

4. Ketone production - far more pronounced in the diabetic (ketoacidosis) than in the starving patient

(who shows “physiological ketosis”)

HIGHYIELD AS@#$%!

DIFFERENCES between DM1 and Starving State

INSULIN: completely absent in DM1low in starving state

BLOOD GLUCOSE LEVELS: hyperglycemia in DM1 (and DM2!!) near normal levels in starving state

KETONE PRODUCTION: HIGH in DM1 (ketoacidosis)elevated, but not as high in starving state

HIGHYIELD AS@#$%!

hwb - jan 2011

Features of diabetes mellitus types I and II

Characteristic Type I Type IIAge of onset pre-or adolescent, usually 35+ yrs, any age! Nature of onset often sudden Slow, insidiousGenetics specific HLA factors* no HLA connection; strongly familial Secondary factors viruses, toxins obesityβ-cell autoimmune?at initial episode not presentInsulin secretion absent or delayed sometimes reduced?Body habitus thin to cachectic normal or obeseSymptoms at onset polyuria, polydypsia often none; no ketoacidosis

hunger, wt loss, ketoacidosis.Long term 2o ret-, nephro-,neuropathy Resemble Type I, often late

Insulin dependency absolute occasionally, often not__________________________________________________________________*HLA = human leukocyte antigen (genes on Chrom 6; gene aberrations here often

associated with DM I & Graves’s disease (hyperthyroidism)

HIGHYIELD AS@#$%!

hwb - jan 2011

Hypoglycemia in IDDM?

can occur if insulin dosage is inaccurately gauged or given.

Frequency of hypoglycemic episodes

very common and may occur in more than 90% of all patients:

seizures, intense sweating, hypothermia → coma are common.

Understand what will happen in an insulin overdose!

All the glucose in the blood will be taken up by GLUT4s in the peripheral tissues causes hypogylcemia=LOW glucose in the BLOOD

due to insulin overdose

HIGHYIELD AS@#$%!

Non-Insulin-Dependent Diabetes Mellitus

NIDDM – Type 2 diabetes

Most common form of the disease. Affects about 90% of all diabetics in the industrialized West.

Major factors contributing to hyperglycemia of DM 2 →

MAJOR FACTORS:

Insulin resistance Hyperglycemia - increased glucose production by liver - decreased glucose uptake by GLUT4 in

tissue (similar to what happens in DM1)

Inadequate insulin secretion

HIGHYIELD AS@#$%!

hwb - jan 2011

Diabetes mellitus Type II (NIDDM)Genetics & environment - major roles

Very strong familial factor to susceptibilityAlmost 100% in identical twins; as environment in West +/- constant, genetic influence the major factor.

Obesity – most powerful risk worldwide

* MODY – mature onset diabetes of the young Monogenic NIDDM subtypeAutosomal dominantSo far – the only form of DM with definite inheritance mode; 5 specific markers have been identified; more possible

HIGHYIELD AS@#$%!

Hyperglycemia Effect (polyol pathway)

cells that do NOT require insulin to import glucose include:

cells of lens, retina, schwann cells, etc.

because entry is insulin independent, large amounts of glucose may enter these cells during hyperglycemia, esp in uncontrolled diabetes…

elevated IC [glucose] and an adequate supply of NADPH cause conversion of

glucose to sorbitol via aldose reductase

sorbitol cannot pass thru cell membranes and thus remains trapped inside the cell

the sorbitol accumulation causes strong osmotic effects resulting in cell swelling which can then lead to damage to the above mentioned cells

this is why you often see cataracts, peripheral neuropathy and vascular problems leading to nephropathy and retinopathy in diabetics…

HIGHYIELD AS@#$%!

hwb - jan 2011

6. Diabetic neuropathy: common; varies with peripheral nerves affected. Not only peripheral nerves affected, but also cranial nerves and autonomic nerves. Symmetrical neuropathy, very common, → loss of sensation in the lower extremities; patient especially prone to injury, leg and foot lesions, → greatly increased incidence of gangrene & necessary amputations. Autonomic dysfunction can affect the GI tract, bladder, heart, vascular tone, even erectile function.

HIGHYIELD AS@#$%!

Starving (low blood glucose) = Glucagon (liver)

and Epinephrine (muscle & liver)

activates adenylyl cyclase → cAMP → PKA active (cAMP-dependent)

if PKA active you get PHOSPHORYLATION of enzymes!!

THUS… in all enzymes involved in raising blood glucose levels, i.e. glycogen degradation, gluconeogenesis, etc. PHOSPHORYLATION ACTIVATES ENZYMES

Well-Fed (high blood glucose) = Insulin

high insulin/glucagon ratio → causes decreased cAMP → reduces [active PKA]

→ NO PHOSPHORYLATION of enzymes!!

THUS… in all enzymes involved in lowering blood glucose levels, i.e. glycolysis, glycogen synthesis, TCA cycle, etc. PHOSPHORYLATION INACTIVATES ENZYMES (REMEMBER the EXCEPTIONS!)

CONCEPT SLIDE

ex: glycogen phosphorylase is ACTIVE when phosphorylated

ex: glycogen synthase is INACTIVE when phosphorylated

INSULIN EFFECTS

CARBOHYDRATE METABOLISM

LIVER: inhibits gluconeogenesis and glycogen breakdown (decreases production of glucose)

MUSCLE & LIVER: increases glycogen synthesis

MUSCLE & ADIPOSE: increases #GLUTs in cell membrane (increases glucose uptake)

LIPID METABOLISM

ADIPOSE TISSUE: responds within minutes, significant reduction in release of Fas

decreased TAG degradation: inhibits hormone-sensitive lipase in adipose (dephosph)

increased TAG synthesis:

increases transport & metabolism of glucose into adipocytesglucose can be converted to glycerol-3-P which is a substrate for TAG

synthesis

increases synthesis of lipoprotein lipase (EC enzyme that degrades TAGs to FAs & glycerol, so that adipose tissue can take up FAs and store them as TAGs

PROTEIN SYNTHESIS

MOST TISSUES: stimulates entry of AA’s into cells and protein synthesis

(high plasma glucose)

To sum it up… build stuff from glucose (glycogen, TAGs) inhibit any pathway that produces glucose (gluconeogenesis, glycogenolysis) take up FAs, AAs from blood to store as fat (TAG), protein

GLUCAGON EFFECTS

CARBOHYDRATE METABOLISM

glycogen breakdown (liver, not muscle), increased gluconeogenesis

LIPID METABOLISM

hepatic oxidation of FAs and subsequent formation of ketone bodies from acetyl-CoAeffect of glucagon on lipolysis in adipose tissue is minimal in humans

PROTEIN METABOLISM

uptake of AAs by liver thus increasing the availability of C skeletons for gluconeogenesisthus… plasma levels of AAs are decreased

GLUCAGON acts to maintain blood glucose during periods of potential hypoglycemia

increases: glycogenolysis gluconeogenesisketogenesisuptake of amino acids

its secretion is stimulated by: low blood glucose, AAs, and epinephrine

its secretion is inhibited by: elevated blood glucose and insulin