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Tissue partnerships muscle: after a meal fates of branched chain AAs (BCAAs)

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 -ketoacids catabolized for energy production (with large energy yield).

hwb - jan 2011

Degradation of intracellular proteins

Lysosomal acidic - enzymes (cathepsins) degrade prots continually; in addition Chemically modified proteins and those tagged by calpains & ubiquitins preferentially degraded Half-life of a protein marked by N-terminal AA residue Egs: if serine life = ~ 20 hrs if aspartate life = ~ 3 mins Aspartates 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)

The process of protein degradation

1st step always - conversion to N-free product Glucogenic AAs converted to TCA / glycolytic intermediates Ketogenic AAs degraded to Acetyl-CoA (final product)

2 are purely ketogenic: leucine & lysine (essential)

4 are both glucogenic and ketogenic: tryptophan, phenylalanine, isoleucine, tyrosine WIF Y? 14 - Purely glucogenic with the exception of alanine & valine, none of these possess a hydrophobic methyl side group (-CH3)
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 amination Activate oxidation: ADP, GDP; Inhibit oxidation: ATP, GTP
Fit 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

OD-ing on Glutamate

Glutamate The key turn-around AA to either glutamine (ATPutilizing synth) or -ketoglutarate (ATPyielding TCA cycle)
hwb - jan 2011

Pyridoxal phosphate (PLP) - a prosthetic group for transaminases:

Lysines -NH2
group binds to the carbon of PLPs 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 (KrebsHenseleit) 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 arginine 5. 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 ATPs) of synthesizing one molecule of urea (yet - consider the alternatives) How calculate 4 ATPs while only 3 ATPs are used directly ? ATP AMP + PPi: equate this with 2 ATPs
hwb - jan 2011

Argininosuccinate synthase

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

A: X LINKED OTC MOST COMMON Features of condition: UREA CYCLE DEFICINEY Orotic acid build-up; hyperammonemia & encephalopathy; feeding problems, vomiting, lethargy or irritability, poor CNS develt, tendency for coma death Condition aggravated by dietary protein Nous 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.
hwb - jan 2011

Failure of the urea cycle, contd

Patient often develops aversion to protein-rich foods In addition to rising NH4+, glutamine is usually elevated, from diversion of extra -ketoglutarate & NH 4+ for glutamine synthesis. Thought: this could limit availability of -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 AAs with their -keto-acids - costly 2. Use of alternative routes of nitrogen excretion Benzoic acid, phenylacetate and phenylbutyrate - these conjugate with 2 N-rich AAs - 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)
hwb - jan 2011

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)
i.e. ALT
hwb - jan 2011 16

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
THFs synthesis from folate requires NADPH in two successive reductions by dihydrofolate reductase

Folate dihydrofolate tetrahydrofolate

hwb - jan 2011 18

Degradation of methionine
coenzyme roles:

pyridoxine (B6)
cystathionine synthase, cystathioninase (see error C&H)

folate (B9)
methyl-THF (from methylene THF);

cobalamin (B12) accepts

methyl group from methylTHF - transfers methyl to homocysteine, remaking methionine
hwb - jan 2011 19


hwb - jan 2011


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 Sadenosylhomocysteine (SAH); this is re-converted to homocysteine and then finally back to methionine Methylation 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, contd

The condensation of homocysteine and serine - makes cystathionine Cystathionine lyase (cystathioninase), now cleaves cystathionine, & creates cysteine and -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 synthase

metabolite build-up always upstream of block; thus

homocysteine, 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 AAs - transaminated in muscle & other extrahepatic tissues, and then catabolized BC--keto acid dehydrogenase resembles pyr. dehydrogenase; defects in PDH can cause maple syrup urine disease
hwb - jan 2011 25

Disorders in metabolism of the branched chain amino acids (BCAAs)

Maple syrup urine disease 1:200,000 incidence much higher in Amish & Mennonite communities (founder effect) Cause: branched chain -ketoacid dehydrogenase (quite nonselective): decarboxylates all 3 branched ketoacids & produces NADH + H+ Rx resembles pyruvate dehydrogenase reaction Signs: Severe mental retardation, acidosis, sweet-smelling urine (resembles burnt caramel), early death is frequent

Treatment: Megadoses of thiamin (why this?) can at times be effective Restrict 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


Phenylalanine is degraded to tyrosine: both are glucogenic and ketogenic

Aromatic ring cannot be synthesized in the human Yet - tyrosine is not essential as it is made during the metabolism of phenylalanine by - 1st -

Phenylalanine hydroxylase (PAH)

Requires O2, & Tetrahydrobiopterin [BH4] as reducer (to form OH group) In active state: tetramer Requires phosphorylation at critical serine(s)
hwb - jan 2011 28

Two causal Deficiencies: Types I & II (lack of I: fumaryl-acetoacetate hydrolase. homogentisate oxidase leads II: tyrosine transaminase (tyr to alkaptonuria, phenylpyruvate) a KEY SIGN IS BLACK Also note homogentisate oxidase URINE) 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

AA Synthesis via Transamination (3):

Transaminase (req. PLP!!)


Transaminase (req. PLP!!)



Transaminase (req. PLP!!)


AA synthesis via REDUCTIVE AMINATION (1):

Glutamate Dehydrogenase

+ NADPH!!! + NH3
Make note

Glutamate + NADP

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):

+ NH3 (in the form of free amonia!!!!) Glutamine Synthetase Reqs ATP!


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

Asparagine Synthetase Reqs ATP!


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

Synthesis of Serine
Transamination via PLP

3PG phosphopyruvate 3-Phosphoserine

(from glycoly sis) AND Requires PLP! + N5-N10-methylene tetrahydrofolate


Serine and Tetrahydrofolate

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
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

hwb - sept '09

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




Synthesis of cysteine from

Methionine (sulfur group of
cysteine comes from Methionine).

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

Three products of terminal reaction: 1. ammonia, 2. -ketobutyrate (to succinyl CoA), 3. cysteine
hwb - sept '09

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 arteriosclerosis Most common cause mutation in gene for cystathionine synthase!! (homocysteine + serine cystathionine) Homocysteines 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

Methyl-THF (B9)

SERINE Cystathionine synthase REQ B6 Homocysteine methyltransferase REQ B12


Cystathionase REQ. B6



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 Deficiency in Phenylalanine Phenylalanine Hydroxylase=

Phenylketonuria (PKU) Phenylalanine Hydroxylase REQ. O2



Tetrahydrobiopterin (BH4)
Dihydropteridine Reductase

Dihydrobiopterin (BH2)



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 eye & renal tubular epithelium where damage most evident of the

Cystine: 2 cysteines in SS- bond; transport/egress mechanism to cytosol is defective. Very poorly soluble: pure cystine 4-sided crystals; cystine-HCl prismatic needles (frequent in cornea) Clinical presentations: Does not involve metabolic breakdown; problem is in tearing, general (kidney) tubular Photophobia, retinal blindness,Transport*** dysfunction (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

Synthesis and Degradation of Porphyrin

Return to the point that heme is synthesized in both erythropoietic and nonerythropoietic 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 HIGH YIELD AS (indirectly via heme-induced inhibition of iron uptake not @#$%! allosteric in an enzymic sense. Enough complete heme means the tissue has sufficient Fe)

Erythropoietin and intracellular Fe availability

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?

hwb - may 2011



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

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

ALA synthase *regulatory step in LIVER (PLP)

ALA Pb poisoning ALA dehydratase or porphobilinogen synthase (Zn2+)

Drugs that affect ALA synthase (inc activity): phenobarbitols (Symptoms of the hydantoins acute hepatic griseofulvin porphyrias are worsened by these drugs)

no light absorption 4 molecules condensed

3 porphobilinogen

HMB synthase AIP

*regulatory step in BONE MARROW Fe2+ inserted into ring Ferrochelatase

CEP - AR! uroporph. III Hydroxymethylbilane = synthase Linear tetramer of Coproporphyrinogen porphobilinogen IIIMITO absorbs light cyclized to uroporphyrinogen Uroporphyrinogen III protoporphyrin can absorb light (ring)


Pb poisoning

Heme synthesis summary.

Common porphyrias and their causes


hwb - may 2011

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 -aminolevulinic acid (ALA)

Two simple & universally available starting metabolites: succinyl CoA and glycine condense in a reaction catalyzed by

(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

ALA synthase

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 synthase LIVER regulation is at first step

Formation of porphobilinogen
2 -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).

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

condensation of the 4 porphobilinogen 2. Uroporphyrinogen IIImolecules synthase can ABSORB LIGHT HIGH YIELD AS @#$%!

1st Synthasecloses the porphyrin ring/isomerization makes chain of 4 porphobilinogens (linear) 2nd Synthase creates ring from this chain; isomerizes it Final product: asymmetric uroporphyrinogen III (see side group arrangement)

hwb - may 2011

No light absorption @ 440 nm by singlet porphobilingen but

Overview: Pathway of porphyrin synthesis

Hydroxymethylbilane, a Tetramer of porphobilinogen absorbs light @ 440 nm


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

hwb - may 2011

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 bodys 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)

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, lead major smelters closed Pb poisoning in ~ 1000 children

Heme synthesis summary.

Common porphyrias and their causes


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 HIGH Classified as hepatic or erythropoietic Generally cause is a defect in heme synthesis;
YIELD AS @#$%!

Student Problem: what happens to the porphyrins & their precursors? what accumulates & what exists at lower concentrations?
hwb - may 2011


ferrochetalase & ALA dehydratase HMB synthase copro and ALA accumulate



porphobilinogen +ALA accumulate patients NOT photosensitive liver enyzme deficiency AUTOSOMAL RECESSIVE patients are photosensitive BM enzyme deficiency


uroporphyrinogen III synthase


uroporphyrinogen decarboxylase

uroporphyrin accumulates most common porphyria patents are photosensitive liver and BM enzymes affected
liver enzyme deficiency patients are photosensitive protoporphyrin accumulates BM enzyme deficiency photosensitive, build up in RBC, BM &


protoporphyrinogen oxidase ferrochetalase

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



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+
Biliverdin Bilirubin Soluble insoluble compound, anti-oxidant (in babies) Bilirubin liver In blood non-covalent association with albumin In liver binds to ligandin enters hepatic circulation Ligandin: glutathione-S-transferase activity 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 Intestine Resident bacteria form urobilins: urobilinogen (colorless), If goes to kideny urobilin (yellow), large colon stercobilin (brown)





Degradation of Heme & formation of bilirubin RBCs life span of ~ 120 days means continual turnover; taken up & degraded by spleens reticulo-endothelial cells 1st - the spleen and 2nd liver.
~ 85% heme destined for degradation comes from RBC, remainder from immature or badly formed RBCs & 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+++)

Step 3: Bilirubin passes to liver

Bilirubins 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)
hwb - may 2011

Step 4: Hemes degradation, contd

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

2 glucuronates added by Bilirubin 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., hyperbilirubinemia NB: unconjugated bilirubin cannot be easily excreted


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+
Biliverdin Bilirubin Soluble insoluble compound, anti-oxidant (in babies) Bilirubin liver In blood non-covalent association with albumin In liver binds to ligandin enters hepatic circulation Ligandin: glutathione-S-transferase activity 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. Intestine Resident bacteria form urobilins: urobilinogen (colorless), If goes to kideny urobilin (yellow), large colon stercobilin (brown)





Major Groupings in Jaundice: Hemolytic Jaundice

Hemolytic Jaundice Liver normally capable of handling a heme load of over 10 times the daily rate of heme production, so generally, able to process all that comes

But on occasion, as in massive lysis of RBCs (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 quantity problem too much bilirubin a

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
hwb - may 2011

Neonatal jaundice
Recall from our previous discussion succession of Hbs 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

Bilirubin is light sensitive becomes polar upon 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 B2) fortification in their formula

hwb - may 2011


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 its 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

Synthesis of Creatine Universal starting materials: Glycine & guanidino group of arginine, & methyl group of Sadenosylmethionine (Ado Met / SAM). Note integration of several sequences via key compounds directly no essential nutrients

what makes up Creatine?

* Synthesized in liver and pancreas in significant amounts * But neither tissue contains creatine kinase So separate sites for creatine synthesis & use of phosphocreatine Uptake & concentration in myocyte by specific Na+-dependent transporter.

hwb - jan 2011


Synthesis of the catecholamines from tyrosine

note 4 Rxs in succession 1st hydroxylation with BH4 & O2, 2nd PLP-dependent decarboxylation, 3rd ascorbate dependent hydroxylation and finally 4th SAM-dependent methylation by phenylethanolamine-N-methyl transferase (PNMT)

hwb - jan 2011


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

Melanin The pigment that colors hair, hide & eyes. Synthesis from tyrosine, via tyrosinase a Cucontaining 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

hwb - jan 2011

Know the main points Activators Enzymes The difference between purine and pyrimidine and not the total reaction Know regulators Know how to salvage purines

Purine nucleotide synthesis de novo Synthesized as the relevant nucleotide on a required scaffold Phosphoribosyl-pyrophosphate (PRPP)
Requirements: Precursors: 3 common AAs: 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 (pyrophosphorylated - 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-ribosylamine

Pyrophosphate 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 pathways 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

hwb - jan 2011

The 3rd step: ends with synthesis of inosine monophosphate (IMP) the stem (parent) purine nucleotide
nine (yes 9!) steps are involved here A derivative of tetrahydrofolate FH4 - (formyltetrahydrofolate) is required here in two key reactions that transfer 1-C groups. Inhibition here blocks purine & therefore nucleic acid synthesis Folate 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 IMP

PRPP platform for assembly of groups. Pyrimidine nucleotide synthesis? NO such platform.

The cells investment? PRPP, 4 ATP, 2 Gln, asp, glycine, 2 formyl-THF, CO2 NO biotin needed!! Note: inhibs & activs; Antibiotics - 2 places in formyl transfers

hwb - jan 2011


4th Step: Conversion of IMP to GMP and AMP note 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 =) 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

1. Hypoxanthine + PRPP inosine monophosphate (IMP)


1. Guanine + PRPP guanosine monophosphate (GMP)


2. Adenine + PRPP adenosine monophosphate (AMP)

Hydrolysis of pyrophosphate to 2 Pis makes reaction irreversible
hwb - jan 2011

Gout uric acid, contd

Refer to previous slide see 2 consecutive reactions involving xanthine (1 as product and 2 as substrate): 1. 2. Hypoxanthine + O2 xanthine + H2O2 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, contd
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.
hwb - jan 2011

Gout, Lesch-Nyhan, contd

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 Parkinsons 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?!

Disorders of Purine metabolism, contd: deficiency of adenosine deaminase (ADA)

Deficiencies of ADA will cause deoxy-adenosine levels to rise; These inhibit ribonucleoside reductase and in turn prevent 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 *C skeleton from aspartate Carbamoyl phosphate *N from glutamine Synthesis: Committed step via cytosolic Carbamoyl phosphate synthetase II \ (CPS II) no biotin required Inhibitor: UTP Activators: ATP, PRPP
No G here (Glycine required for purines)
hwb - jan 2011

Pyrimidine synthesis: note involvement of PRPP to create stem product

hwb - jan 2011


Multifunctional Enzyme
3 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 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

(1st ring formed)

Final step CTP is synthesized by the amination of UTP

has to be UTP form!!!

This is catalyzed by CTP synthetase amido nitrogen donated by glutamine Transfering this costly amido group from glutamine to another compound hydrolysis of anhydrous phosphate (ATP, pyrophosphate, etc)

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 Pis dTTP DNA

Note: these very similar modifications are specific for the phosphorylation status of the acceptor molecule
hwb - jan 2011

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)

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 critical Control of this enzyme: complex yet logical Separate 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 reduced E.g.: ATP tells enzyme that ADP is to be reduced; CTP informs enzyme that CDP will be reduced, etc

hwb - jan 2011


ribonucleotides (RNA) to deoxyribonucleotides (DNA) SUMMARY

the 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 deoxyNTs (dATP) so dont 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

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

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;

KNOW what inhibits each enzyme

linkage of two 5-Fluorouracil inhibits enzymes required: thymidylate synthase dihydrofolate methotrexate inhibits reductase & dihydrofolate reductase thymidylate either of these 2 inhibitors synthase
results in blocking synthesis of dTMP
hwb - jan 2011

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 TH4s 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

Main points of nucleotide metabolism: Summary

Purine nucleotides Start with PRPP 1st corner then added Precursors: 3 AAs: aspartate, glutamine, and glycine (skeleton) ; CO2 Derivatives of folic acid (vitamin B-9) as tetrahydrofolate Very large ATP cost Pyrimidine nucleotides Ring built first Then added to PRPP Precursors: Glutamine (gives NH2) Aspartate (gives 3 C skeleton and NH2) CO2 Modest energetic cost (2 ATPs)
hwb - jan 2011

Summary of main points of nucleotide metabolism, contd

Purine nucleotides IMP yields AMP, GMP Indestructible ring structure Salvage pathway very important Yields uric acid as end product
Pyrimidines OMP yields UMP, and indirectly, CMP and TMP Easily catabolized for energy yield, yields NH3 No salvage pathway

hwb - jan 2011

Main points of nucleotide metabolism: Summary

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

All single carbon groups donated by THF


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

Total body stores: app 80-110 mg; liver is central l 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 Cus; 2-globulin; site of synthesis hepatocyte; -life = 5.5 d.;


Normally 10% cpm apoprotein, secreted w/o Cu, -life ~ 5 hrs; Changes in Cu or cpm synthesis easily effect Wilsons disease
Functions: In Cu-containing metalloenzymes; Generally these use either molecular O2 or an oxygen derivative as a substrate. Examples: cytochrome oxidase, dopamine -hydroxylase, tyrosinase, lysyl oxidase (compare this with lysyl hydroxylase - with its Fe++), and cytoplasmic superoxide dismutase (see Lou Gehrigs disease) *Cu necessary for SOD function
HWB - Sept '09

Some trace metals - a two-edged problem: Deficiency and Toxicity E.g.: Copper 1. Deficiency Menkes 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 AAs Treatment absorbable copper-histidine complex 2. Toxicity Wilsons 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 carryingprotein: Ceruloplasmin* GI uptake with Tranferrin Cu deficiency= Menkes Disease Kinky Hair Syndrome deficiency in ATP dependent Cu transporter Cu excess= Wilsons 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 Gehrigs 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) Egs: 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 foods Avg 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 albumin Storage with metallothionein present - many tissues, synthesis induced by heavy metals : zinc, cadmium, arsenic Zn absorption follows metallothionein levels in intestinal

mucosa.*** (Q!!) Metallothionein(s) protect(s) cell from toxic effects of free, unbound metalHWB S: ions. 9

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 Metallothioneins synthesis induced by heavy metals!!!!! Metallothioneins 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 dismutase Adequate intake ~ 2-5 mg/day. Excess Mn toxic, psychosis and Parkinsonism (manganese madness) resembling Parkinsons 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 AAs & L 171 AAs); 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 low small amounts of ferritin, mostly apoferritin with little bound iron, is also present in blood; released during normal cell turnover

contains 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

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 gene About 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-1 (TPP)
Oxidative decarboxylations, transketolases

B-2 (riboflavin) B-3 (Niacin) B-5 (pantothenic acid) B-6 (pyroxidine PLP)
Transaminations and non-oxidative decarboxylation Carboxylations

B-7 (Biotin) 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 US Q) what do you give alcoholics? TPP supplements Best sources of B-1: pork, whole grains, nuts
hwb - Sept '09

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 -ketoacids Advanced deficiency Beriberi Acutely - Wernicke-Korsakoff syndrome, seen often in alcoholics

Riboflavin B-2 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 dependent Dietary sources: Liver, yeast, eggs, meat, enriched bread, milk Deficiencies: Glossitis, sore throat, moist dermatitis of nose; ****destroyed by natural light (UV); therapy routinely given in hyperbilirubinemia (neonatal hwb - Sept '09 treated with phototherapy jaundice)

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 acid

Precursor of NAD+ & NADP+ - pyrimidine derivatives of nicotinic acid & nicotinamide Dietary forms - hydrolyzed in gut & direct absorption asas nicotinic acid & nicotinamide In Humans - tryptophan small amounts Niacin very inefficient; *** pathway also requires thiamin, riboflavin; In multiple deficiencies (quite common), impairment is strong Niacins conversion to NAD+ and NADP+: Only vitamin that
Synthesis of NADP+
can be made from one of our aa Tryptophan (W)

hwb - jan '10

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

Active form is pyridoxal phosphate= PLP Key Prosthetic group of Aminotransferases (Transamination and Deamination) and NonOxidative Decarboxylases It is also an essential cofactor for glycogen phosphorylase (glycogneloysis)

Folic Acid Vitamin B-9

Supplied only by diet Synthesis - 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) by dihydrofolate reductase, using NADPH

hwb - Sept '09

Homocysteine + Methyl-THF methionine + THF MeTHF donmates Me group to Homocysteine, & is reconstituted as THF

Partnership between methyltetrahydrofolate and cobalamin cobalamine

**** 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 it Yeast, liver, kidney, fish, fruits, green leafy vegetables (Latin folium = leaf) Low levels often seen in late pregnancy Thus (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

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-THF 2. 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 FAs 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 succinylCoA

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

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


Unstable at high 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

Deficiencies uncommon inducible 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

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

B-12 (Cobalamin)
Folic acid (B-9)


o Folate Trap Hyperhomocysteinemia o Intrinsic factor binds B-12 deficiency = pernicious anemia

o Depression and hyperhomocysteinemia o Neural tube defects (MTHFR gene)

B-6 (Pyroxidine active form = PLP)

o Hyperhomocysteinemia o Serotonin production depression

B-1 (TPP) [CNS impairment]

o Wernicks/Korsakoffs encephalopathy o Beriberi

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

Vitamin C
o 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 acid Retinoic acids main target cells: epithelial cells - oxidize retinol, retinal to retinoic acid Retinals function: prosthetic group of rhodopsins visual pigment of rods & cones How light is sensed 1. Its long fatty tail imbeds retinal in retinas plasma membrane; 2. Light absorption changes cis- to trans- (straight) form 3. configuration change sensed by opsin, which holds retinal at its center 4. 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, contd

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 blindness Leading 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 synthesis

Nutritionally 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 KNOW WHERE vitamin D-binding plasma protein.

In (1st) Liver & (2nd) kidney transformed to active 1, 25 dihydroxycholecalciferol (calcitriol) by 2 successive hydroxylations Calcitriol hormone-like action
hwb -jan 2011

Synthesis of Vitamin D
1st step - 25 hydroxylation is NOT rate limiting or regulatory major circulating form - 25-hydroxy-D3. Last step producing calcitriol - 1-hydroxylase - is regulatory tight 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 1,25dihydroxycholecalciferol (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 -Tocopherol

Over 8 related substances, all with vit E activity, isolated from natural sources Most potent & prevalent : -tocopherol double methylated
RDA 10 mg/day for men; 8 mg/day for women Best sources: nuts, seeds, wheat germ & vegetable oils, leafy vegetables Requirement rises as intake of polyunsaturated FAs increases

hwb -jan 2011

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 humans 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 prods As 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

Only 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 K Seen 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 KNOW*: HEMORRHAGIC DISEASE OF THE NEWBORN One other avenue TO USE deficiency of K AND WHAT VITAMIN can cause VITAMIN 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

Vitamin K deficiencies

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

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

RQs for different foodstuffs CH2O 1.0 Fat (TG) 0.7 Protein 0.8
If RQ = Higher value (CH20) = Less energy required to break it (less O2 consumed, less energy needed)down (less O ) 2 RQ is a ratio: Lower value (fat & protein) = C02 More energy to break it down O2 (more O2 consumption)

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/fat Which elicits the higher RQ? B

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


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? A iii. design a diet that yields All Fat 100%= lowest RQ & highest O2 uptake
hwb - jan 2011

Major factors in the livers response to CHO Hepatic GLUT 2 unaffected by insulin HIGH YIELD AS 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 - 2s; 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

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) Summary: Also favors de-phosphorylation of HS-lipase & perilipin (inactive) so - hydrolysis of triacylglycerol in the recently fed state is inhibited.

Insulin is an effective antagonist of lipolysis (& breakdown of energy stores in general); -High Insulin TAGs synthesized after a meal; In contrast, TAGs are degraded during fasting/long-term work via glucagon

hwb - jan 2011

Skeletal muscle: Carbohydrate metabolism

Temporary 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 synthesis if 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

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? HIGH Brain glucose - totally oxidized to CO2 & water YIELD AS

Fat metabolism in brain

Restrictive blood-brain barrier: no FAs 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

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)
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 VLDLs - assembled for distribution of HIGH YIELD AS 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 brains O2 demand in relative terms can vary widely, depending on total metabolic rate of the body
hwb - jan 2011

Second - the insulin-DEPENDENT portion:

Muscle insulin-dependent glucose & FA transporters very active Glycogen synthesis spurred, HIGH YIELD AS triacylglycerols deposited & @#$%! amino acids imported for building blocks for protein synthesis.

Adipose tissue especially insulin-sensitive

Glucose-6-phosphate: essential, as the source of glycerol backbone for triacylglycerol synthesis Of course majority of FAs synthesized de novo come from liver and then are stored in the adipocyte
hwb - jan 2011


Integrating the well-fed state into body weight regulation

Proposed: Lipostatic model for regulation of body weight involves 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
hwb - jan 2011


Trypsin, chymotrypsin, elastase: Active only at neutral pH

carboxypeptidases A & B both are Zn++ metalloenzymes

Well-fed State adipocyte GLUT-4 Muscle GLUT-4 Liver- Glut 2 Pancreas- Glut 2 respiratory quotient (RQ Highest= lots of carbs consume least O2 Lowest= lots of fat; consume most O2

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

Hormones from the pancreas: Insulin, glucagon, somatostatin

Glucagon -cells; Insulin -cells; Somatostatin 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 high 2. Glucagon: X plasma [glucose] from falling too low 3. 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. Corrn: 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

-cells or 2. (indirectly) to dysregulation under sustained high [glucose]s

hwb - jan 2011

GAGS! Main factors cause increased secretion of insulin (mne Glucose (essentially in the well-fed state) Arginine Glucagon Secretin!!

Glucose most powerful stimulator (after uptake) Amino acids especially arginine
Gastrointestinal hormones after the ingestion of food

secretin, especially right

Glucagon - this stimulates the secretion of insulin and

inhibits further release of itself; *** this feature is an especially significant in the Type I diabetic finding

hwb - jan 2011

Metabolic effects of insulin: well-fed state

Carbohydrate Metabolism 3 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

elevated Adipocyte: glucose uptake stimulated to produce glycerol-3-P for triacylglycerol synthesis & storage
hwb - jan 2011

uptake raised, glycogen synthesis

Its BACK! Dont miss it on this block!

Revisit our glucose transporters (GLUTs)

Variant tissue location GLUT1 heart muscle, plasma membrane brain, placenta, erythrocyte GLUT2 liver, kidney, pancreatic -cells, intestine GLUT3 neuron, kidney, placenta GLUT4 * muscle, adipocyte * heart GLUT5 muscle, sperm sarcolemmal . vesicles
hwb - jan 2011

comment In muscle- by hypoxia, independent of insulin

insulin-independent myo-, fat cell activity by insulin dietary fructose

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

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

2. This sets off a rapid chain of events in the -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 tissue BUT 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, quickresponse, carbon
hwb - jan 2011

Glucagon the counter-regulatory hormone

Identified 1920 named 1923 From pancreatic -cells 3,500 MW polypeptide, 29 AAs Both synthesis and release controlled by insulin Primary targets 1. Liver, 2. adipocyte

Principal effect: Liver: elevation of cAMP promote glycogenolysis and inhibit both glycogen synthesis and glycolysis; this activates gluconeogenesis Adipocyte: R cAMP; protein kinase A phosphorylates hormone-sensitive lipase to yield FFAs and glycerol ( hepatic gluconeogenesis)

hwb - jan 2011

Glucagon, contd
From pancreatic -cells (near outer edge of Islet) Primary structure:
NH2 -His-Ser-Gln-Gly-Thr-Phe- Thr-Ser-Asp-Tyr-Ser -Lys-Tyr-Leu-Asp-Ser- Arg-Arg-Ala-Gln-AspPhe-Val-Gln-Trp-Leu- Met-Asn-ThrCOOH note

simple, small & no cysteine 12 AA residues (41 %) are essential or synthesized (Tyr) from essential AAs (40% of our 20 essential)
hwb - jan 2011

Stimulators of its secretion Fall in plasma [glucose] High catecholamines for some time Increased [AAs] to prevent hypoglycemia after an all-protein meal (arginine key role) Acetylcholine Cholecystokinin/pancreazymin Inhibitors of its secretion Somatostatin Insulin Control of output? Mechanism not yet understood: except that -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 energy stores, 2. effects of fat depletion on ingestion, energy expenditure, linear growth, fertility.

Males, [leptin] are - in premenopausal females Other: pregnancy levels double; cannot cross LEPTIN - Promotes the feeling of being full and Note the placenta intake- Produced by adipose inhibits food differences!!!
tissue, like a negative feedback mechanism on eating
hwb - sept '09

Product of adipose tissue - especially white adipose tissue (WAT); also BAT & stomach proportional to fat tissue mass 167 AAs 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

Leptins mechanism & meaning:

Food intake leptin secretion from adipose tissues Message: energy reserves sufficient - stop eating! release of appetite-suppressing (anorexigenic) hormones Effect: 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 reducer

Adiponectin from adipocyte; -oxidation,

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

hwb - sept '09

Adiponectin works through AMPK


Muscle FA uptake -oxidation glucose uptake

Adiponectin= low energy charge STOP SYNTHESIS

AMPK (AMP activated protein kinase)

Liver FA synthesis gluconeogenesis glycolysis

***AMPK inhibits virtually all biosynthetic processes High AMPs message? Low energy charge !
hwb - sept '09

Ghrelin and PYYs roles in control of short-term eating behavior Ghrelin (28 AAs) & PYY3-36 34 AAs) 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):

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

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)
Observe Deceptively 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 - sept '09

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 counterregulatory hormones (epinephrine & esp. glucagon)

Effects: microvascular & macrovascular

Overview: as not painful (at first) most cant 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 AAs (urea cycle) HIGH CHO: glycogenolysis; gluconeogenesis; glycolysis & when [glucose] is low glucose secretion into plasma
YIELD AS @#$%!

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!)
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)
hwb - jan 2011

DIFFERENCES between DM1 and Starving State



completely absent in DM1 low in starving state


hyperglycemia in DM1 (and DM2!!) near normal levels in starving state


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

Features of diabetes mellitus types I and II Characteristic Type I Type II


Age of onset pre-or adolescent, usually 35+ yrs, any age! Nature of onset often sudden Slow, insidious Genetics specific HLA factors* no HLA connection; strongly familial Secondary factors viruses, toxins obesity -cell autoimmune? at initial episode not present Insulin secretion absent or delayed sometimes reduced? Body habitus thin to cachectic normal or obese Symptoms 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 & Gravess disease (hyperthyroidism)

hwb - jan 2011

Hypoglycemia in IDDM?
can occur if insulin dosage is inaccurately gauged or given. HIGH Frequency of hypoglycemic YIELD AS @#$%! 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

hwb - jan 2011

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

Insulin resistance Hyperglycemia - increased glucose production by liver HIGH YIELD AS - decreased glucose uptake by GLUT4 in @#$%! tissue (similar to what happens in DM1) Inadequate insulin secretion

Diabetes mellitus Type II (NIDDM) Genetics & environment - major roles Very strong familial factor to susceptibility Almost 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 subtype Autosomal dominant So far the only form of DM with definite inheritance mode; 5 specific markers have been identified; more possible
hwb - jan 2011


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

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.
HIGH YIELD AS @#$%! hwb - jan 2011

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 ex: glycogen phosphorylase is ACTIVE when phosphorylated 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!)
ex: glycogen synthase is INACTIVE when phosphorylated

CARBOHYDRATE METABOLISM LIVER: inhibits gluconeogenesis and glycogen breakdown MUSCLE & LIVER: increases glycogen synthesis

(high plasma glucose) (decreases production of glucose)

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 adipocytes glucose 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 AAs into cells and protein synthesis
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


glycogen breakdown (liver, not muscle), increased gluconeogenesis

LIPID METABOLISM hepatic oxidation of FAs and subsequent formation of ketone bodies from acetyl-CoA effect 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 gluconeogenesis thus plasma levels of AAs are decreased

GLUCAGON acts to maintain blood glucose during periods of potential hypoglycemia increases: glycogenolysis gluconeogenesis ketogenesis uptake of amino acids its secretion is stimulated by: low blood glucose, AAs, and epinephrine its secretion is inhibited by: elevated blood glucose and insulin