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
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