Biochem: Nitrogen Metabolism 2

Porphyrin

-5 ring molecule
A=acetate
P=propionate
M=methyl
V=Vinl

requires iron to be functional

eg Heme side chian = MVMVMPPM

Biosynthesis of heme 1st step

glycine + succinyl CoA (from TCA)

δ-aminolevulinic acid (ALA) by enzyme δ-ALA synthase.

-inhibited by hemin and heme (product inhibition)

-hemin-drug, reduces heme production

The 2nd step of heme biosynthesis

forms prophobilinogen by enzyme δ-aminolevulinic acid dehydratase (ALAD)

-inhibited by lead (lead also inhibits last step)

-leads to lead poisoning or porphyria

Lead poisoning (Pb replaces Fe) inhibits what steps/enzymes of heme biosynthesis?

1. δ-aminolevulinic acid dehydratase (ALAD) (2nd step)
2. Ferrochelatase (last step), inhibits moving of iron into the ring of heme

-porphyria cutanea tarda-chronic and most common
- causes brownish urine

Porphyrias

- deficiency: any enzyme in heme biosynthesis pathway
-accumulation& increase excretion of porphorins or porphorin precursors

Clinical manifestations:
Sensitive to sun light (photosensitivity)
Skin itches/burns
sunlight produces ROS, start to burn->oxidative stress

Chronic hepatic porphyria: *MOST COMMON

Acute hepatic porphyria: liver type

Erythropoietic porphyria: RBC type

Degradation of Heme

In Macrophages:

Heme ---- Biliverdin ----Bilirubin (B) (unconjugated, UC)

In Liver:

Bilrubin -----Bilirubin diglucuronide (BG; direct bilirubin/conjugated)

In Intestine:

BG--- Bile---- Urobilinogen/Sterocobillin (by intestinal bacteria)

B= "inactive bilrubin" (billirubin = uncojugated billirubin )

BG = direct bilirubin (cojugated billirubin)

*more water- soluable, bilirubin diglucuronide by adding 2 glucoric acid molecules, catalyze by UDP-glucuronosyltransferace

Hyperbilirubemia (high bilirubin in blood) is also called ____.

juandice

UDP glucuronosyltransferase deficiencies

UGT1A1 gene

Crigler-Najjar syndrome Type 1: No enzyme function

Crigler-Najjar syndrome Type 2: 20% of the function

-High levels of unconjugated bilirubin in the blood (unconjugated hyperbilirubinemia) and yellowing of the skin and eyes (jaundice).

Gilbert syndrome: 30% of the function

-Mild unconjugated hyperbilirubinemia, which rarely leads to episodes of jaundice

Crigler-Najjar syndrome Type 1

No enzyme function

Crigler-Najjar syndrome Type 2

20% of the function
-High levels of unconjugated bilirubin in the blood (unconjugated hyperbilirubinemia) and yellowing of the skin and eyes (jaundice).

Gilbert syndrome

30% of the function
-Mild unconjugated hyperbilirubinemia, which rarely leads to episodes of jaundice

UDP glucuronosyltransferase deficiencies also called

bilirubin-UGT, Bilirubin UDP glucuronosyltransferase, bilirubin uridine diphosphate glucuronosyltransferase.

***Reaction: In liver, more water- soluable, bilirubin diglucuronide by adding 2 glucoric acid molecules, catalyze by
UDP-glucuronosyltransferace

Hemolytic Jaundice

B (uncojugated billirubin) high, BG higher but liver may not catch up with conjugation->unconjugated hyperbilirubinemia

lab test? HIGH TOTAL BILIRUBIN

- result from extensive hemolysis (sickle cell anemia, pyruvate kinase or G-6-P deficiency) (hemoglobin overload)

- bilirubin is produced faster then it can be conjugate

(B = bilirubin/ UCB) (CB/BG= conjugated bilirubin) (U= urobilinogen) (S= stercobilin; UDP = uridine diphosphate)

Hepatocellular Jaundice

Liver damage/disease

-BG (conjugated bilirubin) higher, leaked from liver, conjugated hyperbilirubinemia

lab test? HIGH DIRECT BILIRUBIN

HIGH ALA & AST

Enzyme deficiency

-B high, BG low, unconjugated hyperbilirubinemia

lab test? HIGH TOTAL BILIRUBIN

AST/ALT normal unless liver injury involved

(B = bilirubin/ UCB) (CB/BG= conjugated bilirubin) (U= urobilinogen) (S= stercobilin; UDP = uridine diphosphate)

Obstructive Jaundice

-B and BG high in blood, conjugated and unconjugated hyperbilirubinemia

lab test? HIGH DIRECT BILIRUBIN and TOTAL BILIRUBIN

AST/ALT may be high, not always (if liver problem will be high)

- results from obstruction in common bile duct (by cancer, gallbladder stone etc)

-pain in abdomen, pale/clay colored stool, BG high in urine

S: hyperbilirubinemia (liver regurgitates CB into bb), "urinary bilirubin" (CB eventually excrete into urine)

(B = bilirubin/ UCB) (CB/BG= conjugated bilirubin) (U= urobilinogen) (S= stercobilin; UDP = uridine diphosphate)

Neonatal Jaundice

Similar to enzyme deficiency

-B (unconjugated bilirubin)

-UGT enzyme not made in first postnatal week

-unconjugated hyperbilirubinemia

(B = bilirubin/ UCB) (CB/BG= conjugated bilirubin) (U= urobilinogen) (S= stercobilin; UDP = uridine diphosphate)

Lab test for bilirubin

Total and direct bilirubin are directly measured

The van den Bergh reaction:

-Colorimetric estimation of serum bilirubin by coupling with diazotized sulphanilic acid.

1. Direct bilirubin = Bilirubin diglucuronide

(water-soluble bilirubin glucuronides/conjugated form) react rapidly so that is called "direct".

2. Total bilirubin

is measured after solubilized with methanol. (unconjugated bilirubin)

What does this mean?

ALT 20 (normal range, 7-30 IU)
AST 25 (normal range, 9-32 IU)
Bilirubin, total: 10 (normal range 0-1.0 mg/dL)
Bilirubin, direct:.5 (normal range 0-.04 mg/dL)

Hemolytic Jaundice

Alcohol dehydrogenase (ADH)

enzyme for alcohol intoxication and metabolism
(ADH1A, 1B, 1C, 4-7)
-found in liver and stomach
-first step of alcohol metabolism

1B*2->half east asians
1b*3->15-25% of african americans

ADH Reaction

ethanol + NAD+ <---->acetaldehyde + NADH

NADH->fatty liver

acetaldehyde->oxidative stress (ROS), cell injury

Which of the following would be expected to occur after acute alcohol ingestion?

An increase in the NAD+/NADH ratio
A decrease in the NAD+/NADH ratio

decrease NAD+/ NADH ratio

*acute alcohol ingestion results in increase in cytoplasmic and mitochondrial NADH

Ethanol metabolism

ethanol --> acetaldehyde --> acetate (liver)
acetate leaks out to muscle->makes Acetyl CoA using ACS
acetaldehyde can also leak out into blood (toxic)

ethanol-->acetaldehyde uses ADH
acetaldehyde-->acetate uses ALDH

Ethanol metabolism 3 steps

1. In cytoplasm, Ethanol oxidized to produce NADH and Acetaldehyde by alcohol dehydrogenase (ADH)

2. In mitochondria, Acetaldehyde oxidize to produce Acetate and NADH by acetaldehyde dehydrogenase (ALDH)

3. Fate of acetate mediated by ACS (acetyl-CoA synthetase)

Ethanol can also be oxidized by

1. CYP2E1 in microsomes (MEOS: microsomal enzyme oxidation system)

2. Catalase in peroxisomes

Acute effects of ethanol ingestion are due to the elevated NADH/NAD+ ratio, which leads to

- hyperlipidemia (high FA synthesis, inhibition of fatty acid oxidation->fatty liver)

-hypoglycemia (impaired GNG, pyruvate convert to alternate rxn pathways)

- lactic acidosis (high lactate)

- Ketogenesis (break down of FA & ketogenic AA)

- Hyperuricemia (high uric acid)

Alcohol consumption leads to hypoglycemia (mechanism)

The ethanol-mediated increase in NADH causes intermediates of gluconeogenesis to be diverted to alternate rxn pathways:

pyruvate to OAA-->malate (driven by NADH)

pyruvate to lactate (lactic acidosis)

decreases precursors for glucose

-exhaust pyruvate and OAA (increase lactate and malate)

-HIGH levels of cytosolic NADH → Impaired gluconeogenesis (pyruvate convert to lactate, OAA convert to malate) →

Hypoglycemia (decrease glucose)

Which of the following would be expected to occur after acute alcohol ingestion?

The activation of fatty acid oxidation
Lactic acidosis
Deprivation of malate

lactic acidosis

DNA and RNA

DNA and RNA Purines
-Adenine (A)
-Guanine (G)
-can't open/recycle the rings, final metab->uric acid

DNA and RNA Pyrimidines
-Thymine (T) (DNA)
-Cytosine (C)
-Uracil (U) (RNA)
-can recycle pyrimidines

Nucleotide vs. Nuecleoside

nucleotide= base + pentose sugar + P
nucleoside = base +pentose sugar

Phosphate provides energy

Ribose (RNA) vs 2-Deoxyribose (DNA)

ribose = OH @C2
deoxyribose = H @C2 (deoxy=no oxygen)

OH unstable-RNA harder to deal with

Purines and pyrimidines

-dietary uptake low

-synthesized by de novo or salvage pathways

-made from simple molecules (de novo)

-salvaged from existing bases (salvage pathways)

De novo purine synthesis

made from:
- Amino acids (Glutamine, Glycine, and Aspartate)
- Folic acids (N10-formyltetahydrofolate) ....contributes 1 C
- CO2

*purine ring is costructed in LIVER, in series of rxns that donate Carbon and Nitrogen to ribose 5- phosphate (from pentsoe phosphate pathway)

De novo pathway

produces 6 ATP (5 for dATP)

comes from ribose 5 phosphate, from pentose phosphate shunt
-glutamine + PRPP ---->IMP
-folic acid (N10 formyl FH4) important in 2 places
-if folic acid deficient->deficient in G and A nucleotide
-A can be supplemented

First reaction of de novo

R5P to PRPP (first step of de novo synthesis)

RRPP synthase used
inhibit: purine ribonucleotides
activator: Pi (phosphate)

The 2nd reaction of purine de novo synthesis

PRPP to 5-phosphoriboylamine by ...aminotransferase
ACTIVATOR: PRPP
INHIBITOR: AMP, GMP, IMP

N10 formyl FH4 at 2 places
2 drug groups
-PABA analog
-Folic acid analog

PABA
-sulfoamides->antibiotics, inhibit folic acid biosynthesis in bacteria, does not effect humans.

Folic Acid
-used on folic acid, inhibits reaction to block N10 formyl FH4 in 2 places, blocks formyltransferase at both locations, blocks A and G biosynthesis

IMP (Inosine) precursor for A and G

Purine synthesis inhibitors (PABA)

antibiotics

No effect on humans

PABA analogs inhibit bacterial synthesis of folic acid

PABA: para aminobenzoic acid

sulfonamides

inhibits sulfonamides

inhibits N10 formyl FH4 in two placesN10 formyl FH4 in two places

Purine synthesis inhibitors (folic acid analog)

Folic acid analogs

-Methotrexate

-inhibits two transferase reactions

-stops growing cells

-useful in treating cancers, rheumatoid arthritis

IMP reaction

IMP to AMP and GMP
-uses adenylosuccinate synthase and IMP dehydrogenase respectively
-inhibited by AMP and GMP respectively (neg. feedback)

drug mycophenolic acid also inhibits (reversible, non-comp)
-inhibits guanine pathway only and immune system, used to prevent transplant rejection
-when inhibit nucleotide pathway, any proliferation cell is impacted (stem cells, bone marrow, immune cells-T and B)
-if give inhibitor you suppress the immune system and other recycling system (skin, hair, GI side effects)
-main place is to stop cancer

De novo pyrimidine synthesis

Glutamine + CO2 + 2 ATP ---> carbamoyl phosphate
Inhib: CP2, UTP
stim: PRPP

Made from
-Amino acids (Aspartate, Glutamine)
-CO2

FH4/folic acid deficiency-impact T nucleo
if deficient will increase orotic acid if enzyme is inhibited
carbamoyl phosphate comes from urea cycle, increased by UCD, if happens will leak out and also contribute to orotic acid

CPS1-urea cycle deficiency
CPS2-pyrimidine biosynthesis

De novo pyrimidine synthesis (OAU)

Orotic acid urea caused by:

-UMP synthase def->OA high, ammonia normal

-UMP-synthase inhibitors->Gout treatment (Allopurinol) or chemotherapy (6-azauridine)

-OTC def (UCD)->OA high, ammonia high, CP high vs

-CP1 def->OA low, ammonia high, CP low

UMP inhib most common, know the 2 drug treatments above

OTC 2nd leading cause->look at ammonia, if high UCD problem, if OA and CP low then its CPS1 issue not OTC

UMP synth def 3rd cause (not ammonia related, enzyme issue)

Anti-cancer drug: 5-FU (FU cancer)

-block thymidylate synthetase

-dUMP--->dTMP reaction is coupled with folic acid cycle (5, 10 methylene FH4)

-enzyme makes T nucleotide

-uses UMP

5-FU inhibits T synthesis, inhibits cell proliferation

Salvage of bases (purines)

Salvage pathway for Purines

Purines that result from the normal turnover of nucleic acids or small amount obtained from diet can be converted to nucleoside triphosphates and used by the body.

*important for brain

focus on IMP, can make AA (makes AMP or GMP)

Hypoxanthine-make IMP, circle reaction, uses HGRPT (transferase type), if problem causes Lesch Nyhan (same issue for Guanine-->GMP also uses HGPRT)

Adenosine->Inosine using ADA, if def causes SCID

Lesch-Nyhan syndrome

X linked recessive

defect in salvage pathways (enzyme def) (cause)

increased de novo purine synthesis (result not cause)

nucleotide go down, compensate from increase in de novo synthesis

excessive uric acid and neurologic def:

-self mutilation, involuntary movements

HGPRT issue

Severe combined immunodeficiency disease (SCID)

X linked recessive (major cause, 50-60%) leading death in babies

-immune system problem, mutation in y chain subunit of -cytokine receptors (IL2RG)

-defect in cytokine receptor for IL7, can't bind and activate immune reaction

Autosomal recessive (2nd type)

-adenosine deaminase (ADA) problem

-deoxynucleotide (dATP) accumulates, toxic to lymphocytes, decreases B and T cells

-ADA used for degradation pathway to make uric acid, since ADA def only goes to the dATP pathway instead

Degradation in the small intestine

-DNA/RNA to oligonucleotides to purine/pyrimidine
-ribonucleases and deoxyribonucleases (pancreas)
-nucleotidase

(low dietary uptake) we most throw away nucleotides
-we cut them into pieces, can cut nucleases more
-purine is thrown away by uric acid, little bit is used

some plant sources with DNA/RNA can make gout worse and contribute to uric acid

Degradation of purines

-mainly in liver

-AMP/GMP to Xanthine to Uric acid

Allopurinol

-inhibitor of xanthine oxidase

-treat Gout with overproducers of uric acid

can't open purine ring so we partially degrade

two reactions, both use xanthine oxidase->degrades AMP portion->to help form uric acid

Gout

High uric acid: multiple causes (inflammation problem)

-under excretion

-overproduction

acute: use anti inflammatory drugs (first)

long-term: uric acid lowering drugs/diet (after)

Degradation of pyrimidines

pyrimidine ring is opened (purine rings can't be opened)

degraded to highly soluble, b-alanine and b-aminoisobutyrate

Overview

Two different ways to get nucleotides
they can either be made (de novo)
or recycled (salvage pathway)

Disease/Drug Overview

Purines->Adenosine->Inosine w/ ADA (SCIDS if inhibited)

Inosine or Purine->Hypoxanthine & Guanine->Purines w/HGRPT (Lesch-Nyhan if inhibited)

Hypoxanthine & Guanine->Xanthine->Uric acid w/Xanthine oxidase (Gout drugs to inhibit)

Uric acid->excess forms crystals in joints (gout)
excreted in urine->excess forms crystals in kidney (rental stones)