Nitrogen Metabolism I: Biochem MT2

Excess AA ____ be stored->_____ Turnover: _____
~46-56 gm degraded/day need to replace

can't

degraded

400 gm/day

AA degradation links to what?

TCA cycle

acetoacetate

pyruvate

fumarate

aKG

Prop CoA

OAA

Glucogenic AAs

Glucogenic AAs can be converted to "Glucose/Glycogen"

-generates Pyruvate or an intermediate of TCA cycle

Ketogenic AAs

Ketogenic can be converted into ketone bodies

-directly generates Ketone bodies (acetoacetate, acetyl CoA or acetoacetyl CoA)

Ketogenic AA examples

Leu, Lys->Acetoacetate

Ketogenic/Glucogenic examples

Phe, Trp, Tyr->Acetoacetate (Phe and Tyr also gluco for fumarate and Trp gluco for pyruvate)

Ile->ACoA (also gluco for Succ CoA)

Glucogenic (the rest) examples

Phe, Tyr->Fumarate (Phe and Tyr also keto for acetoacetate)

Arg, His, Pro->Glu->aKG
Glu-><-Gln

Ile, Met, Thr, Val->Succ CoA (Ile also keto for ACoA)

Ala, Cys, Gly, Ser, Thr, Trp->Pyruvate (Trp also keto for acetoacetate)

Degradation of branched-chain amino acids (BCAAs)

-Universal fuels (~40% of total amino acids)

-metabolized by thes peripheral tissue (particularly muscle)

-defects cause accumulation of a-keto acids (Maple syrup urine disease)-a-keto acids have same co factors as a

KG (lipoic acid etc)

Maple Syrup Urine Disease

-caused by defect in a keto acid dehydrogenase
-causes brown color, sweet smelling urine

Catabolism of AAs: the Amino nitrogen portion of an AA turns into what and the carbon skeleton to what?

-Amino Nitrogen to urea and glutamine

-the carbon skeleton to intermediates metabolism

Three coenzymes used in AA metabolism

-Pyriodxal phosphate (PLP/B6)
-Tetrahydrofolate (FH4) (folic acid, B vitamin)
-Tetrahydrobiopterin (BH4)

Catabolism of amino acids: Two major reactions

-transamination
-oxidative deamination

Transamination reaction

-transamination is followed by deamination
-transports NH2 from aspartate to a KG to form glutamate
-glutamate can lose NH3 (NH2 plus an H) to the urea cycle

Aspartate Transaminase (AST)

Aspartate + a-keto glutarate w/AST -> OAA + glutamate (can also go other way)

transfers NH2 group from Asp to aKG

same pathway as GOT, can use either one

most transaminases use aKG

Glutamate Oxaloacetate Transaminase (GOT)

Aspartate + a-keto glutarate w/AST -> OAA + glutamate

transfers NH2 group from Asp to aKG

same pathway as AST, can use either one

most transaminases use aKG

Transaminases or Amino transferases require

Pyridoxal phosphate (PLP/B6)

Transaminase (ex. ALT-ALanine amino-transferase) is a marker of ?

liver damage
-more effective than bilirubin as a marker (slow)
-amanita is toxic, can cause liver damage from mushrooms->causes ALT to leak out of the liver cell membrane

Serum transaminase: ALT (GPT) is more specific for

for liver cytoplasmic

AST (GOT) is specific for

mitochondrial & cytoplasmic liver disease

serum [AST]=2x [ALT] in?

alcoholic liver disease

ALT transfers

alanine to alpha-keto glutarate, resulting in the formation of pyruvate and glutamate

Role of glutamate in urea cycle

-1st step is AA w/ TA->glutamate using aKG
-2nd step is moving nitrogen from glutamate to make free ammonium using GDH (oxidative deamination)
-glutamate w/ TA + OAA -> Aspartate (keeps nitrogen, also uses aKG)
-glutamine shuttle from brain (protects brain), can also be from muscle
-alanine shuttle from muscle only

Glutamate dehydrogenase (oxidative deamination)

the deamination of glutamate by glutamate dehydrogenase (GDH) (coenzyme is NADH or NADPH) to form alpha keto-glutarate and NH3

Fate of AA and role of urea cycle

AA-> Carbon path or Nitrogen path
Nitrogen moved by TAs

-make free ammonium or ASP for urea cycle
-when urea cycle knocked out cause hyperammonemia and increased AAs

Urea cycle disorders (UCD) broadly

-hyperammonemia

-urea cycle disorders (UCD)

-UCDs, except for arginase deficiency, present in newborn who are initially well and become hyperammonemia after a period of protein feeding.

Hyperammonemia

-acquired: common: liver disease
-congenital: UCD (type of inborn errors of metabolism, IEM)

Urea cycle disorders (UCD) specific

deficiency of any of the urea cycle enzymes

-Ornithine transcarbamylase (OTC) deficiency (most common; X-linked; other UCDs are AR)

-Citrullinemia (ASS1/enzyme and SLC25A13/transporter mutations)

Urea Cycle Overview

-CPS1: rate limiting

-OTC, ASS: disorders

-Arginine: directly to urea cycle

-Enzymes: ARG1, ASL, ASS, CPS1, NAG, NAGS, OTC

most occurs in mito, citrulline has to be transported out, and orthenine has to be transported in

Urea Cycle Disfunction

OTC most common, X-linked (Orthenine to Citrulline)
all others are autosomal recessive
OTC deficient->accumulate CP, ammonium

ASS mutation affects citrulline transporter (to leave mito), will increase citrulline
ASS deficient/citrulline transport issue->accumulate citrulline, ammonium

all mutations except arginase will increase ammonium (NH4)
even if knock out arginase, can still release arginine, liver release

Essential AA

Carbon skeleton cannot be synthesized

Non-essential AA

can be synthesized

How many essential AAs? How many conditionally essential?

10 essential

6 conditional

Non-essential AA examples

non-essential AA Precursor

Glutamate, Glutamine aKG (common)

Aspartate, Asparagine OAA (TCA)

Serine 3-phosphoglycerate

Glycine serine

Tyrosine phenylalanine (important)

Proline glutamate

Alanine pyruvic acid (pyruvate, glycolysis)

Cysteine serine, methionine (met most)

Essential AA Examples

10 AAs

Phenylalanine 6.6 (most abundant)

Valine 4.4

Tryptophan 1.0 (less abundant)

Threonine 3.2

Isoleucine 3.8

Methionine 2.3

Histidine* 1.5

Arginine*

Leucine 6.4

Lysine 4.6

ideal proportion=ovalbumin

Arginine can be made, still need to eat-conditionally non-essential, Histidine essential for infants

Synthesis of AA

-synthesis by amidation
-synthesis from aKG
-proline synthesis from glutamate
-derived from glycolysis intermediates
-from essential AA

Conditionally essential AAs

6 AAs

Arginine
Cysteine
Glycine
Glutamine
Proline
Tyrosine

essential under catabolic disease (PKU) and other conditions

AAs synthesized by amidation

glutamine (from glutamate)

asparagine (amide donor is glutamine)

Amidation for glutamine

prevention of hyperammonemia

-detoxification of ammonia in brain and liver

-nontoxic storage for NH3

-uses aKG to make Glu, glutamine synthetase for Gln

-Glutamate (Glu) down; Glutamine (Gln) up, NH3 up (during hyperammonemia)

(treatment: can be removed by phenylbutyrate (prodrug) to phenylacetate to phenylacetylglutamine)

glutamine synthetase

an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine

important in the brain

Overview: synthesis of non-essential AA

4 from glycolysis

Serine->Cysteine

Methionine (S)->Cysteine (deficiency is homocystinuria)

Phe->Tyrosine (deficiency is phenylketonuria (PKU))

Cysteine synthesis

from methionine (and serine)

Met cycle->SAM->SAH->homocysteine->cysteine

Folic acid (FH4) coupled with B12 can use methionine for other stuff (B12 convert homocystein->met). When knock out met goes only to homocysteine->cysteine. Overloads cysteine->negative feedback, blocks pathway and accumulates homocysteine

B6 two points: homocysteine->cystathionine and cystathionine->cysteine

If knock out accumulate homocysteine

The coenzymes in methionine metabolism

-pyriodoxal phosphate (PLP/B6)
-tetrahydrofolate (FH4)/B12

Amino acidopathy

-inability to metabolize (urea deficiency)
-inability to detoxify ammonia through urea cycle (AA problem)

Inherited diseases of AA metabolism

(Common)
Cystinuria
Histidinemia
Phenylketonuria

(Rare)
Homocystinuria
Alkaptonuria
Maple syrup urine disease

Aminoacidureas

Cystinuria
Histidinemia
Phenylketonuria
MSUD

Histidinemia

Genetic metabolic disorder of histidine
-histidine can't degrade to glutamine
-enzyme problem with histidase

Cysienuria

genetic disorder of renal AA transport. Cysteine metabolism defect

-AA->COAL (cysteine, ornithine, arginine and lysine; hexagonal kidney or bladder stones)

Phenylketornuria

Genetic metabolic disorder. detected by blood screening

Accumulation of phenylalanine and low tyrosine

CNS problems. Hypopigmentation

Aminoaciduria cont.

Methylmalonyl CoA mutase deficiency (similar to B12 deficiency)

Albinism (missing melanin due to mutations in several genes, including tyrosinase gene)

Alkaptonuria (mutations in homogenistic acid oxidase gene in the tyrosine degradation pathway. Urine turn black when exposed to air. Ochronosis, a buildup of dark pigment in connective tissues such as cartilage and skin)

Hartnup disease (mutations in the neutral AA transporter gene. Shows ataxia and also is associated with niacin deficiency)

Methylmalonyl CoA mutase deficiency

similar to B12 deficiency

Albinism

missing melanin due to mutations in several genes, including tyrosinase gene

Alkaptonuria

mutations in homogenistic acid oxidase gene in the tyrosine degradation pathway. Urine turn black when exposed to air.

Ochronosis, a buildup of dark pigment in connective tissues such as cartilage and skin

Hartnup disease

mutations in the neutral AA transporter gene.

shows ataxia and also is associated with niacin deficiency

Deficiencies in Phe to Tyr (PKU)

common inborn error (prevalence 1:15,000)
two major causes
-PAH deficiency (classic)
-BH4 deficiency (non-classic)

Tyr->fumarate (glucogenic)
Tyr->acetoacetate (ketogenic)

Classic PKU

-IEM

-PAH deficiency (phenylalanine hydroxylase)

-autosomal recessive

-1 in 13,500 to 19,00 births

-CNS deficiencies

--inhibition of LNAAT (large neutral AA transporter)

--elevated Phe (LNAA)->inhibit LNAA transporter (may saturate LNAAT)

--low LNAA is thought to cause intellectual disability

--phenylketones are high, clogged system or high ketones

-hypopigmentation (high phe inhibits tyr for melanin)

-Guthrie test for early diagnosis

PKU cont.

>400 mutations known in PAH gene

lifelong dietary restrict Phe

neonatal screening after 24-48 hours (not immediate)

diagnosis

-elevated serum concentration of phe

-useful lab method->tandem-mass spec (MS-MS), can identify IEM in a single sample

-confirm with 2nd plasma AA analysis

-enzyme analysis not performed to confirm diagnosis, since PAH expressed only in liver (baby liver too small)

Non-classic PKU

BH4 (tetrahydrobiopterin) deficiency, pleiotropic

Biopterin reductase mutation

Phe up, tyr down

impacts Tyr->DOPA and other catecholamines

Classic-tyrosine

Non-classic-serotonin or catecholines cause of biopterin

Homocystinuria

cystathionine beta synthase deficiency.

Low cysteine (low glutathione). High homocysteine (High H2O2, oxid stress).

Homocystinuria symptoms

-1:330,000 autosomal recessive

interferes with collagen cross linking

-dislocated lens

-elongated, thin, arachnodactyly

-scoliosis, osteoporosis

oxidative stress (ROS generated)

intellectual disability

similar but NOT marfan's (not tall and no aortic aneurysm)

Summary: homocysteinuria

high homocysteine

multiple causes:

-cystathionine beta synthase deficiency (most common)

-B12 or folic acid deficiencies lead to high cysteines, leading to feedback inhibition

increased oxidative stress:

-high homocysteine levels increase oxid stress

-low cysteine levels reduce ROS scavenging ability

interferes with collagen cross linking

increased risk of atherosclerosis and dementia

Recommended Uniform Screening Panel (RUSP)

List of conditions that all newborn screening programs should include. Established by United States Secretary of Health and Human Services.

Panel includes (not limited to):

-sickle cell disease

-critical congenital heart disease (most common)

-cystic fibrosis

-classical galactosemia

-severe combined immunodeficiencies

-phenylketonuria (AA problems)

-maple syrup urine disease