Transcript Amino Acids: Disposal of Nitrogen & Urea Cycle

AMINO ACIDS: DISPOSAL OF NITROGEN &
UREA CYCLE
Dr Vivek Joshi, MD
CONTENTS
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Amino acid pool
Protein Degradation
Amino acid degradation
Disposal of Body Nitrogen
 Transamination
 Deamination
 Transport of ammonia
OVERALL NITROGEN
METABOLISM
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Amino Acid pool
 Amino acids-Not stored in
the body.
 Maintain a supply of
amino acids-Amino acid
pool
PROTEIN TURNOVER-PURPOSE
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Affords metabolic flexibility
Protects cells from the accumulation of abnormal proteins
Plays an important role in numerous physiological processes
e.g. Eukaryotic cell cycle control/antigen presentation
Measured in half-lives
 Structural proteins (Collagen)typically have long half-lives
 Regulatory enzymes have half-lives that are typically
measured in minutes
 Proteins with Ser as N-terminal aa -Half life >20 h
 Proteins with shorter half life:
 Asp as N-terminal aa
 Rich in proline (P), glutamate (E),serine (S), and
threonine (T)
PROTEIN DEGRADATION
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Enzyme systems for degradation of
proteins
Energy dependent ubiquitinproteasome pathway
 Cytosolic pathway for
degradation of damaged
/unneeded proteins
 Important in degrading
proteins of intracellular origin
Non energy dependent degrading
enzymes of lysosmes.
 Way by which extracellular
and some intracellular
proteins are degraded
Ubiquitin - Proteasomes pathway
Present in all eukaryotic cells
Cytosolic proteins destined for
degradation are enzymatically
tagged with activated ubiquitin
Ubiquitin-tagged proteins are then
attacked by cytosolic ATPdependent proteases
Amino acid Degradation
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Regardless of the source ,aa not immediately incorporated into a
new protein are rapidly degraded.
AA catabolism involves:
 Removal of the a amino group as ammonia
 Conversion of the ammonia into urea
 Conversion of remaining AA carbon skeleton ( a-keto acid)
into TCA cycle intermediates
Disposal of Body Nitrogen
 Transamination
 Deamination
 Transport of Ammonia
TRANSAMINATION- INTRODUCTION
Most common reaction involving free amino acids
Reversible reaction
Transfer of amino group from an amino acid to a keto acid
to form a newer keto acid and a newer amino acid
respectively.
No release of ammonia
Major process for removing nitrogen from amino acids with
EXCEPTION- Lysine,Threonine,proline
Require Pyridoxal phosphate as an essential coenzyme
Transamination- Introduction
Transamination- General Reaction
Amino transferase/ Transaminase
Catalyses Transamination reaction
Located in the Cytosol and mitochondria of liver, muscle
 Each Aminotransaminases is specific for 1 pair of substrate but
non-specific for the other pair
Aminotransaminases are named after the specific amino group
donor
Major Aminotransaminases
#Aminotransaminases family
Conserve amino group of most of the amino acids
into Glutamate
#Alanine Aminotransaminases(SGPT)Alanine /Pyruvate conversion
#Aspartate Aminotransaminases(SGOT)Aspartate/Oxaloacetate conversion
Amino Transaminase Family
Family
Glutamate
All the amino nitrogen from amino acids that undergo transamination
can be concentrated in glutamate
The acceptor of the amino group is almost always a-keto glutarate
Alanine Amino Transaminase
Alanine-The principal amino acid released from muscle during
starvation
Pyruvate -Form an important substrate for hepatic
Gluconeogenesis.
Aspartate Aminotransaminases
Aspartate which is a source of nitrogen in the UREA cycle.
Oxaloacetate can form Glucose via Gluconeogenesis
DIAGNOSTIC VALUE OF PLASMA AMINOTRANSAMINASES
•Aminotransaminases- Intracellular enzymes with low
levels in plasma
•Elevated plasma amino transaminase- Cell Damage Diagnostic purpose
•Non hepatic diseases
Myocardial infarction and muscle disorders
Serum AST more specific than ALT
•Hepatic diseases
Serum ALT more specific than serum AST
Serum AST more sensitive than serum ALT
ALANINE TRANSAMINASE :INDICATOR OF HEPATIC DAMAGE
Transamination-Pair
Amino Acid
Keto Acid
Glutamate
a ketoglutarate
Alanine
Pyruvate
Aspartate
Oxaloacetate
Transamination- Pyridoxal phosphate (PLP)
AMINOTRANSAMINASES
REQUIRE PYRIDOXAL
PHOSPHATE (PLP) AS AN
ESSENTIAL COFACTOR
Deamination
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Removal of amino group as AMMONIA
Types
 Oxidative Deamination
 Oxidase- L . AA oxidase / D-AA oxidase
 Dehydrogenase –Glutamate Dehydrogenase
 Non Oxidative Deamination
 Hydroxy and Sulfur containing AA
 Catalysed by Dehydratase and Desulfurase
 PLP dependent
OXIDATIVE DEAMINATION BY GLUTAMATE DEHYDROGENASE
In mammals: almost entirely confined to the liver
mitochondrial matrix / Readily reversible
Glutamate –The only amino acid that undergoes rapid oxidative
deamination
Oxidative Deamination by Glutamate
Dehydrogenase
Amino Transaminase
ADP and GDP
Indicative of low cellular energy level
Stimulates glutamate degradation
ATP and GTP
Indicative of ample energy supply
Allosteric activator in the direction of
glutamate synthesis
Transamination is followed by
Deamination: Transdeamination
Glutamate Dehydrogenase
TRANSPORT OF AMMONIA
Nitrogen travels in blood mainly in amino acids,
particularly alanine and glutamine.
 Alanine and glutamine are synthesized in the
peripheral tissues to act as nitrogen carrier
 Glutamine
 Transport of ammonia from peripheral tissue to
liver
 Alanine
 Transport of ammonia from the muscle to
the liver
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Glutamine :Transport of ammonia from peripheral tissue to liver
Glutamine
Provides a non-toxic transport form of ammonia
Transport form of Ammonia from peripheral tissue to the liver
Synthesized by Glutamine synthase -Liver, Muscle and
Brain
Most common free amino acid in human blood plasma.
Major mechanism for detoxification of ammonia in the brain.
50% of circulating amino acid molecules are glutamine, an
ammonia transporter
Glutamine :Transport of ammonia from peripheral tissue to liver
Most of the tissues
TRANSAMINATION
NH3
GLUTAMATE
Glutamine
Synthase
GLUTAMINE
Liver ,Kidney and Intestine
GLUTAMINE
Glutaminase
NH3
GLUTAMATE
Glutamine to Glutamate :Liver, Intestine and Kidney
Glutamine to Glutamate :Liver
Glutamine to Glutamate :Liver
Most of the tissues
TRANSAMINATION
NH3
GLUTAMATE
Glutamine
Synthase
GLUTAMINE
Intestine
GLUTAMINE
Glutaminase
NH3
Portal Blood
GLUTAMATE
Another Source of NH3 in the intestine :Bacterial flora
Glutamine to Glutamate :Intestine
LIVER
Glutamine to Glutamate :Kidney
Glutamine is removed from circulation by the kidneys
Glutamine converted into Glutamate by Glutaminase releasing ammonia
Most of the ammonia is excreted in the urine as NH4+
An important mechanism for maintaining the body’s acid-base balance
Glutaminase
Glutamate
Dehydrogenase
Alanine :Transport of ammonia from muscle to liver
Alanine :Transport of ammonia from muscle to liver
Glucose –Alanine cycle
UREA CYCLE
UREA
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Major end-product of nitrogen catabolism in humans
Principal Non protein nitrogenous waste products (Uric
acid and Creatinine )
Synthesis- liver (Cytosol and mitochondria)
Released in the blood-Cleared by the kidneys
Urea Clearance-Measure of Glomerular filtration
rate(GFR)
UREA CYCLE
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First metabolic pathway to be discovered
Transdeamination of the AA results in release of
NH4 + in the liver
Ammonia is toxic and is converted into non toxic
,water soluble product –UREA.
Mammals are primarily Ureotelic
Birds and reptiles are Uricotelic
UREA
CYCLE
Urea Cycle
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Urea synthesis begins
with reaction of
ammonia with C02
(Bicarbonate) and ATP
to give Carbamoyl
phosphate
Reaction catalyzed by
Carbamoyl phosphate
synthetase I
Reaction requires Mg2+
Carbamoyl phosphate
synthetase II-Pyrimidine
synthesis
Carbamoyl phosphate synthetase-I
Catalyzes the rate-limiting step in urea cycle
Active only in the presence of the allosteric activator Nacetylglutamate(NAG)
N-Acetyl Glutamate is synthesized from Acetyl CoA and
Glutamate by N-Acetyl Glutamate synthase
Binding of NAG to CPS I induces a conformational
change that enhances the affinity of the synthase for ATP
Intrahepatic concentration of NAG increases after a
protein rich meal –Stimulates UREA synthesis.
Know CPS –I …….
UREA CYCLE
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Carbamoyl phosphate combines with ornithine to
form Citrulline –Ornithine trans Carbomylase(OTC)
Citrulline combines with Aspartate to form
Arginosuccinate-Arginosuccinate synthase
Arginosuccinase splits Arginosuccinate into Arginine
and Fumarate
Arginase split Arginine into Urea and Ornithine
Fate of UREA
Renal failure patients- Urease acts on urea-Important source of NH3
Oral neomycin administration-Reduces intestinal Bacteria-Decreased NH3
SUMMARY-UREA CYCLE
Overall Reaction:
Aspartate+NH3 +CO2 +3 ATP
Urea+Fumarate +2 ADP +AMP +2Pi +2 PPi +3 H2O
 4 High energy phosphates –Synthesis of each molecule
of urea
 Source of one nitrogen of urea-Free ammonia
 Source of second nitrogen of urea-Aspartate
 In effect, both nitrogen atoms of urea come from
glutamate, which in turn gathers nitrogen from other
amino acids.
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Summary-UREA cycle
Two nitrogen of the Urea comes from
# Ammonia
# Aspartate
AMMONIA
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Constantly produced in the tissues
Toxic to the central nervous system even in trace
amounts
Amino acids are quantitatively the most important
source of ammonia
Most of the ammonia generated in amino acid
degradation is produced by the oxidative
deamination of glutamate
DISPOSAL OF AMMONIA
Amines/Monoamines
GLUTAMATE
DEHYDROGENASE
Asparagine
L –AA Oxidase
Non –oxidative
Deamination
Ammonia pool
UREA
Glutaminase
Glutamine
Purines/Pyrimidines
Bacterial Urease(25%)
Urea – formation is quantitatively the most important disposal route for
ammonia
HYPERAMMONEMIA
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Normal Ammonia level -5–50
mol/L
Ammonia is normally
detoxified into Urea in the liver
Liver functions compromisedHyperammonemia
(1000mol/L)
Hyperammonemia-Medical
emergency
Hyperammonemia
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Ammonia-Directly Neurotoxic Tremors,slurring of speech and blurring of vision
 Coma and death at high levels
Brain is particularly vulnerable -Depends on the CAC to
maintain its high rate of energy production
Shift in the equilibrium of the glutamate dehydrogenase
reaction toward the direction of glutamate formation
Depletes a-ketoglutarate, an essential intermediate in CAC
 Results in a decrease in cellular ATP production
Hyperammonemia
Major types are:
Acquired hyperammonemia
# Liver diseases
# AcuteViral hepatitis,ischemia,
hepatotoxins
# Chronic- Cirrhosis of liver
Alcoholism,Hepatitis,Biliary
obstruction
(Porto systemic shunting)
Cirrhosis of the
liver
Formation of collateral circulation
around the liver
Portal blood is shunted
directly to the systemic
circulation (no access to
the liver)
Severely impaired
detoxification of ammonia
 Levels of circulating
ammonia
HYPERAMMONEMIA
Hereditary Hyperammonemia
# Inherited deficiency of the enzymes of Urea cycle
# Failure to synthesize urea leads to hyperammonemia during the
first week following birth
# Hyperammonemia Type I- Deficiency of CPS-I
# Hyperammonemia Type II -Deficiency of OTC
- 1;30,000 live births
- Most common of the inherited urea cycle disorders
- X-linked (Predominantly affecting males)
- All others urea cycle disorders are AR
- Presents typically with mental retardation ,few weeks after birth
#Treatment
- Limiting protein in diet
- Orally Phenyl butyrate -Converted to phenyl acetatephenylacetylglutamine -Excreted
- Gene Therapy
HYPERAMMONEMIA
Hyperammonemia I
Hyperammonemia II
CPS –I Deficiency
OTC Deficiency
Blood Glutamine increased
Blood Glutamine increased
BUN Decreased
BUN Decreased
No increase in Orotic acid and
uracil increased in blood
Orotic acid and uracil increased
in blood
Cerebral edema
Cerebral edema
Lethargy ,convulsions, coma ,death
Lethargy ,convulsions, coma ,death
UCD
Enzyme Deficiency
Type I
Hyperammonemia
Carbamoylphosphate
synthetase I
N-acetylglutamate
synthetase
Deficiency
N-acetylglutamate
synthetase
Type 2
Hyperammonemia
Ornithine
transcarbamoylase
Classic
Citrullinemia
Argininosuccinate
synthetase
Argininosuccinic
aciduria
Argininosuccinate lyase
(Argininosuccinase)
Hyperargininemia
Arginase
Symptoms/Comments
With 24h - 72h after birth infant becomes lethargic, needs
stimulation to feed, vomiting, increasing lethargy, hypothermia
and hyperventilation; without measurement of serum ammonia
levels and appropriate intervention infant will die: treament with
arginine which activates N-acetylglutamate synthetase
severe hyperammonemia, mild hyperammonemia associated
with deep coma, acidosis, recurrent diarrhea, ataxia,
hypoglycemia, hyperornithinemia: treatment includes
administration of carbamoyl glutamate to activate CPS I
Most commonly occurring UCD, only X-linked UCD, ammonia
and amino acids elevated in serum, increased serum orotic
acid due to mitochondrial carbamoylphosphate entering cytosol
and being incorporated into pyrimidine nucleotides which leads
to excess production and consequently excess catabolic
products: treat with high carbohydrate, low protein diet,
ammonia detoxification with sodium phenylacetate or sodium
benzoate
episodic hyperammonemia, vomiting, lethargy, ataxia, seizures,
eventual coma: treat with arginine administration to enhance
citrulline excretion, also with sodium benzoate for ammonia
detoxification
episodic symptoms similar to classic citrullinemia, elevated
plasma and cerebral spinal fluid argininosuccinate: treat with
arginine and sodium benzoate
rare UCD, progressive spastic quadriplegia and mental
retardation, ammonia and arginine high in cerebral spinal fluid
and serum, arginine, lysine and ornithine high in urine:
treatment includes diet of essential amino acids excluding
arginine, low protein diet