Lecture outline

Know the:

1.Urea Cycle: Reaction and Significance.

2.The Importance of the Cytosol and Mitochondria.
3.Enzyme Defect and Subsequent Clinical Diagnosis.

27th Mar 2012

Learning Objectives

II.Urea Cycle

A.Reactions of the urea cycle.

1.Synthesis of carbamoyl phosphate.
2. Production of arginine by urea cycle.
3. Cleavage of arginine to produce urea.

B. Origin of ornithine.

C. Regulation of the urea cycle.

D. Function of the urea cycle during fasting.

III. Disorders of the Urea Cycle.

http://www.wiley.com/college/fob/quiz/quiz20/20-8.html

What is Urea

Urea is a diamide, chief nitrogenous waste

product.

O
H2N

NH2

Urea

What are the Precursors of Urea

Ammonia +CO2 + Aspartate are the precursors
of Urea.

Other nitrogenous waste products which includes

uric acid & creatinine.

Summary of Amino Acid Metabolism

Dietary Proteins

Digestion (Stomach Intestine)

Amino Acids in Blood

Membrane
Proteins

Other
N-Containing
Compounds

Amino Acids

Carbon
CO2 + H2O
Energy ATP

Nitrogen
O
H2N-C-NH2

Urea

-Aminobutyrate
Dopamine
Norepinephrine
Epinephrine
Serotonin

Fate of Amino Acid Nitrogen: Urea Cycle

Amino Acids

Nitrogen

CO2+H2O

NH4+
+CO2

COO-

H C- NH2

COOAspartate

Storage

Carbon

Urea
Cycle

Energy
ATP
O
NH2 -C- NH2
Urea

What are the source of amino groups and carbonyl group for
the biosynthesis of Urea?.

Excretion of Nitogenous Waste Products

Amino acids
Most mammals convert
amino-acid nitrogen to
urea for excretion

Most terrestrial
vertebrates

NH4+

The carbon chains are

broken down to molecules
that feed into the TCA
cycle.
Some animals
excrete NH4+ or
uric acid.

Fish & other

aquatic
vertebrates

Birds & reptiles

O
O
H2N-C-NH2

Urea

NH4+

Ammonium
ion

Uric
acid

HN
O

N
H

H
N
N
H

An Overview of Urea Cycle

What Are The Sites & The
Organ In Which Biosynthesis
Of Urea Occurs?
Urea biosynthesis occurs partly
in the cytosol and partly in the
mitochondria of the liver.

Netreaction:
NH3+CO2+2ATP
(HCO3)

CarbamoylPhosphate
+
2ADP+Pi

Mitochondrial
Matrix

The Urea Cycle in the Liver

NH4+ + HCO3- + 2 ATP

Hepatocyte

Carbamoyl
phosphate synthase I (CPS-I)

1
Carbamoyl phosphate

Ornithine
Transcarbamoylase (OTC)

Ornithine

Citrulline

Citrulline
Argininosuccinate
Synthetase

Aspartate
Ornithine

ATP
AMP+ Pi

Argininosuccinate
Argininosuccinate
Lyase
Cytoplasm

4
Arginine

Fumerate

5
Arginase
Urea

What is Normal Value of Urea

Serum urea concentration is 15-40 mg/dL.
Accumulation of urea more than normal value in
blood is referred as
as Uremia.
Urea Excretion: 2030 gm / day.

Is there any Specific functions of Urea

Not yet identified, but probably may involve in
the maintenance of osmotic balance as a
crystalloid.

Osmolality of Plasma
Osmolality is a measure of the solute particles
present in the fluid medium.
Osmolality of Plasma: 285-295 milliosmoles / Kg.
Plasma Osmolality can be computed from the
concentrations (mmol/l) of Na+, K+, urea, & glucose
as follows.
2(Na+) + 2(K+) + Urea + Glucose
The factor 2 is used for Na+ & K+ ions for the
associated anion concentration.
Plasma Osmolality will be altered in lipidaemias, ESRD,
hyperproteinaemia, ketoacidosis, diabetes insipidus,chronic
alcohol intoxication, diuretics & in chronic diarrhoea.

Is any Clinical Conditions Are

related with Low Urea Level
Impairment of liver functions and congenital
deficiencies of urea cycle enzymes will impair the
biosynthesis of urea, which ultimately leads to
accumulation of ammonia in blood and CSF causes
azotemia.

What Is Meant By Azotemia

Accumulation of nitrogenous materials including
ammonia due to impairment in urea biosynthesis
due to,

Urea cycle enzyme deficiencies,

Gastrointestinal tract (GI) bleeding,

Valproic acid therapy and

Renal impairments ect.,.

What Are The Consequences of Uremia

Uremia
hypercapnia
neurotoxicity

will leads
& alkalosis
depending on

to ammonia toxicity,
which may leads to
the severity.

Impaired renal function leads to erythropoietin

deficiency and hence accumulation of CO2.

What Are The Causes of Uremia

Ureamia may occur as a result of impairment in

the renal functions, which occur as a result of

nephrotic syndrome,

heavy metal toxicities,

ESRD and

nitrogen load due to high protein diet.

Importance of Urea Cycle

Removal of CO2.

Removal of Ammonia.
Recycling of TCA cycle intermediates.
Recycling of amino acids & ketoacids.

Involvement of Peripheral Tissues And Liver in the

Ammonia Metabolism

-KG = -Ketoglutaratee
GDH = Glutamate Dehydrogenase.

Molecualr Interconversions in Handling of Ammonia

ALT

NH4+

NADH+H+

NADPH+H+

Glutamate
Dehydrogenase

NADPH

Pyruvate

Aspartate

-Ketoglutarate

Alanine

H2O

AST

NAD
Glutamate

Oxaloacetate

NH4+
Glutamine
Synthetase

ATP
ADP+Pi

Glutamine
The major enzyme responsible for the interconversion of Glutamate into -KG is
Glutamate Dehydrogenase. ALT= Alanine aminotransferase
AST= Aspartate aminotransferase.

Reactions of the Urea Cycle

1. Synthesis of Carbomoyl Phosphate.
2. Production of Arginine by the Urea Cycle.
3. Cleavage of Arginine to Produce Urea.

O
H2N

NH2

Urea

Most terrestrial land animals convert

excess nitrogen to urea, prior to excreting it.

Urea is less toxic than ammonia.
The Urea Cycle occurs mainly in liver.
The 2 nitrogen atoms of urea derived from
NH3 and the amino nitrogen of aspartate.

HCO3 + NH3 + 2 ATP

O
H2N

Carbamoyl Phosphate
Synthase-I
OPO32 + 2 ADP + Pi

Carbamoyl phosphate

Carbamoyl Phosphate Synthase is the committed step of the

Urea Cycle, and is subject to regulation.
The NH3 and HCO3- that will be part of urea are
incorporated first into carbamoyl phosphate (CP).

Glutamate (Glu)
H
H3N+

N-Acetylglutamate
O

COO

CH2

H3C

H
N
H

COO

CH2

CH2

CH2

COO

COO

Carbamoyl Phosphate Synthase-I has an absolute

requirement for an allosteric activator
N-acetylglutamate.
This derivative of glutamate is synthesized from
acetyl-CoA & glutamate when cellular [glutamate] is
high, signaling an excess of free amino acids due to
protein breakdown or dietary intake.

Formation & Degradation of N-Acetylglutamate

(NAG)

NAG

NAG

N-Acetyl glucoseamine (NAG) serves as an allosteric

activator for the enzyme CPS-I for the biosynthesis of

carbamoyl phosphate (CP) with the help of HCO3- and ATP.

NAG, is an allosteric activator of CPS-I.

The Reactions
of the Urea
Cycle
NAG:N-acetyl
glutamate; (in the
formation of urea,
one amino group is
derived from free
NH4+ ion, while the
other is from
aspartate. Carbon is
obtained from CO2.
(*) mitochondrial
enzymes, the rest
of the enzymes are
cytosolic).

Mitochondria

Cytosol

NAG

Role of Glutamate in Urea Production

Amino acids

-Ketoglutarate

Transamination
-Ketoacids

Other reactions

Glutamate

Transamination
-Ketoglutarate

Oxalaoacetate
NH4+
Aspartate

Urea
Cycle

Urea

Role of Glutamate in Amino Acid Synthesis

-Ketoglutarate
Transamination

PLP

GDH

Glutamate
-Ketoacid
PLP
Amino acids
-Ketoglutarate
Glutamate transfers Nitrogen by means of transamination
reactions to ketoacids to form AAs. The nitrogen is
obtained by glutamate either from transamination of other
AAs or from NH4+ by means of the glutamate dehydrogenase

COO

COO

COO

CH2

COO

CH2

CH2

CH2

CH2

CH2

HC

NH3+

COO

COO

COO

HC

NH3+

COO

Aspartate -Ketoglutarate Oxaloacetate Glutamate

Aminotransferase (Transaminase)
Oxaloacetate is converted to aspartate via
transamination (e.g., from glutamate).
Aspartate then reenters Urea Cycle, carrying an amino
group derived from another amino acid.

Regulation of the Urea Cycle

Liver has a vast capacity
,thereby preventing toxic

to convert AA nitrogen to urea

effects from ammonia.

Urea cycle is regulated by substrate (S) availability;

the higher the rate of NH3 production, the higher the
rate of urea formation.
Regulation by the S availability is a general
characteristics of disposal pathways, such as the urea
cycle, which remove toxic compounds from the body.
This is a type of feed-forward regulation, in contrast
to the feed-back regulation characteristic of
pathways that produce functional endproducts.

Regulation of the Urea Cycle

The other type of regulation control the urea cycle:

Allosteric activation of CPS-I by N-acetylglutamate
(NAG) and induction/repression of the synthesis of urea
cycle enzymes.
NAG is formed specifically to activate CPS-I; it has no
other known function in mammals.
The synthesis of NAG from acetyl CoA and glutamate is
stimulated by arginine.
Thus, the arginine level
important reactions are

increase within the liver, 2

stimulated.

The first is the synthesis of NAG, which will increase

the rate at which CP is produced.

D. Functions of Urea Cycle During Fasting

During fasting, the liver maintains blood glucose by
gluconeogenesis by utilizing muscle protein AAs as a
carbon source.

As AA carbons are converted to glucose, the nitrogens

are converted to urea, thus the urinary excretion of
urea is high during fasting.
As fasting progresses, the brain begins to use ketone
bodies, sparing blood glucose.
Less muscle protein is cleaved to provide AAs for
gluconeogenesis,and decreased production of glucose
from AAs for gluconeogenesis.
Hence, Decreased production of glucose from AAs is
accompanied by decreased production of urea.

D.Functions of Urea Cycle During Fasting

The major AA substrate for gluconeogenesis is Alanine,
which is synthesized in peripheral tissues to act as a
nitrogen carrier.
Glucagon release, which is expected during fasting,
stimulates alanine transport into the liver by activating
the transcription of transport systems for alanine.
Two molecules of alanine are required to generate one
molecule of glucose.
The nitrogen from 2 molecules of alanine is converted to
one molecule of urea.

 Hereditary deficiency of any of the Urea Cycle

enzymes leads to hyperammonemia - elevated
[ammonia] in blood.
Total lack of any Urea Cycle enzyme is lethal.
Elevated ammonia is toxic, especially to the brain.
If not treated immediately after birth, severe
mental retardation results.

Genetic Deficiency of Urea Synthesis

May be caused by,
1. Ornithine Transcarbamylase Deficiency (OTC)
2. Citrullinemia.
3. Arginase Deficiency
4. Argininosuccinic Aciduria
5. Carbamyl Phosphate Synthetase (CPS-I) Deficiency
6. N-Acetyl Glutamate Synthetase Deficiency (NAGS)

Genetic Deficiency of Urea Synthesis

Carbamoyl Phosphate Synthetase-I
(CPS-I) Deficiency

[NH4+]; hyperammonemia

Ornithine
Transcarbamylase
(OTC)
[NH4+];
hyperammonemia

Blood Glutamine
BUN is decreased

Blood Glutamine
BUN is decreased

No increase in uracil or orotic acid

Uracil & orotic acid in

blood & urine

Cerbral edema
Lethargy, convulsions, coma,
death

Cerebral edema
Lethargy, convulsions,
coma, death

Metabolic Diseases of the Urea Cycle

Disorders present in infants:
Symptoms: Lethargy, swelling of the brain leads to mental
retardation / brain damage.
Diagnosis: Low blood urea nitrogen (BUN) levels -high levels
of ammonia in the blood elevated circulating glutamine
-other metabolites that accumulate depend on the specific
enzyme defect.
Most common form: Hyperammonemia Type II caused by
Ornithine Transcarbamylase (OTC) Deficiency elevated
Carbamoyl-Phosphate (CP) levels in this deficiency cause
secondary problems in pyrimidine metabolism.

Treatment:
Long term, dietary restriction.
Low protein diet. Supplemented with Arginine.
Short term
Dialysis.
Administration of Nitrogen scavengers.
e.g. Phenylacetate.

Treatment of Hyperammonemia with Phenylacetate:

taking advantage of metabolism
Phenylbutyrate

Glycine + Benzoic Acid

Hippuric Acid
Excreted in Urine

1.Which biomolecule acts as a positive modulator for the first step

of the urea cycle which is catalyzed by carbamoyl phosphate
synthetase-I (CPS-I).
A. Gultamate.
B. Glutamine.
C. N-Acetyl Glutamate (NAG).
D. Aspartate.
E. Ammonium ions (NH4+).
Ans: C.

7. Which of the following is a common compound shared by The

TCA cycle and the urea cycle?
A. -ketoglutarate.
B. Succcinyl Coenzyme A (CoA).
C. Oxaloacetate.
D. Fumarate.*
E. Arginine.
Ans: D.

8. Which of the following enzymes requires adenosine

triphosphate (ATP) to mediate its reactions?
A. Argininosuccinate lyase.
B. Argininosuccinate synthetase.
C. Arginase.
D. Glutaminase.
E. Ornitine transcarbamoylase.
Ans: B.

10. Two days after a full-term normal delivery, a neonate begins

to hyperventilate, develops hypothermia and cerebral edema,
and becomes comatose. Urinalysis reveals high levels of
glutamine and orotic acid. The BUN is below normal. Which
enzyme is most likely to be deficient in this child?
A. Cytoplasmic glutaminase.
B. Cytoplasmic carbamoyl phosphate synthetase.
C. Cytoplasmic orotidylate decarboxylase.
D. Mitochondrial carbamoyl phosphate synthetase.
E. Mitochondrial ornitihine transcarbamoylase.
Ans: E.

11. Transamination reactions are essential for ammonia

assimilation. What cofactor is required to catalyze
transamination reactions?
A. pyridoxal phosphate (PLP).
B. thiamin pyrophosphate.
C. biotin.
D. NAD+.
E. NADPH.
Ans: A.

12. Which of the following enzymes function in the biological

assimilation of ammonia?
A. Glutamate dehydrogenase.
B. Glutamine synthetase.
C. Glutamate synthase.
D. All of the above.***
E. None of the above.
Ans: D.

A 55-year-old man suffers from cirrhosis of the liver. Toxins such

as ammonia are not properly metabolized by the liver and can
now damage structures such as the brain. Which of the following
amino acids covalently binds ammonia and transports and stores
it in a nontoxic form?
A.
B.
C.
D.
E.

Aspartate.
Glutamate.
Serine.
Cysteine.
Histidine.

Ans: B.

14. Which amino acid participates as a pathway intermediate in

the urea cycle?
A.
B.
C.
D.
E.
Ans: B

Lysine.
Arginine.***
Glutamine.
Histidine.
Tyrosine.

Urea Cycle Must Know Key Points

1. Urea cycle occurs partly in .. and partly in the .
Ans: Mitochondria, Cytosol / Cytoplasm.
2. The rate-limiting step (committed step) in the urea cycle is
catalyzed by the enzyme
Ans: Carbamoyl phosphate synthetase-I (CPS-I).
3. The biomolecule acts as a positive modulator in the first step of
the urea cycle is
Ans: N-Acetyl glutamate (NAG).
4. .can be converted to arginine by a series of reactions,
some of which requires urea cycle enzymes.
Ans: Glutamate.

5. The enzymes are responsible for producing the direct donors of

nitrogen into the pathway producing urea, include . And
..
Ans: Aspartate aminotransferase and carbmoyl phosphate
sythetase.
6. The accumulation of glutamine and orotic acid in the blood and
an ultimate ammonia toxicity in the newborn is due to the
deficiency of the urea cycle enzyme.
Ans: Mitochondrial ornitihine transcarbamoylase (OTC).
7. The common nitrogen acceptor for all reactions involving
transaminase is
Ans: -ketoglutarate.
8. The common nitrogen donor for all reactions involving
transaminase is
Ans: Oxaloacetate (OAA).

9. The common compound shared by the TCA cycle and the urea cycle is
Ans: Fumarate.
10. Hyperammonemia caused by a congenital defect of the urea cycle
enzyme..which is characterized by accumulation of excess amount of arginine
in the blood.
Ans: Arginase.
11. The urea cycle enzyme which requires adenosine triphosphate (ATP) to mediate its
reactions is ?.
Ans: Argininosuccinate synthetase.
12. The reaction catalyzed by an urea cycle enzyme ornithine aminotransferase which
requires --------- as a coenzyme, which converts ornithine and -ketoglutarate into
glutamate.
Ans: Pyridoxal phosphate (PLP).

Match the Urea Cycle Disorders With the

Enzyme Defect
No Disorder

Answer Enzyme Defect

Hyperammonemia type I

A. Argininosuccinate synthase

Hyperammonemia type II

B. Ornithine transcarbomylase (OTC)

Citrullinemia

C. Argininosuccinate lyase

Argininosuccinic Aciduria

D. Arginase

Hyperargininemia

E. Carbamoyl-P synthase-I (CPS)

Answer: 1E; 2-B; 3-A; 4-C; 5-D

Genetic Deficiencies in Some of the Urea Cycle Enzymes can be Treated

Pharmacologically by eliminating the amino acids such as glycine and glutamine by
administering,
A. Aspartic acid.
B. Benzoic acid and phenylacetate.
C. GABA.
D. Pyridoxal phosphate.
E. Methionine.
Ans: B. The amide products of these reactions (hippurate and
phenylacetylglutamine) are excreted in the urine. Synthesizing the Gly or Gln
removes ammonia.

The Reaction Catalyzed by Glutamate Dehydrogenase which

reversibly converts glutamate to -ketoglutarate require the
cofactor,
A. ATP
B. NAD
C. NAD(P)+ /NAD(P)H.
D. Biotin
E. Pyridoxal phosphate.
Ans: C. The cofactors require by the glutamate dehydrogenase
is NAD(P)+ /NAD(P)H.