Kwame Nkrumah University of

Science & Technology, Kumasi, Ghana

TRANSPORT OF AMMONIA

Dr Max Efui Annani-Akollor

Dept of Molecular Medicine

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 Presentation Outline
 Introduction
 Ammonia Toxicity
 Nitrogen excretion
 Urea Synthesis
 Urea cycle
 Link between Ornithine and Urea cycles
 Inherited enzymatic defects of the Urea cycles

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 Lecture Objectives
Explain how excess ammonia from peripheral tissues is transported to the liver
Explain how ammonia could be toxic
Describe the urea cycle
Know defects associated with the urea cycle and their outcomes

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 ASSIMILATION OF AMMONIA IN THE BODY
• Reduced N enters the human body as dietary free amino acids, protein and as
ammonia produced by intestinal tract bacteria.

• Principal enzymes, glutamate dehydrogenase and glutamine synthatase, are

found in organisms and effect the conversion of ammonia into the amino acids
glutamate and glutamine, respectively.

• Amino and amide groups from these 2 substances are freely transferred to other
C skeletons by transamination and transamidation reactions.

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 REMOVAL OF N FROM
AMINO ACIDS:
TRANSAMINATION

•Transamination is the process by which an amino group, usually from glu, is

transferred to an -keto acid, forming the corresponding aa plus -KG.
•Transamination provides a route for redistribution of aa N and funnels N from all
free aa into a small number of cpd’s
•These cpd’s are then, either oxidatively deaminated, producing ammonia, or
their amine groups are converted to urea by the urea cycle .
•Transamination reactions are catalyzed by transaminases (aminotransferases).
•Aminotransferases utilize a coenzyme - PLP - which is derived from vit B6
•Aminotransferases exist for all aa acids except thr and lys.
GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHASE REACTION

The Glutamate Dehydrogenase Reaction

The Glutamine Synthase Reaction

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 GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHASE REACTION
•Liver contains both glutamine synthetase and glutaminase which are localized
in different cellular segments.
•The differences in cellular location of these two enzymes allows the liver to
scavenge ammonia that has not been incorporated into urea.
•Urea cycle enzymes are located in the same cells as those that contain
glutaminase.
•Differential distribution of these two hepatic enzymes enables the liver to
control ammonia incorporation into either urea or glutamine.

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 GLUTAMATE DEHYDROGENASE AND GLUTAMINE SYNTHASE REACTION

•Glutamine is the major carrier of NH3 from peripheral tissues to the kidney.
•When acidosis occurs the body will divert more glutamine from the liver to the
kidney. This allows for the conservation of bicarbonate ion since the
incorporation of ammonia into urea in the liver requires bicarbonate.
•When glutamine enters the kidney, glutaminase releases one mole of
NH3ammonia generating glutamate and then glutamate dehydrogenase releases
another mole of ammonia generating -ketoglutarate.
•The ammonia will ionizes to ammonium ion (NH4+) which is excreted. The net
effect is a reduction in the pH.
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TRANSPORT OF AMMONIA TO THE LIVER FOR UREA SYNTHESIS.

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 AMMONIA TRANSPORT
•Ammonia is quite toxic to mammalian tissue
•In peripheral tissues excess ammonia is converted to non toxic compounds
before transport to the liver and kidney
•In many peripheral tissues, ammonia is converted to glutamine in a reaction
catalyzed by glutamine synthetase
•Glutamine is then transported in the blood to the liver where it is converted to
glutamate by glutaminase in the mitochondria

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 AMMONIA TRANSPORT
•In the skeletal muscle, ammonia is transported as alanine via the alanine-
glucose cycle
•Alanine is transported in the blood to the liver where it is converted to pyruvate
and glutamate in a transamination reaction
•Glutamate may be converted to urea where as pyruvate may be converted to
glucose via gluconeogenesis
•The glucose may be transported to the skeletal muscle for its contractile activity
•The alanine-glucose cycle thus imposes the burden of gluconeogenesis on the
liver rather than the muscle
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 AMMONIA TOXICITY
•Ammonia is toxic to mammalian cells for the following reasons:
•Accumulated ammonia reverses the glutamate dehydrogenase reaction thus
depleting the cell of α-ketoglutarate
•This will lead to depressed synthesis of ATP which will thus affect active
transport systems.
•Impaired active transport will lead to loss of electrochemical gradient which is
paramount to the transfer of impulses.
•The accumulation of ammonia may also impede the synthesis of GABA which is
a neurotransmitter.
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 AMMONIA TOXICITY
•This results in further impairment of nerve conduction.
•The accumulated ammonia results in the increased synthesis of glutamine
which may lead to encephalopathy
•Accumulated ammonia may also result in acid-base disturbances
•It may also inhibit amino acid transport and the Na+-K+ ATPase system

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 ALANINE-GLUCOSE CYCLE
•Liver accumulates plasma alanine, reverses the transamination that occurs in
muscle and proportionately increases urea production
•The pyruvate is either oxidized or converted to glucose via gluconeogenesis.
•When alanine transfer from muscle to liver is coupled with glucose transport
from liver back to muscle, the process is known as the glucose-alanine cycle

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 ALANINE-GLUCOSE CYCLE
There are 2 main pathways to production of muscle ala
1) Directly from protein degradation

2) Transamination of pyruvate by alanine transaminase, ALT (also referred to as serum

glutamate-pyruvate transaminase, SGPT).

•Ala is 2nd only to glutamine in prominence as a circulating aa in blood

•Serves a unique role in the transfer of N from peripheral tissue to liver.

•Ala is transferred to the circulation mainly by muscle, in which ala is formed

from pyruvate at a rate proportional to intracellular pyruvate levels
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ALANINE-GLUCOSE CYCLE

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 NITROGEN EXCRETION
•Amino nitrogen may be excreted as ammonia in most aquatic species such as
the bony fishes
•It may be excreted as urea by terrestrial animals
•It may also be excreted as uric acid by birds and reptiles
•These forms of life may thus be referred to as ammonotelic, ureotelic and
uricotelic animals

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 Urea Cycle
•Reactions of the urea cycle occur in both the
mitochondria and cytosol of liver cells.
•Glutamate dehydrogenase, the citric acid cycle enzymes
carbamoyl phosphate synthetase I, and ornithine
transcarbamoylase are localized in the mitochondrion,
whereas the rest of the cycle occurs in the cytosol.
•This means that ornithine must be transported into
mitochondria, and citrulline must be exported to the
cytosol, in order for the cycle to proceed.
•Following synthesis, urea is transported in the
bloodstream to the kidneys, which filter it for excretion.
•Measurements of blood urea nitrogen (BUN) levels
provide a sensitive clinical test of kidney function,
because filtration and removal of urea are impaired in
cases of kidney malfunction.
•Blood ammonia measurements are a sensitive test of
liver function

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 UREA SYNTHESIS
The main site of ureogenesis is the liver - consists of five enzymatic steps
•Step I is the synthesis of carbamoyl phosphate by carbamoyl phosphate
synthetase I (ammonia)
•This reaction occurs in the mitochondria
•This reaction requires N-acetyl glutamate (AGA) as an allosteric activator
•This activator is synthesized from ACoA and Glu by AGA synthase when protein
intake is high
•AGA synthase activity is enhanced by [arginine].

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•Step II- carbamoyl phosphate reacts with L-ornithine to form L-citrulline in a
reaction catalysed by ornithine transcarbamoylase
•This reaction is driven forward by the orthophosphate cleavage
•This reaction occurs in the mitochondria
•Citrulline diffuses from the mitochondria into the cytosol

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•Step III- Asp then condenses with citrulline in the cytosol →argininosuccinate
•This reaction is under the catalysis of argininosuccinate synthetase and is
driven forward by the pyrophosphate cleavage
•Step IV- Argininosuccinate is then converted to fumarate and arginine by
argininosuccinate lyase
•Step V- Arginine is then converted to Urea and ornithine by arginase
Overall reaction for Urea synthesis

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 THE LINK BETWEEN ORNITHINE AND CITRIC ACID CYCLE

•Fumarate produced in the argininosuccinate lyase reaction is transported into

the mitochondria
•In the mitochondria, fumarase and malate dehydrogenase convert it ultimately
into oxaloacetate
•Oxaloacetate has the following metabolic fates
– condense with acetyl CoA and committed into the TCA cycle
– converted to glucose
– converted to aspartate which is an important nitrogen donor in the urea cycle

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 INHERITED ENZYMATIC DEFECTS OF UREA CYCLE
•Infants born with a complete deficiency of urea synthesizing enzymes die soon
after their first protein meal
•Those with partial deficiency of the enzymes may however survive to early
adulthood or later if managed appropriately
•The symptoms of hyperammonaemia include lethargy, stupor, convulsions and
CNS impairment – in severe cases coma and death may result.
•Patients with inherited defects of urea synthesizing enzymes cannot tolerate
protein-rich diets
•They are however provided with diets containing the α-keto analogues of the
essential amino acids
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 REGULATION OF THE UREA CYCLE

•The synthesis of CP and the urea cycle are dependent on the presence of
Nacetylglutamic acid (NAcGlu), which allosterically activates CPS1.
•NAcGlu is an obligate activator of CPS.
• Synthesis of NAcGlu by N-acetylglutamate synthase (NAGS) is stimulated by
both Arg, allosteric stimulator of NAGS, and Glu, a product in the transamination
reactions and one of NAGS's substrates, both of which are elevated when free aa
are elevated.
•Glu not only is a substrate for NAGS but also an activator for the urea cycle.
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 REGULATION OF THE UREA CYCLE

•Glucagon, insulin, and glucocorticoids are major regulators of the expression

of urea cycle enzymes in liver. In contrast, the "urea cycle" enzymes in non-
hepatic cells are regulated by a wide range of pro- and anti-inflammatory
cytokines and other agents.
•Regulation of these enzymes is largely transcriptional in virtually all cell types.

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DEFECTS OF UREA CYCLE

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