Thinking Critically Exercise #6

Role of Extrahepatic Tissue in Arginine Synthesis

FN 4120 - Human Metabolism
March 26th 2018
 1. What role does the urea cycle play in the synthesis of non-essential amino acids?
Include a diagram and explanation of the urea cycle in your answer.

The urea cycle has 5 steps that cycle three non-essential amino acids, ornithine, citrulline and
arginine. The cycle’s predominant function is to remove toxic by products of protein catabolism
from the blood (nitrogen containing ammonia) and produce urea which can be readily excreted
by the kidneys.

The Urea cycle proceeds as follows:

1. ammonia combines with CO2 to form carbamoyl phosphate using carbamoyl phosphate
synthetase I
2. Ornithine and carbamoyl phosphate form citrulline through the action of ornithine
transcarbamoylase (OTC)
3. Aspartate reacts with citrulline through argininosuccinate synthetase to form aginino
succinate
4 Argininosuccinase is cleaved into arginine and fumarate through argininosuccinase
5. Arginase activity forms urea which can be transported to kidneys for excretion and regenerates
orthinine to continue the cycle and combine with carbamoyl phosphate.. The urea cycle is
connected with other metabolic reactions, like gluconeogenesis (Diagram 1) and the TCA cycle
via transamination of glutamate to oxaloacetate to produce aspartate and by other intermediaries
such as fumarate, CO2 production.

Diagram 1
Metabolic integration of nitrogen metabolism (adapted from Stryer, 2007). The urea cycle,
gluconeogenesis and the transamination of oxaloacetate are linked by fumarate and aspartate.
Arginase is one of the five enzymes involved in the urea cycle, the others being ornithine
carbamoyltransferase , argininosuccinate synthetase, argininosuccinate lyase and carbamoyl
phosphate synthetase. These enzymes are also expressed in extra hepatic tissues and are
involved in the biosynthesis of non-essential amino acids and polyamines. Arginase is the most
widespread of these enzymes. The kidney is the most important extrahepatic tissue for
endogenous arginine synthesis, where it is metabolised from citrulline (Melis et Al., 2005).
Citrulline is predominantly metabolised in the intestine from glutamine or arginine (Melis et Al.,
2005). Arginase enzymes are present in kidney and to a lesser extent, brain, mammary glands,
testes, and skin in mammals (Jenkinson et Al., 1996). Of these tissues, activity of the kidney are
the most important, as well as mammary glands during lactation. Extra-hepatic arginase
enzymes are isozymes of the hepatic enzymes, they are involved in the biosynthesis of proline,
glutamate, glutamine, arginine and ornithine (Mezl & Knox, 1977; Jenkinson et Al., 1996; Melis
et Al., 2005).

In summary, The primary function of the urea cycle is to remove ammonia from the blood. This
is especially important during times of surplus and when there is protein catabolism. The liver
therefore plays a central role in amino acid metabolism. It removes ammonia in the blood
through a series of reactions mediated by enzymes. These enzymes are also expressed in other
tissues and play a role in the synthesis of amino acids thereby making them non-essential and
contributing to amino acid metabolism. In the intestine, citrulline is produced through enzymatic
activity ​of ornithine aminotransferase (OAT), carbamoyl phosphate synthetase I (CPS-I), and
ornithine transcarbamoylase (OTC), which results in a net synthesis of citrulline . Citrulline can
then be transported via the circulation to the kidney where it can be converted to arginine.
Glutamine can also be used by intestinal cells to synthesize proline, and urea cycle amino acids:
ornithine, citrulline and arginine (Bertolo & Burrin, 2008). Thus, ​the intestine plays a role in
digestion and absorption of nutrients, along with amino acid synthesis. Similarly, the kidney
also plays a role in amino acid synthesis through expression of the enzyme a​rgininosuccinate
synthetase ​a that are involved in the urea cycle. Arginine is therefore a non-essential amino acid.
During fetal growth characterized by rapid growth and underdeveloped metabolic pathways, or
in diseases where the amino acid metabolic enzymes are deficient, it becomes conditionally
essential where supplementation in the diet is required.

In short, the urea cycle has an important function in recycling carbon skeletons and efficiently
disposing of ammonia, the process takes energy but also produces energy (from carbon
skeletons). The liver takes up glutamine, and glutaminase releases glutamate and ammonia,
some ammonia escapes urea synthesis (a low affinity, high capacity detoxification system), these
substrates are taken up and used for glutamine synthesis. In contrast, glutamine synthesis is a
high affinity/low capacity system, which allows the liver to salvage nitrogen when protein is
restricted and urea synthesis needs to be restricted (Berg, Stryer, Tymozcko, 2007). Extra-hepatic
isoforms of urea cycle enzymes are also involved in the biosynthesis of non-essential amino
acids (proline, glutamate, glutamine, arginine, ornithine). In a wider context, the urea cycle is
integrated into the complete synthesis of all non-essential amino acids when considering the
main energy generating pathways of human metabolism. Glutamate in particular is especially
involved in both the urea cycle and the biosynthesis of non-essential amino acids.
Diagram 3- adapted from Jenkinson et Al. “Reactions and enzymes of the urea cycle and
associated metabolic pathways.” “The urea cycle reaction pathways are indicated by bold arrows.
Enzymes responsible for catalysing principal reactions of the cycle and associated pathways are
numbered. The five key urea cycle enzymes are circled and in bold. Small circles (nearly
impossible to see in reproduced diagram, see hand-drawn diagram) between the two walls of the
mitochondrion represent carriers for glutamate, ornithine and citrulline. The enzymes are 1,
carbamoyl phosphate synthetase I; 2, ornithine carbamoyltransferase; 3, argininosuccinate
synthetase; 4, argininosuccinate lyase; 5, arginase; 6, N-acetylglutamate synthetase; 7, ornithine
aminotransferase; 8, ornithine decarboxylase (cytosolic); 9, transaminase (to
alpha-ketoglutarate); 10, glutamate dehydrogenase; 11, transaminase (to oxaloacetate); 12,
glycine transamidinase; 13, nitric oxide synthase.” (Jenkinson et Al., 1996).

2. Why did citrulline and arginine remain low after liver transplantation?
Citrulline and arginine remain low after liver transplant because the enzymes that catalyze their
synthesis are expressed in other tissues (extrahepatic tissues), specifically the small intestine and
kidney.
Ornithine transcarbamoylase (OTC) -
● Present in the mitochondria of the liver and the intestinal mucosa.
● Catalyzes synthesis of citrulline from ornithine and carbamoyl phosphate which
allows for the removal of toxic ammonia from the blood and is essential in the
urea cycle to convert nitrogenous waste in hepatocytes to urea which can then be
excreted from the body primarily through the kidneys. Citrulline is also essential
to the arginine synthesizing system.
● With OTC deficiency, citrulline production in the small intestine is limited.
● Replacing the liver does not improve the guts ability to produce enzymes that are
lacking before transplant.
● Citrulline is an intermediate in the urea cycle and reacts with aspartate to form
argininosuccinate which then forms arginine through cleavage from
arginosuccinase activity. Thus low levels of citrulline from OTC deficiency in the
small intestine also results in low arginine levels.(Rabier et.al., 1991).

Argininosuccinate synthetase I (ASSI) -

● This enzyme is ubiquitously expressed across tissue types and is involved in both the
production of urea in the liver and nitrous oxide in other tissues from the condensation of
aspartate and citrulline to form the arginine precursor: argininosuccinate.
● ASSI is involved in arginine production for the whole body from either intestine or
kidney. Citrulline is synthesized in the small intestine and further taken up by the kidney
to be synthesized to arginine via ASSI.
● While citrulline is still synthesized in the small intestine in the presence of ASSI
deficiency, it cannot be synthesized in the kidneys to produce arginine.
● With a liver transplant, citrulline will be exclusively used in the urea cycle and therefore,
citrulline will be insufficient in the small intestine and kidneys.
● Thus liver transplantation will only impact the urea cycle and the removal of ammonia
from the blood to produce urea. The kidney and the intestinal tissues will still be deficient
in this enzyme and as a result, arginine levels will still be low. (Rabier et.al., 1991).

While arginine is a non-essential amino acid, with the above deficiencies, arginine becomes
essential because it’s synthesis is substantially limited by the defective arginine synthesizing
system.
3. What is the purpose of the conventional diet/supplement therapies used prior to liver
transplantation? Would you make any specific dietary recommendations for these patients
who had undergone successful liver transplantation? If so, explain.

Maintain positive nitrogen balance (avoid catabolism) while minimizing ammonia production
1. Protein restriction -​ proteins, when ingested by the diet, are broken down into amino
acids by digestion and are efficiently absorbed by the GI tract. Most of these amino acids
are taken up by the liver. Catabolism of amino acids occurs immediately after eating and
metabolism shifts according to the needs of the body. Excess amino acids are catabolized
which involves the removal of the nitrogen containing amino group through deamination.
Because the amino group is not required for synthesis (there is an excess), the amino
group is released as ammonia (NH3). Some foods with proteins also contain their own
sources of ammonia (cheeses, processed meats). Amino acids that are not absorbed by the
enterocytes are also digested in the GI tract by bacteria which produce ammonia that
subsequently enters the bloodstream. Ammonia is toxic to cells and in order to remove it
from the body, the urea cycle which occurs in the liver is necessary. If enzymes in the
urea cycle are deficient (such as in the case of OTc and ASS), the urea cycle can’t
proceed (ornithine can’t react with carbamoyl phosphate and aspartate can’t react with
citrulline respectively), then ammonia build up occurs in blood. By reducing the amount
of protein and therefore amino acids, the likelihood that amino acids will be used for
protein synthesis and generation of non-essential amino acids is greater thereby shuttling
amine groups among carbon skeletons and avoiding ammonia accumulation in the blood.
It is important to also avoid protein catabolism, thus a diet sufficient in calories and
protein to prevent catabolism is also necessary. Avoidance of catabolic stress:
dehydration, starvation, infection is also a helpful strategy.
2.

Supplementation with deficient amino acids (​arginine)-

Amino acid synthesis and essentiality
Due to deficiency of the OTC and ASS enzymes, amino acid synthesis in extra-hepatic tissues is
affected rendering arginine and citrulline in these patients with these metabolic conditions ,
conditionally essential (requiring exogenous source). In the intestine, ornithine and carbamoyl
phosphate are catalyzed by ornithine transcarbamoylase to produce citrulline. When this enzyme
is deficient, citrulline is not produced and can’t be transported to the kidney for conversion to
arginine. Thus citrulline or arginine must be supplemented. When ASS is deficient, citrulline
can’t be converted to arginine in the kidney.
Urea cycle is also affected by deficiency in these substrates
Arginine is an intermediate in the urea cycle which produces both urea (to be excreted by
kidneys) and ornithine. Ornithine is necessary to react with carbamoyl phosphate formed from
the condensation of ammonia from the blood and CO2. Arginine also induces the synthesis of
NAG which is an allosteric activator in the first step of the urea cycle allowing ATP binding to
form carbamoyl-PO4. If arginine is deficient, the urea cycle can’t proceed and ammonia builds
up in the blood and is toxic to cells. In the urea cycle, two nitrogen atoms one from carbamoyl
phosphate and the other from aspartate are eliminated from the body per additional arginine
provided in the diet. Creating an alternate pathway for carrying nitrogen out of the body.
Therefore arginine supplementation helps to remove excess nitrogen (Berg, Stryer, tymoczko,
2007).

Other therapies:
Benzoate and phenylacetate are conjugates that substitute for urea in the disposal of nitrogen.
(Berg, Stryer, Tymozcko, 2007)

Ammonia scavenger medications: lactulose, antibiotics,

Following liver transplant:​ Enzymatic activity in the liver is restored and urea cycle can
proceed. Thus protein restriction can be lessened. However, because deficiencies in enzymes
continue to exist in extrahepatic tissues, supplementation is necessary to ensure synthesis of
amino acids as well as proper functioning of the urea cycle.

(Berg, Stryer, tymoczko, 2007)

4. In TPN the gut is bypassed in supplying nutrients to the body. What effect might this have on
the synthesis of arginine and proline from the precursor glutamate?

A patient who is being treated with TPN, will have issues with carrying out several
reactions for amino acid production. In the small intestine, glutamine is one of the most
important amino acid for the gut. Glutamine is used primarily for energy production but is also
metabolized by enterocytes for conversion to other amino acids such as proline, arginine and
citrulline.
Proline and arginine are both derived from the precursor glutamate. Much of the
glutamate produced in the body happens in the small intestine, as glutamine gets metabolized,
creating ammonia and glutamate. If a patient is receiving TPN, glutamate production would be
decreased in the intestinal mucosa. Typically, glutamine that is produced in the body gets
collected for energy use by the cells of the intestine, but with TPN administration the whole
digestive tract is bypassed. Because of this, glutamate levels are decreased, causing further
inference in production of other amino acids such as alanine, proline and arginine, as glutamate
is their precursor.
Both glutamine and proline have key roles as dietary precursors for arginine synthesis.
The roles of proline and glutamine in arginine metabolism change depending on stage of
development.
Glutamate can also be used to synthesize ornithine, alongside aspartate. Ornithine can be
produced from a semialdehyde by a simple glutamate-dependent transamination. It can then be
used with carbamoyl phosphate to produce citrulline, that can be sent into the portal blood
system and eventually converted to arginine. Proline and arginine production is greatly hindered,
because they are both derived from glutamate.

Hence, to provide more arginine to the adult, the dietary strategy should be based on
feeding dietary precursors, not arginine. However, when the gut is bypassed, as in total
parenteral nutrition feeding, then arginine needs to be fed to meet arginine requirements ​(Bertolo
and Burrin, 2008).
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