MBC 223

 GAMMA GLUTAMYL CYCLE

 THE CONCEPT OF NITROGEN BALANCE
 NITROGEN TURNOVER IN CELLS

GAMMA GLUTAMYL CYCLE

γ-Glutamyl Cycle is an essential pathway for cells to maintain adequate intracellular glutathione
(GSH) levels. Glutathione is the most abundant antioxidant in the human body. It is capable of
preventing damage to important cellular components caused by reactive oxygen species such
as free radicals, peroxides, lipid peroxides, and heavy metals. It is a tripeptide (glutamate,
cysteine and glycine) with a gamma peptide linkage between the carboxyl group of
the glutamate side chain and cysteine. The carboxyl group of the cysteine residue is attached by
normal peptide linkage to glycine.

The enzymes involved in Gamma Glutamyl cycle are:

 γ-glutamyltranspeptidase (GGT)
 γ-glutamyl-cyclotransferase
o Catalyzes the formation of 5-oxoproline from gamma-glutamyl dipeptides and
may play a significant role in glutathione homeostasis. Induces release of
cytochrome c from mitochondria with resultant induction of apoptosis
 5-oxoprolinase
 glutamate-cysteine ligase (GCL)
 glutathione synthase (GS).

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The γ-glutamyl cycle as proposed by Meister begins with the biosynthesis of glutathione. The
biosynthesis involves the cytosolic, non-ribosomal synthesis of this unusual tripeptide by the
action of two ATP-dependant enzymes, glutamate–cysteine ligase (GCL) and glutathione
synthase (GS).The first enzyme, GCL, which catalyzes the formation of γ-glutamyl-cysteine
from glutamate and cysteine in the presence of ATP is the rate limiting step in the biosynthesis.
The second enzyme, GS, involves in the ligation of γ-glutamyl-cysteine to glycine in another
ATP-dependant reaction to yield γ-glutamyl cysteinyl glycine (or glutathione). Following
biosynthesis is the initiation of degradation glutathione. In this cycle, the degradation is carried
out by γ-glutamyl transpeptidase (γ-glutamyl transferase, GGT), the only enzyme known at that
time to degrade glutathione. GGT has a transpeptidation activity that involves in the transfer of
the γ-glutamyl group to the amino acid to form a γ-glutamyl amino acid. In the γ-glutamyl cycle,
the primary GGT activity that was invoked was the transpeptidase activity, and therefore, the
role of the enzyme was to primarily carry out transpeptidation. The transpeptidation reaction
involving the amino acids leads to the eventual transport of the amino acid across the membrane
in the form of γ-glutamyl amino acids. Furthermore, the model also implied that the plasma
membrane bound GGT, which had it active site facing outward facilitated the translocation of the
γ-glutamyl amino acid across the membrane into the cytosol. In the fourth enzymatic step, the γ-
glutamyl amino acid that is released into the cytosol is acted on by the enzyme, γ-glutamyl
cyclotransferase (γ-GCT) to yield 5-oxoproline (pyroglutamic acid) while releasing the amino
acid. The next enzymatic reaction involved the ATP-dependant 5-oxoprolinase enzyme that
acted on 5-oxoproline to yield glutamic acid. In the sixth step, the cysteinylglycine would be
cleaved by cellular peptidases to yield cysteine and glycine. A key player in the entire cycle was
the enzyme GGT. The enzyme is predominantly found on kidney tubules, but is also found in the
choroid plexus and in the ciliary body and the lens. The kidney is where a lot of amino acid
uptake takes place and was one of the reasons that led Meister to propose that amino acid
transport was the key function of the cycle.

CONCEPT OF NITROGEN BALANCE

Nitrogen is a main body component and it is required for both tissue protein synthesis and the
production of several nitrogenous compounds involved in a variety of functions (hormones,
immune mediators, neurotransmitters, antioxidant defenses, etc. Thus, the body’s nitrogen
content should be both quantitatively and qualitatively normal, as well as normally maintained,
to ensure normal body functions. Nitrogen homeostasis(balance) is a highly regulated function.
Nitrogen balance is commonly referred to as the net difference between the intake (and/or the
effective absorption) of nitrogen contained in the diet and its excretion. Since nitrogen is
contained predominantly in proteins, this term pertains mainly to the balance of proteins and of
amino acids

Nitrogen balance is a measure of nitrogen input minus nitrogen output.

Nitrogen Balance = Nitrogen intake - Nitrogen loss

Sources of nitrogen intake include meat, dairy, eggs, nuts and legumes, and grains and cereals.
Examples of nitrogen losses include urine, feces, sweat, hair, and skin. Blood urea nitrogen can
be used in estimating nitrogen balance, as can the urea concentration in urine.

Nitrogen balance occurs when the amount of nitrogen consumed equals that of the nitrogen
excreted in the urine (primarily as urinary urea nitrogen, or UUN), sweat, and feaces.
1. Positive nitrogen balance: This occurs when nitrogen intake exceeds nitrogen excretion. It is
observed during situations in which tissue growth occurs, for example, in childhood, pregnancy,
or during recovery from an emaciating illness.
2. Negative nitrogen balance: This occurs when nitrogen loss is greater than nitrogen intake. It
is associated with inadequate dietary protein; lack of an essential amino acid; or during
physiologic stresses, such as trauma, burns, illness, or surgery.
Requirement for protein in humans
The amount of dietary protein required in the diet varies with its biologic value. The greater the
proportion of animal protein included in the diet, the less protein is required. The RDA for
protein is computed for proteins of mixed biologic value at 0.8g/kg of body weight for adults, or
about 56 g of protein for a 70-kg individual. People who exercise strenuously on a regular basis
may benefit from extra protein to maintain muscle mass, and a daily intake of about 1 g/kg has
been recommended for athletes. Women who are pregnant or lactating require up to 30 g/day in
addition to their basal requirements. To support growth, infants should consume 2 g/kg/day.
Consumption of excess protein: There is no physiologic advantage to the consumption of more
protein than the RDA. Protein consumed in excess of the body’s needs is deaminated, and the
resulting carbon skeletons are metabolized to provide energy or acetyl coenzyme A for fatty acid
synthesis. When excess protein is eliminated from the body as urinary nitrogen, it is often
accompanied by increased urinary calcium, thereby increasing the risk of nephrolithiasis
andosteoporosis
The protein-sparing effect of carbohydrate: The dietary protein requirement is influenced by
the carbohydrate content of the diet. When the intake of carbohydrates is low, amino acids are
deaminated to provide carbon skeletons for the synthesis of glucose that is needed as a fuel by
the central nervous system. If carbohydrate intake is less than 130 g/day, substantial amounts of
protein are metabolized to provide precursors for gluconeogenesis. Therefore, carbohydrate is
considered to be “protein-sparing,” because it allows amino acids to be used for repair and
maintenance of tissue protein rather than for gluconeogenesis.
Protein-energy (calorie) malnutrition
In developed countries, protein-energy malnutrition (PEM) is most commonly seen in patients
with medical conditions that decrease appetite or alter how nutrients are digested or absorbed or
in hospitalized patients with major trauma or infections. PEM may also be seen in children or the
elderly who are malnourished. In developing countries, an inadequate intake of protein and/or
energy is the primary cause of PEM. Affected individuals show a variety of symptoms, including
a depressed immune system with a reduced ability to resist infection. Death from secondary
infection is common. PEM is a spectrum of degrees of malnutrition, and two extreme forms are
kwashiorkor and marasmus
1. Kwashiorkor: Kwashiorkor occurs when protein deprivation is relatively greater than the
reduction in total calories. The main deficiency is protein. Protein deprivation is associated with
severely decreased synthesis of visceral protein. Kwashiorkor is commonly seen in developing
countries in children after weaning at about age 1 year, when their diet consists predominantly of
carbohydrates. Typical symptoms include stunted growth, skin lesions, depigmented hair,
anorexia, enlarged fatty liver, edema, and decreased serum albumin concentration. Edema results
from the lack of adequate blood proteins, primarily albumin, to maintain the distribution of water
between blood and tissues. It may mask muscle loss. Therefore, chronic malnutrition is reflected
in the level of serum albumin.
2. Marasmus: Marasmus occurs when calorie deprivation is relatively greater than the reduction
in protein. It usually occurs in developing countries in children younger than age 1 year when
breast milk is supplemented with watery gruels of native cereals that are usually deficient in both
protein and calories. Typical symptoms include arrested growth, extreme muscle wasting and
loss of subcutaneous fat(emaciation), weakness, and anemia. Individuals with marasmus do not
show the edema observed kwashiorkor.

Protein turnover

When older proteins are broken down in the body, they must be replaced. This concept is called
protein turnover, and different types of proteins have very different turnover rates. Protein
synthesis occurs during the process of translation in ribosomes. Protein breakdown occurs
generally in two cellular locations:

 Lysosomal proteases digest endocytosed proteins

 Cytoplasmic complexes, called proteasomes, digest older or abnormal proteins that have
been tagged with ubiquitin for destruction.

A balance between protein synthesis and protein degradation is required for good health and
normal protein metabolism. More synthesis than breakdown indicates an anabolic state that
builds lean tissues, more breakdown than synthesis indicates a catabolic state that burns lean
tissues. Protein turnover is believed to decrease with age in all senescent organisms including
humans. This results in an increase in the amount of damaged protein within the body.

Four weeks of aerobic exercise has been shown to increase skeletal muscle protein turnover in
previously unfit individuals. A diet high in protein increases whole body turnover in endurance
athletes.

Some bodybuilding supplements claim to reduce the protein breakdown by reducing or blocking
the number of catabolic hormones within the body. This is believed to increase anabolism.
However, if protein breakdown falls too low then the body would not be able to remove muscle
cells that have been damaged during workouts which would in turn prevent the growth of new
muscle cells.

Example protein half-lives

Name Half-Life
Collagen 117 years
Eye lens crystallin >70 years
RFC1 9 hours
RPS8 3 hours
Ornithine decarboxylase 11 minutes