The Biochemistry of Sulphur Amino Acids

Sulphur amino acid metabolism

The sulphur amino acids are methionine and cysteine. Their metabolism is
interlinked. As a result of this metabolism, the sulphur moiety is incorporated into a number
of end-products, three of which, glutathione, taurine and proteins, have important roles in
immune function. Methionine is a nutritionally essential amino acid, due to the inability of
mammals to synthesize from methionine provided that the dietary supply of later is sufficient.
The methyl group of methionine can be removed from and reattached to the carbon skeleton
of the amino acid by a cyclical process referred to as the transmethylation pathway (fig.7.1).
the formation of homocycteine, part way along the transmethylation pathway, is an important
branch point in methabolism of methionine. Homocycteine can be remethylated to form
methionine or can be metabolized by the transulphuration pathway to form cycteine (fig.7.1).
both the remethylation of homocycteine and the formation of cycteine utilize serine. The
latter amino acid forms the carbon skeleton of cycteine and acts as a methyl-group donor to
tetrahydrofolic acid, once the methyl version of the latter compound has donated its methyl
group to homocycteine during the information of methionine.

Methyonine is intimately involved in the synthesis of the polyamines spermine and

spermidine, in which the carbon chain of methionine is donated to a third polyamine,
putrescine, thich is derived from ornithine (fig.7.2). the polyamines are present in high
concentrations in rapidly dividing cells, such as those of an activated immune system. Their
rele is poorly defined but appearsto be important. Polyamines have been likened to
‘molecular grease’, in that they are permissive metabolities, ensuring the fidelity of DNA
transcription and RNA transtition (Grimble and Grimble, 1998). In in vitro studies, cells
depleted \of polyamines exhibit increased error rates in both processes. The first enzyme in
the step from omithine to putrecine is highly induced in rapidly dividing cells.

Methionine also acts as a methyl donor in the synthesis of creatine (fig.7.3), thich is
essential for muscle energy generation through its phosphorylation to creatine phosphate.
Creatine phosphate can transfer its phosphate to ADP to restore cellular ATP supplies during
periods of high metabolic activity.

In addition to incorporation into proteins, cycteine can be incorporared tnto the key
antioxidant glutathione (GSH), or converted to taurine and organic sulphate. The possession
of an SH group by cycteine and GSH allowsteh formation of the S-S bridge between two
moleculesof cyctine or of GSHto form cyctine and oxidized glutathione (GSSG),
respectively, taurine has many roles, including formation of the bile salt taurocholic acid, and
is a putative antioxidant and cell membrane stabilizer. Taurine is the predominant
nitrogenous compound in immune cells.

The synthesis of gluatithone from its three constituent amino acids in mainly limites
to the liver. Two consecutive steps are required to synthesize glutathione, each step
consuming one ATP molecule (Fig. 7.4). the rate-limiting enzyme in the pathway is y-
glutamyl cycteine synthetase (step 1 of fig 7.4). under normal psychological conditions, there
is feedback on the activity of this enzyme by GSH. Thus, conversion of cyctine to GSH is
strongly influences by the rate of utilization/transport is a ‘demand-led’ process, provided
that cyctine is available.

Glutathione is transferred to the blood and transported around the body in both plasma
and cells mainly in its reduces form (GSH)

Thus, apart from protein synthesis, sulphur amino acids are involved as direct and
indirect participants in pathways involved in cells replication and stabilization, antioxidant
defence, assimilation of lipids and energy metabolism, it is not surprising that the availability
of sulphur amino acids has a major impact on immune function.

Control of Sulphur Amino Acid Metabolism

The Km values for the homocysteine transferase enzymes (steps 4 and 5 of fig.7.1)
(which lead to the recycling of methionine) are two orders of magnitude lower than those for
cystathione synthase (step 6 of fig.7.1) and cystathine y-lyase (step 7 of fig 7.1) (which
process methionine towars catabolism via the transulphuration pathway). Thus, at low
intracelullar concentration of methinine, remethylation will be favoured over transulphuration
and methionine will be conserved. When these two pathways were examined in vivo in rats
fed diets containing 3, 15 and 30 g L-methionine kg-1 of diet, the percentage of methionine
metabolized by the twocompeting athways changed (fingkelstein and martin, 1984, 1986).
With increasing dietary methionine intake, substrate flux through the transmethylation
pathway fell and flux through the transphuluration pathway increased.

Examination of the Km values for rate0limiting enymes processing the major cysteine
metabolitiess provides a further insight inot how sulphur amino acid metabolism is influenced
by alteration in the supply of cycteine. The Km for L-cycteinyl-tRNA synthease (step 8 of fig
7.1) (essential for incorporation of cysteine into protein) is less than one-tenth of that for y-
glutamyl cysteine synthetase (step 9 of fig.7.1) (the rate-limiting enzyme for GSH synthesis)
or cysteine diexygenase (step 10 of fig 7.1) (forming cycteine sulphinate, the precursor for
sulphate and taurine). Thus, under conditions of low cysteine availabillity, protein synthesis
will be maintained and sythesis of sulphate, taurine and GSH curtailed.

From the kinetics of the key enzymes in sulphur amino acid metabolism reported
above, it can be seen that, when the diet is low in sulphur amino acids, cellular methionine is
highly conserved. Flux down the transulphuration pathway, which ultimately leads to
methionine catabolism, increase only as dietary methionine intake increases. It an also be
seen that, at how flux rates of substrate down the transulphuration pathway, conservation of
custeine into its main metabolities will be affected, so that protein synthesis will be relatively
maintrained while sulphate and GSH synthesis rates will fall. Synthesis of GSH and suphate
will increase in concert as increasing levels of substrate flow trough the pathway. In a study
in rats, seven molecules of cysteine were incorporated into GSH for every ten invorporated
into protein in liver at adequate sulphur amino intake (Grimble and Grimbe, 1998). At
inadequate to a low intake of sulphur amino acid intake, the ratio fell to < 3 :10. This
response to a low intake of sulphur amino acids will not necessarily be adventageous since
GSH is an important component of antioxidant defence. Thus, at low sulphur amino acid
intakes, antioxidant defences will become weakened. The immune response makes large
demands on these defences and sulphur amino acid metabolism in particular.

Sulphur Amino Acid and Glutathione Metabolism Following Infection and Injury

The immune system has a great capacity for immobilizing invading microbes,
creating a hostile environment for them and bringing about their destruction (fig. 7.5). the
immune system can also become activated, in a similar way to the response to microbial
invasiom, by a wide range oof stimuli and conditions; these include burns, penetrating and
blunt injury, the presence of tumout cells, environmental pollutants,radiations, exposure to
allergens and the presence of chronic inflammatory diseases. The strenght of the response to
this disperate range of stimuli will vary, but it will contain many of the hallmarksof the
response to invading pathogens. The immune response has a high metablic cost, and
inppropriate prolongation of the response will exert a deleterious effect upon the nutritional
status of the host.

The pro-inflammatory cytokines interleukin (IL)-1, IL-6 and tumour necrosis factr
(TNF) –a hae widespread metabolic effects upon the body and stimulate the process of
inflammation. Many of the signs and symptoms expreiences aftrer infection and injury, such
as fever, loss of appetite, weight loss, negative nitrogen, sulphur and mineral balance and
lethargy are caused directly r indirectly by pro-inflammatory cytookines (Fig.7.5). the
indirect effects of cytokines are mediated by actions upon the adrenal glands and endcrine
pancreas, resulting in increased secretion f the catabolic hormones adrenalin, noradrenalin,
glucocorticoids and glucagon. Insulin insensitiity occurs, in addition to thus ‘catabolic state’.
The biochemistry of an infected individual is thus fundamentally changed in a way that will
ensure that the immune system receives nutrients from within the body. Muscle protein is
catabolized to provide amino acids fr synthesizing new cells, GSH and proteins for the
immune response.

Futhermore, amino acids are converted to glucose(apreferred fuel, together with

glutamine, for the immune system). An increase in urinary nitrogen and sulpur excretion
ocurs as a result of this catabolic process. The extent of this process is highlighted by the
significat increase in urinary nitrogen excretion, from 9g day-1 in mily infectiom to 20-30g
day-1 following major burn secere traumatic injury (wilmore,1983). The loss of nitrogen
from the body of an adult during a bacterial infection may be equivalent to 60g of tissue
proein and, in a period of presistent malaria infection, equivalent to over 500g of protein.
However, during the response to infection and injury, the urunary excretion of sulphur
increases to a lesser extent than that of nitrogen (cuthbertson, 1931), suggesting that sulphur
amino acids are preferentially retained and so ‘spread’ from catabolism. Infectin with human
immunodeficiency virus (HIV) infection (Breitkreutz et al 2000). The losses reported were
equivalent to 10g of cycteine day-1, in contrast to losses of approximately 3 g day-1 for
healthy individuals on a ‘westernized diet’. As cysteine is the precursr for both sulphate and
GSH this finding may be linked with the decline in tissue glutathione pools that has been
observed in HIV invection (De Rosa et al 2000). Clearly, such a depletion of antioxidant
defences will not be sustainable over a long period.

Large decreases in plasma glycine, serine and taurine concentrations occour following
infection and injury. These changes may be due to enhanced utilization of a closely related
group of amino acids, namely, glycine, serine methionine and cysteine. Many substances
produced in echanced amunts in respons to pro-inflammantory cytokines are particulary rich
in theseamino acids. These substances include GSH, which comprises glycine, glutamic acid
and cysteine metallothionein (the major zinc-transport protein). Which contains glycine,
serine, cysteine and methionine to a composite percentage of 56% and a range of acute-phase
proteins, which contain up to 25% of these amino acids in teir structure. If an increased
demand for sulphur and related amino acids is created by the inflammatory response, then
provision of additional supplies of these amino acids may assist the response.

Many of the components of antioxidant defence interact to maintain antioxidant status

(see also Hughes, Chapter 9, Prasad, Chapter 10, and McKenzie et al, Chapter 12, this
volume). Glutathione and the enzymes that maintain it in its reduced form are central to
effective antioidant status. Fr example, when oxidants form are central to effective
antioxidant status. For example, when exidants interact with cell membranes, the oxidized
form of vitamin E that results is restored to its reduced form by ascorbic acid by interaction
with the reduced form of glutathione. Subsequently, exidized glutathione formed in the
reaction in reconverted to the recuced form of glutathione formed in the reaction is
reconverted to the reduced form of glutathione by glutathione reductase (Fig.7.6). vitamins E
and C and glutathione are thus intimately linked in antioxidant defence. The interdependence
of the various nutritional components of antioxidant defence is illustrated in a study in which
healthy subjects were given 500mg ascorbic acid day-1 for 6 weeks (johnston et at., 1993). A
47%increase in the glutathione content of red blood cells occurred. Vitamin B6 and
riboflavin, which have no antioxidant properties. per se, also contribute to antioxidant
defences indirectly. Vitamin B6 is the cofactor in the matabolic pathway for the biosynthesis
of cysteine (fig 7.1) cellular cysteine concentration is rate limiting for glutathione synthesis.
Riboflavin is a cofactor for glutathione reductance, which maintains the major part of cellular
glutathione in the reduced form (fig 7.6).

Antioxidant Defences Following Infection and Injury

Although pro-inflammantory cytokines are essential for the nrmal operation of the
immune system, they play a major damaging role in many inflammatory diseases, such as
rheumatoid arthritis, inflammatory bowel disease, asthma, psoriasis and multiple sclerosis,
and in cancer (Tracey and Cerami, 1993’ Grimble, 1996). In conditions such as cerebral
malaria, meningitis and sepsis, they are produced in excessive amounts and are an important
factor in increased mortality (Tracy and Cerami., 1993). In malaria, tubercolosis, sepsis,
cancer, HIV infection and rheumatoid arthritis, inflammatory cytoines bring about a aloss of
lean tissue, which is associated with depleted tissue GSH content and an increased output of
nitrogennous and sulphur-containing excretion products in the urine (see above).

Although the body strives to maintain them, observation in experimental animal and
patients indicate that antioxidant defences become depleted duting infectioon and after injury.
For example, in mice infected with influenza virus, there were 27%, 42% and 45% decreases
in the vitamin C, vitamin E and glutathoine contents of blood, respectively (Hennett et al .,
1992). While values in blood slowly returned to pre-operative values, concentrations in
muscle were still depressed 48 h post-operatively. Furthermore, reduced tissue glutathione
concertation has been noted in hepatitis C, ulcerative colitis and cirrhosis. In patient with
maligant melanoma, metastatic hypernerphroma and metastatic colon cancer, plasma ascorbic
acid concentrations fell from normal to almost undetectable levels within 5 days of
commencement of treatment with IL-2 (Grimble, 1999). Lipid peroxides and increased
thiobarbituric acid reactive substances are present in the blod of patients with septic shock,
asymptomatic HIV infection, chronic hepatitis C, breast cancer, cystic fibrosis, diabetes
melitus and alchoholic liver disease. Peroxides also increase following cancer chemotherapy,
open heart surgery, bone marrow transplantationand haemdialysis (Zimmerman et al., 1989).
The onset of sepsis in patients leads to a transient decrease in the total antioxidant capacityof
blood plasma ( a functional measure of the total antioxidant content ( Cowley et al., 1996).
The capacity returns to normal values over the following 5 days. However, this was not the
case for patients who subsequently died, in whom values remained well below the normal
range.

As well as increasing the risk of direct oxidant damage, a reduction in the strength of
antioxidant defences also indirectly increases the risk of damage to the host via transcription-
factor activation, leading to up-regulation of pro-inflammatory cytokine production (see
below)
Glutathione and the Immune System

Direct effects of glutathione

One of the first indications that glutathione influences aspect of immune function tat
are related to T lymphocytes came from a study in which the GSHcontent of lymphocytes
was measured in a group of healthy volunteers (Kinscherf et al., 1994). The relationship
between cellular glutathione concentrations and cell number was complex, with numbers of
both subsets declining at intracellular glutathione concentration between 30 and 50 nmol mg-
1 protein. When the subjects engaged in a programme of intensive physical exercise daily for
4 weeks, a fall in glutathione concentration occured. This study suggest that immune cell
fuinction may be sensitive to a range of intracellular sulphydryl compounds, including
glutathione and cysteine. In HIV+ individuals and patients with aqcuired immune deficiency
syndrome (AIDS), a reduction in cellular and plasma glutathione has been noted (Staal et
a;.,1992). The depletion in lymphocyte population that occurs in these subject is related to
this phenomenon. In a large, randomized, double blind, placebo-cntroled trial, administratin
of 600 mg day-1 of NAC for 7 months resulted in both anti-inflammantory and
immunoenchancing effects (Breitkreutz et al .,2000). The preciese mechanism underlying the
complex effects of changes in cellular glutathione content are not clear, and whether they are
related t GSH function as an antioxidant or to some other properly is not apparent. However,
a recent study suggests that glutathione promotes IL-12 production by antigen-presenting
cells, so driving T-helper (Th) cells along Th1 pathway of differentiation (peterson et al.,
1998)

Effects of other nutrients that might have an impact on glutatione status

Vitamin B6

Vitamin B6 although having no antioxidant properties, plays an important part in

antioxidant defences, because of its action in the metabolic pathway for the formation of
cysteine, which, as indicated earlier, is the rate-limiting precursor in glutathione synthesis.
Vitamin B6 status has widespread effects upon immune function (Rall and Meydani, 1993).
In human studies, the ability to make antibodies to tetanus and typhoid antigens is not
seriously affected. Various aspects of cellmediated immunity are also influenced by vitamin
B6 deficiency. Deficiency of citamin B6 is rare in humans but can be precipitated with the
anti-tubercolosis (Anti-TB) drug isoniazid. Restoration of vitamin B6 intake to n ormal by
dietary supplements restores immune function. However, intakes that are higher than current
recommended values are required to normalize all immune functions, suggesting that this
vitamin can only restore immune function in this way. It is unclear, at present, whether a
similar situation occurs in younger subjects. Deficiency of the vitamin may limit the
availability of cysteine for glutathione synthesis. In rats, vitamin B6 deficiency resulted in
decreases of 12 and 21% in glutathione concentrations in plasma ans spleen, respectively
(takeuchi at el., 1992). In healthy young women, large doses of vitamin B6 (27 mg day-1 for
2 weeks) resulted in a 50% increase in plasma cysteine content (kang-yoon and kirksey,
1992), presumably by increased flux through the transulphuratin pathway.

Ascorbic acid

Unlike deficiencies in vitamin B6 and E and riboflavin, deficiency of vitamin C des

not cause atrophy of lymphoid tissue. In a study of ultramarathon runners, dietary
supplementation with 600mg day-1 of ascorbic acid reduced the incidence of
upperrespiratory-tract infections after a race by 50% (Peters et al., 1993). When
immunological parameters and antioxidant status were measured in adult males fed 250 mg
day-1 of vitamin C for 4 day, followed by 5 mg day-1 for 32 days, plasma ascorbic acid and
glutathione decreased and impairment of antioxidant status became evident from a doubling
in semen 8 hydroxydeoxyguanoise concentration (a measure of oxidative damage to nucleic
acids) during the second dietary period (jacob et al ., 1992). A fall in vitamin content in
peripheral-blood mononuclear cells was noted and the delayed-type hypersensitivity reaction
to seven recall antigens was significantly reduced in intensity.

Mechanism of thr Effect of Oxidants and Antioxidants on Inflammation and immune

Function

There is a growing body of evidence that antioxidants suppress inflammatory

components of the response to infection and trauma and enchance components related to cell-
mediated immunity. The reverse situation applies when antioxidant defences becme depleted.
The families are nuclear trancription factor kappa B (NFkB) and activator protein 1 (AP1).
These transcription tors act as ‘control switches’ for biological processes, not all of which are
of advantage to the individual. Activation of NFkB can be brought about by a wide range of
stimuli, including pro inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and
viruses and their related products, and UV ionizing radiations. The dissociated lkB is
degraded and the active NFkB is translocated to the nucleus, where it binds to the response
elements in the promoter regions of genes, a similar translocation of AP1, a trancription
factor composed of the proto-oncogenes c-fos and c-jun, from cytosol to nucleus also occurs
in the presence of xidant stress. The activation of a wide range of genes associated with
inflammation and the immune response, including those encoding cytokines, cytokine
receptors, cell-adhesion molecules, acute-phase proteins and groth factors (Schreck et al.,
1991). If antioxidant defences are poor, each encounter with general infections reluts in
cytokine and oxidant production, NFkB activation and an increase in viral replication. It is
thus infrtunate that reduced cellular concentrations of GSH are a common feature of
asymptomatic HIV infection (Staal et al., 1992). The interaction between oxidant stress and
an impaired ability to synthesize glutathione, which results in enchanced inflammation, is
clearly seen in cirrhosis, a disease that reults in high levels of oxidative stress and an
impaired ability to synthesize GSH (Pena et al., 1999). However, not al transcription factor
respond to changes in cell refox stte in the same way. When rats were subjected to depletion
of effective tissue GSH pools by administration in spleen and mesenteric lymph nodes
(Robinson et al., 1993). Modification of glutathione content of lung, liver, spleen and thymus
in young rats, by feeding diets containing a rang of casein ( a protein with a low sulphur
amino acid content) concentrations, changed immune cell numbers in the lung (Hunter and
Grimble ., 1994). However , in animals given an inflammatory challenge (endotoxin), liver
and lung GSH concentration increased directly in relation to dietary protein intake. Thus, it
can be hypothesized that antioxidants exert an immunoenchancing effect, by activating
transcription factors that are strongly assosiated with cell proliferation and an anti-
inflammatry effect, by preventing activation of NFkB by oxidants produced during the
inflammatory response.
Strategies for Modulation Tissue GSH content and improving Immune Function.

While cysteine supplies are the primary determinant of the ability to synthesize GSH,
in sme circumstances an insufficiency in the other two amino acids from which it is made
might limit synthesis. Glutamin, for example, has been shown to maintain hepatic GSH in
animal poisoned with acetaminophen, to enchance gut GSH synthesis when given
intravenously to rats (Cao et al., 1998). In human studies a similar effect on gut GSH
concentrations was noted ( O’Riordain et al., 1996). There are many studies that illustrate the
ability of sulphur amino acid availability to influence tissue GSH concentrations (e.g.
Stipanuk et al., 1992).

Tudies using animal models of inflammation have shown that a low-protein diet will
suppress glutathione synthesis, a situation that in reversef by the provision of cysteine or
methionine (Hunter and Grimble, 1994, 1997). Because cysteine is unstable in its reduced
form, toxic in high doses and mostly degraded in the extracellular compartment, several
compounds have been used to deliver cysteine directly to cells. Recent animal and clinical
trials with NAC and OTZ have demonstrated the ability to the compounds to enchance GSH
status. While not affecting morality rates, NAC shortened hospital length of stay by > 60%.
OTZ increased whole-blood GSH in peritoneal-dialysis patients, normaized tissue GSH in
rats fed a sulphur amino acid-deficient diet and decreased the extent of inflammation in a rat
peritonitis model (Bernard et al., 1997). In a randomized, double-blind, dontrolled study on
asymptomatic HIV+ patients in the presence and absence of anti-retroviral therapy (ART)
have shown that NAC can rise blood GSH, increase natural killer cell activity and enchance
stimulation indicates of T-cells incubated with mitogen or tetanus toin (Simon et al., 1994;
Breitzkreutz et al., 2000). Flow- cytometryc analysis of freshly prepared human peripheral-
blood lymphocytes shows that lipoic acid is able to normalize a sub population of cells with
severely compromised thiol status, rather than increasing the level in all cell above normal
values (Sen et al.,1997)

Taurine and Immune Function

Taurine, along with sulphate, can be regarded as a biochemical end-product of

cysteine metabolism. It has been shown to possess antioxidant properties and to regulate the
release of pro-inflammatory cytokines in hamsters, rats and humans (Grimble, 1994;
Huxtable, 1996; Kontny et al., 2000)

A large decline in lymphocytes, an increase in mononuclear cells and a decrease in

the ability of these cells to produce a ‘respiratory burst’ and to phagocytose bacteria occur.
There was a rise in gamma glbulin concentrations in deficient animals spleen and lymph
nodes showed regression of follicular centres and depletion of mature and immature B
lymphocyte numbers. The changes were reversed by inclusion of taurine in the diets. Studies
in other species have also reported effects of supplementation on imune system and function.
In mice administration of taurine prevented the decline in T-cell number that occurs with
ageing and enchanced the prolifereative responses of T-cells n both young and old mice.