Sulphur Amino Acids and Glutathione: Their Role in Immune Function, Notas de estudo de zootecnia

Baixe Sulphur Amino Acids and Glutathione: Their Role in Immune Function e outras Notas de estudo em PDF para zootecnia, somente na Docsity! 7 Sulphur Amino Acids, Glutathione and Immune Function RoBERT F. GRIMBLE Institute of Human Nutrition, School of Medicine, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK The Biochemistry of Sulphur Amino Acids Sulphur amino acid metabolism The sulphur amino acids are methionine and cysteine. Their metabolism is inter- linked. 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 its carbon skeleton. Cysteine is considered to be semi-essential, in that it is synthesized from methionine pro- vided that the dietary supply of the latter 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 homocysteine, part way along the transmethylation path- way is an important branch point in the metabolism of methionine. Homocysteine can be remethylated to form methionine or can be metabolized by the transulphuration pathway to form cysteine (Fig. 7.1). Both the remethylation of homocysteine and the formation of cysteine utilize serine. This latter amino acid forms the carbon skeleton of cysteine and acts as a methyl-group donor to tetrahydrofolic acid, once the methyl version of the latter compound has donated its methyl group to homocysteine during the formation of methionine. Methionine is intimately involved in the synthesis of the polyamines sper- mine and spermidine, in which the carbon chain of methionine is donated to a third polyamine, putrescine, which 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 role is poorly defined but appears to be important. Polyamines have been likened to 'molecular grease”, in that they are permissive metabolites, ensuring the fidelity of DNA transcription and RNA translation (Grimble and Grimble, 1998). In in vitro studies, cells depleted O CAB International 2002. Nutrition and Immune Function (eds PC. Calder, C.J. Field and H.S. Gill) 133 R.F. Grimble Protein 1 + uethionine Serine THFA Dimethyl mero O) S-AM Glycine xX(e.9.glycine) . s ja Vit Bro Betaine Ê 5-methylene XCHy THFA 5-methyl + (escacósro) cy THFA Choline Homocysteine Ja Serine ; E | Vit.Bo Cystathionine Sulphate e Cysteine 4 8 sulphinate “o Cysteine <-» Protein Taurine 9 Glutathione Fig. 7.1. Outline of sulphur amino acid metabolism. Enzymes: 1, methionine adenosyl transferase; 2, methyl transferase; 3, adenosyl homocysteinase; 4, betaine methyltransferase; 5, S-methyltetrahydrofolate methyl transferase; 6, cystathionine B-synthase; 7, cystathionine w-lyase; 8, L-cysteinyl-tRNA synthetase; 9, y-glutamyl cysteine synthase; 10, cysteine dioxygenase. S-AH, S-adenosyl homocysteine; S-AM, S-adenosyl methionine; THFA, tetrahydrofolic acid. Omithine S-adenosyl methionine Meihionine — —» Putrescine I Spermidine Fig. 7.2. Polyamine biosynthesis. of polyamines exhibit increased error rates in both processes. The first enzyme in the step from ornithine to putrescine is highly induced in rapidly dividing cells. Methionine also acts as a methyl donor in the synthesis of creatine (Fig. 7.3), which is essential for muscle energy generation through its phosphoryla- tion 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, cysteine can be incorporared into the key antioxidant glutathione (GSH), or converted to taurine and inor- ganic sulphate. The possession of an SH group by cysteine and GSH allows the formation of an S-S bridge between two molecules of cysteine or of GSH to form cystine and oxidized glutathione (GSSG), respectively. Taurine has many roles, including formation of the bile salt taurocholic acid, and is a puta- Sulphur Amino Acids, Glutathione and Immune Function 137 Trauma/infection/burns Immune system activation V o Es ( Jmmuno- &, a iti 4 Pro-inflammatory cytokines EN «nutrition, Feedback systems T- and B-cells l N IL-10, al A Antioxidant Heat-shock defence proteins ASicoso / (O Nutrient — Glutamine Pathogen Tests release from killing damage host tissues = Suiphur — Glutathione -— amino synthesis 0 BEN acids aa > / j 2 Antiori x |” Creationota — |” Antioxidant [| hostile | 4— Acute-phase protein ——p | defences 1 *. environment , synthesis * strengthened / Fig. 7.5. The response of the immune system to infection and injury and the effects upon metabolism. the response to microbial invasion, by a wide range of stimuli and conditions; these include burns, penetrating and blunt injury, the presence of tumour cells, environmental pollutants, radiation, exposure to allergens and the presence of chronic inflammatory diseases. The strength of the response to this disparate range of stimuli will vary, but it will contain many of the hallmarks of the response to invading pathogens. The immune response has a high metabolic cost, and inappropriate 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 necro- sis factor (TNF)-a have widespread metabolic effects upon the body and stimu- late the process of inflammation. Many of the signs and symptoms experienced after infection and injury, such as fever, loss of appetite, weight loss, negative nitrogen, sulphur and mineral balance and lethargy are caused directly or indi- rectly by pro-inflammatory cytokines (Fig. 7.5). The indirect effects of cytokines are mediated by actions upon the adrenal glands and endocrine pancreas, resulting in increased secretion of the catabolic hormones adrenalin, noradren- alin, glucocorticoids and glucagon. Insulin insensitivity occurs, in addition to this “catabolic state”. The biochemistry of an infected individual is thus funda- mentally 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 for synthesizing new cells, GSH and proteins for the immune response. 138 R.F. Grimble Furthermore, amino acids are converted to glucose (a preferred fuel, together with glutamine, for the immune system). An increase in urinary nitrogen and sulphur excretion occurs as a result of this catabolic process. The extent of this process is highlighted by the significant increase in urinary nitrogen excretion, from 9 g day! in mild infection to 20-30 g day”? following major burn or severe traumatic injury (Wilmore, 1983). The loss of nitrogen from the body of an adult during a bacterial infection may be equivalent to 60 g of tissue protein and, in a period of persistent malarial infection, equivalent to over 500 g of protein. However, during the response to infection and injury, the urinary excretion of sulphur increases to a lesser extent than that of nitrogen (Cuthbertson, 1931), suggesting that sulphur amino acids are preferentially retained and so 'spared” from catabolism. Infection with human immunodefi- ciency virus (HIV) has been shown to cause substantial excretion of sulphate in the urine during the asymptomatic phase of the disease (Breitkreutz et al., 2000). The losses reported were equivalent to 10 g of cysteine day"? in con- trast to losses of approximately 3 g day-1 for healthy individuals on a “Westernized diet”. As cysteine is the precursor for both sulphate and GSH this finding may be linked with the decline in tissue glutathione pools that has been observed in HIV infection (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 occur following infection and injury. These changes may be due to enhanced utiliza- tion of a closely related group of amino acids, namely, alycine, serine, methion- ine and cysteine. Many substances produced in enhanced amounts in response to pro-inflammatory cytokines are particularly rich in these amino acids. These substances include GSH, which comprises glycine, glutamic acid and cysteine, metallothionein (the major zinc-transport protein), which contains glycine, ser- ine, 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 their structure. If an increased demand for sulphur and related amino acids is cre- ated 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 antioxidant status. For example, when oxidants interact with cell membranes, the oxidized form of vit- amin E that results is restored to its reduced form by ascorbic acid. The dehy- droascorbic acid formed in this process is reconverted to ascorbic acid by interaction with the reduced form of glutathione. Subsequently, oxidized glu- tathione formed in the reaction is reconverted to the reduced form of glu- tathione 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 500 mg ascorbic acid day-1 for 6 weeks (Johnston et al., 1993). A 47% increase in the glutathione content of red blood cells occurred. Vitamin B, and riboflavin, which have no antioxidant properties Sulphur Amino Acids, Glutathione and Immune Function 139 Methionine V Homocysteine Cysteine E E Dehydroascorbio Guutathione Oxidants redue acid esH Glutathione N | 1 t y | f reductase vit E Ascorbic Glutathione oxidized acid assa Fig. 7.6. The interaction between antioxidants in maintaining antioxidant defence. per se, also contribute to antioxidant defences indirectly. Vitamin Bg is the cofactor in the metabolic pathway for the biosynthesis of cysteine (Fig. 7.1). Cellular cysteine concentration is rate limiting for glutathione synthesis. Riboflavin is a cofactor for glutathione reductase, which maintains the major part of cellular glutathione in the reduced form (Fig. 7.6). Antioxidant Defences Following Infection and Injury Although pro-inflammatory cytokines are essential for the normal operation of the immune system, they play a major damaging role in many inflammatory dis- eases, such as rheumatoid arthritis, inflammatory bowel disease, asthma, psoria- sis and multiple sclerosis, and in cancer (Tracey and Cerami, 1993; Grimble, 1996). They are also thought to be important in the development of atheroma- tous plaques in cardiovascular disease (Ross, 1993). In conditions such as cere- bral malaria, meningitis and sepsis, they are produced in excessive amounts and are an important factor in increased mortality (Tracey and Cerami, 1993). Clearly, in these diseases, the cytokines are being produced in the wrong biolog- ical context. In malaria, tuberculosis, sepsis, cancer, HIV infection and rheuma- toid arthritis, inflammatory cytokines bring about a loss of lean tissue, which is associated with depleted tissue GSH content and an increased output of nitroge- nous and sulphur-containing excretion products in the urine (see above). Although the body strives to maintain them, observations in experimental animals and patients indicate that antioxidant defences become depleted during infection 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 glutathione contents of blood, respectively (Hennett et al., 1992). In asymptomatic HIV infec- tion, substantial decreases in glutathione concentrations in blood and lung epithe- lial-lining fluid have been noted (Staal et al., 1992). In patients undergoing elective abdominal operations, the glutathione content of blood and skeletal muscle fell by over 10% and 42%, respectively, within 24 h of the operation (Luo et al., 142 R.F. Grimble One mechanism for the effect of vitamin B, on immune function may be due to the importance of the vitamin in cysteine synthesis, as outlined earlier. Deficiency of the vitamin may limit the availability of cysteine for glutathione synthesis. In rats, vitamin B, deficiency resulted in decreases of 12 and 21% in glutathione concentrations in plasma and spleen, respectively (Takeuchi et al., 1991). In healthy young women, large doses of vitamin B, (27 mg day! for 2 weeks) resulted in a 50% increase in plasma cysteine content (Kang Yoon and Kirksey, 1992), presumably by increased flux through the transulphuration pathway. As cysteine is a rate-limiting substrate for glutathione synthesis, these findings may have implications for the response to pathogens, because of the importance of glutathione in lymphocyte proliferation and antioxidant defence. However, while vitamin B, has cellular effects on the immune system, evidence is lacking of any effect upon the inflammatory response. Ascorbic acid High concentrations of vitamin C are found in phagocytic cells. While the role of vitamin C as a key component of antioxidant defence is well established (Fig. 7.6), most studies have shown only minor effects upon a range of immune functions (see Hughes, Chapter 9, this volume), except in cases where the vita- min may be acting by interacting with GSH metabolism. Unlike deficiencies in vitamins By, and E and riboflavin, deficiency of vitamin C does not cause atro- phy of lymphoid tissue. In a study of ultramarathon runners, dietary supple- mentation with 600 mg day” 1 of ascorbic acid reduced the incidence of upper respiratory-tract infections after a race by 50% (Peters et al., 1993). It is inter- esting to note that strenuous exercise has been shown to deplete tissue glu- tathione content. The interrelationship between glutathione and ascorbic acid may therefore play a role in the effect of exercise on immune function. When immunological parameters and antioxidant status were measured in adult males fed 250 mg day”! of vitamin C for 4 days, followed by 5 mg day-l for 32 days, plasma ascorbic acid and glutathione decreased and impairment of antioxidant status became evident from a doubling in semen 8- hydroxydeoxyguanosine concentration (a measure of oxidative damage to nucleic acids) during the second dietary period (Jacob et al., 1991). A fallin 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 the 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 enhance components related to cell-mediated immunity (see Hughes, Chapter 9, Prasad, Chapter 10, and McKenzie et al., Chapter 12, this volume). The reverse situation applies when antioxidant defences become depleted. Sulphur Amino Acids, Glutathione and Immune Function 143 The oxidant molecules produced by the immune system to kill invading organisms may activate at least two important families of proteins that are sen- sitive to changes in cellular redox state. The families are nuclear transcription factor kappa B (NFkB) and activator protein 1 (AP1). These transcription fac- tors act as “control switches” for biological processes, not all of which are of advantage to the individual. NFxB is present in the cytosol in an inactive form, by virtue of being bound to IxB. Phosphorylation and dissociation of IxB ren- ders the remaining NFkB dimer active. Activation of NFxB can be brought about by a wide range of stimuli, including pro-inflammatory cytokines, hydro- gen peroxide, mitogens, bacteria and viruses and their related products, and UV and ionizing radiations. The dissociated IxB is degraded and the active NFKB is translocated to the nucleus, where it binds to response elements in the promoter regions of genes. A similar translocation of AP1, a transcription factor composed of the proto-oncogenes c-fos and c-jun, from cytosol to nucleus also occurs in the presence of oxidant stress. Binding of the transcription factors is implicated in the activation of a wide range of genes associated with inflamma- tion and the immune response, including those encoding cytokines, cytokine receptors, cell-adhesion molecules, acute-phase proteins and growth factors (Schreck et al., 1991). Unfortunately, NFxB also activates transcription of the genes of some viruses, such as HIV. This sequence of events in the case of HIV accounts for the ability of minor infections to speed the progression of individuals who are infected with HIV towards AIDS, since, if antioxidant defences are poor, each encounter with general infections results in cytokine and oxidant production, NFxB activation and an increase in viral replication. It is thus unfortunate that reduced cellular concentrations of GSH are a common feature of asymptomatic HJWV infection (Staal et al., 1992). Oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. This situation may lead to up-regulation of NF«B activity, since the transcription factor has been shown to be activated in endothelial cells cultured with linoleic acid, the main dietary n-6 polyunsatu- rated fatty acid, an effect inhibited by vitamin E and NAC (Hennig et al., 1996). The interaction between oxidant stress and an impaired ability to syn- thesize glutathione, which results in enhanced inflammation, is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize GSH (Pena et al., 1999). In this study, an inverse relation- ship between glutathione concentration and the ability of monocytes to pro- duce IL-1, IL-8 and TNF-« was observed. Furthermore, treatment of the patients with the GSH pro-drug oxothiazalidine-4-carboxylate (procysteine) (Fig. 7.7) increased monocyte GSH content and reduced IL-1, IL-8 and TNF-« production. Thus, antioxidants might act to prevent NFkB activation by quenching oxidants. However, not all transcription factors respond to changes in cell redox state in the same way. When rats were subjected to depletion of effective tissue GSH pools by administration of diethyl maleate, there was a sig- nificant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (Robinson et al., 1993). In an in vitro study using HeLa cells and cells from human embryonic kidney, both TNF and hydrogen peroxide resulted in 144 R.F. Grimble Methionine NAC oTZ Glutamine Glutathione synthesis «— | Glycine Fig. 7.7. Nutrients and drugs that are important for enhancing glutathione synthesis. NAC, N-acetyl cysteine; OTZ, L-2-0xothiazolidine-4-carboxylate. activation of NFkB and AP1 (Wesselborg et al., 1997). Addition of the antioxi- dant sorbitol to the medium suppressed NFxB activation (as expected) but (unexpectedly) activated AP1. Thus, the antioxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g. NFkB) and cellular proliferation (e.g. AP1). Evidence for this biphasic effect was seen when glutathione was incubated with immune cells from young adults (Wu et al., 1994). A rise in cellular glutathione content was accompanied by an increase in IL-2 production and lymphocyte prolifera- tion and a decrease in production of the inflammatory mediators prostaglandin E, (PGE;) and leucotriene B, (LTB,) Modification of the glutathione content of liver, lung, spleen and thymus in young rats, by feeding diets containing a range of casein (a protein with a low sulphur amino acid content) concentra- tions, changed immune cell numbers in the lung (Hunter and Grimble, 1994). It was found that, in unstressed animals, the number of lung neutrophils decreased as dietary protein intake and tissue glutathione content fell. However, in animals given an inflammatory challenge (endotoxin), liver and lung GSH concentrations increased directly in relation to dietary protein intake. Lung neutrophils, however, became related inversely with tissue glutathione content. Addition of methionine to the protein-deficient diets normalized tissue glutathione content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet of adequate protein content. Thus it can be hypothesized that antioxidants exert an immunoenhancing effect, by activating transcription factors that are strongly associated with cell proliferation (e.g. AP1), and an anti-inflammatory effect, by preventing activa- tion of NFkB by oxidants produced during the inflammatory response. Strategies for Modulating Tissue GSH Content and Improving Immune Function A number of strategies have evolved to raise levels in depleted individuals. As shown in Fig. 7.7, there are three potential ways of enhancing cellular GSH content: administration of the three amino acids (cysteine, glutamic acid and Sulphur Amino Acids, Glutathione and Immune Function 147 (Grimble, 1999). The effect was more marked in cells from old than from voung animals. Taurine has been shown to ameliorate inflammation in trini- trobenzene sulphonic acid-induced colitis. Taurine interacts with hypochlorous acid, produced during the 'oxidant burst” of stimulated macrophages, to produce taurine chloramine (TauCl). This compound may have important immunomodulatory properties and may be responsible for properties that have been ascribed earlier to taurine. TauCl has been shown to inhibit nitric oxide, PGE,, TNF-« and IL-6 production from stimulated macrophages in culture and to inhibit the ability of antigen-present- ing cells to process and present ovalbumin (Grimble, 1999). 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