GLUTATHIONE SYNTHESIS, EFECT OF NUTRITION ON REGULATORY CONTROL MECHANISM

1. GLUTATHIONE SYNTHESIS, EFECT OF NUTRITION ON REGULATORY CONTROL MECHANISM Mohammad Hanafi, MBBS (Syd).,dr.,MS.) Senior Lecturer in Biochemistry Medical Faculty Unair

2. 1.Introducion • Glutathione (γ-glutamylcysteinylglycine, GSH) is a sulfhydryl (-SH) antioxidant, antitoxin, and enzyme cofactor. • Glutathione can be found in animals, plants, and microorganisms. • It is found mainly in the cell cytosol and other aqueous phases of the living system • It one of the most highly concentrated intracellular antioxidants.

3. Introducion (continue)

4. Introducion (continue) • Reduced or oxidized form (GSH GSSG) • In the healthy cell GSSG, the oxidized (electron-poor) form, rarely exceeds 10 percent of total cell glutathione. • Studies have led to the free radical theory of human diseases and to the advancement of nutritional therapies to improve GSH status under various pathological conditions.

5. Introducion (continue) • The adequate provision of sulfur-containing amino acids as well as glutamate or glutamine and glycine (or serine) is critical for the maximization of GSH synthesis. • This article reviews the synthesis of glutathione and the nutritive factors that may modulate the synthesis of glutathione. • Methionine and selenium requirement described in more detail.

6. 2.Glutathion synthesis • The first step of GSH synthesis is rate-limiting and catalyzed by glutamate-cysteine ligase (GCL) formerly referred to as -glutamylcysteine synthetase (GCS). This enzyme absolutely require either Mg++ or Mn++. • GCL, heterodimer made up of the heavy sub unit (73 kDa) has the catalytic activity (GCLC), and is the site of GSH feed back inhibition • The light (30 kDa) or modifier (GCLM) sub unit, alter, or regulate the activity of the holoenzyme

7. Glutamate cysteine ligase (GCL) L-glutamate + L-cysteine + ATP -L-glutamyl-L-cysteine + ADP + Pi GCL is regultate physiologically by : • Feed back competitive inhibition by GSH • The availability of its precurser, L-cysteine.

8. The availability of its precurser, L-cysteine. • The cysteine content of the diet liver Cysteine transport Cystine transport Methionine transport Transsulfuration pathway

9. Cysteine transport • Na+ dependent • High stereospecificity • pH sensitive • Not sensitive to adaptive regulation or insulin and glucagon stimulation • Both inward and outward flows • Influence by the extracellular levels of cysteine, and other system amino acids transporters

10. Cystine transport • Na+ independet • Influence by pH change • Entry of cystine accompanied by an exit of glutamate • High intracellular glutamate concentration functions to stimulate the influx of cystine to maintain an adequate balance between cysteine and glutamate inside the cells • Influence by : insulin, electrophilic agents and O2. • In isolated hepatocytes, treatment with electrophilic agents depletes intracellular GSH

11. Methionine transport • Na+ independent • Not responsive to either adaptive control or hormonal stimulation Cys, Met, and Cys transport Rat hepatocytes cultured for 24 h, the rate of uptake of cysteine is about 3-fold higher than that of methionine and 13-fold higher than that of cystine.

12. Transsulfuration pathway • Ability for the liver cell to convert methionine to cysteine is important since the liver is the major site of methionine catabolism and the major storage organ for GSH • It is absent or insignificant in other GSH-synthesizing systems • Markedly impaired or absent in the fetus and newborn infant, cirrhotic patients, and patients with homocystinemia • the Km of hepatic methionine adenosyltransferase for ATP is high (2 mM) • hypoxic depletion of ATP is more likely to affect GSH synthesis from methionine than cysteine

13. Transsulfuration pathway • Methionine and homocysteine are readily interconvertible • The transsulfuration pathway converts methionine to cysteine, which is then converted to GSH via the GSH synthetic pathway • Methionine can also be resynthesized from homocysteine • The control appears to be exerted at the level of homocysteine: when methionine is needed, homocysteine is remethylated by methionine synthase or betaine-homocysteine methyltransferase; when methionine is in excess, catabolism of homocysteine via the cystathionine synthase reaction is accelerated

14. 1.methionine adenosyltransferase Transsulfuration pathway Folate, vit B12 8. methionine synthase 9.betaine-homocysteine methyltransferase. Vit B6 2.Transmethylation reaction 3.S-adenosylhomocysteine hydrolase 5.gama-cystathionase 4.cystathionine β-synthase 6,γ-glutamylcysteine synthetase (GCL) 7.GSH synthetase

15. Glutamate-cysteine ligase (GCL) activity • Other major determinant of the rate of GSH synthesis • Affecting heavy (GCLC) or both the heavy and light (GCLM) • Transcriptional and post-transcriptional regulation of both subunits Post-transcriptional -both mRNA stabilization -destabilization and -post-translational modification

16. Activity of GCL will be influenced by : • Oxidative stress, insulin and many others increase GCL transcription or activity in variety of cells • Dietary protein deficiency,dexamethasone, and GCL phosphorylation decrease GCS transcription or activity • NO production ↑ NO production loss of GSH ↓ NO production prevent GSH depletion

17. Glutathione synthase (GS) -L-glutamyl-L-cysteine + glycine + ATP GSH + ADP + Pi • The regions of the active site that bind glycine and the cysteinyl moiety of γ-glutamylcysteine are highly specific • It’s activity is modified ADP • GSH synthase is not subject to feedback inhibition by GSH • GSH synthase deficiency γ-glutamylcysteine is converted to 5-oxoproline

18. g-Glu-Cys-Gly S S g-Glu-Cys-Gly Biosynthesis of glutathione GSH synthase (GS) Glutamate-cysteine ligase (GCL) disulfide bond

19. Glutathione distribution • Glutathione synthesis in the body occure mainly in liver • About 80 percent of the GSH synthesized in the liver is exported from the hepatocytes • Most of this is utilized by the kidneys • GSH, to supply cysteine as needed • Circulating GSH is safe; it reacts only slowly with Oxygen, is less susceptible to auto-oxidative degradation than is cysteine • Soluble in the plasma • GSH comes in with the diet (150 mg daily by rough estimate) • Hormones and other vasoactive substances increase GSH efflux into the bile

20. Strategies for Repleting Cellular Glutathione • Oral dosing with GSH An oral bolus of 15 mg/kg to the human to raise plasma GSH two-to five-fold The intestinal lumen absorb GSH via non-energy- requiring, carrier-mediated diffusion, and later export it into the blood (lung alveolar cells, vessel endothelial cells, retinal pigmented epithelial cells, and cells of the kidney's proximal tubule; it seems also to cross the blood-brain barrier; while brain endothelial and nerve cells, red blood cells, lymphocytes - appear incapable of absorbing GSH as the intact tripeptide)

21. L-methionine • Is an essential amino acid • It must first be converted to cysteine • This convertion is inactive in neonates and in certain adults, such as patients with liver disease • The "activated" methionine metabolite known as SAM (S-adenosyl methionine) is effective in raising red cell GSH and hepatic GSH when given orally at 1600 mg per day

22. L-methionine • L-methionine intake is 13 mg per kg or about 0.75 gram daily for adults • During methionine supplementation, intake B6 and folic acid should also be included • Excessive methionine intake, together with inadequate intake of folic acid, vitamin B6, and vitamin B12, can increase the conversion of methionine to homocysteine • Homocysteine is a potentially harmful blood fat that has been linked to atherosclerosis

23. Table 1. Methionine content of 100gr edible food in mg. There are about 15 to 16 eggs in one kg ( at Rungkut market), ie equal to 66.7 - 62.5 gr/egg

24. L-cysteine • Cysteine is probably unsafe for routine oral administration • In the blood it readily auto-oxidizes to potentially toxic degradation products • The auto-oxidation Hydroxyl radical • The cystine produced from cysteine oxidation is taken up into the kidney, and requires energy and enzymatic intervention to be converted to cysteine

25. N-acetyl cysteine (NAC) • NAC is a cysteine precursor; it is well absorbed by the intestine • Metabolized by the liver, cysteine is one of metabolite • It seems not to raise GSH levels if they are already within the normal range • It can raise abnormally low GSH levels back to normal • This is the basis for its use as an antidote to acetaminophen's liver toxicity • Used in Europe for many years as a mucolytic agent • 600 mg was beneficial and innocuous while 1200 mg and 1800 mg per day caused significant adverse effects

26. Alpha lipoic acid • Alpha lipoic acid (ALA) is available as supplement • It is a natural antioxidants and could increase intracellular glutathione through it reducing power

27. Selenium • Is a co-factor for the enzyme glutathione peroxidase • Studies suggest they may play a role in decreasing the risk of certain condition in which glutathione are depleted • Too much selenium can cause toxic effects including gastrointestinal upset, brittle nails, hair lost and mild nerve damage • Upper estimated requirement of 90 μg Se/d • Lower estimated requirement of 39 μg Se/d

28. Table 2. Selenium content of food in μg unit

29. Concluding remark • Advances in molecular biology have led to an explosion of knowledge in understanding how the rate-limiting enzyme GCS is regulated at the molecular level, and prevention of complications that may result from altered GSH synthesis • Protein (or amino acid) deficiency remains a significant nutritional problem in the world to day. In developing country where source of protein is very expensive beyond their budget owing to inadequate income; under nutrition, specially protein deficiency become a great problem

30. Concluding remark (continue) • Thus, new knowledge regarding the efficient utilization of dietary protein or the precursors for GSH synthesis and its nutritional status is critical for the development of effective therapeutic strategies to prevent and treat a wide array of human diseases, especially those condition or diseases that are related to oxidative stress • High methionine content of meat, fish, and dairy product probably meet the daily requirement. In certain condition such as hepatic cirrhosis the ability to convert methionine to SAM is disturbed. Therefore methionine is given in SAM form. SAM is easily available as drug in Europe • Selenium is cofactor for glutathione peroxidase. Suffice to take an action by consuming it even though research still needed to confirm the cost benefits. Food consumed every day contain selenium, probably enough to meet our need