Hyperhomocysteinaemia A Risk Factor Worth Considering

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1 REVIEW ARTICLE JIACM 2003; 4(2): Hyperhomocysteinaemia A Risk Factor Worth Considering Pramood C Kalikiri* At least nine well-known risk factors are known to play a role in the development of coronary artery disease (also known as ischaemic heart disease (IHD)) and cerebrovascular accidents (Stroke): heredity, male gender, advancing age, cigarette smoking, high blood pressure, diabetes mellitus, obesity (especially excess abdominal fat), lack of physical activity, and abnormal blood cholesterol levels (more than 200 mg %). The more of these risk factors a person has, the greater the likelihood of IHD and CVA. Clinical research done over the last decade has shown that an elevated plasma level of the amino acid homocysteine is an independent risk factor for atherosclerosis, including coronary artery disease, cerebrovascular disease, peripheral vascular disease, and venous thromboembolism 1. Molecular species of homocysteine Homocysteine is a sulphur-containing amino acid formed during methionine metabolism. Its concentration in the plasma or serum is about 10 µmol/ L. However, homocysteine exists in various forms; only trace amounts ( 1%) are in the reduced (sulfhydryl) form, the remaining part is oxidised and exists as various disulphides 2. It can dimerise to homocystine, or form disulphide bonds with proteins to form socalled protein-bound homocysteine. About 80% of homocysteine is bound to albumin (via a disulphide bond) in the plasma, whereas the remaining 20% exists as free disulphides. Outline of methionine/homocysteine metabolism Homocysteine is not a dietary constituent and is not incorporated into proteins but is exclusively formed as an intermediary product of methionine metabolism. Through the action of methionine adenosyltransferase, methionine is converted to S-adenosylmethionine (SAM), which is the major biological methyl donor required for numerous cellular processes, including the formation of proteins, nucleic acids, and creatinine. These reactions are catalysed by various methyltransferases that demethylate S- adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). S-adenosyl homocysteine (SAH) is hydrolysed to simultaneously produce homocysteine and adenosine by SAH hydrolase. The SAH hydrolase catalysed step is bidirectional and favours condensation of homocysteine and adenosine 3. Once homocysteine is formed, it may be salvaged to methionine by remethylation, or degraded to cysteine by trans-sulfuration. Remethylation to methionine is in most tissues catalysed by the ubiquitous enzyme, methionine synthase (MS). This enzyme uses vitamin B12 as co-factor, and methyltetrahydrofolate as methyl donor. 5- methyltetrahydrofolate is formed from folic acid by the vitamin B2-dependent enzyme methyl tetrahydrofolate reductase (MTHFR). Reconversion of homocysteine to methionine also contributes to the maintenance of intracellular stores of tetrahydrofolate. Homocysteine may also be converted to methionine by betainehomocysteine methyltransferase using betaine as a methyl donor. This reaction is probably confined to the liver and possibly the kidney. Two vitamin B 6 -dependent enzymes are involved in the trans-sulfuration pathway. The enzyme cystathionine beta synthase (CBS) first condenses homocysteine with serine to form cystathionine, which is then cleaved into cysteine and a-ketobutyrate by cystathionine lyase. Cysteine may be utilised in the synthesis of proteins or as a precursor of the antioxidant glutathione. The trans-sulfuration of homocysteine to cysteine is irreversible, and therefore directs homocysteine to catabolism via cysteine to sulphates as the final product. * PhD - I st Year, Department of Physiology, Louisiana State University Medical Center, New Orleans, LA-70112, USA.

2 Under normal metabolic circumstances, there is a strict balance between homocysteine formation and elimination. Usually about 50% of the homocysteine formed is remethylated to methionine. When protein or methionine intake is in excess, a larger proportion is catabolised by the trans-sulfuration pathway. If there is an increased formation of homocysteine relative to its consumption, homocysteine is excreted from the cells. This can be detected as an increased level of homocysteine in plasma/serum or in the urine. Thus, homocysteine, an intermediate in protein metabolism, is involved in conversion of the amino acid methionine to cysteine or in remethylation to form methionine. Metabolism of homocysteine is by pathways which re-methylate it (and which require vitamin B 12, B 2, and folic acid), or by a trans-sulfuration pathway which requires vitamin B 6. Methionine in the diet is the main source of homocysteine in the blood. Animal proteins provide about three times as much methionine as plant protein. Homocysteine in blood (and elsewhere) is a product of how much methionine is eaten, mainly in protein, and how much is metabolised, which in turn may be affected by amounts of B vitamins and folate available. Aetiology of elevated homocysteine levels Elevated homocysteine levels are often the result of decreased activity of key enzymes involved in conversion of the amino acid methionine to cysteine or in remethylation to form methionine. A plasma homocysteine concentration exceeding 15 µmol per L is now termed hyperhomocysteinaemia 4. Normal levels of fasting plasma homocysteine are considered to be between 5 and 15 µmol/l. Moderate, intermediate, and severe hyperhomocysteinaemia refer to concentrations between 16 and 30, between 31 and 100, and >100 µmol/ L, respectively 5. The most common inherited form of hyperhomocysteinaemia results from an alteration in the gene encoding the enzyme methylene tetrahydrofolate reductase. Less often, the cause of hyperhomocysteinaemia is heterozygous cystathionine b-synthase deficiency. Homozygous form of cystathionine b-synthase deficiency results in homocystinuria, which is a rare but severe condition in which total homocysteine concentrations generally exceed 100 µmol per L, 148 Journal, Indian Academy of Clinical Medicine Vol. 4, No. 2 April-June 2003

3 sometimes even reaching levels upto 500 µmol per L if the disorder is untreated. Individuals with this inherited disorder are known to have severe hyperhomocysteinaemia and a variety of abnormalities, including a high incidence of vascular pathology that may result in early death from myocardial infarction, stroke, or pulmonary thromboembolism. Hyperhomocysteinaemia can be acquired as the result of dietary deficiencies of folate, vitamin B 12 and/or vitamin B 6. These vitamins are necessary co-factors for the optimal function of methylene tetrahydrofolate reductase, methionine synthase, and cystathionine b-synthase. Deficiencies in the absorption or transport of these vitamins can also cause hyperhomocysteinaemia. Pathogenesis of atherosclerosis in hyperhomocysteinaemia The exact mechanisms by which hyperhomocysteinaemia causes atherosclerosis are not completely understood. Animal models of hyperhomocysteinaemia show altered vascular function, including the promotion of smooth muscle cell growth and the development of atherosclerosis. Abnormal homocysteine levels appear to contribute to coronary artery disease by the following mechanisms: (a) causing endothelial dysfunction. A dysfunctional endothelium is an early marker of atherosclerosis and thrombosis 6 ; (b) causing marked impairment of endothelium-dependent vasodilatation in response to acetylcholine and adenosine diphosphate (ADP) 7 ; (c) inducing oxidative stress on endothelial cells which is mediated by hydrogen peroxide 8 ; (d) promoting the growth and proliferation of vascular smooth muscle 9 ; (e) decreasing the bioavailability of Nitric Oxide by forming S-nitrosohomocysteine 10 ; (f ) a direct toxic effect that damages the cells lining the inside of the arteries; (g) interference with clotting factors ; (h) oxidation of low-density lipoproteins (LDL) 4,19-21 ; (i) because the SAH hydrolase catalysed step is bidirectional, elevated homocysteine may cause cardiovascular disease by a reduction in plasma or tissue adenosine levels 22. Adenosine has a wide variety of protective effects on cardiovascular homoeostasis. It causes coronary and cerebral artery vasodilatation, increases blood flow in the microcirculation, inhibits platelet aggregation and decreases proliferation or growth of smooth muscle or mesangial cells 23. Relationship between blood homocysteine levels and severity of coronary artery disease The researchers have found a linear relationship between blood homocysteine levels and severity of the coronary blockages: For every 10% elevation of homocysteine, there was nearly the same (10%) rise in the risk of developing coronary artery disease 24. A similar percentage increase in cholesterol levels would imply about a 20% increased risk for heart disease. This study is important, because it is the first of its size to show a positive correlation with risk of atherosclerosis and also suggests that elevated blood homocysteine levels are as important as high blood cholesterol levels and can operate independently. Studies also suggest a linear relationship between the number of blocked arteries and homocysteine levels (both fasting and postload), regardless of sex, age, or any other risk variables, i.e., more severe the blockages in the coronary arteries, the higher the homocysteine levels. Blood homocysteine assays Most clinical laboratories use assays that report total homocysteine concentrations. Total homocysteine, abbreviated as thcy, is a methodological term and refers to the concentration of homocysteine obtained after plasma/serum has been treated with a reductant which converts the free and bound disulfides into their respective sulfhydryl compounds. Blood should be collected in tubes containing an anticoagulant such as ethylenediamine tetraacetic acid (EDTA), heparin, or sodium citrate. The specimen should be spun (centrifuged) within 30 minutes of collection to avoid a false elevation caused by the release of homocysteine from red blood cells (RBC s), a process that continues at room temperature 25. Once the plasma is separated from cells, it can be stored refrigerated for several weeks or frozen for several months. The total homocysteine concentration can also be measured in serum samples. However, the reference intervals are slightly higher than those for plasma, in part because of the continued release of homocysteine from red blood cells and the delay required for sample clotting before centrifugation. Journal, Indian Academy of Clinical Medicine Vol. 4, No. 2 April-June

4 Because variable changes in homocyst(e)ine levels have been observed postprandially 25, it is customary to obtain measurements in the fasting state. The majority of the assays are based on chromatographic techniques; high performance liquid chromatography (HPLC) with fluorescence detection is the method most commonly used 26. Normal levels of fasting plasma homocysteine are considered to be between 5 and 15 µmol/l. Screening for plasma homocysteine levels is not widely available and may cost $ 200, which is not currently covered by insurance. However, it may be useful in patients with a personal or family history of cardiovascular disease, but in whom the well-established risk factors (smoking, high blood cholesterol, high blood pressure, diabetes, physical inactivity and obesity) don t exist (American Heart Association). Management of patients with hyperhomocysteinaemia Detoxification of excess homocysteine requires methylating factors such as folic acid, vitamin B 12, and trimethylglycine (TMG). Methylation factors donate methyl groups to homocysteine and function to convert (remethylate) homocysteine back into the non-toxic amino acid methionine. Some individuals may also require higher amounts of vitamin B 6, which converts homocysteine to cysteine and sulfate via trans-sulfuration pathway. Regardless of the cause of hyperhomocysteinaemia, most patients should derive some benefit from vitamin supplementation via the conversion of homocysteine back to methionine (remethylation) or to cysteine (trans-sulfuration). Homocysteine levels usually decrease after a few weeks of therapy and normalise within six to eight weeks. In a placebo-controlled study 27, a combination of multiple agents including folic acid (0.65 mg/d), vitamin B 6 (10 mg/d), and vitamin B 12 (0.4 mg/d) was very effective in reducing homocysteine levels in patients with moderate or intermediate hyperhomocysteinaemia. At the time of the initial evaluation, a serum B 12 level should be obtained to ensure that intake of this vitamin is adequate before folic acid supplementation is initiated because the neurological features associated with vitamin B 12 deficiency persist with folic acid supplementation despite improvement in other symptoms and signs of B 12 deficiency. A recent report of the Food and Nutrition Board of the Institute of Medicine has recommended an upper limit of 1 mg/d folic acid on the basis of the possibility that higher doses may mask signs of vitamin B 12 deficiency in some subjects. Vitamin B 12 deficiency can still be detected clinically by the history and physical examination, along with serum methylmalonic acid or vitamin B 12 measurement, factors that are not altered by folic acid supplementation 28. Conclusion Although there is considerable epidemiological evidence for a relationship between plasma homocysteine and coronary artery disease, not all prospective studies have supported such a relationship. Reductions in homocysteine levels may be achieved by proper diet and vitamin supplementation. It has not yet been proved, however, that a reduction in homocysteine levels reduces the risk of coronary artery disease. The results of clinical trials to determine the potential benefit of reducing homocysteine concentrations are not yet available. However, given the substantial risk associated with hyperhomocysteinaemia and the fact that homocysteine levels can be lowered with non-toxic vitamin therapy; the screening of patients at high risk for cardiovascular disease appears to be warranted. Until studies have been completed, it is prudent to advise patients with, or at high risk, for vascular disease, to take enough folic acid and vitamins B 6 and B 12 in their diet. They should eat at least five servings of fruits and green, leafy vegetables daily (American Heart Association). References 1. Clarke R, Daly L, Robinson K et al. Hyperhomocysteinaemia: an independent risk factor for vascular disease. N Engl J Med 1991; 324: Ueland PM. Homocysteine species as components of plasma redox thiol status. Clin Chem 1995; 41: Ueland PM. Pharmacological and biochemical aspects of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacol Rev 1982; 34: Robinson K, Mayer E, Jacobsen DW. Homocysteine and coronary artery disease. Cleve Clin J Med 1994; 61: Kang SS, Wong PWK, Malinow MR. Hyperhomocyst(e)- inaemia as a risk factor for occlusive vascular disease. Ann Rev Nutr 1992; 12: Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: Journal, Indian Academy of Clinical Medicine Vol. 4, No. 2 April-June 2003

5 7. Lentz SR, Sobey CG, Piegors DJ et al. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inaemia. J Clin Invest 1996; 98: Hultberg B, Andersson A, Isaksson A. The cell-damaging effects of low amounts of homocysteine and copper ions in human cell line cultures are caused by oxidative stress. Toxicology 1997; 123: Tsai JC, Wang H, Perella MA et al. Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J Clin Invest 1996; 97: Stamler JS, Osborne JA, Jaraki O et al. Adverse vascular effects of homocysteine are modulated by endothelium derived relaxing factor and related oxides of nitrogen. J Clin Invest 1993; 91: Rodgers GM, Kane WH. Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J Clin Invest 1986; 77: Hajjar KA. Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor. J Clin Invest 1993; 91: Hajjar KA, Mauri L, Jacovina AT et al. Tissue plasminogen activator binding to the annexin II tail domain - direct modulation by homocysteine. J Biol Chem 1998; 273: Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein-c activation by arterial and venous endothelial cells. Blood 1990; 75: Lentz SR, Sadler JE. Inhibition of thrombomodulin surface expression and protein-c activation by the thrombogenic agent homocysteine. J Clin Invest 1991; 88: Hayashi T, Honda G, Suzuki K. An atherogenic stimulus homocysteine inhibits cofactor activity of thrombomodulin and enhances thrombomodulin expression in human umbilical vein endothelial cells. Blood 1992; 79: Nishinaga M, Ozawa T, Shimada K. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells. J Clin Invest 1993; 92: Fryer RH, Wilson BD, Gubler DB et al. Homocysteine, a risk factor for premature vascular disease and thrombosis, induces tissue factor activity in endothelial cells. Arterioscler Thromb 1993; 13: Parthasarathy S. Oxidation of low-density lipoprotein by thiol compounds leads to its recognition by the acetyl LDL receptor. Biochim Biophys Acta 1987; 917: Durand P, Lussier-Cacan S, Blache D. Acute methionine loadinduced hyperhomocysteinaemia enhances platelet aggregation thromboxane biosynthesis, and macrophage derived tissue factor activity in rats. FASEB J 1997; 11: Domagala TB, Libura M, Szczeklik A. Hyperhomocysteinemia following oral methionine load is associated with increased lipid peroxidation. Thromb Res 1997; 87: Ya-Fei Chen, Pin-Lan Li, Ai-Ping Zou. Effect of hyperhomocysteinaemia on plasma or tissue adenosine levels and renal function. Circulation 2002; 106: Biaggioni I, R Mosqueda-Garcia. Adenosine in cardiovascular homeostasis and the pharmacologic control of its activity. In: Laragh JH, Brenner BM, eds. Hypertension, Pathophysiology, Diagnosis, and Management. 2nd ed. New York, NY: Raven; 1995; Verhoef P, Others. Plasma total homocysteine, B vitamins, and risk of coronary atherosclerosis. Arteriosclerosis, Thrombosis, and Vascular Biology 1997; 17: Ueland PM, Refsum H, Stabler SP et al. Total homocysteine in plasma or serum: methods and clinical applications. Clin Chem 1993; 39: Vester B, Rasmussen K. High performance liquid chromatography method for rapid and accurate determination of homocysteine in plasma and serum. Eur J Clin Chem Clin Biochem 1991; 29: Ubbink JB, Becker PJ, Vermaak WJ, Delport R. Results of B- vitamin supplementation study used in a prediction model to define a reference range for plasma homocysteine. Clin Chem 1995; 41: Dickinson CJ. Does folic acid harm people with vitamin B 12 deficiency? QJM 1995; 88: Journal, Indian Academy of Clinical Medicine Vol. 4, No. 2 April-June

Nitrous Oxide induced Elevation of Plasma Homocysteine and Methylmalonic Acid Levels and their Clinical Implications

SHORT COMMUNICATION JIACM 2005; 6(1): 48-52 Abstract Nitrous Oxide induced Elevation of Plasma Homocysteine and Methylmalonic Acid Levels and their Clinical Implications Pramood C Kalikiri*, Reena G Sachan*