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Hyperhomocysteinemia in peripheral arterial disease
(Portuguese PDF version)

Luciene de Souza Venâncio¹, Roberto Carlos Burini², Winston Bonetti Yoshida³

1. Nutritionist. PhD student in General Bases of Surgery, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brazil.
2. Professor, Department of Public Health, Chief of the Center of Nutrition and Metabolism, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brazil.
3. Professor, Department of Surgery and Orthopedics, Faculdade de Medicina de Botucatu, Universidade Estadual Paulista, Botucatu, SP, Brazil.

Correspondence:
Luciene de Souza Venâncio
Rua Chico Padre, 44/31
CEP 18611-310 - Botucatu, SP, Brazil.
Phone: +55 (14) 3882.6570
E-mail: lucienenutri@hotmail.com

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ABSTRACT

Recent studies indicate that a high plasma homocysteine level is an important and prevalent risk factor for cardiovascular, cerebral and peripheral atherosclerotic diseases. Homocysteine is a sulphur-containing amino acid used in several metabolic pathways. Hyperhomocysteinemia can be attributed to the occurrence of genetic defects of some enzymes which are part of the metabolism of homocysteine, to nutritional deficiencies of vitamins and folate, or to others risk factors for atherosclerosis. Some likely biological mechanisms of vascular injury caused by the hyperhomocysteinemia have been suggested, particularly the oxidation of low density lipoprotein (LDL) cholesterol. On the other hand, some studies evidenced that nutritional supplementation with folate results in an efficient reduction in plasma homocysteine concentration. This review will approach the metabolism of homocysteine and its relationship with peripheral arterial disease, and will discuss the causes, pathogenic mechanisms and the possibilities for treatment of the hyperhomocysteinemia.

Key-words: homocysteine, atherosclerosis, vascular diseases.
Palavras-chave: homocisteína, aterosclerose, doenças vasculares.

J Vasc Br 2004;3(1):31-7

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Today, atherosclerosis is the main cause of deaths in Brazil¹ as well as throughout the world.² Atherosclerotic lesions lead to 95% of cardiopathies, 85% of intermittent claudications of lower limb and 75% of cerebrovascular accidents.³ Peripheral arterial disease (PAD), which affects the aorta, its branches and limb arteries, presents a high prevalence, affecting 29% of North-Americans, and is associated with an increase in the rate of morbidity and mortality of cardiovascular and cerebrovascular diseases.⁴ In Brazil, there are no epidemiological data about its incidence, but it is estimated that it is not much different from that of other countries.⁵ It is believed that its prevalence among the general population is being underestimated because the atherosclerotic process remains subclinical and asymptomatic for a long time.⁴

Some risk factors for atherosclerosis are currently well known, such as age, sex, dyslipidemia, smoking behavior, arterial hypertension, diabetes mellitus, obesity and genetic factors or family history of atherosclerosis.⁶ However, usually such risk factors are not identified in patients with PAD. Thus, recent researches are aiming at identifying new factors which can be involved in the genesis of atherosclerosis. Among such factors, homocysteine has been the theme of several articles in international literature.

HEMOCYSTEINE METABOLISM

Homocysteine was discovered by Vincent Du Vigneaud in 1932, when he published a pioneering article about the importance of this amino acid in biochemistry and in nutrition. More recent studies with homocysteine in children evidenced the association between the increase of plasma homocysteine levels and atherosclerotic and thromboembolic processes, such as acute myocardial infarction, cerebrovascular accident and early death.⁷ Recently, the association between a mild increase of plasma homocysteine levels and premature vascular disease in adults was demonstrated.⁸ Homocysteine is a sulphur amino acid, which contains the sulfhydryl group (SH) in its structure. Such amino acid is not part of our daily diet and it does not take part in protein synthesis; it is an intermediate product derived from the intracellular metabolism of methionine.⁹

The metabolism of homocysteine is at the intersection of two pathways: remethylation and transsulfuration (Figure 1). In remethylation, homocysteine receives a methyl group from N⁵ methyltetrahydrofolate (N⁵MTHF) or from betaine in order to form methionine. The formation of N⁵MTHF is dependent upon the enzyme methylenetetrahydrofolate reductase (MTHFR).The reaction with N⁵MTHF occurs in every tissue and is dependent upon vitamin B12. In the transsulfuration pathway, which takes place mainly in the liver and in the kidneys, the enzyme cystathionine beta-synthase (CBS) condenses homocysteine with serine in order to form cystathionine through an irreversible reaction dependent upon pyridoxal phosphate (vitamin B6), forming cysteine at the end. In normal metabolic conditions, there is a rigorous balance between formation and elimination of homocysteine.¹⁰

Figure 1 - Scheme for the metabolic pathways of homocysteine.¹¹

In animal tissues, homocysteine may be present in its reduced form, that is, with a free sulfhydryl group, but in small amounts. The major part is in the form of homocystine (a disulfide oxidation product of homocysteine), homocysteine-cysteine mixed disulfide and protein-bound homocysteine (specially albumin). All these types of homocysteine taken together are named total homocysteine or plasma homocysteine.⁹

CLASSIFICATION OF HYPERHOMOCYSTEINEMIA

Normal concentration of homocysteine in plasma is approximately 10 µmol/L, with values ranging between 5 a 15 µmol/L;¹² values above these characterize hyperhomocysteinemia.¹³ Kang et al.¹⁴ randomly classified hyperhomocysteinemia in severe form (concentrations of over 100 µmol/L), intermediate form (concentrations between 31 and 100 µmol/L) and moderate form (concentrations between 15 and 30 µmol/L). Severe hyperhomocysteinemia is also called homocystinuria.¹⁴

HYPERHOMOCYSTEINEMIA IN PAD

Many epidemiologic studies have demonstrated that increase in plasma homocysteine concentrations may be an additional risk factor for coronary,^15-17 cerebrovascular^18-19 or peripheral^13,14,20,21 vascular diseases, as well as for venous thromboembolism.²² Hyperhomocysteinemia was diagnosed in 28% to 30% of patients with PAD.^23,24 Some authors reported case-control trials in which mean levels of homocysteine in patients with several manifestations of PAD were significantly higher than in control patients; such manifestations included intermittent claudication,²⁵ ileofemoral oclusion,¹³ Leriche syndrome,²⁶ carotid stenosis^27-30 and abdominal aortic aneurysm.^21,31 In our experience, hyperhomocysteinemia was diagnosed in 60% of patients with confirmed PAD, predominantly the moderate form, with a significantly higher prevalence among male patients of over 60 years old.³² Other studies carried out in Brazil also revealed a high prevalence of hyperhomocysteinemia (20%) among Brazilians of Japanese descent who have peripheral atherosclerotic disease, with mean values of homocysteine concentration progressively higher among males and according to the severity of the glycemic status.^33,34 The relevance of hyperhomocysteinemia as an additional risk factor for PAD is widely known, with a risk of 6.8³⁵ to 11³⁶ for its development. Furthermore, hyperhomocysteinemia is associated with an increased risk for early death due to cardiovascular disease,³⁷ and progression of PAD^38,39 and of coronary arterial disease in patients with symptomatic PAD.^38,40 The clinical analysis of plasma homocysteine as a predictor of PAD is considered of great importance, specially in patients with premature atherosclerosis or family history of atherothrombosis without any other risk factors.^32,41

PATHOGENIC MECHANISMS OF HYPERHOMOCYSTEINEMIA

Despite the great amount of epidemiologic data evidencing the relation between hyperhomocysteinemia and an increased risk for cardiovascular diseases (cerebrovascular accident, myocardial infarction, PAD and venous thrombosis), the mechanisms through which hyperhomocysteinemia contribute to atherogenesis and thrombogenesis are still only partially understood. Studies show that one of the mechanisms through which homocysteine leads to vascular lesions is injury to the endothelium. Pioneering in vivo studies carried out by Harker et al. in 1974⁴² and 1976⁴³ with baboons suggested that the endothelial injury was caused by endothelial desquamation, proliferation of smooth muscle cells and intimal thickening, mediated by platelet function, with rapid formation of typical vascular lesions, which are similar to early atherosclerotic lesions in human beings due to the infusion of homocysteine. Recent laboratory studies with human beings and animals suggested that moderate hyperhomocysteinemia would alter the production of endothelium-derived nitric oxide, which is an efficient platelet inhibitor and vasodilator. Homocysteine would affect the synthesis of nitric oxide in a dose-dependent manner inhibiting endothelial nitric oxide synthetase, which would lead to acute vascular events, particularly in individuals with other risk factors.⁴⁴ There are also investigations on the possibility that moderate hyperhomocysteinemia would have an important role in endothelial dysfunction because of oxidative mechanisms. In vitro studies with endothelial cells in culture evidenced that auto-oxidation of homocysteine in plasma would produce oxygen-derivative species, including hydrogen superoxide and peroxide, which would be associated with vascular toxicity, proliferation of smooth muscle cells and oxidation of low-density lipoprotein (LDL) cholesterol. Thus, it could be related to the formation of squamous cells and fatty streaks, which are characteristic of atherosclerotic lesions.^45,46 Other effects of homocysteine would be alterations in anti-thrombotic properties of the vascular endothelium. In vitro studies in cells exposed to homocysteine evidenced an increase in coagulation factors XII and V, reduction in protein C activation, inhibition of tissue plasminogen activator, reduction in bioavailability of nitric oxide and prostacyclin, inhibition of platelet aggregation, increase in activity of von Willebrand factor, inhibition of thrombomodulin expression, induction of tissue factor expression and suppression of heparin sulfate expression in the vascular wall.⁴⁷ All these alterations would cause vascular thrombosis, activating the coagulation cascade and changing the muscle tonus. The various effects of homocysteine described in these studies evidence that a hypothesis to explain the atherothrombogenic effects of homocysteine is still lacking.

CAUSES OF HYPERHOMOCYSTEINEMIA

The most common causes of hyperhomocysteinemia in the general population are related to genetic defects in enzyme or to deficiency of vitamins which are part of the metabolism of homocysteine. Acquired hyperhomocysteinemia is usually secondary to heterozygous deficiencies of cystathionine beta-synthase (CBS) and methylenetetrahydrofolate reductase (MTHFR), with respective prevalence of 1% and 0.5% in the general population.⁴⁸ It would be associated with moderate and intermediate hyperhomocysteinemia.⁴⁹ Heterozygosity for CBS was diagnosed in 30% of patients with symptomatic premature PAD observed by Boers et al.,²³ and 28% of these patients had increased plasma homocysteine levels. Severe hyperhomocysteinemia is secondary to homozigosity for CBS deficiency, with an estimated incidence of 1:335,000 births among the general population. Homozygosity for MTHFR (thermolabile variant) deficiency is present among over 5% of the general population and among 14% to 17% of patients with vascular diseases.⁴⁹ Homozygosity for MTHFR was diagnosed in 16.7% of patients with PAD, who had homocysteine levels mildly increased.⁵⁰ In Brazil, it was found that only 4% out of 296 healthy patients were homozygous for MTHFR. However, the relation of this finding with plasma homocysteine levels was not studied yet.⁵¹

Among non-genetic causes for hyperhomocysteinemia, nutritional status seems to be the most important aspect in regulating homocysteine concentration, specially in terms of deficiencies in vitamins B12, B6 and folate, which are co-factors in the metabolism of homocysteine.⁵² Such deficiencies are highly prevalent and occur in many cases of moderate hyperhomocysteinemia. Plasma homocysteine concentration is inversely related to plasma levels of folate, vitamins B6 e B12⁵³ and to the intake of such vitamins.^35,37,54 On the 20th examination of the Framingham Study, approximately 30% out of 1,160 elderly patients evaluated (ages ranging from 67 to 96 years) had a mild increase in homocysteine levels (> 14 µmol/L), and 67% of such cases of hyperhomocysteinemia were attributed to inadequate plasma concentrations and dietary habits in terms of one or more vitamins of the B complex.⁵⁴ Thus, the diagnosis of vitamin deficiencies is important for the identification of individuals who can have hyperhomocysteinemia in the future.

Some clinical situations may develop with a high plasma homocysteine level, such as reduced renal function, some chronic diseases (as severe psoriasis), hepatic diseases, some types of cancer and Alzheimer disease,⁵⁵ the use of drugs which interact with the metabolism of folate (such as corticoids, cyclosporines, anticonvulsants and diuretics) or with the metabolism of vitamin B12 (such as nitrous oxide).⁸ Some other factors have been related to the increase in plasma homocysteine levels. It is known that homocysteine levels in men and postmenopausal women are generally higher than in premenopausal women,^56,57 and that there is a progressive increase in homocysteine levels with age in both sexes.^35,53,58,59 Another aspect related to the increase of homocysteine concentration to be considered is the influence of the individual's life style. Smoking behavior, sedentary habits, excessive and chronic consumption of alcohol and coffee and the presence of risk factors for arterial vascular disease (including arterial hypertension, high level of total cholesterol and LDL cholesterol, low level of HDL cholesterol and obesity) may be associated with an increase in homocysteine concentrations in adults.^58,60,61 The association of diabetes mellitus types 1 and 2 with hyperhomocysteinemia is still controversial.⁸

PERSPECTIVES FOR TREATMENT

Although the increase in plasma homocysteine levels is associated with endothelial dysfunction, thrombosis and more severe atherosclerosis, there is not a consensus yet about the therapeutic possibilities. A meta-analysis by Clarke et al.⁶² indicated that, among the vitamins studied (B6, B12 and folic acid), the folic acid lead to a reduction of up to 25% in plasma homocysteine levels with a daily dietary supplementation of 50 to 500 µg of folic acid. Another meta-analysis by Boushey et al.³⁵ indicated that an increased folic acid intake of 200 µg/day would lead to a reduction of 4 µmol/L in fasting plasma homocysteine levels in patients with cardiovascular, cerebrovascular and peripheral arterial diseases.

Recently, some studies with animals and human beings are being carried out, specially in the field of cardiology, in order to analyze different and important mechanisms and biological markers in the process of atherosclerosis (amount of oxidized LDL, dilation of brachial artery, intimal hyperplasia, rate of restenosis) with vitamin supplementation (specially folic acid) for controlling plasma homocysteine levels and the biological markers for atherosclerosis.^47,63-71 Results of such studies point to a reduction in homocysteine concentration and to the control of some biological markers of atherosclerosis associated with hyperhomocysteinemia. The authors also considered that vitamin supplementation with folic acid was beneficial, cost-effective and could be adopted as an adjuvant therapy for patients with symptomatic or asymptomatic atherosclerosis.

Based on the above mentioned studies, mainly related to coronary arterial disease, it is suggested that controlled prospective clinical studies of PAD be carried out in order to investigate the precise effects of therapy with vitamin supplements over the reduction of plasma homocysteine levels as well as the risk, the incidence and the natural history of atherosclerosis in preventing the progress or allowing the regression of peripheral atherosclerotic lesions,^3,72-75 specially in Brazil, where there are such particular health and socioeconomic conditions, dietary habits and life style.

Due to easy availability and low cost of the treatment for hyperhomocysteinemia, it is important to identify hyperhomocysteinemia as an additional risk factor for PAD. Based on currently available data, the traditional therapeutic approach may be indicated for patients with hyperhomocysteinemia who may or may not have coronary, cerebrovascular or peripheral vascular disease as well. Following the Recommended Dietary Allowances (RDA),⁷⁶ it is suggested that intake of vitamins B6, B12 and specially folate be prescribed through the ingestion of foods such as whole grain cereals, fresh meat, beans, green leafy vegetables and fruits or through oral supplementation.⁷⁷

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