L. Brattström

Impaired homocysteine metabolism in early-onset cerebral and peripheral occlusive arterial disease Effects of pyridoxine and folic acid treatment☆

Severe homocysteinemia due to genetic defects either of pyridoxal 5-phosphate (PLP)-dependent cystathionine beta-synthase (CBS) or of enzymes in vitamin B12 and folate metabolism is associated with very early-onset vascular disease. Therefore, we studied homocysteine metabolism in 72 patients presenting before the age of 55 years with occlusive arterial disease of cerebral, carotid, or aorto-iliac vessels. Twenty patients (28%) had basal homocysteinemia; and 26 patients (36%) had abnormal increases of plasma homocysteine after peroral methionine loading, which exceeded the highest value for 46 comparable controls and was within the range for 20 obligate heterozygotes for homocystinuria due to CBS deficiency. Basal plasma homocysteine content was strongly and negatively correlated to vitamin B12 and folate concentrations. Plasma PLP was depressed in most patients but there was no correlation between PLP and homocysteine values. In 20 patients, treatment with pyridoxine hydrochloride (240 mg/day) and folic acid (10 mg/day) reduced fasting homocysteine after 4 weeks by a mean of 53%, and methionine response by a mean of 39%. These data show that a substantial proportion of patients with early-onset vascular disease have impaired homocysteine metabolism, which may contribute to vascular disease, and that the impaired metabolism can be improved easily and without side effects.

Folic acid-an innocuous means to reduce plasma homocysteine

With an improved highly reproducible method, we measured total plasma homocysteine (free plus protein-bound) and related amino acids in the fasting state in healthy subjects, before and after treatment with co-factors for homocysteine metabolism: 1 mg cyanocobalamin (n = 14), 5 mg folic acid (n = 13) or 40 mg pyridoxine hydrochloride (n = 15) daily for 14 days. Cyanocobalamin and pyridoxine hydrochloride had no effects on plasma levels of amino acids, but folic acid had a considerable homocysteine-lowering effect. Total plasma homocysteine was reduced in all but two subjects, from 19.9 +/- 4.4 (mean +/- SEM) to 9.5 +/- 1.0 mumol/l (-52%, p less than 0.01). We propose that folic acid in excess acts by enhancing the remethylation of homocysteine to methionine. The finding confirms a previous report by us. Since homocysteine is considered to be an atherogenic amino acid and recent reports suggest that mild to moderate homocysteinaemia is also associated with premature vascular disease, treatment with folic acid might be of use as prophylaxis.

Higher total plasma homocysteine in vitamin B12 deficiency than in heterozygosity for homocystinuria due to cystathionine β-synthase deficiency☆

Homocysteine is an amino acid considered to cause vascular injury, arteriosclerosis, and thromboembolism. Total plasma homocysteine (free and protein-bound) was found to be twice as high in asymptomatic vitamin B12-deficient subjects (23.8 +/- 3.8 mumol/L, means +/- SEM, n = 20) as in controls (11.5 +/- 0.9 mumol/L, P less than .0001, n = 21), and higher than in heterozygotes for homocystinuria due to cystathionine beta-synthase deficiency (13.8 +/- 1.6 mumol/L, P less than .01, n = 14), who were recently shown to be much more common among patients with premature vascular disease than expected. Eight (40%) vitamin B12-deficient and two (14%) heterozygote subjects had significant homocysteinemia (greater than mean +2 SD for controls). After administration of hydroxycobalamin to vitamin B12-deficient subjects, homocysteine levels decreased to normal (-49%, 12.2 +/- 1.5 mumol/L, P less than .0001, n = 20). Thus, if homocysteine does cause vascular injury, theoretically vitamin B12-deficiency might be associated with an increased frequency of vascular disease.

Homocysteine and myocardial infarction.

Five (24%) subjects out of a group of 21 men, 48-58 years old (mean 54), who had suffered their first myocardial infarction (MI) before the age of 55 and with a low risk profile vis-à-vis conventional risk factors in a health screening preceding the MI, had abnormally high total plasma homocysteine values in the fasting state when investigated within 1-7 years (mean 3) after their MI. The patient group was exactly matched with 36 control subjects for sex, age, diastolic blood pressure, smoking, and serum concentrations of cholesterol and triglycerides. Total plasma homocysteine was negatively correlated to both erythrocyte folate and serum vitamin B12, and vitamin concentrations below the median of the normal distribution were found in the five with high plasma homocysteine content, indicating a possible involvement of reduced remethylation of plasma homocysteine to methionine. After methionine loading, in 3 of the patient group (14%) homocysteine levels exceeded mean +2 SD for controls, which may indicate heterozygosity for homocystinuria. Results are consistent with the hypothesis that a high plasma homocysteine content may be a risk factor for MI.

Increased levels of plasma homocysteine are associated with nephropathy, but not severe retinopathy in type 1 diabetes mellitus

The reactive vascular-injuring amino acid homocysteine was measured in plasma samples from 79 well-characterized type 1 diabetic patients and 46 control subjects. Patients with proliferative retinopathy had higher homocysteine levels (15.0 +/- 6.3 mumols l-1; mean +/- SD, p less than 0.001; n = 42) than those with progressive retinopathy during a two-year period (10.4 +/- 1.6 mumols l-1; n = 12), no or minimal retinopathy (10.7 +/- 4.3 mumols l-1; n = 25), and the control subjects (11.0 +/- 3.4 mumols l-1). Within the group of patients with proliferative retinopathy increased homocysteine levels were confined to those patients that had serum creatinine levels greater than 115 mumols l-1 and/or an albumin:creatinine clearance ratio greater than or equal to 0.02 x 10(-3) (17.0 +/- 5.9 mumols l-1; n = 23), whereas those with no or only minimal nephropathy had levels (12.1 +/- 5.5 mumols l-1; n = 18) that were not different from the control group. We conclude that neither type 1 diabetes mellitus nor diabetic retinopathy per se is associated with increased plasma homocysteine levels. In contrast, homocysteine accumulates, probably owing to reduced glomerular filtration, in diabetic patients with advanced nephropathy. This suggests that homocysteine might contribute to the accelerated development of macroangiopathy seen especially in this subgroup of diabetic patients.

Homocysteine in frozen plasma samples. A short cut to establish hyperhomocysteinaemia as a risk factor for arteriosclerosis

Findings in several retrospective studies have supported the hypothesis that hyperhomocysteinaemia may be an independent risk factor for premature arteriosclerotic disease. This prompted us to investigate whether frozen plasma samples could be used to study the question prospectively. Total plasma homocysteine concentrations in 6-16-year-old (10.9 +/- 2.5, mean +/- SD) frozen (-20 degrees C) and fresh samples from the same 76 men were 11.6 +/- 4.9 and 14.1 +/- 4.3 mumol l-1 respectively, the values being significantly correlated (r = 0.58, p < 0.001). The difference was not correlated to storage time. After the first sampling, 13 subjects had survived a stroke, 16 a myocardial infarction, and 51 were still healthy, being used as matched controls. In stroke patients, values from old and fresh samples were 14.2 +/- 5.5 and 16.4 +/- 4.8 mumol l-1, respectively, and tended to be higher (p = 0.06) than in matched controls whose respective values were 11.4 +/- 2.8 and 13.8 +/- 3.8 mumol l-1 (n = 22). No such differences were seen between patients with myocardial infarction and their matched controls. We conclude that total plasma homocysteine can be measured in up to 10-year-old frozen plasma samples, indicating that such samples can be used for prospective studies on the relationship between plasma homocysteine and vascular disease.

Determination of homocysteine in plasma by ion-exchange chromatography

A rapid ion-exchange chromatographic method for determination of total homocysteine in plasma is presented. The method has a coefficient of variation of approximately 5%. A comparison with a slower method previously used in our laboratory showed a high correlation (r = 0.993). The risk of oxidation of homocysteine during the chromatography and the necessary level of dithiotreithol to prevent oxidation are also established.

Plasma homocysteine in women on oral oestrogen-containing contraceptives and in men with oestrogen-treated prostatic carcinoma

The mechanism by which oral oestrogen-containing contraceptives in women and oestrogen treatment of prostatic carcinoma in men increases the risk of vascular disease is unclear. These agents decrease serum concentrations of vitamin B12, pyridoxal 5-phosphate, and folate, all of which are essential for the metabolism of the atherogenic amino acid homocysteine. We found serum vitamin B12 concentrations to be lower in 17 women using oral contraceptives (219 +/- 84 pmol l-1) than in 13 age-matched female controls (385 +/- 129, p less than 0.001), but similar values were obtained in the two groups both for fasting plasma homocysteine concentrations (9.1 +/- 2.4 vs 9.2 +/- 3.6 mumol l-1) and for the increase in these concentrations after methionine loading (19.2 +/- 7.5 vs 17.8 +/- 5.2 mumol l-1). In five men with prostatic carcinoma, high-dose oestrogen treatment decreased serum vitamin B12 concentrations by a mean of 30% (p less than 0.05) within 4 weeks, during which fasting plasma homocysteine concentrations decreased (13.8 +/- 4.5 vs 10.5 +/- 2.8 mumol l-1) and response to methionine loading increased (12.4 +/- 3.4 vs 17.3 +/- 5.1 mumol l-1), though the latter changes were non-significant. Our findings do not support the hypothesis that hyperhomocysteinemia explains cardiovascular risk in women using oral oestrogen-containing contraceptives, or in oestrogen-treated men with prostatic carcinoma.

Asymmetric dimethylarginine and total homocysteine in plasma after oral methionine loading.

Background: Elevation of homocysteine (Hcy) and asymmetric dimethylarginine (ADMA) in plasma are believed to be involved in the pathogenesis of cardiovascular disease (CVD). In humans, oral methionine loading results in acute elevation of plasma Hcy. This is associated with impaired NO‐dependent vasodilatation, a mechanism that may explain the relationship between elevated Hcy and risk of CVD. ADMA, an endogenous competitive inhibitor of NO‐synthase, may be elevated in plasma of patients with CVD. It was proposed that ADMA is synthesized in a methionine‐dependent reaction which also forms Hcy. In this study plasma total homocysteine (tHcy) and ADMA concentrations were measured before and after oral methionine loading of human subjects. Methods: Plasma tHcy and ADMA levels were measured in 12 healthy males (age 32–58 years) before and after oral loading with L‐methionine (100u2005mg/kg body weight in orange juice). Results: At noon, 4u2005h after methionine loading, tHcy and ADMA levels (35.4±10.9 and 0.80±0.13u2005μmol/L, mean ±SD) were significantly higher than the corresponding values obtained at noon the day before (15.6±7.4 and 0.63±0.10u2005μmol/L, both p<0.001). Noon values 4u2005h after methionine loading were also significantly higher than values obtained immediately before the methionine load (13.7±5.9 and 0.66±0.10u2005μmol/L, both p<0.001). Reinvestigation of 8 of 12 subjects showed that at 4 and 8u2005h after compared with levels immediately before methionine loading there was a significant increase in tHcy (28.4±10.2 and 33.45±11.1 vs. 10.8±3.3u2005μmol/L, both p<0.001). However, the corresponding ADMA levels did not increase (0.73±0.17 and 0.76±0.22 vs. 0.70±0.10u2005μmol/L, both not significant). Conclusions: No clear evidence was found to support the supposition that methionine‐induced hyperhomocysteinaemia may be accompanied by elevated levels of ADMA, an endogenous competitive NO‐synthase inhibitor that may represent an alternative pathogenic mechanism for homocysteine‐associated impairment of endothelial NO‐dependent functions.

Influence of hydrolysis on plasma homocysteine determination in healthy subjects and patients with myocardial infarction

After acid hydrolysis, mean plasma homocysteine concentrations, measured as homocysteine disulphides, of about 1000 and 40 mumol/l have recently been reported in 26 survivors of myocardial infarction and 26 matched control subjects, respectively. This finding contrasts sharply with those more than 50 times lower total homocysteine concentrations found by other research groups in non-hydrolysed plasma from survivors of myocardial infarction. Using the same hydrolysis conditions, we could not detect any homocysteine disulphides in plasma hydrolysates from 9 survivors of myocardial infarction and 10 healthy subjects, who had mean total homocysteine concentrations in non-hydrolysed plasma of 16.9 +/- 6.5 and 15.8 +/- 10.3 mumol/l, respectively. The chromatograms contained several peaks, probably representing peptides, which disappeared with more complete hydrolysis and which might have been misinterpreted as homocysteine disulphides in the reported study. Only after reduction of disulphides and by using a sulphydryl-selective extraction procedure were we able to determine mean homocysteine concentrations in hydrolysed plasma to be 26.2 +/- 7.9 mumol/l in the survivors of myocardial infarction and 24.5 +/- 12.2 mumol/l in the healthy reference subjects. Thus, we could not confirm that survivors of myocardial infarction have homocysteine concentrations that are many times higher than found in healthy subjects.