Godfried H.J. Boers

Heterozygosity for Homocystinuria in Premature Peripheral and Cerebral Occlusive Arterial Disease

Premature arteriosclerosis and thromboembolic events are well-known complications of homozygous homocystinuria due to cystathionine synthase deficiency. It is unknown whether heterozygosity for homocystinuria predisposes to premature vascular disease. We explored the frequency of excessive homocysteine accumulation after standardized methionine loading in 75 patients presenting with clinical signs of ischemic disease before the age of 50:25 with occlusive peripheral arterial disease, 25 with occlusive cerebrovascular disease, and 25 with myocardial infarction. In seven patients in each of the first two groups but in none of the patients in the third group, heterozygosity for homocystinuria was established on the basis of pathological homocysteinemia after methionine loading and cystathionine synthase deficiency in skin fibroblast cultures. Because the frequency of heterozygosity for homocystinuria in the normal population is 1 in 70 at the most, we conclude that this condition predisposes to the development of premature occlusive arterial disease, causing intermittent claudication, renovascular hypertension, and ischemic cerebrovascular disease.

Low Circulating Folate and Vitamin B6 Concentrations Risk Factors for Stroke, Peripheral Vascular Disease, and Coronary Artery Disease

BACKGROUND A high plasma homocysteine concentration is a risk factor for atherosclerosis, and circulating concentrations of homocysteine are related to levels of folate and vitamin B6. This study was performed to explore the interrelationships between homocysteine, B vitamins, and vascular diseases and to evaluate the role of these vitamins as risk factors for atherosclerosis. METHODS In a multicenter case-control study in Europe, 750 patients with documented vascular disease and 800 control subjects frequency-matched for age and sex were compared. Plasma levels of total homocysteine (before and after methionine loading) were determined, as were those of red cell folate, vitamin B12, and vitamin B6. RESULTS In a conditional logistic regression model, homocysteine concentrations greater than the 80th percentile for control subjects either fasting (12.1 micromol/L) or after a methionine load (38.0 micromol/L) were associated with an elevated risk of vascular disease independent of all traditional risk factors. In addition, concentrations of red cell folate below the lowest 10th percentile (<513 nmol/L) and concentrations of vitamin B6 below the lowest 20th percentile (<23.3 nmol/L) for control subjects were also associated with increased risk. This risk was independent of conventional risk factors and for folate was explained in part by increased homocysteine levels. In contrast, the relationship between vitamin B6 and atherosclerosis was independent of homocysteine levels both before and after methionine loading. CONCLUSIONS Lower levels of folate and vitamin B6 confer an increased risk of atherosclerosis. Clinical trials are now required to evaluate the effect of treatment with these vitamins in the primary and secondary prevention of vascular diseases.

Maternal hyperhomocysteinemia: A risk factor for neural-tube defects?

The maternal vitamin status, especially of folate, is involved in the pathogenesis of neural-tube defects (NTDs). Maternal folate administration can prevent these malformations. The precise metabolic mechanism of the beneficial effect of folate is unclear. In this study we focus on homocysteine accumulation, which may derive from abnormalities of metabolism of folate, vitamin B12, and vitamin B6. We studied nonpregnant women, 41 of whom had given birth to infants with NTDs and 50 control women who previously had normal offspring. The determinations included the plasma total homocysteine both in the fasting state and 6 hours after the ingestion of a methionine load. In addition, we measured the fasting blood levels of folate, vitamin B12, and vitamin B6. The mean values for both basal homocysteine and homocysteine following a methionine load were significantly increased in the group of women who previously had infants with NTDs. In nine of these subjects and two controls, the values after methionine ingestion exceeded the mean control by more than 2 standard deviations. Cystathionine synthase levels in skin fibroblasts derived from these methionine-intolerant women were within the normal range. Our findings suggest a disorder in the remethylation of homocysteine to methionine due to an acquired (ie, nutritional) or inherited derangement of folate or vitamin B12 metabolism. Increased homocysteine levels can be normalized by administration of vitamin B6 or folate. Therefore, we suggest that the prevention of NTDs by periconceptional folate administration may effectively correct a mild to moderate hyperhomocysteinemia.

Hyperhomocysteinemia: a risk factor in women with unexplained recurrent early pregnancy loss * †

OBJECTIVE To establish the prevalence of hyperhomocysteinemia in women with unexplained recurrent early pregnancy loss. DESIGN In a patient-control study, the methionine-homocysteine metabolism was investigated by a standardized oral methionine-loading test. SETTING Gynecologic outpatient department of university hospital. PATIENTS One-hundred and two women who had been referred to the hospital because they suffered from at least two consecutive unexplained spontaneous abortions (study group) as well as 41 controls who were recruited by public advertisement were selected. INTERVENTIONS Blood samples were collected just before and 6 hours after oral methionine administration to determine plasma total homocysteine concentrations. MAIN OUTCOME MEASURE Plasma total homocysteine concentrations 6 hours after methionine loading. Hyperhomocysteinemia was defined as total homocysteine concentration at 6 hours exceeding the 97.5 percentile level of the controls. RESULTS Hyperhomocysteinemia was diagnosed in 21 women of the study group (21%). In the parous women of the study group, the prevalence of hyperhomocysteinemia was more than two times greater compared with the nulliparous subjects (33% and 14%, respectively). CONCLUSION Hyperhomocysteinemia is a risk factor in women with unexplained recurrent early pregnancy loss.

Cystathionine β‐synthase mutations in homocystinuria

The major cause of homocystinuria is mutation of the gene encoding the enzyme cystathionine β‐synthase (CBS). Deficiency of CBS activity results in elevated levels of homocysteine as well as methionine in plasma and urine and decreased levels of cystathionine and cysteine. Ninety‐two different disease‐associated mutations have been identified in the CBS gene in 310 examined homocystinuric alleles in more than a dozen laboratories around the world. Most of these mutations are missense, and the vast majority of these are private mutations. The two most frequently encountered of these mutations are the pyridoxine‐responsive I278T and the pyridoxine‐nonresponsive G307S. Mutations due to deaminations of methylcytosines represent 53% of all point substitutions in the coding region of the CBS gene. Hum Mutat 13:362–375, 1999.

Hyperhomocysteinaemia and endothelial dysfunction in young patients with peripheral arterial occlusive disease.

Abstract. Hyperhomocysteinaemia, defined as an abnormally high plasma homocysteine concentration after an oral methionine load, is common in young (≤ 50 years) patients with peripheral arterial occlusive disease. It is thought to predispose to atherosclerosis by injuring the vascular endothelium. Treatment with pyridoxine and/or folic acid may lower plasma homocysteine levels. In mildly hyperhomocysteinaemic patients with peripheral arterial occlusive disease, we studied the effect of daily treatment with pyridoxine (250 mg) plus folic acid (5 mg) on homocysteine metabolism (i.e. plasma concentrations in the fasting state and after methionine loading, in 48 patients) and on endothelial function (in 18 patients). Endothelial function was estimated as the plasma concentrations of the endothelium‐derived proteins, von Willebrand factor (vWF), thrombomodulin (TM), and tissue‐type plasminogen activator (tPA). At baseline, fasting homocysteine levels were above normal in 24 of the 48 patients (50%); post‐load levels, by definition, were above normal in 100% of patients. After 12 weeks of treatment, fasting and post‐load levels were normal in 98 and 100% of patients, respectively. Endothelial function was assessed in 18 patients who completed 1 year of treatment. At baseline, median vWF (235%) and TM (57.1 ng mL‐1) levels were above normal. At follow‐up, vWF levels had decreased to 170% (P= 0.01) and TM levels had decreased to 49 ng mL‐1 (P= 0.04). tPA levels were normal at baseline and did not change. Endothelial dysfunction is present in young patients with peripheral arterial occlusive disease and hyperhomocysteinaemia. Pyridoxine plus folic acid treatment normalizes homocysteine metabolism in virtually all patients, and appears to ameliorate endothelial dysfunction.

Treatment of mild hyperhomocysteinemia in vascular disease patients.

Mild hyperhomocysteinemia is recognized as a risk factor for premature arteriosclerotic disease. A few vitamins and other substances have been reported to reduce blood homocysteine levels, but normalization of elevated blood homocysteine concentrations with any of these substances has not been reported. Therefore, we screened 421 patients suffering from premature peripheral or cerebral occlusive arterial disease by oral methionine loading tests for the presence of mild hyperhomocysteinemia. Thirty-three percent of patients with peripheral and 20% of patients with cerebral occlusive arterial disease were identified with mild hyperhomocysteinemia (14% of the men, 34% of the premenopausal women, and 26% of the postmenopausal women). Mildly hyperhomocysteinemic patients were administered vitamin B6 250 mg daily. After 6 weeks methionine loading tests were again assessed to evaluate the effect of treatment. Patients with nonnormalized homocysteine concentrations were further treated with vitamin B6 250 mg daily and/or folic acid 5 mg daily and/or betaine 6 g daily, solely or in any combination. Vitamin B6 treatment normalized the afterload homocysteine concentration in 56% of the treated patients (71% of the men, 45% of the premenopausal women, and 88% of the postmenopausal women). Further treatment resulted in a normalization of homocysteine levels in 95% of the remaining cases. Thus, mild hyperhomocysteinemia, which is frequently encountered in patients with premature arteriosclerotic disease, can be reduced to normal in virtually all cases by safe and simple treatment with vitamin B6, folic acid, and betaine, each of which is involved in methionine metabolism.

A second common variant in the methylenetetrahydrofolate reductase (MTHFR) gene and its relationship to MTHFR enzyme activity, homocysteine, and cardiovascular disease risk

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinemia, an independent and graded risk factor for cardiovascular disease (CVD). We examined the relationship of two polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, the 677C→T and 1298A→C variants, to MTHFR activity, homocysteine concentrations, and risk of CVD in a population of 190 vascular disease patients and 601 apparently healthy controls. The mean specific and residual MTHFR activities were significantly lower in 677CT and 677TT individuals (both P<0.001). The 1298A→C mutation alone showed no effect on MTHFR activities. However, when the 677C→T genotype was taken into account, the 1298A→C mutation also caused a significant decrease in MTHFR activities, which was observed in both the homozygous 1298CC (P<0.001) and the heterozygous 1298AC states (P=0.005). Both the 677TT as the 677CT genotypes were associated with significantly higher fasting and postload homocysteine levels than 677CC (P<0.001 and P=0.003, respectively). The 1298A→C mutation had no effect on fasting or postload homocysteine levels. Since homocysteine itself is considered to be positively associated with the risk of CVD, these findings indicate that the 1298A→C mutation cannot be considered a major risk factor for CVD.

Unique efficiency of methionine metabolism in premenopausal women may protect against vascular disease in the reproductive years.

Premenopausal women develop occlusive artery disease less frequently than postmenopausal women. In coronary heart disease, higher blood levels of homocysteine-cysteine mixed disulphide have been reported. Therefore, in healthy subjects, we studied the role of menopausal status in the transsulphuration of methionine in 10 premenopausal and 10 postmenopausal women. To exclude the role of aging, we compared these results with those in 10 younger and 10 older men of comparable age groups. An oral methionine load (0.1 g/kg of body weight) was administered after overnight fasting. Before and during 8 h, thereafter, serum levels of methionine, homocystine, and homocysteine-cysteine mixed disulphide were measured. In the fasting state, serum methionine levels were similar in the premenopausal women and both groups of men. Postmenopausal women had significantly lower fasting levels. Peak levels and clearances of methionine after loading did not differ between the groups. In the fasting state, homocystine was never detectable; yet, after methionine loading, slight homocystinemia was present in 12 out of 20 men, and was more pronounced in all postmenopausal women. However, homocystinemia did not occur in any of the premenopausal women after loading. Fasting serum homocysteine-cysteine mixed disulphide levels did not differ between both groups of men and postmenopausal women. In premenopausal women, both fasting and postloading disulphide levels were significantly lower than in any other group. We conclude that premenopausal women have a unique efficiency of methionine handling, and thereby are preserved against the accumulation of homocysteine after methionine loading. We speculate that this phenomenon might account for the lower incidence of vascular disease in women in the reproductive life cycle.

Neural tube defects and elevated homocysteine levels in amniotic fluid

OBJECTIVE Our purpose was to study maternal blood and amniotic fluid concentrations of homocysteine and relevant vitamins in relation to neural tube defects. STUDY DESIGN Concentrations of total homocysteine, folate, and vitamins B12 and B6 were measured in maternal blood and amniotic fluid of 27 women carrying a fetus with a neural tube defect and 31 control women carrying a healthy fetus. RESULTS The mean total homocysteine concentration in amniotic fluid of the study group was significantly higher than that of the control group. The mean concentrations of total homocysteine in blood and the vitamins folate, B12, and B6 in, respectively, blood and amniotic fluid were not significantly different between the groups. The mean concentrations of homocysteine and vitamin B6 were significantly lower in amniotic fluid than in blood in both groups, whereas vitamin B12 in amniotic fluid was higher than in blood. CONCLUSION These results support the hypothesis that at least the cause of a subset of neural tube defects could reside in a primary or secondary maternal or fetal derangement of homocysteine metabolism.