David E.L. Wilcken

Common Methylenetetrahydrofolate Reductase Gene Mutation Leads to Hyperhomocysteinemia but Not to Vascular Disease The Result of a Meta-Analysis

BACKGROUND The results of retrospective and prospective case-control studies have clearly established that mild elevations of the plasma homocysteine level are associated with increased risk of coronary, cerebral, and peripheral vascular disease. Recently, a mutation (677C-->T) was identified in the methylenetetrahydrofolate reductase (MTHFR) gene that results in reduced folate-dependent enzyme activity and reduced remethylation of homocysteine to methionine. Mutant homozygotes (TT genotype) constitute approximately 12% of the white population and frequently have mildly elevated circulating homocysteine. Therefore, it seems likely that they would also be at increased risk of vascular disease. A number of studies have investigated this during the past 3 years, and the present article evaluates the results in a meta-analysis. METHODS AND RESULTS We identified 13 studies in which there were measurements of plasma homocysteine in relation to the 3 genotypes (TT, CT, and CC) and 23 case-control studies comprising 5869 genotyped cardiovascular disease patients (mostly coronary artery disease) and 6644 genotyped control subjects. Those bearing the TT genotype had plasma homocysteine concentrations 2.6 micromol/L (25%) higher than those with the CC genotype. However, there was no difference between patients and control subjects either in the frequency of mutant alleles (T) (34.3% versus 33.8%) or the TT genotype (11.9% versus 11.7%). In the analysis of the 23 studies, the relative risk (OR) of vascular disease associated with the TT genotype was 1.12 (95% CI, 0.92 to 1.37). CONCLUSIONS We conclude that although the C677T/MTHFR mutation is a major cause of mild hyperhomocysteinemia, the mutation does not increase cardiovascular risk. Our findings suggest that the mild hyperhomocysteinemia found frequently in vascular disease patients is not causally related to the pathogenesis of the vascular disease.

The effect of weight reduction on left ventricular mass. A randomized controlled trial in young, overweight hypertensive patients.

We compared the effects of weight reduction, metoprolol, and placebo on M-mode echocardiographic measurements of the thickness and mass of the left ventricular wall in a 21-week, randomized controlled trial that enrolled 41 young, overweight patients with hypertension. At the end of the follow-up period, the patients in the weight-reduction group had lost an average of 8.3 kg, and their blood pressure had decreased by an average of 14/13 mm Hg, as compared with 12/8 mm Hg in the metoprolol group and 9/4 mm Hg in the placebo group. In the weight-reduction group, interventricular septal and posterior-wall thickness decreased by 14 percent and 11 percent, respectively, and left ventricular mass decreased by 20 percent (16 percent when adjusted for body-surface area). Decreases in interventricular septal and posterior-wall thickness and in left ventricular mass in the weight-reduction group were significantly greater than those in the placebo group. The changes in thickness of the interventricular septum and the left ventricular mass in the weight-reduction group were also greater than those in the metoprolol group. Changes in weight, independent of changes in blood pressure, were directly associated with changes in left ventricular mass. We conclude that weight reduction decreases left ventricular mass in overweight hypertensive patients and that control of obesity is important not only for the treatment of hypertension but also for the prevention of left ventricular hypertrophy.

Genetic Contribution of the Endothelial Constitutive Nitric Oxide Synthase Gene to Plasma Nitric Oxide Levels

Nitric oxide (NO) has an important physiological role in regulating vascular tone and is also relevant to many pathological processes including hypertension and atherosclerosis. Endothelial constitutive nitric oxide synthase (ecNOS) is the key enzyme in determining basal vascular wall NO production. We used a combination of maximum-likelihood-based statistical genetic methods to explore the contributions of the ecNOS gene and other unmeasured genes to basal NO production measured by its metabolites (NOx: nitrite and nitrate) in 428 members of 108 nuclear families. Our initial quantitative genetic analysis estimated that approximately 30% of the variance in fasting NOx levels is due to genes (chi 2(1) = 16.04, P = .000062). Complex segregation analysis detected the effects of both a single locus and residual polygenes on NOx levels, and measured genotype analysis showed that plasma NOx levels in those homozygous for the rare allele (64.9 +/- 7.8 mumol/L) were significantly higher (P = .000242) than those homozygous for the common allele (30.2 +/- 3.1 mumol/L). The results of the variance component linkage analysis were consistent with linkage of a quantitative trait locus in or near the ecNOS gene to variation in plasma NOx levels (P = .0066). While many environmental factors have been shown to alter transiently plasma NOx levels, our study is the first to identify a substantial effect of the ecNOS locus on the variance of plasma NOx, i.e. basal NO production. This finding may be relevant to atherogenesis and NO-related disorders.

The natural history of vascular disease in homocystinuria and the effects of treatment.

Among 40 patients with homocystinuria due to cystathionine β-synthase deficiency diagnosed in the state of New South Wales, Australia (population 6 million) and followed long-term, there were 10 deaths at ages 2-30 years. Of these 8 were definite vascular deaths, one was a presumed vascular death, and the other was due to an accident and unrelated to homocystinuria. The vascular deaths were all early cases and only one patient, a pyridoxine-responsive 30-year-old woman, had been prescribed adequate treatment although it was uncertain that she was taking it.In 32 patients of mean age 30 years (range 9-66 years) there were 539 patient-years of treatment with pyridoxine, folic acid and hydroxocobalamin. There were 17 pyridoxine-responsive patients and all maintained plasma total free homocyst(e)ine levels <200µmol/L over an average treatment period of 16.6 years. The 15 non-responsive patients received additionally 6-9 g of betaine daily. This resulted in a further 74% mean decline (±14% SD) in plasma total free homocyst(e)ine, persisting during an average (post-betaine) treatment period of 11 years; current mean ± SD levels are 33 ± 17 µmol/L (n=15).There were two vascular events during treatment, one fatal pulmonary embolus (see above) and one myocardial infarction, whereas without treatment, 21 would have been expected, χ2 = 14.22, p = 0.0001, relative risk 0.09 (95% CI 0.02-0.38). There were no events during 258 patient-years of treatment in the 15 pyridoxine-nonresponsive patients (p<0.005 versus expected untreated). Nineteen patients had a total of 19 major and 15 minor operations requiring anaesthetic, and three had successful pregnancies, one whilst receiving betaine. There were no thromboembolic complications.We conclude that treatment which effectively lowers circulating homocyst(e)ine, even to suboptimal levels, markedly reduces cardiovascular risk in patients with cystathionine β-synthase deficiency, and that betaine therapy contributes importantly to this in pyridoxine-nonresponsive patients. Betaine as additional therapy is safe and effective for at least 16 years.

Homocystinuria — The Effects of Betaine in the Treatment of Patients Not Responsive to Pyridoxine

The treatment of homocystinuria that is not responsive to pyridoxine is not usually biochemically or clinically successful, and vascular, ocular, and skeletal complications commonly supervene. Persistent marked homocysteinemia appears to be the most important biochemical disturbance leading to these complications. Ten patients with cystathionine beta-synthase deficiency that was not responsive to pyridoxine and one patient with homocystinuria due to a defect in cobalamin metabolism were treated with 6 g daily of betaine added to conventional therapy, to improve homocysteine remethylation. All patients had a substantial decrease in plasma total homocysteine levels (P less than 0.001) and an increase in total cysteine levels (P less than 0.001). Changes in plasma methionine concentrations were variable. Fasting levels of plasma amino acids became normal in two patients, and in six there was immediate clinical improvement. There were no unwanted effects. We conclude that treatment of homocystinuria that is not responsive to pyridoxine and of disorders of homocysteine remethylation should include betaine in adequate doses to ensure maximum lowering of elevated plasma homocysteine levels.

Folic acid lowers elevated plasma homocysteine in chronic renal insufficiency: Possible implications for prevention of vascular disease

To explore interrelations between folic acid and methionine metabolism in chronic renal insufficiency, we measured plasma amino acids in 21 patients with mean serum creatinine +/- SD of 560 +/- 240 mumol/L, after a ten-hour overnight fast, before and after administration of 5 mg of oral folic acid daily for 15 +/- 6 days. Mean plasma homocysteine was 12.9 +/- 6.8 mumol/L in the patients and 4.2 +/- 0.8 mumol/L in 24 normal controls (P less than .001), and after folic acid administration it declined in the patients to 6.8 +/- 2.8 mumol/L (P less than .0001) in linear proportion (r = .92) to the prefolate homocysteine level. Methionine concentrations were normal in the patients and did not change after folate administration, nor did elevated cysteine and creatinine. Plasma serine was lower (88.3 +/- 17.2 v 121 +/- 25 mumol/L, P less than .41) and declined further to 67.8 +/- 16.4 (P less than .0001) after folate, while prefolate glycine levels increased from 273.3 +/- 61.2 to 313.2 +/- 97.5 mumol/L (P less than .01). Serum and red-cell folate levels were normal in the patients before treatment. The results show that homocysteine levels are increased in chronic renal insufficiency, but may be lowered by folate enhancement of remethylation of homocysteine to methionine. Since elevated plasma homocysteine is associated with premature vascular disease, folic acid may reduce cardiovascular risk in chronic renal insufficiency.

Distribution in Healthy and Coronary Populations of the Methylenetetrahydrofolate Reductase (MTHFR) C677T Mutation

Modest elevations of circulating homocyst(e)ine are common in patients with vascular disease. We explored in normal and coronary artery disease (CAD) populations the distribution of a mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene that results in enzyme thermolability and reduced activity and in homocyst(e)ine elevation to assess its relevance to risk. We identified the C to T substitution at the MTHFR locus and compared the distributions of genotypes in 565 patients aged < or = 65 years without and with angiographically documented CAD and in 225 healthy subjects. In the patients, we also assessed interrelations between genotypes and CAD occurrence and severity, as well as standard risk factors. The frequency of homozygotes for the mutation was the same in patients with and without CAD and in healthy subjects (11.6%, 11.0%, and 10.7%, respectively: P > .5 for each). There was also no excess among the 419 patients with severe disease (ie, one or more vessels with > 50% luminal obstruction) compared with those with no or mild CAD (odds ratio: 1.004; 95% confidence interval: 0.59 to 1.70). Homozygosity for the mutation was also not associated with a history of myocardial infarction or the presence or severity of angina. However, body mass index increased linearly with the presence of the mutant allele (P = .005), and the mutation and hypertension were weakly associated (P = .036). We conclude that the MTHFR genotype is not a risk factor for coronary disease in this Australian population but that the strong association found with body mass index should be explored further.

Sulphur containing amino acids in chronic renal failure with particular reference to homocystine and cysteine‐homocysteine mixed disulphide

Abstract. We measured plasma sulphur amino acids in twenty‐two patients with chronic renal failure and compared the findings with those obtained in twenty‐two normal subjects. In fasting blood (08.00 hours) cysteine‐homocysteine mixed disulphide was significantly increased in the renal patients, mean values (± SD) being 8.2 ± 3.4 and 3.1 ± 1.0 μmol/l respectively (P < 0.001). The increase was positively correlated with reduced renal function, as assessed by serum creatinine (r= 0.62; P < 0.01). Homocystine was detected in nineteen patients, the mean concentration (± SD) being 1.7 ± 0.6 μmol/l; it was not found in any normal subject. Methionine levels were not different but there were significant increases in cystine (P < 0.001) and taurine (P < 0.05) in the patients. Similar values for these amino acids were found in a second blood sample drawn at 16.00 hours. Changes in the other neutral and acidic amino acids measured were in agreement with those reported in chronic azotaemia. We concluded that plasma levels of all the principal sulphur amino acids except methionine are elevated in chronic renal failure emphasizing the importance of the kidney in sulphur excretion. Prolonged accumulation of homocysteine and cysteine‐homocysteine mixed disulphide may be relevant to the development of accelerated vascular disease in patients with chronic renal failure by producing endothelial damage.

NITRIC OXIDE INDUCES AND INHIBITS APOPTOSIS THROUGH DIFFERENT PATHWAYS

Physiological levels of nitric oxide (NO) regulate vascular tone and protect the microvasculature from injury whereas excessive NO may be harmful. The present study explored the effects of NO on human endothelial cell apoptosis. We found that the NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP) inhibited TNFα‐induced endothelial apoptosis and that this was mediated partly through the cGMP pathway. In contrast, high SNAP concentration induced endothelial apoptosis via cGMP‐independent pathways and the cGMP pathway protected against NO‐induced apoptosis. These findings demonstrate that low NO concentrations contribute to human endothelial cell survival, whereas higher NO concentrations are pathological and promote destruction of endothelial cells.

Determinants of severity of coronary artery disease in Australian men and women.

BACKGROUND Factors predicting the occurrence of premature coronary artery disease (CAD) may not be quantitatively the same as those predicting CAD severity, particularly in women, in whom there have been few studies. METHODS AND RESULTS To determine factors predictive of severity of CAD and of angina pectoris, we documented atherogenic variables and the extent of CAD at angiography in 594 consecutively studied men and women aged 65 years or less. Severity was assessed from the number of involved major coronary arteries with significant (> 50%) luminal obstructions and from a coronary disease severity score. We related severity to quantitative and categorical atherogenic variables and assessed severity of angina (no angina, stable angina, or unstable angina) at the time of study in the same way. There were eight variables independently predictive of severity: in descending order of relative importance, male gender, diabetes, smoking dose, ratio of total cholesterol to high-density lipoprotein cholesterol (TC/HDL-C), lipoprotein(a) [Lp(a)], age, positive family history, and hypertension. These correctly classified 43.3% of patients into no-, one-, two-, and three-vessel disease categories and accounted for 25.8% of variance of severity. Among 246 patients not taking lipid-lowering or beta-blocking drugs, these variables (in slightly different order) correctly classified 49.2% of patients and accounted for 36% of the variance. Among men (n = 427), seven significant variables correctly classified 39.3% of patients compared with 54.5% in women (n = 167). For those not taking the above drugs, these proportions were 49.4% and 65.4%, respectively. Among the quantitative variables, total smoking dose was the most predictive independent variable irrespective of current or ex-smoking habit and was more predictive in women than in men; of the lipid variables, high TC/HDL-C (or low HDL-C) and high Lp(a) were consistently highly predictive for all patients and in the subgroup analyses. Patients with unstable angina had higher coronary severity scores and Lp(a) levels and were more likely to have diabetes, hypertension, or a positive family history. CONCLUSIONS We conclude that the quantitative variables most relevant to severity of premature CAD and to its prevention in Australian men and women are total amount of lifetime smoking, TC/HDL-C (or HDL-C), and Lp(a) and that patients with unstable versus stable angina usually have more severe disease and higher Lp(a).