Andrew G. Bostom

The Effect of Folic Acid Fortification on Plasma Folate and Total Homocysteine Concentrations

BACKGROUND In 1996, the Food and Drug Administration issued a regulation requiring all enriched grain products to be fortified with folic acid to reduce the risk of neural-tube defects in newborns. Fortification (140 microg per 100 g) began in 1996, and the process was essentially complete by mid-1997. METHODS To assess the effect of folic acid fortification on folate status, we measured plasma folate and total homocysteine concentrations (a sensitive marker of folate status) using blood samples from the fifth examination (January 1991 to December 1994) of the Framingham Offspring Study cohort for baseline values and the sixth examination (January 1995 to August 1998) for follow-up values. We divided the cohort into two groups on the basis of the date of their follow-up examination: the study group consisted of 350 subjects who were seen after fortification (September 1997 to March 1998), and the control group consisted of 756 subjects who were seen before fortification (January 1995 to September 1996). RESULTS Among the subjects in the study group who did not use vitamin supplements, the mean folate concentrations increased from 4.6 to 10.0 ng per milliliter (11 to 23 nmol per liter) (P<0.001) from the baseline visit to the follow-up visit, and the prevalence of low folate concentrations (<3 ng per milliliter [7 nmol per liter]) decreased from 22.0 to 1.7 percent (P< 0.001). The mean total homocysteine concentration decreased from 10.1 to 9.4 micromol per liter during this period (P<0.001), and the prevalence of high homocysteine concentrations (>13 micromol per liter) decreased from 18.7 to 9.8 percent (P<0.001). In the control group, there were no statistically significant changes in concentrations of folate or homocysteine. CONCLUSIONS The fortification of enriched grain products with folic acid was associated with a substantial improvement in folate status in a population of middle-aged and older adults.

Homocyst(e)ine, Diet, and Cardiovascular Diseases A Statement for Healthcare Professionals From the Nutrition Committee, American Heart Association

Homocysteine is a sulfur-containing amino acid, rapidly oxidized in plasma to the disulfides homocystine and cysteine-homocysteine (Figure 1⇓). Plasma/serum total homocysteine, also termed homocyst(e)ine, is the sum of homocysteine in all 3 components. Figure 2⇓ displays factors involved in the metabolism of homocysteine, including its metabolic relationship to methionine. Although dietary intake of total protein and methionine does not correlate significantly with blood homocyst(e)ine,1 a single dose of oral methionine (100 mg/kg body weight) can elevate homocyst(e)ine levels, and as described further below, this has been used as a diagnostic test to detect disordered homocyst(e)ine metabolism. Because variable changes in homocyst(e)ine levels have been observed postprandially,2 it is customary to obtain measurements in the fasting state. Normal levels of fasting plasma homocyst(e)ine are considered to be between 5 and 15 μmol/L. Moderate, intermediate, and severe hyperhomocyst(e)inemia refer to concentrations between 16 and 30, between 31 and 100, and >100 μmol/L, respectively.3 Figure 1. Molecular species of homocysteine. Figure 2. Simplified outline of methionine/homocysteine metabolism. Vitamin coenzymes and substrates: THF, tetrahydrofolate; B2, riboflavin; B6, vitamin B6 as its biological active form, ie, pyridoxal 5′-phosphate; and B12, methyl cobalamin. Intermediate metabolite: DMG, dimethylglycine. Several vitamins function as cofactors and substrates in the metabolism of methionine and homocysteine (Figure 2⇑). Folic acid and cyanocobalamin (vitamin B12) regulate metabolic pathways catalyzed by the enzymes methylenetetrahydrofolate reductase (MTHFR) and methionine synthase, respectively, whereas pyridoxine (vitamin B6) is a cofactor for cystathionine β-synthase. A number of studies have shown inverse relationships of blood homocyst(e)ine concentrations with plasma/serum levels of folic acid, vitamin B6, and vitamin B12.4 5 6 Administration of supplemental folic acid in doses between 0.2 and 15 mg/d can lower plasma homocyst(e)ine levels without apparent toxicity.7 8 …

Predictive Performance of Renal Function Equations for Patients with Chronic Kidney Disease and Normal Serum Creatinine Levels

Accurate renal function measurements are important for the diagnosis and treatment of kidney disease, proper medication dosing, interpretation of possible uremic symptoms, and decision-making regarding when to initiate renal replacement therapy. Because the use of highly accurate filtration markers to measure renal function has traditionally been limited by cumbersome and costly techniques and the involvement of radioactivity (among other factors), renal function is typically estimated by using specially derived prediction equations. These formulae usually use serum creatinine levels, i.e., a marker of filtration that is insensitive to mild/moderate decreases in GFR. Although attempts have been made to validate certain renal function prediction equations among patients with chronic kidney disease (CKD) with abnormal serum creatinine levels, this is the first study to specifically evaluate the predictive performance of these equations for patients with CKD and serum creatinine levels in the normal range. The results of eight prediction equations for 109 patients with CKD and serum creatinine levels of < or =1.5 mg/dl were compared with standard iohexol GFR values. The most accurate results were obtained with the Cockroft-Gault and Bjornsson equations. The most precise formulae were the Modification of Diet in Renal Disease Study equations, although they were highly biased. Even the most accurate results exhibited levels of error that made them suboptimal for clinical treatment of these patients. These results suggest that measurement of GFR with endogenous or exogenous filtration markers might be the most prudent strategy for the assessment of renal function in the CKD population with normal serum creatinine levels. Further studies are needed to confirm the generalizability of these findings for this patient subgroup.

Nonfasting plasma total homocysteine levels and stroke incidence in elderly persons: the Framingham Study.

Meta-analyses (1, 2) strongly suggest that mildly to moderately elevated circulating levels of the sulfur amino acid homocysteine, either fasting or nonfasting, confer an independent risk for clinical arteriosclerotic outcomes, including stroke. However, just four reports (3-6) have provided somewhat limited, conflicting prospective data on the potential relation between total homocysteine levels and stroke incidence. Only one of these studies (3) used a population-based sample. Samples from two studies were limited to middle-aged men (4, 5): One consisted of U.S. physicians responding to a survey (4), and the other was derived from family practice registries in Great Britain (5). The fourth study (6) was a longitudinal investigation that primarily included women with systemic lupus erythematosus (mean age at baseline, 35 years). No prospective studies have specifically evaluated mild hyperhomocysteinemia as a potential risk factor for stroke in men and women 60 years of age or older, an age group that experiences the most pronounced morbidity and mortality from cerebrovascular disease (7). Accordingly, we examined the association between baseline nonfasting plasma total homocysteine levels and incident stroke in a well-characterized, population-based cohort of elderly women and men who at baseline had not had stroke. Methods The study sample consisted of the original Framingham Study cohort (8). Baseline examinations for the current analyses took place between May 1979 and May 1982, with follow-up occurring through May 1992. Of 2351 persons examined during the baseline period, 1947 had not previously had stroke and had specimens available for measurement of plasma total homocysteine levels. Additional baseline covariables assessed for the current analyses were age, sex, cigarette smoking, diabetes, history of atrial fibrillation, history of coronary heart disease, systolic blood pressure, and creatinine levels. Detailed operational definitions for all these covariables are provided elsewhere (8). Stroke outcome ascertainment and definition methods used in the Framingham Study, including subtype classification, have been described in detail previously (8, 9). Total homocysteine levels were determined by high-performance liquid chromatography with fluorescence detection (10). Nonfasting plasma aliquots were stored at 20 C from the baseline examination period until mid-1997. Data from long-term storage studies conducted at 20 C have confirmed both the biochemical stability and long-term within-person reproducibility of total homocysteine determinations (10). Creatinine levels were measured in nonfasting plasma by the Jaffe method, adapted for autoanalyzers. The skewed total homocysteine data were natural log-transformed, and differences in geometric mean total homocysteine levels according to sex, diabetes, history of atrial fibrillation or coronary heart disease, and smoking status were compared by using unpaired t-tests. The Spearman rho was used to assess unadjusted rank-order correlations between untransformed total homocysteine levels and age, creatinine level, and systolic blood pressure. Unadjusted and adjusted (for age, sex, history of atrial fibrillation or coronary heart disease, diabetes, smoking, systolic blood pressure, and creatinine level) relative risk estimates (hazards ratios with 95% CIs) for total stroke, nonhemorrhagic stroke, and atherothrombotic brain infarction were generated by proportional hazards modeling. Total homocysteine level (natural log-transformed or expressed in quartiles) was the independent variable. All statistical analyses were performed by using SAS software, version 6.12 (SAS Institute, Inc., Cary, North Carolina). Results The study sample (n=1947) consisted of 1158 women (59.5%) and 789 men (40.5%). Four hundred thirteen patients (21.2%) were cigarette smokers, 340 (17.5%) had a history of coronary heart disease, 182 (9.3%) were diabetic, and 81 (4.2%) had a history of atrial fibrillation. Arithmetic means (SD) for key continuous variables were as follows: age, 70 7 years (range, 59 to 91 years); systolic blood pressure, 141 21 mm Hg (range, 86 to 225 mm Hg); creatinine level, 97.2 26.5 mol/L (range, 35.3 to 442 mol/L); and total homocysteine level, 12.65 7.19 mol/L (range, 4.13 to 219.84 mol/L). Geometric mean total homocysteine levels were higher in men than in women (12.35 compared with 11.32 mol/L; P<0.001), in patients with a history of atrial fibrillation than in those without (13.17 compared with 11.66 mol/L; P=0.003), and in patients with a history of coronary heart disease than in those without (12.49 compared with 11.57 mol/L; P<0.001). However, these levels did not differ according to the presence or absence of diabetes (11.83 compared with 11.71 mol/L; P>0.2) or between current cigarette smokers and nonsmokers (11.67 compared with 11.74 mol/L; P>0.2). Weak but significant Spearman correlations were observed between total homocysteine levels and age (r=0.212; P<0.001), creatinine level (r=0.178; P<0.001), and systolic blood pressure (r=0.111; P<0.001). Quartiles of total homocysteine were as follows: quartile 1, 4.13 to 9.25 mol/L; quartile 2, 9.26 to 11.43 mol/L; quartile 3, 11.44 to 14.23 mol/L; quartile 4, 14.24 to 219.84 mol/L. During a median follow-up of 9.9 years, 165 incident total strokes occurred; 153 of these were incident nonhemorrhagic strokes, and 100 were incident atherothrombotic brain infarctions. Five hundred twenty-four persons were censored during follow-up because of death from causes other than stroke. Age, systolic blood pressure, current smoking, diabetes, and history of atrial fibrillation or coronary heart disease were independently predictive of total stroke occurrence (Table 1). Levels of total homocysteine (natural log) as a continuous variable (data not shown) and across quartiles (P<0.001 for linear trend) were associated with all stroke outcomes in unadjusted and multivariable-adjusted proportional hazards analyses (Tables 1 and 2). The interaction term between sex and total homocysteine level (quartile analyses) was nonsignificant (P=0.1); thus, the stroke incidence analyses were not stratified by sex. Further adjustment for creatinine level did not change the results of any of these analyses (data not shown). Table 1. Predictors of Incident Total Stroke in Elderly Women and Men in the Framingham Study Cohort Table 2. Relative Risk Estimates in Elderly Women and Men in the Framingham Study Cohort: Comparison of Each of the Upper Three Quartiles to the Lowest Quartile of Nonfasting Plasma Total Homocysteine Level Discussion Our findings are consistent with previously reported data, derived primarily from elderly female and male participants in the Framingham Study (9, 11), indicating that age, systolic blood pressure, diabetes, cigarette smoking, and history of atrial fibrillation or coronary heart disease were independently predictive of stroke incidence. We report population-based evidence that elevated nonfasting total homocysteine levels are also independently associated with stroke incidence among elderly women and men. Four earlier studies (3-6) have examined the relation between total homocysteine levels and stroke incidence. Alfthan and colleagues (3) did not find an association between total homocysteine levels and incident stroke among Finnish men and women 40 to 64 years of age (total events, 76). Similarly, Verhoef and coworkers (4) found only a weak, nonsignificant association between total homocysteine level and stroke incidence (total events, 109) in a cohort of male physicians whose mean age was 59.7 years (upper quintile compared with lower four quintiles: odds ratio, 1.2 [CI, 0.7 to 2.0]). In contrast, Perry and colleagues (5) reported a robust, independent association between total homocysteine levels (across quartiles) and incident stroke (total events, 107) among British men whose mean age was 54.0 years (quartile 3 compared with quartile 1: odds ratio, 3.3 [CI, 0.9 to 11.5]; quartile 4 compared with quartile 1: odds ratio, 7.4 [CI, 1.9 to 29.0]). More recently, elevated total homocysteine levels were independently linked to the development of stroke outcomes in a cohort of predominantly younger women (mean age, 34.9 years) with systemic lupus erythematosus (6). The two negative studies (3, 4) were characterized by modest stroke event rates and overall exposure to lower total homocysteine levels on the basis of sound nutritional (4) or, possibly, favorable genetic (3) influences. More widespread exposure to elevated total homocysteine levels, due perhaps to worse nutritional status, may have accounted for the strong association between total homocysteine level and incident stroke reported by Perry and colleagues (5). The positive relation between total homocysteine level and stroke occurrence described by Petri and associates in the Hopkins Lupus Cohort (6) was confined to patients with the highest total homocysteine levels (>14 mol/L). Our analyses, which revealed an independent association between total homocysteine level and incident stroke (total events, 165), were performed in an elderly sample characterized, as expected, by a relatively higher stroke event rate (7, 9, 11, 12). This higher rate occurred in conjunction with an increased prevalence of mild hyperhomocysteinemia (that is, in approximately 25% of patients with total homocysteine level>14 mol/L) at the baseline examination. Despite the lack of substantiation by either proven or biologically plausible mechanisms, it has nevertheless been proposed that hyperhomocysteinemia is an epiphenomenon of clinical or even subclinical arteriosclerosis (13, 14). This hypothesis appears untenable in view of the following published findings from both human and animal studies. First, despite the absence of any traditional arteriosclerotic risk factors, 50% of untreated children and young adults with homocystinuria due to cystathionine synthase deficiency ex

The 1298A C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine

A common mutation in methylenetetrahydrofolate reductase (MTHFR), 677C-->T, is associated with reduced enzyme activity, a thermolabile enzyme and mild hyperhomocysteinemia, a risk factor for vascular disease. Recently, a second common mutation (1298A-->C; glutamate to alanine) was reported, but this mutation was suggested to increase homocysteine only in individuals who carried the bp677 variant. To evaluate the functional consequences of this mutation, we performed site-directed mutagenesis and in vitro expression. For in vivo assessment of clinical impact, we examined the 1298A-->C genotypes and plasma homocysteine in 198 individuals from the NHLBI Family Heart Study that had previously been assessed for the 677 substitution. Site-directed mutagenesis of the human cDNA was performed to generate enzymes containing each of the two mutations, as well as an enzyme containing both substitutions. Enzyme activity and thermolability were assessed in bacterial extracts. The activity of the wild-type cDNA was designated as 100%; mutant enzymes containing the 1298 and 677 mutations separately had 68% (+/-5.0) and 45% (+/-10.8), respectively, of control activity while the enzyme containing both mutations had 41% (+/-12.8) of control activity. The 1298 mutation was not associated with a thermolabile enzyme. In the Family Heart Study, fasting homocysteine was significantly higher (P<0.05) in individuals heterozygous for both substitutions, compared to individuals who carried only the 677C-->T variant. This study suggests that two variants in MTHFR should be assessed as genetic risk factors for hyperhomocysteinemia.

High Homocysteine Levels Are Independently Related to Isolated Systolic Hypertension in Older Adults

BACKGROUND The association between homocysteine and isolated systolic hypertension in older adults was evaluated using a case-control design, and the relationship between homocysteine and clinical or subclinical atherosclerosis was explored. METHODS AND RESULTS Cases were 179 adults > or = 60 years with a systolic blood pressure of > or = 160 mm Hg and diastolic blood pressure < 90 mm Hg. One hundred seventy-one control subjects had the same criteria except systolic blood pressures were < 160 mm Hg. All had normal creatinine levels. Homocysteine levels were performed on fasting blood samples that had been stored at -70 degrees C. Atherosclerosis was defined as either a history of clinical disease, an internal carotid stenosis of > or = 40% by duplex scan, or an ankle/arm pressure ratio of < 0.9. The median homocysteine value was 11.5 micromol/L for cases and 9.9 for control subjects (P<.001). After control for potential confounders, homocysteine remained significantly associated with systolic hypertension (P=.019). For the hypertensive group, there was no apparent association between level of homocysteine and prevalence of atherosclerosis. However, among the normotensive group, the prevalence of atherosclerosis went from 22% in the lowest quintile of homocysteine values to 53% in the fifth quintile, with an odds ratio of 4.1 (fifth quintile in comparison to the first, P<.05). After adjustment for age, sex, systolic blood pressure, cholesterol, and smoking, this odds ratio increased to 6.4 (P<.01). CONCLUSIONS Elevated levels of homocysteine may be related to the cause of isolated systolic hypertension in some individuals. In normotensive older adults, homocysteine appears to be an independent risk factor for atherosclerosis.

A prospective investigation of elevated lipoprotein (a) detected by electrophoresis and cardiovascular disease in women. The Framingham Heart Study.

BACKGROUND Sinking prebeta lipoprotein is a putative marker for elevated levels of lipoprotein (a). Although prospective data suggest that increased plasma lipoprotein (a) is an independent risk factor for coronary heart disease in men, no prospective studies are available in women. METHODS AND RESULTS From 1968 through 1975, sinking prebeta lipoprotein was determined by paper electrophoresis in 3103 women Framingham Heart Study participants who were free of prevalent cardiovascular disease. A sinking prebeta lipoprotein band was detectable in 434 of the women (14%) studied. The median follow-up interval was approximately 12 years. Incident cardiovascular disease was associated with band presence using a proportional hazards model that included age, smoking, body mass index, systolic blood pressure, glucose intolerance, low- and high-density lipoprotein cholesterol, and ECG left ventricular hypertrophy. Multivariable adjusted relative risk estimates (with 95% confidence intervals) for outcomes in the band present versus absent groups were as follows: myocardial infarction (82 events), 2.37 (1.48 to 3.81); intermittent claudication (62 events), 1.94 (1.07 to 3.50); cerebrovascular disease (83 events), 1.88 (1.12 to 3.15); total coronary heart disease (174 events), 1.61 (1.13 to 2.29); and total cardiovascular disease (305 events), 1.44 (1.09 to 1.91). A subset analysis indicated that band presence was 50.9% sensitive and 95.4% specific for detecting plasma lipoprotein (a) levels of > 30 mg/dL, the threshold value linked to increased cardiovascular disease risk in men. CONCLUSIONS Sinking prebeta lipoprotein was a valid surrogate for elevated lipoprotein (a) levels in Framingham Heart Study women. Band presence and, equivalently, elevated plasma lipoprotein (a), was a strong, independent predictor of myocardial infarction, intermittent claudication, and cerebrovascular disease. Confirmation of these findings in other longitudinal studies of women is needed.

Elevated Fasting Total Plasma Homocysteine Levels and Cardiovascular Disease Outcomes in Maintenance Dialysis Patients

There is an excess prevalence of hyperhomocysteinemia in dialysis-dependent end-stage renal disease (ESRD) patients. Cross-sectional studies of the relationship between elevated total homocysteine (tHcy) levels and prevalent cardiovascular disease (CVD) in this patient population suffer from severe methodologic limitations. No prospective investigations examining the association between tHcy levels and the subsequent development of arteriosclerotic CVD outcomes among maintenance dialysis patients have been reported. To assess whether elevated plasma tHcy is an independent risk factor for incident CVD in dialysis-dependent ESRD patients, we studied 73 maintenance peritoneal dialysis or hemodialysis patients who received a baseline examination between March and December 1994, with follow-up through April 1, 1996. We determined the incidence of nonfatal and fatal CVD events, which included all validated coronary heart disease, cerebrovascular disease, and abdominal aortic/lower-extremity arterial disease outcomes. After a median follow-up of 17.0 months, 16 individuals experienced at least one arteriosclerotic CVD event. Cox proportional-hazards regression analyses, unadjusted and individually adjusted for creatinine, albumin, and total cholesterol levels, total/HDL cholesterol ratio, dialysis adequacy/residual renal function, baseline CVD, and the established CVD risk factors (ie, age, sex, smoking, hypertension, diabetes/glucose intolerance, and dyslipidemia) revealed that tHcy levels in the upper quartile (> or = 27.0 mumol/L) versus the lower three quartiles (< 27.0 mumol/L) were associated with relative risk estimates (hazards ratios, with 95% confidence intervals for the occurrence of (pooled) nonfatal and fatal CVD ranging from 3.0 to 4.4; 95% confidence intervals (1.1-8.1) to (1.6-12.2). We conclude that the markedly elevated fasting tHcy levels found in dialysis-dependent ESRD patients may contribute independently to their excess incidence of fatal and nonfatal CVD outcomes.

Net uptake of plasma homocysteine by the rat kidney in vivo

Hyperhomocysteinemia is a common finding in dialysis-dependent end-stage renal disease (ESRD) patients, but its etiology and refractoriness to standard homocysteine-lowering B-vitamin therapy are poorly understood. In the absence of actual in vivo data, it has been hypothesized that loss of normal renal parenchymal uptake and metabolism of homocysteine is an important determinant of hyperhomocysteinemia in ESRD, given that urinary homocysteine excretion by healthy kidneys is trivial. We assessed net renal uptake and metabolism of homocysteine using an established rat model for measuring arteriovenous amino acid differences across the rat kidney, along with simultaneous determination of renal plasma flow, urine flow, and urinary homocysteine concentration. Substantial homocysteine uptake and metabolism by normal rat kidneys was demonstrated, and we also confirmed that urinary homocysteine excretion is minimal. These data suggest that loss of the sizable homocysteine metabolizing capacity of the intact kidneys may be an important determinant of the refractory, potentially atherothrombotic hyperhomocysteinemia frequently observed in ESRD.

Hyperhomocysteinemia and traditional cardiovascular disease risk factors in end-stage renal disease patients on dialysis: a case-control study

Hyperhomocysteinemia occurs frequently in end-stage renal disease (ESRD), but its prevalence in comparison with traditional cardiovascular disease (CVD) risk factors is unknown. Fasting total plasma homocysteine, potential determinants of plasma homocysteine (i.e., plasma B-vitamins and serine), total and HDL cholesterol, glucose, and creatinine, were determined in 24 ESRD patients on dialysis, and 24 age, gender, and race matched Framingham Offspring Study controls with normal renal function. Presence of clinical CVD and CVD risk factors was established by standardized methods. Mean plasma homocysteine was markedly higher in the ESRD patients versus controls (22.7 vs. 9.5 mumol/l). ESRD patients were 33 times more likely than controls to have hyperhomocysteinemia (> 15.8 mumol/l) (95% confidence interval, 5.7-189.6). Hyperhomocysteinemia persisted in the ESRD patients despite normal to supernormal B-vitamin status. Plasma serine levels below the tenth percentile of the control distribution were found in 75% of the ESRD patients. Oral serine supplementation caused a 37% increase in mean plasma serine, but had no effect on plasma homocysteine in four ESRD patients with supernormal plasma folate, low plasma serine, and hyperhomocysteinemia. Given its unusually high prevalence, improved management of hyperhomocysteinemia might reduce CVD sequelae in ESRD.