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2 Institute of Reproductive and Child Health, Peking University, Beijing 100083, China; 3 Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115; and 4 Departments of Nutrition and Epidemiology, Harvard School of Public Health, Boston, MA 02115
* To whom correspondence should be addressed. E-mail: jing.ma{at}channing.harvard.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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16.0 µmol/L, a level used to define hyperhomocysteinemia. Within each region, men had higher plasma tHcy concentrations than women (16.1 vs. 10.6 µmol/L in the north, and 10.7 vs. 7.9 µmol/L in the south) and 40% of the northern men had hyperhomocysteinemia. Generally, individuals living in the urban areas had 30% (95% CI, 1.0–1.6) greater odds of having high tHcy levels (16 umol/L) than those living in the rural areas. Low plasma concentrations of folate, vitamins B-12 and B-6, older age, being male, and living in urban areas were all independently associated with elevated tHcy, with low folate as the strongest determinant.
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Plasma tHcy status and its determinants have been studied extensively in developed countries (1,12–15) but seldom in developing countries. Published data suggest that plasma tHcy concentrations vary by age and have significant ethnic and gender differences (13,14,16). Because fruit and vegetable intakes in China rely on local and seasonal productions, the intakes of green-leaf vegetables with high folate content are much higher in the south than the north. Our previous studies demonstrated that a large proportion of Chinese have low folate and vitamin B-12 status with large geographic variations (17,18). Therefore, we hypothesize that hyperhomocysteinemia is prevalent in the same study population and that it may vary by geographic region. In this study, we present the geographic, seasonal, gender, and age variations in plasma tHcy concentrations among apparently healthy Chinese adults aged 35–64 y and evaluate the associations of plasma folate and vitamins B-12 and B-6 with plasma tHcy concentrations.
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Subjects and Methods |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Using face-to-face interviews, we collected information on date of birth, personal history, diet, medication, physical activity, and lifestyle. Subjects who were pregnant or had a severe illness (renal, heart, liver disease, or cancer), based on the medical diagnostic information obtained during the interview, were excluded. Current smoking status was recorded in 4 categories: nonsmoker, smoking 1–10, 11–20, or 21 cigarettes/d. Alcohol consumption (including liquor, beer, and wine) was classified in 3 categories: never use, <1, or 1 drink/d. Vitamin use, including multivitamins and individual vitamins such as thiamin, riboflavin, vitamin B-6, folic acid etc., during the past 3 mo was categorized as user and nonuser. Body height and weight were measured by health workers. BMI was calculated as kg/m2.
In the study, 2471 subjects provided blood samples. Among these, we obtained plasma creatinine data for 2411, folate for 2456, vitamin B-12 for 2407, and tHcy for 2464, with shortages due either to inadequate plasma volume or sample hemolysis. We selected 40% of plasma samples (963) at random for assays of pyridoxal-5'-phosphate (PLP, the active circulating form of vitamin B-6).
The study was approved by the Institutional Review Boards of Peking University Health Science Center. All invited participants provided oral informed consent after hearing the informed consent script read by the investigators.
Biochemistry analyses. Overnight fasting blood samples were drawn and collected in K3EDTA-containing Vacutainer tubes (Becton Dickinson), held at 4°C, and centrifuged at 800 x g for 15 min. The plasma and red blood cells were separated and frozen at –20°C within 1 h of collection. All specimens were transported on dry ice to the central laboratory of the Institute of Reproductive and Child Health, Peking University, and stored at –70°C until the assay was performed. Plasma folate concentrations were determined by a microbial assay (Lactobacillus casei) using a 96-well plate as described by O'Broin et al. (20), plasma vitamin B-12 by the BioRad Laboratories Quantaphase II radioassay, plasma PLP by the tyrosine decarboxylase apoenzyme method (21), and plasma creatinine concentrations by standard techniques adapted for automatic blood chemistry analyzers (HITACHI model 7170). Plasma tHcy measurements were carried out by HPLC with fluorometric detection (22). In the laboratory, the intra and interassay CV across the full range of the concentrations were <9% for folate, <7% for vitamin B-12, <15% for PLP, and <8% for tHcy.
Statistical analyses. We calculated Spearman correlation coefficients to examine the relation between tHcy and creatinine, folate, vitamin B-12, or PLP. The significance of categorical variables was assessed by the chi-square test. Natural logarithmic transformations of plasma tHcy concentrations were used to normalize its distribution, and the geometric means as well as the 95% CI were determined. Student's t tests and 1-way ANOVA were used for comparisons between groups when using continuous variables if the variables were normally distributed. We used the Mann-Whitney U tests for comparisons of nonnormal data. Multivariate analyses of variance were conducted to compare the means of tHcy with Bonferroni correction controlling for region, age, gender, area, season, as well as folate, vitamin B-12, and creatinine. In addition, the polynomial contrasts were used to test for tHcy concentration trends across age. Differences were considered significant at P < 0.05.
Hyperhomocysteinemia was defined as plasma tHcy concentration 16.0 µmol/L (23). Multivariate logistic regression was used to examine the odds ratios (OR) of hyperhomocysteinemia in association with demographic factors, including region, area, season, age, and gender. The associations of hyperhomocysteinemia with BMI, alcohol use, and cigarette smoking status were evaluated controlling for the demographic factors. We classified the participants into 4 categories of folate levels: deficient, and tertiles for the rest whose plasma folate concentrations were not deficient. Vitamins B-12 and B-6 status were classified in the same manner as folate. Vitamin deficiencies were defined as <6.8 nmol/L for plasma folate (17), <185 pmol/L for plasma vitamin B-12 (18), and <30 nmol/L for plasma PLP (24). Plasma creatinine was classified as normal or abnormal (90 µmol/L for women and 110 µmol/L for men) (1). We also performed logistic regression analysis of the odds of hyperhomocysteinemia in association with vitamin supplement use, creatinine status, plasma folate, vitamin B-12, or PLP separately, controlling for region, area, season, gender, and age (model 1). Vitamin supplement use, creatinine, plasma folate, and vitamin B-12 were then added to the multivariate model (model 2). Plasma PLP (for 963 subjects only) was further entered into the multivariate models (model 3) to assess independent associations of these factors. Finally, we evaluated the combined associations of folate and vitamin B-12 status with plasma tHcy, controlling for region, area, season, gender, age, plasma creatinine, and vitamin use. Subjects with missing data were excluded from the logistic regression analysis. The data were analyzed with SPSS 11.0. All P-values were 2-sided at 
= 0.05.
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Results |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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16.0µmol/L, a prevalence significantly higher than that of southerners (7%) (Table 1, Fig. 1).
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16.0 µmol/L. The corresponding proportions were much lower in the south (11% and 2%). Plasma tHcy concentrations were positively associated with age within each region and each gender, except for the men in the north who had similar, high plasma tHcy concentrations in all age groups. The gender difference in tHcy concentrations decreased with age in both regions.
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6-fold higher odds of hyperhomocysteinemia than those living in the south. Also, urban residents had a 30% increased odds of high tHcy concentrations than those in the rural areas, and men had 3.6-fold higher odds of high tHcy than women. However, in a multivariate model with further control for vitamin supplement use, plasma folate, vitamin B-12, and creatinine, all these associations were attenuated toward the null. The prevalence of elevated tHcy tended to increase with age after multivariate adjustment (P = 0.08; Table 4). BMI and alcohol consumption were not associated with hyperhomocysteinemia in either univariate or multivariate analyses. Cigarette smokers (among men only, because very few women smoked in this population) had higher odds of elevated tHcy (OR, 95% CI; 1.4, 1.0–1.9) than nonsmokers but with little dose relation (data not shown) after controlling for region, area, season, and age. Table 5 shows multivariate OR and 95% CI for hyperhomocysteinemia according to plasma folate, vitamin B-12, PLP, and creatinine, and vitamin supplement use. The risk of hyperhomocysteinemia was higher in individuals with low B vitamin status or abnormal creatinine concentrations, controlling for region, area, season, gender, and age (model 1). The risk was lower among vitamin supplement users. The associations remained unchanged when including plasma folate, vitamin B-12, creatinine, and vitamin use in the same model (model 2). In model 3, we further included plasma PLP with smaller sample size because only 40% of the samples were assayed for PLP. Similarly, low levels of plasma folate, vitamin B-12 and vitamin B-6, abnormal creatinine levels, and nonuse of vitamin supplements were strongly associated with elevated tHcy with folate and vitamin B-12 deficiency as the strongest risk factors.
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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16.0 µmol/L. In the north, 28% of adults (40% of men) had tHcy above this level, >4-fold higher than in the south. Marked geographic, gender, and seasonal variations were observed for plasma tHcy concentrations among Chinese adults that could be explained mainly by differences in folate, vitamin B-12, and vitamin B-6 status. The inverse associations of these 3 B vitamins with high tHcy levels were independent of each other. Overall, plasma folate concentrations were higher and tHcy concentrations lower in the spring than in the fall in the south. In the north, folate concentrations were lower and tHcy concentrations higher in the spring, probably reflecting the different seasonal availability of green leaf vegetables in the 2 regions. Men had significantly higher plasma tHcy concentrations than women, consistent with previous results (1,13,14) and in agreement with our findings, which showed men had lower plasma folate and vitamin B-12 concentrations than women among this Chinese population (17,18). After controlling for plasma folate, vitamin B-12, and creatinine, the difference in the plasma tHcy concentration between south and north was no longer significant, but the difference between men and women, and between urban and rural areas remained significant. This suggests that the differences in plasma folate, vitamin B-12, and creatinine between the south and the north were the main cause for the geographic difference of plasma tHcy concentrations in the study population, but they do not fully explain the gender and area differences.
Chinese men had a higher prevalence of cigarette smoking (58%) compared with women (5%) (17). A strong positive association between tHcy concentrations and cigarette smoking was reported by Jacques (15) and from the Hordaland Homocysteine Study (25). We found that cigarette smokers had a higher odds of hyperhomocysteinemia compared with nonsmokers (OR, 95% CI; 1.4, 1.0–1.9) after controlling for region, area, season, and age, but we observed no significant dose relations. Our data showed current smokers had higher odds of folate deficiency than nonsmokers (adjusted OR, 1.9 for plasma folate deficiency, 2.5 for red blood cell folate deficiency), and we found no association of vitamin B-12 status with cigarette smoking status (17,18). After further controlling for plasma folate, the difference in tHcy concentrations between smokers and nonsmokers was substantially attenuated and no longer significant, which suggested that lower blood folate levels might be the main cause of elevated tHcy in smokers compared with nonsmokers.
Our data confirmed the age difference in tHcy concentrations reported previously (1,14). We found an 8% increase in the plasma tHcy concentration in men and 26% in women from age 35–44 y to 55–64 y. The results were consistent with NHANES III, which demonstrated a significant age x sex interaction such that the male-female differences in tHcy concentrations diminished with increasing age (14). In addition, previous observations suggested estrogen is inversely related to plasma tHcy concentrations (26,27), and that tHcy concentrations increase significantly at 50 y of age in women (28). Masse et al. (29) found that plasma tHcy concentrations of fasting, postmenopausal women were twice that of premenopausal women. At each age group, men had significantly higher plasma tHcy concentrations than women, probably due to higher creatine to creatinine systhesis (a function of muscle mass and a major source of homocysteine formation) in men than in women (30).
Individuals living in urban areas had significantly higher median tHcy concentrations than those living in rural areas. This difference in tHcy concentration was not fully explained by the differences in folate and vitamin B-12 status (17,18). Studies have shown that elevated tHcy is also associated with other nutritional or nonnutritional determinants, including low riboflavin intake (15), high caffeine intakes (15,31), low levels of physical activity (31,32), and certain antihypertension medications (15,33).
High plasma tHcy concentrations in the apparently healthy Chinese population observed in our study is in agreement with a Chinese multicenter case-control study of stroke, which showed that tHcy levels were high, with median levels of 12.8 µmol/L among control subjects and 14.7 µmol/L among stroke patients. More than 25% of the controls and >40% of the stroke patients had homocysteine levels 16 µmol/L (34). In that study, high tHcy levels were associated with 2-fold higher risk of stroke, controlling for the conventional CVD risk factors. The south-north difference in B-vitamins and tHcy status parallels the rate of CVD in China, which is 10-fold higher for the incidence of coronary heart disease and 2 to 4-fold higher for the risk of stroke in the north than in the south. These differences cannot be explained by the known CVD risk factors such as smoking, hypertension, BMI, and cholesterol levels (35), and our data suggest a potential ecological link between B vitamin status and the risk of CVD in Chinese adults. Although 4 large secondary prevention trials have recently found no significant effects of folic acid, vitamin B-12, and vitamin B-6 on risks for recurrent CVD or mortality (36–39), one of the possible explanations might be that, if low B vitamin and high tHcy status act in the early process of atherosclerosis development, it could be too late to witness the apparent effect of B vitamin supplements on the recurrence of CVD. Recently published data demonstrated the dramatic improvement in stroke mortality after countrywide folic acid fortification in 1998 in the United States and Canada, but not in England and Wales where fortification was never implemented (40). The proof of a causal relation between tHcy status and CVD would require primary intervention trials.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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5 Abbreviations used: CVD, cardiovascular disease; OR, odds ratio; PLP, pyrodoxal-5-phosphate; tHcy, total plasma homocysteine.
Manuscript received 12 October 2006. Initial review completed 15 October 2006. Revision accepted 26 November 2006.
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LITERATURE CITED |
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