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B Vitamins and Plasma Homocysteine Concentrations in an Urban and Rural Area of Costa Rica

Department of Nutrition, Harvard School of Public Health (M.K.K., H.C.), Boston, Massachusetts
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (J.M.O., J.S.), Boston, Massachusetts
Hanyang University College of Medicine, Seoul, SOUTH KOREA (M.K.K.)

Address reprint requests to: Hannia Campos, PhD, Department of Nutrition, Room 353A, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. E-mail: hcampos{at}hsph.harvard.edu

	ABSTRACT

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Objective: We studied the association between total plasma homocysteine (tHcy) concentrations and folate, B₁₂, and B₆ status in the urban and rural areas of Costa Rica.

Subjects and Methods: We determined plasma tHcy concentrations and assessed dietary folate, B₁₂ and B₆ intake by a food frequency questionnaire in 462 subjects selected by stratified random sampling in the urban and rural areas of Puriscal, Costa Rica. Plasma folate and vitamin B₁₂ concentrations were measured in women.

Results: THcy concentrations were higher (p < 0.01) in the rural compared with the urban area: 12.0 µmol/L vs. 8.9 µmol/L in men, and women 7.3 µmol/L vs. 5.5 µmol/L in women, respectively. The prevalence of hyperhomocysteinemia (greater than 15.0 µmol/L) was twice as high in rural compared with urban men (19.8% vs. 10.8%, p = 0.06) and women (6.6% vs. 3.4%, p = 0.26). Most study subjects (98%) had folate intakes that were less than the recommended 400 µg/day. In women, 31% of those living in the urban area and 40% of those in the rural area had plasma folate concentrations of less than 6.8 nmol/L, an indicator of folate deficiency. In women, age-adjusted mean tHcy concentrations (µmol/L) were higher in the lowest compared with highest quintiles for dietary vitamin B₆ (9.9 vs. 5.4, p < 0.05), B₁₂ (9.2 vs. 4.9, p < 0.01), and folate (7.0 vs. 5.7, p = 0.87). Similar results were found for plasma B₁₂ (9.9 vs. 5.4, p < 0.01) and folate (10.5 vs. 5.0, p < 0.0001).

Conclusions: Residents of the rural area in Puriscal, Costa Rica have higher plasma concentrations of tHcy and lower intake of B vitamins, particularly in women. Because these characteristics are associated with high risk of cardiovascular disease, the efficacy of food fortification program in rural areas should be carefully addressed.

Key words: homocysteine, B vitamins, folate, Hispanic, Costa Rica

	INTRODUCTION

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Cardiovascular disease is the main cause of death in most Latin American countries [1] and in Hispanics living in the United States [2]. The highest age-adjusted mortality rates (per 100,000) from cardiovascular disease are found in men and women living in Argentina (274.8 and 153.5, respectively), whereas, Costa Rica has the highest cardiovascular disease rates in Central America (208.8 and 148.8, respectively). These numbers are comparable to those found in men and women living in industrialized North American countries such as Canada (182.4 and 94.7, respectively) and the United States (223.1 and 136.1, respectively) [1]. Although several population-based studies on cardiovascular disease risk factors are available for Hispanics living in the US [3], population studies in Latin American countries currently experiencing demographic, epidemiologic and nutritional transition are scarce. It has been shown that cardiovascular disease risk factors such as obesity, smoking, saturated fat intake and atherogenic plasma lipoprotein profiles are found more frequently in urban areas than in rural areas of Latin American countries [4–6]. These risk factors are thought to contribute to the increase in cardiovascular disease observed with urbanization. However, the role of micronutrient intake as a potential cardiovascular disease risk factor has not been examined.

Poor dietary habits including deficiencies in several vitamins probably contribute to cardiovascular disease in developing countries [6, 7]. Low levels of folate in the diet or plasma are associated with an increased risk of cardiovascular disease [8–11]. A mechanism for this association may relate to the inverse association between folate, B₆ and B₁₂ status and the concentration of plasma total homocysteine (tHcy), a sulfur-containing amino acid formed during the metabolism of methionine [12, 13]. Selhub and colleagues suggested that inadequate plasma concentrations of one or more B vitamins are major determinants of high concentrations of plasma tHcy, a risk factor for cardiovascular disease [14]. Of concern is the relation between moderate hyperhomocysteinemia and an increased risk for atherosclerosis in the coronary, cerebral, and peripheral vasculature [15, 16]. Moderate hyperhomocysteinemia (greater than 15 µmol/L) [17] occurs in approximately 5% to 7% of the general population in the United States and Europe [13, 15]. It has been estimated that a 5 µmol/L tHcy increment elevates coronary artery disease risk by as much as cholesterol increases of 20 mg/dL (0.5 mmol/L) [18], although a recent comprehensive evaluation of the literature showed that the effects of tHcys on coronary disease are relatively weak [19].

The objective of this study is to examine B vitamins and plasma tHcy concentrations among rural and urban residents of Puriscal, Costa Rica.

	SUBJECTS AND METHODS

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Study Population
The study population was randomly selected from the urban and rural area in the county of Puriscal, Costa Rica. The Puriscal region extends from the middle of Costa Rica to the Pacific coast and covers an area of 800 km². There are approximately 26,000 inhabitants in about 150 sparsely distributed localities. Fewer than 500 people reside in each locality. Although not a true urban center, Santiago, the county’s capital, is referred to as urban by the Center for Census and Statistics of Costa Rica, and their definition and maps were used in this study. The study population has been described in a previous report [20].

Subjects in the urban and rural areas were selected by stratified random sampling to obtain a similar number of participants in each group. Maps and information from the National Census Bureau were used for this procedure. Eligible households were defined as those having one man and one nonpregnant woman 19 to 65 years of age. One hundred thirty such households were selected randomly. Of all the eligible subjects, 85% of the men (n = 222) and 93% of the women (n = 240) agreed to participate and provided a blood sample. Enough sample was available from all men and 240 women for measurements of plasma tHcy concentrations. Because of previous biochemical analysis carried out in this population, there were only 230 women and no men with available sample for measurements of plasma B₁₂ and folate. All subjects gave informed consent by signing a consent form approved at the Institute of Health Research of the University of Costa Rica.

Data Collection
Dietary Assessment and General Characteristics Questionnaire.
Trained fieldworkers visited participants at their homes for data collection. Information on clinical and general characteristics was obtained during an interview. The questionnaire included socioeconomic and demographic characteristics, as well as brief medical history, medication use and smoking habits. Women were classified as being post-menopausal if they reported that they had not had menses for at least one year, as being pre-menopausal if they reported having normal menses and as being peri-menopausal if they reported that they had not had menses for less than one year.

Dietary information was obtained with a semiquantitative food frequency questionnaire (FFQ) during an interview by trained fieldworkers. This FFQ was developed specifically for use in the Costa Rican population [21]. The FFQ inquires about intake of 135 food items including alcohol, vitamin, mineral and food supplements; types of fat used for cooking and frying; consumption of fried foods in and away from home; and food habits related to meal preparation during the past year. Energy and nutrient intakes were computed by multiplying the consumption frequency of each food by the nutrient content of the specific portion [21]; food composition values from the US Department of Agriculture (USDA) database [22, 23] and data from manufacturers and published reports were used. Total daily alcohol intake was calculated on the basis of the individual’s report of the type and amount of alcoholic beverage consumed over the previous year. Daily alcohol intake (g) was calculated based on the average ethanol in a typical portion of each type of beverage in the FFQ. That is 12 g in a 12 oz can of beer, 11 g in a 4 oz glass of wine and 14 g in a standard drink of spirits. We carried out a validation study to compare the FFQ with an assessment of dietary intake by seven 24-hour recalls conducted over seven months of the year to account for seasonal variation [21]. For each subject the months and days on which recalls were conducted were chosen at random, but all seven days of the week were represented. For reproducibility, a second FFQ was administered one year after the first interview. The Pearson partial correlation coefficients for folate, B₁₂ and B₆ were 0.55, 0.42 and 0.41 between the averages of the two FFQ assessments and the seven 24-hour recalls; they were 0.51, 0.46, and 0.46, respectively, between the first and second FFQs (p < 0.0001 for all) [21].

Anthropometric Measurements and Physical Fitness.
Three trained fieldworkers acquainted with standardized methods took all anthropometric measurements. All measurements were performed in duplicate, and the average was used for analysis. The waist was measured at the smallest horizontal trunk circumference and the hip was measured at the largest horizontal circumference around the hip and buttocks, with nonstretching fiberglass or metal tapes. Height was measured with a steel anthropometer. Weight was measured on a Detecto bathroom scale or a Seca Alpha Model 770 digital scale (both calibrated biweekly). Body mass index (BMI) was expressed as weight (kg) divided by height (m) squared. We determined the level of fitness with a modified Harvard step test performed on a portable wooden 40-cm step. Subjects were asked to maintain a rhythm (76 beats/minute for women, 96 beats/minute for men) for three minutes. Pulse rates were taken immediately after the test and at 1, 2, and 3 minutes thereafter. We expressed the fitness score as duration of exercise in seconds divided by the sum of the pulse rates at 1, 2, and 3 minutes after the step test, times 100.

Blood Samples and Blood Pressure.
Fasting blood samples were drawn into tubes containing 0.1% EDTA and stored immediately at 4°C. Most samples were centrifuged at 2,500 rpm for 20 minutes at 4°C to isolate the plasma within six hours of collection. Blood pressure was always measured in the morning while the subject was in a sitting position. All measurements were taken in duplicate by the same registered nurse using a sphygmomanometer. The average of the two measurements was used in the analysis. Hypertension was defined as systolic blood pressure of 140 mmHg or greater, diastolic blood pressure of 90 mmHg or greater, or taking antihypertensive medication [24].

Laboratory
Plasma tHcy concentrations were determined by the method of Araki and Sako [25]. Plasma vitamin B₁₂ was measured with a radioassay kit from Ciba-Corning (Medfield, MA). Plasma folate concentrations were determined by using a radioassay (Ciba-Corning) and a microbial assay as described by Tamura and coworkers [26]. In our data, both methods for measuring plasma folate concentration correlated highly (r = 0.74). We used the values obtained by radioassay because more samples were measured. Interassay coefficients of variation were 8.5% for tHcy, 7% for vitamin B₁₂, and 13% for folate. All samples were analyzed at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University.

Statistical Analyses
All analyses were performed with the SAS software (Version 8.00, SAS Institute Inc., Cary, NC). Because all plasma and intake values for nutrients were skewed, the values were transformed logarithmically to normalize their distributions for statistical analysis. To examine the composition of the diet after accounting for differences in energy requirements among individuals, we adjusted all measurements of nutrient intake by the residual method [27]. The residuals were standardized to a predicted nutrient intake of 8400 kJ/day (2000 kcal/day) for both men and women. We then determined mean values of homocysteine and vitamins in the diet and plasma. We defined high plasma homocysteine concentrations as greater than 15.0 µmol/L [17]. Low plasma concentrations were defined as having less than 6.8 nmol/L (3 ng/mL) for folate and as less than 148 pmol/L (200 pg/mL) for vitamin B₁₂ [28]. Differences between rural and urban men and women for all parameters and differences between age-adjusted homocysteine means by quintile of nutrient were evaluated by Tukey’s multiple comparisons for the least-square of means in a general linear model. Comparisons for plasma folate and vitamin B₁₂ in women were obtained by a two-tailed t test. Categorical variables were evaluated by the Chi-square test or by Cochran-Mantel-Haenszel statistics for the stratified analysis. Because the measurements of BMI and plasma tHcy, folate and vitamin B₁₂ concentrations were positively skewed, logarithmically transformed data were used for the general linear models.

	RESULTS

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Characteristics of the Subjects
The mean age was 41 years (range 19 to 67) for men and women (Table 1). The educational levels of men and women were similar within the same area, but urban men and women were more educated compared to rural residents. On the other hand, men and women in the rural area had higher fitness scores than did those in the urban area. The BMI was higher in women than men and, overall, higher in the urban area (25.96 kg/m²) than the rural area (25.07 kg/m², p = 0.008). The rural-urban differences in BMI within gender were not statistically significant. In contrast, men had a higher waist-to-hip ratio (WHR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and prevalence of cigarette smoking than women, although there were no significant rural-urban differences for these variables. Alcohol consumption (g/day) was significantly higher in the urban area compared with the rural area, but this difference was mostly due to men, since rural-urban differences in women were not statistically significant. Menopausal status and the proportion of oral contraceptive users did not differ between urban and rural women.

View larger version (9K): [in this window] [in a new window]   Fig. 1. Mean plasma total homocysteine concentrations (and 95% confidence intervals) by quintile of plasma folate (left) and vitamin B12 (right) in women from Puriscal, Costa Rica. Means are adjusted to an age of 40 years. Asterisks indicate significant difference from the mean in the highest quintile (** p < 0.01; *** p < 0.001).

	DISCUSSION

 
In the present study, of 462 randomly selected subjects living in Puriscal, Costa Rica, we compared plasma tHcy concentrations with dietary folate, B₆ and B₁₂ intake and (in women only) concentrations of plasma folate and B₁₂. Our data show that mean tHcy concentrations were higher and B vitamin intake was lower in the rural as compared with the urban area. Almost the entire study population consumed less than the recommended daily allowance for folate (400 µg/day). Forty percent of the women in the rural area and 31% in the urban area were classified as plasma-folate deficient, compared to 4% in the US population using the same criteria [29]. We conclude that folate is suboptimal in this population, particularly in subjects living in the rural area where the highest plasma tHcy concentrations were found. The food fortification program in Costa Rica started in 1998 with the fortification of wheat flour (1.5 mg/kg) [30], followed by the fortification of corn flour (1.3 mg/kg) in 2000 [31] and the fortification of milk (40 µg/250 mL) in 2002 [32]. Although this study was carried out before the fortification program started, our data suggest that availability of fortified foods in the rural area should be carefully addressed.

Plasma total homocysteine concentrations depend on age, gender and, in women, possibly menopausal status and oral contraceptive use [33–35]. Consistent with previous studies [35–37], our data showed higher tHcy concentrations in men than women. Furthermore, tHcy concentrations increased with age, but no differences on the basis of menopausal status or use of oral contraceptives were observed (data not shown).

In population studies, the mean plasma tHcy concentrations range between 6 µmol/L in Japan and 13 µmol/L in South Africa [17]. We were surprised to find that the overall plasma tHcy concentrations in Costa Rica, 8.4 µmol/L, were in the lower range, particularly in women (6.4 µmol/L) [37, 38]. Consistent with this observation, plasma tHcy concentrations in Hispanic women participants in NHANES III were lower than in non-Hispanic white women even when plasma folate concentrations were lower among the Hispanics [38]. Thus, in addition to dietary intake, several other environmental and genetic factors probably explain cross-cultural differences in plasma tHcy concentrations. Our data underscore the importance of conducting epidemiological studies in developing countries and minority populations in industrialized countries.

The prevalence of mild hyperhomocysteinemia (>15.0 µmol/L) [17] was higher in the rural area of Costa Rica as compared with the urban area and some Western populations that used the same cut-point [13, 15]. Low intakes of folate and the vitamins B₆ and B₁₂ are associated with high concentrations of plasma tHcy [14]. Thus, the relatively high prevalence of moderate hyperhomocysteinemia in the rural area may be due, in part, to the lower B vitamin intake observed in rural compared to urban residents, particularly in women. Rural-urban difference in tHcy concentrations in women was explained by lower plasma folate concentrations in the rural compared to the urban area. After adjusting for potential confounders (age, physical fitness, education levels, BMI and plasma vitamin B₁₂), the rural and urban difference still remained. When additionally plasma folate concentrations were entered in the model, the rural and urban difference in tHcy concentrations was markedly attenuated and no longer statistically different. It is interesting to note that the average plasma folate concentrations in the Costa Rican women (7.8 nmol/L) were comparable to the lowest quintile of women participants in the Framingham Offspring Study (7.9 nmol/L) [39], who also reported a total folate intake (257 mg/day) that is comparable to women in this study. The observed folate values in Costa Rica were lower than in Hispanic women in the NHANES III (11.7 nmol/L), although plasma B₁₂ concentrations were higher in the Costa Ricans (467 pmol/L vs. 351 pmol/L, respectively) [38].

Consistent with other populations [14], we found that in women, decreased intake of vitamins B₆ and B₁₂ and lower plasma folate and B₁₂ concentrations were associated with increased plasma tHcy. The correlation between plasma tHcy and plasma folate concentrations was stronger than that between plasma tHcy and folate intake assessed by a validated FFQ [21], a finding consistent with observations in Western countries [9]. The lower correlations observed with folate assessment by an FFQ could be due to measurement error associated with the use of dietary questionnaires to estimate intake. Another explanation for the low correlations observed with the FFQ may be attributed to the estimated folate content of foods in nutrient databases. The bioavailability of naturally occurring folate in foods is low (approximately 50% to 70%) due to hydrolysis and its extreme susceptibility to destruction by heating, cooking and oxidation [40, 41]. Therefore, the content of folate in the diet may be difficult to determine accurately.

We did not find significant correlations between B vitamin intake and plasma tHcy concentration in men. It is possible that self-reported intake in men is less accurate than in women [42]. The range of homocysteine concentration in plasma also differs by gender even when controlling for dietary intake, suggesting sex differences in tHcys metabolic regulation. It is possible that the level of intake necessary to lower plasma tHcy in men is higher than in women. Since dietary supplements are uncommon in this population, and this study was conducted before food fortification programs, high B vitamin intakes are very difficult to attain.

Moderately high plasma homocysteine concentrations have been independently associated with risk for coronary heart disease [18], most likely in a dose-dependent manner [43]. Intervention studies have shown that supplementation with folate and vitamins B₁₂ and B₆ can lower tHcy concentrations [44–48]. Most important, however, is the observation of an association between increased intake of folate per se and decreased risk of cardiovascular disease in prospective epidemiological studies [8–11].

	CONCLUSION

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Residents of the rural area in Puriscal, Costa Rica have higher plasma concentrations of tHcy and lower intake of B vitamins, particularly in women. Because these characteristics are associated with high risk of cardiovascular disease, the efficacy of food fortification program in rural areas should be carefully addressed.

	ACKNOWLEDGMENTS

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
The authors thank Xinia Siles and the field workers of Proyecto Salud Coronaria, San José, Costa Rica, for their help with data collection.

	FOOTNOTES

TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
This study was supported by National Institutes of Health grant HL49086.

Received June 3, 2002. Accepted October 7, 2002.

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TOP FOOTNOTES ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 

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