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1 From the Laboratory of Membrane Biochemistry and Biophysics, Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD (KDS, RJP, SB, MM, and NS); the Food Composition Laboratory, Beltsville Human Nutrition Research Center, US Department of Agriculture, Beltsville, MD (RJP and VPF); the Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (HR); and the Departments of Obstetrics & Gynecology, School of Medicine (JJ, MB-A, JEW, RJS, and JHH) and Psychology (JHH), Wayne State University, Detroit, MI
2 Supported by the National Institute on Alcohol Abuse and Alcoholism research grant N01-AA83019 to JHH. 3 Address reprint requests to N Salem Jr, LMBB, NIAAA, 5625 Fishers Lane, Room 3N-07, Rockville, MD 20852. E-mail: nsalem{at}niaaa.nih.gov.
ABSTRACT
Background:African American women and socioeconomically challenged women are at risk of compromised folate status and, thus, of folate-related birth defects. Data are limited on circulating folate concentrations in pregnant African American women after folic acid fortification of the food supply was implemented.
Objective:The objective was to determine the influence of smoking and alcohol consumption on plasma 5-methyltetrahydrofolic acid (5-MTHFA) concentrations in pregnant African American women.
Design:Alcohol consumption, smoking exposure, and other characteristics of pregnant African American women reporting to an inner-city antenatal clinic were assessed. At 24 wk of gestation, blood samples and food-frequency intake data were collected. Plasma 5-MTHFA concentrations were determined by liquid chromatography–mass spectrometry for 116 subjects and examined in a correlational study design.
Results:Dietary folate and markers of alcohol consumption were positively associated, whereas exposure to smoke was negatively associated with plasma 5-MTHFA. More than one-half of the participants in this population failed to meet the recommended dietary allowance for dietary folate equivalents of 600 µg/d during pregnancy.
Conclusions:Most inner-city African American women are not meeting the recommended dietary allowance for dietary folate during pregnancy, and smoking may further compromise their folate status. Programs to reduce smoking and raise awareness about the importance of folate and multivitamin supplementation during pregnancy need to target this population.
Key Words: 5-Methyltetrahydrofolic acid • folate • folic acid • fortification • African American women • pregnancy • nutrition • smoking • alcohol • electrospray mass spectrometry • human plasma • polyunsaturated fatty acids
INTRODUCTION
In 1996 the federal government mandated that grain-based foods manufactured in the United States be fortified with folic acid (140 µg/100 g). This fortification was considered the most effective method of increasing folate intake in women of childbearing age (1). Fortification of grain products with folic acid increases folate concentrations in middle-aged and older populations (2, 3) and in women of childbearing age (4, 5). The implementation of folic acid fortification has lowered the incidence of neural tube defects (NTDs) in the United States by 20% on the basis of birth certificate data (6) and by >50% when the incidence of NTDs in stillbirths and terminated pregnancies were included in the analysis (7). Congenital anomalies of the central nervous system, including spina bifida and anencephaly, have not decreased in Detroit African Americans after folic acid fortification (3.0 per 1000 live births in 1995 compared with 3.1 per 1000 live births in 2000) (8).
In women of childbearing age, African American women have lower folate concentrations in serum and erythrocytes than do white, Hispanic (4, 5), and Asian (4) women in the United States. These lower values coincide with lower intakes of dietary folate equivalent (DFE), after energy adjustment, in pregnant African American women than in white women (9). Socioeconomically disadvantaged women also have lower folate status than advantaged women (4). Low socioeconomic status has been associated with a cluster of health-detrimental behaviors, including poor diet, smoking, and lack of physical exercise (10). Other methods of increasing folate status such as multivitamin supplementation by inner-city African American women are difficult and require substantial effort because awareness about folate and NTDs is low (11).
Active and passive exposures to tobacco smoke are associated with lower serum and erythrocyte folate concentrations (12), and smoking during pregnancy decreases maternal plasma folate concentrations (13). Exposure to smoke is associated with both lower intake of dietary folate and lower blood folate status. The association of smoke exposure to folate status persists after adjusting for differences in intake, suggesting nondietary as well as dietary influences (12). Smoking and alcohol consumption have been positively correlated in African American women (14). Halsted et al (15) reviewed the negative effects of chronic alcohol intake on folate homeostasis in alcoholics and in animal models, but the effects may be complicated by the folate content of some alcoholic beverages, specifically beer (16). The effects of alcohol on folate metabolism during pregnancy in humans remain unclear (17).
The primary purpose of this study was to examine the influence of diet, smoking, and alcohol consumption in pregnant African American women at risk of alcohol-related birth defects on plasma concentrations of 5-methyltetrahydrofolic acid (5-MTHFA), the predominant circulating folate metabolite. We expected these results to provide insight into the effectiveness of folic acid fortification on the intakes of DFE and the concentrations of plasma 5-MTHFA in a population known to be at risk of both folate deficiency and of giving birth to infants with complex disorders of the nervous system. In addition, this study provides highly accurate and specific determinations of plasma 5-MTHFA by a stable-isotope liquid chromatography–mass spectrometry method, the advantages of which have been discussed previously (18).
SUBJECTS AND METHODS
Subjects and study design
Pregnant African American women (n = 116) presenting between February 1999 and January 2001 at the antenatal clinic at Wayne State University in Detroit were recruited on the basis of reported alcohol intake (described in "Alcohol and smoking exposure"). Women with high-risk pregnancies were excluded from the study. All procedures and protocols received prior approval by the Wayne State University Human Investigations Committee, and informed consent was obtained during the initial clinical visit.
A structured interview at the first antenatal visit determined eligibility and assessed demographic characteristics, alcohol intake, and smoking exposure (19). Socioeconomic status was measured with the use of a modified Hollingshead index (20). At the regular obstetrical visit at 24 wk of gestation, a 15-mL fasting blood sample was collected by venipuncture. Specimens were collected into heparinized tubes, kept cold (4 °C) until centrifuged (2000 x g for 5 min at 4 °C) to separate plasma and erythrocytes, and frozen at –75 °C until analyzed. Nutritional status was assessed with the use of a validated food-frequency questionnaire (21) modified to quantify selected dietary fats. Quantification was based on the US Department of Agriculture National Nutrient Database for Standard Reference, release 14 (22). Intakes of individual nutrients were adjusted for total energy intake by the nutrient residual method (23) to reduce measurement error (24). The energy adjusted nutrient intakes were used for statistical analyses and are herein described as "adjusted" throughout. DFEs were calculated according to the method recommended by the Food and Nutrition Board (25). Briefly, for natural food sources of folate, 1 µg folate = 1 µg, DFE; for synthetic vitamin preparations, 1 µg folic acid = 2 µg DFEs; and, for mixed food and supplement products, µg DFE is calculated by food folate (in µg) x 1 plus folic acid (in µg) x 1.7. All participants were advised about nutrient supplementation during pregnancy and received a prescription for a prenatal vitamin.
Alcohol and smoking exposure
At-risk drinking was determined by several proven screening tests, including the Michigan Alcoholism Screening Test (MAST) and the Tolerance, Annoyed or Angry, Cut down or quit, Eye opener (T-ACE) questionnaire (26-28). Quantitative alcohol intakes were determined by 14-d recalls from the time of conception and at the first prenatal visit. Recall information generated estimates of alcohol intakes as grams of absolute alcohol per day around the time of conception (AAD0) and at the time of the first prenatal visit (AAD1), grams of absolute alcohol per drinking day (AADD0; AADD1), and proportion of drinking days over those 14 d (PROPDD0; PROPDD1). Cigarette smoking by both the mother and the father were determined by maternal recall of the number of cigarettes smoked per day around the time of conception and at the first prenatal visit. All women with AAD0 14.2 g (0.5 oz) were recruited into the study, plus a random 8% sample of the remaining patients were recruited. This selection strategy oversampled the high-risk drinking women (19, 29).
Sample analyses
Liquid chromatography–mass spectrometry was used to determine the plasma 5-MTHFA concentrations as described previously (18). Before extraction, 13C5-5-MTHFA (10 ng) was added to 0.5 mL plasma as an internal standard. The analyte was isolated with the use of solid-phase extraction (Strata phenyl column 100 mg/mL; Phenomenex, Torrance, CA), washed with 0.03 M K2HPO4, and eluted with 0.5 mL HPLC mobile phase (acetonitrile:methanol:water, 26:14:60). Extract (40 µL) was injected onto a C18 HPLC column (150 x 4.6 mm; Phenomenex) with the use of a binary pumped Agilent 1100 HPLC (Palo Alto, CA) interfaced to an ion trap mass spectrometer (Finnigan LCQ, San Jose, CA), and samples were analyzed by electrospray ionization in the positive ion mode.
Statistical analyses
Pearson's correlations (two-tailed) were used to determine the bivariate associations of plasma 5-MTHFA with energy-adjusted DFEs and selected maternal variables. As the accuracy of DFE estimates are questionable, bivariate correlations to adjusted DFEs were also determined according to rank by Spearman's correlation coefficients. Adjusted DFE, food folate, fortified folate, and plasma 5-MTHFA were grouped according to adjusted DFE quartiles for trend analyses and mean comparisons by one-way analysis of variance (ANOVA) with a priori comparisons between the lowest and highest quartiles. Associations with 5-MTHFA were also evaluated with the use of multiple linear regression analyses. A parsimonious model with all included independent variables having P values < 0.10 and a controlled model with potential confounders included as independent variables were generated. Variables were included on the basis of information from the literature, influence on the model R2 value, and degree of collinearity with other variables. Adjusted DFE was entered as both continuous data and after being assigned the appropriate rank. Associations with specific types of alcoholic beverages were also examined by linearregression. In addition, participants were grouped according to levels of periconceptional smoking (>0 or 0) and drinking (AAD0 > 0 or AAD0 = 0) to examine demographic characteristics. The subgroups were analyzed by a two-factor ANOVA with interaction for drinking and smoking with Holm's (30) post hoc comparisons of individual means. Data are presented as the mean ± SD with P value < 0.05 accepted as significant. Ad hoc bivariate correlations between maternal characteristics with father's smoking were also determined. The Mantel-Haenszel statistical procedure was used to compare percentages of drinking fathers. All statistical analyses were completed with SPSS for WINDOWS statistical software (release 11.5.1; SPSS Inc, Chicago).
RESULTS
Maternal characteristics
The analyses of plasma 5-MTHFA were completed for samples from 116 subjects with an age range of 16–38 y. Mean gestational age at the first prenatal visit was 16.4 ± 6.5 wk. The means ± SDs for selected demographic characteristics, alcohol intake variables, smoking exposure variables, and selected dietary intakes are shown in Table 1. Demographic characteristics were examined by two-factor ANOVA for categorized drinking and smoking status with interaction (data not shown). No significant effects of categorized drinking and smoking were observed on plasma 5-MTHFA. A significant interaction (P < 0.05) between smoking and drinking was detected for education with women who smoke and drink having the lowest acquired education (11.2 ± 1.3 grade acquired) and the nonsmoking drinkers having significantly higher acquired education (12.4 ± 2.0 grade acquired). Drinking women scored significantly higher (4.2 ± 1.0) than nondrinking women (3.6 ± 1.1) on the Hollingshead index, indicating lower socioeconomic status. The percentage of drinking fathers was higher for drinking women (77%) than for nondrinking women (27%), and the amount of father's smoking was almost 3 times greater for women who smoked (9.4 ± 10.4 cigarettes/d) than for nonsmoking women (3.2 ± 6.1 cigarettes/d). Smoking women also ate fewer carbohydrates (45.8 ± 8.0%) and more fat (38.9 ± 5.4% of energy) than nonsmoking women (carbohydrates: 48.9 ± 5.9% of energy; fat: 36.3 ± 4.4% of energy). Several significant associations of these various maternal characteristics and exposures with adjusted DFE intakes and plasma 5-MTHFA were determined by Pearson's correlations (along with Spearman's correlations for adjusted DFE intakes) and are presented in Table 1. Pre-pregnancy maternal smoking and maternal smoking during pregnancy were negatively associated with adjusted DFE as assessed by Spearman's correlations ( = –0.22, P = 0.017 for both). The association was slightly weaker when assessed by Pearson's correlations (r = –0.18, P = 0.049 for pre-pregnancy smoking; r = –0.18, P = 0.051 for smoking during pregnancy). Adjusted DFE was also negatively associated with dietary fat intake and positively associated with dietary vitamin B-12 and carbohydrate intake in both correlation analyses. Vitamin B-6 intake was also positively associated with adjusted DFE by the Spearman's correlational test only. Plasma 5-MTHFA was positively correlated to various screening test scores that indicate at-risk drinking and negatively correlated to the amount of father's smoking.
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TABLE 1. Characteristics and dietary intakes of pregnant African American women and correlations with adjusted dietary folate equivalents (DFE) and plasma 5-methyltetrahydrofolic acid (5-MTHFA)1
Associations with plasma 5-MTHFA
Plasma 5-MTHFA was positively associated with adjusted DFE (r = 0.29, R2 = 0.09, P = 0.001) by linear regression (Figure 1). This association was lower but remained significant when adjusted DFE was ranked and regressed against plasma 5-MTHFA (r = 0.25, R2 = 0.06, P = 0.007) or when adjusted DFE and plasma 5-MTHFA were examined by Spearman's correlation test ( = 0.19, P = 0.045). On the basis of the initial linear regression relation, the plasma 5-MTHFA concentration of 18.2 ng/mL (95% CI: 4.23, 32.28 ng/mL) would be predicted from a DFE intake of 600 µg/d, the current recommended dietary allowance (RDA) for DFE for pregnant women (25). The means, ranges, and CIs for adjusted DFE intakes, food folate, fortified folate, and plasma 5-MTHFA according to quartiles of adjusted DFE are shown in Table 2. About 57% of the participants reported adjusted DFE < 600 µg/d. The 57th percentile of plasma 5-MTHFA was 19.4 ng/mL. Mean differences between the lowest and highest quartile of adjusted DFE, food folate, and fortified folate were significant (P < 0.001) by a priori contrasts after significant one-way ANOVA result. Trend analyses confirmed that the adjusted folate intakes and plasma 5-MTHFA increased from the lowest to highest quartile (Table 2). Without fortification, almost the entire sample (98th percentile) would fail to consume the recommended 600 µg DFE/d, as indicated by adjusted food folate intake (Table 2). Fortification contributed an estimated 198 µg DFE/d as indicated by adjusted fortified folate intake. Contributions from daily multivitamin use are minimal because compliance by urban African American women educated about folic acid and NTDs has been previously reported to be only 9% (31).
FIGURE 1.. Relation between adjusted dietary folate equivalent intakes and plasma 5-methyltetrahydrofolic acid in pregnant African American women. Dietary folate intakes were adjusted with the use of the nutrient residual model and were linearly correlated to plasma folate by linear regression: r = 0.29, R2 = 0.09, P = 0.001 (n = 116). RDA, recommended dietary allowance; EAR, estimated average requirement.
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TABLE 2. Various folate intake variables and plasma 5-methyltetrahydrofolic acid (5-MTHFA) concentrations of pregnant African American women by quartiles of adjusted dietary folate equivalents (DFE)1
Plasma 5-MTHFA was positively correlated with adjusted DFE, MAST score, and total energy intake and negatively correlated with father's smoking (Table 3). The multiple regression analyses were restricted to 77% of the sample (n = 89), largely because of missing data about father's smoking. These associations persisted after control for maternal education level, total pregnancies, and body mass index. Mother's age, socioeconomic status, maternal smoking, alcohol intakes, and other screening tests were also considered. The effect of alcohol consumption around the time of conception (AAD0) approached significance (standardized ß = 0.17, P = 0.08) when substituted for MAST score in the unadjusted model from Table 3 when a single outlier was excluded from the analysis (standardized ß = 0.11, P = 0.25 with outlier included). This substitution did not significantly change the associations of adjusted DFE, total energy, and father's smoking. No significant changes were observed in these results when the adjusted DFE intakes were ranked and substituted into either model.
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TABLE 3. Multiple linear regression results with plasma 5-methyltetrahydrofolic acid concentrations set as the dependent variable and dietary folate equivalents, smoking, drinking, and other maternal characteristics set as independent variables1
Maternal smoking was negatively, but not significantly, associated with changes in plasma 5-MTHFA (standardized ß = –0.12, P = 0.23) when entered in place of father's smoking. The stronger association between father's smoking and maternal plasma 5-MTHFA, compared with the association between mother's smoking and maternal plasma 5-MTHFA, was unexpected. Ad hoc Pearson's correlation coefficients between various maternal characteristics and father's smoking were determined. Father's smoking was positively correlated with mother's smoking (r = 0.27, P = 0.009) and frequency of maternal drinking (PROPDD0; r = 0.27, P = 0.009) and negatively correlated with maternal plasma 5-MTHFA (r = –0.21, P = 0.043).
Dietary sources of food folate and folic acid as determined by food-frequency questionnaire
The percentage contribution of individual foods to food folate intake, folic acid intake, and DFEs from food as determined by food-frequency questionnaires are presented in Table 4. Cold breakfast cereal contributed 26.9 ± 12.0% of the DFE intake from food, 45.7 ± 20.5% of the folic acid intake, and 4.2 ± 2.8% of the food folate intake. With the exception of orange juice, the foods providing the greatest contribution to folic acid intake were also the greatest contributors to the DFE intake. Orange juice provided the greatest percentage of food folate at 26.3 ± 18.0% that translated into 11.9 ± 9.6% of the DFE intake from food sources. After orange juice, the next 3 biggest contributors to food folate intake were milk, French fries, and breads, respectively.
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TABLE 4. Percentage contribution of individual food sources on the food-frequency questionnaire to food folate intake, folic acid intake, and dietary folate equivalents (DFE) from food1
Types of alcoholic beverages
After age, smoking, body mass index, total energy, and adjusted DFE were controlled for, alcohol consumption from alcoholic coolers at the first prenatal visit was negatively associated with plasma 5-MTHFA as a separate variable (ß = –1.90, P = 0.042) and with other alcohol beverages included as independent variables (ß = –1.86, P = 0.050). Beer was positively related to plasma 5-MTHFA in the various models but failed to reach statistical significance. Around the time of conception, the percentages of the total alcohol consumed for the beverage categories were 28% (beer), 13% (wine), 7% (coolers), and 51% (liquor). On average, women reported drinking 93% less alcohol at their first prenatal visit than they reported drinking around the time of conception. The percentages of distribution of each individual alcohol type consumed at the time of the first prenatal visit were 26% (beer), 37% (wine), 7% (coolers), and 30% (liquor).
DISCUSSION
In pregnant African American women at risk of alcohol-related birth defects, adjusted DFE intake was a positive and the most significant indicator of plasma folate status. The adjusted DFEs in this sample suggest that daily folate intake for at-risk pregnant women in this community is below the current RDA of 600 µg/d for more than 57% of the women and below the estimated average requirement (EAR) of 520 µg/d for 34% of the women. The estimated contribution of fortified folate in this population was 198 ± 98 µg/d (
Lower blood folate concentrations in smokers is often attributed to lower dietary folate intakes (25), and in this study, too, a negative correlation was observed between pre-pregnancy maternal smoking and adjusted DFE. However, the negative association between father's smoking and maternal plasma 5-MTHFA persisted when DFE intake was included in the model, suggesting a nondietary mechanism, such as higher folate turnover, may be involved. A persistent negative association of smoke exposure after adjusting for dietary folate has been reported previously (12).
Alcohol consumption has been associated with lower blood folate status in chronic alcoholics (15), but it has been associated also with higher erythrocyte folate concentrations in drinking, pregnant women (16). In the present study, at-risk drinking, as indicated by screening tools, was associated with higher plasma 5-MTHFA both in bivariate models and in multivariable linear regression models with adjustments for potential confounds. Positive correlations between plasma folate concentrations and quantitative alcohol variables (ie, AAD0, AADD0, and PROPDD0) were not significant. The positive association between alcohol intake and blood folate concentrations may be a result of folate content in beer (16), although the consumption of spirits has also been positively associated with erythrocyte folate in women (39). Neither beer nor liquor consumption in the current study was significantly correlated with plasma concentrations of 5-MTHFA, although the coefficients were positive in all the regression models examined. The consumption of alcoholic coolers at the first prenatal visit was negatively correlated to plasma folate. The negative association of alcoholic coolers suggests a negative effect of ethanol on plasma folate status.
In African Americans, the prevalence of the C677T and A1298C mutation in methylene tetrahydrofolate reductase is low (4, 40). Therefore, folate status is largely determined by diet and environmental factors. In the current study, dietary folate, exposure to smoke, and indexes of at-risk drinking are significant predictors of plasma concentrations of 5-MTHFA in pregnant African American women. Dietary folate fortification contributed 200 µg DFE/d, but more than one-half of the pregnant women in this study failed to meet the RDA for folate of 600 µg DFE/d. Only 18% of inner-city African American women of childbearing age have heard of NTDs and only 9% know folate could prevent NTDs (11). For mothers in Michigan, folic acid awareness is lower among black women, women with unplanned pregnancies, and women with no high school education (41). Education programs can augment knowledge about NTDs and folate and can raise daily multivitamin use in pregnant African American women (31).
On the basis of the current results, fortification of food with folic acid alone may not be sufficient to provide the RDA for urban African American women during pregnancy; indeed, it does not appear that fortification has been capable of reducing the number of congenital central nervous system defects in African Americans living in the Detroit area (8). The results of this study suggest that targeted programs for education on folate use, as well as compliant, daily multivitamin supplementation during pregnancy, are required in this population and that the assessment of both supplementation compliance and concentrations of folate fortification of foods should continue (32). This study also highlights the need to control for lifestyle factors, including amount and type of alcohol consumption and active and passive tobacco exposure, when examining dietary folate and circulating folate concentrations in humans.
ACKNOWLEDGMENTS
We express our appreciation to Rudolf Moser of Merck Eprova AG for supplying a pure standard of 5-MTHFA. We are indebted to the Population and Provide Data Unit at the Vital Records and Health Data Development Section, Epidemiology Services Division, Bureau of Epidemiology, Michigan Department of Community Health for providing data on birth defects for African Americans in Detroit from 1993 to 2000. We thank the technical staff (E Russell, T Martin, L DiCerbo, et al) and the participants for their commitment to this study.
KDS was responsible for data management, interpretation, and statistical analysis and was the primary writer of the manuscript. RJP and VPF were responsible for the 5-MTHFA analysis. RJP made significant contributions to data interpretation and manuscript preparation. SB and MM contributed to data management and interpretation. MM was responsible for the study design. MB-A was responsible for study coordination and management and analyses of the data. JJ provided statistical advice and analyses. HR provided quantitative interpretation of the nutrition surveys. JEW made decisions for clinical exclusion and delivered the infants. RJS was the director of antenatal research and assisted in the study design, data interpretation, and manuscript preparation. JHH was a coprincipal investigator, secured partial funding, was responsible for the study design and management, and contributed to manuscript preparation. NS was a coprincipal investigator, provided funding, and contributed to the study design and management, data interpretation, and manuscript preparation. None of the authors had a conflict of interest.
REFERENCES