Maternal Supplemental and Dietary Zinc Intake and the Occurrence of Neural Tube Defects in California

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1 American Journal of Epidemiology Copyright O 1999 by The Johns Hopkins University School of Hygiene and Public Health All rights reserved Vol.150, No. 6 Printed In USA. Maternal Supplemental and Dietary Zinc Intake and the Occurrence of Neural Tube Defects in California Ellen M. Velie, 1 Gladys Block, 1 Gary M. Shaw, 2 Steven J. Samuels, 3 Donna M Schaffer, 4 and Martin Kulldorff 8 ' The authors investigated the association between maternal preconceptional supplemental and dietary zinc intake and risk of neural tube defects (NTDs) in a population-based case-control study conducted between 1989 and 1991 in California. Cases were 430 NTD-affected fetuses/infants, and controls were 429 randomly selected non-malformed infants. Mothers reported their preconceptional use of vitamin, mineral, and food supplements, and completed a 98-item food frequency questionnaire. Increased total preconceptional zinc intake was associated with a reduced risk for NTDs (quintile 5 vs. quintile 1, odds ratio (OR) = 0.65, 95% confidence interval (Cl) 0.43, 0.99). Phytate intake, a constituent of the diet known to impede zinc absorption, appeared to modify the zinc - NTD association. In addition, increased servings of animal products, the most bioavailable food source of zinc, was associated with a reduced risk for NTDs (quintile 5 vs. quintile 1, OR = 0.49, 0.32, 0.76). Risk estimates for zinc intake were changed little after controlling for multiple sociodemographic factors and total folate intake, but were attenuated after controlling for nutrients highly correlated with dietary sources of zinc, such as protein. In sum, the analyses indicate that risk of NTDs in infants and fetuses decreased with increasing maternal preconceptional zinc intake. However, it remains unclear whether increased zinc intake, or another nutrient or combination of nutrients highly correlated with zinc intake in the diet, is causally associated with reduced NTD risk. Am J Epidemiol 1999; 150: abnormalities; anencephaly; diet; food habits; meat; proteins; spina brfida cystica; zinc Neural tube defects (NTDs) occur in about one in every 1,000 births in the United States and contribute substantially to childhood morbidity and fetal and infant mortality (1). Although the cause of most NTDs remains unknown, low maternal socioeconomic status and maternal nutritional factors, including inadequate folate nutriture, elevated body mass index (BMI), and inadequate zinc nutriture have been implicated in some populations (\-4). Zinc intake is essential for normal fetal growth and development (4). Zinc is a constituent of over 200 enzymes and other proteins, hormones, and neuropep- Received for publication September 11, 1998, and accepted for publication January 22, Abbreviations: BMI, body mass index; Cl, confidence interval; NTD, neural tube defect; OR, odds ratio; RDA, recommended dietary allowance. 1 Department of Epidemiology, University of California, Berkeley, Berkeley, CA. 2 California Birth Defects Monitoring Program, Emeryville, CA. 3 Department of Community and International Health, University of California, Davis, CA. 4 Division of Research, The Kaiser Permanente Medical Care Program of Northern California, Oakland, CA. 6 Division of Cancer Prevention, Biometry Branch, National Cancer Institute, Bethesda, MD. Reprint requests to Dr. Ellen M. Velie, National Cancer Institute, Nutritional Epidemiology Branch, Building EPS, Room 7026, 6120 Executive Plaza Blvd., MSC 232, Bethesda, MD tides (4). It facilitates gene transcription and is necessary for cell division, development, and differentiation (5). Moreover, although it is known that zinc homeostasis is tightly regulated through excretion and absorption over a wide range of dietary zinc intakes, it does not appear to be released from muscle tissue and bone, where it is primarily stored, when dietary zinc intake is low (6). Therefore, inadequate maternal zinc intake for even a short period may reduce circulating zinc levels, and thus possibly reduce the availability of this nutrient to the developing fetus during a critical time in neural tube development. In both animals and humans, inadequate zinc intake has been associated with NTDs (4). In laboratory animals, severely reduced zinc intake for 1-2 days early in gestation was associated with the development of NTDs (7). In humans, women with an extremely rare genetic disorder of zinc metabolism, acrodermatitis enteropathica, are at high risk for NTDs (8). Also, ecologic observations have linked chronic low dietary zinc intake to increased prevalence of NTDs in Egypt and the Middle East, and among Sikhs (9). Human observational studies that have examined the association between maternal zinc tissue levels and NTDs have shown conflicting results, in part due to their inadequate ability to measure maternal zinc status (4). There is no reliable biologic marker or functional 605

2 606 Velie et al. index of zinc status (10). Therefore, although dietary intake is difficult to accurately ascertain, estimated usual zinc intake from the diet and supplements, in combination with factors known to affect availability, may be the best indicator of maternal zinc status for epidemiologic studies (11). Approximately 70 percent of dietary zinc (not including supplements) consumed by women of childbearing age in the United States comes from animal sources (11), but the bioavailability of zinc from different foods varies widely (6). Zinc from animal sources is believed to be most bioavailable (12). Zinc from a legume- and whole grain-based diet, although able to provide quantities of zinc similar to amounts from a diet containing animal products, appears to be substantially less bioavailable because of the presence of fiber and phytate (13). Phytate is a naturally occurring substance found in plants, with highest levels in beans, tubers, seeds, nuts, and cereal grains (13). Other nutrients, such as calcium, iron and possibly folic acid, may also reduce zinc availability, while protein and products of protein digestion are believed to enhance zinc availability (6,14-17). Zinc from supplements and fortified cold breakfast cereals is believed to be readily absorbed, but to have reduced availability in the presence of competing nutrients, particularly the phytate in cold breakfast cereals (12). The adequacy of zinc nutriture in the US population is unclear (11). While the recommended dietary allowance (RDA) is 12 mg/day (18), the third National Health and Nutrition Examination Survey (19) found that the average dietary zinc intake (not including supplements) of US women aged years is 9.6 mg/day. Further, in the United States, virtually every study that has evaluated dietary intake of pregnant women has shown that mean zinc intake is less than the RDA of 15 mg per day for pregnant women (4). Lacto-vegetarians who use dairy and soybean products as their major sources of protein, and populations with a diet that consists primarily of grains, snack foods, and peanut butter, or a diet comprised primarily of grains, corn, and legumes, are believed to be at highest risk for zinc deficiency (13,20). We examined data from a large population-based case-control study to investigate a possible association between having a NTD-affected pregnancy and maternal periconceptional zinc intake, as well as whether a possible association is more pronounced once factors known to affect the bioavailability of zinc, such as dietary phytate intake, are taken into account. MATERIALS AND METHODS Study population The California Birth Defects Monitoring Program identified infants and fetuses with NTDs and a random sample of control births in all California counties except Riverside, Ventura, and Los Angeles (21). Eligibility was restricted to women who delivered singleton live or stillborn infants between June 1, 1989 and May 31, 1991, or women who electively terminated an NTD-affected fetus between February 1, 1989 and January 31, Women were excluded if they did not speak English or Spanish or did not report California as their residence at delivery. Infants or fetuses with a NTD (anencephaly, spina bifida cystica, craniorachischisis, or iniencephaly) were ascertained through review of medical records, including ultrasonography, at all hospitals and genetic centers (excluding military facilities) in the study area. The review attempted to ascertain all singleton livebom, stillborn (>19 weeks gestation), electively terminated fetuses, and clinically recognized spontaneously aborted fetuses diagnosed with a reportable NTD. A total of 624 singleton infants or fetuses were so diagnosed and their mothers eligible for interview. Controls were infants born alive without a reportable congenital anomaly (22). They were selected from each area hospital (excluding military facilities) in proportion to the hospital's contribution to the total population of infants born alive in the study area and time period. A total of 612 singleton infants were selected. Interviews were completed with 1,089 women (549 cases and 540 controls). Among both case and control mothers, 12 percent could not be located or refused to participate. Women who were not interviewed were similar to participants with respect to maternal race/ethnicity, age, delivery status, and NTD phenotype of their fetus/infant Because the dietary questionnaire used in these analyses was only developed to capture the diets of Whites, Hispanics, and Blacks, 23 women of other races/ethnicities (primarily South East Asian, Filipino, and Indian immigrants, comprising 30 case and 40 control mothers) were considered ineligible for the study. Twelve interviewed women (11 case mothers and one control mother) had a previous NTD-affected pregnancy and were also ineligible. Of the remaining eligible women, 66 did not complete the food frequency questionnaire (41 case mothers and 25 control mothers) and 82 did not complete a usable dietary questionnaire (37 case mothers and 45 control mothers), as assessed by dietary software error checks (24). The final sample thus included 430 cases (>74 percent of eligible) and 429 controls (>75 percent of eligible). In-person interview The primary source of study data was in-person interviews with the mother. Interviews in English or

3 Zinc Intake and Neural Tube Defects 607 Spanish took place after an average of 4.9 months for case mothers and 4.6 months for control mothers from the actual or projected date of term delivery. Recall was assisted with a calendar on which the interviewer marked relevant dates. Study participants were asked about their medical, reproductive, and family history, as well as their use of vitamins and food supplements in the 3 months before conception and in each trimester of pregnancy. To ensure accuracy of recording, brand name information was elicited, and photographs were taken of multivitamin supplement containers. When nutrient content information of supplements was unknown, nutrient values were imputed based on the mode of values for similar supplements used by other study women with picture information available. Dietary assessment Average daily dietary nutrient and food group intake was assessed by the National Cancer Institute/Block 98-item food frequency questionnaire (23). The questionnaire was translated into Spanish. The study women completed the questionnaire in English or Spanish with an interviewer present to answer questions or to administer the questionnaire if the women had difficulty. The questionnaire asked about intake over the 3-month period before conception. Although the first 30 days of pregnancy is the critical time for neural tube development, the women were asked about their intake during the preconceptional period because we were concerned that if women were asked about their usual diet during early pregnancy they might be more likely to recall a diet after the critical first 30 days. Furthermore, preconceptional dietary habits are Likely to continue into the first 30 days postconception, before women often know that they are pregnant. Finally, intake in the preconceptional period is Likely to be the source of stored nutrients that are released during the first 30 days of pregnancy. The average daily intakes of nutrients and food groups were computed by analytic software developed for the survey instrument (23). Phytate values were developed by the authors (EV and GB) with data from Dr. Suzanne Murphy (unpublished), and from Harland and Oberleas (13). For all nutrients examined, the total average daily preconceptional nutrient intake value was defined as nutrient intake from supplements combined with nutrient intake from diet. Servings of animal products included red meat, poultry, seafood, eggs, and dairy products. Categories of total, animal, and plant and cold breakfast cereal sources of zinc, and servings of animal products, were based on quintile distributions in the control population. Statistical analysis Odds ratios and their 95 percent confidence intervals were computed using logistic regression with STATA statistical software (25). Confounding was assessed by comparing crude and adjusted odds ratios. Nested models were compared by the Likelihood ratio test. Effect modification was assessed in bivariate analyses by the Mantel-Haenszel test for heterogeneity, and in multivariate analyses with the LikeLihood ratio test. Nonparametric Spearman correlation coefficients were calculated to examine the association between nutrient intakes. Continuous variables were examined in quintiles and as Linear and quadratic terms and were included in multivariate analyses in the form that optimized model fit (as assessed by the model chi-square value) taking the number of parameters into account. Maternal age was included as a quadratic, and BM1, total folate, total calcium, dietary protein, dietary methionine, and total energy were included as linear terms, and total zinc in quintiles. Quintiles were used for zinc in order to derive odds ratios for arbitrary dose categories. To evaluate potential interaction, factors were examined as dichotomous variables, because sample size did not permit further categorization (grouped above and below the median for phytate, fiber and iron, and by the first two and last three quintiles for zinc). RESULTS Sample Compared with mothers of non-malformed infants, mothers with a NTD-affected pregnancy were more likely to be Latinas bom outside of the United States, Spanish-speaking, less educated, younger, primigravidous, to have a greater BMI, and more likely to have had a preconceptional infection and lower average daily folate and methionine intakes (table 1). The top dietary source of zinc in this population (cases and controls) was beef, followed by fortified cold breakfast cereals, milk, cheese, and eggs. The top dietary sources of phytate were "corn tortillas/corn bread," beans, cereal products (e.g., cold breakfast cereals and bread), "salty snacks" (e.g., corn or potato chips), and french fries. Zinc intake and NTD risk Mean intakes of preconceptional zinc from all sources were higher in controls than cases (table 2). In table 3, associations between zinc intake from all sources and NTD risk are reported. Estimates were adjusted for maternal race/ethnic origin, education,

4 608 Velie et al. TABLE 1. Characteristics of mothers In a case-control study of zinc Intake neural tube defects In California, * and the occurrence of Mo. of controls (n = 429) No. of cases (n = 430) Odds ratio 95% Ethnicity White US-born Latina Foreign-born Latina Black Language* English Spanish Education <High school diploma High school diploma or vocational certificate 2- or 4-year college graduate Age (years) < >34 Qravidity Body mass index (kg/m 2 ) < >29.0 Preconceptional infection None Infection PreconceptionaJ cigarettes smoked (cigarettes/day) None 1-19 >19 PreconceptionaJ alcohol intake (drinks/day) None <1 21 Preconceptional vitamin useh None Some PreconceptionaJ total folate intake (mcg/day)# ^ >709.9 Preconceptional methionine intake (mg/day)# 51, , , , , , ,522.5 >2, * Missing data not Included. Column percents do not always add to 100. t Cl, confidence interval;, referent category. t Language completed dietary questionnaire. Any reported infection in 3 months before conception. H Includes use of any vitamins (food supplements not included) in 3 months prior to conception. # Qulntiles defined by controls. Retf 0.74, , , , , , , , , , , , , , , , , , , , , , , , , , , , , 0.85

5 Zinc Intake and Neural Tube Defects 609 TABLE 2. Distribution of maternal preconceptional zinc Intake among cases and controls In acasecontrol study of neural tube defects In California, Controls Cases Source of zinc intake Mean(SD») % of total Mean(SD) % of total Total sample, n = 859 (429 controls/430 cases)t Supplemental Dietary animal Dietary plant & fortified breakfast cereal Total Supplemental users, n= 142 (70 controls/72 cases)t Supplemental Dietary animal Dietary plant & fortified breakfast cereal Total Non-supplemental users, n = 694 (359 controls/ 358 cases) Dietary animal Dietary plant & fortified breakfast cereal Total dietary 3.8(19.6) 7.7 (3.8) 6.3 (4.7) 17.7(20.5) 22.5 (43.5) 7.1 (3.3) 6.7 (4.6) 36.3 (43.7) 7.8 (3.8) 6.2 (4.8) 13.9 (6.7) (6.2) 7.1 (3.5) 5.7 (4.6) 15.2(9.1) 14.1 (8.0) 7.3 (3.3) 6.1 (3.7) 27.4(10.0) 7.0 (3.5) 5.7 (4.8) 12.7 (6.6) * SD, standard deviation. t Total zinc includes zinc from supplemental and dietary sources. Data for supplemental zinc intake (dose or period of use) were not available for 11 control and 12 case mothers. Two control women took high dose zinc supplements and had supplemental intakes of 150 and 350 mg/day. age, and BMI. There was essentially no difference in NTD risk between women who did and did not use zinc-containing supplements preconceptionally, although there was a non-statistically significant suggestion of an increased NTD risk among the subset of women who reported <12 mg/day. (Among greater than 40 percent of women who reported preconceptional supplemental zinc intakes of <12 mg/day, the supplemental source was powdered food supplements, which is often used for dieting.) For all other sources of zinc examined, in both crude and adjusted analyses, as zinc intake increased, NTD risk decreased. Tests for linear trend were significant for all but dietary plant and fortified breakfast cereal zinc sources (p < 0.2) and adjusted estimates for total zinc intake (p = 0.067), and were most pronounced for the most bioavailable sources of zinc, dietary animal zinc intake (p < 0.009), and weekly servings of animal products (p < 0.001). We also examined the association between zinc intake and NTD risk in the subset of women who did not use zinc-containing supplements in the period 3 months before conception {n = 694). For all sources of dietary zinc, in crude analyses, nearly identical associations to those seen for the complete sample were observed. In adjusted analyses, the protective effect of zinc intake became more pronounced. Odds ratios (95 percent confidence intervals (CI)) for increasing quintiles of dietary zinc intake (compared with the lowest quintile), adjusted for dietary folate, maternal race/ethnic origin, education, age, and BMI were 0.70 (0.43, 1.14), 0.68 (0.40, 1.14), 0.49 (0.28, 0.84), and 0.26 (0.13, 0.52) (chi-square test for linear trend, p = 0.004). Similar associations between total zinc intake (lowest two quintiles, ^12.4 mg/day, vs. highest three quintiles, >12.4 mg/day) and separate NTD phenotypes were observed. For all cases combined, risk for NTDs was 0.66 (95 percent CI 0.50, 0.87). For anencephaly, it was 0.68 (95 percent CI 0.47, 0.98) and for spina bifida 0.64 (95 percent CI 0.47, 0.88). Adjustment for correlated nutrients Nutrients are highly correlated in the diet. We found the following correlation coefficients between zinc and nutrients: between total zinc and nutrients found in supplements and fortified breakfast cereals: total folate, 0.81; total iron, 0.89; and total calcium, 0.67; between total zinc and nutrients generally found only in the diet (and not supplements): protein, 0.69; methionine, 0.64; and energy, 0.58; between dietary animal zinc and nutrients found in supplements and fortified breakfast cereals: total folate, 0.34; total iron, 0.48; and total calcium, 0.68; between dietary animal zinc and nutrients generally found only in the diet: protein, 0.91; methionine, 0.92; and energy, The associations between total and dietary animal zinc intake and NTD risk adjusted for nutrient covariates of interest are presented in table 4. We first examined estimates adjusted for non-nutrient covariates and

6 610 Velieetal. TABLE 3. Maternal preconceptlonal supplemental and dietary zinc and animal product Intake and risk of neural tube defects In a case-control study In California, * Supplemental and dietary intake Total zinc (mg/day)* >23.4 Supplemental zinc (mg/day)* No use < Dietary zinc (mg/day) >18.2 Dietary animal zinc (mg/day)h >10.2 Dietary plant and breakfast cereal zinc (mg/day) ^ >8.4 Animal products (servings/week)# ^ >6.9 Cases (n = 430) No % ORf Crude analysis (n = 859) 95%Clf t 0.49, , , , 0.99 p = , , , , , , 0.84 p= , , , , 0.88 p = , , , ,1.13 p = , , , , 0.76 p < Adjusted analysis!: ( n = 784) OR , , , ,1.11 p = , , , , , , 0.71 p = , , , , 0.81 p = , , , , 1.13 p = , , , ,0.80 p < Total zinc includes zinc from supplemental and dietary sources. Data for supplemental zinc intake (dose or period of use) were not available for 11 control mothers and 12 case mothers. 97% of women who used zinccontaining supplements preconception did so in combination with other nutrients (as a food supplement, a multivitamin, a prenatal vitamin, or a mineral supplement). t OR, odds ratio; Cl, confidence interval;, referent category. i Odds ratios were adjusted for race/ethnic origin, education, mother's age, and body mass index. Participants with missing values for any covariate were removed from analyses (n = 75). p value for continuous linear terms from logistic regression analyses. Dietary animal zinc includes zinc from animal sources only. # Animal products include red meat poultry, seafood, eggs, and dairy products. then added each nutrient of interest one at a time in separate analyses. Adjustment for maternal race/ethnic origin, education, age, and BMI had little effect on zinc estimates (table 4). Singular adjustment for total folate, iron, and energy intake, in addition to nonnutrient covariates, also had little effect on zinc estimates (table 4). In contrast, adjustment for methionine, protein, and calcium intake attenuated both total and

7 Zinc Intake and Neural Tube Defects 611 TABLE 4. Maternal preconceptional total and dietary animal zinc Intake and risk of neural tube defects adjusted for demographic and nutritional covariates In a case-control study In California, (n = 784)* Zinc Intake Total zinc (mg/day) <S >23.4 Dietary animal zinc (mg/day) >10.2 ORt Crude analysis f t 0.45, , , ,1.00 p = , , , , 0.89 p = Adjusted analysis (base) OR , , , , 1.11 p = , , , , 0.81 p = Adjusted + fotatet Adjusted + methkxiinej Adjusted + protein} OR , , , , 1.34 p = , , , , 0.83 p = OR , , , , 1.59 p= , , , , 2.81 p = OR , , , , 1.71 p= , , , , 2.80 p = Participants with missing values for any covariate were removed from analyses (n = 75). t OR, odds ratio; Cl, confidence interval;, referent category. t Odds ratios were adjusted for race/ethnic origin, education, mother's age, and body mass index; folate, methionine, and protein were included where noted as continuous linear terms. p value for continuous linear terms from logistic regression analyses. dietary animal zinc intake estimates, and the confidence intervals after adjustment included 1.0 (table 4, data shown for methionine and protein). Risk estimates for both total and animal zinc intake after adjustment for non-nutrient covariates and protein were almost unchanged after additional adjustment for folate and methionine. Potential effect modifiers Compared with women with low zinc and high phytate intake, reduced risks were observed for all other combinations of zinc and phytate intake (table 5), although sample size was limited and odds ratios included one for two of these three comparisons. In multivariate analyses, the interaction of total zinc and dietary phytate was statistically significant (p = 0.011). In analyses examining the combined effect of protein and total zinc intake, when protein intake was low, total zinc intake at moderate to high levels was not as protective (data not shown), but, in multivariate analyses, the interaction was not statistically significant (p = 0.712). In stratified analyses, we examined all potential modifiers of the association between total zinc (<!12.4 mg/day vs. >12.4 mg/day) and NTD risk (i.e., maternal ethnicity, language completed interview, education, age, BMI, gravidity, preconceptional infection, packs of cigarettes smoked, alcohol consumed, total folate, dietary methionine, dietary protein, total cal- TABLE 5. Maternal preconceptional total zinc and dietary phytate Intake and adjusted risk of neural tube defects in a case-control study in California, (n = 784)* Zinc Intake (mg/day) < ORt t 0.38, 1.03 (122/130)* 0.34, 1.02 (79/69) Phytate intake (mgftiay) OR t (36/65) 0.25, 0.66 (158/125) * Odds ratios were adjusted for race/ethnic origin, education, mother's age, and body mass index. Participants with missing values for any covariate were removed from analyses (n = 75). t OR, odds ratio; Cl, confidence interval;, referent category. t No. of controls/no, of cases. cium, energy, dietary phytate, dietary fiber, and total iron (data not shown, see reference 26)). In crude analyses, tests for heterogeneity revealed some evidence for heterogeneity in NTD risk (p < 0.20) associated with zinc intake for the maternal characteristics of race/ethnic origin, education, BMI, and gravidity. In multivariate analyses, interaction terms for total zinc intake (in quintiles) were significant for the following characteristics: race/ethnic origin (p = 0.163), BMI (dichotomized) (p = 0.012), and gravidity (p = 0.197). The protective association between increased total zinc intake and NTD risk was most pronounced in women who were foreign-bom Latina, who had a BMI (kg/m 2 ) <26.0, and who were multigravidous.

8 612 Velieetal. In table 6, we report the association between total and animal zinc intake and NTD risk stratified by maternal race/ethnic origin and BMI. Higher total and animal zinc intake was associated with lower NTD risk in all race/ethnic groups, but was most pronounced in foreign and US-born Latinas. In addition, higher total and animal zinc intake was associated with lower NTD risk in underweight and normal weight women (BMI ^26.0), but in overweight women (BMI >26.0) there was no such association. DISCUSSION Our results are consistent with the hypothesis that inadequate maternal zinc intake is associated with increased NTD risk. We observed that increased maternal preconceptional zinc intake from all sources and from the most bioavailable source, animal products, was associated with decreased NTD risk. Further, phytate intake appeared to modify the zinc/ntd association in the hypothesized direction, i.e., low total zinc intake in combination with high phytate intake was associated with the highest risk for NTDs. Moreover, the association between total zinc intake and NTD risk did not appear to be mediated either by total folate intake or by sociodemographic factors. However, zinc intake estimates were attenuated after controlling for nutrients highly correlated with dietary sources of zinc, such as protein. Therefore, it remains unclear whether increased zinc intake, or another nutrient or combination of nutrients highly correlated with zinc intake in the diet, is causally associated with reduced NTD risk. Related literature To our knowledge, this is the first study to examine the association between both dietary and supplemental sources of zinc intake and NTD risk. One clinical trial of supplemental trace minerals (including 7.5 mg of zinc) (27) did not observe a decrease in NTD risk with maternal preconceptional supplementation, but it had an extremely limited sample size (n = 6 NTD-affected pregnancies) and the usual dietary zinc intake of women was not reported. One study examined supplemental zinc intake and NTD risk, without dietary intake (28), and reported no association. Other studies (29-31) examined intake of dairy or animal products (high in bioavailable zinc), without supplemental intake, and found that mothers of controls had significantly higher mean intakes of dairy or animal products than mothers of NTD cases. Zinc supplementation In analyses that compared women who used zinccontaining supplements preconceptionally with women TABLE 6. Maternal preconceptlonal total and dietary animal zinc Intake and adjusted risk of neural tube defects by maternal race/ethnic origin and body mass Index In a case-control study In California, (n = 784) ^n«*«h i rt irt 11 I-I Zinc Intake Total zinc (mg/day)* >23.4 Test for trendu Dietary animal zinc (mg/day) >10.2 Test for trendfl ORt, t White (n = 432) f f 0.63, , , , , , , , By race/ethnic origin US-bom Latina Iin =118) OR* , , , , , , , Fofe(gn-bom Latina ( n= 195) ORt , , , , , , , , OR By body mass Index (kjjftn*) S26.0 (n = 594) 0.43, , , , , , , , OR >26.0 (n=190) 0.33, 2.00, , , , , , , * Participants with missing values for any covariate were removed from analyses (n = 75). t OR, odds ratio; Cl, confidence interval;, referent category. $ Odds ratios were adjusted for education, mother's age, and body mass index. Odds ratios were adjusted for maternal race/ethnic origin, education, and mother's age. H p value for continuous linear terms from logistic regression analyses.

9 Zinc Intake and Neural Tube Defects 613 who did not use such supplements, we observed no association between supplement use and NTD risk (table 3). However, the examination of zinc intake from supplemental sources independent of dietary sources is problematic. Among women who did not use zinc-containing supplements preconception, mean dietary zinc intake was statistically significantly higher among controls compared with cases (13.9 vs mg/day, p = 0.008), while it was essentially equal among supplement users (13.8 vs mg/day, p = 0.346) (table 2). Thus, the observed associations for zinc intake from supplemental sources only may be attributable to the higher dietary zinc intake in control mothers compared with case mothers in the referent group. In addition, nearly all women who reported use of a zinc-containing supplement in our population did so in combination with other vitamins (97 percent of preconceptional zinc vitamin users) and so we cannot disentangle the independent contribution of zinc from supplements on NTD risk. Similar analyses for use of folate containing supplements preconception, for example, also showed no association. Total zinc intake and adjustment for correlated nutrients Folic acid intake from supplements and higher levels of circulating red blood cell and plasma folate have been associated with decreased NTD risk in clinical trials and other studies (2, 27, 32). Since in observational studies women who take multivitamin supplements or eat fortified breakfast cereals have increased intakes of multiple nutrients, including zinc and folate, it is again difficult to disentangle the independent effect of zinc on NTD risk. With combined information from all sources of intake, in our population, 40 percent of women with a zinc intake in the lowest quintile did not have a folate intake in this quintile. This allowed us to examine the effect of low zinc intake on NTD risk independent of folate intake. Increased total zinc intake was associated with reduced NTD risk after adjustment for total folate intake and other covariates (quintile 5 vs. quintile 1, odds ratios (95 percent CIs) of 0.73 (0.47, 1.14), 0.59 (0.37, 0.95), 0.42 (0.25, 0.70), and 0.68 (0.34, 1.34)) (table 4). In contrast, increased total folate intake was not associated with reduced NTD risk after adjustment for total zinc intake and other covariates (quintile 5 vs. quintile 1, odds ratios (95 percent CIs) of 0.86 (0.55, 1.36), 0.68 (0.42, 1.10), 1.13 (0.68, 1.87), and 1.00 (0.50, 2.03)). We were not able to fully differentiate the independent association between zinc and NTD risk from that of nutrients highly correlated with dietary sources of zinc in multivariate analyses. Zinc estimates were attenuated after adjustment for protein, methionine, and calcium, although evidence of a total zinc-ntd association persisted (table 4). Several interpretations of these findings are plausible. First, protein, methionine, and calcium could have served as markers for zinc intake (particularly the most bioavailable zinc from animal sources), because they are highly correlated with animal zinc intake. Second, these nutrients could have substantially confounded the zinc-ntd association observed. Methionine, in particular, has been hypothesized to play a role in neural tube development based on animal models and its association with folate metabolism (33). Finally, protein, methionine, and calcium may be needed in combination with zinc, for improved zinc bioavailability or neural tube development. In light of the correlation of these nutrients, as well as the measurement error present in dietary intake instruments that may differ by nutrient (34), we cannot disentangle in these multivariate analyses which single nutrient or combination of these nutrients is most associated with NTD risk. Potential effect modifiers In this population, several factors appeared to modify the NTD risk associated with zinc intake, in the hypothesized direction, to suggest a causal association between zinc and NTD risk. Phytate is known to have a significant impact on zinc bioavailability (13). Women with low total zinc intake, in combination with high phytate intake, were at highest risk for NTDs after adjustment for potential confounders (table 5). The simultaneous presence of phytate, calcium, and zinc in the gut is also hypothesized to affect zinc absorption (16, 17, 20). Studies in rats show that when zinc intake is held constant, growth decreases as phytate intake increases particularly when calcium intake is also elevated (16). Although sample size was limited, we observed the highest NTD risk in women with a high phytate to zinc molar ratio in combination with a high molar calcium intake (data not shown). This effect persisted, although it was reduced, after adjustment for several sociodemographic factors. In stratified analyses, we examined the effect of infections and alcohol intake, which have been hypothesized to reduce zinc absorption, on NTD risk (6, 35). In our data, neither maternal infection or alcohol consumption substantially modified the zinc/ntd association. However, few women reported highly elevated alcohol intakes, and maternal infection measures were self-reported and crude. Additional exploration of these factors is warranted, particularly in populations with potentially compromised nutritional status and increased susceptibility to chronic infections.

10 614 Velieetal. Zinc intake was not associated with NTD risk in overweight women (BMI >26.0) or primigravidous women and appeared particularly protective in women who were foreign-born Latina and multigravidous. However, sample sizes were small in some cells so these findings must be considered largely exploratory. Our finding of no protective effect of increased zinc intake in overweight women is consistent with that of Goldberg et al. (36). Those authors did not observe a protective effect of zinc supplementation on low birth weight in obese women, although they did observe a protective effect in all other women. Our finding of a protective effect of increased zinc intake in multigravidous but not primigravidous women may be related to a higher prevalence of zinc deficiency in multigravidas from previous childbirths and lactation. The more pronounced protective effect of increased zinc intake in foreign-born Latinas may be attributable to increased zinc need due to a particularly elevated phytate intake from legume consumption (37). Conclusion Among this study's strengths is that it utilized a population-based sample drawn from over 700,000 births. Multiple sources of case ascertainment were used, including information on elective terminations. Termination data are particularly important when the frequency of prenatal diagnosis varies among demographically diverse populations, as it does in California (38). Furthermore, the food frequency questionnaire used to assess usual dietary intake has been validated in other settings and detailed questions about vitamin, mineral, and food supplements were asked. Limitations of the present study must also be considered. Some clinically recognized NTD-affected pregnancies may have been missed and could introduce selection bias if these missed cases had higher or lower zinc intake levels than ascertained cases (38). Selection bias might also have been introduced if nonrespondents or women with unusable dietary data differed from women with available data with respect to characteristics associated with NTD risk and zinc intake. Additionally, as in all case-control studies, recall bias may be present. In particular, women may have tended to report their diet at the time of interview rather than preconceptionally. For example, because control mothers with healthy infants were more hkely to be lactating at the time of interview, they may have been more likely to over-report their preconceptional milk intake. Milk contains animal zinc and protein and therefore could contribute to an explanation for our findings. However, in analyses stratified by median servings of milk, the zinc effect persisted in both low and high milk drinkers. Case mothers may have been more meticulous in completing the food frequency questionnaire than control mothers but equal percents of case and control mothers reported "additional foods" consumed, which suggests comparable carefulness. Further, to assess potential reporting bias, all women were asked whether they believed that what they eat can cause birth defects, and whether periconceptional vitamins cause, prevent, or have no effect on whether a child is born with a birth defect. To both questions, case and control mothers' responses were similar. Limitations of the food frequency questionnaire used to assess nutrient intake have been described (39, 40). Although the instrument used in this study was not internally validated, previous validation studies (41-43) have demonstrated that it provides reasonable estimates of usual dietary intake for diets consumed by women in the distant past. The questionnaire has also been used in surveys with Hispanic populations and has produced dietary findings that are comparable with data collected using other survey methods (44), but it has not been well validated in Spanish-speaking populations. One study showed that a similar food frequency instrument administered to a low-income, predominantly Spanish-speaking population in Texas compared well with 24~hour recall assessments of intake (45). In our study, Spanish-speaking women were more likely than English-speaking women to obtain considerable assistance from interviewers (40 percent vs. 5 percent), and to have completed unusable questionnaires (16 percent vs. 10 percent). We would expect error in accurately ascertaining dietary intake in Spanish-speaking or Latina populations to be nondifferential by case status and to lead to an attenuation in risk estimates. However, the NTD risk associated with low total zinc was present in both English- and Spanish speaking women, as well as in white, US-born and foreign-born Latinas (table 6). To summarize, our data provide some evidence that increased maternal zinc intake, and in particular zinc from animal sources, may be associated with decreased NTD risk. We cannot disentangle in this observational study whether zinc, protein, a specific constituent of protein, a combination of zinc and protein, or another constituent of the diet highly correlated with zinc and protein is causally associated with decreased NTD risk. However, the biologic and animal evidence, when combined with our findings that factors that affect zinc bioavailability, such as phytate intake, appear to modify the zinc/ntd association, suggest that increased zinc intake is associated with reduced NTD risk in our population. Other studies, particularly with a larger sample size to more closely examine phytate, protein, and other potential effect

11 Zinc Intake and Neural Tube Defects 615 modifiers of a zinc/ntd association, are needed to confirm our findings. Although we cannot disentangle the specific nutrient constituent of the diet that is most protective for NTDs, our data suggest that women with the lowest intakes of zinc and animal products are at increased risk for NTDs. We believe nutrition programs targeted to women before conception should not only emphasize the importance of an adequate intake of folate, but also the importance of a nutrient-rich, balanced diet with an adequate intake of zinc and protein. ACKNOWLEDGMENTS Supported in part with funds from the Grossman Doctoral Student Dissertation Award, University of California, Berkeley. The authors thank the interview staff of the California Birth Defects Monitoring Program as well as Dr. Suzanne Murphy for providing access to the data base she created of the phytate contents of foods. They also thank Drs. Virginia Ernster, Z. I. Sabry, Barbara Sutherland, Lowell Sever, Lorraine Halinka Malcoe, Karen Todoroff, Karen Woodson, and Tamara Zemlo for helpful comments on earlier versions of this manuscript. REFERENCES 1. Elwood J, Little J, Elwood J. Epidemiology and control of neural tube defects. New York: Oxford University Press, Medical Research Council Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council vitamin study. Lancet 1991 ;338: Shaw G, Velie E, Schaffer D. Risk of neural tube defect-affected pregnancies among obese women. JAMA 1996;275: Tamura T, Goldenberg R. Zinc nutriture and pregnancy outcome. Nutr Res 1996;16: Vallee B, Falchuk K. The biochemical basis of zinc physiology. Physiol Rev 1993;73: Hunt S, Groff J, eds. Advanced nutrition and human metabolism. New York; West Publishing Co, Hurley L, Swenerton H. Congenital malformations resulting from zinc deficiency in rats. Proc Soc Exp Biol Med 1966; 123: Hambidge KM, Neldner KH, Walravens PA. Zinc, acrodermititis enteropathica, and congenital malformations. Lancet 1975; 1: Sever LE. Zinc and human development: a review. Hum Ecol 1975;3: Aggett P. Workshop on "Assessment of Zinc Status." Proc Nutr Soc 1991;50: Mares-Perlman JA, Subar AF, Block G, et al. Zinc intake and sources in the US adult population: J Am Coll Nutr 1995; 14: Zheng J, Mason JB, Rosenberg EH, et al. Measurement of zinc bioavailability from beef and ready-to-eat high-fiber breakfast cereal in humans: applications of a whole-gut lavage technique. Am J Clin Nutr 1993;58: Harland B, Oberleas D. Phytate in foods. World Rev Nutr Diet 1987;52: Wood R, Zheng J. High dietary calcium intakes reduces zinc absorption and balance in humans. Am J Clin Nutr 1997; 65: Whittaker P. Iron and zinc interactions in humans. Am J Clin Nutr 1998;68:442S-446S. 16. Davies N, Carswell A, Mills C. The effect of variation in dietary calcium intake on the phytate-zinc interaction in rats. In: Mills C, Bremmer I, Chesters J, eds. Trace Elements in Man and Animals - TEMA 5. Proceedings of the Fifth International Symposium on Trace Elements in Man and Animals. Aberdeen, Scotland: Commonwealth Agricultural Bureaux, 1985: Hunt J, Matthys L, Johnson L. Zinc absorption, mineral balance, and blood lipids in women consuming controlled lactoovovegetarian and omnivorous diets for 8 wk. Am J Clin Nutr 1998;67: National Research Council. Recommended dietary allowances. 10th ed. Washington, DC: National Academy Press, Alaimo K, McDowell MA, Briefel RR, et al. Dietary intake of vitamins, minerals, and fiber of persons aged 2 months and over in the United States: Third National Health and Nutrition Examination Survey, phase 1, Atlanta, GA: Centers for Disease Control and Prevention, Bindra G, Gibson R, Thompson L. Phytate x calcium/zinc ratios in Asian immigrant lacto-ovo vegetarian diets and their relationship to zinc nutriture. Nutr Res 1986;6: Shaw G, Schaffer D, Velie EM, et 1. Periconceptional vitamin use, dietary folate, and the occurrence of neural tube defects. Epidemiology 1995;6: Croen L, Shaw G, Jensvold N. Birth defects monitoring in California: a resource for epidemiological research. Paediatr Perinat Epidemiol 1991;5: Block G, Hartman AM, Dresser CM, et al. A data-based approach to diet questionnaire design and testing. Am J Epidemiol 1986; 124: National Cancer Institute EMS Inc. and Block Dietary Data Systems. Diet user's guide. Bethesda, MD: National Cancer Institute, Stata Statistical Software. Release 5.0. College Station, TX: StataCorp, Velie EM. The increased risk of neural tube defects among Mexicans in California: possible nutritional explanations with special emphasis on maternal periconceptional zinc nutriture. PhD dissertation. Berkeley, CA: Department of Epidemiology, University of California, Berkeley, June Czeizel A, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327: Milunsky A, Jick H, Jick SS, et al. Multivitamin/folic acid supplementation in early pregnancy reduces the prevalence of neural tube defects. JAMA 1989;262: Choi N, Klaponski FA, Ateah E, et al. Some epidemiological aspects of central nervous system malformations in Manitoba. In: Klingburg M, Abramavici A, Chemke J, eds. Drugs and fetal development. New York: Plenum Press, 1972: Sanford M, Kissling G, Joubert P. Neural tube defects etiology: new evidence concerning maternal hyperthermia, health and diet. Dev Med Child Neurol 1992;34: Freil J, Frecker M, Fraser F. Nutrition patterns of mothers of children with neural tube defects in Newfoundland. Am J Med Genet 1995;55: Scott J, Weir D, Kirke P. Folate and neural tube defects. In: Bailey L, ed. Folate in health and disease. New York: Marcel Dekker, 1995: Coehlo CND, Klein N. Methionine and neural tube closure in cultured rat embryos: morphological and biochemical analyses. Teratology 1990;42: Marshall JR, Hastrup JL. Mismeasurement and the resonance of strong confounders: uncorrelated errors. Am J Epidemiol 1996; 143: Keen C, Taubeneck MW, Daston GP, et al. Primary and sec-