Plasma Folate, Vitamin B12, and Homocyst(e)ine Concentrations in Preeclamptic and Normotensive Peruvian Women

Sixto E. Sanchez1, Cuilin Zhang2, M. Rene Malinow3, Suzie Ware-Jauregui1, Gloria Larrabure4 and Michelle A. Williams2,5,6

1 Dos de Mayo Hospital, Lima, Peru.
2 Department of Epidemiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA.
3 Oregon Regional Primate Research Center, Beaverton, OR.
4 Materno-Perinatal Institute, Lima, Peru.
5 Center for Perinatal Studies, Swedish Medical Center, Seattle, WA.
6 Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The authors measured maternal third trimester plasma folate, vitamin B12, and homocyst(e)ine concentrations among 125 women with preeclampsia and 179 normotensive women in Lima, Peru (1997–1998), to determine whether these analytes were associated with the occurrence of preeclampsia. Logistic regression procedures were used to calculate maximum likelihood estimates of odds ratios and 95% confidence intervals. Relative to women in the upper quartile of the control distribution of maternal plasma folate concentrations, women with values in the lowest quartile experienced a 1.6-fold increased risk of preeclampsia (odds ratio = 1.6; 95% confidence interval: 0.8, 3.2). There was no evidence of an increased risk of preeclampsia associated with low plasma vitamin B12 concentrations. The unadjusted relative risk of preeclampsia increased across successively higher quartiles of plasma homocyst(e)ine level (odds ratios were 1.0, 1.0, 1.5, and 2.9, respectively, with the lowest quartile used as the referent; p for linear trend = 0.0004). After adjustment for maternal age, parity, gestational age, use of prenatal vitamins, whether the pregnancy had been planned, and educational attainment, the relative risk between extreme quartiles was 4.0 (95% confidence interval: 1.8, 8.9). These findings are consistent with earlier reports suggesting that hyperhomocyst(e)inemia in pregnancy may be a risk factor for preeclampsia.

folic acid; homocysteine; homocystine; pre-eclampsia; pregnancy; risk factors; vitamin B 12

Abbreviations: CI, confidence interval; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Homocyst(e)ine or total homocysteine is the sum of free and bound, reduced, and oxidized forms of the amino acid homocysteine. The metabolism of homocysteine, a by-product of the essential amino acid methionine, involves either transsulfuration to cysteine or remethylation to methionine. The transsulfuration pathway requires vitamin B6 and the enzyme cystathionine-ß-synthase. Defects in the cystathionine-ß-synthase gene result in the inborn error of metabolism homocystinuria (1Go). The major pathway for remethylation requires methylenetetrahydrofolate reductase (MTHFR), methionine synthase, vitamin B2 (2Go), vitamin B12, and folate (3Go). Mutations in the MTHFR gene and dietary deficiencies for vitamin B6, vitamin B12, or folate are associated with elevated homocyst(e)ine concentrations (4Go). Hyperhomocyst(e)inemia can usually be corrected by supplementation with B vitamins including folic acid (5Go, 6Go).

Hyperhomocyst(e)inemia is an independent risk factor for vascular disease, including atherosclerosis and thrombosis (7GoGo–9Go). Elevated plasma homocyst(e)ine levels are also seen in women with preeclampsia, both in the acute phase of the disorder (10GoGo–12Go) and postpartum (13Go). Sorensen et al. (14Go) reported recently that high maternal serum homocyst(e)ine concentrations in the second trimester are associated with an increased risk of subsequent preeclampsia. The emerging evidence of an association between elevated homocyst(e)ine levels and preeclampsia is consistent with other data implicating diffuse endothelial cell dysfunction in the etiology of the disorder (15GoGoGoGo–19Go).

Few investigators have evaluated the relation between maternal folate and vitamin B12 status in relation to risk of preeclampsia. Results from a recent meta-analysis of preeclampsia risk in relation to maternal folate and vitamin B12 status underscored the need for additional research designed to assess the relation between preeclampsia risk and these two micronutrients (20Go). The goal of the present study was to examine whether maternal plasma folate, vitamin B12, and homocyst(e)ine concentrations, measured in the third trimester of pregnancy, are associated with the risk of preeclampsia. Because of the strong positive association of preeclampsia with nulliparity and increased maternal prepregnancy body mass index (12Go, 14Go, 21Go, 22Go), interactions of these factors with elevated homocyst(e)ine were also examined.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This case-control study was conducted at the Materno-Perinatal Institute and the Dos de Mayo Hospital in Lima, Peru, from June 1997 through January 1998. Both institutions are operated by the Peruvian government and are primarily responsible for providing maternity services to low income women residing in Lima. The ethical committees of the Dos De Mayo Hospital and the Materno-Perinatal Institute of Lima and the Human Subjects Committee of the University of Washington Medical Center approved this investigation.

Cases eligible for inclusion were those women with a diagnosis of preeclampsia. Potential preeclampsia cases were identified by daily monitoring of all new admissions to antepartum wards, emergency room wards (intensive care units), and labor and delivery wards of the study hospitals. Study subjects were recruited during their hospital stay. Study personnel made periodic visits to specific wards in a fixed order for the purpose of identifying potential cases and controls for the present study. Preeclampsia was defined as 1) a persistent (i.e., lasting more than 6 hours) 15-mmHg rise in diastolic blood pressure or 30-mmHg rise in systolic blood pressure or 2) persistent blood pressure of at least 140/90 mmHg and a urinary protein concentration of 30 mg/dl or more (or a score of 1 or higher on a urine dipstick test). Nulliparity was not a criterion for diagnosis for this investigation. Ninety-seven percent of eligible case patients who were approached and asked to participate in the study elected to do so (193 of 199 subjects).

Controls were women with pregnancies uncomplicated by pregnancy-induced hypertension or proteinuria. Each day during the enrollment period, controls were searched for from among normotensive pregnant women admitted to one of the study hospitals for normal labor and delivery, third trimester bleeding, urinary tract infection, fetal growth restriction, or preterm labor on the same day as a case. Potential controls were approached in the order in which research personnel identified them. Controls were frequency-matched to cases according to maternal age (within 5 years) and gestational age at admission (within 2 weeks). Of the 204 controls approached, 196 (96 percent) agreed to participate in the study.

A standardized structured interview questionnaire was used to collect information regarding maternal sociodemographic, medical, reproductive, and lifestyle characteristics during in-person interviews. All interviews were conducted in the hospital. Maternal and infant records were reviewed to collect detailed information concerning antepartum, labor, and delivery characteristics and conditions of the newborn. Information about maternal prepregnancy weight was also abstracted from medical records. Maternal anthropometric measures (height, weight, and mid-arm circumference) were taken at the end of the interview. Maternal prepregnancy body mass index was determined using maternal prepregnancy weight abstracted from medical records and the height measured during the interview. Blood samples were collected in 10-ml Vacutainer tubes (Becton Dickinson, Rutherford, New Jersey) containing ethylenediaminetetraacetic acid and were immediately transported, in a cooler with wet ice, to the Blood Bank Laboratory of Dos de Mayo Hospital. Upon arrival at the laboratory, plasma was separated by refrigerated centrifugation and divided into 1.0- to 2.0-ml aliquots, placed in cryovials, and stored at -70°C. Specimens were shipped on dry ice and blue ice to the United States for biochemical analyses.

Blood samples were available for 180 cases and 196 control subjects. After exclusion of six cases and three control subjects with chronic hypertension diagnosed prior to pregnancy or during the first 20 weeks of the index pregnancy, 174 preeclampsia cases and 193 normotensive control subjects remained for study. Because maternal plasma homocyst(e)ine concentrations are known to increase after parturition (23Go), we further excluded 49 preeclampsia cases and 14 controls for whom blood samples were drawn after delivery or during the intrapartum period, thus leaving 125 cases and 179 controls for this research. Ninety-four percent of cases and 89 percent of control subjects were fasting at the time of blood collection.

Plasma homocyst(e)ine concentrations were measured by high performance liquid chromatography and electrochemical detection as described previously, with minor modification (24Go). The interassay coefficient of variation was 7.2 percent. Plasma folate and vitamin B12 concentrations were measured by radioimmunoassay (Chiron Diagnostics Corporation, East Walpole, Massachusetts). The interassay coefficients of variation for folate and vitamin B12 were <4.5 percent and <6.3 percent, respectively. All laboratory analyses were performed without knowledge of pregnancy outcome.

Plasma samples used for this research had been stored for a period of 1–2 years. It is unlikely that a storage period of such duration altered the concentration of homocyst(e)ine. Isrealsson et al. (25Go) found that homocyst(e)ine concentrations in stored plasma (kept at -20°C) remained stable for up to 10 years and were strongly correlated with concentrations measured from fresh plasma.

The frequency distributions of maternal sociodemographic characteristics and medical and reproductive histories were examined according to case/control status. To estimate the relative associations between preeclampsia and levels of plasma folate, vitamin B12, and homocyst(e)ine, respectively, we categorized each subject according to quartiles determined by the distribution of analyte concentrations in normotensive controls. Using the highest quartile category (for folate and vitamin B12) or the lowest quartile category (for homocyst(e)ine) as the referent group, we estimated odds ratios and their 95 percent confidence intervals. The Mantel extension test for linear trend in proportions (26Go) was used in univariate analyses to test for a linear component of trend in risk between preeclampsia and specific analytes. Logistic regression procedures were used to calculate maximum likelihood estimates for the coefficients, and their standard errors were used to calculate odds ratios and 95 percent confidence intervals, adjusted for confounders (27Go). In multiple logistic regression models, significance for linear trend in risk of preeclampsia with increasing (or decreasing) concentrations of the analytes was assessed by treating the four quartiles as a continuous variable after assigning a score to each quartile (27Go). To assess confounding, we entered variables into a logistic regression model one at a time and then compared the adjusted and unadjusted odds ratios (27Go). Final logistic regression models included covariates that altered unadjusted odds ratios by at least 10 percent, as well as those covariates with a priori interest (e.g., maternal age and parity). Effect modification by parity and maternal prepregnancy obesity was evaluated in stratified analyses and by including appropriate interaction terms in logistic regression models. All reported p values are two-tailed, and confidence intervals were calculated at the 95 percent level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Table 1 shows the characteristics of the study participants. Mean maternal plasma folate concentrations were 11 percent lower in cases than in controls (11. 9 ± 0.4 (standard error) nmol/liter vs. 13.2 ± 0.50 nmol/liter; p = 0.068). Preeclampsia cases had 13 percent higher mean vitamin B12 concentrations (253.72 ± 1.9 pg/liter vs. 224.2 ± 5.4 pg/liter; p < 0.001) and 19 percent higher mean homocyst(e)ine concentrations (10.0 ± 0.6 µmol/liter vs. 8.4 ± 0.1 µmol/liter; p = 0.028) than controls (not shown in table).


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TABLE 1. Distribution of case women with preeclampsia and normotensive control subjects according to selected characteristics, Lima, Peru, 1997–1998

 
Relative to women in the upper quartile of the control distribution of maternal plasma folate concentrations, women with values in the lowest quartile experienced a 1.8-fold increased risk of preeclampsia (odds ratio (OR) = 1.8; 95 percent confidence interval (CI): 0.8, 3.2) (table 2). After adjustment for potential confounding by maternal age, nulliparity, gestational age at blood collection, use of prenatal vitamins, whether the pregnancy was planned, and maternal educational attainment, the association between extreme quartiles was slightly attenuated (OR = 1.6; 95 percent CI: 0.8, 3.2). Compared with women in the highest quartile, women in the second and third quartiles experienced no increased risk of preeclampsia (adjusted odds ratios were 0.9 and 1.2, respectively). There was no evidence of an increased risk of preeclampsia associated with low plasma vitamin B12 concentrations.


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TABLE 2. Odds ratios for preeclampsia according to quartiles of maternal plasma folate, vitamin B12, and homocyst(e)ine concentrations, Lima, Peru, 1997–1998

 
The relative risk of preeclampsia increased significantly across increasing quartiles of maternal plasma homocyst(e)ine concentration (for linear trend in risk across quartiles, p < 0.0001). Women in the highest quartile experienced a threefold increased risk of preeclampsia as compared with women in the lowest quartile. After adjustment for potential confounding by maternal age, nulliparity, gestational age at blood collection, use of prenatal vitamins, whether the pregnancy was planned, and maternal educational attainment, the relative risk between extreme quartiles increased slightly (OR = 4.0; 95 percent CI: 1.8, 8.9). Women in the upper fifth percentile of the control distribution of homocyst(e)ine, compared with women in the lowest quartile of the distribution, had a relative risk for preeclampsia similar to that seen for women in the upper quartile (OR = 3.90; 95 percent CI: 1.19, 12.78) (not shown in table).

Table 3 shows the results from analyses of the effects of combinations of maternal prepregnancy obesity (i.e., upper quartile of the distribution of prepregnancy body mass index among controls (>=25.3 kg/m2)) and parity and elevated homocyst(e)ine levels (i.e., upper quartile of the distribution among controls (>=9.1 µmol/liter)) on the occurrence of preeclampsia. Compared with nonobese women without homocyst(e)ine elevations, obese women with elevated homocyst(e)ine experienced a fivefold increased risk of preeclampsia (OR = 5.1; 95 percent CI: 1.9, 14.1). The excess risk of preeclampsia associated with being obese and having elevated homocyst(e)ine concentrations was approximately equal to the sum of the excess relative risk for each factor considered independently. Hence, in this population, there was no evidence of a greater-than-additive effect between the two characteristics and the occurrence of preeclampsia. In analyses of the interactive effect of nulliparity and elevated homocyst(e)ine concentrations on the occurrence of preeclampsia, results suggested a greater-than-additive relation.


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TABLE 3. Evaluation of joint associations between maternal prepregnancy obesity and parity and elevated plasma homocyst(e)ine level in relation to the risk of preeclampsia, Lima, Peru, 1997–1998

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this case-control study, low maternal plasma folate levels and high homocyst(e)ine concentrations were associated with an increased risk of preeclampsia. Women with homocyst(e)ine concentrations above 9.1 µmol/liter experienced a fourfold increase in risk of preeclampsia as compared with women with values below 5.3 µmol/liter. There was no evidence of an increased risk of preeclampsia associated with low plasma vitamin B12 concentrations.

Previous epidemiologic studies relating maternal plasma folate, vitamin B12, and homocyst(e)ine concentrations to preeclampsia risk are limited. Results from studies of maternal serum or plasma folate concentration as a risk factor for preeclampsia have been null (10GoGo–12Go, 28Go). Similarly, previous investigators have reported that maternal plasma or serum vitamin B12 concentrations were not associated with preeclampsia risk (10Go). However, results from prior studies of maternal serum or plasma homocyst(e)ine concentrations during pregnancy or postpartum have been positive (10GoGo–12Go, 23Go). Results from one published prospective study demonstrated that maternal serum homocyst(e)ine concentrations >=5.5 µmol/liter (measured at 16 weeks' gestation, on average) were associated with a 3.2-fold increased risk of preeclampsia (14Go).

Several potential limitations of our study merit consideration when interpreting reported results. Because of the retrospective design of our study, we cannot determine whether the observed elevations in homocyst(e)ine and attenuated plasma folate concentrations preceded preeclampsia, or whether the differences may be attributed to disease-related alterations in folate and homocysteine metabolism. For instance, we cannot exclude the possibility that preeclampsia-related renal dysfunction accounts for some or all of the homocyst(e)ine elevations noted among cases versus controls in our study. However, results from the only prospective study that we know of (14Go) suggest that elevations in homocyst(e)ine precede the clinical manifestation of preeclampsia by approximately 8–16 weeks. We did not determine allele status for the MTHFR gene for preeclampsia cases and controls in this present study. Results from some (29Go, 30Go) studies of the 677 C->T MTHFR polymorphism, though not all (31Go), suggest that carriers of the mutant allele are more likely to develop preeclampsia than are noncarriers. Differential misclassification of maternal plasma folate, vitamin B12, and homocyst(e)ine concentrations is unlikely, as all laboratory analyses were conducted without knowledge of participants' pregnancy outcome status. We did not have information on participants' folate and vitamin B12 intakes, so inferences concerning maternal dietary habits in relation to preeclampsia risk are limited. This is a particularly important limitation of our study, as there is some concern that physiologic changes common to pregnancy may make plasma vitamin B12 an unreliable measure of maternal status for that nutrient. Prospective studies which allow for the assessment of maternal dietary intake, nutritional supplement use, and plasma folate, vitamin B12, and homocyst(e)ine concentrations will afford a greater specificity with respect to assessing the extent to which maternal diet and/or metabolism of folic acid and other B vitamins contributes to the pathogenesis of preeclampsia. Lastly, although we controlled for multiple confounding factors, we cannot with certainty conclude that the odds ratios reported are unaffected by residual confounding.

Diffuse endothelial cell dysfunction resulting in vascular permeability is thought to be important in the pathogenesis of preeclampsia (15GoGoGo–18Go). Results from clinical and other studies (32Go, 33Go) support this hypothesis. Several lines of evidence suggest plausible biologic mechanisms for the association between elevated homocyst(e)ine, endothelial cell dys-function, and atherothrombogenesis (3Go, 34Go). Results from several in vitro studies have indicated that homocysteine added to cultured cells causes endothelial cell damage in a dose-dependent manner (35GoGo–37Go). A positive association between elevated concentrations of von Willebrand factor, an important marker of endothelial cell dysfunction/activation, and homocyst(e)ine in humans has been reported (38Go). In a human umbilical vein, endothelial cells released von Willebrand factor in a dose-dependent manner when incubated in different concentrations of homocysteine (39Go). Importantly, von Willebrand factor concentrations have been shown to be attenuated with treatment of elevated homocyst(e)ine with folic acid and vitamin B12 (38Go, 40Go).

Endothelial dysfunction induced by homocyst(e)ine elevations may also be mediated by the generation of reactive oxygen species (3Go). Reactive oxygen species including superoxide and hydrogen peroxide are produced during the auto-oxidation of homocysteine and hydrogen peroxide (34Go). Notably, homocyst(e)ine has been shown to attenuate the expression of cellular glutathione peroxidase by endothelial cells, and this effect is known to promote lipid peroxidation by reactive oxygen species elaborated during the oxidation of homocysteine (34Go, 41Go). Elevated homocyst(e)ine is also thought to promote endothelial cell dysfunction and subsequent atherogenesis via its role in increasing nitric oxide production in vascular smooth muscle cell by activating the transcription factor NF-{kappa}ß (42Go).

Nulliparity is a well known though poorly understood risk factor for preeclampsia (21Go, 22Go). Our finding of an interaction between nulliparity and elevated homocyst(e)ine in relation to preeclampsia risk is consistent with that reported previously (12Go). However, the underlying biologic mechanism for the observed interaction is unknown at present.

Maternal mortality (43Go) and serious maternal morbidity, including acute renal failure and pulmonary edema resulting from hypertensive pregnancies, represent a troublesome area of modern perinatology. Hypertensive disorders of pregnancy, including preeclampsia and eclampsia, continue to be among the leading causes of maternal mortality. Perinatal complications are now among the top 10 (seventh) leading causes of overall mortality worldwide (44Go). Emerging evidence (20Go, 45Go) and results from our present study suggest that folic acid and other B vitamins may be important in the pathogenesis of preeclampsia.


    ACKNOWLEDGMENTS
 
This research was supported in part by awards from the National Institutes of Health (T37-TW00049, R01-32562, and RR00163-35).

The authors thank Mohammed Adem, Eric Graf, Mirtha Grande, Elena Sanchez, Hong Tang, Nelly Toledo, and Barbara Upson for their skillful technical assistance.


    NOTES
 
Reprint requests to Dr. Michelle A. Williams, Box 357236, School of Public Health and Community Medicine, University of Washington, 1959 Northeast Pacific, Seattle, WA 98195 (e-mail: mwilliam{at}u.washington.edu).


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Received for publication November 2, 1999. Accepted for publication April 28, 2000.