Perinatal Reference Intervals for Plasma Homocysteine and Factors Influencing Its Concentration

Moderately increased plasma total homocysteine (tHcy) concentrations have been associated with an increased risk of atherothrombotic vascular events (1). Disturbances in homocysteine metabolism have also been reported as a possible risk factor for early pregnancy loss and congenital birth defects, such as neural tube defects, as well as for maternal obstetric complications (2). Reference intervals for healthy maternal and newborn populations are scarce; in particular, we could not find data on large samples of women at delivery. Raijmakers et al. (3) reported tHcy results for samples collected at delivery or 4 h before caesarian section on 35 women. Böhles et al. (4) and Malinow et al. (5) measured tHcy at delivery in 60 and 35 women, respectively, and Bjørke Monsen et al. (6) reported results for 169 samples collected between 96 to 108 h after birth. Available data on newborns include the results from one large study in Italy (7) and a few smaller studies (3)(4)(5)(6), among which only one is from North America; it included 35 women and their newborns (5). Mean tHcy values were quite different among these studies. Genetic, nutritional, and lifestyle factors are believed to influence tHcy concentrations (8)(9). Among the genetic factors, methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C gene polymorphisms are potentially important. Supplementation of the diet with folate as well as smoking and caffeine consumption are among other factors that can affect tHcy concentrations. None of the studies cited above took the effect of these factors into account. The goals of our study are (a) to provide reference values for tHcy measured within 48 h of delivery from a large unselected sample of women who gave birth to babies born at or above the 10th percentile for gestational age and sex; (b) to provide similar values for their newborns; and (c) to study the impact on maternal as well as newborn tHcy concentrations of common MTHFR genetic polymorphisms as well as nutritional factors. We performed a hospital-based case-control study of intrauterine growth restriction in which all live-born singleton cases seen over a 2-year period (mid-1998 and mid-2000) were matched for sex, gestational age, and race to a live newborn control whose weight was at or above the 10th percentile based on gestational age and sex, as determined by Canadian population standards (10). Cases and controls were born at the same hospital and generally within a few days of each other. We report here on 468 mothers and their 438 babies who were the controls in our case-control study and for whom a blood sample could be obtained and tHcy determined. The study included a total of 472 mother–newborn controls. The project was approved by the ethics committee of the hospital, and informed consent was obtained from all mothers. Venous maternal blood was obtained within 48 h of delivery (median time of collection, 25 h). Placental blood was collected from the umbilical veins of the newborns. Citrate buffer (0.5 mol/L, pH 4.3) was used as the anticoagulant (0.5 mL of citrate for 4.5 mL of blood), and samples were kept at 4 °C until centrifugation, which took place within 6 h (median, 0.58 h) for maternal samples and within 24 h for newborns (median, 6.06 h). This difference reflects the fact that the maternal specimen was obtained by the research nurse on duty, whereas the specimen from the newborn was obtained by the delivery room personnel at all hours. Under these collection and storage conditions, tHcy concentrations (in μmol/L) were stable for at least 24 h (11). After centrifugation, samples were stored at −70 °C until analyzed by HPLC as reported previously (12) with modifications. Briefly, 60 μL of 0.15 mol/L N-acetyl-l-cysteine and 30 μL of 100 g/L tri-n-butylphosphine in dimethylformamide were added to 240 μL of plasma (reducing step). After incubation at 4 °C for 30 min, the proteins were precipitated with 300 μL of 0.6 mol/L perchloric acid containing 1 mmol/L EDTA. The tubes were vortex-mixed and centrifuged for 1 min at 10 000g. The derivatization step was performed as follows. A 50-μL aliquot of the supernatant was transferred into another tube and mixed with 10 μL of 1.55 mol/L NaOH, 125 μL of borate buffer (125 mmol/L, pH 9.5, containing 4 mmol/L EDTA), and 50 μL of a 7-fluorobenzofurazane-4 sulfonic acid (SBD-F) solution (freshly prepared in the same borate buffer). The mixture was incubated at 60 °C for 1 h, and then a 50-μL aliquot was mixed with 50 μL of the elution buffer. One-half of the resulting mixture (50 μL) was injected on a Kromasil C18 column (150 × 4.6 mm; 5-μm bead size; Phenomenex). SBD-F derivatives were eluted isocratically with a mobile phase containing 0.2 mol/L sodium acetate buffer (pH 4) and methanol (98:2 by volume). The HPLC analysis was performed at 1.5 mL/min. Fluorescence detection was on a Shimadzu RF551 Fluorescence Spectrometer (Shimadzu Corporation). Data were collected and analyzed with Gold software (Ver. 6.0) from Beckman-Coulter. To determine the role of maternal smoking and eating habits (for foods rich in folic acid) as well as that of vitamin supplementation on tHcy concentrations, mothers were interviewed face-to-face after delivery. For nutritional factors, we asked about quantities (in units such as cups, cans, glasses, and so forth) consumed in a day or a week and averaged the results over a specific pregnancy trimester. In addition, MTHFR C677T and A1298C gene polymorphisms were determined for newborns and their mothers. PCR-allele-specific oligonucleotide hybridization assays were used. Data were analyzed with a descriptive purpose. The 95% reference interval was used (13). Univariate linear regression was used to estimate regression coefficients and their 95% confidence intervals (CIs). The regression coefficient (β) is interpreted as the amount of change in the mean homocysteine concentration with one unit of change in the explanatory variable. For example, for tHcy in newborns, if the regression coefficient for the mother smoking 10 cigarettes a day during pregnancy was 0.60, this would imply a predicted increase of 0.60 in the newborn tHcy concentration for every 10 cigarettes smoked daily by the mother. We used the consumption data from the third trimester. A log transformation of homocysteine was used, but results were not different in their interpretation from those obtained with the original units. We therefore retained the latter for ease of interpretation. The unpaired t-test was used to compare means. The reference population is defined here as mothers who gave birth to babies with a birth weight above the 10th percentile according to gestational age. Mean maternal age was 29.5 years (SD, 5.78 years); 70.5% of mothers were white, 23.3% were black, 2.7% were Asian, and 3.4% were Amerindian. The mean body mass index (prepregnancy weight in kg divided by height in m2) was 23.1 (SD, 5.2), and the mean weight gain during pregnancy was 14.4 kg (SD, 5.6 kg). Among newborns, 53.6% were girls (more girls are affected with intrauterine growth restriction, and these controls were matched for sex), 41.8% were born before the 39th week (reflecting matching on gestational age), and mean birth weight was 3208.1 g (SD, 734.5 g; range, 795-4810 g). The mean values and CIs for tHcy concentrations in mothers and newborns as well a reference interval for these measures are shown in Table 1. As shown in Table 1, the number of hours after delivery upwardly influenced the maternal tHcy concentration, whereas time between sample collection and centrifugation did not. Only vitamin intake (yes/no; β = −0.57; 95% CI, −1.02 to −0.13) and consumption of lentils (2 cups/week; β = −0.52; 95% CI, −0.87 to −0.17) significantly reduced maternal tHcy concentrations; results were unchanged when controlled for delay between delivery and blood collection. However, maternal smoking (10 cigarettes/day; β = 0.60; 95% CI, 0.30–0.90) and caffeine intake (1 cup/day; β = 0.27; 95% CI, 0.08–0.45) increased the tHcy concentration in newborns, whereas vitamin intake (β = −0.87; 95% CI, −1.27 to −0.51) and consumption of lentils (β = −0.32; 95% CI, −0.61 to −0.03) decreased it. We also considered consumption of tea, alcohol, and other folate-rich foods, such as chick peas, red kidney beans, or black turtle beans, but none had an effect on either maternal or newborn concentrations. Noticeably, the MTHFR polymorphisms had no significant effects on tHcy concentrations. Postpartum and fetal umbilical vein reference intervals for plasma tHcy. Time, in hours, between delivery and blood collection. Time, in hours, between blood collection and centrifugation of specimen. t-Test comparing “within 1 h” and “between 1 and 6 h” = 0.48 (P = 0.63). t-Test comparing “within 6 h” and “between 6 and 24 h” = 1.77 (P = 0.08). Postpartum and fetal umbilical vein reference intervals for plasma tHcy. Time, in hours, between delivery and blood collection. Time, in hours, between blood collection and centrifugation of specimen. t-Test comparing “within 1 h” and “between 1 and 6 h” = 0.48 (P = 0.63). t-Test comparing “within 6 h” and “between 6 and 24 h” = 1.77 (P = 0.08). The stability of homocysteine in specimens during the time between collection and storage is an important factor to consider in a large epidemiologic study, in which technical conditions can hardly match those of a strict experimental study. That homocysteine was stable in our samples was reflected in the results for both the mothers and the newborns, which showed no statistically significant differences (overlapping CIs and t-test results with P values >0.05) in the mean tHcy values according to time between specimen collection and centrifugation (Table 1). In Canada, vitamin supplementation before conception and during pregnancy is strongly encouraged by physicians and public health authorities (14), and folic acid fortification of enriched flour, although not mandatory, has been permitted since 1998 (15), a period that overlaps that of the study period. These reasons as well as the fact that we studied a group of women with normal pregnancy outcomes may explain why the MTHFR polymorphisms did not have a significant impact on tHcy concentrations. When we compared our mean tHcy concentrations for mothers at delivery with those from other studies (3)(4)(5)(6)(16), only the results from the American study of Malinow et al. (5) were close (5.43 ± 1.40 μmol/L). In the other studies, carried out in Europe, concentrations were higher. This same statement applies to newborn data except for a recent large Italian study (7) in which the mean value (4.9 μmol/L) was closest to that of our study; however, the dispersion of results in that study was greater (±2.7 μmol/L). Several genetic and nutritional factors could explain the discrepant results, but with the exception of the former study (7), they could also be explained by a lack of precision in the estimates because of generally small sample sizes. In conclusion, maternal and newborn tHcy concentrations seem lower in North America than in Europe, probably because of food fortification. Nutritional and lifestyle factors seem to have a greater influence on homocysteine concentrations than do common MTHFR polymorphisms. This project was supported by Grant MA-14705 from the Canadian Institutes of Health Research. C.I.R. holds a Canada Research Chair (James McGill Professorship) from McGill University.

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