Adequacy of vitamin B6 supplementation during pregnancy: a prospective study13

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1 dequacy of vitamin B6 supplementation during pregnancy: a prospective study13 Lawrence Lumeng,4 M.D., Robert E. Cleary,5 M.D., Rosemary Wagner,6 M.Sc., Pao-Lo Yu,7 Ph.D., and Ting-Kai Li,8 M.D. BSTRCT This prospective study assesses the effect of 2.5, 4, and 1 mg of pyridoxine supplementation during pregnancy on maternal and fetal plasma levels of pyridoxal 5 -phosphate (PLP) and on the degree of coenzyme saturation (activation factor) of aspartate aminotransferase and alanine aminotransferase (aegot and aegpt) in maternal erythrocytes. More than 4 mg of pyridoxine supplementation daily was required for most pregnancies to maintain maternal plasma PLP levels within the range observed during the first trimester and in the nonpregnant state. The plasma PLP concentrations in maternal and cord blood were highly correlated and indicated a dependence of fetal vitamin B, nutrition on maternal circulating PLP. Measurements of aegot and aegpt were not as reproducible as plasma PLP assays and were less sensitive and quantitative indicators. In the majority of subjects, the changes in aegot and aegpt with time correlated poorly with the changes in plasma PLP. However, when the data were analyzed without regard for their dependence on time, they demonstrated a negative, linear correlation between aegot and log plasma PLP and between aegpt and log plasma PLP for the group on 2.5 mg of pyridoxine and for all the subjects combined. Finally, the dietary records showed that most of the subjects consumed less than 2 mg of vitamin B, daily from their food. The results indicate that the current Recommended Dietary llowance for vitamin B, during pregnancy (2.5 mg) is too low and that supplementation of this vitamin in an amount more than 4 mg daily is recommended. m. J. C/in. Nuir. 29: , It is generally recognized that there is an increased requirement for vitamin B6 during pregnancy (1-4) and that the vitamin is essential for normal fetal development and postnatal growth (5-1). The current recommendation of the National cademy of Sciences-National Research Council (NS- NRC) for the daily dietary allowance of vitamin B6 during pregnancy is 2.5 mg (4), an increase of.5 mg over that for nonpregnant women. However, two recent studies based on plasma pyridoxal 5 -phosphate (PLP) measurements (11, 12) have suggested that maternal vitamin B6 nutrition may become abnormal during the second and third trimesters of pregnancy even when subjects are supplemented daily with 2. to 2.5 mg of pyridoxine. Since the study of Hamfelt and Tuvemo (11) was performed in a Swedish population and our previous study (12) was a cross-sectional analysis at the time of delivery, we have conducted a prospective study designed to define more precisely the amount of pyridoxme supplementation that is needed during pregnancy in merican women. The effects of 2.5,4, and 1mg of pyridoxine supplementation on maternal and fetal vitamin B6 nutritional status were compared by measuring the level of PLP in plasma and the percent stimulation in vitro of erythrocyte alanine aminotransferase (EGPT) and aspartate ami- 1 From the Departments of Medicine, Obstetrics and Gynecology, and Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana Supported in part by the United States Department of griculture, Grant and by the United States Public Health Service, Grant RR -75. ddress reprint requests to: Ting-Kai Li, M.D., Department of Medicine, Indiana University School of Medicine, 11 West Michigan Street, Indianapolis, Indiana Clinical Investigator, Veterans dministration Hospital and ssociate Professor of Medicine and Biochemistry. ssociate Professor of Obstetrics and Gynecology. #{149} Chief Nutritionist, Clinical Research Center. ssociate Professor of Medical Genetics. 8 Professor of Medicine and Biochemistry. Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, The merican Journal of Clinical Nutrition 29: DECEM BER 1976, pp Printed in U.S..

2 VITMIN B. SUPPLEMENTTION DURING PREGNNCY 1377 notransferase (EGOT) activities by exogenous PLP. dditionally, the amount of pyridoxine consumed in the diet was assessed by dietary record. The reliability of plasma PLP as a direct measure of vitamin B6 nutrition has been documented recently by a number of investigators (13-17). The strengths and weaknesses of erythrocytic GOT and GPT activity measurements as indicators have been the subject of several recent publications (17-19). Materials and Methods Thirty-three pregnant subjects who were in good health were randomly admitted to the study from the private obstetrical population of the University Medical Center. In each instance, the procedure and risk were carefully explained to each patient and informed, written consent was obtained before any vitamin was administered or blood sample obtained. ll the patients were instructed by a metabolic research dietician to consume a diet of 1,8 to 2,4 kcal containing 1 g of protein. The total amount of food and beverages consumed during a 3-day consecutive period was recorded by each of the subjects on at least two occasions. The amount of dietary vitamin B6 ingested was computed according to standard tables provided by the U.S. Department of griculture (2). The subjects were randomly assigned to three groups and instructed to take daily one of three prenatal vitamin preparations containing either 2.5, 4, or 1 mg of vitamin B,. These vitamin preparations, custom-formulated specifically for the purposes of this study by courtesy of Mead Johnson and Company, Evansville, Indiana, also contained: vitamin, 8, lu: vitamin D, 4 IU: vitamin E. 3 IU: vitamin C. 12 mg; folic acid,.8 mg; thiamine, 1.7 mg: riboflavin, 2 mg: niacin, 2 mg: vitamin B12, 8 g: Ca, 26 mg: 1, 15 zg: Fe. 6 mg, and Mg, 115mg. Twenty-six of the 33 subjects completed the study. Two patients left the area before completion of the study. bortion, premature labor, inadequate dietary records, and missing more than three prenatal visits were the reasons that the other five subjects were excluded. Venous blood samples were collected in vacutainer tubes containing either ethylene diaminetetra-acetic acid (EDT) or heparin at 6-week intervals from each subject. In addition, maternal venous and cord bloods were obtained at the time of delivery. The blood samples collected in EDT,\ sere centrifuged within 3 mm of venipuncture and the PLP in plasma was measured enzymaticallv with tyrosine decarboxylase apoenzyme as described previously (21). The between-run reproducibility of the method was ±4.7% (1 SD, n = 2) and recovery of added, authentic PLP was 9 8.5%. The erythrocytes in the blood samples collected in heparin were washed with cold saline, hemolyzed with twice its volume of distilled water and frozen. Erythrocytic GOT and GPT activities were then determined on the thawed hemolysates with and without added PLP,.2 msi. The assays were performed as described by Cabaud et al. (22) except that. I M triethanolamine HCI was employed as the buffer instead of phosphate. The ratio of the stimulated activity in the presence of added PLP to the intrinsic activity without added PLP is the activation factor, aegot or aegpt. The between-run reproducibilities of aegot and aegpt determinations in hemolysates were ± 1.8% and ± 16.8%, (1 SD, n = 34), respectively. The upper limits of normal for aegot and segpt are 1.5 and respectively (18, 23). Results Ten subjects in the 2.5 mg group, six in the 4 mg group, and 1 subjects in the 1 mg group completed the study. Plasma PLP levels in maternal blood.. 9, With 2.5 mg of pyridoxine supplementation, the mean concentration of PLP in maternal plasma increased from an initial value of 12.7 ± 3.5 to a high of 19. ± 6.5 ng/ml (±SE) by the following measurement period (Fig. 1). The extent of change was variable between individuals: seven of the subjects exhibited this rise and three did not. fter the mean plasma PLP level had peaked between the 13th and 18th weeks, it decreased T 1 I I I Term WEE #{149} FIG. I. Pyridoxal phosphate concentrations in plasma during pregnancy of 1 women receiving 2.5 mg of pyridoxine supplementation. Five of the 6 serial samples are not shown because three patients missed these prenatal visits. KS Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, 216

3 1378 LUMENG ET L progressively during the remaining course of pregnancy. The mean level of plasma PLP at term, 3.8 ±.8 ng/ml, was significantly less than the initial mean value (P <.5). Seven subjects in the latter half of their third trimester and a similar number of subjects at the time of delivery exhibited abnormally low plasma PLP concentrations, i.e. <4.7 ng/ml, the lower limit of normal for nonpregnant women of comparable age (24). These results confirmed the observations of Hamfelt and Tuvemo (11) and of Cleary et al. (12) that supplementation with 2.5 mg of pyridoxine daily is not sufficient to maintain normal maternal vitamin B6 nutrition in most pregnancies. Figure 2 shows the results for the subjects placed on 4 mg of pyridoxine supplementation. The pattern of change in mean plasma PLP values did not differ significantly (P >.5) from that exhibited by the subjects on 2.5 mg of pyridoxine daily (Fig. 1 and Table I). bnormal plasma PLP levels developed in two of the subjects during the last trimester and in four of the subjects at term. The results for the 1 mg group (Fig. 3 and Table 1) were quite different from those of the other two groups. Plasma PLP values rose in all the subjects after initiation of supplementation. This was reflected by a rise in mean PLP from an initial value of 19.9 ± 7.6 to 4.4 ± 7.6 ng/ml when measured 6 weeks,, U,, #{149} #{149} WEEKS #{149} S - I < Term FIG. 2. Pyridoxal phosphate concentrations in plasma during pregnancy of 6 women receiving 4 mg of pyridoxinc supplementation. Three of 36 serial samples are not S.E. shown because three patients missed these prenatal visits. TBLE 1 Statistical comparison of the concentration of PLP in maternal plasma during the course of pregnancy in women who received different doses of pyridoxine supplementation daily Weeks of pregnancy 2.5 mg versus4mg Statist ical comparison, P values 4 mg versus 1mg #{176} NS, not significant, P > mg versus IOmg 7-12 NS8 NS NS NS <.5 < NS <.2 NS 25-3 NS <.5 < NS <.1 <.1 Term NS <.5 <.1 later. lthough PLP concentrations also fell during the second and third trimesters, the mean values did not differ significantly from the initial mean value. t the time of delivery, only one of the 1 subjects exhibited abnormally low plasma PLP value. Thus 1 mg of pyridoxine supplementation appears to be adequate to normalize maternal vitamin B6 nutrition in most pregnant subjects. Plasma PLP values in cord blood Supplementation with 1 mg of pyridoxine daily also significantly altered the plasma PLP concentrations in cord blood (Fig. 4). The mean PLP value in cord plasma from these subjects was twice (P <.2) that for subjects taking 2.5 mg but was not significantly different from that for subjects supplemented with 4 mg of pyridoxine. In addition to this relationship, there was also a highly significant correlation (P <.1) between maternal and cord plasma PLP levels (Fig. 5). Thus the state of vitamin B6 nutrition in the mother directly affects the plasma level of PLP in the fetus. Erythrocytic amino! ransferase activation factors in maternal blood and their correlation with plasma PLP values Whereas the plasma PLP values exhibited consistent patterns of change during the course of pregnancy, the changes in aegot and aegpt with time were quite variable. There was no significant correlation in the majority of the individuals between plasma PLP levels and aegot and aegpt as Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, 216

4 VITMIN B, SUPPLEMENTTION DURING PREGNNCY 1379 dependent variables of time. The data were therefore analyzed by treating all paired observations without regard for the duration of pregnancy (Figs. 6 and 7). The decreasing order of sensitivity of these indicators of vitamin B6 status was shown by the incidence with which they were abnormal: 22% for PLP, 14% for aegpt, and 5% for aegot. Most of the abnormal values in PLP, aegot, and aegpt were accounted for by the subjects in the 2.5 mg of supplementation group. By regression analysis, significant but low levels of linear correlations were found between aegot and log PLP and between aegpt and log PLP for the group on 2.5 mg (r = and -.26, respectively) and for all the subjects combined (r = and -.214, respectively). C.a, $ S.E. C 4, ( B6 SUPPLEMENT, mg/day FIG. 4. The effect of pyridoxine supplementation on the plasma concentrations of pyridoxal phosphate in cord blood at the time of delivery.... a C C-) Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, I Term WEEKS FIG. 3. Pyridoxal phosphate concentrations in plasma during pregnancy of 1 women receiving 1 mg pyridoxinc supplementation. Three of 6 serial samples are not shown because two patients missed these prenatal visits. MTERNL PLSM PLP ng/ml FIG. 5. The relationship of plasma pyridoxal phosphate concentrations in cord and maternal blood at the time of delivery.

5 138 LUMENG ET L. Dietary intake of pyridoxine The dietary records of the patients also provided interesting data (Table 2). Only one of the 26 subjects who completed the study consumed the Recommended Dietary llowance of 2.5 mg of pyridoxine daily from their diet. Eighteen of the subjects ingested 1.9 mg or less. Discussion In a previous cross-sectional study (12), we demonstrated by plasma PLP assay that pyridoxine supplementation in dosages less than 2.5 mg daily is inadequate to maintain normal maternal vitamin B6 nutrition during pregnancy. We also provided evidence that the PLP metabolism in the fetus is dependent to a large extent on the maternal supply of this B6 vitamer. In that study, it was not possible to obtain a reasonable assessment of the dietary habits or the degree of cooperation of the subjects in taking their daily vitamins, but these factors have been monitored in the present prospective study. The results reported here substantiate our previous conclusions and confirm the report of #{176} I- V., Ui o ool o 9 io lo, o.u PYRIDOXINE SUPPLEME?. r o 2.Smg/d #{163} 4 mg/d #{149} lomg/d #{149} #{163} S #{163} #{149}#{149} #{149} S #{149} #{149} US t #{149} L Hamfelt and Tuvemo (11). dditionally, based on plasma PLP measurements, it appears that more than 4 mg of pyridoxine is required to restore normal vitamin B6 nutrition in the mother during pregnancy (Figs. 1 to 3). The assessment of the dietary intake of vitamin B6 in our population of pregnant women is of some interest. ll of them could be classified as belonging to the upper middle and middle socioeconomic class. lthough all were carefully instructed to consume a diet of 1,8 to 2,4 kcal containing 1 g of protein by a dietician, only one of the 26 subjects consumed 2.5 mg of vitamin B6, the Recommended Dietary llowance for this vitamin. This finding is consistent with the reports of Borsook (25) and of Thompson et al. (26) which indicate that the nutrient intake of most pregnant women is inadequate and vitamin-mineral supplementation is required. This problem is even more serious in pregnant teenagers as pointed out by a recent study by Kaminetsky et al. (27). In our previous and present studies, particular emphasis has been placed on plasma PLP measurements. Plasma PLP can be Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, Plasma PLP, ng/ml FIG. 6. The correlation of plasma pyridoxal phosphate levels with aegot values in maternal blood. The upper limit of normal for aegot is 1.5.

6 VITMIN B, SUPPLEMENTTION DURING PREGNNCY ol 1 PYRIDOXINE SUPPLEMENT 2.Smg/d 4 mg/d 5 1 mg/d 5. V., Ui I Q S Ocl 1 5, 1 o #{163} S S o #{176} 15 #{149}S S oc S US 55 #{149} S S 5S S S Plasma PLP, ng/mi 5 1 FIG. 7. The correlation of plasma pyridoxal phosphate levels with aegpt values in maternal blood. The upper limit of normal for aegpt is TBLE 2 The mean daily intake of vitamin B, in the pregnant subjects vitamin B, No. of patients mg/day l assayed accurately and reproducibly by the tyrosine decarboxylase apoenzyme method (17, 21, 28). In addition, PLP is the active coenzyme form and the major B6 vitamer in plasma and tissues (29). PLP in circulation exists bound principally to albumin and its concentration represents a dynamic equilibrium between hepatic synthesis on the one hand and extraction and/or degradation by other tissues on the other (3). Thus, from the physiological viewpoint, it can be anticipated that the measurement of plasma PLP is a quantitatively meaningful indicator of the state of vitamin B6 nutrition. Indeed, a number of studies have supported this expectation and have demonstrated significant correlation between the plasma and tissue contents of this B6 vitamer (13-16). In the clinical evaluation of vitamin B6 nutritional status, it is desirable to corroborate the reduction of the vitamin level in biological fluids with other biochemical tests which reveal metabolic change (e.g., tryptophan load test or aegot and aegpt). Unfortunately, at the present, all these tests have limitations. The tryptophan load test is sensitive, but it is readily altered by hormonal changes (31-33). Measurements of aminotransferase activation factors (a) in erythrocytes lack reproducibility and sensitivity (17). Since PLP is bound very tightly to these am inotransferases, undersaturation occurs only under conditions of extreme vitamin depletion. In our recent experiments in rats (16), undersaturation of EGOT was detected only when growing animals were supplied with less than 4 g of dietary pyridoxine daily and were severely retarded in growth. Similarly, in the recent report by Brown et al. (15), the appearance of abnormal aegot lagged significantly behind the development of low Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, 216

7 1382 LUMENG ET L. plasma PLP levels in women who received a vitamin B6-deficient diet. lthough we did find a significant correlation between plasma PLP measurements and activation factors of GOT and GPT in red cells, the level of significance was low (Figs. 6 and 7). Furthermore, it was difficult to relate the pattern of change in plasma PLP levels in the individual subjects tu the aegot and aegpt values that tended to vary widely. This latter finding is in agreement with the results reported by Hamfelt and Tuvemo (11). Based on abnormal PLP levels in plasma (34), whole blood (17), and leukocytes (35), and on altered aegot and aegpt (36), there is now good evidence that a vitamin B, deficiency state or at least a state of increased requirement for the vitamin exists in most pregnant women. Since the vitamin B6 nutritional status of the fetus may be dependent on maternal plasma PLP, and since animal studies have shown that vitamin B6 is necessary for normal fetal development and growth, it is our conclusion that pyridoxine supplementation should be instituted to maintain maternal vitamin B6 status within the range observed in normal nonpregnant women. t the present, this opinion is not shared universally. Some argue that what is normal for the nonpregnant state may not necessarily be normal for pregnancy, but data that bear directly on this argument are scarce. lthough convulsive seizures resulting from vitamin B6 deficiency in neonates have been well documented (1), Hillman et al. (37) did not find that the routine administration of pyridoxine during pregnancy was necessarily beneficial. However, in their study, parameters such as birth weight, length, and the occurrence of prematurity were employed to assess clinical outcome. In the future more specific criteria for evaluating the state of neonatal development, e.g., the neurological assessment outlined by Prechtl and Beintema (38) and the Brazelton behavioral assessment scale (39), should be employed to discern the implications of pyridoxine adequacy for fetal development. The authors wish to acknowledge the generosity of Mead Johnson and Company, Evansville, Indiana for providing the vitamin preparations. We are grateful to Ronald Minter and Squire Heard for their skilled technical assistance. References 1. CONTRCTOR, S. F., ND B. SHNE. Pyridoxal kinase in the human placenta and foetus through gestation. Clin. Chim. cta 25: 465, CONTRCTOR, S. F., ND B. SHNE. Blood and urine levels of vitamin B, in the mother and fetus before and after loading ofthe mother with vitamin B,. m. J. Obstet. Gynecol. 17: 635, BRIN, M. bnormal tryptophan metabolism in pregnancy and with the oral contraceptive pill. II. Relative levels of vitamin B, vitamers in cord and maternal blood. m. J. Clin. Nutr. 24: 74, Food and Nutrition Board, National Research Council. Recommended Dietary llowances, ed. 8. Washington, D.C.: National cademy of Sciences, DKSHINMURTI, K., ND M. C. STEPHENS. Pyridoxine deficiency in the neonatal rat. J. Neurochem. 16: 1515, DVIS, S. D., T. NELSON ND T. H. SHEPRD. Teratogenicity of vitamin B, deficiency: omphalocele, skeletal and neural defects, and splenic hypoplasia. Science 169: 1329, MooN, W-H. Y., ND. KIRKSEY. Cellular growth during prenatal and early postnatal periods in progeny of pyridoxine-deficient rats. J. Nutr. 12: 123, LTON-MCKEY, M. G., ND B. L. WLKER. Graded levels of pyridoxine in the rat diet during gestation and the physical and neuromotor development ofoffspring. m. J. Clin. Nutr. 26: 42, Dvis, S. D. Immunodeficiency and runting syndrome in rats from congenital pyridoxine deficiency. nature 251: 548, BESSEY,.., D. J. D. DM ND. E. HNSEN. Intake of vitamin B, and infantile convulsions: a first approximation of requirements of pyridoxine in infants. Pediatrics 2: 33, HMFELT,., ND T. TUVEMO. Pyridoxal phosphate and folic acid concentration in blood and erythrocyte aspartate aminotransferase activity during pregnancy. Clin. Chim. cta 41: 287, CLERY, R. E., L. LUMENG ND T-K. Li. Maternal and fetal plasma levels of pyridoxal phosphate at term: adequacy of vitamin B, supplementation during pregnancy. m. J. Obstet. Gynecol. 121: 25, WCHSTEIN, M., J. D. KELLNER ND J. M. ORTIz. Pyridoxal phosphate in plasma and leukocytes of normal and pregnant subjects following B, load tests. Proc. Soc. Exptl. Biol. Med. 13: 35, HMFELT,. Pyridoxal phosphate concentration and aminotransferase activity in human blood cells. Clin. Chim. cta 16: 19, BROWN, R. R., D. P. ROSE, J. E. LEKLEM, H. LINKSWILER ND R. NND. Urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B,. m. J. Clin. Nutr. 28: 1, RYN, M. P., L. LUMENG ND T-K. Li. Validation of plasma pyridoxal 5-phosphate measurements as an indicator of vitamin B6 nutritional status. Federation Proc. 35: 66, Downloaded from ajcn.nutrition.org at PENNSYLVNI STTE UNIV PTERNO LIBRRY on May 11, 216

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