Maternal and fetal plasma levels of pyridoxal phosphate at term: Adequacy of vitamin B, supplementation during pregnancy ROBERT
E.
CLEARY,
LAWRENCE
LUMENG,
TING-KAI
LI,
Indianapolis,
M.D. M.D.*
M.D.
Indiana
The effect of different amounts of vitamin B, supplementation during pregnancy on maternal and fetal plasma levels of pyrido.xal phosphate (PLP) at term has been studied. Ten of 13 subjects given 2 to 2.5 mg. of vitamin B, daily exhibited plasma PLP levels lower than 4.7 ng. per milliliter (the lower limit of normal for nonpregnant subjects). In contrast, only 4 of 1 I subjects given a supplement of 10 mg. of uitamin B, daily had plasma PLP less than this value. The mean plasma PLP level (64.4 ng. per milliliter) of 10 cord blood samples from newborn infants whose mothers exhibited plasma PLP levels greater than 4.7 ng. per milliliter was significantly higher (P < 0.005) than that (34.2 ng. per milliliter) from 14 newborn infants whose mothers had abnormally lowered plasma PLP concentrations. In cord plasma, an average venous-arterial gradient of 10.6 ng. per milliliter was observed, indicating that the fetus retains and/or degrades PLP. These results suggest that more than 2 to 2.5 mg. of vitamin B, supplement daily is required for most pregnancies to restore normal vitamin B, nutrition in the mother and, perhaps, also in the fetus.
T H E R E Q U I R E M E N T for vitamin B, (pyridoxine) has been reported to be increased during pregnancy because of the high metabolic demands of the growing fetus. I-4 The 1968 recommendation of the National Academy of Sciences-National Research Council for the daily dietary allowance of vitamin B, during pregnancy is 2.5 mg., an increment of 0.5 mg. over that for nonpregnant women.” In order to ensure adequacy of intake, supplementation of this vitamin has been widely prescribed in From the Department of Obstetrics and and the Department of Medicine, Indiana University School of Medicine, Veterans Administration Hospital. Supported in part by Grant United States Department Received Reuised Accepted
for
publication
May
1, 1974.
May
antepartum care. Most prenatal vitamins currently marketed in this country contain 2 to 2.5 mg. of pyridoxine, although the amount varies from preparation to preparation, ranging from 1 to 25 mg. How much vitamin B, is needed to meet fetal requirements is unknown at present. However, there is considerable evidence that a substantial number of pregnant women on presumably “normal” diets develop biochemical abnormalities consistent with vitamin B, deficiency,S-’ which are correctable by the administration of pyridoxine.“, G The investigations by Wachstein and Gudaitis” which employed the tryptophan load test as the principal indicator of the state of vitamin B, nutrition suggested that a daily supplement of 10 mg. of pyridoxine was necessary for most pregnant women. In view of the hormonal effects upon the enzymes of the tryptophan-niacin pathway, R, g the reliability of the tryptophan load test as an accurate indicator of vitamin B, nutrition in pregnancy has been questioned. However, a recent study of Hamfelt and TUvemo,” who measured the plasma concentration of pyridoxal phosphate (PLP) and the saturability of erythrocytic aspartate aminotransferase as indicators
Gynecology and
the
12-14-1001-44, of Agriculture.
December
27,
1973.
IO, 1974.
Reprint requests: Dr. Robert E. Cleary, Department of Obstetrics and Gynecology, Indiana University Medical Center, 1100 W. Michigan St., Indianapolis, Indiana 46202. *Clinical Hospital.
Investigator,
Veterans
Administration
25
26
Cleary,
Lumeng,
and
Li
2.5 mg. or 10 mg. of vitamin B, supplement during pregnancy. The relationship between maternal and fetal plasma PLP levels in paired samples obtained at the time of delivery was assessed. In addition, the plasma concentrations of PLP in the umbilical arter). and vein were measured to define the degree of degradation and/or utilization of PLP by the fetus. Plasma PLP is generally considered a reliable indicator of vitamin B, nutrition since PLP represents a substantial portion of vitamin B, compounds in tissues and circulationl” Its concentration correlates well with other indicators of vitamin B, nutrition l’s Iii and its level can be reproducibly measured by a sensitive enzymatic assay.16
.
0)
, I
PdO.05
I P~O.001 I
I
p’o.02
,
Fig. 1. The plasma PLP concentrations of nonpregnant control female subjects receiving no vitamin supplement, pregnant subjects at the time of delivery who had been receiving 10 mg. of pyridoxine per day, and pregnant subjects at the time of delivery who had been receiving 2 to 2.5 mg. of pyridoxine supplement daily.
of vitamin B, nutrition, has also indicated that a daily supplement of 2 mg. of pyridoxine is insufficient and, in agreement with the results of Wachstein and Gudaitis,l a 10 mg. daily supplement may be required to normalize both maternal and fetal PLP values. The study by Hamfelt and TuvemoG was done in Sweden. Since there may be differences in dietary habits, it cannot be concluded with certainty that their findings are applicable also to the American population. Pyridoxal phosphate, the coenzyme form of vitamin B,, is essential for numerous metabolic pathways, including nucleic acid and protein synthesis in the growing fetus. lo Studies by Contractor and Shane”, 12 indicate that the placenta has a high activity of pyridoxal kinase which catalyzes the synthesis of PLP and suggest that PLP is the major vitamin B, compound which is transported across the placenta, apparently against a concentration gradient. Whether a venous-arterial difference in PLP concentrations exists between the umbilical vein and artery has not actually been determined. This knowledge is of particular importance since the supply and metabolism of PLP in the fetus may be strongly dependent upon that of the mother. In this communication, the plasma levels of PLP in nonpregnant control subjects receiving no vitamin supplements were compared with plasma PLP values of subjects at term who have been given either 2 to
Material
and
method
Healthy pregnant women were randomly admitted to the study from the private obstetric population of the University Medical Center. Thirteen paired samples of cord and maternal blood were collected from subjects who daily ingested a prenatal vitamin supplement containing 2 to 2.5 mg. of vitamin B,.” Eleven paired samples were obtained from those who took a preparation containing 10 mg.+ In addition, blood samples from the umbilical artery and vein was collected separately from 16 newborn infants whose mothers had been receiving a daily supplement of 2 mg. of vitamin B,. All blood samples were collected in 2 mM. ethylenediaminetetra-acetic acid at the time of delivery. The samples were centrifuged within 30 minutes of collection and then assayed immediately or stored at -loo C. and assayed within 2 days of collection. The plasma concentration of PLP was measured enzymatically with the use of tyrosine decarboxylase apoenzyme as described previously.‘” The reproducibility of the method was k4.7 per cent (1 S. D.) and the recovery of authentic PLP was 90+8.5percent (1 SD.). Results The plasma concentrations of PLP from 58 nonpregnant control female subjects, ages 20 to 34 years, who were not receiving vitamin supplement; 11 pregnant subjects at the time of delivery who had been receiving 10 mg. of vitamin B, per day; and 13 pregnant subjects at the time of delivery who had been receiving 2 to 2.5 mg. of pyridoxine supplement *En-Cebrin
F., Eli Lilly
& Co., Box 618, Indianapolis,
Indiana 46206, or Natalins l-FA, Mead Johnshn Laboratories, Div. of Mead Johnson & Co., 2404 Pennsylvania St., Evansville, Indiana 47721. +Natalins-RX, Mead Johnson Laboratories.
Volume Number
121 1
Plasma
daily are shown in Fig. 1. The mean plasma PLP level for the control subjects as reported previously,17 was 10.5 t 4.1 ng. per milliliter (1 S. D.), and none of the subjects had values lower than 4.7 ng. per milliliter.* The mean levels of plasma PLP of subjects given 10 and 2 to 2.5 mg. of vitamin B, daily were significantly lower 7.5 i 4.5 ng. per milliliter (P < 0.05) and 3.7 t 1.5 ng. per milliliter (P < 0.001)) respectively, than the mean of the control population. Four of the subjects receiving 10 mg. of vitamin B, daily had plasma concentrations of PLP less than 4.7 ng. per milliliter, whereas 10 of 13 subjects receiving 2 to 2.5 mg. of vitamin B, daily exhibited PLP levels less than normal. Therefore, these results confirm the observations of Hamfelt and Tuvemo6 and suggest that a vitamin B, supplement of 2 to 2.5 mg. daily is not sufficient to normalize maternal vitamin B, nutrition in most pregnant subjects at term. The relationship between maternal and cord plasma PLP levels was also studied. The mean concentrations of PLP in the plasma from the umbilical cords of neonates whose mothers were given 10 and 2 to 2.5 mg. of vitamin B, supplements were 58.4 and 37.1 ng. per milliliter, respectively. The difference was significant at the level of P < 0.05. Since this study did not control dietary intake, it seemed more meaningful to compare the plasma levels of PLP in the cord blood of newborn infants of mothers whose plasma PLP concentrations were normal with those whose maternal levels were abnormally low (Fig. 2). When the mothers exhibited normal plasma PLP levels, the mean cord level was 64.4 ng. per milliliter. In contrast, when the plasma PLP concentrations of the mothers were abnormally low, the mean cord value was 34.2 ng. per milliliter (P < 0.005). Thus, the state of vitamin B, nutrition in the mother significantly affects the plasma level of PLP in the fetus. In order to understand further the economy of PLP nutrition during pregnancy, the venous-arterial differences in PLP concentrations between the umbilical vein and artery in 6 paired samples were measured. In all instances, the PLP concentrations in the umbilical vein were higher (P < 0.05)) and an average venous-arterial difference of 10.6 ng. per milliliter was observed (Table I). Therefore, these data indicate that the fetus takes up and/or degrades PLP that is transported across the placenta. *The value the lower limit ages of 20 and
transformation
of 4.7 ng. per milliliter has been assigned of normal for female subjects between the 34. It is the mean -2 S. D. after logarithmic of each of the plasma PLP values.
levels
of
pyridoxal
phosphate
Fig. 2. The relation of maternal and cord plasma concentrations collected at the time of delivery. Table I. Venous and arterial plasma of PLP in the umbilical cord Cord Sample No. 1 2 3 4 5 6 Mean *P
Venous (ng./ml.) 38.9 28.1 43.6 25.8 69.2 53.1
plasma
I
33.3 21.4 27.5 22.2 42.8 47.8
PLP
concentrations
concentrations Arterial (ng./ml.)
27
of PLP
I
Venous-arterial difference (ng./ml.) 5.6 6.7 16.1 3.6 26.4 5.3 10.6*
< 0.05, difference between the means of paired data.
Comment Plasma PLP is generally considered a reliable and sensitive indicator of the state of vitamin B, nutrition.18 It has been previously demonstrated that plasma PLP is significantly correlated with the cellular content of PLP and with functional tests of vitamin B, sufficiency or deficiency, e.g., the saturability of aspartate and alanine aminotransferases with PLP, during pregnancy and in experimentally produced vitamin B, deficiency in human subjects.l”t lo In this study, comparison of the plasma PLP values of subjects at term with those of nonpregnant women of comparable ages indicates that a daily supplement of pyridoxine in excess of 2 to 2.5 mg. will be required to correct the altered vitamin B, nutrition during pregnancy in a majority of women. A similar conclusion was reached previously by
28
Cleary,
Lumeng,
and
Januarg 1. I!Fi Am. J. Ohstet. C+nrr
E.
CLEARY,
LAWRENCE
LUMENG,
TING-KAI
LI,
Indianapolis,
M.D. M.D.*
M.D.
Indiana
The effect of different amounts of vitamin B, supplementation during pregnancy on maternal and fetal plasma levels of pyrido.xal phosphate (PLP) at term has been studied. Ten of 13 subjects given 2 to 2.5 mg. of vitamin B, daily exhibited plasma PLP levels lower than 4.7 ng. per milliliter (the lower limit of normal for nonpregnant subjects). In contrast, only 4 of 1 I subjects given a supplement of 10 mg. of uitamin B, daily had plasma PLP less than this value. The mean plasma PLP level (64.4 ng. per milliliter) of 10 cord blood samples from newborn infants whose mothers exhibited plasma PLP levels greater than 4.7 ng. per milliliter was significantly higher (P < 0.005) than that (34.2 ng. per milliliter) from 14 newborn infants whose mothers had abnormally lowered plasma PLP concentrations. In cord plasma, an average venous-arterial gradient of 10.6 ng. per milliliter was observed, indicating that the fetus retains and/or degrades PLP. These results suggest that more than 2 to 2.5 mg. of vitamin B, supplement daily is required for most pregnancies to restore normal vitamin B, nutrition in the mother and, perhaps, also in the fetus.
T H E R E Q U I R E M E N T for vitamin B, (pyridoxine) has been reported to be increased during pregnancy because of the high metabolic demands of the growing fetus. I-4 The 1968 recommendation of the National Academy of Sciences-National Research Council for the daily dietary allowance of vitamin B, during pregnancy is 2.5 mg., an increment of 0.5 mg. over that for nonpregnant women.” In order to ensure adequacy of intake, supplementation of this vitamin has been widely prescribed in From the Department of Obstetrics and and the Department of Medicine, Indiana University School of Medicine, Veterans Administration Hospital. Supported in part by Grant United States Department Received Reuised Accepted
for
publication
May
1, 1974.
May
antepartum care. Most prenatal vitamins currently marketed in this country contain 2 to 2.5 mg. of pyridoxine, although the amount varies from preparation to preparation, ranging from 1 to 25 mg. How much vitamin B, is needed to meet fetal requirements is unknown at present. However, there is considerable evidence that a substantial number of pregnant women on presumably “normal” diets develop biochemical abnormalities consistent with vitamin B, deficiency,S-’ which are correctable by the administration of pyridoxine.“, G The investigations by Wachstein and Gudaitis” which employed the tryptophan load test as the principal indicator of the state of vitamin B, nutrition suggested that a daily supplement of 10 mg. of pyridoxine was necessary for most pregnant women. In view of the hormonal effects upon the enzymes of the tryptophan-niacin pathway, R, g the reliability of the tryptophan load test as an accurate indicator of vitamin B, nutrition in pregnancy has been questioned. However, a recent study of Hamfelt and TUvemo,” who measured the plasma concentration of pyridoxal phosphate (PLP) and the saturability of erythrocytic aspartate aminotransferase as indicators
Gynecology and
the
12-14-1001-44, of Agriculture.
December
27,
1973.
IO, 1974.
Reprint requests: Dr. Robert E. Cleary, Department of Obstetrics and Gynecology, Indiana University Medical Center, 1100 W. Michigan St., Indianapolis, Indiana 46202. *Clinical Hospital.
Investigator,
Veterans
Administration
25
26
Cleary,
Lumeng,
and
Li
2.5 mg. or 10 mg. of vitamin B, supplement during pregnancy. The relationship between maternal and fetal plasma PLP levels in paired samples obtained at the time of delivery was assessed. In addition, the plasma concentrations of PLP in the umbilical arter). and vein were measured to define the degree of degradation and/or utilization of PLP by the fetus. Plasma PLP is generally considered a reliable indicator of vitamin B, nutrition since PLP represents a substantial portion of vitamin B, compounds in tissues and circulationl” Its concentration correlates well with other indicators of vitamin B, nutrition l’s Iii and its level can be reproducibly measured by a sensitive enzymatic assay.16
.
0)
, I
PdO.05
I P~O.001 I
I
p’o.02
,
Fig. 1. The plasma PLP concentrations of nonpregnant control female subjects receiving no vitamin supplement, pregnant subjects at the time of delivery who had been receiving 10 mg. of pyridoxine per day, and pregnant subjects at the time of delivery who had been receiving 2 to 2.5 mg. of pyridoxine supplement daily.
of vitamin B, nutrition, has also indicated that a daily supplement of 2 mg. of pyridoxine is insufficient and, in agreement with the results of Wachstein and Gudaitis,l a 10 mg. daily supplement may be required to normalize both maternal and fetal PLP values. The study by Hamfelt and TuvemoG was done in Sweden. Since there may be differences in dietary habits, it cannot be concluded with certainty that their findings are applicable also to the American population. Pyridoxal phosphate, the coenzyme form of vitamin B,, is essential for numerous metabolic pathways, including nucleic acid and protein synthesis in the growing fetus. lo Studies by Contractor and Shane”, 12 indicate that the placenta has a high activity of pyridoxal kinase which catalyzes the synthesis of PLP and suggest that PLP is the major vitamin B, compound which is transported across the placenta, apparently against a concentration gradient. Whether a venous-arterial difference in PLP concentrations exists between the umbilical vein and artery has not actually been determined. This knowledge is of particular importance since the supply and metabolism of PLP in the fetus may be strongly dependent upon that of the mother. In this communication, the plasma levels of PLP in nonpregnant control subjects receiving no vitamin supplements were compared with plasma PLP values of subjects at term who have been given either 2 to
Material
and
method
Healthy pregnant women were randomly admitted to the study from the private obstetric population of the University Medical Center. Thirteen paired samples of cord and maternal blood were collected from subjects who daily ingested a prenatal vitamin supplement containing 2 to 2.5 mg. of vitamin B,.” Eleven paired samples were obtained from those who took a preparation containing 10 mg.+ In addition, blood samples from the umbilical artery and vein was collected separately from 16 newborn infants whose mothers had been receiving a daily supplement of 2 mg. of vitamin B,. All blood samples were collected in 2 mM. ethylenediaminetetra-acetic acid at the time of delivery. The samples were centrifuged within 30 minutes of collection and then assayed immediately or stored at -loo C. and assayed within 2 days of collection. The plasma concentration of PLP was measured enzymatically with the use of tyrosine decarboxylase apoenzyme as described previously.‘” The reproducibility of the method was k4.7 per cent (1 S. D.) and the recovery of authentic PLP was 90+8.5percent (1 SD.). Results The plasma concentrations of PLP from 58 nonpregnant control female subjects, ages 20 to 34 years, who were not receiving vitamin supplement; 11 pregnant subjects at the time of delivery who had been receiving 10 mg. of vitamin B, per day; and 13 pregnant subjects at the time of delivery who had been receiving 2 to 2.5 mg. of pyridoxine supplement *En-Cebrin
F., Eli Lilly
& Co., Box 618, Indianapolis,
Indiana 46206, or Natalins l-FA, Mead Johnshn Laboratories, Div. of Mead Johnson & Co., 2404 Pennsylvania St., Evansville, Indiana 47721. +Natalins-RX, Mead Johnson Laboratories.
Volume Number
121 1
Plasma
daily are shown in Fig. 1. The mean plasma PLP level for the control subjects as reported previously,17 was 10.5 t 4.1 ng. per milliliter (1 S. D.), and none of the subjects had values lower than 4.7 ng. per milliliter.* The mean levels of plasma PLP of subjects given 10 and 2 to 2.5 mg. of vitamin B, daily were significantly lower 7.5 i 4.5 ng. per milliliter (P < 0.05) and 3.7 t 1.5 ng. per milliliter (P < 0.001)) respectively, than the mean of the control population. Four of the subjects receiving 10 mg. of vitamin B, daily had plasma concentrations of PLP less than 4.7 ng. per milliliter, whereas 10 of 13 subjects receiving 2 to 2.5 mg. of vitamin B, daily exhibited PLP levels less than normal. Therefore, these results confirm the observations of Hamfelt and Tuvemo6 and suggest that a vitamin B, supplement of 2 to 2.5 mg. daily is not sufficient to normalize maternal vitamin B, nutrition in most pregnant subjects at term. The relationship between maternal and cord plasma PLP levels was also studied. The mean concentrations of PLP in the plasma from the umbilical cords of neonates whose mothers were given 10 and 2 to 2.5 mg. of vitamin B, supplements were 58.4 and 37.1 ng. per milliliter, respectively. The difference was significant at the level of P < 0.05. Since this study did not control dietary intake, it seemed more meaningful to compare the plasma levels of PLP in the cord blood of newborn infants of mothers whose plasma PLP concentrations were normal with those whose maternal levels were abnormally low (Fig. 2). When the mothers exhibited normal plasma PLP levels, the mean cord level was 64.4 ng. per milliliter. In contrast, when the plasma PLP concentrations of the mothers were abnormally low, the mean cord value was 34.2 ng. per milliliter (P < 0.005). Thus, the state of vitamin B, nutrition in the mother significantly affects the plasma level of PLP in the fetus. In order to understand further the economy of PLP nutrition during pregnancy, the venous-arterial differences in PLP concentrations between the umbilical vein and artery in 6 paired samples were measured. In all instances, the PLP concentrations in the umbilical vein were higher (P < 0.05)) and an average venous-arterial difference of 10.6 ng. per milliliter was observed (Table I). Therefore, these data indicate that the fetus takes up and/or degrades PLP that is transported across the placenta. *The value the lower limit ages of 20 and
transformation
of 4.7 ng. per milliliter has been assigned of normal for female subjects between the 34. It is the mean -2 S. D. after logarithmic of each of the plasma PLP values.
levels
of
pyridoxal
phosphate
Fig. 2. The relation of maternal and cord plasma concentrations collected at the time of delivery. Table I. Venous and arterial plasma of PLP in the umbilical cord Cord Sample No. 1 2 3 4 5 6 Mean *P
Venous (ng./ml.) 38.9 28.1 43.6 25.8 69.2 53.1
plasma
I
33.3 21.4 27.5 22.2 42.8 47.8
PLP
concentrations
concentrations Arterial (ng./ml.)
27
of PLP
I
Venous-arterial difference (ng./ml.) 5.6 6.7 16.1 3.6 26.4 5.3 10.6*
< 0.05, difference between the means of paired data.
Comment Plasma PLP is generally considered a reliable and sensitive indicator of the state of vitamin B, nutrition.18 It has been previously demonstrated that plasma PLP is significantly correlated with the cellular content of PLP and with functional tests of vitamin B, sufficiency or deficiency, e.g., the saturability of aspartate and alanine aminotransferases with PLP, during pregnancy and in experimentally produced vitamin B, deficiency in human subjects.l”t lo In this study, comparison of the plasma PLP values of subjects at term with those of nonpregnant women of comparable ages indicates that a daily supplement of pyridoxine in excess of 2 to 2.5 mg. will be required to correct the altered vitamin B, nutrition during pregnancy in a majority of women. A similar conclusion was reached previously by
28
Cleary,
Lumeng,
and
Januarg 1. I!Fi Am. J. Ohstet. C+nrr
