B6 requirement
in oral contraceptive
I. Assessment by pyridoxal level and tra n sfe rase activity in e ryth rocytes1’2 Teresa
R. Boss#{233},3B.Sc.,
M.Sc.
and
Elizabeth
A. Donald,4
B.Sc.,
M.S.,
Ph.D.
ABSTRACT Eight college-age women using estrogen-containing oral contraceptives (OC) were fed a low vitamin B6 diet (0.36 mg/day) for 42 days. During the first 10 days (adjustment period) the diet was supplemented with 1.7 mg pyridoxine hydrochloride bringing the total intake to 2.06 mg/day. Following depletion, repletion was done in three consecutive steps: intakes of 0.96, 1.56, and 5.06 mg were consumed for 8, 9, and 7 days, respectively. Continuous 24-hr urine collections were made throughout the study and fasting blood samples were drawn periodically. Vitamin B6 nutriture was assessed by erythrocyte pyridoxal level, erythrocyte alanine aminotransferase and erythrocyte aspartic aminotransferase activity; and stimulation of these enzyme systems with pyridoxal phosphate. Results were compared with data obtained from non-OC users who consumed a similar diet. The data obtained suggest that 0.96 mg vitamin B6 was not adequate to meet the needs of OC users. Predepletion levels had been reached in almost all subjects at an intake of 1.5 mg/day. Assessed by the parameters studied, an intake between 1.5 and 5.0 mg/day of vitamin B6 was adequate to meet the needs of OC users; this compares with 1.5 mg/day previously suggested for the nonuser. Am. J. Clin. Nutr. 32: 1015-1023, 1979.
The vitamin B6 requirement of women using estrogen-progestogen-containing oral contraceptives (OC) has been principally studied by measuring the changes occurring in the urinary excretion of tryptophan metabolites. Twenty to 40 mg vitamin B6 daily will correct such abnormalities (1-3). Salkeld et al. (4) pointed out, however, that whereas 80 to 100% of OC users are known to exhibit abnormal excretion of tryptophan metabolites following a load dose of tryptophan, only 50% of his sample showed evidence of reduced aspartic aminotransferase activity (EAsp-AT) in erythrocytes. Recently, Brown et al. (5) and Leklem et al. (6), using a multiparameter assessment of vitamin B6 needs, found that 2.0 mg vitamin B6 restored plasma pyridoxal phosphate (PLP) levels and alanine (E-Ala-AT) and aspartic (E-Asp-AT) aminotransferase activities to initial levels. Other parameters were measured in urine and all, except the xanthurenic acid (XA) level, were restored. They concluded that the use of estrogen-progestogen-containing OC only mildly affect the requirement for vitamin B6 and could only be considered of doubtful clinical significance. The American
Journal
of Clinical
Nutrition
32: MAY
The study reported here was designed to use a multiparamater approach to assess the vitamin B6 nutritional status of young women using OC; to determine whether they require more vitamin B6 than nonusers, and if the need is greater to establish what this requirement might be. A description of subjects, diet, and methods used; and requirement as assessed by erythrocyte pyridoxal level (E-PL) and E-Asp-AT and E-Ala-AT activities are included in this report. Results obtained from measuring levels of tryptophan metabolites, total vitamin B6 and pyridoxic acid in urine are described elsewhere (7). Materials
and methods
Subjects Eight
young
women
using
OC served
mental
group and another eight, not using controls. Volunteers were selected from college-age population. Each subject was plete medical examination by a physician ‘From
the Faculty
of Home
Economics,
as the experi-
OC, served as an interested given a comto ensure that University
of Alberta, Edmonton, Alberta, Canada, T6G 2M8. 2Supported in part by a grant from the Medical Research Council, Ottawa, 3Sessional Lecturer.
1979, pp. 1015-1023.
Printed
Canada. 4Professor.
in U.S.A.
1015
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The vitamin users
BOSSE
1016
AND
Oral contraceptives Four of the eight experimental subjects had used OvraI5 for 1, 6, 7, and 13 months; three had used either Norinyl-l,’ Orthonovum l/2 or Orthonovum l/50 for 3, 12, or 25 months, respectively; and one subject had used a sequential-type, Norquen,5 for 14 months. Diet Food used in the basal diet supplied energy, protein, most of the needed vitamins and minerals. The diet similar to one used in a previous study (8). The food served as three meals that were weighed, prepared, eaten in the metabolic unit. When necessary a meal packed and consumed at home. The basal diet provided 1650 kcal, 65 g protein, 670 mg calcium, 1.9 mg macin, 4290 IU vitamin A, and 73 mg ascorbic acid per day. Nutrient content was calculated using USDA Handbook no. 8 (9). Calcium, iron, thiamin, and riboflavin were supplemented in capsule form so that the total intake of these nutrients met the 1968 Recommended Dietary Allowances (RDA) (10). The diet contained 0.36 mg vitamin B6. It was similar to one used in a previous study which had been analyzed to contain 0.34 mg vitamin B6 per day (8). The original diet was modified by the addition of two extra slices of bread and 20 g apply jelly per day thus increasing the vitamin B6 content to 0.36 mg. Butter, sugar, shortbread cookies, candy, and gingerale were consumed ad libitum to provide energy for maintenance of initial body weight. Each subject kept a daily record of amounts eaten so that total energy intake could be calculated. and was was and was
Experimental
design
The study consisted of three phases. The first 10 days constituted the predepletion phase, when both experimental and control subjects consumed the basal diet plus a pyridoxine hydrochloride (PN-HCI) supplement of 1.7 mg/day. This brought the total daily intake of the vitamin to 2.06 mg, thus meeting the adult R.DA for vitamin B6 (10). Only the experimental subjects underwent the depletion and repletion phases. The depletion phase began on day 11 when the vitamin B6 supplement of 1.7 mg/day was withdrawn, thus resulting in a net daily intake of 0.36 mg. Depletion continued for 32 days at which time the repletion phase began. On days 43, 51, and 60 the basal diet was supplemented with 0.6, 1.2, and 4.7 mg of PN-HC1, respectively, for total daily intakes of the vitamin of 0.96, 1.56, and 5.06 mg for 8, 9, and 7 days, respectively. Metabolic
Results Prestudy
samples of approximately 14 ml each from experimental subjects by veni-
physical
and
clinical
analyses
The prestudy physical and clinical examinations showed all subjects to be in good health. Hemoglobin levels for the control and experimental groups averaged 14.2 ± 0.3 and 13.2 ± 0.3 g/lOO ml, respectively. Serum AspAT activity levels used to assess liver function were normal and urinary glucose was negative. Hemoglobin
and
hematocrit
Hemoglobin and hematocrit levels in the experimental group were determined after 1, 10, 17, 24, 31, 38, 42, 49, 58, and 65 days on the study; and after 1 and 9 days on the study in the control group. The mean hemoglobin level in the control subjects was 14.8 g/lOO ml compared with 14.2 g/lOO ml for experimental subjects. The vitamin B6 level in the diet had little effect on hemoglobin levels for mean values ranged from 13.4 to 14.7 g/lOO ml throughout the study. The mean hematocrit levels in control and experimental subjects were 41.8 and 40.4 mi/l#{174} ml, respec5Ovral contained 0.05 mg n-norgestrel, Norinyl-l: norethindrone;
studies
Fasting blood were withdrawn
puncture after 1, 10, 17, 24, 31, 38, 42, 49, 58, and 65 days on the study and from control subjects after 1 and 9 days. Vacutainer tubes containing 0.07 ml of 15% EDTA were used. Duplicate analysis for microhematocrit and hemoglobin were done on fresh whole blood. Plasma and erythrocytes were separated by centrifugation. The determination of E-PL involved extraction of the vitamin from tissue, chromatographic separation of B6 VItamiflS, followed by microbiological assay of PL. The methods of Toepfer and Lehmann (1 1) and a modification of the microbiological procedure of Atkin Ct al. (12) were used. The determination of E-Ala-AT activity was based on the colorimetric procedure of Heddle et al. (13) for whole blood and modified for erythrocytes by Woodring and Storvick (14). In vitro percent stimulation by PLP was determined according to Woodring and Storvick (14). E-Asp-AT activity was determined spectrophotometrically using a premeasured kit (Boehringer Mannheim Company). The procedure followed was sireilar to that described by Donald et aL (8). The PLP stimulated activity of the enzyme systems were determined according to the method of Cheney et al. (15). Urine was collected and analyzed for several tryptophan metabolites, vitamin B6, and 4-pyridoxic acid (7).
Orthonovum
mg ethinyl estradidiol 0.05 mg mestranol 1/2:
0.50mg norethindrone; Orthonovum tranol + 1.00 mg norethindrone; mestranol + 2.0 mg norethindrone.
0.10
mg
+
+ 0.25 1.00mg
mestranol
1/50:0.05 Norquen:
+
mg mes0.08 mg
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they were in good physical health, had normal menstrual histories and liver function. Informed consent was obtamed from each participating subject. Subjects ranged in age from 18 to 23 years with mean ages of 20 and 21 years for the experimental and control subjects, respectively. Body weight of the experimental subjects ranged from 54.5 to 69.5 kg with a mean of 63.2 kg, whereas control subjects ranged from 56.8 to 68.2 kg with a mean of 61.4 kg.
DONALD
VITAMIN
B6 REQUIREMENT
IN OC USERS
tively. tuated
Like hemoglobin levels, they flucvery little, with a range of 39.0 to 41.4 mi/i#{174} ml for experimental subjects. Both hemoglobin and hematocrit values obtained
study
compared
favorably
with
obtained in a similar study conducted adult women who were not using OC Pyridoxal
levels
e’
I
those with (8).
28.7
spectively
rl.440.H
in erythrocytes
and
(Table
32.2
ng/ml
1). These
pkd levels
cells,
re-
were
not
significantly different from each other. Withdrawal of the PN-HC1 supplement from cxperimental subjects caused a significant decrease in E-PL from 28.7 to 14.0 ng/ml pkd cells (P < 0.001) (Table 1). By the end of the depletion phase, only 49% of the original EPL level was retained. While an intake of 0.96 mg vitamin B6 in the first repletion phase partially restored the E-PL level to the predepletion level, an intake of 2.06 mg in the second phase produced a mean level of 29.9 ng/ml pkd cells, similar to the predepletion level of 28.7 ng/ml pkd cells. Consumption of 5.06 mg vitamin B6 per day resulted in a significant increase (P < 0.025) in E-PL to 39.2 ng/ml pkd cells. At the end of the third repletion phase mean E-PL was 136% of the predepletion level. E-PL levels in OC users were compared with levels found in subjects eating a similar diet, but not using OC (8). E-PL in the present study was determined using Saccharomyces carisbergensis as the test organism whereas results reported previously (8) were determined using Tetrahymena pyrformis. Values found in the latter study were much higher than ones reported in the literature or in the present study. To compare the results of the two studies more efficiently, the E-PL level at the
beginning
considered in subsequent cent
change
as
H
O.O000.O.O
When subjects consumed the supplemented diet containing 2.06 mg vitamin B6 per day for the 9 days comprising the predepletion phase, E-PL in OC users and nonusers
averaged
00
C
of the
100 and
periods from this
depletion
amounts were value
phase
r1H+l
I -0
(4
00
“
.E
(6
‘6r
Fa, a
(6
41
C
F-
ri
.< (6
(6
4
0
00
o
000
re,
(4
0-.
4.
.
O
0
‘1.
‘t
41
0-
00-
o -ri
.
00-.0000
4
N
r’
41 a4 a4
was
determined
calculated (Fig. 1).
as per-
Although the degree of depletion achieved with OC users was not as great as that achieved with nonusers (8), the rates of change, as reflected by the slopes of the curves illustrated in Figure 1, are similar in both studies. Restoration of E-PL to a similar
a 0
.
0
..2
DpL),L)
o .
c c;I
. ‘U
z ‘U
0 z 4 I
Us.rs
OC
0
#{149}#{149}Non Us.rs
z ‘U
$ Vitamin
0
So
Intak.,
mglday
‘U
a-
30 DAYS
FIG.
1. Effect
of
vitamin
B6 intake
on
ON percent
change
degree required a greater intake of vitamin B6 in OC users than in nonusers. To reach 135% of the undepleted mean value required vitamin intakes of 0.96, 1.56, and 5.06 mg for 8, 9, and 7 days, respectively, in OC users, whereas, to reach 136% of the undepleted value in nonusers required 0.94, 1.54, and
in E-PL
50
40
DPLtTION-REP1ET
ION
concentration
60
DIET
in users
and
nonusers
(8)
of OC.
The fmal degree of depletion in nonusers was also much more severe as mean E-PL levels had dropped to 14% of the predepletion level before repletion began, whereas, repletion began when levels had decreased to only 49% of predepletion values in OC users.
0.00 1). At the height of depletion E-Ala-AT activity was 45% of the mean undepleted level. During the three phases of repletion, enzyme activity increased from 61.7 to 129.6 sg pyruvate per milliliter pkd RBC/hr (Table 1, Fig. 2). Mean E-Ala-AT activity was increased to 94% of the initial activity with individual values restored to original levels in four of the eight subjects. Although the mean E-Ala-AT activity on the last day of the study was still below the predepletion value the difference between the two means was not significant.
E-Ala-A
E-Asp-A
1.62
mg
When
for
7, 3, and
11 days,
respectively.
T
both users and nonusers of OC consumed 2.06 mg of the vitamin for 10 and 9 days, respectively, the amount of Ala-AT present in the red blood cell (RBC) was 138.0 and 104.0 ng pyruvate per milliliter pkd RBC/hr, respectively. These levels were not significantly different from each other. Consumption of a diet containing 0.36 mg of the vitamin for 32 days by subjects using OC caused a gradual decrease in enzyme levels from the predepletion level of 138.0 to 61.7 g pyruvate per milliliter pkd RBC/hr (Table 1, Fig. 2). This change was significant (P
VITAMIN
B6 REQUIREMENT VITAMIN
I
I
2.06
B6
IN OC USERS
INTAKE,
I
0.36
MG/
DAY
0.96
I
1.56
1019
I
5.06
‘I)
a. a.
‘U
0
0
z
(U
0 U 4
5-
a-
4
(U
5-
5-
Sn
4 >
5-
z(U a.
U
0
(U
a.
DAYS
FIG.
2. Effect with in vitro PLP
of of
vitamin eight
B6 intake adult
young
OC users for vitamin B6. Rose reported random fluctuations activity. In vitro stimulation
of enzyme
E-AIa-AT women using on
ON
activity OC.
et al. (3) also in E-Asp-AT
systems
PLP was added, in vitro, to erythrocyte samples from experimental subjects to stimulate the activity of both E-Ala-AT and EAsp-AT systems. Results are reported in Table 1 and Figures 2 and 3. Percent stimulation of E-Ala-AT found during the predepletion phase for OC users were similar to values reported by others for normal subjects (3, 14-17). Overall, values for percentage stimulation gradually increased during the depletion period (Fig. 2). The amount of stimulation obtained at the height of depletion was significantly different (P < 0.05) from the amount on the last day of the predepletion period. The increase in stimulation would indicate that although erythrocyte levels of the coenzyme were diminishing, as evidenced by decreasing basal activity of EAla-AT, the level of apoenzyme remained unchanged. Supplementation of the basal diet with vitamin B6 caused a decrease (P < 0.05) in percentage stimulation (Fig. 2) to a level
DIET
(mean
that
± SE) and percentage
was
not
significantly
stimulation
(broken
different
than
line)
levels
found in the predepletion phase. Only four of six subjects however, achieved values equal to or below predepletion levels. These findings indicate that E-Ala-AT is sensitive to a dietary deficiency of vitamin B6 but is slow to respond to in vivo supplementation. In vitro stimulation of E-Asp-AT with PLP produced fluctuations that corresponded to the unstimulated enzyme activity; where EAsp-AT declined, the percent stimulation increased (Fig. 3). As with unstimulated E-AspAT activity, PLP stimulation of this enzyme system showed no consistent response to level of vitamin B6 intake. Discussion To determine the requirement for a particular nutrient the basal or normal level of that nutrient or some parameter that reflects its status is established, body stores of the nutrient are depleted and the body is then repleted with known levels until the basal level is again reached. This classical method was the general procedure followed in the present study.
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I
1020
BOSSE VITAMIN
2.06
I
AND 6
DONALD
INTAKE,
MG/DAY
I 0.96 I
0.36
1.56
I
5.06
I
a. a.
‘U
U
0
z
(U
0 U
5-
4 a.
4
0
5Sn I-
z
0 4
(U
U
z
‘U
Sn
a.
(U
0
FIG. 3. Effect of vitamin B6 intake PLP of eight adult women using OC.
on
E-Asp-AT
DAYS ON DIET activity (mean
The mean E-PL level in OC users was 28.7 ng/ml pkd cells which, although slightly lower, was not significantly different from 32.2 ng/ml pkd cells found in nonusers. This relationship of PL in users and nonusers was similar to that reported by Miller et al. (18) for vitamin B6 levels in whole blood; 7.7 versus 6.0 ng/ml, respectively, and Brown et al. (5) for plasma; 11.7 and 9.2 ng/ml, respectively. These findings indicate that lower blood levels of the vitamin are found in those using OC, even though the difference was not significantly less. E-PL levels decreased with depletion and gradually increased with supplementation (Table 1). Intakes of 0.96 mg of PN-HC1 for 7 days resulted in an increase to 74% of initial levels; 1.56 mg PN-HC1 for 8 days restored predepletion levels in five of eight subjects and increased mean levels to 104% of predepletion values. Levels were not restored in the three subjects who had the highest predepletion values. This delay in achieving predepletion levels could not be attributed to the type of OC used or the duration of use. An intake of 5.06 mg vitamin B6 for 6 days restored EPL levels in all subjects to at least predepletion levels so that the mean level was 136% of the undepleted concentration. Comparison of these results with results
± SE) and percentage
stimulation
with
in vitro
obtained in a similar study with young women not using OC (8) indicate that a comparatively higher level of PN-HC1 was required to increase E-PL concentrations of OC users
to the
same
degree
as nonusers
(Fig.
1).
Each group was followed over 21 days of repletion. Comparing the total response to repletion, E-PL concentrations in OC users responded with less sensitivity. A total intake of approximately 50 mg vitamin B6 by users, resulted in an increase from 14.0 to 39.2 ng/ ml pkd cells, whereas, an intake of 30 mg increased E-PL from 46 to 454 ng/ml pkd cells in nonusers. These represented increases of 180 and 887% over the mean depleted values for users and nonusers, respectively. These observations could reflect an increased requirement for vitamin B6 by OC users. Based on the criterion of complete restoration of E-PL
to starting
values
after
depletion
of
erythrocyte stores of vitamin B6, this requirement would be between 1.5 and 5 mg vitamin B6 per day, compared to the requirement of 1.5 mg/day for normal young women (8). These results agree with those obtained by Brown et al. (5) who measured PLP in plasma rather than E-PL. They found that supplementation with 2.0 mg for 2 weeks restored plasma levels in both OC users and nonusers to starting values but OC users showed no
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Sn
VITAMIN
further
increase
with
B6 REQUIREMENT
2 additional
supplementation, whereas, to increase in nonusers.
weeks
levels
of
continued
activity decreased to 45% of initial activity in OC users during depletion and increased to a mean of 47% of initial activity after 7 days of vitamin B6 intake at 0.96 mg daily.
Mean
activity
increased
to 77%
of the
initial level with 1.56 mg vitamin B6 per day for 8 days. None of the individual values reached respective initial levels. An intake of 5.06 mg daily resulted in 94% of the mean initial activity being reached with individual values restored in four of eight subjects. Using
E-Ala-AT
activity
B6 level close OC users. Slowness enzyme
as criterion,
to 5.0 mg would in restoration have been
a vitamin
be needed
by
of original levels reported by others
of
in non-OC users (19, 20). Results from the present study do not agree with results from nonusers following the same regimen (8). In that study, levels of E-Ala-AT did not reflect dietary changes. Different analytical methods to assess E-Ala-AT were used in each study this
and
could
account
for
the
differing
results.
The rate of change in E-Ala-AT activity during depletion and repletion phases could be influenced by the amount of alanine present for transamination. Rose et al. (21) showed that plasma alanine levels were similar in users and nonusers of OC, however, plasma levels were significantly lower in OC users after administration of a load-dose of alanine. Although the alanine level in the blood was not assessed in this study, based on Rose’s
findings,
a deficiency
of this
amino
acid would not be expected in OC users. Slow restoration of E-Ala-AT activity to predepletion levels by in vivo PN-HC1 suggests that the decreased activity produced by vitamin B6 deprivation might be due to apoenzyme depletion, since it is known that apoenzyme synthesis is vitamin B6 dependent (22). If a deficiency of apoenzyme did exist, addition of the coenzyme in vitro as PLP would produce little if any additional enzymatic activity. In the present study, however, addition of 50 g PLP to an in vitro system resulted in an increasing percent stimulation as the
deficiency
progressive
decline
that
the
due
presumably
decreasing
progressed,
with
followed
repletion,
by
indicating
E-Ala-AT
activity
was
to deficiency
of the
coen-
a
zyme only (Fig. and final values
2). Although for percent
not significantly subjects achieved
different, values
the mean stimulation only equal
four to or
initial were of six below
their predepletion levels. E-Ala-AT is sensitive to a dietary deficiency of vitamin B6 but is slow to respond to in vivo supplementation. These data suggest that some adult women using OC may require an amount of vitamin B6 in excess of that required by normal adult women. The lack of in vivo response of E-Ala-AT activity during repletion might be caused by redistribution of the coenzyme to some other enzyme system thus leaving insufficient amounts for the E-Ala-AT system. Brin and Thiele (23) suggested that Asp-AT apoenzyme in rat tissues and plasma has a greater affinity for vitamin B6 than does Ala-AT. Cavil and Jacobs (24) found that human EAsp-AT combined more strongly with the coenzyme while E-Ala-AT appeared to be more sensitive to vitamin B6 deficiency. Erythrocytes from subjects studies by Cinnamon and Beaton (20) also appeared to contain Asp-AT possessing a greater affinity for the coenzyme than did Ala-AT. E-Asp-AT activity in OC users showed no systematic response to dietary deprivation or repletion with vitamin B6. This enzyme system has also been found by others (8, 25) to be relatively unresponsive. This lack of response may be due to a strong affinity by the enzyme for the coenzyme. In some studies (3, 17, 26) a significantly higher basal activity level was found in OC users than nonusers. In vitro stimulation of the E-Asp-AT system with PLP caused changes in activity, similar in amount but opposite in direction, to
the
others
nonstimulated
(20,25)
have
responses.
found
Although
that changes in the stimulated activity could be used to assess vitamin B6 nutriture, in the present study neither changes in the nonstimulated nor stimulated activities were reliable indicators. Some of the first reports (1, 27) concerning the effect of OC on the requirement for vitamin B6 suggested routine administration of 30 to 50 mg vitamin B6 per day to OC users. Later studies by Woodring and Storvick (14) and Rose et al. (17) found that oral supplementation of 40 to 50 mg/day of PN-HC1 to OC users caused an increase in E-Ala-AT and E-Asp-AT activity. Rose et al. (17) cx-
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E-Ala-AT
1021
IN OC USERS
1022
BOSSE
AND
pressed
The authors gratefully acknowledge the technical assistance of Barbara Lowell-Davis, Carol Williamson, and Lois Labelle. The cooperation of the subjects is greatly appreciated, for without them, this study could not have been done.
References 1. LUHBY,
A. L., M. AND H.
BRIN,
M.
P.
GORDON,
M.
DAvis,
Vitamin B6 metabolism in users of oral contraceptive agents. I. Abnormal urinary xanthurenic acid excretion and its correction by pyridoxine. Am. J. Cliii. Nutr. 24: 684, 1971. MURPHY
2. Piuca.,
Effects
S.
SPIEGEL.
A., D. P. Rosa
AND
P. A.
TO5ELAND.
vitamin B6 deficiency and oral contraceptives on the spontaneous urinary excretion of 3-hydroxy-anthranillic acid. Am. J. Clin. Nutr. 25: 494, 1972. 3.
of
dietary
RosE, D. P., R.
STRONG,
P. W.
ADAMS
AND
P.
E.
Experimental vitamin B6 deficiency and the effect of oestrogen containing oral contraceptives on tryptophan metabolism and vitamin B6 requirements. Clin. Sci. 42: 465, 1972. HARDING.
4.
5.
7.
DONALD, E. A., AND 1. R. Bosst. The vitamin B6 requirement in oral contraceptive users: II. Assessment by tryptophan metabolites, vitamin Be and pyridoxic acid levels in urine. Am. 3. Clin. Nutr. 32: 1024, 1979.
8.
DONALD,
acid
Suw, R. M., K. KNORR AND W. R. KORNER. The effect of oral contraceptives on vitamin B6 status. Cliii. Chiin. Acta 49: 195, 1973. Bitoww, R. R., D. P. Rosa, J. E. Laiaas, H. LINKSWILER AND R. ANAND. Urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythrocyte ami-
notransferase levels in oral contraceptive users receiving controlled intakes of vitamin Be. Am. 3. Cliii. Nutr. 28: 10, 1975. 6. LEKLEM, 3. E., R. R. BRowN, D. P. Rosa, H. LINKSWILER AND R. A. AaaND. Metabolism of tryptophan and niacin in oral contraceptive users receiving controlled intakes of vitamin B6. Am. 3. Clin. Nutr. 28: 146, 1975.
M. F. Swi
E. A., L. D. MCBEAN, M. H. W. SIMPSON, AND H. E. ALy. Vitamin B. requirement
of young adult women. Am. J. Clin. Nutr. 24: 1028, 1971. 9. WAn, B. K., AND A. L. Maiuuu, Composition of Foods, Agriculture Handbook no. 8, Washington, D.C.: United States Department of Agriculture, 1963. 10. National Research CounciL Recommended Dietary Allowances (rev. ed. 7). Washington, D.C.: National Academy of Sciences, 1968, pubL no. 1694. I 1. Toama, E. W., AND 3. LEHMANN. Procedure for chromatographic separation and microbiological assay of pyridoxine, pyridoxal, and pyridoxamine in food extracts. 3. Am. Assoc. Agric. Chem. 44: 426, 1961. 12. ATKIN, A., A. S. SCHULTZ, W. L. WIwAM5 AND C. N. Faay. Yeast microbiological methods for the determination ofvitamins. md. Eng. Chem. 15: 141, 1943. 13. HEDDLE, 3. G., E. W. MCHENRY AND 0. H. BEATON. Penicillamine and vitamin B6 interrelationships in the rat. Can. 3. Biochem. Physiol. 41: 1215, 1963. 14. WOODRING, M. J., AND C. A. STORVICK. Effect of pyridoxine supplementation on glutamic-pyruvic transamunase and in vitro stimulation in erythrocytes ofnormal women. Am. 3. Cliii. Nutr. 23: 1385, 1970. 15. CHENEY, M., Z. I. SABRY AND 0. H. BEATON. Erythrocyte glutamic-pyruvic transaminase activity in man. Am. J. Cliii. Nutr. 16: 337, 1965. 16. SAuBERUcH, H. E., J. E. CANHAM, E. M. BAKER, N. R.AICA AND R. H. HEI1J.IAN. Biochemical assessment of the nutritional status of vitamin B. in the human. Am. J. Clin. Nutr. 25: 629, 1972. 17. RosE, D. P., R. STRONG, J. FouD AND P. W. ADAMS. Erythrocyte aminotransferase activities in women using oral contraceptives and the effect of vitamin B6 supplementation. Am. 3. Clin. Nutr. 26: 48, 1973. 18. Miu.na, L. T., E. M. BENSON, M. A. EDWARDS AND J. YOUNG. Vitamin Re metabolism in women using oral contraceptives. Am. 3. Cliii. Nutr. 27: 797, 1974. 19. BAYSAL, A., B. A. JOHNSON AND H. LINK5WILER. Vitamin B6 depletion in man: blood vitamin B., plasma pyridoxal-phosphate, serum cholesterol, serum transaminases and urinary vitamin B. and 4pyridoxic acid. J. Nutr. 89: 19, 1966. 20. CINNAMON, A. D., AND 3. R. BEATON. Biochemical assessment of vitamin B. status in man. Am. 3. Clin. Nutr. 23: 696, 1970. 21. Rosa, D. P., 3. E. LEKLEM, R. R. BROWN AND C. Pomn.. Effect of oral contraceptives and vitamin B. supplements on ahanine and glycine metabolism. Am. 3. Clin. Nutr. 29: 956, 1976. 22. OREENGARD, 0., AND M. GORDON. The co-factor mediated regulation of apoenzyme levels in animal tissues. 3. Biol. Chem. 238: 3708, 1963. 23. BRDi, M., AND V. F. THIELE. Relationships between vitamin B55-vitamer content and the activities of two transamunase enzymes in rat tissues at varying intake
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concern that cofactor induction of catabolizing enzymes could result in low plasma levels of amino acids in OC users, thus, if PN-HC1 supplementation was necessary, the lowest level needed to meet vitamin B6 requirements would be desirable. However, Rose et al. (21) have recently reported that administration of 25 mg/day of PN-HC1 for several weeks did not decrease plasma alanine levels. Routine supplementation of OC users with high levels of PN-HC1 does not seem to be needed. Based on results of PL levels and Ala-AT activity in erythrocytes found in this study the vitamin B6 need for OC users would be between 1.5 and 5 mg/day, likely closer to 1.5 than 5 mg as restoration of original levels has been achieved in most subjects with vitamin B6 intakes of 1.5 mg. Nonusers needed 1.5 mg/day, as previously reported (8). These amounts can be easily met in the usual North American diet. U amino
DONALD
VITAMIN
B. REQUIREMENT
ciency. 26.
ALY,
1023
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15: 67, 1964. AND
Oral contraceptives and vitamin 3. Cliii. Nutr. 24: 297, 1971. 27.
TOSELAND,
oral
P. A.,
contraceptives.
M. H. W.
SIMPSON.
B, metabolism.
S. A. PRICE. Tryptophan Brit. Med. J. 1: 777, 1969.
AND
Am. and
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levels of vitamin B.. 3. Nutr. 93: 213, 1967. 24. CAVILL, I. A. J., AND A. JAcoBs. Erythrocyte transaminase activity in iron deficiency anemia. Scand. 3. HaematoL 4: 249, 1967. 25. RAICA, N., JR., AND H. E. SAUBERLICH. Blood cell transaminase activity in human vitamin B. defi-
IN OC USERS