Vitamin B6 requirements of women using
The American Journal of Clinical Nutrition 28: MAY 1975, pp. 535-541. Printed in U.S.A. 535
oral contraceptive&’ 2
J. E. Lekiem, Ph.D., R. R. Brown, Ph.D., D. P. Rose, M.D., Ph.D.,
and H. M Linkswiler, Ph.D.
ABSTRACT Fifteen women who used combined estrogen-progestogen oral contraceptives
and nine control women were given a vitamin B6 -deficient diet for 4 weeks and the same dietsupplemented with 0.8, 2.0, or 20.0 mg of pyridoxine hydrochloride for an additional 4 weeks.
At weekly intervals a variety of indices of vitamin B6 nutrition were measured to determine
rates of depletion and repletion. The tryptophan load test (2.0 g) was significantly different inthe contraceptive users. However, other indices, including urinary cystathionine (3.0 g
L-methionine load), urinary 4-pyridoxic acid, plasma phosphate, and erythrocyte alanine and
aspartate aminotransferases, were not significantly different. Since altered tryptophanmetabolism persisted in contraceptive users even when other indices of vitamin B6 nutritionwere normal, we suggest that the use of oral contraceptives specifically affects tryptophan
metabolism by some means other than through a vitamin B6 deficiency. Am. J. Clin. Nutr.
28: 535-541, 1975.
Women using estrogen-containing oral con-traceptives excrete elevated amounts of trypto-
phan metabolites after a tryptophan load testcompared with women not taking oral contra-ceptives (1-3). Elevated levels of 3-hydroxy-anthranilic acid were also observed in basal
urines from such subjects (4). When pyridoxinewas administered to such subjects the excretionof tryptophan metabolites was decreasedtoward normal levels, but in some subjects largeamounts of the vitamin may be required (3).These data have been widely interpreted asindicating that the use of oral contraceptive
drugs causes an increased requirement for
vitamin B6, although other explanations for the
altered tryptophan metabolism may be pos-sible. To evaluate the effect of oral contracep-
tive usage on the requirement for vitamin B6, a
variety of indices of vitamin B6 nutrition were
measured to determine the rate at which
control and oral contraceptive using womenbecame depleted of this vitamin while ingesting
a diet low in vitamin B6. The same indices wereused to measure the rate at which these subjects
became repleted when supplemented with
physiological levels of pyridoxine.
Methods and materials
The experimental details of these studies con-cerning the selection of subjects, diets used, and
indices of vitamin B6 nutrition measured werereported previously (5). In brief, 9 healthy controlwomen (average age 22.3 ± 1.9 years), and 15 women(23.2 ± 3.1 years) who had used oral contraceptiveagents for at least 6 months were given a diet thatcontained only 0.19 mg of pyridoxine equivalents perday (6). Oral contraceptive users started the diet onday 1 1 of a 21-day pill sequence. Control subjectsstarted 14 days after onset of the previous menses. Forthe first 4 days, while baseline studies were made, thediet was supplemented daily with 0.8 mg of pyri-doxine hydrochloride (PN-HC1). This supplement wasthen withdrawn and both groups of subjects consumedonly the deficient diet for 28 days. After thisdepletion period, subjects were supplemented witheither 0.8, 2.0, or 20 mg/day of PN-HCI for another28 days. Before release from the study, subjects weresupplemented for a final 4 days with 100 mgPN-HC1/day. During the baseline period, and atweekly intervals throughout the study, several indicesof vitamin B6 nutrition were measured in each subject.The indices measured included urinary tryptophanmetabolites before and after a 2.0 g oral load ofL-tryptophan (7, 8), urinary cystathionine after a load
‘From the Division of Clinical Oncology, Uni-versity of Wisconsin Medical School and Departmentof Nutritional Sciences, University of WisconsinCollege of Agricultural and Life Sciences, Madison,Wisconsin 53706.
� Supported in part by Wisconsin College ofAgricultural and Life Sciences, Contract NIH, HICHD72-2782 with the National Institute of Child Healthand Human Development, and Grant No. CA-13302from the National Cancer Institute, Public HealthService, Bethesda, Maryland 20014.
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LEKLEM El AL.
At comparable times of depletion and at
equal levels of PN -HC1 supplementation, the
mean excretion of tryptophan metabolites was
consistently greater than that of controls, andsuggests that the requirement for vitamin B6
may be slightly greater in oral contraceptiveusers than in controls. However, it should be
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of 3.0 g of L-methionine (9, 10), urinary 4-pyridoxicacid (5), plasma pyridoxal phosphate (5), and erythro-cyte activities of alanine arninotransferase and aspar-tate aminotransferase (5). In order to study metabo-lism along the kynurenine pathway and to circumventany variations in activity of tryptophan oxygenase,loading doses of L-kynurenine sulfate (200 mg/dose)were given initially, at the time of maximumdeficiency, and after repletion with pyridoxine for 4weeks.
Results
Measurement of urinary tryptophan metabo-lites after tryptophan loading prior to starting
the deficient diet showed that the oral contra-
ceptive users excreted elevated levels of kynure-nine , acetylkynurenine , 3-hydroxykynurenineand xanthurenic acid compared with the con-trol subjects (8). These differences persisted
and increased during the period of vitamin B6depletion. This is summarized in Fig. 1 , which
shows the yield of tryptophan metabolitesexcreted. During depletion, both groups of
subjects excreted increasingly large amounts of
metabolites, with the oral contraceptive users
excreting consistently larger amounts than the
controls. Supplementation of control subjects
with 0.8 mg of PN-HC1/day dramatically
reduced the excretion within 1 week, and by
the third week of repletion the excretion level
had stabilized at essentially the predepletion
values. The oral contraceptive users supple-
mented with 0.8 mg of PN-HC1/day alsoexhibited a marked reduction in excretion of
tryptophan metabolites. However, even after 4weeks of supplementation, the excretion was
still slightly higher than the starting (predeple-
tion) values for these subjects and was
markedly higher than the control subjects at
the same time. The low value observed in oral
contraceptive users at day 47 and the inflection
at day 19 were a consistent fmding whichcoincided with the 7-day period during whichoral contraceptive use was interrupted.
Supplementation of the control subjects with2.0 mg of PN-HC1 daily restored tryptophan
metabolism to normal ranges within 1 to 2
weeks. Excretion by oral contraceptive userswas also promptly decreased by daily supple-
ments of 2.0 mg of PN-HC1, with excretion
values considerably lower than the predepletion
values for these subjects, but stifi not at control
levels. Supplementation of oral contraceptive
users with 20 mg/day promptly restored excre-
tion to control levels.
pointed out that because of large individualvariations and limited numbers of subjects,
many of these differences do not achieve
statistical significance. Details of the excretion
of individual tryptophan metabolites are pub-
lished elsewhere (8).The urinary excretion of cystathionine after
a 3.0 g oral load of L-methionine is shown in
Fig. 2. The pattern is different from that of thetryptophan metabolite yield in that predeple-
tion and week 1 levels of cystathionine were
similar in both groups. However, apparent
differences occurred at later times, and during
FIG. 1 . Yield of metabolites excreted after a 2.0 g
tryptophan load by control subjects and oral contra-captive users (O.C.). During the first 4 days thedeficient diet (-B6) was supplemented with 0.8 mgpyridoxine hydrochloride. During the second +B6period the diets were supplemented with the numberof milligrams of pyridoxine hydrochloride indicated inthe figure. The shaded bars below the figure designatethe 7-day period when oral contraceptive use wasdiscontinued. There were 9 control subjects and 15contraceptive users. During the repletion period 6controls were supplemented with 0.8 mg and 3 weregiven 2.0 mg of pyridoxine hydrochloride. Of the oralcontraceptive users, 5 were supplemented with 0.8 mg,6 with 2.0 mg, and 4 with 20 mg. The metabolitesincluded in the yield value were kynurenine, N’-acetylkynurenine, 3-hydroxykynurenine, anthraniic acidglucuronide, o-aminohippuric acid, kynurenic acid,and xanthurenic acid.
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VITAMIN B6 REQUIREMENTS IN ORAL CONTRACEPTIVE USERS 537
FIG. 2. Urinary excretion of cystathionine after a3.0 g oral load of L-methionine in oral contraceptiveusers (O.C.) and control women. During the -B6period the diet contained the equivalent of 0.19 mg ofpyridoxine. During the +B6 period the diet wassupplemented daily with 0.8, 2.0, or 20 mg ofpyridoxine hydrochloride. Cystathionine wasmeasured by an amino acid analyzer (modifiedBeckman model 1 20) using a modified buffer gradientsystem (9).
the first 2 weeks of repletion with PN-HC1 theexcretion by oral contraceptive users remained
above that of controls at comparable times.Again, because of wide variations betweenindividuals and because of the small number of
subjects, these differences were not of signifi-cance statistically.
Excretion of urinary 4-pyridoxic acid de-
creased at similar rates in both groups of
subjects (Fig. 3) and repletion rates duringsupplementation with PN�HCl were also quite
similar. These data suggest that use of oral
contraceptives has no measurable effect onabsorption, utilization or conversion of pyri-doxine to 4-pyridoxic acid (5).
Plasma pyridoxal phosphate (PLP) levels of
oral contraceptive users were slightly lowerthan control values initially and remainedslightly lower throughout the depletion period(Fig. 4). During repletion with PN-HC1 no
differences between groups were found. Supple-ments of 0.8 mg PN-HC1/day were not enough
to restore PLP values to predepletion levels in
either group. However, supplements of 2.0
mg/day for 2 and 3 weeks resulted in PLP
levels appreciably higher than starting values in
both groups (5).
To evaluate metabolic effects of oral contra-
ceptive use on the tryptophan metabolic path-
way independent of any effects on the activity
of tryptophan oxygenase, 200 mg loads of
L-kynurenine sulfate monohydrate were given
before deficiency, at peak deficiency, and after
pyridoxine supplementation. The excretions ofkynurenine , 3-hydroxykynurenine , acetylkyn-urenine, and xanthurenic acid are shown in Fig.
5. Excretions of kynurenine and 3-hydroxykyn-
urenine were slightly higher in the oral contra-
ceptive group than in controls at all three times
studied and were highest in both groups when
at the peak of deficiency. Excretions of
acetylkynurenine and xanthurenic acid were
essentially unchanged. Because of sizable indi-vidual variations, none of these differences were
significant but they suggest that the use of oral
contraceptives has an effect on tryptophan
metabolism at one or more steps beyondkynurenine in addition to any effects they may
have on tryptophan oxygenase activity.
Activity of erythrocyte alanine aminotrans-
CONTROLS O.C.
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FIG. 3. Urinary excretion of 4-pyridoxic acid bycontrol subjects and oral contraceptive users (O.C.).During the first 4 days, the deficient diet (-B6) wassupplemented with 0.8 mg of PN#{149}HC1.During the-B6 period, the basal diet containing 0.19 mgequivalents of pyridoxine was not supplemented withB6. This diet was supplemented in the +B6 periodwith 0.8 or 2.0 mg PN.HC1/day. The shaded barsindicate the 7-day period each month when oralcontraceptive use was interrupted. Pyridoxic acid wasassayed as previously described (7).
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1 and controls were found initially and activity of� both groups decreased similarly during the
� depletion period. Daily supplements of 0.8 mg
, of PN-HC1 slowly increased the activity but not
� to predepletion levels. With 2.0-mg supple-
ments, the activity of both groups had risen to
� approximately predepletion levels. The final
� points shown were after 3 or 4 days of
� supplementation with 100 mg PNHC1/day.
� This level of supplement resulted in normal or
� supernormal levels.
_L.��- L �i The above erythrocyte preparations were7 8 also assayed after fortification in vitro with
- -, saturating levels of PLP (Fig. 6). The percentstimulation by PLP increased as the deficiency
progressed, and did so to the same extent in
controls and oral contraceptive users. Duringdeficiency, in vitro stimulation by PLP did notrestore activity to predepletion levels, suggest-
ing that the decreased activity was due, in part,to loss of apoenzyme. Dietary supplementationwith PN- HC1 again decreased the percent
stimulation with no consistent differences be-tween comparable groups.
Similar unstimulated and PLP-stimulated as-says were done for erythrocyte aspartate
CONTROL
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2 6 7 8
538 LEKLEM El AL.
3 4 5 6
WEEK OF STUDY
4--- ---Be - -64 --FIG. 4. Concentration of plasma pyridoxal phos-
phate (PLP) in controls and oral contraceptive users.Footnotes are the same as in Fig. 3. PLP was assayedwith tyrosine apodecarboxylase using L-tyrosine-
1-’ 4C as substrate.
YN�B1�� HK-�-�XR
‘23 23 23 23
Period of Study
FIG. 5. Excretion of kynurenine (KYN), acetyl-kynurenine (AK), 3-hydroxykynurenine (HK), andxanthurenic acid (XA) by control subjects and oralcontraceptive users (O.C.) (given an oral load of 200mg of L-kynurenine sulfate) prior to vitamin B6depletion (period 1), at the peak of deficiency (period2) and after repletion with pyridoxine (period 3). Theabbreviated metabolic pathway serves to identity themetabolites in each group of bars. Metabolites weremeasured as previously described (7).
ferase (not fortified with PLP in vitro) at
weekly intervals throughout the study is shown
in Fig. 6. Details were reported elsewhere (5).No differences between oral contraceptive users
3 4 5
WEEK OF STUDY
FIG. 6. The upper figure shows changes inerytkrocyte alanine aminotransferase basal activity(without addition of PLP in vitro) in controls (CONT.)and oral contraceptive users (O.C.) during vitamin B6depletion and repletion. The daily supplements ofPN-HC1 (in mg) are shown on each curve. The lowerfigure shows the percent stimulation observed in theactivity of this enzyme when saturated in vitro byadded PLP.
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VITAMIN B6 REQUIREMENTS IN ORAL CONTRACEPTIVE USERS 539
aminotransferase , and the findings paralleled
those of the alanine enzyme although therelative decreases induced by vitamin B6 de-
ficiency were not as great. Details of thesestudies have been presented elsewhere (5).
Discussion
The excretion of tryptophan metabolites byoral contraceptive users, after the tryptophan
load test, was significantly different fromsimilarly loaded controls prior to induction of
vitamin B6 deficiency. This confirmed previous
observations of altered tryptophan metabolismin oral contraceptive users (1-3). Other indicesof vitamin B6 nutrition measured before vita-
mm B6 depletion were not clearly different inoral contraceptive users. Thus, urinary cysta-
thionine and urinary 4-pyridoxic acid were notdifferent from controls; and plasma PlY and
the erythrocyte aminotransferases, while
slightly lower in oral contraceptive users, were
not statistically different. When the rates ofchange of these indices were compared during
dietary depletion of vitamin B6 in both groups,the excretion of tryptophan metabolites and
cystathionine suggested that the oral contra-ceptive group might be depleting at a slightlyfaster rate than the controls. Other indices,
including 4-pyridoxic acid excretion, plasmaPly, and erythrocyte aminotransferases
changed at virtually the same rate in both
groups during depletion and reached the samenadir at the time of maximum deficiency.
Supplementation with pyridoxine (0.8mg/day) resulted in very similar restorationrates of plasma PLP, erythrocyte alanine amino-
transferase and urinary 4-pyridoxic acid . How-ever, correction of urinary excretion of cysta-thionine and tryptophan metabolites was
slightly slower in the oral contraceptive group,although not significantly so. Similarly, reple-
tion rates between groups supplemented with2.0 mg of PN�HCl were not significantlydifferent. The data clearly indicate that a daily
supplement of 0.8 mg of PN-HC1 for 28 days isnot enough to replete either controls or oral
contraceptive users. However, 2.0 mg/day for 4
weeks restored all indices in both groups totheir respective predepletion levels and, in some
cases, to ultranormal levels.
The detailed data (8) show that the excretion
of tryptophan metabolites by the oral contra-
ceptive group was stifi elevated above controlvalues after supplementation with 2.0 mg, at
which time all other indices were within normal
control ranges. This suggests that the oral
contraceptives may have some relatively spe-
cific effect on the metabolism of tryptophanwhich is independent of vitamin B6 levels. This
effect may be primarily on the activity of
tryptophan oxygenase, since studies in rats have
shown direct as well as adrenal-mediated effectsof estrogens on the activity of this enzyme
(11). However, the present observations in
subjects given small kynurenine loads, which
bypass any tryptophan oxygenase effects, sug-
gest that the usage of contraceptive hormones
also may have an effect elsewhere in the
pathway of kynurenine metabolism. Previous
studies (12, 13) indicate that steroids or steroid
conjugates can affect the activity of thePLP-dependent enzymes of the kynurenine
pathway, i.e., kynureninase and kynurenine
aminotransferase. Thus, it seems most likely
that the altered tryptophan metabolism of oral
contraceptive users is the summation of hormo-
nal effects on tryptophan oxygenase and
kynureninase rather than the production of a
general vitamin B6 deficiency. This interpreta-
tion is in agreement with the recent report by
Lumeng et al. (14) in which urinary xan-thurenic acid and plasma PLP levels were
compared in oral contraceptive users and
controls. They found that xanthurenic acid
excretion was elevated in most contraceptiveusers even though plasma PlY levels were, in
most cases, not correspondingly low. However,
in contrast to the present study, they reported
that about 20% of oral contraceptive users inthe 20- to 34-year-age range had subnormal
levels of plasma PLP. Further, plasma PLY
levels decreased in subjects during the first
months of oral contraceptive use but tended
toward normal with continued usage. Since no
dietary information was presented, the possi-
biity of contraceptive-induced changes in diet
must be considered. Salkeld et a!. (15) foundthe erythrocyte aspartate aminotransferase
stimulation test to be abnormal in almost 50%
of oral contraceptive users, but observed noeffect of duration of contraceptive usage on
this index. Rose et al. (16) found low urinary
4-pyridoxic acid levels and increased hydroxy-
kynurenine-to-hydroxyanthranilate ratios in
about 19% of oral contraceptive users althoughthe mean 4-pyridoxic acid value for the whole
group was not significantly different from
controls.
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LEKLEM El AL.
3.0 g load of L-methionine), urinary 4-pyri-doxic acid, plasma pyridoxal phosphate, and
erythrocyte alanine and aspartate aminotrans-
ferases. In addition, 200 mg oral loads of
L-kynurenine sulfate were given initially, at the
peak of deficiency, and after pyridoxine reple-
tion.No significant differences were observed
540
The activities of erythrocyte aminotrans-ferases, particularly the PLY activation thereof,
are considered reliable indices of vitamin B6
nutrition (17). In the present study these
indices changed as expected with the induction
of a vitamin B6 deficiency, but there were noconsistent or significant differences betweencontrol subjects and oral contraceptive users.
Rose et al. (18) found an increased in vitro PLY
stimulation of erythrocyte alanine aminotrans-ferase in about 1 5% of contraceptive users.
The brief review of the literature presented
above indicates that ample evidence exists tosuggest that the use of estrogen-containing oral
contraceptives may produce a vitamin B6deficiency in certain subjects. In the present
study in which precise dietary control was
maintained, it was not possible (with the
exception of the tryptophan load test) to
demonstrate clear-cut differences in vitamin B6requirements between control subjects and oral
contraceptive users when a variety of indices of
vitamin B6 nutrition were measured. Perhaps
because of the limited number of subjects, we might not have obtained experi-
mental subjects susceptible to contraceptive-
induced vitamin B6 deficiency. Perhaps with amuch larger number of subjects some of the
minor differences would have reached statistical
significance. However, it is possible to say that
the use of oral contraceptives per se does not
significantly or consistently increase the re-
quirement for vitamin B6 in the majority ofwomen using these agents. However, a small
subgroup of women may well exist who are
particularly susceptible to steroid-induced vita-
min B6 deficiency, and in these women other
metabolic abnormalities may result (19).
Summary
Fifteen women who used estrogen-containingoral contraceptives and nine control women
who had never used these agents were given adiet deficient in vitamin B6 (containing theequivalent of 0.19 mg of pyridoxine/day).
After 4 weeks, this diet was supplemented dailywith 0.8, 2.0 or 20.0 mg of pyridoxine
hydrochloride for an additional 4 weeks.
Initially, and at weekly intervals, measurementswere made of several indices of vitamin B6
nutrition, including urinary tryptophan me-
tabolites (before and after a 2.0 g load of
L-tryptophan), urinary cystathionine (after a
between oral contraceptive users and controls
in the above measured indices with the excep-tion of the tryptophan load test, although veryminor differences occurred in several of theindices. The data suggest that the use of oralcontraceptives may specifically affect trypto-phan metabolism by some means other thanthrough a vitamin B6 deficiency, since alteredtryptophan metabolism persisted even whenother indices of vitamin B6 nutrition were
normal. The amount of vitamin B6 (as pyri-doxine) needed to maintain normal levels ofthese indices (except for tryptophan metabo-lism) was between 0.8 and 2.0 mg/day. Thedata suggest that if the use of oral contracep-tives of the combined estrogen-progestogen
type does alter the requirement for vitamin B6,
the effect is a minor one and of doubtful
clinical significance to the majority of women
taking these steroid preparations. LI
We gratefully acknowledge the competent anddedicated technical assistance of Joan Chesters, JaneMueller, Rekha Anand, Pat Stauber, Heidi Kan, andRichard Arend throughout the conduct of this studyand of Kay Deighton and Suzi Pertzborn for assistancein preparation of the manuscript.
References
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2. PRICE, J. M., M. J. THORNTON AND L. M.MUELLER. Tryptophan metabolism in womenusing steroid hormones for ovulation control. Am.J.Chin. Nutr. 20: 452, 1967.
3. LUHBY, A. L., M. BRIN, M. GORDON, P.DAVIS, M. MURPHY AND H. SPIEGEL. VitaminB6 metabolism in users of oral contraceptiveagents. I. Abnormal urinary xanthurenic acidexcretion and its correction by pyridoxine. Am. J.Chin. Nutr. 24: 684, 1971.
4. PRICE, S. A., D. P. ROSE AND P. A. lOSE-LAND. Effects of dietary vitamin B, deficiencyand oral contraceptives on the spontaneousurinary excretion of 3-hydroxyanthraniic acid.Am. J. Chin. Nutr. 25: 494, 1972.
5. BROWN, R. R., D. P. ROSE, J. E. LEKLEM, H.LINKSWILER AND R. ANAND. Urinary 4-pyri-doxic acid, plasma pyridoxal phosphate, and
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VITAMIN B6 REQUIREMENTS IN ORAL CONTRACEPTIVE USERS 541
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6. SWAN, P., J. WENTWORTH AND H. LINK-SWILER. Vitamin B6 depletion in man: Urinarytaurine and sulfate excretion and nitrogen bal-ance. J. Nutr. 84: 220, 1964.
7. PRICE, J. M., R. R. BROWN AND N. YESS.Testing the functional capacity of the tryptophan-niacin pathway in man by analysis of urinarymetabohites. In: Advances in Metabolic Disorders,edited by R. Levine and R. Luft. New York:Academic, 1965, vol. 2, p. 159.
8. LEKLEM, J. E., R. R. BROWN, D. P. ROSE, H.LINKSWILER AND R. A. AREND. Metabolismof tryptophan and niacin in oral contraceptiveusers receiving controlled intakes of vitamin B6.Am. J. Chin. Nutr. 28: 146, 1975.
9. THOMSON, A. R., AND B. J. MILES. Ion-ex-change chromatography of amino acids: Improve-ments in the single column system. Nature 203:483, 1964.
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11. BRAIDMAN, I. P., AND D. P. ROSE. Effects ofsex hormones on three glucocorticoid-inducibleenzymes concerned with amino acid metabolismin rat liver. Endocrinology 89: 1250, 1971.
12. MASON, M., AND B. MANNING. Effects ofsteroid conjugates on availability of pyridoxalphosphate for kynureninase and kynurenineaminotransferase activity. Am. J. Chin. Nutr. 24:
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14. LUMENG, L., R. E. CLEARY AND 1.-K. LI.Effect of oral contraceptives on the plasmaconcentration of pyridoxal phosphate. Am. J.Cliri. Nutr. 27: 326, 1974.
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18. ROSE, D. P., R. STRONG, J. FOLKARD AND P.W. ADAMS. Erythrocyte aminotransferase activ-ities in women using oral contraceptives and theeffect of vitamin B6 supplementation. Am. J.Chin. Nutr. 26: 48, 1973.
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