Vitamin B6 requirements of women using

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 diet
supplemented 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 in
the contraceptive users. However, other indices, including urinary cystathionine (3.0 g

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L-methionine load), urinary 4-pyridoxic acid, plasma phosphate, and erythrocyte alanine and
aspartate aminotransferases, were not significantly different. Since altered tryptophan
metabolism persisted in contraceptive users even when other indices of vitamin B6 nutrition
were 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- indices of vitamin B6 nutrition measured were
traceptives excrete elevated amounts of trypto- reported previously (5). In brief, 9 healthy control
women (average age 22.3 1.9 years), and 15 women
phan metabolites after a tryptophan load test (23.2 3.1 years) who had used oral contraceptive
compared with women not taking oral contra- agents for at least 6 months were given a diet that
ceptives (1-3). Elevated levels of 3-hydroxy- contained only 0.19 mg of pyridoxine equivalents per
anthranilic acid were also observed in basal day (6). Oral contraceptive users started the diet on
day 1 1 of a 21-day pill sequence. Control subjects
urines from such subjects (4). When pyridoxine
started 14 days after onset of the previous menses. For
was administered to such subjects the excretion the first 4 days, while baseline studies were made, the
of tryptophan metabolites was decreased diet was supplemented daily with 0.8 mg of pyri-
toward normal levels, but in some subjects large doxine hydrochloride (PN-HC1). This supplement was
amounts of the vitamin may be required (3). then withdrawn and both groups of subjects consumed
only the deficient diet for 28 days. After this
These data have been widely interpreted as
depletion period, subjects were supplemented with
indicating that the use of oral contraceptive either 0.8, 2.0, or 20 mg/day of PN-HCI for another
drugs causes an increased requirement for 28 days. Before release from the study, subjects were
vitamin B6, although other explanations for the supplemented for a final 4 days with 100 mg
PN-HC1/day. During the baseline period, and at
altered tryptophan metabolism may be pos-
weekly intervals throughout the study, several indices
sible. To evaluate the effect of oral contracep- of vitamin B6 nutrition were measured in each subject.
tive usage on the requirement for vitamin B6, a The indices measured included urinary tryptophan
variety of indices of vitamin B6 nutrition were metabolites before and after a 2.0 g oral load of
measured to determine the rate at which L-tryptophan (7, 8), urinary cystathionine after a load
control and oral contraceptive using women
became depleted of this vitamin while ingesting From the Division of Clinical Oncology, Uni-
versity of Wisconsin Medical School and Department
a diet low in vitamin B6. The same indices were
of Nutritional Sciences, University of Wisconsin
used to measure the rate at which these subjects College of Agricultural and Life Sciences, Madison,
became repleted when supplemented with Wisconsin 53706.
physiological levels of pyridoxine. Supported in part by Wisconsin College of
Agricultural and Life Sciences, Contract NIH, HICHD
72-2782 with the National Institute of Child Health
Methods and materials
and Human Development, and Grant No. CA-13302
The experimental details of these studies con- from the National Cancer Institute, Public Health
cerning the selection of subjects, diets used, and Service, Bethesda, Maryland 20014.

The American Journal of Clinical Nutrition 28: MAY 1975, pp. 535-541. Printed in U.S.A. 535
536 LEKLEM El AL.

of 3.0 g of L-methionine (9, 10), urinary 4-pyridoxic At comparable times of depletion and at
acid (5), plasma pyridoxal phosphate (5), and erythro- equal levels of PN -HC1 supplementation, the
cyte activities of alanine arninotransferase and aspar-
mean excretion of tryptophan metabolites was
tate aminotransferase (5). In order to study metabo-
lism along the kynurenine pathway and to circumvent consistently greater than that of controls, and
any variations in activity of tryptophan oxygenase, suggests that the requirement for vitamin B6
loading doses of L-kynurenine sulfate (200 mg/dose) may be slightly greater in oral contraceptive
were given initially, at the time of maximum
users than in controls. However, it should be
deficiency, and after repletion with pyridoxine for 4
weeks. pointed out that because of large individual
variations and limited numbers of subjects,
Results many of these differences do not achieve
Measurement of urinary tryptophan metabo- statistical significance. Details of the excretion
lites after tryptophan loading prior to starting of individual tryptophan metabolites are pub-
the deficient diet showed that the oral contra- lished elsewhere (8).
ceptive users excreted elevated levels of kynure- The urinary excretion of cystathionine after
nine , acetylkynurenine , 3-hydroxykynurenine a 3.0 g oral load of L-methionine is shown in
and xanthurenic acid compared with the con- Fig. 2. The pattern is different from that of the

Downloaded from ajcn.nutrition.org by guest on September 15, 2015

trol subjects (8). These differences persisted tryptophan metabolite yield in that predeple-
and increased during the period of vitamin B6 tion and week 1 levels of cystathionine were
depletion. This is summarized in Fig. 1 which , similar in both groups. However, apparent
shows the yield of tryptophan metabolites differences occurred at later times, and during
excreted. During depletion, both groups of
subjects excreted increasingly large amounts of
metabolites, with the oral contraceptive users
I
excreting consistently larger amounts than the
controls. Supplementation of control subjects
a0
with 0.8 mg of PN-HC1/day dramatically C
reduced the excretion within 1 week, and by U)
I
the third week of repletion the excretion level I-
-J
had stabilized at essentially the predepletion 0

values. The oral contraceptive users supple-

Ii

mented with 0.8 mg of PN-HC1/day also

U.
0
exhibited a marked reduction in excretion of
0
-i
tryptophan metabolites. However, even after 4 Ui

weeks of supplementation, the excretion was

still slightly higher than the starting (predeple-
DLI OF STUDY
tion) values for these subjects and was +8,4- --8, -$4-------------+6, -,

markedly higher than the control subjects at

the same time. The low value observed in oral FIG. 1 . Yield of metabolites excreted after a 2.0 g
contraceptive users at day 47 and the inflection tryptophan load by control subjects and oral contra-
at day 19 were a consistent fmding which captive users (O.C.). During the first 4 days the
deficient diet (-B6) was supplemented with 0.8 mg
coincided with the 7-day period during which
pyridoxine hydrochloride. During the second +B6
oral contraceptive use was interrupted. period the diets were supplemented with the number
Supplementation of the control subjects with of milligrams of pyridoxine hydrochloride indicated in
2.0 mg of PN-HC1 daily restored tryptophan the figure. The shaded bars below the figure designate
metabolism to normal ranges within 1 to 2 the 7-day period when oral contraceptive use was
discontinued. There were 9 control subjects and 15
weeks. Excretion by oral contraceptive users
contraceptive users. During the repletion period 6
was also promptly decreased by daily supple- controls were supplemented with 0.8 mg and 3 were
ments of 2.0 mg of PN-HC1, with excretion given 2.0 mg of pyridoxine hydrochloride. Of the oral
values considerably lower than the predepletion contraceptive users, 5 were supplemented with 0.8 mg,
6 with 2.0 mg, and 4 with 20 mg. The metabolites
values for these subjects, but stifi not at control
included in the yield value were kynurenine, N-acetyl
levels. Supplementation of oral contraceptive kynurenine, 3-hydroxykynurenine, anthraniic acid
users with 20 mg/day promptly restored excre- glucuronide, o-aminohippuric acid, kynurenic acid,
tion to control levels. and xanthurenic acid.
 VITAMIN B6 REQUIREMENTS IN ORAL CONTRACEPTIVE USERS 537

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
I

C.,J
both groups (5).
To evaluate metabolic effects of oral contra-
Ui
-J
0 ceptive use on the tryptophan metabolic path-
way independent of any effects on the activity
Ui
z of tryptophan oxygenase, 200 mg loads of
z L-kynurenine sulfate monohydrate were given
0
I before deficiency, at peak deficiency, and after
4I-
pyridoxine supplementation. The excretions of
U)

C., kynurenine , 3-hydroxykynurenine , acetylkyn-

urenine, and xanthurenic acid are shown in Fig.
4
z 5. Excretions of kynurenine and 3-hydroxykyn-
urenine were slightly higher in the oral contra-

Downloaded from ajcn.nutrition.org by guest on September 15, 2015

ceptive group than in controls at all three times
studied and were highest in both groups when
at the peak of deficiency. Excretions of
2 3 4 5 6 7 8
acetylkynurenine and xanthurenic acid were
WEEK OF STUDY essentially unchanged. Because of sizable indi-
-B, +8,
4- .#
vidual variations, none of these differences were
FIG.2. Urinary excretion of cystathionine after a significant but they suggest that the use of oral
3.0 g oral load of L-methionine in oral contraceptive
contraceptives has an effect on tryptophan
users (O.C.) and control women. During the -B6
period the diet contained the equivalent of 0.19 mg of
metabolism at one or more steps beyond
pyridoxine. During the +B6 period the diet was kynurenine in addition to any effects they may
supplemented daily with 0.8, 2.0, or 20 mg of have on tryptophan oxygenase activity.
pyridoxine hydrochloride. Cystathionine was Activity of erythrocyte alanine aminotrans-
measured by an amino acid analyzer (modified
Beckman model 1 20) using a modified buffer gradient
system (9).

CONTROLS O.C.
the first 2 weeks of repletion with PN-HC1 the 0.8 #{149}- 0.8 0--- .7
#{149} 2.0 #{163}- 2.0 0---
excretion by oral contraceptive users remained
above that of controls at comparable times.
.5

Again, because of wide variations between

individuals and because of the small number of
0
subjects, these differences were not of signifi- 0 .3

cance statistically. >-

-----
Excretion of urinary 4-pyridoxic acid de-
creased at similar rates in both groups of - ------- - i

subjects (Fig. 3) and repletion rates during

9 25 3 39 47 53 59
supplementation with PNHCl were also quite DAY OF STUDY
similar. These data suggest that use of oral +8, -B6 +8

contraceptives has no measurable effect on

absorption, utilization or conversion of pyri- FIG. 3. Urinary excretion of 4-pyridoxic acid by
control subjects and oral contraceptive users (O.C.).
doxine to 4-pyridoxic acid (5).
During the first 4 days, the deficient diet (-B6) was
Plasma pyridoxal phosphate (PLP) levels of supplemented with 0.8 mg of PN#{149}HC1.During the
oral contraceptive users were slightly lower -B6 period, the basal diet containing 0.19 mg
than control values initially and remained equivalents of pyridoxine was not supplemented with
slightly lower throughout the depletion period B6. This diet was supplemented in the +B6 period
with 0.8 or 2.0 mg PN.HC1/day. The shaded bars
(Fig. 4). During repletion with PN-HC1 no
indicate the 7-day period each month when oral
differences between groups were found. Supple- contraceptive use was interrupted. Pyridoxic acid was
ments of 0.8 mg PN-HC1/day were not enough assayed as previously described (7).
538 LEKLEM El AL.

-
6.- PLP 1 and controls were found initially and activity of
CONTROLS OC. both groups decreased similarly during the
0.8 0---
0.8.-
2.0- 20 0---
depletion period. Daily supplements of 0.8 mg
I 2 , of PN-HC1 slowly increased the activity but not
a.
U)
0 to predepletion levels. With 2.0-mg supple-
i0-
a. ments, the activity of both groups had risen to
8
approximately predepletion levels. The final
x0
0 6-
points shown were after 3 or 4 days of
supplementation with 100 mg PNHC1/day.
0. 4-

4
This level of supplement resulted in normal or
In2 supernormal levels.
4,
-J
a. _L.- L i The above erythrocyte preparations were
0 2 3 4 5 6
7 8 also assayed after fortification in vitro with
WEEK OF STUDY
-
4---

FIG. 4. Concentration
- ---Be

of plasma
-
pyridoxal
-64

phos-
- -, saturating
stimulation
progressed,
levels

and
of PLP (Fig.
by PLP increased
did so to
6). The percent

extent the in
as the
same
deficiency

Downloaded from ajcn.nutrition.org by guest on September 15, 2015

phate (PLP) in controls and oral contraceptive users. controls and oral contraceptive During users.
Footnotes are the same as in Fig. 3. PLP was assayed deficiency, in vitro stimulation by PLP did not
with tyrosine apodecarboxylase using L-tyrosine-
restore activity to predepletion levels, suggest-
1- 4C as substrate. ing that the decreased activity was due, in part,
YNB1 HK--XR to loss of apoenzyme.
with PN- HC1 again
Dietary
decreased
supplementation
the percent
stimulation with no consistent differences be-
tween comparable groups.
Similar unstimulated and PLP-stimulated as-
CONTROL
says were done for erythrocyte aspartate

q 23

23 23 23 23

Period of Study

FIG. 5. Excretion of kynurenine (KYN), acetyl-

kynurenine (AK), 3-hydroxykynurenine (HK), and
xanthurenic acid (XA) by control subjects and oral
contraceptive users (O.C.) (given an oral load of 200
mg of L-kynurenine sulfate) prior to vitamin B6
2 3 4 5 6 7 8
depletion (period 1), at the peak of deficiency (period
2) and after repletion with pyridoxine (period 3). The WEEK OF STUDY

abbreviated metabolic pathway serves to identity the FIG. 6.

The upper figure shows changes in
metabolites in each group of bars. Metabolites were erytkrocyte alanine aminotransferase basal activity
measured as previously described (7). (without addition of PLP in vitro) in controls (CONT.)
and oral contraceptive users (O.C.) during vitamin B6
ferase (not fortified with PLP in vitro) at
depletion and repletion. The daily supplements of
PN-HC1 (in mg) are shown on each curve. The lower
weekly intervals throughout the study is shown
figure shows the percent stimulation observed in the
in Fig. 6. Details were reported elsewhere (5). activity of this enzyme when saturated in vitro by
No differences between oral contraceptive users added PLP.
 VITAMIN B6 REQUIREMENTS IN ORAL CONTRACEPTIVE USERS 539

aminotransferase , and the findings paralleled which time all other indices were within normal
those of the alanine enzyme although the control ranges. This suggests that the oral
relative decreases induced by vitamin B6 de- contraceptives may have some relatively spe-
ficiency were not as great. Details of these cific effect on the metabolism of tryptophan
studies have been presented elsewhere (5). which is independent of vitamin B6 levels. This
effect may be primarily on the activity of
Discussion tryptophan oxygenase, since studies in rats have
The excretion of tryptophan metabolites by shown direct as well as adrenal-mediated effects
oral contraceptive users, after the tryptophan of estrogens on the activity of this enzyme
load test, was significantly different from ( 11). However, the present observations in
similarly loaded controls prior to induction of subjects given small kynurenine loads, which
vitamin B6 deficiency.This confirmed previous bypass any tryptophan oxygenase effects, sug-
observations of altered tryptophan metabolism gest that the usage of contraceptive hormones
in oral contraceptive users (1-3). Other indices also may have an effect elsewhere in the
of vitamin B6 nutrition measured before vita- pathway of kynurenine metabolism. Previous
mm B6 depletion were not clearly different in studies (12, 13) indicate that steroids or steroid

Downloaded from ajcn.nutrition.org by guest on September 15, 2015

oral contraceptive users. Thus, urinary cysta- conjugates can affect the activity of the
thionine and urinary 4-pyridoxic acid were not PLP-dependent enzymes of the kynurenine
different from controls; and plasma PlY and pathway, i.e., kynureninase and kynurenine
the erythrocyte aminotransferases, while aminotransferase. Thus, it seems most likely
slightly lower in oral contraceptive users, were that the altered tryptophan metabolism of oral
not statistically different. When the rates of contraceptive users is the summation of hormo-
change of these indices were compared during nal effects on tryptophan oxygenase and
dietary depletion of vitamin B6 in both groups, kynureninase rather than the production of a
the excretion of tryptophan metabolites and general vitamin B6 deficiency. This interpreta-
cystathionine suggested that the oral contra- tion is in agreement with the recent report by
ceptive group might be depleting at a slightly Lumeng et al. (14) in which urinary xan-
faster rate than the controls. Other indices, thurenic acid and plasma PLP levels were
including 4-pyridoxic acid excretion, plasma compared in oral contraceptive users and
Ply, and erythrocyte aminotransferases controls. They found that xanthurenic acid
changed at virtually the same rate in both excretion was elevated in most contraceptive
groups during depletion and reached the same users even though plasma PlY levels were, in
nadir at the time of maximum deficiency. most cases, not correspondingly low. However,
Supplementation with pyridoxine (0.8 in contrast to the present study, they reported
mg/day) resulted in very similar restoration that about 20% of oral contraceptive users in
rates of plasma PLP, erythrocyte alanine amino- the 20- to 34-year-age range had subnormal
transferase and urinary 4-pyridoxic acid How- . levels of plasma PLP. Further, plasma PLY
ever, correction of urinary excretion of cysta- levels decreased in subjects during the first
thionine and tryptophan metabolites was months of oral contraceptive use but tended
slightly slower in the oral contraceptive group, toward normal with continued usage. Since no
although not significantly so. Similarly, reple- dietary information was presented, the possi-
tion rates between groups supplemented with biity of contraceptive-induced changes in diet
2.0 mg of PNHCl were not significantly must be considered. Salkeld et a!. (15) found
different. The data clearly indicate that a daily the erythrocyte aspartate aminotransferase
supplement of 0.8 mg of PN-HC1 for 28 days is stimulation test to be abnormal in almost 50%
not enough to replete either controls or oral of oral contraceptive users, but observed no
contraceptive users. However, 2.0 mg/day for 4 effect of duration of contraceptive usage on
weeks restored all indices in both groups to this index. Rose et al. (16) found low urinary
their respective predepletion levels and, in some 4-pyridoxic acid levels and increased hydroxy-
cases, to ultranormal levels. kynurenine-to-hydroxyanthranilate ratios in
The detailed data (8) show that the excretion about 19% of oral contraceptive users although
of tryptophan metabolites by the oral contra- the mean 4-pyridoxic acid value for the whole
ceptive group was stifi elevated above control group was not significantly different from
values after supplementation with 2.0 mg, at controls.
540 LEKLEM El AL.

The activities of erythrocyte aminotrans- 3.0 g load of L-methionine), urinary 4-pyri-

ferases, particularly the PLY activation thereof, doxic acid, plasma pyridoxal phosphate, and
are considered reliable indices of vitamin B6 erythrocyte alanine and aspartate aminotrans-
nutrition (17). In the present study these ferases. In addition, 200 mg oral loads of
indices changed as expected with the induction L-kynurenine sulfate were given initially, at the
of a vitamin B6 deficiency, but there were no peak of deficiency, and after pyridoxine reple-
consistent or significant differences between tion.
control subjects and oral contraceptive users. No significant differences were observed
Rose et al. (18) found an increased in vitro PLY between oral contraceptive users and controls
stimulation of erythrocyte alanine aminotrans- in the above measured indices with the excep-
ferase in about 1 5% of contraceptive users. tion of the tryptophan load test, although very
The brief review of the literature presented minor differences occurred in several of the
above indicates that ample evidence exists to indices. The data suggest that the use of oral
suggest that the use of estrogen-containing oral contraceptives may specifically affect trypto-
contraceptives may produce a vitamin B6 phan metabolism by some means other than
deficiency in certain subjects. In the present through a vitamin B6 deficiency, since altered

Downloaded from ajcn.nutrition.org by guest on September 15, 2015

study in which precise dietary control was tryptophan metabolism persisted even when
maintained, it was not possible (with the other indices of vitamin B6 nutrition were
exception of the tryptophan load test) to normal. The amount of vitamin B6 (as pyri-
demonstrate clear-cut differences in vitamin B6 doxine) needed to maintain normal levels of
requirements between control subjects and oral these indices (except for tryptophan metabo-
contraceptive users when a variety of indices of lism) was between 0.8 and 2.0 mg/day. The
vitamin B6 nutrition were measured. Perhaps data suggest that if the use of oral contracep-
because of the limited number of subjects tives of the combined estrogen-progestogen
, we might not have obtained experi- type does alter the requirement for vitamin B6,
mental subjects susceptible to contraceptive- the effect is a minor one and of doubtful
induced vitamin B6 deficiency. Perhaps with a clinical significance to the majority of women
much larger number of subjects some of the taking these steroid preparations. LI
minor differences would have reached statistical
We gratefully acknowledge the competent and
significance. However, it is possible to say that dedicated technical assistance of Joan Chesters, Jane
the use of oral contraceptives per se does not Mueller, Rekha Anand, Pat Stauber, Heidi Kan, and
significantly or consistently increase the re- Richard Arend throughout the conduct of this study
and of Kay Deighton and Suzi Pertzborn for assistance
quirement for vitamin B6 in the majority of
in preparation of the manuscript.
women using these agents. However, a small
subgroup of women may well exist who are
particularly susceptible to steroid-induced vita- References
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metabolic abnormalities may result (19). tryptophan metabolism in man. Chin. Sci. 31:
265, 1966.
2. PRICE, J. M., M. J. THORNTON AND L. M.
Summary MUELLER. Tryptophan metabolism in women
Fifteen women who used estrogen-containing using steroid hormones for ovulation control. Am.
J. Chin. Nutr. 20: 452, 1967.
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who had never used these agents were given a DAVIS, M. MURPHY AND H. SPIEGEL. Vitamin
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equivalent of 0.19 mg of pyridoxine/day). agents. I. Abnormal urinary xanthurenic acid
excretion and its correction by pyridoxine. Am. J.
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Chin. Nutr. 24: 684, 1971.
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hydrochloride for an additional 4 weeks. LAND. Effects of dietary vitamin B, deficiency
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were made of several indices of vitamin B6
Am. J. Chin. Nutr. 25: 494, 1972.
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tabolites (before and after a 2.0 g load of LINKSWILER AND R. ANAND. Urinary 4-pyri-
L-tryptophan), urinary cystathionine (after a doxic acid, plasma pyridoxal phosphate, and
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erythrocyte aminotransferase levels in oral contra- 786, 1971.

ceptive users receiving controlled intakes of 13. ROSE, D. P., AND R. R. BROWN. The influence
vitamin B6 . Am. J. Chin. Nutr. 28: 10, 1975. of sex and estrogens on liver kynureninase and
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