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

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_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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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

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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

1. ROSE, D. P. The influence of oestrogens ontryptophan metabolism in man. Chin. Sci. 31:265, 1966.

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

erythrocyte aminotransferase levels in oral contra-ceptive users receiving controlled intakes ofvitamin B6 . Am. J. Chin. Nutr. 28: 10, 1975.

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.

10. PARK, Y. K., AND H. LINKSWILER. Effect ofvitamin B6 depletion in adult man on theexcretion of cystathionine and other methioninemetabolites. J. Nutr. 100: 110, 1970.

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:

786, 1971.13. ROSE, D. P., AND R. R. BROWN. The influence

of sex and estrogens on liver kynureninase andkynurenine aminotransferase in the rat. Biochim.Biophys. Acta 184: 412, 1969.

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.

15. SALKELD, R. M., K. KNORR AND W. F.KORNER. Ihe effect of oral contraceptives onvitamin B6 status. Chin. Chim. Acta 49: 195,1973.

16. ROSE, D. P., R. STRONG, P. W. ADAMS AND P.E. HARDING. Experimental vitamin B6 defi-ciency and the effect of oestrogen-containing oralcontraceptives on tryptophan metabolism andvitamin B6 requirements. Chin. Sci. 42: 465,1972.

17. SAUBERLICH, H. E., J. E. CANHAM, E. M.BAKER, N. RAICA, JR. AND Y. F. HERMAN.Biochemical assessment of the nutritional statusof vitamin B6 in the human. Am. J. Chin. Nutr.25: 629, 1972.

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.

19. ROSE, D. P., AND P. W. ADAMS. Oral contracep-tives and tryptophan metabolism: Effects ofoestrogen in low dose combined with a progesta-gen (megestrol acetate) given alone. J. Chin.Pathol. 25: 252, 1972.

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