T HE ABNORMAL TRYPTOPHAN METABOLISM

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1 TIlE AMERICAN JOURNAL OF CLINICAL NUTRITION Vol. 20, No. 5, May, 1967, pp Prinled in U.S.A. Tryptophan Metabolism in Women Using Steroid Hormones for Ovulation Control J. M. PRICE,3 M.D., PH.D., MADELINE J. THORNTON, M.D., AND Lots M. MUELLER T HE ABNORMAL TRYPTOPHAN METABOLISM associateti with pregnancy has been witlely studied since 1951 when Sprince et al. (1) ailti Vantlelli (2) m-eported the cxcietion of abnormally large amounts of xanthuienic aciti by pregnant women. Vandelli s study (2), and those of Wachstein anti Gudaitis (3) and Wachstein anti Lobeh (4), showed that vitamin B6 would correct this metabolic abnormality in )regnant women. Tile excretion of several tryptophan metabohites by pregnant women before and after tryptophan loading was reported by Brown et al. (5) who found that tile atiministration of a multiple vitamin preparation had no effect on the abnormal tryptophan metabolism of the pregnant women in their study unless vitamin B6 was included in the supplementation. The latter investigators found that 6 mg of vitamin B6 in tile form of pyridoxine hydrochloritle failed to completely correct tile abnormal tryptophan metabolism of pregnant women although thlis quantity of pyrithoxine probably is From Division of Clinical Oncology aimd Dcpartument of Gynecology and Obstetrics, University of Wisconsin Medical School, Madisolm, Wisconsin This research was in part I)y Public 2 supported Health Service Grant AM from the National Immstitute of Arthritis almd Metabolic Diseases, by the American Cancer Society, and by time Elsa U. Pardee Foundatiolm. Americalm Cammcer Society-Charles S. Hayden Foummdation Professor of Stmrgery in Cancer Research. considerably more than the daily Iequirement for atlumlt human subjects (6). Brown et al. (5) pointetl out that althioughi vitamum B6 would partially commcct thus abnormal pa ttcin of urinary tiy)topha ii metal)olites, the faihmre of the abnot-mal pattel-il to be comiupletely corrected by the adnh nistnation of pymitloxine sumpplemen ts and tile observation of a slightly different pattern of urinary tiyptophan metabolites from that seemi in a dietary deficiency of vitamin B11 suggested thlat there might be en(locri lid factors associa teti wi th pregnancy which were partially responsible for this (hisol(ier of tmyptopiuan nictabolism. Further evitlence that tl-y)to)han metal)- ohism in women is affected by hom-mones associated withu 1eh)rotltlction was obtamed when it was observed that iii nonjregntmit controls the amount of huydroxykyn urenine excreted was significantly less iliiuilediately after menstruation thlan the amount exci-eted at the anticipated time of ovulation or just prior to the time of menstruation (5). These results suggested that there may be an interrelationship between the female hlol-mones associateti with reproduction and the role of vitamin B11 in the metabolism of tryptophan. There is considerable cvitlence for an intcrm-elationsllip between steroid hormones, particularly the cortical steroids, anti enzyme activity (7). Although them-c is much literature concerning the interrelationship of hormones anti vitamin 452

2 Tryptophan Metabolism and Steroid Hormones 453 B6 in mctabolisnu (8), there is relatively little tiata to relate vitamin B6 specifically with female hormones. Nelson et al. (9) found that pyridoxine-deficient rats which usually failed to carry a litter to parturition could be induced to do so in about 90% of the cases by the injection of 1 g of estrone and 4 mg of progesterone but tlot by either hormone separately. They Pb0h)5etI tiltt theii results indicated an inadequacy C)f the secretion of the ovarian hormones in pyridoxine deficiency. A later study, which showed that pitui tary gonadotropins maintained pregnancy in about 25% of pyridoxine-deficient anirnals, led Nelson et al. (10) to suggest that dysfumiction of both the pituitary and the ovary is involved in the hormonal madequacies of vitamin B6-tieficient animals. Recently, Rose (1 1) found that young womemu wilo were using a combination of progesterone and estrogen for ovulation control had a very abnornual xanthurenic aci(l excretion after a loading dose of tryptophan. The results ieported here are an extension of the work reported by Rose. MATERIALS AND METHODS Ten subjects were selected for study as outpatients. These young women were found to be in good general health, and the steroid hormones were prescribed solely for ovulation control. The subjects ingested 2.5 mg of norethynodrel and 0.1 ing of mestranol (Enovid-E)4 from the 5th day of the menstrual cycle through the 24th day. Most of tile subjects had taken the drug for several cycles at the time of study. The subjects collected a 24-hr urine specimen before and after the administration of 2 g of L- tryptophan. They were then given 25 mg of pyridoxine hydrocilloritie four times daily during the 48 hr required to repeat the tryptophan loading test. The control subjects were 18 healthy women who were not taking drugs. The urinary metai)olites of tryptophan which were determined were imidican, anthranilic acid glucu- The Enovid-E used in these studies was kindly provided through the courtesy of Dr. J. William Crosson, G. D. Searle & Co., P. 0. Box 5110, Chicago, Illinois ronide, o-aminohippuric acid, kynurenic acid, acetylkynurenine, kynurenine, 3-hydroxykynurenine, xanthurenic acid, and N-methyi-2-pyridone-5-carboxamide. The urine specimens were analyzed for these tryptophan metabolites and for 4-pyridoxic acid according to methods de- eloped in these laboratories and the usual precautions were taken to avoid dietary factors or drugs which might vitiate the results (12). RESULTS The ingestion of the steroid hormones had no significant effect on the basal cxcretion of urinary tryptophan metabolites (Table i). None of the mean values for basal metabolite excretion differed by more than 2 standard deviations from the mean of the controls. After the administration of the loading dose of tryptophan, however, the subjects taking Enovid-E cxcreted a mean level of 697 mo1es of xanthurenic acid compared with a mean level of 2 moles excreted by the controls. Xanthurenic acid was the chief unnary tryptophan metabolite followed in order of decreasing quantitative significance by hydroxykynurenine, kynurenine, kynurenic acid, and acetylkynurenine, all of which were excreted in quantities greater than 2 standard deviations above the mean value for the control subjects. The other metabolites of tryptophan were excreted in essentially normal quantities by the subjects ingesting Enovid-E. When the experimental subjects were given 100 mg/day of supplemental pynidoxine hydrochloride, theit- tryptophan metabolism was essentially normal (Table II). The only metabolites excreted by the subjects ingesting Enovid-E in quantities greater than 2 standard deviations above the mean for the control subjects were the basal excretion of xanthurenic acid (13.4 1zmoles) and the post-tryptophan excretion of anthranilic acid glucuronide (,moles). As might be expected, the excretion of 4-pynidoxic acid increased to very high values following the administration

3 454 Price ct al. TABLE I Avet-age Urinary Excretion of Tryptophan Metabolites by 10 Subjects Ingesting Enovid-E and 18 Female Controlsb 4-PA Basal EnovidEC 419 ±158 Femalecontrols 371 Post-tryptophan ±145 Enovid.E 488 ±151 Femalccontrols 500 ±270 ±2.2 ±1.2 ± I8.0 ± ±6.0 ± ± ±4.0 ±4.0 Values are expressed as average inicrornoles of metabolite excreted (± standard deviation) per 24 hr ±39.1 ± C The following abbreviations are used : ISA, indoxyl sulfate (indican) ; AAG, anthranilic acid glucuronide; o-ah, o-aminohippuric acid; KA, kynurenic acid; ACK, acetylkynurenine; KYN, kynurenine; HKYN, hydroxykynurenine; XA, xanthurenic acid; PYR, N-methyl-2-pyridone-5-carboxamide; 4-PA, 4-pyridoxic acid; AA, anthranilic acid; HAA, hydroxyanthranilic acid. B, mdicates known sites of action of the coenzyme, pyridoxal phosphate. Legend at the top of the table indicates the metabolic interrelationships of the urinary products, and abbreviations in parentheses indicate intermediates which were not measured. Underlined values are those which were more than 2 standard deviations above the mean for the female controls. b The data for control women were taken from a recent publication (12). Subjects ingesting Enovid-E. Values underlined differ from the mean values for controls by more than 2 standard deviations. of the large quantities of pyridoxine hydrochloride. DISCUSSION These results demonstrate that the administration of Enovid-E produced a profound abnormality of tryptophan metabolism iii human subjects ingesting the drug in the usual prescribed dosage. Supplementation with large doses of pyridoxine hydrochloride almost completely corrected the disorder of tryptophan metabolism. These results are in agreement with those of Rose (1 1) who studied only the excretion of xanthurenic acid by women taking similar preparations. The disorder of tryptophan metabolism in women ingesting these steroid hormones resembled but was not identical to that described previously ± ± ± 27.5 ±190 ±14.1 ± ± ± ± ±8.2 ±26.5 ± ± ±36.1 in pregnant women (5). The pregnant ±1.04 ± ±1.32 women failed to respond completely to pyridoxine supplementation although they were given a smaller dose of the vitamin than that given the subjects taking Enovid-E. In the present report, the administration of steroid hormones with chiefly progestational but some estrogenic activity had a metabolic effect which was correctable by the administration of vitamin B6. However, Nelson et al. (9) found that pyridoxinc-deficient rats would maintain gestation to the 17th day in only 10% of pregnancies, while similar animals injected with 1.0 g of estrone plus 4 mg of progesterone remained pregnant to the 17th day of gestation in 90% of pregnancies. Thus, an effect of a pyridoxine deficiency in rats was corrected by the administration ±

4 Tryptophan Metabolism and Steroid Hormones 455 TABLE II Average Urinary Excietion of Tryptophan Metabolitesa by 10 Subjects Ingesting Enovid-E + B6 and 18 Female Controls (AA) (HAA) + A ISA AAG o-ah K. ACK -KYN -IHK\N---+XA P1 R 4-PA Basal Post Enovid-E + B6 430 ±161 remaic controls (nobt) 371 -tryptophan ±145 Enos,id.EC + B6 512 ±181 Female controls (nob6) 500 ±270 ±1.7 ± ± ± ±11.2 Values are expressed as average micromoles of metabolite excreted (± standard deviation) per 24 hr. ±3.0 ± ± ±19.1 For abbreviations used see Table m. The data for control women were taken from a recent publication (12). The controls were not given supplements of pyridoxine. Subjects ingesting Enovid-E plus 25 mg of pyridoxine hydrochloride four times daily. Values underlined differ from the mean values for controls by more than 2 standard deviations. of hormones similar to those shown to produce a functional pyridoxine deficiency in the women in the present study. Mason and Gullekson (13, 14) found that estradiol disulfate was a powerful inhibitor of the kynurenine transaminase of rat kidney, which they attributed to its competition with pynidoxal phosphate for binding sites on the apoenzyme. Estradiol disulfate also inhibited the reactivation of nuuscle phosphorylase a by pyridoxal phospiiate (13). In their studies, tightly bound pynidoxal phosphate had been removed from the muscle phosphorylase a and the degree of reactivation in the presence of estradiol disulfate depended upon the concentration of pyridoxal phosphate in the system. Diethylstilbestrol disulfate displayed similar inhibitory properties on kynurenine transaminase at a concentration as low as 5 X l0 M. Estrone sulfate and pregnanediol ghmcuronide inhibited 10.2 ± ± ±6.0 ± ± ±18.7 ±14.4 ± ± ±3.1 ± ± ±44.5 ± ± ±36.1 the transaminase only at much higher concentrations and several nonanionic steroids 163 ±21 ± ± ±1.16 such as estradiol, diethylstilbestrol and estrone were not inhibitory even in saturated solutions (14). Diethylstilbestrol disulfate also protected kynurenine transaminase during dialysis and from the proteolytic effects of chymotrypsin. These interesting results indicate a possible clue to the interrelationships between vitamin B6 anti steroid hormones used in the present studies. The large number of metabolic reactions depending on pynidoxine for proper function and the widespread use of steroid hormones in the regulation of ovulation of women should serve as an appropriate warning for those conducting studies in human metabolism or nutrition. In every study care should be taken to determine whether or not the subjects are ingesting drugs of this type; and, if so, whether or not the drugs have a metabolic effect that would influence the results.

5 456 Price et al. SUMMARY Ten women using steroid hormones for ovulation control were studied for urinary excretion levels of tryptophan metabolites before and after the ingestion of a 2-g loading tlose of tryptophan. The subjects took 2.5 mg of norethynodrel and 0.1 mg of mestranol (Enovid-E) daily from the 5th tilrough the 24th day of the menstrual cycle. After the initial study, the subjects were given 25 mg of pyridoxine hydrochloride four times daily during the 48 hr required to repeat the tryptophan loading test. Basal excretion levels (before tryptophan loading) of urinary tryptophan metabolites were normal, but after the loading dose of the amino acid the pattern of tryptophan metabolites was very abnormal as compared with that of the 18 female controls. The mean excretion level for xanthurenic acid after tryptophan loading was 697 moles for the subjects ingesting Enovid-E compared with about 30 moles for the controls. The other metabolites that were excreted in increased amounts were, in order of decreasing quantities, 3-hydroxykynureni ne, kynurenine, kynurenic acid, and acetylkynurenine. Pyridoxine supplementation almost completely corrected the metabolic disorder. These results appear to demonstrate another metabolic interrelationship between steroid hormones and pyridoxine. The widespread use of steroid hormones for ovulation regulation should be considered in human metabolic studies with females, especially if pyridoxine-requiring enzyme systems are involved. We are ilidebted to Miss Mary Hostak, Miss Jacqueline Hazen, Miss Barbara Fleurdelys, Miss Carole Aebischer, and Mrs. JoAnne Gulledge for assistance with the analyses. REFERENCES 1. SPRINCE, H., R. S. Lowy, C. E. F0L50ME AND J. S. BEHRMAN. Studies on the urinary excretion of xanthurenic acid during normal and abnormal pregnancy: a survey of the excretion of xanthurenic acid in normal nonpregnant, normal pregnant, pre-eclamptic, and eclamptic women. Am. J. Obstet. Gynecol. 62: 84, VANDELLI, I. The rise of vitamin B6 (pyridoxine) for suppressing the elimination of xanthurenic acid in pregnant and non-pregnant wonien following the oral intake of a measured quantity of tryptophan. Acta Vitaminol. 5: 55, WACHSTEIN, M., AND A. GuDAITI5. Disturbance of vitamin B0 metabolistmm in pregnancy. II. The influence of various almsoulmts of pyridoxine hydrochloride upon the abnormal tryptophane load test in pregnant wonmen. J. Lab. Clin. Med. 42: 98, \VACHSTEIN, M., AND S. LOBEL. Abnormal tryptophan metabolites in human pregnancy and their relation to deranged vitamin B6 mnetabolism. Proc. Soc. Exptl. Biol. Med. 86: 624, BROWN, R. R., M. J. THORNTON AND J. M. PRICE. The effect of vitamnm supplementation on the urinary excretion of tryptophan metabolites by pregtmant women. j. Cli,m. Invest. 40: 617, Yrss, N., J. M. PRICE, R. R. BROWN, P. B. SWAN AND H. LINKSWILER. Vitamin B0 depletion in man: urinary excretion of tryptophan metabolites. J. Nutr. 84: 229, ROSEN, F. AND C. A. NICHOL. Corticosteroids and enzyme activity. Vitamins Hormones 21: 135, Hsu, J. M. Interrelations between vitamin B6 and hormones. f itamins Hormones 21: 113, NELSON, SI. M., W. R. LIoNs AND H. M. EVANS. Maintenance of pregnancy in pyridoxine-deficient rats when injected with estrone and progesterone. Endocrinology 48: 726, NELSON, M. M., W. R. LYONS AND H. M. EVANS. Comparison of ovarian and pituitary hormones for maintenance of pregnancy in pyridoxinedeficient rats. Endocrinology 52: 585, RosE, D. P. Excretion of xanthurenic acid in the urine of women taking progestogen-oestrogen preparations. Nature 210: 196, PRIcE, J. M., R. R. BROWN AND N. YEss. Testing the functional capacity of time tryptophan-niacin pathway in man by analysis of urinary tnetabolites. In: Advances in Metabolic Disorders, edited by R. LEVINE AND R. LUFT. New York: Academic, vol. 2, p MASON, M., AND E. H. GULLEKSON. Inhibition of pyridoxal pimosphate-dependent enzymes by the sulfate esters of estradiol, estrone and diethylstilbestrol. J. Am. Chem. Soc. 81: 1517, MASON, M., AND E. H. GULLEKSON. Estrogenenzyme interactions: inhibition and protection of kynurenine transaminase by the sulfate esters of diethylstilbestrol, estradiol, and estrone. J. Biol. Chem. 235: 1312, 1960.