QUANTITATIVE- STUDIES ONHUMANURINARY METABOLITESOF TRYPTOPHANAS AFFECTEDBY ISONIAZID AND

DEOXYPYRIDOXINE1

BY J. M. PRICE,2R. R. BROWN,ANDFRANKC. LARSON

(From the Cancer Research Hospital, Medical Sch6ol, Uuiversity of Wisconsin, and theVeterans Admintration Hespitd, Madison, Wisc.)

(Submitted for publication May 21, 1957; accepted July 25, 1957)

Peripheral neuropathy has been observed as acomplication of the treatment of tuberculosis withisonicotinic acid hydrazide (isoniazid) (1, 2), es-pecially when large doses have been employed (3,4). This complication of isoniazid therapy hasbeen largely eliminated by the simultaneous ad-ministration of pyridoxine (3, 5).

In pyridoxine deficiency, rats (6) and humans(7) have been found to excrete abnormally largeamounts of the tryptophan metabolite xanthurenicacid. The administration of the pyridoxine an-tagonist, deoxypyridoxine, to humans (8) or rats(9) has been shown to produce a severe disturb-ance of tryptophan metabolism in these species asmanifested by the excretion of large amounts ofxanthurenic acid following the administration of a"loading" dose of tryptophan. These observationshave prompted studies of the urinary excretion ofxanthurenic acid as an index of the antipyridoxineactivity of isoniazid. Studies with humans (3)and rats (10) ingesting isoniazid have revealed anincreased excretion of xanthurenic acid after in-gestion of tryptophan. Rosen (11) reported noincrease in xanthurenic acid excretion when iso-niazid was administered to pyridoxine-deficientrats, even though the rats showed other signs ofpyridoxine deficiency. In the studies of Biehl andVilter (3) the administration of large doses ofisoniazid resulted in a considerable increase in uri-nary excretion of vitamin B8, about a 40 per centincidence of clinical evidence for pyridoxine de-ficiency, but an irregular elevation of xanthurenicacid excretion following a tryptophan load. Thus

1Supported in part by grants from the AmericanCancer Society, upon recommendation of the Committeeon Growth of the National Research Council; the Wis-consin Division of the American Cancer Society; theElsa U. Pardee Foundation; and the National LeagueBaseball Club of Milwaukee, Inc.

2 Scholar in Cancer Research of the American CancerSociety.

it appeared that the clinical evidence for pyridoxinedeficiency in rats or humans given isoniazid wasmore striking than the xanthurenic acid excretionstudies would suggest (3, 11).

The present studies were undertaken in an ef-fort to determine whether or not the simultaneousquantitative measurement of several urinary me-tabolites of tryptophan would reveal additional evi-dence of a disturbance in the metabolism of thisessential amino acid in patients ingesting isoniazid.The extensive paper chromatographic studies ofDalgliesh (12) revealed that pyridoxine-deficientrats excreted not only xanthurenic acid, but largeamounts of several other intermediary metabolitesof tryptophan. Methods have been developed forthe quantitative determination of several of theseintermediary metabolites of tryptophan in urine(13-15). The application of these methods inquantitative studies of tryptophan metabolism innormal human subjects has been reported (14, 16).

The present detailed studies on tryptophan me-tabolism were made repeatedly over a period ofseveral weeks during which time the dosage ofisoniazid was increased until abnormal tryptophanmetabolism was observed. Once the metabolicpattern became abnormal, pyridoxine and threeother members of the vitamin B-complex, eachof which has been suggested to play some role intryptophan metabolism, were administered in anattempt to determine which of these vitamins mightbe required to overcome the effects of isoniazid onthis metabolic pathway. A preliminary report ofthese studies has been made (17).

EXPERIMENTAL

Analytical procedures. The methods for the deter-minations of urinary kynurenic acid, xanthurenic acid,N-methyl-2-pyridone-5-carboxamide (pyridone), anthra-nilic acid glucuronide, N-a-acetylkynurenine, kynurenine,o-aminohippuric acid, and 3-hydroxykynurenine werethose previously described (13-15). Paper chromatog-

1600

TRYPTOPHANMETABOLISM IN MAN

FIG. 1. MILLIMOLES OF METABOLITES ExcRETED IN THE24-HOUR PERIOD FOLLoWING INGESTION OF TRYPTOPHAN

Subject 1 (weight, 77 Kg.) was receiving 300 mg. ofisoniazid daily at the start of the experiment. The dosewas increased to 600 mg. after two weeks and was givenat a level of 750 mg. daily from the start of the sixthweek until the end of the experiment. B-2 = riboflavin;B-6 = pyridoxine hydrochloride.

raphy (14, 18) was utilized to verify the quantitative de-terminations of aromatic amines in every urine studied.

Subjects. The studies were conducted on six malepatients with tuberculosis from the Veterans Administra-tion Hospital, but the detailed data presented were ob-tained from three of these subjects. Subject 2 was usedfor three of the studies and the others were used for onestudy each. Subjects 1 and 2 were Negroes, and sub-ject 3 was white. The subjects all had pulmonary tu-

1.2 THIAMINERIBOFLAVIN

0 NIACIN< ~~~~~~~PYRIDOXINE

W~ ~ ~~~EK

E0.80

F KYNURENINE 1ETLXOU.4 KYNURENINE-a

3 5 7 9WEEKS

FIG. 2. MILLIMOLES, OF ME~T~ABoLTs ExcRETED IN THE24-HOUR PERIOD FOLLOWINGINGESTION OF TRYPTOPHAN

Subject 2 (weight, 57 Kg.) was not on therapy at thestart of the experiment. Isoniazid was started at alevel of 300 mg. daily after the initial study and increasedto 450, 600, 750 mg. daily after the first, second, andthird week, respectively, and continued throughout thestudy at a level of 750 mg. daily. X. A. = xanthurenicacid; K. A. = kynurenic acid.

FIG. 3. MILLIMOLES OF METABOLITES ExcRiun IN THE24-HouR PERIOD FoLLowING INGESTION OF TRYTOPHAN

Subject 3 (weight, 51 Kg.) was started on 300 mg. ofisoniazid daily after the initial, zero-time study and thedose was increased to 450, 600, and 900 mg. daily afterthe first, second, and third week, respectively. This studywas discontinued because the patient left the hospitalafter the fourth week. Note that the scale of this graphis different from Figures 1 and 2.

berculosis but were in a stationary phase of their dis-ease. The subjects were ambulant and afebrile. Genito-urinary tuberculosis was specificially excluded.

Each patient was thoroughly instructed concerningthe proposed experiments. At weekly intervals (except

5 7WEEKS

FIG. 4. MmImoLs OF METAoLrrs EXcRETED IN THE24-HOUR PERIOD FOLLowING INGESTION OF TRYPTOPHAN

Subject 2 was given 900 mg. of isoniazid daily startingat zero time, but had not received any chemotherapy for18 weeks prior to the time this study was started. Nopre-treatment study was done, but the patient had a normalpre-treatment response to the 2 Gm. of tryptophan in theother experiments in which he participated (Figures 2and 5). X. A. =xanthurenic acid; K. A. = kynurenicacid.

1601

J. M. PRICE, R. R. BROWN,AND FRANK C. LARSON

ksI-

0oI-aai

3 5WEEK7 9 if

FIG. 5. MILIMOLES OF METABOLITES EXCRETEDIN THE24-HOUR PERIOD FOLOwINGINGESTION OF TRYPTOPHAN

Deoxypyridoxine was given to subject 2 at a level of75, 150, 225, 300, and 375 mg. daily starting after 0, 1,2, 3, and 5 weeks, respectively, and the latter dose wascontinued throughout the study. X. A.= xanthurenicacid; K. A. = kynurenic acid.

for Figure 1) the following procedure was carried out:a 24-hour urine collection (in amber bottles under tolu-ene) was started at 7 armL on Monday, and the subjectingested 2.0 Gm. (9.8 mM) of L-tryptophan at 7 a.m. onTuesday. A second 24-hour urine collection was com-pleted by 7 a.m. on Wednesday and all changes in medi-cation were made at this time.

The tryptophan was administered as a single dose infour 0.5-Gm. gelatin capsules (Figures 1 and 2) or asfour 0.5-Gm. tablets 3 (Figures 3 through 5). Thedeoxypyridoxine4 was given in gelatin capsules and theisoniazid and vitamins were standard pharmaceuticalpreparations. The deoxypyridoxine and isoniazid wereadministered in three equal doses each day and in amountsindicated in the legends for the figures. Thiamine wasgiven at a dosage of 300 mg., and the other vitamins atlevels of 75 mg., in three equal portions daily. Once avitamin supplement was started it was continued through-out the remainder of the study. The urine samples werealways analyzed the week they were collected in orderto obtain the data in time so that it could be used inselecting the next dose of drug or other supplement.

RESULTS

The data obtained have been summarized inFigures 1 through 5. An abbreviated outline ofthe metabolic pathway from tryptophan to nico-tinic acid has been presented in Figure 6. The

8 The tryptophan was compressed into 0.5-Gm. tabletsfor these studies by Dr. Rodney P. Gwinn and his as-sociates at Abbott Laboratories, North Chicago, Illinois.

' The deoxypyridoxine was generously supplied by Dr.Elmer Severinghaus of Hoffmann-La Roche, Inc., Nuty,New Jersey.

positions of the metabolites under consideration inthese studies of the pathway have all been includedin the diagram. The data for pyridone, anthranilicacid, anthranilic acid glucuronide, and o-amino-hippuric acid were not shown on the graphs be-cause the values remained at normal or less thannormal levels throughout the studies. The basalexcretions (the 24-hour periods before the ad-ministration of tryptophan) of the metabolites rep-resented in Figures 1 through 5 were very lowcompared with excretions after ingestion of the9.8 mMsupplements of tryptophan. Therefore,the basal values were not subtracted from the datapresented in the graphs. Thus the data in Figures1 through 5 represent the millimoles of metabolitesexcreted in the 24-hour period following ingestionof the 9.8 mMof tryptophan (Figure 1). Un-less otherwise indicated all values below referto the 24 hours after tryptophan.

Although subject 1 (Figure 1) was receiving300 mg. of isoniazid daily at the start of the ex-periment, the urinary excretion of metabolites wasnormal for the first two studies. After three weeks

c CH2HCOOH07 NH2TRYPTOPHAN1,

ANTHRANILCACID

0O-C-NHCH2COOH

NH2O-AMiNO141PPURICACID

OwIV 0A.A-r8CH..IHCOOH 0r .i-H29COOH ~~N COCH3 r-O-C6H9506

V'NH2 NH2 0 NH2 3 NH 6KYNURENINE ANTHRANIUCACIDOH GLUCURONIDEO ¢IDCOOH

-NCH2 NH2CO KYNURENIC ACID

3-HYDROXYKYNUREN XCOOHJINH

\XHCOON OH 7 CH3O NH2 XANTHURENIC ACID N-ME .2-PYRIDONE-OH 5-CAMBOXAMIDE

3-HYDROXYANTHRANILIC NCOO"ACID ' NICOTINIC ACID

FIG. 6. AN AzmxvLATED DTAGRAMOF THE METABOLICPATHWAYFRoM TRYPTOPHANTO NICOTINIC ACID ANDN-METHYL-2-PYRIDONE-5-CARBOXAMIDE, THE CHIEF URI-NARYNIACIN METABOLITE IN MAN

Pyridoxine, in the form of pyridoxal phosphate, hasbeen reported to function in the conversion of: 1) ky-nurenine to anthranilic acid (36); 2) 3-hydroxykynure-nine to 3-hydroxyanthranilic acid (37); 3) kynurenine tokynurenic acid (37); and 4) 3-hydroxykynurenine toxanthurenic acid (37). Niacin in the form of triphos-phopyridine nucleotide was required for the hydroxylationof kynurenine to hydroxykynurenine (18) and riboflavinalso has been suggested to play a role in this reaction(24-26).

1,62

TRYPTOPEANMETABOLISM IN MAN

of ingestion of 600 mg. of isoniazid daily, the ex-cretion of kynurenine and acetylkynurenine wereincreased to abnormal levels after ingestion oftryptophan. After the isoniazid intake was in-creased to 750 mg. daily the yield of kynurenine,xanthurenic acid and acetylkynurenine rose to in-creasingly abnormal levels. The vitamin supple-ments were started after the seventeenth week andan additional vitamin was added weekly. Afterthe pyridoxine was added to the vitamin supple-ment the response to the test dose of tryptophanwas restored to normal. The effects of the otherthree vitamin supplements were of doubtful sig-nificance, though there was a slight drop in kynu-renine excretion when the niacin supplement wasstarted.

The second experiment (Figure 2) was essen-tially a repetition of the first study and gave com-parable results. Subject 2 did not receive anyisoniazid when the initial study was done. Thissubject usually excreted more acetylkynureninethan xanthurenic acid, while the opposite was truewith subject 1. The subject was kept on supple-ments of thiamine, riboflavin, and nicotinamide fortwo weeks before the pyridoxine was started, sincethe results obtained with the first subject suggestedthat a slight decrease in kynurenine occurred af-ter niacin was started. Again the addition of ni-acin slightly decreased the yield of kynurenine.The pyridoxine supplements restored the trypto-phan metabolism to normal.

By the time the next study was undertaken(Figure 3) a method for the determination of3-hydroxykynurenine was available. This subjectexcreted an abnormally high level of 3-hydroxy-kynurenine in the initial study. A daily ad-ministration of 450 to 600 mg. of isoniazid resultedin a severe disturbance of tryptophan metabolismand the last study at four weeks (900 mg. isoniaziddaily) revealed a remarkable increase in the ex-cretion of 3-hydroxykynurenine. This one metab-olite accounted for over 25 per cent of the 9.8 mMsupplement of tryptophan, and the kynurenine ac-counted for over 10 per cent of the amino acidgiven. Acetylkynurenine and xanthurenic acidwere excreted in approximately equal quantities,and as in all studies on isoniazid the kynurenicacid excretion was less than normaL

In the next study (Figure 4) the subject wasstarted on 900 mg. of isoniazid daily at zero time

and was maintained at that dosage for elevenweeks. Within one week, when the first studywas made, the hydroxykynurenine accounted forabout 17 per cent of the dose of tryptophan. Aftersix weeks on an intake of 900 mg. of isoniazid dailythe pyridoxne supplements were started, andthereafter the response to the 9.8 mMdose oftryptophan was normal. The addition of the otherthree vitamins had no detectable influence on theresponse to tryptophan.

The effect on deoxypyridoxine on the fate of in-gested tryptophan (Figure 5) was similar to thatof isoniazid except that xanthurenic acid excretionwas considerably greater and was almost equal tothat of kynurenine. Unlike the observations dur-ing isoniazid treatment the administration of de-oxypyridoxine was accompanied by abnormallyhigh kynurenic acid excretion. Again, althoughthe addition of thiamine, riboflavin and niacin sup-plements had little, if any, effect on the metabolismof the test dose of tryptophan, pyridoxine restoredthe urinary excretion of metabolites to normal

Another subject was started on deoxypyridoxine(subject 3) at a level of 150 mg. daily. Withinone week the response to tryptophan ingestionwas similar to that in Figure 5 at the same dosageof deoxypyridoxine (2 weeks). The first supplyof deoxypyridoxine was exhausted at this pointand a new batch was used. The second week (endof one week on the new supply) the response totryptophan was about half that of the previousweek. Although the intake of deoxypyridoxinewas gradually increased to 450 mg. daily the ex-cretion of the metabolites of tryptophan fell toalmost normal by the seventh week, indicatingthat the second lot of deoxypyridoxine probablycontained some substance with vitamin Bs ac-tivity.,

The other studies on isoniazid which were notpresented were all incomplete for some reason,usually because the subject developed some addi-tional illness. The incomplete results were in gen-eral agreement with the data presented (Figures1 through 4).

None of the subjects experienced any symp-

5Hoffmann-La Roche, Inc., reported that samples ofthe two lots of deoxypyridoxine have now been testedmicrobiologically for antipyridoxine activity, and the firstbatch of drug had about 20 times the activity of thesecond.

1603

J. M. PRICE, R. R. BROWN,AND FRANK C. LARSON

toms that could be attributed to the ingestion ofeither isoniazid or deoxypyridoxine.

Although, as stated previously, the basal excre-

tions of the various metabolites (the 24-hour pe-

riods prior to tryptophan supplementation) were ofinsignificant quantities compared with the excretionfollowing ingestion of tryptophan, they were al-tered somewhat by isoniazid or deoxypyridoxine.Thus, kynurenic acid was usually excreted at basallevels of 7 to 10 pM daily and the ingestion ofisoniazid or deoxypyridoxine decreased theselevels to 3 to 8 MM per day. Basal values forkynurenine excretion ranged from 8 to 12 ,Mdaily but invariably increased to 35 to 45 ,uM dailyat the times when the response to the tryptophanload yielded quantities of 700 to 1,000 ,M of thismetabolite. The pre- and post-tryptophan valuesfor urinary o-aminohippuric acid and anthranilicacid glucuronide were very similar to those ob-served in studies on normal human subjects (14),and the volatile amine from the acetylkynureninefraction (14) was usually comparable to the valuesfound on direct diazotization and coupling of thisfraction, which indicated that little, if any, freeanthranilic acid was excreted.

Although the pyridone excretion usually re-

mains elevated for several days after the ingestionof tryptophan (14, 16) or niacin (19) the amountexcreted the first day provides some indication ofniacin formation from tryptophan. In general,the subjects excreted less than the expectedamounts of pyridone following ingestion of trypto-phan. Subject 1, however, invariably excretedmore than the expected amounts of pyridone fol-lowing ingestion of tryptophan, so that in thesestudies no conclusions could be made concerningthe effects of isoniazid on the conversion of trypto-phan to niacin. When the nicotinamide supple-ments were given to these subjects the pyridoneexcretion increased to very high levels, whichsuggested that isoniazid and deoxypyridoxine hadlittle, if any, effect on the conversion of niacin tothe pyridone.

DISCUSSION

In these studies the subjects were maintainedon a general hospital diet and no attempt was madeto regulate the food intake. The subjects main-tained their weights or gained slightly during theexperiments. Previous work has indicated that

the urinary excretion of these metabolites oftryptophan (except for the pyridone) was aboutthe same on self-selected or constant dietary in-take (16). The magnitude of the disturbance oftryptophan metabolism observed in the presentstudies makes it appear unlikely that a carefullyregulated diet would have contributed much to theresults. However, a larger dietary intake ofpyridoxine probably would have made it neces-sary to administer higher doses of isoniazid anddeoxypyridoxine to achieve effects similar tothose observed.

All of the subjects used in these studies hadessentially normal tryptophan metabolism priorto the ingestion of isoniazid or deoxypyridoxineexcept for subject 3 who, for unknown reasons,excreted an abnormal amount of 3-hydroxykynu-renine. Abnormal tryptophan metabolism hasbeen found in patients with tuberculosis (20, 21).However, Dalgliesh and Tekman (22) reportedthat large quantities of kynurenine and hydroxy-kynurenine were excreted by patients, includingthose with tuberculosis, only if there was an ele-vation of body temperature. The fact that thesubjects used in the present studies did not havean elevation of the body temperature and hadnormal tryptophan metabolism in the absence ofthe drugs corroborates these findings.

Biehl and Vilter (3) reported that not all tu-berculous patients receiving isoniazid therapy ex-creted abnormal quantities of xanthurenic acidafter tryptophan loading. The present studies,however, leave little doubt that large doses ofisoniazid produced a profound disturbance in themetabolism of a test dose of tryptophan. Normalsubjects excreted an additional 3 to 5 ,uM of acetyl-kynurenine and 15 to 30 !&M of kynurenine duringthe first 24 hours after ingestion of 9.8 mMofL-tryptophan (14, 16). In subjects ingestingisoniazid, however, the yield of acetylkynureninereached 20 to 55 times, and of kynurenine 20 to 30times, these quantities. Xanthurenic acid excre-tion was not nearly as useful as the excretion ofkynurenine or hydroxykynurenine in detecting in-ability to metabolize tryptophan in a normal man-ner, and kynurenic acid excretion was usuallynormal or less than normal in these subjects.There appear to be some clinical conditions wherethe urinary excretion of xanthurenic acid may benormal or less than normal after ingestion of a

1604

TRYPTOPHANMETABOLISMIN MAN

"loading dose" of tryptophan, while kynurenicacid, kynurenine, and other metabolites may befound in very abnormally high levels (23). Thusthe determination of a single urinary metaboliteof tryptophan cannot be relied upon as an indica-tion of abnormal tryptophan metabolism.

Pyridoxine appeared to correct the metabolismof tryptophan without any definite evidence for theneed of additional thiamine, riboflavin, or niacin.Niacin in the form of triphosphopyridinenucleotidehas been shown to be required for the hydroxyla-tion of L-kynurenine to 3-hydroxy-L-kynurenine(18). There was some evidence that niacin ad-ministration slightly decreased the excretion ofkynurenine, but since hydroxykynurenine was notdetermined in these studies (Figures 1 and 2) itwas not known whether or not these observa-tions indicated increased hydroxylation of kynu-renine. Considerable hydroxykynurenine was ex-creted by the subjects ingesting the largest dosesof isoniazid (Figures 3 and 4) which would indi-cate that in this one reaction the isoniazid failedto reveal significant antiniacin activity.

Riboflavin supplements were administered tothese subjects because it has been reported (24-27) that this vitamin may be involved in themetabolism of tryptophan. Dalgliesh (28) foundthat rats deficient in both pyridoxine and thiaminefailed to excrete the urinary metabolites of trypto-phan which were detected in pyridoxine deficiency,and suggested that thiamine may be involved inthe formation of formylkynurenine from trypto-phan. In the present experiments there was nodemonstrable effect of riboflavin supplements ontryptophan metabolism. A slight increase in ky-nurenine excretion occurred after the thiaminesupplements were started, but this was observedunder conditions where the kynurenine excretionwas increasing, probably because of the adminis-tration of the drugs.

In previous studies (16) conducted in a man-ner similar to those reported here, but with nor-mal subjects on a constant dietary regimen, it wasfound that the increase in urinary tryptophanmetabolites after supplementation with 9.8 mMof the amino acid would account for about 2 percent of the administered tryptophan. Slightly lessthan half of the tryptophan was accounted for asthe pyridone metabolite of niacin. Such resultsmight be interpreted to indicate that very little

tryptophan enters the kynurenine pathway undernormal conditions. In the present experimentsas much as 40 per cent of the -9.8 mMdose oftryptophan was accounted for in the urine in theform of kynurenine and its metabolites. Thus,subjects ingesting isoniazid either converted morethan normal amounts of tryptophan to kynurenineand related substances, or they were unable toconvert these intermediates to the usual endproducts at a normal rate.

The enzyme system tryptophan peroxidase,which initiates the conversion of tryptophan tokynurenine, has been shown to be adaptive (29).Administration of substances like kynurenine, his-tamine, phenylalanine, cortisone, and especiallytryptophan itself have been found to increase theactivity of tryptophan peroxidase in rat liver (29).Thus it may be possible that patients ingestingisoniazid convert more than the normal quantityof tryptophan to kynurenine. However, sincethere is so much evidence to indicate that isoniazidhas an antagonistic effect on pyridoxine in man(3, 5) and the metabolites found in large quanti-ties in the urine in the present studies were identi-cal with those detected in the urine of pyridoxine-deficient rats (12), it appears reasonable to ex-plain the present findings in terms of incompletemetabolism of the kynurenine produced. Dalglieshand Tabechian (30) have recently discussed themetabolic fate of tryptophan and have suggestedthat considerable tryptophan may normally enterthis pathway and may be converted to 3-hydroxy-anthranilic acid and then to non-aromatic products.This conclusion was reached by eliminating othermetabolites of kynurenine, such as kynurenic andxanthurenic acids, as likely precursors of unknownmajor metabolites because they appeared to berelatively inert from a metabolic standpoint, while3-hydroxyanthranilic acid has been shown to bemetabolized rapidly (31-33). Although it isnow known that kynurenic acid and xanthurenicacid cannot be regarded as metabolically inert,since both have been shown to be dehydroxylatedin the 4-position (34, 35), there is little evidenceto suggest that these quinoline derivatives are ex-creted in quantities adequate to account for themajority of supplements of L-tryptophan. Theresults of the present study, therefore, cannot beregarded as in disagreement with the explanationoffered by Dalgliesh and Tabechian (30) for the

1605

J. M. PRICE, R. R. BROWN,AND FRANK C. LARSON

normal metabolic fate of the major part of atryptophan supplement.

The differences in the patterns of the urinarymetabolites produced by isoniazid and deoxypyri-doxine were striking. Xanthurenic and kynurenicacids were both excreted in abnormally largequantities during ingestion of deoxypyridoxine,while less than normal amounts of kynurenic acidand only moderately increased quantities of xan-thurenic acid were excreted under the influenceof isoniazid. Since kynurenine and hydroxyky-nurenine were produced in large amounts in thepresence of each drug it would appear that invivo isoniazid was a better inhibitor of kynureninetransaminase than deoxypyridoxine.

SUMMARY

The influence of isoniazid and deoxypyridoxineon the urinary excretion of tryptophan metabolitesby tuberculous patients was studied. The subjectsall had essentially normal tryptophan metabolismprior to treatment with these drugs.

During treatment with isoniazid or deoxypyri-doxine, 3-hydroxykynurenine was excreted in theurine in quantities adequate to account for from10 to 25 per cent of a 9.8 mM(2 Gm.) supple-ment of L-tryptophan. Kynurenine was often ex-creted in quantities equivalent to 5 to 10 per centof the trpophan administered, while N-a-acetyl-kynurenine and xanthuremic acid were also ex-creted in abnormally large amounts. The ex-cretion of kynurenic acid was slightly less thannormal during isoniazrid treatment and abnormallyhigh when deoxypyridoxine was administered.Normal or slightly less than normal amounts ofo-aminohippuric acid, anthranilic acid glucuronide,anthranilic acid and N-methyl-2-pyridone-5-car-boxamide were found in the urine after ingestionof tryptophan during treatment with either drug.

Pyridoxine administration resulted mi a returnto normal tryptophan metabolism even when theingestion of isoniazid or deoxypyridoxine wascontinued. Supplemental thiamine, riboflavin, orniacin had little, if any, effect on the abnormaltryptophan metabolism produced by these drugs.

ACKNOWLEDGMENT

The authors are indebted to Mrs. Mary Bryan, Mrs.Marian Prahl, and Mrs. Masako Kaihara for assistancewinth the analyical pro

REFERENCES

1. Jones, W. A., and Jones, G. P., Peripheral neurop-athy- due to isoniazid: report of two cases. Lamnet,1953, 1, 1073.

2. Biehl, J. P., and Skavlem, J. H., Toxicity of isoniazid.Am. Rev. Tuberc., 1953, 68, 296.

3. Biehl, J. P., and Vilter, R W., Effect of isoniazidon vitamin B., metabolism; its possible significancein producing isoniazid neuritis. Proc. Soc. Exper.Biol. & Med., 1954, 85, 389.

4. Lubing, H. N., Peripheral neuropathy in tuberculo-sis patients treated with isoniazid. Am. Rev.Tuberc., 1953, 68, 458.

5. Carlson, H. B., Anthony, E. M., Russell, W. F., Jr.,and Middlebrook, G., Prophylaxis of isoniazidsevropathy with pyridoxine. New England J.Med., 1956, 255, 118.

6. Lepkovsky, S., Roboz, E., and Haagen-Smit, A. J.,Xanthurenic acid and its rote in the tryptophanemetabolism of pyridoxine-deficient rats. J. Biol.Ches., 1943, 149, 195.

7. Greenberg, L. D, Bohr, D. F., McGrath, H., andRinehart, J. F., Xanthurenic acid excretion in thehuman subject on a pyridoxine-deficient diet. Arch.Biochem., 1949, 21, 237.

8. Glazer, H. S., Mueller, J. F., Thompson, C., Hawkins,V. R., and Vilter, R. W., A study of urinary ex-cretion of xanthurenic acid and other tryptophanmetabolites in human beings with pyridoxine de-ficiency induced by desoxypyridoxine. Arch. Bio-chem., 1951, 33, 243.

9. Porter, C. C., Clark, I., and Silber, R. H., The ef-fect of pyridoxine analogues on tryptophane me-tabolism in the rat. J. Biol. Cheow, 1947, 167, 573.

IO. Boone, I. U., Turney, D. F., and Langham, W. H.,Isoniazid in vitamin B,-deficient rats. J. Lab. &Clin. Med., 1955, 46, 549.

11. Rosen, F., Effect of isonicotinic acid hydrazide onmacin and pyridoxine metabolism in rats. Proc.Soc. Exper. BioL & Med., 1955, 88, 243.

12. Dalgliesb, C E, The relation between pyridoxin andtryptophan metabolism, studied in the rat. Bio-chem. J., 19S2, 52, 3.

13. Price, J. M., The determination of N-mithyl-2-pyri-done-5-carboxamide in lumn un J. Biol.Chess., 1954, 211, 117.

14. Brown, R. R., and Price, J. M., Quantitative studieson metabolites of tryptophan in the urine of thedog, cat, rat, and man. J. Biol. Chem., 1956, 219,985.

15. Brown, R. R., The isolation and det ion ofurinary hycoxykynurenine. J. Biol. Ches., 1957,227, 649.

16. Price, J. M.,, Brown, R. R., aEd Ellis, Mk. E*, Quan-titative studies on the urinary excretion of trypto-phan metabolites by humans ingesting a con-stant diet. J. Nutrition, 1956, 60, 323.

17. Price, J. M., Brown, R R., and Larson, F. C., Quan-titutive stdies on the metabolism of tryptophan

1606

TRYPTOPHANMETABOLISM IN MAN

in patients receing isoiazid therapy. Fe a-tion Proc., 1956i IS, 330.

18. deCastro, F. T., Price, J. M., and Bzowq, R. R.o,Reduced triphosphopyridinenucleotide requirementfor the enzymati formaion of 3-hydroxykymure-nine from L-kynurenine. J. Am. Chem. Soc.,1956, 78, 2904.

19. Walters, C. J., Brown, R. R., Kaihara, 'M., andPrice, J. M., The excretion of N-methyl-2-pyri-done-5-carboxamide by man following lgeston ofseveral known or potential precursors. J. Biol.Chem, 1955, 217, .489.

20. Maklno, K., Satoh, K., Fjild, T., and Kawaguchi, K,Relation of 3-hydroxykynurenne to the Ehrlichdiazo reaction of urine in severe tuberculosis. Na-ture, 1952, 170, 977.

21. Musajo, L., Spada, A., and Coppini, D, Isolation of3-hydroxyanthranilic acid from pathological hu-man urine after administration of L-tryptophan. 3.BioL. Chem., 1952, 196, 185.

22. Dalgliesb C. E., and Tekman, S., The excretion ofkynurenine and 3-hydroxykynurenine by man.Biochem. J., 1954, 56, 458.

23. Price, J. M., Brown, R. R., Rukavina, J. G., Mendel-son, C., and Jchnson, S. A. M., Scleroderma(Acrosclerosis). IL Trytophan metabolism beforeand during treatment by chelation (EDTA). 3.Invest. Dermat., In press.

24. Porter, C. C., Clark, I., and Silber, R. H., The effectof B vitamin deficiencies on tryptophan metabo-lism' in the rat: Arch. Biohe., 1948, 18 339.

25. Henderson, L. M., Weinstock, I. M., and ama,G. B., Effect of deficiency of B vitamins on themetabolism of tryptophan by the rat.- . Biot.Chem., 1951, 189, 19.

26. Charconnet-Hardingr F., Daigliesh, C. E., and Neu-berger, A., The relation between riboflavin andtryptophan metabolsm, studied in the rut. Bio-chem. J., 1953, 53,. 513.

27, MaOM,- f,. The metabolism of tryptophan in ribo-9avinrdeficient -rats. J. Biol. Chem., 1953, 201,

A'. Dalgfiesh, C. E., The relation between thiamine, bio-ti, ad tryptopha metabolism, studied in the rat.Biochem. J., 1955, 61, 32&.

29. Knox,' W. -E, Two mechaIims. which increase in-v*e liver trtaphat peroxidase activity: spe-

cific enzyme adaptation and stimulation of thepituitary-adrena ssemn Brit. J. Exper. Path.,1951, 32, 462.

30. Dalglieph, C. E., and Tahizn, H., Comparison ofthe metabolism of uniformly "C-labelled L-phenyla-lanine, L-tyrosine, and L-tryptophan in the rat.Biochem. J., 1956, 62, 625.

31. Brany, H G., Thorpe, W. V., and Wood, P. B, Thefate of certain organic acids aWd amis IS te rab-bit. 12. Aminohydroxybenzoic acids. Biochem. J.,1951, 48, 394.

32. Henderson, L. M., Quinolinic acid metabolism. II.Rephacment of nicotinic acid for the growth ofthe rat and Neurospora. J. Biol. Chem., 1949, 18,677.

33. Bokman, A. H, and Schweigert, B. S., 3-Hydroxy-anthranilic acid metabolism. III. Molar conversionto quinolinic acid. J. BioL. Chem., 1950, 186, 153.

34. Takahashi, H., Kai-hara, M., and Price, J. M., Theconversion of kynurenic acid to quinaldic acd byhumm. and rats, J. BioL Chem., 1956, 223, 705.

35. Takahashi, Hs and Price, J. M*, Urinary excretionof 8-hydroxyquinaklic acid after ingestion ofxanthurenic acid. Federation Proc., 1957, 16, 259.

36. Braunshtein, A. E., Goryacenkcova, E. V., andPaskhim,. T. S., Enzymic fortio of alaninefrom L-kyaurein and Ltryptephan, and therole of vitamin B. in this process. Biokhimiya-1949,. 14, 163.

37. Wiss, 0., Der enzymatische Abbau des Kynureninsund 3-Oxy-Kynurenins im terischen Orgamismus.Hoppe Seyler's Ztschr. f. physiol. Chem., 1953,293, 106k

1607