[CANCER RESEARCH 33, 1071 1077, May 1973]

Studies on Tryptophan Metabolism in Patients with BladderCancer1

S. Gailani, G. Murphy, G. Kenny, A. Nussbaum, and P. Silvernail

Departments of Medicine [S.G., A.N., P.S.] and Urology [G.M., G.K.], Roswell Park Memorial institute, Buffalo, New York 14203

SUMMARY

Measurement, after 2 g L-tryptophan load p. o., ofurinary excretion of some of the metabolites of thetryptophan-niacin pathway was performed on 36 patientswith bladder cancer. Assay of hepatic tryptophan pyrrolaseactivity was done on 21 patients, and assay of kynureninasewas done on all patients. Of these patients, increasedexcretion of kynurenine was found in 12, 3-hydroxykynure-nine was found in 25, kynurenic acid was found in 9,xanthurenic acid was found in 4, and acetylkynurenine wasfound in 10. There was no correlation between tryptophanpyrrolase activity and the level of the urinary excretion oftryptophan metabolites, while patients with low kynureninase activity tended to excrete increased quantitiesof these metabolites. Depressed kynureninase activityand increased excretion of tryptophan metabolites weremore marked in the more advanced Stage Dt and D2bladder cancer. Measurements of excretion of the tryptophan metabolites 6 to 12 months after eradication ofthe disease in 19 patients showed a statistically significant decrease in the excretion of kynurenine, while increased excretion of 3-hydroxykynurenine persisted in9 of the patients. The excretion of the other metaboliteswas within normal range in all the patients postopera-tively. Similar measurements on two other patientswith known evidence of métastasesshowed an increasein the excretion of the various metabolites in the postoperative compared to the preoperative measurements.

INTRODUCTION

Most of the known industrial and experimental bladdercarcinogens are aromatic amines or their metabolites (13).A search for endogenous bladder carcinogens led toextensive studies of urinary excretion of tryptophan metabolites in patients with bladder cancer (2, 3, 14). This workwas given impetus by the finding of Dunning et al. (9) thatalmost 100% of Fischer line 344 rats developed bladdercancer if they were fed supplemental DL-tryptophan in adiet containing 0.06% 2-acetylaminofluorene, whereas nobladder tumor developed when the diet was not supplemented with tryptophan.

1This investigation was supported by USPHS Research GrantCA-5834 from the National Cancer Institute.

Received May 25, 1972; accepted February 15, 1973.

One of the metabolic pathways of tryptophan proceedsthrough a series of enzymatic degradations into kynurenineand several other intermediary metabolites to nicotinic acid(Chart 1). A key enzyme in this metabolic sequence istryptophan pyrrolase, a hepatic heme-containing enzymethat catalyzes the oxidation of tryptophan to formylky-nurenine. Several of the enzymes in this pathway utilizepyridoxal phosphate as a cofactor, and of these kynureninase, which catalyzes the conversion of kynurenineto anthranilic acid and of 3-hydroxykynurenine to 3-hy-droxyanthranilic acid, would appear to play a key role.

Brown et al. (3) measured the urinary excretion of someof the metabolites of the tryptophan-niacin pathway after 2g L-tryptophan load in 41 patients with bladder cancer; 20of the patients excreted increased amounts of kynurenine,3-hydroxykynurenine, acetylkynurenine, and kynurenicacid compared to normal controls. Subsequently, it wasdemonstrated that cholesterol pellets impregnated with8-hydroxyquinaldic acid, 3-hydroxykynurenine, 3-hydrox-yanthranilic acid, xanthurenic acid, or the 8-methyl etherof xanthurenic acid produced a high incidence of bladdercancer when implanted into the bladder of mice (4). Thefinding that some of the abnormalities of tryptophanmetabolite excretion in patients with bladder cancer mightpersist after eradication of the neoplasm (17) led to thespeculation that abnormal tryptophan metabolite excretionin bladder cancer patients might represent a metabolicaberration in some of those patients that led to increasedexcretion of carcinogenic tryptophan metabolites withsubsequent increased incidence of bladder cancer.

Altman and Greengard (1) noted a linear correlationbetween the level of hepatic tryptophan pyrrolase activityand the urinary excretion of kynurenine in patients with avariety of diseases, most of whom had rheumatoid arthritis. Tryptophan pyrrolase is inducible by tryptophan orcortisol, and conceivably increased activity of this enzymein some of the patients with bladder cancer is responsiblefor the elevated excretion of tryptophan metabolites. Apossible alternative or additional factor might be a decrease in activity of some of the enzymes involved in thispathway, e.g., kynureninase, that would lead to increasedaccumulation of certain metabolites. In this investigation,an attempt was made to correlate hepatic tryptophanpyrrolase and kynureninase activity in patients with bladder cancer with the urinary excretion of some of thetryptophan metabolites after 2 g L-tryptophan load.

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Gallarti, Murphy, Kenny, Nussbaum, and Silvernail

TRYPTOPHAN-NIACIN PATHWAY

CH2-CH-COOH

NH2

I Tryptophan pyrrolase

L-N- formylkynurenine

k-C-CH2-CH-COOH

W2 NH-CO-CHj

<9 N- Acetylkynurenine

OH

COOHChart I. Abbreviated pathway for metabolic

conversion of tryptophan to nicotinic acid.CO-NH-CH2-COOH

NHJ0-Aminohippuric Acid

3-Hydroxykynurenine

Kynureninase4 (Be)

-COOH'N

OH

Xanthurenic acid

jj-COOH _ rj'^i-COOH

3-Hydroxyanthranilic acid

MATERIALS AND METHODS

Thirty-six patients with an established diagnosis ofbladder cancer, who were admitted to Roswell ParkMemorial Institute for investigation for possible totalcystectomy and construction of ileal conduit, were subjected to the following investigative procedures after an informed consent was obtained.

A Menghini needle biopsy aspiration of the liver wasobtained on the 1st 15 patients in this series. A smallportion of the specimen was sent for histological examination and the remaining 10 to 20 mg were used for assay oftryptophan pyrrolase.

L-Tryptophan, 2 g, blended in ginger ale, was given at 9a.m. or within 2 hr after the liver biopsy specimen wasobtained (not later than 10:30 a.m.). This was followed bya 24-hr urine collection in ajar containing 20 ml of glacialacetic acid. Since all the patients had indwelling cathetersas part of their preoperative management, completeness ofthe urinary collection was assured. The urine was keptrefrigerated until delivery to the laboratory where analiquot was obtained and frozen until the day of analysis.Patients with grossly bloody or infected urines and thosereceiving medications such as glucocorticoids, estrogens, orsulfanomides that might influence the excretion of tryptophan metabolites or interefere with the tests were excluded.The urinary excretion of kynurenine, 3-hydroxykynure-nine, kynurenic acid, xanthurenic acid, and acetylkynure-nine was measured by the methods described by Price et al.(15). The same urinary metabolites also were measured in10 patients with cancer other than cancer of the bladder

or lymphoma and in 5 normal men aged 20 to 30 years.Measurement of tryptophan metabolites after tryptophanloading was repeated on 21 of the bladder cancer patients 6to 21 months after total cystectomy; 19 of the patients wereclinically free of evidence of cancer, while 2 had definiteevidence of métastases.

In those patients who had laparotomy, a liver specimenweighing about 0.5 g was obtained early in the operativeprocedure (usually within 20 min of the beginning of theoperation) for assay of tryptophan pyrrolase and kynu-reninase.

Assay of tryptophan pyrrolase activity by the micro-method of Altman and Greengard (1) was first attempted.In this method the 100,000 x g supernatant of 10 to 20 mgliver homogenate was placed in a microcuvet with a 1-cmlight path and was incubated at 37°with tryptophan in the

presence of methemoglobin and rat liver mitochondrialpreparation. Tryptophan pyrrolase activity was calculatedfrom the rate of increase of absorbance at 360 nm(absorption peak of kynurenine) in a Gilford spectropho-tometer. The enzyme activity was expressed as Amólesofkynurenine formed per hr per 100 mg of soluble protein.Assay of the 1st 3 human liver specimens by this method inour laboratory produced very little or no change inabsorbance at 360 nm. Rat liver homogenate preparationprepared as described above and incubated in a water bathat 37°with shaking produced 3- to 4-fold in absorbance at

360 nm compared to incubation of a similar preparation at37°but without shaking. This observation led us to try a

similar method of incubation on human liver preparationswith a resulting substantial increase in spectrophotometric

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Tryptophan Metabolism in Bladder Cancer

absorbance at 360 nm. The supernatant of homogenates ofhuman liver biopsies obtained during laparotomy from 4patients with cancer other than cancer of the bladder wasprepared as described by Altman and Greengard (1);0.2-ml samples of the supernatant of the liver homogenates(obtained from 10 mg of liver) were placed in flasks andincubated with tryptophan, methemoglobin, and rat mito-chondrial preparations in a water bath at 37°with shaking.

Two such samples and a blank preparation containingthese reagents but without the addition of liver weretransferred to microcuvets prior to and after 5, 10, 15, and30 min of incubation, and the spectrophotometric absorbance at 360 nm was measured. Increased absorbancebecame apparent after 5 min of incubation and progressedwith time. Chart 2 indicates the linear increase in absorbance that was observed in 4 individual liver samples duringthe initial 30-min incubation period. No appreciablechange of absorbance occurred in the blank preparations.Tryptophan pyrrolase activity calculated from the changein absorbance between 0 and 30 min in the 4 linearpreparations was 3.2, 3.32, 3.4, and 4.9 ¿¿moleskynurenineper 100 mg protein per hr. When enzyme activity wascalculated from the difference in absorbance reading at 10and 30 min of incubation, comparable results were obtained in 2 specimens, while in the other livers the resultswere higher by 10 and 15%. The results reported in theseexperiments for human liver tryptophan pyrrolase weredistinctly higher than those reported by Altman andGreengard (1). The reason for this is unknown but may berelated to the modification of the method. All subsequentcalculations of tryptophan pyrrolase activity in this studywere based on change in absorbance at 360 nm after 30 minof incubation.

Kynureninase was assayed in the earlier samples by themethod of Dalgliesh et al. (6). Kynureninase was determined by the disappearance of kynurenine from thereaction vessel compared to a control incubation. Becausekynureninase activity was frequently low in the livers ofpatients with bladder cancer, the method of de Castro et al.(7) was used for the assay of this enzyme in the last 16samples. In this method the supernatant of a liver homoge-nate was incubated in the presence of excess L-kynurenineand pyridoxal phosphate and the anthranilic acid formedmeasured.

o9n

uo

200

ISO

O 5 IO15 30 O 5 IO 15 30 O 5 IO15 30 O 5 IO 15 30

INCUBATION TIME IN MIN

Chart 2. Change in absorbance at 360 nm during assay of tryptophanpyrrolase in 4 human liver specimens.

700

600

LÜ

500

o 400O

ci 300^ÃœJ

o 200

100 I

* 1•¿�S"l

lK 30H-K KA XA AcK

Chart 3. Urinary excretion of kynurenine (K), 3-hydroxykynurenine(30H-K), kynurenic acid (KA), xanthurenic acid (XA), and acetylkynure-nine (AcK) after 2 g L-tryptophan load in patients with bladder cancer (O)and cancer of other organs (•)and in normal controls (A).

Protein was measured by the method of Lowry et al.(11).

RESULTS

The urinary excretion of tryptophan metabolites, following a 2-g dose of L-tryptophan, was measured in 36 patientswith bladder cancer prior to operation (Chart 3). On thebasis of data obtained from the literature (3, 5) and thelimited number of normal controls that we studied, theupper limits of normal for the excretion of tryptophanmetabolites after a 2-g L-tryptophan load were set at 70^moles/24 hr for kynurenine, xanthurenic acid, and ace-tylkynurenine and at 100 //moles/24 hr for 3-hydroxykynurenine and kynurenic acid. In the 36 patients withbladder cancer, there was increased excretion of kynurenine in 12, 3-hydroxykynurenine in 25, kynurenic acid in 9,xanthurenic acid in 4, and acetylkynurenine in 10. Thepattern of excretion of the various metabolites in the 15patients who received the tryptophan immediately afterneedle biopsy of the liver was similar to those who were notsubjected to the procedure (Chart 4). Similar measurements were performed on 10 patients with cancer otherthan cancer of the bladder or lymphoma; there wasincreased excretion of kynurenine in 4, 3-hydroxykynurenine in 6, kynurenic acid in 1, xanthurenic acid in 2, andacetylkynurenine in 3 (Chart 3).

Increased excretion was seen in any stage of bladdercancer, but values in excess of 450 ¿imoles/24hr (sum ofthe 5 tryptophan metabolites measured in the urine) wereseen only in the more extensive Stage D, and D2 disease(Chart 5).

Tryptophan pyrrolase activity was assayed in needlebiopsy specimens of the liver of 15 patients with bladdercancer. In 10 of these patients, the enzyme was subsequently measured in a surgical biopsy specimen of the liver.In 6 other patients, the enzyme was assayed in samples ofliver obtained by surgical biopsy only (Chart 6). The results

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Gailani, Murphy, Kenny, Nussbaum, and Silvernail

700

111zero

OX>^C/)UJ0a.600500400300200100•

•¿�•

•¿�.

•¿�«••?

;•• i r r«¡i" M ¿tt¿K 30H-K KA XA Ac K

Chart 4. Tryptophan metabolite excretion in patients with bladdercancer who received 2 g L-tryptophan shortly after needle biopsy of theliver (O) and in those who were not subjected to needle biopsy of the liver(•).Abbreviations as in Chart 3.

1000

900

800

u 70O

E* 60°

500CVJ

Ì2400

3OO

200

IOO

B Di DzNORMAL ACONTROLS

BLADDER CANCER-STAGE OF DISEASE

Chart 5. Correlation of the stage of bladder cancer with the 24-hrurinary excretion of the sum of the 5 tryptophan metabolites measuredafter 2 g L-tryptophan load. •¿�.O, excretion of the sum of thosemetabolites in the normal controls.

shown in Chart 7 indicate that there was no correlationbetween tryptophan pyrrolase activity in either the needlebiopsy or the surgical biopsy specimens and the 24-hrurinary excretion of kynurenine. Similarly, there was nocorrelation between the activity of this enzyme and theexcretion of the other metabolites or the sum of excretionof these metabolites. Moreover, there was no correlationbetween tryptophan pyrrolase activity and the stage ofbladder cancer.

Tryptophan pyrrolase was measured in 5 liver specimensobtained at laparotomy on patients with neoplasms otherthan cancer of the bladder or lymphoma. The range ofenzyme activity in these liver specimens was 1.98 to 4.4Amóleskynurenine per 100 mg protein per hr with amedian of 2.94; this is similar to the range of enzymeactivity found in the liver of patients with bladder cancer.

Kynureninase was assayed in 24 surgical biopsy specimens obtained from patients with bladder cancer by themethod of Dalgliesh et al. (6). The values ranged from0.118 to 0.56 ¿tmoleof kynurenine metabolized per 10 mgprotein per hr. Kynureninase tended to be lower in patientswith Stage D! and D2 disease compared to those with lessextensive disease (Chart 8). Twenty of these patients weregiven 2 g L-tryptophan, and the subsequent 24-hr urinespecimens were assayed for tryptophan metabolites. Pa

rr¡6

ÃœJ5

foQ 3

tr

ItoLU

NEEDLE SURGICALBIOPSY BIOPSY

Chart 6. Tryptophan pyrrolase activity in needle and surgical liverbiopsies of bladder cancer patients. Lines connect samples done on thesame patients.

3|4

¡|3Q- 2

<Ã?̄ 2i z

S 2t- tn

3 20 40 60 80 100 120 140 160 180 200 220

S_ KYNURENINE (jMOLES/24 HOUR URINE

Chart 7. Lack of correlation between tryptophan pyrrolase activity,surgical (•)and needle biopsy (O) liver specimens, and 24-hr urinaryexcretion of kynurenine after 2 g L-tryptophan load in patients withbladder cancer.

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Tryptophan Metabolism in Bladder Cancer

0.6

?, 0.5

Z E

0.4

i O-3

| 0.2

ì 0.1

B C

STAGE OF DISEASE

Di

Chart 8.measured by

Hepatic kynureninase in patients with bladder cancerthe method of Dalgliesh el al.

tients with low kynureninase activity tended to excretelarger amounts of kynurenine. A better correlation couldbe demonstrated when the sum of the amounts of kynurenine, 3-hydroxykynurenine, kynurenic acid, and acetylky-nurenine excreted was compared with kynureninase activity (Chart 9). Patients with low kynureninase excretedlarger amounts of these metabolites compared to thosewith high enzyme activity.

Kynureninase was assayed by the method of de Castro etal. (7) in the liver of 16 patients with bladder cancer. Inthese patients, the enzyme activity ranged from 0.062 to1.24 Amólesanthranilic acid per 10 mg protein per hr(Chart 10). Patients with Stage D2 bladder cancer had thelowest activity. In 14 of these patients urinary tryptophanmetabolites were measured, and again, patients with lowkynureninase activity excreted increased amounts of tryptophan metabolites. Kynureninase measured by the lattermethod on 8 liver specimens obtained from patients withneoplasms other than cancer of the bladder or lymphomaranged from 0.063 to 0.12 /¿moleper 10 mg protein perhr.

Kynureninase was measured by the methods of bothDalgliesh et al. and de Castro et al. in 4 liver samples(Table 1). There was good correlation between the 2methods, although the absolute activities were different.

Five of the 36 patients with bladder cancer in this serieshad recurring bladder papillomas over a period of 4 to 9years, and 6 patients had 3 to 8 local recurrences of cancerafter excision over a period of 2 to 24 years. In 10 of thesepatients, the urinary excretion of tryptophan metaboliteswas within the normal range, a finding which fails tosupport a role for the increased excretion of these metabolites in the etiology of the recurring tumors.

Measurement of the urinary excretion of tryptophanmetabolites was repeated in 19 patients 6 to 12 monthsafter the eradication of their disease (Chart 11). All of thesepatients were clinically free of bladder cancer. The excretion of kynurenine was significantly lower in the postoperative period (p < 0.01). There was no statistical differencebetween the pre- and postoperative excretion of the othermetabolites (p > 0.05), although the excretion of kynu

renic acid, xanthurenic acid, and acetylkynurenine waswithin the normal range in all the postoperative specimens.However, 9 out of 19 patients continued to excrete morethan 100 Amólesof 3-hydroxykynurenine per 24 hr. Similar

8 °-6r

C 0.5

Iz E

0.4

0.3

£ 0.2

i 0.1

*_ 100 200 300 400 500 60O 70O

pMOLES/24 HOURURINE

Chart 9. Correlation of hepatic kynureninase (Dalgliesh el al.) and thesum of 24-hr urinary excretion of kynurenine, 3-hydroxykynurenine,kynurenic acid, and acetylkynurenine after 2 g L-tryptophan load inpatients with bladder cancer.

KQ 0.14

0.12

» 0.10LÜE(/) Oz à 0-08z yLU «

Z -I>- z

0.06

0.04

0.02

OD| Dz OTHER

CANCERS

STAGE OF DISEASE - BLADDER CANCER

Chart IO. Hepatic kynureninase measured by the method of de Castroel al. in patients with various stages of bladder cancer and in patients withcancer of other organs.

Table 1Kynureninase measured by methods of Dalgliesh et al. and de Castro et al.

in 4 liver specimens

Dalgliesh et al.(¿/molekynurenine utilized/

10mg protein/hr)

de Castro et al.(/itnole anthranilic acid/

10mg protein/hr)

0.170.290.340.470.0640.0840.100.12

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Galloni, Murphy, Kenny, Nussbaum, and Silvernail

350

300

I 250

I 200

150

100

50

K 30H-K KA XA AcKChart I \. Pre- (O) and postoperative (•)excretion of kynurenine (K),

3-hydroxykynurenine (30H-K), kynurenic acid (KA), xanthurenic acid(XA), and acetylkynurenine (AcK) after 2 g L-tryptophan load in patientswith bladder cancer who became clinically free of cancer. Similar pre- (A)and postoperative (A) measurements in patients who developed definitepostoperative métastases.

measurements were done on 2 other patients 8 and 10months after total cystectomy at a time when there wasdefinite evidence of recurrence of the neoplastic disease; incomparison to the preoperative period both patientsshowed an increase in the excretion of the various metabolites, including kynurenine.

DISCUSSION

The finding of frequent increased urinary excretion oftryptophan metabolites of the kynurenine pathway inpatients with bladder cancer and the persistence of theseabnormalities in some of the patients after eradication ofthe disease were taken as evidence that an aberration oftryptophan metabolism in some patients leads to anincreased excretion of carcinogenic tryptophan metabolitesand consequent high incidence of bladder cancer (13). Theabsence of similar abnormalities in 16 patients withindustrial bladder cancer was used as additional evidence insupport of the latter hypothesis (3). On the other hand, thedemonstration by several workers of similar although notalways identical abnormalities in tryptophan metabolismin a variety of clinical states including lymphoma andcancer of organs other than the bladder, porphyria,scleroderma, rheumatoid arthritis, and pregnancy raisedthe possibility that these abnormalities were most probablythe result of the disease state rather than its cause (12).

In this study, the high incidence of increased excretion oftryptophan metabolites in patients with bladder cancer wassimilar to that reported by Brown et al. (3). Similar to theobservations made in patients with Hodgkin's disease (5)

and breast cancer (8), these abnormalities were morepronounced in patients with the more advanced D¡and D2stages of the disease. Moreover, many of the patients whoexhibited marked abnormalities died within 1 year.

The normal excretion of kynurenine, kynurenic acid,xanthurenic acid, and acetylkynurenine postoperatively indisease-free patients could support the argument that the

abnormal tryptophan metabolism in bladder cancer patients is secondary to the disease process. However, theargument is weakened by the persistence of increased3-hydroxykynurenine excretion in 9 of these patients. Thelow kynurenine and high 3-hydroxykynurenine excretionpostoperatively might be the result of increased reabsorp-tion of kynurenine compared to 3-hydroxykynurenine bythe ileal conduit; in experiments with rat ileum theabsorption of kynurenine was much more efficient thanthat of 3-hydroxykynurenine (16). The persistence of veryhigh kynurenine excretion postoperatively in 2 other patients who had definite evidence of disease activity tends toexclude the reabsorption of kynurenine by the ileal conduitas the only cause for the restoration of kynurenineexcretion to normal values. The persistence of increased3-hydroxykynurenine might be the result of other clinicalproblems from which the patients suffered.

The greater increase in the excretion of tryptophanmetabolites in advanced breast cancer compared to themore limited disease was interpreted by De George andBrown (8) as evidence supporting the suggestion of Altmanand Greengard (1) that increased tryptophan pyrrolaseactivity is responsible for this effect. This enzyme isinduritale by glucocorticoids, and it is conceivable that thestress of cancer could result in increased tryptophanpyrrolase activity and consequent increase in the excretionof tryptophan metabolites. The introduction of minormodifications to the micromethod of tryptophan pyrrolaseassay resulted in a much higher level of this enzyme thanthat reported by Altman and Greengard (1). The range oftryptophan pyrrolase activity in the present series was 1.98to 4.9 Amóles kynurenine per 100 mg protein per hrcompared to 0.26 to 1.2 Amóleskynurenine per 100 mgprotein per hr in the series of Altman and Greengard (1).Perhaps the higher activity in this series was the result ofstress caused by cancer and hospitalization in our patients.However, the demonstration in our laboratory of a 3- to4-fold increase in tryptophan pyrrolase activity in rat liverpreparations assayed by our modification implies that thedifference in the assay methods was responsible for thehigher values for tryptophan pyrrolase in this series.Contrary to what has been reported in patients withrheumatoid arthritis and several other diseases (1), therewas no correlation between tryptophan pyrrolase activityand the urinary kynurenine excretion in the cancer patientsstudied in this series. A similar lack of correlation wasfound between the activity of the enzyme and the excretionof the other metabolites. This lack of correlation could notbe attributed to a possible difference of 15% in activity thatmight occur if the increase in absorbance was calculatedafter 30 min of incubation rather than by using the linearpart of the increase in absorbance during the 1st 30 min tocalculate this value.

The frequent observation of elevated kynurenine and3-hydroxykynurenine in the urine of patients with bladdercancer led us to measure the level of hepatic kynureninasein patients with bladder cancer. Kynureninase catalyzes theconversion of kynurenine into anthranilic acid and 3-hydroxykynurenine into 3-hydroxyanthranilic acid (10).

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We observed low activity of kynureninase in many patientswith bladder cancer, particularly in the patients with themore advanced disease. This hypoactivity was not causedby vitamin B6 deficiency, since an excess amount ofpyridoxal phosphate was added to the assay system invitro. Patients with low kynureninase tend to excrete largerquantities of tryptophan metabolites in the urine. Thus,kynureninase may play a significant although not the solerole in the control of the level of various metabolites of thetryptophan-niacin pathway. Low activity of kynureninasealso was observed in patients with other neoplasms.

REFERENCES1. Altman, K., and Greengard, O. Correlation of Kynurenine Excretion

with Liver Tryptophan Pyrrolase Levels in Disease and afterHydrocortisone Induction. J. Clin. Invest., 45: 1527-1534, 1966.

2. Benassi, C. A., Perissinotto, B., and Allergri, G. The Metabolism ofTryptophan in Patients with Bladder Cancer and Other UrologicalDiseases. Clin. Chim. Acta, 8: 822-831, 1963.

3. Brown, R. R., Price, J. M., Satter, E. J., and Wear, J. B. TheMetabolism of Tryptophan in Patients with Bladder Cancer. ActaUnióIntern. Contra Cancrum, 16: 299 303, I960.

4. Bryan, G. T., Brown, R. R., and Price, J. M. Mouse BladderCarcinogenicity of Certain Tryptophan Metabolites and Other Aromatic Nitrogen Compounds Suspended in Cholesterol. Cancer Res..24: 596 602, 1964.

5. Chabner, B. A., DeVita, V. T., Livingston, D. M., and Oliverio, V. T.Tryptophan Metabolism and Plasma Pyridoxal Phosphate in Hodg-kin's Disease. New Engl. J. Med., 282: 838-843, 1970.

6. Dalgliesh, C. E., Knox, W. E., and Neuberger, A. IntermediaryMetabolism of Tryptophan. Nature, 168: 20 23, 1951.

Tryptophan Metabolism in Bladder Cancer

1. de Castro, F. T., Brown, R. R., and Price, J. M. The IntermediaryMetabolism of Tryptophan by Cat and Rat Tissue Preparations. J.Biol. Chem., 225: 777-784, 1957.

8. De George, F. V., and Brown, R. R. Differences in TryptophanMetabolism between Breast Cancer Patients with and without Cancerat Other Sites. Cancer, 26: 767-770, 1970.

9. Dunning, W. F., Curtis, M. R., and Maun, M. E. The Effect ofAdded Dietary Tryptophan on the Occurrence of 2-Acetylamino-fluorene-induced Liver and Bladder Cancer in Rats. Cancer Res., 10:454-459, 1950.

10. Knox, W. E. The Relation of Liver Kynureninase to TryptophanMetabolism in Pyridoxine Deficiency. Biochem. J., 53: 379 385,1953.

11. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.Protein Measurement with Folin Phenol Reagent. J. Biol. Chem.,193: 265-275, 1951.

12. Musajo, L., and Bennassi, C. A. Aspects of Disorders of theKynurenine Pathway of Tryptophan Metabolism in Man. Advan.Clin. Chem., 7: 63-135, 1964.

13. Price, J. M. Bladder Cancer. Can. Cancer Conf., 6: 224 243, 1966.14. Price, J. M., and Brown, R. R. Studies on the Etiology of Carcinoma

of the Urinary Bladder. Acta Unió Intern. Contra Cancrum, 18:684-688, 1962.

15. Price, J. M.. Brown, R. R., and Yess, N. Testing the FunctionalCapacity of the Tryptophan-Niacin Pathway in Man by Analysis ofUrinary Metabolites. Advan. Metabolic Disorders, 2: 159-225, 1965.

16. Rose, D. P., Yi-Min, S. Y., and Brown, R. R. Transport and FurtherMetabolism of Intermediate Compounds of the Tryptophan Nico-tinic Acid Ribonucleotide Pathway by Rat Small Intestine. Biochim.Biophys. Acta, 163: 93 100, 1968.

17. Yoshida, O., Brown, R. R., and Bryan. G. T. Relationship betweenTryptophan Metabolism and Heterotopic Recurrences of HumanUrinary Bladder Tumor. Cancer, 25: 773-780, 1970.

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1973;33:1071-1077. Cancer Res   S. Gailani, G. Murphy, G. Kenny, et al.   CancerStudies on Tryptophan Metabolism in Patients with Bladder

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