Quantitative Analysis of Serotonin Biosynthesis in

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1 INFECTION AND IMMUNITY, Aug. 1974, p Copyright 1974 American Society for Microbiology Vol. 10, No. 2 Printed in U.S.A. Quantitative Analysis of Serotonin Biosynthesis in Endotoxemial KATHERINE M. MORRIS2 AND ROBERT J. MOON Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan Received for publication 28 March 1974 Endotoxin-poisoned mice converted significantly greater quantities of tryptophan to serotonin than did normal mice. The percentage increase, approximately 10 to 15%, was the same whether mice were given only trace quantities of tryptophan or tryptophan load (20 mg of L-tryptophan). The increased serotonin synthesis was accompanied by a decreased flow of tryptophan into the kynurenine pathway. These results indicate that excess serotonin synthesis occurs not only at dose levels of tryptophan which are toxic for endotoxin-poisoned mice but also at physiological levels of tryptophan. This latter observation may have some importance in the overall pathophysiology of endotoxin shock. Endotoxin-poisoned mice frequently die in convulsions within 4 to 8 h after tryptophan load (1, 13, 15). Since this hyper-reactivity to tryptophan can be prevented by pretreatment with cyproheptadine, an antiserotonin drug, we have felt that overproduction of serotonin might be at the basis of the hyper-reactivity. Repeated efforts in our laboratory to reliably quantitate serotonin production by traditional fluorimetric procedures have failed, primarily because we have been unable to completely remove contaminating metabolic products of tryptophan which interfere with the assay. For this reason we developed a procedure to monitor the rate of serotonin synthesis from tryptophan by monitoring the amount of radioactive metabo excreted from mice given D, L-tryptophan (benzene ring-14c). The method is based on the assumption that the metabolic profile of tryptophan metabo in urine approximates the in vivo distribution of the amino acid between the kynurenine and serotonin pathways and hence by determining the quantity of both kynurenine and serotonin metabo in urine we can accurately judge in vivo pathway flow. The details of our methodology will be published elsewhere (K. M. Morris and R. S. Moon, Anal. Biochem., submitted for publication). Development of this procedure has also allowed us to analyze the rate of serotonin synthesis from tryptophan in normal and endotoxinposioned animals given only trace quantities (<1 Ag) of radioactive tryptophan. This latter 'Journal article no from the Michigan Agricultural Experimental Station. 2 Present address: Department of Bacteriology. University of California at Los Angeles, Los Angeles. Calit approach is of particular interest because it quantitatively determines whether endotoxin poisoning alters the rate of serotonin production on a physiological level and hence becomes more directly pertinent to endotoxin pathophysiology and endotoxin shock. Although there has been considerable debate as to the significance of serotonin in the pathophysiology of endotoxin shock (6-8, 14, 15), it is well established that release of preformed serotonin occurs relatively soon after endotoxin poisoning (7, 8). Hence, if serotonin plays a role in the pathophysiology of endotoxin shock, significant quantities of the biogenic amine would have to be synthesized de novo. The primary objective of this paper is to describe the quantitative flow of tryptophan between the kynurenine and serotonin pathways in vivo and to determine whether this distribution is significantly altered in endotoxinpoisoned mice given a 20-mg tryptophan load. MATERIALS AND METHODS Mice. Eighteen to 20-g female CF-1 mice (Carworth Farms, Portage, Mich.) were used throughout the study. They were housed six per cage with wood chips as bedding. Purina laboratory chow (Ralston Purina Co., St. Louis, Mo.) and water were provided ad libitum. All mice were starved for at least 7 h prior to endotoxin administration. Endotoxin. Heat-killed cells of Salmonella typhimurium, strain SR-11 (13), served as the source of endotoxin in all experiments. The mean lethal dose (LD50) of this preparation for mice, determined according to the method of Reed and Muench (18), was approximately 4 x 109 heat-killed organisms. Endotoxin was diluted in nonpyrogenic physiological saline 340

2 VOL 10, 1974 SEROTONIN BIOSYNTHESIS IN ENDOTOXEMIA and was administered intraperitoneally 10 h prior to radioactive tryptophan. Chemicals and radioisotopes. L-Tryptophan was purchased from Nutritional Biochemicals Co. (Cleveland, Ohio). D,L-Tryptophan (benzene ring- 14C), specific activity 95 mci/mmol, was purchased in 50-,gCi amounts from Amersham/Searle (Des Plaines, Ill.). To reduce degradation, it was diluted to 0.5 MCi/ml (1.1 x 106 dpm per ml) in 100 ml of sterile saline (Baxter Laboratories, Morton Grove, Ill.), sampled in 5-ml volumes, frozen in dry ice, relyophilized, and stored at -70 C. The isotope was reconstituted to original volume in deionized distilled water just prior to use. For tryptophan load studies, 20 mg of unlabeled L-tryptophan was dissolved in 1 ml of the labeled solution just prior to injection. All injections of tryptophan were given intraperitoneally at 9 a.m. Collection of urine. The total amount of radioactivity excreted in urine was determined by collecting urine on filter paper disks as described previously (16). For calculation of disintegrations per minute per microliter and identification and quantitation of urinary tryptophan metabo, urine was collected from mice in metabolism cages. The disintegrations per minute per microliter were determined by spotting duplicate 10-,gliter samples of urine on strips of Whatman no. 1 filter paper, counting in a Packard 2420 liquid scintillation spectrometer, and making appropriate calculations using a counting efficiency of 62%. Identification of serotonin and kynurenine metabo. The methods used for identification and quantification of urinary tryptophan metabo have been described in detail by K. M. Morris and R. J. Moon (Anal. Biochem., submitted for publication). Briefly, 20 to 60 uliters of urine from individual mice are spotted on thin-layer plates for ascending twodimensional chromatography in methyl acetate, isopropanol, and ammonium hydroxide (45:35:20) and butanol, acetic acid, and water (12:3:5). R, values of all radioactive tryptophan metabo present in the urine sample were calculated from autoradiographs of the thin-layer plates. Data from fluorescence, color reactions with several spray reagents, and diethylaminoethyl-cellulose chromatography (4) aided in identification of excreted metabo (K. M. Morris and R. J. Moon, Anal. Biochem., submitted for publication). Quantitation of urinary serotonin and kynurenine metabo. The radioactive spots observed by autoradiography were carefully scraped from thinlayer plates with a small spatula, placed in scintillation vials, and crushed. A 10-ml amount of toluene, 2, 5-diphenyloxazolyl, and 1, 4-bis-(5-phenyloxazolyl)-benzene scintillation cocktail and 5 ml of ethyleneglycolmonomethylether were added and the samples were counted in a Packard model 2420 Tri-Carb liquid scintillation spectrometer. The percentage of total counts per minute recovered from the urine sample was calculated for each spot on the chromatogram. No more than 1% of the original isotope remained on the plate. Statistical methods. Since the absolute quantities of individual metabo varied extensively among the plates as a direct consequence of the amount of 341 radioactivity spotted, all data were calculated as a function of a percentage of the total radioactivity recovered. The statistical differences between percentages of individual metabo were determined by the White rank test (20). For the statistical comparison of the total kynurenine or serotonin pathway metabo excreted, the total percentage of metabo for a given pathway was determined by adding the percentages of individual metabo from each pathway on a single plate, and the percentage differences among plates were compared by using the rank-order test. RESULTS Quantity of radioactivity recovered from urine of normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-'4c) without and with tryptophan load. Within 1 h after intraperitoneal injection of labeled tryptophan without load, normal mice excreted 21.6% of the isotope in their urine (Table 1). The amount gradually increased to a total of 29.2% after 6 h. Endotoxin-poisoned animals excreted 6% of the isotope during the first hour and only 17% by 6 h. All decreases were statistically significant at P < If the data are expressed as disintegrations per minute per microliter of urine instead of the total isotope excreted, differences between normal and endotoxin-poisoned mice were significant only at the 3-h time period (Table 1). In mice given a tryptophan load, the differences in total urinary excretion between normal and endotoxin-poisoned mice were significant at 1, 2, and 3 but not 6 h (Table 2). At 1 h the disintegration per minute per microliter count was significantly higher in normal mice with load but not at any other time period. Relative percentage of serotonin and kynurenine pathway metabolite in urine of normal and endotoxin-poisoned mice given D, L- tryptophan (benzene ring-14c) without and with load. The relative quantity of serotonin pathway metabo was significantly increased in endotoxin-poisoned mice without load at all time periods (Fig. 1). The average increase was approximately 6%. The quantity of kynurenine pathway metabo was decreased on an average of 10% in endotoxin-poisoned mice throughout the 6-h experimental period. In mice given a tryptophan load, excretion of serotonin metabo was significantly increased in endotoxin-poisoned animals at all time periods except 2 h after tryptophan (Fig. 2). The average increase was greater than 10% at all time periods studied. On an average, 9% more kynurenine pathway metabo were excreted in normal than in endotoxin-poisoned mice given tryptophan load. The values be-

3 342 MORRIS AND MOON INFECTr. IMMUNITY TABLE 1. Time (h) Quantity of radioactivitv recovered from urine of normal and endotoxin-poisoned mice given D, L-trvptophan (benzene ring-14c) without load Normala Endotoxin Total dpm (%) dpm peruliter X SD Total dpm (') dpm per liter + SD 1 238,990 (21.6) ,412 (6.0) ,105b (23.7) i ,487 (9.9) ± ,807b (27.2) 351.1c i ,650 (12.1) ± ,243b (29.2) i ,505 (17.0) ± a All values represent averages of data from at least five mice. SD, Standard deviation. bp < c p < TABLE 2. Time (h) Quantity of radioactivitv recovered from urine of normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-"c) with load Normala Endotoxin Total dpm (%) dpm per uliter ± SD Total dpm (%) dpm per gliter + SD 1 250,695b (22.8) 1,681.5c ,464 (9.5) ,442c (28.2) ± ,707 (14.0) i ,440b (28.5) i ,071 (15.3) ± ,566 (25.1) ,219 (19.1) a All values represent averages of data from at least five mice. SD, Standard deviation. P < cp < tween the two groups were statistically different at 1, 2, and 3 but not 6 h. Quantitative enumeration of individual serotonin and kynurenine pathway metabo after 1 and 6 h in normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-'4c) without load. Tables 3 and 4 show the relative percentages of individual serotonin and kynurenine pathway metabo at 1 and 6 h after labeled tryptophan was injected into animals without load. Five serotonin pathway metabo, including 5-hydroxyindoleacetic acid, 5-hydroxytryptophol, and 5-hydroxytryptamine-0-glucuronide, were enumerated. The glucuronides of 5-hydroxytryptophol and 5- hydroxyindoleacetic acid migrated closely together on the thin-layer plates and were quantitated as one spot. Although no differences in the individual serotonin pathway metabo were detected after 1 h in normal compared with endotoxin-poisoned animals, the relative percentage of serotonin metabo was significantly increased (Table 3). This increase was also observed after 6 h, and at this period a statistically significant difference in the amount of 5-hydroxyindoleacetic acid excreted was also observed (Table 4). The six kynurenine metabo quantitated were kynurenine, 3-hydroxykynurenine, kynurenic acid, xanthurenic acid, kynurenine-0-sulfate, and N-acetylkynurenine. As noted previously, the percentage of kynurenine pathway metabo was significantly decreased in urine of endotoxin-poisoned mice after 1 h but not after 6 h. At 1 h statistical differences were noted in the relative percent of kynurenine and N-acetylkynurenine excreted. The remaining kynurenine pathway metabo were not statistically decreased but the total was significantly decreased in endotoxinpoisoned compared with normal mice (Table 3). After 6 h the relative percentages of 3-hydroxykynurenine and xanthurenic acid were significantly decreased in urine of endotoxin-poisoned mice, and yet the total kynurenine metabo were not significantly different after 6 h (Table 4). The percent of excreted label recovered as tryptophan ranged from 14.8% in endotoxinpoisoned mice to 21% in normal mice after 1 h (Table 3). At 6 h the values were 27.7% in normal mice and 34.1% in endotoxin-poisoned animals (Table 4). None of the differences were statistically significant. Unidentified metabo accounted for 15 to 30% of the total label excreted in urine. Quantitative enumeration of individual serotonin and kynurenine pathway metabo after 1 and 6 h in normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-'4c) with a 20-mg trypophan load. Ta-

4 VOL. 10, 1974 SEROTONIN BIOSYNTHESIS IN ENDOTOXEMIA 343 bles 5 and 6 compare the relative percentages of individual serotonin and kynurenine pathway metabo in urine of normal and endotoxinpoisoned mice given tryptophan load. At the 1-h time period only the percent of 5-hydroxyindoleacetic acid was significantly increased in endotoxin-poisoned compared with normal mice a.35 - O: 1:0 x2 aj 20 cr w c20 IO Z 25- O.- 0 U)10 * NORMAL DENDOTOXIN U HOURS FIG. 1. Relative percentage of serotonin urenine pathway metabo in urine of n endotoxin-poisoned mice given D, L-tryptol zene ring-14c) C a 'c35- w e al c30- c tz 0 ic25- z C.) Id a. x r20- o51 o60 5- O- TABLE 3. Relative percentage of individual serotonin and kynurenine pathway metabo in urine of normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-'4c) without load after I h 5-Hydroxyindoleacetic b acid 5HT-O-glucuronide HTOH- and 5HIAA-O glucuronide 5-Hydroxytryptophol 0.0 ± ± 0.9 Total serotonin metabo c 18.0 ± 2.7 Kynurenine d 1.4 ± Hydroxykynurenine 6.9 ± ± 0.9 Kynurenic acid 11.7 ± ± 1.9 Xanthurenic acid 11.5 i ± 0.9 Kynurenine-O-sulfate 1.8 ± ± 0.5 N-acetylkynurenine 8.8 ± 3.0d 21.7 ± 3.6 Total kynurenine metab c 34.6 ± 3.3 o Tryptophan ± 4.9 Other metabo 17.7 ± ± 6.1 Total metabo 98.0 ± ± 4.3 aall values represent averages of data from between four and seven mice. 56 b Average percentage of radioactivity recovered ± standard deviation. Cp l and kyn- d P < Iormal and phan (ben- (Table 5). At 6 h the percentage of all serotonin metabo was significantly increased in en- * NORMAL ENDOTlOXIN dotoxin-poisoned mice (Table 6). For the kyn- I I I HOURS FIG. 2. Relative percentage of serotonin and kynurenine pathway metabo in urine of normal and endotoxin-poisoned mice given D, L-tiyptophan (benzene ring-14c) with a 20-mg L-t,yptophan load. urenine pathway after 1 h with load, kynurenic acid was significantly decreased in endotoxinpoisoned mice and the total radioactivity excreted was also significantly reduced by 13%. (Table 5). At 6 h kynurenine and kynurenine-osulfate were reduced (P < 0.01 and P < 0.05, - respectively) in endotoxin-poisoned animals with load (Table 6). The quantity of tryptophan excreted in animals given a tryptophan load varied between 32 and 37%. There were no statistical differences observed in the quantity of tryptophan excreted. Likewise, unidentified metabo were between 16 and 18% and again showed no statistical differences between normal and endotoxin-poisoned animals. Total recovery was approximately 99%. DISCUSSION Tryptophan labeled with '4C in benzene ring provided an invaluable tool for the accomplishment of our primary objective. Because of the position of the label, it is retained in all of the metabo of the serotonin pathway and all

5 344 MORRIS AAlND MOON INFECT. IMMUNITY TABLE 4. Relative percentage of individual serotonin TABLE 6. Relative percentage of individual serotonin and kynurenine pathway metabo in urine of and kynurenine pathway metabo in urine of normal and endotoxin-poisoned mice given normal and endotoxin-poisoned mice given D, L-trvptophan (benzene ring-'4c) without load after D, L-trvptophan (benzene ring-14c) with load after6h 6 h 5-Hydroxyindoleacetic 4.3 ± Hydroxyindoleacetic b 13.4 i 4.1 acid acid 5HT-O-glucuronide c HT-O-glucuronide HTOH- and 5HIAA-O b 3.1 ± 0.5 5HTOH- and 5HIAA glucuronide O-glucuronide 5-Hydroxytryptophol 0.0 ±f ± Hydroxytryptophol Total serotonin metabo t 1.3c Total serotonin metab c o Kynurenine e Kynurenine 3.1 _ Hydroxykynurenine ± Hydroxykynurenine c Kynurenic acid Kynurenic acid Xanthurenic acid Xanthurenic acid b Kynurenine-O-sulfate b Kynurenine-O-sulfate i 0.2 N-acetylkynurenine 7.3 ± N-acetylkynurenine Total kynurenine metab c 26.4 ± 6.8 Total kynurenine me o tabo Tryptophan Tryptophan Other metabo 18.6 _ Other metabo Total metabo Total metabo aall values represent averages of data from between four and seven mice. baverage percentage of radioactivity recovered 4 standard deviation. P < cp < TABLE 5. Relative percentage of individual serotonin and kynurenine pathway metabo in urine of normal and endotoxin-poisoned mice given D, L-tryptophan (benzene ring-14c) with load after 1 h 5-Hydroxyindoleacetic acid b HT-O-glucuronide HTOH- and 5HIAA-O- 1.1 ± ±0.4 glucuronide 5-Hydroxytryptophol Total serotonin metabo c Kynurenine Hydroxykynurenine Kynurenic acid lb Xanthurenic acid Kynurenine-0-sulfate N-acetylkynurenine i 4.9 Total kynurenine metabo t 2.5c Tryptophan Other metabo Total metabo a All values represent averages of data from between four and seven mice. b Average percentage of radioactivity recovered standard deviation. P < C p < a All values represent averages of data from between four and seven mice. "Average percentage of radioactivity recovered _ standard deviation. P < cp < but the terminal metabo of the kynurenine pathway. Injection of tryptophan was delayed until 10 h after endotoxin administration, since this is a time when CF-1 mice are particularly hyper-reactive to tryptophan load (13). When endotoxin-poisoned mice were given benzene-ring-labeled tryptophan without load, they excreted significantly less tryptophan and metabo than normal mice throughout the 6-h time period (Table 1). Tryptophan load did not significantly alter the quantity of label appearing in urine (except after 1 h in normal mice) despite the fact that 20,000 times more tryptophan was administered. Interestingly, the amount of label per unit volume was similar in both normal and endotoxin-poisoned mice without and with load, except at the 6-h period in normal mice. These data suggest a passive rather than an active mechanism for the renal filtration of tryptophan metabo. The decrease observed in urine of endotoxin-poisoned mice is probably due to the decreased blood flow to the kidneys due to vascular shock (11, 17). The decreased excretion of radioactivity in endotoxin-poisoned mice was accompanied by a significant increase in the percentage of serotonin metabo and a decrease in kynurenine metabo excreted (Fig. 1 and 2). The shift in

6 VOL. 10, 1974 SEROTONIN BIOSYNTHESIS IN ENDOTOXEMIA 345 metabolism from the kynurenine to the serotonin pathway in endotoxin-poisoned mice occurred in animals both without and with load. The data both without and with load are particularly significant in their own right. The fact that poisoned animals produce more serotonin from tryptophan when not given a tryptophan load suggests that serotonin production is increased throughout the course of the poisoning, even in the absence of tryptophan load. Such an event may have significance in the overall pathogenesis of endotoxemia. The fact that we also observed significant increases in serotonin synthesis in endotoxin-poisoned animals given a tryptophan load has particular relevance to the mechanisms of the hyper-reactivity of endotoxin-poisoned mice to tryptophan. These data are the first quantitative evidence supporting the theory that tryptophan is funneled into serotonin synthesis in endotoxin-poisoned animals. The precise metabolic reason for this funneling remains unclear. Several authors have postulated that tryptophan oxygenase activity regulates the flow of tryptophan into the kynurenine pathway. Our data suggest that this is not the case since over a 20,000-fold concentration gradient the amount of substrate entering the kynurenine pathway remains fairly constant. Further, it seems unlikely that decreased flow through the kynurenine pathway in endotoxin-poisoned mice can be attributed to the depression of tryptophan oxygenase since there is a 40% decrease in enzyme activity in the poisoned animal (17) but only a 10% decrease in flow through the kynurenine pathway. We conclude from these data that although increased synthesis of serotonin from tryptophan does occur in endotoxin-poisoned mice either with or without tryptophan load, the enzymatic basis for these changes, if in fact one exists, is not established. In all, 11 kynurenine metabo were isolated from urine of normal and endotoxin-poisoned mice given labeled tryptophan without and with tryptophan load. In addition to the compounds described in the results, 5-hydroxyindoleacetic acid-0-sulfate, anthranilic acid, 3-hydroxyanthranilic acid, N-methylnicotinamide, 0-aminophenol, and quinolinic acid were also tentatively identified, but they appeared infrequently and in quanitities too low to be measured confidently. Six serotonin metabo were isolated from urine of mice given labeled tryptophan. Endotoxin-poisoned mice consistently excreted significantly more 5-hydroxyindoleacetic acid than normal mice. At 2, 3, and 6 h the glucuronide conjugates of serotonin were also significantly increased in endotoxin-poisoned mice (Table 6). Glucuronides are known to be detoxification products in mammals (17). 5-Hydroxyindoleacetic acid, which has been used as an indicator for metabolic changes in several diseases (9, 12, 17), seems to be a valid marker for endotoxemia as well. It is interesting to note that, in certain instances (cf. Tables 3 to 6), the total quantity of metabo from either the kynurenine or serotonin pathway is significantly altered between normal and endotoxin-poisoned mice even though very little significant change is noted between individual tryptophan metabo. These data emphasize the fact that subtle changes can occur in the pathway flow of metabo in vivo which are clearly statistically significant but may not be noted if only one or a few metabo are monitored. The high excretion rate of unmetabolized tryptophan was not expected. In animals given a tryptophan load, some tryptophan excretion may be expected, but greater than 30% seems excessive. In animals given the isotope alone, 17% of the label was selectively excreted. Although mice have the enzymes to convert D- tryptophan to L-tryptophan (11), this mechanism may have been inoperative, especially in experiments with tryptophan load. This problem should not alter the interpretation of our data on pathway flow since evaluation of the relative distribution of label among pathways only took into account the amount of metabolite which entered the pathway. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Berry, L. J., and D. S. Smythe Effects of bacterial endotoxins on metabolism. VI. The role of tryptophan pyrrolase in response of mice to endotoxin. J. Exp. Med. 118: Burtis, C. A., and K. S. Warren Identification of urinary constituents isolated by anion-exchange chromatography. Clin. Chem. 14: Burtis, C. A., G. Goldstein, and C. D. 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