‘ l x r \ { M 1’ W M ‘ I W “ {M If“; 1’ UI‘ ‘ w W TRYPTGPHAN OXYGEi‘éASE ACTWITY MD SENSE‘HWTY T0 TRYPTOF‘HAN IN ADRENALECTOMIZED MICE ~I 1 ! -_‘©_1 ‘ 1030 I H Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY 'MELBA JEAN BERNE 1970 LIB R A R Y Michigan State University ,...¢L:.‘~- . In a... ..... _ w: am‘mm av 4“ H0“ & SDNS' BUUKBINIJERY INC. .IJFT" ABSTRACT TRYPTOPHAN OXYGENASE ACTIVITY AND SENSITIVITY TO TRYPTOPHAN IN ADRENALECTOMIZED MICE BY Melba Jean Beine A study was undertaken to examine the possible correlation between depressed tryptophan oxygenase activity and sensitivity of adrenalectomized mice to tryptophan. Adrenalectomized mice were injected with 20 mg of L-trypto- phan, alone, in combination with 5 mg or 0.5 mg of allopurinol, or in combination with l LDSO endotoxin. Sensitivity to tryptophan was estimated by increased numbers of deaths within 8 hours following tryptophan and/or by increased deaths in #8 hours. Tryptophan oxygenase activity, significantly lower in adrenalectomized mice than in intact mice, was further depressed with allopurinol; however, adrenalectomized mice were not sensitive to trypotophan when the amino acid was given alone or in combination with allopurinol. This sug- gests that lowered tryptophan oxygenase activity pg£_§3_ does not increase sensitivity of adrenalectomized mice to tryptophan. Significant numbers of adrenalectomized mice Melba Jean Beine died within 8 hours following trypotophan when given con- 0 At these times, tryptophan oxygenase activity was elevated currently or delayed u or 10 hours after 1 LDS endotoxin. above the levels due to endotoxin alone, but were signifi- cantly lower than following only tryptophan, especially when compared with tryptophan-treated intact mice. Follow- ing delayed injection of tryptophan, tryptophan oxygenase activity attained only approximately control levels. These results suggest that endotoxin not only results in impaired ability to clear tryptophan by tryptophan oxygenase, but also has other effects on the host so that the response of adrenalectomized mice to tryptophan is altered. TRYPTOPHAN OXYGENASE ACTIVITY AND SENSITIVITY TO TRYPTOPHAN IN ADRENALECTOMIZED MICE BY Melba Jean Beine A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1970 DEDICATION To my parents, to my Sisters, and to my major professor. ii ACKNOWLEDGMENTS The author wishes to thank Dr. Robert J. Moon for his guidance and friendship throughout this investigation. I am also grateful to Sister Joyce Hibbert for constant encouragement and for her help in typing this theSiS 0 iii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . vi INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . 11 Mice. . . . . . . . . . . . . . . . . . . . . . . . . ll Adrenalectomy procedure . . . . . . . . . . . . . . . ll Tryptophan oxygenase assay. . . . . . . . . . . . . . 12 EDdOtOXin o o o o o o a o o o o o o o o o o o o o o o 13 Chemicals . . . . . . . . . . . . . . . . . . . . . . 1h RESIJLTS O O O O O O O O O O O O O O O O O O 0 O O O O O 15 Effect of single or repeated injections of tryptophan on tryptophan oxygenase activity and survival of adrenalectomized mice . . . . . . . 15 Effect of allopurinol, alone or in combination with tryptophan, on tryptophan oxygenase activity and survival of adrenalectomized mice. . . . . . . . . . . . . . . . . . . . . . . . 18 Effect of endotoxin, alone or in combination with tryptophan, on tryptophan oxygenase activity and survival of adrenalectomized mice. . . . . . . . . . . . . . . . . . . . . . . . 22 DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O O 30 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . 36 LITERATURE CITED 0 O O O O O O O 0 O O O O O O O O O O O 37 iv LIST OF TABLES Table Page 1. Survival of adrenalectomized mice given a single injection of 20 mg L-tryptophan or 2 injections of 20 mg L-tryptophan 2 hours apart. . . . . . . . . . . . . . . . . l7 2. Survival of adrenalectomized mice following various doses of allopurinol . . . . . . . . . 2O 3. Survival of adrenalectomized mice following administration of 5 mg or 0.5 mg allopurinol in combination with 20 mg L-tryptophan . . . . 23 u. Determination of LD 0 doses of endotoxin for normal and ad enalectomized mice . . . . . 2h 5. Survival of adrenalectomized mice following 1 LD 0 endotoxin, given alone or in combination with 20 mg L-tryptophan. . . . . . 26 LIST OF FIGURES Figure l. Tryptophan oxygenase activity in adrenal- ectomized and normal mice given single or repeated injection of 20 mg of L- tryptophan. . . . . . . . . . . . . . . 2. Tryptophan oxygenase activity in adrenal- ectomized mice given 5 mg or 0.5 mg of allopurinol and in normal mice given 5 mg allopurinol. . . . . . . . . . . . 3. Tryptophan oxygenase activity in adrenal- ectomized and normal mice given concur- rent injection of 5 mg of allopurinol ' and 20 mg of L-tryptophan . . . . . . . u. Tryptophan oxygenase activity in adrenal- ectomized and normal mice given 1 LDSO endOtOXin O O O O O O O C O O O O O O O 5. Tryptophan oxygenase activity in adrenal- ectomized mice given 20 mg of L-tryptophan concurrent with or u or 10 hours after 1 LDSO endOtOXino o o o o o o o o o o o o 0 vi Page 16 19 21 27 28 INTRODUCTION Dramatic changes in host metabolism are among the numerous parameters altered by endotoxin poisoning (5-8). An enzyme involved in amino acid metabolism which has been extensively studied in this context is trypotophan oxygenase. This enzyme is an adaptive liver enzyme which regulates the conversion of tryptophan to formylkynurenine in the meta- bolic pathway leading to the biosynthesis of nicotinamide adenine dinucleotide. Tryptophan oxygenase (L-tryptophan:oxygen oxi- doreductase, EC 1.13.1.12) activity is decreased both in adrenalectomized animals and in endotoxin-poisoned animals (6,7,33,3h,hl). Adrenalectomized rats and endotoxin-poisoned mice show sensitivity to tryptophan when the amino acid is given at a time when tryptophan oxygenase activity is lowered (6,33,3h,hl). Increased sensitivity to tryptophan is defined as death within 8 hours following injection of tryptophan and/or increased deaths in M8 hours when compared with appropriate control groups. Such sensitivity may reflect a reduced ability to metabolize tryptophan through the kynurenine pathway, resulting in funnelling of excess tryptophan into alternate metabolic pathways producing potentially toxic products, including serotonin. The aim of this project was to determine whether or not a correlation exists between depressed tryptophan oxygenase activity and the sensitivity of adrenalectomized mice to tryptophan and endotoxin. Treatments involved administration of L-tryptophan alone, or concurrent with, or u or 10 hours after 1 LD5O endotoxin. Similar observa- tions were made in mice injected with tryptophan and either 5 mg or 0.5 mg allopurinol. The latter compound has been shown to decrease tryptophan oxygenase activity activity ignzlzg, as well as in 11339 (3,12,13,31), and was used to simulate the endotoxin-induced depression of the enzyme activity. LITERATURE REVIEW Tryptophan oxygenase is an iron porphyrin enzyme which exists in both the ferrous and ferric states (60), as indicated by its absorption spectrum and inhibition reactions characteristic of iron porphyrin enzymes, such as light-reversible carbon monoxide inhibition, and inhi- bition by cyanide or ferricyanide. Inhibition of tryptophan oxygenase activity by cyanide and ferricyanide or by cata— lase could be lessened or prevented with ascorbic acid or peroxide (60). That the inhibition of tryptophan oxygenase by these compounds is reversible with reducing agents indi- cates the enzyme is active when in the reduced ferrous state and inactive as the oxidized ferric form (h2,60). Isolation and purification of tryptophan oxygenase indicate the existance of 3 forms: an apoenzyme (26), as well as an oxidized and reduced holoenzyme (60). Both the apoenzyme and oxidized holoenzyme require further activation to become catalytically active as the reduced holoenzyme. The overall ln_zlt£g activation process requires the pre- sence of the substrate L-tryptophan, the prosthetic group hematin, and a reducing agent, such as ascorbic acid or H202(19,20,25,26,3h,36,37,h2,60). A sequential series of 3 h events occurs in the substrate-mediated activation process (U2). Inactive apotryptophan oxygenase formed from d2_2213 synthesis is conjugated with its prosthetic group hematin to form the oxidized holoenzyme. This step requires the presence of L-tryptophan or certain analogues and can be inhibited by globin or thiol reagents. A second reaction involves the reduction of the oxidized holoenzymes to the catalytically active reduced holoenzyme. The reduction pro- cess involves ascorbic acid or H202 and has a specific requirement for L-tryptophan. Reversible loss of the active, reduced holotryptophan oxygenase to the inactive, oxidized holoenzyme can occur by oxidation in air following removal of L-tryptophan. Tryptophan oxygenase from tryptophan-treated rats is more saturated with hematin than the enzyme from untreated or hydrocortisone-treated rats (18). This increased satura- tion with hematin significantly increases the ratio of holo- to apoenzyme following tryptophan treatment (25); this ratio is relatively unchanged by hydrocortisone treatment (18). Further elevation of tryptophan oxygenase activity beyond activation of latent enzyme occurs following administration of corticosteroids or tryptophan as a result of d2 BEER apoenzyme synthesis (1h,15,18,2l,22,27,29,33,35,hh-h7). Both corticosteroids and tryptophan increase the amount of apoenzyme present, as determined by radioactive label incorporation into proteins and RNA (18), by the 5 effects of inhibitors of protein synthesis (26,29,h6), and by specific immunological reactions (21,22). Both cortisone and tryptophan increase lLAC-glycine incorporation into liver proteins which correspond with changing tryptophan oxygenase activities, but only cortisone stimulates 32P-orthophosphate incorporation into RNA at a time corresponding to its effects on tryptophan oxygenase activity. Following tryptophan administration, there is no increase in RNA labeling while the enzyme activity' is rising. These results imply that the mechanism of cortisone-mediated elevation of tryptophan oxygenase activity involves stimulation of RNA synthesis, whereas tryptophan-mediated elevation involves a different process, namely, decreased enzyme degradation. These con- clusions were confirmed by the action of puromycin and actinomycin D on cortisone-mediated and tryptophan-mediated increases of tryptophan oxygenase activity. Both types of induction were greatly diminished by puromycin, an inhibitor of protein synthesis at the translation level. Only hormonal induction was inhibited by actinomycin D, a compound which exhibits DNA-dependent RNA polymerase, thus inhibiting RNA synthesis. The decreased rate of enzyme degradation caused by tryptophan, as well as some of its analogues, is effective invi_tr_o_(l5,18,l+7), as well as law (ML,L|.6,LL7). EH22. stabilization can be measured by comparing the effect of L- tryptophan or its analogues on the loss of tryptophan oxygenase activity normally occurring on incubation without tryptophan, or on loss of activity following inactivation with heat, ethanol, urea or trypsin (R7). Ig.zixg.stabili- zation of tryptophan oxygeanse in rats was indicated by retention of pro-labeled tryptophan oxygenase levels fol- lowing tryptophan administration, whereas in control rats without tryptophan, there was a rapid loss of total counts precipitated by the specific anti-tryptophan oxygenase anti- serum. Significant influence of corticosteroids on tryptophan oxygenase activity is also implied since the control levels of tryptophan oxygenase activity are significantly lower in adrenalectomized mice (6) and rats (33-35) than in intact animals. Additional factors may influence tryptophan oxygenase activity, such as stimulation of the hypothalmus (R9), presence of pituitary growth hormone (16,38), tryp- tophan analogues (15,23,h1,h7) or tryptophan metabolites (10,23). Previous reports have noted increases in tryptophan oxygenase activity by purines (11,13,31) and by 3', 5'-cyclic AMP (12). The probable action of purines is thought to occur through their conversion to hypoxanthine. This is indicated in that blocking the conversion of cyclic AMP to hypoxanthine with theophylline prevents activation of tryptophan oxygenase by cyclic AMP (12). It has recently been suggested that xanthine oxidase may be operative in the regulation of tryptophan oxygenase activity (12,31). Xanthine oxidase, present in soluble (rat) liver prepara- tions, activates tryptophan oxygenase when its substrate hypoxanthine is present. Removal of xanthine oxidase from the soluble fraction of rat liver preparations with specific antibody resulted in loss of tryptophan oxygenase by purines (31). Because activation of purines was not dependent on the presence of hematin (12) or hemoglobin (31), xanthine oxidase apparently activates tryptophan oxygenase by causing the reduction of the inactive holoenzyme, possibly due to the production of H202, one of the products of xanthine oxidase reaction (12). Inhibition of xanthine oxidase with allopurinol inhibits tryptophan oxygenase activity in_zitrg_(3,l2,3l). In ELIE: allopurinol was found to be potent inhibitor of tryptophan oxygenase, in that, u hours after an injection of allopurinol (20mg/kg) to normal rats, tryptophan oxygenase activity was inhibited 90-95% (3,31). Mice were similarly inhibited by lower doses (3). Allopurinol-mediated inhibi- tion of tryptophan oxygenase activity is not due to a direct effect on the enzyme, in that allopurinol does not inhibit tryptophan oxygenase activity in a purified preparation of the apoenzyme, thus implying allopurinol does not inhibit conjugation of the apoenzyme with hematin (31). Nor does allopurinol significantly inhibit hydrocortisone-mediated synthesis of tryptophan oxygenase, as measured by precipitation of the enzyme with specific anti-tryptophan oxygenase antibodies (31). Addition of ascorbic acid pre- vented inhibition of tryptophan oxygenase by allopurinol (12), thus implying allopurinol inhibits tryptophan oxygenase activity at the step of the reduction of the oxidized holoenzyme, possibly by the inhibition of xanthine oxidase activation of tryptophan oxygenase. Tryptophan oxygenase activity is depressed in endotoxin-poisoned mice (l,6,7.hl). Because tryptophan oxygenase is the first enzyme in the pathway leading to the formation of pyridine nucleotides, its depression implies a block in the biosynthesis of such compounds, the consequence of which could be of biological significance in endotoxin poisoning. This concept is consistent with evidence that administration of NAD or nicotinamide concurrent with endo- toxin protects mice against endotoxin lethality (6). A decrease in the levels of total oxidized pyridine nucleotide found in the liver of endotoxin-poisoned mice could be pre- vented by concurrent administration of nicotinamide which, when given alone, approximately doubled the control levels of total oxidized pyridine nucleotides in 17 hours. Cortisone, known to elevate tryptophan oxygenase activity, (19,20,35) protects mice against endotoxin lethality (7,2h,37,hl) and also maintains the levels of total oxidized pyridine nucleotides (7). The protective role of cortisone, as well as its ability to elevate tryp- tophan oxygenase activity, is dependent on the time of administration relative to the administration of endotoxin (6,7). Thus, 5 mg cortisone, given concurrent with l LD5O of endotoxin, increased survival of mice and elevated tryptophan oxygenase activity; however, if given more than one hour after endotoxin, cortisone failed to protect mice and failed to maintain tryptophan oxygenase activity at control levels. Certain inhibitors of protein synthesis, namely actinomycin D, ethionine, 2-thiouracil, and 8-azaguanine, were found to potentiate endotoxin lethality and to prevent cortisone protection against endotoxin (7). These same compounds prevented cortisone elevation of tryptophan oxygenase activity in normal mice and prevented cortisone maintainance of tryptophan oxygenase activity at control levels in endotoxin-poisoned mice when endotoxin, cortisone and inhibitor were given concurrently. In the above instances, maintainance of tryptophan oxygenase activity at control levels during endotoxin-poisoning was associated with protection against the lethal effects of endotoxin. Although tryptophan elevated tryptophan oxygenase activity in untreated mice, it failed to maintain control levels of activity when given concurrent with l LDSO endotoxin. Concurrent injections of tryptophan and endo- toxin also failed to protect against lethality (6,hl). Elevation of tryptophan oxygenase activity with oC-methyl- tryptophan did not increase survival of endotoxin-poisoned 10 mice. Correspondingly, depression of tryptophan oxygenase activity with 5-hydroxytryptophan did not increase suscepti- bility of mice to endotoxin (41). These results imply that tryptophan oxygenase peruse is not directly related to sur- vival of endotoxin-poisoned mice. A delayed injection of tryptophan, given A hours after endotoxin, at a time when tryptophan oxygenase activity was below control levels, potentiated the lethal effects of endotoxin, resulting in convulsive death, such that the LDSO became LDlOO (6,h1). This effect was prevented by prior treatment with cyproheptadine, an anti-serotonin drug (hl). Convulsive deaths did not occur and cyproheptadine had no effect on mortality when endotoxin and tryptophan were given concurrently. Protection against convulsive death with cyproheptadine, at a time when tryptophan oxygenase activity was lowered by endotoxin, suggests that tryptophan is converted in excess to serotonin, possibly as a result of the lowered tryptophan oxygenase activity. Further impli- cations of funnelling of tryptophan into serotonin synthesis come from protection by cyproheptadine against the hypo- thermia produced in mice kept at 15 C given serotonin alone or against increased hypothermia produced by tryptophan in endotoxin-poisoned mice (RC). Although tryptophan oxygenase probably does not influence survival of mice given endotoxin, it may be a factor in convulsive death seen in endotoxin- poisoned mice given tryptophan. MATERIALS AND METHODS .Migg: Eighteen to 20 gram, female CF-l mice (Car- worth Farms, Portage, Michigan) were used in all experi- ments. They were housed, 10 per cage, with pine wood chips as bedding. Food (Purina mouse chow, Ralston Purina Co., St. Louis, Missouri) and water were available ad_libitum, unless otherwise noted. Adrenalectomized mice received 0.9% saline as drinking water. Adrenalectomy_procedures: Hair was removed from the backs of mice using electric hair clippers. Mice were anesthesized with 0.25 mg Nembutal (sodium pentobarbital) injected subcutaneously in 0.1 ml of 0.85% NaCl, followed 10-15 minutes later by light etherization. After etheriza- tion, the mice were restrained on a board with tape. The back of the mouse was disinfected with 0.2% iodine (Rexall). A 2-cm incision was made through the skin over the back bone, using small surgical scissors. The skin was pulled to the right side and a l-cm incision was made through the muscle layer, beginning near the rib cage and extending down at a slight angle. ffluaadrenal gland was removed using two pairs of straight point surgical forceps. The 11 12 skin was then pulled to the left side and the procedure repeated, removing the other adrenal gland. No stitches were used to close the muscle layer incisions. The medial dorsal incision was closed with two stitches of silk surgi- cal thread, gauge 000, using a h-O suture needle. Completeness of adrenalectomy was determined 2h-h8 hours prior to experimentation utilizing the water-retention test of Beatty (2). Mice were used for experimentation 6-8 days after surgery. Tryptophan oxygenase assay: Tryptophan oxygenase was assayed by the procedure of Knox and Auerbach (35) as modified in our laboratory to insure activation of all latent enzyme. Mice were fasted overnight prior to experi- mentation, normal mice for 12-18 hours and adrenalectomized mice for 8-12 hours. Mice were sacrificed by cervical dislocation and liver removed. Approximately 1 gram of mouse liver (wet weight) was homogenized in 7 ml ice cold homogenizing medium, composed of 0.1M M KCl, 0.0025 M NaOH, and 0.01 M L-tryptophan, the latter served as substrate for the reaction. Two ml of each homogenate were dried in crucibles to determine dry weights. Additional 2-ml aliquots of each homogenate were added to two 25-ml Erlen- meyer flasks (a and b). Each reaction flask contained 3.0 ml distilled water, 2.0 ml Na2HP0 buffer, pH 7.0, 'KH PO LL 2 LL and 1.0 ml methemoglobin (h mg/ml). Methemoglobin (recrys- talized bovine hemoglobin) was purchased from Nutritional 13 Biochemical Corp., Cleveland, Ohio. The flasks were incu- bated at 37 C for 30 minutes on a Forma Thermo-Shaker (Forms Scientific, Inc., Marietta, Ohio), after which the reaction in flask b of each pair was stopped by the addition of U ml of 15% metaphosphoric acid. The reaction in flask a was allowed to proceed for an additional 60 minutes, then stopped as stated above. The reaction mixture was filtered and 6.0 ml of each filtrate was neutralized by the addition of 2.0 ml of 1.5 N NaOH. The optical density of the filtrate was determined at 360 nm with a Beckman model B spectrophoto- meter (Beckman Instruments, Inc., South Pasadena). This wavelength is the maximum for kynurenine which was read as the product of tryptophan oxygenase since all formylkynurenine was converted immediately to kynurenine by the enzyme formy- lase present in excess in the homogenate. The amount of kynurenine formed per hour was calculated from the difference in optical density between flask a (stopped after 90 minutes reaction time) and flask b (stopped after 30 minutes). The quantity of product was estimated from a standard curve and the results expressed in umoles kynurenine per gram dry weight of liver per hour (quK/gm/hr). Endotoxin: Heat-killed cells of Salmonella typhimurium, strain SR-ll, suspended in isotonic saline served as the source of endotoxin. Heat-killed cells were prepared in the following manner. Overnight cultures (18 hr) were grown in 500ml brain heart infusion broth to a 1h concentration of approximately 109 cells/ml. The cells were concentrated by centrifugation at 6000 x g for 5 min- utes in a Sorvall SS-l table centrifuge. Thirty ml aliquots of the culture were centrifuged, the supernant decanted, and additional culture added. This procedure was repeated until all the culture was centrifuged. The cells were washed twice with non-pyrogenic isotonic saline (Baxter Laboratory, Morton Grove, Illinois) and resuspended in saline to approxi- mately 10 times the original concentration. The pooled cells were heat killedcby autoclaving at 6 lb pressure and 222 F for 6 minutes. The preparation was judged sterile by lack of growth in N8 hours on subculture in brain heart infusion broth. The LD of this preparation for both nor- 50 mal and adrenalectomized mice were determined according to x the method of Reed and Munch (N3). The LD50 dose for nor- mal mice was a l:h dilution of the final preparation and that for adrenalectomized mice was a 1:1000 dilution. Chemicals: Nembutal (sodium pentobarbital), 50 mg/ ml, was purchased from a local supply house. L-tryptophan was purchased from Nutritional Biochemical Corp., Cleveland, Ohio. Allopurinol, "Zyloprim" brand, Burroughs Wellcome and Co., Tuchahoe, New York, was obtained through the courtesy of Dr. Stanley T. Bloomfield, Medical Department. This latter preparation was injected as a suspension stabilized with 0.01% Methocel (Dow Chemical 00., Midland, Michigan). RESULTS Effect of single or repeated igjections of tryptophan on tryptophan oxygenase activity and survival of adrenalecto- mized mice: The control level of tryptophan oxygenase activity was significantly lower (P<0.0l) in adrenalecto- mized mice than in normal mice (Figure l). Tryptophan oxygenase activity reached a maximum in adrenalectomized mice in 2 hours after injection of 20 mg of L-tryptophan and returned to control levels within 6 hours. Tryptophan induction of the enzyme in normal mice is greater in both magnitude and duration, as shown by a reference curve in Figure 1. Further elevation of tryptophan oxygenase activity in adrenalectomized mice could be attained with a second injection of tryptophan given 2 hours after the first, at a time indicated by an arrow in Figure 1. Maximum activity occurred 2 hours after the second injection; the activity returned to control levels within 8 hours. The maximum activity attained after 2 injections was significantly higher (P<0.0l) than after 1 injection. No deaths occurred following a single injection of 20 mg of L-tryptophan in adrenalectomized mice. Although 15 l6 was UmNflEopooncosUm CH hufl>flpow ommcmwhxo chQOBQhaB .H oasmflm 5803).: Lots 9501 we or m 0 v N O p b . h - lb 15 l.nv roe . ION L. 520 c038? Ucm O IOm UmflEOuUQDCOLUd. l BELOZ O .chQOpmhmpnq mo we om Mo cowpooncfi oopmomoa no oaMch cm>fiw mafia Hmeaoq Ju/wfi/an pconMficwfim haamoapmfipwpm poz w m> n 17 nb\m N\N m\w popwa mn30£ m chQOthApIA we om mafia cwnmothApnq we om soa\oa oa\oa oa\oa macaw messengaspuq ms om 3 ma. m mASom pcoepwoae Hmpcosfinomxm Umpoowcw prOp \ mao>fi>95m * .pawmw mason m cwnmopmhApuq we om mo msofipoomCH N no :wSQOpmhapnq we om mo coapoonsfl mamcwm s co>wm cows UoNHEopooncoLUw mo Hw>w>95m .H canoe l8 2 of 7 adrenalectomized mice died following 2 injections of L-tryptophan given 2 hours apart, the numbers were not statistically different from appropriate controls (Table 1). Single or repeated injections of L-tryptophan were not lethal for normal mice (30). Effect of allopurinol, alone or in combination with tryptophan, on tryptophan oxygenase activity and survival of adrenalectomized mice: Five or 0.5 mg of allopurinol depressed tryptophan oxygenase activity in adrenalectomized mice to approximately the same levels within 2 hours (Figure 2). The depressed activity persisted in both cases for at least 12 hours. A similar depression was seen in normal mice given 5 mg ofallopurinol. Approximately 50% of the adrenalectomized mice died in E8 hours following subcutaneous injection of 5 mg of allopurinol (Table 2). Only 1 of 10 adrenalectomized mice died following injection of 1 mg of allopurinol. There were no deaths among the group given 0.5 mg of allopurinol. Concurrent injection of 5 mg of allopurinol and 20 mg of L-tryptophan elevated tryptophan oxygenase activity in adrenalectomized mice (Figure 3). The maximum enzyme activity attained 2 hours after this treatment was not sig- nificantly different from that attained after 20 mg of L- tryptophan alone. The enzyme activity returned to control levels by h hours and was below control levels by 6 hours. These results show no significant inhibition of tryptophan- l9 .Hocfiasmoaas mg m co>wm oofis staoc CH was HOCflASQoHHw ho we m.o so we m co>flw ooHS oosfisopooascosom CH hpfi>fipow omwcomhxo cosmopmhae .m oaswfim _o£.3oo__o Lots mono: ma Q v... w“? 0.7 m w v .N O n p _ — h — lb iliTl. \lfl?\¢ 1‘ nTll .1) 6:22.26 08 md 53> ocquUochLo/x 4 65.2326 9: m 53> ocquuochmLD/x I .0c_._:ao__o 9: m 53> .9502 o 1.0 I Q r.ON Ju/wfi/an 20 oa\oa oaxoa QH\OH oaxo oa\oa OH\OH oaxm oa\o oa\oa menom panache“ prop \ meo>fi>edm % Hocfiezmoaaw we m.o Hocfiasmoaaw we H Hocfinsmoaas we m psoEpwoAB prcoewaomwm .HOCfiQSQOHHw Mo momoo meofiew> wcHSoHHom oofle woufieOpooHscoaow mo Hm>fi>hdm .m capes 21 .co>fiw ohm csemopmbepuq mo we om wowzoaaow mo>950 monopowmm .csnmoum%Apuq Mo we om was Homagemoaaw mo we m 90 coapooncfi psoAASocoo co>fiw oOfle Hmeeoc was doufieopoodwsoeow ca hufi>fipow omwcowhxo cwnmopmhee .m oaswwm 9501 . w y N 0 W1. - p (5 AU ’| £2 sundown?“ m2u _o:_._:oo__fi>eem * .chQOpmhepnq we om Spas cofiuwcwneOo cw Hocfiesmoaaw we m.o go we m mo cowpruchHepw wnazoaaom oofie UoNHEOpooncoepw mo Hw>H>eem .m oHnme 2h NOH K d.H No sea A o.m m: mos a s o: OHOH a :.H OH oaoa a a.m o Hs>H>ASm pcooaom o\o ooomua ox: oooaua oofie ooufieopooamcoao< os\: and 0H\H mua oa\o opsHfiocs oofie Hweeoz Awmwaefodv pcoepwone meo>a>nzm * Hancoeaeomfim Hweao: pom cakonooco no momoo ommq .ooae UoNHeOpoonconvw was ho COfipGCHEAopoQ .: oanwe 25 approximately 7 x 109 bacterial cells per 0.5 ml dose. The LDSO for adrenalectomized mice was a l to 1000 dilution of the endotoxin preparation, corresponding to approximately 2.9 x 107 bacterial cells per 0.5 ml. This dose was equiva- lent to a 1/250th LD50 dose for normal mice. Adrenalectomized mice showed increased sensitivity to tryptophan when the amino acid was given concurrent with or u or 10 hours after 1 LD50 endotoxin, as estimated by significant increases in deaths 8 hours after tryptophan was injected (Table 5). There was no significant alteration in the number of deaths after h8 hours in either of the three cases, as compared with endotoxin-poisoned mice. Adrenalectomized mice given 1 LD50 endotoxin showed a transient increase in tryptophan oxygenase activity, fol- lowed by depressed activity which returned to near control levels within 18 hours (Figure h). A similar effect was noted in normal mice; however, the activity remained depressed at least 18 hours. Figure 5 shows the effect of an injection of 20 mg of L-tryptophan given to adrenalectomized mice at various times following administration of 1 LD50 endotoxin. When tryptophan was given concurrent with endotoxin or delayed h or 10 hours, tryptophan oxygenase activity was elevated above the level following endotoxin alone. Following con- current injections, the enzyme activity remained signifi- cantly elevated for at least 8 hours above the activity 26 unmeamficwfim hHHonpmewpm poz u .w.w.z .w.m.z w m> n mo.ovm o m> M .m.m.z w m> a Ho.ovm o m> o .m.m.z m m» s moo.ovm e m» n msSon 0H pophw chQOpmhauuq hom\aa mom\ma om\oH om\om we om mafia saxopoosm Hom\mH om\:a som\mH om\eH som\o om\OH om\0H nom\mH mom\oa oom\mH oom\om som\om maSoe : nophw chQOthepna we om mean CHKOpoocm haonAQSOQoo swamopmhepuq we om mean saxopooem osoas camouocco ommq H w: wH NH w medom UopoonCH prop \ meo>fi>ndm % pacepwoee Hancoeaeomxm .cwemopmhApuq we om an“: coprCflneoo CH no ocoaw so>fiw .cfiNOpocco ommq H wcfizoaaom ooae woufieopooawcoaow Mo Hs>fi>e3m .m wands 27 .CHKOHOUCo omQH H co>Hw ooHe Hmeeoc pew UoNHeopooHscoaom CH th>Hpom omwsowhxo cmemopmhee .: oestm Exosbocm cots PEOI m.. we .3. NH Or w m V N O —711 P1, .7 b _ _ b P \_ I. .Imv I OF T 1 omquUochLHX. I . ION BELOZ o Ju/wfi/Mwn .CHNOHooco omQH H songs mASOQ OH ao : no nuHS psoneSocoo cm£m0pmhele 90 we om co>Hw ooHe ooNHeOpooncerw cH th>Hpow ommcowhxo sw£m0pmhee .m oastm EXOHOUcm Lmfic mLDOI m? 9 3 NH OH we or w w v w w v N O . - h — n h — n — _ F L I O 28 I $2 I ION Ju/wfilan I 53898» was £onon 4 Exoaoocm I 5:83»: o 1‘ 29 due to either endotoxin or tryptophan alone. Delayed injection of tryptophan h hours after 1 LD50 endotoxin resulted in elevation of tryptophan oxygenase activity to approximately control levels in 2 hours after tryptophan; this level was maintained for at least 8 hours. Similar induction of tryptophan oxygenase activity to control levels followed injection of 20 mg tryptophan 10 hours after 1 LD5O endotoxin. These initial increases in tryptophan oxygenase activity decreased as the time span lengthened between injections of endotoxin andatryptophan, not only with respect to the level following endotoxin alone, but also with respect to levels following injection of tryptophan alone. These increases attained only control levels, except for concur- rent injection of endotoxin and tryptophan, which elevated tryptophan oxygenase activity to a level similar to that following tryptophan alone. DISCUSSION Studies by Knox (33,3h) indicate adrenalectomized rats are extremely sensitive to tryptophan, in that all died within h-8 hours following an injection of 100 mg L-tryptophan / 100 gm body weight. A similar dose of tryptophan given as a single or repeated injection does not have this effect on adrenalectomized mice. Tryptophan oxygenase activity can be induced by substrate in both adrenalectomized rats (33,3h) and adrenalectomized mice (5, Figure 1); however the enzyme remains significantly lower at all times when compared with normal animals, suggesting the participation of endogenous corticosteroids on the enzyme response on normal animals. That this enzyme is effected by corticosteroids has been well established (lh,33,35,h5). Adrenalectomized mice are sensitive to 5 mg of allo- purinol, a dose relatively nontoxic for normal mice. Decreas- ing doses of 1 mg and 0.5 mg of allopurinol were successively less toxic for adrenalectomized mice. A probable explanation of the toxicity of allopurinol is that allopurinol-mediated inhibition of xanthine oxidase causes an accumulation of xanthine beyond urine saturation levels which, in small 30 31 animals, results in crystallization of xanthine in renal tubules (30). It has been found that allopurinol exhibits a progressively greater toxicity, the smaller the animal being tested, from man to dog to rat to mouse. Just as survival following allopurinol is dose- dependent, (30) the response of tryptophan oxygenase to allopurinol is also (3). Twenty-four hours after an injec- tion of 10 or 20 mg/kg body weight of allopurinol, tryptophan oxygenase activity in rats was 60% of the control level, but that with smaller doses, the activity returned to normal in 8-10 hours. Tryptophan oxygenase activity in mice, inhibited with 2 or 8 mg/kg allopurinol, returned to control levels within 20-2h hours. Allopurinol-mediated inhibition of tryptophan oxygenase activity persists in both normal and adrenalectomized mice at least 18 hours following injec- tion of 5 mg allopurinol and in adrenalectomized mice at least 12 hours after 0.5 mg allopurinol (Figure 2). Concurrent injection of allopurinol and tryptophan elevated tryptophan oxygenase activity in both normal and adrenalectomized mice; however, these increases in 2 hours are not significantly different than following tryptophan alone. Julian and Chytil (31) have shown that in normal rats concurrent injection of allopurinol (20 mg/kg) and L-tryptophan (1.0 mg/kg) resulted in significant (50-60%) inhibition of tryptophan oxygenase activity in h hours. A possible explanation for the discrepency in the amount 32 of inhibition at a time of maximum tryptophan oxygenase activity lies in the dose of tryptophan used. The dose used for rats was 1.0 mg/kg body weight, whereas that for mice in these experiments was 20 mg/mouse, equivalent to 1000 mg/kg body weight. Thus, with a thousand-fold increase in the amount of tryptophan administered, one might expect less inhibition of tryptophan oxygenase activity by allopurinol. Inhibition of tryptophan oxygenase activity with allopurinol did not increase the susceptibility of adrena- lectomized mice to tryptophan (Table 3). This suggests that lowered tryptophan oxygenase activity per 22 does not increase sensitivity of adrenalectomized mice to tryptophan. A similar effect was noted in normal mice (Moon, submitted for publication). Adrenalectomized animals are more susceptible to stress than normal animals, presumably due to their inability to respond with adrenal hormones (5,6,9,33,3h). Thus, it is not surprising that the LD5O endotoxin for adrenalectomized mice was l/250th the dose required for normal mice (Table h). Significant numbers of endotoxin-poisoned adrenal- ectomized mice given tryptophan died within 8 hours after administration of the amino acid (Table 5). Many of these mice were in convulsions prior to death. Although the cause of convulsive death is not known, several possible explanations have been offered. One is based on the 33 observation that endotoxin depletes liver glycogen and blood sugar levels (5,7,8,hl,h8). Normal endotoxin- poisoned mice given tryptophan are more hypoglycemic than mice given endotoxin alone (Moon, unpublished data). Thus one factor in convulsive death seen in endotoxin-poisoned mice given tryptophan may result from extreme hypoglycemia. Another possible explanation suggests that excess serotonin is produced from tryptophan due to inhibition of tryptophan oxygenase. This explanation is favored by evidence of pro- tection against endotoxin-induced convulsive death in normal mice by cyproheptadine, an anti-serotonin drug (kl). Following 1 LD50 endotoxin, tryptophan oxygenase activity in adrenalectomized mice was initially elevated, then depressed below control levels. The activity returned to near normal levels in 18 hours. A similar response was observed in normal mice; however, the activity remained lowered for at least 18 hours. When tryptophan was given concurrently or u or 10 hours after endotoxin, its ability to elevate tryptophan oxygenase activity in adrenalectomized mice at each of these times was not completely destroyed by endotoxin treatment. The increases in activity decreased as the time between endotoxin treatment and administration of tryptophan lengthened, implying a progressive inhibition of tryptophan oxygenase by endotoxin. The time relationship between tryptophan induction of tryptophan oxygenase activity and convulsive deaths in 3h adrenalectomized mice was similar to that observed in adrenalectomized rats (33,3h): tryptophan oxygenase activity was elevated above control levels prior to and at the time of death. The evidence that the enzyme was elevated during this time suggests that either the activity of tryptophan oxygenase does not relate to survival of endotoxin-poisoned adrenalectomized mice given tryptophan or that the elevated activity is not accompanied by a corresponding increase in the efficiency of tryptophan oxygenase. Kim and Miller (32) have shown that an increase in tryptophan oxygenase activity in intact or adrenalectomized rats was not associated with increased conversion of luc- tryptophan to 1“”002 unless the substrate load was increased. Others (18) have shown that there is an increased amount of active holotryptophan oxygenase, as well as apoenzyme, following administration of tryptophan. In contrast, there was little increase in the amount of active holoenzyme fol- lowing cortisone treatment, although this resulted in high levels of apoenzyme. Because the elevated tryptophan oxygenase activity in endotoxin-poisoned adrenalectomized mice was induced with tryptophan, rather than with corti- sone, the latter possibility that increased tryptophan oxygenase activity was not accompanied by increased effi- ciency of clearing tryptophan, is lessened. Because depression of tryptophan oxygenase activity with allopurinol did not increase susceptibility of adrena- lectomized mice to tryptophan and because tryptophan 35 oxygenase activity was elevated by tryptophan in endotoxin- poisoned adrenalectomized mice at the time of death, tryp- tophan oxygenase activity apparently has little influence on survival following such treatment. This has been pre- viously suggested (Ml). However, in spite of the elevation of tryptophan oxygenase by tryptophan in endotoxin-poisoned adrenalectomized mice, the overall increases in activity are greatly lessoned when compared to the capacity of normal mice given tryptophan (cf. Figure 1). This implies that the elevated activity may not be sufficient to handle the 20 mg dose of L-tryptophan, and thus, excess tryptophan may be funnelled into other pathways, including the one to serotonin. Endotoxin-poisoned adrenalectomized mice were sensi- tive to both concurrent and delayed injections of tryptophan, whereas normal mice were sensitive to tryptophan only when tryptophan oxygenase activity was depressed with endotoxin (hl). This suggests the possibility that endotoxin has effects on the host, in addition to those on tryptophan oxygenase, which are important in the response of such animals to tryptophan. These latter effects are not under- stood at this time. SUMMARY AND CONCLUSIONS Adrenalectomized mice do not exhibit increased sensitivity to tryptophan when the amino acid is given alone or in combination with allopurinol. These mice, die in greater numbers within 8 hours when tryptophan is given concurrently or delayed h or 10 hours after 1 LD5O endotoxin. At these times, tryptophan oxygenase is elevated above the levels following endotoxin alone, but is significantly lower than levels following tryptophan alone, especially in refer- ence to the levels in tryptophan-treated intact mice. This suggests a reduced ability to metabolize tryptophan by tryptophan oxygenase, leading to possible funnelling into other pathways. Lowered tryptophan oxygenase activity per 33 is not responsible for increased sensitivity to trypto- phan, in that allopurinol inhibition of tryptophan oxygenase activity did not sensitize adrenalectomized mice to trypto- phan. Thus, endotoxin may exert other effects on the host which alter its response to tryptophan. 36 LI TERATURE CITED LITERATURE CITED Agarwal, M. K., W} W. Hoffman, and F. Rosen. 1969. The effect of endotoxin and thorotrast on inducible enzymes in the isolated, perfused rat liver. Biochim. Biophys. Acta. 1115250-258. Beatty, C. H., R. M. Boak, and R. D. Peterson. 195A. 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Occur- rence in animals hyperreactive to endotoxin. Proc. Soc. Exptl. Biol. Med. 139;N13-N17. Shimazu, T. 196N. Role of the hypothalmus in the induction of tryptophan pyrrolase activity in rabbit liver. J. Biochenh 55:163-171. Tanaka, T. and W. E. Knox. 1959. The nature and mechanism of tryptophan pyrrolase (peroxidase- oxidase) reaction of Pseudomonas and of rat liver. IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII LILL|LLLLLILLLLLLLILLLLLLILLLLLILLL |LLLLL LlLlLLLI ILLLLILL