METABOLISM OF TESTOSTERONE BY LIVERS OF DIFFERENT SPECIES OF ANIMALS* Salt Lake City)

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1 METABOLISM OF TESTOSTERONE BY LIVERS OF DIFFERENT SPECIES OF ANIMALS* BY LEO T. SAMUELS, MAX L. SWEAT, BLAINE H. LEVEDAHL, M. M. POTTNER, AND M. L. HELMREICH (From the Department of Biological Chemistry, University of Utah College of Medicine, Salt Lake City) (Received for publication, August, 949) In previous papers (4) it has been demonstrated that the metabolism of testosterone by liver tissue involves at least two enzyme systems, one requiring diphosphopyridine nucleotide (DPN) and the other citrate as cofactor. The former compound is involved in the oxidation of the alcohol group on C7 to a ketone; the latter increases the rate of destruction of the cr,p conjugation in Ring A formed by the A4 double bond and the ketone group on C3. The variation in testosterone metabolism has been investigated in a wider series of species, and evidence of a phylogenetic development of the enzyme systems has been found. Methods Except in the case of the steer and the human, liver tissue was removed in the laboratory immediately after death by decapitation. Human liver tissue was removed during surgery and immediately immersed in icecold Locke s solution. It was then transported to the laboratory as rapidly as possible. Steer liver was obtained at the slaughterhouse, cooled, and also brought to the laboratory within a short time. The tissue was immediately sliced with a razor or minced with a mechanism made up of multiple razor blades. It was then suspended in the buffer solution of testosterone and incubated in an atmosphere of oxygen, as previously described. Enzymic action was stopped by boiling the contents of the flask under a reflux for 2 minutes, after which extraction and analysis were carried out as in previous studies (5). 6 per cent DPN was procured from the Schwarz Laboratories. It was added directly to the flasks to give a final concentration of.6 M DPN. Sodium citrate was used at a concentration of. M. The concentration of molecules containing the c &unsaturated ketone group in Ring A was estimated by the ultraviolet absorpton band at 24 *This investigation was supported by grants from the National Cancer Institute of the National Institutes of Health, United States Public Health Service, the American Cancer Society through the Committee on Growth of the National Research Council, and Ciba Pharmaceutical Products, Inc., Summit, New Jersey. 23

2 Buffer I DPN + citrate (iii ) 5 M M58) z (it 5) E ii (ii 92) ia (f: 76) 2 (9824) (FL 66) AJhlal Fish... Frog.... Turtle... Rattlesnake.... Chicken.... Steer... Rat fedt... (( fasted 48 hrs.?.... I fed,..... I d... Rabbit t t d( TABLE I Destruction of Testosterone by Livers of DijTerent Species 3riginal estoster me colt centration Y Per flask 576 Y Per gm. per hr. Buffer only Y Per gm. per hr. I Buffer + DPN i Buffer + citrate 7 y per gm. per hr. (ii 92) (iii 7 (:: 69) (ii 42) (ii 38 (ifi 62) (:I ) (6324) (48s 588 )( (Z227) (O 29 I( (tk) (i: 23) (% 266 ) (ii 54) (i: 3) (iii 62 I( 2 6 >367 24) (II 2) (>365>367 ) ) (55 88) ( ) (865) (5 67 L,* I( d from by guest on January 6, 29 IIK&osteroids formed Y Per gm. per hr ) ) (ii 62) 6 67) (k 97) ) (LOO) a.8 destruction 7 Per m. per hr.* (:S 76 (; & 62 (% (: ( , &259,. I( ) ) ) 7K&osteroids formed (t 27 I( (;: 28 (FL I( (E 53 > (ii 42 7 a,9 destructior (E 43 >273 >252> (74 24 >365 >358> ( (26 266

3 $ El CIuinea pig i Dog, CT.... Monkey.... Human (Las)::Lll) (ELO4) (k 37) (4662) (2248) 37 (696) (% 68) (223226) (845) 5 (29436) (ii 94) 68 (5@ 54 (38 2 ( ) (274) (526) (24 33) ) (974) (427) (28 34) ) (479) (47. (282287) (828) ( ) (k 65) 246 ( ) (iti 52) $ (284 6) (992) R J (gsllo4) (6 37) (26242) $ ig 3 3 l ( ) (886) Except in the case of the fish and turtle, in which data from three incubations are combined in each case, each line represents 5 E a single incubation. The number of livers in all cases except the two mentioned represents the numbers which were minced together to furnish sufficient sample for the series. The numbers in parentheses indicate the range of values obtained from individual.p g incubation flasks. Each flask contained 25 ml. of buffer and.5 to.oo gm. of liver mince. In most cases the results from three flasks have been averaged to give the mean value. Testosterone concentration is given because, over the range involved, destruc tion varies almost directly as substrate concentration. This must be considered in comparing the rates of destruction. * Micrograms of steroid per gm. of liver mince per hour. t These rats were from the same litters. d from by guest on January 6, 29

4 234 METABOLISM OF TESTOSTERONE BY LIVER rnp. The l7ketone group was determined by the absolute alcohol method of Callow, Callow, and Emmens (6). Spectrophotometric readings were made at 44, 52, and 6 rnp wavelengths. These were corrected for background absorption on the assumption that it showed a constant fall over this range. When the three wavelengths mentioned were used, the brown color of testosterone in the quantities employed gave no significant value after correction. Results The results of studies of the various species are summarized in Table I. The livers of fish, amphibians, and reptiles all destroyed the CL,@ structure in testosterone at a slow rate, but no evidence of oxidation at C7 was found. The addition of either DPN or citrate did not affect the result significantly. The enzyme system involved, therefore, apparently does not require these compounds as cofactors. A typical series of experiments on one form of reptile, the turtle, is shown in Table II. While the activity of slices was approximately twice that of the minced tissue from any one individual, the reaction to the addition of cofactors was the same. There did not seem to be any acceleration in the presence of DPN or citrate. The variation between turtles of the same sex was as great as that between turtles of opposite sexes. With birds and mammals, however, enzyme systems requiring each of these cofactors appear. If DPN was added to minces or slices of livers from members of these orders, 7ketosteroids were formed in all animals except the rabbit. In the latter, 7ketosteroids were produced in the presence of the liver tissue but the nucleotide seemed to have no effect on t,his process. In addition to the effect on C7, the presence of DPn increased the destruction of the conjugated system in Ring A as measured by the decrease in absorption at 24 rnp, again with the exception of the rabbit. This would appear to be due to the ability of an enzyme to reduce the ketonic group on C3 more readily when there is a ketone rather than an alcohol on C7. When the 7ketosteroids formed by chicken liver, which has a relatively high concentration of this oxidative system, were treated with succinic anhydride, all were found in the watersoluble fraction. They must, therefore, have had an alcohol group as well as the 7ketone. The most likely assumption is that the ketone on C3 was reduced to an alcohol and formed Alandrostenol3one7. As can be seen in Table I, in all species except the steer the number of conjugated groups disappearing equals or exceeds the number of ketonic groups formed at the other end of the molecule. This was true both before and after the addition of exogenous DPN. The problem of the role

5 SAMUELS, SWEAT, LEVEDAHL, POTTNER, AND HELMREICH 235 of the 7ketosteroids has been analyzed in the case of chicken liver. This tissue forms relatively large amounts of alcoholic 7ketosteroids from testosterone without added cofactor. In Table III the effect of time on the proportions of the different fractions is demonstrated. The nonconjugated non7ketosteroids represent the difference between Columns and 2. When incubation time was increased, the concentration of the 7ketosteroids remained relatively constant but the amount in Column 3 increased. These observations could The than Temperature No. and sex C _ TABLE II Metabolism of Testosterone by Turtle Liver? is given as micrograms per gm. per hour Average I Preparation Mince Slices Mince Slices Mince Slices Mince Mince Slices I BufIer only* d groups destroyed l Buffer + Buffer +. Y. Y DPN citrate %P groups groups cr.8 groups destroyed destroyed destroyed Buffer + C. Y DPN +.?d citrate *The buffer solution contained KC.56 M, MgCla.2 M, NaCl.8 M, NarHPOdNaHzPO, buffer (ph 7.4).4 M, glucose. per cent. Approximately gm. of mince or slices was used per flask together with y of testosterone and 25 ml. of buffer solution. The extracts from all incubations were analyzed for 7ketosteroids but none were found. 4 best be explained if the alcoholic 7ketosteroids were themselves intermediates which underwent a reduction at C7. The sequential relationship of the formation of 7ketosteroids and their breakdown was investigated by comparing the effect of chicken and rat livers. In the absence of added DPN rat livers form little 7ketosteroids but break down many more cu,p groups. Testosterone was first incubated with mince from one species of liver and the products were then incubated with liver mince of the other species. Analyses for LIP,P conjugation and 7ketosteroids were made at the end of each incubation. The

6 236 METABOLISM OF TESTOSTERONE BY LlVER results are shown in Table IV. It is evident that bird liver was unable to form 7ketosteroids from the products of the action of rat liver on testosterone. The small amounts present could be accounted for by conversion of the residual testosterone. When the order was reversed, how TABLE Effect of Incubation with Chicken Liver Mince on Di$erent Parts of Testosterone Molecule The tissue was incubated with 4 y of testosterone in glucose buffer. III Incubation time Em. min TABLE groups destroyed IV () Nonconjugated nonl7ketosteroids formed (3) ger CCRI per cenl per cent Balance Sheet for Reincubation Experiment in Which Both Rat and Chicken Livers Were Used in Series The 7ketosteroids formed by chicken liver were destroyed by rat liver. Incubation st 2nd Testosterone added recovered destroyed Ketosteroids formed... Testosterone present, start.. 7Ketosteroids present, start. Testosterone recovered... 7Ketosteroids recovered..... Testosterone destroyed Ketosteroids destroyed... I formed Rat liver followed by chicken liver Chicken liver follo~~~ejy rat Y Y ever, rat liver was able to convert the 7ketosteroids formed by the bird liver into nonketonic unconjugated compounds. This would seem to confirm the previous evidence that the formation of 7ketosteroids might be an intermediate step. When citrate was added to any of the tissue preparations of birds or mammals, there was a significant, often a marked, increase in the destruc

7 SAMUELS, SWEAT, LEVEDAHL, POTTNER, AND HELMREICH 237 tion of the a,/3 structure. As shown previously (3), this appears to be a specific effect of citrate. There was no effect in the other orders of animals. In addition, there was usually a definite decrease in 7ketosteroid formation if these would otherwise have been formed. One exception was the monkey, in which there was little effect on the 7ketosteroids but a marked increase in ru,p bonds destroyed. This second effect of citrate appears to be unrelated to the first, as can be seen in Table I. A comparison of the results when both citrate and DPN were added with those when either was used alone indicates that the effects were roughly the algebraic sum of the individual influences. The destruction of ar,p groups was greater than with either alone, while the concentration of 7ketosteroids was intermediate. Within mammalian species themselves there was considerable variation in the activity of the different enzyme systems. The liver from only one herbivore was investigated, that of the steer. In Table I it will be seen that there was little or no destruction of the conjugated system or oxidation at C7 by the mince alone. The addition of DPN led to a marked formation of 7ketosteroids but no significant change in Ring A. Addition of citrate brought about the change in cr,/3 structure but no oxidation at the other end of the molecule. If both cofactors were added, changes occurred in both positions. The relative inactivity of steer liver minces and extracts, together with the apparently single action in the presence of each cofactor alone, has been repeatedly observed. This would seem to indicate either a low level of these cofactors in the original tissue or a very rapid destruction during preparation. Apparently the enzyme system which does not require either of these cofactors and which is present in the livers of the lower orders is not found in steer liver, or else some as yet unknown cofactor is absent. Rabbit liver seems to show a more fundamental difference from the liver of other mammals. While the unsupplemented liver both destroyed the or,p structure and formed 7ketosteroids, the addition of DPN had no influence on the rate of either reaction. Citrate increased the destruction of cr,p groups and depressed the formation of 7ketosteroids, but the simultaneous addition of DPN had no significant additional effect. These observations would all be true if triphosphopyridine nucleotide rather than DPN were the cofactor in this system. The definitive experiment with added triphosphopyridine nucleotide has not yet been done. DISCUSSION The evidence here presented would indicate that, associated with the development of homeothermic control, there was an increase in the complexity of the enzyme systems degrading the gonadal steroids.

8 238 METABOLISM OF TESTOSTERONE BY LIVER In the poikilotherms body temperature is ordinarily lower than that of the homeotherms and chemical reactions, both synthetic and degradative, would be slower in the presence of the same concentrations of substrate and catalyst. The turnover of such active compounds as the sex steroids would therefore be less, and the need for complex and rapid adjustment rare. Moreover, the changes in temperature of the organism due to season and environment themselves regulate the production of hormones. A simple system of inactivation would be sufficient. In the homeotherms, however, production of sex steroids is both more rapid and more complex. Cyclic phenomena occur under conditions of a constant internal physical environment. In turn more rapid inactivation mechanisms have been developed. These, however, require labile energy transfer systems. Thus, while destruction may be more rapid, it also becomes subject to the supply of these systems. A more rapid, more adaptable, but also more labile system has thus evolved. SUMMARY The ability of liver minces and slices from various species and orders of animals to destroy testosterone has been investigated, both in the absence and in the presence of added diphosphopyridine nucleotide and citrate. In fish, amphibians, and reptiles there appears to be an enzyme system which slowly destroys the conjugated double bond system in Ring A but does not form 7ketosteroids. No system, the activity of which was increased by DPN or citrate, was identified. In birds and mammals, systems requiring DPN and citrate are present. The DPNcatalyzed reaction leads to the formation of 7ketosteroids, while that requiring citrate acts on the (Y,P conjugation in Ring A without forming 7ketosteroids. The formation of 7ketosteroids in the presence of DPN is also decreased by citrate. The relative concentrations of the two systems requiring cofactors, as well as cofactor availability, vary between different species. The chicken appears to have a relatively high concentration of the DPNactivated system, man and the guinea pig an intermediate balance, and the rat and dog a relatively high activity of the citrateactivated enzyme. Steer liver shows little activity unless cofactors are added, but destruction occurs in the presence of either of the two compounds studied which play this r&e. This would indicate a relative unavailability of these substances, at least under the conditions of preparation of the tissue. Rabbit tissue seems to use some cofactor other than DPN in the oxidation of C7. The relation of the development of the systems activated by DPN

9 SAMUELS, SWEAT, LEVEDAHL, POTTNER, AND HELMREICH 239 and citrate to the appearance of homeothermic mechanisms is discussed. A tentative outline of the possible order of enzymic reactions is also given. BIBLIOGRAPHY. Samuels, L. T., McCaulay, C., and Sellers, D. M., J. Bid. Chem., 66, 477 (947). 2. Sweat, M. L., and Samuels, L. T., J. Biol. Chem., 73, 433 (948). 3. Sweat, M. L., and Samuels, L. T., J. Biol. Chem., 76, (948). 4. Samuels, L. T., and Pottner, M., Federalion Proc., 6, 287 (947). 5. Samuels, L. T., J. Biol. Chem., 66, 47 (947). 6. Callow, N. H., Callow, R. K., and Emmens, C. W., Biochem,.I., 32, 32 (938).

10 METABOLISM OF TESTOSTERONE BY LIVERS OF DIFFERENT SPECIES OF ANIMALS Leo T. Samuels, Max L. Sweat, Blaine H. Levedahl, M. M. Pottner and M. L. Helmreich J. Biol. Chem. 95, 83: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites references, of which can be accessed free at tml#reflist