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Observations on the oxidation of methionine-S35 to S35O4 and taurine-S35 in the X-irradiated rat
ARCHIVES
OF BIOCHEMfSTRY
AND
BIOPHYSICS
82,
362-369 (1959)
Observations on the Oxidation of Methionine-S35 to W04 and Taurine436 in the X-Irradiated RatI.2 Robert E. Kay and Cecil Entenman From the Biological Radiological
and Medical Sciences Division, United States Naval Defense Laboratory, San Framisco, California Received
December
8, 1958
Increases in the urinary excretion of taurine by the x-irradiated rat were first reported by Kay et al. (1, 2) and later confirmed by Mefferd (3), Aebi et aE. (4), and Pentz (5). In addition, Kay et al. (6) showed that following x-irradiation there is an increase in urinary sulfate and urea excretion. In view of the known x-ray-induced stimulation of adrenal activity (7-9) and the established catabolic effects of adrenal cortical hormones on protein metabolism, it appeared possible that the excess taurine, urea, and sulfate excreted could be derived from protein breakdown brought about by adrenal stimulation. Indeed, in regard to urea and sulfate this appeared to be the case, since adrenalectomy prior to x-irradiation prevented the excessive excretion of these compounds (6). On the other hand, adrenalectomy did not prevent the excessive urinary excretion of taurine following x-irradiation (6). Extensive studies (6, 10) carried out on organectomized and partially shielded rats showed that the abdomen must be irradiated and the pancreas present to obtain excessive taurine excretion. However, the pancreas was not the source of the excessive taurine, and numerous experiments failed to establish any one organ in the abdominal area as the source (10). Since, the excessive urinary taurine excreted following x-irradiation was apparently not derived from any one particular organ or from protein breakdown due to adrenal stimulation, it appeared that the excessive taurine excreted might be the result of an alteration in the metabolism of sulfur-containing compounds. Therefore, the metabolism of methionine was studied in the x-irradiated rat. Methionine was selected as the compound to be studied, since it is one of the most abundant sulfur-containing 1 This work was supported, in part, by funds provided by the Bureau of Medicine and Surgery, U. S. Navy Department. * The opinions or assertions contained herein are private ones of the authors and are not to be construed as official, or reflecting the views of the Navy Department. 362
OXIDATION
OF
363
METHIONINE
compounds found in biological material and is capable of being converted readily to other sulfur-containing compounds. METHODS
Animals Female Sprague-Dawley rats 10 weeks of age weighing 208-214 g. were selected from groups of rats born during the same week. All rats were maintained ad Zibitum on Purina laboratory chow and tap water. Twenty-four hours prior to the time of irradiation, the food was removed and the animals were allowed only water until the time of sacrifice.
Radiation of Animals In each experiment the rats were x-irradiated in groups of six with a single dose of 600 r. (250 kvp. 15 ma., 0.5 mm. Cu, 1 mm. Al filter, 1.5 mm. Cu half-value layer) at a rate of 25 r./min. and a target-to-skin distance of 40 in. The rats were held in acetate-plastic boxes during the period of irradiation and rotated under the x-ray beam (11). Control rats were treated in the same manner.
Collection of Urine and Incubation
of Liver Slices
When 24-hr. urine collections were made, the rats were placed in individual stainless steel metabolism cages and the urine and cage washings were collected by a method previously described (2). When liver slices were obtained, the rats were sacrificed by a blow on the head 4 hr. after the time of x-irradiation. Immediately after sacrifice the liver was removed, weighed, and placed in iced bicarbonateRinger solution (12). A lobe of appropriate size was selected and sliced in a St)adieRiggs microtome (13). Slices of 0.5 mm. in thickness and weighing about 200 mg. were obtained. These slices were blotted on filter paper, and duplicate 400 f 10 mg. samples of the slices were incubated at 37.5”C. for 3 hr. under an atmosphere of 95% 02 and 5% CO2 . At the end of the incubation period 4 ml. of trichloroacet,ic acid was added to the incubation mixture.
Isolation and Determination
of Urinary Sulfate and Taurine
The amount of sulfate excreted in the urine was determined as follows: 10 ml. of urine plus washings were transferred to a 1 X 6 cm., Dowex 50 ion-exchange column (8X, 200-400 mesh, hydrogen form) and the compounds were eluted with distilled water. The first 20 ml. of eluant were collected and evaporated to a volume of 10 ml. A 4-ml. aliquot was treated with benzidine hydrochloride by the method of Kahn and Leiboff (14), and the benzidine sulfate formed was isolated and determined by the method of Andersen (15). The taurine content of the urine plus washings was determined by chromatographing 30 ~1. of the solution on paper according to the method of Kay et al. (16).
Isolation of Taurine and Sulfate from Liver Preparation The contents of the incubation flask were washed into a glass homogenizing tube with small amounts of 10% trichloroacetic acid, and the volume was made to 10 ml. The tissue was homogenized and the homogenate centrifuged. Five milliliters of the supernatant was pipetted into a test tube, and the trichloroacetic acid was extracted
364
KAY AND ENTENMAN
with ethyl ether. The aqueous solution was evaporated to dryness and the residue taken up in 1.1 ml. of distilled water. One milliliter of the concentrated solution was transferred to a 1 X 6 cm., Dowex 50 ion-exchange column (8X, 200-400 mesh, hydrogen form), and the sulfate and taurine were eluted with 8 ml. of distilled water. Five milliliters of the eluant was pipetted into a test tube and evaporated to dryness. The residue was taken up in 0.2 ml. of distilled water. Thirty microliters of the concentrated solution was applied to a paper chromatogram, and the taurine was isolated and determined by the method of Kay et al. (16). The sulfate present in 1.0 ml. of the eluant from the Dowex 50 column was precipitated as beneidine sulfate by the addition of 2 mg. of ammonium sulfate and benzidine hydrochloride reagent (14).
Isotope Measurement The taurine isolated on the paper chromatograms was eluted with 71% ethanof and mounted as a thin layer on copper disks. The activity was counted with a thin mica end-window Geiger tube, and an appropriate correction for decay was made. The 536content of the benzidine sulfate was determined by mounting the precipitate on filter-paper disks, counting, and correcting for mass absorption and decay by the method of Henriques et al. (17). The amount of activity injected into the rat or added to the bath was determined by two methods. In the first method the activity was diluted by a factor of 5 or 50, and 10 ~1. of the diluted sample was mounted as an infinitely thin layer on a copper disk. The activity was counted, and the results obtained were used when computing the per cent conversion of methionine-W to taurine-P6. In the second method the methionine was oxidized to sulfate, and the sulfate was isolated as benzidine sulfate. The benzidine sulfate was mounted on filter-paper disks, and the activity was determined and corrected for self-absorption (17). The activity found by this method was used to calculate the per cent conversion of methionine536 to sulfate-W and the per cent excretion of injected sulfate-P The ratio of the counts/min. obtained by the direct mounting technique to the counts/min. obtained by mounting the sulfate as bensidine sulfate was 1.28:1. The injected dose given in the test was obtained by oxidizing the methionine to sulfate, mounting, and counting as benzidine sulfate. EXPERIMENTAL
AND RESULTS
The E$ect of Whole-Body X-Irradiation on the Urinary Excretion of Free Sulfate-P and Taurine-Sa6 Following Injection of Methionine-S36 The rate of oxidation of a substance is often reflected in the rate of excretion of its oxidation products in the urine. Therefore, in order to determine if the oxidation of methionine is enhanced following x-irradiation, the rates of excretion of the end products of methionine oxidation, taurine and sulfate, were investigated. Twelve rats were fasted for 24 hr. and divided into two groups of six rats each. One group was exposed to 600 r. of total body x-irradiation, and the other group was sham-irradiated. Immediately after the time of x-irradiation, all of the rats were injected intraperitoneally with 0.20 ml, of distilled water containing 20 Mmoles L-methionine including 1,640,000 counts/min. L-methionine-S36. Urine was
OXIDATION
Urinary
Excretion of Taurine-P
OF
TABLE I and Sulfate-W
of Methionine-S35
365
METHIONINE
following Zntraperitoneal Rats
Injection
into X-Zrradiated
Inorganic sulfate
Taurine Measurement 600 r. Total amount excreted,
0 r.
600 r.
0 r.
16.4 f
0.74~ 7.04 f
0.67
32.4 f
1.8
23.5 f
2.0
3.49 i
0.10
0.15
25.6 f
1.09
17.8 f
1.28
rng.124 hr.
Total activity excreated, y. I.D.b/2.4
2.60 f
hr.
Specific
activity,
To 0.212 f
0.0140.372 f
0.0300.789 f
0.0230.758 =I=0.038
Z.D./mg.
0 The averages obtained from six rats plus or minus the standard error of the mean. b The term I.D. stands for injected dose of radioactivity.
collected for 24 hr. and the amount of activity excreted as sulfate-W and taurine-S35 as well as the total amount of taurine and sulfate excreted was determined. The results appear in Table I. Both sulfate and taurine excretions were increased in the x-irradiated rat. Taurine excretion increased by 133 % and sulfate excretion by 37 %. The increase in the excretion of P5 as sulfate by x-irradiated rats was approximately the same as the increase in total sulfate excretion, and thus the specific activity of the sulfate excreted by the x-irradiated rat was not different from that excreted by the unirradiated rat. On the other hand, the increase in excretion of W as taurine by the x-irradiated rat was only 34% as compared to the 133% increase in total taurine excretion. Therefore, although slightly more taurine-LY6 was excreted by the x-irradiated rat, the specific activity of the taurine excreted was only 57 % of the control value. The E$ect of X-Irradiation on the Urinary Excretion of Intraperitoneally Injected S3504 The results of the above experiments suggest that either more methionine is oxidized to sulfate or that the handling of sulfate by the kidneys is albered after x-irradiation. If kidney function is altered, then the source of the sulfate is not a factor and injected inorganic sulfate should be excreted at a greater rate following x-irradiation. To investigate this possibility the following experiment was performed. Twelve rats were fasted for 24 hr. and divided into two equal groups. One group was exposed to 600 r. of whole-body x-irradiation and the other group was sham-irradiated. Immediately after the time of irradiation the rats were given by intraperitoneal injection 0.25 ml. of a solution containing 5 pmoles of sodium sulfate
366
KAY
AND
ENTENMAN
TABLE Urinary Excretion
Treatment
600 r. 0 r.
II
of Sulfate-W
following Intraperitoneal NadP604 into X-Irradiated Rats
Av. total inorganic sulfate, mgJ2.J hr. 24.9 zk 0.P 19.4 f 1.7
Injection
y0 injected radioactivity in 24 hr. 71.9 f 1.8 69.8 zk 2.7
of
excreted”
per mg. of inorganic sulfate 2.90 f 3.52 f
0.12 0.14
0 Values are the average obtained from six rats plus or minus the standard error of the mean.
including 70,540 countq’min. of NazS3504. The rats were placed in individual metabolism cages, and the urine was collected for 24 hr. The total amount of inorganic sulfate and S3604present in the urine was determined, and the specific activity of the sulfate was calculated. The results are given in Table II. The total inorganic sulfate excreted by the x-irradiated rats was greater than that excreted by the controls, but the total S3604excretion did not increase after x-irradiation. As a result, the specific activity of the inorganic sulfate excreted by the x-irradiated rat was less than the control value. Neglecting possible changes in the absorption of sulfate following x-irradiation, the data suggest that the kidneys do not handle inorganic sulfate in a different manner following x-irradiation. The E$ect of Whole-Body X-Irradiation on the Conversion of MethionirmS35 to Taurine-S35 and Sulfate-S35 by Liver Slices The results of the preceding experiments suggest that the increase in sulfate excretion following x-irradiation may be due to an increase in the rate of methionine oxidation, The liver appears to be a primary site for the oxidation of methionine and its metabolic products to taurine and sulfate (18, 19). Thus, if an increase in the oxidation of methionine is responsible for the increased sulfate excretion following x-irradiation, one might expect that the oxidation of methionine by surviving rat liver slices from x-irradiated rats would be enhanced. This possibility was investigated. Twelve rats were fasted 24 hr. and divided into groups of six rats each. One group of rats was exposed to 600 r. of whole-body x-radiation, and the other group was sham-irradiated. Four hours after the time of x-irradiation, the rats were scarificed and liver slices were incubated in 5 ml. of bicarbonate-Ringer solution containing 10 pmoles L-methionine including 829,400 counts/min. L-methionine-S 35. The conversion of methionine-P6 to taurine-P5 and sulfate-Ss6 was determined. The results of the experiment (Table III) show that the rate of conversion of methionine-S36 to taurine-
OXIDATION
OF
TABLE Conversion
367
METHIONINE
III
of Methionine-W to Taurine-835 and Inorganic Lizler Slices from X-Irradiated Rats
Treatment
m Values are the average of the mean.
by
Per cent Sz5 added as methionine-W recovered as Taurinea
600 r. 0 r.
Sulfate-W
3.69 f 3.34 f obtained
0.43 0.17
Inorganica 12.2 f 12.6 f
sulfate 0.104 0.84
from six rats plus or minus the standard
S35and sulfate-S35 by liver slices from x-irradiated that of unirradiated rats.
error
rats is not different from
DISCTJSSION
Increases in the urinary excretion of both taurine and inorganic sulfate have been found following x-irradiation (6). Since these compounds are common end products of the oxidation of sulfur-containing substances, it appeared that the excess taurine and sulfate excreted might be derived from the same source. However, adrenalectomy prior to x-irradiation prevented the excess excretion of inorganic sulfate, but not taurine (6). This finding suggested that the excess sulfate excreted after x-irradiation arose from a different source than the excess taurine. The data in the present study show that this is the case. The rate of appearance in the urine of inorganic sulfate-S35 derived from injected methionine-S35 is greater in x-irradiated rats than in unirradiated rats, but the specific activity of the urinary sulfate is the same in both x-irradiated and unirradiated rats. Thus, the excess sulfat,e-S35 found in the urine of the x-irradiated rat is derived entirely from an increase in the rate of methionine-S35 oxidation, or it is due to an alteration in kidney function. An increase in the oxidation of methionine appears to be the most probable source of the excessive inorganic sulfate, since intraperitoneally injected inorganic sulfate does not appear in the urine at a more rapid rate following x-irradiation. However, if the excess sulfate found in the urine following x-irradiation is due to an increase in methionine oxidation, it is apparently the result of an increase in an extrahepatic function, since oxidation of methionine to inorganic sulfate and taurine by liver slices is not enhanced by exposure of the rat. t.o x-irradiation. Because the protein catabolic effects of the adrenals are essentially extrahepatic (20) and excess sulfate does not appear in the urine of the adrenalectomized x-irradiated rat (6), it seems likely that the excess
368
KAY AND ENTENMAN
sulfate found in the urine of the x-irradiated rat is derived from extrahepatic catabolism related to adrenal activity. Although the rate of appearance in the urine of taurine-S35 derived from injected methionine-S35 is somewhat greater than in the unirradiated rat, the specific activity of the urinary taurine is only 57 % of the control value. This shows that the bulk of the excesstaurine found in the urine following x-irradiation is probably derived from a source other than the oxidation of methionine. In addition, the data indicate that the increase in urinary taurine is probably not due to an alteration in kidney function. If the increase were due to a change in kidney function the source of the taurine should not be a factor. However, labeled urinary taurine derived from methionine-EY5is increased 34 %, whereas total urinary taurine is increased 133 %. SUMMARY
The bulk of the excess taurine appearing in the urine of rats following x-irradiation does not appear to be derived from the oxidation of methionine, whereas the excess inorganic sulfate found in the urine is due to an increase in methionine oxidation or to an alteration in kidney function. An increase in methionine oxidation is more probable, since intraperitoneally injected sulfate-S 36 does not appear in the urine at a more rapid rate. However, the increase in methionine oxidation does not appear to be the result of increased hepatic oxidation since oxidation of methionine by liver slices is not enhanced. REFERENCES 1. KAY, R. E., AND ENTENMAN, C., Federation Proc. 13, 520 (1954). 2. KAY, R. E., HARRIS, D. C., AND ENTENMAN, C., Am. J. Physiol. 186, 175 (1955). 3. MEFFERD, R. B., AND MARTENS, H. H., Science 122,829 (1955). 4. AEBI, H., BERNAYS, L., FLUCEIGER, H., SCHMIDLI, B., AND ZUPPINGER, A., Helv. Physiol. et Pharmacol. Acta 13, C49 (1955). 5. PENTZ, E. I., J. Biol. Chem. 231, 165 (1958). 6. KAY, R. E., EARLY, J. C., AND ENTENMAN, C., Radiation Research 6, 98 (1957). 7. WEXLER, B. C., PENCHARZ, R., AND THOMAS, S. F., Am. J. Physiol. 183,71 (1955). 8. NIMS, L. F., AND SUTTON, E., Am. J. Physiol. 177, 51 (1954). 9. FRENCH, A. B., MIGEON, C. J., SAMUELS, L. T., AND BOWERS, J. Z., Am. J. Physiol. 183, 469 (1955). 10. KAY, R. E., AND ENTENMAN, C., U. S. Naval Radiological Defense Laboratory Report, TR-239, May 27, 1958. Il. BOND, V. P., SWIFT, M. N., ALLEN, A. C., AND FISHLER, M. C., Am. J. Physiol. 161, 323 (1950). 12. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques and Tissue Metabolism.” Burgess Publ. Co., Minneapolis, Minn., 1949. 13. STADIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 164,687 (1944). 14. KOHN, B. S., AND LEIBOFF, S. L., J. Biol. Chem. 80, 626 (1928).
OXIDATION
OF
METHIONINE
369
15. ANDERSEN, L., Acta &em. Stand. 7,689 (1953). 16. KAY, R. E., HARRIS, D. C., AND ENTENMAN, C., Arch. Biochem. Biophys. 63, 14 (1956). 17. HENRIQUES, F. C., JR., KISTIAKOWSKY, G. B., MARGNETTI, C., AND SCHNEIDER, W. G., In& Eng. Chem., Anal. Ed. 16, 349 (1946). 18. ELDJARN, L., Stand. J. Clin. & Lab. Invest. 6, Suppl. No. 13 (1954). 19. AWAPARA, J., J. Biol. Chem. 203, 183 (1953). 20. SILBER, R. H., AND PORTER, C. C., Endocrinology 62, 618 (1953).
OF BIOCHEMfSTRY
AND
BIOPHYSICS
82,
362-369 (1959)
Observations on the Oxidation of Methionine-S35 to W04 and Taurine436 in the X-Irradiated RatI.2 Robert E. Kay and Cecil Entenman From the Biological Radiological
and Medical Sciences Division, United States Naval Defense Laboratory, San Framisco, California Received
December
8, 1958
Increases in the urinary excretion of taurine by the x-irradiated rat were first reported by Kay et al. (1, 2) and later confirmed by Mefferd (3), Aebi et aE. (4), and Pentz (5). In addition, Kay et al. (6) showed that following x-irradiation there is an increase in urinary sulfate and urea excretion. In view of the known x-ray-induced stimulation of adrenal activity (7-9) and the established catabolic effects of adrenal cortical hormones on protein metabolism, it appeared possible that the excess taurine, urea, and sulfate excreted could be derived from protein breakdown brought about by adrenal stimulation. Indeed, in regard to urea and sulfate this appeared to be the case, since adrenalectomy prior to x-irradiation prevented the excessive excretion of these compounds (6). On the other hand, adrenalectomy did not prevent the excessive urinary excretion of taurine following x-irradiation (6). Extensive studies (6, 10) carried out on organectomized and partially shielded rats showed that the abdomen must be irradiated and the pancreas present to obtain excessive taurine excretion. However, the pancreas was not the source of the excessive taurine, and numerous experiments failed to establish any one organ in the abdominal area as the source (10). Since, the excessive urinary taurine excreted following x-irradiation was apparently not derived from any one particular organ or from protein breakdown due to adrenal stimulation, it appeared that the excessive taurine excreted might be the result of an alteration in the metabolism of sulfur-containing compounds. Therefore, the metabolism of methionine was studied in the x-irradiated rat. Methionine was selected as the compound to be studied, since it is one of the most abundant sulfur-containing 1 This work was supported, in part, by funds provided by the Bureau of Medicine and Surgery, U. S. Navy Department. * The opinions or assertions contained herein are private ones of the authors and are not to be construed as official, or reflecting the views of the Navy Department. 362
OXIDATION
OF
363
METHIONINE
compounds found in biological material and is capable of being converted readily to other sulfur-containing compounds. METHODS
Animals Female Sprague-Dawley rats 10 weeks of age weighing 208-214 g. were selected from groups of rats born during the same week. All rats were maintained ad Zibitum on Purina laboratory chow and tap water. Twenty-four hours prior to the time of irradiation, the food was removed and the animals were allowed only water until the time of sacrifice.
Radiation of Animals In each experiment the rats were x-irradiated in groups of six with a single dose of 600 r. (250 kvp. 15 ma., 0.5 mm. Cu, 1 mm. Al filter, 1.5 mm. Cu half-value layer) at a rate of 25 r./min. and a target-to-skin distance of 40 in. The rats were held in acetate-plastic boxes during the period of irradiation and rotated under the x-ray beam (11). Control rats were treated in the same manner.
Collection of Urine and Incubation
of Liver Slices
When 24-hr. urine collections were made, the rats were placed in individual stainless steel metabolism cages and the urine and cage washings were collected by a method previously described (2). When liver slices were obtained, the rats were sacrificed by a blow on the head 4 hr. after the time of x-irradiation. Immediately after sacrifice the liver was removed, weighed, and placed in iced bicarbonateRinger solution (12). A lobe of appropriate size was selected and sliced in a St)adieRiggs microtome (13). Slices of 0.5 mm. in thickness and weighing about 200 mg. were obtained. These slices were blotted on filter paper, and duplicate 400 f 10 mg. samples of the slices were incubated at 37.5”C. for 3 hr. under an atmosphere of 95% 02 and 5% CO2 . At the end of the incubation period 4 ml. of trichloroacet,ic acid was added to the incubation mixture.
Isolation and Determination
of Urinary Sulfate and Taurine
The amount of sulfate excreted in the urine was determined as follows: 10 ml. of urine plus washings were transferred to a 1 X 6 cm., Dowex 50 ion-exchange column (8X, 200-400 mesh, hydrogen form) and the compounds were eluted with distilled water. The first 20 ml. of eluant were collected and evaporated to a volume of 10 ml. A 4-ml. aliquot was treated with benzidine hydrochloride by the method of Kahn and Leiboff (14), and the benzidine sulfate formed was isolated and determined by the method of Andersen (15). The taurine content of the urine plus washings was determined by chromatographing 30 ~1. of the solution on paper according to the method of Kay et al. (16).
Isolation of Taurine and Sulfate from Liver Preparation The contents of the incubation flask were washed into a glass homogenizing tube with small amounts of 10% trichloroacetic acid, and the volume was made to 10 ml. The tissue was homogenized and the homogenate centrifuged. Five milliliters of the supernatant was pipetted into a test tube, and the trichloroacetic acid was extracted
364
KAY AND ENTENMAN
with ethyl ether. The aqueous solution was evaporated to dryness and the residue taken up in 1.1 ml. of distilled water. One milliliter of the concentrated solution was transferred to a 1 X 6 cm., Dowex 50 ion-exchange column (8X, 200-400 mesh, hydrogen form), and the sulfate and taurine were eluted with 8 ml. of distilled water. Five milliliters of the eluant was pipetted into a test tube and evaporated to dryness. The residue was taken up in 0.2 ml. of distilled water. Thirty microliters of the concentrated solution was applied to a paper chromatogram, and the taurine was isolated and determined by the method of Kay et al. (16). The sulfate present in 1.0 ml. of the eluant from the Dowex 50 column was precipitated as beneidine sulfate by the addition of 2 mg. of ammonium sulfate and benzidine hydrochloride reagent (14).
Isotope Measurement The taurine isolated on the paper chromatograms was eluted with 71% ethanof and mounted as a thin layer on copper disks. The activity was counted with a thin mica end-window Geiger tube, and an appropriate correction for decay was made. The 536content of the benzidine sulfate was determined by mounting the precipitate on filter-paper disks, counting, and correcting for mass absorption and decay by the method of Henriques et al. (17). The amount of activity injected into the rat or added to the bath was determined by two methods. In the first method the activity was diluted by a factor of 5 or 50, and 10 ~1. of the diluted sample was mounted as an infinitely thin layer on a copper disk. The activity was counted, and the results obtained were used when computing the per cent conversion of methionine-W to taurine-P6. In the second method the methionine was oxidized to sulfate, and the sulfate was isolated as benzidine sulfate. The benzidine sulfate was mounted on filter-paper disks, and the activity was determined and corrected for self-absorption (17). The activity found by this method was used to calculate the per cent conversion of methionine536 to sulfate-W and the per cent excretion of injected sulfate-P The ratio of the counts/min. obtained by the direct mounting technique to the counts/min. obtained by mounting the sulfate as bensidine sulfate was 1.28:1. The injected dose given in the test was obtained by oxidizing the methionine to sulfate, mounting, and counting as benzidine sulfate. EXPERIMENTAL
AND RESULTS
The E$ect of Whole-Body X-Irradiation on the Urinary Excretion of Free Sulfate-P and Taurine-Sa6 Following Injection of Methionine-S36 The rate of oxidation of a substance is often reflected in the rate of excretion of its oxidation products in the urine. Therefore, in order to determine if the oxidation of methionine is enhanced following x-irradiation, the rates of excretion of the end products of methionine oxidation, taurine and sulfate, were investigated. Twelve rats were fasted for 24 hr. and divided into two groups of six rats each. One group was exposed to 600 r. of total body x-irradiation, and the other group was sham-irradiated. Immediately after the time of x-irradiation, all of the rats were injected intraperitoneally with 0.20 ml, of distilled water containing 20 Mmoles L-methionine including 1,640,000 counts/min. L-methionine-S36. Urine was
OXIDATION
Urinary
Excretion of Taurine-P
OF
TABLE I and Sulfate-W
of Methionine-S35
365
METHIONINE
following Zntraperitoneal Rats
Injection
into X-Zrradiated
Inorganic sulfate
Taurine Measurement 600 r. Total amount excreted,
0 r.
600 r.
0 r.
16.4 f
0.74~ 7.04 f
0.67
32.4 f
1.8
23.5 f
2.0
3.49 i
0.10
0.15
25.6 f
1.09
17.8 f
1.28
rng.124 hr.
Total activity excreated, y. I.D.b/2.4
2.60 f
hr.
Specific
activity,
To 0.212 f
0.0140.372 f
0.0300.789 f
0.0230.758 =I=0.038
Z.D./mg.
0 The averages obtained from six rats plus or minus the standard error of the mean. b The term I.D. stands for injected dose of radioactivity.
collected for 24 hr. and the amount of activity excreted as sulfate-W and taurine-S35 as well as the total amount of taurine and sulfate excreted was determined. The results appear in Table I. Both sulfate and taurine excretions were increased in the x-irradiated rat. Taurine excretion increased by 133 % and sulfate excretion by 37 %. The increase in the excretion of P5 as sulfate by x-irradiated rats was approximately the same as the increase in total sulfate excretion, and thus the specific activity of the sulfate excreted by the x-irradiated rat was not different from that excreted by the unirradiated rat. On the other hand, the increase in excretion of W as taurine by the x-irradiated rat was only 34% as compared to the 133% increase in total taurine excretion. Therefore, although slightly more taurine-LY6 was excreted by the x-irradiated rat, the specific activity of the taurine excreted was only 57 % of the control value. The E$ect of X-Irradiation on the Urinary Excretion of Intraperitoneally Injected S3504 The results of the above experiments suggest that either more methionine is oxidized to sulfate or that the handling of sulfate by the kidneys is albered after x-irradiation. If kidney function is altered, then the source of the sulfate is not a factor and injected inorganic sulfate should be excreted at a greater rate following x-irradiation. To investigate this possibility the following experiment was performed. Twelve rats were fasted for 24 hr. and divided into two equal groups. One group was exposed to 600 r. of whole-body x-irradiation and the other group was sham-irradiated. Immediately after the time of irradiation the rats were given by intraperitoneal injection 0.25 ml. of a solution containing 5 pmoles of sodium sulfate
366
KAY
AND
ENTENMAN
TABLE Urinary Excretion
Treatment
600 r. 0 r.
II
of Sulfate-W
following Intraperitoneal NadP604 into X-Irradiated Rats
Av. total inorganic sulfate, mgJ2.J hr. 24.9 zk 0.P 19.4 f 1.7
Injection
y0 injected radioactivity in 24 hr. 71.9 f 1.8 69.8 zk 2.7
of
excreted”
per mg. of inorganic sulfate 2.90 f 3.52 f
0.12 0.14
0 Values are the average obtained from six rats plus or minus the standard error of the mean.
including 70,540 countq’min. of NazS3504. The rats were placed in individual metabolism cages, and the urine was collected for 24 hr. The total amount of inorganic sulfate and S3604present in the urine was determined, and the specific activity of the sulfate was calculated. The results are given in Table II. The total inorganic sulfate excreted by the x-irradiated rats was greater than that excreted by the controls, but the total S3604excretion did not increase after x-irradiation. As a result, the specific activity of the inorganic sulfate excreted by the x-irradiated rat was less than the control value. Neglecting possible changes in the absorption of sulfate following x-irradiation, the data suggest that the kidneys do not handle inorganic sulfate in a different manner following x-irradiation. The E$ect of Whole-Body X-Irradiation on the Conversion of MethionirmS35 to Taurine-S35 and Sulfate-S35 by Liver Slices The results of the preceding experiments suggest that the increase in sulfate excretion following x-irradiation may be due to an increase in the rate of methionine oxidation, The liver appears to be a primary site for the oxidation of methionine and its metabolic products to taurine and sulfate (18, 19). Thus, if an increase in the oxidation of methionine is responsible for the increased sulfate excretion following x-irradiation, one might expect that the oxidation of methionine by surviving rat liver slices from x-irradiated rats would be enhanced. This possibility was investigated. Twelve rats were fasted 24 hr. and divided into groups of six rats each. One group of rats was exposed to 600 r. of whole-body x-radiation, and the other group was sham-irradiated. Four hours after the time of x-irradiation, the rats were scarificed and liver slices were incubated in 5 ml. of bicarbonate-Ringer solution containing 10 pmoles L-methionine including 829,400 counts/min. L-methionine-S 35. The conversion of methionine-P6 to taurine-P5 and sulfate-Ss6 was determined. The results of the experiment (Table III) show that the rate of conversion of methionine-S36 to taurine-
OXIDATION
OF
TABLE Conversion
367
METHIONINE
III
of Methionine-W to Taurine-835 and Inorganic Lizler Slices from X-Irradiated Rats
Treatment
m Values are the average of the mean.
by
Per cent Sz5 added as methionine-W recovered as Taurinea
600 r. 0 r.
Sulfate-W
3.69 f 3.34 f obtained
0.43 0.17
Inorganica 12.2 f 12.6 f
sulfate 0.104 0.84
from six rats plus or minus the standard
S35and sulfate-S35 by liver slices from x-irradiated that of unirradiated rats.
error
rats is not different from
DISCTJSSION
Increases in the urinary excretion of both taurine and inorganic sulfate have been found following x-irradiation (6). Since these compounds are common end products of the oxidation of sulfur-containing substances, it appeared that the excess taurine and sulfate excreted might be derived from the same source. However, adrenalectomy prior to x-irradiation prevented the excess excretion of inorganic sulfate, but not taurine (6). This finding suggested that the excess sulfate excreted after x-irradiation arose from a different source than the excess taurine. The data in the present study show that this is the case. The rate of appearance in the urine of inorganic sulfate-S35 derived from injected methionine-S35 is greater in x-irradiated rats than in unirradiated rats, but the specific activity of the urinary sulfate is the same in both x-irradiated and unirradiated rats. Thus, the excess sulfat,e-S35 found in the urine of the x-irradiated rat is derived entirely from an increase in the rate of methionine-S35 oxidation, or it is due to an alteration in kidney function. An increase in the oxidation of methionine appears to be the most probable source of the excessive inorganic sulfate, since intraperitoneally injected inorganic sulfate does not appear in the urine at a more rapid rate following x-irradiation. However, if the excess sulfate found in the urine following x-irradiation is due to an increase in methionine oxidation, it is apparently the result of an increase in an extrahepatic function, since oxidation of methionine to inorganic sulfate and taurine by liver slices is not enhanced by exposure of the rat. t.o x-irradiation. Because the protein catabolic effects of the adrenals are essentially extrahepatic (20) and excess sulfate does not appear in the urine of the adrenalectomized x-irradiated rat (6), it seems likely that the excess
368
KAY AND ENTENMAN
sulfate found in the urine of the x-irradiated rat is derived from extrahepatic catabolism related to adrenal activity. Although the rate of appearance in the urine of taurine-S35 derived from injected methionine-S35 is somewhat greater than in the unirradiated rat, the specific activity of the urinary taurine is only 57 % of the control value. This shows that the bulk of the excesstaurine found in the urine following x-irradiation is probably derived from a source other than the oxidation of methionine. In addition, the data indicate that the increase in urinary taurine is probably not due to an alteration in kidney function. If the increase were due to a change in kidney function the source of the taurine should not be a factor. However, labeled urinary taurine derived from methionine-EY5is increased 34 %, whereas total urinary taurine is increased 133 %. SUMMARY
The bulk of the excess taurine appearing in the urine of rats following x-irradiation does not appear to be derived from the oxidation of methionine, whereas the excess inorganic sulfate found in the urine is due to an increase in methionine oxidation or to an alteration in kidney function. An increase in methionine oxidation is more probable, since intraperitoneally injected sulfate-S 36 does not appear in the urine at a more rapid rate. However, the increase in methionine oxidation does not appear to be the result of increased hepatic oxidation since oxidation of methionine by liver slices is not enhanced. REFERENCES 1. KAY, R. E., AND ENTENMAN, C., Federation Proc. 13, 520 (1954). 2. KAY, R. E., HARRIS, D. C., AND ENTENMAN, C., Am. J. Physiol. 186, 175 (1955). 3. MEFFERD, R. B., AND MARTENS, H. H., Science 122,829 (1955). 4. AEBI, H., BERNAYS, L., FLUCEIGER, H., SCHMIDLI, B., AND ZUPPINGER, A., Helv. Physiol. et Pharmacol. Acta 13, C49 (1955). 5. PENTZ, E. I., J. Biol. Chem. 231, 165 (1958). 6. KAY, R. E., EARLY, J. C., AND ENTENMAN, C., Radiation Research 6, 98 (1957). 7. WEXLER, B. C., PENCHARZ, R., AND THOMAS, S. F., Am. J. Physiol. 183,71 (1955). 8. NIMS, L. F., AND SUTTON, E., Am. J. Physiol. 177, 51 (1954). 9. FRENCH, A. B., MIGEON, C. J., SAMUELS, L. T., AND BOWERS, J. Z., Am. J. Physiol. 183, 469 (1955). 10. KAY, R. E., AND ENTENMAN, C., U. S. Naval Radiological Defense Laboratory Report, TR-239, May 27, 1958. Il. BOND, V. P., SWIFT, M. N., ALLEN, A. C., AND FISHLER, M. C., Am. J. Physiol. 161, 323 (1950). 12. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques and Tissue Metabolism.” Burgess Publ. Co., Minneapolis, Minn., 1949. 13. STADIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 164,687 (1944). 14. KOHN, B. S., AND LEIBOFF, S. L., J. Biol. Chem. 80, 626 (1928).
OXIDATION
OF
METHIONINE
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15. ANDERSEN, L., Acta &em. Stand. 7,689 (1953). 16. KAY, R. E., HARRIS, D. C., AND ENTENMAN, C., Arch. Biochem. Biophys. 63, 14 (1956). 17. HENRIQUES, F. C., JR., KISTIAKOWSKY, G. B., MARGNETTI, C., AND SCHNEIDER, W. G., In& Eng. Chem., Anal. Ed. 16, 349 (1946). 18. ELDJARN, L., Stand. J. Clin. & Lab. Invest. 6, Suppl. No. 13 (1954). 19. AWAPARA, J., J. Biol. Chem. 203, 183 (1953). 20. SILBER, R. H., AND PORTER, C. C., Endocrinology 62, 618 (1953).