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The metabolism of methionine sulfoximine
The Metabolism of Methionine Sulfoximinel Jay S. Roth2 Promn the William Hahnewlann
Goldman Zsotope Laboratory, Medical College and Hospital, Received
February
Division of Biological Chemistr?/, Philadelphia, Pennqlvania 18, 1954
INTRODUCTION Previous studies on the distribution and excretion of S36-labeled Lmethionine sulfoximine (MSI) in rats (1,2) indicated that approximately 60% of an injected dose was excreted by 24 hr. after the injection. In this report, studies have been made of the distribution of the Sa5in the various sulfur fractions in the urine in an effort to determine the extent to which MS1 is metabolized by the rat. MATERIALS AND METHODS Male Wistar rats weighing approximately 200 g. were injected intraperitoneally with 100 mg. MSI-EYE/kg. This amount did not produce toxic symptoms although the injected animals did not eat very much during the first 24 hr. after the injection. The MS1 had a specific activity of 5.72 X lo5 counts/min./m@;., and it and all samples were assayed in a gas-flow counter with standard geometry. The rats were housed in stainless steel metabolism cages, two to a cage, and urine was collected each 24 hr. using HCl as a preservative. The animals were fed water and Fox chow meal ad libitum. The urine samples were made up to 100 ml.; phosphate was precipitated as MgNHdPOa , and the sulfur was fractionated according t,o the procedure of Fiske as described by Hawk, Oser, and Summerson (3) with several modifications. The precipitated benzidine sulfate was filtered on sintered-glass filter disks of medium porosity 20 mm. in diameter and 10 mm. high. -4fter drying, t,hese could be placed directly in the gas-flow counter. When counted, the disks were placed in a beaker of boiling water, and the benzidine sulfate was titrated with 0.02 N NaOH. Where necessary, corrections have been made in the counm for coincidence, decay, and self-absorption. In the first experiment, eight rats were utilized and urine was collected only 1 This project was supported by a grant from the Navy Department, Office of Naval Research. 2 Present address: Strangeways Research Laboratory, Cambridge, England. 269
270
J. S. ROTH
Excrelion
Total inorganic SO,-
Sample
Rats Rats Rats Rats
1, 3, 5, 7,
TABLE I of P6 During 8.J Hours by Rats Injected with L-Methionine SuljoximineGP
2 4 6 8
Activity countsfmin.
1LJ 12.7 11.1 13.3
‘“%
26,008’ 25,098 22,286 20,607
I sas
lY7 13.9 15.5 16.5
Activity counlr/min.
S”’ as sor %
490,730 415,465 468,982 519,153 Average
5.3 6.0 4.8 4.0 5.0
(1Twenty-milliliter sample of urine used for analysis. b Corrected for blank. Blank consisted of rat urine to which a similar quantity of MSI-W had been added. during the first 24 hr. The results, shown in Table I, indicated that approximately 5% of the S3” excreted during the first day had been oxidized to sulfate. Since the injected MS1 consists of two isomers, only one of which may be biologically active, the material excreted during the first 24 hr. after injection could represent mostly the inactive form which was not metabolized. On subsequent days larger percentages of W might be found in the oxidized form. To test this possibility, the procedure was repeated on six rats, and urine samples were collected on the 2d, 3d, 4th and 5th days. As the first experiment indicated that large deviations were not to be expected in the individual urines, the samples from each day’s collections were pooled and duplicate analyses were carried out on each pooled sample. The results are given in Table II. Examination of the data in this table indicates that an increasing percentage of Sa6was excreted as sulfate from the second to the fifth day of the experiment. By the fifth day, however, the total radioactivity reached a very low value which seemed to be leveling off. It is possible that Sa6excreted as sulfate at this time represented sulfur which had been incorporated into protein
Sulfur Partition sample
Imf go
TABLE II in the Urine of Rats Injected with L-Methionine Sulfozimine-P
Activity
1
Total. inorrzx I
Activity
Ether&Activity I
___-__---~----counts/ #sin.
mg.
countsl nrie.
+w.
counts/ niin.
4.7. 9080 5.8 1394 6.9 590 6.5 434
4.8 6.0 7.5 6.7
9160 1475 660 464
0.1 0.2 0.6 0.2
80 81 70 30
w.
2ndday 3rd day 4th day 5th day
0 Each value is the average of duplicate of urine.
Or: yFsc sot
Activity
counts/
Mb-.
??A
‘Sk’ sor mi?.
Activity
f;‘& 1
COWS/
min.
1.3 106,540 6.1 109,700 8.4 1.4 9,575 7.4 11,050 13.4 1.3 2,922 8.8 3,582 18.4 1.2 971 7.9 1,435 32.4
determinations
on a 5.0-ml. sample
METABOLISM OF METHIONINE
SULFOXIMIiVE
271
and was then being metabolized at a constant rate. The amount of it, however, was not very significant. Of the S36 injected, about 92’% of the tot,al artivity had been recovered in the urine by the sixth day. Very little ethereal sulfate-S36 w-as found.
DISCUSSION
The results indicate that only a very small fraction of an injected dose of MS1 is metabolized, at least by oxidation to sulfate. As preliminary paper chromatography studies indicated (2) that only small amounts of MS1 can be demonstrated in the urine, it is possible that this compound is detoxified by combination with glutamic acid, as has recently been shown to be the case with a very similar compound, methionine sulfone (4). This latter compound is also oxidized only to a small extent to sulfate. SUMMARY
h single dose of L-methionine sulfoximine-S3s was injected into rats and the distribution of S35determined in the various sulfur fractions of the urine at 24 hour intervals for 5 days. The amount of SP as sulfate increased from 5 % the first day to 32 % the fifth. By the fifth day, however, the total excretion of F5 had dropped to low levels, thus the total amount of L-methionine sulfoximine-S36 that was oxidized to sulfate was not much greater than 5 to 6%. REFERENCES 1. ROTH, J. S., WASE, A., AND REINER, L., Science 116, 256 (1952). 2. ROTH, ,J. S., EICHEL, H. J., AND WASE, A., J. Biol. Chem. 200,647 (1953). 3. HAWK, I’. B., OSER, B. L., AND SUMMERSON, W. H., Practical Physiological Chemistry, 12th ed., p. 887. Blakiston, Philadelphia, 1947. 1. WIXGO, W. J., SMITH, R. iz., AND WOOD, J., Arch. Biochem. and Biophgs. 47,3Oi (1953).
Goldman Zsotope Laboratory, Medical College and Hospital, Received
February
Division of Biological Chemistr?/, Philadelphia, Pennqlvania 18, 1954
INTRODUCTION Previous studies on the distribution and excretion of S36-labeled Lmethionine sulfoximine (MSI) in rats (1,2) indicated that approximately 60% of an injected dose was excreted by 24 hr. after the injection. In this report, studies have been made of the distribution of the Sa5in the various sulfur fractions in the urine in an effort to determine the extent to which MS1 is metabolized by the rat. MATERIALS AND METHODS Male Wistar rats weighing approximately 200 g. were injected intraperitoneally with 100 mg. MSI-EYE/kg. This amount did not produce toxic symptoms although the injected animals did not eat very much during the first 24 hr. after the injection. The MS1 had a specific activity of 5.72 X lo5 counts/min./m@;., and it and all samples were assayed in a gas-flow counter with standard geometry. The rats were housed in stainless steel metabolism cages, two to a cage, and urine was collected each 24 hr. using HCl as a preservative. The animals were fed water and Fox chow meal ad libitum. The urine samples were made up to 100 ml.; phosphate was precipitated as MgNHdPOa , and the sulfur was fractionated according t,o the procedure of Fiske as described by Hawk, Oser, and Summerson (3) with several modifications. The precipitated benzidine sulfate was filtered on sintered-glass filter disks of medium porosity 20 mm. in diameter and 10 mm. high. -4fter drying, t,hese could be placed directly in the gas-flow counter. When counted, the disks were placed in a beaker of boiling water, and the benzidine sulfate was titrated with 0.02 N NaOH. Where necessary, corrections have been made in the counm for coincidence, decay, and self-absorption. In the first experiment, eight rats were utilized and urine was collected only 1 This project was supported by a grant from the Navy Department, Office of Naval Research. 2 Present address: Strangeways Research Laboratory, Cambridge, England. 269
270
J. S. ROTH
Excrelion
Total inorganic SO,-
Sample
Rats Rats Rats Rats
1, 3, 5, 7,
TABLE I of P6 During 8.J Hours by Rats Injected with L-Methionine SuljoximineGP
2 4 6 8
Activity countsfmin.
1LJ 12.7 11.1 13.3
‘“%
26,008’ 25,098 22,286 20,607
I sas
lY7 13.9 15.5 16.5
Activity counlr/min.
S”’ as sor %
490,730 415,465 468,982 519,153 Average
5.3 6.0 4.8 4.0 5.0
(1Twenty-milliliter sample of urine used for analysis. b Corrected for blank. Blank consisted of rat urine to which a similar quantity of MSI-W had been added. during the first 24 hr. The results, shown in Table I, indicated that approximately 5% of the S3” excreted during the first day had been oxidized to sulfate. Since the injected MS1 consists of two isomers, only one of which may be biologically active, the material excreted during the first 24 hr. after injection could represent mostly the inactive form which was not metabolized. On subsequent days larger percentages of W might be found in the oxidized form. To test this possibility, the procedure was repeated on six rats, and urine samples were collected on the 2d, 3d, 4th and 5th days. As the first experiment indicated that large deviations were not to be expected in the individual urines, the samples from each day’s collections were pooled and duplicate analyses were carried out on each pooled sample. The results are given in Table II. Examination of the data in this table indicates that an increasing percentage of Sa6was excreted as sulfate from the second to the fifth day of the experiment. By the fifth day, however, the total radioactivity reached a very low value which seemed to be leveling off. It is possible that Sa6excreted as sulfate at this time represented sulfur which had been incorporated into protein
Sulfur Partition sample
Imf go
TABLE II in the Urine of Rats Injected with L-Methionine Sulfozimine-P
Activity
1
Total. inorrzx I
Activity
Ether&Activity I
___-__---~----counts/ #sin.
mg.
countsl nrie.
+w.
counts/ niin.
4.7. 9080 5.8 1394 6.9 590 6.5 434
4.8 6.0 7.5 6.7
9160 1475 660 464
0.1 0.2 0.6 0.2
80 81 70 30
w.
2ndday 3rd day 4th day 5th day
0 Each value is the average of duplicate of urine.
Or: yFsc sot
Activity
counts/
Mb-.
??A
‘Sk’ sor mi?.
Activity
f;‘& 1
COWS/
min.
1.3 106,540 6.1 109,700 8.4 1.4 9,575 7.4 11,050 13.4 1.3 2,922 8.8 3,582 18.4 1.2 971 7.9 1,435 32.4
determinations
on a 5.0-ml. sample
METABOLISM OF METHIONINE
SULFOXIMIiVE
271
and was then being metabolized at a constant rate. The amount of it, however, was not very significant. Of the S36 injected, about 92’% of the tot,al artivity had been recovered in the urine by the sixth day. Very little ethereal sulfate-S36 w-as found.
DISCUSSION
The results indicate that only a very small fraction of an injected dose of MS1 is metabolized, at least by oxidation to sulfate. As preliminary paper chromatography studies indicated (2) that only small amounts of MS1 can be demonstrated in the urine, it is possible that this compound is detoxified by combination with glutamic acid, as has recently been shown to be the case with a very similar compound, methionine sulfone (4). This latter compound is also oxidized only to a small extent to sulfate. SUMMARY
h single dose of L-methionine sulfoximine-S3s was injected into rats and the distribution of S35determined in the various sulfur fractions of the urine at 24 hour intervals for 5 days. The amount of SP as sulfate increased from 5 % the first day to 32 % the fifth. By the fifth day, however, the total excretion of F5 had dropped to low levels, thus the total amount of L-methionine sulfoximine-S36 that was oxidized to sulfate was not much greater than 5 to 6%. REFERENCES 1. ROTH, J. S., WASE, A., AND REINER, L., Science 116, 256 (1952). 2. ROTH, ,J. S., EICHEL, H. J., AND WASE, A., J. Biol. Chem. 200,647 (1953). 3. HAWK, I’. B., OSER, B. L., AND SUMMERSON, W. H., Practical Physiological Chemistry, 12th ed., p. 887. Blakiston, Philadelphia, 1947. 1. WIXGO, W. J., SMITH, R. iz., AND WOOD, J., Arch. Biochem. and Biophgs. 47,3Oi (1953).