Acetylation of methionine sulfoxide and methionine sulfone by the rat

304

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 26787

A C E T Y L A T I O N OF M E T H I O N I N E S U L F O X I D E AND M E T H I O N I N E S U L F O N E BY T H E RAT

R O B E R T C. S M I T H

Department of Animal and Dairy Sciences, Agricultural Experiment Station, Auburn University, Auburn, Ala. 3683° (U.S.A.) (Received August 3oth, i97 I)

SUMMARY

About 12% of the radioactivity of EMe-14CJmethionine sulfoxide injected into rats was excreted in the urine in 24 h. N-Acetyhnethionine sulfoxide contained about 65% of the radioactivity excreted. The N-acetylmethionine sulfoxide was identified b y its co-chromatography on paper with the chemically synthesized compound and its deacetylation to methionine sulfoxide b y acid hydrolysis. About 55% of the radioactivity of [Me-14C]methionine sulfone injected into rats was excreted in the urine in 24 h. N-Acetylmethionine sulfone contained about 70 % of the radioactivity and methionine sulfone contained most of the rest of the radioactivity.

INTRODUCTION

Chemical treatments and heat incident to processing of food proteins can result in a loss of available methionine 1,2. The oxidation of methionine to methionine sulfoxide could result in reduced availability of methionine if the sulfoxide was not efficiently reduced to methionine in rats*. Growth experiments using diets containing mixtures of amino acids in place of casein have shown that when methionine was completely replaced b y methionine sulfoxide, there was a significant reduction in weight gain over a IT-day experimental period*. When methionine sulfoxide replaced half of the methionine, there was no significant effect on weight gain. It is possible that only part of the methionine sulfoxide consumed is reduced to methionine and used by the normal pathways of methionine metabolism; the remainder m a y be metabolized through other pathways or be excreted unchanged. Methionine sulfoxide was reported to be excreted in the urine of rats fed a diet containing a 3% excess of methionine a. It was also excreted in the urine in severe infantile liver disease where there was a 3o-5o-fold increase in the methionine level in the blood 4. The rat can effectively metabolize ethionine to ethionine sulfoxide and N-acetylethionine sulfoxideS, °. In the present investigation, rats were injected with EMe-l*C]methionine sulfoxide and EMe-l~C]methionine sulfone and the identification of the acetylated products in the urine was undertaken. Biochim. Biophys. Acta, 261 (1972) 3o4-3o9

ACETYLATION OF METHIONINE SULFOXlDE

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MATERIALS AND METHODS

Treatment of the rats Female Charles River rats were fed Purina Laboratory Chow. They were housed in wire-bottom cages in an air-conditioned room. When they weighed about 15o g, they were injected intraperitoneally with 2 ml of the radioactive compound while lightly anaesthetized with diethyl ether. The rats were placed in individual metabolism cages and urine was collected in erlenmeyer flasks containing 2 ml of toluene as a preservative, Paper and column chromatography The urine (o.2 ml) was streaked over a 5-cm distance on Whatman 3MM filter paper and chromatographed. The solvents used were : (I) tert.-butanol-methylethylketone-water-diethylamine (IO : IO: 5 : I, by vol.), (2) butanol-acetic acid-water (12:3:5, by vol.) ; (3) butanol-pyaidine-water (I : I : I, by vol.) ; (4) isobutyric acid-NH4OH-water (66: i :33, by vol.). Methionine sulfoxide and N-acetylmethionine sulfoxide were detected by spraying the chromatograms with a platinic iodide solution 7. Methionine sulfone was detected with ninhydrin and N-acetylmethionine sulfone by its radioactivity. The radioactivity on the chromatograms was detected with a Packard Model 72oo radiochromatogram scanner or on radioautograms prepared with Dupont Cronex 2 DE film. Separation of the radioactive compounds was done on a Dowex-I column, formate form e. The pH of the urine was adjusted to about 9 with I.O M NH~OH, the urine was applied to the column, and tt-/e column was washed with water to remove radioactive material not adsorbed to the column. The column was then eluted with a gradient of formic acid u p to I.O M; in some experiments the compounds were eluted sequentially with o.oi, o. i, and i.o M formic acid. Chemicals [Me-14C]Methionine (specific activity, 55.1 mC/mmole) was obtained from International Chemical and Nuclear Corp. [Me-l~ClMethionine sulfoxide and [Me-I*C]methionine sulfone were synthesized by the methods described by GREENSTEIN AND WlNITZ s. N-Acetylmethionine sulfoxide was synthesized from methionine sulfoxide by acetylation with acetic anhydride or from N-acetylmethionine by oxidation with hydrogen peroxide e. KOBAYASHI9 and MORIHARA1° have reported the synthesis of N-acetylmethionine sulfoxide by these methods. The product from each reaction had the same RF values in Solvents 1- 4 and gave a positive reaction with the platinic iodide reagent but was ninhydrin negative. The N-acetylmethionine sulfoxide prepared was not stable. On storage at refrigerator temperature it gradually was converted to methionine sulfoxide, N-acetylmethionine, and traces of methionine. N-Acetyl [Me-x~Clmethionine sulfone was synttiesized from [Me-14C]methionine sulfone by acetylation with acetic anhydride by the same procedure used to synthesize N-acetylmethionine sulfoxide 6. The product was a single radioactive compound that was ninhydrin negative.

Biochim. Biophys. Acta, 261 (1972) 3o4-3o9

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R.C. SMITH

RESULTS

Excretion of radioactivityfrom [Me-l*C]methionine sulfoxide Rats injected with I00 mg of methionine sulfoxide containing I0 juC of [Me-I'C] methionine sulfoxide excreted about 12% of the radioactivity in 24 h (Fig. I). The urine was chromatographed on paper in Solvent I and a scan of the radioactivity made (Fig. 2). One major radioactive compound was present in the urine that accounted for

~80 rr 1.#

O

G

×

z

,~2c r~

12

w o

i

i

i

6

0

f

12 24 3 6 4 8 HOURS

Fig. I. Cumulative u r i n a r y excretion of radioactivity b y r a t s injected with [Me-X4C],methionine sulfoxide (.) or [Me-i*C]methionine sulfone (o). Fig. 2. R a d i o c h r o m a t o g r a m scan of a 24-h urine sample from a r a t injected w i t h [Me-14C]me thionine sulfoxide. MS, methionine sulfoxide; AMS, N - a c e t y l m e t h i o n i n e sulfoxide. The solvent was tert.-butanol-methylethylketone-water-diethylamine (io: io: 5 : i, b y vol.).

about 65% of the radioactivity excreted. When the urine was chromatographed on Dowex-I, it was found that about 35% of the radioactivity was not adsorbed oll the column. This material has not been identified. The remainder of the radioactivity was eluted with I.O M formic acid, but was not eluted with o. I M formic acid. This compound after chromatography on paper was ninhydrin negative, but gave a positive reaction with the platinic iodide reagent. It chromatographed with the same RF value as chemically synthesized N-acetylmethionine sulfoxide in four solvent systems (Table I). After acid hydrolysis, the radioactive compound gave a positive reaction with both ninhydrin and platinic iodide and had the same RF values as methionine sulfoxide (Table I). On the basis of the chromatographic, chemical, and radiological data, it was concluded that the major product in the urine of rats injected with large doses of methionine sulfoxide was N-acetylmethionine sulfoxide. It accounted for about 8% of the radioactivity injected. TABLE I PAPER CHROMATOGRAPHY

OF U R I N A R Y ~ r - A C E T Y L M E T H I O N I N E

SULFOXIDE

F o r composition of Solvents 1- 4 see MATERIALS AND METHODS, Paper chromatography.

Compound N - A c e t y l m e t h i o n i n e sulfoxide, from urine N - A c e t y l m e t h i o n i n e sulfoxide, a u t h e n t i c N - A c e t y l m e t h i o n i n e sulfoxide, after acid hydrolysis Methionine sulfoxide, a u t h e n t i c Methionine, a u t h e n t i c

Biochim. Biophys. Acta, 261 (i972) 304-309

RF value in Solvent z

2

3

4

0.23 0.23

0.48 0.48

0.37 0.39

0.65 0.65

o. 13 o.13 0.39

O.2I

0.28 0.28 0.57

0.51 o.51 0.70

O.2I

0.52

ACETYLATION OF METHIONINE SULFOXIDE

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6

×

6

Z

~-

E

4o u 2

Fig. 3. R a d i o c h r o m a t o g r a m scan of a 24-h urine sample f r o m a r a t in ected w i t h [Me-14e]me thionine sulfone. M sulfone, methionine sulfone. The solvent was tert.-butanol-methylethylketonew a t e r - d i e t h y l a m i n e (lO : I o : 5 : I, b y vol.).

Excretion of radioactivityfrom [Me-14CJmethioninesulfone In some of the earlier experiments it was observed that rats injected with [Me-14Clmethionine sulfoxide contaminated with EMe-l*Clmethionine sulfone excreted a radioactive compound that chromatographed with the same RF value as methionine. Since it was possible that methionine sulfone might also be metabolized by the rat, it was decided to determine the urinary metabolites of rats injected with methionine sulfone. Rats injected with IOO mg of methionine sulfone and 5/uC of ~Me-14C]methionine sulfone excreted about 55% of the radioactivity in the first 24 h (Fig. I). The urine (o.2 ml) was chromatographed in Solvent I and a scan of the radioactivity made (Fig. 3). Two maj or radioactive peaks were observed that contained most of the radioactivity. The urine was chromatographed on Dowex-I and the urinary compound with the lower RF value was eluted with o.I M formic acid. I t chromatographed with the same RF as chemically synthesized methionine sulfone (Table II). After acetylation with acetic anhydride, this compound had the same RF value as the other radioactive compound excreted in the urine. The second compound was eluted from the Dowex-I column with I.O M formic acid. I t was ninhydrin-negative and chromatographed with the same RF values as chemically synthesized N-acetylmethionine sulfone in the four solvents (Table II). After acid hydrolysis this compound was ninhydrin-positive and had the same RF values as methionine sulfone. From the data obtained b y these procedures it was concluded that rats injected with methionine sulfone excrete about 30% of the radioactivity in the urine as methionine sulfone and 70% as N-acetylmethionine sulfone. TABLE II PAPER CHROMATOGRAPHY OF URINARY N-ACETYLMETHIONINE

SULFONE AND METHIONINE

SULFONE

F o r composition of Solvents i - 4 see MATERIALS AND METHODS, Paper chromatography.

Compound N - A c e t y l m e t h i o n i n e sulfone, f r o m urine N - A c e t y l m e t h i o n i n e sulfone, a u t h e n t i c N - A c e t y l m e t h i o n i n e sulfone, after acid hydrolysis Methionine sulfone, a u t h e n t i c Methionine sulfone, from urine Methionine sulfone, from urine, after acetylation

RF value in Solvent: z

2

3

4

0.39 0.39

0.56 0.56

0.45 0.45

0.58 0.58

o.25 o.25 0.25

0.24 0.24 0.24

0.40 0.39 o.39

o.51 o.51 o.5o

o. 39

o.56

o.45

o.58

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R.C. SMITH

DISCUSSION

There has been interest in the metabolism of methionine sulfoxide in animals because of the possibility that it is formed during various heat and chemical treatments of fish meal and other sources of protein. These treatments could result in a reduced availability of methionine because of its conversion to the sulfoxide.In short-term experiments, rats did not grow as well when fed a protein-free diet in which the methionine in the amino acid mixture used was replaced by methionine sulfoxide ~. It was suggested that this was probably because the methionine sulfoxide was not completely reduced to methionine. This could be because the methionine sulfoxide is not reduced to methionine at a rate fast enough to prevent some of it from being excreted. Since N-acetylmethionine sulfoxide was present in the urine of rats injected with methionine sulfoxide, it is possible that this pathway m a y account for some of the non-utilization of methionine sulfoxide. Since acetylation of drugs can occur in the kidney, it is possible that the methionine sulfoxide is carried to the kidney and acetylated before excretion. N-Acetylmethionine was utilized b y rats when it was used to supplement the methionine level of 12% casein diets 11. High doses of N-acetylmethionine resulted in excretion of the compound in the urine. The rats injected with methionine sulfone excreted a greater percentage of the radioactivity than rats injected with methionine sulfoxide. This is probably because the rats can use methionine sulfoxide to meet their dietary methionine requirement, but they cannot use methionine sulfone 1~. The methionine sulfoxide is probably reduced to methionine for protein synthesis, to form S-adenosylmethionine for transmethylations, or catabolized to COs. The methionine sulfone is relatively inert and much of it is excreted unchanged or acetylated to N-acetylmethionine sulfone and excreted. The formation of the sulfoxides and N-acetyl derivatives of alkylated thio amino acids is a prevalent pathway for the metabolism of these compounds in the rat. Rats injected with S-ethylcysteine or S-ethylcysteine sulfoxide excreted N-acetylethylcysteine in the urine a3. Rats injected with ethionine or ethionine sulfoxide excreted N-acetylethionine sulfoxide in the urine~, 6. Rats injected with S-methylcysteine excreted some of it as N-acetyl-S-methylcysteine sulfoxide 1~. Rats injected with Sethyl-, S-propyl-, S-butyl-, S-pentyl-, and S-hexylcysteine excreted the N-acetylated derivatives of these compounds in the urine x~. Microsomes from rat liver oxidized ~-thiocarboxylic acids to the corresponding sulfoxides 16. S-Propylcysteine was converted to its sulfoxide by microsomal preparations from rat liver ~7. Rats fed a diet containing excess methionine excreted methionine sulfoxide in the urine 3. Methionine sulfoxide was in the urine of humans dosed with methioninelL and in the plasma and urine of homocystinuric children and children with hypermethioninemia4, lg. N-Acetylmethionine sulfoxide m a y have also been present in the urine under these conditions, but was not detected because it does not give a positive ninhydrin reaction. The formation of sulfoxides and N-acetylated compounds is probably a mechanism for detoxifying these compounds when they are present in excessive amounts. MILLER et al. ~ reported that when rats were fed a diet containing methionine sulfoxide in place of methionine, the methionine levels in the plasma, as assayed by a microbiological method, were 10-20 times those of rats fed the same diet with methionine. The compound supporting the growth of the bacteria was not methionine, methionine sulfoxide or methionine sulfone. There were no unaccounted ninhydrin-posiBiochim. Biophys. Acta, 26I (I972) 304-309

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tive peaks in the ion-exchange chromatograms. It is possible that N-acetylmethionine sulfoxide is the compound that was present in the plasma since it does support the growth of methionine auxotrophs (R. C. SMITH, unpublished observations), it is ninhydrin negative, and is a metabolite of methionine sulfoxide. ACKNOWLEDGMENT

The author wishes to thank Mrs. Cheryl Rodgers for her technical assistance. This investigation was supported by Public Health Service Research Grant No. HE-o2615 from the National Heart Institute and Hatch and State funds of Auburn University Experiment Station. REFERENCES I 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18 19

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Biochim. Biophys. Acta, 261 (1972) 3o4-3o9