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Excessive urinary excretion of methionine in mutant mice lacking d -amino-acid oxidase activity
Excessive Urinary Excretion of Methionine in Mutant Mice Lacking D-Amino-Acid Oxidase Activity Ryuichi
Konno,
Kazumasa
Isobe,
Akira
Niwa,
and
Yosihiro
Yasumura
Thin-layer chromatography and amino acid analysis showed that mutant (ddY/DAO-) mice lacking D-amino-acid oxidase activity excreted about 3.5 times more methionine in urine than did normal (ddY/DAO+) mice. High-performance liquid chromatography using a chiral column showed that approximately 82% of urinary methionine of the ddY/DAOmice had the o-configuration. Analysis revealed that the mouse diet used contained 0.04% free methionine and that approximately 45% of methionine was the D-iSOf?Ier. When the ddY/DAOmice were given a diet containing a low level of supplementary oL-methionine or a diet without o-methionine. they excreted the normal levels of methionine. These results indicate that the ddY /DAOmice were unable to metabolize o-methionine and excrete it in urine. o 1988 by Grune & Stratton, Inc.
D
-AMINO-ACID oxidase (EC 1.4.3.3) oxidizes a variety of D-amino acids (stereoisomers of naturally occurring L-amino acids) to the corresponding a-keto acids. This enzyme is widely distributed in vertebrates (for a review see Meister’). However, its physiologic function has been unknown since its discovery in 1935* because its substrates are very rare in vertebrates. Although some investigators recently suggested that the enzyme had substrates other than D-amino acids,3‘5 it is not clear that these compounds are the physiologic substrates. We have been studying the physiologic role of D-aminoacid oxidase using animals that have a mutant form of this enzyme. In a survey for mutants we noticed that mutant mice lacking D-amino-acid oxidase activity excreted more methioninelike substance in urine compared with normal mice.6 However, identification of the substance and the reason why they excreted it in urine remained unknown. These problems were examined in this work. MATERIALS
AND
METHODS
Normal ddY/DAO+ mice and mutant ddY/DAO- mice lacking o-amino-acid oxidase activity’ were raised on a commercial diet (type NMF, Oriental Yeast Co, Ltd, Tokyo). According to a company catalog, the diet contained 0.6% methionine including both protein-bound and free forms. Three or four adult mice were housed in a cage with bedding of wood shavings. Food and water were given ad libitum. They were maintained at 24 + 2°C with a 12-hour dayjnight cycle. To examine the effects of diets on the urinary methionine levels, the ddY/DAO- mice were given Rodent Laboratory Chow 5001 (the methionine content: 0.43%; Ralston Purina Co, St Louis) or a modified NMF diet (Oriental Yeast Co, Ltd) in which supplementary DL-methionine in the NMF diet was replaced with Lmethionine. They were maintained for at least 2 weeks on the new diet before urine collection.
of Urine
Mice were kept in individual metabolism cages. Urine was collected overnight while they were given food and water ad libitum. One milliliter of the urine was mixed with 1 mL of 10% trichloroacetic acid for deproteinization. After the mixture was kept in ice-cold water, it was centrifuged at 8,000 xg for 20 min. The supernatant solution was applied to a column (I .8 x 4 cm) of Dowex 5OW-X2, 200-400 mesh, H form (Muromachi Kagaku Kogyo Kaisha, Ltd. Tokyo). The resin was washed with 100 mL of distilled water to remove the trichloroacetic acid and unbound materials. Amino acids bound to the resin were eluted with 30 mL of 3 mol/L ammonium hydroxide. The eluate was collected and evaporated to dryness under reduced pressure. The dried residue was dissolved in 0.2 N citrate buffer (pH 2.2) and applied to an automated amino acid analyzer (Model KLA-5, Hitachi, Ltd, Tokyo). The methionine concentration was normalized by the urinary creatinine content. of Configuration
of Urinary
Methionine
The urine of several ddY/DAO- mice was combined. Deproteinization, ion-exchange chromatography and evaporation of eluate containing amino acids were done as described above. The dried residue was redissolved in a small volume of distilled water and applied to a thin-layer plate of silica gel (LK 6, Whatman Chemical Separation Inc, Clifton, NJ). The LK 6 plate was used instead of the TLC plate silica gel 60 because it had a preadsorbent layer for sample application and allowed application of a large volume of sample for preparative thin-layer chromatography. The sample was chromatographed with n-butanol:acetic acid:water. The plate was visualized under an ultraviolet lamp, and an area corresponding to standard methionine was marked and scraped off. Methionine was extracted with a small volume of distilled water. The solution was passed through 0.22 hrn filter (Corning Glass Works, Corning, NY) and applied to a high-performance liquid chromatograph (Waters Associates, Milford, MA). A Chiralpak WH column (0.46 x 25 cm) (Daicel Chemical Industries, Ltd. Tokyo) with a chiral stationary
Chromatography
Free amino acids in urine were examined by thin-layer chromatography. Fresh urine was collected by letting the mice urinate spontaneously or by massaging them gently on the bladder. Creatinine concentration in the urine was determined with an assay kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan). Urine containing 4 rg creatinine was applied to a thin-layer plate of silica gel (TLC plates silica gel 60, E Merck AG, Darmstadt, FRG) or cellulose (Avicel SF, Funakoshi Yakuhin, K. K., Tokyo). Ascending development was carried out using n-butanol: acetic acid: water Merabolism,
Amino Acid Analysis
Analysis
Mice and Diet
Thin-Layer
(3:1:1, w/w) or phenol: water (3:l, w/w). After approximately five hours, the plate was dried at IOO‘C for 10 min and sprayed with 0.2% ninhydrin in ethanol as described by Horton et al8
Vol 37, No 12 Oxember),
1988:
pp 1139-l
142
From the Department of Microbiology and the Section of Physicochemical Analysis, Dokkyo University School of Medicine, Tochigi, Japan. Address reprint requests to Ryuichi Konno, PhD, Department of Microbiology, Dokkyo University School of Medicine, Mibu. Tochigi 321-02, Japan. o 1988 by Grune & Stratton, Inc. 0026-0495/88/3712-0006$03.00/0 1139
1140
KONNO
ET AL
phase able to separate the D- and L-isomer of methionine was used. The mobile phase was 0.25 mmol/L CuSO., and the flow rate was 1.5 mL/min. Methionine was detected at 230 nm. D-Methionine obtained from Sigma Chemical Co (St Louis) and L-methionine from Ajinomoto Co, Ltd (Tokyo) were used as the standard.
to chromatograph urine on a silica gel plate, a ninhydrinpositive material comigrating with methionine was also observed in the urine of ddY/DAO- mice. These results suggested that the extra substance found in the urine of ddY/DAO- mice was methionine.
Analysis
Amino
of Free Methionine
in the Mouse Diet
Pellets of the NMF diet were powdered with a mortar and pestle. Five grams of the powder was mixed with 50 mL of distilled water. Five micromoles of DL-norfeucine (Wako Pure Chemical Industries, Ltd) was added as an internal standard to check the recovery of amino acids. Free amino acids were extracted by keeping the mixture in boiling water for approximately 1 hour with occasional shaking. The slurry was passed through cheesecloth and the filtrate was centrifuged at 500 xg for 30 min. The supernatant solution was deproteinized and subjected to ion-exchange chromatography. The eluate was evaporated and the dried residue was resuspended in the citrate buffer. A part of the solution was used for determination of the amino acid concentration, and the rest was applied to a LK 6 thin-layer plate. After thin-layer chromatography methionine was extracted and its configuration was examined with a high-performance liquid chromatograph as described above. RESULTS
Thin-Layer
Chromatography
of Urine
When the urine of normal ddY/DAO+ mice and mutant ddY/DAO- mice lacking D-amino-acid oxidase activity was chromatographed with n-butanol:acetic acid:water on a silica gel thin-layer plate, urine of both male and female ddY/DAO- mice was observed containing a ninhydrinpositive, fast-migrating substance, which was faint in that of the normal mice (Fig 1,) The substance had a similar Rf to authentic methionine. Similar results were obtained when a cellulose thin-layer plate was used in place of a silica gel plate. Furthermore, when phenol:water was used as a solvent
Acid Analysis
of Urine
Amino acid analysis showed that the urine of normal ddY/DAO+ mice contained 0.679 * 0.135 pmol free methionine/mg creatinine (mean it SD, n = 5), whereas that of the mutant ddY/DAO- mice contained 2.409 + 0.478 pmol free methionine/mg creatinine (mean 2 SD, n = 5). The urine of ddY/DAO- mice contained 3.5 times more methionine than that of the ddY/DAO+ mice. Although these results were obtained in male mice, female mice also gave similar results. Configuration
of Urinary
Methionine
Isomer composition of methionine found in the urine of ddY/DAO- mice was examined with a high-performance liquid chromatograph. Separation of standard D- and L-isomer of methionine is shown in Fig 2A. Surprisingly, 81.5% + 12.0% (mean k SD, n = 6) of methionine in the urine of ddY/DAO- mice had the o-configuration (Fig 2B). Analysis
of Free Methionine
in the Mouse Diet
Becausematerials found in urine are known to be derived from diets in many cases,the mouse diet used was examined for methionine content and its isomer composition. Free amino acids were extracted from type NMF diet (lot
A
C
u -Oriqicl
12
3
4
5
Fig 1. Thin-layer chromatogram of the urine of normal ddY / DAD+ mice and mutant ddY/DAOmica lacking o-amino-acid oxidasa activity. The urine was chromatographed with n-butanohacatic acidwater on a silica gal plate. 1 and 2: the ddY/DAO+ mice, male and female, respectively. 3 and 4: the ddY/DAOmica, male and female, respectively. 5: standard methionina, 1.6 pg.
Fig 2. Separation of o- and L-methionine through a chiral column. (A) Standard methionine, o-isomer: L-isomer = 2:l. (6) A mathionina fraction purified from the urine of ddY/DAOmice. (C) A methionine fraction extracted from the mouse NMF diet. Ordinate: absorbance at 230 nm.
o-METHIONINURIA
IN MUTANT
1141
MICE
620856) and measured with an amino acid analyzer. The diet contained 0.040% 5 0.002% (mean f SD, n = 5) free methionine. Methionine was further purified by thin-layer chromatography, and its isomer composition was examined by high-performance liquid chromatography. One of the chromatograms is shown in Fig 2C. Nearly one-half (45.6% + 6.7%) (mean + SD, n = 6) of free methionine extracted from the diet had the o-configuration. These results indicated that the mouse diet was supplemented with or-methionine. Change of the Mouse Diet
We thought that the urinary level of methionine might be decreasedif ddY/DAO- mice were given a diet containing a low level of supplementary o-methionine. Because Ralston Purina Company told us that their Rodent Laboratory Chow 5001 contained a very low level of D-methionine (an estimated value, 0.0037%), the male ddY/DAO- mice were given this diet. As expected, the urinary level of methionine became low (0.553 + 0.277 Mmol/mg creatinine, mean + SD, n = 5). However, because there was a possibility that ingredients other than supplementary or,-methionine affected the urinary level of methionine, we asked Oriental Yeast Company to make us a modified NMF diet in which supplementary oL-methionine was replaced with L-methionine. When the male ddY/DAO- mice were given this diet, the urinary level of methionine was also decreased (0.416 + 0.126 hmol/mg creatinine, mean f SD, n = 5). Therefore, it was concluded that o-methionine supplemented in the mouse diet caused the mutant ddY/DAOmice lacking D-amino-acid oxidase activity to excrete the high level of methionine in urine. DISCUSSION
L-Methionine is one of the essential amino acids. Many investigators have shown that D-methionine is well utilized in place of its L-isomer in chicks, mice, rats, rabbits, dogs, and Pigs,‘.9.‘owhich is one reason for supplementation of DL-
methionine in commercial animal diets. The D-isomer is considered to be oxidized by D-amino-acid oxidase to cw-keto-y-methiolbutyric acid, which is subsequently reaminated by transaminase to form the L-isomer. The mutant ddY/DAO- mice lacking D-amino acid oxidase activity were, therefore, unable to metabolize D-methionine absorbed from their diet and excrete it in urine. When humans and monkeys are given o-methionine, they also excrete a large amount of the D-amino acid in urine.“-‘4 Because they have the substantial levels of D-amino-acid oxidaseactivity, 15-*’it is unclear why they utilize the o-amino acid poorly. The normal ddY/DAO+ mice given the NMF diet excreted more methionine in urine than did the mutant ddY/DAO- mice given Rodent Laboratory Chow 5001 or the modified NMF diet. Even the normal mice might be excreting some degree of D-methionine, but the concentration of urinary methionine was too low to determine its isomer composition, The NMF diet used in this study is a closed formula diet. According to an Oriental Yeast Company catalog, it contains 0.6% methionine, including both protein-bound and free methionine. The manufacturer does not discloseits exact composition, Our analysis showed that the diet (lot 620856) contained 0.04% free methionine and that nearly one-half (46%) was o-isomer. These values may differ, depending on the lot, because natural ingredients vary in methionine content and, therefore, the manufacturer adjusts the amount of supplementary DL-methionine to make a total content of 0.6% methionine. However, supplementation of about 0.018% o-methionine in the NMF diet seemshigh compared to 0.0037% D-methionine in Rodent Laboratory Chow 5001. Free methionine extracted from the mouse diet contained more L-isomer than D-isomer (54% v 46%). They should theoretically be 50:50 if the diet was supplemented with DL-methionine. This deviation would be because natural ingredients contained some free L-methionine, and/or L-methionine residues were released from proteins and peptides during extraction and purification of methionine from the diet, or it would simply reflect an experimental error.
REFERENCES
1. Meister A: Biochemistry of the Amino Acids, vol 1 (ed 2). San Diego, Academic, 1965, pp 220-224.297-304 2. Krebs HA: CXCVII. Metabolism of amino-acids. III. Deamination of amino-acids. Biochem J 29:1620-1644, 1935 3. Hamilton GA, Buckthal DJ, Mortensen RM, et al: Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amin acid oxidase. Proc Nat1 Acad Sci USA 76~26252629, 1979 4. Fitzpatrick PF, Massey V: Thiazolidine-2-carboxylic acid, an adduct of cysteamine and glyoxylate, as a substrate for D-amino acid oxidase. J Biol Chem 257:1166-1171, 1982 5. Moreno CM: Theoretical approaches to o-amino acid oxidase. J Theor Biol 119:369-378, 1986 6. Konno R, Yasumura Y: A simple and rapid method to screen for mutant mice lacking D-amino acid oxidase activity. Lab Anim Sci 38:292-295, 1988
7. Konno R, Yasumura Y: Mouse mutant deficient in o-amino acid oxidase activity. Genetics 103:277-285, 1983 8. Horton D, Tanimura A, Wolfrom ML: Two-dimensional thinlayer chromatography of amino acids on microcrystalline cellulose. J Chromatogr 23:309-3 12, 1966 9. Cho ES, Stegink LD: D-Methionine utilization during parenteral nutrition in adult rats. J Nutr 109:1086-1093, 1979 10. Cho ES, Andersen DW, Filer LJ Jr, et al: o-Methionine utilization in young miniature pigs, adult rabbits, and adult dogs. JPEN 4:544-547, 1980 11. Efron ML, McPherson TC, Shih VE, et al: r)-Methioninuria due to DL-methionine ingestion. An artefact detected by a mass screening program for errors of amino acid metabolism. Am J Dis Child 117:104-107, 1969 12. Stegink LD, Schmitt JL, Meyer PD, et al: Effect of diets fortified with DL-methionine on urinary and plasma methionine levels in young infants. J Pediatr 79:648-655, 197 1
1142
13. Printen KJ, Brummel MC, Cho ES, et al: Utilization of D-methionine during total parenteral nutrition in postsurgical patients. Am J Clin Nutr 32:1200-1205, 1979 14. Stegink LD, Moss J, Printen KJ, et al: D-Methionine utilization in adult monkeys fed diets containing DL-methionine. J Nutr 110:1240-1246, 1980 15. Dunn JT, Perkoff GT: D-Amino acid oxidase activity in human tissues. Biochim Biophys Acta 73:327-331, 1963 16. Neims AH, Zieverink WD, Smilack JD: Distribution of o-amino acid oxidase in bovine and human nervous tissues. J Neurochem 13:163-168,1966 17. Barker RF, Hopkinson DA: The genetic and biochemical
KONNO
ET AL
properties of the D-aIIIin0 acid oxidases in human tissues. Ann Hum Genet 41:27-42, 1977 18. Konno R, Yasumura Y: Activity and substrate specificity of D-amino acid oxidase in kidneys of various animals. Dobutsugaku Zasshi (Zoo1 Mag, Tokyo) 90:368-373, 1981 19. Goldfischer S, Collins J, Rapin I, et al: Pseudo-Zellweger syndrome: Deficiencies in several peroxisomal oxidative activities. J Pediatr 108:25-32, 1986 20. Vamecq J, Draye J-P, Van Hoof F, et al: Multiple peroxisoma1 enzymatic deficiency disorders. A comparative biochemical and morphologic study of Zellweger cerebrohepatorenal syndrome and neonatal adrenoleukodystrophy. Am .I Path01 125524-535, 1986
Konno,
Kazumasa
Isobe,
Akira
Niwa,
and
Yosihiro
Yasumura
Thin-layer chromatography and amino acid analysis showed that mutant (ddY/DAO-) mice lacking D-amino-acid oxidase activity excreted about 3.5 times more methionine in urine than did normal (ddY/DAO+) mice. High-performance liquid chromatography using a chiral column showed that approximately 82% of urinary methionine of the ddY/DAOmice had the o-configuration. Analysis revealed that the mouse diet used contained 0.04% free methionine and that approximately 45% of methionine was the D-iSOf?Ier. When the ddY/DAOmice were given a diet containing a low level of supplementary oL-methionine or a diet without o-methionine. they excreted the normal levels of methionine. These results indicate that the ddY /DAOmice were unable to metabolize o-methionine and excrete it in urine. o 1988 by Grune & Stratton, Inc.
D
-AMINO-ACID oxidase (EC 1.4.3.3) oxidizes a variety of D-amino acids (stereoisomers of naturally occurring L-amino acids) to the corresponding a-keto acids. This enzyme is widely distributed in vertebrates (for a review see Meister’). However, its physiologic function has been unknown since its discovery in 1935* because its substrates are very rare in vertebrates. Although some investigators recently suggested that the enzyme had substrates other than D-amino acids,3‘5 it is not clear that these compounds are the physiologic substrates. We have been studying the physiologic role of D-aminoacid oxidase using animals that have a mutant form of this enzyme. In a survey for mutants we noticed that mutant mice lacking D-amino-acid oxidase activity excreted more methioninelike substance in urine compared with normal mice.6 However, identification of the substance and the reason why they excreted it in urine remained unknown. These problems were examined in this work. MATERIALS
AND
METHODS
Normal ddY/DAO+ mice and mutant ddY/DAO- mice lacking o-amino-acid oxidase activity’ were raised on a commercial diet (type NMF, Oriental Yeast Co, Ltd, Tokyo). According to a company catalog, the diet contained 0.6% methionine including both protein-bound and free forms. Three or four adult mice were housed in a cage with bedding of wood shavings. Food and water were given ad libitum. They were maintained at 24 + 2°C with a 12-hour dayjnight cycle. To examine the effects of diets on the urinary methionine levels, the ddY/DAO- mice were given Rodent Laboratory Chow 5001 (the methionine content: 0.43%; Ralston Purina Co, St Louis) or a modified NMF diet (Oriental Yeast Co, Ltd) in which supplementary DL-methionine in the NMF diet was replaced with Lmethionine. They were maintained for at least 2 weeks on the new diet before urine collection.
of Urine
Mice were kept in individual metabolism cages. Urine was collected overnight while they were given food and water ad libitum. One milliliter of the urine was mixed with 1 mL of 10% trichloroacetic acid for deproteinization. After the mixture was kept in ice-cold water, it was centrifuged at 8,000 xg for 20 min. The supernatant solution was applied to a column (I .8 x 4 cm) of Dowex 5OW-X2, 200-400 mesh, H form (Muromachi Kagaku Kogyo Kaisha, Ltd. Tokyo). The resin was washed with 100 mL of distilled water to remove the trichloroacetic acid and unbound materials. Amino acids bound to the resin were eluted with 30 mL of 3 mol/L ammonium hydroxide. The eluate was collected and evaporated to dryness under reduced pressure. The dried residue was dissolved in 0.2 N citrate buffer (pH 2.2) and applied to an automated amino acid analyzer (Model KLA-5, Hitachi, Ltd, Tokyo). The methionine concentration was normalized by the urinary creatinine content. of Configuration
of Urinary
Methionine
The urine of several ddY/DAO- mice was combined. Deproteinization, ion-exchange chromatography and evaporation of eluate containing amino acids were done as described above. The dried residue was redissolved in a small volume of distilled water and applied to a thin-layer plate of silica gel (LK 6, Whatman Chemical Separation Inc, Clifton, NJ). The LK 6 plate was used instead of the TLC plate silica gel 60 because it had a preadsorbent layer for sample application and allowed application of a large volume of sample for preparative thin-layer chromatography. The sample was chromatographed with n-butanol:acetic acid:water. The plate was visualized under an ultraviolet lamp, and an area corresponding to standard methionine was marked and scraped off. Methionine was extracted with a small volume of distilled water. The solution was passed through 0.22 hrn filter (Corning Glass Works, Corning, NY) and applied to a high-performance liquid chromatograph (Waters Associates, Milford, MA). A Chiralpak WH column (0.46 x 25 cm) (Daicel Chemical Industries, Ltd. Tokyo) with a chiral stationary
Chromatography
Free amino acids in urine were examined by thin-layer chromatography. Fresh urine was collected by letting the mice urinate spontaneously or by massaging them gently on the bladder. Creatinine concentration in the urine was determined with an assay kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan). Urine containing 4 rg creatinine was applied to a thin-layer plate of silica gel (TLC plates silica gel 60, E Merck AG, Darmstadt, FRG) or cellulose (Avicel SF, Funakoshi Yakuhin, K. K., Tokyo). Ascending development was carried out using n-butanol: acetic acid: water Merabolism,
Amino Acid Analysis
Analysis
Mice and Diet
Thin-Layer
(3:1:1, w/w) or phenol: water (3:l, w/w). After approximately five hours, the plate was dried at IOO‘C for 10 min and sprayed with 0.2% ninhydrin in ethanol as described by Horton et al8
Vol 37, No 12 Oxember),
1988:
pp 1139-l
142
From the Department of Microbiology and the Section of Physicochemical Analysis, Dokkyo University School of Medicine, Tochigi, Japan. Address reprint requests to Ryuichi Konno, PhD, Department of Microbiology, Dokkyo University School of Medicine, Mibu. Tochigi 321-02, Japan. o 1988 by Grune & Stratton, Inc. 0026-0495/88/3712-0006$03.00/0 1139
1140
KONNO
ET AL
phase able to separate the D- and L-isomer of methionine was used. The mobile phase was 0.25 mmol/L CuSO., and the flow rate was 1.5 mL/min. Methionine was detected at 230 nm. D-Methionine obtained from Sigma Chemical Co (St Louis) and L-methionine from Ajinomoto Co, Ltd (Tokyo) were used as the standard.
to chromatograph urine on a silica gel plate, a ninhydrinpositive material comigrating with methionine was also observed in the urine of ddY/DAO- mice. These results suggested that the extra substance found in the urine of ddY/DAO- mice was methionine.
Analysis
Amino
of Free Methionine
in the Mouse Diet
Pellets of the NMF diet were powdered with a mortar and pestle. Five grams of the powder was mixed with 50 mL of distilled water. Five micromoles of DL-norfeucine (Wako Pure Chemical Industries, Ltd) was added as an internal standard to check the recovery of amino acids. Free amino acids were extracted by keeping the mixture in boiling water for approximately 1 hour with occasional shaking. The slurry was passed through cheesecloth and the filtrate was centrifuged at 500 xg for 30 min. The supernatant solution was deproteinized and subjected to ion-exchange chromatography. The eluate was evaporated and the dried residue was resuspended in the citrate buffer. A part of the solution was used for determination of the amino acid concentration, and the rest was applied to a LK 6 thin-layer plate. After thin-layer chromatography methionine was extracted and its configuration was examined with a high-performance liquid chromatograph as described above. RESULTS
Thin-Layer
Chromatography
of Urine
When the urine of normal ddY/DAO+ mice and mutant ddY/DAO- mice lacking D-amino-acid oxidase activity was chromatographed with n-butanol:acetic acid:water on a silica gel thin-layer plate, urine of both male and female ddY/DAO- mice was observed containing a ninhydrinpositive, fast-migrating substance, which was faint in that of the normal mice (Fig 1,) The substance had a similar Rf to authentic methionine. Similar results were obtained when a cellulose thin-layer plate was used in place of a silica gel plate. Furthermore, when phenol:water was used as a solvent
Acid Analysis
of Urine
Amino acid analysis showed that the urine of normal ddY/DAO+ mice contained 0.679 * 0.135 pmol free methionine/mg creatinine (mean it SD, n = 5), whereas that of the mutant ddY/DAO- mice contained 2.409 + 0.478 pmol free methionine/mg creatinine (mean 2 SD, n = 5). The urine of ddY/DAO- mice contained 3.5 times more methionine than that of the ddY/DAO+ mice. Although these results were obtained in male mice, female mice also gave similar results. Configuration
of Urinary
Methionine
Isomer composition of methionine found in the urine of ddY/DAO- mice was examined with a high-performance liquid chromatograph. Separation of standard D- and L-isomer of methionine is shown in Fig 2A. Surprisingly, 81.5% + 12.0% (mean k SD, n = 6) of methionine in the urine of ddY/DAO- mice had the o-configuration (Fig 2B). Analysis
of Free Methionine
in the Mouse Diet
Becausematerials found in urine are known to be derived from diets in many cases,the mouse diet used was examined for methionine content and its isomer composition. Free amino acids were extracted from type NMF diet (lot
A
C
u -Oriqicl
12
3
4
5
Fig 1. Thin-layer chromatogram of the urine of normal ddY / DAD+ mice and mutant ddY/DAOmica lacking o-amino-acid oxidasa activity. The urine was chromatographed with n-butanohacatic acidwater on a silica gal plate. 1 and 2: the ddY/DAO+ mice, male and female, respectively. 3 and 4: the ddY/DAOmica, male and female, respectively. 5: standard methionina, 1.6 pg.
Fig 2. Separation of o- and L-methionine through a chiral column. (A) Standard methionine, o-isomer: L-isomer = 2:l. (6) A mathionina fraction purified from the urine of ddY/DAOmice. (C) A methionine fraction extracted from the mouse NMF diet. Ordinate: absorbance at 230 nm.
o-METHIONINURIA
IN MUTANT
1141
MICE
620856) and measured with an amino acid analyzer. The diet contained 0.040% 5 0.002% (mean f SD, n = 5) free methionine. Methionine was further purified by thin-layer chromatography, and its isomer composition was examined by high-performance liquid chromatography. One of the chromatograms is shown in Fig 2C. Nearly one-half (45.6% + 6.7%) (mean + SD, n = 6) of free methionine extracted from the diet had the o-configuration. These results indicated that the mouse diet was supplemented with or-methionine. Change of the Mouse Diet
We thought that the urinary level of methionine might be decreasedif ddY/DAO- mice were given a diet containing a low level of supplementary o-methionine. Because Ralston Purina Company told us that their Rodent Laboratory Chow 5001 contained a very low level of D-methionine (an estimated value, 0.0037%), the male ddY/DAO- mice were given this diet. As expected, the urinary level of methionine became low (0.553 + 0.277 Mmol/mg creatinine, mean + SD, n = 5). However, because there was a possibility that ingredients other than supplementary or,-methionine affected the urinary level of methionine, we asked Oriental Yeast Company to make us a modified NMF diet in which supplementary oL-methionine was replaced with L-methionine. When the male ddY/DAO- mice were given this diet, the urinary level of methionine was also decreased (0.416 + 0.126 hmol/mg creatinine, mean f SD, n = 5). Therefore, it was concluded that o-methionine supplemented in the mouse diet caused the mutant ddY/DAOmice lacking D-amino-acid oxidase activity to excrete the high level of methionine in urine. DISCUSSION
L-Methionine is one of the essential amino acids. Many investigators have shown that D-methionine is well utilized in place of its L-isomer in chicks, mice, rats, rabbits, dogs, and Pigs,‘.9.‘owhich is one reason for supplementation of DL-
methionine in commercial animal diets. The D-isomer is considered to be oxidized by D-amino-acid oxidase to cw-keto-y-methiolbutyric acid, which is subsequently reaminated by transaminase to form the L-isomer. The mutant ddY/DAO- mice lacking D-amino acid oxidase activity were, therefore, unable to metabolize D-methionine absorbed from their diet and excrete it in urine. When humans and monkeys are given o-methionine, they also excrete a large amount of the D-amino acid in urine.“-‘4 Because they have the substantial levels of D-amino-acid oxidaseactivity, 15-*’it is unclear why they utilize the o-amino acid poorly. The normal ddY/DAO+ mice given the NMF diet excreted more methionine in urine than did the mutant ddY/DAO- mice given Rodent Laboratory Chow 5001 or the modified NMF diet. Even the normal mice might be excreting some degree of D-methionine, but the concentration of urinary methionine was too low to determine its isomer composition, The NMF diet used in this study is a closed formula diet. According to an Oriental Yeast Company catalog, it contains 0.6% methionine, including both protein-bound and free methionine. The manufacturer does not discloseits exact composition, Our analysis showed that the diet (lot 620856) contained 0.04% free methionine and that nearly one-half (46%) was o-isomer. These values may differ, depending on the lot, because natural ingredients vary in methionine content and, therefore, the manufacturer adjusts the amount of supplementary DL-methionine to make a total content of 0.6% methionine. However, supplementation of about 0.018% o-methionine in the NMF diet seemshigh compared to 0.0037% D-methionine in Rodent Laboratory Chow 5001. Free methionine extracted from the mouse diet contained more L-isomer than D-isomer (54% v 46%). They should theoretically be 50:50 if the diet was supplemented with DL-methionine. This deviation would be because natural ingredients contained some free L-methionine, and/or L-methionine residues were released from proteins and peptides during extraction and purification of methionine from the diet, or it would simply reflect an experimental error.
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