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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:2001-2005, August 2005

Nutritional Methodology

Direct Detection and Evaluation of Conversion of D-Methionine into L-Methionine in Rats by Stable Isotope Methodology1

Hiroshi Hasegawa2, Yoshihiko Shinohara, Kenji Akahane and Takao Hashimoto

Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432–1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

2To whom correspondence should be addressed. E-mail: hasegawa{at}ps.toyaku.ac.jp.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The stereoselective kinetics of methionine enantiomers in rats was investigated to evaluate the fraction that converted from D-methionine to the L-enantiomer using a stable isotope methodology. After bolus i.v. administration of D- or L-[2H3]methionine, their plasma concentrations and that of endogenous L-methionine were determined by a stereoselective GC-MS method. L-[2H3]Methionine appeared rapidly after administration of D-[2H3]methionine, whereas D-[2H3]methionine was not detected after administration of L-[2H3]methionine. The fraction of conversion of D-[2H3]methionine into L-[2H3]methionine was estimated using the area under the plasma concentration vs. time curve of L-[2H3]methionine on D-[2H3]methionine administration and total clearance of L-[2H3]methionine on L-[2H3]methionine administration, and that fraction was >90%. This result demonstrates that almost all i.v. administered D-methionine is converted into the L-enantiomer in vivo.

KEY WORDS: • D-methionine • chiral inversion • GC-MS • stable isotope • D-amino-acid oxidase

Utilization of D-amino acids for growth has been confirmed repeatedly in different species of animals; indeed, several D-amino acids were used for growth (1). In these nutritional studies, a widely accepted method involves the comparison of the growth rate of animals fed a control diet with that of animals fed a diet containing the D-amino acid in place of the corresponding L-enantiomer. The efficiency of D-amino acid was estimated indirectly from the dosage of the D-amino acid required to achieve the same growth rate in the control animal. Among the D-amino acids, D-methionine was the amino acid most effectively used by animals in place of the L-enantiomer (2). Because the utilization of exogenous D-amino acid depends on whether it can be efficiently transformed to the L-isomer, it is thought that almost all D-methionine is converted into the L-enantiomer (3,4). However, little information is available on the extent to which D-methionine is converted into the L-enantiomer in vivo because the L-methionine formed is indistinguishable from endogenous L-methionine.

One of the unique advantages for the use of a stable isotope-labeled compound as a tracer is that an endogenous compound and its exogenously administered labeled analog can be measured separately using GC-MS. Our recent use of stable isotope-labeled D-leucine and the stereoselective GC-MS method proved to be a powerful methodology for examining the pharmacokinetic behavior of exogenously administered D-leucine and studying the conversion of D-leucine to the L-enantiomer (5). It became apparent that ~30% of exogenously administered D-leucine was converted stereospecifically into the L-enantiomer.

Successful application of stable isotope methodology to the metabolic investigation for D-methionine depends on the availability of compounds labeled at a predesigned position. We chose a deuterium-labeled D-methionine at the S-methyl group (D-[2H3]methionine) as a tracer. When the exogenously administered D-[2H3]methionine is converted into the L-enantiomer, the L-[2H3]methionine formed is likely metabolized and excreted in the same way as endogenous L-methionine (Fig. 1). The S-C2H3 label of L-[2H3]methionine is removed to form nonlabeled L-homocysteine during transmethylation. Then, L-homocysteine can be remethylated to reform L-methionine by accepting a methyl group from either 5-methyltetrahydrofolate or betaine. The reformed L-methionine does not retain the deuterium label in the S-methyl group. Therefore, it has become feasible to investigate the extent of conversion of D-[2H3]methionine into the L-enantiomer without considering the transmethylation cycle.



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  		FIGURE 1 Proposed metabolic profiles of D- and L-methionine. The numbers represent the following enzymes or sequences: (1) D-amino-acid oxidase; (2) transaminases; (3) methionine adenosyltransferase; (4) S-adenosylmethionine-dependent transmethylation; (5) adenosylhomocysteine hydrolase; (6) betaine-homocysteine methyltransferase or methionine synthase.

 
The purpose of this study was to clarify what fraction of the i.v. administered D-methionine in rats is converted into the L-enantiomer in vivo using stable isotope methodology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Chemicals. D-Methionine and L-methionine were purchased from Wako Pure Chemicals. DL-[S-methyl-2H3]Methionine (DL-[2H3]methionine, 99.2 atom 2H %) and DL-[3,3,4,4-2H4]methionine (>99 atom 2H %) were purchased from CDN isotopes. DL-[3,3,4,4,S-methyl-2H7]Methionine (DL-[2H7]methionine) was prepared from DL-[3,3,4,4-2H4]methionine in our laboratory as described previously (6). D-[2H3]Methionine and L-[2H3]methionine were prepared from DL-[2H3]methionine according to the method for the optical resolution of DL-[2H7]methionine (6). The enantiomeric purities of D-[2H3]methionine and L-[2H3]methionine were determined by chiral HPLC using a Crownpak CR column (150 x 4 mm i.d., Daicel Chemicals) and 0.07% HClO4 as a mobile phase; the respective enantiomeric purities were >99.8% enantiomeric excess. (S)-(+)-{alpha}-Methoxy-{alpha}-trifluoromethylphenylacetyl chloride [(+)-MTPA-Cl]3 and 10% HCl in methanol were purchased from Tokyo Kasei. A strong cation-exchange solid-phase extraction column BondElut SCX (H+ form, size 1 mL/100 mg) was purchased from Varian. Chloroform stabilized with amylene was purchased from Cica-Merck. All other chemicals and solvents were of analytical reagent grade and used without further purification.

    Stock solutions. Stock solutions of D-methionine (101.13 µmol/L), L-methionine (100.73 µmol/L), DL-[2H3]methionine (199.65 µmol/L) and DL-[2H7]methionine (99.40 µmol/L) were prepared in methanol. There was no detectable decomposition after storage of these solutions at 4°C for >6 mo. All analyses were performed by diluting the stock solutions with methanol.

    Sample preparation. DL-[2H7]Methionine (0.994 nmol) was added to 50 µL of rat plasma in a polypropylene microtube (1.5 mL) as an analytical internal standard. The plasma sample was deproteinized and extracted with ethanol (1 mL) with mixing on a vortex for ~0.5 min. After centrifugation at 1000 x g for 10 min, the supernatant was transferred into another polypropylene microtube and evaporated at 40°C under a stream of nitrogen. The residue was dissolved in 1 mL of 40 mmol/L HCl and then applied to a BondElut SCX cartridge. The cartridge was prewashed and activated with 3 mL of methanol, 3 mL of a mixture of methanol and 0.1 mol/L HCl (1:1, v:v), and 3 mL of 0.1 mol/L HCl. The cartridge was washed with 1 mL of water and 1 mL of methanol, and then eluted with 0.5 mL of 10% HCl in methanol into a PTFE-lined screw-cap conical centrifuge tube (100 x 16 mm i.d.). The eluent was heated directly at 60°C for 1 h. After removal of the solvent under a stream of nitrogen, the residue was reconstituted in 100 µL of 2% (+)-MTPA-Cl in chloroform, shaken for 30 s on a vortex mixer, and left at room temperature for 1 h. After the reaction mixture was washed with water (1 mL x 2), the solvent was evaporated at room temperature under a stream of nitrogen. The residue was dissolved in 20 µL of ethyl acetate and a 1–2 µL of the solution was subject to GC-MS.

    GC-MS with selected ion monitoring (SIM). GC-MS-SIM analyses were performed on a Shimadzu QP1000EX quadrupole GC-MS equipped with a data processing system. A methylsilicone bonded-phase fused-silica capillary column SPB-1 (15 m x 0.25 mm i.d.) with a 0.25-µm film thickness (Supelco) was connected directly into the ion source. Helium was used as the carrier gas at a column head pressure of 8 kPa. A split-splitless injection system Shimadzu SPL-G9 operating in the splitless mode was used with a septum purge flow rate of 1.0 mL/min and a split vent flow rate of 30 mL/min. The purge activation time was 2 min after injection. The initial column temperature was set at 120°C. After the sample injection, it was maintained for 2 min, increased at 15°C/min to 250°C, and held at 250°C for 1 min. The temperature of the injector was 280°C. The MS was operated in chemical ionization mode with isobutane as the reactant gas at a pressure of 2.5–6 mPa. The ionization voltage and ionization current were 200 eV and 150 µA, respectively. The ion source temperature was 280°C. Because the MTPA-methyl ester (OMe) derivatives of methionine, [2H3]methionine, and [2H7]methionine produce strong molecular-related ions [M+H]+ at m/z 380, 383, and 387, respectively, SIM was performed on their molecular-related ions. The MTPA-OMe derivatives of D- and L-methionine underwent baseline separation within 10 min and eluted in that order on GC.

    Calibration curves and quantification. Each of standards containing known amounts of DL-[2H3]methionine (0.050, 0.075, 0.100, 0.199, 0.399, 0.998, 1.497, 1.996, 3.993, 9.982, and 14.973 nmol) or L-methionine (0.252, 0.504, 1.007, and 2.015 nmol) was added to 50 µL portions of rat blank plasma. DL-[2H7]Methionine (0.994 nmol) was added to the samples as an internal standard. The sample was prepared each time. The samples were purified and derivatized according to the procedure described in sample preparation.

The peak area values were determined at m/z 380 for MTPA-OMe derivatives of L-methionine, m/z 383 for those of D- and L-[2H3]methionine, and m/z 387 for those of D- and L-[2H7]methionine, and the peak area ratios (L-methionine:L-[2H7]methionine, D-[2H3]methionine:D-[2H7]methionine, and L-[2H3]methionine:L-[2H7]methionine, respectively) were calculated. The curves were obtained by an unweighted least-squares linear fitting of the peak area ratios vs. the mixed molar ratios on each analysis of unknown samples. Plasma concentrations were calculated by comparing the peak area ratios obtained from the unknown samples with those obtained from the standard mixtures.

The intra- and interday precision values of the assay for D-[2H3]methionine spiked to rat plasma in the range of 1.0–99.8 µmol/L were <4 and 3%, respectively. Similarly, the intra- and interday precision values for L-[2H3]methionine were <3 and 4%, respectively.

    Animals. The experimental protocols were approved by the Institutional Animal Care Committee of Tokyo University of Pharmacy and Life Science. Male Sprague-Dawley rats aged 7 wk were obtained from Tokyo Laboratory Animal Center. They were housed in stainless steel cages in an air-conditioned room maintained at 23 ± 1°C and 55 ± 5% humidity with a 12-h dark:light cycle. All rats were acclimated for at least 7 d, during which time they had free access to water and food (CE-2, Clea Japan).

    Dose experiments. After overnight food deprivation, rats (body weight, 250–350 g) were anesthetized with sodium pentobarbital (50 mg/kg weight, i.p.). D-[2H3]Methionine (35 µmol/kg body weight) or L-[2H3]methionine (10 or 35 µmol/kg body weight) dissolved in saline (0.5 mL dosing solution/kg body weight) was administered into the femoral vein. Heparinized blood samples (150 µL) were obtained from the jugular vein 10 min before and 0.5, 1, 3, 5, 10, 15, 20, 30, 60, 90, 120, 180, 240, 300, and 360 min after dosing. The blood was centrifuged to separate plasma at 1000 x g for 10 min. The plasma was stored at –20°C until analysis.

    Data analysis. Pharmacokinetic parameters were calculated by model-independent analysis using a macroprogram MOMENT(EXCEL) (7) running on Microsoft Excel. The half-life (t1/2) of the terminal elimination phase of the plasma concentration vs. time curve was estimated using a regression equation. The area under the plasma concentration vs. time curve (AUC) and the area under the first-moment plasma concentration vs. time curve (AUMC) were calculated by the trapezoidal method, and were extrapolated to infinity using the last detectable plasma concentration and the terminal elimination rate constant. Mean residence time (MRT), total clearance (CL), and the apparent volume of distribution at steady state (VdSS) were calculated using the equations MRT = AUMC/AUC, CL = Dose/AUC, and VdSS = CL · MRT, respectively.

The fraction of conversion of D-[2H3]methionine into L-[2H3]methionine (FD->L) after administration of D-[2H3]methionine is calculated as follows:

(1)

where CLDL is the metabolic clearance associated with conversion of D-[2H3]methionine into L-[2H3]methionine and AUCD is the AUC of D-[2H3]methionine. According to mass balance, the rate of change of L-[2H3]methionine in the body is represented as follows:

(2)

where CLL is the total clearance of L-[2H3]methionine and CD and CL(<-D) are the respective plasma concentrations of D-[2H3]methionine and L-[2H3]methionine. Because no L-[2H3]methionine is present in the body at zero or at infinite time, integrating Eq. (2) between these time limits yields the following:

(3)

where AUCL(<-D) is the AUC of L-[2H3]methionine after administration of D-[2H3]methionine. Substitution for CLDL, according to equation [3], into Eq. (1) yields the following:

(4)

The application of Eq. (4) does not depend on where L-[2H3]methionine is formed or whether the conversion is irreversible (8–10).

    Statistical analysis. Values are means ± SD. The statistical analysis was carried out using StatView version 5 (SAS Institute). Comparison between t1/2 of D-[2H3]methionine and the formed L-[2H3]methionine after administration of D-[2H3]methionine was made by paired t test. To determine the dose dependency of L-[2H3]methionine, the pharmacokinetic parameters for L-[2H3]methionine after administration of L-[2H3]methionine itself were analyzed by unpaired t test. Differences with P < 0.05 were considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
The plasma concentration vs. time profiles of D-[2H3]methionine, L-[2H3]methionine, and endogenous L-methionine after a bolus i.v. administration of D-[2H3]methionine (35 µmol/kg body weight) to rats are shown in Figure 2. Administered D-[2H3]methionine disappeared biexponentially from the plasma with an elimination t1/2 of 130.0 ± 23.4 min (Table 1). L-[2H3]Methionine appeared quickly in the plasma, reached a maximum concentration (12.6 ± 4.0 µmol/L) at 1min after administration, and then gradually decreased with a t1/2 of 125.3 ± 33.4 min. Plasma concentration of endogenous L-methionine was almost constant during 6-h periods after administration.



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  		FIGURE 2 Plasma concentration vs. time curves of D-[2H3]methionine, L-[2H3]methionine, and endogenous L-methionine in rats after a bolus i.v. administration of D-[2H3]methionine (35 µmol/kg body weight). Values are means ± SD, n = 6.

 

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  		TABLE 1 Pharmacokinetic parameters of D-[2H3]methionine and the formed L-[2H3]methionine after i.v. administration of D-[2H3]methionine (35 µmol/kg body wt) to rats1

 
After a bolus i.v. administration of L-[2H3]methionine (10 or 35 µmol/kg body weight) to rats (n = 6 each), plasma concentration of L-[2H3]methionine declined rapidly in a biexponential manner (Fig. 3). The t1/2 and CLL of L-[2H3]methionine did not differ between the 2 dose amounts (Table 2), suggesting linearity within 10–35 µmol/kg body weight. Administration of L-[2H3]methionine produced no detectable concentration of D-[2H3]methionine in the plasma. Plasma concentration of endogenous L-methionine was almost constant during 6-h periods after administration.



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  		FIGURE 3 Plasma concentration vs. time curves of L-[2H3]methionine and endogenous L-methionine in rats after a bolus i.v. administration of L-[2H3]methionine 10 µmol/kg body weight (A) or 35 µmol/kg body weight (B). Values are means ± SD, n = 6.

 

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  		TABLE 2 Pharmacokinetic parameters of L-[2H3]methionine after i.v. administration of L-[2H3]methionine to rats

 
The fraction of conversion of D-[2H3]methionine into L-[2H3]methionine (FD->L) after administration of D-[2H3]methionine was calculated according to Eq. (4). Using the mean CLL values of L-[2H3]methionine after administration of 10 or 35 µg/kg body weight of L-[2H3]methionine, the respective values of FD->L were 95.7 and 91.7%.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The stereoselective behavior of methionine enantiomers was studied after bolus administration of deuterium-labeled methionine enantiomers in rats so that we could investigate the chiral inversion of D-methionine to L-methionine. Our present study indicates directly that L-[2H3]methionine appeared quickly in the plasma after administration of D-[2H3]methionine to rats. The t1/2 values of the formed L-[2H3]methionine did not differ from those of administered D-[2H3]methionine, indicating that the conversion of D-[2H3]methionine into the L-enantiomer is the rate-limiting step.

There are 2 approaches for determining the fraction of conversion of D-[2H3]methionine into L-[2H3]methionine. One is based on an estimation of the ratio between the rate constant for conversion of D-[2H3]methionine into the L-enantiomer and the overall elimination rate constant of D-[2H3]methionine. It requires a fitting of the observed set of plasma concentration data of D-[2H3]methionine and the L-enantiomer to a kinetic compartment model with a first-order rate constant. The other approach is based on estimation of the ratio between the AUC of L-[2H3]methionine formed after administration of D-[2H3]methionine and CL of L-[2H3]methionine after administration of L-[2H3]methionine itself as shown in Eq. (4). This method does not depend on where L-[2H3]methionine is formed or whether D-[2H3]methionine is eliminated by other routes, although i.v. administration of D- and L-[2H3]methionine is necessary. We chose the latter methodology for estimating the fraction of D-[2H3]methionine to the L-enantiomer because we could not calculate the rate constants by computational fitting. Therefore, the pharmacokinetics of L-[2H3]methionine itself was also studied after bolus injection of either 10 or 35 µmol/kg body weight. No detectable amount of D-[2H3]methionine was found in plasma after administration of L-[2H3]methionine. The values of t1/2 and CLL of L-[2H3]methionine after i.v. administration of 10 µmol/kg body weight did not differ from those of L-[2H3]methionine after administration of 35 µmol/kg body weight, suggesting linearity within the range from 10 to 35 µmol/kg body weight.

This is the first study to evaluate the extent to which D-methionine is converted into the L-enantiomer in vivo. Of the i.v. administered dose of D-[2H3]methionine, >90% was converted into L-[2H3]methionine. This result supports the idea that D-methionine possesses a nutritive value that is nearly equal to that of L-methionine in rats. D-Methionine is thought to be converted to the L-enantiomer by 2 steps (11–13). The initial step is an oxidative deamination by D-amino-acid oxidase to form {alpha}-keto-{gamma}-methiolbutyric acid (Fig. 1). Subsequently, {alpha}-keto-{gamma}-methiolbutyric acid is stereospecifically reaminated by transaminases to form L-methionine. Because the fraction of conversion of D-methionine into the L-enantiomer is thought to be the product of the fraction of deamination and that of reamination, the present result suggests that both steps proceed >90% yield, respectively.

D-Amino-acid oxidase oxidizes the neutral and basic amino acids to the corresponding {alpha}-keto acids. In mammals, D-amino-acid oxidase is localized mainly in the kidney, liver, and brain (14). Our previous study (15) showed that mutant mice lacking D-amino-acid oxidase activity could not convert D-[2H7]leucine to the corresponding {alpha}-keto acid, [2H7]{alpha}-ketoisocaproic acid, and L-[2H7]leucine. Because mutant mice may have the ability to convert {alpha}-ketoisocaproic acid into L-leucine, their conversion failure is due to a defect in D-amino-acid oxidase activity. Approximately 70% of the administered dose of D-[2H7]leucine was converted into [2H7]{alpha}-ketoisocaproic acid in rats (10). Because the Km value for D-methionine by D-amino-acid oxidase was 1.7 mmol/L compared with 6.3 mmol/L of D-leucine in vitro (16), it is likely that the exogenously administered D-[2H3]methionine is almost completely converted into the corresponding {alpha}-keto acid.

The transamination between {alpha}-keto-{gamma}-methiolbutyric acid and L-methionine is a reversible reaction, catalyzed by enzymes such as glutamine transaminase, asparagine transaminase, and leucine transaminase (17–19). Cooper and Meister (17) indicated that the reaction by glutamine transaminase in vivo favors the synthesis of new amino acids from {alpha}-keto acids. Their conclusion provides a meaningful explanation for our observation of a high reamination flux in the direction of conversion of {alpha}-keto-{gamma}-methiolbutyric acid into L-methionine.

{alpha}-Keto-{gamma}-methiolbutyric acid also may be decarboxylated to form ß-methylthiopropionic acid or excreted into urine. In vitro experiments with rat homogenates (20) and mitochondria (21) showed that L-methionine was metabolized to ß-methylthiopropionic acid. Kaji et al. (22) reported that {alpha}-keto-{gamma}-methiolbutyric acid and ß-methylthiopropionic acid were excreted into urine after administration of D-methionine to humans. Our previous study showed that the fraction of conversion of the i.v. administered [2H7]{alpha}-ketoisocaproic acid into L-[2H7]leucine in rats was ~40% (10). This relatively low conversion might depend on the irreversible decarboxylation of {alpha}-ketoisocaproic acid (23). However, the high conversion ratio of i.v. administered D-[2H3]methionine to the L-enantiomer in the present study suggests that the reamination of {alpha}-keto-{gamma}-methiolbutyric acid to give L-methionine is a favorable reaction rather than the urinary excretion or further degradation of {alpha}-keto-{gamma}-methiolbutyric acid in rats. This result is consistent with the observations of Sugiyama and Muramatsu (12) and London and Gabel (13).

In summary, the present stereoselective GC-MS method combined with stable isotope methodology has made it possible to evaluate the kinetics of methionine enantiomers. Almost all of an i.v. administered dose of D-[2H3]methionine was converted stereospecifically into the L-enantiomer.


   FOOTNOTES
 
1 Supported by a grant provided by the Promotion and Mutual Aid Corporation for Private Schools of Japan. Back

3 Abbreviations used: AUC, area under the plasma concentration vs. time curve; AUCL(<-D), the AUC of L-[2H3]methionine after administration of D-[2H3]methionine; AUMC, the area under the first-moment time curve; CL, total clearance; FD->L, the fraction of conversion of D-[2H3]methionine to L-[2H3]methionine; MRT, mean residence time; MTPA, {alpha}-methoxy-{alpha}-trifluoromethylphenylacetyl; SIM, selected ion monitoring; OMe, methyl ester; VdSS, the apparent volume of distribution at steady state. Back

Manuscript received 21 March 2005. Initial review completed 23 April 2005. Revision accepted 20 May 2005.


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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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