Role of renal D-amino-acid oxidase in pharmacokinetics of D-leucine

Hiroshi Hasegawa,1 Takehisa Matsukawa,1 Yoshihiko Shinohara,1 Ryuichi Konno,2 and Takao Hashimoto1

1Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392; and 2Department of Microbiology, Dokkyo University School of Medicine, Mibu, Tochigi 321-0293, Japan

Submitted 2 September 2003 ; accepted in final form 9 March 2004


    ABSTRACT
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 ABSTRACT
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D-Amino acids are now recognized to be widely present in mammals. Renal D-amino-acid oxidase (DAO) is associated with conversion of D-amino acids to the corresponding {alpha}-keto acids, but its contribution in vivo is poorly understood because the {alpha}-keto acids and/or L-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of D-amino acids to their {alpha}-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of D-[2H7]leucine to mutant mice lacking DAO activity (ddY/DAO) and normal mice (ddY/DAO+), elimination of D-[2H7]leucine and formation of {alpha}-[2H7]ketoisocaproic acid ([2H7]KIC) and L-[2H7]leucine in plasma were determined. The ddY/DAO mice, in contrast to ddY/DAO+ mice, failed to convert D-[2H7]leucine to [2H7]KIC and L-[2H7]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of D-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of D-[2H7]leucine and formation of [2H7]KIC and L-[2H7]leucine. Renal mass reduction slowed down the elimination of D-[2H7]leucine. The fraction of conversion of D-[2H7]leucine to [2H7]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of D-[2H7]leucine observed in rats suggested that kidney was the principal organ responsible for converting D-leucine to KIC.

{alpha}-ketoisocaproic acid; kidney; nephrectomy


ALL AMINO ACIDS, EXCEPT FOR GLYCINE, occur as optically active isomers. Amino acids used in protein synthesis are of the L-configuration, whereas D-amino acids rarely occur in proteins. Recent progress in chromatography on the separation of DL-amino acids reveals that significant amounts of several free D-amino acids are present in higher animals (5). A number of studies have indicated that D-amino acids play essential roles in several physiological functions (2, 9, 17). Because D-amino acids have also been found in foods and beverage drinks (3), it is considered that quite a few D-amino acids are taken into the body via these diets. Thus it has become important to understand how their levels are controlled.

D-Amino-acid oxidase (DAO; EC 1.4.3.3 [EC] ) is a flavoenzyme that catalyzes the oxidation of D-amino acids to the corresponding {alpha}-keto acids (15). These {alpha}-keto acids are stereospecifically converted to their corresponding L-amino acids by transaminases. Almost all higher animals have DAO in their kidney, liver, and brain, although the mouse is an exception and does not have the enzyme in the liver (11). Because DAO is present at the highest activity in the kidney compared with the other organs, injury to the kidney may cause accumulation of D-amino acids. Increasing evidence that plasma levels of D-amino acids were significantly higher in patients with renal diseases than in healthy subjects (1, 18) suggests that renal DAO plays a prominent role in elimination of D-amino acids. However, little information is available on the contribution of renal DAO to the elimination of D-amino acids, because the {alpha}-keto acid and L-amino acid formed are indistinguishable from the endogenous {alpha}-keto acid and L-amino acid.

One of the unique advantages of the use of a stable isotope-labeled compound as a tracer is that an endogenous compound and its exogenously administered labeled analog are separately measurable by using gas chromatography-mass spectrometry (GC-MS). Our recent use of stable isotope-labeled D-leucine (D-[2H7]leucine) has proved a powerful methodology for examining the pharmacokinetic behavior of exogenously administered D-leucine and studying the conversion of D-leucine to the corresponding {alpha}-keto acid, {alpha}-ketoisocaproic acid (KIC), and L-leucine (6–8, 16). It became apparent that ~30% of an administered dose of D-[2H7]leucine in rats was converted to the L-enantiomer through [2H7]KIC as an intermediate.

The purpose of this study was to explore the hypothesis that D-leucine is predominantly metabolized by renal DAO to KIC. To confirm the role of DAO, we investigated by using the stable isotope tracer technique whether mutant mice lacking DAO activity (ddY/DAO) are able to convert D-leucine to KIC and/or L-leucine. We further investigated the pharmacokinetics of exogenously administered D-leucine in five-sixths-nephrectomized rats to establish the role of renal DAO.


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Chemicals. Optically pure D- and L-[4,5,5,5,6,6,6-2H7]leucine (D-[2H7]leucine and L-[2H7]leucine, respectively; >98 atom % 2H and >99.8% e.e., each) and sodium [4,5,5,5,6,6,6-2H7]2-oxo-4-methylpentanoate ([2H7]KIC Na, >98 atom % 2H) were prepared from DL-[2H7]leucine in our laboratory as described previously (7, 16). DL-[2,3,3-2H3]leucine and sodium [5,5,5-2H3]2-oxo-4-methylpentanoate ([2H3]KIC Na) were purchased from Isotec (Miamisburg, OH). (S)-(+)-{alpha}-methoxy-{alpha}-trifluoromethyl-phenylacetyl chloride [(+)-MTPA-Cl, >99% {alpha}-[4,5,5,5,6,6,6-2H7]ketoisocaproate] and 10% HCl in methanol were purchased from Tokyo Kasei (Tokyo, Japan). N-phenyl-phenylene-1,2-diamine was purchased from Aldrich (Milwaukee, WI).

Animals. The experimental protocols were approved by the Institutional Animal Care Committee of Tokyo University of Pharmacy and Life Science. Mutant ddY/DAO mice lacking DAO activity were described before (10). Male ddY/DAO mice and normal ddY/DAO+ mice (7 wk old) were chosen for the experiment. Male Sprague-Dawley rats (7 wk old) were obtained from Tokyo Laboratory Animal Center (Tokyo, Japan). These animals were maintained in an air-conditioned room at 23 ± 1°C and 55 ± 5% humidity on a 12:12-h dark-light cycle. All animals were allowed free access to water and food (CE-2; Clea Japan, Tokyo, Japan).

Surgery. Rats (n = 6) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and the right kidneys were removed. At 3 days after the first surgery, the anterior and posterior apical segmental branches of the left kidney were removed under anesthesia. Rats (n = 6) underwent sham operations with decapsulation of both kidneys at the same intervals as nephrectomy. After surgery, animals were allowed free access to water and food for 3 days.

Dose experiments. The ddY/DAO and ddY/DAO+ mice (n = 6 each) were fasted overnight. D-[2H7]leucine was dissolved in saline (70 µmol/ml) and administered (35 µmol/kg body wt) into the tail vein. The mice were killed by cervical dislocation at 5 or 60 min after the administration, and a blood sample (300 µl) was collected from the inferior vena cava. The blood was centrifuged to separate plasma at 3,000 rpm for 15 min. The plasma was stored at –20°C until analysis.

After an overnight fast, five-sixths (5/6)-nephrectomized and sham-operated rats (n = 6 each) were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). D-[2H7]leucine (35 µmol/kg body wt) was administered into the femoral vein. 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 the administration. The blood was centrifuged to separate plasma at 3,000 rpm for 10 min. The plasma was stored at –20°C until analysis.

Sample preparation for GC-MS-selected ion monitoring. DL-[2H3]leucine (1 nmol) and [2H3]KIC (0.1 nmol) were added to plasma samples (50 µl each) as analytic internal standards for the measurement of D-[2H7]leucine, L-[2H7]leucine, [2H7]KIC, D-leucine, L-leucine, and KIC. All the plasma samples were then subjected to GC-MS-selected ion monitoring (SIM) analysis, and the plasma concentrations of D-[2H7]leucine, L-[2H7]leucine, [2H7]KIC, D-leucine, L-leucine, and KIC were determined by the isotope dilution method as described previously (8).

GC-MS-SIM. The GC-MS-SIM analysis was conducted on a Shimadzu (Kyoto, Japan) QP1000EX gas chromatograph-mass spectrometer equipped with a data processing system. The operating conditions were the same as those described in previous publications (6, 16).

For leucine enantiomer analysis, the GC-MS conditions were as follows. The initial column temperature was set at 120°C. After the sample injection, temperature was maintained for 2 min, increased at 15°C/min to 190°C, and held at 190°C for 3 min. The mass spectrometer was operated in chemical ionization mode at 200 eV with isobutane as the reactant gas. SIM was performed on the quasimolecular ions of the (+)-MTPA methyl ester (-OMe) derivatives of leucine [2H0, mass-to-charge ratio (m/z) 362; 2H3, m/z 365; 2H7, m/z 369]. (+)-MTPA-OMe derivatives of D- and L-leucine underwent baseline separation and were eluted in this order. The lower limit of quantification (LOQ) for the method was ~200 pmol/ml plasma for [2H7]leucine. The intra- and interday precision values of the assay for D-[2H7]leucine spiked to rat plasma in the range of 0.5 to 50 nmol/ml were <5 and 4%, respectively. Similarly, the intra- and interday precision values for L-[2H7]leucine were <9 and 5%, respectively.

For KIC analysis, the GC-MS conditions were as follows. The initial column temperature was set at 120°C. After the sample injection, temperature was maintained for 2 min, increased at 30°C/min to 250°C, and held at 250°C for 2 min. The mass spectrometer was operated in electron impact ionization mode at 70 eV. SIM was performed on the molecular ions of the N-phenyl-quinoxalinone derivative of KIC (2H0, m/z 278; 2H3, m/z 281; 2H7, m/z 285). The LOQ for the method was ~50 pmol/ml plasma for [2H7]KIC. The intra- and interday precision values of the assay for [2H7]KIC spiked to rat plasma in the range of 0.1 to 10 nmol/ml were <7 and 4%, respectively.

Kinetic analysis. The areas under the curves (AUC)0–360 min of D-[2H7]leucine, [2H7]KIC, and L-[2H7]leucine were calculated by the trapezoidal rule. The half-life of D-[2H7]leucine was determined from the slope obtained in the last four or five time points. Extrapolation of the AUC was based on the last plasma concentration and the terminal slope. Systemic plasma clearance was determined as dose/AUC0–{infty}.

The fraction of conversion of D-[2H7]leucine to [2H7]KIC was estimated by the ratio between the rate constant for conversion of D-[2H7]leucine to [2H7]KIC and the overall elimination rate constant of D-[2H7]leucine. To assess these rate constants, the observed set of plasma D-[2H7]leucine and [2H7]KIC mean data was fitted to a kinetic model shown in Fig. 1 with first-order rate constant. The differential equations for the model are as follows

(1)

(2)

(3)
where X1, X2, and X3 are the amounts of D-[2H7]leucine in the plasma compartment, D-[2H7]leucine in the peripheral compartment, and [2H7]KIC in the plasma compartment, respectively; k21 and k12 are the intercompartmental distribution rate constants, k31 is the rate constant for the conversion of D-[2H7]leucine to [2H7]KIC; k01 is the elimination rate constant of D-[2H7]leucine other than the conversion of D-[2H7]leucine to [2H7]KIC; and k03 is the elimination rate constant of [2H7]KIC. Equations 13 were numerically integrated by a personal computer using the Runge-Kutta method, and the values for the rate constants (k01, k21, k12, k31, and k03) were obtained by a least square estimation using the steepest descent method. The distribution volume of KIC was assumed to be the same as that of D-leucine.



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Fig. 1. Two-compartment model for D-leucine kinetics. KIC, {alpha}-ketoisocaproic acid.

 
Statistical analysis. Differences between means were evaluated with Student's t-test and Welch's t-test. P < 0.05 was considered statistically significant.


    RESULTS
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Metabolism of D-[2H7]leucine in mutant mice lacking DAO activity. After a bolus intravenous administration of D-[2H7]leucine to mutant ddY/DAO mice and normal ddY/DAO+ mice, the plasma concentrations of the labeled and nonlabeled leucine enantiomers and KIC were determined by GC-MS-SIM (Figs. 2 and 3). As shown in Fig. 4, the plasma levels of D-[2H7]leucine at 5 min after the administration were similar in ddY/DAO+ mice (49.4 ± 15.5 nmol/ml) and ddY/DAO mice (60.8 ± 5.1 nmol/ml). However, a significant difference in those at 60 min was observed (32.8 ± 5.7 vs. 53.8 ± 3.4 nmol/ml, P < 0.0005). Formations of [2H7]KIC and L-[2H7]leucine were observed in ddY/DAO+ mice, whereas no detectable amounts of [2H7]KIC and L-[2H7]leucine were present in the plasma from ddY/DAO mice (Figs. 24).



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Fig. 2. Representative selected ion monitoring (SIM) profiles for labeled and nonlabeled leucine enantiomers in plasma at 5 min after administration of D-[2H7]leucine (35 µmol/kg body wt iv) to normal ddY/DAO+ or mutant [D-amino-acid oxidase-deficient (ddY/DAO)] mice. Masses at m/z 362, 365, and 369 were monitored for {alpha}-methoxy-{alpha}-trifluoromethylphenylacetyl methyl ester [(+)-MTPA-OMe] derivatives of leucine, [2H3]leucine, and [2H7]leucine enantiomers, respectively. (+)-MTPA-OMe derivatives of D-leucine and L-leucine were observed at retention time (tR) 7.08 min and tR 7.20 min, respectively. In tracings at m/z 362, a peak for (+)-MTPA-OMe derivative of endogenous L-isoleucine was also observed at tR 7.25 min.

 


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Fig. 3. Representative SIM profiles for labeled and nonlabeled KIC in plasma at 5 min after administration of D-[2H7]leucine (35 µmol/kg body wt iv) to normal ddY/DAO+ or mutant ddY/DAO mice. Masses at m/z 278, 281, and 285 were monitored for N-phenyl-quinoxalinone derivatives of KIC, [2H3]KIC, and [2H7]KIC, respectively. N-phenyl-quinoxalinone derivative of KIC was observed at tR 6.63 min. In tracings at m/z 278, a peak for N-phenyl-quinoxalinone derivative of endogenous {alpha}-keto-{beta}-methylvaleric acid was also observed at tR 6.53 min.

 


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Fig. 4. Plasma concentrations of D-[2H7]leucine, [2H7]KIC, and L-[2H7]leucine at 5 and 60 min after administration of D-[2H7]leucine (35 µmol/kg body wt iv) to normal ddY/DAO+ and mutant ddY/DAO mice. Values represent mean ± SD (n = 6). *Not detected; **P < 0.0005 compared with normal mice.

 
No detectable amounts of endogenous D-leucine were observed in the plasma from ddY/DAO+ mice, whereas considerable amounts of endogenous D-leucine were found in ddY/DAO mice, which were ~5% of total endogenous leucine (Fig. 5). On the other hand, there were no differences in endogenous KIC and L-leucine concentrations between ddY/DAO+ and ddY/DAO mice (Fig. 5).



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Fig. 5. Plasma concentrations of endogenous D-leucine, KIC, and L-leucine at 5 and 60 min after administration of D-[2H7]leucine (35 µmol/kg body wt iv) to normal ddY/DAO+ and mutant ddY/DAO mice. Values represent means ± SD (n = 6). No statistical differences were found between ddY/DAO+ and ddY/DAO mice except for D-leucine in the plasma. *Not detected.

 
Effect of renal mass reduction on pharmacokinetics of D-[2H7]leucine in rats. After a bolus intravenous administration of D-[2H7]leucine to sham-operated and 5/6-nephrectomized rats, plasma concentration of D-[2H7]leucine, [2H7]KIC, and L-[2H7]leucine were examined at various times over 6 h (Fig. 6). Endogenous leucine enantiomers and KIC were measured simultaneously. D-[2H7]leucine disappeared biexponentially in both rats (Fig. 6A), but the systemic plasma clearance was significantly lower in 5/6-nephrectomized rats than in sham-operated rats (1.2 ± 0.3 vs. 6.4 ± 2.1 ml·min–1·kg–1, P < 0.005; Table 1).



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Fig. 6. Plasma concentration time profiles for D-[2H7]leucine (A), [2H7]KIC (B), and L-[2H7]leucine (C) in sham-operated and 5/6-nephrectomized (5/6-Nx) rats after administration of D-[2H7]leucine (35 µmol/kg body wt iv). Values represent mean ± SD (n = 6). Simulated curves obtained from calculated rate constants are represented by the lines in (A) and (B).

 

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Table 1. Pharmacokinetic parameters for D-[2H7]leucine, [2H7]KIC, and L-[2H7]leucine in sham-operated and 5/6-nephrectomized rats

 
The appearance of [2H7]KIC into the plasma was very rapid (Fig. 6B). In sham-operated rats, the plasma concentration of [2H7]KIC had already reached maximum concentration (2.7 ± 0.6 nmol/ml) in the first plasma sample taken 0.5 min after administration of D-[2H7]leucine. In 5/6-nephrectomized rats, the plasma concentration of [2H7]KIC reached maximum (0.3 ± 0.2 nmol/ml) at 1 min after the administration. The AUC0–360 min values of [2H7]KIC in 5/6-nephrectomized rats were markedly decreased to 28% compared with those in sham-operated rats (24 ± 12 vs. 86 ± 18 min·nmol·ml–1, P < 0.0005).

The appearance of L-[2H7]leucine into the plasma was also rapid in both rats (Fig. 6C). In the plasma at 1 min after administration of D-[2H7]leucine, the concentration of L-[2H7]leucine reached maximum (5/6 nephrectomized, 0.9 ± 0.8 nmol/ml vs. sham operated 4.7 ± 1.7 nmol/ml). The AUC0–360 min values of L-[2H7]leucine in 5/6-nephrectomized rats were significantly decreased to 22% compared with those in sham-operated rats (123 ± 45 vs. 551 ± 182 min·nmol·ml–1, P < 0.005).

In both groups of rats, the plasma concentration of endogenous L-leucine was almost constant for 6 h after administration of D-[2H7]leucine (Fig. 7A), and no detectable amounts of endogenous D-leucine were found. As shown in Fig. 7B, the plasma concentration of endogenous KIC in 5/6-nephrectomized rats was almost constant for 6 h. On the other hand, KIC in sham-operated rats tended to fall gradually, reaching the minimum value of 16.8 ± 5.3 nmol/ml at 15 min after the administration, and then increased steadily, which was consistent with our previous study in normal rats (8).



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Fig. 7. Plasma concentration time profiles for endogenous L-leucine (A) and KIC (B) in sham-operated and 5/6-Nx rats after administration of D-[2H7]leucine (35 µmol/kg body wt iv). Values represent mean ± SD (n = 6). For definitions of constants, see MATERIALS AND METHODS.

 

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Mutant mice lacking DAO activity were found in the ddY strain and had been established as inbred strains through brother-sister mating (10, 12, 14). The mutant ddY/DAO mice have a missense mutation (G541A) in the DAO gene, which causes the loss of enzyme activity (20). The ddY/DAO mice, in contrast to normal ddY/DAO+ mice, could not utilize D-phenylalanine for growth in place of its L-isomer. However, both ddY/DAO and ddY/DAO+ mice utilized phenylpyruvic acid, the corresponding {alpha}-keto acid, in place of L-phenylalanine for growth. From these nutritional experiments, Konno and Yasumura (13) suggested that DAO is indispensable for the first step in the conversion of D-amino acid to the L-isomer.

The present study directly shows that ddY/DAO mice, in contrast to ddY/DAO+ mice, failed to convert D-[2H7]leucine to [2H7]KIC and L-[2H7]leucine. Because ddY/DAO mice are considered to have the ability to convert KIC to L-leucine, their conversion failure is due to the defect of DAO activity. This result clearly revealed that DAO was indispensable for the process of chiral inversion of D-leucine and that the sequential conversion through KIC as an intermediate was the only pathway for conversion of D-leucine to the L-enantiomer.

Considerable amounts of endogenous D-leucine were detected in the plasma of ddY/DAO mice, which was consistent with the report by Hamase et al. (4). We have determined the amounts of D-leucine in the diet (CE-2) by use of GC-MS-SIM and found that a considerable amount of D-leucine (20.0 nmol/g) is present. To our knowledge, there is no literature that demonstrates the presence of the biosynthesis of D-leucine in higher animals. Thus dietary D-leucine might be one of the possible sources.

In the present study, we found that renal mass reduction by partial nephrectomy slowed down the elimination of D-[2H7]leucine in rats. Systemic plasma clearance of D-[2H7]leucine in 5/6-nephrectomized rats was one-sixth of that in sham-operated rats. It should be noted that the decrease in plasma clearance of D-[2H7]leucine in 5/6-nephrectomized rat might be due to decreased elimination of D-[2H7]leucine other than the conversion of D-[2H7]leucine to [2H7]KIC, such as in urinary excretion. In an attempt to distinguish between the conversion and other elimination, rate constants were calculated on the basis of the kinetic model shown in Fig. 1. In sham-operated rats, the rate constants for the conversion of D-[2H7]leucine to [2H7]KIC (k31) and the elimination of D-[2H7]leucine other than the conversion of D-[2H7]leucine to [2H7]KIC (k01) were 0.02 and 0.006 min–1, respectively (Table 2). On the other hand, k31 in 5/6-nephrectomized rats (0.0016 min–1) was one-twelfth of that in sham-operated rats, whereas k01 in 5/6-nephrectomized rats (0.0049 min–1) was four-fifths of that in sham-operated rats. These results showed that the renal mass reduction mainly caused a decrease of the conversion of D-[2H7]leucine to [2H7]KIC.


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Table 2. Rate constants for the kinetic analysis model on intravenous administration of D-[2H7]leucine to sham-operated and 5/6-nephrectomized rats

 
The fraction of conversion of D-[2H7]leucine to [2H7]KIC was obtained by the ratio between the total elimination rate constants of D-[2H7]leucine and the rate constant for the conversion, that is, k31/(k01 + k31). The value in sham-operated rats was estimated to be 0.77, which was almost consistent with the value in our previous study in normal rats (8). On the other hand, the fraction in 5/6-nephrectomized rats was markedly decreased to 0.25 (32%), which resulted from the DAO activity in the remnant kidney and other organs such as liver and brain. The decrease of renal DAO contents by nephrectomy may compensate DAO activity by inducing DAO. Nagata et al. (19) observed that DAO was not inducible in adult mice even with a high dose of D-alanine. Thus, assuming that DAO in liver and brain was not induced by nephrectomy, the DAO activity in kidney (x) and that in the other organs (y) were estimated by the following two simultaneous equations: x + y = 0.77 and x/6 + y = 0.25, and were calculated to be 0.62 and 0.15, respectively. Namely, the ratio of conversion in kidney occupied ~80% of overall conversion. These results suggested that the kidney was the principal organ responsible for converting D-leucine to KIC in vivo.

In conclusion, the present study shows that renal DAO plays the principal role in the metabolism of D-leucine in vivo. Because urinary excretion may also play a role in the elimination of D-leucine, it is important to establish the quantitative roles of metabolism by DAO compared with urinary excretion in both intact and 5/6-nephrectomized rats. We are now in the process of extending this approach to elucidate the overall role of the kidney in the elimination of D-leucine.


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This work was supported in part by grants from The Japan Private School Promotion Foundation and The Science Research Promotion Fund, The Promotion and Mutual Aid Corporation for Private Schools of Japan, and The Seki Minato Foundation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Hasegawa, Dept. of Pathophysiology, School of Pharmacy, Tokyo Univ. of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan (E-mail: hasegawa{at}ps.toyaku.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Brückner H and Hausch M. Gas chromatographic characterization of free D-amino acids in the blood serum of patients with renal disorders and of healthy volunteers. J Chromatogr B 614: 7–17, 1993.[CrossRef]
  2. D'Aniello A, Fiore MMD, Fisher GH, Milone A, Seleni A, D'Aniello S, Perna AF, and Ingrosso D. Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J 14: 699–714, 2000.[Abstract/Free Full Text]
  3. Friedman M. Chemistry, nutrition, and microbiology of D-amino acid. J Agric Food Chem 47: 3457–3479, 1999.[CrossRef][ISI][Medline]
  4. Hamase K, Inoue T, Morikawa A, Konno R, and Zaitsu K. Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity. Anal Biochem 298: 253–258, 2001.[CrossRef][ISI][Medline]
  5. Hamase K, Morikawa A, and Zaitsu K. D-Amino acids in mammals and their diagnostic value. J Chromatogr B 781: 73–91, 2002.
  6. Hasegawa H, Matsukawa T, Shinohara Y, and Hashimoto T. Simultaneous determination of the enantiomers of leucine and [2H7]leucine in plasma by capillary gas chromatography-mass spectrometry. J Chromatogr B 735: 141–149, 1999.[CrossRef][ISI]
  7. Hasegawa H, Matsukawa T, Shinohara Y, and Hashimoto T. Assessment of the metabolic chiral inversion of D-leucine in rat by gas chromatography-mass spectrometry combined with a stable isotope dilution analysis. Drug Metab Dispos 28: 920–924, 2000.[Abstract/Free Full Text]
  8. Hasegawa H, Matsukawa T, Shinohara Y, and Hashimoto T. Kinetics of sequential metabolism from D-leucine to L-leucine via {alpha}-ketoisocaproic acid in rat. Drug Metab Dispos 30: 1436–1440, 2002.[Abstract/Free Full Text]
  9. Hashimoto A, Oka T, and Nishikawa T. Anatomical distribution and postnatal changes in endogenous free D-aspartate and D-serine in rat brain and periphery. Eur J Neurosci 7: 1657–1663, 1995.[ISI][Medline]
  10. Konno R, Isobe K, Niwa A, and Yasumura Y. Excessive urinary excretion of methionine in mutant mice lacking D-amino-acid oxidase activity. Metabolism 37: 1139–1142, 1988.[CrossRef][ISI][Medline]
  11. Konno R, Sasaki M, Asakura S, Fukui K, Enami J, and Niwa A. D-Amino-acid oxidase is not present in the mouse liver. Biochim Biophys Acta 1335: 173–181, 1997.[ISI][Medline]
  12. Konno R and Yasumura Y. Mouse mutant deficient in D-amino acid oxidase activity. Genetics 103: 277–285, 1983.[Abstract/Free Full Text]
  13. Konno R and Yasumura Y. Involvement of D-amino-acid oxidase in D-amino acid utilization in the mouse. J Nutr 114: 1617–1621, 1984.[ISI][Medline]
  14. Konno R and 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.[Medline]
  15. Krebs HA. Metabolism of amino acids. III. Deamination of amino acids. Biochem J 29: 1620–1644, 1935.
  16. Matsukawa T, Hasegawa H, Shinohara Y, and Hashimoto T. Gas chromatographic-mass spectrometric determination of {alpha}-ketoisocaproic acid and [2H7]{alpha}-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine. J Chromatogr B 751: 213–220, 2001.[CrossRef][ISI]
  17. Mothet JP, Parent AT, Wolosker H, Brady RO Jr, Linden DJ, Ferris CD, Rogawski MA, and Snyder SH. D-Serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 97: 4926–4931, 2000.[Abstract/Free Full Text]
  18. Nagata Y, Akino T, Ohno K, Kataoka Y, Ueda T, Sakurai T, Shiroshita K, and Yasuda T. Free D-amino acids in human plasma in relation to senescence and renal diseases. Clin Sci (Lond) 73: 105–108, 1987.[Medline]
  19. Nagata Y, Yamada R, Nagasaki H, Konno R, and Yasumura Y. Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice. Experientia 47: 835–838, 1991.[ISI][Medline]
  20. Sasaki M, Konno R, Nishio M, Niwa A, Yasumura Y, and Enami J. A single-base-pair substitution abolishes D-amino-acid oxidase activity in the mouse. Biochim Biophys Acta 1139: 315–318, 1992.[ISI][Medline]