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Vol. 30, Issue 12, 1436-1440, December 2002
-Ketoisocaproic Acid in Rat
Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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D-Leucine is considered to be converted into the
L-enantiomer by two steps: oxidative deamination to form
-ketoisocaproic acid (KIC) and subsequent stereospecific reamination
of KIC. We investigated the pharmacokinetics of leucine enantiomers and
KIC in rats to evaluate how deamination of D-leucine,
reamination of KIC, and decarboxylation of KIC were affected to the
overall extent that converted D-leucine into the
L-enantiomer. After intravenous administrations of
D-[2H7]leucine,
L-[2H7]leucine, or
[2H7]KIC, their plasma concentrations
together with endogenous L-leucine and KIC were determined
by gas chromatography-mass spectrometry. The rapid appearances
of [2H7]KIC and
L-[2H7]leucine were observed
after administration of
D-[2H7]leucine, whereas no
detectable amount of
D-[2H7]leucine was found after
administrations of [2H7]KIC or
L-[2H7]leucine. The fraction of
conversion from D-[2H7]leucine
into [2H7]KIC (FD
KIC) was
estimated by using the area under the curve (AUC) of
[2H7]KIC on the
D-[2H7]leucine administration
[AUCKIC(
L) was 40.2%. The fraction of conversion from
D-leucine to the L-enantiomer
(FDL) was considered to be the product of
FDKIC and FKICL, indicating that 28.2%
of D-[2H7]leucine was metabolized
to L-[2H7]leucine via
[2H7]KIC. These results suggested that the
relatively low conversion of D-leucine into the
L-enantiomer might depend on irreversible decarboxylation
of KIC. Regardless of [2H7]KIC,
FDL was also calculated directly using
AUCL(L values, suggesting
that almost all of the formation of
L-[2H7]leucine from
D-[2H7]leucine occurred via
[2H7]KIC as an intermediate.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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D-Amino acids play
prominent roles in microorganisms but have not been thought to occur in
substantial quantity or to have any important function in mammals.
However, recent progress in chromatography on the separation of
DL-amino acids reveals that significant amounts of several
D-amino acids are present in a variety of mammals
(Friedman, 1999
). For example, D-serine is observed in the
forebrain and is thought to function as a coagonist as the glycine site
of the N-methyl-D-aspartate receptor
(Mothet et al., 2000). D-Aspartic acid is
observed in the neuroendocrine and endocrine organs and is thought to
be the regulator of hormones such as testosterone and melatonin
(D'Aniello et al., 1996; Takigawa et al., 1998). However, the
physiological roles of aliphatic D-amino acids
have not been investigated, although the presence of
D-proline (Nagata et al., 1992) and
D-leucine (Inoue et al., 2000) in mammals was observed.
Use of D-amino acids for the purpose of growth or
maintenance of nitrogen equilibrium was repeatedly confirmed by
different species of animals for about 40 years, and it was found that
several D-amino acids were used for growth (Meister, 1965;
Friedman, 1999). In these studies, a widely accepted method involves a
comparison of growth rate of animals fed a control diet with that of
animals fed a diet containing the D-amino acid instead of
the corresponding L-enantiomer. The fraction that converted
from D-amino acid to the L-enantiomer was
estimated from the dosage of the D-amino acid required to
achieve the same growth rate in control animal. However, the technique
is very laborious and cannot readily be applied to human subjects.
One of the most unique advantages for the use of stable isotopically
labeled compounds as tracers is that an endogenous compound and its
exogenously administered labeled analog are separately measurable by
using mass spectrometry (Matthews and Bier, 1983; Shinohara et al.,
2001). This methodology would be useful for studying the
pharmacokinetics of naturally occurring substances in humans. Our
recent use of stable isotope-labeled D-leucine (D-[2H7]leucine),
in conjunction with gas chromatography-mass spectrometry (GC-MS),1 has proven a
powerful methodology for examining the pharmacokinetic behavior of
exogenous D-leucine and studying the inversion of D-leucine to L-leucine (Hasegawa et al., 1999,
2000). After an intravenous administration of
D-[2H7]leucine
to rats, about 30% of an administered dose of
D-[2H7]leucine
was converted into the L-enantiomer.
D-Leucine is considered to be converted into the
L-enantiomer by two steps. The initial step is an oxidative
deamination by D-amino acid oxidase to form
-ketoisocaproic acid (KIC). Subsequently, KIC is stereospecifically
reaminated by branched chain amino acid aminotransferase to form
L-leucine. Direct conversion of D-leucine into
the L-enantiomer may also occur. Racemase is a principal enzyme catalyzed interconversion between L- and
D-enantiomer. Although no racemase was thought to be
produced in mammals, serine racemase has recently been purified from
rat brain (Wolosker et al., 1999). However, the conversion of
D-leucine into the L-enantiomer is unlikely to
be attributable to the action of a racemase (Hasegawa et al., 2000).
Thus, the fraction that converted from D-leucine into the
L-enantiomer is considered to be the product of the
fraction that converted from D-leucine into KIC and the
fraction that converted from KIC into L-leucine. KIC may be
decarboxylated by branched chain -keto acid dehydrogenase to form
isovaleryl-CoA, resulting in irreversible loss of leucine (Frick et
al., 1981; Toth et al., 2001). How deamination of
D-leucine, reamination of KIC, and decarboxylation of KIC
are affected to the overall extent of D-leucine that is converted into the L-enantiomer has been a
fascinating question that has not yet been clearly answered. To address
the question, we chose to study KIC kinetics in rat and have already
developed a procedure for determining stable isotopically labeled and
endogenous KIC in plasma by GC-MS (Matsukawa et al., 2001). The purpose
of the present study focuses on determining the fractions that
converted from D-leucine into KIC and the subsequent
conversion into L-leucine in rat using stable isotope methodology.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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 each) were prepared
from
DL-[4,5,5,5,6,6,6-2H7]leucine
(Isotec, Miamisburg, OH) in our laboratory (Hasegawa et al.,
2000). Sodium
[4,5,5,5,6,6,6-2H7]2-oxo-4-methylpentanoate
([2H7]KIC Na, >98 atom
% 2H) was prepared by oxidative deamination of
L-[2H7]leucine
by L-amino acid oxidase using the method of Meister (1952)
with minor modification.
DL-[2,3,3-2H3]Leucine
and sodium
[5,5,5-2H3]2-oxo-4-methylpentanoate
([2H3]KIC Na) were
purchased from Isotec.
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 weeks were obtained from Tokyo Laboratory Animal Center (Tokyo, Japan). 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 days, during which time they were allowed free access to water and food (CE-2; Nihon Clea, Tokyo, Japan).
Dose Experiments.
After an overnight fast, each rat, weighing 250 to 330 g, was
anesthetized with pentobarbital (50 mg/kg weight i.p.). Each rat was
then administered intravenously
D-[2H7]leucine,
L-[2H7]leucine,
or [2H7]KIC (35 µmol/kg
weight, each) dissolved in saline (0.5 ml of dosing solution/kg weight)
from 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 3000 rpm for 10 min. The plasma
was stored at 
20°C until analysis.
, 2000; Matsukawa et al., 2001) with
minor modifications.
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GC-MS-SIM.
GC-MS-SIM analyses were conducted on a QP1000EX gas chromatograph-mass
spectrometer (Shimadzu, Kyoto, Japan) and were performed under the
conditions described previously (Hasegawa et al., 1999, 2000; Matsukawa
et al., 2001).
Data Analysis.
Pharmacokinetic parameters were calculated by model-independent
analysis using a macro-program MOMENT(EXCEL) (Tabata et al., 1999;
http://bunseki02.pharm.kyoto-u.ac.jp/download.html) running on
Microsoft Excel (Microsoft, Redmond, WA) as described previously (Hasegawa et al., 2000).
B) is calculated as follows:
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(1) |
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(2) |
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(3) |
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(4) |
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(5) |
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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After intravenous administrations of D-[2H7]leucine, L-[2H7]leucine, or [2H7]KIC (35 µmol/kg weight, each) to rats, their plasma concentrations together with endogenous leucine enantiomers and KIC were determined by GC-MS-SIM. The results are presented in Fig. 2.
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After administration of D-[2H7]leucine (Fig. 2A), the substrate disappeared biexponentially from the plasma, with a distribution half-life of 0.91 ± 0.08 min and a terminal half-life of 84.8 ± 15.6 min. The appearance of [2H7]KIC into the plasma was very rapid. In the first blood samples, taken 0.5 min after administration of D-[2H7]leucine, the plasma concentration of [2H7]KIC already reached a maximum concentration (3.2 ± 1.8 nmol/ml). Thereafter, the plasma levels of [2H7]KIC gradually decreased with a terminal half-life of 105.2 ± 13.8 min. L-[2H7]Leucine also appeared quickly in the plasma, reached a maximum concentration (2.9 ± 0.4 nmol/ml) at approximately 3 min, and then gradually decreased with a terminal half-life of 124.4 ± 26.4 min.
After administration of [2H7]KIC (Fig. 2B), plasma concentration of [2H7]KIC was observed to decline in apparently triexponential manner with a terminal half-life of 143.3 ± 39.1 min and reached below the limit of quantitation (50 pmol/ml) by 300 min. Plasma concentration of L-[2H7]leucine already reached a maximum concentration (28.7 ± 6.6 nmol/ml) at 0.5 min after administration and then L-[2H7]leucine declined more slowly than that of [2H7]KIC. Administration of [2H7]KIC produced no detectable amounts of D-[2H7]leucine.
After administration of L-[2H7]leucine (Fig. 2C), plasma concentration of L-[2H7]leucine was observed to decline in apparently triexponential manner with a terminal half-life of 214.5 ± 52.3 min. Plasma concentration of [2H7]KIC already reached a maximum concentration (8.2 ± 1.8 nmol/ml) at 0.5 min after administration. Administration of L-[2H7]leucine produced no detectable amounts of D-[2H7]leucine.
In each administration, no detectable amount of endogenous D-leucine was found. Plasma concentration of endogenous L-leucine was almost constant during 6-h periods after administration, whereas plasma concentration of endogenous KIC tended to fall temporarily by 30 min and then recovered by 6 h.
Table 1 shows the AUC values for
D-[2H7]leucine,
[2H7]KIC, and
L-[2H7]leucine
after administrations of
D-[2H7]leucine,
[2H7]KIC, or
L-[2H7]leucine.
The fraction of
D-[2H7]leucine
that converted into
[2H7]KIC
(FDKIC) was estimated according to eq. 5 using
the mean AUC value of
[2H7]KIC on the
D-[2H7]leucine
administration [AUCKIC(L) was calculated using
AUCL(
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The GC-MS and stable isotopically labeled compounds as tracers make it possible to provide a precise analytical method for both the parent and the metabolites with high sensitivity and selectivity. Thus, plasma requirements for measurements could be reduced to 50 µl/sample, or 0.8 ml of total plasma per 16 samples per a rat for the pharmacokinetic study. With the intravenous administration of D-[2H7]leucine, [2H7]KIC, or L-[2H7]leucine, plasma concentrations of D-[2H7]leucine, L-[2H7]leucine, and [2H7]KIC together with their endogenous compounds could be followed up to 360 min. The advantage of using stable isotope method was significant in assessing the conversion process.
This is the first study to evaluate the conversion of
D-leucine into KIC in vivo. Approximately 70% of the
administered dose of
D-[2H7]leucine
was converted into
[2H7]KIC (Fig. 3). On the
other hand, no detectable amount of
D-[2H7]leucine
was found in plasma after
[2H7]KIC and
L-[2H7]leucine
administrations. D-Leucine is considered to be converted into KIC in the following two steps: dehydrogenation by
D-amino acid oxidase to yield the corresponding imino acid
and the subsequent nonenzymatically hydrolysis to yield KIC (Greenstein
and Winitz, 1961). Because the latter is an irreversible reaction, KIC
does not convert to D-leucine in rats. The remaining 30%
of administered dose of
D-[2H7]leucine
was eliminated by distinct pathway. Increased excretion of
D-amino acids in urine was reported after administration of the racemic or D-amino acids (Cho and Stegink, 1979;
Lehmann et al., 1983; Stegink et al., 1986; Darling et al., 1999).
Lehmann et al. (1983) reported that about 30% of the oral dose of
D-[2H]phenylalanine in human was
excreted into urine. However, in our preliminary study only 1% of
administered dose of
D-[2H7]leucine
was excreted as an intact form in urine over 24 h. This result
suggests the existence of other metabolic pathway, although we do not
have the direct data to substantiate this speculation.
After administration of
D-[2H7]leucine,
endogenous KIC was temporarily diminished by 25%, whereas there was no
significant change in the endogenous L-leucine
concentration. Administration of L-leucine in human is
associated with decrease of -keto-
-methylvaleric acid and
-ketoisovaleric acid, which are the corresponding -keto acids of
isoleucine and valine, respectively (Schauder, 1985). L-Leucine has been shown to stimulate the activity of
branched chain -keto acid dehydrogenase that decarboxylated all
branched chain -keto acids irreversibly (Frick et al., 1981). The
decrease in the concentration of endogenous KIC in the present study
might be attributed to enhance decarboxylation by
[2H7]KIC and
L-[2H7]leucine
formed. However, the decrease seems to make no significant contribution
on estimating the extent of conversion because similar tendency in
variation of KIC concentration was observed in administrations of
[2H7]KIC or
L-[2H7]leucine.
The fraction that converted from KIC into L-leucine
(FKICL) was 40.2%. Several investigators have
studied the interconversion of KIC and L-leucine (Nissen
and Haymond, 1981; Matthews et al., 1982; Kang and Walser, 1985;
Schauder, 1985; Imura et al., 1988; Thompson et al., 1988; Shiota et
al., 1989; Cobelli et al., 1991; Hoerr et al., 1991; Chinkes et al.,
1996). The nutritional efficiency of KIC relative to
L-leucine in rat was determined by double-labeled radioisotope methodology (Kang and Walser, 1985; Shiota et al., 1989).
After oral administration of
L-[3H]leucine plus
[1-14C]KIC to rat, the efficiency of KIC to
L-leucine could be estimated from measurement of the ratio
between 14C/3H into the
leucine of whole-body protein and
14C/3H injected and the
value was 39%, which seems to be consistent with our assessment. The
inefficiency of KIC as a protein precursor relative to
L-leucine was attributable to systemic oxidation by measurement of labeled CO2 exhalation after
administration of L-[1-14C]leucine
and [1-14C]KIC (Imura et al., 1988). Therefore,
the relatively low conversion of
[2H7]KIC into
L-[2H7]leucine
in the present study might depend on irreversibly rapid decarboxylation
of [2H7]KIC.
The fraction that converted from D-leucine into the
L-enantiomer (FDL) was considered
to be the product of FDKIC and
FKICL, indicating that 28.2% of the dose of
D-[2H7]leucine
was metabolized into
L-[2H7]leucine
via [2H7]KIC. Regardless
of [2H7]KIC, on the other
hand, FDL was calculated directly using AUCL(L values, suggesting that almost all of the
formation of
L-[2H7]leucine
from
D-[2H7]leucine
occurred via [2H7]KIC as
an intermediate. Other pathways do not seem to be involved in the
formation of
L-[2H7]leucine
from
D-[2H7]leucine
in rats. Thus, D-amino acid oxidase would be an
indispensable enzyme for conversion of D-leucine.
In summary, the present stable isotope methodology has made it possible to evaluate the pharmacokinetics of leucine enantiomers and KIC and an average of 27% of an administered dose of D-[2H7]leucine was stereospecifically converted into the L-enantiomer via [2H7]KIC. The relatively low conversion might depend on irreversible rapid decarboxylation of KIC.
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Footnotes |
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Received July 29, 2002; accepted September 9, 2002.
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.
Address correspondence to: Hiroshi Hasegawa, Ph.D., Department of Pathophysiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail: hasegawa{at}ps.toyaku.ac.jp
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Abbreviations |
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Abbreviations used are:
GC-MS, gas
chromatography-mass spectrometry;
KIC, -ketoisocaproic acid;
SIM, selected ion monitoring;
FAB, fraction of conversion
from compound A to metabolite B;
CL, clearance;
AUCB(
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-keto acid dehydrogenase in rat adipose tissue.
J Biol Chem
256:
2618-2620-ketoisocaproate relative to leucine, assessed isotopically.
Am J Physiol
249:
E355-E359-ketoisocaproic acid and [2H7]-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine.
J Chromatogr B
751:
213-220[CrossRef].-ketoisocaproate during a L-[1-13C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment.
Metabolism
31:
1105-1112[CrossRef][Medline].-keto acids.
J Biol Chem
197:
309-317-ketoisocaproate in vivo.
Am J Physiol
241:
E72-E75-ketoisocaproic acid during continuous infusion of labelled leucine.
Eur J Clin Invest
18:
639-643[Medline].This article has been cited by other articles:
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),
[2H7]KIC (
),
L-[2H7]leucine (
), endogenous
KIC (
), and endogenous L-leucine (
) in male rats
after intravenous bolus administration of
D-[2H7]leucine (A),
[2H7]KIC (B), or
L-[2H7]leucine (C) (35 µmol/kg
weight each; mean ± S.D., n = 6).
