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Department of Pediatrics, Kyoto Prefectural University of Medicine, Kajii-cho Hirokoji Kawaramachi Kamigyo-ku, Kyoto 602, Japan
2To whom correspondence should be addressed at Department of Pediatrics, Social Insurance Kyoto Hospital, Shimofusa-cho Koyama Kita-ku, Kyoto 603-8151, Japan. E-mail: zenro{at}ped.kpu-m.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-butyrobetaine and
acylcarnitines using tandem mass spectrometry. Hepatocytes from rats
treated with 20 mmol/L of pivalate for 4 wk had greater
L-[14C] carnitine uptake than those of
unsupplemented rats after 5, 10, 30 and 90 min. Addition of 1 mmol/L of
pivalate or 1 mmol/L of pivaloylcarnitine to control cell suspensions
did not affect L-[14C] carnitine uptake. The
Km values for L-[14C]
carnitine uptake for pivalate-treated rats were significantly lower
than control (2.9 ± 0.7 mmol/L for pivalate-treated rats, 6.2
± 1.1 mmol/L for controls). The concentration of free carnitine
was not reduced in the liver of pivalate-treated rats, whereas the
concentrations of acetylcarnitine and -butyrobetaine were
significantly lower than controls. In the heart and muscle the
concentration of free carnitine was significantly lower and that of
-butyrobetaine was higher than controls. These results suggest that
carnitine transport from plasma into the liver and synthesis in the
liver are accelerated in rats with secondary carnitine deficiency
induced by the administration of pivalate.
KEY WORDS: • pivalate • carnitine • hepatocytes • tandem mass spectrometry • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Several reports exist of secondary CN deficiency and
disturbance of ß-oxidation induced by antibiotics esterified with
pivalate (Abrahamsson et al. 1994
, Holme et al. 1989
, Melegh et al. 1987
). Our previous study
revealed that pivalate greatly reduced the plasma CN concentration in
rats; however, liver CN concentrations were maintained at levels that
supported gluconeogenesis and ketogenesis (Nakajima et al. 1996
).
To maintain the concentrations of CN in the tissues, two major
mechanisms exist: one is absorption from food and the other is the
biosynthesis of CN [hydroxylation of -butyrobetaine (BB)] which is
localized chiefly in the liver (Feller and Rudman 1988
).
Both absorbed and synthesized CN are transferred to the tissues and
excreted in the free form or as acylcarnitines in urine.
Some reports exist suggesting that CN is transported into the tissues
via an active transport system (Rebouche 1986
,
Vary and Neely 1982
). CN import from the plasma is one
of three or four mechanisms by which tissue carnitine concentrations
are maintained or altered. Another is loss of CN by diffusion or
facilitated export (Sandor et al. 1985
). However, to
date, the transport system of CN into the liver has not been elucidated
in animals with secondary CN deficiency. Furthermore, the statuses of
acylcarnitines and BB in pivalate-treated rats are unclear.
The purpose of this study was to test the hypothesis that the differences in CN concentration between the liver and plasma in secondary CN-deficient rats are due to accelerated CN import into the liver and CN biosynthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Wistar rats (Japan Animals, Osaka, Japan) weighing 190–200 g were
kept for 4 wk in stainless wire-mesh cages with a 12-h light/dark
cycle and were given free access to a commercial diet previously
described (Nakajima et al. 1996
) and water containing 20
mmol/L of pivalate (adjusted to pH 7.0 with 1 mol/L NaOH) according to
Bianchi and Davis (1991)
. Control rats were subjected to
the same conditions, except their water was not supplemented with
pivalate. This experimental procedure was permitted by the Committee
for Animal Research, Kyoto Prefectural University of Medicine.
Isolation of hepatocytes.
Parenchymal liver cells were prepared and purified according to the
procedure of Seglen with minor modification (Seglen 1972
). After anesthesia with sodium pentobarbital (50 mg/kg,
i.p.), the liver was perfused for 10 min in a noncirculating
system with a medium containing 137 mmol/L of NaCl, 5.4 mmol/L of KCl,
0.8 mmol/L of MgSO4, 0.3 mmol/L of
Na2HPO4, 8 mmol/L of HEPES, 0.6 mmol/L of EGTA,
25 mmol/L of NaHCO3 (adjusted to pH 7.4 by 1 mol/L of
NaOH). The perfusion was then continued for an additional 12 min with a
medium containing 137 mmol/L of NaCl, 5.4 mmol/L of KCl, 0.8 mmol/L of
MgSO4, 0.3 mmol/L of Na2HPO4, 8
mmol/L of HEPES, 25 mmol/L of NaHCO3, 2 mmol/L of
CaCl2, and 0.16 g/L of collagenase (adjusted to pH 7.4 by 1
mol/L NaOH). The liver was minced and suspended in the
Krebs-Henseleit buffer containing 10 g/L of bovine serum albumin
and 8 mmol/L of HEPES. The digested tissue was strained through cotton
gauze, and isolated cells were centrifuged at 50 x g
for 2 min to separate parenchymal from nonparenchymal cells such as
Küpffer cells. The parenchymal cells in the pellet were
resuspended and washed twice with the Krebs-Henseleit buffer. Cells
prepared in this manner had greater than 85% viability by Trypan blue
exclusion method.
Transport assay.
A transport assay of CN was initiated in a 50-mL Falcon centrifugation tube (Becton Dickinson and Company, Franklin Lakes, NJ) after incubation for 15 min at 37°C by the addition of 1 mmol/L of [14C] CN to three mL of cell suspension (3.5 x 109 cells/L). To investigate the direct effect of pivalate or pivaloylcarnitine, 1 mmol/L of pivalate or 1 mmol/L of pivaloylcarnitine was added, respectively, to cell suspensions of control rats. Each reaction was terminated by the addition of 15 mL of ice-cold Krebs-Henseleit buffer. After separation of the medium from the cell pellets by centrifugation for 5 s at 700 x g, the cell pellets were washed with 10 mL of ice-cold buffer and then recentrifuged for 5 s at 700 x g. Cell pellets were extracted by the addition of 1 mL of 0.39 mol/L trichloroacetic acid, and radioactivity was measured by liquid scintillation spectrometry (Packard TRI-CARB 460, Packard Instrument Co., Downers Grove, IL). The Km value was determined by a simple regression for double reciprocal plots of data of concentration curve employing a statistical application (StatView 4.0; Abacus Concepts, Inc., Berkeley, CA).
Analyses of free CN, BB and acylcarnitines in the tissues.
The concentrations of free CN, acetylcarnitine, C5 acylcarnitine and BB
in the liver, heart and skeletal (psoas) muscle were analyzed using
tandem mass spectrometry with liquid secondary ion mass spectrometry
according to Millington et al. (1991)
with minor
modification. The tissues were resected immediately after blood
drainage from inferior vena cava of rats under anesthesia with
pentobarbital. The resected tissues were stored in a deep freezer at
-30°C. Quantitative analysis was achieved by use of stable
isotope-labeled internal standards including d9-CN,
d3-acetylcarnitine, d9-isovalerylcarnitine and d9-BB. Tissues were
homogenized with 10 vol of phosphate buffer (pH 7.4). Homogenate (100
µL) was added with d9 or d3-labeled CN, acylcarnitine, and BB as
internal standards and 300 µL of methanol for deproteinization. The
specimen was centrifuged with an Eppendorf centrifuge 5414 at 10000
x g for 10 min, and the supernatant was filtered by
centrifugal filtration (ULTRA FREE-MC, Millipore Corporation,
Bedford, MA). The supernatant was dried under a nitrogen stream. The
dry residue was derivatized with 100 µL of 1.37 mol/L of hydrochloric
acid in methanol at 65°C for 15 min. The sample was dried again under
a nitrogen stream, and the final residue was dissolved in 50 µL of
methanol/glycerol (1:1), plus sodium octylsulfate (10 g/L) as a matrix.
QUATTRO (Fisons-VG instruments, Danvers, MA) was used for tandem mass
spectrometry. Collision-induced dissociation was performed by argon
gas, and precursor ion scannings were performed for the analyses of CN,
BB, acylcarnitines (m/z 117, 101 and 99, respectively)
(Millington et al. 1989
, Okochi et al. 1996
). For quantification, ratios of peak height of
corresponding internal standards were used after smoothing, subtracting
background and centering.
Chemicals.
Pivalate, HEPES, EGTA and bovine serum albumin were purchased from
Nacalai Tesque Inc. (Tokyo, Japan). Collagenase was purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan).
L-[14C] CN hydrochloride (3700 MBq/L) was
purchased from DUPONT/NEN Products (Wilmington, DE). L-CN
and BB were purchased from Sigma (St. Louis, MO). D9-CN and d9-BB were
synthesized according to the method of Ingalls (1982)
.
Pivaloylcarnitine was synthesized in our laboratory from pivalate
hydrochloride and L-CN. D9-isovalerylcarnitine and
d3-acetylcarnitine were the gifts from Costa et al. (1997)
.
Statistical methods.
Values were reported as mean ± SEM. Statistical analyses employed ANOVA followed by the least significant difference test (LSD) for the transport assay and Mann-Whitney U-test for comparison of the concentrations of free CN, acetylcarnitine, pivaloylcarnitine and BB in tissue (StatView 4.0, Abacus Concepts, Inc., Berkeley, CA). Differences with P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The CN uptake of hepatocytes from rats treated with pivalate for 4 wk
was significantly higher than those of controls after 5, 10, 30 and 90
min (Fig. 1
).CN uptake into hepatocytes of controls supplemented with 1 mmol/L of
pivalate and 1 mmol/L of pivaloylcarnitine were not different from
unsupplemented hepatocytes.
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Hetapocytes of pivalate-treated rats had significantly higher
affinity for carnitine than did those of controls (Fig. 2
).Double reciprocal plots of the data indicated the
Km value of CN transport into the hepatocytes in
pivalate-treated rats was significantly lower than control (2.9
± 0.7 mmol/L, 6.2 ± 1.1 mmol/L, respectively).
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Hepatic free CN did not differ between pivalate-treated rats and
controls (Table 1
).However, acetylcarnitine and BB were lower than in controls
(P < 0.05). A C5 acylcarnitine peak was detected in
the liver of pivalate-treated rats (Fig. 3A
).This peak represented mainly pivaloylcarnitine, because the C5
acylcarnitine peak was very small in controls. In the heart and
skeletal muscle of pivalate-treated rats free CN and
acetylcarnitine concentrations were lower, and BB was significantly
higher than in the controls. A large pivaloylcarnitine peak was
detected in the heart but not the skeletal muscle of
pivalate-treated rats (Fig. 3B
and 3C)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, our previous study revealed the following
discrepancy: administration of pivalate reduced the plasma CN
concentration to 10–20% of controls, whereas it reduced the tissue CN
levels to only 70–80% (Nakamjima et al. 1996
). The
mechanisms that maintain free CN level in liver are as follows: (i) the
import from plasma into the hepatocytes and (ii) biosynthesis of CN
from BB.
We investigated the active transport system of CN in isolated rat
hepatocytes. Christiansen and Bremer (1976)
reported
that both CN and BB are actively transported, and the carrier has a
high affinity for BB (Km = 0.5 mmol/L) and
a lower affinity for CN (Km = 5.6 mmol/L).
In the present experiment, the Km value of
controls was 6.2 mmol/L, which was almost consistent with their report.
The Km for CN in pivalate-treated rats was
2.9 mmol/L. These data suggest that in the secondary CN deficiency
induced by pivalate administration, affinity for CN import was
increased in hepatocytes, and increased CN import into the liver in
maintained physiological levels.
Liver is a major site of CN synthesis in rats. In rats with secondary
CN deficiency, CN synthesis and/or the import of BB, the precursor of
CN, may be accelerated. We measured the concentrations of free CN,
acylcarnitines and BB in three tissues using tandem mass spectrometry,
one of the best methods for analysis of acylcarnitines because of its
high sensitivity and selectivity (Millington et al. 1989
). Pivaloylcarnitine peaks were detected only in the liver
and heart, indicating that pivaloylcarnitine was synthesized in these
organs but not in the skeletal muscle. The depletion of BB in the liver
suggested that BB was consumed to maintain the normal hepatic level of
free CN for ß-oxidation. The synthesis of BB likely was accelerated
in the heart and skeletal muscle of pivalate-treated rats because
BB concentrations were higher than in controls. In the liver, the
concentration of actylcarnitine was much lower in pivalate-treated
rats than in controls. This evidence suggests that both acetyl-CoA
and pivaloyl-CoA are substrates for carnitine acetyltransferase
(CAT) and pivaloyl-CoA competitively inhibits the formation of
acetylcarnitine. Diep et al. (1995)
stated that the
heart and the brown adipose tissue played important roles in
pivaloylcarnitine formation because of the lower activity of CAT in the
liver. Ruff and Brass (1991)
demonstrated that pivalate
was activated to pivaloyl-CoA within 10 min of its introduction to
the hepatocytes, and pivaloyl-CoA accumulation resulted in a
greater than 95% reduction in hepatocyte CoA concentration.
Pivaloyl-CoA is a poor substrate for CAT because of the presence of
the tertiary carbon at the branch point (Melegh et al. 1990
). However, in the current study, marked accumulation of
pivaloyl-CoA was enough to reduce the formation of acetylcarnitine
in the liver.
We suggest that the CN transport system into hepatocytes and CN synthesis are accelerated in the liver of pivalate-treated rats, and these mechanisms maintain the concentration of free CN in the liver.
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FOOTNOTES |
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3 Abbreviations used: BB, -butyrobetaine; CAT,
carnitine acetyltransferase; CN, carnitine; LSD, the least significant
difference test; ND, not detected.
Manuscript received January 22, 1999. Revision accepted May 28, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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