Introduction

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It is well known that L-carnitine plays an important role in the transport of long-chain fatty acids across the mitochondrial inner membrane for -oxidation and energy metabolism (Bremer, 1962; Fritz and Yue, 1964). Primary chronic L-carnitine deficiency may cause encephalopathy through hypoketonemia and hyperammonemia, and cardiomyopathy or hepatic encephalopathy in combination with skeletal myopathy (Breningstall, 1990; Scholte et al., 1990). Furthermore, L-carnitine and acetylcarnitine may have a role in the clinical treatment of acute myocardial infarction (Iliceto et al., 1995) and Alzheimer's disease (Parnetti, 1995).

In 1988, we found that homozygous mutant mice, named juvenile visceral steatosis (JVS) mice, have systemic L-carnitine deficiency and develop fatty liver, hyperammonemia, and hypoglycemia (Koizumi et al., 1988). There have been some in vitro studies on the mechanism of L-carnitine deficiency in JVS mice. Horiuchi et al. (1994) examined kidney slice preparations and concluded that the primary deficiency of JVS mice is most probably related to a reduction in reabsorption of L-carnitine. Kuwajima et al. (1996) reported that at the endogenous L-carnitine concentration (50 µM), the L-carnitine transport activity of fibroblasts obtained from the heart of JVS mice was only 18% of that of normal mice. Our kinetic analysis using embryonic fibroblasts derived from normal and JVS mice suggested that JVS mice lack the high-affinity carnitine transporter, which has Na⁺ and temperature dependence (Hashimoto et al., 1998). These in vitro results suggest that the JVS mouse would be a useful animal model of primary L-carnitine transporter deficiency.

Therefore, the next step should be to elucidate the precise mechanism of the systemic L-carnitine deficiency by means of kinetic studies of the tissue distribution and elimination of L-carnitine in the whole animal. Such studies should also help to confirm the relationship between the development of disease and the effect of L-carnitine and to establish the role of transcellular transport in determining the characteristic tissue distribution of L-carnitine in JVS mice. In the present study, we investigated the disposition kinetics of L-carnitine after i.v. and p.o. administration in normal and JVS mice and confirmed the existence of an L-carnitine transporter in vivo.

	Experimental Procedures

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Materials. L-[methyl-³H]Carnitine hydrochloride (L-[³H]carnitine, 79 Ci/mmol, radiochemical purity, 99.6%) was purchased from Amersham International Ltd. (Buckinghamshire, UK). Acetyl-L-[methyl-³H]carnitine hydrochloride ([acetyl-³H]carnitine, 65 Ci/mmol, radiochemical purity, 97.5%) was purchased from Moravek Biochemicals Inc. (Brea, CA). All other chemicals were of reagent grade and were used without further purification. Endogenous unchanged L-carnitine was determined by using a commercial kit (Free Carnitine Kit; Kainos Co., Tokyo, Japan).

Animal Experiments. JVS mice were originally found among mice of the C3H.OH strain in our laboratory (Koizumi et al., 1988). The autosomal recessive mutant gene, jvs, was then backcrossed into C57BL/6 (CLEA, Tokyo, Japan), and this strain, C57BL/6-jvs, was used in this study.

Assay for Total Radioactive Carnitine (L-[³H]Carnitine, [acetyl-³H]Carnitine, and Other [acyl-³H]Carnitines). Plasma (20 µl) and urine samples (200 µl) were mixed with 8 ml of scintillation cocktail (ACS II; Amersham Corp., Arlington Heights, IL) and maintained at room temperature for 15 h. Tissue samples (0.05-0.2 g) were dissolved in 1 ml of Soluene-350 (Packard Co., Canberra, Australia) by incubation at 50°C for 15 h. The dissolved samples were mixed with 8 ml of scintillation cocktail, neutralized with 1 N HCl, and then left at room temperature for 15 h. The radioactivity was counted in a liquid scintillation counter (LSC-5100; Aloka, Japan).

Separation of L-[³H]Carnitine and [acyl-³H]Carnitines. According to the method of Gudjonsson et al. (1985), thin-layer chromatography (TLC) was used to separate unchanged L-[³H]carnitine and [acyl-³H]carnitines in plasma, urine, and various tissues. An equal volume of 10% trichloroacetic acid was immediately added to the plasma, urine, and tissue samples. Tissues were immediately homogenized with a cutting homogenizer and then sonicated. The samples were centrifuged at 12,000 rpm for 5 min, and the resultant supernatant was spotted onto silica gel Kieselgel 60F₂₅₄ plates (Merck); ascending chromatography was performed using the solvent methanol/chloroform/water/25% NH₄OH/concentrated formic acid in the ratio of 55:50:10:7.5:2.5 (v/v). L-[³H]Carnitine and [acetyl-³H]carnitine standards were also spotted onto 0.25-cm sections of each lane of the TLC plate. After development, were added to 8 ml of scintillation cocktail, the mixture was vortexed well, and the radioactivity was counted. The peaks of L-[³H]carnitine and [acetyl-³H]carnitine for samples of plasma, urine, and various tissues were determined from the authentic R_f values. The individual extraction efficiency for L-[³H]carnitine and [acetyl-³H]carnitine was approximately similar over 95%.

Data Analysis. The steady-state distribution volume and the total body clearance were estimated according to model-independent moment analysis as described by Yamaoka et al. (1978). The data were analyzed using Student's t test to compare the unpaired mean values of two sets of data. The number of determinations (n) is noted in each table and figure. A value of p  .05 was taken to indicate a significant difference between sets of data.

	Results

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Plasma Concentration of Endogenous L-Carnitine. The concentrations of endogenous L-carnitine in plasma of normal and JVS mice before fasting were 4.49 ± 0.32 and 1.33 ± 0.66 µg/ml (mean ± S.E., n = 9), respectively. The value for JVS mice was significantly lower than that for normal mice (p < .001). In normal and JVS mice after overnight starvation, the plasma concentrations were 2.75 ± 0.27 and 2.63 ± 0.90 µg/ml (n = 5), respectively. The plasma concentration in normal mice was significantly decreased by starvation, whereas that of JVS mice tended to be increased.

Plasma Concentration-Time Course of Total Radioactive Carnitine. The plasma concentration-time courses of total radioactive carnitine after an i.v. or a p.o. administration of 250 ng/kg of L-[³H]carnitine in normal and JVS mice are shown in Fig. 1. After i.v. administration, the behavior of total radioactive carnitine in plasma was biphasic, but there was a marked difference between the two types of mice. At the distribution phase, the concentration in JVS mice was significantly higher than that in normal mice. The linear terminal elimination of JVS mice was remarkably faster than that of normal mice. On the other hand, although the plasma concentration of total radioactive carnitine gradually increased after p.o. administration, after 1 h, it was significantly lower in JVS mice than in normal mice.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Plasma concentration-time courses of total radioactive carnitine after a single i.v. or p.o. administration of L-[3H]carnitine (250 ng/kg) in normal and JVS mice. Each point represents mean ± S.E. of five mice; i.v. () and p.o. () administration in normal mice; i.v. () and p.o. () administration in JVS mice. *, **Significantly different from normal mice at p < .05 and p < .01, respectively.

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Tissue Concentration of Total Radioactive Carnitine. The concentrations of total radioactive carnitine in various tissues at 4 h after a single i.v. administration of L-[³H]carnitine are shown in Fig. 2. The tissue concentrations of total radioactive carnitine in normal mice were spleen > kidney > liver > heart > lung > gut > muscle > brain. The concentrations in the tissues, except for the brain, of JVS mice were significantly lower than those of normal mice. The concentration in the brain was very low, and there was no significant difference between the two types of mice.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Tissue concentration of total radioactive carnitine at 4 h after an injection of L-[3H]carnitine (250 ng/kg i.v.) in mice. Each bar represents mean ± S.E. of five mice. , Normal mice; , JVS mice. *, **, ***Significantly different from normal mice at p < .05, p < .01, and p < .001, respectively.

Metabolism of L-[³H]Carnitine. Figure 3A shows the TLC of authentic unchanged L-[³H]carnitine and [acetyl- ³H]carnitine. The peaks of the two drugs were well separated, and the R_f values for unchanged and [acetyl-³H]carnitine were 0.32 and 0.5, respectively. Figure 3, B and C, shows the chromatograms of plasma samples at 4 h after the i.v. injection of 250 ng/kg L-[³H]carnitine in normal and JVS mice, respectively. The first and second peaks were identified as unchanged carnitine and acetylcarnitine, respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   TLC of L-[3H]carnitine and [acetyl-3H]carnitine. Chromatogram A was obtained with blank plasma containing added L-[3H]carnitine and [acetyl-3H]carnitine. Chromatograms B and C were obtained from plasma samples at 4 h after an injection of L-[3H]carnitine (250 ng/kg, i.v.) in normal and JVS mice, respectively. 1, L-[3H]Carnitine; 2, [acetyl-3H]carnitine.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Time courses of unchanged L-[3H]carnitine, [acetyl-3H]carnitine, and other [acyl-3H]carnitines in plasma as percentages of total radioactive carnitine after an injection of L-[3H]carnitine (250 ng/kg i.v.) in normal and JVS mice. Each point represents mean (%) of four mice. , Unchanged carnitine; , acetylcarnitine; , other acylcarnitines.

Plasma Concentration-Time Course of Unchanged L-[³H]Carnitine. Figure 5 shows the plasma concentration-time courses of unchanged L-[³H]carnitine after an i.v. dose of 250 ng/kg L-[³H]carnitine in normal and JVS mice. The ratios of unchanged L-[³H]carnitine to the total radioactive carnitine gradually decreased until 60 min in both cases, and thereafter the ratios were fairly constant at 0.27 for normal mice and 0.46 for JVS mice.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Plasma concentration-time courses of unchanged L-[3H]carnitine after an injection of L-[3H]carnitine (250 ng/kg i.v.) in normal () and JVS () mice. Each point represents mean ± S.E. of four mice. *, **Significantly different from normal mice at p < .05 and p < .01, respectively.

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Tissue Distribution of Unchanged L-[³H]Carnitine. Table 3 shows the percentage of unchanged L-[³H]carnitine concentration with respect to total radioactive carnitine for each tissue at 4 h after the i.v. dose of 250 ng/kg in normal and JVS mice. Unchanged carnitine in every tissue except for muscle accounted for more than 80% of total radioactivity, whereas in muscle, exogenously administered carnitine was present to the extent of about 50 to 60% as the unchanged form and about 40% as acetylcarnitine in both types of mice.

Urinary Excretion of L-[³H]Carnitine. Figure 6 shows the urinary excretion of total radioactive carnitine for 4 h after a single i.v. dose of 250 ng/kg L-[³H]carnitine in normal and JVS mice. The cumulative amounts of total radioactive carnitine for 4 h in normal and JVS mice were 0.22 and 2.25 ng, respectively. The values of urinary recovery of total radioactive carnitine were about 4% and 45% of the administered L-[³H]carnitine in normal and JVS mice, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Accumulation of total radioactive carnitine in urine for 4 h after an injection of L-[3H]carnitine (250 ng/kg i.v.) in mice. Each point represents mean ± S.E. of three mice. , Normal mice; , JVS mice

View larger version (13K): [in this window] [in a new window]   Fig. 7.   TLCs of L-[3H]carnitine and [acetyl-3H]carnitine. Chromatograms were obtained with urine samples accumulated for 4 h after an injection of L-[3H]carnitine (250 ng/kg i.v.) in normal (A) and JVS (B) mice. 1, L-[3H]Carnitine; 2, [acetyl-3H]carnitine.

	Discussion

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The weanlings of JVS mice cannot live unless L-carnitine is supplied to maintain a plasma level 3 to 5 µg/ml L-carnitine. However, the chronic fatty liver is not improved by this L-carnitine supplement. The JVS mice seem to be congenitally carnitine deficient.

We demonstrated that the bioavailability of L-[³H]carnitine in JVS mice was about half of that in normal mice (Fig. 1, Table 1). Gudjonsson et al. (1985) examined the small intestinal absorption of carnitine using single-pass perfusion techniques in rats and found a partially saturable absorption process. McCloud et al. (1996) reported that the uptake of L-carnitine in Caco-2 cells involves a carrier-mediated system that is dependent on temperature, Na⁺, and energy but independent of pH. Therefore, JVS mice seem to lack the carrier-mediated membrane transport system for L-carnitine in the gastrointestinal tract. Moreover, we observed that the distribution of total radioactive carnitine into several tissues, except for the brain, after a single i.v. injection of L-[³H]carnitine (250 ng/kg) was reduced in JVS mice (Fig. 2 and Table 4). Because the concentrations of radiolabeled L-carnitine in the body after the injection of L-[³H]carnitine were estimated to be 3 log orders of magnitude lower than those of endogenous carnitine, the disposition kinetic data obtained in this study should reflect the behavior of endogenous carnitine.

L-Carnitine is known to be converted to acylcarnitines in tissues (Rebouche and Paulson, 1986; Hokland, 1988). After an i.v. injection of L-[³H]carnitine, the fraction of unchanged L-[³H]carnitine in plasma from normal mice was decreased rapidly, concomitantly with the appearance of [acetyl-³H]carnitine. The plasma concentration ratio of L-[³H]carnitine and [acetyl-³H]carnitine was about 1:2 at 1 h. The plasma concentration of unchanged L-[³H]carnitine in JVS mice decreased more slowly than that in normal mice, and the concentration ratio of unchanged L-[³H]carnitine and [acetyl-³H]carnitine was about 1:1 at 2 h after administration. Exogenously administered L-carnitine was present to the extent of more than 80% as the unchanged form in most tissues, but the muscle contained about 50% unchanged form and 40% acetylated form (table 3). In our preliminary in vitro experiments, in which homogenates of brain, heart, liver, and muscle, and plasma were incubated with L-[³H]carnitine, [acetyl-³H]carnitine was clearly produced in the muscle, but its formation was negligible in plasma and other tissues (data not shown). L-Carnitine may be metabolized to acetylcarnitine mainly in the muscle. In both types of mice, acylcarnitines other than acetylcarnitine were negligible (below 10% of total radioactivity) (Figs. 3 and 4). Consequently, after the i.v. injection of L-[³H]carnitine, the moment analysis of the plasma concentration of unchanged L-[³H]carnitine exhibited lower Vd_ss, higher CL_tot, and higher k_e values in JVS mice than in normal mice (Fig. 5 and Table 2). The lower Vd_ss value of unchanged L-[³H]carnitine in JVS mice suggests that the tissue distribution is reduced in JVS mice. Actually, the K_{p, app} values of unchanged L-[³H]carnitine for all major tissues, except for the brain, in JVS mice were significantly lower than those in normal mice (Table 4). The Vd_ss values in normal and JVS mice were almost equal to the distribution volumes of 5.7 and 1.6 liters/kg, respectively, which were calculated using the K_{p, app} values. These data indicate that the tissue distribution of L-carnitine is very low in JVS mice compared with normal mice.

Based on studies using cell culture systems (Bohmer et al., 1977; Martinuzzi et al., 1991; Stieger et al., 1995) and heterologous gene expression technology (Berardi et al., 1995), it has been suggested that transcellular transport of L-carnitine involves a carrier-mediated transport mechanism. Our findings on the specific characteristics of tissue distribution of carnitine in normal and JVS mice support the idea that a carnitine transporter contributes to the tissue distribution of carnitine. This in vivo disposition kinetic study indicated that JVS mice lack or have a decreased transporter function in the whole body. However, there was only a slight difference in the distribution of unchanged L-[³H]carnitine into the brain between the two types of mice, and the K_{p, app} value was about 1. Mroczkowska et al. (1996) reported that the uptake of carnitine by brain capillary endothelial cells is not related to any Na⁺-dependent transport process. This seems to support our in vivo results, suggesting that no carnitine transporter is present at the blood-brain barrier.

The higher k_e value of unchanged L-[³H]carnitine in JVS mice than in normal mice was reflected by the larger CL_tot and lower Vd_ss values in JVS mice (Fig. 5 and Table 2). The urinary excretion of total radioactive carnitine in JVS mice was about 10-fold higher than that in normal mice (Fig. 6). In urine from JVS mice, unchanged L-[³H]carnitine and [acetyl-³H]carnitine were detected as major peaks on TLC. In urine from normal mice, radioactivity was detected at high R_f values, presumably due to long-chain [acyl-³H]carnitines but not unchanged carnitine or acetylcarnitine (Fig. 7). This indicates that normal mice reabsorbed both unchanged L-carnitine and acetylcarnitine, whereas JVS mice did not, suggesting a defect of the carnitine transporter in the kidney, as reported by Horiuchi et al. (1994).

Various values of the Michaelis constant (K_m) of the high-affinity site on the carnitine transporter have been reported in isolated, perfused rat heart (24 µM) (Vary and Neely, 1982), human heart cultured cells (4.8 µM) (Bohmer et al., 1977), human muscle cultured cells (0.5-10 µM) (Martinuzzi et al., 1991), and rat kidney brush-border membrane vesicles (17.4 µM) (Stieger et al., 1995). In this study, the plasma concentration of endogenous L-carnitine after overnight starvation was about 17 and 16 µM in normal mice and JVS mice, respectively. These values are similar to the reported K_m values of carnitine, implying that the operation of the carnitine transport system could be properly evaluated in our in vivo experiments.

There is an increasing number of reports dealing with primary carnitine deficiency in humans. It has been suggested that symptoms can be a consequence of acceleration of urinary excretion, decrease in tissue uptake, and abnormality of mitochondrial function (Stanley et al., 1990; Bennett et al., 1996; Charmers et al., 1997; Rinaldo et al., 1997). Our present study on the disposition kinetics of carnitine in normal and JVS mice supports the idea that a lack or functional deficiency of the transcellular carnitine transporter system exists in JVS mice, as suggested previously on the basis of in vitro kinetic studies using embryonic fibroblasts derived from normal and JVS mice.

In conclusion, our results suggest that the JVS mice will be a useful experimental model in which to investigate the relationship between carnitine deficiency diseases and carnitine transport characteristics.