Juvenile visceral steatosis (JVS) mice have been reported to have systemic carnitine deficiency, and the carnitine concentration in the liver of JVS mice was markedly lower than that of controls (11.6 +/- 2.6 versus 393.5 +/- 56.4 nmol/g of wet liver). To evaluate the role of carnitine in mitochondrial beta-oxidation in liver, we examined the effects of carnitine on ketogenesis in perfused liver from control and JVS mice. In control mice, ketogenesis was increased by the infusion of 0.3 mM oleate, but not by L-carnitine. In contrast, although ketogenesis in JVS mice was not increased by the infusion of oleate, it was increased 2.5-fold by the addition of 1000 microM L-carnitine. Addition of 50, 100, and 200 microM L-carnitine increased ketogenesis in a dose-dependent manner. The infusion of 0.3 mM octanoate or butyrate increased ketogenesis in a carnitine-independent fashion in both control and JVS mice. These findings suggest that endogenous long chain fatty acids from accumulated triglycerides may be used as substrates in the presence of carnitine in JVS mice. The relationship between ketogenesis and free carnitine concentration was examined in livers from JVS mice. Ketogenesis increased as free carnitine levels increased until concentrations exceeded about 100 nmol/g of wet liver (340 microM). The free carnitine concentration required for half-maximal ketone body production in liver of JVS mice was 45 microM (13 nmol/g of wet liver), which corresponds to a K(m) value of carnitine palmitoyltransferase I. We conclude that carnitine is a rate-limiting factor for beta-oxidation in liver only when the carnitine level in liver is very low.

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Recently, several cases of short-chain acyl-CoA dehydrogenase (SCAD) deficiency (McKusick 201470) in humans have been described. Symptoms of this disorder are variable (Turnbull et al 1984;Amendt et al 1987;Coates et al 1988) and metabolic characteristics are not well understood. A mutant mouse (BALB/cBYJ) with no SCAD activity in liver, which is the first animal model of fatty acid oxidation disorders, has been reported (Wood et al 1989). To investigate the metabolic characteristics in these mice, we performed a series of experiments using liver perfusion techniques and HPLC. MATERIALS AND METHODSMale BALB/cBYJ (J) mice and BALB/cA (A) mice (both from Nihon Clea, Osaka, Japan) received laboratory chow and water ad libitum. The perfusion technique for rat livers has been described elsewhere (Scholz et al 1973). Livers were perfused with Krebs-Henseleit bicarbonate buffer (pH 7.4, 37~ saturated with an oxygen-carbon dioxide mixture (95 : 5) in a non-recirculating system modified for mouse liver. Lactate was used as a substrate for glucose production, and butyrate, octanoate and oleate as substrates for ketogenesis. Samples of effluent perfusate were collected and analysed for glucose, 3-hydroxybutyrate and acetoacetate by standard enzymatic techniques.For acyl-CoA analysis, livers were freeze-clamped and stored at -80~ until analysed. A piece of frozen liver (0.3-0.5 g) was immediately weighed and then extracted with 1.5 ml of ice-cold 1 mol/L trichloroacetic acid. Valeryl-CoA (30 nmol) was added as internal standard. Acid-insoluble residue was removed by centrifugation and the supernatant was washed with 1 ml of diethyl ether five times to remove trichloroacetic acid. The aqueous sample was passed through a filter (pore size 0.5 #m) and an aliquot (10/~1) was injected into an HPLC system with a Sim-Pack CLC-ODS column (4.6mm x 250ram; Shimazu, Kyoto). Mobile phases were 0.1 mol/L KH2PO 4, pH 5 (buffer A) and 0.1 mol/L KHEPO 4 containing 40% acetonitrile, pH 5 (buffer B). A dual pump system was used and the composition was changed from 353

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