Characterization of carnitine and fatty acid metabolism in the long-chain acyl-CoA dehydrogenase-deficient mouse

Abstract

In the present paper, we describe a novel method which enables the analysis of tissue acylcarnitines and carnitine biosynthesis intermediates in the same sample. This method was used to investigate the carnitine and fatty acid metabolism in wild-type and LCAD-/- (long-chain acyl-CoA dehydrogenase-deficient) mice. In agreement with previous results in plasma and bile, we found accumulation of the characteristic C14:1-acylcarnitine in all investigated tissues from LCAD-/- mice. Surprisingly, quantitatively relevant levels of 3-hydroxyacylcarnitines were found to be present in heart, muscle and brain in wild-type mice, suggesting that, in these tissues, long-chain 3-hydroxyacyl-CoA dehydrogenase is rate-limiting for mitochondrial beta-oxidation. The 3-hydroxyacylcarnitines were absent in LCAD-/- tissues, indicating that, in this situation, the beta-oxidation flux is limited by the LCAD deficiency. A profound deficiency of acetylcarnitine was observed in LCAD-/- hearts, which most likely corresponds with low cardiac levels of acetyl-CoA. Since there was no carnitine deficiency and only a marginal elevation of potentially cardiotoxic acylcarnitines, we conclude from these data that the cardiomyopathy in the LCAD-/- mouse is caused primarily by a severe energy deficiency in the heart, stressing the important role of LCAD in cardiac fatty acid metabolism in the mouse.

Figure 1. Tissue and plasma levels of free carnitine and acetylcarnitine in wild-type and LCAD^−/− mice

Grey bars and white bars represent wild-type and LCAD^−/− mice respectively. Values are means±S.D. for six mice, where each tissue was analysed in duplicate. Only one plasma analysis per mouse was performed due to the small amount of sample available. *P<0.05; #P<0.01.

Figure 2. Tissue and plasma acylcarnitine levels in wild-type and LCAD^−/− mice

Grey bars and white bars represent wild-type and LCAD^−/− mice respectively. Values are means±S.D. for six mice, where each tissue was analysed in duplicate. Only one plasma analysis per mouse was performed due to the small amount of sample available. *P<0.05; #P<0.01.

Figure 3. Hydroxyacylcarnitine levels in heart, muscle and brain in wild-type and LCAD^−/− mice

(A) Grey bars and white bars represent wild-type and LCAD^−/− mice respectively. Values are means±S.D. for six mice, where each tissue was analysed in duplicate. All the differences between wild-type and LCAD^−/− mice were determined to be significant (P<0.01). (B) Parent scan of m/z 85, showing the acylcarnitine spectrum of wild-type and LCAD^−/− mice in heart. Note the presence of high levels of hydroxylacylcarnitines in the wild-type heart and their dramatic reduction in LCAD^−/− mice. The peak at m/z 445 corresponds to the internal standard (I.S.), [²H₃]C₁₆-acylcarnitine.

Figure 4. Carnitine biosynthesis intermediates in plasma and tissues of wild-type and LCAD^−/− mice

Grey bars and white bars represent wild-type and LCAD^−/− mice respectively. Values are means±S.D. for six mice, where each tissue was analysed in duplicate. Only one plasma analysis per mouse was performed due to the small amount of sample available. *P<0.05; #P<0.01.

Figure 5. Western blot analysis of VLCAD in heart, muscle, kidney and liver of three wild-type and three LCAD^−/− mice

Each lane represents 100 μg of tissue homogenate.