Maria S.K. Stoll

Mitochondrial long chain fatty acid β-oxidation in man and mouse

Several mouse models for mitochondrial fatty acid beta-oxidation (FAO) defects have been developed. So far, these models have contributed little to our current understanding of the pathophysiology. The objective of this study was to explore differences between murine and human FAO. Using a combination of analytical, biochemical and molecular methods, we compared fibroblasts of long chain acyl-CoA dehydrogenase knockout (LCAD(-/-)), very long chain acyl-CoA dehydrogenase knockout (VLCAD(-/-)) and wild type mice with fibroblasts of VLCAD-deficient patients and human controls. We show that in mice, LCAD and VLCAD have overlapping and distinct roles in FAO. The absence of VLCAD is apparently fully compensated, whereas LCAD deficiency is not. LCAD plays an essential role in the oxidation of unsaturated fatty acids such as oleic acid, but seems redundant in the oxidation of saturated fatty acids. In strong contrast, LCAD is neither detectable at the mRNA level nor at the protein level in men, making VLCAD indispensable in FAO. Our findings open new avenues to employ the existing mouse models to study the pathophysiology of human FAO defects.

Quantification of carnitine and acylcarnitines in biological matrices by HPLC electrospray ionization-mass spectrometry.

BACKGROUND Analysis of carnitine and acylcarnitines by tandem mass spectrometry (MS/MS) has limitations. First, preparation of butyl esters partially hydrolyzes acylcarnitines. Second, isobaric nonacylcarnitine compounds yield false-positive results in acylcarnitine tests. Third, acylcarnitine constitutional isomers cannot be distinguished. METHODS Carnitine and acylcarnitines were isolated by ion-exchange solid-phase extraction, derivatized with pentafluorophenacyl trifluoromethanesulfonate, separated by HPLC, and detected with an ion trap mass spectrometer. Carnitine was quantified with d(3)-carnitine as the internal standard. Acylcarnitines were quantified with 42 synthesized calibrators. The internal standards used were d(6)-acetyl-, d(3)-propionyl-, undecanoyl-, undecanedioyl-, and heptadecanoylcarnitine. RESULTS Example recoveries [mean (SD)] were 69.4% (3.9%) for total carnitine, 83.1% (5.9%) for free carnitine, 102.2% (9.8%) for acetylcarnitine, and 107.2% (8.9%) for palmitoylcarnitine. Example imprecision results [mean (SD)] within runs (n = 6) and between runs (n = 18) were, respectively: total carnitine, 58.0 (0.9) and 57.4 (1.7) micromol/L; free carnitine, 44.6 (1.5) and 44.3 (1.2) micromol/L; acetylcarnitine, 7.74 (0.51) and 7.85 (0.69) micromol/L; and palmitoylcarnitine, 0.12 (0.01) and 0.11 (0.02) micromol/L. Standard-addition slopes and linear regression coefficients were 1.00 and 0.9998, respectively, for total carnitine added to plasma, 0.99 and 0.9997 for free carnitine added to plasma, 1.04 and 0.9972 for octanoylcarnitine added to skeletal muscle, and 1.05 and 0.9913 for palmitoylcarnitine added to skeletal muscle. Reference intervals for plasma, urine, and skeletal muscle are provided. CONCLUSIONS This method for analysis of carnitine and acylcarnitines overcomes the observed limitations of MS/MS methods.

Enhanced acyl-CoA dehydrogenase activity is associated with improved mitochondrial and contractile function in heart failure

AIMS Heart failure is associated with decreased myocardial fatty acid oxidation capacity and has been likened to energy starvation. Increased fatty acid availability results in an induction of genes promoting fatty acid oxidation. The aim of the present study was to investigate possible mechanisms by which high fat feeding improved mitochondrial and contractile function in heart failure. METHODS AND RESULTS Male Wistar rats underwent coronary artery ligation (HF) or sham surgery and were immediately fed either a normal (14% kcal fat) (SHAM, HF) or high-fat diet (60% kcal saturated fat) (SHAM+FAT, HF+FAT) for 8 weeks. Mitochondrial respiration and gene expression and enzyme activities of fatty acid-regulated mitochondrial genes and proteins were assessed. Subsarcolemmal (SSM) and interfibrillar mitochondria were isolated from the left ventricle. State 3 respiration using lipid substrates octanoylcarnitine and palmitoylcarnitine increased in the SSM of HF+FAT compared with SHAM+FAT and HF, respectively (242 +/- 21, 246 +/- 21 vs. 183 +/- 8, 181 +/- 6 and 193 +/- 17, 185 +/- 16 nAO min(-1) mg(-1)). Despite decreased medium-chain acyl-CoA dehydrogenase (MCAD) mRNA in HF and HF+FAT, MCAD protein was not altered, and MCAD activity increased in HF+FAT (HF, 65.1 +/- 2.7 vs. HF+FAT, 81.5 +/- 5.4 nmoles min(-1) mg(-1)). Activities of short- and long-chain acyl-CoA dehydrogenase also were elevated and correlated to increased state 3 respiration. This was associated with an improvement in myocardial contractility as assessed by left ventricular +dP/dt max. CONCLUSION Administration of a high-fat diet increased state 3 respiration and acyl-CoA dehydrogenase activities, but did not normalize mRNA or protein levels of acyl-CoA dehydrogenases in coronary artery ligation-induced heart failure rats.

Validated Method for the Quantification of Free and Total Carnitine, Butyrobetaine, and Acylcarnitines in Biological Samples

A validated quantitative method for the determination of free and total carnitine, butyrobetaine, and acylcarnitines is presented. The versatile method has four components: (1) isolation using strong cation-exchange solid-phase extraction, (2) derivatization with pentafluorophenacyl trifluoromethanesulfonate, (3) sequential ion-exchange/reversed-phase (ultra) high-performance liquid chromatography [(U)HPLC] using a strong cation-exchange trap in series with a fused-core HPLC column, and (4) detection with electrospray ionization multiple reaction monitoring (MRM) mass spectrometry (MS). Standardized carnitine along with 65 synthesized, standardized acylcarnitines (including short-chain, medium-chain, long-chain, dicarboxylic, hydroxylated, and unsaturated acyl moieties) were used to construct multiple-point calibration curves, resulting in accurate and precise quantification. Separation of the 65 acylcarnitines was accomplished in a single chromatogram in as little as 14 min. Validation studies were performed showing a high level of accuracy, precision, and reproducibility. The method provides capabilities unavailable by tandem MS procedures, making it an ideal approach for confirmation of newborn screening results and for clinical and basic research projects, including treatment protocol studies, acylcarnitine biomarker studies, and metabolite studies using plasma, urine, tissue, or other sample matrixes.

Functional redundancy of mitochondrial enoyl-CoA isomerases in the oxidation of unsaturated fatty acids

Mitochondrial enoyl‐CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild‐type (WT) and Eci1‐deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1‐deficient mice displayed normal blood β‐hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil‐rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1‐deficient mice is mild, possibly caused by the presence of a second enoyl‐CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1‐deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12‐fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1‐deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.—van Weeghel, M., te Brinke, H., van Lenthe, H., Kulik, W., Minkler, P. E., Stoll, M. S. K., Sass, J. O., Janssen, U., Stoffel, W., Schwab, O. K., Wanders, R. J. A., Hoppel, C. L., Houten, S. M. Functional redundancy of mitochondrial enoyl‐CoA isomerases in the oxidation of unsaturated fatty acids. FASEB J. 26, 4316–4326 (2012). www.fasebj.org