Hypertrophic cardiomyopathy is a typical manifestation of very long-chain acyl-CoA dehydrogenase deficiency (VLCADD), the most common long-chain β-oxidation defects in humans; however in some patients cardiac function is fully compensated. Cardiomyopathy may also be reversed by supplementation of medium-chain triglycerides (MCT). We here characterize cardiac function of VLCAD-deficient (VLCAD(-/-)) mice over one year. Furthermore, we investigate the long-term effect of a continuous MCT diet on the cardiac phenotype. We assessed cardiac morphology and function in VLCAD(-/-) mice by in vivo MRI. Cardiac energetics were measured by (31)P-MRS and myocardial glucose uptake was quantified by positron-emission-tomography (PET). Metabolic adaptations were identified by the expression of genes regulating glucose and lipid metabolism using real-time-PCR. VLCAD(-/-) mice showed a progressive decrease in heart function over 12 months accompanied by a reduced phosphocreatine-to-ATP-ratio indicative of chronic energy deficiency. Long-term MCT supplementation aggravated the cardiac phenotype into dilated cardiomyopathy with features similar to diabetic heart disease. Cardiac energy production and function in mice with a β-oxidation defect cannot be maintained with age. Compensatory mechanisms are insufficient to preserve the cardiac energy state over time. However, energy deficiency by impaired β-oxidation and long-term MCT induce cardiomyopathy by different mechanisms. Cardiac MRI and MRS may be excellent tools to assess minor changes in cardiac function and energetics in patients with β-oxidation defects for preventive therapy.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the ACADM gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%–60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new ACADM genotypes associated with asymptomatic disease variants.

Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long-term increase in corticosterone levels. Thus, male mice from both sub-strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice.

Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD−/−) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD−/− mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD−/− mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD−/− mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD−/− mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.

Background: Sulfur carrier proteins Rhd_2599, TusA, and DsrE2 occur in many sulfur oxidizing prokaryotes. Results: Rhd_2599, TusA, and possibly DsrE2 are involved in cytoplasmic sulfur trafficking during dissimilatory sulfur oxidation. Conclusion: Sulfur transfer from persulfide intermediates to dissimilatory sulfite reductase involves Rhd_2599, TusA, and possibly DsrE2. Significance: Proteins involved in dissimilatory sulfur oxidation have been identified.

Although the MCT-supplemented diet has been reported to prevent the development of cardiomyopathy and skeletal myopathy in fatty acid oxidation disorders, our data show that long-term MCT supplementation results in a severe clinical phenotype similar to that of nonalcoholic steatohepatitis and the metabolic syndrome.

Medium-chain acyl-coenzyme-A dehydrogenase (MCAD) catalyzes the first step of mitochondrial beta-oxidation for medium-chain acyl-CoAs. Mutations in the ACADM gene cause MCAD deficiency presenting with life-threatening symptoms during catabolism. Since fatty-acid-oxidation disorders are part of newborn screening (NBS), many novel mutations with unknown clinical relevance have been identified in asymptomatic newborns. Eighteen of these mutations were separately cloned into the human ACADM gene, heterologously overexpressed in Escherichia coli and functionally characterized by using different substrates, molecular chaperones, and measured at different temperatures. In addition, they were mapped to the three-dimensional MCAD structure, and cross-link experiments were performed. This study identified variants that only moderately affect the MCAD protein in vitro, such as Y42H, E18K, and R6H, in contrast to the remaining 15 mutants. These three mutants display residual octanoyl-CoA oxidation activities in the range of 22 % to 47 %, are as temperature sensitive as the wild type, and reach 100 % activity with molecular chaperone co-overexpression. Projection into the three-dimensional protein structure gave some indication as to possible reasons for decreased enzyme activities. Additionally, six of the eight novel mutations, functionally characterized for the first time, showed severely reduced residual activities < 5 % despite high expression levels. These studies are of relevance because they classify novel mutants in vitro on the basis of their corresponding functional effects. This basic knowledge should be taken into consideration for individual management after NBS.

Newborn screening identifies patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency with disease-specific acylcarnitine profiles. We here present a patient who died at 16 months during a gastrointestinal infection because of undiagnosed VLCADD. The primary acylcarnitine profile on newborn screening performed at 55 h of life revealed C14-acylcarnitine values and ratios within the 1st percentile VLCAD disease range and C12-acylcarnitine values and ratios within the 10th percentile disease range. The acylcarnitine cumulative percentiles in neonatal dried blood spots analyzed by tandem mass spectrometry have been obtained by participants of the Region 4 Stork collaborative project. A secondary screen was requested by the screening laboratory as a result of the initial screen and was normal on day 8 of life. With the initial acylcarnitines only within the 1st-10th percentile disease range, newborn screening for VLCAD deficiency was in the end considered normal. The most important lesson learned is that acylcarnitine profiles from healthy newborns during catabolism and VLCAD-deficient patients can in certain cases not be distinguished by any means. With a known high incidence of false positive cases for VLCADD on newborn screening, it finally remains unknown, whether forced anabolism in the first days of life may result in normal acylcarnitine profiles in VLCAD-deficient patients resulting in missed cases and false negatives on newborn screening. Our observations are of great significance since they demonstrate the limitations of acylcarnitine analysis as screening tool for VLCAD-deficiency.