I. E. M. Luyt-Houwen

Riboflavin-responsive complex I deficiency

Three patients from a large consanguineous family, and one unrelated patient had exercise intolerance since early childhood and improved by supplementation with a high dosage of riboflavin. This was confirmed by higher endurance power in exercise testing. Riboflavin had been given because complex I, which contains riboflavin in FMN, one of its prosthetic groups, had a very low activity in muscle. Histochemistry showed an increase of subsarcolemmal mitochondria. The low complex I activity contrasted with an increase of the activities of succinate dehydrogenase, succinate-cytochrome c oxidoreductase and cytochrome c oxidase. Isolated mitochondria from these muscle specimens proved deficient in oxidizing pyruvate plus malate and other NAD(+)-linked substrates, but oxidized succinate and ascorbate at equal or higher levels than controls. Two years later a second biopsy was taken in one of the patients, and the activity of complex I had increased from 16% to 47% of the average activity in controls. In the four biopsies, cytochrome c oxidase activity correlated negatively with age. We suspect that this is due to reactive oxygen species generated by the proliferating mitochondria and peroxidizing unsaturated fatty acids of cardiolipin. Three of the four patients had low blood carnitine, and all were found to have hypocarnitinemic family members.

Defects in oxidative phosphorylation. Biochemical investigations in skeletal muscle and expression of the lesion in other cells

Mitochondria are very vulnerable to genetic and environmental damage. If a patient is suspected of having a mitochondrial disease, elevated blood lactate, lowered blood free carnitine, abnormal urinary organic acids and carnitine esters and tissue histopathology may help with the diagnosis. For biochemical assessment of the defect, muscle is the tissue of choice even when involvement of other organs like heart or brain is more prominent.We have studied isolated muscle mitochondria and homogenates from muscle biopsies in 250 patients, and have detected in more than one third mitochondrial defects in oxidative phosphorylation, dehydrogenases, non-redox enzymes catalyzing synthesis of fuel molecules and in the carnitine system. Several patients showed more than one defect.We have selected eight patients to illustrate how a relatively simple series of investigations in both isolated mitochondria and homogenate can be used for the identification of defects in oxidative phosphorylation in a small amount of muscle (200 mg or more). Identification of the defect(s) is important since it may provide the basis for rational treatment. A minority of the patients recovered partly or completely, which is unique in treatment of inborn errors of subcellular organelles.An important aspect of mitochondrial dysfunction is the tissue specificity. The defect may be systemic but is often clinically expressed in only one or a few tissues. Rarely, tissue-specific defects can be understood on the basis of tissue-specificity of mitochondrial (iso-)enzymes. Mitochondrial deficiencies of all biotin enzymes and most CoA-linked enzymes are expressed in fibroblasts; most respiratory chain defects are not.When mitochondrial ATP synthesis has been compromised by a mitochondrial defect, secondary lesions may be generated by changes in mitochondrial protein synthesis, activated proteases and phospholipases, increased matrix CoA and resulting carnitine deficiency, decrease in Krebs cycle intermediates and increased free radical formation and lipid peroxidation.

The source of malonyl-CoA in rat heart. The calcium paradox releases acetyl-CoA carboxylase and not propionyl-CoA carboxylase.

The formation of malonyl-CoA in rat heart is catalyzed by cytosolic acetyl-CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl-CoA carboxylase, which is also able to catalyze the carboxylation of acetyl-CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl-CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl-CoA carboxylase was almost fully retained. Acetyl-CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.The formation of malonyl‐CoA in rat heart is catalyzed by cytosolic acetyl‐CoA carboxylase. The existence of this enzyme in heart is difficult to prove by the abundant occurrence of mitochondrial propionyl‐CoA carboxylase, which is also able to catalyze the carboxylation of acetyl‐CoA. We used the calcium paradox as a tool to separate cytosolic components from the remaining heart, and found that acetyl‐CoA carboxylase activity was preferentially released, like lactate dehydrogenase and carnitine, while propionyl‐CoA carboxylase was almost fully retained. Acetyl‐CoA carboxylase activity was determined after activation by citrate ion and Mg2+. The activity decreased to 64% by 48 h of fasting.

Assessment of deficiencies of fatty acyl-CoA dehydrogenases in fibroblasts, muscle and liver

Deficiency of medium-chain acyl-CoA dehydrogenase (MCAD; McKusick 201450) and long-chain acyl-CoA dehydrogenase (LCAD; McKusick 201460) cause systemic diseases and can be assessed in (cultured) cells and tissues. Short-chain acyl-CoA (SCAD) deficiency (McKusick 201470) is not always expressed in fibroblasts (Turnbull et al 1990). When it is systemic, its assessment is complicated by overlapping specificity of SCAD and MCAD (Coates et al 1988)

Familial AMP deaminase deficiency with skeletal muscle type I atrophy and fatal cardiomyopathy

Muscular AMP deaminase deficiency was found in two sibs suffering from a skeletal myopathy, characterized by type I fibre atrophy and a dilated cardiomyopathy. The family history suggests an autosomal dominant inheritance of this disorder.

Vitamin-responsive pyruvate dehydrogenase deficiency in a young girl with external ophthalmoplegia, myopathy and lactic acidosis

An 8-year-old girl had external ophthalmoplegia, bilateral ptosis, facial muscle weakness, perceptive hearing loss, and pain behind the sternum. The CT-scan of brain showed a small subinsular infarct. The girl was small for age. Serum lactate (4.2mmol/L) and pyruvate (0.092mmol/L) and CSF lactate (4.1 mmol/L) and pyruvate (0.151 mmol/L) were increased. Serum creatine kinase was slightly increased to 151 U/L, while CSF protein was not increased. There was no deficiency of folate. A biopsy of m. quadriceps showed no ragged red fibres. The distribution of glycogen and fat droplets was normal. Also the fibre type distribution was normal. Mitochondrial DNA, isolated from muscle by Agsteribbe and Ruiters (Scholte et al 1990) had the normal size and was undeleted.

The role of the carnitine system in myocardial fatty acid oxidation: carnitine deficiency, failing mitochondria and cardiomyopathy.

The carnitine system functions in the transport of activated acyl groups over the mitochondrial inner membrane, and is needed for oxidation of long-chain fatty acids by all mitochondria. The rate of cardiac fatty acid oxidation is determined by availability of fatty acids, oxygen and the activity of carnitine palmitoyltransferase I, which is regulated by a variety of factors. It is inhibited by malonyl-CoA, which in rat heart was found to be synthesized by acetyl-CoA carboxylase. It is also inhibited by long-chain acylcarnitine. Linoleoylcarnitine was found to be a better inhibitor than palmitoylcarnitine. The concentration of carnitine in human heart, muscle and other tissues is much higher than is needed for the optimal beta-oxidation rate. In contrast to controls, we found in several myopathic patients that extra carnitine (from 1/2 to 5 mM) caused a considerable increase in beta-oxidation rate of isolated muscle mitochondria. In some of these patients we detected medium-chain acyl-CoA dehydrogenase deficiency. Patients with primary carnitine deficiency caused by a renal carnitine leak often show cardiomyopathy, which completely disappears under carnitine therapy. Cardiomyopathy may also be the cause of secondary carnitine deficiency resulting from a mitochondrial defect in acyl-CoA metabolism, or by the mitochondrial defect itself, which may be induced by drugs or viral attack, or be the result of a genetic error. In cardiomyopathic patients with a (subclinical) myopathy, study of isolated mitochondria and homogenate from skeletal muscle may reveal a mitochondrial dysfunction, which, in some patients, is treatable by dietary measures and supplementation with vitamins, CoQ and/or carnitine. When the cause of cardiomyopathy is not known, determination of plasma carnitine and carnitine supplementation of hypocarnitinemic patients is of great therapeutic value.

Glutaric Aciduria Type II: Multiple Defects in Isolated Muscle Mitochondria and Deficient β-Oxidation in Fibroblasts

A further patient with glutaric aciduria type II (GAII, McKusick) is described. He seems to be the first reported patient with an intermediate form of this disease. A deficiency was found in the carnitine stimulated mitochondrial β-oxidation of isolated muscle mitochondria and fibroblasts. He responded to chronic treatment with carnitine and riboflavin.

Neonatal cardiomyopathy and lactic acidosis responsive to thiamine

SummaryA congestive cardiomyopathy was diagnosed in a girl at the age of 4 weeks. In the weeks following she developed general muscle hypotonia and plasma lactate increased to 8.5 mmol/L. Biochemical investigations of a muscle biopsy at the age of 3 months showed a deficiency in the oxidation of all substrates tested: pyruvate plus malate, 2-ketoglutarate and palmitate plus malate. After freezing and thawing of the homogenate and the addition of essential cofactors, the oxidation of the ketoacids normalized. The oxidation defect in the untreated homogenate can be explained by a deficiency in one of the cofactors (such as thiamine pyrophosphate, NAD+ or CoASH), or by a defect in the oxidative phosphorylation. Treatment with thiamine and carnitine resulted in a decrease in blood lactate to normal levels and a dramatic clinical improvement. Suspension of thiamine caused deterioration of her clinical condition and lactic acidaemia. The thiamine therapy was then continued. The girl is now 6 years old and in perfect health.