Kim Bartlett

Clear correlation of genotype with disease phenotype in very-long-chain acyl-CoA dehydrogenase deficiency.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the initial rate-limiting step in mitochondrial fatty acid beta-oxidation. VLCAD deficiency is clinically heterogenous, with three major phenotypes: a severe childhood form, with early onset, high mortality, and high incidence of cardiomyopathy; a milder childhood form, with later onset, usually with hypoketotic hypoglycemia as the main presenting feature, low mortality, and rare cardiomyopathy; and an adult form, with isolated skeletal muscle involvement, rhabdomyolysis, and myoglobinuria, usually triggered by exercise or fasting. To examine whether these different phenotypes are due to differences in the VLCAD genotype, we investigated 58 different mutations in 55 unrelated patients representing all known clinical phenotypes and correlated the mutation type with the clinical phenotype. Our results show a clear relationship between the nature of the mutation and the severity of disease. Patients with the severe childhood phenotype have mutations that result in no residual enzyme activity, whereas patients with the milder childhood and adult phenotypes have mutations that may result in residual enzyme activity. This clear genotype-phenotype relationship is in sharp contrast to what has been observed in medium-chain acyl-CoA dehydrogenase deficiency, in which no correlation between genotype and phenotype can be established.

A new method for the detection of hydrogen in breath and its application to acquired and inborn sugar malabsorption

An electrochemical device for the measurement of hydrogen in end-expired air is described and compared with an existing gas chromatographic method. The application of both these methods to subjects with acquired and inborn defects of sugar malabsorption is documented. Our simplified method of breath collection and the rapid estimation of hydrogen concentration provides a reliable, convenient and well-tolerated means of detecting sugar malabsorption.

A combined defect of three mitochondrial carboxylases presenting as biotin-responsive 3-methylcrotonyl glycinuria and 3-hydroxyisovaleric aciduria.

A child with a history of episodes of metabolic acidosis was found to excrete 3-hydroxyisovaleric acid and 3-methylcrotonylglycine. These metabolites disappeared following the administration of biotin. The specific activities of propionyl CoA carboxylase, 3-methylcrotonyl CoA carboxylase and pyruvate carboxylase were found to be low in skin fibroblasts cultured in the absence of added biotin. With the addition of biotin, the specific activity of all three carboxylases returned to normal, that of 3-methylcrotonyl CoA carboxylase ahowing the greatest sensitivity to biotin.

Plasma coenzyme Q(10) in children and adolescents undergoing doxorubicin therapy.

The objective of this study was to test the hypothesis that doxorubicin treatment for cancer in childhood and adolescence causes a dose-related decrease in the concentration of plasma coenzyme Q(10). The concentration of plasma coenzyme Q(10) was measured before and after administration of doxorubicin in six patients, and before and after chemotherapy in six patients undergoing treatments that did not include doxorubicin. There was a significant increase in the concentration of plasma coenzyme Q(10) in post-treatment samples compared to pre-treatment samples in patients treated with doxorubicin (P=0.008; n=32), whereas there were no significant changes in plasma coenzyme Q(10) concentrations in patients treated with chemotherapy that did not include doxorubicin. (P=0.770; n=30). We hypothesise that the increase in plasma coenzyme Q(10) that was observed in patients undergoing doxorubicin treatment is due to release of coenzyme Q(10) from apoptotic or necrotic cardiac tissue. We conclude that the cardiotoxicity due to doxorubicin therapy does not involve acute myocardial depletion of coenzyme Q(10).

Control of mitochondrial beta-oxidation: sensitivity of the trifunctional protein to [NAD(+)]/[NADH] and [acetyl-CoA]/[CoA]

Isolated human mitochondrial trifunctional protein was incubated with 2-hexadecenoyl-CoA, CoA and NAD+ and the resultant CoA esters measured. Steady state with respect to the concentrations of the intermediates 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA and the rate of formation of the product tetradecanoyl-CoA was reached within 4 min. Flux was greatly enhanced by the addition of Tween 20 (0.2% v/v) which stimulated 3-ketoacyl-CoA thiolase activity by over 7-fold. When 3-ketoacyl-CoA thiolase was not stimulated, 3-hydroxyhexadecanoyl-CoA was the prominent CoA ester accumulated, presumably due to inhibition of 3-hydroxyacyl-CoA dehydrogenase activity by accumulated 3-ketoacyl-CoA, analogous to the inhibition of short-chain 3-hydroxyacyl-CoA dehydrogenase by 3-ketoacyl-CoA. When [NAD+]/[NADH] was varied at a fixed total [NAD++NADH], the overall flux was only inhibited by [NAD+]/[NADH] less than 1. In contrast, when [acetyl-CoA]/[CoA] was varied at a fixed total [CoA], much greater sensitivity was observed.

Intermediates of myocardial mitochondrial β-oxidation: possible channelling of NADH and of CoA esters

Adult rat heart mitochondria were isolated and incubated with [U-14C]hexadecanoyl-CoA or unlabelled hexadecanoyl-CoA. The accumulating CoA and carnitine esters and [NAD+]/[NADH] ratio were measured by HPLC or tandem mass spectrometry. Despite minimal changes in the intramitochondrial [NAD+]/[NADH] ratio, 2, 3-unsaturated and 3-hydroxyacyl esters were observed as well as saturated acyl-CoA and acylcarnitine esters. In addition to acetylcarnitine, significant amounts of butyryl-, hexanoyl-, octanoyl- and decanoylcarnitines were detected and measured. Rat myocardial beta-oxidation is subject to control at the level of 3-hydroxyacyl-CoA dehydrogenase but this control is not due to a simple lack of oxidised NAD. We hypothesise a pool of NAD in contact between the trifunctional protein of beta-oxidation and complex I of the respiratory chain, the turnover of which is responsible for some of the control of beta-oxidation flux. In addition, short- and medium-chain acylcarnitine esters were detected whereas only small amounts of long-chain acylcarnitines were present. This may imply the presence of a mitochondrial carnitine octanoyl transferase or may reflect channelling of long-chain CoA esters so that they are not available for carnitine palmitoyl transferase II activity.

Tissue Specific Differences in Intramitochondrial Control of β-Oxidation

It has become clear that there are important tissue specific differences in the control and regulation of oxidation and that these differences may largely reside with the entry of acyl moieties into the mitochondrion by the carnitine palmitoyltransferase (CPT) /translocase system. CPT I has wide tissue-specific variations in sensitivity to its physiological inhibitor, malonyl-CoA,1 which is probably due to the presence of different isozymes. However, it is possible that there are additional intramitochondrial controls which vary amongst tissues. The individual reactions of the oxidation of long-chain fatty acids within the mitochondrion have been known for many years although their functional and topological relationship is less well known. Recently, it has been found that the long-chain activities of three of the enzymes reside on a single membrane-bound protein (the trifunctional protein,4,5) and, in addition, there exists a fourth acyl-CoA dehydrogenase which is similarly membrane bound. Hence, the possibility of functional organization of oxidation enzyme activities, associated with the inner mitochondrial membrane, must be considered. The organization of oxidation enzymes within the mitochondrion has previously been postulated as a result of the classic studies of Stanley and Tubbs, amongst others, leading to the “leaky hosepipe” model of oxidation. Their work also led to the view that the acyl-CoA dehydrogenases were the “rate-limiting step” for oxidation, as only saturated acyl groups accumulated under well oxygenated conditions. These workers measured acyl groups resulting from hydrolysis of CoA and car-

Thiamine deficiency mimicking acute rejection following cardiac transplantation.

We describe a recipient of an orthotopic cardiac transplant who developed severe ventricular dysfunction following an episode of sepsis. He had normal cardiac biopsies and responded to treatment with thiamine. Causes other than rejection should be considered in patients who have received a cardiac transplant and who present in cardiac failure.