Combined malonic and methylmalonic aciduria with normal malonyl-coenzyme A decarboxylase activity: a case supporting multiple aetiologies.

We identified a patient who excreted large amounts of methylmalonic acid and malonic acid. In contrast to other patients who have been described with combined methylmalonic and malonic aciduria, our patient excreted much larger amounts of methylmalonic acid than malonic acid. Since most previous patients with this biochemical phenotype have been reported to have deficiency of malonyl-CoA decarboxylase, we assayed malonyl-CoA decarboxylase activity in skin fibroblasts derived from our patient and found the enzyme activity to be normal. We examined four isocaloric (2000 kcal/day) dietary regimes administered serially over a period of 12 days with 3 days devoted to each dietary regimen. These diets were high in carbohydrate, fat or protein, or enriched with medium-chain triglycerides. Diet-induced changes in malonic and methylmalonic acid excretion became evident 24-36 h after initiating a new diet. Total excretion of malonic and methylmalonic acid was greater (p < 0.01) during a high-protein diet than during a high-carbohydrate or high-fat diet. A high-carbohydrate, low-protein diet was associated with the lowest levels of malonic and methylmalonic acid excretion. Perturbations in these metabolites were most marked at night. On all dietary regimes, our patient excreted 3-10 times more methylmalonic acid than malonic acid, a reversal of the ratios reported in patients with malonyl-CoA decarboxylase deficiency. Our data support a previous observation that combined malonic and methylmalonic aciduria has aetiologies other than malonyl-CoA decarboxylase deficiency. The malonic acid to methylmalonic acid ratio in response to dietary intervention may be useful in identifying a subgroup of patients with normal enzyme activity.

Evaluation of gene therapy for citrullinaemia using murine and bovine models.

Citrullinaemia is an autosomal recessive disorder caused by the deficiency of argininosuccinate synthase. The deficiency of this enzyme results in an interruption in the urea cycle and the inability to dispose of excess ammonia derived from the metabolism of protein. The only treatment for this disorder has been dietary restriction of protein and supplementation with medications allowing for alternative excretion of excess nitrogen. Gene therapy offers the possibility of a long-term cure for disorders like citrullinaemia by expressing the deficient gene in the target organ. We have explored the use of adenoviral vectors as a treatment modality for citrullinaemia in two animal models, a naturally occurring bovine model and a murine model created by molecular mutagenesis. Mice treated with adenoviral vectors expressing argininosuccinate synthase lived significantly longer than untreated animals (11 days vs 1 day; however, the animals did not exhibit normal weight gain during the experiment, indicating that the therapeutic effectiveness of the transducing virus was suboptimal. It is speculated that part of the failure to observe better clinical outcome might be due to the deficiency of arginine. In the bovine model, the use of adenoviral vectors did not result in any change in the clinical condition of the animals or in the level of plasma ammonia. However, the use of 15N isotopic ammonia allowed us to assess the flux of nitrogen through the urea cycle during the experiment. These studies revealed a significant increase in the flux through the urea cycle following administration of adenoviral vectors expressing argininosuccinate synthase. We conclude that the use of adenoviral vectors in the treatment of citrullinaemia is a viable approach to therapy but that it will be necessary to increase the level of transduction and to increase the level of enzyme produced from the recombinant viral vector. Future experiments will be designed to address these issues.

Patient selection may affect gene therapy success. Dominant negative effects observed for ornithine transcarbamylase in mouse and human hepatocytes.

We have achieved significant improvement of ornithine transcarbamylase deficiency (OTCD) in a mouse model through adenoviral-mediated gene transfer of the human ornithine transcarbamylase cDNA. Substantial reduction in orotic aciduria was observed within 24 h of treatment. Metabolic correction was later associated with phenotypic correction and moderate increase in enzymatic activity. In an effort to identify the level of gene expression required to achieve wild-type levels of enzyme activity we uncovered a dominant negative effect of the endogenous mutant protein on the activity of the delivered recombinant wild-type protein. This phenomenon is relevant to homomultimeric protein defects such as OTCD, represent a challenging category of disorders for gene therapy. Thus, although our findings indicate that adenoviral-mediated gene transfer may have potential as a short-term treatment for OTCD in humans and may be effective especially during catabolic crisis, the observations in this study suggest that careful patient selection based on mutation class may be essential for initial OTCD gene therapy trials, and perhaps, for other homomultimeric enzyme deficiencies being considered as gene therapy targets.

Lipogenic activity of catfish liver. Lack of response to dietary changes and insulin administration.

1. Some of the physical, chemical and kinetic properties of catfish liver lipogenic enzymes (acetyl-CoA carboxylase and fatty acid synthetase) were investigated. 2. The liver lipogenic enzymes of catfish exhibited maximal activity at 37 degrees C, even though these fish usually live at temperatures not above 24 degrees C. 3. The activity of the lipogenic enzymes of catfish liver was always low, regardless of the proportions of lipids or carbohydrates in the diet and could not be raised by insulin administration. 4. Under the conditions of the experiments, catfish liver fatty acid synthetase produced more stearate than palmitate and no myristate.