Pyruvate Dehydrogenase Complex Deficiency: A Novel Presentation

Abstract

A 3y/o male presented with a progressive neurologic disorder characterized by hypotonia, ophthalmoplegia and ataxia. Postmortum studies at 4 1/2y revealed Leigh's disease. Biochemical findings included elevation of blood lactate (L) (ranging from 1.9 to 7.OmM, normal mean f SD 1.0+0.4mM), pyruvate (P) (0.11 to 0.25, normal 0.11+0.03mM) and L/P ratio (14 to 35, normal 10 to 18). 0-OH-butyrate (BOH), acetoacetate and L were intermittently present in urine. Plasma amino acids were normal. CSF L (4.7mM) and P (0.24mM) were elevated. Increased urinary excretion of glycine and alanine was present. No increase in blood L or P was noted after IV glucose (100mg/kg). During an 18 hr fast blood BOH increased to 200OuM (normal 91+81vM, n=4) with little change in blood L, P, or glucose levels. Skeletal muscle biopsy (premortum) showed absent CO on histochemical staining. Fibroblasts from patients with isovaleric L d e m i a (IVA), a disorder of leucine catabolism, have deficient activity of isovaleryl-CoA dehydrogenase (IVDH), as demonstrated by Rhead and Tanakj (PNAS 77:580, 1980) with a tritium release assay using [2,3-Hlisovaleryl-CoA as substrate. They found 12% of control activity using mitochondria isolated from 5 IVA lines. However, in crude homogenatef of IVA cells they measured 50-60% of normal activity, while 12-4~]leucine oxidation by the intact cells was 1-2% of normal. While this demonstrated a specific deficiency of mitochondria1 IVDH activity in IVA cells, the magnitude of residual dehydrogenating activity could not be accurately determined. To resolve this problem, we improved the assay by using Normal ASB and the mutant enzyme in Maroteaux-Lamy syndrome (MLS) have been shown to be immunologically indistinguishable when compared using rabbit anti-ASB antiserum. In the present study a monoclonal antibody library was obtained by fusion of spleen cells from mice immunized with purified ASB and X63NSl/lAg-4 (NSl) mice myeloma cells. Seventy-six hybridoma lines with ASB antibody specificity were obtained, of which 64 were subcloned and further studied. Of these, 56 reacted with both control and mutant ASB. Five reacted only with the active form of ASB and not with either the mutant form or with partially denatured (enzymatically inactive) control ASB. Three reacted only with heat denatured or urea treated control ASB and with the mutant enzyme but not with the active form. The specificity of these antibodies toward ASB was documented using Western blotting. Several of these hybridoma lines were injected into Pristane sensitized mice, and the specific monoclonal antibodies werc purified from the ascites fluid. A solid phase radioimmunoassay was developed using IV-16-8 antibody (reacts with control and mutant ASB and has high binding affinity to polyvinyl chloride), 1251-labeled V-4-7 (reacts with both mutant and control ASB), and 1251-labeled V-18-8 (reacts specifically with active ASB). Using this assay the ratio between active and inactive ASB was determined in liver, fibroblast and leukocyte homogenates from con...