ATP Synthesis in Lipoamide Dehydrogenase Deficiency Ann Saada,* Iris Aptowitzer,* Gaby Link,† and Orly N. Elpeleg* *Metabolic Disease Unit, Shaare-Zedek Medical Center, Jerusalem 91031, Israel; and †Department of Human Nutrition and Metabolism, Hebrew University, Hadassah Medical School, Jerusalem, Israel Received January 24, 2000 Lipoamide dehydrogenase deficiency is an inborn error of several metabolic pathways, including pyru- vate metabolism, Krebs cycle, and branched-chain amino acid degradation. The clinical course is vari- able, ranging from infantile neurodegenerative dis- ease to recurrent episodes of liver failure or myoglo- binuria starting later in life. In contrast, residual enzymatic activity in muscle tissue spans over a nar- row range. Despite the recent elucidation of the un- derlying molecular pathology in most patients, rela- tionships between the genotype and the biochemical and clinical phenotype remain unclear. In order to find a suitable assay for the prediction of clinical out- come and assessment of treatment, we have evaluated enzymatic activities and energetic states in fibroblasts from lipoamide dehydrogenase-deficient patients rep- resenting three different phenotypes and genotypes. Direct relationships between clinical parameters such as age of onset and disease severity and biochemical characteristics, including lipoamide dehydrogenase activity, pyruvate dehydrogenase complex activity, and ATP production ratio in fibroblasts, were identi- fied. Clinical parameters were not reflected by lactate/ pyruvate ratio. ATP production rate was in direct re- lationship with the severity of the neurological involvement; the patient with reduced ATP synthesis to 30% of the control mean had a severe neurodegen- erative disease, whereas ATP synthesis values above 45% were associated with a more favorable course. Incubation of the patients’ fibroblasts with dichloro- acetate coupled with thiamin resulted in slight but significant improvement of the cell energetic state. © 2000 Academic Press Lipoamide dehydrogenase (LAD) (EC 1.6.4.3) is the third catalytic subunit of the pyruvate dehydrogenase complex (PDHc) and is also shared by -ketoacid de- hydrogenase and branched chain-ketoacid dehydroge- nase complexes. Deficiency of LAD is associated with metabolic disturbances including lactic acidemia, Krebs cycle dysfunction and impaired branched-chain amino acid degradation (1). The clinical presentation of LAD deficiency is variable, presenting in infancy with neurological disease of varying severity, or later in life with recurrent episodes of liver failure or myoglobin- uria (1–3). The molecular basis of LAD deficiency in the pa- tients with the neurodegenerative course has been elu- cidated (4 –7). Homozygosity for the G229C mutation was associated with onset in early childhood, recurrent liver failure and only minor neurological involvement (8). Compound heterozygosity for the Y35X/G229C mu- tations was associated with neonatal presentation, re- current liver failure, and neurological disease (1). Nonetheless genotype–phenotype correlation remained questionable since patients homozygous for some mis- sense mutations were more severely affected than pa- tients who were compound heterozygotes for a non- sense and a missense mutation. Furthermore, despite low activities of LAD and PDHc in muscle and lympho- cyte homogenates, neither residual activity nor immu- noblot assays could predict clinical outcome (3, 8, 9). Since energy depletion is believed to be a major conse- quence of LAD deficiency, we have focused on the en- ergetic state of the patients’ fibroblasts and investi- gated its value in the prediction of the neurological outcome. We have also studied its usefulness as a tool for the assessment of therapy. PATIENTS AND METHODS Patients. Fibroblast cell lines of 3 patients were included: patient 1 was an adult who presented with recurrent vomiting, liver dys- function and myoglobinuria and was homozygous for the G229C mutation (8, 10). Patient 2 presented in the neonatal period with recurrent liver failure and severe muscle hypotonia and was a com- pound heterozygote for the Y35X and the G229C mutations (2, 8, 11). Patient 3 had suffered from severe neurodegenerative disease since early infancy and was homozygous for the D479V mutation (4). Methods. Tissue culture medium and supplements were pur- chased from Biological Industries (Beit Haemek, Israel). Radioactive pyruvate was from NEN Du Pont (Dreiech Germany). All other chemicals were from Sigma Chemical Company (St. Louis, MO). Fibroblast cell cultures were established from forearm skin biop- sies. Cells grown to confluence in MEM tissue culture medium sup- plemented with 10% fetal calf serum (FCS), glutamine, penicillin Biochemical and Biophysical Research Communications 269, 382–386 (2000) doi:10.1006/bbrc.2000.2310, available online at http://www.idealibrary.com on 382 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.