Biotin-responsive multiple carboxylase deficiency is an inherited disorder that is characterized by deficient activity of propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, pyruvate carboxylase and acetyl-CoA carboxylase. The activities of these carboxylases can be corrected with biotin. Biotin metabolism in mammals involves holocarboxylase synthetase (HCS) and biotinidase. HCS fixes biotin to the apocarboxylases yielding active holocarboxylases. Biotinidase cleaves biotin from biocytin or biocytin peptides. Patients with MCD can be divided into the neonatal form occurring in the first few weeks of life and a juvenile form in which the onset is late in infancy. We have reported that the enzymatic cause of the neonatal form is defective activity of HCS. Biotinidase deficiency has been reported in patients with the juvenile form. We propose a series of integrated studies of biotin metabolism involving MCD and HCS. We will purify and characterize HCS from rat liver. We will establish simpler, more reliable assays for HCS based on the rate of fixation of radioactive biotin to the apocarboxylases or to their peptide fragments. We will seek a synthetic substrate containing the common sequence of Met-Lys-Met at the active site of all carboxylases. An antibody will be produced against rat HCS and used in immunological studies. Kinetic studies will be performed on rat HCS and HCS of human fibroblasts and leucocytes of normal subjects and patients with MCD to explore the defect at the molecular level. We have synthesized the natural substrate 14C-biocytin to be used to measure biotinidase activity in human serum normals and patients with MCD. These integrated studies on biotin metabolism in MCD should provide improved methodology for differential diagnosis of infants presenting with the clinical symptoms of MCD. It remains possible that there are other forms of multiple carboxylase deficiency unrelated to HCS or biotinidase deficiency. This systematic study of a substantial sample of patients available should elucidate this issue. The results of this endeavor should lead to a better understanding of the mechanisms of MCD at the molecular level and to the design of more rational schedules of treatment of patients with MCD with biotin.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Biochemistry Study Section (BIO)
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University of California San Diego
Schools of Medicine
La Jolla
United States
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