title: Biochemical and immunochemical study of seven families with 3-ketothiolase deficiency: diagnosis of heterozygotes using immunochemical determination of the ratio of mitochondrial acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase proteins.
abstract: The possibility of identifying heterozygotes of 3-ketothiolase deficiency, an inborn error of metabolism caused by a defect of mitochondrial acetoacetyl-CoA thiolase (T2), was tested in seven unrelated families by using enzymatic assay of thiolase activity and immunoblot analysis. The ratio of acetoacetyl-CoA thiolase activities, in the presence and absence of K+ ion (+K/-K ratio), in fibroblasts from 15 normal controls was around 2.0 (1.8 to 2.4), whereas the +K/-K ratio in eight patients was always 1.0. The ratio for the 13 obligate carriers ranged from 1.4 to 1.9, causing a minor overlap with control. Identification of heterozygote cells by immunoblot analysis, using anti-T2 antibody alone as a probe, was difficult, as previously reported. We therefore carried out immunoblot analysis, using as probes a mixture of anti-T2 antibody and the antibody against mitochondrial 3-ketoacyl-CoA thiolase (T1), another mitochondrial thiolase, and determined the ratio of the intensities of the T2 and T1 bands (T2/T1 ratio) using a densitometer. When the T2/T1 ratio was calculated, there was no overlap between the heterozygotes and normal controls. Hence, the heterozygotes can be unambiguously identified using this method. The thiolase activities and T2/T1 proteins in immunoblotting were detectable in peripheral lymphocytes, rectal mucosa, amniocytes, and liver. Thus, the postnatal diagnosis of 3-ketothiolase deficiency can be readily made using lymphocytes or rectal mucosa. The applicability of these methods in amniocytes indicates that prenatal diagnosis of this disease should be possible.
authors.affiliation: Department of Pediatrics, Gifu University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: A
authors.initials: A
authors.lastname: Sakai
authors.forename: T
authors.initials: T
authors.lastname: Fukao
authors.forename: A
authors.initials: A
authors.lastname: Wakazono
authors.forename: T
authors.initials: T
authors.lastname: Kuwahara
authors.forename: T
authors.initials: T
authors.lastname: Orii
authors.forename: T
authors.initials: T
authors.lastname: Hashimoto
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000032:analysis; D000102:Acetyl-CoA C-Acyltransferase / Q000032:analysis / Q000172:deficiency* / Q000235:genetics; D005260:Female; D006580:Genetic Carrier Screening; D006720:Homozygote; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008661:Metabolism, Inborn Errors / Q000175:diagnosis / Q000201:enzymology / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology; D011247:Pregnancy; D011296:Prenatal Diagnosis; D014018:Tissue Distribution
chemical_list: D000102:Acetyl-CoA C-Acyltransferase; D000101:Acetyl-CoA C-Acetyltransferase