3-Methylcrotonyl-CoA Carboxylase

Principle Of Method: [14C]NaHCO3 is used as a carboxylating agent using the substrate specific for each carboxylase, propionyl-CoA, 3-methylcrotonyl-CoA or sodium pyruvate. At the end of the incubation the excess [14C]NaHCO3 is converted to 14CO2 by addition of trichloroacetic acid and the 14CO2 is absorbed by NaOH in a fume chamber. Precipitated protein is measured by centrifugation and the supernatant fluid contained labelled product is counted.

Uses And Limitations Of The Method: These assays are used to diagnose propionic acidaemia , 3-methylcrotonyl-glycinuria, pyruvate carboxylase deficiency and multiple carboxylase deficiency after elevated urinary, plasma or CSF metabolites have been found that lead to the specific suspicion. Diagnosis of multiple carboxylase deficiency is complicated by possible responsiveness to biotin, but should be possible using white blood cells if the patient is not receiving biotin supplements.

For Diagnosis Using Fibroblasts The Cells Must Be Cultured In Low-Biotin Medium.

Prenatal diagnosis of the diseases is possible, but the laboratory only has experience for propionic acidaemia (PAA). The other diseases are all very rare. For PAA direct analysis of the enzyme in chorionic villi is possible, or analysis in cultured amniotic cells is carried out. If amniocentesis is performed elevated metabolites should be tested for in cell-free amniotic fluid by GC-MS.

Specimen Requirements: For preliminary testing, blood. 5 ml lithium heparin (orange capped tube) unseparated and unfrozen. Send at room temperature to arrive at the laboratory within 24h of venepuncture. For follow-up, fibroblasts cultured from a skin biopsy may be needed. Biopsy material should be collected aseptically into a sterile bottle containing tissue culture medium (available from the laboratory), and sent at room temperature to arrive within 24 hours.

Biopsies for tissue culture should not be frozen.

Fibroblast cultures established in other laboratories should be sent in plastic 25 cm2 flasks filled with medium.

The Laboratory Recommends Use Of A Courier Service Or Royal Mail Special Delivery For Sending All Specimens To The Laboratory.

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