Assays / Genetic Enzymes/ [¹⁴C]-Citrulline Incorporation

Principle Of Method: A monolayer of confluent cells or fragments of intact chorionic villi are incubated overnight in arginine- and serum-free medium to deplete them of their arginine stores. This medium is then replaced with a similar medium containing [¹⁴C]-citrulline, and the amount of label incorporated into protein during a 6h incubation is determined. Normal healthy cells incorporate label during this period, which is not converted to urea because these cell types lack urease. Cells deficient in either of the enzymes argininosuccinate synthetase or argininosuccinate lyase (which are deficient in the urea cycle disorders citrullinaemia and argininosuccinic aciduria, respectively) incorporate very low amounts of radioactivity because citrulline cannot be incorporated into protein via arginine as a result of the block.

Uses And Limitations Of The Method: The method is used to confirm diagnosis of citrullinaemia or argininosuccinic aciduria after abnormal metabolites suggestive of one of these disorders have been demonstrated by amino acid analysis. It will not distinguish between the two, however, and the final diagnosis rests on the abnormal amino acid profile. The method is especially useful for prenatal diagnosis of the disorders using chorionic villi or amniotic cells. In the case of the former, it is the practice of the laboratory to assay villi both directly and after culture because sometimes relatively high residual activity is found in the direct assay for affected fetuses. In the case of the latter, amino acid analysis of amniotic supernatant fluid (second trimester) is carried out to look for elevation of citrulline or presence of argininosuccinic acid and its anhydrides as a preliminary test before assay of the cells. When chorionic villi are used directly for prenatal diagnosis it is important for the laboratory to receive through the referring obstetrician a small specimen of normal control villi from another patient since material cannot be stored for running as a control. The method is not offered for heterozygote detection of either disease.

Specimen Requirements: Fibroblasts cultured from a skin biopsy are 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 at the laboratory 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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