Assays / Genetic Enzymes / Dihydroxyacetone Phosphate Acyl Transferase (DHAP-AT)

Principle Of Method: The substrate [¹⁴C]-dihydroxyacetone phosphate is prepared in a preliminary step by the action of glycerol 3-phosphate dehydrogenase on [¹⁴C]-glycerol 3-phosphate. The [¹⁴C]-dihydroxyacetone phosphate is then reacted with palmitoyl-CoA in the main enzyme reaction to produce palmitoyl-[¹⁴C]-dihydroxyacetone phosphate. This is partitioned into chloroform and then spotted on to filter paper discs. The discs are washed with successively decreasing concentrations of ice-cold trichloroacetic acid to remove traces of the substrate, and then dried and counted.

Uses And Limitations Of The Method: The method is the most useful enzyme assay for diagnosis of generalised peroxisomal disorders (Zellweger syndrome, neonatal adrenoleucodystrophy and infantile Refsum disease), both post-natally and pre-natally. It can also be used for diagnosis of rhizomelic chondrodysplasia punctata (RCDP), although the degree of deficiency is not as great in this disease as in the generalised disorders. It does not allow differential diagnosis of the three generalised disorders, and is not suitable for heterozygote detection because DHAP-AT is not the primary deficiency in these conditions. Other tests for generalised disorders should also be carried out on suspected positive patients for confirmation of the diagnosis. These include catalase latency in fibroblasts and plasma or fibroblast very long chain fatty acids levels. This will allow distinction to be made between generalised disorders and isolated DHAP-AT deficiency, a rare metabolic error with a phenotype similar to RCDP.

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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