Principle Of Method:
The transfer of an acetyl group from acetyl-CoA to 14C(U)glucosamine is measured by determining the amount of radioactivity eluted from a Dowex-50 (H+) column. Unreacted [14C]-glucosamine remains bound. The glucosamine in this reaction serves as a substitute for glucosamine at the non-reducing end of heparan sulphate molecules which is the natural substrate for the enzyme. Use of glucosamine greatly facilitates assay of the enzyme. An alternative fluorogenic substrate, 4-methylumbelliferyl-a-D-glucosaminide is available but the method is not yet running in the SAS laboratory.
Uses And Limitations Of The Method:
Deficiency of the N-acetyltransferase (full name acetyl-CoA:a-glucosaminide N-acetyltransferase) is the primary defect in Sanfilippo disease type C (MPS IIIC). Its natural function is to acetylate a-linked glucosamine residues in heparan sulphate after they have been de-N-sulphated by heparan sulphamidase so that a-N-acetylglucosaminidase can then cleave them. In its absence there is storage of heparan sulphate in the tissues and excess excretion in the urine. The deficiency can be shown in white cells or fibroblasts, and the assay is usually run after excess heparan sulphaturia has been found. MPS IIIC is rare in the UK but native-born cases are known. It is probably rather more common in continental Europe. Prenatal diagnosis is possible by analysis of chorionic villi or cultured amniotic cells; when amniocentesis is performed investigation of the GAG pattern in the supernatant fluid may assist in the diagnosis. Heterozygote detection is not offered by the laboratory.
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.