PRINCIPLE OF METHOD: N-acetylneuraminic acid (NANA) is measured in samples with and without prior hydrolysis in sulphuric acid, using a colorimetric method based on reaction with thiobarbituric acid (TBA). Values obtained after hydrolysis correspond to the total NANA in the sample (ie including NANA in covalent linkage plus that which is free), and values obtained without hydrolysis correspond to the free NANA only. The covalently bound is obtained from the difference total – free. A pH-adjustment step is included to remove some contaminating compounds (particularly deoxyribose) that also react with TBA.
USES AND LIMITATIONS OF THE METHOD: The main use of the method is for diagnosing the free NANA (or sialic acid) storage diseases, including infantile N-acetylneuraminic acid storage disease, Salla disease and sialuria. Tissue and urine from patients with the first two of these diseases contain elevated amounts of free NANA but normal amounts of bound. Urine from patients with sialuria contains very high amounts of free NANA. In cells levels are also increased but the sialic acid is present in the cytosolic rather than the lysosomal fraction. Elevated amounts of bound NANA (but normal free) are found in sialidosis, galactosialidosis, I-cell disease and, to a lesser extent, mucolipidosis type III, and this will provide further evidence for diagnosis of these diseases. In practice, however, these last-named conditions are more conveniently diagnosed by enzymatic analysis.
A limitation of the method is that it will not detect elevated levels of free NANA in fibroblasts from later-onset patients (Salla disease), although these patients can be shown to have elevated levels in their urine.
SPECIMEN REQUIREMENTS: Urine. 10-20 ml in a sterile container without preservative. Freeze if not sending immediately but can be transported to the laboratory at room temperature. 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 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.