Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10.When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality.

Fabry disease, Lysosomal storage disorders, Metabolic diagnosis, Mucopolysaccharidosis, Newborn screening, Pompe disease,
Molecular Genetics and Metabolism
Erasmus MC: University Medical Center Rotterdam

Oemardin, L.F, Boer, A.M, Ruijter, G.J.G, van der Ploeg, A.T, de Klerk, J.B.C, Reuser, A.J.J, & Verheijen, F.W. (2011). Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots. Molecular Genetics and Metabolism, 102(1), 44–48. doi:10.1016/j.ymgme.2010.09.008