4-Methylumbelliferyl-alpha-D-N-sulphoglucosaminide (MU-alpha-GlcNS) was synthesized and shown to be a substrate for the lysosomal heparin sulphamidase. Sanfilippo A patients' fibroblasts (n = 42) and lymphocytes (n = 1) showed 0-3% of mean normal heparin sulphamidase activity; in total leukocytes from patients (n = 8) sulphamidase activity was clearly deficient. In fibroblasts from obligate heterozygotes for Sanfilippo A, the sulphamidase activity was reduced in 9 out of 10 cases. Heparin sulphamidase desulphates MU-alpha GlcNS to MU-alpha GlcNH2 and further hydrolysis during a second incubation is required to liberate 4-methylumbelliferone, which can be measured. Yeast alpha-glucosidase, which has low but sufficient alpha-glucosaminidase activity, was used to hydrolyse the reaction intermediate MU-alpha GlcNH2 to release 4-methylumbelliferone and free glucosamine.

Both the alpha- and beta-anomers of 4-methylumbelliferyl-D-glucosaminide were synthesized and shown to be substrates for the lysosomal acetyl-CoA:glucosaminide N-acetyltransferase. Using the beta-anomer, fibroblasts and leukocytes from 11 different Sanfilippo C patients showed < 1% of mean normal N-acetyltransferase activity. Heterozygotes showed intermediate activities. The enzymatic liberation of the fluorochrome from 4-methylumbelliferyl-beta-D-glucosaminide requires the sequential action of the N-acetyltransferase and beta-hexosaminidase. Normal beta-hexosaminidase activity caused complete hydrolysis of the reaction intermediate 4-methylumbelliferyl-beta-D-N-acetylglucosaminide formed by the N-acetyltransferase. In cell extracts with a beta-hexosaminidase deficiency, however, a second incubation in the presence of excess beta-hexosaminidase is needed to avoid underestimation of the N-acetyltransferase activity.

No abstract

A new fluorogenic substrate, 4-methylumbelliferyl beta-D-glucosaminide, was used for the assay of acetyl CoA:glucosaminide N-acetyltransferase in chorionic villi, cultured villus cells, and amniocytes. Optimal conditions for the assay and the ranges of enzyme activity were established for the various types of fetal cells. This simple fluorometric assay provides a reliable method for early prenatal diagnosis of Sanfilippo disease type C which is more convenient than current methods using radiolabelled substrates. The method was applied to amniotic fluid cells and fetal fibroblasts from an at-risk pregnancy in which an affected fetus was diagnosed by two-dimensional electrophoresis of glycosaminoglycans in the amniotic fluid.

Introduction. Cardiovascular diseases (CVD) are a leading cause of morbidity and mortality. The role of occupational hazards in the CVD prevalence remains to be clarified. Material and methods. Here we report the results of the study of risk factors and CVD prevalence in 590 workers at the largest copper production plants in the Sverdlovsk region, exposed to heavy metals in the workplace. The workers` health information was obtained during a regular medical examination in 2018. The lead concentration increase to 1.3-1.8 occupational exposure limits was registered in the working areas of the concentrating mill (for bunkerman) and copper smelting workshops (transporter, smelter, converter, non-ferrous metal spreader, repairman, electrician). Results. We studied the exposure indices (Pb level in blood), the response markers (reticulocyte count, erythrocytes basophilic stippling, coproporphyrin, and aminolevulinic acid in the urine), and their correlation to a working tenure. Based on this analysis, we attributed CVD risk factors and cardiovascular diseases to the occupation, in order to potentially modify some of those risk factors and ultimately inform the risk management. Hypertension occurred in 57% of the examined workers, which is higher than in the general population. We calculated relative risk, confidence intervals and attributable fraction. We developed a predictive mathematical model (stepwise logistic regression) to predict high-stage hypertension and identified the risk factors associated with its development. Conclusions. Correlation analysis revealed direct correlations between stages 2 and 3 hypertension and a working tenure over 20 years. We think it’s reasonable to consider the documented CVDs as related to the toxic effects of heavy metals (lead and cadmium).