A new fluorimetric enzyme assay for the diagnosis of Niemann–Pick A/B, with specificity of natural sphingomyelinase substrate

Abstract

6-Hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMUPC) was shown to be a specific substrate for the determination of acid (lysosomal) sphingomyelinase (ASM; gene SMPD1). Fibroblasts (n = 27) and leukocytes (n = 8) from both the A and B types of Niemann-Pick disease showed < 6% and < 10% of mean normal ASM activity, respectively. Niemann-Pick A or B patients bearing the Q292K mutation had apparently normal ASM activity with our new artificial substrate. These patients with false-normal sphingomyelinase activity, however, could readily be detected by determining the extent of inhibition of enzymatic hydrolysis of the artificial substrate HMU-PC by an unlabelled natural substrate, in particular lysosphingomyelin. This approach is generally applicable. Our novel assay for ASM combines the ease of a rapid and robust enzyme assay using a fluorogenic substrate with the specificity of an ASM assay using a natural substrate. Such assays are obviously more convenient to the diagnostic laboratory, since radiolabelled substrates are not required.

Sunitinib and SU11652 Inhibit Acid Sphingomyelinase, Destabilize Lysosomes, and Inhibit Multidrug Resistance

“…Total cellular acid sphingomyelinase activity was measured with 6-hexadecanoylamino-4-methylumbelli-ferylphosphorylcholine (HMU-PC) assay as described previously (30). The total cysteine cathepsin activity (zFRase) was measured using benzyloxycarbonyl-Phe-Arg-aminotrifluoromethylcoumarin (zFR-AMC; Enzyme System Products) probe as described (27), and the N-acetyl-glucosaminidase (NAG) activity was measured using NAG substrate solution (Sigma-Aldrich) as described (6).…”

Th1 cytokines accentuate but Th2 cytokines attenuate ceramide production in the stratum corneum of human epidermal equivalents: An implication for the disrupted barrier mechanism in atopic dermatitis

“…Acid SMase activity was measured according to the method of Van Diggelen et al [28]. Reaction mixtures consisted of 10 ml enzyme solution, 10 ml 1.35 mM 6-hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMU-PC) (Moscerdam, Oegstgeest, Netherlands) as a substrate in 0.1 M Na-acetate/0.1 M acetic acid buffer, pH 5.2, containing 0.2% (w/v) Na-taurocholate (Sigma).…”

Galactocerebrosidase assay on dried-leukocytes impregnated in filter paper for the detection of Krabbe disease

“…We should mention that, similar to the fluorometric assay in leucocytes [3], we still have to face the limited affinity of GALC for artificial fluorescent substrates [14,15]. Difficult cases may need to be further investigated with the GALC activity assay using radiolabeled substrate and or genetic analysis of the GALC gene.…”