Improved sensitivity of an acid sphingomyelinase activity assay using a C6:0 sphingomyelin substrate

Abstract

Short-chain C6-sphingomyelin is an artificial substrate that was used in an acid sphingomyelinase activity assay for a pilot screening study of patients with Niemann–Pick disease types A and B. Using previously published multiplex and single assay conditions, normal acid sphingomyelinase activity levels (i.e. false negative results) were observed in two sisters with Niemann–Pick B who were compound heterozygotes for two missense mutations, p.C92W and p.P184L, in the SMPD1 gene. Increasing the sodium taurocholate detergent concentration in the assay buffer lowered the activity levels of these two patients into the range observed with other patients with clear separation from normal controls.

Acid sphingomyelinase deficiency: The clinical spectrum of 2 patients who carry the Q294K mutation and diagnostic challenges

“…Harzer et al have postulated that the substitution of a positively charged amino acid for one that is uncharged, such as the substitution of lysine for glutamine in the Q294K mutation, produced a defective enzyme (with reduced affinity for SPM) but with an unexpectedly high affinity for the fluorometric HNP substrate [ 13 ] Other SMPD-1 variants can give rise to pseudo normal ASM assay results. This was observed in 2 ASMD type B patients (sisters) who were compound heterozygotes for p.C92W and p.P184L mutations when ASM activity was estimated using a synthetic short-chain C6-SPM substrate [ 15 ]. In our experience, ASMD diagnosis in patient 1 was delayed by pseudo normal ASM assay readouts obtained using a synthetic fluorometric (HNP) substrate.…”

“…Van Diggelen et al advocate that ASM assays should be carried out using synthetic substrates in the presence and absence of natural substrate SPM [ 14 ]. Synthetic substrate fatty acyl chain length and the type and concentration of surfactant used can influence ASM assay sensitivity and the propensity for false-negative readouts [ 15 ].…”

“…Q294K (also referred to as Q292K) [ 11 , 12 ] is a common chronic neurovisceral ASMD phenotype variant that is strongly linked with neurological involvement [ 4 , 5 ]. However, patients may be underdiagnosed when pseudo normal or even enhanced ASM activity is reported by ASM assays using synthetic fluorometric substrates [ [13] , [14] , [15] , [16] ].…”

Contribution of tandem mass spectrometry to the diagnosis of lysosomal storage disorders

“…Various improvements have been described: For sphingolipidoses, synthetic substrates chemically close to natural ones (with free fatty acid of variable and unphysiological chain length) giving more hydrophobic ceramide products and IS (ceramide species with free fatty acid of different chain lengths) are generally used, avoiding falsenegative results (NPAB, Ghomashchi et al 2015). Careful attention must be paid to the concentrations of various components (effectors, inhibitors of other enzymes) in the enzyme measurement mixture: i.e., (i) acarbose to inhibit glucosidases other than lysosomal acid α-glucosidase, (ii) N-acetyl-galactosamine to inhibit α-galactosidase B, (iii) high concentration of taurocholate for acid sphingomyelinasem e a s u r e m e n t (since two NPAB patients were missed with low concentrations, Chuang WL et al 2015).…”