Enzymes In Dried Blood Spots Research Articles

Multiplex tandem mass spectrometry enzymatic activity assay for the screening and diagnosis of Mucolipidosis type II and III

Mucolipidosis type II and III (MLII/III) is caused by defects in the mannose-6-phosphate system, which is essential to target most of the lysosomal hydrolases to the lysosome. MLII/III patients present with marked elevations in the activities of most lysosomal enzymes in plasma, but their profiles in dried blood spots (DBS) have not been well described. In the current study, we measured the activities of 12 lysosomal enzymes in DBS, among which acid sphingomyelinase, iduronate-2-sulfatase, and alpha-N-acetylglucosaminidase were significantly elevated in MLII/III patients when compared to random newborns. This sets the stage for using DBS to diagnose MLII/III. Furthermore, given an increasing number of lysosomal storage disorders are being included in the recommended uniform screening panel, our results also indicate that population-based newborn screening for MLII/III can be implemented with minimal efforts.

Characteristics of PPT1 and TPP1 enzymes in neuronal ceroid lipofuscinosis (NCL) 1 and 2 by dried blood spots (DBS) and leukocytes and their application to newborn screening

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay.A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS.

Detecting multiple lysosomal enzymes in dried blood spots by tandem mass spectrometry

Objective Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry (MS/MS). Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders(LSDs)of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment. Results The intraassay and interassay precisions ranged from 1.7% to 11.8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999. The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L·h), 0.09 μmol/(L·h), 0.12 μmol/(L·h) and 0.16 μmol/(L·h). The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51 μmol(L·h), 1.99-22.22 μmol/(L·h), 1.68-41.59 μmol/(L·h) and 2.36-19.21 μmol/(L·h). The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPSⅠ patients can be effectively detected by this MS/MS method. Conclusions A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.(Chin J Lab Med, 2016, 39: 761-765) Key words: Lysosomal storage diseases; Tandem mass spectrometry; Dried blood spot testing; Galactosylceramidase; Alpha-Glucosidases; Alpha-Galactosidase; Iduronidase