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Analysis of Acid Sphingomyelinase Activity in Dried Blood Spots Using Tandem Mass Spectrometry
Elisa Legnini,Joe J. Orsini,Adolf Mühl,Britt Johnson,Angela Dajnoki,Olaf A Bodamer 대한진단검사의학회 2012 Annals of Laboratory Medicine Vol.32 No.5
Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP. Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS)method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening. Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6- sphingomyelin (C29H59N2O6P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C22H43NO3)]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product). Results: ASM activities were stable for up to 2 months at or below 4˚C. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intraand inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 μmol/h/L (mean 10.6) with a SD of 5.06 μmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 μmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 μmol/h/L). Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.
Joe, M.,Moon, M.W.,Oh, J.,Lee, K.H.,Lee, K.R. Pergamon Press ; Elsevier Science Ltd 2012 Carbon Vol.50 No.2
The morphological evolution of an amorphous carbon film deposited by energetic carbon atoms of 75eV with various angles of incidence was investigated by molecular dynamics simulation. Normal or near-normal incidence of carbon atoms resulted in a smooth surface of the deposited film. In contrast, a bump-like surface structure emerged and led to rough surfaces at grazing incidences, in agreement with the experiments. The bifurcated growth mode was explained by the impact-induced transport of atoms on the growing surface. The downhill transport of atoms on a sloping surface dominates at normal incidence, which suppresses the evolution of surface irregularities to form a rough surface. However, the dominance of uphill transport at a grazing incidence made the surface irregularities grow to a seed structure, which provided the shadowing effect during carbon deposition. This mechanism mediates initial seed formation and subsequent roughening together with shadowing effects under grazing incidence.
Joe, M.M.,Karthikeyan, B.,Chauhan, P.S.,Shagol, C.,Islam, Md.R.,Deiveekasundaram, M.,Sa, T. Gauthier-Villars ; Elsevier Science Ltd 2012 European journal of soil biology Vol.50 No.-
The objective of this study was to compare the efficiency of Azospirillum brasilense MTCC125 flocculated cells with standard grown cells under in vitro conditions and in association with maize (Zea mays L.) under field conditions. The results revealed that A. brasilense flocculated cells had higher survivability in alginate bead (37 x 10<SUP>6</SUP> CFU g<SUP>-1</SUP> dry wt.) and disinfected soil (11 x 10<SUP>6</SUP> CFU g<SUP>-1</SUP> dry wt.) after 12 months of storage in room temperature. A. brasilense flocculated cells offered a higher degree of resistance to dehydration in terms of reduction in water availability (a<SUB>w</SUB>) and their survivability was 12% higher in soil and 27% higher in alginate compared to standard grown cells. The adhesion efficiency of A. brasilense flocculated cells was 54% higher to hydrophobic polystyrene and 101% higher to maize roots when compared to standard grown A. brasilense cells. Furthermore, flocculated cells had better spermoplane survivability (48%) and spermosphere colonization (73%) along with a concomitant enhancement on the germination percentage (11%) and vigor index (23%) of maize. Field studies with A. brasilense flocculated cells conducted under normal irrigated conditions and by withholding irrigation at 25, 50, and 75% available water-holding capacity (AWHC) showed a significant increase in plant height (19%), plant dry weight (16%), grain yield (31%), stover yield (17%) and nitrogen uptake (18%) compared with standard grown cell treatment.