Analysis of glucocerebrosidase activity in dry blood spots using tandem mass spectrometry.
BACKGROUND
Gaucher disease (GD) is due to deficiency of acid-β-glucosidase (ABG) and comprises a clinical spectrum with variable age of onset and severity. We evaluated a tandem mass spectrometry (MS/MS) method to measure ABG activity for high through-put screening.


METHODS
ABG activity was measured in 3.2 mm punches from dry blood spots (DBS). Each punch was incubated for 21 h with the substrate D-Glucosyl-β1-1'-N-dodecanoyl-D-erythro-sphingosine [C12-glucocerebroside (C(36)H(69)NO(8))] and internal standard N-myristoyl-D-erythro-sphingosine [C14-ceramide (C(32)H(63)NO(3))]. The product and internal standard were quantified using MS/MS.


RESULTS
ABG activities in anonymized newborn screening samples from NY State were (mean) 22.0 μmol/h/L±(SD) 13.8 μmol/h/L (n=2088, median 19.9 μmol/h/L, 95%CI 22.59-21.41 μmol/h/L). The enzymatic activity in DBS from 10 treatment naïve adult Gaucher patients was less than 4.2 μmol/h/L. ABG activity was stable for 3 months at room temperature a 20% activity reduction was observed. Inter- and intra-run imprecisions were 8% and 13.7%, respectively. The limit of detection was 0.75 μmol/h/L and limit of quantification was 1.25 μmol/h/L.


CONCLUSIONS
The measurement of ABG activities in DBS using MS/MS is suitable for high-throughput analysis of at-risk individuals and potentially for newborn screening for GD.
Department of Pediatrics, University Children's Hospital, University of Padua, Italy.
Elisa
E
Legnini
Elisa
E
Legini
Joseph J
JJ
Orsini
Christina
C
Hung
Monica
M
Martin
Amanda
A
Showers
Maurizio
M
Scarpa
X Kate
XK
Zhang
Joan
J
Keutzer
Adolf
A
Mühl
Olaf A
OA
Bodamer
D000328:Adult; D001800:Blood Specimen Collection / Q000379:methods*; D016022:Case-Control Studies; D057075:Enzyme Assays / Q000379:methods*; D004795:Enzyme Stability; D005776:Gaucher Disease / Q000097:blood / Q000175:diagnosis / Q000201:enzymology; D005962:Glucosylceramidase / Q000097:blood* / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D005962:Glucosylceramidase