Development and validation of leukocyte enzyme activity assay by LC-MS/MS for diagnosis of Krabbe disease and other lysosomal storage diseases Hsuan-Chieh (Joyce) Liao1,2, Laura Mitchell1, Katerina Sadilkova1, Rhona Jack1, Jane Dickerson1, Anna Scott1,2,* 1Department of Laboratories, Seattle Children's Hospital, 2Department of Lab Medicine, University of Washington, Seattle, WA

Introduction Results Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. The Enzyme reaction products were chromatographically resolved from substrate breakdown products diagnosis for Krabbe disease includes measurement of GALC enzymatic activity by radioisotope with a 3.5 minute liquid chromatography gradient (Fig. 1). Intra- and inter-assay imprecision was assay or by the accumulation of the metabolite psychosine. To improve the current diagnostic determined by 11 replicates of samples containing low and high concentration (CV<15%). Carryover workflow and assay performance, we developed and validated a leukocyte enzyme assay using was determined by assaying triplicates of cell lysate-free cocktails directly after injection of high liquid chromatography-tandem mass spectrometry (LC-MS/MS), which we hope will improve the enzyme activity sample (less than 0.1%) (Table 1). Linearity was validated using fractions (0-100%) diagnostic approach to Krabbe disease. of normal activity cell lysate combined with Krabbe cell lysate (R2=0.99). Very small differences in GALC enzymatic activity at the low end (3% of normal cell lysate) could be observed in a Method statistically significant way (Fig. 3). Enzyme activity from three, known affected patients ranged from We used galactosyl-ceramide with a 7-carbon heptanoyl chain attached to the sphingosine 0.01-0.07 (nmol/hr/mg protein); two disease carriers had enzyme activity from 0.14-0.40. The backbone as our substrate (GALC-S, Fig. 1). The internal standard (GALC-IS) was isotope labeled reference interval was established from 63 residual, unaffected samples and was 0.17-5.97 heptanoyl-ceramide, which was chemically identical to the GALC product (GALC-P, Fig.1), but (1.44±1.44) (Table 2). contained five deuterons in the heptanoyl chain. Leukocytes were extracted from whole blood samples, and total protein was quantified by the BCA method. Commercially available reagents Table 1: Validation results for the quantification of GALC assay (NeoLSDTM MSMS kit, PerkinElmer) were incubated with cell lysates (Fig. 2). After incubating Intra-assay (N=11) CV (%) substrate with cell lysates, liquid-liquid extraction with ethyl acetate was used to purify GALC-P and Low control 12.5 GALC-IS from the reaction. Liquid chromatography using a Waters CSH C18, 2.1 x 50 mm column High control 9.1 and Acquity UPLC system was used to resolve the substrate and product. A Waters Xevo TQS Inter-assay (N=11) tandem mass spectrometer was used for mass detection in positive ion MRM mode. Low control 12.6 High control 14.9 Fig. 1: Chromatography of GALC assay Fig. 4: Chromatography of other lysosomal enzymes Instrumental precision (N=11) Low control 4.9 High control 1.9 GALC-S GALC Substrate Stability GLA-S Pre-extraction WBC < 72 hours Post-reconstitution at 4°C > 2 month Others Anticoagulant ACD or EDTA; Unacceptable: Heparin GALC Internal standard

GAA-S Carry-Over (%) <0.1 Matrix effect Not found ABG-S

ASM-P Figure 3: Linearity of GALC activity IDUA, S and P Table 2: Enzyme activities from affected and carrier patients GAA-P GLA-P ABG-P ASM-S GALC-P

Substrate ion source nmole/hr/mg % of mean breakdown product Enzymatic product assay blank <0.01 0 S: substrate, P: product, GALC: Galactosylcerebrosidase (Krabbe); IDUA: Iduronidase (MPS1); GLA: Galactosidase (Fabry); GAA: Glucosidase (Pompe); ABG: Glucocerebrosidase (Gaucher); ASM: Boiled WBC <0.01 0.23 Sphingomyelinase (Niemann-Pick A/B) Affected patient 1 0.01 0.74 Affected patient 2 0.03 2.22 Fig. 2: Sample work up Affected patient 3 0.07 5.19 Carrier 1 0.14 10.37 Carrier 2 0.40 29.63 Normal (N=65, 95%CI) 0.17-5.97 12.6-442

Conclusion and discussion A simple LC-MS/MS assay was developed, which can measure trace residual GALC activity in leukocytes and aid in the diagnosis of Krabbe disease. The multiplexed substrate mixture allows for built-in sample quality control. Future work will include the optimization of assay conditions for the other analytes in the multiplexed cocktail of enzyme substrates that will enable a streamlined Reference: Clin Chem. 2017 August ; 63(8): 1363–1369 workflow for multiple clinical conditions (Fabry, Pompe, Gaucher, MPS type I, Niemann-Pick Contact information: [email protected] or [email protected] diseases, Fig. 4)