Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

Figure 1.

LC-MS/MS chromatography of the (a) ARSA substrate; (b) ARSA enzymatic product; and (c) ARSA internal standard channel. The asterisk in the ARSA enzymatic product channel was from substrate breakdown in the heated ESI source. The x-axis is time (min) and the y-axis is ion counts in the MRM channel after being normalized to the maximum signal (100%).

Figure 2.

The amount of ARSA enzymatic product formed as a function of the amount of protein in the leukocyte lysate used. Error bars are standard deviations based on triplicate measurement.

Figure 3.

ARSA activity as a function of the fraction of ARSA-containing lymphoblast lysate (GM14603) added to ARSA-deficient lymphoblast lysate (GM23097). A total of 2 μg protein was used per assay. Error bars are standard deviations based on the triplicate measurements. The insert is an expansion of the plot at the lower end.

Figure 4.

ARSA activity in DBS from 4 MLD patients (median: 0.007 μM/h, range: 0.005–0.011 μM/h), 1 MSD patient (0.087 μM/h), and 7 healthy adults (0.63 μM/h, range: 0.39–1.30 μM/h), after immuno-precipitation purification. The horizontal bar indicates the median of each group.

Figure 5.

ARSA activity after size-exclusion chromatography purification in CDC Quality Control DBS samples, including base pool (0.023 ± 0.003 μM/h), low (0.048 ± 0.007 μM/h), medium (0.21 ± 0.02 μM/h), and high (0.37 ± 0.02 μM/h) controls, each representing 0, 5%, 50% and 100% to high control, respectively. Each point was measured in 20 replicates. Error bars were standard deviations based on the 20 measurements.

Figure 6.

(a) ARSA activity after size-exclusion chromatography purification in DBS from 34 MLD patients (median: 0.0015 μM/h, range: 0–0.18 μM/h), 3 MSD patients (median: 0.032 μM/h, range: 0.028–0.076 μM/h), 10 healthy adults (median: 0.80 μM/h, range: 0.45–1.3 μM/h) and 294 random newborns (median: 0.27 μM/h, range: 0.082–0.65 μM/h). Fresh DBS from patients and healthy adults were used. (b)ARSA activity in aged DBS (stored at room temperature for 1 month before processing) from 18 MLD patients (median: 0.003 μM/h, 0–0.043 μM/h), 2 MSD patients (0.021 and 0.035 μM/h), and 205 random newborns (median: 0.21 μM/h, 0.073–0.56 μM/h. The horizontal bar indicates the median of each group.