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Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, ...
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A New Analytical Bench Assay for the Determination of Arylsulfatase A Activity Toward Galactosyl-3-Sulfate Ceramide: Implication for Metachromatic Leukodystrophy Diagnosis Francesco Morena,† Ilaria di Girolamo,† Carla Emiliani,† Angela Gritti,‡ Alessandra Biffi,‡ and Sabata Martino*,† †

Department of Experimental Medicine and Biochemical Science, Section of Biochemistry and Molecular Biology, University of Perugia, Via del Giochetto, 06126 Perugia, Italy ‡ San Raffaele Scientific Institute, San Raffaele Telethon Institute for Gene Therapy, Milano, via Olgettina 58, 20132 Milano, Italy S Supporting Information *

ABSTRACT: Here, we present the design and validation of a new assay for the diagnosis of metachromatic leukodystrophy. The method is highly specific, simple, reproducible, and straightforward. In our spectrophotometric method, the determination of arylsulfatase A (ARSA) activity toward the natural substrate, galactosyl-3-sulfate ceramide (or sulfatide), is performed using neat sulfatide without chemical modification. This confers to the assay high analytical specificity. The hydrolyzed sulfatide is monitored upon inclusion of the colorimetric reagent Azure A. The nonhydrolyzed sulfatide-Azure A is recovered and measured at a wavelength of λ = 650 nm. Thus, ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide from the total sulfatide used in the enzyme reaction (sulfatide-Azure A present in a parallel assay performed in the absence of ARSA). Within a clinical context, our method definitely discriminated between healthy subject samples and metachromatic leukodystrophy patient samples, and, therefore, it is suitable for diagnostic applications and for monitoring the efficacy of therapeutic treatments in patients or animal models. fluorogenic derivatives12−15 or on fluorogenic/colorimetric artificial substrates.16−18 Notwithstanding their specificity, radiolabeled- and fluorogenic-sulfatide-based assays require several steps prior to the final detection, thus compromising the assay reproducibility and accuracy.9−11,13 Of note, accuracy could be also prejudiced by the need to synthesize the radiolabeled sulfatide, since both the 35 S-radiolabeled sulfatide19 and the 14C-radiolabeled sulfatide20 are not generally commercially available. Preparation procedures are tedious, time-consuming, and necessitate either a relatively large amounts of radioactivity to yield a high-quality product or a moderately large amounts of protein as source of enzyme activity, raising major sensitivity issues in light of the limited availability of specimens from patients, particularly if in the pediatric age. More recently, electrospray ionization (ESI) mass spectrometry assay for the determination of the hydrolysis of sulfatide by ARSA has been developed.21 The assay is sensitive and highly specific; although similar to the above-mentioned methods, requires specialized laboratory analyses to be carried out, thereby limiting its application potential.

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etachromatic leukodystrophy (MLD) (Omim No. 250100) is a rare inherited autosomal recessive lysosomal storage disorder caused by the deficiency of arylsulfatase A (ARSA) (EC.3.1.6.8). ARSA catalyzes the first step in the degradation pathway of galactosyl-3-sulfate ceramide (alias: cerebroside sulfate or sulfatide), which is one of the major membrane lipids of myelin sheaths. Because of the abolishment of ARSA activity, the desulfation reaction does not occur and the undegraded cerobroside sulfate accumulates in the lysosomes, leading to a progressive demyelination of the nervous system of patients. Death occurs within a few years from the onset of symptoms.1−3 Currently, a therapeutic option for patients with late infantile MLD is a Phase I−II clinical trial based on the bone marrow transplantation of hematopoietic stem cell transduced with the lentiviral vector to transfer a functional ARSA4 and a Phase I−II enzyme replacement therapy trial to evaluate the efficacy of the rhARSA administration in MLD patients.5 MLD diagnosis is performed by either the biochemical evaluation of the absence of the enzymatic activity6,7 or by molecular characterization of the ARSA gene through complete sequencing of the coding region.8 Currently, ARSA activity is measured through a variety of biochemical assays based on radiolabeled sulfatide9−11 or © 2013 American Chemical Society

Received: July 30, 2013 Accepted: December 2, 2013 Published: December 2, 2013 473

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corresponding concentration. Linear regression analysis using GraphPad was used to generate the standard curve. The QC samples (blank sulfatide-AA) used in both the validation study and each experimental run of the study were prepared in the same manner as the calibration standards to obtain final concentrations of 21 nmol. Optimum pH. The optimum pH was evaluated using 0.012 mU ARSA in the presence of sulfatide (21 nmol) at different pH values: 3.0, 3.5, 4.0, 4.25, 4.5, 5.0, 5.5, and 6.0. Reactions were carried out at different times of incubation at 37 °C (see the Results section for details). Results were expressed as the mean of five independent experiments each in triplicate. Optimal Time of Incubation. We determined the optimal reaction time by plotting a curve of the rate of sulfatide hydrolyzed by a series of fixed ARSA mU (0.0015, 0.003, 0.006, 0.012, 0.024, 0.048 mU) at different times of incubation: 6, 12, 18, 24, and 36 h. Results were expressed as the mean of five independent experiments, each of which was performed in triplicate. Determination of Km,Vmax, and Kcat. Km and Vmax were determined by the Lineweaver−Burk method.23 Kcat and Kcat/ Km were deduced by the Michaelis−Menten equation (see details in the Supporting Information). The kinetic parameters were defined using the following concentrations of sulfatide: 0.052, 0.104, 0.208, 0.312, 0.416, 0.500, 0.625, 0.750, and 0.833 mM (corresponding to 2.5, 5, 10, 15, 20, 25, 31, 37.5, and 41.6 nmol of sulfatide, respectively). We performed enzymatic reactions using 0.012 mU from purified ARSA incubated for 18 h at 37 °C. Results were expressed as the mean of five independent experiments, each performed in triplicate. Method Validation. As validation procedures, we evaluated linearity, accuracy, sensitivity, selectivity, intra-run and inter-run precision, and reproducibility. Linearity. Linearity was assessed by plotting a curve of the rate of sulfatide hydrolyzed by a series of ARSA mU (0.0015, 0.003, 0.006, 0.012, 0.024, and 0.048 mU) at fixed selected incubation times: 6, 12, 18, 24, and 36 h. Results were expressed as the mean of five independent experiments, each performed in triplicate. Accuracy. The absence of stained sulfatide within the upper phase and the total recovery of the sulfatide in the lower phase were estimated to assess the assay accuracy. The absence of sulfatide-AA was estimated by measuring the absorbance of the upper phase of serial working standards obtained from the stock standards of sulfatide (83.3 and 66.6 nmol) and used for the generation of the sulfatide-AA calibration curve. As reference, the upper phase of the Azure A blank assay was also measured. The trueness and bias were evaluated from 10 replicates, either using three concentrations of sulfatide (16, 21, and 29 nmoles) in sulfatide-AA blank assay or in the presence of three concentrations of ARSA (0.003, 0.012, and 0.024 mU) and 21 nmol of sulfatide. Determinations were according to the standard procedures (http://www.fao.org/docrep/w7295e/ w7295e09.htm; also see the Supporting Information). Sensitivity. We determined the lowest and highest limit of detection of the sulfatide assay by plotting a curve of the rate of sulfatide hydrolyzed in tubes containing various concentrations of WT and MLD proteins extract (1−40 μg). Results were expressed as the mean of five independent experiments, each performed in triplicate.

Thus, there is a need for a more suitable and easy method to monitor the ARSA activity toward the cerebroside sulfate, even in small amounts of cell or tissue protein extracts. Here, we report the design and validation of a new spectrophotometric assay for the determination of ARSA activity toward the hydrolysis of the natural cerebroside sulfate. The assay is very specific, reproducible, highly sensitive, and simple, such that it can be performed in all laboratories. Moreover, this method is the first colorimetric bench assay that uses neat sulfatide without chemical modifications and is capable of measuring the ARSA activity toward the sulfatide directly, using a spectrophotometer at λ = 650 nm.

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EXPERIMENTAL SECTION Sulfatide and Azure A Preparation. Sulfatide (Matreya ≥98% TLC, bovine) was dissolved in a 2:1:0.1 chloroform:methanol:water mixture and dried at room temperature overnight, after which time it was redissolved in Buffer A (100 μL of dimethyl sulfoxide (DMSO) and ARSA enzymatic buffer (100 mM sodium acetate/acetic acid, pH 4.5) containing 20 mM MnCl2 and taurodeoxycholate (100 μg/50 μL)) to a final concentration of 5 mM. Azure A was dissolved in 95 mL of distilled water and 5 mL of 50 mN sulfuric acid. The dye solution was stored in a dark bottle and was stable for at least 10 days.22 Preparation of Standard and Quality Control. Azure A Calibration Curve. A series of working standards were obtained from the stock standards (100 mg and 80 mg of Azure A dissolved in 47.5 mL distilled water and 2.5 mL of 50 mN sulfuric acid) by serial dilution with the same buffer over the range of 5−100 mg. An amount of 0.17 mL of dye solution Azure A22 was added to 0.85 mL of a 1:1 chloroform:methanol mixture and 0.85 mL of 50 mN H2SO4. The mixture was mixed by inversion, tubes were centrifuged at 300g for 10 min at room temperature, and absorbance of the lower phase was measured at a wavelength of λ = 650 nm on a Microplate Reader (Model GDV-DV-990BV6). The calibration curve was constructed using different concentrations of Azure A (five replicates of each concentration). The absorbance at λ = 650 nm was plotted against the corresponding dye concentration. Linear regression analysis using GraphPad was used to generate the standard curves. The quality control (QC) samples (blank Azure A) used in both the validation study and each experimental run were prepared in the same manner as the calibration standards to obtain final concentrations of 0.4 mg/mL. Sulfatide Calibration Curve. A series of working standards were obtained from the stock standards of sulfatide (83.3 and 66.6 nmol) by serial dilution with Buffer A over the range of 0.52−83.3 nmol. Fifty microliters (50 μL) of a solution created by mixing an equal volume of Buffer A with the above amount of sulfatide in Buffer A (25 μL + 25 μL, respectively) was added to 0.85 mL of a 1:1 chloroform:methanol, 0.85 mL of 50 mN H2SO4, and 0.17 mL of dye solution Azure A (final concentration of 0.4 mg/mL).22 After shaking, tubes were centrifuged at 300g for 10 min at room temperature; the upper phase and the lower phase (containing the sulfatide-Azure A [sulfatide-AA]) were measured at λ = 650 nm on a Microplate Reader (Model GDV-DV-990BV6). Calibration curve was constructed using different concentrations of sulfatide, five replicates of each concentration. The absorbance at λ = 650 nm was plotted against the 474

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Figure 1. Schematic of the assay for hydrolysis of cerebroside sulfate by ARSA. The procedure consists of three main steps. Step 1, incubation of equal volumes (25 μL + 25 μL) of unknown sample and sulfatide preparation at 37 °C for 18 h; Step 2, addition of C:M (1:1) and Azure A (this step allows (i) the staining of all sulfate moieties linked to nonhydrolyzed sulfatide, and (ii) the recovery of the lower phase containing the nonhydrolyzed sulfatide-Azure A (sulfatide-AA)); and Step 3, measurement of nonhydrolyzed sulfatide-AA at λ = 650 nm. Assays are performed in the presence of ARSA (blue arrow) and in the absence of the enzyme (black arrow, blank substrate). The amount of sulfatide hydrolyzed by ARSA (in nmol) were calculated by subtracting the value measured in the presence of the enzyme, with respect to the blank substrate. Measurements are referred to the calibration curve of sulfatide-AA (see Figure S1A in the Supporting Information).

Selectivity. The interfering effect of protein extract and other molecules (e.g., DNA,24 hyaluronic acid,25 lipids, sphingosine22) was estimated to assess the assay selectivity. The effect of proteins was evaluated by plotting a curve of the Azure A absorbance measured using serial proteins extract concentration (10−100 μg) in the Azure A blank assay. The effect of DNA was evaluated by plotting a curve of absorbance of DNA-Azure A measured using serial (μg) purified DNA (gently provided by Dr. M. Di Cristina, University of Perugia) either in the Azure A blank assay or in the sulfatide-AA blank assay. The effect of lipids was evaluated by plotting a curve of absorbance of lipds-Azure A measured using lipid extract from tissue samples either in the Azure A blank assay or in the sulfatide-AA blank assay. Lipids were extracted by the method of Folch et al.26 Because of the potential interfering of sphingosine,22 we also measured the effect of a serial concentration of purified sphingosine (Matreya, synthetic,

98+% TLC) either in the Azure A blank assay or in the sulfatide-AA blank assay. Sphingosine was prepared as described by Bandhuvula et al.27 Finally, we monitored the potential effect of hyaluronic acid (Sigma−Aldrich, No. 53747). A stock solution of hyaluronic acid (0.3% w/v) in distilled water was prepared as described according to the manufacturer’s recommendation. The effect of hyaluronic acid was evaluated by plotting a curve of absorbance of hyaluronic acid-Azure A, using a serial concentration of pure hyaluronic acid either in the Azure A blank assay or in the sulfatide-AA blank assay mixture. Intra-run and Inter-run Precision Accuracy. Four different concentrations of ARSA enzyme were used to assess the intrarun and inter-run precision, using the coefficient of variation obtained from 10 replicates in 10 consecutive runs. Reproducibility. The reproducibility of the assay on discriminating healthy subjects and MLD patients was 475

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Figure S2B in the Supporting Information). This procedure increased the ARSA specific activity at least 70-fold. Optimum pH. We performed the assay at different pH covering the range of 3.0−6.0. We found a peak of activity at pH 4.5 (Figure 2A, representative pH curve obtained with 0.012 mU of ARSA at 18 h of incubation).

evaluated by the Fisher’s test using the R software (http:// www.r-project.org/). In routine analysis, each analytical set includes a blank Azure A, a blank sulfatide-AA, a blank of unknown sample containing Azure A without sulfatide, and the unknown samples containing sulfatide and Azure A for the ARSA activity determination. Other Analytical Methods. Other analytical methods include patients, animal model, tissue and cell preparation, cell transduction with ARSA-encoding lentiviral vector, tissue and cell extract preparation, ARSA activity assay toward the fluorogenic substrate, DEAE-cellulose chromatography, trueness and bias determination, Kcat/Km determination, and statistical analysis. These other methodologies are detailed in the Supporting Information.

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RESULTS Rational Design. Enzyme reactions were performed in a final volume of 50 μL by mixing an equal volume of ARSA sample with 21 nmol of sulfatide at 37 °C for 18 h (see Figure 1, blue arrow, Step 1). As a reference assay, 21 nmol of sulfatide at 37 °C for 18 h were mixed with an equal volume of Na/ acetate buffer (Figure 1, black arrow, Step 1). Reactions were stopped by adding 0.85 mL of a 1:1 chloroform:methanol mixture, 0.85 mL of 50 mN H2SO4, and 0.17 mL of dye solution Azure A22 (final concentration of 0.4 mg/mL). After shaking, tubes were centrifuged at 300g × 10 min at room temperature (Figure 1, blue and black arrow, Step 2). The Azure A binds either to sulfate-residues of the nonhydrolyzed sulfatide or to free sulfate-groups hydrolyzed by ARSA from the sulfatide. After centrifugation, the upper phases were discarded and the lower phases, containing the nonhydrolyzed sulfatideAA (blue arrow) and the blank sulfatide-AA (black arrow), were recovered (see Figure 1, Step 2) and measured at λ = 650 nm on a Microplate Reader (Model GDV-DV-990BV6) (see Figure 1, Step 3). The amount of sulfatide hydrolyzed (in nmol) were calculated by subtracting the value measured in the assay performed in the presence of the ARSA enzyme (blue arrow) from the value obtained from the assay carried out in the absence of the enzyme (black arrow). Absorbance was evaluated by using a sulfatide-AA calibration curve as a reference (see Figure S1A in the Supporting Information). As a reference, the Azure A calibration curve is also reported (see Figure S1B in the Supporting Information). Optimization of Kinetics Parameters. Ad hoc kinetics parameters for the enzyme reaction were determined by using different mU (0.0015, 0.003, 0.006, 0.012, 0.024, 0.048 mU) of partial purified ARSA, each at different incubation times: 6, 12, 18, 24, and 36 h. Enzyme Preparation. We used DEAE-cellulose chromatography to separate ARSA from the other protein extracts, according to our previous protocol.16 As an enzyme source, we used human mononuclear cells (PBMCs) freshly isolated from peripheral blood of healthy donors. In our experimental condition, arylsulfatase B (ARSB) (EC.3.1.6.12), which has similar ARSA biochemical properties (http://www.brendaenzymes.info/), is eluted with the void volume of the column, while the ARSA activity retained by the column is eluted by a linear gradient of NaCl 50−250 mM as confirmed by the specific assay in the presence of the AgNO3, an ARSA inhibitor (see Figure S2A in the Supporting Information) and the absence of ARSA activity in PBMCs from MLD patients (see

Figure 2. Ad hoc kinetics parameters for the enzyme reaction: (A) optimum pH, depicted using a representative pH curve obtained with 0.012 mU of ARSA at 18 h of incubation; (B) the reported Lineweaver−Burk plot; and (C) incubation time, depicted by a timedependent profile of the enzyme reaction using different amounts of partially purified ARSA (0.0015, 0.003, 0.006, 0.012, 0.024, 0.048 mU) at different incubation times (6, 12, 18, 24, and 36 h). All results were expressed as ± SEM (standard error of mean) of five independent biological samples, each performed in triplicate (p < 0.005).

Km, Vmax, and Kcat. We found a Km value of 0.099 mM (4.71 nmol), a Vmax value of 8.96 nmol/h (Figure 2B), a Kcat value of 0.087 s−1, and a Kcat/Km ratio of 8.81 × 102 M−1 s−1. Time of Incubation. We observed a comparable time-course behavior curve for each enzyme concentration with an increasing trend of activity in the order 0.03 ARSA mU → 0.006 ARSA mU → 0.012 ARSA mU → 0.024 ARSA mU 476

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Figure 3. Method validation. (A) Linearity was assessed by plotting a curve of the rate of sulfatide hydrolyzed by a series of ARSA mU (0.0015, 0.003, 0.006, 0.012, 0.024, 0.048 mU) at fixed selected incubation times of 6 h (R2 = 0.9824), 12 h (R2 = 0.998), 18 h (R2 = 0.9937), 24 h (R2 = 0.9922), and 36 h (R2 = 0.9743). (B) To measure accuracy, the upper phase was measured at λ = 650 nm on a Microplate Reader (Model GDV-DV990BV6). In the upper phase, blank Azure A and blank sulfatide-AA have comparable values. (C, D) To measure the sensitivity, we determined the lowest and highest limit of detection of the sulfatide assay by plotting a curve of the rate of sulfatide hydrolyzed in tubes containing various quantities of (C) WT and MLD proteins tissue extract and (D) human PBMCs of normal donors (NDs). Enzyme reactions were linear in the range of 3−10 μg of proteins for tissue extracts (panel C) and 5−15 μg of proteins for cell extracts (panel D). (E−J) To examine the selectivity, the interfering effect of proteins was evaluated using serial tissue extract protein concentrations (10−100 μg) in the Azure A blank assay ((E) lower phase and (F) upper phase). The effect of lipids (panel G) and sphingosine (panel H) was evaluated by measuring the absorbance using serial lipid extract (μg) or pure sphingosine (μmol), either in the Azure A blank assay or in the sulfatide-AA blank assay. The effect of DNA (panel I) was evaluated by measuring the absorbance of serial-purified DNA (μg) either in the Azure A blank assay or in the sulfatide-AA blank assay. The effect of hyaluronic acid (panel J) was evaluated by measuring absorbance of serial pure hyaluronic acid (μg) either in the Azure A blank assay or in the sulfatide-AA blank assay. All results were expressed as ±SEM of five independent biological samples, each performed in triplicate (p < 0.005). [Note that sulfatideAA = sulfatide-Azure A.]

sensitivity, intra-run and inter-run precision, and reproducibility. Assay Linearity. Enzyme reactions were linear in the range of 0.003−0.024 mU at each incubation time (see Figure 3A). These data indicated that 0.003 mU is the lowest quantity of enzyme mU requested for a valid assay. Assay Accuracy. We found comparable absorbance values between the upper phase of Azure A blank standard and either

The levels of sulfatide hydrolyzed reached a peak at 18 h of incubation, although the levels of sulfatide hydrolysis were highest at the limits of detection (LODs)/limits of quantification (LOQs), even after 6 and 12 h of incubation (Figure 2C). Sulfatide Assay Validation. As standard assay validation procedures, we evaluated the linearity, accuracy, selectivity, 477

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the upper phases of a serial sulfatide-AA blank standards (Figure 3B) or of a serial protein extracts samples (Figure 3F) thereby indicating the absence of nonhydrolyzed sulfatide-AA within the upper phase. The degree of accuracy evaluated in the blank sulfatide-AA assay using three different sulfatide concentrations gave trueness values of 98.6%, 96.83%, and 97.06% for 21, 29, and 16 nmol of used sulfatide, respectively, and a corresponding bias of −1.4%, −3.17%, and −2.94% (Table 1). The trueness

Table 2. Intra-run and Inter-run Sulfatide Assay Precision Accuracya

Table 1. Sulfatide-AA Assay Accuracy, showing Trueness and Bias Values of the Assay in the Absence and Presence of the ARSA Enzymea Without ARSA Enzyme (n = 10) sulfatide-AA concentration (nmol)

trueness (%)

0.003 0.012 0.024

trueness (%) 98.29 98.84 98.69

sulfatide hydrolyzed

0.003 0.006 0.012 0.024

3.76 4.79 5.74 6.91

0.003 0.006 0.012 0.024

3.78 4.78 5.69 6.88

standard deviation

coefficient of variation, CV (%)

Within the Run (n = 10) 0.05 0.05 0.03 0.04 Between Run (n = 10) 0.08 0.08 0.06 0.07

1.27 1.02 0.56 0.53 2.11 1.63 1.07 1.02

a Four different concentrations of ARSA enzyme were used to assess the intra-run and inter-run precision by coefficient of variation (CV) obtained from 10 replicates in 10 consecutive runs. (See method section for details).

bias (%)

21 98.6 −1.4 29 96.83 −3.17 16 97.06 −2.94 With the ARSA Enzyme (n = 10), for a Sulfatide-AA Concentration of 21 nmol ARSA mU

ARSA mU

protein extract samples (Figure 3F) is comparable to blank Azure A and blank sulfatide-AA (Figure 3B). We observed an inhibition effect of the lipid extract in the sulfatide-AA blank standard assay over 200 μg (Figure 3G), whereas we confirmed the highest inhibition of pure sphingosine on the sulfatide-AA blank mixture (Kean et al.;22 see Figure 3H). No interfering contribution of the DNA-Azure A color was observed, even at high DNA concentration either in the sulfatide-AA blank standard or in the Azure A blank standard (Figure 3I). Finally, no interfering contribution of the hyaluronic-Azure A staining was observed either in the sulfatide-AA blank standard or in the Azure A blank standard (Figure 3J). Assay Reproducibility. The F-test, evaluated between ND (N = 15) and MLD patients (N = 5), gave a p-value 30 μg in the reaction mixture (Figure 3E), whereas in the upper phase 478

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Figure 4. ARSA assay applications. (A, B) ARSA activity in PBMCs (panel A) and fibroblasts (panel B) isolated from MLD patients and normal donors (ND). (C) Basal levels of ARSA activity toward its natural substrate in several biological samples from normal human tissues (frontal cortex [hFC], cerebellum [hCereb], periventricular white matter [hPeriv WM]); normal nonhuman primates (total brain [NHP Brain]); and WT mice (total brain [mBrain], cerebellum [mCereb], spinal cord [mSC], liver [mLiver]). (D) ARSA activity after DEAE-chromatography of hHSCs derived from untransduced normal donors (ND-UT), untransduced MLD patients (MLD-UT), and LV.ARSA-transduced MLD (MLD-T). (E) ARSA activity after DEAE-chromatography of mNPCs derived from WT-UT, MLD-UT, and MLD-T mice. Similar amount of enzyme mU (0.003) hydrolyses comparable nmol of sulfatide in MLD-T, ND-UT hHSCs, and WT-UT mNSCs, respectively. Results were expressed as ± SEM of at least five independent experiments, each performed in triplicate (p < 0.005). [Blue symbols denote sulfatide assay performed on different tissues; red symbols represent blank samples (assay performed in the absence of sulfatide).]

prior to stop the enzyme reaction. The nonhydrolyzed sulfatide-AA is recovered and measured in a spectrophotometer at λ = 650 nm. Thus, the ARSA activity toward the sulfatide is obtained by subtracting the nonhydrolyzed sulfatide-AA from the total sulfatide-AA used in the enzyme reaction (sulfatide present in a parallel assay performed in the absence of ARSA). Of note, with respect to other established assays, our method requires a single step prior to the final detection (see Table S1 in the Supporting Information). This makes the assay more rapid, easy standardizable, and highly precise, as established by the inter-run and intra-run assay CV measurements. Moreover, the use of neat sulfatide without chemical modifications confers to the assay high analytical specificity. The complete recovery of nonhydrolyzed sulfatide within the lower phase and the trueness and bias values indicated that the sulfatide recovered by the extraction procedure is not significantly different from the sulfatide concentration used in the method. The high sensitivity of our assay (3−15 μg of protein extract), compared to other assays (see Table S1 in the Supporting Information), makes the method useful for the

4E) derived from MLD patients and As2−/− mice, respectively. Importantly, these results show that the transgenic enzyme hydrolyzes comparable nmoles of sulfatide to that measured in ND hHSCs (Figure 4D) and WT mNSCs (Figure 4E).

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DISCUSSION ARSA activity toward the cerebroside sulfate is currently evaluated in conventional assays using a radiolabeled sulfatide or fluorescent-tagged derivatives as substrates.10−15 Notwithstanding being specific, these methods exhibit poor limits of detection and reproducibility and require relatively large quantities of protein as the source of enzyme activity. Here, we report the development and validation of a new analytical spectrophotometric assay to measure ARSA activity toward its natural substrate. The assay is carried out using neat sulfatide as a substrate. Because of the absence of chemical modifications (the presence of fluorogenic groups or radioactive residues), the hydrolyzed sulfatide is monitored using a colorimetric reagent (Azure A)22 that is added to the mixture 479

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ACKNOWLEDGMENTS Part of this work has been supported by EUPRIM-Net (under the EU Contract No. RII3-026155 of the 6th Framework Programme) and the Telethon Foundation (through Project No. TGT11B02 to A.G.).

analysis of a small amount of biological samples, thereby representing an advantage for the diagnostic investigation. The selectivity of the method is demonstrated by (i) the absence of matrix (e.g., proteins, lipids) staining interfering effect within the assay condition; (ii) the absence of any noisy effect of DNA and hyaluronic acid that, based on recent studies,24,25 could interact with Azure A; (iii) the absence of interfering of other potential compounds present within the sample extract, as revealed by the comparable absorbance value of a blank sample (assay performed in the absence of sulfatide) and the blank Azure-A. Thus, we could exclude the interference of sphingosine22 that we confirmed could inhibit the sulfatide staining. Taking into account the variability of enzyme activity in normal donors and patients, our assay returned activities that were highest for ND-derived cells and lowest for patient’s cells, compared to the range of values previously reported in the literature10−13 or routinely generated through conventional methods in laboratory facilities specialized in prenatal diagnostics. Thus, our assay allows for detection of ARSA activity specifically in cells and tissues from NDs, defining a reference threshold of activity that must be restored in target tissues for a successful therapeutic treatment and clearly identifying samples from MLD patients, in which ARSA activity is undetectable. In light of the potential use of engineered hematopoietic and/or neural stem cells for the treatment of MLD, the assay’s performance was validated in stem cell types of human (hHSCs) and murine (mNSCs) origin. Results indicated that the assay can be used to monitor the efficacy of viral-mediated gene therapy approaches.4,29

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CONCLUSIONS Our study demonstrates the advantages of the new spectrophotometric assay for the determination of the ARSA activity toward the hydrolysis of sulfatide. The assay is ideal for performing accurate diagnosis of MLD disease but also for monitoring the follow-up during a therapeutic treatment. Moreover, because of the relatively easy manufacture, the method could be suitable for high-throughput diagnostic screening. ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +39 0755857439. Fax +390755857443. E-mail: sabata. [email protected]. Author Contributions

F.M. and I. di G. performed experiments and analyzed data. A.G. and A.B. performed gene therapy experiments and analyzed and discussed data. C.E. analyzed data. S.M. designed assay, supervised experiments, analyzed data, and wrote the manuscript. All authors have given their approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 480

dx.doi.org/10.1021/ac4023555 | Anal. Chem. 2014, 86, 473−481

Analytical Chemistry

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