Gaucher and Niemann–Pick diseases—enzymatic diagnosis in dried blood spots on filter paper: retrospective diagnoses in newborn-screening cards

doi:10.1016/S0009-8981(01)00798-7

Clinica Chimica Acta

Volume 317, Issues 1–2, March 2002, Pages 191-197

https://doi.org/10.1016/S0009-8981(01)00798-7 Get rights and content

Abstract

Background: Gaucher disease (GD) and Niemann–Pick (NP) disease are caused by deficient activity of the lysosomal enzymes acid β-d-glucosidase (ABG) and acid sphingomyelinase (ASM), respectively. For diagnosis, these enzymes are usually measured in the extracts of leukocytes or cultured fibroblasts. Chitotriosidase (CTE), a chitinolytic enzyme, is markedly increased in the plasma of Gaucher patients. We describe methods for the assay of acid β-d-glucosidase, acid sphingomyelinase, chitotriosidase, and α-N-acetyl-galactosaminidase (NAGA) as a control enzyme in blood spots that were dried onto filter paper. Methods: To tubes containing a 3 mm-diameter blood spot, we added elution liquid and substrate solution. After incubation at 37 °C, the amount of hydrolyzed product was compared with a calibrator to allow the quantification of enzyme activity. We examined 80 healthy controls, 54 Gaucher patients, 8 Niemann–Pick patients, 27 obligate carriers, and the newborn-screening cards (NSC) from a case of Gaucher and a case of Niemann–Pick disease. Results and conclusion: The described methodology is useful to identify Gaucher and Niemann–Pick patients and controls, using samples that are sufficiently stable to be transported to the testing laboratory by mail. The diagnosis of both diseases on a newborn-screening card was clearly established. The newborn-screening card has been added to the biological materials that allow the identification of patients with Gaucher and Niemann–Pick diseases.

Introduction

Gaucher disease (GD) and Niemann–Pick (NP) disease are the most frequent lysosomal storage diseases with visceral storage without dysmorphic features. GD is characterized by a deficiency of the lysosomal acid β-d-glucosidase (ABG), and the resultant accumulation of its substrate, glucosylceramide, in macrophages (Gaucher cells). The demonstration of deficient ABG activity in peripheral blood leukocytes does not allow the differentiating of their five possible clinical presentations: GD type 1 (GD1) chronic non-neuronopathic, GD type 2 (GD2) acute neuronopathic, GD type 3 sub-acute neuronopathic, the GD perinatal-lethal form, and the GD cardio-vascular form [1]. A deficiency of lysosomal acid sphingomyelinase (ASM), usually measured in leukocytes or fibroblast cultures, is the primary defect in the two clinical types (A, neuronopathic, and B, non-neuronopathic) of NP diseases [2]. In the NP type C/D patients, the ASM activity is in the normal range. High plasma concentrations of chitotriosidase (CTE), a chitinolytic enzyme secreted by activated macrophages, is a useful marker for the diagnosis of GD. A decrease in the plasma concentration of chitotriosidase has been demonstrated in response to enzyme replacement therapy [3].

We describe methods for the assay of ABG [4], ASM [4], [5], CTE [3], and α-N-acetyl-galactosaminidase (NAGA) as control enzyme [6] in dried blood spots (DBS) on filter paper.

Section snippets

Materials and methods

4-Methylumbelliferyl-α-N-acetylgalactosaminide was provided by Moscerdam Substrates (Rotterdam, The Netherlands). The other 4-methylumbelliferyl derivatives, taurodeoxycholate, Triton-X 100, bovine albumin, sodium taurocholate, and sphingomyeline were from Sigma (St. Louis, MO). Ultima Gold-XR® and ¹⁴C sphingomyeline were obtained from Packard Instrument (Argentine) and from Amersham Pharmacia Biotech (Buckinghamshire, England), respectively.

After obtaining informed consent from the patients or

Determination of ABG activity

A 3-mm diameter blood spot (≅3.6 μl of blood) was placed into a 2-ml test tube. Forty microliters of 0.75% (w/v) sodium taurodeoxycholate in distilled water and 30 μl of 0.4 mol/l citrate-phosphate buffer (pH 5.2) as elution liquid, and 50 μl of 0.02 mol/l 4-methyl-umbelliferyl-β-d-glucopyranoside in distilled water as substrate were added. After gentle mixing, the tubes were incubated for 20 h at 37 °C in a shaking-water bath. The reaction was terminated by the addition of 300 μl of 0.13 mol/l

Determination of CTE activity

We essentially used the same procedure, with the following differences: elution liquid, 20 μl of 0.25 mol/l sodium acetate buffer (pH 5.5); substrate, 20 μl of 0.19 mmol/l 4-methylumbelliferyl-β-d-N, N′, Nʺ-triacetylchitotrioside in distilled water; incubation time, 30 min.

Determination of NAGA activity

We essentially used the same procedure, with the following differences: elution liquid, 20 μl of distilled water; substrate, 40 μl of 1 mol/l 4-methyl-umbelliferyl-2-acetamido-2-deoxy-α-d-galactopyranoside in 0.2 mol/l citrate-phosphate buffer (pH 4.7); incubation time, 20 h.

Determination of ASM activity

Twenty microliters of a 2:1 (v/v) chloroform/methanol solution, containing 23.4 μg of sphingomyeline and 0.02 μCi ¹⁴C sphingomyeline was dried under nitrogen at room temperature into a 2-ml test tube. Eighty microliters of distilled water, containing 25 μg of sodium taurocholate and 125 μg of Triton X-100, 20 μl of 2.5 mol/l sodium acetate buffer (pH 5.5), and a 3 mm-diameter blood spot were added. After vortex-mixing for 3 min, the tubes were incubated for 30 h at 37 °C in a shaking

Results and discussion

The activities of ABG and ASM were linear with regard to incubation times over the biological range. The effect of variable incubation times on the activity of CTE is shown in Fig. 1A. The short period of the selected incubation provided a clear separation between controls and untreated GD1 samples, with a marked elevation of the CTE activity.

The NAGA activity was chosen as control enzyme for its similar properties during storage and transportation compared with ABG and ASM. The enzymatic

Conclusions

Our laboratory made the diagnosis of lysosomal diseases in DBS samples [8], [9], [10]. DBS analysis offers several advantages over the whole blood samples [11]. The presented methodology is reliable and sensitive for measuring the enzymatic activities in DBS. It is possible to simultaneously measure >50 samples in duplicate for each assay. Reagents are commercially available and there are no special or costly equipments needed. Micro-plate adaptation, method automation, and a quality control

Acknowledgments

We gratefully acknowledge the cooperation of the members of the Asociación Gaucher Argentina and his authorities (Mrs. M.A. Ibáñez and Mr. M. Giancristiano), Drs. A. Feliú Torres and R. Kohan (Buenos Aires, Argentine), A. Lemes (Montevideo, Uruguay), R. Giugliani (Porto Alegre, Brazil), M. Beck (Mainz, Germany), and J. Chinsky (Baltimore, USA), for providing DBS from affected and/or carrier patients. We thank Drs. S. Rosenberg (Sao Paulo, Brazil), J. Clarke (Toronto, Canada), Y. Marsdens

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