Methods and compositions for assaying the activity of one or more lysosomal enzymes

Patent metadata

Field	Value
Publication number	US-2017159103-A1
Application number	US-201615210766-A
Country	US
Kind code	A1
Filing date	Jul 14, 2016
Priority date	Nov 8, 2011
Publication date	Jun 8, 2017
Grant date	—

Abstract

Methods and liquid compositions for assaying the activity of one or more lysosomal enzymes in a sample are provided. In some embodiments, the assay is a multiplexed assay for the activities of a plurality of lysosomal enzymes in the sample. The compositions and methods can comprise or employ: one or more metal cations effective for precipitating sulfate ions, one or more metal cations effective for precipitating phosphate ions, a maltase glucoamylase inhibitor, a beta-N-acetylhexosaminidase inhibitor, and one or more surfactants.

First claim

1 . A method for assaying enzymatic activities of one or more lysosomal enzymes, comprising:
(a) contacting a sample with a first solution to provide a solution comprising one or more lysosomal enzymes; (b) adding an enzyme substrate for each lysosomal enzyme to be analyzed to the solution comprising the enzymes and incubating the substrates with the enzymes in an enzyme reaction solution for a time sufficient to provide a solution comprising an enzyme product for each lysosomal enzyme present in the sample, wherein the enzyme reaction solution comprises:
(i) one or more metal cations effective for precipitating sulfate ions;
(ii) one or more metal cations effective for precipitating phosphate ions;
(iii) a maltase glucoamylase inhibitor;
(iv) a beta-N-acetylhexosaminidase inhibitor; and
(v) one or more surfactants;
(c) optionally quenching the enzyme reaction; and (d) determining the quantities of the enzyme products.

2 . The method of claim 1 further comprising adding an internal standard for each lysosomal enzyme to be analyzed before, after, or simultaneously with the addition of substrates.

3 . The method of claim 1 , wherein the sample is a blood or tissue sample.

4 . The method of claim 1 , wherein the sample is a dried blood spot.

5 . The method of claim 2 , wherein determining the quantities of the enzyme products comprises determining the ratio of each product to its internal standard by mass spectrometric analysis.

6 . The method of claim 5 , wherein the mass spectrometric analysis is tandem mass spectrometric analysis.

7 . The method of claim 6 , wherein determining the quantities of the products comprises tandem mass spectrometric analysis in which the parent ions of the products and their internal standards are generated, isolated, and subjected to collision-induced dissociation to provide product fragment ions and internal standard fragment ions.

8 . The method of claim 7 , wherein determining the quantities of the products comprises comparing the peak intensities of the product fragment ions and internal standard fragment ions to calculate the amount of the products.

9 . The method of claim 1 further comprising using the quantities of the products to determine whether the dried blood sample is from a candidate for treatment for a condition associated with one or more lysosomal enzyme deficiencies.

10 . The method of any one of claims 1 - 8 , wherein the one or more lysosomal enzymes comprises an enzyme selected from the group consisting of:
(a) α-glucosidase (GAA); (b) α-galactosidase (GLA); (c) α-L-iduronidase (IDUA); (d) β-glucocerebrosidase (ABG); (e) β-galactocerebrosidase (GALC); (f) sphingomyelinase (ASM); (g) iduronate 2-sufatase (ID2S); (h) N-acetylgalactosamine 6-sulfatase (GAL6S); and (i) N-acetylgalactosamine 4-sulfatase (GAL4S).

11 . The method of any one of claims 1 - 8 , wherein the one or more lysosomal enzymes comprise:
(a) α-glucosidase (GAA); (b) α-galactosidase (GLA); (c) α-L-iduronidase (IDUA); (d) β-glucocerebrosidase (ABG); (e) β-galactocerebrosidase (GALC); (f) sphingomyelinase (ASM); (g) iduronate 2-sufatase (ID2S); (h) N-acetylgalactosamine 6-sulfatase (GAL6S); and (i) N-acetylgalactosamine 4-sulfatase (GAL4S).

12 . The method of claim 5 , wherein determining the quantities of the enzyme products comprises conducting the solution comprising the enzyme product to a mass spectrometer by liquid chromatography.

13 . The method of claim 5 , wherein determining the quantities of the enzyme products comprises conducting the solution comprising the enzyme product to a mass spectrometer by flow injection.

12 . The method of any one of claims 1 - 8 , wherein the enzyme reaction solution further comprises a buffer.

13 . An aqueous composition, comprising:
(a) one or more metal cations effective for precipitating sulfate ions; (b) one or more metal cations effective for precipitating phosphate ions; (c) a maltase glucoamylase inhibitor; (d) a beta-N-acetylhexosaminidase inhibitor; and (e) one or more surfactants.

14 . The composition of claim 13 further comprising a buffer.

15 . The composition of claim 14 , wherein the buffer is selected from the group consisting of phosphate, carboxylate, sulfate, sulfonate, and sulfate monoester buffers.

16 . The composition of claim 13 , wherein the one or more surfactants are selected from the group consisting of cationic, anionic, neutral, and non-ionic surfactants.

17 . The composition of claim 13 , wherein the metal cation effective for binding sulfate ions is selected from the group consisting of Ba 2+ , Ce 3+ , Hg + , Pb 2+ , Ra 2+ , Sr 2+ , Bi 3+ , Cd 2+ , Ca 2+ , and Mg 2+ .

18 . The composition of claim 13 , wherein the metal cation effective for binding phosphate ions is Ba 2+ , Ce 3+ , Hg + , Pb 2+ , Ra 2+ , Sr 2+ , Bi 3+ , Cd 2+ , Ca 2+ , and Mg 2+ .

19 . The composition of claim 13 , wherein the maltase glucoamylase inhibitor is acarbose.

20 . The composition of claim 13 , wherein the beta-N-acetylhexosaminidase inhibitor is 2-acetamido-2-deoxy-D-glucono-1,5-lactone.

21 . The composition of claim 13 having a pH from about 2 to about 9.

22 . The composition of claim 13 further comprising one or more substrates for a lysosomal enzyme.

23 . The composition of claim 22 , wherein the substrate is a substrate for a lysosomal enzyme selected from the group consisting of:
(a) α-glucosidase (GAA); (b) α-galactosidase (GLA); (c) α-L-iduronidase (IDUA); (d) β-glucocerebrosidase (ABG); (e) β-galactocerebrosidase (GALC); (f) sphingomyelinase (ASM); (g) iduronate 2-sufatase (ID2S); (h) N-acetylgalactosamine 6-sulfatase (GAL6S); and (i) N-acetylgalactosamine 4-sulfatase (GAL4S).

24 . The composition of claim 22 further comprising one or more internal standards for a lysosomal enzyme.

25 . The composition of claim 24 , wherein the internal standard is an internal standard for a lysosomal enzyme selected from the group consisting of:
(a) α-glucosidase (GAA); (b) α-galactosidase (GLA); (c) α-L-iduronidase (IDUA); (d) β-glucocerebrosidase (ABG); (e) β-galactocerebrosidase (GALC); (f) sphingomyelinase (ASM); (g) iduronate 2-sufatase (ID2S); (h) N-acetylgalactosamine 6-sulfatase (GAL6S); and (i) N-acetylgalactosamine 4-sulfatase (GAL4S).

Assignees

• Univ Washington

Inventors

• Gelb Michael H
• Turecek Frantisek
• Spacil Zdenek
• Scott C Ronald
• Barcenas Rodriguez Mariana Natali

Classifications

• C12Q1/40 Primary

  involving amylase · CPC title

• C12Q1/34 Primary

  involving hydrolase · CPC title

• C12Q1/44

  involving esterase · CPC title

• G01N30/7233

  interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04) · CPC title

• G01N33/573

  for enzymes or isoenzymes · CPC title