Composition and method of assaying for ketone bodies

• US 5,510,245 A
• Filed: 01/18/1994
• Issued: 04/23/1996
• Est. Priority Date: 09/08/1992
• Status: Expired due to Term

• Current Assignee: Bayer Corporation (Bayer AG)
• Original Assignee: Bayer Corporation (Bayer AG)
• Inventors: Magers, Thomas A.
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/182,405
• Time in Patent Office: 826 Days
• Field of Search: 435/26, 435/25, 435/14, 435/4, 436/128, 436/904, 436/119, 436/63, 436/74
• US Class Current: 435/26
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: Y10S 436/904 Oxidation - reduction indicators

Abstract

A composition, test device and method of determining the presence or concentration of ketone bodies, and specifically D-β-hydroxybutyrate, in a test sample are disclosed. The test device includes a test pad comprising a suitable carrier matrix incorporating an indicator reagent composition capable of interacting with D-β-hydroxybutyrate to produce a detectable or measurable response. In addition, a new and improved indicator reagent composition, comprising a) an indicator dye that is responsive to thiols, such as a substituted isobenzothiazolone, Ellman'"'"'s reagent or a derivative of Ellman'"'"'s reagent; b) D-β-hydroxybutyrate dehydrogenase; c) lipoamide dehydrogenase; d) D,L-lipoamide; and e) nicotinamide adenine dinucleotide, is incorporated into the carrier matrix to provide an accurate and sensitive assay of a test sample for D-β-hydroxybutyrate (DHBA) in particular, and for ketone bodies in general. The improved method and composition are especially useful in the assay of whole blood, blood serum, blood plasma and urine for ketone bodies.

Claims

1. A method of determining the presence or concentration of D-β

-hydroxybutyrate in a test sample consisting essentially of;

(a) contacting the test sample with a composition consisting essentially of D-β

-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, a disulfide reductase system consisting essentially of a single disulfide substrate and a disulfide reductase, and a thiol-responsive indicator dye; and

(b) determining the presence or concentration of D-β

-hydroxybutyrate in the test sample from the intensity and degree of a color change of the composition.

2. The method of claim 1 wherein the intensity and degree of the color change is determined visually or instrumentally.

3. The method of claim 1 wherein the presence or concentration of D-β

-hydroxybutyrate is determined by a dry phase assay.

4. The method of claim 1 wherein the test sample is a biological fluid.

5. The method of claim 4 wherein the biological fluid is whole blood, blood plasma, blood serum or urine.

6. The method of claim 5 wherein the presence or concentration of D-β

-hydroxybutyrate in urine is determined in the range of from 0 mmol/L to about 60 mmol/L.

7. The method of claim 5 wherein the presence or concentration of D-β

-hydroxybutyrate in whole blood, blood plasma or blood serum is determined in the range of from 0 mmol/L to about 10 mmol/L.

8. The method of claim 1 wherein the D-β

-hydroxybutyrate dehydrogenase is present in an amount ranging from about 50 units to about 5000 units.

9. The method of claim 1 wherein the nicotinamide adenine dinucleotide is present in a concentration ranging from about 10 mM to about 500 mM.

10. The method of claim 1 wherein the disulfide substrate is present in a concentration ranging from about 10 mM to about 200 mM, and the disulfide reductase is present in an amount ranging from about 100 units to about 2000 units.

11. The method of claim 1 wherein the thiol-responsive dye is present in a concentration ranging from about 10 mM to about 200 mM.

12. The method of claim 1 wherein the disulfide reductase system includes D,L-lipoamide as the disulfide substrate and lipoamide dehydrogenase as the disulfide reductase.

13. The method of claim 1 wherein the thiol-responsive:

dye is Ellman'"'"'s reagent, an amide derivative of Ellman'"'"'s reagent or an ester derivative of Ellman'"'"'s reagent, wherein amide and ester derivative of Ellman'"'"'s reagent is represented by the structural formula ##STR33## wherein R is the residue of an alcohol or an amine;

or a combination thereof.

14. The method of claim 1 wherein the thiol-responsive dye is a 2,2'"'"'-dipyridyl disulfide or a 4,4'"'"'-dipyridyl disulfide.

15. The method of claim 1 wherein the thiol-responsive dye is an isobenzothiazolone compound having the formula:

##STR34## wherein at least one of the R₁ and R₂ substituents is selected from the group consisting of nitro, arylazo, substituted arylazo, benzylideneamino and substituted benzylideneamino; and

the R₃ substituent is selected from the group consisting of alkyl, carboxyalkyl, hydroxyalkyl, aminoaryl, heteroaryl, carboxyheteroaryl, hydroxyheteroaryl, aminoheteroaryl, hydroxy, alkoxy, amino and substituted derivatives thereof.

16. The method of claim 15 wherein when the R₁ substituent is nitro, arylazo, substituted arylazo, benzylideneamino or substituted benzylideneamino, the R₂ substituent is hydrogen.

17. The method of claim 15 wherein when the R₂ substituent is nitro, arylazo, substituted arylazo, benzylideneamino or substituted benzylideneamino, the R₁ substituent is hydrogen.

18. The method of claim 1 wherein the composition has a pH in the range of from about 7 to about 8.5.

19. A method of determining the presence or concentration of d-β

-hydroxybutyrate in a test sample consisting essentially of;

(a) contacting the test sample with an indicator reagent composition consisting essentially of;

from about 50 units to about 5000 units of D-β

-hydroxybutyrate dehydrogenase;

from about 10 mM to about 500 mM of nicotinamide adenine dinucleotide;

a disulfide reductase system consisting essentially of from about 10 mM to about 200 mM D,L-lipoamide and from about 100 units to about 2000 units of lipoamide dehydrogenase; and

from about 10 mM to about 200 mM of a thiol-responsive dye; and

(b) determining the presence or concentration of D-β

-hydroxybutyrate in the test sample from the intensity and degree of a color change of the composition.

20. The method of claim 19 wherein the thiol-responsive dye is selected from the group consisting of Ellman'"'"'s reagent, 3-N-(3-dimethylaminopropyl)carboxamido-4-nitrophenyl disulfide, 2,2'"'"'-dithiobis(5-nitropyridine), N-3-(dimethylaminopropyl)-5-nitroisobenzothiazol-3-one, N-(3-dimethylaminopropyl)-5,7-dinitroisobenzothiazol-3-one, N-(3-dimethylaminopropyl)-5-(4-hydroxyphenylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(4-nitro-2-methylphenylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(2,4-dinitrobenzylideneamino)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-phenylazoisobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(2-hydroxy-1-naphthylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(2-hydroxy-5-methylphenylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(2-hydroxy-4-nitro-5-methylphenylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(4-hydroxy-1-naphthylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(4-hydroxy-3-methyl-1-naphthylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(4-hydroxy-5-aza-1-naphthylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(2-amino-1-naphthylazo)isobenzothiazol-3-one;

N-(3-dimethylaminopropyl)-5-(4-methoxy-1-hydroxy-2-naphthylazo)isobenzothiazol-3-one; and

combinations thereof.

21. A method of determining the concentration of total ketone bodies in a biological fluid consisting essentially of:

(a) contacting the biological fluid with a composition consisting essentially of D-β

-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, a disulfide reductase system consisting essentially of a single disulfide substrate and a disulfide reductase, and a thiol-responsive indicator dye;

(b) determining a concentration of D-β

-hydroxybutyrate in the biological fluid from a color transition of the composition in response to D-β

-hydroxybutyrate present in the biological fluid; and

(c) correlating the concentration of D-β

-hydroxybutyrate in the biological fluid to the concentration of total ketone bodies in the biological fluid.

22. A method of determining the presence or concentration of D-β

-hydroxybutyrate in a liquid sample consisting essentially of;

(a) contacting the liquid sample with an analyte detection device comprising a reagent test pad having incorporated therein a composition consisting essentially of D-β

-hydroxybutyrate dehydrogenase, nicotinamide adenine dinucleotide, a disulfide reductase system consisting essentially of a single disulfide substrate and a disulfide reductase, and a thiol-responsive indicator dye; and

(b) examining the analyte detection device for a color transition in response to the D-β

-hydroxybutyrate content in the liquid sample.

23. An analyte detection device to determine the presence or concentration of D-β

-hydroxybutyrate in a liquid test sample consisting essentially of;

a support strip;

a reagent test pad operatively attached to the support strip; and

a composition incorporated into the reagent test paid said composition consisting essentially of;

(a) D-β

-hydroxybutyrate dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a disulfide reductase system consisting essentially of a single disulfide substrate and a disulfide reductase; and

(d) a thiol-responsive indicator dye.

24. A method of monitoring or detecting diabetes mellitus by determining the concentration of D-β

-hydroxybutyrate in a biological fluid, said method consisting essentially of;

(1) contacting the biological fluid with an analyte detective device consisting essentially of a test pad including a reagent composition capable of undergoing a color transition in response to the concentration of D-β

-hydroxybutyrate in the biological fluid, said reagent composition consisting essentially of;

(a) D-β

-hydroxybutyrate dehydrogenase,

(b) nicotinamide adenine dinucleotide,

(c) a disulfide reductase system consisting essentially of a single disulfide substrate and a disulfide reductase, and

(2) examining the analyte detective device for the color transition in response to the D-β

-hydroxybutyrate concentration in the test sample; and

(3) determining concentration of D-β

-hydroxybutyrate in the biological fluid from the intensity and degree of the color transition.

25. The method of claim 24 wherein the biological fluid is whole blood, blood serum, blood plasma or urine.

26. A composition capable of exhibiting a sufficient color transition upon contacting a test sample to demonstrate the presence or concentration of D-β

-hydroxybutyrate in the test sample, said composition consisting essentially of;

(a) D-β

-hydroxybutyrate dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a disulfide reductase system consisting essentially of oxidized glutathione and glutathione reductase; and

(d) a thiol-responsive indicator dye.

27. A composition capable of exhibiting a sufficient color transition upon contacting a test sample to demonstrate the presence or concentration of D-β

-hydroxybutyrate in the test sample, said composition consisting essentially of;

(a) D-β

-hydroxybutyrate dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a disulfide reductase system consisting essentially of oxidized thioredoxin and thioredoxin reductase; and

(d) a thiol-responsive indicator dye.

28. A composition capable of exhibiting a sufficient color transition upon contacting a test sample to demonstrate the presence or concentration of D-β

-hydroxybutyrate in the test sample, said composition consisting essentially of;

(a) D-β

-hydroxybutyrate dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a disulfide reductase system consisting essentially of CoAS-S glutathione and CoAS-S glutathione reductase; and

(d) a thiol-responsive indicator dye.

29. A composition capable of exhibiting a sufficient color transition upon contacting a test sample to demonstrate the presence or concentration of D-62 -hydroxybutyrate in the test sample, said composition consisting essentially of:

(a) D-β

-hydroxybutyrate dehydrogenase;

(b) nicotinamide adenine dinucleotide;

(c) a disulfide reductase system consisting essentially of asparagusate and asparagusate reductase; and

(d) a thiol-responsive indicator dye.