Assay and method for quantitating carbonic anhydrase activity and assessing red blood hemolysis

Patent metadata

Field	Value
Publication number	US-2016355867-A1
Application number	US-201615174445-A
Country	US
Kind code	A1
Filing date	Jun 6, 2016
Priority date	Jun 4, 2015
Publication date	Dec 8, 2016
Grant date	—

Abstract

A method and assay for using carbonic anhydrase (CA), particularly CA-I or CA-II, as a biomarker of hemolysis. The method and assay detect hemolysis by determining a percentage erythrocyte hemolysis in a specimen or sample of blood based upon quantification of carbonic anhydrase present in the extracellular portion of the blood. The method and test serve to optimize therapeutic efficacy for treatments of hemolysis.

First claim

Having described the invention the following is claimed:

1 . A method of measuring the amount or activity of carbonic anhydrase enzyme, in a bodily sample, the method comprising:
mixing a bodily sample including RBC lysate with a first physiological CO 2 /HCO 3 − solution; mixing the first physiological CO 2 /HCO 3 − solution with a second CO 2 /HCO 3 − solution having a dissimilar pH; and measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the enzyme.

2 . The method of claim 1 , wherein an increase in the rate compared to a control rate is indicative of an increase in enzyme or enzyme activity.

3 . The method of claim 1 , wherein the first physiological CO 2 /HCO 3 − solution and the second CO 2 /HCO 3 − solution are mixed in a stopped flow device.

4 . The method of claim 1 , wherein the rate at which the pH equilibrates is measured by adding a fluorescent pH indicator dye to the first physiological CO 2 /HCO 3 − solution and/or the second physiological CO 2 /HCO 3 − solution and measuring a change of fluorescence of the dye.

5 . The method of claim 4 , wherein the pH indicator dye is pyranine.

6 . The method of claim 1 , wherein the bodily sample includes red blood cells and an increase in enzyme or enzyme activity is indicative of increased red blood cell hemolysis.

7 . The method of claim 6 , wherein the red blood cells are from stored blood.

8 . The method of claim 1 , wherein the first physiological CO 2 /HCO 3 − solution is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered solution.

9 . The method of claim 8 , the first physiological CO 2 /HCO 3 − solution having a pH of about 7 and the second physiological CO 2 /HCO 3 − solution having a pH of about 8.4 prior to mixing.

10 . The method of claim 9 , the first physiological CO 2 /HCO 3 − solution and the second physiological CO 2 /HCO 3 − solution having a temperature of about 10° C.

11 . A method for determining red blood cell hemolysis in a bodily sample, the method comprising:
mixing a bodily sample including red blood cells (RBC) and/or RBC lysate with a first physiological CO 2 /HCO 3 − solution; mixing the first physiological CO 2 /HCO 3 − solution with a second CO 2 /HCO 3 − solution having a dissimilar pH; measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the carbonic anhydrase enzyme in the bodily sample; and correlating the rate of pH equilibration with carbonic anhydrase concentration to determine a percentage RBC hemolysis present in the bodily sample.

12 . The method of claim 11 , wherein an increase in the rate of pH equilibration compared to a control rate is indicative of an increase in the percentage of red blood cell hemolysis in the bodily sample.

13 . The method of claim 11 , wherein the first physiological CO 2 /HCO 3 − solution and the second CO 2 /HCO 3 − solution are mixed in a stopped flow device.

14 . The method of claim 11 , wherein the rate at which the pH equilibrates is measured by adding a fluorescent pH indicator dye to the first physiological CO 2 /HCO 3 − solution and/or the second physiological CO 2 /HCO 3 − solution and measuring a change of fluorescence of the dye.

15 . The method of claim 14 , wherein the pH indicator dye is pyranine.

16 . The method of claim 14 , wherein the bodily sample includes red blood cells from stored blood.

17 . The method of claim 11 , wherein the first physiological solution is a hydroxyethyl)-1-piperazineethanesulfonic acid) solution.

18 . The method of claim 11 , the first physiological CO 2 /HCO 3 − solution having a pH of about 7 and the second physiological CO 2 /HCO 3 − solution having a pH of about 8.4 prior to mixing.

19 . The method of claim 18 , the first physiological CO 2 /HCO 3 − solution and the second physiological CO 2 /HCO 3 − solution having a temperature of about 10° C.

20 . A method for determining red blood cell hemolysis in a bodily sample, the method comprising:
mixing a bodily sample including red blood cells (RBC) and/or RBC lysate with a first physiological CO 2 /HCO 3 − solution, the first physiological CO 2 /HCO 3 − solution having a pH of about 7; mixing the first physiological CO 2 /HCO 3 − solution with a second CO 2 /HCO 3 − solution having a pH of about 8.4; measuring the rate at which the pH of the newly mixed solution equilibrates under the influence of the carbonic anhydrase enzyme in the bodily sample; and correlating the rate of pH equilibration with carbonic anhydrase concentration to determine a percentage RBC hemolysis present in the bodily sample.

Assignees

• Univ Case Western Reserve

Inventors

• Geyer Ryan R
• Zhao Pan
• Parker Mark D
• Boron Walter F

Classifications

• G01N2333/988

  Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase · CPC title

• C12Y402/01001

  Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase · CPC title

• C12Q1/527 Primary

  involving lyase · CPC title

• G01N33/84

  involving inorganic compounds or pH · CPC title

• G01N33/80

  involving blood groups or blood types {or red blood cells (white blood cells G01N33/56972)} · CPC title