Biomarkers of oxidative stress

Abstract

The present invention relates to a method for detecting cardiovascular oxidative stress in an individual, comprising detecting in a blood sample from the individual modification of a cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K + pump protein or of an equivalent cysteine in a homologue or variant thereof. The invention further relates to a kit for detecting cardiovascular oxidative stress in an individual, the kit comprising at least one agent for detecting the presence of a modification in a cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K + pump protein or of an equivalent cysteine in a homologue or variant thereof, wherein said modification is a result of oxidation.

1 . A method for detecting cardiovascular oxidative stress in an individual, said method comprising detecting in a blood sample from said individual modification of a cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or variant thereof. 
     
     
         2 . The method of  claim 1  which is for determining the prognosis of an individual with risk factors for cardiovascular disease, wherein the level of modification of said cysteine detected in the individual's blood is indicative of the severity of cardiovascular oxidative stress in said individual. 
     
     
         3 . The method of  claim 2 , wherein the individual is treated, or the individual's existing treatment regime is altered as necessary, according to the severity of oxidative stress detected. 
     
     
         4 . The method of  claim 1  which is for determining whether an individual is at risk of heart failure or has undiagnosed heart failure, wherein the level of modification of said cysteine detected in the individual's blood is indicative of the risk of heart failure in said individual or indicative of whether the individual is suffering heart failure. 
     
     
         5 . The method of  claim 4 , wherein prophylactic or interventional therapy is administered to said individual on determination of imminent or existing heart failure. 
     
     
         6 . The method of  claim 1  which is for monitoring the recovery of an individual from cardiovascular oxidative stress or for monitoring the effectiveness of treatment of an individual suffering from cardiovascular oxidative stress, wherein a change in the level of modification of said cysteine detected in the individual's blood compared to a previously detected level is indicative of the individual's recovery or of the effectiveness of said treatment. 
     
     
         7 . The method of  claim 6  which is for monitoring recovery of said individual from impending or ongoing cardiovascular disease or for monitoring the effectiveness of treatment of said individual from impending or ongoing cardiovascular disease. 
     
     
         8 . The method of  claim 6  which is for monitoring the recovery of an individual from strenuous physical exercise. 
     
     
         9 . The method of any one of  claims 1  to  8 , wherein said modification comprises a glutathione moiety bound to said cysteine as a result of said oxidation. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the level of modification of said cysteine detected in said individual is compared to the level of modification of said cysteine detected in a control individual or control population of individuals. 
     
     
         11 . The method of  claim 9 , wherein said control individual or control population of individuals is selected from subjects not suffering from cardiovascular pathophysiology, subjects with impending or existing heart failure, or rested subjects. 
     
     
         12 . The method of any one of  claims 1  to  8 , wherein the level of modification of said cysteine detected in said individual is compared to the level of modification of said cysteine in said individual at an earlier time point. 
     
     
         13 . The method of any one of  claims 1  to  11 , wherein the blood sample is processed to isolate the erythrocytes. 
     
     
         14 . The method of any one of  claims 1  to  12 , wherein the erythrocytes are processed to release at least said cysteine as the modified or unmodifed cysteine for analysis, optionally as a moiety in a fragment of the complete β1 subunit or as a moiety in the complete β1 subunit. 
     
     
         15 . The method of  claim 14 , wherein said process further comprises fractionation of the processed erythrocytes by any one of techniques selected from centrifugation, electrophoresis, isoelectric focusing, filtration, affinity binding or other binding chemistries, ion exchange chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, and adsorptive/absorptive techniques, or any combination thereof. 
     
     
         16 . The method of  claim 14  or  claim 15 , wherein said β 1  subunit is fragmented to release a fragment comprising at least said modified or unmodified cysteine using a chemistry that does not affect the modified or unmodified cysteine. 
     
     
         17 . The method of any one of  claims 13  to  16 , wherein detecting modification of said cysteine comprises contacting said processed sample with a first binding agent which is specific for said modification. 
     
     
         18 . The method of  claim 17 , wherein said first binding agent is an antibody or a modification-binding fragment thereof. 
     
     
         19 . The method of  claim 18 , wherein said antibody or fragment thereof specifically binds a glutathione moiety when said glutathione moiety is bound to said cysteine residue. 
     
     
         20 . The method of  claim 18  or  claim 19 , wherein the antibody is a conjugated antibody. 
     
     
         21 . The method of any one of  claims 18  to  20 , wherein the antibody is a polyclonal antibody. 
     
     
         22 . The method of any one of  claims 18  to  20 , wherein the antibody is a monoclonal antibody. 
     
     
         23 . The method of any one of  claims 17  to  22 , wherein said detecting also comprises contacting said sample with a second binding agent which is specific for said β 1  subunit or at least a fragment thereof, which subunit or fragment comprises said modified cysteine. 
     
     
         24 . The method of  claim 23 , wherein either said first binding agent or said second binding agent is bound to a solid surface. 
     
     
         25 . The method of  claim 24 , wherein the binding agent not bound to the solid surface is conjugated to a detectable moiety. 
     
     
         26 . The method of any one of  claims 13  to  16 , wherein detecting the extent of modification of said cysteine comprises one or more enzymic or non-enzymic reactions which provide a detectable result specific for said modification. 
     
     
         27 . The method of  claim 26 , which comprises further modification of said cysteine or the modification thereof, or removal of the modification. 
     
     
         28 . The method of any one of  claim 13  to  16 ,  26  or  27 , wherein detecting modification of said cysteine comprises HPLC, colorimetric of fluorometric spectrophotometry, mass spectrometry, gas chromatography, or flow cytometry. 
     
     
         29 . The method of any one of  claims 1  to  28 , for assessing the efficacy of an agent for treatment of cardiovascular oxidative stress in an individual, the method comprising administering said agent to an individual having cardiovascular oxidative stress and detecting the level of a modified cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or variant thereof in a blood sample from said individual and comparing said detected level of modified cysteine to a level of modified cysteine detected for said individual at an earlier time point, wherein a decrease in the level of modified cysteine in a blood sample from said individual after administration of said agent compared to the level prior to said administration is indicative of an agent capable of treating impending or existing heart failure. 
     
     
         30 . The method of  claim 29 , comprising comparing said detected level of modified cysteine to a level of modified cysteine detected for a blood sample obtained from said individual prior to administration of said agent. 
     
     
         31 . The method of any one of  claims 1  to  28 , for assessing the efficacy of an agent for preventing cardiovascular oxidative stress in an individual, the method comprising administering the agent to an individual, and detecting the level of a modified cysteine at position 45 of the β 1 -subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or a variant thereof in a blood sample from said individual before and after exposing said individual to a cardiovascular oxidative challenge, wherein no change or a minor change in the level of modified cysteine detected in said individual is indicative of an agent effective for preventing cardiovascular oxidative stress. 
     
     
         32 . The method of any one of  claims 29  to  31  wherein the method is conducted as part of a research trial or clinical trial of a candidate agent for the treatment or prevention of cardiovascular oxidative stress. 
     
     
         33 . The method of any one of  claims 29  to  32 , wherein said agent is for treatment of impending or existing heart failure. 
     
     
         34 . The method of any one of  claims 1  to 33, wherein the individual is human. 
     
     
         35 . A kit for detecting cardiovascular oxidative stress in an individual, the kit comprising at least one agent for detecting the presence of a modification in a cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or variant thereof, wherein said modification is a result of oxidation. 
     
     
         36 . The kit of  claim 35 , which comprises multiple agents associated with detection of said modified cysteine. 
     
     
         37 . The kit of  claim 35  or  claim 36 , wherein the at least one agent comprises at least a first binding agent which is specific for said modification. 
     
     
         38 . The kit of  claim 37 , wherein the at least one agent is an antibody or a modification-binding fragment thereof. 
     
     
         39 . The kit of  claim 38  wherein the antibody is a conjugated antibody or a modification-binding fragment thereof. 
     
     
         40 . The kit of  claim 38  or  claim 39 , wherein the antibody is a polyclonal antibody or a modification-binding fragment thereof. 
     
     
         41 . The kit of  claim 40  wherein the polyclonal antibody is a rabbit polyclonal antibody. 
     
     
         42 . The kit of  claim 38  or  claim 39 , wherein the antibody is a monoclonal antibody. 
     
     
         43 . The kit of any one of  claims 38  to  42 , wherein the antibody binds to glutathione when bound to a cysteine residue at position 45 within the human β1 subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or variant thereof, wherein said glutathionylation is a result of oxidation. 
     
     
         44 . The kit of any one of  claims 37  to  43 , which also comprises a second binding agent which is specific for at least a fragment of said β 1  subunit, which fragment comprises said modified cysteine. 
     
     
         45 . The kit of  claim 44 , wherein either said first binding agent or said second binding agent is bound to a solid surface. 
     
     
         46 . The kit of  claim 45 , wherein the binding agent not bound to the solid surface is conjugated to a detectable moiety. 
     
     
         47 . The kit of  claim 35  or  claim 36 , comprising one or more reagents which provide a detectable result specific for said modification. 
     
     
         48 . The kit of any one of  claims 35  to  47 , which comprises one or more calibrated standards wherein the standard comprises a known level of modified cysteine. 
     
     
         49 . The kit of any one of  claims 35  to  48  which comprises one or more additional components selected from the group consisting of (i) one or more reference sample(s); (ii) one or more detectable moieties; (iii) one or more substance(s) for immobilising an agent for detecting a modified cysteine on a solid support; (iv) a solid support material; (v) one or more container(s) for collection and/or storage of a blood sample; (vi) one or more reagent(s) for use in preparation of a blood sample; and (viii) instructions for use of the kit or a component(s) thereof in a method for determining the level of a modified cysteine at position 45 of the β1-subunit of the human erythrocyte ATP-dependent Na + K +  pump protein or of an equivalent cysteine in a homologue or variant thereof in a blood sample.