Marker useful for detection and measurement of free radical damage and method

Abstract

The present invention teaches a marker useful for detection and measurement of free radical damage. Specifically, the invention takes advantage of alterations which occur to the N-terminus of the albumin molecule, a circulating protein in human blood, in the presence of free radicals. These alterations effect the ability of the N-terminus of the albumin molecule to bind metals. Methods for detecting and quantifying this alteration include evaluating and quantifying the cobalt binding capacity of an albumin-containing sample, analysis and measurement of the ability of albumin to bind exogenous cobalt, detection and measurement of the presence of copper in a purified albumin sample and use of an immunological assay specific to the altered form of serum albumin which occurs following free radical damage. Also taught by the present invention is the use of the peptide Asp Ala His Lys and the compound Asp-Ala-His-Lys-R, wherein R is any chemical group capable of producing a detectable signal when a metal ion capable of binding to the N-terminus of naturally-occurring albumin is bound to the compound, for detection and quantitation of the marker. Methods of the present invention also include use of the marker as a “biochemical tag,” thereby allowing for sensitive detection and measurement of the efficacy of clinical drugs and therapeutics which result in the generation of free radicals or which act to limit free radical damage. The marker also acts as a “biological tag” of a process implicated in a wide array of diseases and conditions and, accordingly, may be used to monitor and assess such diseases and conditions. Finally, the invention provides antibodies, immunoassays, and kits for use in detecting or quantitating the marker.

1 . A method of monitoring or assessing treatment of a disease or condition in which free radicals play a role comprising: 
 obtaining a first biological sample containing albumin from a patient suffering from the disease or condition;    treating the patient;    obtaining one or more additional biological samples of the same type from the same patient at one or more times after the treatment; and    determining if there is a change in the quantity of a modified albumin present in the first sample as compared to the subsequent sample(s), the modified albumin having a reduced ability to bind metal ions at its N-terminus, the quantity of the modified albumin in each sample being indicative of the amount of free radical damage in that sample, and a change in the quantity of the modified albumin in the first sample as compared to the subsequent sample(s) being indicative of a change in the amount of free radical damage and, therefore, of the effectiveness or ineffectiveness of the treatment, the determination being made by: 
 a) contacting each of the biological samples with an excess quantity of a metal ion salt, the metal ion being capable of binding to the N-terminus of unmodified human albumin, to form a mixture of bound metal ions and unbound metal ions;  
 b) determining the amount of bound metal ions for each biological sample, the amount of bound metal ions providing a measure of the quantity of the modified albumin in the sample; and  
 c) determining if there is a change in the amount of bound metal ions between the first and subsequent sample(s).  
   
     
     
         2 . The method of  claim 1 , wherein the treatment is selected from the group consisting of surgery, radiation therapy, chemotherapy, reperfusion of an ischemic tissue or organ, and administration of a free radical scavenger.  
     
     
         3 . The method of  claim 1 , wherein said sample is serum or plasma.  
     
     
         4 . The method of  claim 1 , wherein said sample is purified albumin.  
     
     
         5 . The method of  claim 1 , wherein said metal ion salt is a salt of a transition metal ion of Groups 1b-7b or 8 of the Periodic Table of the elements.  
     
     
         6 . The method of  claim 1 , wherein said metal ion salt is a salt of a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag.  
     
     
         7 . The method of  claim 1 , wherein said metal ion salt is a salt of cobalt.  
     
     
         8 . The method of  claim 1 , wherein step (b) is conducted using atomic absorption or atomic emission spectroscopy.  
     
     
         9 . The method of  claim 1 , wherein step (b) is conducted using an immunological assay.  
     
     
         10 . A method of monitoring or assessing a disease or condition in which free radicals play a role comprising: 
 obtaining a first biological sample containing albumin from a patient suffering from the disease or condition;    obtaining one or more additional biological samples of the same type from the same patient at one or more times after obtaining the first sample; and    determining if there is a change in the quantity of a modified albumin present in the first sample as compared to the subsequent sample(s), the modified albumin having a reduced ability to bind metal ions at its N-terminus, the quantity of the modified albumin in each sample being indicative of the amount of free radical damage in that sample, and a change in the quantity of the modified albumin in the first sample as compared to the subsequent sample(s) being indicative of a change in the amount of free radical damage and, therefore, of the status of the disease or condition, the determination being made by: 
 a) contacting each of the biological samples with an excess quantity of a metal ion salt, the metal ion being capable of binding to the N-terminus of unmodified human albumin, to form a mixture of bound metal ions and unbound metal ions;  
 b) determining the amount of bound metal ions for each biological sample, the amount of bound metal ions providing a measure of the quantity of the modified albumin in the sample; and  
 c) determining if there is a change in the amount of bound metal ions between the first and subsequent sample(s).