Enzyme method for detecting lysophospholipids and phospholipids and for detecting and correlating conditions associated with altered levels of lysophospholipids

Abstract

The present invention is an enzymatic method and diagnostic kits for detecting and quantifying the presence of one or more lysophospholids in a sample of bodily fluid taken from a test subject. The method uses enzymes in a two step assay and may be used to detect disease conditions associated with altered levels of lysophospholipids and to correlate such conditions with altered levels of lysophospholipids.

what is claimed:  
     
         1 . A method to detect disease in a patient comprising: 
 digesting a phospholipid in a sample of bodily fluid from the subject with a first enzyme to produce substrate;    reacting the substrate with a second enzyme in an enzymatic cycling reaction to produce a detectable product;    determining the concentration of phospholipid by measuring the detectable product; and    correlating the concentration of phospholipid to the disease condition by comparison to a normal concentration.    
     
     
         2 . The method of  claim 1 , wherein said first enzyme is selected from the group consisting of phospholipase B, lysophospholipase, phospholipase A 1 , and phospholipase A 2 .  
     
     
         3 . The method of  claim 1 , wherein the second enzyme is selected from the group consisting of glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate oxidase, glycerokinase and glycerol dehydrogenase.  
     
     
         4 . The method of  claim 1 , wherein the substrate is glycerol-3-phosphate.  
     
     
         5 . The method of  claim 1 , wherein the detectable product is hydrogen peroxide.  
     
     
         6 . The method of  claim 5 , wherein the step of determining the concentration of phospholipid by measuring detectable product comprises measuring an increase in hydrogen peroxide by colorimetry.  
     
     
         7 . The method of  claim 1 , wherein the detectable product is NADH.  
     
     
         8 . The method of  claim 7 , wherein said step of determining the concentration of the phospholipid by measuring the detectable product comprises measuring oxidation of NADH.  
     
     
         9 . The method of  claim 1 , wherein the step of reacting the substrate in an enzyme cycling reaction comprises reacting G-3-P with glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase.  
     
     
         10 . The method of  claim 1 , wherein, the sample of bodily fluid is selected from the group consisting of plasma, serum, urine, saliva, ascites, cerebral spinal fluid and pleural fluid.  
     
     
         11 . The method of  claim 1 , further comprising the step of extracting lipids from the sample of bodily fluid.  
     
     
         12 . The method of  claim 1 , further comprising the step of comparing the concentration of phospholipid with an earlier concentration from the same subject.  
     
     
         13 . The method of  claim 1 , wherein an increase or decrease in the concentration of phospholipid relative to normal subjects indicates the presence of the disease condition.  
     
     
         14 . The method of  claim 1 , wherein the disease condition is gynecological cancer or ovarian cancer.  
     
     
         15 . The method of  claim 1 , wherein the disease condition is a blood disorder associated with alteration in the level of phospholipid.