Method of detecting isolevuglandin phospholipid adducts
A method of measuring isolevuglandin and/or levuglandin adducts associated with oxidative injury in a subject includes obtaining a bodily sample from a subject suspected of including isolevuglandin and/or levuglandin phospholipid adducts, hydrolyzing isolevuglandin and/or levuglandin phospholipid adducts from the sample with an enzyme that forms isolevuglandin and/or levuglandin phospholipid derivatives, and determining the amount of isolevuglandin and/or levuglandin phospholipid derivatives by mass spectrometry.
1. A method of measuring isolevuglandin and/or levuglandin ethanolamine phospholipid adducts associated with oxidative injury in a subject, the method comprising:
obtaining a bodily sample suspected of including isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from a subject,
selectively hydrolyzing isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from the sample with a phospholipase A2 (PLA2) enzyme that forms isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives, and
determining the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives by mass spectrometry, wherein the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives is determinative of the level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the sample and indicative of the level of oxidative injury in the subject, and wherein an increased level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject compared to a control level is indicative of an increased level of oxidative injury in the subject.
2. The method of claim 1 , wherein the isolevuglandin and/or levuglandin ethanolamine phospholipid adducts are extracted from the bodily sample.
3. The method of claim 2 , wherein isolevuglandin and/or levuglandin ethanolamine phospholipid adducts are extracted from the bodily sample in the presence of at least one of a chelating agent or antioxidant to inhibit oxidation of the bodily sample.
4. The method of claim 3 , the chelating agent or antioxidant comprising at least one of ethylenediaminetetraacetic acid (EDTA) or butylated hydroxytoluene (BHT).
5. The method of claim 1 , the bodily sample comprising at least one of blood, plasma, sera, saliva, mucous, synovial fluid, cerebrospinal fluid, urine, stool, cells, a cellular extract, a tissue sample, or a tissue biopsy.
6. The method of claim 1 , the bodily sample comprising liver tissue, and the method measuring ethanol induced oxidative liver injury in a subject.
7. The method of claim 1 , wherein the control level comprises a level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in a normal or healthy subject or tissue.
8. The method of claim 1 , wherein the control level comprises a previously measured level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject or tissue of the subject.
9. A method of measuring isolevuglandin and/or levuglandin ethanolamine phospholipid adducts associated with oxidative injury in a subject, the method comprising:
obtaining a bodily sample suspected of including isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from a subject,
extracting lipids from said bodily sample,
selectively hydrolyzing isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from the extracted sample with a phospholipase A2 (PLA2) enzyme to form isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives, and
determining the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives by mass spectrometry, wherein the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives is determinative of the level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the sample and indicative of the level of oxidative injury in the subject, and wherein an increased level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject compared to a control level is indicative of an increased level of oxidative injury in the subject.
10. The method of claim 9 , wherein isolevuglandin and/or levuglandin ethanolamine phospholipid adducts are extracted from the bodily sample in the presence of at least one of a chelating agent or antioxidant to inhibit oxidation of the bodily sample.
11. The method of claim 10 , the chelating agent or antioxidant comprising at least one of ethylenediaminetetraacetic acid (EDTA) or butylated hydroxytoluene (BHT).
12. The method of claim 9 , the bodily sample comprising at least one of blood, plasma, sera, saliva, mucous, synovial fluid, cerebrospinal fluid, urine, stool, cells, a cellular extract, a tissue sample, or a tissue biopsy.
13. The method of claim 9 , the bodily sample comprising liver tissue, and the method measuring ethanol induced oxidative liver injury in a subject.
14. The method of claim 9 , wherein the control level comprises a level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in a normal or healthy subject or tissue.
15. The method of claim 9 , wherein the control level comprises a previously measured level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject or tissue of the subject.
16. A method of measuring oxidative damage in a subject, the method comprising:
obtaining a bodily sample suspected of including isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from a subject,
extracting lipids from said bodily sample in the presence of chelating agent or antioxidant,
selectively hydrolyzing isolevuglandin and/or levuglandin ethanolamine phospholipid adducts from the extracted sample with a phospholipase A2 (PLA2) enzyme to form isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives, and
determining the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives by mass spectrometry, wherein the amount of isolevuglandin and/or levuglandin 2-lysoethanolamine phospholipid derivatives is determinative of the level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the sample and indicative of the level of oxidative damage in the subject, and wherein an increased level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject compared to a control level is indicative of an increased level of oxidative injury in the subject.
17. The method of claim 16 , the chelating agent or antioxidant comprising at least one of ethylenediaminetetraacetic acid (EDTA) or butylated hydroxytoluene (BHT).
18. The method of claim 16 , the bodily sample comprising at least one of blood, plasma, sera, saliva, mucous, synovial fluid, cerebrospinal fluid, urine, stool, cells, a cellular extract, a tissue sample, or a tissue biopsy.
19. The method of claim 16 , the bodily sample comprising liver tissue, and the method measuring ethanol induced oxidative liver damage in the liver of the subject.
20. The method of claim 16 , wherein the control level comprises a level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in a normal or healthy subject or tissue.
21. The method of claim 16 , wherein the control level comprises a previously measured level of isolevuglandin and/or levuglandin ethanolamine phospholipid adducts in the subject or tissue of the subject.
22. The method of claim 1 , the isolevuglandin and/or levuglandin ethanolamine phospholipid adducts comprising isolevuglandin and/or levuglandin phosphatidylethanolamine adducts.
23. The method of claim 9 , the isolevuglandin and/or levuglandin ethanolamine phospholipid adducts comprising isolevuglandin and/or levuglandin phosphatidylethanolamine adducts.
24. The method of claim 16 , the isolevuglandin and/or levuglandin ethanolamine phospholipid adducts comprising isolevuglandin and/or levuglandin phosphatidylethanolamine adducts.