Isoprostanes are a newly discovered class of compounds formed in vivo as a consequence of non-enzymatic oxidation of polyunsaturated fatty acids. The isoprostanes produced by free radical-catalyzed peroxidation of arachidonic acid, so called F2-isoP, were the first to be introduced by Mihelich. The discovery of isoprostanes has important evidences for medicine. Their occurrence has been established as the most predictable marker of oxidative stress, while the measurement of their concentration in biological samples is the most recommended approach to the assessment of redox status in vivo, providing a crucial tool to explore the role of oxidative processes in the pathogenesis of human disease. The elevated levels of F2-isoP have been observed in many conditions associated with increased abundance of reactive oxygen species such as asthma, neurodegenerative diseases (Huntington, Alzheimer or multiple sclerosis), alcoholic and non-alcoholic liver diseases, pulmonary disease or diabetes.
Figure 1 Biological function for isoprostanes.
Except for F2-isoP, there exist other types of isoprostanes found in vivo, including F3-isoP formed from eicosapentaenoic acid (EPA) and the most structurally diverse products of docosahexaenoic acid (DHA) non-enzymatic oxidation. The EPA- and DHA-derived isoprostanes seem to have multiple biological roles, so F2-isoP remains the primary indicators of oxidative stress.
The isoprostanes are usually measured in plasma or urine by well-established gas chromatography–mass spectrometry (GC-MS) and enzyme-linked immunosorbent assays (ELISA). Even if the GC-MS approach is sensitive, the procedure is usually tedious and time-consuming due to the fact that a derivatization step is required prior to separation. Typically, isoprostanes are converted to pentafluorobenzyl esters by treatment with pentafluorobenzyl bromide. The main disadvantage of commercially available ELISA is that a cross-linking reaction can be observed and each assay kit can only measure just one isomer of F2-isoprostanes. However, when sensitivity and selectivity issues in the analysis of biomolecules arise, among various chromatographic methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is optimal to overcome such problems.
The advantages of LC-MS/MS method include relatively higher sensitivity and selectivity and short run times, especially in the case of such samples as e.g., plasma that contain a large amount of coeluting interferences. LC-MS/MS method has been proven to be very useful for simultaneous measurement of different isomers of F2-isoprostanes. What’s more, to achieve accurate quantitation at extremely low concentrations, there is the possibility of using nonradioactive isotope-labeled standards to compensate for the loss of analyte during sample preparation, which has been the most important step for eliminating the matrix effect for analysis of biomarkers. With experienced scientists at Creative Proteomics, we have developed a reliable and reproducible method using highly sensitive LC-MS/MS method for the rapid identification and quantification of diverse isoprostanes in different sample types, which can satisfy the needs of academic and industrial study in your lab.
- Identification and quantification of diverse isoprostanes by LC-MS/MS.
- Normal Volume: 100ul plasma; 50mg tissue; 2e7 cells
- Minimal Volume: 50uL plasma; 30mg tissue; 5e6 cells
- A full report including all raw data, MS/MS instrument parameters and step-by-step calculations will be provided (Excel and PDF formats).
- Analytes are reported as μM or μg/mg (tissue), and CV's are generally<10%.
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Staffed by experienced biological scientists, Creative Proteomics can provide a wide range of services ranging from the sample preparation to the metabolite extraction, characterization, identification and quantification. We are willing to provide our customer the outstanding isoprostanes analysis service with accurate and reliable results, quick turnaround, saving you time and resources! Please feel free to contact us to discuss the wide range of services we can perform.