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      Quantitative analysis of lipids: a higher-throughput LC–MS/MS-based method and its comparison to ELISA


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          Lipids such as prostaglandins, leukotrienes and thromboxanes are released as a result of an inflammatory episode in pain (central and peripheral).

          Methodology & results:

          To measure these lipids as potential mechanistic biomarkers in neuropathic pain models, we developed a higher-throughput LC–MS/MS-based method with simultaneous detection of PGE2, PGD2, PGF2α, LTB4, TXB2 and 2-arachidonoyl glycerol in brain and spinal cord tissues. We also demonstrate that the LC–MS/MS method was more sensitive and specific in differentiating PGE2 levels in CNS tissues compared with ELISA.


          The ability to modify the LC–MS/MS method to accommodate numerous other lipids in one analysis, demonstrates that the presented method offers a cost–effective and more sensitive alternative to ELISA method useful in drug discovery settings.

          Lay abstract:

          In humans, lipids carry out various functions such as energy production and storage, insulation, digestion and absorption and hormone production. Out of the several lipids, prostaglandins, thromboxanes and leukotrienes play a critical role in cardiovascular diseases, allergic reactions and inflammation. Thus, it is important to monitor their levels as potential mechanistic biomarkers to effectively diagnose and treat the underlying diseases. We have successfully used a highly specific and higher-throughput mass spectrometric method to quantify these lipids in brain cells as well as in brain and spinal cord tissues from rats (pain model) and compared the data obtained in the traditional ELISA.

          Most cited references26

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          A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.

          A peripheral mononeuropathy was produced in adult rats by placing loosely constrictive ligatures around the common sciatic nerve. The postoperative behavior of these rats indicated that hyperalgesia, allodynia and, possibly, spontaneous pain (or dysesthesia) were produced. Hyperalgesic responses to noxious radiant heat were evident on the second postoperative day and lasted for over 2 months. Hyperalgesic responses to chemogenic pain were also present. The presence of allodynia was inferred from the nocifensive responses evoked by standing on an innocuous, chilled metal floor or by innocuous mechanical stimulation, and by the rats' persistence in holding the hind paw in a guarded position. The presence of spontaneous pain was suggested by a suppression of appetite and by the frequent occurrence of apparently spontaneous nocifensive responses. The affected hind paw was abnormally warm or cool in about one-third of the rats. About one-half of the rats developed grossly overgrown claws on the affected side. Experiments with this animal model may advance our understanding of the neural mechanisms of neuropathic pain disorders in humans.
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            Anti-inflammatory therapy in chronic disease: challenges and opportunities.

            A number of widespread and devastating chronic diseases, including atherosclerosis, type 2 diabetes, and Alzheimer's disease, have a pathophysiologically important inflammatory component. In these diseases, the precise identity of the inflammatory stimulus is often unknown and, if known, is difficult to remove. Thus, there is interest in therapeutically targeting the inflammatory response. Although there has been success with anti-inflammatory therapy in chronic diseases triggered by primary inflammation dysregulation or autoimmunity, there are considerable limitations. In particular, the inflammatory response is critical for survival. As a result, redundancy, compensatory pathways, and necessity narrow the risk:benefit ratio of anti-inflammatory drugs. However, new advances in understanding inflammatory signaling and its links to resolution pathways, together with new drug development, offer promise in this area of translational biomedical research.
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              The role of inflammation in CNS injury and disease.

              For many years, the central nervous system (CNS) was considered to be 'immune privileged', neither susceptible to nor contributing to inflammation. It is now appreciated that the CNS does exhibit features of inflammation, and in response to injury, infection or disease, resident CNS cells generate inflammatory mediators, including proinflammatory cytokines, prostaglandins, free radicals and complement, which in turn induce chemokines and adhesion molecules, recruit immune cells, and activate glial cells. Much of the key evidence demonstrating that inflammation and inflammatory mediators contribute to acute, chronic and psychiatric CNS disorders is summarised in this review. However, inflammatory mediators may have dual roles, with detrimental acute effects but beneficial effects in long-term repair and recovery, leading to complications in their application as novel therapies. These may be avoided in acute diseases in which treatment administration might be relatively short-term. Targeting interleukin (IL)-1 is a promising novel therapy for stroke and traumatic brain injury, the naturally occurring antagonist (IL-1ra) being well tolerated by rheumatoid arthritis patients. Chronic disorders represent a greater therapeutic challenge, a problem highlighted in Alzheimer's disease (AD); significant data suggested that anti-inflammatory agents might reduce the probability of developing AD, or slow its progression, but prospective clinical trials of nonsteroidal anti-inflammatory drugs or cyclooxygenase inhibitors have been disappointing. The complex interplay between inflammatory mediators, ageing, genetic background, and environmental factors may ultimately regulate the outcome of acute CNS injury and progression of chronic neurodegeneration, and be critical for development of effective therapies for CNS diseases.

                Author and article information

                Future Sci OA
                Future Sci OA
                Future Science OA
                Future Science Ltd (London, UK )
                March 2017
                16 January 2017
                : 3
                : 1
                : FSO157
                [1 ]Molecular Pharmacology, Bioanalysis & Operations, Lundbeck Research USA, 215 College Road, Paramus, NJ, USA
                [2 ]Neuroinflammation Disease Biology Unit, In Vitro Biology, Lundbeck Research USA, 215 College Road, Paramus, NJ, USA
                Author notes
                *Author for correspondence: adarsh.gandhi@ 123456fda.hhs.gov
                © Adarsh Gandhi

                This work is licensed under a Creative Commons Attribution 4.0 License

                : 30 June 2016
                : 18 October 2016
                Research Article

                elisa,higher-throughput,lc–ms/ms,lipids,neuropathic pain
                elisa, higher-throughput, lc–ms/ms, lipids, neuropathic pain


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