Oxysterols are oxygenated cholesterol molecules that have distinct cell-signaling properties. They are inhibitors of SREBP-2 (sterol response element binding protein-2), which affects cholesterol biosynthesis. Oxysterols are also ligands for the liver X receptors, which regulates cholesterol, glucose and fatty acid homeostasis, allosteric modulators of the NMDA (N-methyl-D-aspartate) receptor, and serve as ligands for proteins within the Hedgehog signaling pathway. Research has shown that the oxysterols may play roles in multiple human pathologies, including ALS; Huntington’s, Alzheimer’s, and Parkinson’s diseases; and some forms of cancer. For a general or a comprehensive review, see Biochemical Society Transactions (2019) 47, 517-526 or Physiological Reviews (2000), 80(1) 361-554, respectively.
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Absolute quantitation is an analytical technique designed to measure, as accurately as possible, the concentration of an analyte within a given matrix. The term “absolute” is a bit of a misnomer—there are many sources of error that affect the final number, including systematic error, matrix-related effects and the nature and purity of the standards used in the experiment. Indeed, most quantitative assays are defined by parameters such as the limit of quantitation (LOQ), the dynamic range of analysis, accuracy, and precision. These values provide context for the term “absolute,” and are used to describe the quantitative power of a validated assay.
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The ability to quickly and accurately measure the total content of individual phospholipid and lysophospholipid classes is important for the characterization of liposomal formulations, nutraceutical preparations, and any other type lipid sample/extract. A traditional approach to quantitation has been separation of the phospholipid classes by TLC followed by quantitation of lipid phosphorus by a colorimetric assay. However, this method is not quick, and accuracy depends on the quality of chromatography and extraction efficiency of the phospholipids from silica. An attractive alternative for quantitation is 31P-NMR.
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The field of lipidomics aims to quantitatively define lipid classes at the molecular species level in biological systems. Mass spectrometry is the primary means by which lipidomics analysis is performed, but there are significant challenges associated with this technique, including isobaric interference, differential ionization and fragmentation of lipids and the need for complex internal standard strategies for quantitation. Lipidomics studies have evolved tremendously over the last 10 years, progressing from sum composition analysis by shotgun analysis to untargeted analysis by HRAM.
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One of the biggest challenges we face in the study of lipids by mass spectrometry is dealing with the overwhelming number of lipid isomers and isobars and trying to address isotopic overlap in a way that enables correct identification and quantitative results. Due to the polymeric nature of lipids, these struggles are the rule and not the exception, and they present unique challenges to lipid analysis.
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Have you ever wondered about claims there are perhaps 150,000 or more different lipid molecular species? Then, when you look in text books, our catalog, or even at your own data, do you get the sense there are only hundreds of species?
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There are two significant challenges in the field that still must be better addressed before lipidomics can become “mainstream: Isobaric Interference and Quantitation (the subject of this post)!
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