are integral building blocks in the cellular membranes of animals and have important functions in signaling, regulation, and metabolism ( 1 ). To date, the majority of studies have focused on cholesterol, the most abundant sterol in mammals that serves as both a precursor and product to a host of important molecules, including steroid hormones, bile acids, oxysterols, and intermediates in the cholesterol biosynthetic pathway ( 2-4 ). Although cholesterol has gained signifi cant notoriety due to the compound's negative impact on health, other sterols, such as plant-derived phytosterols, are thought to offer potential human health benefi ts by lowering circulating cholesterol levels ( 5, 6 ).Novel functions for sterols and secosteroids continue to be identifi ed. For example, the cholesterol metabolites 25-hydroxycholesterol and 7 ␣ ,25-dihydroxycholesterol have recently been shown to play important signaling roles in the immune system ( 7-9 ). 24-hydroxycholesterol has been shown to be involved in cholesterol turnover in the brain, and it plays a role in memory ( 10 ) and glucose metabolism ( 11 ). Alterations in vitamin D levels arising from the lack of sun exposure and/or use of sunscreen are postulated to have a negative impact on health ( 12, 13 ). These fi ndings together with the large number of sterols suggest that there may be undiscovered roles for sterols in biology, and they highlight the need for continued research into the biochemical pathways associated with these compounds.The extraction and analysis of sterols in human plasma present a unique challenge due to their virtual insolubility, sequestration within lipoproteins, and dramatic differences in the levels of individual sterols. Cholesterol is the most abundant sterol, with circulating levels on the order of 1 to 3 mg/ml, whereas 25-hydroxycholesterol is a millionfold less abundant at 1 to 3 ng/ml ( 14, 15 ). The circulating form of sterols in humans is primarily as steryl esters, in which a fatty acid is esterifi ed to carbon 3 of the sterol; Abstract We describe the development of a method for the extraction and analysis of 62 sterols, oxysterols, and secosteroids from human plasma using a combination of HPLC-MS and GC-MS. Deuterated standards are added to 200 l of human plasma. Bulk lipids are extracted with methanol:dichloromethane, the sample is hydrolyzed using a novel procedure, and sterols and secosteroids are isolated using solid-phase extraction (SPE). Compounds are resolved on C 18 core-shell HPLC columns and by GC. Sterols and oxysterols are measured using triple quadrupole mass spectrometers, and lathosterol is measured using GC-MS. Detection for each compound measured by HPLC-MS was ʜ 1 ng/ml of plasma. Extraction effi ciency was between 85 and 110%; day-to-day variability showed a relative standard error of <10%. Numerous oxysterols were detected, including the side chain oxysterols 22-, 24-, 25-, and 27-hydroxycholesterol, as well as ring-structure oxysterols 7 ␣ -and 4  -hydroxycholesterol. Intermediates from the cholesterol b...
The PGD test detected bacterial contamination in 1:3069 (9 of 27,620) doses released as negative by prestorage culture in PLTs as young as 3 days old. Three contaminated doses, two clinically insignificant, had nonreactive PGD tests, while 0.51% of tests were false positives. Application of this test on day of issue can interdict contaminated units and prevent transfusion reactions.
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