Oxysterols in plasma

Oxysterols in plasma are usually analyzed by GC-MS. Dzeletovic et al. developed a classical methodology which is still widely adopted today [63]. Oxysterols are found in plasma mostly esterified with fatty acids but also with sulfuric acid and as an acetal with glucuronic acid [64,65]. The protocol developed by Dzeletocvic et al. focused on fatty acids esters, so an initial base hydrolysis step was performed with KOH in ethanol. Following neutralization with phosphoric acid and a Folch-like extraction into CHCI3, oxysterols were separated from other less polar sterols on a silica cartridge. Derivatization was performed with pyridine hexameth-yldisilazane trimethylchlorosilane 3 2 1 (v/v/v) and analysis of the TMS ethers by GC-EI-MS. Nine oxysterols were identified in plasma using this protocol includ-... 

Studies conducted by Barenghi eta.1. (1990) and Lodge etal. (1993) independently have demonstrated the facile, multicomponent analysis of a wide range of PUFA-derived peroxidation products (e.g. conjugated dienes, epoxides and oxysterols) in samples of oxidized LDL by high-field H-NMR spectroscopy. Figure 1.9 shows the applications of this technique to the detection of cholesterol oxidation products (7-ketocholesterol and the 5a, 6a and 5/3,60-epoxides) in isolated samples of plasma LDL pretreated with added coppcr(Il) or an admixture of this metal ion with H2O2, an experiment conducted in the authors laboratories. 

Vine, D.F., Mamo, J.C.L., Beilin, L.J., Mori, T.A., Croft, K.D. 1998. Dietary oxysterols are incorporated in plasma triglyceride-rich lipoproteins, increase their susceptibility to oxidation and increase aortic cholesterol concentration in rabbits. J. Lipid Res. 39, 1995-2004. 

The most abundant oxysterols in human plasma are 27-, 24-, and 7a-hydroxycholesterol (Fig. 5), and most of these are esterified to fatty acids at the 3p position by lecithinxho-lesterol acyltransferase [19]. Both unesterified and esterified oxysterols partition in... 

Weiner MF, Vega GL, Diaz-Arrastia R, Moore C, Madden C, Hudak A, Liitjohann D (2008) Plasma 24S-hydroxycholesterol and other oxysterols in acute closed head injury. Brain Inj 22 611-615... 

K. D. (1998) Dietary Oxysterols Are Incorporated in Plasma Triglyceride-Rich Proteins, Increase Their Susceptibility to Oxidation and Increase Aortic Cholesterol Concentration of Rahhits, J. Lipid Res. 39,1995-2004. 

Vaya, J., M. Aviram, S. Mahmood et al. 2001. Selective distribution of oxysterols in atherosclerotic lesions and human plasma lipoproteins. Q e adic 34 (5) 485-97. 

Table 2 Content of oxysterols in isolated lipoprotein fractions VLDL, LDL, HDL), lipoprotein-free plasma (LFP), and unfractionated plasma (plasma)...

FIGURE 14.8 Sample preparation protocol for the analysis of oxysterols in brain, CSF, or plasma. 

Possible structures of bile alcohol (oxysterol) conjugates found in plasma of a child with neonatal hepatitis of unknown etiology. The location of the hydroxyl groups and conjugating groups are not known however, previous studies of children have shown sulfation... 

Griffiths, W.J., Crick, P.J., Wang, Y., Ogundare, M., Tuschl, K., Morris, A.A., Bigger, B.W. and Clayton, P.T. (2013) Analytical strategies for characterization of oxysterol lipidomes Liver X receptor ligands in plasma. Free Radic. Biol. Med. 59, 69-84. 

Emanuel, H.A. Hassel, C. A., Addis, P.B., Bergmann, S.D., Zavoral, J.H. 1991. Plasma cholesterol oxidation products in human subjects fed a meal rich in oxysterols. J. Food Sci. 56, 843-847. 

The oxysterol 7-ketocholesterol is an important COP involved in atherosclerotic lesions and macrophage foam cells (275). There is no direct evidence in humans that COPs contribute to atherogenesis, but it has been found that COP levels are elevated in LDL subfractions that are considered potentially atherogenic (276). In addition, raised levels of 7p-hydroxycholesterol may be associated with an increased risk of atherosclerosis. Arterial injury by COPs causes endothelial dysfunction and arterial wall cholesterol accumulation (277). Even under normocholesterolemic conditions, COPs can cause endothelial dysfunction, increased macromolecular permeability, and increased cholesterol accumulation. These are all factors believed to be involved in the development of atherosclerotic lesions. The atherogenic potential of COPs has been demonstrated by in vitro cell culture (73, 278), as well as in animal feeding studies (279). Japanese quail fed either purified cholesterol or oxidized cholesterol exhibited greater plasma and liver cholesterol concentrations in association with increased severity of atherosclerotic lesions when fed the oxidized cholesterol (279). 

There is experimental evidence that suggests that some oxysterols, but not pure cholesterol, are the prime cause of atherosclerotic lesion formation (162). Upon cholesterol feeding, a strong relationship was seen between plasma oxysterols and aortic wall oxysterols. One may speculate that the deposition of pure lipids, such as cholesterol and its esters, may be merely a secondary process in response to oxysterol-induced endothelial cell injury. Cell injury/dysfunction and the subsequent disruption of endothelial barrier function by oxysterols (163, 164) could initiate the early events in atherosclerosis. Such injury could allow increased uptake... 

Figure 10. The total cholesterol level in blood plasma of rabbits fed with oxidized or non-oxidized cholesterol (1), intact animals (2), animals fed with purified cholesterol (3), animals fed with cholesterol preparation which contain about 5% oxysterols.

Essential non-steroidal isoprenoids, such as dolichol, prenylated proteins, heme A, and isopentenyl adenosine-containing tRNAs, are also synthesized by this pathway. In extrahepatic tissues, most cellular cholesterol is derived from de novo synthesis [3], whereas hepatocytes obtain most of their cholesterol via the receptor-mediated uptake of plasma lipoproteins, such as low-density lipoprotein (LDL). LDL is bound and internalized by the LDL receptor and delivered to lysosomes via the endocytic pathway, where hydrolysis of the core cholesteryl esters (CE) occurs (Chapter 20). The cholesterol that is released is transported throughout the cell. Normal mammalian cells tightly regulate cholesterol synthesis and LDL uptake to maintain cellular cholesterol levels within narrow limits and supply sufficient isoprenoids to satisfy metabolic requirements of the cell. Regulation of cholesterol biosynthetic enzymes takes place at the level of gene transcription, mRNA stability, translation, enzyme phosphorylation, and enzyme degradation. Cellular cholesterol levels are also modulated by a cycle of cholesterol esterification mediated by acyl-CoA cholesterol acyltransferase (ACAT) and hydrolysis of the CE, by cholesterol metabolism to bile acids and oxysterols, and by cholesterol efflux. 

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