Sterols and Oxysterols

Both 2-chloro fatty aldehydes and fatty alcohols can be quantitatively analyzed by classical GC-MS approaches after derivatization with pentafluorobenzoyl hydrox-ylamine and pentafluorobenzoyl chloride, respectively [40, 41]. Bioactive 2-chloro FA species can be readily ionized in the negative-ion mode of ESl-MS as described in Chapter 2 to form deprotonated molecular ions. Product-ion ESI analysis of these deprotonated molecular ions after CID shows the following fragmentation pattern [45]  

Therefore, bioactive 2-chloro FA species are readily identified and quantified by LC-MS/MS in the MRM mode utilizing these characteristic fragment ions [40,41]. 

Numerous sterols and oxysterols are present in nature [46], produced through enzymatic or nonenzymafic reactions. The majority of these compounds play many essential roles in biological systems. For example, numerous sterols are involved in the biosynthesis and metabolism of cholesterol [47,48]. GC-MS is used to be the widely used tool for analysis of these sterols and their metabolites after derivatization [48, 49]. Nowadays, LC-MS and LC-MS/MS after ESI become as the popular tool of choice [49]. 

FRAGMENTATION PATTERNS OF OTHER BIOACTIVE LIPID METABOLITES 

It should be recognized that since the majority of these sterol compounds are relatively hydrophobic, direct ionization of these compounds by ESI is not sensitive. Moreover, many of the sterol species carry at least one hydroxyl group. Detection of quasimolecular ions of these sterol species as the form after loss of water is very common. To enhance the sensitivity and stabilize the original structure of a sterol species, appropriate derivatization is commonly employed for characterization and elucidation of sterol structures. Readers who are interesting in this area of work may consult the work of Dr Griffiths and colleagues [50-53]. 

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Spann, N. J. Glass, C. K., Sterols and oxysterols in immune cell function,... 

Isoprenoid synthesis is regulated by sterol and non-sterol components of the biosynthetic pathway, oxysterols, and also by physiological factors. The cholesterol content of the... 

Additionally, cholesterol oxidation can occur in in vivo by enzymes or by lipid peroxidation. Enzymatic formation of oxysterols can be divided into two classes (a) direct enzymatic action on cholesterol or another related sterol and (b) enzymatic activity leading to the formation of radicals, which in turn attack cholesterol. All reactions of the first type seem at present to be cytochrome P-450 dependent [16]. 

Add plasma in 70% ethanol (1.5 mL) to the column, and allow to flow at 0.25 mL/min (if necessary, apply back pressure via the Leur-lock syringe). Collect the flow through and combine with column wash of 1.5 mL of 70% ethanol in a 15 mL polypropylene centrifuge tube (Corning Inc. Amsterdam, The Netherlands). Steroids, oxy-sterols, and bile acid elute in this fraction (SPEl-Fr-1). Elute less polar oxysterols from the column... 

Oxysterols are reported to act as potent inhibitors of growth in mammalian cell systems because of their interference with sterol biosynthesis (13). Upon superficial examination, brassinosteroids could be regarded as just highly oxygenated sterols. Therefore, two oxysterols (24-ketolanosterol and 7-... 

Squalene is converted into the first sterol, lanosterol, by the action of squalene epoxidase and oxidosqualene lanosterol cyclase. The catalytic mechanism for the cyclase s four cyclization reactions was revealed when the crystal stmcture of the human enzyme was obtained (R. Thoma, 2004). Oxidosqualene lanosterol cyclase is considered an attractive target for developing inhibitors of the cholesterol biosynthetic pathway because its inhibition leads to the production of 24,25-epoxycholesterol (M.W. Huff, 2005). This oxysterol is a potent ligand activator of the liver X receptor (LXR) and leads to expression of several genes that promote cellular cholesterol efflux, such as ABCAl, ABCG5, and ABCG8 (Section 4.1). Thus, inhibitors of oxidosqualene lanosterol cyclase could be therapeutically advantageous because they would reduce cholesterol synthesis and promote cholesterol efflux (M.W. Huff, 2005). 

Excess cholesterol can also be metabolized to CE. ACAT is the ER enzyme that catalyzes the esterification of cellular sterols with fatty acids. In vivo, ACAT plays an important physiological role in intestinal absorption of dietary cholesterol, in intestinal and hepatic lipoprotein assembly, in transformation of macrophages into CE laden foam cells, and in control of the cellular free cholesterol pool that serves as substrate for bile acid and steroid hormone formation. ACAT is an allosteric enzyme, thought to be regulated by an ER cholesterol pool that is in equilibrium with the pool that regulates cholesterol biosynthesis. ACAT is activated more effectively by oxysterols than by cholesterol itself, likely due to differences in their solubility. As the fatty acyl donor, ACAT prefers endogenously synthesized, monounsaturated fatty acyl-CoA. 

Fig. 4. The bile acid biosynthetic pathways. The classical pathway operates entirely in the liver and cholesterol 7a-hydroxylase (CYP7A1) initiates the pathway. In other tissues, the entry of cholesterol into the alternate pathways is facilitated by sterol 27-hydroxylase (CYP27A1), cholesterol 24-hydroxylase (CYP46A1), and cholesterol 25-hydroxylase (CH25H). The oxysterols generated by these enzynies are 7a-hydroxylated by oxysterol 7a-hydroxylases CYP7B1 and CYP39A1, and the products enter the latter steps of the classical pathway.

The existence of an alternate pathway for the synthesis of bile acids was suspected because it was possible for oxysterols to be converted into bile acids (N. Wachtel, 1968). It is now recognized that a variety of oxysterols produced by an assortment of cell types can be converted into bile acids. The production of these oxysterols is catalyzed by several sterol hydroxylases sterol 27-hydroxylase (CYP27A1) (J.J. Cali, 1991), cholesterol 25-hydroxylase (CH25H) (E.G. Lund, 1998), and cholesterol 24-hydroxylase (CYP46A1) (E.G. Lund, 1999). Cholesterol 25-hydroxylase is not a cytochrome P-450 monooxygenase, unlike the two other enzymes. Almost all of the 24-hydroxycholesterol that ends up in the liver originates from the brain, and it has been suggested that the production of... 

Oxysterols have diverse roles in cholesterol efflux, a critical topic in foam cell biology. On the one hand, cells incubated with 7-ketocholesterol and 25-hydroxycholesterol have decreased cholesterol efflux. Possible mechanisms include inhibition of membrane desorption of cholesterol or phospholipids or, as mentioned above, inhibition of lysosomal sphingomyelinase leading to lysosomal sequestration of cholesterol (M. Aviram, 1995). On the other hand, the conversion of cholesterol by macrophage sterol 27-hydroxylase to 27-hydroxycholesterol and 3[l-hydroxy-5-cholestenoic acid, which are efficiently effluxed from cells, has been proposed to promote sterol efflux from foam cells (1. Bjorkhem,... 

A number of oxysterols, which suppress HMGR when added to cell cultures, are natural precursors of cholesterol or products of its metabolism to bile acids or steroid hormones [18]. These sterols include the bile acid intermediates 7a- and 26-hydroxycholesterol, the steroid hormone precursors 20a- and 22R-hydroxycholesterol. 24(s)-hydroxycholesterol (cerebrosterol) was foimd by Kandutch et al [18] that other oxysterol metabolites capable of repressing the reductase may also be produced. Kandutch et al [18] foimd several unidentified oxysterols capable of suppressing activity in extracts of cultured cells. Inhibitory oxysterols are, therefore, produced in all cells that synthesize cholesterol as an end product, as well as in cells that synthesize cholesterol and metabolize it to other steroid products. 

The expression cholesterol oxidation products (COP) or oxysterols refers to a group of sterols similar in structure to cholesterol but containing an additional hydroxyl, ketone, or epoxide group, on the sterol nucleus or a hydroxyl group on the side chain of the molecule. Table 6.2 presents the names of most prominent COP formed in foods, plasma, and tissues. 

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