Cholesterol enzymatic oxidation

A steroid very closely related structurally to cholesterol is its 7 dehydro derivative 7 Dehydrocholesterol is formed by enzymatic oxidation of cholesterol and has a conju gated diene unit m its B ring 7 Dehydrocholesterol is present m the tissues of the skin where it is transformed to vitamin D3 by a sunlight induced photochemical reaction... 

ALTERNATE PROTOCOL 2 ENZYMATIC MEASUREMENT OF CHOLESTEROL Test combination kits for enzymatic determination of cholesterol in food are now commercially available. For the determination of total cholesterol, esterified cholesterol is hydrolyzed to free cholesterol and fatty acid under mild alkaline conditions. Cholesterol oxidase oxidizes free cholesterol to cholest-4-en-3-one to generate hydrogen peroxide, which further oxidizes methanol to formaldehyde. Formaldehyde then reacts with acetyl acetone in the presence of NH4+ ions to form yellow lutidine dye, which is subsequently determined spectrophotometric al 1 y. 

The structural relationship between phytosterols and BRs has been proposed from the biosynthetic points of view. All naturally occurring BRs possess carbon skeletons identical to those of common phytosterols (e.g., campesterol, 24-methylene-cholesterol, isofiicosterol, sitosterol, and cholesterol). Thus, BRs may be speculatively regarded as the enzymatic oxidation products of phytosterols with the corresponding carbon skeletons, as is the case of the biosynthesis of other steroid hormones (e.g., ecdysteroids (25) and 1,25-dihydroxyvitamin D3 (26)). Although BL has recently been proved to be biosynthesized from CS in crown gall cells of Catharanthus roseus (27), a major part of the biosynthesis of BRs is remained to be investigated. Experiments using radio-labeled precursors are required to clarify the biosynthesis of BRs in a suitable plant system. 

The major bioactive products of fatty acid metabolism relevant to atherosclerosis are those that result from enzymatic or non-enzymatic oxidation of polyunsaturated long-chain fatty acids. In most cases, these fatty acids are derived from phospholipase A2-mediated hydrolysis of phospholipids (Chapter 11) in cellular membranes or lipoproteins, or from lysosomal hydrolysis of lipoproteins after internalization by lesional cells. In particular, arachidonic acid is released from cellular membrane phospholipids by arachidonic acid-selective cytosolic phospholipase Aj. In addition, there is evidence that group II secretory phospholipase Aj (Chapter 11) hydrolyzes extracellular lesional lipoproteins, and lysosomal phospholipases and cholesterol esterase release fatty acids from the phospholipids and CE of internalized lipoproteins. Indeed, Goldstein and Brown surmised that at least one aspect of the atherogenicity of LDL may lie in its ability to deliver unsaturated fatty acids, in the form of phospholipids and CE, to lesions (J.L. Goldstein and M.S. Brown, 2001). 

Dietary intake of n-6 fatty acids such as linoleic acid, and n-3 fatty acids, such as the fish oils eicosapentanoic acid and docosahexaenoic acid, lowers plasma cholesterol and antagonizes platelet activation, but the fish oils are much more potent in this regard [26]. In particular, n-3 fatty acids competitively inhibit thromboxane synthesis in platelets but not prostacyclin synthesis in endothelial cells. These fatty acids have also been shown to have other potentially anti-atherogenic effects, such as inhibition of monocyte cytokine synthesis, smooth muscle cell proliferation, and monocyte adhesion to endothelial cells. While dietary intake of n-3 fatty acid-rich fish oils appears to be atheroprotective, human and animal dietary studies with the n-6 fatty acid linoleic acid have yielded conflicting results in terms of effects on both plasma lipoproteins and atherosclerosis. Indeed, excess amounts of both n-3 and n-6 fatty acids may actually promote oxidation, inflammation, and possibly atherogenesis (M. Toberek, 1998). In this context, enzymatic and non-enzymatic oxidation of linoleic acid in the sn-2 position of LDL phospholipids to 9- and 13-hydroxy derivatives is a key event in LDL oxidation (Section 6.2). 

This chapter considers the nutritional consequences of polyunsaturated lipids in the diet on free radical reactions in biological systems and diseases. The term lipid peroxidation is now used broadly to include both non-enzymatic and enzymatic oxidative reactions of free fatty acids, phospholipids, triacylglycerols, cholesterol and cholesteryl esters, lipoproteins, proteins,... 

SCHEME 12.7 Enzymatic oxidation of cholesterol to cholestenone by cholesterol oxidase. 

Randolph, T., Clark, D., Blanch, H., etal. (1988). Enzymatic oxidation of cholesterol aggregates in snpercritical carbon dioxide. Science, 239, pp. 387-390. 

Enzymatic oxidation in a supercritical fluid medium has also been demonstrated for the formation of cholest-4-ene-3-one from cholesterol in supercritical carbon dioxide by Randolph et aL [32]. Many cholesterol oxidases showed catalytic activity. That isolated from Streptomyces spp. gradually degraded, but gave reaction rates comparable with those sustained in aqueous systems whilst it was yet catalytic. That from Gloecysticum chrysocrea is chemically stable at 35" C and 100 bar, and yielded reaction rates 75 times faster than in water (5 x 10 phosphate, pH 7, and 5% v/v propanol [34]) which were further increased by the addition of aggregating cosolvents. 

Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Belkner et al. [32] demonstrated that 15-LOX oxidized preferably LDL cholesterol esters. Even in the presence of free linoleic acid, cholesteryl linoleate continued to be a major LOX substrate. It was also found that the depletion of LDL from a-tocopherol has not prevented the LDL oxidation. This is of a special interest in connection with the role of a-tocopherol in LDL oxidation. As the majority of cholesteryl esters is normally buried in the core of a lipoprotein particle and cannot be directly oxidized by LOX, it has been suggested that LDL oxidation might be initiated by a-tocopheryl radical formed during the oxidation of a-tocopherol [33,34]. Correspondingly, it was concluded that the oxidation of LDL by soybean and recombinant human 15-LOXs may occur by two pathways (a) LDL-free fatty acids are oxidized enzymatically with the formation of a-tocopheryl radical, and (b) the a-tocopheryl-mediated oxidation of cholesteryl esters occurs via a nonenzymatic way. Pro and con proofs related to the prooxidant role of a-tocopherol were considered in Chapter 25 in connection with the study of nonenzymatic lipid oxidation and in Chapter 29 dedicated to antioxidants. It should be stressed that comparison of the possible effects of a-tocopherol and nitric oxide on LDL oxidation does not support importance of a-tocopherol prooxidant activity. It should be mentioned that the above data describing the activity of cholesteryl esters in LDL oxidation are in contradiction with some earlier results. Thus in 1988, Sparrow et al. [35] suggested that the 15-LOX-catalyzed oxidation of LDL is accelerated in the presence of phospholipase A2, i.e., the hydrolysis of cholesterol esters is an important step in LDL oxidation. 

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