Of sterols

In milk fat, cholesterol is associated with Hpoproteins in the milk fat globule. It is also a component of animal membranes and controls rigidity and permeabihty of the membranes. Cholesterol has interesting surface properties and can occur in Hquid crystalline forms. Plants contain sterols such as P-sitosterol [83-46-5] (4b) or stigmasterol [83-48-7] (4c). Their functions in plant metaboHsm are not yet well understood. Analysis of sterols has proven useful for detection of adulteration of edible fats (9). 

This represents the first large-scale appHcation of a fluoroaryl organometaOic. Other silicon-containing aryl fluorides such as pentafluorophenyldimethyl silanes, CgF Si(CH2)2X (X = Cl NH2 N(0211 )2), are offered commercially as Flophemsyl reagents for derivati2ation of sterols in chromatographic analysis (166). 

IPP react with each other, releasiag pyrophosphate to form another allyl pyrophosphate containing 10 carbon atoms. The chain can successively build up by five-carbon units to yield polyisoprenes by head-to-tad condensations alternatively, tad-to-tad condensations of two units can yield squalene, a precursor of sterols. Similar condensation of two C2Q units yields phytoene, a precursor of carotenoids. This information is expected to help ia the development of genetic methods to control the hydrocarbon stmctures and yields. 

Bacterial removal of sterol side chains is carried out by a stepwise P-oxidation, whereas the degradation of the perhydrocyclopentanophenanthrene nucleus is prevented by metaboHc inhibitors (54), chemical modification of the nucleus (55), or the use of bacterial mutants (11,56). P-Sitosterol [83-46-5] (10), a plant sterol, has been used as a raw material for the preparation of 4-androstene-3,17-dione [63-05-8] (13) and related compounds using selected mutants of the P-sitosterol-degrading bacteria (57) (Fig. 2). 

Initial steroid research involved isolation of sterols and bile acids from natural sources. DeFourcroy is generally credited with the discovery of cholesterol [57-88-5] (2) in 1789 (3). In 1848, choHc acid [81-25-4] (3) was isolated from the saponification of ox bile and its elementary composition deterrnined as... 

The rate of side-chain cleavage of sterols is limited by the low solubiUty of substrates and products and thek low transport rates to and from cells. Cyclodextrins have been used to increase the solubiUties of these compounds and to assist in thek cellular transport. Cyclodextrins increase the rate and selectivity of side-chain cleavage of both cholesterol and P-sitosterol with no effect on cell growth. Optimal conditions have resulted in enhancement of molar yields of androsta-l,4-diene-3,17-dione (92) from 35—40% to >80% in the presence of cyclodextrins (120,145,146,155). 

Tricyclohexaprenol, a possible forerunner of sterols in the evolution of biomembranes, was synthesized by construction of the cyclic network in one step using cation-olefin tricyclization and subsequent stereocontroUed attachment of the Cio appendage to ring C. 

For the determination of sterols and their esters the chromatogram is immersed in a 10% aqueous copper(II) sulfate solution and then heated to 105°C for 30 min. In this case green-yellow fluorescent chromatogram zones are visible in long-wavelength UV light (X = 365 nm) [6]. 

The analysis of sterols, sterols esters, erythrodiol and uvaol, and other minor components of oils and fats, is usually carried out by normal-phase HPLC-HRGC by using a loop-type interface and the concurrent eluent evaporation technique, as reported in the papers cited by Mondello et al. (48) (up to 1995) and in more recent papers (49, 50). More recently, reversed-phase LC-GC methods have been... 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

Some examples of sterols and steroids are given in Figure 9.1. Also included in this Figure are some examples of bile salts. You should realise that the structures shown are only a few of the many hundreds of compounds which occur in nature. All of these compounds include the steroidal ring structure which is numbered as shown below. 

In the following sections we will explain some applications of enzymes (and cells) in the transformation of sterols and steroids. You should realise, however, that for each process a decision has to be made whether to use an enzyme-mediated transformation or to use a chemical reaction. In many instances the biotransformation process is foe most attractive but, as we will see later, this is not always the case. 

Figure 9.2 Generalised metabolic sequences of sterol side chain degradation by micro-organisms.

Figure 9.3 The figure shows the degradation of the side chain of sterols which have substitutions at C-19. Removal of the C-19 methyl group (eg 19 norchoiesta-1,3,5 (10) triene-3-ol) also prevents ring breakdown. Note, however, hydroxylation of C-19 does not prevent all ring modifications.

Table 9.3 Yieids of 1,4 androstadiene-3,17, dione using a variety of sterols and steroids as substrates and employing cultures of A. simplex (data from Martin CKA Sterols in Biotechnology Vol 6a. Edited by Kiesllch K 1984 Verlag Chemle, Weinheim).

Table 9.4 Some examples of mutants used industrially for the removal of the side chain of sterols.

Many examples of microbial hydroxylation of sterols/steroids have been reported. These hydroxylations usually involve mixed function oxidases which utilise molecular oxygen and cytochrome P-450. The reaction can be represented by ... 

In this chapter, we have examined the use of cells and enzymes to chemically transform lipids. We have had to be selective and have predominantly focused attention on the transformation of sterols and steroids. We first explained why these compounds were commercially important and why they only occur in low concentrations in natural systems. We pointed out that a very large number of reaction types are possible, but those which have found greatest use include stereospedfic hydroxylations, alcohol/ketone interconversion, hydrolysis, conjugation and isomerisation. 

Also, phosphorylation of Akt results in activation of sterol regulatory-element binding protein 1 (SREBP1), a key transcription factor involved in regulation of lipogenic enzymes. In addition, some of the effects of insulin on cell proliferation and survival may be explained by an Akt-dependent inhibition of apoptosis through phosphorylation and inactivation of proa-poptotic proteins (e.g., BAD, Caspase 9). 

WONG c K, KEUNG w M (1997) Daidzein sulfoconjugates are potent inhibitors of sterol sulfatase (EC 3.1.6.2). Biochem Biophys Res Commun. 233 579-83. 

Since carotenoids are derived for the central isoprenoid pathway (Fig. 13.3), the regulation of their formation must involve a co-ordinated flux of isoprenoid imits into this branch of the pathway as well as into others such as the biosynthesis of sterols, gibberellins, phytol and terpenoid quinones. An imderstanding of the complexities of regulation of the pathway is necessary in order to target the regulatory steps for genetic manipulation. 

Although not very commonly used in the separation of nentral hpids, two-dimensional systems have been nsed to separate hydrocarbons, steryl esters, methyl esters, and mixed glycerides that move close to each other in one-dimensional systems. Complex neutral lipids of Biomphalaria glabrata have been first developed in hexane diethyl ether (80 20), dried, and the plates have been turned 90°, followed by the second development in hexane diethyl ether methanol (70 20 10) for complete separation of sterol and wax esters, triglycerides, free fatty acids, sterols, and monoglycerides [54]. 

Chloroform-methanol extracts of Borrelia burgdorferi were used for the identification of lipids and other related components that could help in the diagnosis of Lyme disease [58]. The provitamin D fraction of skin lipids of rats was purified by PTLC and further analyzed by UV, HPLC, GLC, and GC-MS. MS results indicated that this fraction contained a small amount of cholesterol, lathosterol, and two other unknown sterols in addition to 7-dehydrocholesterol [12]. Two fluorescent lipids extracted from bovine brain white matter were isolated by two-step PTLC using silica gel G plates [59]. PTLC has been used for the separation of sterols, free fatty acids, triacylglycerols, and sterol esters in lipids extracted from the pathogenic fungus Fusarium culmorum [60]. 

PTLC was used to enrich the polar fraction of deep-fried potato chips and vegetable oils used in industrial frying operahons. After PTLC, capillary GC, GC-MS, and NMR were used to quantify sterols and sterol oxides in fried-potato products, as well as the composition of sterols in the oil used for frying [72]. 

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