Sterols and steryl esters

Although sterols and steryl esters constitute some of the simplest lipid molecules, complications arise when these molecules must be isolated and identified from complex biological matrices. Both GC/MS and LC/MS have been effective (Evershed, 1994 Moving, 1995). However, problems arise when the multitude of combinations of ring and fatty chain peroxidation products in the steryl esters must also be considered. 

Recently, LC/MS has made significant contributions in the analysis of sterols and steryl esters. Takatsu and Nishi (1993) have employed discharge-assisted LC/TS/MS for the determination of total serum cholesterol. The method incorporates stable isotope dilution using [3,4- C] cholesterol as an internal standard. [MM — H20] ions were monitored by the SIM method. Satisfactory agreement between the analytical result and the certified value of the National Institute of Standards and Technology standard reference material was obtained with a relative standard deviation of 0.6%. The method does not require sterol derivatization. Yang et al. (1992) used FAB/MS to identify cholesteryl sulphate m/z 465) as the [M — H] ion recovered from the appropriate TLC fraction. 

Interesting applications of GC-MS and LC-MS methods may be found in studies of the diverse marine oxylipids (Gerwick, 1996, and references cited therein). 

Sevanian et al. (1994) applied GLC and LC/TS/MS for the analysis of plasma cholesterol-7-hydroperoxides and 7-ketocholesterol. Analysis of human and rabbit plasma identified the commonly occurring oxidation products, yet dramatic increases in 7-ketocholesterol and cholesterol-5p, 6P-epoxide were observed. The study failed to reveal the presence of choles-terol-7-hydroperoxides, which were either too unstable for isolation, metabolized or further decomposed. The principal ions of cholesterol oxides monitored by LC/TS/MS were m/z 438 (cholestane triol) m/z 401 (cholesterol-7-hydroperoxide) m/z 401 (7-ketocholesterol) m/z 367 (7a-hydroxycholesterol) m/z 399 (cholesta-3,5-dien-7-one) and m/z 385 (choles-terol-5a,6a-epoxide). The major ions were supported by minor ions consistent with the steroid structure. Kamido et al. (1992a, b) synthesized the cholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as stable secondary oxidation products of cholesteryl arachidonate and linoleate, respectively. These compounds were identified as the 3,5-dinitrophenylhydrazone (DNPH) derivatives by reversed-phase LC/NICI/MS. These standards were used to identify cholesteryl and 7-ketocholesteryl 5-oxovaleroyl and 9-oxononanoyl esters as major components of the cholesteryl ester core aldehydes generated by copper-catalysed peroxidation of low-density lipoprotein (LDL). In addition to 9-oxoalkanoate (major product), minor amounts of the 8, 9, 10, 11 and 12 oxo-alkanoates were also identified among the peroxidation products of cholesteryl linoleate. Peroxidation of cholesteryl arachidonate yielded the 4, 6, 7, 8, 9 and 10 oxo-alkanoates of cholesterol as minor products. The oxysterols resulting from the peroxidation of the steroid ring were mainly 7-keto, 7a-hydroxy and 7P- 

Bortolomeazzi et al. (1994) used GC/EI/MS with an ion trap to identify the thermal oxidation products of cholesteryl acetate as the 7P-hydroperoxy and 7a-hydroperoxy cholesteryl acetate, 7keto-cholesteryl acetate, the a and P isomers of 7-hydroxycholesteryl acetate, the a- and P-5,6-epoxy isomers and several derivatives arising from the loss of acetate and water. Dzeletovic et al. (1995b) have observed that saponification during sample preparation did not hydrolyse all of the oxysterol esters completely and that separation of oxysterols from cholesterol by HPLC was tedious and incomplete. They developed a stable isotope dilution GC/EI/MS SIM method for the determination of cholesterol oxidation products in human plasma. Nine oxysterols were determined by using deuterium-labelled internal standards. 

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Note Silica gel, kieselguhr and polyamide layers can be used as stationary phases. Not all acids are stained on RP layers. Amino layers yield a pale blue background. The detection limits are in the pg range for carboxylic acids [1], thioglycolic and dithioglycolic acids [2] and for antithyroid pharmaceuticals [4] they are about 5 ng per chromatogram zone for sterols and steryl esters [6]. 

Standard methods of analysis of total sterol content of oils involve saponification of the oil, followed by extraction and isolation of total sterols from the unsaponihable fraction by thin layer chromatography (TLC) (AOCS, 1998). Quantification of individual sterols involves silylation of the sterol fraction and analysis by gas chromatography (GC). Sterols and steryl esters in oils and fats can be analysed by LC-GC after silylation or acylation of the free sterols (Artho et al., 1993). An alternative approach to the analysis of intact steryl esters involves separation of sterols and steryl esters by solid phase... 

Bile Acids, Sterols, and Steryl Esters Many of these compounds have high melting points, making them unsuitable for melt applications. Exceptions are the salicylic acid-cholic acid system formed by a melt-granulation technique reported by Froemming and Vetter.f The system provided a sustained release in acid media (pH 1-3.5). 

Ferrari R.Ap. W. Esteves K.D. Mukherjee E. Schulte. Alteration of sterols and steryl esters in vegetable oils during industrial refining./. Agric. Food Chem. 1997, 45, 4753-4757. 

Weber, N. and Mukheijee, K.D., Plant sterols and steryl esters in functional foods and nutracuticals, in Nutraceutical and Specialty Lipids and Their Co-Products, Shahidi, F, Ed., CRC Press, Taylor Francis Group, Boca Raton, FL, 2006, pp. 483—508. 

Content of Free Sterols, Esterified Sterols and Steryl Esters in Oils from Brassica campestris and B, napus Seeds ... 

Free sterols and steryl esters released by parasites... 

H611 W, Goller I 1982 Free sterols and steryl esters in the trunkwood of Picea abies (L.) Karst. Z Pflanzenphysiol 106 409-418... 

TOTAL STEROL CONCENTRATION (yg sterol and steryl ester/mg dry weight cells)... 

Mercer, Studies on the sterols and steryl esters of the intracellular organelles of maize shoots. Biochem. J. 110 II9... 

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]. 

The overall metabolism of vitamin A in the body is regulated by esterases. Dietary retinyl esters are hydrolyzed enzymatically in the intestinal lumen, and free retinol enters the enterocyte, where it is re-esterified. The resulting esters are then packed into chylomicrons delivered via the lymphatic system to the liver, where they are again hydrolyzed and re-esterified for storage. Prior to mobilization from the liver, the retinyl esters are hydrolyzed, and free retinol is complexed with the retinol-binding protein for secretion from the liver [101]. Different esterases are involved in this sequence. Hydrolysis of dietary retinyl esters in the lumen is catalyzed by pancreatic sterol esterase (steryl-ester acylhydrolase, cholesterol esterase, EC 3.1.1.13) [102], A bile salt independent retinyl-palmitate esterase (EC 3.1.1.21) located in the liver cell plasma hydrolyzes retinyl esters delivered to the liver by chylomicrons. Another neutral retinyl ester hydrolase has been found in the nuclear and cytosolic fractions of liver homogenates. This enzyme is stimulated by bile salts and has properties nearly identical to those observed for... 

The intestinal absorption of dietary cholesterol esters occurs only after hydrolysis by sterol esterase steryl-ester acylhydrolase (cholesterol esterase, EC 3.1.1.13) in the presence of taurocholate [113][114], This enzyme is synthesized and secreted by the pancreas. The free cholesterol so produced then diffuses through the lumen to the plasma membrane of the intestinal epithelial cells, where it is re-esterified. The resulting cholesterol esters are then transported into the intestinal lymph [115]. The mechanism of cholesterol reesterification remained unclear until it was shown that cholesterol esterase EC 3.1.1.13 has both bile-salt-independent and bile-salt-dependent cholesterol ester synthetic activities, and that it may catalyze the net synthesis of cholesterol esters under physiological conditions [116-118], It seems that cholesterol esterase can switch between hydrolytic and synthetic activities, controlled by the bile salt and/or proton concentration in the enzyme s microenvironment. Cholesterol esterase is also found in other tissues, e.g., in the liver and testis [119][120], The enzyme is able to catalyze the hydrolysis of acylglycerols and phospholipids at the micellar interface, but also to act as a cholesterol transfer protein in phospholipid vesicles independently of esterase activity [121],... 

Sterol acyltransferase (SGTase) and steryl ester hydrolase (SEHase)... 

Bouvier-Nave, R and Benveniste, P. (1995) Sterol acyl transferase and steryl ester hydrolase activities in a tobacco mutant which overproduces sterols. Plant Sci, 110, 11-9. 

Table I. Relative percentages of sterols in free sterol (FS) and steryl ester sterols...

Lanolin steryl ester determinations Lanolin is the wool grease secreted by the sheep sebaceous glands, and represents a complex mixture of high molecular mass lipids, including fatty acids and alcohols, sterols, hydroxyacids, diols, and aliphatic and steryl esters. Lanolin is widely used in cosmetic and pharmaceutical formulations. The study of minor lipids... 

We investigated the ratio of free sterol to steryl ester in auxotrophic cells. Although the principal sterol of yeast is ergosterol, we have found that cholesterol can satisfy the sterol growth requirement of adapted yeast sterol auxotrophs and is readily esterified. We chose to use cholesterol for these experiments because of its stability against oxidative degradation and availability as a high purity radiolabeled compound. 

Figure 2 Relationship between free sterol concentration and steryl ester content of yeast cells cultured aerobically.

Qualitative thin layer chromatography of neutral lipids revealed one dominant spot of free sterols and more faint spots of triacylglycerols, free fatty acids and steryl esters (not shown here). Total mol phospholipids decreased after one stress period to 83 % of the control and decreased after three stress periods to 50 %. The phospholipid composition and how this changes with stress is shown in Tab. 1. Very little change after 24 h stress. After three stress periods however, the PC/PE mol ratio changed from 1.8 (control) to 1.2. In addition, the percentage of FI increased significantly. Tab. 2 shows the fatty acid coposition of individual phospholipids from the PMEF after different amounts of water-deficit stress. 

Sterols are minor constituents of most fats. Those of animal origin contain cholesterol and traces of other sterols, whereas plants contain phytosterols, of which p-sitosterol is the most common. Sterols occur in the free form or, after esterification to fatty acids through the 3-OH group, as steryl esters. The presence of sterols in archaeological residues can be a useful indicator of a plant or animal origin or an indicator of both if cholesterol and phytosterols are detected in the same sample. That noted, cholesterol is a potential contaminant of all archaeological samples subjected to handling. 

Abidi, S. L. (2004). Capillary electrochromatography of sterols and related steryl esters derived from vegetable oils. J. Chromatogr. A 1059, 199—208. 

This enzyme [EC 3.1.1.13] (also known as cholesterol esterase, sterol esterase, cholesterol ester synthase, and triterpenol esterase) catalyzes the hydrolysis of a steryl ester to produce a sterol and a fatty acid anion. This class represents a group of enzymes exhibiting broad specificity. They act on esters of sterols and long-chain fatty acids, and may also bring about the esterification of sterols. These enzymes are typically activated by bile salts. See also Esterases D. P. Hajjar (1994) Adv. Enzymol. 69, 45. 

Figure D1.6.6 latroscan TLC-FID chromatograms of (A) a lipid fraction enriched with neutral lipids isolated from cod flesh and stored in ice (B) neutral lipids spiked with authentic 1 -0-palmityl-glyceryl ether dipalmitate (GE), coinciding in position with authentic highly unsaturated acids such as 22 6n-3 (C) hydrogenated neutral lipids spiked with GE. The solvent system was 97 3 1 (v/v/v) hexane/diethyl ether/formic acid for 40 min. Abbreviations O, origin SF, solvent front FFA, free fatty acid PL, phospholipids SE, steryl ester ST, free sterol TG, triglyceride. Reproduced from Ohshima et al. (1987) with permission from AOCS Press.

Sitosterol and stigmasterol were the major components of celery seed oil. The other components were cholesterol, brassicasterol, campesterol, 97-campesterol, 95-avenasterol, 97-stigmasterol and 9 7-avenasterol (Zlatanov and Ivanov, 1995). Oil bodies isolated from celery cell suspension cultures contained at least 60% of the total steryl ester present in the cells. Free sterols comprised < 0.5% of the total lipid in the oil body. Sterylesters constituted 4.5% of the total lipid of celery oil bodies. The proportion of precursor 4-methylsterols in the free sterol fraction of celery was greater in the oil body (Dyas... 

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