Gerhard Maerker

Cholesterol autoxidation-current status

Several oxidation products of cholesterol have been reported to have biological activity in animals. Cholesterol oxidizes readily in solution, in aqueous dispersion and in foods when it is exposed to air, elevated temperatures, free radical initiators, light, or a combination of these. The main outline of the pathway for cholesterol oxidation is fairly well understood and involves initial formation of allylic hydroperoxides. HPLC and GC techniques are available for measuring the concentration of cholesterol oxides in animal-derived lipids. Biologically active cholesterol oxides have been reported to be present in egg products, dairy products, frying oils and other foods. Their concentration may be subject to process control.

Cholesterol oxides I. Isolation and determination of some cholesterol oxidation products

Cholesterol in purified triolein was subjected to saponification by two procedures employing hot alkali and was found to give rise to oxidation products despite precautions. The isomeric 5,6-epoxycholestanols and 7-hydroxycholesterols were stable under saponification conditions, but 7-ketocholesterol and 6-ketocholestanol were destroyed by hot alkali. The oxidation of cholesterol during saponification was not inhibited by the addition of BHT. The determination of a number of standard cholesterol oxides by direct on-column GC was demonstrated with good resolution. Their order of elution was different from that of the TMS-derivatized products.

Quantitation of cholesterol oxidation products in unirradiated and irradiated meats

A method to detect 7-ketocholesterol, cholesterol-5β,6β-epoxide, cholesterol-5α,6α-epoxide, 4-cholesten-3-one, 4,6-cholestadien-3-one and 4-cholestene-3,6-dione in unirradiated and irradiated beef, pork and veal was developed by use of chloroform-methanol-water extraction, solid-phase extraction, column separation, thin-layer chromatography and gas chromatography. This method recovered 78–88% of the cholesterol oxidation products and detected the cholesterol oxidation products at 10 ppb or higher. Irradiation of the meats to a dose of 10 kGy increased these compounds, except 4,6-cholestadien-3-one for all three types of meat, over unirradiated, and except cholesterol-5α,6α-epoxide and 4-cholesten-3-one for the pork. All the cholesterol oxidation products in the unirradiated meats increased during storage at 0–4°C for 2 wk with some exceptions for the pork. The increases of cholesterol oxidation products in stored irradiated meats were greater than those in the unirradiated.

Process for the preparation of branched chain fatty acids and esters

Complex mixtures of branched chain fatty acids or esters in which the yield of monomeric branched chain products are substantially increased and the yield of polymeric products substantially decreased are prepared by heating monounsaturated fatty acids or esters in the presence of certain combinations of catalysts.

Cholesterol oxides II. Measurement of the 5,6-epoxides during cholesterol oxidation in aqueous dispersions

Cholesterol in aqueous dispersion with sodium stearate or Triton surfactants was aerated at various pH values at 50 and 80 C. Analysis of the reaction mixtures by TLC during the oxidation produced qualitatively similar patterns regardless of pH or temperature. Major oxidation products observed were 7-ketocholesterol, the isomeric 7-hydroperoxy- and 7-hydroxycholesterols, the isomeric 5,6-epoxycholestanols and 3β,5α,6β-trihydroxycholestanol. The concentrations of 3β,5α,6β-trihydroxycholestanol and an unknown compound increased greatly at the lower pH values.Recovery of the 5,6-epoxide isomers by preparative TLC followed by capillary GC allowed theα- andβ-epoxide isomers to be quantitated. Oxidations at pH 8 and 12 produced increasing amounts of the epoxides with time, without significant changes in theα/β-epoxide ratio. However, oxidations at the acidic pH values of 5.5 and 3 showed large changes in theα/β-epoxide ratio during the oxidation. Measurement of the hydrolysis rates of the 5,6-epoxides at pH 5.5 showed that theβ-epoxide isomer is more labile than theα-isomer by a factor of 2.5. The rate constant for the hydrolysis of theα-epoxide isomer was 5.3 × 10−7 sec−1 and that of theβ-isomer 13 × 10−7 sec−1.

Reaction of cholesterol 5,6-epoxides with simulated gastric juice.

Cholesterol 5α,6α-epoxide (α-epoxide) and cholesterol 5β,6β-epoxide (β-epoxide) were individually suspended in simulated gastric juice (pH 1.2) at 37 C, and their reaction was followed by gradient high performance liquid chromatography (HPLC) with flame ionization (FID) detection. Both epoxides reacted rapidly in the aqueous acid medium. The α-epoxide formed 6β-chlorocholestane-3β,5α-diol (α-chlorohydrin) and 5α-cholestane-3β,5,6β-triol (triol), while the β-epoxide formed 5α-chlorocholestane-3β,6β-diol (β-chlorohydrin) and triol. The isomeric chlorohydrins reacted further to form the triol. In mildly alkaline aqueous medium, each chlorohydrin reverted to the epoxide from which it was formed. The data suggest that both epoxides, which have been reported to have adverse health effects in animals, would be largely hydrolyzed in the stomach and to the triol, which also has been reported to have biological activity. The data furher suggest that residual chlorohydrins surviving stomach residence can be expected to revert to epoxide in the more alkaline intestinal environment.

Unusual product ratios resulting from the gamma-irradiation of cholesterol in liposomes

Cholesterol in aqueous suspensions of multilamellar vesicles (MLV) was exposed to gamma-irradiation (0.5–10 kGy) at 0–4°C. Cholesterol oxidation products resulting from the irradiation were isolated by dry column extraction followed by preparative thin-layer chromatography (TLC) and were quantitated by on-column gas chromatography (GC). The ratio of 7-ketocholesterol/cholesterol 5,6-epoxides generated by irradiation was less than one, much lower than the ratio of ten commonly produced by autoxidation. Irradiation also produced relatively higher amounts of 7-hydroxycholesterol than did autoxidation. These unique product ratios may be suitable indicators of past exposure to irradiation.

Determination of oxirane content of derivatives of fats

Although oxirane functions react readily with a large variety of chemical reagents, no single analytical method has been universally successful in measuring the oxirane content of all epoxides. This failure is ascribable to the manner in which the chemical reactivity of the three membered cyclic ethers is modified by molecular structure and by the presence of nearby substituents.A survey of the various types of published analytical methods is given, but major emphasis is placed upon a discussion of procedures applicable to the analysis of epoxy derivatives of fats. Most of these latter methods depend upon the addition of some reagent, HX, to the epoxide ring with simultaneous cleavage of a carbon-oxygen bond.

Effect of low-dose γ-radiation on individual phospholipids in aqueous suspension

A series of individual phospholipids (phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and phosphatidylglycerols) containing either saturated or unsaturated fatty acid chains was irradiated at 9.66 kGy and 0–4°C in aqueous suspension. The phospholipids were analyzed by normal-phase high-performance liquid chromatography on a silica column with an evaporative light scattering detector. Phospholipid disappearance and production of two radiolytic products, phosphatidic acid and the lysophospholipid, after irradiation were quantitated from calibration curves of synthetic standards. Dipalmitoylphosphatidic acid and monopalmitoylphosphatidylcholine from irradiated dipalmitoylphosphatidylcholine were identified by liquid secondary-ion mass spectrometry.

A-ring oxidation products from γ-irradiation of cholesterol in liposomes

Abstractγ-Irradiation of cholesterol in multilamellar vesicles (MLV) at 0–4°C causes oxidation of the A-ring. Two A-ring oxides formed in considerable amounts are cholest-4-en-3-one (10) and cholest-4-ene-3,6-dione (12) in addition to the usual B-ring oxides. Lesser amounts of cholesta-4,6-dien-3-one (11) are also generated. Compounds 10 and 12 were detected and measured in cholesterol irradiated at less than 0.5 kGy in liposomes containing saturated or unsaturated phospholipids. Lesser amounts of 10 and 12, as well as lesser amounts of other cholesterol oxides, were formed when a major constituent of the MLV was dilinoleoylphosphatidylcholine. Autoxidation of cholesterol in MLV also gave rise to small amounts of 10, 11 and 12.