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.     

Cholesterol oxidation in heated lard enriched with two levels of cholesterol

Cholesterol oxidation in lard containing two levels of added cholesterol was monitored using capillary gaschromatography. Loss of cholesterol and formation of cholesterol oxidation products (COPs) were measured. Lard samples with 10 times (Test I) and 2 times (Test II) the amount of cholesterol originally found in each batch of lard were heated at 180°C for 10 hr a day for 240 and 160 hr, respectively. Cholesterol steadily decreased throughout the heating period in both tests. Cholesterol loss followed a first-order reaction rate, with a rate constant (k) of −1.18×10⁻³ h⁻¹ for Test I and −9.45×10⁻³ h⁻¹ for Test II. The COPs accumulated during both heating tests. But the amount of COPs formed did not total the amount of cholesterol lost. During heating, thermal degradation of cholesterol likely occurred, and those products were not detected. During cooling, hydroperoxides formed, which further oxidized into the COPs that were detected. The 7-ketocholesterol and 5α,6α-epoxycholesterol were the predominant COPs formed. The isomeric 7α-and 7β-hydroxycholesterols also accumulated in the heating tests. The 3β,5α,6β-cholestantriol was found in very small amounts and the 25-hydroxycholesterol was not detected. Presented in part at the 80th AOCS Annual Meeting, Cincinnati, OH, in May, 1989.     

Cholesterol autoxidation in phospholipid membrane bilayers

Lipid peroxidation in unilamellar liposomes of known cholesterol-phospholipid composition was monitored under conditions of autoxidation or as induced by a superoxide radical generating system, γ-irradiation or cumene hydroperoxide. Formation of cholesterol oxidation products was indexed to the level of lipid peroxidation. The major cholesterol oxidation products identified were 7-keto-cholesterol, isomeric cholesterol 5,6-epoxides, isomeric 7-hydroperoxides and isomeric 3,7-cholestane diols. Other commonly encountered products included 3,5-cholestadiene-7-one and cholestane-3β,5α,6β-triol. Superoxide-dependent peroxidation required iron and produced a gradual increase in 7-keto-cholesterol and cholesterol epoxides. Cholesterol oxidation was greatest in liposomes containing high proportions of unsaturated phospholipid to cholesterol (4∶1 molar ratio), intermediate with low phospholipid to cholesterol ratios (2∶1) and least in liposomes prepared with dipalmitoylphosphatidylcholine and cholesterol. This relationship held regardless of the oxidizing conditions used. Cumene hydroperoxide-dependent lipid peroxidation and/or more prolonge oxidations with other oxidizing systems yielded a variety of products where cholesterol-5β,6β-epoxide, 7-ketocholesterol and the 7-hydroperoxides were most consistently elevated. Oxyradical initiation of lipid peroxidation produced a pattern of cholesterol oxidation products distinguishable from the pattern derived by cumene hydroperoxide-dependent peroxidation. Our findings indicate that cholesterol autoxidation in biological membranes is modeled by the peroxide-induced oxidation of liposomes bearing unsaturated fatty acids and suggest that a number of cholesterol oxidation products are derived from peroxide-dependent propagation reactions occurring in biomembranes.     

Parameters influencing cholesterol oxidation

The purpose of this study was to investigate the effects of temperature, oxidation time, presence of water, pH, type of buffer and form of substrate used on cholesterol oxidation. Microcrystalline cholesterol films, both solid and melted, and aqueous suspensions of film fragments were used as substrates. Use of dispersing agents was avoided. Quantitative analysis of the unaltered substrate and the products of its autoxidation was carried out by gas chromatography over the course of oxidation. Solid cholesterol films were found to be resistant to autoxidation in the dry state. However, when heated at 125°C, a sudden increase in oxidation rate occurred at a point coinciding with the visible melting followed by a plateau of the oxidation rate. All of the autoxidation products formed underwent further decomposition. Film fragments of cholesterol oxidized at a faster rate in aqueous suspensions than when oxidized in the dry state. In aqueous suspensions, the differences in the resistance of cholesterol to oxidation were not significant within the pH range 6.0–7.4, except for the early stages of oxidation. The 7-ketocholesterol/7-hydroxycholesterol ratio dropped significantly with increasing pH. However, at all pH levels tested, this ratio remained relatively constant during the 6 h of heating. While the 7β-hydroxycholesterol/7α-hydroxycholesterol ratio was not affected by pH in the range of 6.0–7.4, at pH 7.4 a high preference was observed for the cholesterol β-epoxide over its α-isomer. The β/α-epoxide ratio decreased with time of heating and with decreasing pH. The data show that the physical state of the substrate exerts a major influence on the oxidative behavior of cholesterol.     

Gamma-irradiation of individual cholesterol oxidation products

Cholesterol and seven of its oxidation products in aqueous suspensions of multilamellar vesicles or sonicated aqueous suspensions were subjected individually to γ-radiation (10 KGy) at 0–4°C in air, N₂ or N₂O. All compounds underwent some changes under the influence of radiation. β-Epoxide (cholesterol 5β,6β-epoxide) and, to a much lesser extent, α-epoxide (cholesterol 5α,6α-epoxide) were converted in low yield to 6-ketocholestanol (5α-cholestan-3β-ol-6-one). 7β-Hydroxycholesterol (cholest-5-ene-3β,7β-diol) and, to a lesser extent, 7α-hydroxycholesterol (cholest-5-ene-3β,7α-diol) gave low yields of 7-ketocholestanol (5α-cholestan-3β-ol-7-one). The latter compound also was obtained by irradiation of 7-ketocholesterol (cholest-5-ene-3β-ol-7-one). 6-Ketocholestanol and 7-ketocholestanol are potential biomarkers for irradiated meat and poultry.     

α-tocopherol oxidation mediated by superoxide anion (O 2 − ). II. Identification of the stable α-tocopherol oxidation products

The present paper describes the identification of two stable end products of α-tocopherol oxidation that were previously detected among the products of the reaction of α-tocopherol with superoxide anion (O ₂ ⁻ ) under aprotic conditions. One compound, previously designated compound A, was identified astrans-7-hydroxy-trans-8,8a-epoxy-α-tocopherone, and the other, designated compound B, was identified ascis-7-hydroxy-cis-8,8a-epoxy-α-tocopherone. It was also observed that under protic conditions (10% water in acetonitrile) the reaction of α-tocopherol with O ₂ ⁻ did not produce compounds A and B, but rather α-tocopheryl quinone, α-tocopherol dimer, α-tocopherol dihydroxy dimer, and the previously designated compound C. Compound C was identified in the present study as α-tocopheryl-quinone-2,3-epoxide.     

Biosynthesis of sterols and ecdysteroids in Ajuga hairy roots

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously ²H- and ¹³C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and ¹³C₂-acetate established the mechanism of the biosynthesis of the two C₂₉-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3α-, 4α-, and 4β-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5β-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5α,6α-epoxide. Moreover, 7-dehydrocholesterol, 3β-hydroxy-5β-cholest-7-en-6-one (5β-ketol), and 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol→7-dehydrocholesterol→7-dehydrocholesterol 5α,6α-epoxide→5β-ketol→5β-ketodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3β-Hydroxy-5β-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.     

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.     

Effects of quenching mechanisms of carotenoids on the photosensitized oxidation of soybean oil

The effects of 0, 1.0 × 10”⁻⁵, 2.5 × 10⁻⁵, and 5.0 × 10⁻⁵ M β-apo-8'-carotenal, β-carotene, and canthaxanthin on the photooxidation of soybean oil in methylene chloride containing 3.3 × 10⁻⁹ M chlorophyll b were studied by measuring peroxide values and conjugated diene content. β-Apo-8'-carotenal, β-carotene, and canthaxanthin contain 10,11, and 13 conjugated double bonds, respectively. The peroxide values and conjugated diene contents of oils containing the carotenoids were significantly lower (P<0.05) than those of control oil containing no carotenoid. As the number of conjugated double bonds of the carotenoids increased, the peroxide values of soybean oils decreased significantly (P<0.05). The quenching mechanisms and kinetics of the carotenoids in the photosensitized oxidation of soybean oil were studied by measuring peroxide values. The steady-state kinetics study showed that carotenoids quenched singlet oxygen to reduce chlorophyll-sensitized photooxidation of soybean oil. The singlet-oxygen quenching rate constants ofβ- apo-8'-carotenal, β-carotene, and canthaxanthin were 3.06 × 10⁹, 4.60 × 10⁹, and 1.12 × 10¹⁰ M⁻¹sec⁻¹, respectively.     

Synthesis of deuterium labeled cholesterol and steroids and their use for metabolic studies

A simple method is described for the preparation of [6,7,7⁻²H₃] sterols and steroids. The synthesis starts with a Δ⁵-sterol or steroid and involves preparation of the 6-oxo-3α,5α-cyclosteroid, base exchange in the presence of deuterium oxide to introduce two deuteriums at the C-7 position and sodium borodeuteride reduction of the 6-oxo group to introduce the third deuterium atom at C-6. Rearrangement of the [6,7,7⁻²H₃]6α-hydroxy-3α,5α-cyclosteroid then gives the desired [6,7,7^-2H₃]-Δ⁵ sterol or steroid. [6,7,7⁻²H₃]Cholesterol, [6,7,7⁻²H₃]pregnenolone and [6,7,7⁻²H₃]3β-hydroxyandrost-5-en-17-one were synthesized in this fashion and [6,7,7⁻²H₃]progesterone was prepared from the [6,7,7⁻²H₃]pregnenolone. Three examples of the use of these deuchromatography-mass spectrometry. The chrysophyte alga,Ochromonas malhamensis, was shown to be capable of introducing an extra methyl or ethyl group at C-24 of the side chain of [6,7,7⁻²H₃]cholesterol to yield brassicasterol and poriferasterol, respectively. The ovary of the echinoderm,Asterias rubens, was demonstrated to metabolize [6,7,7⁻²H₃]progesterone to yield mainly the 5α-isomers of pregnane-3,20-dione and 3β-hydroxypregnan-20-one. However, the 5β-isomers of these compounds were also detected as minor products for the first time as progesterone metabolites in this animal. Isolated oocytes of the frog,Xenopus laevis, produced a number of metabolites of [6,7,7⁻²H₃]progesterone. In this report, two of them were shown to be 17α-hydroxy-pregn-4-en-3,20-dione and 20α-hydroxypregn-4-en-3-one. Presented at the “Sterol Symposium” of the American Oil Chemists' Annual International Conference, New Orleans, LA, May 1981.     

Isolation and quantitative determination of sterol oxides in plant-based foods: Soybean oil and wheat flour

Soybean oil and wheat flour were analyzed for the content of sitosterol oxides. The method involved chromatography on a Lipidex-5000 column and enrichment on a disposable NH₂-column, yielding a sterol fraction and a sterol oxide fraction. Each fraction was separated as trimethylsilyl-ethers on a methyl silicone capillary column. Analysis of crude and freshly refined soybean oil showed no detectable levels of the isomeric 5,6-epoxysitosterols, the epimeric 7-hydroxysitosterols and 5,6-dihydroxysitosterol at the detection limit of 0.2 ppm. Storage of a refined soybean oil for one year at 4°C caused no significant increase in the level of free sitosterol oxides when compared to the freshly refined soybean oil. Analysis of three wheat flours (at 2, 8 and 36 months) revealed that the samples contained variable levels of 5α,6α-epoxysitosterol (5.4–55 ppm in the lipids), 5β,6β-epoxysitosterol (0.2–29 ppm), 7α-hydroxysitosterol (9.3–118 ppm) and 7β-hydroxysitosterol (9.7–126 ppm).     

Studies on phytosterol oxides. I: Effect of storage on the content in potato chips prepared in different vegetable oils

Potato chips fried in palm oil, sunflower oil, and high-oleic sunflower oil were studied for the content of different phytosterol oxides during 0 to 25 weeks of storage in the dark. Oxidation products of sitosterol (24α-ethyl-5-cholesten-3β-ol) and campesterol (24α-methyl-t-cholesten-3β-ol) were synthesized to help identify the phytosterol oxides. The oxides of phytosterols were analyzed by preparative thin-layer chromatography, solid-phase extraction, capillary column gas chromatography (GC), and GC-mass spectrometry. Epimers of 7-hydroxysitosterol and 7-hydroxycampesterol; 7-ketositosterol and 7-ketocampesterol; epimers of 5,6-epoxy-sitosterol and 5,6-epoxy-campesterol; 24α-ethylcholestane-3β,5,6β-triol (dihydroxysitosterol) and 24α-methylcholestane-3β,5,6β-triol (dihydroxycampesterol) were detected and quantitated in the samples of chips fried in different vegetable oils. Potato chips fried in palm oil had the lowest level of total sterol oxides, ranging from 5 to ca. 9 ppm in the lipids from time 0 to 25 wk of storage. The level of total sterol oxides in chip samples fried in sunflower oil ranged from 46 to 47 ppm, and the lipids in samples fried in high-oleic sunflower oil ranged from 35 to 58 ppm from 0 time to 25 wk of storage. During 25 wk of storage no considerable increase in sterol oxides was observed in the samples of chips fried in palm oil and sunflower oil. The chip samples fried in high-oleic sunflower oil had slightly higher levels of sterol oxides after 10 and 25 weeks of storage. In addition to the levels of individual sterol oxides, a new method for enrichment of phytosterol oxides from the unsaponifiables and full-scan mass spectra of various oxidation products of sitosterol and campesterol are reported in this paper. Part of the results were presented at the 86th Annual Meeting of the AOCS, May 7–11, 1995, San Antonio, TX.     

On the structure,biosynthesis, function and phylogeny of isoarborinol and motiol

The solid-state conformations of the C-3 acetates of two isomeric hopanoids—1, isoarborinol (D∶C-friedo-B¹∶A¹-3β,5α,8α,10β,13β,14α,17β,18α,21β) and 2, motion (D∶C-friedo-B¹∶A¹-neogammacer-7(8)-en-3β-ol[3β,5α,9α,10β,13α,14β,17α,18β,21α])—have been determined by X-ray crystallography. The data show that whereas both molecules are planar, 1 orients into a chair-halfchair-chair-chair-halfchair conformation while 2 orients into a chair-sofa-twist-halfchair-halfchair conformation. To explain the biogenesis of 1 and 2 from squalene oxide, a step-wise mechanism is proposed which proceeds through the protosteroid cation (for 1) and dammarenyl cation (for 2). After ring enlargement from the corresponding 13(17)bond followed by concerted 1,2-migrations and loss of the 11β-H and 7β-H as protons, respectively, a 9,11-double bond (in 1) and a 7,8-double bond (in 2) is introduced into the nucleus. The mechanism is discussed in relation to the classical view of a non-stop cyclization process where, for example, squalene oxide folds in a chair-chair-chair-chair-boat conformation to give a cyclized product (motiol) presumably with the same conformational disposition as the cyclizing material. The three-dimensional geometry of 1 and 2 was found to be structurally dissimilar from sterols. For instance, 1 and 2 are shorter and volumetrically smaller molecules than cholesterol, and this may explain their diminished importance as membrane inserts compared with sterols in eukaryote evolution.     

The sterols ofCucurbita moschata (“calabacita”) seed oil

From the sterol fraction of seed oil from commercialCucurbita moschata Dutch (“calabacita”) Δ⁵ and Δ⁷ sterols having saturated and unsaturated side chain were isolated by chromatographic procedures and characterized by spectroscopic (¹H and¹³C-nuclear magnetic resonance, mass spectrometry) methods. The main components were identified as 24S-ethyl 5α-cholesta-7,22E-dien-3β-ol (α-spinasterol); 24S-ethyl 5α-cholesta-7,22E, 25-trien-3β-ol (25-dehydrochondrillasterol); 24S-ethyl 5α-cholesta-7, 25-dien-3β-ol; 24R-ethylcholesta-7-en-3β-ol (Δ⁷-stigmastenol) and 24-ethyl-cholesta-7, 24(28)-dien-3β-ol (Δ^7,24(28)-stigmastadienol).Lipids 31, 1205–1208 (1996).     

The steric requirements for sterol inhibition of tetrahymanol biosynthesis

Many naturally occurring sterols are accumulated and metabolized byTetrahymena pyriformis. In most cases, the sterols are desaturated to give^Δ5,7,22-derivatives. Compounds with an ethyl, but not with a methyl, substituent at C-24 are dealkylated. Exposure of the ciliates to the appropriate sterol sharply curtails the synthesis of the native pentacyclic triterpenoid alcohols, tetrahymanol and diplopterol. An analysis with modified sterols has revealed several additional features that are required for desaturation at C-7,8 and C-22,23 and for inhibition of tetrahymanol biosynthesis. The presence of atrans-17(20)-double bond, which eliminates free rotation at C-20 and fixes C-22 to the right of the nucleus, does not interfere with desaturation, while thecis- or left-handed isomer is not metabolized. Thecis-Δ¹⁷⁽²⁰⁾-isomer is, however, an effective inhibitor of tetrahymanol biosynthesis, although less so that thetrans-counterpart. When a methyl or hydroxyl group at C-20 protrudes to the front of the molecule in the right-handed conformation, metabolism is reduced or abolished. Shortening (by one C-atom) or lengthening of the sterol side chain has little effect on the ability of the compounds to inhibit tetrahymanol biosynthesis or to support growth, as long as the overall length of the side chain does not exceed seven carbons from C-20. The presence of a 7α-, 7β-, 20α-, 20β-, or a 25-hydroxy group in the cholesterol molecule sharply inhibits desaturation and curtails the effectiveness of the compound as an inhibitor of tetrahymanol biosynthesis. The 7- or 22-keto derivatives seem to act in a fashion similar to the hydroxy derivatives, but these compounds show greater inhibition of growth. 20-Methylcholesterol, however, is a potent inhibitor of synthesis, which suggests that the polarity of the substituent of C-20 is more important than bulk. Many sterols can effectively replace tetrahymanol in the membranes of these ciliates. However, several of the compounds, which inhibit synthesis, appear to be physiologically inappropriate, and poor growth results. An example of the latter class is 20-methylcholesterol. Finally, a class of sterols, represented by 20α-hydroxycholesterol and 7-ketocholesterol, does not severly inhibit tetrahymanol synthesis but leads to growth inhibition and surface abnormalities. These sterols apparently lead to a disordered membrane, even in the presence of tetrahymanol.     

Characterization of epoxy carotenoids by fast atom bombardment collision-induced dissociation MS/MS

The characterization and structure of epoxy carotenoids possessing 5,6-epoxy, 5,8-epoxy and 3,6-epoxy end groups conjugated to the polyene chain were investigated using highenergy fast atom bombardment collision-induced dissociation MS/MS methods. In addition to [M-80]⁺, a characteristic fragment ion of an epoxy carotenoid, product ions resulting from the cleavage of C−C bonds in the polyene chain from the epoxy end group, such as m/z 181 (b ion) and 121 (c ion), were detected. On the other hand, diagnostic ions of m/z 286 (e-H ion) and 312 (f-H ion) were observed, not in the 5,6-epoxy or 5,8-epoxy carotenoid but in the 3,6-epoxy carotenoid. These fragmentation patterns can be used to distinguish 3,6-epoxy carotenoids from 5,6-epoxy or 5,8-epoxy carotenoids. The structure of an epoxy carotenoid, 3,6-epoxy-5,6-dihydro-7′,8′-didehydro-β,β-carotene-5,3′-diol (8), isolated from oyster, was characterized using FAB CID-MS/MS by comparing fragmentation patterns with those of related known compounds.     

Ascorbate-enhanced lipid peroxidation in photooxidized cell membranes: Cholesterol product analysis as a probe of reaction mechanism

Cholesterol was used as an in situ probe for studying mechanisms of lipid peroxidation in isolated erythrocyte membranes subjected to different prooxidant conditions. The membranes were labeled with [¹⁴C]cholesterol by exchange with prelabeled unilamellar liposomes and photosensitized with hematoporphyrin derivative. Irradiation with a dose of blue light resulted in thiobarbituric acid-detectable lipid peroxidation that was increased markedly by subsequent dark incubation with 0.5–1.0 mM ascorbate (AH⁻). Ascorbate-stimulated lipid peroxidation was inhibited by EDTA, desferrioxamine (DOX) and butylated hydroxytoluene (BHT), suggesting that the process is free radical in nature and catalyzed by membrane-bound iron. Thin layer chromatography and radiometric scanning of extracted lipids from photooxidized membranes revealed that the major oxidation product of cholesterol was the 5α-hydroperoxide (5α-OOH), a singlet oxygen adduct. Post-irradiation treatment with AH⁻/Fe(III) resulted in an almost-total disappearance of 5α-OOH and the preponderance of free radical oxidation products, e.g. 7-ketocholesterol, the epimeric 7α-/7β-hydroperoxides (7α-/7β-OOH) and their respective alcohols (7α-/7β-OH). EDTA, DOX and BHT inhibited the formation of these products, while catalase and superoxide dismutase had no effect. These results are consistent with a mechanism involving 1-electron reduction of photogenerated hydroperoxides to oxyl radical, which trigger bursts of free radical lipid peroxidation. Though generated in this system, partially reduced oxygen species, viz. superoxide, hydrogen peroxide and hydroxyl radical, appear to be relatively unimportant in the autoxidation process. Presented at the symposium “Free Radicals Antioxidants, Skin Cancer and Related Diseases” at the 78th AOCS Annual Meeting in New Orleans, LA, May 1987.     

Incorporation oftrans long-chain n−3 polyunsaturated fatty acids in rat brain structures and retina

During heat treatment, polyunsaturated fatty acids and specifically 18∶3n−3 can undergo geometrical isomerization. In rat tissues, 18∶3 Δ9c, 12c, 15t, one of thetrans isomers of linolenic acid, can be desaturated and elongated to givetrans isomers of eicosapentaenoic and docosahexaenoic acids. The present study was undertaken to determine whether such compounds are incorporated into brain structures that are rich in n−3 long-chain polyunsaturated fatty acids. Two fractions enriched intrans isomers of α-linolenic acid were prepared and fed to female adult rats during gestation and lactation. The pups were killed at weaning. Synaptosomes, brain microvessees and retina were shown to contain the highest levels (about 0.5% of total fatty acids) of thetrans isomer of docosahexaenoic acid (22∶6 Δ4c, 7c, 10c, 13c, 16c, 19t). This compound was also observed in myelin and sciatic nerve, but to a lesser extent (0.1% of total fatty acids). However, the ratios of 22∶6trans to 22∶6cis were similar in all the tissues studied. When the diet was deficient in α-linolenic acid, the incorporation oftrans isomers was apparently doubled. However, comparison of the ratios oftrans 18∶3n−3 tocis 18∶3n−3 in the diet revealed that thecis n−3 fatty acids were more easily desaturated and elongated to 22∶6n−3 than the correspondingtrans n−3 fatty acids. An increase in 22∶5n−6 was thus observed, as has previously been described in n−3 fatty acid deficiency. These results encourage further studies to determine whether or not incorporations of suchtrans isomers into tissues may have physiological implications. Presented in part at the 32nd International Conference on the Biochemistry of Lipids, 1991, Granada, Spain. Delta nomenclature (Δ) is used fortrans polyunsaturated fatty acids to specify the position and geometry of ethylenic bonds. Polyunsaturated fatty acids containingtrans double bonds are abbreviated giving the locations of thetrans double bonds only; e.g., 20∶5 Δ17t 20∶5 Δ5c,8c,11c,14c,17t; 22∶5 Δ19t, 22∶5 Δ7c,10c,13c,16c,19t; 22∶6 Δ19t 22∶6 Δ4c,7c,10c,13c,16c,19t.     

Effects of hydrocarbon structure on fatty acid,fatty alcohol,and β-hydroxy acid composition in the hydrocarbon-degrading bacterium <Emphasis Type="Italic">Marinobacter hydrocarbonoclasticus</Emphasis>

The lipids of the gram-negative bacterium Marinobacter hydrocarbonoclasticus grown in a synthetic seawater medium supplemented with various hydrocarbons as the sole carbon source were isolated, purified, and their structures determined. The hydrocarbons were normal, iso, anteiso, and mid-chain branched alkanes, phenylalkanes, cyclohexylalkanes, and a terminal olefin. According to the sequential procedure used for lipid extraction, three pools were isolated: unbound lipids extracted with organic solvents (corresponding to metabolic lipids and to the main part of membrane lipids), OH⁻ labile lipids [mainly ester-bound in the lipopolysaccharides (LPS)], and H⁺ labile lipids (mainly amide-bound in the LPS). Each pool contained FA, fatty alcohols, and β-hydroxy acids. The proportions of these lipids in the unbound lipid pools were 84–98%, 1.1–11.6%, and 0.1–3.6% (w/w), respectively. The chemical structures of the lipids were strongly correlated with those of the hydrocarbons fed; analytical data suggested a metabolism essentially through oxidation into primary alcohol, then into FA and degradation via the β-oxidation pathway. Subterminal oxidation of the hydrocarbon chains, α-oxidation of FA or double-bond oxidation in the case of the terminal olefin, were minor, although sometimes substantial, routes of hydrocarbon degradation. Cyclohexyldodecanedid not support growth, likely because of the toxicity of cyclohexylacetic acid formed in the oxidation of the alkyl side chain. In the OH⁻ and H⁺ labile lipid pools, β-hydroxy acids, the lipophilic moiety of LPS, generally dominated (28–72% and 64–98%, w/w, respectively). The most remarkable feature of these cultures on hydrocarbons was the incorporation in LPS of β-hydroxy acids with C_odd, ω-unsaturated, iso, or anteiso alkyl chains in addition to the specific β-hydroxy acid of M. hydrocarbonoclasticus, 3-OH-n-12∶0. These β-hydroxy acids were tolerated insofar as their geometry and steric hindrance were close to those of the 3-OH-n-12∶0 acid.     

AY-9944 inhibition of sterol biosynthesis inChlorella emersonii

WhenChlorella emersonii, a green alga, was cultured in the presence of 20 ppm AY-9944, a number of sterols accumulated which appear to be intermediates of sterol biosynthesis in this organism. The sterols isolated include 14α-methyl-ergost-8-en-3β-ol, 14α-methyl 24S-stigmast-8-en-3β-ol, 14α-methyl ergosta-8,24(28)-dien-3β-ol and 4α, 14α-dimethyl 24S-stigmast-8-en-3β-ol. Smaller quantities of several other sterols were found in addition to the normally occurring Δ⁷, chondrillasterol and Δ⁷. Control cultures were found to contain, in addition to the normally occurring sterols, smaller quantities of most of the sterols isolated from AY-9944 inhibited cultures. AY-9944 is a specific inhibitor of Δ⁷ in cholesterol biosynthesis in animals. However, sinceC. emersonii terminates sterol biosynthesis one step prior to the Δ⁷ step, AY-9944 apparently inhibits sterol biosynthesis prior to this step in this organism. The accumulation of 14α-methyl sterols in treated cultures suggests that AY-9944 is an effective inhibitor of the 14α-methyl removal inC. emersonii. Scientific Article No. A1865, Contribution No. 4775 of the Maryland Agricultural Experiment Station.