Dealkylation of various 14-alkylsterols by the nematodeCaenorhabditis elegans

The metabolism of 4 dietary 24-alkylsterols was investigated in the free-living nematodeCaenorhabditis elegans. The major unesterified sterols ofC. elegans in media supplemented with either campesterol, 22-dihydrobrassicasterol or stigmasterol included cholesta-5,7-dienol, cholesterol, cholest-7-enol, and 4α-methylcholest-8(14)-enol. Dietary stigmastanol yielded cholest-7-enol, cholestanol, cholest-8(14)-enol, and 4α-methylcholest-8(14)-enol as major unesterified sterols. Esterified sterols comprised less than 22% of the total sterol. Removal of a C-24 ethyl substituent of sterols was neither hindered by the presence of a Δ²²-bond in the sterol side chain nor was it depedent on unsaturation in ring B of the steroid nucleus.C. elegans reduced a Δ²²-bond during its metabolism of stigmasterol; it did not introduce a Δ²²-bond during stigmastanol metabolism.C. elegans was capable of removing a C-24 methyl substituent regardless of its stereochemical orientation. Metabolic processes involving the steroid ring system of cholesterol (C-7 dehydrogenation, Δ⁵-bond, 4α-methylation, Δ⁸⁽¹⁴⁾-isomerization inC. elegans were not hindered by the presence of a 24-methyl group; various 24-methylsterol metabolites from campesterol were detected, mostly 24-methylcholesta-5,7-dienol. In contrast, no 24-ethylsterol metabolites from the dietary ethylsterols were found. More dietary 24-methylsterol remained unmetabolized than did dietary 24-ethylsterol. A 24α-ethyl group and a 24β-methyl group were dealkylated to a greater extent byC. elegans than was a 24α-methyl group, perhaps reflecting the substrate specificity of the dealkylation enzyme system, or suggesting different enzymes altogether.     

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.     

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to γ-linolenic acid in rat liver microsomes

The effects of antibodies against microsomal electron-transport components on the in vitro activity of Δ⁶-desaturation of linoleic acid to γ-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the Δ⁶-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b₅ and NADPH-cytochrome c reductase, only the antibody against cytochrome b₅ demonstrated a marked ability to inhibit the Δ⁶-desaturase activity. This evidence supports a participation of cytochrome b₅ in the Δ⁶-desaturation of linoleic acid and suggests a pathway analogous to the Δ⁹-desaturation of stearyl-CoA.     

Growth hormone and liver mitochondria: Effects on phospholipid composition and fatty acyl distribution

Effects of growth hormone on phospholipid composition and fatty acyl distribution were studied in liver mitochondria of hypophysectomized rats. After hypophysectomy, only cardiolipin showed a 25% decrease. Its fatty acyl distribution, which consisted mainly of linoleic acid (55–60%) and oleic acid (20%), was unchanged. In phosphatidylcholine and phosphatidylethanolamine fractions the contents of docosahexaenoic and arachidonic acids were decreased with a concomitant increase in linoleic acid content. These changes could be accounted for by small but significant decreases in the activities of Δ⁹-desaturase (sucrose-induced), Δ⁵-desaturase and mitochondrial elongation enzymes. The activities of Δ⁶-desaturase NADH cytochrome b₅ ferri-reductase, cytochrome b₅, NADH cytochrome c reductase and microsomal elongation enzymes remained virtually unchanged. Injection of bovine growth hormone daily for seven days restored cardiolipin and fatty acyl distribution and the enzyme activities. From these and other results, we conclude that growth hormone-dependent increase of respiratory activity of liver mitochondria may be partly mediated by the hormonal effects on membrane lipid distribution.     

Subterminal hydroxylation of lauric acid by microsomes from a marine fish

Microsomes from the liver of sea bass (Dicentrarchus labrax) were shown to hydroxylate lauric acid at subterminal positions. The cytochrome P-450 system converted lauric acid to several mono-hydroxylated metabolites including ω-1 hydroxylaurate, which was the major metabolite (44% of total products). In addition, ω-2, ω-3, ω-4 and a small amount (2.3%) of ω hydroxylaurates were found. Reaction products were identified using thin-layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS). Oxidation reactions were dependent upon O₂ and NADPH, and did not occur with boiled microsomes or in the presence of a mixture of CO/O₂. Hydroxylation proceeded linearly up to 20 min at 28°C for protein concentrations below 380 μg. Treatment of fish with benzo(α)pyrene (BP) (20 mg/kg) drastically increased xenobiotic metabolism (ECOD, EROD and BPMO activities), but no difference in laurate hydroxylase activity was observed between untreated and treated fish. Starvation strongly enhanced laurate hydroxylase activity, and resumption of feeding reduced by half this increase of activity. In all of the experiments we did not observe any modification of the regioselectivity of lauric acid hydroxylation by this microsomal in-chain hydroxylating system. We suggest that cytochrome P-450 enzymes involved in lauric acid and xenobiotics metabolism are regulated independently.     

Comparative studies of metabolism of 4-desmethyl, 4-monomethyl and 4,4-dimethyl sterols inManduca sexta

To investigate the metabolism and possible deleterious effects of 4-methyl and 4,4-dimethyl steroids inManduca sexta, the 4,4-dimethyl sterols lanosterol and cycloartenol, the 4-methyl sterol obtusifoliol and the 4,4-dimethyl pentacyclic triterpenoid α-amyrin were fed in an artificial agar-based diet at various concentrations. Utilization and metabolism of these four compounds were compared with sitosterol, stigmasterol, brassicasterol, ergosterol and 24-methylenecholesterol, 24-alkyl sterols that are readily dealkylated and converted to cholesterol inManduca and in most phytophagous insects. None of the 4-methylated compounds significantly inhibited development except at very high dietary concentrations. The Δ²⁴-bonds of lanosterol and cycloartenol were effectively reduced by theManduca Δ²⁴-sterol reductase enzyme, as is the Δ²⁴-bond of desmosterol which, in most phytophagous insects, is an intermediate in the conversion of sitosterol, stigmasterol and other C₂₈ and C₂₉ phytosterols to cholesterol. On the other hand, the 24-methylene substituent of obtusifoliol was not dealkylated. Each of the 4-desmethyl C₂₈ and C₂₉ sterols was readily converted to cholesterol, and a significant amount of 7-dehydro-cholesterol was derived from ergosterol metabolism. The reason for the differences in substrate specificity of these sterols is not clear, but the information may be useful in the development of new, specific, mechanism-based inhibitors of sterol metabolism.     

Sterol utilization and metabolism by Heliothis zea

Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvea did not develop on a sterol-less medium. Δ⁵-Sterols with a hydrogen atom, a methylene group, an E-or Z-ethylidene group, or an α- or β-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and Δ⁵-sterols doubly substituted in the side chain at C-24 with an α-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3β-cholestanol with a 9β, 19-cyclopropyl group, gem dimethyl group at C-4, a Δ^5,7-bond or Δ⁸-bond, or by side-chain modified sterols that possessed a Δ²⁵⁽²⁷⁾-24β-ethyl group, Δ²³⁽²⁴⁾-24-methyl group, or 24-ethyl group, or Δ²⁴⁽²⁵⁾-24-methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae) treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 μg per insect, whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 μg per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24-dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).     

Changes in lipid composition of liver microsomes and fatty acyl-CoA desaturase activities induced by medium chain triglyceride feeding

Changes in fatty acid composition, microsomal Δ⁹- and Δ⁶-desaturase activities and liver contents of cholesterol and phospholipids were studied in rats fed medium chain triglyceride-supplemented diets. Weanling rats were divided into four groups and fed for three weeks a basal diet with different 10%-fat supplements: corn oil, medium chain triglyceride-corn oil, olive oil and medium chain triglyceride-olive oil. The highest relative content of saturated fatty acids corresponded to corn oil-fed animals. Both monounsaturated fatty acid content and Δ⁹-desaturase activity were higher in the animals fed olive oil diets than in corn oil-fed rats. The long chain polyunsaturated fatty acids of the n−3 series were increased in the olive oil and medium chain triglyceride-olive oil-fed groups probably due to the lower linoleic/α-linolenic ratios found in these two diets. The cholesterol/phospholipid molar ratio was unaffected by diet and the unsaturation index was only slightly changed in the four groups. Thus, some mechanism may be operative under these conditions to maintain the homeostasis of the membrane.     

Hydroxylation of fatty acids and alcohols by hepatic microsomal cytochrome P-450 system from the Mongolian gerbil

The liver microsomes of the Mongolian gerbilMeriones unguiculatus catalyzed the hydroxylation of various saturated fatty acids (C₈−C₁₈), alcohols (C₁₂ and C₁₆) and hydrocarbon (C₁₂) to the corresponding ω- and (ω-1)-hydroxy derivatives. Lauric acid was hydroxylated most effectively among saturated fatty acids and the order of activity as hydroxylation substrates was C₁₂>C₁₄>C₁₃>C₁₆>C₁₀>C₁₈>C₈. The specific activity of laurate hydroxylation (5.99 nmol/mg microsomal protein/min) in gerbil liver microsomes was higher than that observed in other species. 1-Dodecanol was also hydroxylated very effectively (4.58 nmol/mg microsomal protein/min) by gerbil liver microsomes, but in general the hydroxylation rates for fatty alcohols were much lower than those for the corresponding acids. It was found from both inhibitor and cofactor studies that the enzyme catalyzing the hydroxylation of fatty acids and alcohols in the liver microsomes of the Mongolian gerbil was a typical cytochrome P-450-linked monooxygenase, and at least two different cytochrome P-450 species were involved in the hydroxylation. Presented in part at the AOCS annual meeting (a joint meeting with the Japan Oil Chemists' Society), Honolulu, Hawaii, May 1986.     

Sterols of cucurbitaceae: The configurations at C-24 of 24-Alkyl-Δ5-,Δ7- and Δ8-sterols

The major sterols of the seeds ofBenincasa cerifera, Cucumis sativus, Cucurbita maxima, C. pepo andTrichosanthes japonica and of the mature plant tissues (leaves and stems) ofCitrullus battich, Cucumis sativus andGynostemma pentaphyllum of the family Cucurbitaceae were 24-ethyl-Δ⁷-sterols which were accompanied by small amounts of saturated and Δ⁵-and Δ⁸-sterols. The 24-ethyl-Δ^7,22,Δ^7,25(27) and Δ^7,22,25(27)-sterols constituted the predominant sterols for the seed materials, whereas the 24-ethyl-Δ⁷ and Δ^7,22-sterols were the major ones for the mature plant tissues. The configurations of C-24 of the alkylsterols were examined by high resolution¹H NMR and¹³C NMR spectroscopy. Most of the 24-methyl- and 24-ethylsterols examined which lack a Δ²⁵⁽²⁷⁾-bond (i.e., 24-methyl-, 24-methyl-Δ²²-, 24-ethyl- and 24-ethyl-Δ²² sterols) were shown to occur as the C-24 epimeric mixtures in which the 24α-epimers predominated in most cases. The 24-ethylsterols which possess a Δ²⁵⁽²⁷⁾ (i.e., 24-ethyl-Δ²⁵⁽²⁷⁾-and 24-ethyl-Δ^7,22,25(27)-sterols) were, on the other hand, composed of only 24β-epimers. The Δ⁸-sterols identified and characterized were four 24-ethyl-sterols: 24α-and 24β-ethyl-5α-cholesta-8,22-dien-3β-ol, 24β-ethyl-5α-cholesta-8,25(27)-dien-3β-ol and 24β-ethyl-5α-cholesta-8,22,25(27)-trien-3β-ol. This seems to be the first case of the detection of Δ⁸-sterols lacking a 4-methyl group in higher plants, and among the four Δ⁸-sterols the latter two are considered to be new sterols. The probable biogenetic role of the Δ⁸-sterols and the possible biosynthetic pathways leading to the 24α- and 24β-alkylsterols in Cucurbitaceae are discussed.     

Metabolism of sterols of varying ring unsaturation and methylation byCaenorhabditis elegans

The metabolism of three dietary 4,4-desmethylsterols and two 4α-methylsterols was investigated in the free-living nematodeCaenorhabditis elegans. Dietary cholestanol was converted mostly to lathosterol. Dietary lathosterol, 7-dehydrocholesterol, 4α-methylcholest-7-enol and 4α-methylcholest-8(14)-enol each remained largely unchanged. An absolute requirement for a substantial quantity of 7-dehydrocholesterol inC. elegans did not exist.C. elegans was unable to remove a 4α-methyl group or introduce a double bond at C-5 and also demonstrated the lack of a Δ⁷-reductase. Its nutritional sterol requirement was satisfied by cholestanol, lathosterol or 7-dehydrocholesterol; growth was comparable to that obtained previously in media containing Δ⁵-sterols. However, the two 4α-methylsterols appeared to be unsatisfactory sterol nutrients. The possible physiological importance of 4α-methylsterols is discussed briefly.     

Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14α-demethylation (P45014DM) in hamster liver

To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450, lanosterol 14α-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL-4B, hydroxylapatite, DEAE-5PW, and CM-Sephrose CL-6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein howed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14α-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14α-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital-and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals.     

Reduction of blood and liver cholesterol in the rat by straight and branched chain alkyl amines

The activities of a branched chain and several straight chain amines (C₁₂ to C₁₈ chain length), and the azasteroid 25-aza-5α-cholestane were compared with those of 20,25-diazacholesterol dihydrochloride, which is a potent hypocholesterolemic agent in the rat. These amines and azasteroids inhibit the Δ²⁴-sterol reductase system in the tobacco hornworm,Manduca sexta (L.), and also block the conversion of C₂₈ and C₂₉ plant sterols to cholesterol, with a resulting accumulation of desmosterol. The effects of these compounds in the rat were determined on body weight gain, cholesterol, desmosterol, and lipid composition of blood, feces, liver, and epididymal fat pad weight. The two azasteroids and the branched chain amine, N,N-dimethyl-3, 7,11-trimethyldodecanamine, had the greatest effect, reducing total plasma lipids and plasma sterols to approximately 40–50% of the levels in control rats and produced a concomitant increase in plasma and liver desmosterol. The branched chain dodecanamine caused a reduction in both feed consumption and body weight gain. The branched and straight chain dodecanamines also severely reduced epididymal fat pad weight. Our results demonstrate that the simple azasteroid, 25-aza-5α-cholestane, is a more potent inhibitor of cholesterol biosynthesis than the diazasterol and that the Δ²⁴-sterol reductase system in a mammal can be inhibited by simple, nonsteroidal, acyclic amines.     

Synthesis of Δ5,22-cholestadien-3β-ol from Δ5,7,22-cholestatrien-3β-ol by a liver enzyme

The rat liver enzyme system, which catalyzes reduction of Δ^5,7,24-cholestatrien-3β-ol to cholesterol (Δ⁵-cholesten-3β-ol), converted radiolabeled Δ^5,7,22-cholestatrien-3β-ol to Δ^5,22-cholestadien-3β-ol, but not to cholesterol. This enzyme system thus contains membrane-bound Δ⁷ and Δ²⁴-reductases and no Δ²²-reductase. Kinetic and competition studies showed that the enzymes system contains a single Δ^5,7-sterol Δ⁷-reductase, which is not influenced by unsaturtion at the Δ²²-position of the sterol side chain. The identity of Δ^5,22-cholestadienol was established by chromatographic, spectral and chemical analyses. Use of the enzyme system and readily available Δ^5,7,22-cholestatrienol provides a facile procedure for specific production of Δ^5,22-cholestradien-3β-ol in quantity.     

Sterols of the phylum Zygomycota: Phylogenetic implications

The sterol composition of 42 fungal species representing six of the eight orders of the Zygomycota was determined using gas-liquid chromatography-mass spectrometry to assess whether the distribution of major sterols in this phylum has taxonomic or phylogenetic relevance. Ergosterol, 22-dihydroergosterol, 24-methyl cholesterol, cholesterol, and desmosterol were detected as the major sterols among the species studied. Ergosterol was the major sterol of the Dimargaritales, Zoopagales, and 13 of the 14 Mucorales families included in this study. Desmosterol appeared to be the characteristic sterol of the Mortierellaceae (Mucorales). 24-Methyl cholesterol was the major sterol of the Entomophthorales genera Entomophthora, Conidiobolus and Basidiobolus, but cholesterol was the sole sterol detected in Delacroixia coronatus. The Kickxellales species analyzed in this study were characterized by 22-dihydroergosterol as the major sterol. These results suggest that certain orders of the Zygomycota may be distinguished on the basis of major sterol. Also, if sterol structure has phylogenetic implications, then orders might be arranged in the order Kickxellales (C₂₈Δ^5,7) → Dimargaritales, Zoopagales and Mucorales (C₂₈Δ^5,7,22) on the basis of evolution of the predominant and presumably most competent sterol, ergosterol. Although the Entomophthorales would be expected to be more primitive than the above orders based on the predominance of C₂₈Δ^5,, it is not apparent from these data that members of the Zygomycota with ergosterol or its precursors as major sterols evolved from this taxon or the Chytridiomycota.     

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.     

Characterization of chilean hazelnut (Gevuina avellana mol) seed oil

The fatty acid composition, tocopherol and tocotrienol content, and oxidative stability of petroleum benzene-extracted Gevuina avellana Mol (Proteaceae) seed oil were determined. Positional isomers of monounsaturated fatty acids were elucidated by gas chromatography-electron impact mass spectrometry after 2-alkenyl-4,4-dimethyloxazoline derivatization. This stable oil (Rancimat induction period at 110°C: 20 h) is composed of more than 85% monounsaturated fatty acids and about equal amounts (6%) of saturated and polyunsaturated (principally linoleic) fatty acids. Unusual positional isomers of monounsaturated fatty acids, i.e., C_16:1 Δ¹¹, C_18:1 Δ¹², C_20:1 Δ¹¹, C_20:1 Δ¹⁵, C_22:1 Δ¹⁷, and presumably C_22:1 Δ¹⁹ were identified. The C_18:1 Δ¹² and C_22:1 Δ¹⁹ fatty acids are described for the first time in G. avellana seed oil. While only minute quantities of α-, γ-tocopherols and β-, γ- and δ-tocotrienols were found, the oil contained a substantial amount of α-tocotrienol (130 mg/kg). The potential nutritional value of G. avellana seed oil is discussed on the basis of its composition.     

Δ9 desaturase activity in normal mouse liver and hepatoma SS1K

The activity of Δ9 desaturase was determined in the microsomal fraction of normal mouse liver and hepatoma SS1K in the presence of the 105,000 × g supernatant. Neither hepatic nor hepatoma soluble fractions were able to modify the low desaturating capacity. Two enzymes from the microsomal electron transport chain associated with Δ9 desaturase, namely NADH-cytochrom b₅ reductase and NADH-cytochrome C reductase were also measured. The results indicate that the low Δ9 desaturase activity in hepatoma SS1K could be related to the reduced amount of desaturase.     

Growth support and metabolism of phytosterols inParamecium tetraurelia

The basis of the growth requirement ofParamecium for one of several structurally similar phytosterols is not known. Previous research has indicated that selective esterification of only growth-promoting sterols may be a key. In this study, it was found that under certain conditions sterols that fail to support growth (e.g., cholesterol) can be esterified in large amounts inParamecium. We found no compelling evidence to support the hypothesis that steryl esters serve a specialized role in the fatty acid metabolism of the cell. Octadecenoic acid, essential for cell growth, was the major fatty acid in both steryl esters and triglycerides. It was also shown thatP. tetraurelia can dehydrogenate Δ⁰ and Δ⁷, as well as Δ⁵-3β-hydroxy sterols, to yield the conjugated 5,7-diene derivative. These results indicate the presence of a Δ⁵, in addition to a Δ⁷, desaturase of the sterol nucleus in this ciliate. Two C₂₄ α-ethyl sterols, Δ²²-stigmasterol (Δ²²) and stigmastanol (Δ⁰), were shown for the first time to promote growth. Finally, we found that non-growth-promoting sterols may compose a high percentage of the free sterols of the surface membrane without adversely affecting cell growth or viability. These data support the conclusion that the growth requirement for select phytosterols inParamecium does not involve the structural or functional role of “bulk” sterols in cell membranes.     

Storage lipid accumulation and acyltransferase action in developing flaxseed

Investigations of storage lipid synthesis in developing flaxseed (Linum usitatissimum) provide useful information for designing strategies to enhance the oil content and nutritional value of this crop. Lipid content and changes in the FA composition during seed development were examined in two cultivars of flax (AC Emerson and Vimy). The oil content on a dry weight basis increased steadily until about 20 d after flowering (DAF). The proportion of α-linolenic acid (α-18∶3, 18∶3cisΔ^{9, 12, 15}) in TAG increased during seed development in both cultivars while the proportions of linoleic acid (18∶2cisΔ^{9, 12}) and saturated FA decreased. The developmental and substrate specificity characteristics of microsomal DAG acyltransferase (DGAT, Ec 2.3.1.20) and lysophosphatidic acid acyltransferase (LPAAT, EC 2.3.1.51) were examined using cultivar AC Emerson. The maximal acyltransferase specific activities occurred in the range of 8–14 DAF, during rapid lipid accumulation on a per seed basis. Acyl-CoA of EPA (20∶5cisΔ^5,8,11,14,17) or DHA (22∶6cis ^{4,7,10,13,16,19}) were included in the specificity studies. DGAT displayed enhanced specificity for α-18∶3-CoA, whereas the preferred substrate of LPAAT was 18∶2-CoA. Both enzymes could use EPA- or DHA-CoA to varying extents. Developing flax embryos were able to take up and incorporate these nutritional FA into TAG and other intermediates in the TAG-formation pathway. This study suggests that if the appropriate acyl-CoA-dependent desaturation/elongation pathways are introduced and efficiently expressed in flax, this may lead to the conversion of α-18∶3-CoA into EPA-CoA, thereby providing an activated substrate for TAG formation.