Identification of plant sterols in plasma and red blood cells of man and experimental animals

Direct gas liquid chromatography (GLC) of total plasma lipids showed small peaks (0.5–1.5% of total free sterol area) corresponding to free C₂₈ and C₂₉ sterols in ca. 50% of some 3,000 normal subjects and patients with hyperlipemia. Comparable proportions of similar peaks were present in the sterol fraction isolated from the red blood cells of many of these subjects. The maximum levels of these components in the plasma and red blood cells of domestic and laboratory animals were up to 10 times higher than those seen in man. Detailed gas chromatography/mass spectrometry analyses of the plasma lipids from a much more limited number of subjects and animals showed that the GLC peaks corresponding to the free C₂₈ and C₂₉ sterols were largely due to the plant sterols campesterol, stigmasterol, and β-sitosterol. In all instances, variable amounts (0.05–0.2% of the total free sterol area) of 7-dehydrocholesterol, desmosterol, lanosterol, and cholesterol α-oxide were also detected. While the total content and composition of the plasma plant sterols appeared to vary greatly among the subjects, it never exceeded 2% of total sterol in the normal subjects and patients examined. There was no evidence for a significant increase in the plant sterol content of the plasma of patients with hypercholesterolemia or hypertriglyceridemia.     

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.     

Distribution of sterols in the fungi I. Fungal spores

The freely extractable sterols of spores ofLinderina pennispora, Spicaria elegans, Penicillium claviforme, Aspergillus niger, Ustilago nuda, U. maydis, Puccinia graminis, andP. striiformis were examined using mass spectrometric techniques. Each species contained at least 3–5 detectable sterol components in the 4-desmethyl sterol fraction, and, when present, ergosterol was generally the most abundant sterol produced by an individual species. Smaller relative concentrations of fungisterol (ergost-Δ⁷-enol) di- and tetraunsaturated C₂₈ sterols also were found. In some species, fungisterol was the most abundant sterol. In uredospores of rust fungi, stigmast-Δ⁷-enol (C₂₉) was predominant and was accompanied by lower relative concentrations of a diunsaturated C₂₉ sterol and fungisterol. Cholesterol was found only in the teliospores of the corn smut fungus (U. maydis). Application of glass capillary columns to the separation of yeast sterols by gas liquid chromatography is illustrated. One of eight papers presented in the symposium “Phytosterols,” AOCS Spring Meeting, New Orleans, April 1973.     

Analysis of molluscan sterols: Colorimetric methods

The wide variety of sterols normally found in extracts of bivalve molluscs leads to high variability in analytical data obtained with colorimetric (chole)sterol methods. Total sterol levels in oyster (Crassostrea virginica) extracts were determined using the Liebermann-Burchard reagent, an acid-FeCl₃ reagent and a cholesterol oxidase procedure. The data from the latter two agreed to within 5.4% and yielded about 30% higher estimates of sterol content than the Liebermann-Burchard test. Gas-liquid chromatographic data also are compared. Several pure sterols, selected because of their presence in oyster sterol fractions or because of their structural similarities to such sterols, were examined using each of the three procedures. Sterols, differing from cholesterol only with regard to the side chain, reacted 80–102% as well as cholesterol with the acid-FeCl₃ reagent and cholesterol oxidase. The Liebermann-Burchard reaction was more specific for cholesterol. The colorimetric cholesterol oxidase method is recommended for the estimation of total molluscan sterol content.     

The content and composition of sterols and sterol esters in sunflower and poppy seed oils

The composition and proportion of free sterols and sterol esters in crude sunflower and poppy seed oils were determined, using preparative thin layer chromatography followed by gas chromatography with cholesterol as an internal standard. Free sterols and sterol esters were also isolated in a liquid fraction obtained by low temperature crystallization (−80 C) of the oils and enriched with minor lipid classes. This enrichment procedure provided a liquid fraction suitable for studies of minor components in the oils. However, selectivity towards sterol esters was observed since sterols esterified to very long chain fatty acids (C₂₀–C₂₄) were preferentially retained in the precipitate. The proportions of free and esterified sterols were found to be 0.34 and 0.28%, respectively, in the sunflower oil, whereas the corresponding figures for poppy seed oil were 0.33% and 0.05%. Sunflower oil was characterized by a relatively high percentage of Δ7-sterols, preferentially obtained in the esterified fraction, and by very long chain saturated fatty acids of sterol esters. The sterols in poppy seed oil were composed almost entirely of campesterol, stigmasterol, sitosterol and Δ5-avenasterol, although their percentage distributions were remarkably different in the free and esterified fraction.     

Fatty acid and sterol specificity of cholesterol esterifying enzyme in developing rat brain

The properties and fatty acid and sterol specificity of cholesterol-esterifying enzyme (EC 3.1.1.13) in rat brain were studied. The enzyme utilized free fatty acid for esterification, and activity was maximal at pH 5.6. Exogenous ATP and CoA did not stimulate the incorporation of free fatty acids into sterol esters. Substrates dispersed in Tween 20 or Triton X-100 were just as effective as the substrates dissolved in acetone solution, while dispersion in propylene glycol or sodium taurocholate was not as effective. Snake venom phospholipase A₂ (EC 3.1.1.4) increased the esterification of cholesterol in the absence of added fatty acid. The fatty acid specificity data indicated that oleic and palmitic acids were the preferred fatty acids. Little or no esterification occurred in the presence of long chain fatty acids (C₂₀–C₂₄). Esterification of cholesterol with palmitate or stearate was not affected by the presence of oleic acid in the mixture. Thus, the nonrequirement of the brain-esterifying enzyme for a bile acid or for an amphiphile such as an unsaturated fatty acid suggests that micellar solubilization of the substrate is not essential for activity. Although the brain enzyme catalyzed the esterification of desmosterol, cholesterol was the preferred substrate. Neither lanosterol (C₂₉ sterols) nor Δ7-dehydrocholesterol was esterified to any significant extent. The presence of low concentrations of desmosterol increased cholesterol esterification slightly, while there was a concentration-dependent inhibition of demosterol esterification by cholesterol. These data on fatty acid and sterol specificity of the esterifying enzyme correlate well with the composition of sterol esters present in developing rat brain.     

The fatty acids of wax esters and sterol esters from vernix caseosa and from human skin surface lipid

Separation of sterol esters from wax esters in the lipids of vernix caseosa and adult human skin surface was accomplished by column chromatography on MgO. The fatty acids of the sterol esters and wax esters of both samples were separated into saturates and monoenes, and examined in detail by gas liquid chromatography (GLC). The saturated fatty acids of the wax esters of vernix caseosa and of adult human skin surface were remarkably similar. They ranged in chain length from at least C₁₁ to C₃₀, six skeletal types being present: straight even, straight odd, iso, anteiso, other monomethyl branched and dimethyl branched. A large number of patterns of monoenes were observed, each pattern consisting of desaturation of a specific chain at Δ6 or Δ9 plus its extension or degradation products. The mole per cent of the total Δ6 and Δ9 patterns of wax ester fatty acid monoenes of vernix caseosa were 87% and 12%, respectively, and 98% and 1%, respectively, for adult human skin surface lipid. The sterol ester fatty acids of vernix caseosa were much different from those of adult human skin surface: vernix caseosa saturates were largely branched and of lengths greater than C₁₈, whereas the saturates of adult human surface lipid resembled the wax ester fatty acids. Of the vernix caseosa monoene patterns, the mole per cent was 30% Δ6 and 70% Δ9, whereas of the adult human skin surface sterol ester fatty acids 89% were Δ6 and 11% Δ9. Chain extension was particularly pronounced in the sterol ester fatty acid monoenes of vernix caseosa amounting to 7–8 C₂ units in some cases. The fatty acids of the sterol esters of both vernix caseosa and adult human skin surface appear to be derived from the sebaceous gland and from the keratinizing epidermis, but those of the wax esters are from the sebaceous glands only.     

Characterization of skin-surface lipids from the monkey (Macaca fascicularis)

Skin-surface lipids from the monkeyMacaca fascicularis are composed of sterol esters (38%), cholesterol (4%) and two types of wax diesters, identified as Type II (IIa and IIb, 17% and 40%, respectively). Type IIa contained diesters of 1,2-alkanediols esterified with two molecules of long-chain (C₁₄−C₃₄) fatty acids having straight and branched chains. In the diesters IIa, fatty acids shorter than C₁₉ predominated in position 1, and fatty acids longer than C₂₀ predominated in position 2. Type IIb contained diesters of 1,2-alkanediols esterified with C₄ and C₅ branched-chain fatty acids (predominantly isovaleric acid) at position 1 and long-chain (C₁₄−C₂₇) acids, having straight and branched chains, at position 2. The shortchain acids were converted to 2-nitrophenylhydrazides and analyzed by high-performance liquid chromatography (HPLC). Ammonia chemical ionization (CI)-gas chromatography (GC)-mass spectrometry (MS) resolved the intact diesters IIb into 12 peaks corresponding to molecular weights ranging from 597 to 748, and showed that the molecular species, such as C₂₁−C₁₆−C₅ (diol, fatty acid in position 2, fatty acid in position 1), C₂₂−C₁₆−C₅ and C₂₃−C₁₆−C₅, were prevalent. The fatty acids from both diesters were mostly (>98%) saturated. The 1,2-alkanediols from both diesters consisted of C₁₆−C₂₆ saturated straight- and branched-chain components. The acyl groups of sterol esters contained 86% C₁₄−C₃₄ branched-chain acids. The unsaturated fatty acids (5.4%) belonged to a straight-chain monoenoic series having extremely long chains (C₁₈−C₃₄). The branched-chain structures in the fatty acids and diols were iso and anteiso. These results show the species-specific profile for the skin-surface lipid synthesis.     

Hydrocarbons and alcohols of basking shark and pig liver lipids

Four samples of the unsaponifiables of basking shark liver oil were adsorbed on alumina and eluted to yield Fractions 1–5, inclusive. Analyses by temperature programmed GC and by silica gel chromatography showed hydrocarbons in the first four fractions with squalene increasing to Fraction 3 and the pristane level being highest in Fraction 1. Aside from pristane and squalene, other hydrocarbons occurred at levels of 420–750 mg% in the oils on a weight basis, of which about 60% constituted a series of n-paraffins (relative carbon number range: 15.0–38.0) together with smaller amounts of at least one branched chain saturated group. Unsaturated hydrocarbons eluted mainly after squalene. The oils contained up to 460 mg% sterol and 78–270 mg% alcohols of C₁₀ to C₃₀, the ratio of saturated to unsaturated members being about 1.6. The composition of the unsaponifiable lipids of pig liver was quite different from that of the marine oils. It contained 10.6% sterol in addition to 400 mg% alcohols, the latter consisting of 81.8% saturated components (C₁₂ to C₃₁; ratio of saturated: unsaturated members, 4.4). The hydrocarbons comprised 450–700 mg% of the unsaponifiable mixture and squalene, paraffins and additional unsaturated components occurred at levels of 20.6, 24.4 and 11.9 mg%, respectively. The saturated hydrocarbons were high in normal homologs of relative carbon number range, 15 to 36; pristane could not be detected.     

Unsaponifiable matter of green and blue-green algal lipids as a factor of biochemical differentiation of their biomasses: II. Terpenic alcohol and sterol fractions

The composition of the terpenic alcohol and sterol fractions of the unsaponifiables of ten algal species, fiveMyxophyceae and fiveChlorophyceae, is discussed. The major component of the terpenic fraction is phytol, a diterpenic alcohol. Minor amounts of straight chain and triterpenic alcohols are also present. Practically all the species examined contain ten components in the sterol fraction: cholesterol, brassicasterol, Δ⁵-ergostenol, poriferasterol, Δ⁷-ergostenol, clionasterol, chondrillasterol, Δ⁵-avenasterol, Δ⁷-chondrillastenol, and an unidentified component. Identification of the sterols was made by gas chromatography-mass spectrometry, and a 24 S configuration was assumed. The prokaryotic blue-green algae are characterized by a higher content in cholesterol (3.5–14%) than the eucaryotic green algae (0–2.5). Also, brassicasterol, poriferasterol, clionasterol, and Δ⁵-avenasterol are more abundant in blue-green algae. Δ⁷-Ergostenol, chondrillasterol, and Δ⁷-chondrillastenol predominate, on the contrary, in green algae.     

The incorporation of fatty acids of different chain length into liver and biliary lipids in the perfused rat liver

In an attempt to correlate the incorporation of fatty acids (FA) of different chain length into liver and biliary lipids’ isolated rat livers were perfused for 2 h with Krebs-Ringer bicarbonate containing 1% albumin and 10 μmol of [1-¹⁴C]-labeled FA: C₂’ C₈’ C₁₀’ C₁₂’ C₁₆’ and C_18∶1. One to 1.36 μmol of medium-chain fatty acids (MCFA’ C₈’ C₁₀’ and C₁₂) and 6.6 μmol of long-chain FA (LCFA) were incorporated into liver lipids’ 40% of the latter into phosphatidylcholine (PC). ¹⁴C-acetate (13 nmol) was incorporated into biliary cholesterol; ¹⁴C-MCFA contributed only 3.2–5 nmol; LCFA did not lead to newly synthesized cholesterol. Newly synthesized liver PC (2.75 to 3.25%) and newly synthesized liver cholesterol (6.5 to 10%) were secreted into bile. The specific radioactivity of biliary PC after infusion of all-saturated FA was 3.8–6.8 times higher than that of liver PC; for C_18∶1 it was only 1.7-fold. The specific radioactivity of biliary cholesterol’ as compared to liver cholesterol’ was 12 times higher for C₂ and five times higher for MCFA. This indicates that a considerable proportion of the newly synthesized lipids was secreted into bile prior to significant mixing with preexisting liver PC and cholesterol pools. liver PC contained 8% of unchanged ¹⁴C−C₁₂; while ¹⁴C−C₁₀ was not detected. Biliary PC’ in contrast’ contained 18% of unchanged ¹⁴C−C₁₂ and 3% ¹⁴C−C₁₀. These results suggest that after prolonged infusion of medium-chain triacylglycerols/longchain triacylglycerols to patients’ biliary PC may become enriched with MCFA. In addition’ the oxidation of these FA may provide C-2 units which increase cholesterol synthesis.     

Comparative studies on the fatty acid composition of moderately and extremely thermophilic bacteria

The fatty acids of three strains of extremely thermophilic bacteria and three strains of moderately thermophilic bacteria were examined by gas liquid chromatography. All the thermophiles contained straight, iso, and ante-iso branched fatty acids. Iso C_17∶0 acid was abundant in both the moderately thermophilic strains (10–33%) and the extremely thermophilic strains (50–61%). The pair of fatty acids iso C_15∶0 and iso C_17∶0 was the predominant pair in both the moderately (34–64%) and extremely (76–87%) thermophilic strains. The pair of fatty acids ante-iso C_15∶0 and ante-iso C_17∶0 was present in larger amount in moderately (25–34%) than in extremely (8.5–15%) thermophilic strains. No hydroxy cyclopropane, or unsaturated fatty acids were found. One extreme thermophile,Flavobacterium thermophilum HB-8 was grown at 6 different culture temperatures from 49–82 C, and the changes of its fatty acid composition were studied. The ratios of iso C_17∶0/iso C_15∶0 and ante-iso C_17∶0/ante-iso C_15∶0 were much greater at higher culture temperatures, indicating chain elongation.     

A method for the separation of seed oil steryl esters and free sterols: Application to peanut and corn oils

A method for separating and quantitating seed oil steryl esters and free sterols was developed using a combination of preparative column, thin layer (TLC), and gas liquid chromatography (GLC). Cholesteryl heneicosanoate and cholesterol served as internal standards. The method was applied to corn-oil samples (Mazola, Kroger) obtained from the local market and peanut-oil samples prepared in the laboratory from commercial varieties of peanuts (Florunner, Starr). Concentration (mg/100 g oil; mean ± SD) of steryl esters and free sterols in the 4 oils were: Mazola, 1420±40 and 370±8; Kroger, 950±40 and 320±4; Florunner, 74±0.5 and 150±3; and Starr, 51±0.5 and 130±2. Sitosterol was the major sterol in both the free sterol and steryl ester fractions of all oils and together with campesterol, stigmasterol and Δ⁵-avenasterol made up 90–95% of all sterols. Steryl esters of peanut oil contained higher proportions of linoleic acid and long-chain acids (C₂₀–C₂₄) than did whole oil. Corn-oil steryl esters also contained a higher proportion of linoleic acid than did whole oil. Squalene was the major hydrocarbon of all oils with the remaining hydrocarbon fraction consisting of a mixture of compounds. Presented at the AOCS meeting, Toronto, May 1982.     

The content and composition of sterols and sterol esters in low erucic acid rapeseed (Brassica napus)

The low temperature crystallization technique for the enrichment of “minor” components, such as sterols and sterol esters, from vegetable oils was applied to low erucic acid rapeseed oils. The recovery of free sterols and sterol esters was estimated by use of¹⁴C-cholesterol and¹⁴C-cholesterol oleate. 80% of the free sterols and 45% of the sterol esters were recovered in the liquid fraction, while in two studies total recoveries were 95% and 99%, respectively. This technique showed some selectivity toward the sterol bound fatty acids when compared to direct preparative thin layer chromatography (TLC) of the crude oil. Gas liquid chromatography (GLC) analysis of the free and esterified sterols as TMS-derivatives showed very little selectivity in the enrichment procedure. The fatty acid patterns of the sterol esters demonstrated, however, a preference in the liquid fraction for those sterol esters which have a high linoleic and linolenic acid content. The content of free sterols was 0.3–0.4% and that of sterol esters 0.7–1.2% of the rapeseed oils in both winter and summer types of low erucic acid rapeseed (Brassica napus) when the lipid classes were isolated by direct preparative TLC of the oils. The free sterols in the seven cultivars or breeding lines analyzed were composed of 44–55% sitosterol, 27–36% campesterol, 17–21% brassicasterol, and a trace of cholesterol. The esterified sterols were 47–57% sitosterol, 36–44% campesterol, 6–9% brassicasterol, and traces of cholesterol and Δ5-avenasterol. The fatty acid patterns of these esters were characterized by ca. 30% oleic acid and ca. 50% linoleic acid, whereas these acids constitute 60% and 20%, respectively, of the total fatty acids in the oil. Little or no variation in sterol and sterol ester patterns with locality within Sweden was observed for the one cultivar of summer rapeseed investigated by the low temperature crystallization technique.     

A cross-species comparison of neutral lipid composition of milk fat of prosimian primates

The fatty acid composition of milk fat is known to be affected by dietary and genetic differences, while the milk triacylglycerol structure is believed to be attuned to the needs of the subsequent lipolysis during gastrointestinal passage. The availability of milk samples from eight species of prosimian primates, whose milk triacylglycerol structure had not been analyzed, offered an opportunity to further assess these ideas. The milk samples were collected by manual expression and the lipids extracted with chloroform/methanol (2∶1, vol/vol). The lipid classes were resolved by thin-layer chromatography, and the neutral lipids subjected to detailed analyses by capillary gas-liquid chromatography of fatty acids and molecular species of triacylglycerols using nonpolar and polarizable liquid phases. The milk samples were found to differ greatly in total fat content (4–73%) and in the composition of the neutral liqid classes and molecular species. The concentration of triacylglycerols ranged from 88–95%, free fatty acids from 0.5–10%, alkyldiacylglycerols from 0.5–5.0%, and diacylglycerols, monoacylglycerols and free and esterified cholesterol made up the remainder. The fatty acid chain length ranged from C₈−C₂₄, with palmitic (16–31%) and oleic (13–40%) acids being the major components in most of the species. In all instances, the molecular association of the fatty acids differed from random distribution by a higher proportion of the monoacid (trioleoyl) and diacid (dipalmitoyloleoyl) glycerols. The phylogenetic influences on neutral milk lipid composition, however, remained unclear, as some of the differences between closely related species were greater than those between more distantly related ones. Triacylglycerol structures are abbreviated by listing their three constituent fatty acids in sequence, e.g., PPP, LaOL.     

Molecular species of wax esters in jaw fat of atlantic bottlenose dolphin,Tursiops truncatus

The jaw fat of the Atlantic bottlenose dolphin (Tursiops truncatus) contains unusual wax esters which can be separated into short chain (<C₂₄) and long chain (>C₂₄) fractions by thin layer chromatography. The short chain wax esters (28 wt. %) have been characterized as a 72∶24∶4 mixture of isovaleroyl, isobutoryl, and 2-methylbutyrol, esters of C₁₄–C₁₈ n- and iso-alcohols. The intact <C₂₄ esters have been resolved into individual molecular species by gas liquid chromatography on open-tubular polyester columns. The long chain wax esters (12 wt. %) contain C₁₀–C₂₂ n- and iso-acids esterified to the same C₁₄–C₁₈ n- and iso-alcohols. Gas liquid chromatography of the intact, hydrogenated >C₂₄ esters on a short JXR column has characterized them according to carbon number and the number of methyl branches they contain.     

The lipids of the common house cricket,Acheta domesticus L. III. Sterols

Sterols constitute 1.95% of the total extractable lipids ofAcheta domesticus L., of which 18% are esterified. The free sterols consist of cholestane-3β-ol (0.5%), Δ⁵-cholestene-3β-ol (83.5%), Δ⁷-cholestene-3β-ol (2.3%) Δ^5,7-cholestadiene-3β-ol (3%), Δ^5,22-cholestadiene-3β-ol (4%), Δ^5,7,22-cholestatriene-3β-ol (0.2%), campestane-3β-ol (0.03%), Δ⁵-campestene-3β-ol (1.0%), Δ⁷-campestene-3β-ol (trace), Δ^5,7-campestadiene-3β-ol (0.2%), stigmastane-3β-ol (0.09%), Δ⁵-stigmastene-3β-ol (2.1%), Δ⁷-stigmastene-3β-ol (0.04%), Δ^5,7-stigmastadiene-3β-ol (0.4%), Δ^5,22-stigmastadiene-3βol (0.1%). The same sterols are present in the esterified sterol fraction. Δ⁷-Sterols and Δ^5,7-sterols are present in significantly larger amounts in the esterified fraction than in the free sterol fraction. By a comparison with the sterols of the cricket food, it is clear thatA. domesticus is capable of removing methyl and ethyl groups from C-24 of sterols of the campestane and stigmastane type. The ability to introduce a Δ⁷ double bond into saturated and Δ⁵-sterols is indicated, and it is suggested that Δ⁷-sterols of the C₂₇, C₂₈, and C₂₉ sterol series may be intermediates in the conversion of Δ⁵-sterols to Δ^5,7-sterols. Associate Professor, Department of Chemistry, University of Michigan, Ann Arbor, Mich.; Alfred P. Sloan Foundation Fellow, 1968–68. Public Health Service Predoctoral Fellow, 1968–67.     

Skin surface lipids of the dog

The skin surface lipid of the dog has been reported to contain a high proportion of diol diesters having a lower mobility on thin layer chromatography than diesters from other species in spite of containing similar fatty acid and diol components. In the present study, dog skin surface lipid was separated by preparative thin layer chromatography into sterol esters (42%), wax diesters (32%), free sterols (9%), polar lipids (7%), and unidentified components (10%). The diesters contained 1,2-diols, each esterified with one long chain fatty acid and one isovaleric acid moiety. The diols were principally branched chain C₂₁ and C₂₂ compounds while the long chain fatty acids esterified with them were mainly C₂₀ and C₂₁ branched compounds. The fatty acids from the sterol esters were mostly saturated, branched chain C₁₉ to C₂₃, together with 7% of straight chain monoenoic acids, principally C₂₁ and C₂₂. There were only trace amounts of free sterols other than cholesterol, while the esterified sterols contained 96% cholesterol and 4% lathosterol.     

General characteristics of Pinus spp. Seed fatty acid compositions, and importance of Δ5-olefinic acids in the taxonomy and phylogeny of the genus

The Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) contents and profiles of gymnosperm seeds are useful chemometric data for the taxonomy and phylogeny of that division, and these acids may also have some biomedical or nutritional applications. We recapitulate here all data available on pine (Pinus; the largest genus in the family Pinaceae) seed fatty acid (SFA) compositions, including 28 unpublished compositions. This overview encompasses 76 species, subspecies, and varieties, which is approximately one-half of all extant pines officially recognized at these taxon levels. Qualitatively, the SFA from all pine species analyzed so far are identical. The genus Pinus is coherently united—but this qualitative feature can be extended to the whole family Pinaceae—by the presence of Δ5-UPIFA with C₁₈ [taxoleic (5,9–18∶2) and pinolenic (5,9,12–18∶3) acids] and C₂₀ chains [5,11–20∶2, and sciadonic (5,11,14–20∶3) acids]. Not a single pine species was found so far with any of these acids missing. Linoleic acid is almost always, except in a few cases, the prominent SFA, in the range 40–60% of total fatty acids. The second habitual SFA is oleic acid, from 12 to 30%. Exceptions, however, occur, particularly in the Cembroides subsection, where oleic acid reaches ca. 45%, a value higher than that of linoleic acid. α-Linolenic acid, on the other hand, is a minor constituent of pine SFA, almost always less than 1%, but that would reach 2.7% in one species (P. merkusii). The sum of saturated acids [16∶0 (major) and 18∶0 (minor) acids principally] is most often less than 10% of total SFA, and anteiso-17∶0 acid is present in all species in amounts up to 0.3%. Regarding C₁₈ Δ5-UPIFA, taxoleic acid reaches a maximum of 4.5% of total SFA, whereas pinolenic acid varies from 0.1 to 25.3%. The very minor coniferonic (5,9,12,15–18∶4) acid is less than 0.2% in all species. The C₂₀ elongation product of pinolenic acid, bishomo-pinolenic (7,11,14–20∶3) acid, is a frequent though minor SFA constituent (maximum, 0.7%). When considering C₂₀ Δ5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11–20∶2 and sciadonic acids are ≤0.3 and ≤1.9%, respectively, whereas in the latter subgenus, they are most often ≥0.3 and ≥2.0%, respectively. The highest values for 5,11–20∶2 and sciadonic acids are 0.5% (many species) and 7.0% (P. pinaster). The 5,11,14,17–20∶4 (juniperonic) acid is present occasionally in trace amounts. The highest level of total Δ5-UPIFA is 30–31% (P. sylvestris), and the lowest level is 0.6% (P. monophylla). Uniting as well as discriminating features that may complement the knowledge about the taxonomy and phylogeny of pines are emphasized.     

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.