Jojoba oil wax esters and derived fatty acids and alcohols: Gas chromatographic analyses

HCl-catalyzed ethanolysis followed by saponification readily surmounts the resistance of long chain wax esters to direct hydrolysis by alkali. Additionally, choosing ethyl instead of methyl esters allows baseline separations between long-chain alcohols and corresponding esters in gas liquid chromatographic (GLC) analysis of total alcohol and acid components before saponification. Liquid wax esters were analyzed on a temperature-programmed 3% OV-1 silicone column. Geographical and genetic effects on the variability of jojoba oil composition were investigated with five different seed samples. Major constituents in jojoba seed oil from shrubs in the Arizona deserts, as indicated by GLC analyses of oil, ethanolysis product, isolated fatty alcohols and methyl esters of isolated fatty acids, were C₄₀ wax ester 30%, C₄₂ wax ester 50% and C₄₄ wax ester 10%; octadecenoic acid 6%; eicosenoic acid 35%, docosenoic acid 7%, eicosenol 22%, docosenol 21% and tetracosenol 4%. Oil from smaller leaved prostrate plants growing along California’s oceanside showed a slight tendency toward higher molecular size than oils from the California desert and Arizona specimens. The wax esters are made up of a dispro-portionately large amount of docosenyl eicosenoate and are not a random combination of constituent acids and alcohols.Lunaria annua synthetic wax ester oil was used as a model for evaluating the analytical procedures. Presented at the AOCS Meeting, Chicago, September 1970 No. Utiliz, Res. Dev. Div., ARS, USDA.     

Molecular analysis of intact preen waxes of Calidris canutus (Aves: Scolopacidae) by gas chromatography/mass spectrometry

The intact preen wax esters of the red knot Calidris canutus were studied with gas chromatography/mass spectrometry (GC/MS) and GC/MS/MS. In this latter technique, transitions from the molecular ion to fragment ions representing the fatty acid moiety of the wax esters were measured, providing additional resolution to the analysis of wax esters. The C₂₁−C₃₂ wax esters are composed of complex mixtures of hundreds of individual isomers. The odd carbon-numbered wax esters are predominantly composed of even carbon-numbered n-alcohols (C₁₄, C₁₆, and C₁₈) esterified predominantly with odd carbon-numbered 2-methyl fatty acids (C₇, C₉, C₁₁, and C₁₃), resulting in relatively simple distributions. The even carbon-numbered wax esters show a far more complex distribution due to a number of factors: (i) Their n-alcohol moieties are not dominated by even carbon-numbered n-alcohol moieties are not dominated by even carbon-numbered n-alcohols esterified with odd carbon-numbered 2-methyl fatty acids, but odd and even carbon-numbered n-alcohols participate in approximately equal amounts; (ii) odd carbon-numbered methyl-branched alcohols participate abundantly in these wax ester clusters; and (iii) with increasing molecular weight, various isomers of the 2,6-, 2,8-, and 2,10-dimethyl branched fatty acids also participate in the even carbon-numbered wax esters. The data demonstrate that there is a clear biosynthetic control on the wax ester composition although the reasons for the complex chemistry of the waxes are not yet understood.     

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.     

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.     

Wax esters in the cystacanths ofPolymorphus minutus (Acanthocephala)

The lipids of the cystacanth of the acanthocephalanPolymorphus minutus have been analyzed. Wax esters constituted nearly 90% of the total cystacanth lipids. The wax ester fraction contained approximately 10% steroid ester; the rest was long chain alcohols C₁₂ to C₂₀, largely saturated, esterified with fatty acids C₁₂ to C₂₂, mostly unsaturated, with C₁₈ predominating. Corresponding quantities of wax esters were not found in the adult parasite. Cholesterol was identified as the only steroid present in the cystacanth.     

Solubility and heat stability of fat-soluble derivatives of vitamin B6

Summary The heat stability of several fatty acid esters of pyridoxine and their solubility in fats was tested. Short chain fatty acid esters of pyridoxine, such as pyridoxine triacetate, and long chain saturated fatty acid esters of the vitamin, such as pyridoxine tripalmitate were almost insoluble or had a rather limited solubility in fats. An improvement in solubility was observed when pyridoxine was partially esterified with palmitic acid, fully esterified with a long chain unsaturated fatty acid, such as linoleic acid, or when it was esterified with a fatty acid of intermediate chain length (C₆, C₈ or C₁₀). Free pyridoxine and the acetate were destroyed by heat when mixed in a fat and heated at 205–210°C. A profound improvement in heat stability in pyridoxine was noted when one palmitoyl group was introduced, and the ester became completely stable when it was fully esterified with a saturated fatty acid of C₁₀ or longer chain length. Portion of a thesis presented by T. Sakuragi as partial fulfillment of the requirement for the degree of Doctor of Philosophy in Food Technology. This work was supported by research grant No. A-257 from the National Institute of Health, U. S. Public Health Service, Department of Health, Education and Welfare. Presented at the 29th annual meeting of the American Oil Chemists’ Society, Oct. 10–12, 1955, Philadelphia, Pa.     

Melting points of synthetic wax esters

Saturated, monoenoic and dienoic wax esters, C₂₆−C₄₀, have been synthesized from even-numbered fatty alcohols and acids. In homologous series of saturated esters, the increments of melting points follow a regular trend except for those esters which have an acid moiety two carbon atoms shorter than the alcohol moiety. These wax esters have melting points higher than interpolation would predict. Monoenoic wax esters with the double bond in the alcohol chain have melting points about 10 C higher than their isomers with the double bond in the acid chain.     

Distinctive medium chain wax esters,triglycerides, and diacyl glyceryl ethers in the head fats of the pacific beaked whale,Berardius bairdi

Lipids were extracted from the mandibular fat body (jaw), the fatty forehead (melon), and the dorsal blubber of a Pacific beaked whale (Berardius bairdi) and separated into lipid classes by preparative thin layer chromatography. The head fats were mixtures of wax esters and triglycerides with a very small amount of diacyl glyceryl ether. The blubber fat contained 97% was ester and 3% triglyceride. Gas liquid chromatography (GLC) of the intact lipid classes indicated an unusually low C₂₆–C₃₀ range for most of the jaw and melon wax esters compared to the more normal C₃₂–C₄₀ molecules found in the blubber. Distinctive lower molecular weight C₂₄–C₄₀ triglycerides occurred in the head fats vs. the usual C₄₄–C₅₈ range in the blubber. Most diacyl glyceryl ethers were in the C₃₅–C₄₆ range, below the molecular weight of hexadecyldipalmitoyl glyceryl ether (C₄₈). GLC of the derived fatty acid methyl esters showed that the lower molecular weight neutral lipids in the head fats were due to high levels of iso-10∶0, n−10∶0, iso-11∶0, iso-12∶0, n−12∶0, and iso-13∶0 acids. The wax ester fatty alcohols and the alkoxy chains of the glyceryl ethers were mostly the C₁₄–C₂₀ chain lengths commonly observed in marine organisms. The distinctive medium chain neutral lipids in the jaw and melon fats of this whale may be related to the postulated acoustical role of these tissues in echolocation.     

Occurrence of wax esters and 1-O-alkyl-2,3-diacylglycerols in goat epididymal sperm plasma membrane

Two unusual lipid classes were detected by thin-layer chromatography in the neutral lipids derived from goat cauda-epididymal sperm plasma membrane. The lipids were identified as wax esters and 1-O-alkyl-2,3-diacylglycerols based on chromatographic properties, identity of their hydrolysis products, and infrared/¹H nuclear magnetic resonance spectral evidence. The membrane containedca. 3 and 5 μg/mg protein of wax esters and alkyldiacylglycerols, respectively. The relative proportions of wax esters and alkyldiacylglycerols in the total neutral lipids were 1.5% and 2.4%, respectively. The lipids contained fatty acids with chain lengths of C₁₄ to C₂₂. The major fatty acids of the wax esters were 14∶0, 16∶0, 16∶1ω7, 18∶0 and 18∶1ω9. The fatty acids in alkyldiacylglycerol were 16∶0, 18∶0, 22∶5ω3 and 22∶6ω3. Alkyldiacylglycerol was particularly rich in docosahexaenoic acid 22∶6ω3) representing 30% of the total fatty acids. The alcohols of wax ester were all saturated with C₂₀–C₂₉ carbon chains. The deacylated products derived from alkyldiacylglycerols were identified as hexadecyl, octadecyl and octadec-9′-enyl glycerol ethers.     

Esterified alkan-1-ols and alkan-2-ols in barley epicuticular wax

The epicuticular wax on all barley organs is characterized by the presence of long chain esters which have alkan-1-ols as their alcohol moiety (mainly C₃₈−C₄₈). A second type of long chain ester in which alkan-2-ols serve as the alcohol moiety has been identified in the wax from all organs except the awns and leaf blades (mainly C₃₃−C₃₅). Utilizing Silica Gel H column chromatography, a 95% separation of the two ester types was achieved. Unlike alkan-1-ols, the alkan-2-ols (primarily C₁₃ and C₁₅) do not occur free in the wax. Esters isolated from spike minus awn wax consisted of 62% alkan-2-ol and 38% alkan-1-ol containing esters. The isomeric composition of each ester was determined with the aid of gas liquid chromatography-mass spectrometry. In the mass spectra of the alkan-2-ol containing esters, mass ions were absent and the relative intensities of the RCO₂H₂ ⁺, RCO₂H⁺, [R′-1]⁺, and RCO⁺ ions were markedly different from those characteristic for alkan-1-ol containing esters. Since esterified alkan-2-ols occur only in those waxes having β-diketones, a close biosynthetic relationship between these two lipid classes is suggested.     

Cuticular lipids of insects: V. Cuticular wax esters of secondary alcohols from the grasshoppersMelanoplus packardii andMelanoplus sanguinipes

Wax esters of secondary alcohols constitute 18–20% of the cuticular lipid extract ofMelanoplus packardii and 26–31% of the cuticular lipids ofMelanoplus sanguinipes. The total number of carbons in the wax esters range from 37–54 with 41 predominating in both species. The fatty acids ofM. packardii wax esters are 16∶0, 18∶0, 14∶0, 20∶0 and 12∶0 in decreasing quantity. The fatty acids ofM. sanguinipes wax esters are 18∶0, 20∶0, 16∶0 22∶0, 14∶0, 19∶0 and 17∶0 in decreasing quantity. The secondary alcohols from the wax esters ofM. packardii are C₂₅, C₂₃ and C₂₇ in decreasing quantity, and the secondary alcohols of theM. sanguinipes are C₂₃, C₂₅, C₂₁, C₂₇, C₂₄, C₂₂ and C₂₆ in decreasing quantity. Each secondary alcohol consists of two to four isomers with the hydroxyl group located near the center of the chain. Montana Agriculture Experiment Station, Journal Series No. 332.     

Fatty Acid Composition in Ergosteryl Esters and Triglycerides from the Fungus Ganoderma lucidum

Free and esterified ergosterols are detected almost solely in fungi and are often employed as a biomarker of living fungi. In this work, the fatty acid composition and δ¹³C values of major fatty acids in triglycerides and ergosteryl esters from the fungus Ganoderma lucidum were analyzed by gas chromatography–mass spectrometer and gas chromatography–isotopic ratio mass spectrometer, respectively. The results showed that the fatty acid profiles varied in triglycerides and ergosteryl esters. The percentage of saturated fatty acids in ergosteryl esters was remarkably higher than that in triglycerides, where C_18:1^Δ9c was the predominant fatty acid and constituted 61.26 % of the total fatty acids. In contrast, C_16:0 was the predominant fatty acid and constituted 71.88 % of the total fatty acids in ergosteryl esters. The study suggests that, after fungal death, free ergosterols in the cell membrane of the dead fungus were esterified with preferentially saturated fatty acids, mainly C_16:0, from triglycerides and then stored in lipid particles for a longer period while free ergosterol markedly decreased. The δ¹³C values of C_16:0, C_18:0, C_18:1 and C_18:2 in ergosteryl esters exhibit a pronounced depletion in ¹³C compared with that in triglycerides within the range of ?1.3 to ?0.9 ‰, supporting the above inference. It is again suggested that free ergosterol in the cell membrane should be used as an indicator of living fungi, and ergosteryl esters in the lipid particles should not be included in the measurement of living fungal biomass.     

Composition of wax esters,triglycerides and diacyl glyceryl ethers in the jaw and blubber fats of the Amazon River dolphin (Inia geoffrensis)

The lower jaw fat of the Amazon River dolphinInia geoffrensis contains 52.8% wax ester, 44.7% triglyceride and 2.5% diacyl glyceryl ether, while its dorsal blubber fat is >98% triglyceride. Examination of the intact lipids, the derived fatty acids and the derived fatty alcohols by gas chromatography reveals that the blubber triglycerides show characteristics of freshwater fish fats, but the jaw fat lipids have several distinctive features. Jaw fat wax esters, triglycerides and diacyl glyceryl ethers are all rich in C₁₀, C₁₂ and C₁₄ fatty acids and contain no polyunsaturated acids. The fatty alcohols in the wax esters are over 90% saturated. The major carbon numbers in the jaw fat triglycerides (C₃₈–C₄₆) are considerably lower than those of the blubber triglycerides (C₄₈–C₅₄). The possible adaptation of the jaw lipids for use in the underwater echolocation process of this dolphin is discussed.     

Cholesteryl ester compounds containing alkyl branched acyl groups — their preparations,properties and applications

Cholesterol having a reactive hydroxyl group at C₃-position can react with fatty acids to give the corresponding cholesteryl esters. Most of the natural cholesteryl esters consist of straight alkyl chain fatty acids with a high melting point. In oleochemistry it is well known that alkyl branched fatty acids, which are derived from petroleum or the fat and oil industry, have low melting points (mp.) and are chemically more stable if they are saturated acids. We designed alkyl branched fatty acid cholesteryl esters by means of common esterification and found some esters having a low mp. (mostly as a liquid). They had no irritative effect on both animal and human skin. They showed characteristic emulsification properties, namely the formation of either O/W or W/O emulsion coexistence together with other lipid components. Applying them onto human skin, they were able to penetrate towards the stratum corneum and improve the water-retaining ability and the barrier function of the stratum corneum. Based on these properties we have been applying the alkyl branched fatty acid cholesteryl ester, especially the methyl branched isostearic acid cholesteryl ester (IS-CE), to a shampoo and a rinse as hair cosmetics, skin care cosmetics and bath-additive products in the past decade.     

Lipids of freshwater dolphinSotalia fluviatilis: Comparison of odontocete bioacoustic lipids and habitat

The melon and jaw lipids of the freshwater dolphinSotalia fluviatilis are composed mainly of isovaleroyl wax esters and diisovaleroyl triglycerides. The blubber fat contains only a trace of wax ester and is mostly tri- (long chain) and monoisovaleroyl triglycerides. Detailed gas liquid chromatographic analyses of the intact wax esters and triglycerides and of the derived fatty acids and fatty alcohols indicate common compositional patterns in the wax esters and triglycerides of the respective head lipids. Both odd and even long chain (C₁₂−C₁₆) iso-structures are prominent in the melon and jaw lipids, but only higher odd chain length iso-acids are major components in the blubber.Sotalia fluviatilis (family Delphinidae) andInia geoffrensis (family Platanistidae) share the same freshwater habitat in the upper Amazon River, and both utilize echolocation to navigate and to find food. Comparison of their respective bioacoustical lipid compositions shows distinctive types of head fats,Sotalia being rich in iso-5∶0 andIndia lacking iso-5∶0. This indicates that isovaleric acid per se has no obligatory role in dolphin echolocation.     

Gas-liquid chromatography of triglycerides from erucic acid oils and fish oils

By critically selecting optimum operating conditions, quantitative gas-liquid chromatography of triglycerides has been extended to molecules containing substantial amounts of C₂₀, C₂₂, and C₂₄ fatty acids. The triglycerides of four erucic acid oils (water cress, rapessed, nasturtium, andLunaria annua) and two fully hydrogenated fish oils (menhaden and tuna) have been quantitatively analyzed by this technique. The average fatty acid chain length calculated from the triglyceride composition of each oil agreed closely with that determined by GLC of its respective methyl esters. Several conclusions about the triglyceride composition of the fats analyzed are discussed. Winner, AOCS Bond Award. Presented at the AOCS Meeting in Cincinnati, October 1965.     

The Di- and triesters of the lipids of steer and human meibomian glands

Three groups of diesters have been isolated and identified in the lipids of steer meibomian glands. The first group, designated as α Type I, with the abbreviated formula FA-αOHFA-FA1c, consisted of α-hydroxy fatty acids esterified to fatty acids and fatty alcohols in the approximate molar ratio 1∶1∶1. The second group, designated as ω Type I-St, with the abbreviated formula FA-ωOHF A-St, consisted of ω-hydroxy fatty acids esterified to fatty acids and sterols in the approximate molar ratio 1∶1∶1. The third group, designated as α,ω Type II, with the abbreviated formula FA-α,ωdiol-FA, consisted of α,ω-diols esterified to 2 moles of fatty acids. The sum of the different diesters comprised about 9% of total steer meibomian lipids. Capillary GLC of the fatty acids of αType I diesters showed the fatty acids to be a family with a two-cluster profile, one at C₁₂ to C₂₀ and the other at C₂₁ to C₃₁, with anteiso chains predominating. Fatty acids from ωType I-St and α,ωType II diesters gave mainly a one-cluster profile in the short long chain, C₂₃ to C₃₀, with anteiso chains predominating, while the α-hydroxy fatty acids were short chain C₁₃ to C₁₈ acids with C₁₆ predominating. The sterols in diesters ωType I-St were cholesterol (∼60%), Δ7 cholestenol (∼35%) and an unidentified compound (∼5%) with a GLC retention time slightly longer than Δ7 cholestenol on SE-30 phase. The ω-hydroxy fatty acids and α,ω-diols both were of exceedingly long chain lengths, C₂₉−C₃₈, and showed similar GLC profiles. Two types of triesters comprising approximately 1% of total steer meibomian lipids have been isolated but incompletely characterized. In terms of molar ratios, one group of triesters gave fatty acids:ω-hydroxy fatty acids:α-hydroxy fatty acids:sterols + fatty alcohols as approximately 1∶1∶1∶1. The other contained fatty acids, α-hydroxy fatty acids and α,ω-diols in what appears to be a complex mixture of several triesters. Diesters ωType I and α,ωType II also were found in human meibum. Hitherto these two diesters have not been found in any animal tissue.     

Neutral lipids of chickpea flour and protein isolates

The neutral lipids composition of defatted chickpea flour and two types of protein isolates has been studied. The main compounds in neutral lipids are triacylglycerols, free fatty acids, and diacylglycerols. Other compounds present are wax esters, free fatty alcohols, and free sterols. The main fatty acids in neutral lipids are C_18:2 and C_18:1 among the unsaturated, and C_16:0 and C_18:0 among the saturated acids. Free and esterified alcohols range from C_16:0 to C_28:0, the majority being those with an even number of carbon atoms. Sterols observed are β-sito-sterol, campesterol, stigmasterol, and δ-5-avenasterol. Triacyl-glycerols are partially hydrolyzed, and the amounts of unsaturated sterols and unsaturated fatty acids are reduced as a result of the chemical treatment during production of the protein isolates.     

The metabolism of neutral lipids in the spur dogfish,Squalus acanthias

Cell free studies have shown that liver and intestine are the major sites of synthesis of triacyl glycerols inSqualus acanthias. The liver and to a lesser extent the intestine and stomach are major sites of wax ester synthesis. Muscle does not synthesize either triacyl glycerols or wax esters significantly. In vivo studies have shown that intravenously injected (³H) fatty alcohol is massively oxidized to (³H) fatty acid, the bulk of which appears in muscle. Liver appears to export both free fatty acids and triacyl glycerols to serum and thence to muscle. Free fatty acids, triacyl glycerols, wax esters and cholesteryl esters are all turned over within 48 hr inSqualus serum. The turnover of triacyl glycerols greatly exceeds the turnover of alkyl diacyl glycerols.     

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.