Cuticular lipids of insects

Cuticular lipids cover nearly all parts of insect and are the chief agent for restricting water loss. The structure of insect epicuticles is reviewed, with emphasis on the role of cuticular lipids and the metabolism of cuticular lipids. The chemical composition of insect cuticular lipids are discussed with particular emphasis on the types in which hydrocarbons, was esters and fatty alcohols predominate. What little evidence is available on the biosynthesis of insect cuticular lipids is discussed. One of six papers to be published from the Symposium on Natural Waxes, presented at the AOCS Meeting, San Francisco, April 1969.     

Cuticular lipids of insects: VIII. Alkanes of the mormon cricketAnabrus simplex

The cuticular hydrocarbons of the Mormon cricket,Anabrus simplex, are all saturated and consist of n-alkanes (29+%), 3-methylalkanes (12%), internally branched monomethylalkanes (26+%), and dimethylalkanes (28+%). The principal n-alkane is the C₂₉ component, with a range from C₂₃ to C₃₃. The 3-methylalkanes range from C₂₈ to C₃₂, and the internally branched monomethyl-and dimethylalkanes range from C₂₉ to C₃₉. When the branched alkanes ofA. simplex are compared to those from other insects in the order Orthoptera, interesting patterns of methyl branching are observed. Scientific Journal Series 631, Agricultural Experiment Station, University of Montana, Bozeman, Montana 59715.     

Cuticular lipids of insects: I. Hydrocarbons ofLeucophae maderae andBlatta orientalis

The major hydrocarbons found in the surface lipids of the cockroachesLeucophaea maderae andBlatta orientalis have been identified asn-heptacosane, 11-methylheptacosane, 13-methylheptacosane and 3-methylheptacosane. The hydrocarbons from the two genera of cockroaches are qualitatively identical and quantitatively similar.     

Incorporation of oxygen-18 into secondary alcohols of grasshopperMelanoplus sanguinipes

Incorporation of¹⁸O₂ and H₂ ¹⁸O into the secondary alcohol moiety of wax esters in the grasshopperMelanoplus sanguinipes was determined by mass spectrometry. Results of this study show that oxygen-18 from¹⁸O₂ was incorporated into the secondary alcohol, whereas no incorporation of oxygen-18 from H₂ ¹⁸O was detected. The data suggest that the reaction, which hydroxylates n-alkanes at or near the center of the carbon chain, involves a mixed function oxidase type enzyme.     

Cuticular lipids of insects: II. Hydrocarbons of the cockroachesPeriplaneta australasiae,Periplaneta brunnea andPeriplaneta fuliginosa

The major cuticular hydrocarbons of the cockroachesPeriplaneta australasiae, Periplaneta brunnea andPeriplaneta fuliginosa aren-tricosane,cis-9-tricosene, 3-methyltricosane, 11-methyltricosane and 13-methylpentacosane. There are as yet unexplained quantitative differences between the hydrocarbon compositions of males and females ofP. australasiae andP. fuliginosa, cis-9-tricosene being a major hydrocarbon of the males only. A series of mono-methyl internally branched hydrocarbons ranging in chain length from 23 to 26 carbons with the methyl branch on the 13th carbon from one end was observed. Minor quantities of other hydrocarbons have been identified.     

Fatty alcohols in capelin,herring and mackerel oils and muscle lipids: II. A comparison of fatty acids from wax esters with those of triglycerides

The fatty acids recovered from the triglycerides and wax esters of common northwest Atlantic copepods are compared with the fatty acids of wax esters recovered intact from certain fish skin and body lipid, and from commercial fish oils. The fish species, herring, capelin and mackerel, all feed on copepods, and many resemblances of the copepod lipid fatty acids to those of a previous analysis of similar copepods suggest that the basic dietary fat input for these fish may be quite constant. The two copepod fatty acid analyses differed quantitatively in triglyceride 20∶1 and 22∶1 and also in 20∶5ω3 and 22∶6ω3, confirming the primary role of the wax esters in copepods. Selectivity factors are discussed in comparing the copepod wax ester fatty acids with the fatty acids of the wax esters recovered intact from the fish lipids and oils. The basic role of copepods in supplying all types of fatty acids to fish depot fats is considered to be strongly supported by these findings.     

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.     

Studies of biosynthesis of waxes by developing jojoba seed: III. Biosynthesis of wax esters from Acyl-CoA and long chain alcohols

Acyl-CoA: alcohol transacylase activity was demonstrated in cell-free homogenates of developing jojoba seeds. The optimal pH was 8.0–8.1. Under optimal conditions, wax formation had a nearly linear relationship with extract concentration; the time course of wax formation was also linear up to 30 min.cis-11-Eicosenol was the most effective alcohol substrate whereas tetradecanol, octadecanol, dodecanol,cis-9-octadecanol, andcis-13-eicosenol gave progressively lower activities. Either saturated or unsaturated acyl-CoA with 18 or 20 C-atoms had similar activity. The enzyme was fairly stable at 0 C, less stable at RT and labile above 30 C. Differential centrifugation showed that the 12,000 × g fat pad was the most active in wax formation; to maximize the activity, a 12,000 × g supernatant appeared to be necessary. This factor in the supermatant was thermolabile and nondialyzable.     

Kinetics of the formation of symmetrical wax esters from the corresponding alcohols with the use of hydrobromic acid and hydrogen peroxide

The primary aliphatic alcohols n-octanol, n-decanol, and n-dodecanol have been converted to their corresponding symmetrical esters by using HBr and H₂O₂ in the absence of a solvent. The reaction was carried out at 30, 40, and 50°C and at mole ratios of alcohol to HBr of 1∶0.1, 1∶0.2, 1∶0.3, and 1∶0.5. The rate of the reaction was found to increase with increase in the reaction temperature and concentration of HBr. The maximal conversion of n-octanol was 72% at 40°C and a mole ratio of n-octanol to HBr of 1∶0.5. The kinetics of the reaction have been established, and the reaction was found to be first-order with respect to alcohol and bromine concentration in the organic phase, and second-order with respect to both. The second-order rate constants for n-octanol, n-decanol, and n-dodecanol are 27.08, 32.58, and 37.42 mL mol⁻¹ min⁻¹, respectively, at 40°C. The activation energy for the esterification reaction of n-octanol was found to be 16.32 kcal mol⁻¹.     

Physico‐chemical properties of wax esters synthesised from corresponding alcohols using hydrobromic acid and hydrogen peroxide action

Symmetrical wax esters were prepared directly from the C₁₄–C₂₂ alcohols using HBr and H₂O₂. Conversion of alcohol up to 98% was obtained. Physical properties such as melting point, refractive index, viscosity and specific gravity were determined for these wax esters at different temperatures. The physical properties of the synthetic wax esters were compared with those of some commercial samples of wax esters. The physical properties of the wax esters can be manipulated by starting with commercially available mixtures of alcohols.     

Separation of wax esters from steryl esters by chromatography on magnesium hydroxide

Chromatography of stearyl oleate and cholesteryl oleate on thin layer plates coated with magnesium hydroxide-celite, 1:1, or magnesia-celite, 1:1, showed that magnesium hydroxide had better resolving power for the separation of these wax ester and steryl ester model compounds than did magnesia, an adsorbent which has been used previously for this separation. By means of high pressure liquid chromatography on magnesium hydroxide, wax esters and steryl esters from the skin surface lipids of human, rat and monkey were separated completely and without hydrolysis.     

Fatty acids and fatty alcohols of wax esters in the orange roughy: Specific textures of minor polyunsaturated and branched-chain components

Open-tubular gas chromatography was carried out on fatty acids and alcohols obtained from wax esters of the orange roughy,Hoplostethus atlanticus, caught at sea off New Zealand. The major (above 5%) components were 16∶1(n−7), 18∶1(n−9) and (n−7), 20∶1(n−9) and (n−7), and 22∶1(n−11, n−13) as fatty acids, and 16∶0, 18∶0, 18∶1(n−9), 20∶1(n−9) and (n−7), and 22∶1(n−11, n−13) as fatty alcohols. The total percentages of the minor components were 10% in the acids and 26% in the alcohols. The 22∶1/20∶1 ratio of the fatty alcohols obtained in this study was less than 1.0, although the ratio for the Atlantic orange roughy has been reported as being greater than 1.0. The contents of polyenes were as low as 2.48% in the acids and 0.95% in the alcohols, but their compositions showed some specific features. The percentages of the C₁₆−C₂₂ dienes in the total polyenes were remarkably high, 57.7% of these acids and 53.1% of these alcohols. The most important dienes were 18∶2(n−6) in the acids and 20∶2(n−6) in the alcohols.     

Enzymatic synthesis of wax esters from rapeseed fatty acid methyl esters and a fatty alcohol

Wax esters were transesterified from fatty acid methyl esters of rapeseed and a fatty alcohol (1-hexadecanol, 16:0). The amounts of both the substrates were fixed to 0.1 mmol and an immobilized enzyme, Lipozyme, was used as catalyst. The experiment was performed following a statistic central composite design with five variables. The enzyme/lipid ratio was varied between 0.3–0.9 of the substrate weight and the enzyme was equilibrated to different water activities varying from 0.11 to 0.44. A temperature range of 50–80°C was investigated and the reaction time lasted up to 40 min. A solvent, isooctane, constituted 0–30% of the substrate weight. The first experimental series was performed in small closed test tubes. In the second series the caps of the test tubes were off to evaporate the methanol produced during the reaction. The highest initial reaction rate was 9.6 g_{wax esters}/g_enzyme · h. It appeared when: the enzyme/lipid ratio was low, 0.3, the temperature was high, 80°C; no isooctane was present; and the water activity was below 0.11. The initial reaction rate was independent of the caps on the test tubes. With the large amount of enzyme the yield of wax esters was above 70% after 10 min in both experimental series. In the reaction with caps, the reaction reached equilibrium at 83% after 20 min at 80°C. However, without caps the continuous evaporation of methanol increased the equilibrium constantly, and after 40 min at 80°C a yield of 90% was reached.     

Separation of wax esters from olive oils by high-performance liquid chromatography

To detect adulteration of olive oil with solvent-extracted oils, the determination of the wax ester content has become more important during recent years. Hence, a greater number of wax ester analyses need to be performed by quality control laboratories. The most common method in use requires a liquid chromatographic (LC) separation of the less polar fraction, which contains the wax esters, from the glyceride matter on a hand-filled silica gel column. The aim of this project was to verify the possibility of replacing LC with high-performance liquid chromatography by taking advantage of the greater reliability and repeatability of this technique, as well as of the possibility of making the separation automatic. The paper describes how to perform the analysis and the statistical test that was carried out; furthermore, a comparison has been made with the usual method and results are in good agreement.     

Arylsulfonate esters of fatty alcohols: IV. Effects on cholesterol catabolism

Hypercholesterolemic rats, fed 1% cholesterol and 0.5% glycocholate, were treated with arylsulfonates in various ways to observe the pattern of cholesterol elimination. Dietary linoleyl p-toluene-sulfonate (LTS) hastened return to normocholesterolemia and lowered hepatic cholesterol either with or without continued cholesterol feeding. LTS administered via the portal vein significantly lowered plasma cholesterol in 48 hr; ethyl linoleate and monoolein produced no lowering. LTS administered via the portal vein to glycocholate-infused rats increased the biliary excretions of label from [4-¹⁴C]cholesterol administered intracardially and also increased total bile acid excretion 21% without increased bile volume when compared to similar injection of ethyl linoleate. No change in biliary excretion of cholesterol was seen. Bile acid kinetics were studied by using isotopic dilution techniques. Cholate turnover was enhanced by feeding oleyl p-toluenesulfonate (OTS) and oleylp-(n-decyl)-benzenesulfonate (ODS) as suggested by a 16–35% decrease in half-life in both normal and hypercholesterolemic rats. Rats consuming a grain-based colony diet had a 54% increase in cholate synthesis when OTS was included in the diet. The composition of bile was changed when either OTS or ODS was fed; an increase in chenodeoxycholate was noted. This change was gradual with OTS but rapid with ODS and paralleled enhanced decay of chenodeoxycholate specific radioactivity in response to treatment. ODS and OTS also increased¹⁴CO₂ expiration from oral [26-¹⁴C] cholesterol in hypercholesterolemic rats. Dietary OTS and ODS elevated hepatic free cholesterol in hypercholesterolemic rats; ODS also elevated plasma free cholesterol and increased cholesteryl ester hydrolase activity in the liver. The data suggest that arylsulfonates stimulate cholesterol catabolism, in addition to the reported inhibition of cholesterol absorption [Lipids 12:819 (1977)]. Published as Journal Paper No. 6699, A.E.S., Purdue University.     

Sperm oil replacements: Synthetic wax esters from selectively hydrogenated soybean and linseed oils

Synthetic wax esters with properties similar to those of sperm whale oil have been prepared entirely from soybean and linseed oils. the synthesis required: (a) selective hydrogenation of the oils with copper-on-silica gel catalyst, (b) hydrogenolysis of fatty acids to fatty alcohols with copper-cadmiumchromium catalyst, and (c) esterification of hydrogenolysis products to yield predominantly long chain fatty esters which contained unsaturation in both the alcohol and acid moieties. Similarity of physical and chemical properties indicate that these wax esters are possible replacements for sperm oil. After sulfurization, the wax esters also have potential as extreme pressure lubricant additives.     

Temperature-enhanced alumina HPLC method for the analysis of wax esters,sterol esters,and methyl esters

Previous attempts at separating nonpolar lipid esters (including wax esters, sterol esters, and methyl esters) have achieved only limited success. Among the several normal-phase methods tested, a single recent report of a method employing an alumina column at 30°C with a binary gradient system was the most promising. In the current study, modification of the alumina method by increasing the column temperature to 75°C improved the separation of standards of wax esters and sterol esters. Elevated column temperature also enhanced the separation of FAME with differing degrees of unsaturation. Evidence was also presented to indicate that the method similarly separated phytosterol esters, based on their levels of unsaturation. With the increased interest in phytosterol- and phytostanol esterenriched functional foods, this method should provide a technique to characterize and compare these products.     

Organogelation of plant oils and hydrocarbons by long-chain saturated FA,fatty alcohols,wax esters,and dicarboxylic acids

Conversion of oils into gels generally involves altering the chemical characteristics of the liquid. We describe here the gelling of vegetable oils, essential oils, and hydrocarbons at ambient temperature, without changing the chemical characteristics of the liquids, using saturated FA having carbon chain lengths of 10 to 31. The gelling ability of the added FA increased linearly with their chain lengths. Structure-function studies demonstrated that the carboxyl group, position of an additional hydroxyl group, and acyl chain length played an important role in gelation. Long-chain saturated fatty alcohols, wax esters, and dicarboxylic acids also had the ability to gel plant oils and hydrocarbons.     

Jojoba wax: Its esters and some of its minor components

A lack of reliability in the usual determinations of fatty acids and fatty alcohols of jojoba wax prompted us to propose an original method of hydrolysis and extraction, making it possible to better determine the composition of fatty acids and alcohols of the wax. High-performance liquid chromatography fractionation of the wax allowed isolation of four main classes of esters (which differed by their partition number). The detailed study of these ester classes emphasized the way acids and alcohols are connected, and fourteen distinct esters were thus identified. Some triacylglycerols, free fatty alcohols and other minor components of jojoba wax were found and quantitated. Seven sterols were identified, four for the first time.     

One-step methodology for the synthesis of FA picolinyl esters from intact lipids

Picolinyl derivatives are used for structural determination of FA by GC-MS. Although they provide reliable diagnostic fragments, the usual multistep methodologies applied for their preparation require TAG hydrolysis or acid chloride formation prior to picolinyl synthesis. These reaction conditions may result in the presence of artifact molecules in the samples and thus compromise analytical quality and accuracy. To address these problems, a rapid, simple and quantitative methodology for the synthesis of FA picolinyl esters from intact lipids was developed. It involves their transesterification under basecatalyzed conditions using 3-potassiooxamethylpyridine in methylene chloride. The catalyst was prepared by proton exchange between potassium tert-butoxide and anhydrous 3-hydroxymethylpyridine. Mild reaction conditions allowed complete derivatization of TAG and phospholipids in 2 min at room temperature, and of FAME in 15 min at 45°C. The proposed procedure, which can be used on a routine basis, was applied to Ipomoae imperialis seed lipids and used to confirm occurrence of γ-linoleic acid at a level of 0.9%.