Eutectic interactions between saturated and unsaturated chain cholesteryl esters: comparison of calculated and observed phase diagrams

Binary phase behavior of saturated chain with unsaturated chain cholesteryl esters is evaluated by analysis of the phase diagrams in terms of ideal solution theory. Cholesteryl palmitate, which crystallizes in the bilayer structure, forms a eutectic with either cholesteryl oleate or cholesteryl linoleate and, as indicated by low angle X-ray data, the components are nearly totally fractionated in the solid state. The fit of the two experimental liquidus curves by a calculation of freezing point depression for an ideal solution indicates that the molecular interactions are nonspecific in the binary liquid state. Cholesteryl caprylate and cholesteryl oleate, both of which crystallize as the monolayer II form, also form a eutectic. X-ray data again indicate nearly total fractionation. The liquidus curve is reasonably well matched by calculation of ideal freezing point depression. However, dissimilar molecular volumes can cause the melt-cholesteric transition line to deviate from an ideal concentration dependence. Possible fractionation mechanisms for cholesteryl esters in arterial lesions are thereby indicated. For example, when the molecules have greatly different volumes, clustering can occur in the liquid crystalline state. Even when the molecular volumes are similar, the saturated component can solidify in regions where it is relatively abundant, because of the incompatibility of two crystal structures with greatly different layer structures.     

Acyl unsaturation and cholesteryl ester miscibility in surfaces. Formation of lecithin-cholesteryl ester complexes

The surface behavior of a series of cholesteryl esters was studied in mixtures with a model phospholipid, 1-palmitoyl-2-oleoyl phosphatidylcholine. The cholesteryl esters were representative of the predominant forms occurring naturally and qualitative similarities in their phase behavior permits generalization of their surface properties. Quantitative differences, however, show that the availability of cholesteryl esters in all surface states is dependent on the structure of the acyl moiety. All except cholesteryl stearate were surface-active and formed preferred packing arrays, i.e., complexes, with the lecithin at compositions grouped around cholesteryl ester mol fractions of 0.015. Exceptions were cholesteryl arachidonate and docosahexaenoate, with complex stoichiometries of 0.034 and 0.032, respectively. Lecithin had the same apparent area in all complexes, 56.5 +/- 1.04 A2, which was larger than that of uncomplexed lecithin, 53.3 +/- 0.7 A2. This implies that the conformation or orientation of the two polyunsaturated species in complexes is markedly different than the others studied. The areas and hydrations of all uncomplexed cholesteryl esters were similar. Because mixing of complex with uncomplexed cholesteryl ester deviated positively from ideality, the apparent molecular areas of the uncomplexed cholesteryl esters ranged from 161 +/- 22 (complex-rich) to 107 +/- 15 A2 (cholesteryl ester-rich). The similarity of the monolayer phase complex stoichiometries and the bilayer miscibilities of cholesteryl oleate suggests a correspondence between states. If so, the availability of cholesteryl arachidonate or docosahexaenoate in bilayers should be approximately twice that of other naturally occurring cholesteryl esters.     

Co-solubility of saturated cholesteryl esters: a comparison of calculated and experimental binary phase diagrams

Based on ideal solution theory, phase diagrams are calculated for binary compositions of cholesteryl esters and compared to experimental data from pairwise combinations in a saturated acyl chain series from caprylate to arachidate, which encompasses three crystal packing motifs in the solid state. Within a crystal structure class, nearly ideal co-solubility is found for binary solids, where the acyl chain lengths of the pure components differ by one methylene group. Beyond this chain length difference, nonideal solutions occur until fractionation occurs at e.g., six methylene unit increments between the components. The observed liquidus lines of the eutectic are near the theoretical curves when the combinations of two compounds packing in the same crystal structure fractionate. Fractionation also is found when liquids composed of two esters which favor different crystal structures are solidified from the melt, no matter what the chain length difference is; the liquidus curves for re-heated solids, however, are not necessarily predicted by the Schr?der equation. In general, co-miscibility can be found in mesophases formed from compounds with two different crystal structures.     

The chemical synthesis of ecdysone 22-long-chain fatty acyl esters in high yield

Eight ecdysone 22-long-chain fatty-acyl esters (laurate, myristate, palmitate, stearate, oleate, linoleate, linolenate and arachidate) have been chemically synthesised in high yield. Ecdysone was first converted to the 2,3-acetonide derivative and then acylated selectively at C-22 with the appropriate acyl anhydride. The protecting acetonide group is then removed by mild acid treatment to yield the ecdysone 22-acyl ester. Reaction conditions have been optimised to maximise the overall yield (ca. 70%). The ecdysone acyl esters and their 2,3-acetonide derivatives have been characterised by 1H- and 13C-NMR and high-resolution FAB-mass-spectrometry.     

Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa,with its application to cleaning lipid-stained contact lenses

With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.     

Properties of cholesteryl esters in pure and mixed monolayers

The surface properties of cholesteryl palmitate, stearate, linoleate, linolenate, arachidonate, and acetate were investigated. Long-chain esters were not surface-active and force-area (pi-A) isotherms were not obtained. Unsaturated cholesteryl esters were oxidized at the air-water interface and these oxidized lipids gave expanded pi-A isotherms. Cholesteryl acetate had an equilibrium spreading pressure of 14.0 dynes/cm and formed a stable monolayer indistinguishable from cholesterol below that surface pressure. Cholesteryl linoleate formed mixed monolayers with surface-active lipids, and the amount of cholesteryl linoleate in the monolayer depended both on its solubility in the other lipid and on the surface pressure. Even at moderate surface pressures cholesteryl linoleate was extruded from the monolayer into a bulk phase. Cholesteryl acetate exhibited the well-known condensing effect of cholesterol in mixed monolayers with egg lecithin.     

3种红枣的挥发性化学成分的乙醇提取及测定

新鲜成熟的去核油枣、木枣及团枣用95％乙醇浸泡,低温蒸除乙醇、膏状物用乙醚萃取,用气相色谱－质谱联用仪测定乙醚溶液。结果表明,3种新鲜成熟的红枣的挥发性成分主要为棕榈烯酸乙酯、棕榈酸乙酯、肉豆蔻乙酯、亚油酸乙酯、油酸乙脂、月桂酸乙酯、硬脂酸乙酯、苹果酸二乙酯、葵酸乙酸等16种酯类,以及乙酸和棕榈烯酸等2种酸和3－羟基－2－丁酮。不同品种的红枣中所含酯类的多少及含量的高低均有所差异。在同一种红枣的果肉中检测到16种酯类尚属首次报道。     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Crystal structure of cholestanyl caprylate and binary phase behavior with cholesteryl caprylate

The crystal structure of cholestanyl n-octanoate (caprylate) (C35H62O2) is monoclinic with space group A2 and cell dimensions a = 10.103(7), b = 7.646(7), c = 87.63(7) A, beta = 90.51(6) degrees; Z = 8 [two molecules (A, B) in asymmetric unit], V = 6769 A3, Dc = 1.010 g cm-3. Integrated X-ray intensities for 3798 reflections with I greater than 2 sigma (I) were measured with a rotating anode diffractometer at room temperature. The structure was determined using direct methods. Block diagonal least squares refinement gave R = 0.111. Molecules A and B have almost fully extended conformations, but differ significantly in the rotation about the ester bond and in the C17 chains. The molecular packing in the crystal structure of cholestanyl caprylate consists of stacked bilayers each having d002 = 43.8 A in thickness and within each bilayer, cholestanols pack with cholestanols and caprylate chains pack with caprylate chains. The crystal structure is very similar to that of cholesteryl myristate but is quite different from that of cholesteryl caprylate. The phase equilibria of the cholestanyl caprylate/cholesteryl caprylate binary system have been shown to involve limited mutual solubility of the two components and to have a eutectic point at 73% cholestanyl caprylate. The cholesteric mesophase is monotropic at all compositions except for a narrow range near the eutectic point where it is enantiotropic.     

Synthesis of saturated long chain fatty acids from sodium acetate-1-C14 by Mycoplasma

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-1-C(14). The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromatographic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester derivative, which migrated in a position intermediate to methyl myristate-1-C(14) and methyl palmitate-1-C(14). This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate.     

Effect of substrate composition and concentration on aortic cholesterol ester hydrolase activity.

Cholesterol ester hydrolase activity of pig aorta has been examined under optimum experimental conditions for hydrolysis of different cholesterol esters. The enzyme specific activity values were in the numerical order of substrates hydrolyzed: cholesteryl linoleate larger than or equal to linolenate greater than palmitate larger than or equal to stearate greater than oleate. The results are discussed in relation to the arterial accumulation of cholesterol esters.     

Phase behavior and crystalline structures of cholesteryl ester mixtures: a C-13 MASNMR study.

Cholesteryl esters are a transport and storage form of cholesterol in normal physiology but also a significant lipid in atherosclerotic plaques. To understand better the molecular properties of cholesteryl esters in tissues and plaques, we have studied the polymorphic and mesomorphic features of pure and mixed cholesteryl esters by solid state C-13 NMR with magic angle sample spinning (MASNMR). The temperature-dependent properties of two single components (cholesteryl linoleate (CL, C18:2) and cholesteryl linolenate (CLL, C18:3)), four binary systems (cholesteryl palmitate (CP, C16:0) with CL, CLL or cholesteryl oleate (CO, C18:1), and CO/CL), one ternary system (CO/CP/CL), and one quaternary system (CO/CP/CL/CLL) were studied. The mixing ratios were based on the composition of an atherosclerosis plaque dissected from a cholesterol-fed New Zealand white rabbit. C-13 MASNMR determined the phase transition temperatures, identified the phases present in all systems, and provided novel information about molecular structures. For example, solid CL exhibited a disordered structure with multiple molecular conformations, whereas pure CLL had a crystalline structure different from the three most commonly characterized forms (MLII, MLI, BL). In binary mixtures, the crystalline structure of each cholesteryl ester species was identified by its own characteristic resonances. It was found that CP always existed in its native BL form, but CL and CO were influenced by the composition of the mixture. CL was induced to form MLII crystals by the coexisting CP (55 wt%). When CO was cooled from the isotropic phase, it existed as a mixture of MLII and an amorphous form. The presence of CP significantly accelerated the conversion of the amorphous form to the MLII form. For the ternary mixture co-dried from chloroform, CL cocrystallized with CO in the MLII form and CP existed in BL form. Addition of a small amount of CLL slightly increased the heterogeneity of the solid mixture, but had little effect on the crystal structures or the phase transitions. C-13 MASNMR represents a powerful method for physical characterization of cholesteryl ester mixtures reflecting the composition of biological samples.     

Cholesteryl esters on the body surfaces of the camel tick,Hyalomma dromedarii (Koch, 1844) and the brown dog tick,Rhipicephalus sanguineus (Latreille, 1806)

Cholesteryl esters were found to constitute a major component of the lipids coating the body cuticle of females of the camel tick, Hyalomma dromedarii and the brown dog tick, Rhipicephalus sanguineus. One or more cholesteryl esters, alone or in combination, have been shown to serve as the mounting sex pheromone of several species of ixodid ticks. Consequently, knowledge of these compounds is important for an understanding of the mating behavior of these ticks. Based on thin layer chromatography, cholesterol and cholesteryl esters were the most abundant neutral lipids found on the body surfaces of fed females of these two species. Analysis using HPLC demonstrated significant quantities of the following compounds, tentatively identified as cholesteryl esters (expressed in micrograms per female equivalent), in H. dromedarii: Cholesteryl acetate 18.2; cholesteryl laurate, 6.8; cholesteryl linoleate, 24.8; cholesteryl oleate, 12.9; cholesteryl palmitate, 0.3; and cholesteryl stearate 1.7.In contrast, the same method revealed only 3 cholesteryl esters in extracts of females of R. sanguineus: Cholesteryl acetate, 2.0; cholesteryl linoleate, 8.5; and cholesteryl oleate, 3.0. In both species, two unidentified peaks, with the spectral characteristics of cholesteryl esters, were also observed. Identification of the cholesteryl esters was confirmed: by (1) positive bioassay results with conspecific (H. dromedarii) males and heterospecific (Dermacentor variabilis) males; (2) similarity of ultraviolet spectra between identified sample peaks and authentic standards; and (3) demonstration of cholesterol and the corresponding free fatty acid following enzymatic digestion of each of the HPLC-separated fractions containing the different cholesteryl esters. Comparisons with the cholesteryl ester composition of the mounting sex pheromone of other metastriate Ixodidae are discussed. These findings, along with studies reported previously, suggest that differences in the mounting sex pheromones of ixodid ticks are an important factor in minimizing heterospecific matings in nature.Supported by Cooperative Agreement No. 263-0152-A-00-2207 with the U. S. Agency for International Development to the Department of Infectious Diseases. College of Veterinary Medicine, University of Florida, Gainesville, FL and a subcontract, ODURF No. 536521, to the Department of Biological Sciences, Old Dominion University, Norfolk, VA     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

Phase behavior of cholesteryl ester dispersions which model the inclusions of foam cells

In order to understand the phase behavior of the approximately 1-micron-diameter droplets which occur in the cytoplasm of cholesterol-enriched cells, differential scanning calorimetry has been utilized to elucidate the factors controlling the rate of crystallization of cholesteryl esters. The kinetics of the thermotropic transitions between liquid, liquid-crystal, and crystal states which occur in mixtures of cholesteryl oleate and cholesteryl palmitate present in monodisperse, phospholipid-stabilized, emulsion droplets have been determined and are compared to the characteristics of these transitions in bulk mixtures. Cholesteryl palmitate is observed to crystallize in undercooled phospholipid-stabilized dispersions of cholesteryl palmitate/cholesteryl oleate (50/50 w/w) at temperatures up to 50 degrees C lower than it does in bulk mixtures of the same cholesteryl ester composition. It is postulated that this difference between crystallization temperatures is due primarily to the presence of impurities present in bulk mixtures which act as catalysts that promote crystallization. It is suggested that phospholipid-stabilized dispersions of cholesteryl palmitate/cholesteryl oleate are more appropriate models than bulk mixtures of these cholesteryl esters for studying the kinetic and thermodynamic basis of the phase behavior in cholesteryl ester rich inclusions characteristic of foam cells and atherosclerotic plaque. The thermotropic phase behavior of these dispersions can be satisfactorily analyzed by using the equations of homogeneous nucleation theory. The interfacial tension between the crystal nucleus and the surrounding fluid cholesteryl ester is about 10 erg/cm2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solvent-free enzymatic production of high quality cetyl esters

A solvent-free biocatalytic process for the synthesis of high quality cetyl laurate, myristate, palmitate and stearate has been optimized. This enzymatic procedure follows the fundamental principles of the Green Chemistry and lead to sustainable products, which can be labeled as natural and conform to the principal requirements for its use in high value-added goods. The four esters selected are the main components of spermaceti, a mixture of waxes very appreciated in cosmetic and pharmacy because of its physical properties and emolliency, which was formerly extracted from the head of the sperm whales. In this paper, the influence of the amount of biocatalyst, the commercially available Novozym^® 435, and the temperature were studied in an open-air batch reactor before carrying out the synthesis in a high performance vacuum reactor with dry nitrogen input to shift the equilibrium towards product formation. Under optimal conditions, conversion was higher than 98.5 %. The characterization of the enzymatic cetyl esters puts in evidence that these are ultra-pure compounds, which have similar properties to the ones obtained through the conventional industrial processes with the extra benefit of being environmentally friendly.     

Lipid structure and the behavior of cholesteryl esters in monolayer and bulk phases.

The behavior of cholesteryl esters at the air-buffer interface was studied as a function of molecular area and the presence of noncholesterol-containing lipids (colipids). The data obtained indicate that cholesteryl esters with other than long, saturated acyl groups can be present in surface phases up to packing densities approximately those in natural membranes. Their apparent molecular areas in such phases, which are largely determined by colipid structure, suggest their orientation with the ester function toward the interface. The extent of miscibility in the surface phase is also a strong function of colipid structure. Reversibility of the monolayer to bulk phase transition is determined exclusively by the acyl structure of the cholesteryl ester. Of the esters examined, only those with cis unsaturation collapsed reversibly. Our data predict that cholesteryl esters should be present in small, but finite amounts on the surface of arterial lipid deposits and that a prerequisite for the removal of such deposits is that the bulk lipid phase be in a liquid or liquid crystalline state.     

Crystal structure and microstructure of cholesteryl oleyl carbonate

The crystal structure as well as the microstructure, i.e., size and strain, of crystallites of cholesteryl oleyl carbonate was determined from X-ray powder diffraction data. The X-ray line broadening was analyzed through the refinement of TCH-pseudo-Voigt function parameters (isotropic effects) and the refinement of multipolar functions, i.e., symmetrized cubic harmonics (anisotropic effects). The crystal structure turns out to be primitive monoclinic, space group Pc, type I monolayer having two molecules per unit cell with parameters: a = 18.921 ± 0.006 Å, b = 12.952 ± 0.003 Å, c = 9.276 ± 0.002 Å and β = 91.32 ± 0.03°. The average size of a well ground specimen of crystallites was 60 nm. The average micro-strain, e.g., 45 × 10⁻⁴ has been tentatively attributed to fatty chain conformational disorder. The unit cell parameters, including the lamellar thickness, of COC crystal is very closely similar to those of another, structurally similar cholesterol ester, e.g., cholesteryl oleate (CO) crystal, space group P2₁, type II monolayer. Type I monolayer structure has been established for COC on the basis of the intensity calculations of the XRD profiles of both CO and COC. The dipolar and structural disorder in a 4:1 molar, binary mixture of CO and COC can be accommodated in an induced smectic phase with a lamellar thickness, which is nearly equal to that of pure CO or pure COC.     

Transformations of steroids and the steroidal alkaloid,solanine, by Phytophthora infestans

The following steroids and steroidal alkaloids have been incubated with the blight fungus Phytophthora infestans: androst-4-ene-3,17-dione, cholesterol, cholesteryl acetate, cholesteryl myristate, cholesteryl palmitate,cholesteryl stearate, dehydroisoandrosterone, 6α-hydroxy-androst-4-ene-3,17-dione, 6β-hydroxyandrost-4-ene-3,17-dione, 11α-hydroxyprogesterone, pregnenolone, progesterone, sitosterol, sitosteryl acetate, solanidine, solanine, stigmasterol, stigmasteryl acetate and testosterone. No hydroxylation was observed, but the fungus is able to oxidize alcohol functions at C-3β, C-6α, C-11β and C-17β to carbonyl. In addition, hydrolysis of acetate to hydroxyl at C-3β, and of solanine to solanidine, was observed. The relationship between metabolism and the nature of substitution at C-17β is discussed.     

Changes in ester compounds and higher alcohols of awamori during aging

The total contents of ethyl acetate, ethyl caproate, ethyl caprylate, isoamyl caproate, ethyl caprate, isoamyl caprylate, ethyl laurate, isoamyl caprate, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate and β-phenethyl acetate in awamori ranged from 70.67 to 569.72 mg per l a t 100 per cent alcohol. The ratio of ethyl acetate content to the total content of 14 ester compounds (ethyl acetate/total esters) ranged from 0.59 to 0.86, and that of ethyl caprylate to the total esters was from 0.98 ×10⁻² to 8.26 × 10⁻². Ethyl acetate was the main component of ester compounds followed in descending order by ethyl caprate, ethyl palmitate and ethyl caprylate. Of the higher alcohols, awamori contained only β-phenethyl alcohol in significant quantity.On aging in kame 13 of 16 ester compounds tended to decrease distinctly and the remaining 3 components to show no distinct change, while 3 of 5 higher alcohols tended to increase distinctly. On aging in non-porous containers, however, 3 of 16 ester compounds decreased distinctly, and 3 of 5 higher alcohols increased distinctly. In the process of aging, esters underwent hydrolysis in kame but not in non-porous containers.