O. Fennema

Isolation and characterization of cholesterol-5B,6B-oxide from an aerated aqueous dispersion of cholesterol

An unknown autoxidation product in an aerated cholesterol sol was isolated by preparative thin layer chromatography. This compound was identified as cholesterol-5β,6β-oxide by gas liquid chromatography along with infrared and mass spectrometry.

Gas hydrates in aqueous-organic systems. II. Concentration by gas hydrate formation.

Summary The ice-like nature of gas hydrates would appear to make them of potential value for concentrating aqueous fluids. The concentration process is similar to freeze concentration, but higher and possibly more economical operating temperatures could be used. Hydrates of CH3Br and CCl3F were utilized for concentrating apple, orange, and tomato juices. CCl3F was limited to 15 to 20% (v/v), since larger quantities resulted in solidification of the substrate, and smaller quantities lessened the concentration effect. No difficulty was encountered in removing approximately 80% of the water from the substrates. A basket-type centrifuge was used to separate the hydrate crystals from the fluid phase. The purity of the crystals was further improved by washing. The concentration process diminished the color and flavor of most substrates, and frequently imparted a slightly bitter aftertaste. Differences were noted in the suitability of substrates and hydrate formers for use in the concentration process.

Gas hydrates in aqueous-organic systems: I. Preliminary studies*†

Summary Gas hydrates are ice-like compounds, many of which are capable of existing at temperatures well above 0°C, providing the pressure is sufficient. They consist of small “guest” molecules, such as halogenated, short chain hydrocarbons, which are physically entrapped in hydrogen-bonded eages of HOH molecules (the “host”). This study involved hydrates of CH 3 Br and CCl 3 F. Stirring speed affected the rate of hydrate formation, but had no effect on the total amount formed. Gradual addition of the hydrate former failed to alter the rate or amount of hydrate formation as compared to the single addition method. Closed reaction vessels and low air temperatures were helpful in obtaining maximum hydrate formation. Increases in the volume per cent of the hydrate former resulted in greater amounts of hydrate and decreased efficiency (less hydrate per gram of hydrate former). It was possible to form gas hydrates in aqueous systems containing sizable quantities of carbohydrates, proteins, or lipids. Various solutes were found to depress hydrate decomposition temperature and freezing point to the same extent.

Effect of gas hydrates and hydrate formers on invertase activity

Abstract Gas hydrates are inclusion compounds of the clathrate or cage type, many of which are stable above 0 °. The object of this study was to determine the effects of some gas hydrates and hydrate formers on invertase activity. It was shown that invertase activity: (1) was not significantly influenced by the increase in solute concentration that accompanied hydrate formation, (2) was not significantly influenced by the presence of crystalline hydrates of CCl3F or propane, (3) was decreased significantly and irreversibly by exposure to liquid CCl3F, and (4) was decreased greatly and irreversibly by gradual decomposition of either CCl3F or propane hydrate. Since hydrate decomposition results in a molecular dispersion of the hydrate former in water, intimate contact would no doubt occur between the hydrate former (CCl3F or propane) and invertase, and this is the probable cause of the decrease in enzyme activity.

Steroids in bovine muscle and adipose tissue.

Thin layer and gas liquid chromatography, (GLC) were employed as complementary techniques to investigate naturally-occurring steroids in the unsaponifiable matter of bovine muscle and adipose tissue. Three GLC liquid phases, differing in selective partition properties, were used to effectively identify unknown steroids. The results indicate that cholesterol and minor amounts of desmosterol, Δ7-cholestenol, lanosterol, dihydrolanosterol, dehydromethostenol, Δ8-methostenol, Δ7-methostenol, cholestanol and possibly ergosterol were present in the bovine tissues. The minor steroids, with the exception of cholestanol and ergosterol, are steroid precursors in cholesterol biosynthesis. Common hormonal steroids were not found in the unsaponifiables of the tissues.

Methodology for determining unfreezable water in protein suspensions by low-temperature NMR

Abstract The conventional reference standard (4.5 m LiCl, 0.01 m MnCl 2 ) for measuring unfreezable water by NMR was found to be unsuitable at temperatures below −32 °C because of partial freezing. An aqueous solution of 24.0% LiCl, 0.10% MnCl 2 , had suitable NMR properties and did not freeze at temperatures down to −50 °C. This solution had a water concentration of 48.5 m at −35 °C. The amount of unfrozen interfacial water, I , in an aqueous solution of 10% lysozyme, 0.19 m NaCl, 0.001 m KCl, 0.05 m m acetate, pH 4.9, was measured at −35 °C by NMR. During holding of the sample for 4 days at −35 °C, the I value decreased by 27%, approaching an asymptotic value, I A , of 0.33 g water/g lysozyme. The reduction of interfacial water (slow freezing of transition water) was consistent with first-order reaction kinetics.

Rate and extent of enzymatic lipolysis at subfreezing temperatures

Abstract Little attention has been given to the effects that various freezing treatments have on rates of enzyme-catalyzed reactions in frozen systems and to the relationship between subfreezing temperatures and the ultimate extent to which a given reaction proceeds. Both of these aspects were explored using a model system consisting of lipase in an emulsion of tributyrin in water. The ultimate extent to which tributyrin was hydrolyzed decreased from 5.4% at −2 °C to 4.0% at −12 °C. Hydrolysis proceeded almost to completion at temperatures above 0 °C. Rapid freezing to −80 °C produced a substantially slower initial reaction rate at −8 °C than rapid freezing to −20 °C, or than slow freezing, regardless of the temperature nadir.

Gas hydrates in aqueous-organic systems: VII. Linear crystallization velocities of the hydrates of ethylene oxide and tetrahydrofuran

Abstract Linear crystallization velocities of hydrates (aqueous clathrates) of type I ethylene oxide and type II tetrahydrofuran were determined in simple solutions and polyacrylamide gels supercooled from 3.1 to 24 °C. At any given degree of supercooling, all hydrate-forming solutions crystallized much more slowly than pure water, and the hydrate of tetrahydrofuran crystallized more slowly than the hydrate of ethylene oxide. Ethylene oxide hydrate formed at a velocity greater than tetrahydrofuran hydrate even when the solutions were designed to bind approximately equal numbers of water molecules during hydrate formation. In all instances, the presence of polyacrylamide gel decreased the velocity of hydrate formation.

Gas hydrates in aqueous-organic systems. IV. Formation and detection of ethylene oxide hydrate in tissue.

Abstract Formation of gas hydrate (aqueous clathrate or clathrate hydrate) crystals in intact tissue was studied using water-soluble ethylene oxide as a hydrate former. The presence of internal hydrate crystals was assessed by the characteristics of time-temperature plots obtained during warming of treated samples, by microscopic examination of treated samples, and by visual observation of hydrate formation in a device containing treated tissue embedded in three layers of polyacrylamide gel. On the basis of these tests, it was concluded that ethylene oxide hydrate can be formed in either plant or animal tissue if a sufficient quantity of ethylene oxide is allowed to diffuse into the tissue prior to initiation of crystallization.

Gas hydrates in aqueous-organic systems: III. Hydrate formation in polyacrylamide gel*†

Summary The formation of gas hydrates in polyacrylamide gels was attempted using hydrate formers with different solubilities in water. Hydrate crystals of highly water-insoluble trichlorofluoromethane formed only at the gel-hydrate former interface. Hydrate crystals of highly water-soluble ethylene oxide formed abundantly within the gel, and the depth of penetration depended on the equilibration time prior to initiation of crystallization. Equilibration for 166 hrs resulted in the formation of ethylene oxide hydrate throughout a 5-cm column of gel. All ethylene oxide samples were observed to recrystallize following the initial crystallization process. Hydrate crystals of highly watersoluble sulfur dioxide and slightly soluble dichlorofluoromethane also formed within the gel. The sulfur dioxide hydrate crystals were present in quantities similar to those obtained with ethylene oxide, whereas the dichlorofluoromethane hydrate crystals were far less abundant. Several of the experiments were successfully repeated using an agar substrate. In the case of dichlorofluoromethane, the quantity of hydrate crystals in the gel was influenced by the method of formation. This was not true of ethylene oxide hydrate.