O. S. Privett

Quantitative analysis of lipids by thin-layer chromatography.

A procedure is described for the quantitative analysis of neutral and phospholipids by thinlayer chromatography (TLC) employing densitometry. The chromatophates are prepared with the usual solvent systems. The spots are charred under standard conditions and analyzed with a Photovolt Corp. densitometer equipped with a special stage designed for holding 20×20 cm chromatoplates. Each spot on the chromatoplate gives a peak of density values which is used for quantitative analysis.Radioactive lipids are analyzed by autoradiography by the densitometry of radiograms of chromatoplates developed from X-ray films.The precision of the method is demonstrated on model mixtures of mono, di-and triglycerides, neural and phospholipids and C14 labeled lipids.Results of the analysis of several samples of rat liver lipids compared closely to those obtained by silicic acid column chromatography.

Analytical fractionation of complex lipid mixtures: DEAE cellulose column chromatography combined with quantitative thin layer chromatography

A quantitative chromatographic procedure for the fractionation of complex lipid mixtures is described. The method utilizes diethylaminoethyl (DEAE) cellulose column chromatography followed by thin layer chromatography (TLC). Spots produced in TLC are charred with sulfuric acid-potassium dichromate and heat and are then measured by quantitative densitometry. Results obtained with beef brain and beef heart mitochondrial lipids are presented, and the close correspondence between column isolation procedures and the new procedure is demonstrated. Methods utilizing only column chromatography, column chromatography and TLC, and one- and two-dimensional TLC without column chromatography are compared.

LIPID ANALYSIS BY QUANTITATIVE THIN-LAYER CHROMATOGRAPHY.

Techniques for the quantitative analysis of lipids using thin-layer chromatography (TLC) are reviewed. The general procedures are divided into two groups on the basis of whether or not the methods involve the recovery of substances from chromatoplates.Recovery methods are elaborated under detection of spots, recovery of substances and quantification. Methods are described for the recovery of labile compounds from chromatoplates and for the determination of the structures of triglycerides and lecithins.Methods for the direct quantitative analysis of spots on chromatoplates are reviewed. These include measurements of spot size, reflectance, absorbance of transmitted light, and fluorescence. Details of the photodensitometric method, particularly, spot visualization and instrumentation are described. The analysis of lipid classes using a combination of DEAE cellulose chromatography and TLC by the densitomery of charred spots is illustrated.

Studies on the lipid composition of developing soybeans

Studies are reported on changes in fatty acid and lipid class composition in developing soybeans picked at intervals from ca. nine days after flowering to maturity. In the early stages of development of the bean, the lipid was virtually devoid of triglyceride and the major constituents consisted of glycolipids and phospholipids. As the bean developed, there was a rapid synthesis of triglyceride that paralleled the deposition of lipid. Simultaneously, unknown substances which occurred in relatively large amounts in the neutral, as well as the glycolipid and phospholipid, fractions of the immature bean diminished to less than 2% of the total lipid in the mature bean. The glycolipids and phospholipids also increased as the bean developed but at a much slower rate than the triglycerides and became minor components in the mature bean. The major component of the phospholipids in the immature bean was phosphatidic acid. It decreased as the phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol, as well as triglyceride, increased. The major component of the glycolipid fraction in the early stages of the development of the bean had the same migration pattern on two-dimensional thin layer chromatography as phosphatidic acid and gave a positive test for phosphorus; it also gave a positive test for glycolipids and was separated completely from phosphatidic acid and other phospholipids by column chromatography. It also decreased as the bean developed. Changes also occurred in the fatty acid composition of the developing bean. The percentage of saturated fatty acids decreased rapidly in the early stages of the development of the bean; oleic and linoleic increased rapidly as the bean developed. Linolenic acid increased rapidly to a maximum concentration in the early stages of the development of the bean and then gradually decreased as the bean matured.

Determination of mono-, di-, and triglycerides by molecular distillation and thin-layer chromatography

Analysis of mixtures of mono-, di-, and triglycerides by molecular distillation and thin-layer chromatography is described.Mono- and diglycerides undergo appreciable acyl migration through the effect of heat during molecular distillation. Nevertheless this technique may be used for the quantitative analysis of mixtures of mono-, di-, and triglycerides, provided there are no substances present which catalyze, disproportionation.Thin-layer chromatographic (TLC) analysis of mono-, di-, and triglycerides is fast and simple and can be carried out on a micro-scale with a high degree of accuracy and precision. It also is extremely sensitive, permitting the quantitative estimation of as little as 0.1% of a single component in a mixture.

A comparison of acyltransferase activities in vitro with the distribution of fatty acids in lecithins and triglycerides in vivo

The location and configuration of a double bond in a fatty acid influences the rate of its acyltransferase-catalyzed esterification to form lecithin and its distribution in vivo between the primary and secondary positions of triglycerides and lecithins.Saturated acids of shorter chain length are transferred at rates similar to the long chain unsaturated acids.The positional distributions of acids in the diglyceride units of liver triglycerides appear to be similar to that found in the lecithins.Acyltransferase activities measured in vitro have a considerable predictive value in terms of the ultimate distribution of fatty acids in glycerolipids in vivo.

Structure of hydroperoxides obtained from autoxidized methyl linoleate

SummaryA sample of debromination methyl linoleate has been autoxidized to a peroxide value of 671 m.e./kg. at approximately 0°C. in the dark. An essentially pure concentrate of methyl octadecadienoate monohy-droperoxide was quantitatively separated; infrared and ultraviolet spectral studies were made on the peroxide concentrate and on the corresponding hydroxyl derivative obtained by reducing the peroxides with stannous chloride.The infrared data showed no conjugated peroxides having geometric configurations other than cis, trans; the same data also showed that the peroxide concentrate contained at least 90% conjugated cis,trans forms. Calculations based on ultraviolet spectrophotometric methods also indicated that the peroxides were at least 90% conjugated. The remaining 10% of the sample is most likely nonconjugated diene hydroperoxide. Since analogous cis, cis conjugated dienes have not been isolated and their infrared and ultraviolet properties are unknown, their presence here in small amounts is possible. Ultraviolet and infrared spectra of the reduced compounds conform closely to those of the peroxides except for reduction in the intensity of the OH bond at 2.88 μ.The infrared absorption spectra of the C−H structure and carbonyl groups of an essentially pure conjugated cis, trans methyl octadecadienoate monohydroperoxide were recorded, using a LiF prism.The infrared absorption spectra of the C−H strucfraction isolated from methyl linoleate autoxidized in the dark at 24°C. indicated that appreciable amounts of conjugated trans, trans hydroperoxides were present, in addition to those of the cis, trans type. It is possible that the conjugated cis, trans isomers were formed originally but were labile at the higher temperature and in the presence of catalysts (e.g., peroxides) were transformed to the thermodynamically more stable conjugated trans, trans isomer.

A method for the structural analysis of triglycerides and lecithins

The structural analysis of lecithins and triglycerides is described. The procedure is carried out on 2–5 mg of sample by a combination of reductive ozonolysis and thin-layer chromatography (TLC). The ozonides as well as the aldehyde “cores” derived from reduction of the ozonides are separated by TLC and analyzed quantitatively by densitometry. The constituent saturated fatty acids of the separated aldehyde “cores” are methylated and analyzed by gasliquid chromatography (GLC). The scope of the method is demonstrated by the analysis of several synthetic and natural triglycerides and several lecithins isolated from natural sources.

Toxicity of fatty ozonides and peroxides.

Studies on the acute toxicity of the ozonides and hydroperoxides of methyl linoleate are reported. High purity preparations of these compounds were injected intravenously or administered orally to adult male rats. The lethal dose by iv injection of these compounds was virtually the same −0.07 mmol/100 g body wt. No deaths were caused in a 24 hr period by single oral dosages of these compounds of ca. 10-fold that causing death by the iv route. The major effect of these compounds was on the lungs. The lungs became enlarged from edema and accumulation of fluid, and the animals died of lung congestion and injury similar to the effects of ozone toxicity. There was no destruction of vitamin E in the tissues of animals given lethal dosages of ozonides or peroxides intravenously, but significant changes occurred in fatty acid composition of the lipids of the serum and lung. Arachidonic acid increased at the expense of linoleic and oleic acids in these tissues. Only small amounts of peroxidic and TBA positive substances were detected in lung and serum, indicating that the injected ozonides and hydroperoxides were destroyed in the tissues.

Determination of the specific positions ofcis andtrans double bonds in polyenes.

AbstractA method is described for the determination of the positions and geometric configurations of double bonds in polyunsaturated fatty acids. The procedure consists of three steps:1)Partial reduction of the double bonds with hydrazine under conditions which give high yields of monoenes.2)Isolation of thecis- and thetrans-monoene fractions by thin-layer chromatography (TLC) directly or in the form of their ozonide derivatives. In the former technique, selective argentation is employed, in the latter, silicic acid adsorption.3)Determination of the structure of the monoenes via reductive ozonolysis. The position of the double bonds is determined from the structures of the monoenes. Since thecis-monoenes are separated from thetrans-monoenes the geometric configuration of each double bond is determined.The method also provides a direct determination of the spacings of the internal double bonds and it may be employed for the determination of the structures of mixtures of fatty acids in conjunction with direct ozonolysis procedures. The various ramifications of the method are demonstrated on pure fatty acids and model mixtures thereof.