Gene Kritchevsky

Lipid composition of beef brain, beef liver, and the sea anemone. Two approaches to quantitative fractionation of complex lipid mixtures.

Two new schemes for fractionation of complex lipid mixtures are presented. Their use for the study of lipids of beef brain, beef liver, and the sea anemone are described. Apparatus and techniques for working in an inert atmosphere, evaporation of solutions in the cold under nitrogen, use of infrared spectroscopy for examination of lipids and their hydrolysis products, preparation and clution of diethylaminoethyl (DEAE) cellulose and silicic acid-silicate columns and general column combinations that can be used to fractionate complex lipid mixtures are considered in detail. The first scheme, employing DEAE cellulose columns followed by thin layer and paper chromatographic examination of the fractions, was applied to liver lipids. The many components, some of them new lipids not previously detected, are clearly seen with this technique but are not seen when paper or thin layer chromatography alone or silicic acid chromatography are used.The second scheme employing DEAE for initial fractionation, followed by complete separation on silicic acid and silicic acid-silicate columns, was applied to lipids of the sea anemone and beef brain. Typical lecithin and phosphatidyl ethanolamine were isolated, but sphingomyelin was not found. A new sphingolipid, ceramide aminoethylphosphonate, with a free amino group and a direct carbon to phosphorus bond was isolated and characterized. The methods used for quantitative isolation, the infrared spectra, and the amounts of cholesterol, ceramide, cerebroside, galactosylglyceride, sulfatide, sphingomyelin, lecithin, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, triphosphoinositide, phosphatidic acid, cardiolipin, and ganglioside of beef brain are presented. Finally, the types of lipid-nonlipid interactions disclosed by column chromatography and their potential application to biological problems are discussed.

Quantitative analysis of brain and spinach leaf lipids employing silicic acid column chromatography and acetone for elution of glycolipids

AbstractsQuantitative elution of acidic and neutral glycolipids of brain and spinach leaves from silicic acid columns with acetone was demonstrated. Cerebrosides and sulfatides of brain and sulfolipid and glycosyl diglycerides of spinach leaves were eluted quantitatively with acetone while prospholipids remained on the column. The observations provide the basis for an analytical procedure employing column and quantitative thin-layer chromatography (TLC). Sephadex column chromatography is utilized for separation of lipids from nonlipids; silicic acid column chromatography for separation into neutral lipid, glycolipid and phospholipid fractions; and quantitative TLC for analysis of lipid classes of each column fraction.

Laboratory contaminants in lipid chemistry: Detection by thin-layer chromatography and infrared spectrophotometry and some procedures minimizing their occurrence.

Many sources of contamination for lipid preparations exist in the laboratory. These contaminants can be detected using thin-layer chromatography (TLC) and infrared spectroscopy. Numerous components that are potential contaminants and can lead to false analyses were demonstrated by TLC in laboratory soaps, cleaners, hand creams and lotions, hair tonics, laboratory greases, floor waxes, oil vapors, tobacco smoke, hydrocarbon phases for gas-liquid chromatography, etc. Procedures preventing introduction of contaminants are presented including descriptions of equipment and precautions to eliminate or minimize contamination. These are useful in isolation of pure polar and nonpolar lipids.

Species variations in phospholipid class distribution of organs: I. Kidney, liver and spleen

Improved procedures for preparation of lipid extracts and determination of phospholipids by phosphorus analysis of spots separated by thin layer chromatography (TLC) were employed to determine the phospholipid class distributions of vertebrate (human, bovine, rat, mouse, frog) kidney, liver and spleen. The absence of significant changes arising from postmortem enzymatic degradation was demonstrated by analysis of lipids of organs after standing for different times postmortem. Intraspecies variability was evaluated by separate analysis of several rat organs. Accuracy of analytical results was insured by demonstrating the absence of spot overlap by two-dimensional TLC and low values for standard deviations. The values for kidney and liver demonstrate little or no species variability, whereas values for spleen indicate two groups which differ in cellular composition. The findings for kidney and liver are in keeping with data obtained from heart, skeletal muscle, lung and highly purified subcellular particulates which indicate that, among vertebrates, there is little or no species variability of phospholipid class distribution of organs and most subcellular particulates.

DETERMINATION OF POLAR LIPIDS: QUANTITATIVE COLUMN AND THIN-LAYER CHROMATOGRAPHY.

The structures of the polar lipid classes of plants and animals are presented, their nomenclature discussed, and suggestions are presented for clarification of nomenclature. The three general types of quantitative chromatographic procedures (column chromatography, thin-layer chromatography, and combinations of column and thin-layer chromatography) available for polar lipids are reviewed and a new quantitative two-dimensional thin-layer chromatographic procedure is presented. Useful quantitative procedures employing columns of cellulose, silicic acid, silicic acid mixed with silicate, magnesium silicate, and ion exchange celluloses are presented. New findings with diethylaminoethyl cellulose columns are described. New quantitative procedures employing silicic acid, magnesium silicate, or diethylaminoethyl cellulose column chromatography with quantitative thin-layer chromatography are described.

Lipids in the Nervous System of Different Species as a Function of Age: Brain, Spinal Cord, Peripheral Nerve, Purified Whole Cell Preparations, and Subcellular Particulates: Regulatory Mechanisms and Membrane Structure

Publisher Summary The importance of lipid metabolism in health and disease has made a better knowledge of lipid metabolism and enzymology necessary. The lipid composition of whole organs and isolated subcellular particulates has become of great importance to those who study cell membranes. Membranology has become the common meeting ground for many chemists and biologists with different types of specialized training and experience. The lipid composition of the major organs of some species of higher animals has been defined with considerable accuracy. The study of subcellular articulates requires their isolation and characterization as well as their lipid analysis. This chapter presents the data that is relevant to the changes with age and species variations of the lipid composition of the nervous system. It also presents a scheme for membrane biosynthesis and a model for membrane structure; it considers the factors that determine the lipid class composition of membranes.

Lipid class composition of normal human brain and variations in metachromatic leucodystrophy, Tay-Sachs, Niemann-Pick, chronic gaucher’s and Alzheimer’s diseases

Procedures suitable for obtaining representative samples of whole brain and of total grey and white matter of brain are presented and discussed. A procedure is described for the quantitative determination of lipid class distribution of human brain specimens utilizing in sequence : a cellulose column to separate gangliosides and nonlipid material from the remaining lipids, diethylaminoethyl (DEAE) cellulose column chromatography to separate the lipid classes into manageable groups, and finally quantitation of the lipid classes by thin-layer chromatography (TLC). TLC is made quantitative by correlating the amt of charring of spots on chromatograms with the amt of lipid present by means of transmission densitometry. The use of two-dimensional TLC for the analysis of brain lipids and its application to the study of pathological brain specimens is also described.The application of these procedures to the study of metachromatic leucodystrophy, Tay-Sachs, Niemann-Pick, and Alzheimer’s diseases and senile cerebral cortical atrophy is described and data are presented. In two cases of Alzheimer’s disease, a large reduction in fresh weight and total lipid of brain were found; the lipid class distribution of whole brain in one case and of total grey and total white matter in another were essentially normal. The lipid class distributions of the brain in metachromatic leucodystrophy, Tay-Sachs disease, and Niemann-Pick disease were shown to be similar to that of normal infant brain except that one sphingolipid was greatly increased in each disease (sulfatide in metachromatic leucodystrophy, one ganglioside in Tay-Sachs disease, and sphingomyelin in Niemann-Pick disease).

Simplified procedure for preparation of35S-labeled brain sulfatide

A simplified procedure for the preparation of35S-labeled brain cerebroside sulfates has been developed. The labeled sulfatides are synthesized in vivo after intracerebral administration of inorganic35S-sulfate in developing rats. The animals are sacrificed three days later and brain homogenates extracted with chloroform-methanol. The extract is subjected to mild alkaline treatment and washed with water. The organic phase is chromatographed on triethylaminoethyl-cellulose from which sulfatides are eluted with chloroform-methanol containing potassium acetate. Radioactive fractions are pooled, concentrated, and potassium acetate removed by dialysis against water. Alternatively, salts are removed by passing radioactive fractions through Sephadex G-25. After evaporating to near dryness, the radioactive cerebroside sulfates are dissolved in a small volume of chloroform-methanol and stored at −20 C. The35S-sulfatides are essentially free of lipid contaminants, and 97% of the radio-activity corresponds with sulfatides on chromatographic analysis.

Identification of elementary sulfur and sulfur compounds in lipid extracts by thin-layer chromatography

Elementary sulfur, long chain thiols and sulfides in lipid mixtures can be separated and identified by thin layer chromatography (TLC), preparation of derivatives, development of typical flourescent colors with Rhodamine 6G under ultraviolet light, and colors with other spray reagents. Silica gel mixed with magnesium silicate and the same adsorbent plus silver nitrate are used for polar stationary phase and silver nitrate complexing chromatography, respectively.Elementary sulfur yields a purple fluorescent spot with Rhodamine 6G in contrast to the yellow fluorescent of most lipids. Compounds isolated by means of TLC were further identified by spectroscopic methods. The sulfur bacterium (Chromatium sp.), and the Orgueil carbonaceous meteorite were analyzed by the new technique. Elementary sulfur was identified in both samples, but the lipid compositions of the bacteria and meteorite were found to be entirely different. The meteorite lipids and hydrocarbons were also different from the abiological hydrocarbons synthesized in a Miller high frequency spark discharge experiment.The new analytical technique is suitable for the analysis of recent biological matter, petroleum, bitument and organic matter from marine sediments.

Detection of phthalate esters as contaminants of lipid extracts from soil samples stored in standard soil bags

the column should be performed as much as possible in the absence of heat and light, and in a nitrogen atmosphere. Also the runs are performed under nitrogen. The individual fractions (10 ml) were analyzed by silver nitrateTLC and GLC using the same experimental conditions described for cholesterol and desmosterol in the previous paper (1) . Comparisons with recrystallized cholesterol, desmosterol, and 7-dehydrocholesterol (Calbiochem.) standards, showed that the sterols were quantitatively eluted in pure form. The structure of 7-dehydrodesmosterol was determined, and the identity of the other sterols confirmed, by means of an LKB 9000 gas chromatograph-mass spectrometer. The recovery of the sterols was determined on the pooled fractions of each sterol acetate on GLC, using cholestane as the internal standard (4) . Sterols differing only in double bond position, e.g. desmosterol and 7-dehydrocholesterol, are completely resolved by the present method. This separation is particularly useful because these two sterols are poorly separated on GLC and a preparative separation on TLC with AgNOs impregnated plates can not be easily applied because of the chemical instability of 7-dehydrocholesterol when exposed to air and l ight .