Elmer Stotz

Analysis of human plasma phosphatides by paper chromatography

Abstract The individual phosphatides of human plasma were analyzed qualitatively and quantitatively by paper chromatography. The phosphatides which were always present were lecithin, sphingomyelin, lysolecithin phosphatidylethanolamine and a component having the properties of inositol phosphatide. Phosphatidylserine was not detected by this procedure and hence is at most a trace constituent in plasma. Very small amounts of other unidentified phosphatides also occur in plasma. One of these may be lysophosphatidylethanolamine. A study was made of the plasma phosphatides of persons having recent cardiac infarctions. There is suggestive evidence that plasma lysolecithin is significantly diminished in some of these persons. The advantages and limitations of the paper chromatographic method for the analysis of plasma phosphatides are discussed.

The hydrolysis of lecithins by snake venom phospholipase

Abstract [ 32 P]labeled lysolecithins were prepared from rat liver by the action of snake venoms on the corresponding lecithins. The lysolecithins were oxidized under mild conditions with permanganate. Two lyco-compounds were formed after this oxidation. Acid hydrolysis of the lyso-compounds yielded at least four [ 32 P]-labeled water-soluble phosphate compounds. Similar results were obtained with egg lecithin. Beef and pig heart lecithins (mixture of the diester and plasmalogen forms) were subjected to hydrolysis with snake venom. Under conditions where egg lecithin was completely degraded to lysolecithin the beef and pig lecithins were only partially hydrolyzed (62–64%). The lecithins which resisted hydrolysis in the early time interval were essentially all the plasmalogen type. Hence the snake venom degrades the diester lecithin at a faster rate than the plasmalogen analogue. However, after several days exposure to the venom the plasmalogens are completely hydrolyzed to the corresponding lysoplasmalogens. Reduction of the lysoplasmalogens with hydrogen and subsequent acid hydrolysis gave a 70% theoretical yield of a -glycerol ether. These studies show that snake venom phospholipase A can hydrolyze either the a or β-linked fatty acid in lecithins to yield a - and β-lysolecithins.

The in vitro incorporation of 32P-labeled orthophosphate into the phosphatides of isolated rat liver mitochondria☆

Abstract 1. 1. The incorporation of 32 P i into the lipides of intact rat liver mitochondria occurs mainly into phosphatides which resemble but are not identical to phosphatidic acids and which constitute only a very small fraction of the total lipide P. Evidence is presented which indicates that one of these highly labeled phosphatides is an α,β-conjugated ester of glycerophosphate. The incorporation into the common phosphatides such as lecithin and phosphatidyl ethanolamine is extremely small in these systems. 2. 2. Glycerol, Mg, and several nucleotides such as ATP and CTP were found to stimulate the incorporation into at least 3 phosphatides one of which contains glycerophosphate. The microsomal-rich supernatant fluid also had a stimulating effect. On the other hand, cyanide, dinitrophenol, malonate, and a variety of metal ions and surface agents were found to inhibit the system. 3. 3. The identification of the mitochondrial lipides was made by chemical, infrared spectral, and paper and column chromatographic analysis. 4. 4. In contrast to intact mitochondria, the acetone powders of these particles were far less effective in incorporating 32 P i into the phosphatides. However, a striking difference was found inasmuch as in these latter systems the incorporation occurred predominantly in lecithin and phosphatidyl ethanolamine and none occured in the fast moving glycerophosphatide. 5. 5. A discussion is given of the experimental findings.

The structure and abundance of rat tissue cardiolipins

Abstract Cardiolipins of rat tissues were isolated by column chromatography and analyzed. The P content ranged from 2.61% to 3.64%. The ester:P ratio varied from 1.63 to 2.09, and the glycerol:P ratios varied from 1.34 to 1.68. The fatty acid spectrum of the tissue cardiolipins showed some, like heart, kidney and liver to be highly unsaturated and others like brain, lung and testis to be highly saturated. Analysis of the fatty acids released by phospholipase A demonstrated that certain fatty acids are preferentially β-linked, but on the whole, the fatty acid distribution is much more random than that of other phospholipids, like the lecithins. All the rat tissue cardiolipins are degraded by hot acetic acid to yield diglycerides, very small amounts of monoglycerides, and other P-containing products. However, only with rat-liver cardiolipin, beef-heart cardiolipin, and a synthetic cardiolipin are appreciable amounts of water-soluble P released. On treatment with sodium methoxide at 0° all rat tissue cardiolipins give rise to 4–6 lyso-derivatives resulting from the sequential loss of one or more fatty acids. All cardiolipins are eventually completely degraded to fatty acid methyl esters, a water-soluble P compound and smaller amounts of other products including free fatty acids and vitamin A. In the case of rat-liver cardiolipin, alkaline hydrolysis yields vitamin A but not in stoichiometric amount. All cardiolipins react with [14C]acetic anhydride to yield a radioactive acetylated derivative. The results of this investigation show that whereas beef-heart cardiolipin has a structure consistent with the recently synthesized cardiolipin, the cardiolipins of rat tissues are not consistent with this structure. New provisional structures for the rat tissue cardiolipins are proposed in which one of the phosphate groups bears a third moiety (such as vitamin A) as a tertiary phosphate ester or the phosphate group bears a fatty acid as a mixed carboxylic acid-phosphoric acid anhydride.

Time study of incorporation in vivo of 32P-orthophosphate into phosphatides of rat tissues☆

The in vivo incorporation of labeled orthophosphate into the phosphatides of rat tissues was investigated by the use of paper-chromatographic methods. The time-course curves from 2 to 96 hours are given. A discussion of the experimental data is presented.

The application of chromatographic methods to study the incorporation of 32P-labeled orthophosphate into the phosphatides of rat liver homogenates.

Abstract The incorporation of 32 P-labeled orthophosphate into the phosphatides of rat liver homogenates was studied by paper and column chromatographic methods. The results show that the degree of incorporation is small and that the lipides having the highest activity correspond chromatographically to phosphatidic acids but still remain to be characterized. On the other hand, the common and abundantly occuring phosphatides such as lecithin and phospatidyl ethanolamine show very little incorporation. The incorporation is an aerobic dependent process and is stimulated by glycerol. By column chromatography it appears that phosphatides behaving like phosphatidal cholines occur in rat liver.

Hydrolysis of lecithins by venom phospholipase A. I. Structure of the enzymically formed lysolecithins.

Abstract Rat-liver [ 32 P]lecithin was hydrolyzed by snake venom phospholipase A to give quantitative yield of [ 32 P]lysolecithins. The lysolecithins were oxidized in buffered aqueous solutions in the pH range 2.8 to 7.0, with bromine or permanganate to yield a keto-lysolecithin derivative. The extent and rate of oxidation is pH dependent. Bromine oxidation is more rapid at pH 6.8 than at pH 5.5 or 2.8. Permanganate oxidation is rapid at pH 5.5 but very slow at pH 6.8. During the oxidation partial concomitant hydrolysis occurs. The keto-lysolecithin can be separated and measured by paper chromatography and thus permits the determination of the α-lysolecithin isomer. The amount of this isomer was found to vary between 54–94% depending on the pH at which the bromine oxidation is carried out. Definitive evidence for the formation of the lysolecithinic acid has not yet been obtained. Passage of lysolecithin through silicic acid causes ester migration from the beta to the alpha position of glycerol. This is reflected by an increase in the keto-lysolecithin upon bromine oxidation. A novel reaction occurs when lysolecithin is treated with bromine in chloroform or methanol. Brominolysis rather than oxidation takes place with the formation of lysophosphatic acids and choline. A discussion of these findings is presented.

The separation of ionic forms of phosphatidylserine by column chromatography

Abstract Two ionic forms of brain phosphatidylserine have been separated by column chromatography on silicic acid. The free acid form of this phosphatide is readily eluted with chloroform-methanol 4:1 whereas the sodium salt form required a more polar solvent for elution. The two forms of phosphatidylserine were identified by chemical, spectral, and chromatographic analyses.

Spectrophotometric Characteristics and Assay of Biological Stains

I. The chemical and spectrophotometric methods adopted in the Stain Commission laboratories for the certification of stains are described. Spectrophotometric examination is by the Beckman spectrophotometer in which the color density at the absorption maximum is determined as well as a ratio of color densities designed to detect abnormal symmetry of the absorption curve. A somewhat modified titanous chloride assay is described; eerie sulfate is used to determine the normality of the unstable titanous chloride, and the oxidant solution, which is stable, is standardized against Bureau of Standards arsenious oxide. The general approach used for re-study of the stains by these methods and the criteria necessary for adoption of the spectrophotometric method of assay are described.II. This report gives the details of the spectrophotometric examination and assay of the individual stains in the thiazin group, namely methylene blue, thionin, methylene violet, toluidine blue O, and azures A, B, and C. The spectropho...

Hydrolysis of lecithins by venom phospholipase A II. Fatty acid chain length preference of the enzyme

Abstract Phospholipase A of snake venom shows chain length preference with respect to the hydrolysis of lecithin fatty acids. With pig heart, egg, and rat-liver lecithins the C-22, C-20 and C-18 acids are hydrolyzed in preference to the C-16 acids. It also appears that the C-22 and C-20 acids are hydrolyzed in preference to both the C-16 and C-18 acids.