Inflammation in acne vulgaris: leukocyte attraction and cytotoxicity by comedonal material.

The chemoattraction of comedonal material for leukocytes was evaluated. Material from open comedones attracted mononculear leukocytes but did not attract polymorphonuclear leukocytes. At higher concentrations, comedonal material was cytotoxic for leukocytes of both types. Of the comedonal components tested, free fatty acids produced the greatest cytotoxicity. The attraction and killing of leukocytes by comedonal components may be the mechanisms for the initiation or the enhancement (or both) of inflammation in acne vulgaris.

Effect of hypophysectomy of cholesteryl ester synthesis and hydrolysis in testes and on serum lecithin-cholesterol acyltransferase activity in rats.

Studies are reported of the effect of hypophysectomy on cholesterol esterase activity of testicular tissue and serum lecithin-cholesterol acyltransferase (LCAT) activity in rats. The testes of male Sprague-Dawley rats of 200-255 g were excised from animals sacrificed at 3, 7, and 15 days after hypophysectomy. Assays for cholesterol-esterifying and hydrolytic activities of the testicular tissues of these animals, compared to control animals, showed that hypophysectomy decreased both cholesteryl ester synthesis and hydrolysis. Hydrolytic activity was affected to a greater extent than esterifying activity. LCAT activity was significantly decreased by hypophysectomy compared to that of control animals. Although serum LCAT and testicular cholesterol esterase activities were decreased, the overall effect of hypophysectomy produced an increase in the level of serum cholesterol and cholesteryl esters. It is suggested that the role of essential fatty acids (EFA) in testicular function is related to the utilization of cholesteryl esters in androgen synthesis.

Studies of effects of trans fatty acids in the diet on lipid metabolism in essential fatty acid deficient rats.

Effects of diets containing mixtures of safflower oil, hydrogenated coconut oil with elaidate of linolelaidate on growth, fatty acid composition, serum lecithin: cholesterol acyl transferase (LCAT) and postheparin plasma lipoprotein lipase activities in essential fatty acid (EFA) deficient rats were determined. Addition of trans fatty acids to the diet lowered the growth response to linoleic acid. Both elaidate and linolelaidate accumulated in the serum and liver, imparied the conversion of oleic acid to eicosatrienoic acid and linoleic acid to arachidonic acid, and the incorporation of eicosatrienoic acid into cholesteryl esters. Trans fatty acids also influenced the fatty acid composition of testicular lipids, but much lower amounts of these acids accumlated in tests than in liver or serum. Serum lecithin:cholesterol acyl transferase activity was elevated by an EFA deficiency, was unaffected by dietary elaidate, but was significantly decreased by linolelaidate. These effects were nullified by the addition of safflower oil to the diet. Postheparin plasma extrahepatic and hepatic lipase activities were also affected by an EFA deficiency, and by the addition of elaidate or linolelaidate alone or in combination with safflower oil to the diets of EFA deficient rats. It is suggested that trans fatty acids exhibit particular effects on the metabolism of lipids in addition to aggravation of an EFA deficiency.

Fatty acid synthesis from 2-14C-acetate in rat testis mitochondrial and cytosol fractions in vitro.

An in vitro system for acetate incorporation into fatty acids by the mitochondrial and the cytosol fractions of rat testis is described. The rate of incorporation of acetate into fatty acids was twice as fast with the mitochondrial as with the cytosol fraction; both systems were stimulated in the presence of adenosine triphosphate, reduced nicotinamide adenine dinucleotide phosphate, coenzyme A, and MgC1(2). The optimum pH was between 7.0-7.5 for the mitochondrial fraction and between 6.5-8.0 for the cytosol fraction. Radio gas chromatography showed that palmitic acid was the most highly labeled acid, followed by stearic acid, in the mitochondrial fraction in accord with the pathway of de novo fatty acid synthesis. Some of the labeled acetate was also incorporated into the 16:1 and 18:1 fatty acids of this fraction. Distribution of radioactivity among the mitochondrial lipid classes was highest in the phospholipids and monoglycerides, followed by diglycerides and cholesterol; little radioactivity was present in the triglyceride fraction. These observations are in accord with studies of the incorporation of labeled metabolites into testicular lipids following intratesticular injection and indicate the validity of the in vitro system for studies of specific reactions occurring in vivo.

A simplified procedure for the quantitative extraction of lipids from brain tissue.

A method is described for the quantitative extraction of lipid from brain tissue with chloroform/methanol (C/M) that eliminates secondary purification of the lipid extract by dextran-gel chromatography or aqueous washing of the organic extract. Nonlipid substances that generally contaminate C/M lipid extracts are separated by pre-extraction of the tissue with dilute (0.25%) aqueous acetic acid. The residual tissue is extracted twice with 40 volumes of C/M (1:1, v/v). Approximately 97% of the lipid is recovered in these extractions. A third extraction which yields ca. 1% more lipid is performed if the process is discontinued at this stage in a shortened version of the method. The remainder of the lipid is recovered after treatment of the tissue with 1 N HC1 by two additional extractions, the first with 40 volumes of C/M (1:2, v/v) and the second with 40 volumes of methanol. The method, which was demonstrated with pig brain, gave a complete extraction of the lipid, including gangliosides, free of nonlipid substances.

Effects of dietary trans acids on the biosynthesis of arachidonic acid in rat liver microsomes.

Effects of dietary trans acids on the interconversion of linoleic acid was studied using the liver microsomal fraction of rats fed a semipurified diet containing fat supplements of safflower oil (SAFF), hydrogenated coconut oil (HCO) at 5 and 20 at and 20% levels or a 5% level of a supplement containing 50.3% linolelaidic and 24.3% elaidic acids devoid of cis,cis-linoleic acid (TRANS). Growth rate was suppressed to greater extent with the animals fed the 20% than the 5% level of the HCO-supplemented diets and still further by the TRANS diet compared to the groups fed the SAFF diets. Food intake was greater in the groups fed the HCO than the SAFF-supplemented diets, demonstrating the marked effect of an essential fatty acid (EFA) deficiency on feed efficiency. In contrast to an EFA deficiency produced by the HCO supplement, which stimulated the in vitro liver microsomal biosynthesis of arachidonic acid, diets containing the TRANS supplement exacerabated the EFA deficiency and depressed 6-desaturase activity of the liver microsomal fraction. The liver microsomal fraction of the animals receiving this supplement also was more sensitive to fatty acid inhibition of the desaturation of linoleic acid than those obtained from animals fed either the SAFF or HCO diets. It is suggested that dietary trans acids alter the physical properties of the 6-desaturase enzyme system, suppressing its activity, which increases the saturation of the tissue lipids and, in turn, the requirement for EFA or polyunsaturated fatty acids.

Changes in the structure of soybean triglycerides during maturation.

Soybeans of the Hawkeye variety were picked at eleven periods from 30 to 111 days after flowering and extracted with chloroform-methanol. The triglyceride fraction of five pickings, selected 35 to 91 days after flowering (when synthesis of lipid was most active), were isolated by silicic acid thin layer chromatography (TLC) and species composition determined using argentation TLC and lipase hydrolysis. The triglyceride content of the total lipid increased from 6.5% at 30 days after flowering to 85% in the mature bean (111 days). The major changes in fatty acid composition of the triglycerides occurred during the first 52 days after flowering. During this period linolenic acid decreased from 34.2% to 11.7%, the percentages of linoleic and oleic acids increased, stearic remained fairly constant and palmitic decreased slightly. Large quantitative changes occurred in the molecular species of the triglycerides of the bean during maturation; some triglycerides containing linolenic acid could not be detected approximately 66 days after flowering. Although changes occurred in the percentage and amount of each triglyceride species, the positional distribution of fatty acids remained virtually unchanged throughout maturation. Linolenic acid was distributed fairly uniformly between the ß-position and the α-positions, linoleate favored esterification in the ß-position, and oleate the α-positions. Most of the stearic and palmitic acids were esterified in the α-positions. The consistency of the positional arrangement of the fatty acids indicated that the mode of glyceride synthesis was established very early during maturation and molecular species composition was controlled by the fatty acids available for synthesis.

Determination of the structure of lecithins via the formation of acetylated 1,2-diglycerides.

A detailed procedure for quantitative determinations of molecular species of lecithins is described and applied to several lecithins isolated from natural sources. The method is based on the conversion of lecithin to acetylated 1,2-diglycerides and analysis of these compounds by methodology used for the determination of triglyceride structure.The preparation of the acetylated 1,2-diglycerides was carried out via hydrolysis with phospholipase C and acetylation of the resultant, 1,2-diglycerides with pyridine-acetic anhydride. Preparation of acetylated 1,2-diglycerides from lecithin by acetolysis with acetic acid-acetic anhydride was shown to be accompanied by intermolecular as well as intramolecular rearrangement of the fatty acids.The structure of the acetylated 1,2-diglycerides was determined by a combination of argentation-TLC and pancreatic lipase hydrolysis using internal standards for quantification. The method was applied to lecithins isolated from milk serum, egg, soybean, safflower seed and wheat germ lipids.

A simple, rapid micromethod for the determination of the structure of unsaturated fatty acids via ozonolysis.

A micromethod for the localization of double bonds in unsaturated fatty acids via ozonolysis employing pyrolytic cleavage of ozonides in the presence of a hydrogenation catalyst is described. Cleavage of the ozonides is carried out in a gasliquid chromatographie instrument in a small glass tube, containing the catalyst, inserted in the top of the column opposite the in input heaters at 225C. Ozonides of methyl esters of straight chain unsaturated fatty acids are cleaved through the action of the catalyst to aldehyde fragments which are swept simultaneously into the column for analysis.The double bond positions are deduced from the chain length of the fragments. The method is demonstrated on methyl oleate, linoleate, linolenate and arachidonate.

Phospholipase a properties of several snake venom preparations.

The hydrolytic properties of the venoms of seven species of snakes,Crotalus adamanteus, Ancistrodon contortrix, Naja naja, Bothrops atrox, Ophiophagus hannah, Crotalus atrox andVipera russeli, were studied with purified lecithins and mixtures of lecithins of known fatty acid and class composition as substrates.The relative rates of hydrolysis of the fatty acids by the above venoms were studied by analysis of the products of the reaction at intervals during the course of the reaction. Of the seven venoms studied, that ofOphiophagus hannah was the only one which did not give some degree of preferential rate of hydrolysis of individual fatty acids.In general, saturated fatty acids were liberated faster than unsaturated fatty acids; differences in the rates of the hydrolysis of individual saturate and unsaturated fatty acids were also observed. Individual classes of lecithin were also hydrolyzed at different rates. For the determination of the distribution of the fatty acids between the alpha- and beta-position of lecithin, the reaction should be carried to completion. If the reaction requires a prolonged time to go to completion, it should be carried out under nitrogen to prevent autoxidation.

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

A 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.

An electrostatic precipitator for preparative gas-liquid chromatography.

The effect of the operating variables of electrostatic precipitators on the recovery and structure of methyl esters and related aerosol forming compounds collected in preparative gas-liquid chromatography was studied.Aerosol formation was prevented by AC or DC voltages of 5000 to 12000 volts. AC was more effective than DC but caused changes in structure which were detectable by both thin-layer and gas-liquid chromatographic methods of analysis.An apparatus of simple construction and operation was designed for the collection of methyl esters and its use demonstrated with several model compounds.

Evaluation of mathematical distribution methods for the determination of triglyceride composition.

The triglyceride composition of a number of animal and vegetable fats was determined directly by means of selective argentation and lipase hydrolysis, and compared to that given by the 1,3-random,2-random method of analysis described by Vander Wal [JAOCS37, 18 (1960)].Exceptions to the basic assumption of the 1,3-random,2-random method that the fatty acids in the 2-position are distributed randomly are reported.The analyses of some fats determined by the 1,3-random,2-random method agreed closely with those determined by the direct method, but the overall results indicated that methods based on mathematical distribution patterns generally are not as precise as direct methods.

A new system for lipid analysis by liquid chromatography-mass spectrometry.

A simple system for interfacing liquid chromatography with mass spectrometry for the analysis of lipids is described. The system is based on the moving chain transport principle and employs an endless stainless steel belt of perforated construction that gives it superior surface properties and capacity to entrain solvent. The entire column eluent is collected on the belt which transports it into an evaporator where the solvent is removed. The solute, which remains as a residue on the belt, is transported into a reactor where it is converted to hydrocarbons by reaction with hydrogen at 400-450 C. The hydrocarbons, which are characteristic of the structures of the parent compounds, are swept into an outlet tube where ca. 15% are drawn into the ion source of a mass spectrometer operating in the chemical ionization mode using methane as the reagent gas. The spectrum is recorded on an oscillographic recorder for identification purposes. Detection and quantitative analysis is performed by single ion monitoring of the most intense ion using a conventional analog recorder. The system is domonstrated by application to a standard mixture of tripalmitin, cholesteryl palmitate, and cholesterol separated on a 3.2 x 250 mm silica column, and exhibited a sensitivity of ca. 1 nanogram per component injected on the column.

Ifluence of dietary fatty acids on membrane properties and enzyme activities of liver mitochondria of normal and hypophysectomized rats.

Studies are reported on the effects of diets containing fatty supplements with (A) a high concentration of arachidonate (46% concentrate of ethyl arachidonate), (B) a high concentration of linoleate (corn oil), and (C) an essential fatty acid deficient, fully saturated fat (hydrogenated coconut oil) upon lipid composition, membrane permeability, and enzyme activities of liver mitochondria of normal and hypophysectomized rats. The fatty supplements produced differences in the fatty acid composition of the liver mitochondria; hypophysectomy, in addition, influenced the neutral and phospholipid composition. Permeability, indicated by swelling properties, correlated generally with the degree of unsaturation and essential fatty acid content of the lipid of the mitochondria of the normal animals. The fatty supplements also influenced the enzyme acitivites of the mitochondria of the normal animals. The mitochondria of the hypophysectomized animals were less responsive to the differences in the dietary fat in both their swelling properties and enzyme activities. Although the relationship was complex, it appeared that the hypophysis was involved in the functions of essential fatty acids in liver mitochondria.

Interrelationship between dietary trans Fatty acids and the 6- and 9-desaturases in the rat.

Studies are reported on the effects of dietary trans fatty acids on the 6- and 9-acyl desaturase activities in the liver microsomes of rats fed essential fatty acid (EFA)-deficient and non-EFA-deficient diets. In experiment I, weanling male rats were fed a semisynthetic diet with either 10% safflower oil (SAF) or 10% hydrogenated coconut oil (HCO). At the age of one year, half of the dietary fat was replaced by a supplement containing elaidate, linolelaidate and cis, trans-trans,cis-18:2 (TRANS) for 12 weeks. In experiment II, male rats which were kept from weaning on a 10% (SAF + TRANS, or 5% HCO + 5% TRANS. Feeding TRANS depressed the 6-desaturase activity in the liver microsomes, especially in the EFA-deficient rats (HCO + TRANS group of experiment I). Unlike the 6-deaturase activity, the 9-desaturase activity was not inhibited by the dietary trans fatty acids and was significantly stimulated in the non-EFA-deficient rats (SAF + TRANS group of experiment I and HCO + TRANS groups of experiment II). This was evidence by incubation reaction and by comparisons of fatty acid consumptions and microsomal fatty acid levels, showing extra biosynthesis of 16:1 and 18:1 when TRANS was fed. The biosynthesis of essential (n-6) fatty acids was depressed by the TRANS supplement in EFA-deficient as well as in non-EFA-deficient animals.

Effects of dietary saturated and trans fatty acids on cholesteryl ester synthesis and hydrolysis in the testes of rats.

Studies were made of the enzymic synthesis and hydrolysis of cholesteryl esters in rat testes. Weanling rats were fed for 14 weeks diets containing 5% by wt of hydrogenated coconut oil (HCO), a concentrate of ethyl elaidate and linolelaidate (TRANS), devoid of essential fatty acids (EFA), or safflower oil (SAFF). Cholesterol esterifying activity was localized in the soluble fraction, and cholesteryl ester hydrolase activity was distributed in both particulate and soluble fractions obtained from tissue homogenates. The optimum pH was 6.0 for esterification and 6.9-7.0 for hydrolysis. Neither esterifying nor hydrolytic activity was affected by freezing and thawing, but both reactions were inhibited by heat or sonication. The animals of both the HCO and TRANS groups had developed an EFA deficiency before they were sacrificed. The EFA deficiency produced upon feeding the HCO diet had no apparent effect on the synthesis and hydrolysis of cholesteryl esters in rat testes. The TRANS diet influenced the development of the testes as judged by their size, and cholesterol esterifying and cholesteryl ester hydrolyzing activities were suppressed in the testes of the animals of this group. A major difference in the effects of the HCO and TRANS diets on the lipids of the tests was the relatively minor amount of eicosatrienoic acid (20:3) and the elevated level of docosapentaenoic acid (22:5) in the cholesteryl esters of the testicular lipids of the TRANS group.

Isolation and analysis of age-related fluorescent substances in rat testes.

Fluorescent substances were extracted from rat testicular tissue with 2,2-dimethoxypropane (DMP) and analyzed by 2-dimensional thin layer chromatography (TLC). One substance that accumulated with increasing age of the animals was isolated and analyzed quantitatively by spectrophotofluorometry using quinine sulfate as a standard. This substance, which was designed as an age-related fluorescent substance (ARFS), exhibited an excitation maximum at 355 nm and an emission maximum at 490 nm. Its fluorescence was quenched by metal chelators and at alkaline pH, indicating it contained a conjugated Schiff base structure. Quantitative analysis of this substance in the testes of rats 1, 2, 11 and 20 months of age showed that it increased linearly with age. The relation of this substance to aging also was indicated by its detection in animals of different ages fed diets of both low and high unsaturation.

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation.

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.

Studies of lipid class and fatty acid profiles of rat mammary tumors induced by 7, 12-dimethylbenz(a)anthracene.

The lipid class and fatty acid composition profiles of mammary glands of female rats fed a nutritionally adequate diet are compared to those of tumors induced in the mammary glands by intravenous injection of dimethylbenz(a)anthracene of animals fed the same diet. Ca. 95% of the lipids of the mammary glands of the control group of animals consisted of triglycerides; glycolipids and phospholipids were present in only minor amounts. In contrast, the lipids of the mammary tumors contained much lower amounts of neutral lipids and higher concentrations of phospholipids. The glycolipid fraction was a minor component of both tissues but differed greatly in composition. The composition of the phospholipid and neutral lipid fractions, particulary the latter, of the mammary tumors also differed from that of the mammary glands of the control animals. The neutral lipids of the tumor tissues contained elevated levels of free fatty acids and cholesterol and much lower concentrations of triglyceride compared to the mammary gland lipids. Differences also were observed in the fatty acid composition of tumor and mammary gland lipid. The greatest differences occurred in the concentrations of polyunsaturated fatty acids which were generally much higher in the tumor lipids.