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 composition of different areas of murine brain: Effects of lipid extraction procedures

The effects of various chemical extraction procedures on the determination of lipid composition of rat and mouse brain have been investigated. Tissue extractions with formic acid/acetone or perchloric acid both resulted in significant losses of total phospholipids and cholesterol. Perchloric acid extraction also degraded, almost quantitatively, ethanolamine plasmalogens to lysophosphatidylethanolamine. Our findings have thus demonstrated that conventional procedures used for extraction of brain tissue for analysis of choline and acetylcholine content cannot also be used for concurrent/simultaneous extraction of phospholipids and cholesterol from the same tissue.     

Lipid and fatty acid composition of turkey liver,skin and depot tissue

Total lipid and phospholipid contents of liver, skin and depot fat from yearling hen turkeys have been studied. Liver lipid averaged 88.5 mg/g wet tissue; skin, 385.0 and depot fat, 753.5. Phospholipids comprised 32.05% of total lipid of liver, but only 0.81% of skin and 0.46% of depot fat. Fatty acids of liver differed from those of skin or depot fat by larger amounts of 16∶0, 18∶0, 20∶4, 22∶0 and 24∶0, and smaller amounts of 16∶1, 18∶1, 18∶2, 18∶3 and 20∶0. Similarity existed between skin and depot fat. Journal Paper No. J-6473 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 1696.     

New data on the total lipid, cholesterol and fatty acid composition of raw and grilled beef longissimus dorsi

Simultaneous analyses of total lipids, cholesterol and fatty acids were carried out on raw and grilled beef longissimus dorsi trimmed of external fat. Cholesterol was determined by high performance liquid chromatography and the fatty acids by gas chromatography. Mean total lipid (g/100 g) ranged from 2.1 to 2.6 for raw beef and 3.5 to 4.0 for grilled beef steaks. Cholesterol levels (mg/100 g) ranged from 40 to 43 for raw beef and 67 to 70 for grilled beef steaks. The main intramuscular fatty acids of the raw and grilled meat were 14:0, 16:0, 16:1n7, 18:0, 18:1n9, 18:1n7 and 18:2n6. Grilled lean beef steaks had significantly higher contents of the principal fatty acids and most of the minor fatty acids. The higher values for the three components in the grilled meat were due to loss of moisture during grilling. There was no significant difference between the apparent and true retentions values, both indicating no significant loss or degradation of total lipids, cholesterol and fatty acids during grilling.     

Analytical13C NMR: A rapid,nondestructive method for determining the cis,trans composition of catalytically treated unsaturated lipid mixtures

High resolution natural abundance 13C Fourier transform nuclear magnetic resonance (NMR) has been found to be an effective tool for the rapid and direct determination of the cis/trans composition in partially hydrogenated and isomerized unsaturated lipids. With the eis and trans allylic carbon resonances as representative probes for double bond stereo-chemistry. Evaluation of the cis/trans composition of complex, positionally isomerized mixtures can be made without the necessity of carrying out detailed analyses of multishift olefinic carbon resonances. Migration of double bonds in monoenes and polyenes and formation of conjugated unsaturation in cata-lytically treated fats are discussed and assessed as possible sources of error in the evaluation of cis/trans isomer ratios. Carbon spin lattice relaxation times T₁ were measured for both cis and trans allylic reson-ances in isomeric mixtures of varying composition to assure quantitative intensity relationships. 13C NMR compositional analysis of complex mixtures is demonstrated.     

Relative susceptibility of microsomes from lung,heart, liver,kidney, brain and testes to lipid peroxidation: Correlation with vitamin E content

Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely among different tissues and species. In rats and rabbits, lung microsomes peroxidized at a 25- to 50-fold lower rate than liver, kidney, testes and brain microsomes. Heart microsomes peroxidized at a rate slightly greater than, but most similar to, lung microsomes. Comparison of tissue homogenates also revealed the unique resistance of lung and heart to lipid peroxidation. The ratio of vitamin E to peroxidizable polyunsaturated fatty acids in lung and heart microsomes was several-fold higher than in microsomes from the other tissues studied, which accounted for the relative resistance of lung and heart to lipid peroxidation. Liposomes of extracted rat lung microsomal lipid were also resistant to peroxidation and the amount of vitamin E contained in the lung lipid extract was sufficient to confer the same degree of resistance when incorported into an equivalent amount of rat liver lipid. Higher rates of peroxidation in mouse lung microsomes relative to rabbit, rat and human lung microsomes were similarly correlated with a lower ratio of vitamin E to peroxidizable fatty acids in mouse lung microsomes. These data provide strong support for the role of vitamin E as the major cellular antioxidant, especially in the highly oxygenated tissues of heart and lung, and demonstrate the utility of the microsomal system in characterizing tissue differences in susceptibility to peroxidative membrane decomposition.     

Myelin subfractions isolated from mouse brain: Analysis of the lipid composition at three developmental stages

Lipids were examined in whole myelin and 8 myelin subfractions isolated from mouse brain at 18–24, 44–48 and 80–90 days of age. Relative to protein, total lipid was lowest in whole myelin isolated from the oldest animals as well as from subfractions isolated at greater sucrose densities, thus partially accounting for the observed myelin subfraction distribution pattern which shifted during development from an average peak density banding between 0.55 and 0.65 M sucrose to one banding between 0.60 and 0.70 M sucrose. Whole myelin and each myelin subfraction isolated at one age contained nearly the same ratio of sterol and phospholipid to galactolipid; these ratios decreased uniformly during development suggesting enrichment with galactolipid in all myelin subfractions. Sulfatide, as percentage of total galactolipid, was relatively constant during development and appeared to be slightly enriched in the denser myelin subfractions. The findings suggest that regardless of the origin(s) of the subfractions, an age-related mechanism exists in the central nervous system which modifies myelin lipid composition relatively uniformly.     

Dietary sandalwood seed oil modifies fatty acid composition of mouse adipose tissue, brain, and liver

Sandalwood (Santalum spicatum) seed oil, which occurs to about 50% of the weight of the seed kernels, contains 30–35% of total fatty acids (FA) as ximenynic acid (XMYA). This study was designed to obtain basic information on changes in tissue FA composition and on the metabolic fate of XMYA in mice fed a sandalwood seed oil (SWSO)-enriched diet. Female mice were randomly divided into three groups, each receiving different semisynthetic diets containing 5.2% (w/w) fat (standard laboratory diet), 15% canola oil, or 15% SWSO for 8 wk. The effects of SWSO as a dietary fat on the FA composition of adipose tissue, brain, and liver lipids were determined by analyses of FA methyl ester derivatives of extracted total lipid. The FA compositions of the liver and adipose tissue were markedly altered by the dietary fats, and mice fed on a SWSO-enriched diet were found to contain XMYA but only in low concentration (0.3 3%) in these tissues; XMYA was not detected in brain. Oleic acid was suggested to be a principal XMYA biotransformation product. The results were interpreted to suggest that the metabolism of XMYA may involve both biohydrogenation and oxidation reactions.     

Molecular species composition of phosphatidylinositol from the brain,retina, liver and muscle of cod (Gadus morhua)

The molecular species composition of phosphatidylinositol (PI) purified from four different tissues from cod was found to show large tissue-specific differences. In brain 18∶0/20∶5 was the most abundant species (40.8%) followed by 18∶1/20∶5 (13.5%). In retina, 24–26% each of PI was the 16∶0/22∶6 and 18∶0/20∶4 species with 16–18% each of 18∶0/20∶5 and 18∶0/22∶6. In liver, almost half of the PI was 18∶0/20∶4 with 18% 18∶1/20∶4. In contrast, muscle contained almost 40% of 18∶0/22∶6 with 10–14% each of 18∶0/20∶4, 18∶0/20∶5 and 18∶1/22∶6. Molecular species are abbreviated as follows:e.g., 18∶0/20∶4 PI is 1-stearoyl-2-eicosatetraenoyl-sn-glycero-3-phosphoinositol.     

Lipid composition of erythrocytes and thrombocytes of a subantarctic seabird, the king penguin

Phospholipid (PL) compositions and fatty acid (FA) patterns of PL were determined in the erythrocytes and blood thrombocytes of a seabird, the king penguin, living in the subantarctic area and feeding on prey rich in n−3 polyunsaturated FA. Results were compared between birds in three different physiological states (breeding and molting adults, chicks) to those reported for other birds. In erythrocytes, the ratios of cholesterol to PL and of sphingomyelin to phosphatidylcholine (PC) were lower than in other birds. The PL distribution was similar to those previously reported in the hen and pigeon. In contrast to other birds, cardiolipin levels were unexpectedly high (4%). Very long chain n−3 FA were abundant (13–27%) in phosphatidylethanolamine (PE), phosphatidylserine and PC, probably in relation to the natural diet of these birds. Among n−3 FA, 22∶6n−3 was the most abundant in all PL (2−20%), whereas the highest levels of arachidonic acid were observed in PE (14%). In thrombocytes, the PL distribution and FA composition of the main PL (PC, PE) differed from those of erythrocytes, and in particular, levels of n−3 FA (9–12%) were 1.5–2 times lower. The highest levels of arachidonic acid were found in phosphatidylinositol (24%). The lipid profile of penguin erythrocytes could contribute to the efficiency of blood circulation and oxygen delivery in microvascular beds, thus favoring diving capacity of these animals. Our observations do not support the hypothesis of a common origin of avian thrombocytes and erythrocytes.     

Molecular species composition of the major diacyl glycerophospholipids from muscle,liver, retina and brain of cod (Gadus morhua)

The molecular species composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) from white muscle, liver, retina and brain of cod (Gadus morhua) were determined by high-performance liquid chromatography of the respective 1,2-diacylglycerol 3,5-dinitrobenzoyl derivatives. A minimum of 69 diacyl species was identified. In muscle and liver saturated fatty acid/polyunsaturated fatty acid (PUFA) and monounsaturated fatty acid/PUFA molecular species were predominant, particularly 16∶0/20∶5 and 16∶0/22∶6 in PC, 16∶0/22∶6 and 18∶1/22∶6 in PE and 18∶0/22∶6 and 18∶1/22∶6 in PS. Didocosahexaenoyl species were major components of PC, PE and PS from retina, comprising 29.3, 71.8 and 59.7% of the respective totals. Didocosahexaenoyl species were also abundant in PE and PS from brain, accounting for 13.8 and 24.0% of the totals, respectively. DiPUFA species were important in muscle, totalling 21.2% in PC and 38.3% in PE. PC from all tissues had the largest amounts of species containing only saturated or monounsaturated fatty acids, accounting for 59.8% of PC from brain, including 12.8% of 18∶1/24∶1 plus 24∶1/18∶1.     

Studies on drug-induced lipidosis: VII. Effects of bis-β-diethylaminoethylether of hexestrol,chloroquine, homochlorocyclizine,prenylamine, and diazacholesterol on the lipid composition of rat liver and kidney

4,4′-Bis (β-diethylaminoethoxy)-α,β-diethyldiphenylethane (DH), which had been shown to induce a type of lipidosis resembling Niemann-Pick disease, was given to rats at a dose of 20, 50, 100, and 150 mg/kg body weight per day for 1 or 2 weeks. An enlargement of the liver with marked increases in free cholesterol, total phospholipids, and phosphatidylinositol took place by administration of a larger dose. The increase in bis (monoacylglyceryl) phosphate (BMGP), which is peculiar to this kind of drug-induced lipidosis, was dependent upon the dose of the drug as well as the length of time. Similar changes were also observed in kidney. Among several other drugs tested, chloroquine and diazacholesterol brough on as much increase in BMGP as treatement with DH.     

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).     

Changes in muscle lipid composition and resistance adaptation to temperature in the freshwater crayfish,Austropotamobius pallipes

The phospholipid and fatty acid composition of muscle lipid extracts from crayfish acclimated to 4 C and 25 C (18 hr-light photoperiod) were analyzed. The phospholipid content and class distribution, and cholesterol content were unaffected by the acclimation treatment. Unsaturation of muscle phosphoglycerides was higher in cold acclimated crayfish. Serine/inositol phosphoglycerides from cold-acclimated animals showed somewhat higher proportions of mono-and polyunsaturated fatty acids, whereas choline and ethanolamine phosphoglycerides were less affected. This was correlated with a decreased resistance of cold-acclimated crayfish to lethal high temperature. Acclimation at 4 C under an 8 hr-light photoperiod caused an increased fatty acid unsaturation of the total phospholipid fraction compared to the 4 C, 18 hr-light photoperiod acclimated animals. The resistance of 4 C acclimated crayfish to lethal high temperature, however, was unaffected by daylength treatment. The resistance of freshwater crayfish to lethal high temperature is not simply related to the degree of saturation of the muscle phospholipids. It is suggested that a breakdown in the integrity of a bulk-lipid bilayer is not involved in the process of heat death; rather, that a membrane-bound protein factor, whose thermal sensitivity is modified by changes in its phospholipid environment during temperature adaptation but not during photoperiod adaptation, is the primary site of heat injury.     

Effect of vitamin E deficiency on the lipid class and fatty acid composition of rat brain gray and white matter

Three-week old male Sprague-Dawley rats were placed on a control or vitamin E-deficient diet for 9 months. The total lipid and cholesterol contents of brain gray and white matter areas in the vitamin E-deficient group did not differ from controls. The concentration of cerebrosides was lower in white matter but higher in gray matter of deficient animals. However, sulfatide was significantly (P<0.001) higher in white and gray matter of deficient animals compared with controls. Lysolecithin was not found in vitamin E-deficient gray matter but was present in control gray matter lipids. No marked differences were found in the concentrations or relative amounts of sphinogomyelin, phosphatidyl choline, phospholipids of gray or white matter of vitamin E-deficient rats as compared to controls. In addition no remarkable differences were found in the fatty acid composition of total lipid extracts of gray or white matter from vitamin E-deficient rats when compared with controls. Presented in part at the Biochemistry/Biophysics Meeting, Minneapolis, 1974, and the Sixth Meeting of the American Society for Neurochemistry, Mexico City, 1975.     

Effect of early postnatal dietary sterculate on the fatty acid composition of rat liver and brain lipids

Pregnant rats were fed a high carbohydrate diet containing either 1% trilinolein or 1% trilinolein with 0.2% methyl sterculate from 18 day gestation to 21 day postpartum. The pups were weaned at 21 days and continued on the same diet for an additional 10 days. The microsomal stearyl CoA desaturase activities of the liver were effectively inhibited. Liver triglycerides showed increases in the saturated fatty acids concentrations at the expense of the corresponding monoenes. The concentration ofcis 6–7 octadecenoic acid was elevated. In liver phospholipids, the concentration of stearic acid was increased without a corresponding decrease in the oleic acid content. A drastic decrease in the nervonic acid (24∶1, n−9) concentration of liver sphingomyelin was observed. The lipids of the brain did not contain sterculic acid, and brain desaturase activity was unaffected. There was no significant change in the concentration of monoenoic acids from 16∶1 to 22∶1. However, nervonic acid was decreased by 32%. These results suggest that brain nervonic acid may be derived from a precursor other than oleic acid.     

Fatty acid composition of unfertilized and fertilized eggs of the sea urchin,Arbacia punctulata

Fatty acid compositions of polar lipids and triacylglycerols isolated from eggs of the sea urchin,Arbacia punctulata, were examined before and 15 min after insemination. The main fatty acids present in polar lipids and triacylglycerols were palmitate, palmitoleate, eicosaenoate, arachidonate and eicosapentaenoate, while palmitate and palmitoleate predominated in triacylglycerols. There were no significant changes in weight percentrage of individual fatty acids of either polar lipids or triglycerides of eggs after insemination.     

Electrodeposition of Sn-Bi lead-free solders: Effects of complex agents on the composition, adhesion, and dendrite formation

The influences of complex agents, including citric acid, ethylenediaminetetraacetic acid (EDTA), and polyethylene glycol (PEG) on the electrochemical co-deposition of Sn-Bi alloys were systematically investigated through the linear sweeps voltammetric (LSV) analyses. The onset deposition potential of Bi ions is obviously shifted to a negative value, close to that of Sn ions by the simultaneous addition of the above three compounds into the plating solution. Based on the LSV results, deposits plated from the typical solutions with the Sn⁴⁺/Bi³⁺ ratio of 1, pH of 6.0, and various combinations of complex agents were characterized by scanning electron microscopic (SEM), energy-dispersive spectroscopic (EDS), and X-ray diffraction (XRD) analyses. The 30Sn-70Bi alloy (in wt%) is successfully plated from the typical plating bath containing 0.4 M citric acid, 1.0 M EDTA, and 0.2 M PEG, suggesting the possibility in plating the Sn-Bi alloys with their composition around the eutectic point (42Sn-58Bi). The adhesion of deposits and the formation of dendrites are respectively improved and inhibited by the synergistic effects of these three complex agents. Contrary to this combination, nearly pure Bi deposits are obtained from the typical plating solutions containing complex agents in the other combinations.