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Lipids such as cholesterol, triacylglycerols, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we describe the development and validation of a high-performance thin layer chromatography (HPTLC) method allowing the separation and quantification of free cholesterol, cholesteryl esters, nonesterified fatty acids, and triacylglycerols. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform., (© 2020 AOCS.)

Sebocytes are specialized epithelial cells that rupture to secrete sebaceous lipids (sebum) across the mammalian integument. Sebum protects the integument from UV radiation, and maintains host microbial communities among other functions. Native glandular sebum is composed primarily of triacylglycerides (TAG) and wax esters (WE). Upon secretion (mature sebum), these lipids combine with minor cellular membrane components comprising total surface lipids. TAG and WE are further cleaved to smaller molecules through oxidation or host enzymatic digestion, resulting in a complex mixture of glycerolipids (e.g., TAG), sterols, unesterified fatty acids (FFA), WE, cholesteryl esters, and squalene comprising surface lipid. We are interested if fatty acid methyl ester (FAME) profiling of bat surface lipid could predict species specificity to the cutaneous fungal disease, white nose syndrome (WNS). We collected sebaceous secretions from 13 bat spp. using Sebutape(®) and converted them to FAME with an acid catalyzed transesterification. We found that Sebutape(®) adhesive patches removed ~6× more total lipid than Sebutape(®) indicator strips. Juvenile eastern red bats (Lasiurus borealis) had significantly higher 18:1 than adults, but 14:0, 16:1, and 20:0 were higher in adults. FAME profiles among several bat species were similar. We concluded that bat surface lipid FAME profiling does not provide a robust model predicting species susceptibility to WNS. However, these results provide baseline data that can be used for lipid roles in future ecological studies, such as life history, diet, or migration.

The chemical characterization of the free fatty acid (FFA) fractions of the cuticular lipids of Calliphora vicina larvae and pupae was performed by separating the FFA fraction using high-performance liquid chromatography with laser light scattering detection (HPLC-LLSD) and quantitatively analyzing the FFA using gas chromatography-electron impact mass spectrometry (GC-MS). Thirty-two saturated and unsaturated FFA were identified and quantified in the insect lipids. Cuticular FFA profiles of C. vicina larvae and pupae were compared. Cuticular FFA of larvae and pupae accounted for 70.8 and 77.8 % of the total lipids, respectively. The cuticular lipids of C. vicina larvae contained 24 FFA ranging from 8:0 to 24:0, whereas the cuticular lipids of pupae contained 32 FFA ranging from 6:0 to 26:0. The cuticular lipids of the larvae contained 16 saturated, five monounsaturated, one diunsaturated, and two polyunsaturated FFA. The cuticular lipids of the pupae contained 18 saturated, nine monounsaturated, two diunsaturated, and three polyunsaturated FFA. The major cuticular FFA in C. vicina larvae and pupae was 18:1 (47.6 and 41.7 %, respectively). The highest amounts of total cuticular FFA were detected in larvae of C. vicina (1.7 mg/g of the insect body). The quantities of total cuticular FFA in pupae were smaller (1.4 mg/g of the insect body).

A non-aqueous flow injection method for determining free fatty acid (FFA) content in corn and sunflower oil samples was developed. A single-line manifold system was built by modification of an HPLC for flow injection analysis (FIA). Without pre-treatment, oil samples were injected into a n-propanol solution containing KOH and phenolphthalein (PHP). The main parameters, such as flow rate of carrier phase, length, geometry, inner diameters of the coils and reagent concentration were all optimized. The proposed FIA method was validated for precision, accuracy, linear region, limit of detection (LOD) and limit of quantification (LOQ). The intra- and inter-day measurements of the precision of the method were found to be within the limits of acceptance criteria (RSD < 1%), and were rugged when the method was performed by a different analyst. The linear concentration range was calculated as 0.09-1.50 and 0.07-1.40 FFA% for corn and sunflower oils, correspondingly. The LOD and LOQ were found to be 7.53 × 10(-4)-2.28 × 10(-3) oleic acid % and 7.11 × 10(-4)-2.23 × 10(-3) oleic acid % for corn and sunflower oils, respectively. The results were compared with those obtained by the AOCS (Ca-5a-40) method using statistical t and F tests, and a significant difference was not observed between the methods at a 95% confidence level. The proposed method is suitable for quality control of routine applications due to its simplicity, high sample throughput, and economy of solvents and sample, offering considerable promise as a low cost analytical system that needs minimum human intervention over long periods of time.

Sphingolipids are important components of cell membranes that may also serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. The aim of this study was to examine the effect of 5 weeks of aerobic training on key enzymes and intermediates of ceramide metabolism in skeletal muscles. The experiments were carried out on rats divided into two groups: (1) sedentary and (2) trained for 5 weeks (on a treadmill). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), neutral and alkaline ceramidases (nCDase and alCDase) and the content of sphingolipids was determined in three types of skeletal muscle. We also measured the fasting plasma insulin and glucose concentration for calculating HOMA-IR (homeostasis model assessment) for estimating insulin resistance. We found that the activities of aSMase and SPT increase in muscle in the trained group. These changes were followed by elevation in the content of sphinganine. The activities of both isoforms of ceramidase were reduced in muscle in the trained group. Although the activities of SPT and SMases increased and the activity of CDases decreased, the ceramide content did not change in any of the studied muscle. Although ceramide level did not change, we noticed increased insulin sensitivity in trained animals. It is concluded that training affects the activity of key enzymes of ceramide metabolism but also activates other metabolic pathways which affect ceramide metabolism in skeletal muscles.

The aim of the present study was to identify the effects of dehydroepiandrosterone (DHEA) on hepatic lipid metabolism parameters and lipogenic gene mRNA expression in broiler chickens. A total of 72 1-day-old broiler chicks received a common basal diet with DHEA added at either 0 (control), 5 or 20 mg/kg feed. In the present study, the hepatic triglyceride (TG) concentration was significantly lower in male and female broilers that had bed administered DHEA than in control birds. In contrast, DHEA administration caused a marked rise in the hepatic non-esterified fatty acid (NEFA) concentration in both male and female broilers and also increased lipase (HL) activity in male broilers, while in female birds, no significant differences were observed in HL activity. The expression of peroxisome proliferators-activated receptor alpha (PPARalpha) and carnitine palmitoyl transferase I (CPTI) mRNA was decidedly enhanced following treatment with DHEA, and a similar tendency was also observed in the expression of acyl-Coenzyme A oxidase 1 (ACOX1). However, no significant differences were observed in the expression of either sterol regulatory element binding protein-1c (SREBP-1c) or acetyl CoA carboxylase (ACC) mRNA, except for a decline in the expression of ACC in females treated with 5 mg DHEA/kg. Numerous peroxisomes without a core and an increased number of peroxisomes were evident during morphological observations of broiler livers, in animals that had been treated with DHEA. Overall, the results of the present study indicated that DHEA accelerated lipid catabolism by direct regulation of hepatic lipid metabolism and by induction of relevant gene expression.

This paper discusses a rapid GC-FID technique for the simultaneous quantitative analysis of FFA, MAG, DAG, TAG, sterols, and squalene in vegetable oils, with special reference to palm oil. The FFA content determined had a lower SE compared with a conventional titrimetric method. Squalene and individual sterols, consisting of beta-sitosterol, stigmasterol, campesterol, and cholesterol, were accurately quantified without any losses. This was achieved through elimination of tedious conventional sample pretreatments, such as saponification and preparative TLC. With this technique, the separation of individual MAG, consisting of 16:0, 18:0, and 18:1 FA, and the DAG species, consisting of the 1,2(2,3)- and 1,3-positions, was sufficient to enable their quantification. This technique enabled the TAG to be determined according to their carbon numbers in the range of C44 to C56. Comparisons were made with conventional methods, and the results were in good agreement with those reported in the literature.

Lipid analysis was performed on freshly ovulated eggs (n = 5) of the oviparous lizard Bassiana duperreyi. The fresh weight of the whole egg contents was 132.0 +/- 4.3 mg (mean +/- SE) of which lipid constituted 21.9 +/- 1.1% (w/w). Triacylglycerol formed an exceptionally high proportion (85.4 +/-0.5%, w/w) of the total lipid, whereas phospholipid, free cholesterol, cholesteryl ester, and free fatty acid, respectively, contributed 11.2 +/- 0.3, 1.4 +/- 0.1, 1.3 +/- 0.1, and 0.6 +/- 0.1% of the total lipid mass. Linoleic and alpha-linolenic acids were the major polyunsaturates of the triacylglycerol fraction, respectively, forming 16.3 +/- 0.1 and 8.3 +/- 0.1% (w/w) of the fatty acids. Linoleic acid was the major fatty acid (29.0 +/- 0.1%) of the total phospholipid, which also contained substantial amounts of arachidonic (6.4 +/- 0.1%) and eicosapentaenoic (3.0 +/- 0.1%) acids, but a relatively low proportion (1.6 +/- 0.1%) of docosahexaenoic acid. Phosphatidylcholine formed the major phospholipid class (73.8 +/- 2.3%) w/w of total phospholipid) and was enriched in linoleic acid, whereas phosphatidylethanolamine, which formed 20.4 +/- 1.9%(w/w) of total phospholipid, contained higher proportions of arachidonic and docosahexaenoic acids.

Alterations in the lipid and fatty acid composition of brush border membrane (BBM) of small intestine were studied in well-fed, starved, and refed rats. The ratios of cholesterol/phospholipid (mol/mol), sphingomyelin/phosphatidylcholine (mol/mol), protein/lipid (w/w), and free fatty acids (w/w) decreased whereas the total phospholipid (w/w) ratio and the double-bond index increased in BBM of the intestine of the starved rat compared to that of the well-fed rat. Analyses of fatty acids showed higher percentage of stearic and arachidonic acids whereas oleic and linoleic acids decreased under starvation. The acyl chain of starved rat BBM was less ordered compared with that of well-fed rat BBM. On refeeding, these changes were restored to well-fed levels. The change in membrane state under starvation is associated with alterations in the lipid and fatty acid composition of BBM and may be responsible for functional changes that occur under nutritional stress.

An enzymatic assay for the determination of nonesterified fatty acid concentrations in milk and plasma is described. The procedure is semiautomated for use with a plate luminometer or plate spectrophotometer and enables routine batch processing of large numbers of small samples (< or =5 microL). Following the activation of nonesterified fatty acids (NEFA) by acylCoA synthetase, the current assay utilizes UDP-glucose pyrophosphorylase to link inorganic pyrophosphate to the production of NADH through the reactions catalyzed by phosphoglucomutase and glucose-6-phosphate 1-dehydrogenase. With this assay sequence the formation of NADH from NEFA is complete within 50 min at 37 degrees C. Enzymatic spectrophotometric techniques were unsuitable for NEFA determination in human milk due to the opacity of the sample. The use of the NADH-luciferase system has overcome this problem, allowing the enzymatic determination of NEFA in human milk. Sample collection and treatment procedures for milk and plasma have been developed to prevent enzymatic lipolysis and to limit interference from enzymes present in milk. The recovery of palmitic acid added to milk and plasma samples was 94.9+/-2.9 and 100+/-4.5%, respectively. There was no difference (P = 0.13) in plasma NEFA concentrations determined by the current method and a commercially available enzymatic spectrophotometric technique (Wako NEFA-C kit). Plasma NEFA concentrations determined by gas chromatography were 28% higher compared to both the Wako NEFA-C kit and the current method.

Previously, we demonstrated that several vegetable oils that included low-erucic rapeseed oil markedly shortened the survival time (by approximately 40%) of stroke-prone spontaneously hypertensive (SHRSP) rats as compared with perilla oil, soybean oil, and fish oil. We considered that a factor other than fatty acids is toxic to SHRSP rats, because the survival time-shortening activity could not be accounted for by the fatty acid compositions of these oils. In fact, a free fatty acid (FFA) fraction derived from lipase-treated rapeseed oil was found to be essentially devoid of such activity. A high-oleate safflower oil/safflower oil/perilla oil mixture exhibited a survival time-shortening activity comparable to that of rapeseed oil, but the activity of this mixed oil was also reduced by lipase treatment. A partially hydrogenated soybean oil shortened the survival time by approximately 40%, but a FFA fraction derived from lipase-treated partially hydrogenated soybean oil shortened it by 13% compared with soybean oil. Fatty acid compositions of the rapeseed oil and a FFA fraction derived from lipase-treated rapeseed oil were similar, but those of hepatic phospholipids of rats fed the oil and FFA were slightly but significantly different. These results support the interpretation that the survival time-shortening activity exhibited by some vegetable oils is due to minor components other than fatty acids, and that an active component(s) were produced in or contaminated soybean oil during the partial hydrogenation processes.

Determination of free fatty acids (FFA) in hepatopancreas of mussel, Mytilus coruscus, contaminated by diarrhetic shellfish poisoning (DSP) toxins, was carried out by high-performance liquid chromatography-fluorometry as their 9-anthryldiazomethane derivatives. The FFA in the hepatopancreas of mussels during May 30-June 14 were 443-666 micrograms/g. A marked increase in FFA content was observed on June 20 (2197 micrograms/g hepatopancreas) and June 27 (6322 micrograms/g hepatopancreas). The proportion of polyunsaturated fatty acids (PUFA), including 20:4n-6, 20:5n-3, and 22:6n-3, in mussel FFA increased during the experimental period. Diatoms almost completely dominated the phytoplankton community at the experimental site, suggesting that diatoms were the origin of the PUFA accumulated in mussels. The toxicity of FFA in mussel hepatopancreas on June 27 was sufficiently high as to interfere with the mouse bioassay by injection of an extract of shellfish, the official method for DSP toxin analysis.

Diazomethane is widely used for the selective methylation of nonesterified fatty acids in the presence of other lipids. However, when the reaction is carried out directly with plasma or serum, substantial methanolysis of phospholipid acyl groups occurs. Because of the importance of rigorous selectivity in the assay of unesterified fatty acids which are present only in trace amounts in cells and body fluids, we have investigated the diazomethane procedure in detail and reached the following conclusions: (i) When diazomethane reacts with lipid extracts in organic solvent, no ester hydrolysis occurs. (ii) In the presence of serum or plasma, diazomethane reacts with water and inorganic salts, causing the solution to become basic (CH2N2 + NaCl + HOH-->Ch3Cl + Na+ + OH- + N2); methoxide ions are formed from methanol (CH3OH + OH(-)-->CH3O- + HOH) causing extensive methanolysis (CH3O- + RO-CO-R'-->CH3O-CO-R' + RO-). An analogous reaction takes place with ethanol. All esters of glycerol are transesterified in aqueous salt solution by this mechanism. It is therefore essential to prepare a lipid extract prior to the assay of unesterified fatty acids when using the diazomethane procedure.

The sterol composition of free sterol and steryl ester fractions of the fish parasite Paratenuisentis ambiguus was determined. In addition, the fatty acid composition of various neutral lipid classes, i.e., wax esters, steryl esters, triacylglycerols and free fatty acids, as well as the composition of the 1-O-alkyl moieties of total ether glycerolipids of the parasite, were investigated. The results of these studies were compared with those obtained on the intestinal tract tissue of its host, the eel (Anguilla anguilla). Cholesterol is the major sterol in both P. ambiguus and A. anguilla. However, the sterols of P. ambiguus contain high proportions (> 20%) of other sterols, such as campesterol and various dehydrosterols. [e.g., 7-dehydrocholesterol and cholesta-5,22(E)-dienol]. The presence of these minor sterols agrees with the known biotransformations of exogenous sterols in various helminths. Considerable differences are found in the fatty acid composition of neutral lipid fractions, as well as the total lipid extract from the endoparasite as compared to the host tissue. In particular, eicosapentaenoic acid (20:5n-3), other polyunsaturated fatty acids, such as 20:4n-6, 22:5n-3 and 22:6n-3, as well as long-chain saturated fatty acids, such as 20:0, are generally enriched in the neutral lipid fractions of the parasite as compared to those of infected eel intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

The fatty acid composition of milk fat is known to be affected by dietary and genetic differences, while the milk triacylglycerol structure is believed to be attuned to the needs of the subsequent lipolysis during gastrointestinal passage. The availability of milk samples from eight species of prosimian primates, whose milk triacylglycerol structure had not been analyzed, offered an opportunity to further assess these ideas. The milk samples were collected by manual expression and the lipids extracted with chloroform/methanol (2:1, vol/vol). The lipid classes were resolved by thin-layer chromatography, and the neutral lipids subjected to detailed analyses by capillary gas-liquid chromatography of fatty acids and molecular species of triacylglycerols using nonpolar and polarizable liquid phases. The milk samples were found to differ greatly in total fat content (4-73%) and in the composition of the neutral lipid classes and molecular species. The concentration of triacylglycerols ranged from 88-95%, free fatty acids from 0.5-10%, alkyldiacylglycerols from 0.5-5.0%, and diacylglycerols, monoacylglycerols and free and esterified cholesterol made up the remainder. The fatty acid chain length ranged from C8-C24, with palmitic (16-31%) and oleic (13-40%) acids being the major components in most of the species. In all instances, the molecular association of the fatty acids differed from random distribution by a higher proportion of the monoacid (trioleoyl) and diacid (dipalmitoyloleoyl) glycerols. The phylogenetic influences on neutral milk lipid composition, however, remained unclear, as some of the differences between closely related species were greater than those between more distantly related ones.

Rats were fed (for 2 or 6 wk) purified diets containing lard (LD) or menhaden oil (MO) at two levels of dietary fat, i.e., at 11.5 and 20.8% of energy in the low fat (LF) and the medium fat (MF) diets, respectively. Following the diet period, rats were sacrificed after either an overnight fast or after uninterrupted ad libitum feeding. The studies were designed to investigate the dependence of our previously reported effects of MO, i.e. the reduction of plasma free fatty acid (FFA) levels and accumulation of hepatic triacylglycerols, on the dietary fat concentration and the nutritional state of the animal at the time of sacrifice. Reductions in plasma triacylglycerol and cholesterol levels in MO-fed relative to LD-fed rats were observed under all conditions. FFA levels were consistently reduced by MO-feeding at both dietary fat concentrations, but only when blood was sampled from ad libitum fed rats. Under these conditions there was a significant positive relationship between plasma FFA and triacylglycerol concentrations. Reduction in plasma FFA levels may be an additional mechanism associated with the triacylglycerol-lowering effect of fish oil (FO). The LF and MF MO diets caused a rise in plasma glucose levels with no significant change in insulin concentration, indicating that the reduction of FFA by MO was not related to changes in insulin concentration or insulin sensitivity. The MO diets had no effect on skeletal muscle or epididymal adipose tissue lipoprotein lipase activity, demonstrating that catabolism of triacylglycerol-rich lipoproteins contributes little, if any, to the MO-dependent reductions of plasma triacylglycerol and FFA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan-induced diabetes in rats results in elevated levels of nonesterified fatty acids (NEFA) in whole sciatic nerve and its endoneurium. Increases in NEFA levels are more pronounced in whole diabetic nerve (40% over control) than in its endoneurial portion (20-30%). Alterations in the composition of phospholipid fatty acids are observed as well, including an increase in linoleate (18:2n-6) in endoneurial phosphatidylethanolamine and a decrease in arachidonate (20:4n-6) in both phosphatidylethanolamine and phosphatidylinositol of diabetic nerve.

The activity of the overt form of rat liver mitochondrial carnitine palmitoyltransferase or CPT0 (EC 2.3.1.21) towards different fatty acid substrates was studied. The following non-esterified fatty acids (NEFA) and their CoA esters in the presence of 1% bovine serum albumin (BSA) were tested: 16:0, 18:0, 18:1, 18:2, 18:3 omega 3, 20:4, 20:5 omega 3 and 22:6 omega 3. The data fit a square hyperbolic model for enzyme catalysis (p less than 0.001, non-linear regression). Asymptotic Vmax and K0.5, substrate concentration at one-half Vmax, were calculated using total concentrations of acyl-CoA, or unbound concentrations of NEFA. BSA was found to act as a true substrate reservoir for NEFA in that the dissociation of the NEFA-BSA complex was 10-330 times faster than the CPT0 reaction. Regardless of form (NEFA or CoA ester), 18:3 omega 3 gave the highest, while 22:6 omega 3 and 18:0 gave the lowest rates of acylcarnitine synthesis. Except for 18:3 omega 3 and 18:2, Vmax for NEFA was generally lower than for acyl-CoA, with the greatest differences observed for 20:4, 20:5 omega 3 and 22:6 omega 3, suggesting that acyl-CoA synthesis may also be important in the control of the entry of these fatty acids into the mitochondria. The data provide an enzymatic rationale for the relatively low content of 18:3 omega 3 in esterified lipid.

A method combining data on fatty acid composition into subsets is used to illustrate general relative competitive selectivities in the metabolic and transport events that maintain fatty acid compositions in tissue lipids and to minimize differences among tissues or species in the amount of individual fatty acids. Fatty acid compositions of triglycerides and phospholipids in several tissues of the rat were maintained with simple relationships between the exogenous n-3 and n-6 dietary polyunsaturated fatty acids and the endogenous n-7 and n-9 types of fatty acid. The general pattern of fatty acids in triglycerides was similar for liver, plasma and adipose tissue, averaging about 30% as saturated acids, 67% as 16- and 18-carbon unsaturated acids and only about 2% as 20- and 22-carbon highly unsaturated acids. The tissues maintained a linear relationship between the amount of 18-carbon polyunsaturated fatty acids in the diet and in the tissue triglycerides, with the proportionality constant for 18:3n-3 being 60% of that for 18:2n-6. The total phospholipids of liver, plasma and red blood cells maintained about 45% of the fatty acids in the form of saturated fatty acids and 20-30% as 20- and 22-carbon highly unsaturated fatty acids irrespective of very different proportions of n-3, n-6 and n-9 types of fatty acids. In all three tissues, the 20-carbon highly unsaturated fatty acids of the n-3, n-6 and n-9 type were maintained in a competitive hyperbolic relationship with apparent EC50 values for dietary 18:2n-6 and 18:3n-3 near 0.1% of dietary calories. The consistent quantitative relationships described in this study illustrate an underlying principle of competition among fatty acids for a limited number of esterification sites. This approach may be useful in predicting the influence of diet upon tissue levels of the substrates and antagonists of eicosanoid biosynthesis.

Myocardial nonesterified fatty acids (NEFA) increase markedly within the first two days after the induction of insulin-dependent diabetes mellitus in rats by intravenous injection of alloxan. After initial variability, NEFA levels in diabetic hearts remain constant at approximately 450 nmol/g tissue (16 nmol/mumol lipid P), which is about three times higher than that in control hearts. Nonesterified linoleic acid is significantly increased in diabetic heart whereas both arachidonic and docosahexaenoic acids are decreased compared to controls.

A systematic approach has been developed for the collection and analysis of gas chromatographic (GC) data from multiple fatty acid profiles. The approach was applied to a series of polar and nonpolar tissue lipids generated in animal feeding studies to allow a comparison of mean fatty acid profiles as a function of either dietary regimen or tissue location. The magnitude of the studies, sufficiently large to minimize error from animal variabilities, mandated the use of computer assistance. Nevertheless, manual input was essential due to the complexity of the GC patterns, and was invoked for peak assignment and report editing. The approach discussed here allowed for the consolidation and statistical analysis of data from over 30,000 GC peaks, and generated results in both tabular and graphic formats. It should be extendable to other chromatographic studies of lipid components.

The use of thin layer chromatography (TLC) for separation (using silica gel coated quartz rods) and subsequent flame ionization for detection (FID) was examined to determine whether this method could be used for the quantitation of lipids. However, response factors (RF) for various lipids were different and depended upon several variables including the amount of material analyzed. For example, RF were 3-fold greater when 10 micrograms of tripalmitin was analyzed as compared to 1 microgram of the same material. The amount of lipid detected by FID was also dependent upon the rate at which it passed through the flame. During analysis of methylpentadecanoate, detector response increased with scan speed, while at all speeds it was completely removed from the rod. On the other hand, depending upon the amount of cholesterol or phospholipid analyzed, the response either increased, remained unchanged or decreased with scan speed. During a fast scan, detector response was reduced because some material remained on the rod. Thus, the detector response is influenced by sample volatility. In conclusion, there appears to be a complex relationship between detector response and the amount of heat available per microgram of sample. Since we could not find a direct correlation between detector response and sample quantity, it would be difficult to use TLC-FID as a tool for quantitating the components of a lipid mixture.

The lipids of Neurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.

This study compared to effect of environmental temperature on sebum composition in 2 breeds of cattle, British (SH) and Brahman (GB), which differ in their abilities to tolerate heat. By long-term exposure of both breeds to environmental temperatures of 24 C and 32 C and the more heat-tolerant GB breed to 38 C, it was possible to make breed comparisons at (a) different body temperatures, i.e., when all animals were exposed to the same environmental temperature, and (b), at the same body temperature, i.e., when the 2 breeds were exposed to different ambient temperatures. The composition of sebum excreted to saturation level on the skin surface was determined. At the same body temperatures, the amounts of fatty acids in each lipid class were higher in GB than in SH animals except during hyperthermia when the amounts of triglyceride fatty acids were similar in both breeds. The total amounts of individual fatty acids except 14:1, 16:1, 20:0 and 14:OH were higher in both breeds at 32 C than at 24 C. The GB cattle excreted more essential fatty acids (EFA) than the SH cattle at 24 C and at 32 C. There was a significant genotype by environment interaction in the amount of EFA excreted in triglyceride decreased whereas the amount excreted in wax esters increased with rising body temperature.

The neutral lipid and phospholipid compositions of purified sinusoidal (fat-storing, endothelial and Kupffer) cells, parenchymal cells and liver homogenates were determined by thin layer chromatography. In addition, the retinoid content of the same purified cell populations was determined by high performance liquid chromatography. From each cell type, both a lipid droplet fraction and a pellet fraction (containing the majority of the remaining cell organelles) were prepared by differential centrifugation. Electron microscopic analysis showed that lipid droplets isolated from fat-storing cells were larger (up to 8 microns) than those isolated from parenchymal cells (up to 2.5 microns). Moreover, the parenchymal lipid droplets seemed to be surrounded by a membranous structure, while the fat-storing lipid droplets seemed not to be. Both fat-storing and parenchymal cells contained high concentrations of neutral lipids, 57.9 micrograms and 71.0 micrograms/10(6) cells, respectively, while endothelial and Kupffer cells contained only 8.6 micrograms and 13.8 micrograms/10(6) cells of neutral lipids, respectively. Sixty-five percent of fat-storing cell lipid droplet fractions comprised esters of retinol and cholesterol. This combined ester fraction contained mainly retinyl esters. In addition, considerable quantities (20%) of triglycerides were present. Parenchymal cell lipid droplet fractions comprised triglycerides (62%) and cholesteryl esters (up to 30%). The pellet fractions prepared from all four cell types consisted mainly of cholesterol (41-67%) and free fatty acids (20-28%). The phospholipid content was much higher in parenchymal cells than in the sinusoidal liver cell types. The relative proportions of the four major phospholipid classes were comparable in all liver cell types analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

The lipid and fatty acid composition of a strain of Streptomyces antibioticus has been studied as a function of culture age and glycine concentration in the growth medium. Under all conditions, the main polar lipids were phosphatidylethanolamine, cardiolipin and phosphomannoinositides in order of decreasing abundance; no ornithinolipids were detected. Acylglucoses and menaquinones were found among the nonpolar lipids. The main fatty acids present were anteiso 15:0 and anteiso 17:0. The lipid composition of the cells varied with the age of the culture, but no uniform pattern of variation was found in the cultures grown on different amounts of glycine. Among the cells harvested at the end of the exponential phase of growth, those grown on 2% glycine give the highest yield of protoplast formation. These cells were found to contain low amounts of nonpolar lipids and of phosphatidylethanolamine, and high proportions of anteiso fatty acids. We propose that the membrane bilayer of these cells, because of its peculiar lipid composition, is particularly stable and fluid. As a consequence, the yield and stability of derived protoplasts should be increased.

In the course of our study on the lipids of the rat sciatic nerve, the analysis of the neutral lipids allowed us to detect and characterize cholesteryl esters present at a relatively high level (5%). Among the phospholipids, ethanolamine phosphoglyceride is the most abundant fraction and contains neraly all the plasmalogens (20% of total lipid phosphorus). The glycolipids consist of five different fractions; the cerebrosides with hydroxy fatty acids account for 38% of total glycolipids. Monogalactosyl diglyceride represents 7% of total glycolipids.

The detailed composition of cellular lipid of more than 23 species of yeast has been determined quantitatively by thinchrography on quartz rods, a method previously used for estimating cellular lipids of seven species of yeast. That data was fortified by neutral and phospholipid quantitations on 30 species of yeast cells. Most of the test organisms contained 7-15% total lipid and 3-6% total phospholipid per dry cell weight, except for the extremely high accumulation of triglycerides in two species of Lipomyces. Qualitatively, 30 species of yeast cells contained similar neutral lipid constituents (triglyceride, sterol ester, free fatty acid, and free sterol) and polar lipid components (phosphatidyl choline, phosphatidyl ehtanolamine, phosphatidyl serine, phosphatidyl inositol, cardiolipin, and ceramide monohexoside) without minor constituents. Based on the quantitative composition of neutral lipids, the 30 species of yeast were divided into two groups , the triglyceride predominant group and the sterol derivative group. These groupings were fairly well overlapped from the standpoint of the distribution characteristics of fatty acid. The relative polar lipid compositions also grossly resembled each other. Only one exception of polar lipid composition in yeast cells was found in Rhodotorula rubra species which contained phosphatidyl ethanolamine as the most abundant phospholipid. Fatty acid distribution patterns in yeast cells consistently coincided with other reports concerning fatty acid composition of yeast cells. Correlation of lipid composition and classification of yeasts are suggested and discussed.

Values are presented for whole brain free fatty acid levels of rats sacrificed by decapitation vs focused microwave irradiation. Free fatty acids were quantitated by specific colorimetric analysis. Within ca. 1 min of sacrifice by either decapitation or microwave, rat whole brain free fatty acid concentrations ranged from ca. 80-100 mug/g fresh tissue. If the brain remained in the head for a total of 5 min after decapitation, free fatty acid levels increased by over 100%. The free fatty acids at this time were enriched with arachidonic acid. The increase in free fatty acid levels following decapitation was completely absent in rats sacrificed by the microwave irradiation. This microwave technique could be a valuable tool in determining free fatty acid and other heat stable compounds in brain tissue.

Prior to fertilization, mammalian sperm must undergo the acrosome reaction, which involves modifications of the plasma and outer acrosomal membranes followed by vesiculation and release of the membranes. The membrane fraction that was released from caudal boar sperm undergoing an in vitro acrosome-like reaction was isolated and characterized with respect to density, marker enzymes and lipid composition. This membrane had a lower phospholipid/protein ratio (mg/mg) than the sperm plasma membrane, whereas both membranes had similar molar sterol/phospholipid ratios. The major phospholipid was sphingomyelin, followed by phosphatidylethanolamine and phosphatidylcholine, whereas in the plasma membrane the order was reversed; the two major phosphoglycerides contained alkylacyl and alkenylacyl species in addition to the diacyl species. The released membrane also contained lower amounts of cholesterol sulfate and unsaturated fatty acids than the plasma membranes. These results, in combination with our studies on the changes of the sperm membranes during maturation and acrosome reaction, will allow a better understanding of the mechanism of the sperm acrosome reaction.

In three steers which were given grass silage for six months, the content of phytanic acid (i.e. 3,7,11,15-tetramethyl-hexadecanoic acid) in plasma lipid increased to about 8% of the total fatty acids, whereas after this time the proportion in the total fatty acids of liver and heart lipids was about 1%, and only 0.1% in those of kidney lipids; the acid was present in trace amounts in adipose-tissue triglycerides and was apparently absent from brain lipids. In eight lactating cows which were given grass silage for about 3 months, the content of phytanic acid in the total long chain fatty acids of milk and of plasma was 0.7% and 13%, respectively. In the plasma lipids of both steers and lactating cows, phytanic acid constituted a substantial proportion of the total fatty acids of the triglycerides and phospholipids; the acid was present in lowest proportion in the cholesteryl esters.

The lipid composition of Morris hepatoma 5123c was analyzed together with that of liver and blood plasma from both normal and tumor-bearing rats. The results showed that the liver of tumor-bearing rats contained higher amounts of glycerides, choelsteryl esters, free fatty acids and phospholipids than the liver of normal rats. In the blood plasma of tumor-bearing rats, there was an increase of free cholesterol and triglycerides; this latter difference, however, was not statistically significant. Acyl chain changes in the liver of tumor-bearing rats consisted of an increase of palmitic and oleic acids and a decrease of stearic and arachidonic acids in phosphatidylinositol. Morris hepatoma 5123c contained a lower amount of triglycerides than the livers (both host and normal) and showed a significant decrease of total phospholipids when compared to the host liver. The major acyl chain changes found in Morris hepatoma 5123c compared with both normal and host rat livers were: a) a higher percentage of arachidonic acid together with a lower proportion of palmitic acid in cholesteryl esters; b) an increase of stearic and arachidonic acids and a decrease of palmitic acid in triglycerides; and c) a higher level of palmitic and oleic acids associated with a lower percentage of stearic and C22 polyunsaturated acids in phosphatidylcholine.

Binding antibodies to surface membranes stimulated incorporation of fatty acids (FA) into phospholipids of L cells. Antibodies stimulated at least a 3.4-fold greater incorporation of arachidonic acid into phosphatidylinositol than into any other class of phospholipid when compared on a molar basis (p less than 0.003). This enhanced incorporation was selective, depending on the character of the FA, because antibodies stimulated the incorporation of arachidonic acid at least 2.4-fold more than oleic acid, palmitic acid or stearic acid (p less than 0.001). Surprisingly, an antibody-stimulated incorporation of palmitic acid into sphingomyelin (SM) was at least 2.2-fold greater than that into any other class of phospholipid (p less than 0.001) and the antibody-stimulated incorporation of palmitic acid into SM was at least 60-fold greater than that of arachidonic acid, stearic or oleic acid (p less than 0.001). Nontoxic doses of ethylenediamine tetraacetic acid (EDTA), dexamethasone, 4-bromophenacylbromide and indomethacin inhibited the antibody-stimulated incorporation of arachidonic acid into cellular phospholipids, principally phosphatidylinositol (PI), and similarly inhibited the antibody stimulation of DNA synthesis. We conclude that when antibody binds to surface antigens on L cells, a rapid and selective incorporation of fatty acids into certain cellular phospholipids occurs, possibly mediated by calcium-dependent phospholipases. Degradation products of arachidonic acid, i.e., prostaglandins, may be important in these antibody stimulation events, as well. These early changes in phospholipid metabolism may serve as an important signal or mechanism for the subsequent stimulation of DNA synthesis in L cells.

Total lipids from whole pig adrenal glands as well as from their mitochondria, microsomes, liposomes, and cell sap were extracted and fractionated first into neutral lipids and phospholipids. The highest percentage of neutral lipids was found in the cell sap, and the lowest in the microsomal fraction. Neutral lipids were subfractionated into cholesteryl esters, free cholesterol, triglycerides, and free fatty acids. Cholesteryl esters were distributed throughout the liposomes. Free fatty acids represented a substantial part of cell sap lipids, but were present also in the mitochondria, microsomes, and liposomes. Fatty acids of all fractions were analyzed by gas liquid chromatography. Free fatty acids and cholesteryl ester fatty acids from all cellular fractions were similar in composition and were characterized by considerable quantities of linoleic and arachidonic acid. Triglycerides were characterized by an increased percentage of palmitic and a low content of arachidonic acid. Phosphatidyl choline, phosphatidyl ethanolamine, diphosphatidyl glycerol, and sphingomyelin plus phosphatidyl inositol were isolated from the lipids by preparative thin layer chromatography, and their fatty acids analyzed by gas liquid chromatography. Phosphatidyl choline and phosphatidyl ethanolamine from mitochondria, microsomes, and cell sap were very similar in respect of their fatty acid composition. Sphingomyelin plus phosphatidyl inositol was characterized by a high content of C22:2omega6. Diphosphatidyl glycerol was present in mitochondria and in the cell sap.

Analysis of human milk was conducted to determine if free fatty acids occur naturally or as a consequence of artifactual lipolysis after milk expression. Five mothers provided triplicate early morning milk samples on day 43 of lactation. Following extraction, lipid classes were separated by preparative thin layer chromatography and quantified by capillary gas liquid chromatography. Fresh milk samples collected with 20 volumes chloroform-methanol (1:1, v/v) were analogous in total free fatty acid level and profile of fatty acids to a duplicate sample collected with 0.4M EDTA and immediately frozen at -10 C. Low milk levels of free fatty acids appear to exist naturally. During days 4-37 of lactation, four serial milk samples from 15 mothers were collected and frozen with 0.4M EDTA. The concentration of free fatty acids in colostrum (0.03-0.5%, w/w) was lower than for subsequent days (0.3-2.5%, w/w). Additional samples were collected with and without a lipase inhibitor (0.4M EDTA) and subjected to routine collection and storage procedures. Significantly different fatty acid profile and higher levels of free fatty acids in milk collected without a lipase inhibitor added indicate that domestic freezing and/or thawing ruptures the fat globule membrane, allowing sn-1-stereospecific serum stimulated lipoprotein lipase contact with its triglyceride substrate. Standard procedures for collection of human milk for gavage fed infants appears to stimulate artifactual lipolysis of milk triglyceride and subsequent release of free fatty acids. The proposed relationship between dietary free fatty acids and prolonged, unconjugated hyperbilirubinemia in the newborn is discussed with regard to the significance of preintestinal lipolysis.

We have examined hepatic lipid profiles in a mouse model for Reye's Syndrome (RS) in which young animals are exposed to nontoxic doses of an industrial pesticide emulsifier and subsequently are infected with sublethal doses of mouse-adapted human Influenza B (Lee) virus (FluB). The purpose of this study was to determine whether liver lipid content was altered in the mice, the time course of any changes, and whether lipid changes were consistent with liver pathology. Neonatal mice exposed dermally to the emulsifier, Toximul MP8 (Tox), had significantly elevated levels of hepatic cholesterol, with otherwise normal lipid composition. Subsequent inoculation of the mice with FluB significantly increased mortality rate. The combined Tox + FluB treatment had several significant effects on liver lipids, including a transient increase in phospholipid (PL) content, a reduction in neutral glycerides and persistently high cholesterol levels. Abnormalities in fatty acid profiles included an apparent elevation in medium chain fatty acids and increased ratios of PL arachidonic to docosahexaenoic acids. Histologically, there was no evidence of fat accumulation in the liver; however, hepatic mitochondria had severe structural abnormalities characteristic of RS. These studies demonstrate that chemical-dependent enhancement of viral virulence is associated with significant alterations of hepatic lipids. We believe that these abnormalities are related to mitochondrial structural damage in RS despite the absence of hepatic steatosis.