Neutral Lipid Profiles Research Articles

Early Embryonic Lethality Caused by Disruption of the Gene for Choline Kinase α, the First Enzyme in Phosphatidylcholine Biosynthesis

Choline kinase alpha (CK-alpha) is one of two mammalian enzymes that catalyze the phosphorylation of choline to phosphocholine in the biosynthesis of the major membrane phospholipid, phosphatidylcholine. We created mice lacking CK-alpha with an embryonic stem cell line containing an insertional mutation in the gene for CK-alpha (Chka). Embryos homozygous for the mutant Chka allele were recovered at the blastocyst stage, but not at embryonic day 7.5, indicating that CK-alpha is crucial for the early development of mouse embryos. Heterozygous mutant mice (Chka(+/-)) appeared entirely normal in their embryonic development and gross anatomy, and they were fertile. Although choline kinase activity was decreased by approximately 30%, the amount of phosphatidylcholine in cells and the levels of other enzymes involved in phosphatidylcholine biosynthesis were unaffected. Phosphatidylcholine biosynthesis measured by choline incorporation into hepatocytes was also not compromised in Chka(+/-) mice. Enhanced levels of choline and attenuated levels of phosphocholine were observed in both the livers and testes of Chka(+/-) mice. Triacylglycerol and cholesterol ester were elevated approximately 2-fold in the livers, whereas neutral lipid profiles in plasma were similar in Chka(+/-) and wild-type (Chka(+/+)) mice. Thus, Chka is an essential gene for early embryonic development, but adult mice do not require full expression of the gene for normal levels of phosphatidylcholine.

High performance thin layer chromatographic analysis of neutral lipids in the urine of BALB/c mice infected with Echinostoma caproni

Silica gel high performance thin layer chromatography-densitometry was used to determine the neutral lipid profile in the urine of BALB/c mice experimentally infected with adults of Echinostoma caproni. An approximate 30 worm burden per host induced certain changes in the neutral lipid profile of the mice at 2 to 10 weeks post-infection (PI) relative to the uninfected controls. Infection caused a significant increase (Student's t test, P < 0.05) in the methyl oleate fraction at 6 and 7 weeks PI relative to the uninfected controls. Neutral lipid profiles of urine may serve as an indicator of experimental infection of echinostomes in mice.

Lipids of human meibum: mass-spectrometric analysis and structural elucidation

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MS(n)). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MS(n) detection of the analytes (HPLC API MS(n)). The major precursor ions were isolated and subjected to further sequential fragmentation in MS(n) experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M - H(2)O + H](+)) and oleic acid (OA; m/z 283; [M + H](+)) were found. The other major compounds of the general molecular formula C(n)H(2n-2)O(2) were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas C(n)H(2n-4)O(2) and C(n)H(2n)O(2). Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MS(n) analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.

High Performance Thin Layer Chromatographic Analysis of Neutral Lipids in the Feces of BALB/c Mice Infected with Echinostoma caproni

High performance thin layer chromatography was used to determine neutral lipid profiles in the feces of BALB/c mice infected with adults of Echinostoma caproni (Trematoda). An approximate 25 worm burden per host induced changes in the neutral lipid profile of the mice at 1 to 5 weeks post infection (PI) relative to the uninfected controls. Infection caused a significant decrease (Student's t‐test, P<0.05) in the triacylglycerol fraction at 3 weeks PI and a significant increase (Student's t‐test, P<0.05) in the free sterol fraction at 2 weeks PI relative to the uninfected controls. Neutral lipid profiles of feces may serve as an indicator of infection by intestinal trematodes in animals and humans.

Influence of Aging on the Polar and Neutral Lipid Profiles in Human Meibomian Gland Secretions

To determine whether aging is associated with significant alterations in the polar and neutral lipid profiles in human meibomian gland secretions. Meibomian gland secretions were collected from both eyes of younger and older men and women. Samples were processed for high-performance liquid chromatography or mass spectrometry and for the analysis of associated spectra of fragment ions. Subjects also underwent slitlamp evaluations of the eyelid. Aging is associated with numerous significant alterations in the lipid profiles of human meibomian gland secretions. Analysis of polar and neutral lipid patterns identified ions that were significantly different in secretions of younger vs older men and women, as well as ions that varied significantly only between men and women. Correlation coefficients within, but not between, groups were high. Aging was accompanied by increased opacity of meibomian gland secretions and by eyelid and eyelid margin changes. Aging is associated with significant sex-related alterations in the polar and neutral lipid profiles of human meibomian gland secretions. The observed changes may contribute to the age-related increase in the prevalence of dry eye syndromes.

Ischemic and Endotoxin Pre-Conditioning Reduce Lung Reperfusion Injury-Induced Surfactant Alterations

Pulmonary ischemia/reperfusion injury represents a common clinical phenomenon after lung transplantation, pulmonary embolism, and cardiac surgery with extracorporeal circulation. We investigated the influence of ischemic and endotoxin pre-conditioning on gas exchange and surfactant properties in a canine model of ischemia/reperfusion injury. Twenty-six foxhounds underwent 3 hours of warm ischemia of the left lung, followed by 8 hours of reperfusion. Ischemic pre-conditioning was performed for either 5 minutes (IPC-5) or by 2 10-minute ischemic periods (IPC-10), before ischemia. For endotoxin pre-conditioning, dogs were pre-treated by a daily intravenous application of increasing amounts of endotoxin for 6 days. No pre-conditioning was performed in the controls. Bronchoalveolar lavage was performed before ischemia/reperfusion injury (baseline) and after the 8-hour reperfusion period in the non-injured right and in the reperfused left lung. Bronchoalveolar lavage fluids were analyzed for the phospholipid-protein ratio, the content of large surfactant aggregates, the phospholipid and neutral lipid profile, the surfactant protein (SP) content, and for biophysical activity. Severe surfactant alterations were observed in the ischemia/reperfusion-injured left lung, with increased protein concentrations and depressed concentrations of large surface aggregates, SP-B, dipalmitoylated phosphatidylcholine, and phosphatidylglycerol. Endotoxin pre-conditioning and IPC-5 were both capable of greatly preventing the ischemia/reperfusion injury-related deterioration of surfactant properties. IPC-10 exerted no effects. Endotoxin pre-conditioning and IPC-5, but not IPC-10, also prevented loss of gas exchange. Ischemic and endotoxin pre-conditioning may protect against impairment of gas exchange in ischemia/reperfusion injury by restoring physiological surfactant properties.

Neutral lipid classes of black cumin (Nigella sativa L.) seed oils

n-Hexane (H) and chloroform:methanol (CM) were used to extract the oils of Nigella sativa L. seeds. A combination of column and thin-layer chromatography procedures on silica gel were performed to fractionate the main neutral lipid classes of seed oils. The fatty acid pattern of neutral lipid (NL) fractions, triacylglycerol (TAG) and sterol content were determined. NLs constituted 97.2% and 96.1% of total H and CM extract, respectively. TAGs were the major neutral lipid class (80.8–83.1% of the total NLs), while the NL profile was characterized by high levels of free fatty acids (14.3–16.2% of the total NLs). Linoleic acid (C18:2) was the predominant fatty acid followed by oleic (C18:1) and palmitic (C16:0) acids in all examined classes. A good resolution of the TAG critical pairs was accomplished employing a gas liquid chromatograph equipped with a flame ionization detector. The separation of the TAG critical pairs C48:0, C50:1, C52:2 and C54:3 [equivalent carbon number (ECN)=48] as well as C42:0, C48:3 and C54:6 (ECN=42) was accomplished without the aid of any interacting ion such as silver. Six TAG species were determined, but two of them, C54:3 (ECN=48) and C54:6 (ECN=42) were present to the extent of 74% or above of the total TAG content. The 4-desmethylsterols isolated from the unsaponifiable fractions were β-sitosterol (1135–1182 µg/g oil) as the main component followed by Δ5-avenasterol (925–1025 µg/g oil), and Δ7-avenasterol (615–809 µg/g oil). Stigmasterol, campesterol, and lanosterol were detected in small amounts.

Comparison of neutral lipid profile of various trilaminar outer cell wall (TLS)-containing microalgae with emphasis on algaenan occurrence

The neutral lipid profiles of nine species of thin trilaminar outer wall (TLS)-containing freshwater and marine microalgae from the class of Chlorophyceae were studied with emphasis on the relationship between the lipid content and the occurrence of insoluble non-hydrolysable biopolymer (i.e. algaenan). All the freshwater microalgae produce a highly aliphatic algaenan. In sharp contrast, no algaenan was isolated from the two marine microalgae, Chlorella marina and Chlorella minutissima marina, supporting the absence of a close relationship between the presence of TLS and the occurrence of algaenan. High molecular weight straight-chain hydrocarbons (C 23–C 29) were identified in most of the algaenan-producing microalgae and in the algaenan-devoid C. minutissima marina, whereas only low molecular weight hydrocarbons were detected in algaenan-producing Scenedesmus subspicatus and in algaenan-devoid C. marina. Sterols, phytol and fatty alcohols were the major constituents of the polar fraction of the neutral lipids of all the microalgae investigated. High molecular weight saturated or mono-unsaturated alcohols were detected in C. emersonii and in all the microalgae belonging to the genus Scenedesmus. High amounts of saturated C 30 and C 32 α,ω-diols were also detected in S. subspicatus, S. armatus and S. pannonicus. Three classes of lipids were encountered in very small amounts in the medium polarity fraction of the neutral lipids of the microalgae investigated: (i) Monoesters composed predominantly of saturated C 16 or C 18 fatty acids and saturated C 8, C 16 or C 18 alcohols and (ii) long-chain methyl ketones from C 25 to C 31 were detected in several species and (iii) methyl esters of fatty acids ranging from C 16 to C 28 were identified in all the microalgae. Attempts to use the neutral lipid composition and particularly the unusual long-chain lipids, as specific indicators of the occurrence of algaenan in TLS-containing microalgae were unsuccessful.

Further optimization of culture method for rat keratinocytes: titration of glucose and sodium chloride.

The present study further improved the serum-free method of culturing rat keratinocytes. To obtain the best growth of rat keratinocytes, we modified our previous serum-free medium (MCDB153 based medium), particularly the amounts of glucose and sodium chloride (NaCl). Titration experiments showed the optimal concentration to be 0.8 mM for glucose and 100 mM for NaCl. This modification eliminated the requirement for albumin, which had been essential for colony formation when our previous medium was used. Titration of glucose and NaCl, followed by adjustment of essential amino acids and growth factors, produced a new formulation. More satisfactory and better growth was achieved with the new medium than with the previous medium. Accumulation of monoalkyldiacylglycerol (MADAG) was consistently noted in this study, representing the unusual lipid profile. A tendency toward normalization was, however, noted with the neutral lipid profile of keratinocytes cultivated in the new medium: lower production of MADAG was obtained with the new formulation, rather than the previous one.

HIGH PERFORMANCE THIN-LAYER CHROMATOGRAPHIC ANALYSIS OF NEUTRAL LIPIDS IN THE MARINE SNAILS ILYANASSA OBSOLETUS AND LITTORINA LITTOREA INFECTED WITH LARVAL TREMATODES

High performance thin-layer chromatography analysis was used to analyze neutral lipids in the digestive gland-gonad (DGG) complex of the marine snails Ilyanassa obsoletus and Littorina littorea infected with larval trematodes. The results were compared with the DGGs of snails not infected with larval trematodes. The most abundant neutral lipid in both infected and non-infected snails was triacylglycerols, with percentages ranging from 2.8% in I. obsoletus snails infected with unidentified armatae cercariae to 24.7% in I. obsoletus snails infected with Zoogonius rubellus. L. littorea snails infected with an unidentified armatae cercaria contained 6 to 7 times the amount of cholesteryl ester than uninfected snails or those infected with Cryptocotyle lingua cercariae. I. obsoletus snails infected with Z. rubellus or an unidentified armatae cercaria contained 2 to 3 times more free sterol than the uninfected controls. Larval trematode parasitism was found to alter the neutral lipid profiles of the marine snails I. obsoletus and L. littorea.

Thin-layer chromatography and histochemistry analyses of neutral lipids in Helisoma trivolvis infected with four species of larval trematodes.

Thin-layer chromatography (TLC) and histochemistry studies were done on the neutral lipid content of the digestive gland gonad (DGG) complex of Helisoma trivolvis infected with four species of larval trematodes. Two of the species, Ribeiroia sp. and Zygocotyle lunata, contained rediae, and the two others, Spirorchis sp. and an armatae xiphidiocercaria, contained sporocysts. The DGG infected by each species had its own distinct neutral lipid profile as determined by TLC. All profiles differed from that of the uninfected DGG. Densitometric TLC studies showed some quantitative differences in free sterols in infected versus uninfected DGGs. Visual observations of the chromatograms showed that all four species caused a marked elevation in the triacylglycerol fraction in the DGG as compared with the uninfected controls. Oil Red O (ORO) histochemistry studies showed that levels of neutral lipids were increased in the DGGs of infected versus uninfected samples. These histochemistry studies showed a variable distribution of neutral fat, ranging from its absence in the cercariae of Z. lunata and the armatae xiphidiocercaria to ORO-positive droplets in the excretory system of Ribeiroia sp. Rediae and sporocysts contained ORO-positive material in the body wall and in the space between cercariae.

Automated Gas Chromatographic Method for Neutral Lipid Carbon Number Profiles in Marine Sample

A gas chromatographic (GC) lipid-profiling method is optimized for analysis of neutral lipids in highly unsaturated marine samples. Chemometrics are employed to optimize autoinjection procedures and column temperature programs. The use of large solvent plug sizes, fast injection rates, hydrogen carrier gas with high flow rates, and short capillary columns enhanced recoveries of high molecular weight neutral lipids. A hydrogenation step was required to avoid discrimination of the flame ionization detector (FID) responses in highly unsaturated lipids. Analyses of fatty acids, which are moieties of the neutral lipids, support the neutral lipid profile data. The identification of major sterols is confirmed by gas chromatography with mass spectrometry (GC-MS). The values of summed molecular species from each neutral lipid class determined by GC are linearly correlated with neutral lipid subclasses determined by Chromarod thin-layer chromatography with flame ionization detection (TLC-FID). Automated high temperature GC on hydrogenated samples is found to offer a precise tool for the measurement of highly unsaturated neutral lipid compounds, which are critical in understanding physiological characteristics of cold water species. The method is applied to a range of marine samples including algae, bivalves, polychaetes, fish eggs, and fish larvae and reveals, for the first time, neutral lipid carbon number distributions in marine species from cold oceans.

Modified epidermal lipid composition in air-exposed culture of non-bullous congenital ichthyotic erythroderma (NBCIE) keratinocytes.

Emerged epidermal cultures on dead de-epidermized dermis (DED) constitute an excellent model for in vitro reproduction of dermatoses linked to a keratinocyte defect. We used such cultures for studies of non-bullous congenital ichthyotic erythroderma (NBCIE). Keratinocytes of normal and pathological origin were expanded in submerged cell cultures and frozen keratinocytes from the resulting cell bank were subsequently used for seeding on DED. Lipid extracts from 14 day emerged cell cultures were assayed qualitatively and quantitatively using thin layer chromatography and compared with the neutral and non-polar lipid profiles obtained from normal epidermis extracts and with those from the plantar stratum corneum of healthy donors and untreated NBCIE patients. The ichthyotic cultures were found to contain significantly elevated levels of n-alkanes, as were the lipid extracts from the patients' plantar horny layer. Our results demonstrate that a major marker of the NBCIE epidermis can be reproduced under the emerged culture conditions. They also indicate that the characteristic n-alkane increase in NBCIE is indeed endogenous and not merely related to possible contamination from topical treatments.

Lipids in the broodsac of Leucochloridium variae (Digenea, Leucochloridiidae) and its snail host Succinea ovalis

Thin-layer Chromatographic analysis was used to examine lipophilic pigments and neutral lipids in the broodsac of Leucochloridium variae and in the tissues of its snail host, Succinea ovalis. Beta-carotene and lutein were not detected in either the parasite or the host on a C-18 reversed phase layer developed in a solvent system of petroleum ether-acetonitrile-methanol (2:4:4). This chromatographic system was able to detect 10 ng of a beta-carotene standard and 100 ng of a lutein standard. The Mangold solvent system on a silica gel plate showed the presence of triacylglycerols, free sterols, and sterol esters as the major neutral lipids in both snail and parasite tissues. As seen in a previous sporocyst-snail relationship, the qualitative neutral lipid profiles of both host and parasite are similar.

Thin Layer Chromatographic Analysis of Neutral Lipids in the Digestive Gland-Gonad Complex of Biomphalaria Glabrata Snails Maintained on Various Diets

Abstract Thin layer chromatography on channeled, high performance preadsorbent silica gel plates was used to study the neutral lipid composition of six diets/i.e., hen's egg albumen, hen's egg yolk, leaf lettuce, Tetramin fish food, lettuce/Tetramin (1:1), and Spirulina, and the digestive gland-gonad complex of Biomphalaria qlabrata snails maintained on these diets. Differences in the neutral lipid profiles between the diet and the DGG suggest that factors other than dietary uptake, presumably metabolic, play a role in determining the lipid composition of the snail.

A comparison of the surfactant associated lipids derived from reptilian and mammalian lungs

The lungs of the central netted dragon Ctenophorus nuchalis are bag-like, with most of the gas exchange region located in the anterior third. Although the faveoli are much larger than the mammalian alveoli, the lizard at 37°C has approximately 70 times more surfactant phospolipid per unit area of respiratory surface than does a similar sized mammal. However, when expressed as per wet lung weight, lizards and rats possessed similar amounts of phospholipids. Dipalmitoylphosphatidylcholine was the principal phospholipid in both species. However, major differences existed in the phospholipid, neutral lipid and fatty acid profiles. Whereas the lizard contained neither phosphatidylglycerol nor phosphatidyl-ethanolamine it had more cholesterol, esterified cholesterol, acylglycerides and unsaturated fatty acids. Although the ratio of saturated: unsaturated fatty acids was similar in rats and lizards, palmitic acid predominated in the former. The composition of lizard surfactant suggests that it would adsorb rapidly at reduced body temperature.

A correlative lipid and lipolytic study of the pancreas and small intestine of the guinea-pig

1. 1. With the aid of a variety of TLC preparations the phospholipid and neutral lipid profiles of guinea-pig pancreas and small intestine has been established. 2. 2. Changes in these profiles, subsequent to incubation of these tissues and prior to lipid extraction, has provided evidence relating to their respective lipolytic enzyme capabilities, mainly in the phospholipase group.

Seasonal changes in the lipids of the sea anemone, Metridium senile (L.)

Total lipids were extracted from freshly collected Metridium senile (L.) in September, November, February, and June. The neutral lipid profile as well as total content was determined in each of these months. With the exception of June, the larger anemones contained less lipid as a percentage of the wet weight than did the smaller animals. In June the anemones contained the highest level of total lipid and a large part of this was sterol. In February, total lipid was also higher as a result of the increased wax ester content. Triglyceride levels remained relatively constant throughout the year while the wax ester content was found to vary both with season and temperature. Both triglyceride and wax ester levels were low in June, when sterol content was high, suggesting sexual reproduction at this time of the year. It is postulated that triglycerides serve as the primary lipid energy reserve in Metridium, while wax esters function in a secondary capacity, being called upon in times of metabolic stress such as sexual reproduction.

Determination of plasma lipid profiles by automated gas chromatography and computerized data analysis.

Plasma or serum [ 0.1-1.0 ml] was digested with phospholipase C and total lipid extracts were prepared and silylated in the presence of tridecanoylglycerol as internal standard. The neutral lipid and free fatty acid profiles were determined by means of an automated GLC system equipped with an unheated on-column inlet, time actuated liquid injector, programmed heating, cooling and equilibration cycles, and an electronic peak area integrator. The separations were accomplished on a 50 cm x 2 mm i.d. steel column packed with 3% OV-1 on100-120 mesh Gas Chrom Q using nitrogen as a carrier gas in the temperature range 175-350 degrees C. The tube number, peak retention time and peak area were recorded on a punched paper tape, which was subsequently read into a computer via a time-share terminal. The composition of the sample was calculated in relation to the internal standard using a modification of a commercially available computer program and the results were expressed as mg or mole % and characteristic molar ratios of lipid classes. In addition to estimates for total cholesterol and triglyceride, the method provides a detailed account of individual or small groups of molecular species of various lipid classes, which is a major advantage over other automated methods of plasma lipid analyses.