A complete separation of lipids by three-directional thin layer chromatography

A novel three-directional thin layer chromatography (TLC) method is reported by which all the polar and neutral lipids are isolated on a single TLC plate. Following resolution of the phospholipids by two-directional TLC, lipids are visualized by ultraviolet light after spraying with 2′,7′-dichloro-fluorescein. A line is drawn across the plate, parallel to the second direction of development, separating the resolved phospholipids and the neutral lipids concentrated along the solvent front. The TLC plate is then chromatographed in the reverse direction of the second development to resolve the neutral lipids. By exposing the lipids to HCl fumes after the first development, the plasmalogen content of the lipids may also be determined. This new technique is rapid and lends itself to qualitative and quantitative analyses of total lipids. Contribution no. 1163 from the Animal Research Centre.     

Determination and occurrence of oxofatty acids in fats and oils

A relatively simple method is detailed for the routine isolation and estimation of oxofatty acids (OFA) in lipids. The lipid in cyclohexane is transmethylated in a two-phase, 3.5 min procedure, and the carbonyls in the methyl ester fraction are derivatized with 2,4-dinitrophenylhydrazine (DNPH) in the presence of monochloroacetic acid (MCA). The derivatives are fractionated on alumina, and the OFA fraction is obtained and evaluated spectrophotometrically. A large variety of animal, plant, and marine lipids contained OFA ranging from <1 to >50 μmoles/g. Data also show that (a) OFA are formed in naturally oxidizing fats and oils, and (b) strongly acidic conditions can cause elaboration of OFA in hydroperoxidized fats and oils.     

Studies on lipid and fatty acid compositions of puffer livers from Indian coastal waters with seasonal variation

The puffer fishes Chelonodon patoca, Sphaeroides oblongus, Lagocephalus lunaris, and L. inermis of Indian coastal waters are wasted in huge quantity. The livers of these fishes were investigated for their lipid contents and fatty acid compositions in different seasons. It was found that monsoon season is the suitable time to obtain the maximal lipids (40.1–48.8%) from their livers, an amount similar to cod liver lipid content (39.5–55.0%). The fatty acids were mostly saturates and monoenes (60–70%). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations (7–12%) were high during monsoon season. Neutral lipids were the predominant lipid class (>80%) and comprised triglycerides (277–674 mg/g) and cholesterol (0.6–3.1 mg/g). Quality indices of puffer liver lipids, e.g., specific gravity, refractive index, acid value, iodine value, saponification value and unsaponifiable matter, were evaluated. Puffer liver lipids were quantitatively and qualitatively comparable to other commercially important marine fish oils. The overall study suggests the possibility of future commercial utilization of liver lipids from puffer, an unconventional, cheap, and easily available source.     

The analysis of lipids via HPLC with a charged aerosol detector

Because most lipid extracts are a mixture of saturated and unsaturated molecules, the most successful strategies for the quantitative analysis of lipids have involved the use of so-called “mass” or universal detectors such as flame ionization detectors and evaporative light scattering detectors. Recently a new type of HPLC “mass” detector, a charge aerosol detector (CAD), was developed and is now commercially available. This detection method involves nebulizing the HPLC column effluent, evaporating the solvents charging the aerosol particles, and measuring the current from the charged aerosol flux. In the present study, the CAD was evaluated with several normal phase and reverse phase HPLC methods commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, diacylglycerols, glycolipids, phospholipids, and sterols. Lower molecular weight lipids such as free FA had smaller peak areas (50–80% lower). FAME were not detected by the CAD, probably because they were completely evaporated and did not form aerosol particles. The minimum limits of detection of the CAD with lipids varied with different mobile phase solvents. Using solvent systems that were predominantly hexane, the minimum limits of detection of triacylglycerols, cholesterol esters, and free sterols were about 1 ng per injection and the mass-to-peak area ratio was nearly linear from the range of about 1 ng to about 20 mg per injection. Three other solvents commonly used for HPLC lipid analysis (methanol, isopropanol, and acetonitrile) caused higher levels of background noise and higher minimum limits of detection. These experiments indicate that the CAD has the potential to become a valuable tool for the quantitative HPLC analysis of lipids. Long-term studies are needed to evaluate full instrument performance.     

Characteristics of Oil from Hulless Barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) Bran from Tibet

The bran of hulless barley (Hordeum vulgare L.) from Tibet was investigated. This paper reports on the physicochemical characteristics, lipid classes and fatty acids of the oil from the bran. The petroleum (60–90 °C) extract of hulless barley bran was found to be 8.1%. The investigated physiochemical parameters included density at 40 °C (0.96 g/cm³), refractive index at 40 °C (1.41), melting point (30.12 °C), acid value (11.6 mg KOH/g), peroxide value (19.41 μg/g), saponification value (337.62 mg KOH/g), iodine value (113.51 mg iodine/g) and unsaponifiable matter (4.5% of total lipids).The amount of neutral lipids in the crude oil was the highest (94.55% of total lipids), followed by glycolipids (4.20% of the total lipid) and phospholipids (1.25% of the total lipid). Linoleic acid (75.08% of total fatty acids) followed by palmitic acid (20.58% of total fatty acids), were the two major fatty acids in the oil. The results show that the oil from the hulless barley bran could be a good source of valuable essential fatty acids.     

The distribution of lipids in the germ,endosperm, pericarp and tip cap of amylomaize,LG-11 hybrid maize and waxy maize

The quantitative distribution of 23 acyl lipid classes and unsaponifiable matter in kernels of amylomaize, LG-11 hybrid maize and waxy maize is described. LG-11 and waxy maize were normal (oil content) varieties, containing 4.9% and 5.1% lipid, respectively, while amylomaize (9.3% lipid) was a high oil variety. The distribution of kernel lipids was 76–83% in germ, 1–2% in pericarp, 1% in tip cap, 1–11% in starch, and 13–15% in aleurone plus the nonstarch fraction of the starchy endosperm. Germ contained 39–47% lipid, which was nostly triglyceride (TG), with some steryl esters (SE) and diglycerides (DG), and small amounts of glycolipids (GL) and phospholipids (PL). Aleurone lipids appeared to be TG with some free fatty acids (FFA) and SE. The other nonstarch lipids in starchy endosperm were FFA with very small amounts of SE, DG, GL and PL. The starches had a little surface lipid (FFA) and true (internal) starch lipid (FFA, lyso-PL) in quantities roughly related to amylose content (amylomaize =ca. 73% amylose, 1.0% lipid; LG-11=23% amylose, 0.7% lipid; waxy maize =<5% amylose, 0.2% lipid). Pericarp lipids (0.8–2.5%) were mainly unsaponifiable matter, the acyl lipids being TG, SE, DG and FFA. Tip cap lipids (2.5–2.9%) had more TG, GL and PL than pericarp lipids, but were otherwise similar. Pericarp lipids and endosperm nonstarch lipids appeared to have suffered extensive degradation at some time during kernel development or after harvesting, while lipids in starch, germ and tip cap were evidently unaffected. FFA and lyso-PL are regarded as normal components of maize starch (rather than degradation products) and may occur as amylose inclusion complexes.     

Estimation of polyunsaturated fatty acid content in lipids of aquatic organisms using thin-layer chromatography on a plain silica gel plate

We have designed a rapid method for the separation of polyunsaturated fatty acids (PUFA, ≥trienes) from non-PUFA, and for estimation of total amounts of PUFA in lipids of aquatic organisms. Lipids from thirty-one species, including marine and fresh water fishes, shell fishes, marine algae, and other aquatic animals, and from terrestrial organisms, were transesterified with sodium methoxide in methanol. The resulting fatty acid methyl esters were separated by thin-layer chromatography on commercially available plain silica gel plates with a developing solvent ofn-hexane/ethyl ether/acetic acid (95∶5∶1, by vol). All of the methyl esters from aquatic organisms tested separated into two spots, whereas those from terrestrial sources, except for linseed oil, showed a single unresolved spot. The upper and lower spots were scraped separately from the plate, and their fatty acid compositions were determined by gas-liquid chromatography. The lower spot was composed of PUFA having more than two double bonds, whereas components of the upper spot were saturated, monoenoic, and the greater part of the dienoic fatty acids. When the spots on the silica gel plate were stained with Coomassie brilliant blue, the amounts of PUFA in aquatic organisms could be estimated satisfactorily using a scanning densitometer. Presented in Japanese at the general Meeting of JSSF held in Mie University, Tsu-city, Japan, October 1994.     

The effect of relative humidity gradient on water vapor permeance of lipid and lipid-hydrocolloid bilayer films

The water vapor (WV) permeance of lipid and lipid-hydrocolloid films exposed to relative humidity (RH) gradients of 100–0%, 100–50%, 100–65% and 100–80% RH were determined. The lipids used were beeswax (BW) or a blend of BW and acetylated monoglycerides (AG). Hydrocolloids used were methylcellulose, carboxymethylcellulose or ethylcellulose (EC). All films, except those containing EC, exhibited increased water vapor permeance as the RH gradient was reduced by raising the low-end RH. This increase in permeance was apparently caused by hydration and swelling across the entire film thickness, thus facilitating water movement through the film. Because of its hydrophobicity, EC likely lessened this swelling. Knowledge of the WV properties of edible films at relatively small gradients in the upper half of the RH spectrum, such as those used in this study, is useful because these conditions are far more common to foods than are the 100–0% gradients that are often used when evaluating films. Even though the WV permeance of BW and BW/AG films increased greatly at the 100–80% RH gradient, as compared to gradients ranging from 100–65% to 100–0%, they still possess WV barrier properties sufficient to be useful for foods.     

Detection and Semi-Quantification of Lipids on High-Performance Thin-Layer Chromatography Plate Using Ceric Ammonium Molybdate Staining

It is desirable to quickly check the composition of lipids in small size samples, but achieving this is challenging using the existing staining methods. Herein, a highly sensitive and semi-quantitative method is developed for analysis of lipid samples with ceric ammonium molybdate (CAM) staining. The CAM detection method is systematically evaluated with a wide range of lipid classes including phospholipids, sphingolipids, glycerolipids, fatty acids (FA), and sterols, demonstrating high sensitivity, stability, and overall efficiency. Additionally, CAM staining provides a clean yellow background in high performance thin-layer chromatography (HPTLC) which facilitates quantification of lipids using image processing software. Lipids can be stained with CAM reagent regardless of their head group types, position of the carbon–carbon double bonds, geometric isomerism, and the variation in the length of FA chain, but staining is mostly affected by the degree of unsaturation of the FA backbone. The mechanism of the CAM staining of lipids is proposed on principles of the reduction–oxidation reaction, in which Mo(VI) oxidizes the unsaturated lipids into carbonyl compounds on the HPTLC plate upon heating, while itself being reduced to Mo(IV). This method is applied for the separation, identification, and quantification of lipid extracts from porcine brain. Practical applications: The CAM staining method that is developed in this work shows a high sensitivity for diverse lipid classes following HPTLC separation. HPTLC with CAM staining is a promising method for quick assessment of the identity and quantity of diverse lipid classes in lipid extracts of small size biological samples.     

In Vitro Simultaneous Transfer of Lipids to HDL in Coronary Artery Disease and in Statin Treatment

The exchange of lipids with cells and other lipoproteins is a crucial process in HDL metabolism and for HDL antiatherogenic function. Here, we tested a practical method to quantify the simultaneous transfer to HDL of phospholipids, free-cholesterol, esterified cholesterol and triacylglycerols and to verify the lipid transfer in patients with coronary artery disease (CAD) or undergoing statin treatment. Twenty-eight control subjects without CAD, 27 with CAD and 25 CAD patients under simvastatin treatment were studied. Plasma samples were incubated with a donor nanoemulsion prepared by ultrasonication of the constituent lipids and labeled with radioactive lipids; % lipids transferred to HDL were quantified in the HDL-containing supernatant after chemical precipitation of non-HDL fractions and the nanoemulsion. The assay was precise and reproducible. Increase of temperature (4–37 °C), of incubation period (5 min to 2 h), of HDL-cholesterol concentration (33–244 mg/dL) and of mass of nanoemulsion lipids (0.075–0.3 mg/μL) resulted in increased lipid transfer from the nanoemulsion to HDL. In contrast, increasing pH (6.5–8.5) and albumin concentration (3.5–7.0 g/dL) did not affect lipid transfer. There was no difference between CAD and control non-CAD with regard to the lipid transfer, but statin treatment reduced the transfer to HDL of all four lipids. The test herein described is a valid and practical tool for exploring an important aspect of HDL metabolism.     

Quantitative high-performance liquid chromatography analysis of plant phospholipids and glycolipids using light-scattering detection

Application of the evaporative light-scattering principle to quantitative high-performance liquid chromatography (HPLC) analyses of plant membrane lipids has received little study. Light-scattering detection response curves were generated for nine classes of plant membrane phospholipid and glycolipids. Quantitative results obtained by HPLC/light-scattering detection and conventional lipid analytical methods (thin-layer chromatography and lipid-P assay) were in close agreement, confirming the reliability of HPLC/evaporative light-scattering detection (ELSD) analyses. Only three of the nine plant lipid classes gave linear detector response functions above 10 μg injected lipid mass. This finding contradicts earlier precepts involving light-scattering detection of lipids. At a given mass, appreciable variation in ELSD signal intensity and detection limit was found to exist among the various plant membrane lipid classes. The variation in detector response among plant lipid classes is an important consideration in achieving accurate quantitative results in plant lipid analyses.     

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

Comparison of lipid composition ofCandida guilliermondii grown on glucose,ethanol and methanol as the sole carbon source

The carbon and energy source for aerobically grown cultures ofCandida guilliermondii profoundly influenced the neutral lipid content and the fatty acid composition of the individual lipid components. Methanol (0.80%, w/v) grown cells cultivated at 30 C in presence of 0.025% ammonium sulfate contained 12% total lipids, 67% of which was neutral lipids. Glucose (0.74%, w/v) or ethanol (0.53%, w/v) grown cells contained 21–22% total lipids, 80% of which was neutral lipids, under the same conditions. Methanol-grown cells contained a decreased 18∶1 acid (52–54% of total fatty acids) and an increased 18∶2 acid (23–25%), as compared with glucose- or ethanol-grown cells which contained 57–66% 18∶1 acid and 8–14% 18∶2 acid, in both neutral and polar lipid fractions. The relationship between methanol metabolism and desaturation of fatty acid in yeast was discussed.     

Production of sophorose lipids byTorulopsis bombicola from safflower oil and glucose

The production of sophorose lipids increased with increasing concentrations of both safflower oil and glucose, and was profoundly influenced by the concentration of yeast extract. A high concentration of sophorose lipids (about 135 g/L) was obtained (in a 1-L Bellco stirred reactor) when the medium consisted of 10% glucose, 10.5% safflower oil, 0.1% urea, and 0.25–0.3% yeast extract. A similar yield of sophorose lipids also was obtained in a 20-L bioreactor. About 50% of the apolar sophorose lipid 1′,4″-lactone 6′,6″-diacetate (SL-1) was found in the mixture of sophorose lipids produced under these conditions.     

Simultaneous Quantification of Plant Glyceroglycolipids Including Sulfoquinovosyldiacylglycerol by HPLC–ELSD with Binary Gradient Elution

Membrane lipids of photosynthetic organisms consist of glycerophospholipids and glyceroglycolipids. We investigated a method for the simultaneous quantitative analysis of neutral and acidic lipids using HPLC–ELSD, and quantified monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). Ten complex lipid classes were separated with a binary gradient system consisting of chloroform and methanol–acetone–water–acetic acid (30:60:9:1, v/v/v/v) with 0.3% triethylamine (pH 4), and were eluted within 16 min. The contents of SQDG in ten edible plants ranged from 3 to 101 mg/100 g, and were positively correlated to the neutral glyceroglycolipids contents.     

Electron spin resonance spectroscopy for determination of the oxidative stability of food lipids

Evaluation of the oxidative stability of food lipids based on the tendency of formation of radicals is shown to be possible using electron spin resonance (ESR) spectroscopy and the spin-trapping technique. Induction time can be determined for mildly accelerated conditions (50°C for lipid fraction from mayonnaise enriched with fish oil), and the length of the induction time decreases during storage and γ-tocopherol depletion. The protection by ethylenediaminetetraacetic acid against initiation of lipid oxidation is also detected in the new assay. For more oxidatively stable lipids (butter, rapeseed oil, dairy spread) the mildly accelerated conditions can be used in the assay, provided that difference in signal height for fixed times replaces determination of induction time. ESR spin trapping provides a sensitive method for evaluating the oxidative stability of food lipids. Detection of radicals in the lipid as an early event in oxidation allows mild conditions to be used, and future experiments should also include sensory evaluation in relation to determination of practical shelf life.     

Analysis of cardiac membrane phospholipid peroxidation kinetics as malondialdehyde: Nonspecificity of thiobarbituric acid-reactivity

When exposed to xanthine oxidase (superoxide)-dependent, iron-promoted Fenton chemistry, purified cardiac membranes evidenced, by the thiobarbituric acid (TBA) test, a virtually instantaneous peroxidative response with a maximal linear rate of 5.8 nmol malondialdehyde (MDA)-equivalents/mEquivalents lipid ester reacted/min. Yet when the lipids purified from these same membranes and reconstituted into liposomes were peroxidized under identical reaction conditions, the TBA test indicated that a pronounced (∼20-min) lag period preceded a maximal peroxidation rate of only 2.1 nmol MDA-equivalents/ mEquivalents lipid ester reacted/min. After 120 min of peroxidation, the cardiac membranes yielded some 300 nmol TBA-reactive MDA-equivalents/mEquivalent ester, whereas the isolated membrane lipids evidenced ∼40% less TBA-reactivity. To verify that these quantitative and kinetic differences in membrane (phospho)-lipid peroxidation occurred with removal of the lipids from their membrane milieu, the MDA produced during both cardiac membrane peroxidation and the peroxidation of the lipids derived therefrom was isolated as its free anion by ion-pair high-pressure liquid chromatography. As quantified spectrophotometrically, true MDA production during myocardial membrane peroxidation was identical in kinetics and in amount to the production of TBA-reactive substance from the peroxidized isolated membrane lipids. These results demonstrate that significant non-MDA. TBA-reactive species are generated during the peroxidation of cardiac membranes, especially before the maximal rates of bona fide MDA production. As a direct consequence, artifactual levels and kinetics of membrane lipid peroxidation do result. Presented in part at the 78th American Oil Chemists' Society Annual Meeting, New Orleans, LA (May, 1987). Recipient of an Outstanding Paper Presentation Award (1).     

Choline and Ethanolamine Decompose Lipid Hydroperoxides into Hydroxyl Lipids

Analysis of lipid hydroperoxides and hydroxyl lipids in food and biological systems has historically been difficult due to the instability and diversity of these compounds as well as the complexity of the associated purification and derivatization processes. A rapid, high throughput and robust method based on mass spectrometry using multiple reaction monitoring mode of LC–ESI–MS/MS has been developed for the simultaneous quantitative analysis of fatty acid hydroperoxides and hydroxyl fatty acids without purification or derivatization. The method has been used to demonstrate that functional groups of phospholipids, choline and ethanolamine, are capable of decomposing lipid hydroperoxides into their corresponding hydroxyl lipids. In model systems where choline and ethanolamine were added, the amounts of lipid hydroperoxide isomers decreased while the amounts of hydroxyl lipids increased. In similar model systems containing normal food antioxidants such as tocopherols and butylated hydroxytoluene, and in the control without antioxidants, the amounts of lipid hydroperoxide and hydroxyl lipids did not change significantly under current experimental conditions. Data also demonstrated that phospholipids have a much lower hydroperoxide decomposition ability than choline and ethanolamine, and that the antioxidant activity of phospholipids is probably due to the ability of choline, ethanolamine and other amines from phospholipid degradation to convert primary oxidation products to the corresponding hydroxyl lipids.     

Mass determination of lipids with automated thermogravimetric analytical procedure

An automated thermogravimetric system for the microdetermination of lipids is described. A study of the analytical variability experienced with this method revealed that samples can be assayed with considerable accuracy (±3%) down to 100–200 μg, while samples less than this range exhibit variations of ±7–10%. In the absence of other more sensitive methods, even this experimental error is acceptable. In addition, multiple applications of very dilute samples to achieve a lipid mass with an acceptable coefficient of variation is possible. No significant variations were observed with samples assayed on the same day and on different days, indicating a high degree of control of nonoperator errors. A careful application of the method to different lipid classes revealed no significant weighing losses under the experimental conditions of this study, except for cholesterol; however, application of the method to mixtures of lipids containing significant amounts of cholesterol did not reveal abnormal errors.     

Dry column method for the quantitative extraction and simultaneous class separation of lipids from muscle tissue

A method for lipid isolation is presented that is alternative to the traditional chloroform/methanol extraction methods. This new method allows lipid isolation by solvent elution of a dry column composed of a tissue sample, anhydrous sodium sulfate, and Celite 545 diatomaceous earth groud together. To isolate total lipids, the dry column is eluted with a mixture of dichloromethane/methanol (90∶10, v/v). Alternatively, the lipids may be isolated and simultaneously separated into neutral and polar fractions by a sequential elution procedure; neutral lipids free of polar lipids are eluted first with dichloromethane, followed by elution of polar lipids with the dichloromethane/methanol (90∶10) mixture. The two dry column methods-isocratic or sequential elution-were compared with the traditional chloroform/methanol methods by gravimetric, thin layer chromatographic and phosphorus analyses. Presented in part at the AOCS 70th Annual Meeting, San Francisco, May 1979. Agricultural Research, Science and Education Administration, U.S. Department of Agriculture.