Studies of hyperlipidemia in drug-induced diabetic rats by high-performance liquid chromatography

A hyperlipidemic condition is often associated with diabetes. The possibility that specific serum lipids (i.e., individual triglycerides or cholesterol esters) may be altered in the diabetic state was investigated. Serum lipids from both controls and streptozotocin- and alloxan-treated rats were separated into approximately twenty chromtographic fractions by reversed-phase high-performance liquid chromatography; a number of individual triglycerides and cholesterol esters were identified. The methodology described allowed subtle changes in individual lipid components to be detected. Only minor variations in the cholesterol and cholesterol ester fractions were observed between the control and diabetic samples. While not uniform throughout, elevations in the triglyceride fractions occurred in the diabetics. Also, differences in triglyceride content were found to exist between the groups of streptozotocin- and alloxan-treated animals.     

Separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography.

The separation of acidic and neutral lipids by aminopropyl-bonded silica gel column chromatography is presented. Total lipid extracts from Escherichia coli and human spermatozoa were loaded onto pre-packed aminopropyl-bonded silica gel columns and the lipids separated into four fractions. Non-polar lipids including cholesterol esters, triglycerides, diglycerides, monoglycerides and cholesterol, were eluted with 4 ml of isopropanol-chloroform (1:2, v/v) (fraction 1); free fatty acids were eluted with 4 ml of 2% acetic acid in diethyl ether (fraction 2); neutral polar lipids, including phosphophatidylethanolamine, phosphatidylcholine, sphingomyelin and neutral glycolipids, were eluted with 4 ml of methanol (fraction 3); and, finally, polar acidic lipids, including phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine, seminolipid lipid A and acidic glycosphingolipids, were eluted with 4 ml of chloroform-methanol-0.8 M sodium acetate (60:30:4.5, v/V/V) (fraction 4). The recoveries for the different lipids ranged between 89 and 98% and the intra-assay variation, expressed as the standard deviation, was less than 5%.     

Enzymic determination of cerebrospinal fluid lipid fractions

Colorimetric peroxidase-coupled procedures for the determination of several cerebrospinal fluid (CSF) lipid classes are described. These methods were modified to increase the effectiveness of each cerebrospinal fluid lipid assay by using the sample as the primary diluent for a highly concentrated reagent in an inverse concentration technique. Direct enzymic assays for the determination of CSF cholesterol (free and total), choline phospholipids, and triglycerides were adapted from existing assays to require less than 0.5 ml of sample per assay. This made determinations of the several lipid analytes possible even when samples were from pediatric specimens. In a study model, 51 pediatric CSF samples were analyzed for these lipid constituents. Mean values and standard deviations were determined. Within and between-run studies were performed by sampling from a pool of cerebrospinal fluid specimens. Within-run coefficients of variation for the several proposed procedures were less than 3% while the between-run findings for all of the procedures were less than 5%.     

Metabolites of the pathogenic fungusVerticillium dahliae III. The lipid composition ofVerticillium dahliae

Summary The compositions of the neutral lipids of the mycelium and of the culture liquid from the pathogenic fungusVerticillium dahliae Kleb. have been investigated for the first time. The fatty-acid compositions of the main lipid fractions and the glyceride composition of the triglyceride fraction have been determined. The main acids of all the fractions are the 16:0 and 18:1 acids and in the triglyceride fraction, additionally, the 18:2 acid. Diisobutyl phthalate has been found in the combined neutral lipids of the fungal mycelium.V. I. Lenin Tashkent State University, Institute of the Chemistry of Plant Plant Substances, Academy of Sciences of the Uzbek SSR, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 431–435, July–August, 1976.     

A quantitative densitometric method for the rapid separation and quantitation of the major tissue and lipoprotein lipids by high-performance thin-layer chromatography. I. Sample preparation, chromatography, and densitometry

A rapid method for the separation and quantitation of the major lipids of tissues and lipoproteins by automated high-performance thin-layer chromatography is presented. Solvent systems for one-dimensional separation of neutral lipids, of cholesteryl esters, and of phospholipids are described. Separated lipids are measured following treatment with methanolic sulphuric acid containing manganese chloride and scanned in fluorescence or absorption mode. Absolute quantitation is obtained by the use of an internal standard and by references to standards for each lipid run on the same plates as samples. The method described here is particularly suitable for the rapid quantitation of small amounts of lipid (0.01-0.02 nmol per sample), for example in tissue culture studies; 100 micrograms of fibroblast or macrophage protein are sufficient for complete lipid analysis. The coefficients of variation due to the sample preparation, application to the plates and densitometry are in the range 7.2-9.1%. The method was compared with enzymatic determinations for cholesterol and gave correlation coefficients of 0.95 for total cholesterol and 0.91 for unesterified cholesterol. Phospholipid estimation was compared with large-plate thin-layer chromatography and phosphorus analysis and gave correlation coefficients of 0.90 for phosphatidylcholine and 0.89 for sphingomyelin.     

N.m.r. determination of wax esters in marine lipids

The proton n.m.r. spectra of lipids containing triglycerides and wax esters dissolved in CDCl₃ are characterized by seven sets of signals. The areas of the signals of terminal methyl group and of methylene protons of both wax ester and triglyceride were integrated. These were used to calculate the content of wax esters in lipids or oils. The rapid n.m.r. procedure is directly usable for natural lipids containing as low as 3 mg of wax esters in 50-mg samples with an error of about 7%. The method described compares favorably with t.l.c. determination.     

Die Zusammensetzung der Neutrallipide aus normalen Hirnen alter Menschen

The chemical composition of lipids from six human brains (60–73 years) is reported. The total lipids out of cortex, white matter, diencephalon and cerebellum, pons, and medulla oblongata have been isolated and the neutral lipids have been separated in cerebrosides, sphingomyelines, and lecithins. The highest amount of pure lipids is found in the white matter, the lowest in the cortex. The relation of neutral lipids to acid lipids as well as the amount of cholesterol are about equal for all regions. The white matter shows more cerebrosides and sphingomyelines than the cortex, the opposite being the case for lecithins. The differences are strongly significant. The fatty acids out of the different pure lipid fractions have been analysed as esters by gas chromatography. Stearic and lignoceric acid, and cerebronic and hydroxy nervonic acid respectively are main components of cerebrosides, with only little differences for the different brain regions. The fatty acids of sphingomyelines consist mainly of stearic and nervonic acid; in the white matter these two acids are present about in the same quantity, whereas stearic acid dominates in the cortex and the other sections. Lecithins contain above all palmitic and oleic acid. The amount of the latter in the white matter is higher than that of palmitic acid.     

气相色谱法测定费托合成水相产物中的低碳醇、醛、酮化合物

建立了铁基催化剂费托合成反应水相产物中低碳(C₁~C₈)醇、醛、酮的气相色谱测定方法.对色谱分离条件进行了优化,确立了以乙醇为基准物质并结合各组分校正因子的定量方法;考察了方法的精密度和准确度,并对费托合成反应水相产物样品进行了测定.结果表明,乙醇在不同的含量范围内呈现良好的线性关系,相关系数均大于0.99.费托合成水相产物中的加标回收率在93.4%~109.6%之间,准确性可以满足实际分析的需要.实际费托合成水相产物的分析结果表明,费托合成水相产物中主要的低碳醇、醛、酮的总质量分数约为3%~12%,乙醇含量最高(约为1.7%~7.3%),且正构醇、异构醇和醛酮类化合物所占的总比率依次降低.该方法简单、快速,对费托合成水相产物中重要组分的分析有重要意义.     

Convenient method for the gas chromatographic analysis of hexosamines in the presence of neutral monosaccharides and uronic acids

A convenient gas chromatographic method has been devised for the analysis of hexosamines in the presence of neutral and acidic sugars, which involves sequential derivatization reactions of nitrous acid deamination, mercaptalation, and trimethylsilylation. This method allows rapid, simultaneous determination of 0.1-1 micromole samples of hexosamines with coefficients of variation of less than 3%.     

A new gas chromatographic methodology for the estimation of the composition of binary gas mixtures

The relatively new technique of reversed-flow gas chromatography (RFGC) is used to determine the diffusion coefficients of pure gases into gas mixtures (D(mix)(exp)). The pure gases are CO and CO(2), and the mixtures consist of H(2) and He in various volume percentage compositions. A linear regression analysis of D(mix)(exp) of CO and CO(2) in various mixtures of H(2) and He against the percentage composition (X(H2) or X(He)) of the mixtures at different temperatures results in an empirical equation relating D(mix)(exp) to the corresponding theoretical values of the diffusion coefficients of CO and CO(2) in the pure gases H(2) and He, as they are calculated from the Fuller-Schettler-Giddings equation. The empirical equation shows that the diffusion coefficient of an analyte gas in a gas mixture is the partial sum of its diffusion coefficients in the component gases, therefore making possible the determination of the mole fractions of the components of the mixture. The found percentage volume compositions are very close to those determined independently by routine gas chromatography, indicating that the proposed RFGC methodology could be successfully applied to the accurate determination of the volume composition of binary gas mixtures.     

Determination of lipids in infant formula powder by direct extraction methylation of lipids and fatty acid methyl esters (FAME) analysis by gas chromatography.

Fatty acid methyl esters (FAMEs) of pure triglyceride standards, oils, and fat from dry matrixes were formed by transesterification using sodium methoxide in methanol-hexane. FAMEs were produced by direct addition of sodium methoxide-hexane to samples and heating to simultaneously extract and transesterify acyl lipids. FAMEs were quantitated by capillary gas chromatography (GC) over a fatty acid concentration range of 0 to 1.7 mg/mL (r > or = 0.9997). Total fat was calculated as the sum of individual fatty acids expressed as triglyceride equivalents, in accordance with nutrition labeling guidelines. Saturated, polyunsaturated, and monounsaturated fats were calculated as sums of individual free fatty acids. Absolute recoveries determined from individual fatty acids in test samples ranged from 69.7 to 106%. Recoveries (relative to the C13:0 internal standard) for individual fatty acids in test samples ranged from 95 to 106%. Reproducibility was constant at each fatty acid level in the reaction mixture (n = 5, coefficient of variation [CV] < 2%). Absolute recovery determined from the sum of total fatty acids in standard reference material (SRM) 1846 (powdered infant formula) was 96.4%. Analysis of SRM 1846 gave results that agreed closely with the certified fat and fatty acid values. Analysis of commercial infant formula gave results that were comparable to those obtained with AOAC Method 996.01. The direct extraction methylation procedure is rapid, and the transesterification of acyl lipids to form FAMEs is complete within 15 min. Classical saponification and refluxing are not required. This method provides FAMEs free of interferences and easily quantitated by GC or confirmed by GC/mass spectrometry (MS). Unambiguous MS identification of individual FAMEs derived from pure standards, SRM 1846, and powdered infant formula product was obtained.     

Development and application of an analytical method for the determination of storage lipids, fatty acids and fatty alcohols in Calanus finmarchicus

A method was developed for the determination of the major storage lipids, wax ester and triglycerides, in the copepod Calanus finmarchicus. A variation of the Folch method was used to extract the lipid. The method was scaled down to enable the extraction of either pooled (-1 mg) or individual (approximately 200 microg) copepods. The major lipid classes were identified using TLC and quantified using HPLC coupled with evaporative light scattering detection. Analysis of laboratory reference materials indicated that this method underestimated the minor triglyceride component, but gave a good estimate of the major wax ester component. The fatty acid and fatty alcohol composition of the C. finmarchicus were determined following trans-esterification of the lipid extract in methanol. Fatty acids and fatty alcohols were initially identified by comparison with authentic standard and by mass spectroscopy. Using GC with flame ionisation detection the normalised area percentage of the fatty alcohols and fatty acid methyl esters was determined simultaneously in one run for either pooled or individual copepod samples. These methods were applied to C. finmarchicus collected from the Irminger Sea, North Atlantic in 2001 and 2002.     

Analysis of Vegetable Oils by High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatographic procedure is proposed to monitor the fatty-acid and triglyceride composition of sunflower-seed oil. The procedure uses a column packed with Diasfer-110-C₁₈ (6 m), a 2 : 8 mixture of acetonitrile and acetone as an eluent, and refractometric detection. Analyses that use simple normalization and normalization with correction factors that take into account the difference between calculated refraction coefficients of different triglycerides are compared. It is shown that the proposed procedure can be used for the quantitative determination of total triglycerides in various vegetable oils as well.     

Quantification of fatty acids as methyl esters and phospholipids in cheese samples after separation of triacylglycerides and phospholipids

Determination of the individual fatty acid composition of neutral- and phospholipids as well as the phospholipid content of dairy food and other foodstuffs are important tasks in life sciences. For these purposes, a method was developed for the separation of lipids (standards of triolein and diacylphosphatidylcholines as well as three cheese samples) by solid-phase extraction using a self-packed column filled with partly deactivated silica. Non-halogenated solvents were used for the elution of the lipid classes. Cyclohexane/ethyl acetate (1:1, v/v) served for the elution of neutral lipids, while polar lipids were eluted with three solvents (ethyl acetate/methanol, methanol, and methanol/water) into one fraction. The separated lipid fractions were transesterified and the individual fatty acids were quantified by using gas chromatography coupled to electron ionization mass spectrometry (GC/EI-MS) in the selected ion monitoring (SIM) mode. The recovery rate for standard phosphatidylcholines was ∼90% and cross-contamination from neutral lipids was negligible. The method was applied to cheese samples. Quantitative amounts of individual fatty acids in the phospholipid fraction were <0.002-0.29% of total lipids from camembert, <0.002-0.12% of total lipids from mozzarella, and <0.002-0.18% of total lipids in a goat cream cheese. Differences in the fatty acid pattern of neutral and polar lipids were detected. The quantity of the fatty acids determined in the phospholipid fraction was divided by the factor 0.7 in order to convert the fatty acid content into the phospholipid content of the cheese samples. This factor is based on the contribution of 16:0 to dipalmitoylphosphatidylcholine (DPPC). The resulting DPPC equivalents (DPPC_eq) were found to be representative for the average contribution of fatty acids to all classes of phospholipids in dairy products. Using this approach, the phospholipid content of lipids from mozzarella, camembert, and goat cream cheese was 0.60%, 1.42% and 0.79%, respectively.     

Fat content for nutritional labeling by supercritical fluid extraction and an on-line lipase catalyzed reaction

A method using sequential supercritical fluid extraction (SFE) and enzymatic transesterification has been developed for the rapid determination of total nutritional fat content in meat samples. SFE conditions of 12.16 MPa and 50°C were utilized to extract lipid species from the sample matrix. The enzymatic transesterification of the lipids by methanol was catalyzed by an immobilized lipase isolated from Candida antarctica. Conversion of the triglycerides to fatty acid methyl esters was monitored by supercritical fluid chromatography, while the fatty acid content of the extract was determined by capillary gas chromatography (GC). Total fat, saturated fat and monounsaturated fat contents were calculated from the GC data and compared to values from traditional extraction and lipid determination methods. Both off-line SFE and automated SFE followed by on-line GC analysis using two different instruments were utilized in this study. The enzymatic-based SFE method gave comparable results to the organic solvent extraction-based method followed by conventional BF₃-catalyzed esterification.     

Lipid content and fatty acid composition in lemon wax

Lipids were extracted from lemon wax and fractionated into four classes on a silicic acid glass packed column by thin-layer chromatography (TLC). The free fatty acids, the fatty acid composition and the amount of each separated lipids were determined by capillary column gas chromatography (GC). Total lipids (TL) were 60 mg per 100 g raw weight and the ratio of nonpolar lipids (NPLs): glycolipids (GLs): phospholipids (PLs) was about 47:2:2. The main free fatty acids in lemon wax were hexadecanoic acid, cis-9-octadecenoic acid and cis,cis-9,12-octadecadienoic acid, while in the lipid fractions the main fatty acids were hexadecanoic acid in all the fractions, cis-cis-9,12-octadecadienoic and decanoic acids in triglyceride (TG) fraction, dodecanoic and cis-9-octadecenoic acids in diglyceride (DG) fraction and tetradecanoic, octadecanoic and cis-9-octadecenoic acids in GL and PL fractions. The ratio of unsaturated to saturated fatty acids showed a remarkable difference among these four lipid fractions. In PL and GL fractions this ratio was similar, 47.7% and 47.1% respectively, and in TG fraction it was 42.4% while in DG fraction this value was 23.5%.     

High-performance liquid chromatographic procedure for the determination of di-(2-ethylhexyl)phthalate in human blood specimens. Problems of variable-extraction yield and the use of standard addition for calibration

A high-performance liquid chromatographic procedure was developed for the determination of di-(2-ethylhexyl)phthalate (DEHP) concentrations in human whole blood samples. The solvent extraction of DEHP was found to be highly variable between samples obtained from different subjects (coefficient of variation of 30.4%). The recovery of DEHP following extraction with ethyl acetate was negatively correlated with serum lipid content, as expressed by the sum of serum cholesterol and triglyceride concentrations (r = -0.864). The technique of standard addition of DEHP allowed a single-point calibration of DEHP extractability in individual blood samples, and provided an accurate estimation of DEHP concentration (coefficient of variation of approximately 6% in replicate samples). The potential for intersample variability in the solvent extraction of other highly lipid-soluble compounds should be considered.     

Development of a Nuclear Magnetic Resonance Method and a Near Infrared Calibration Model for the Rapid Determination of Lipid Content in the Field Pea (Pisum sativum)

Pisum sativum is a leguminous crop suitable for cultivation worldwide. It is used as a forage or dried seed supplement in animal feed and, more recently, as a potential non-traditional oilseed. This study aimed to develop a low-cost, rapid, and non-destructive method for analyzing pea lipids with no chemical modifications that would prove superior to existing destructive solvent extraction methods. Different pea accession seed samples, prepared as either small portions (0.5 mm²) of endosperm or ground pea seed powder for comparison, were subjected to HR-MAS NMR analyses and whole seed samples underwent NIR analyses. The total lipid content ranged between 0.57–3.45% and 1.3–2.6% with NMR and NIR, respectively. Compared to traditional extraction with butanol, hexane-isopropanol, and petroleum ether, correlation coefficients were 0.77 (R² = 0.60), 0.56 (R² = 0.47), and 0.78 (R² = 0.62), respectively. Correlation coefficients for NMR compared to traditional extraction increased to 0.97 (R² = 0.99) with appropriate correction factors. PLS regression analyses confirmed the application of this technology for rapid lipid content determination, with trends fitting models often close to an R² of 0.95. A better robust NIR quantification model can be developed by increasing the number of samples with more diversity.     

Lipids ofViburnum opulus seeds

Lipid components of the seeds ofViburnum opulus (Caprifoliaceae family) were investigated. The neutral lipids consist of eight classes, the glycolipids consist of three classes, and the phospholipids contain seven classes. The fatty-acid contents of all of the acyl-containing lipids were determined. The 18∶2 fatty acid is the main component of all the lipid fractions. The content of saturated acids is greater in the glycolipids and phospholipids. The lipophilic components, higher fatty alcohols and sterols, were identified.