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.     

Extraction of Algal Lipids and Their Analysis by HPLC and Mass Spectrometry

Algae are a promising source of biofuel but claims about their lipid content can be ambiguous because extraction methods vary and lipid quantitation often does not distinguish between particular lipid classes. Here we compared methods for the extraction of algal lipids and showed that 2-ethoxyethanol (2-EE) provides superior lipid recovery (>150–200 %) compared to other common extraction solvents such as chloroform:methanol or hexane. Extractions of wet and dry algal biomass showed that 2-EE was more effective at extracting lipids from wet rather than dried algal pellets. To analyze lipid content we used normal-phase HPLC with parallel quantitation by an evaporative light scattering detector and a mass spectrometer. Analysis of crude lipid extracts showed that all major lipid classes could be identified and quantified and revealed a surprisingly large amount of saturated hydrocarbons (HC). This HC fraction was isolated from extracts of bioreactor-grown algae and further analyzed by HPLC/MS, NMR, and GC/MS. The results showed that the sample consisted of a mixture of saturated, straight- and branched-chain HC of different chain lengths. These algal HC could represent an alternative biofuel to triacylglycerols (TAG) that could feed directly into the current petroleum infrastructure.     

Analysis of lipid classes by solid-phase extraction and high-performance size-exclusion chromatography

An improved method to analyze lipid classes of edible oils and fats by solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) is presented. A mixture of lipid standards was fractionated by the solid-phase extraction procedure (NH₂ phase) into polar and nonpolar fractions; these were then submitted to analysis by HPSEC. The size-exclusion chromatographic columns were three styrene/divinylbenzene columns with pore sizes of 100 Å and 50 Å. Light-scattering was used for the detection system, and the parameters of the detector were optimized to minimize the difference between the responses of the compounds studied. With this procedure it was possible to separate the following lipid classes: triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids, sterols, sterol esters, tocopherols and carotenoids. Quantitative analysis was studied for a light-scattering detector with several lipid standards of different molecular weights and unsaturation levels.     

Sterols,Fatty Alcohols,and Triterpenic Alcohols in Commercial Table Olives

This work supplies information on the lipids, unsaponifiable matter, sterols, and fatty and triterpenic alcohols in table olives. The mean lipid contents, unsaponifiable values, concentration of sterols and total alcohols (aliphatic and triterpenic alcohols) were 16.15 g/100 g edible portion (e.p.), 4.53 g/100 g lipid, 28.68 mg/100 g e.p. and 13.28 mg/100 g e.p., respectively. The overall mean content of cholesterol was 0.5 mg/100 g e.p., with a minimum of 0.08 mg/100 g e.p. in Manzanilla olives stuffed with “piquillo” pepper, and a maximum of 4.9 mg/100 g e.p. in Manzanilla olives stuffed with marinated anchovy strips. Table olives contain higher concentrations of phytosterols than olive oil. The chemometric analysis showed that lipids, unsaponifiable matter, sterols, and fatty and triterpenic alcohol contents in table olives were slightly affected by processing and that some misclassification was possibly related to maturation. There were also noticeable differences between cultivars.     

Neutral lipids in non‐starch lipid and starch lipid extracts from Portuguese sourdough bread

This research effort was aimed at assessing the changes in extractable neutral lipids (NL) throughout the baking process of Broa, a Portuguese traditional sourdough bread. NL were accordingly isolated, purified and quantitated – starting from non‐starch lipid (NSL) and starch lipid (SL) extracts of maize and rye flours, as well as fermented dough and bread. NSL accounted for the major fraction of extracted lipids; furthermore, the NSL/SL ratio evolved throughout processing in agreement with the phenomena prevailing during dough preparation, fermentation, and baking. An analytical method based on resolution by normal‐phase HPLC coupled with detection by evaporative light scattering was accordingly developed for quantitation of the aforementioned NL classes. Distinct NL classes correlated well with the stage of bread making. The main NL in NSL were triacylglycerols (ca. 75% of the total), but relatively high concentrations of sterol esters and diacylglycerols were also found. Conversely, free fatty acids were the dominant component of SL, whereas monoacylglycerols and free sterols were comparable to those in NSL.     

The effect of lyophilization on the solvent extraction of lipid classes,fatty acids and sterols from the oysterCrassostrea gigas

The lipid class compositions of adult Pacific oysters [Crassostrea gigas (Thunberg)] were examined using latroscan thin-layer chromatography/flame-ionization detection (TLC/FID), and fatty acid compositions determined by capillary gas chromatography and gas chromatography/mass spectrometry (GC/MS). The fatty acid methyl esters were separated using argentation TLC and also analyzed as their 4,4-dimethyloxazoline derivatives using GC/MS. Major esterified fatty acids inC. gigas were 16∶0, 20∶5n−3, and 22∶6n−3. C₂₀ and C₂₂ nonmethylene interrupted (NMI) fatty acids comprised 4.5 to 5.9% of the total fatty acids. The NMI trienoic fatty acid 22∶3(7,13,16) was also identified. Very little difference was found in the proportions of the various lipid classes, fatty acids or sterols between samples of adult oysters of two different sizes. However, significant differences in some of the lipid components were evident according to the method of sample preparation used prior to lipid extraction with solvents. Lyophilization (freeze drying) of samples led to a significant reduction in the amounts of triacylglycerols (TG) extracted by solvents in two separate experiments (7.0 and 52.5% extracted). Extracts from lyophilized samples had less 16∶0, C₁₈ unsaturated fatty acids, and 24-ethylcholest-5-en-3β-ol, while C₂₀ and C₂₂ unsaturated fatty acids comprised a higher proportion of the total fatty acids. There was no significant change in the amounts of polar lipids, total sterols, free fatty acids or hydrocarbons observed in extracts from lyophilized samples relative to extracts from nonlyophilized samples. Addition of water to the freezedried samples prior to lipid extraction greatly improved lipid yields and resulted in most of the TG being extracted.     

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.     

Simple and Efficient Profiling of Phospholipids in Phospholipase D-modified Soy Lecithin by HPLC with Charged Aerosol Detection

Dietary phosphatidylinositol (PI) can be synthesized via phospholipase D (PLD)-catalyzed transphosphatidylation of phosphatidylcholine (PC), abundant in soy lecithin, with myo-inositol. However, a generated mixture of phospholipid (PL) classes poses a challenge for analysis. Our current work on Streptomyces PLD engineering requires a robust analytical method for profiling of PI and related PLs derived from the transphosphatidylation reactions. Therefore, we optimized an HPLC-based method with charged aerosol detector (CAD) for PL quantification. PLs were separated on a normal phase silica column by a gradient elution system using two solvents containing chloroform/methanol/1 M formic acid–triethylamine buffer in different ratios. Retention times of the PL standards and LC–MS under identical conditions were used to identity PL classes. PL standards gave linear response in 100- and 10-fold (lyso-PI) concentration range. The method provided a simple, sensitive, repeatable, and precise analysis of PI, PC, phosphatidylethanolamine, phosphatidic acid, and lyso forms of PC and PI. Compared to the similar existing method, introduction of CAD provided a three- to fivefold decrease at the lower end and a two- to fivefold increase at the upper end of the dynamic range. High precision, high sensitivity, and low limits of detection and quantification further underline the benefits of CAD in PL analysis.     

The use of iodine staining for the quantitative analysis of lipids separated by thin layer chromatography

The method described for quantitative estimation of lipids separated on thin layer chromatography plates exploits the observation that most lipids can be stained by iodine vapor and that, in “controlled” conditions, the intensity of this staining is proportional to the actual amount of lipid in the spot. The method consists of i) exposing the developed plate to iodine vapor; ii) spraying it with a suitable solvent to prevent halogen evaporation; iii) collecting the stained lipids by scraping the spots off the plate; and iv) determining by a rate-sensing method the absorbed iodine. The final determination is performed by measuring spectrophotometrically (at 410 nm) the rate of decolorization of a solution of Ce(IV) by As(III) in strong acidic conditions. The reaction rate, which is positively related to the concentration of iodine, is derived from the slope of the absorbance change plotted vs time. Providing that standards and samples are stained simultaneously, a quantitative estimation of lipid components of a mixture is possible in a reasonable time with excellent accuracy and reproducibility. In our hands, the method has been successfully applied to several common phospholipids, long chain fatty acids, cholesterol and deoxycholate, and triacylglycerols, in the range of 5–60 μg.     

Analysis of ubiquinone and tocopherol levels in normal and hyperlipidemic human plasma

The type and amount of lipophilic antioxidants in plasma of hyperlipidemic patients are of great importance since they play a central role in preventing deleterious oxidation of blood lipids and proteins. Isolation and quantitation of lipophilic antioxidants from hyperlipidemic plasma samples meet great obstacles because of increased levels of various intermediary lipid products. This study was designed to develop a rapid and efficient extraction and separation procedure for simultaneous analysis of ubiquinone-9 and-10 as well as α-, δ-, and γ-tocopherol isomers. The levels of ubiquinone-10, α- and γ-tocopherol were analyzed in human plasma samples using high-performance liquid chromatography. Lipid extraction was performed by petroleum ether/methanol/water. After phase separation, ubiquinone was reduced to ubiquinol by sodium borohydride and the lipids were separated on a C18 column. A binary gradient with solvents containing lithium perchlorate was used, and an electrochemical detector was employed for quantitation. This procedure was also efficient for the analysis of antioxidant lipids in samples containing a large number of accumulated and interfering lipid intermediates. Thus, the procedure described here is useful for efficient and rapid quantitation of ubiquinones and tocopherols in human plasma samples, especially those originating from hyperlipidemic patients.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.     

Characterization of Fenugreek (<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis>) Seed Lipids

The composition and content of lipids, fatty acids, triacylglycerols, tocopherols and sterols in nine fenugreek genotypes were analyzed. Lipid content in fenugreek seeds ranged from 5.8 to 15.2%. Major fatty acids were: linoleic acid (45.1–47.5%), α-linolenic (18.3–22.8%), oleic (12.4–17.0%), palmitic (9.8–11.2%) and stearic (3.8–4.2%) acids. The ratios of n-6 to n-3 fatty acids were between 2.1 and 2.7. Similar fatty acid distribution was observed in all analyzed samples with some deviations. α-Tocopherol was the predominant component found in the fenugreek lipid antioxidants, and it constituted over 84% of the total amounts of tocopherols. It amounts ranged from 620 to 910 mg/kg lipids. β-Sitosterol was the major sterol in all samples, varying from 14,203 to 18,833 mg/kg of lipids. Campesterol and cycloartenol were other major sterols, and these compounds including β-sitosterol constituted 56–72% of all sterols. Fenugreek seed lipids consisted predominantly triunsaturated (56.9–66.5%) and diunsaturated (32.2–41.6%) triacylglycerides. Among these components trilinolein (LLL; 12.9–20.5%) dominated followed by PLL (14.0–20.4%), LnLnO (7.8–17.7%), PLO (5.7–11.6%), OLL (6.9–10.6%), LLLn (3.2–9.6%), and LnLnL (3.5–7.6%). Results of the study show that fenugreek seed lipids may be a source of a nutraceutical ingredient for food applications.     

A study on cashew nut oil composition

Eight samples of cashew nut oil were assayed, and the component triacylglycerols, fatty acids and several unsaponifiable compounds were analyzed by gas chromatography (GC) and high-performance liquid chromatography (HPLC). Total lipid amount, unsaponifiable percentage, fatty acids, sterols, triterpene alcohols and tocopherols are reported here. The combination of GC and HPLC enhanced the resolution of compound classes.     

Sterol and fatty acid composition of neutral lipids ofParatenuisentis ambiguus and its host eel

The sterol composition of free sterol and steryl ester fractions of the fish parasiteParatenuisentis 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 bothP. ambiguus andA. anguilla. However, the sterols ofP. 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. The analysis of ether glycerolipids revealed that 1-O-hexadecyl (16∶0) and 1-O-hexadecenyl (16∶1) moieties were present in similar proportions in the ether lipids of bothP. ambiguus and eel intestine, whereas 1-O-octadecyl (18∶0) moieties are more prominent in the parasite and 1-O-octadecenyl (18∶1) moieties in the eel. The results of these studies show thatP. ambiguus has specific mechanisms for the regulation of the sterol and fatty acid composition of its neutral lipids. Dedicated to Professor Helmut K. Mangold on the occasion of his 70th birthday.     

Determination of the adulteration of butter

The adulteration of butter is a serious problem due to economic advantages taken by replacing expensive milk fat with cheaper oil without informing the customers. The authentication of milk fat methods include analysis of bulk components, especially triacylglycerols, fatty acids, sterols and tocopherols. Fatty acid and sterol composition was analysed by using GC‐MS. TAG and tocopherol profiles were examined by HPLC with diode array (DAD) and fluorescence detectors (FLDs). In addition, identification of selected TAG of butter fat was conducted by LC‐atmospheric pressure chemical ionisation (APCI)/MS technique. The lipid composition of 16 different butters available on Polish market were investigated. The cholesterol content in butter fat ranged from 176.8 to 264.8 mg/100 g of fat and in two samples of milk fat β‐sitosterol was found. The total saturated fatty acid (SFA) content in milk fat was 67.1–73.5%, monounsaturated fatty acid 24.5–30.5% and polyunsaturated fatty acid was 1.2–2.0%. Abnormalities in fatty acid profiles, e.g. high concentration of linoleic fatty acid, were found in two butters. These abnormalities were also determined in TAG profiles. The examination of tocopherols in butter fat confirmed that two products were adulterated by the addition of plant oils because they contained δ‐tocopherol which is typical for plant origin foodstuffs. The methods described are useful for investigating milk fat adulterations, and the most efficient are analysis of sterols and tocopherols composition. Practical applications: The described methods are useful for investigating adulteration of milk fat. Traditional strategies rely on examination of fatty acids methyl esters and TAG; these methods have some disadvantages. Due to the variability of fatty acid composition of milk fat and because TAG analysis is complex and time consuming, FA analysis is not an efficient approach for butter authentication. The most efficient method for butter authentication is qualitative and quantitative analysis of sterols and tocopherols. This analysis will determine if components of plant origin were used for butter production.     

Advantages of total lipid hydrogenation prior to lipid class determination on chromarods-SIII

The changes in detector responses of lipid classes for standards and natural lipid samples after hydrogenation were investigated with the TLC/FID Iatroscan system using Chromarods-SIII (Newman-Howells Associates, Ltd., Midwales, U.K.). Samples included lipids of human plasma and erythrocytes, egg lipids, a fish oil concentrate and triacylglycerols of sea scallop, as well as standards (mono-, di- and triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, lysophosphatidylcholines, free fatty acid, and sterol). The duration of hydrogenation was increased from 45 min to 90 min for some samples to bring hydrogenation to completion. Since the detector response of a lipid class is known to depend on its relative position on the Chromarod, different solvent systems which would vary the migration rate were investigated. The increases in response after hydrogenation, given as relative percent increase (RI), were significant for all compounds examined (p<0.05), and ranged from 7 to 45%. Among the triacylglycerols, the fish oil and the sea scallop triacylglycerols with wide ranges of fatty acid composition had the highest RI while the other lipid classes did not differ from each other. Differences in the solvent systems did not affect the RI except for fish oil triacylglycerols. The precision of peak area measurements was usually better after hydrogenation. The combination of greater sample stability, greater response, improved resolution, and simpler choice of standards make hydrogenation a significant advance in TLC/FID quantification.     

Lipid and fatty acid composition and energy partitioning during embryo development in the shrimp Macrobrachium borellii

Energy partitioning, composition of lipids and fatty acids, and their utilization by embryos were determined in the lecithotrophic shrimp Macrobrachium borellii during seven development stages. The biochemical composition at stage I is represented by lipids, proteins, and carbohydrates, with 29.3, 28.7, and 0.2% dry weight, respectively. The former two were identified as the major energy-providing components, contributing 131 and 60 cal/100 mg egg, dry weight, respectively. The overall conversion efficiency (CE) was 45.0% (calculated as percentage of vitelline energy transformed into embryonic tissues). Lipids were the most important energy reserve (CE 39.3%), followed by proteins (CE 57.1%), both being simultaneously utilized during development while carbohydrates were synthesized de novo (CE 587.5%). Variation in the lipid class composition of embryos and vitellus showed an accumulation of triacylglycerols (TAG) and phospholipids (PL) up to stage IV, a more active accumulation and selective utilization phase (stages V and VI), and a consumption and de novo synthesis period until hatching. Structural lipids (PL and cholesterol) and pigment astaxanthin were selectively conserved in embryos, but TAG, hydrocarbons, and esterified sterols were preferentially depleted. Monounsaturated fatty acids (FA) were the major group in TAG, whereas polyunsaturated FA (PUFA) were the major group in PL after organogenesis. Certain PUFA such as 22∶6n−3 and 20∶5n−3 were selectively accumulated in PL.     

Lipid components of borage (Borago officinalis L.) seeds and their changes during germination

The changes in composition of total and neutral lipids (NL) as well as glycolipids (GL) and phospholipids (PL) of borage (Borago officinalis L.) seeds, germinated in the dark at 25^°C for 10 d, were studied. Total lipids constituted 34.0% of the dry matter of borage seeds. During germination, the content of total lipids was decreased by 95%. NL accounted for 95.7% of total lipids prior to germination and were composed of triacylglycerols (TAG; 99.1%), diacylglycerols (DAG; 0.06%), monoacylglycerols (MAG;0.02%), free fatty acids (FFA;0.91%), and sterols (0.02%). The content of TAG was significantly (P≤0.05) decreased, while that of other components, such as MAG and FFA, significantly (P≤0.05) increased during germination. However, the content of DAG did not change. GL and PL accounted for 2.0 and 2.3% of total lipids, respectively, and their contents significantly (P≤0.05) increased as germination proceeded. The thin layer chromatography-flame-ionization detection studies showed that phosphatidylcholine (PC; 69.7%) was the major PL present. The total content of phosphatidylserine (PS) and phosphatidylethanolamine (PE), which were coeluted, was 18.2%; phosphatidic acid (PA) was present at 11.2% of the total PL fraction. Lysophosphatidylcholine was detected at 0.9%. The proportion of PC, PS, and PE significantly (P≤0.05) decreased during germination, but that of PA increased (P≤0.05) markedly. The fatty acid composition of lipid fractions changed as germination proceeded. The predominant fatty acids of total lipids, NL, and GL were linoleic and linolenic acids, while those of PL were linoleic and palmitic acids. The present study demonstrated that the overall changes of lipids seen in borage seeds during germination agree well with results for other oilseeds. Changes in lipid compositions during germination result from the formation of tissues and metabolic interconversion of lipid classes. Rapid changes in lipid composition during seed germination may enhance the nutritional value of the sprouts.     

Lipid composition of perilla seed

The composition of lipids and oil characteristics from perilla [Perilla frutescens (L.) Britt.] seed cultivars are reported. Total lipid contents of the five perilla seed cultivars ranged from 38.6 to 47.8% on a dry weight basis. The lipids consisted of 91.2–93.9% neutral lipids, 3.9–5.8% glycolipids and 2.0–3.0% phospholipids. Neutral lipids consisted mostly of triacylglycerols (88.1–91.0%) and small amounts of sterol esters, hydrocarbons, free fatty acids, free sterols and partial glycerides. Among the glycolipids, esterified sterylglycoside (48.9–53.2%) and sterylglycoside (22.1–25.4%) were the most abundant, while monogalactosyldiacylglycerol and digalactosyldiacylglycerol were present as minor components. Of the phospholipids, phosphatidylethanolamine (50.4–57.1%) and phosphatidylcholines (17.6–20.6%) were the major components, and phosphatidic acid, lysophosphatidylcholine, phosphatidylserine and phosphatidylinositol were present in small quantities. The major fatty acids of the perilla oil were linolenic (61.1–64.0%), linoleic (14.3–17.0%) and oleic acids (13.2–14.9%). Some of the physicochemical characteristics and the tocopherol composition of perilla oil were determined.     

Reevaluation of the neutral lipids ofTilletia controversa andTilletia tritici

Differential scanning calorimetry of whole teliospores and lipid extracts ofTilletia controversa Kühn andT. tritica Tul. indicated that the lipid composition of teliospores was different than earlier reported. An exothermic peak at −40 to −45°C and an endotherm at −25 to −15°C indicated that the majority of lipids were triacylglycerols (TAG). Hot isopropanol was used to inactivate lipases during lipid extraction. Thin-layer chromatographic analysis of extracted lipids showed that free fatty acids (FFA) were not present in great quantities unless water was present during lipid extraction. As measured by gas chromatography. FFA accounted for 1–5% of the lipid content in teliospores ofTilletia spp. The TAG content of teliospores was 60–80% of total lipids.