Novel N-Substituted Amino Acid Hydrazone-Isatin Derivatives: Synthesis,Antioxidant Activity,and Anticancer Activity in 2D and 3D Models In Vitro

A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-N′-(2-oxoindolin-3-ylidene)propanehydrazide and (N′,N‴)-1,1′-(methylenebis(4,1-phenylene))bis(5-oxo-N′-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid.     

Biological Activities of Toninia candida and Usnea barbata Together with Their Norstictic Acid and Usnic Acid Constituents

The aim of this study was to investigate the chemical composition of acetone extracts of the lichens Toninia candida and Usnea barbata and in vitro antioxidant, antimicrobial, and anticancer activities of these extracts together with some of their major metabolites. The chemical composition of T. candida and U. barbata extracts was determined using HPLC-UV analysis. The major phenolic compounds in these extracts were norstictic acid (T. candida) and usnic acid (U. barbata). Antioxidant activity was evaluated by free radical scavenging, superoxide anion radical scavenging, reducing power and determination of total phenolic compounds. Results of the study proved that norstictic acid had the largest antioxidant activity. The total content of phenols in the extracts was determined as the pyrocatechol equivalent. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration using the broth microdilution method. The most active was usnic acid with minimum inhibitory concentration values ranging from 0.0008 to 0.5 mg/mL. Anticancer activity was tested against FemX (human melanoma) and LS174 (human colon carcinoma) cell lines using the microculture tetrazolium test. Usnic acid was found to have the strongest anticancer activity towards both cell lines with IC₅₀ values of 12.72 and 15.66 μg/mL.     

In vitro hydrolysis of natural and synthetic γ-linolenic acid-containing triacylglycerols by pancreatic lipase

The present study compared thein vitro hydrolysis of two 18:3n-6-rich oils—evening primrose oil (EPO) and borage oil (BO)—and different synthetic 18:3n-6-containing triacylglycerols (TG). Incubation of EPO and BO with pancreatic lipase lipolyzed 18:3n-6 from the TG species. The rate of lipolysis of TG species containing two or three molecules of 18:3n-6, which comprised 36% of total 18:3n-6 in BO and only 7% in EPO, was significantly slower than those containing only one molecule of 18:3n-6. This was found especially in those with two molecules of linoleic acid, which constituted 20% of total 18:3n-6 in BO, whereas over 80% were present in EPO. In a separate study, various synthetic 18:3n-6-containing TG were also subjected toin vitro hydrolysis by pancreatic lipase. Results showed that release of 18:3n-6 from thesn-1/sn-3 positions was significantly slower when two other stereospecific positions in the same TG molecule were occupied by either palmitic acid (16:0) or monounsaturated (18:1 and 20:1) fatty acids than when occupied by 18:2n-6. The rate of hydrolysis ofsn-2-γ-linolenyl-sn-1(3)-diacylglycerol to formsn-2-mono-γ-linolenyl glycerol was also significantly slower when both thesn-1 andsn-3 positions in TG molecules were occupied by either saturated fatty acids (16:0 and 18:0) or long-chain monounsaturated fatty acids than when occupied by 18:2n-6. These findings suggest that the stereospecific position of 18:3n-6 in TG molecules and the constituent of its neighboring fatty acids modulated availability of 18:3n-6 from 18:3n-6-containing TG or 18:3n-6-rich oils.     

Generation and remodeling of highly polyunsaturated molecular species of rat hepatocyte phospholipids

Freshly isolated rat hepatocytes were incubated for 20 min with [U-¹⁴C]glycerol in the presence or absence of unlabeled linoleic (18∶2n-6), arachidonic (20∶4n-6), or docosahexaenoic (22∶6n-3) acid, added as albumin complex in 10% ethanol. Most of the radioactivity (≈95%) recovered in hepatocyte lipids was present in phosphatidylcholine (PC), phosphatidylethanolamine (PF), and triacylglycerol (TAG). The presence of exogenous fatty acids resulted in (i) higher incorporation of [U-¹⁴C]glycerol, (ii) higher percentage of label in TAG, and (iii) enhanced formation of PC and PE molecular species bearing the exogenous fatty acid at both the sn-1 and sn-2 positions of glycerol. In each case, these molecular species contained 60 to 70% of the label in that lipid class. Further incubation of the cells for 40 and 80 min in the absence of labeled substrate and exogenous fatty acids resulted in a redistribution of label among PC and PE molecular species due to deacylation-reacylation at the sn-1 position of glycerol.     

Macromolecular Conjugates of 4- and 8-Aminoquinoline Compounds

Some members of 4-aminoquinolines and 8-aminoquinolines have been found to provide adjuvant effects when used in combination with anti-cancer drugs. The clinical co-administration of active anti-cancer drugs with other drugs acting as potentiating agents has shown considerable merits when compared to a single-drug administration. Anti-cancer drugs are often toxic when delivered straight, but the bio-reversible drug conjugation of anticancer drugs to water-soluble macromolecular carriers has proved to enhance the therapeutic effectiveness of anticancer drugs. Following facilitated pharmacokinetics pathways, the conjugates, acting as pro-drugs, will release the active drug species in the transformed target cells and their designs are geared towards reducing pharmacological barriers of toxicity, drug resistance and poor bioavailability encountered with currently used anti-cancer drugs. This paper describes the synthesis of water-soluble macromolecular carriers containing 4- and 8-aminoquinolines that are bio-reversibly anchored with cytotoxic drugs. The conjugates and co-conjugates are isolated as water soluble solids and characterized by NMR-spectroscopy.     

Laccase-initiated reaction between phenolic acids and chitosan

Phenolic acids are known to possess antioxidant activities whilst chitosan is a biocompatible polymer with antibacterial activity against a broad spectrum of bacteria. Merging both types of molecules could therefore provide several potential applications. In this work, antioxidant properties of phenolic acid–functionalized-chitosan were investigated after being prepared from structurally-different phenolic acids (caffeic and gallic acids) and chitosan using the laccase from Trametes versicolor as the reaction initiator. A laccase-mediated oxidation kinetic of phenolic acids was monitored by UV–vis spectroscopy and cyclic voltammetry, as well as spin-trapping electron paramagnetic resonance spectroscopy (ST-EPR). The pH was shown to have a significant effect on the degree of phenolic acid self-polymerization, indicating the involvement of phenolate anions within the formations of coupled polyphenol products, and their functionalities, i.e. antioxidant activity. All the phenolic acid-functionalized-chitosans displayed greatly improved ABTS radical cation scavenging capacities, compared with the untreated chitosan.     

<Emphasis Type="Italic">sn</Emphasis>-Position determination of phospholipid-linked fatty acids derived from erythrocytes by liquid chromatography electrospray ionization ion-trap mass spectrometry

The sn-position of FA in membrane lipids has an influence on the physiological function of cells, is predictive for diseases, and therefore is useful for diagnostics. The current study compares the compositions of acyl chain substituents in the sn-1 and sn-2 positions of the glycerol backbones of phospholipids derived from human erythrocytes by using RP-HPLC coupled with on-line electrospray ionization ion trap MS. Preferential loss of the acyl group in the sn-1 position was used to determine the degree of regiospecific preference exhibited by the phospholipid molecules. The identities of the molecular species and the positions of the acyl substituents were identified using product-ion spectra of major precursor ions selected from the mass spectra averaged across peaks in the total ion chromatogram. Saturated FA were found to be located mainly in the sn-1 position of the glycerol backbones of erythrocyte phospholipids, whereas PUFA were found primarily in the sn-2 position. All measured phospholipids revealed palmitic acid (16∶0) at the sn-1 position. Linoleic acid (18∶2n−6) and arachidonic acid (20∶4n−6) were found to be attached exclusively to the sn-2 position of the backbone, whereas eicosadienoic (20∶2n−6) and eicosatrienoic acid (20∶3n−9) occurred in both positions of the backbone of PC. Oleic (18∶1n−9), linoleic (18∶2n−6), and octadecatrienoic (18∶3) acids of PE and PS were linked to both positions. Lignoceric acid (24∶1n−9) was found to be strictly localized at the sn-2 position, whereas nervonic (24∶1n−9) acid of PS was associated with both positions of the backbone. A detailed analysis of the blood cell membrane lipids by MS might be helpful to characterize postprandial kinetics of pharmacological or dietary lipid applications, as well as environmental influences on cell membranes.     

Identification of triacylglycerols in the enzymatic transesterification of rapeseed and butter oil

A blend of rapeseed and butter oil was transesterified using immobilized Thermomyces lanuginosus lipase (Lipozyme® TL IM) as catalyst. The reaction was followed by reversed-phase HPLC and the triacylglycerol peaks were tentatively identified from their elution times by calculating equivalent carbon numbers. Further identification was made using HPLC-electrospray tandem mass spectrometry. A few of the triacylglycerols detected were typical combinations of fatty acids originating from rapeseed oil, such as α-linolenic acid, and short-chain fatty acids from butter oil. Due to the regioselectivity of the lipase, the transesterification reaction involved mainly fatty acids in the sn-1 and sn-3 positions. However, significant changes in the fatty acid composition in the sn-2 position were detected after 6 h.     

Identification of phenolic compounds and antioxidant activity of guava dehydrated by different drying methods

Abstract

The influence of different drying techniques on guava was investigated, including phenolic components and antioxidant activities. Through drying processes, total phenolic content (TPC) increased and formation of small molecular phenolic acids (multi-methoxy benzoic acid and sinapic acid) was promoted. UPLC-ESI-QTOF-MS determination showed flavanol compounds, hydrolyzable tannins, ellagic acid conjugates and cinnamic acid derivatives were four predominant phenolics of guava. Drying treatments caused degradation of catechin and its derivatives. Contrarily, drying treatments contributed to higher contents of procyanidin trimers. Moreover, thermal drying treatments led to degradation of macromolecular tannins and formation of smaller molecular tannins and ellagic acid conjugates, while simultaneously reduced the stabilities of most intrinsic ellagic acid conjugates. Furthermore, drying processes increased the yield of cinnamic acid dihexose, probably generating from lignin or phenolics–carbohydrate complex. Freeze drying and hot air drying showed better performance on retention of TPC and enhancement of antioxidant activity (AA).     

Positional distribution of 24∶6(n−3) in triacyl-sn-glycerols from flathead flounder liver and flesh

This paper presents the positional distribution of very long-chain fatty acids, 24∶6(n−3), in triacyl-sn-glycerols (TG) of flathead flounder (Hippoglossoides dubius). Each of the liver and flesh TGs was subjected to the stereospecific analysis. The liver TGs contained 24∶6(n−3) at concentrations of 1.5, 1.2 and 1.7 mole % in thesn-1,sn-2 andsn-3 positions, respectively, and the flesh TGs had 9.0, 7.8 and 7.1 mole % in thesn-1,sn-2 andsn-3 positions, respectively. This fatty acid was distributed almost evenly among the three positions of the TGs. No preference for thesn-2 position was observed in contrast to the general tendency for the distribution of longer-chain polyunsaturated fatty acids, such as 22∶6(n−3), 22∶5(n−3) and 20∶5(n−3). There was essentially no difference in the positional distributions of the liver and flesh TGs. The results obtained in this study give new fundamental information to the investigation of very long-chain fatty acids.     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.     

Synthesis and Characterization of Novel n-9 Fatty Acid Conjugates Possessing Antineoplastic Properties

The present study enumerates the synthesis, spectroscopic characterization, and evaluation of anticancer potential of esters of two n-9 fatty acids viz., oleic acid (OLA) and ricinoleic acid (RCA) with 2,4- or 2,6-diisopropylphenol. The synthesis strategy involved esterification of the hydroxyl group of diisopropylphenol (propofol) to the terminal carboxyl group of n-9 fatty acid. The synthesized propofol-n-9 conjugates having greater lipophilic character were tested initially for cytotoxicity in-vitro. The conjugates showed specific growth inhibition of cancer cell lines whereas no effect was observed in normal cells. In general, pronounced growth inhibition was found against the human skin malignant melanoma cell line (SK-MEL-1). The anticancer potential was also determined by testing the effect of these conjugates on cell migration, cell adhesion and induction of apoptosis in SK-MEL-1 cancer cells. Propofol-OLA conjugates significantly induced apoptosis in contrast to propofol-RCA conjugates which showed only weak signals for cytochrome c. Conclusively, the synthesized novel ester conjugates showed considerable moderation of anti-tumor activity. This preliminary study places in-house synthesized conjugates into the new class of anticancer agents that possess selectivity toward cancer cells over normal cells.     

Positional analysis of triglycerides and phospholipids rich in long-chain polyunsaturated fatty acids

Four sources of long-chain polyunsaturated fatty acids (LCP) differing in their chemical structure (triglycerides or phospholipids) and in their origin (tuna triglycerides, fungal triglycerides, egg phospholipids, and pig brain phospholipids) were analyzed to determine the distribution of the component fatty acids within the molecule. Lipase and phospholipase A₂ hydrolysis was performed to obtain 2-monoacylglycerols and lysophospholipids, respectively, which allowed us to determine the distribution of fatty acids between the sn-2 and sn-1,3 positions of triglycerides or between the sn-1 and sn-2 position of phospholipids. Fatty acids in the LCP sources analyzed were not randomly distributed. In tuna triglycerides, half of the total amount of 22∶6n−3 was located at the sn-2 position (49.52%). In fungal triglycerides, 16∶0 and 18∶0 were esterified to the sn-1,3 (92.22% and 91.91%, respectively) 18∶1 and 18∶2 to the sn-2 position (59.77% and 62.62%, respectively), and 45% of 20∶3n−6 and only 21.64% of 20∶4n−6 were found at the sn-2 position. In the lipid sources containing phospholipids, LCP were mainly esterified to the phosphatidylethanolamine fraction. In egg phospholipids, most of 20∶4n−6 (5.50%, sn-2 vs. 0.91%, sn-1) and 22∶6n−3 (2.89 vs. 0.28%) were located at the sn-2 position. In pig brain phospholipids, 22∶6n−3 was also esterified to the sn-2 (13.20 vs. 0.27%), whereas 20∶4n−6 was distributed between the two positions (12.35 vs. 5.86%). These results show a different fatty acid composition and distribution of dietary LCP sources, which may affect the absorption, distribution, and tissue uptake of LCP, and should be taken into account when supplementing infant formulas.     

Phenolic Acids and Antioxidant Capacity of Distillers Dried Grains with Solubles (DDGS) as Compared with Corn

Three sets of ground corn and the corresponding distillers dried grains with solubles (DDGS) were collected from three commercial plants and analyzed for individual phenolic acids and antioxidant capacity. This study was undertaken to investigate the influence of processing on phenolic acids content and antioxidant capacity of corn and the corresponding processed DDGS samples. The five phenolic acids identified in corn and DDGS were vanillic, caffeic, p-coumaric, ferulic, and sinapic acids. Ferulic and p-coumaric acids accounted for about 80% of the total identified and quantified phenolic acids. The phenolic acids profile of DDGS was comparable to that of corn. The content of total phenolic acids per gram basis, in DDGS was 3.40 fold higher and antioxidant capacity was 2.58 fold more than that of corn. These observations suggest that there was little degradation in individual phenolic acids content during dry grind processing. Furthermore, significant variation in measured individual and total phenolic acids, and antioxidant capacity among processing plants existed for both corn and DDGS. Results from this study will be valuable to bioethanol manufacturers and the feed industry.     

Monitoring the oxidation of docosahexaenoic acid in lipids

The oxidation of free DHA, DHA mixed with PC, and DHA incorporated into PC, PE, or TG was evaluated to determine which lipid provided DHA with the best protection against oxidation. DHA was either situated at the sn-1 position, sn-2 position, or both positions of the phospholipid, whereas the TG contained DHA at all positions. All lipids were incubated as bulk lipids, in chloroform, or as an emulsion in contact with air at 25–30°C for 28 d. Since DHA, which is highly sensitive to oxidation, has a great impact on our health and is desired as a food additive, the stability of this FA is of great importance. This study was mainly focused on the primary oxidation products, which were monitored as eight monohydroperoxy-DHA isomer groups, the total amount of polyhydroperoxides, and the PV. However, a measure of secondary oxidation products, the carbonyl value, was also monitored. We found that DHA was most protected against hydroperoxide formation when it was incorporated at one position of either PC or PE. In these lipids, hydroperoxide formation at carbon atoms 4, 7, 8, and 11 was completely prevented. DHA mixed with PC was also protected, although to a lesser extent, and all hydroperoxide isomers were detected. In contrast, PC and TG containing DHA at all positions should be avoided, since they were highly oxidized.     

Acylglycerol structure of genetic varieties of peanut oils of varying atherogenic potential

Detailed investigation was made of the triacylglycerol structure of three varieties of peanut oils of varying atherogenic activity. By means of chromatographic and stereospecific analyses, it was shown that all the oils had markedly nonrandom enantiomeric structures with the long chain saturated fatty acids (C₂₀−C₂₄) confined exclusively to thesn-3-position, whereas the palmitic and oleic acids were distributed about equally between thesn-1-andsn-3-positions, with the linoleic acid being found preferentially in thesn-2-position. On the basis of detailed studies of the molecular species of the separatesn-1,2-,sn-2,3- andsn-1,3-diacylglycerol moieties, it was concluded that the fatty acids in the three positions of the glycerol molecule are combined with each other solely on the basis of their relative molar concentrations. As a result, it was possible to calculate the composition of the molecular species of the peanut oil triacylglycerols (including the content of the enantiomers and the reverse isomers) by means of the 1-random 2-random 3-random distribution. In general, the three peanut oils possessed triacylglycerol structures which where closely similar to that derived earlier for a commercial peanut oil of North American origin. Since their oil has exhibited a high degree of atherogenic potential, it was anticipated that the present oils would likewise be atherogenic, which has been confirmed by biological testing. However, there are certain differences in the triacylglycerol structures among these oils, which can be correlated with the variations in their atherogenic activity. The major differences reside in the linoleic/oleic acid ratios in the triacylglycerols, especially in thesn-2-position, and in the proportions in which these acids are combined with the long chain fatty acids. On the basis of the characteristic structures identified in the earlier analyzed atherogenic peanut oil, the peanut oil of South American origin would be judged to possess the greatest atherogenic potential and this has been borne out by biological testing.     

Effects of different medium-chain fatty acids on intestinal absorption of structured triacylglycerols

To study the effect of the chain length of medium-chain fatty acids on the intestinal absorption of long-chain fatty acids, we examined the lymphatic transport of fat following administration of five purified structured triacylglycerols (STAG) containing different medium-chain fatty acids in the sn-1, 3 positions and long-chain fatty acids in the sn-2 position in a rat model. Significant amounts of medium-chain fatty acids were found in lymph samples after intragastric administration of 1,3-dioctanoyl-2-linoleyl-sn-glycerol (8∶0/18∶2/8∶0), 1,3-didecanoyl-2-linoleyl-sn-glycerol, and 1,3-didodecanoyl-2-linoleyl-sn-glycerol. The accumulated lymphatic transport of medium-chain fatty acids increased with increasing carbon chain length. The recoveries of caprylic acid (8∶0), capric acid (10∶0), and lauric acid (12∶0) were 7.3±0.9, 26.3±2.4, and 81.7±6.9%, respectively. No significant differences were observed for the maximal intestinal absorption of linoleic acid (18∶2n−6) when the chain length of medium-chain fatty acids at the primary positions was varied, and the absorption of 18∶2 and oleic acid (18∶1) from 8∶0/18∶2/8∶0 and 1,3-dioctanoyl-2-oleyl-sn-glycerol was similar. We conclude that the chain length of the medium-chain fatty acids in the primary positions of STAG does not affect the maximal intestinal absorption of long-chain fatty acids in the sn-2 position in the applied rat model, whereas the distribution of fatty acids between the lymphatics and the portal vein reflects the chain length of the fatty acids. Presented in part at the 3rd ISSFAL Conference, Lyon, France, June 1–5, 1998.     

Antioxidative properties of phenolic acids and interaction with endogenous minor components during frying

The ability of selected phenolic acids to improve the frying performance of canola oil was evaluated in a frying test. The frying performance of the oil was assessed by analysis of total polar components (TPC), level of 4‐hydroxynonenal (HNE), and the rate of formation of volatile carbonyl compounds (VCC). All the tested phenolic acids; ferulic acid (FA), caffeic acid (CA), dihydrocaffeic acid (HCA), gallic acid (GA), and vanillic acid (VA) significantly increased the frying performance of canola oil triacylglycerols (CTG). At the end of the frying test, the amount of TPC in CTG was 22.9 ± 1.0% compared to a maximum of 18.8 ± 0.8% in CTG fortified with the phenolic acids. Similarly, the level of HNE was reduced by up to 45% when it was supplemented with phenolic acids. The results showed that ethyl ferulate (EF) was a better antioxidant than FA under frying conditions; HCA offered a slightly better protection than CA; and the cinnamic acid derivative, FA was better than VA, its benzoic acid analogue. A significant synergy was observed between phenolic acids and the sterol fraction isolated from canola oil. The observed synergy was attributed to the possible formation of steryl phenolates during the frying test. Practical applications: The poor thermal stability of polyunsaturated oils limits their application for prolonged frying. PUFA offer important health benefits and can improve nutritional value of fried foods. Contrary to the commonly applied synthetic antioxidants, the phenolic acids tested in this study often are part of endogenous oil components present in oilseeds and also in some oils, and are known for their positive health benefits. Thus, the simple phenolic acids, especially the cinnamic acid derivatives may be applied as potent antioxidants to protect oils during thermal processes used for food production.     

Stereospecific analysis of triacyl-sn-glycerolsvia resolution of diastereomeric diacylglycerol derivatives by high-performance liquid chromatography on silica

The compositions of positionssn-1, 2 and 3 of triacylglycerols can be determined by partial hydrolysis with ethyl magnesium bromide, derivatization of the total products with (S)-(+)-1-(1-naphthyl)ethyl isocyanate and isolation of the diacyl-sn-glycerol urethane derivatives by chromatography on solid-phase extraction columns containing an octadecylsilyl phase. The diastereomericsn-1,2-and 2,3-diacylglycerol derivatives are separated by high-performance liquid chromatography on silica for determination of their fatty acids by gas chromatography. Each step in the process has been evaluated rigorously. The compositions of all three positions can be calculated with good accuracy from the analyses of these compounds and that of the total triacylglycerols. Although the 1,3-sn-diacylglycerol derivatives can also be isolated easily, they do not give reliable results for the composition of positionsn-2 because acyl migration occurs during their generation. The stereospecific analysis procedure has been applied to some plant and animal triacyl-sn-glycerols of commercial and scientific interest, containing predominantly C₁₆ and C₁₈ fatty acids,i.e. safflower, sunflower, olive and palm oils, tallow, egg and rat adipose tissue. The method is not at present suited to the analysis of more complex triacylglycerols, such as milk fat or fish oils, and problems associated with these are discussed.