Hydrolysis of neutral lipid substrates by rat hepatic lipase

Rat hepatic lipase, an enzyme whose involvement in the catabolism of lipoproteins remains poorly defined, has both neutral lipid and phospholipid hydrolyzing activity. We determined the substrate specificity of hepatic lipase for 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol in the Triton X-100 mixed micellar state, and compared these results to those obtained previously in our laboratory for the phospholipid substrates phosphatidic acid (PA), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). V_max values were determined by diluting the substrate concentration in the surface of the micelle by Triton X-100. The V_max values obtained were 144 μmol/min/mg for 1-oleoyl-sn-glycerol, 163 μmol/min/mg for 1,2-dioleoyl-sn-glycerol, and 145 μmol/min/mg for 1,3-dioleoyl-sn-glycerol. These values were higher than those obtained earlier for phospholipids which were 67 μmol/min/mg for PA, 50 μmol/min/mg for PE and 4 μmol/min/mg for PC. In addition, the mole fraction of lipid substrate at half maximal velocity (K) in the surface dilution plot was lower for the neutral lipid substrates as compared to those obtained for the phospholipid substrates. When the hydrolysis of 1,3-dioleoyl-sn-glycerol mixed micelles was studied as a function of time, cleavage at thesn-1 andsn-3 positions occurred at the same rate, suggesting that hepatic lipase is not stereo-selective with respect to 1,3-diacyl-sn-glycerol substrates. To determine if the presence of one lipid could affect the hydrolysis of the other, all possible dual combinations of 1-oleoyl-sn-glycerol, 1,2-dioleoyl-sn-glycerol, and 1,3-dioleoyl-sn-glycerol, in the same micelle were made and the hydrolysis rate of each substrate was determined. Interaction occurred only for the 1,2-dioleoyl-sn-glycerol/1,3-dioleoyl-sn-glycerol mixture where the hydrolysis of 1,2-dioleoyl-sn-glycerol was slightly inhibited and that of 1,3-dioleoyl-sn-glycerol slightly activated compared to the predicted theoretical rate. These findings demonstrate that when presented in similar physical states, the neutral lipid substrates tested were hydrolyzed at a higher rate by hepatic lipase than the phospholipid substrates.     

Stereospecificity of monoacylglycerol and diacylglycerol acyltransferases from rat intestine as determined by chiral phase high-performance liquid chromatography

Using chiral phase high-performance liquid chromatography of diacylglycerols, we have redetermined the ratios of 1,2-/2,3-diacyl-sn-glycerols resulting from acylation of 2-monoacylglycerols by membrane bound and solubilized triacylglycerol systhetase of rat intestinal mucosa. With 2-oleoyl[-³H]glycerol as the acyl acceptor and oleoyl-CoA as the acyl donor, 97–98% of the diacylglycerol product was 1,2(2,3)-dioleoyl-sn-glycerol, 90% of which was thesn-1,2-and 10% thesn-2,3-enantiomer. The remaining diacylglycerol (less than 3%) was thesn-1,3-isomer. The overall yield of acylation products was 70%, of which 60% were diacylglycerols and 40% triacylglycerols. With 2-oleylglycerol ether as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, 90% of the diradylglycerol was 1-oleoyl-2-oleyl-sn-glycerol and 10% was the 2-oleyl-3-oleoyl-sn-glycerol. The diradylglycerols made up 96% and the triradylglycerols 4% of the radioactive product. With 1-palmitoyl-sn-glycerol as the acyl acceptor and [1-¹⁴C]oleoyl-CoA as the acyl donor, the predominant reaction product was 1-palmitoyl-3-oleoyl-sn-glycerol. The 3-palmitoyl-sn-glycerol was not a suitable acyl acceptor. Both 1,2- and 2,3-diacyl-sn-glycerols were substrates for diacylglycerol acyltransferase as neither isomer was favored when 1,2-dioleoyl-rac-[2-³H]glycerol was used as the acyl acceptor. There was a marked decrease in the acylation of the 1(3)-oleoyl-2-oleyl-sn-glycerol to the 1,3-dioleoyl-2-oleyl-sn-glycerol. It is concluded that neither monoacylglycerol nor diacylglycerol acyltransferase exhibit absolute stereospecificity for acylglycerols as fatty acid acceptors.     

Glyceroamidothiophosphates of cholecalciferol (Vitamin D3)

The hexamethyltriamide of phosphorous acid activated by the addition of iodine at the optimum molar ratio 1.05∶0.05 was used as a phosphorylating reagent to synthesize cholecalciferyl-3-0-(N,N-dimethylamido)thiophosphate derivatives of 1,3-benzylidene-rac-glycerol, 1,2-isopropylidene-rac-glycerol, 1,3-dioleoyl-rac-glycerol, and 1,2-dioleoyl-rac-glycerol in a one-pot procedure in high overall yields (81–86%). The compounds represent new model types of phospholipid structures which, in addition to glycerol and a steroid fragment, contain a biologically important linkage other than an oxygen-phosphorus bond.     

Parameters affecting diacylglycerol formation during the production of specific-structured lipids by lipase-catalyzed interesterification

Diacylglycerols (DAG) are important intermediates in lipase-catalyzed interesterification, but a high DAG concentration in the reaction mixture results in a high DAG content in the final product. We have previously shown that a high DAG concentration in the reaction mixture increases the degree of acyl migration, thus adding to the formation of by-products. In the present study we examined the influence of water content, reaction temperature, enzyme load, substrate molar ratio (oil/capric acid), and reaction time on the formation of DAG in batch reactors. We used response surface methodology (RSM) to minimize the numbers of experiments. The DAG content of the product was dependent on all parameters examined except reaction time. DAG formation increased with increasing water content, enzyme load, reaction temperature, and substrate ratio. The content of sn-1,3-DAG was higher than that of sn-1,2-DAG under all conditions tested, and the ratio between the contents of the former compounds and the latter increased with increasing temperature and reaction time. The water content, enzyme load, and substrate ratio had no significant effect on this ratio. The DAG content was positively correlated with both the incorporation of acyl donors and the degree of acyl migration.     

Absorption of synthetic,stereochemically defined acylglycerols in the rat

The stereochemistry of fat digestion and absorption was investigated in rats with thoracic duct fistulas, after feeding synthetic triacylglycerol or alkyldiacylglycerol. After feeding 1,2-dilauroyl-3-oleoyl-sn-glycerol, dilauroyloleoylglycerol and lauroyldioleoylglycerol were the most abundant chyle triacylglycerols. Positional analysis of the fatty acid distribution and the absence of optical activity indicated that the following structures dominated:rac-1,2-dilauroyl-3-oleoylglycerol andrac-1,3-dioleoyl-2-lauroylglycerol. Therefore, the triacylglycerol resynthesized from 2-lauroylglycerol (precursor to 60% of chyle triacylglycerol) and other precursors was essentially racemic. Chyle phospholipids contained largely endogenous fatty acids, and the proportion of lauric acid was very low. A racemic mixture of 1,2-di[³H] oleoyl-3-tetradecyl-sn-glycerol and 1-tetradecyl-2,3-di[¹⁴C] oleoyl-sn-glycerol was absorbed to a lower degree than triacylglycerol. The appearance of oleic acid with different labels in chyle and intestinal lipids did not differ, indicating the absence of stereospecificity in fat digestion. Possible explanations for the low absorption are discussed.     

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO₄ and MgCl₂ stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg²⁺ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.     

Binary Phase Behavior of Diacid 1,3-Diacylglycerols

Fifteen phase diagrams were prepared using data from differential scanning calorimetry analysis of binary blends of representative diacid 1,3-DAG. The behavior observed in binary phase diagrams is related to the difference in T _m (ΔT _m) between system components—eutectic for ΔT _m < 26 °C and monotectic for ΔT _m > 30 °C. Binary blends were prepared using six diacid 1,3-DAG: 1,3-hexanoyl-lauroyl-rac-glycerol, 1,3-hexanoyl-palmitoyl-rac-glycerol, 1,3-hexanoyl-oleoyl-rac-glycerol, 1,3-lauroyl-palmitoyl-rac-glycerol, 1,3-lauroyl-oleoyl-rac-glycerol and 1,3-palmitoyl-oleoyl-rac-glycerol. Diacid 1,3-DAG were synthesized using representative FA: hexanoic (6:0)—short-chain FA; lauric (12:0)—medium-chain FA; palmitic (16:0)—long-chain FA; and oleic (18:1)—mono-unsaturated FA. In addition to the aforementioned phase diagrams, the physical chemistry of 1,3-hexanoyl-lauroyl-rac-glycerol, 1,3-hexanoyl-oleoyl-rac-glycerol and 1,3-lauroyl-oleoyl-rac-glycerol is reported.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of fat,FA, and cholesterol on sphingomyelin hydrolysis induced by rat intestinal alkaline sphingomyelinase

Dietary sphingomyelin (SM) may have regulatory effects on cell proliferation and tumorigenesis in the colon. Alkaline sphingomyelinase (SMase) is the major enzyme responsible for hydrolysis of SM in the gut. Previously we purified the enzyme and showed that the presence of glycerophospholipids inhibited SM hydrolysis induced by alkaline SMase in vitro. In the present work, we studied the effects of TG, DG, FA, ceramide, and cholesterol on SM hydrolysis catalyzed by purified alkaline SMase. The results showed that both TG (triolein and tristearin) and DG (1,2-dioleoyl-sn-glycerol and 1,2-distearoyl-rac-glycerol) inhibited the activity of alkaline SMase. 1-Mono-oleoyl-rac-glycerol, 1-monostearoyl-rac-glycerol, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid stimulated the activity of alkaline SMase at 0.4–0.8 mM concentrations but inhibited the enzyme at higher concentrations. There was no difference between the effects induced by saturated and unsaturated FA. A short-chain FA such as lauric acid had a stronger stimulatory effect at low concentrations and weaker inhibitory effect at high concentrations than long-chain FA. Choosing linoleic acid as an example, we found that FA had similar effects on both alkaline SMase and neutral SMase. Cholesterol and ceramide when mixed with FA to increase its solubility in bile salt micelles inhibited SMase activity. In conclusion, glycerides, FA, ceramide, and cholesterol influence SM hydrolysis catalyzed by intestinal alkaline SMase. The presence of lipids in the diet may thus influence the course of SM digestion in the gut and thereby the exposure of colon to SM metabolites.     

Crystallization and Polymorphism of 1,3-Acyl-Palmitoyl-<Emphasis Type="Italic">rac</Emphasis>-Glycerols

Crystallization and melting behavior, small-angle X-ray scattering, X-ray powder diffraction and infra-red absorbance were measured for nine 1,3-acyl-palmitoyl-rac-glycerols (1,3-acetoyl-, -butyroyl-, -hexanoyl-, -octanoyl-, -decanoyl-, -lauroyl, -myristoyl- and -oleoyl-palmitoyl-rac-glycerol and 1,3-dipalmitoyl-glycerol). All but one of the prepared 1,3-diacylglycerols (1,3-DAG) were β-stable with 1,3-acetoyl-palmitoyl-rac-glycerol being the exception (β′-stable). Small-angle X-ray scattering indicates that molecules in β-tending diacid 1,3-DAG adopt a herringbone-type configuration similar to monoacid 1,3-DAG. In this configuration acyl chains of the same length associate and regular chain-end matching between terminal methyl groups delineate lamellae. In contrast, molecules in crystalline 1,3-acetoyl-palmitoyl-rac-glycerol are oriented similar to those of 1(3)-monoacylglycerol. Interestingly, DSC curves indicate five of the nine diacid compounds have meta-stable forms—suggesting these forms are quite common for diacid 1,3-DAG. Meta-stable forms are observed in the melting curve when the difference in length between acyl chains is large (1,3-acetoyl-, -butyroyl- and -hexanoyl-palmitoyl-rac-glycerol), and in the crystallization curve when the difference is moderate (1,3-decanoyl- and -lauroyl-palmitoyl-rac-glycerol).     

Stereospecific analysis of TAG from sunflower seed oil

Stereospecific analysis of TAG from a sunflower seed oil of Tunisian origin was performed. The TAG were first fractionated according to chain length and degree of unsaturation by RP-HPLC. The four major diacid- and triacid-TAG fractions were palmitoyldilinoleoyl-glycerol, dioleoyllinoleoylglycerol, oleoyldilinoleoylglycerol, and palmitoyloleoyl-linoleoyl-glycerol, amounting to 7.2, 16.6, 29.5, and 12 mol%, respectively. The TAG of the four fractions were individually submitted to stereospecific analysis, using a Grignard-based partial deacylation, separation of sn-1,2(2,3)-DAG from sn-1,3-DAG by boric acid-impregnated silica gel TLC plates, conversion of the sn-1,2(2,3)-DAG to their 3,5-dinitrophenylurethane (DNPU) derivatives, fractionation of DNPU derivatives by RP-HPLC, resolution of the DNPU-DAG by HPLC on a chiral column, transmethylation of each sn-DNPU-DAG fraction, and analysis of the resulting FAME by GC. The data obtained were used to determine the triacyl-sn-glycerol composition of the main TAG of the oil. Fifteen triacyl-sn-glycerols were identified and quantified, representing, along with the monoacid-TAG, trilinoleoylglycerol and trioleoylglycerol, more than 90% of the total oil TAG. The two major triacyl-sn-glycerols were trilinoleoyl-glycerol and 1-linoleoyl-2-linoleoyl-3-oleoyl-glycerol (18.6 and 18.5% of the total, respectively). Results clearly identified linoleic acid as the major FA at the sn-2 position, whereas oleic and palmitic acids were the major FA at the sn-3 position. The sn-1 position was occupied to nearly the same extent by linoleic and oleic acids, and to a greater extent by palmitic acid, which was practically absent at the sn-2 position.     

Analysis of diastereomeric DAG naphthylethylurethanes by normal-phase HPLC with on-line electrospray MS

Normal-phase HPLC resolution of sn-1,2(2,3)- and x-1,3-DAG generated by partial Grignard degradation from natural TAG was carried out with both (R)-(−) and (S)-(+)-1-(1-naphthyl)ethylurethane derivatives. The diastereomeric sn-1,2- and sn-2,3-DAG derivatives were resolved using two Supelcosil LC-Si (5 μm, 25 cm × 4.6 mm i.d.) columns in series and an isocratic elution with 0.37% isopropanol in hexane at a flow rate of 0.7 mL/min. The DAG were detected by UV absorption at 280 nm and were identified by electrospray ionization MS in the positive ion mode following postcolumn addition of chloroform/methanol/30% ammonium hydroxide (75∶24.5∶0.5, by vol) at 0.6 mL/min. Application of the method to a stereospecific analysis of the molecular species of TAG of rat VLDL showed that the TAG composition of VLDL circulating under basal conditions differs markedly from that of VLDL secreted by the liver during inhibition of serum lipases. The inhibition of serum lipases resulted in a significant proportional decrease in 16∶0 and PUFA and an increase in 18∶0 and oligoenoic FA in the sn-1-position, whereas the FA compositions in the sn-2- and sn-3-positions were much less affected.     

Triacylglycerol composition and structure in genetically modified sunflower and soybean oils

Changes in composition were examined in oils extracted from genetically modified sunflower and soybean seeds. Improvements were made to the analytical methods to accomplish these analyses successfully. Triacylglycerols (TAG) were separated on two 300 mm × 3.9 mm 4μ Novapak C18 high-performance liquid chromatography (HPLC) columns and detected with a Varex MKIII evaporative light-scattering detector. Peaks were identified by coelution with known standards or by determining fatty acid composition of eluted TAG by capillary gas chromatography (GC). Stereospecific analysis (fatty acid position) was accomplished by partially hydrolyzing TAG with ethyl magnesium bromide and immediately derivatizing the resulting diacylglycerols (DAG) with (S)-(+)-1-(1-naphthyl)ethyl isocyanate. The derivatized sn-1,2-DAG were completely resolved from the sn-2,3-DAG on two 25 mm × 4.6 mm 3 μ silica HPLC columns. The columns were chilled to −20°C to obtain baseline resolution of collected peaks. The distribution of fatty acids on each position of the glycerol backbone was derived from the fatty acid compositions of the two DAG groups and the unhydrolyzed oil. Results for the sn-2 position were verified by hydrolyzing oils with porcine pancreatic lipase, isolating the resulting sn-2 monoacylglycerols by TLC, and determining the fatty acid compositions by GC. Results demonstrated that alterations in the total fatty acid composition of these seed oils are determined by the concentration of TAG species that contain at least one of the modified acyl groups. As expected, no differences were found in TAG with fatty acid quantities unaffected by the specific mutation. In lieu of direct metabolic or enzymatic assay evidence, the authors’ positional data are nevertheless consistent with TAG biosynthesis in these lines being driven by the mass action of available acyl groups and not by altered specificity of the acyltransferases, the compounds responsible for incorporating fatty acids into TAG.     

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[¹⁴C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[¹⁴C]rici-noleoyl-sn-glycero-3-phosphocholine, ¹⁴C-labeled 2-linoleoyl-phosphatidylcholine (PC), and ¹⁴C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A₂ strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[¹⁴C]ricinoleoyl-PC and [¹⁴C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC. Out results demonstrate the proposed pathway: 2-oleoyl-PC→2-ricinoleoyl-PC→ricinoleate →triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.     

Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

Separation of the enantiomers of 1-alkyl-2-acyl-rac-glycerol and of 1-alkyl-3-acyl-rac-glycerol by high performance liquid chromatography on a chiral column

High performance liquid chromatographic separations of two enantiomeric pairs of 1-alkyl-2-acyl-rac-glycerol (1-alkyl-2-acyl- and 3-alkyl-2-acyl-sn-glycerols) and 1-alkyl-3-acyl-rac-glycerol (1-alkyl-3-acyl- and 3-alkyl-1-acyl-sn-glycerols) as 3,5-dinitrophenylurethanes (3,5-DNPUs) were carried out on a chiral stationary phase, N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(S)-valine chemically bonded to γ-aminopropyl silanized silica (Sumipax OA-4100). Good separation of the enantiomers of 1-hexadecyl-2-hexadecanoyl-rac-glycerol was easily achieved within 10 min using hexane/ethylene dichloride/ethanol (80∶20∶1, v/v/v) as a mobile phase. Separation of the enantiomers of 1-hexadecyl-3-hexadecanoyl-rac-glycerol was more difficult and required about 80 min to achieve satisfactory peak resolution (0.8) using hexane/ethylene dichloride/ethanol (250∶20∶1, v/v/v) as a mobile phase. Presented at the American Oil Chemists' Society 79th Annual Meeting, Phoenix, AZ, May, 1988.     

Critical analysis of phospholipid hydrolyzing activities in ripening tomato fruits. Study by spectrofluorimetry and high-performance liquid chromatography

Using the spectrofluorimetric method described by Wittenaueret al. [Wittenauer, L.A., Shirai, K., Jackson, R.L., and Johnson, J.D. (1984)Biochem. Biophys. Res. Commun. 118, 894–901] for phospholipase A₂ (PLA₂) measurement, we have detected a phospholipase activity in Ailsa Craig and in mutantrin tomatoes at their normal harvest time (mature green stage). This activity in Ailsa Craig tomatoes increased at the beginning of fruit ripening (green-orange stage) and then decreased slowly. The decrease in activity, however, was greater when ripening occurred after tomato picking at normal harvest time than when ripening occurred on tomato plants. This phospholipase activity was always higher inrin tomatoes than in normal ones. Thin-layer chromatography of compounds obtained after incubation of tomato extract demonstrated a decrease in the substrate 1-acyl-2-{6[(7-nitro-2,1,3, benzoxadiazol-4-yl)amino]-caproyl}-sn-glycero-3-phosphocholine (C₆-NBD-PC), and an increase in one product (NBD-aminohexanoic acid), but failed to detect the second product (1-acyl-sn-glycero-3-phosphocholine). We, therefore, developed a new one-step method for separation and quantification of a mixture of phospholipids and other lipids, using straight-phase-high-performance liquid chromatography with light-scattering detection. This method detected another fatty acid-releasing activity in enzyme extract from green-orange tomatoes. This lipolytic enzyme (or family of enzymes) slowly produced free fatty acids when 1-oleoyl-sn-glycero-3-phosphocholine was added as substrate. The production of fatty acids was stoichiometric and more rapid when 1-oleoyl-sn-glycero-3-phosphate and 1-oleoyl-sn-glycerol were used as substrates. On the other hand, the same tomato extract was unable to hydrolyze 1,2-dioleoyl-sn-glycero-3-phosphate and 1,2-dioleoyl-sn-glycerol. Crude tomato extract exhibited lipid acyl hydrolase activity according to the definition of Galliard [Galliard, T. (1979), inAdvances in the Biochemistry and Physiology of Plant Lipids (Appelqvist, L.A., and Liljenberg, C. eds.), pp. 121–132, Elsevier, Amsterdam]. But in order to demonstrate whether tomato extract contains PLA₂ activity and/or lysophospholipase activity, further work on purified tomato extract will be necessary.     

High-performance liquid chromatography of the triacylglycerols ofVernonia galamensis andCrepis alpina seed oils

The triacylglycerols ofVernonia galamensis andCrepis alpina seed oils were characterized because these oils have high concentrations of vernolic (cis-12,13-epoxy-cis-9-octadecenoic) and crepenynic (cis-9-octadecen-12-ynoic) acids, respectively. The triacylglycerols were separated from other components of crude oils by solid-phase extraction, followed by resolution and quantitation of the individual triacylglycerols by reversed-phase high-performance liquid chromatography with an acetonitrile/methylene chloride gradient and flame-ionization detection. Isolated triacylglycerols were characterized by proton and carbon nuclear magnetic resonance and by capillary gas chromatography of their fatty acid methyl esters. The locations of the fatty acids on the glycerol moieties in the oils were obtained by lipolysis. TheVernonia galamensis oil contained 50% trivernoloyl and 21% divernoloyllinoleoyl glycerols along with 20% triacylglycerols with one vernolic and two other fatty acids. TheCrepis alpina oil contained 36% tricrepenynoyl and 33% dicrepenynoyllinoleoyl glycerols, 17% triacylglycerols with two crepenynic and one other fatty acid and 7% triacylglycerols with one crepenynic acid and two other fatty acids. Vernolic acid was found at both the 1(3)- and 2-glycerol carbons but was more abundant at the 1,(3)-position in theVernonia galamensis oil. Crepenynic acid was found at both glycerol carbon positions but was more abundant at the 2-position in theCrepis alpina oil. Visiting scientist from Technical Research Institute, Snow Brand Milk Company, Ltd., Saitana, Japan.     

Evidence of Strict Stereospecificity in the Structure of sn‐1,2‐Diacyl‐3‐Acetyl‐Glycerols from Euonymus maximowiczianus Seeds Using Nuclear Magnetic Resonance Spectroscopy

Asymmetric, optically active sn‐1,2‐diacyl‐3‐acetyl‐glycerols (AcDAG) have been known to scientists for several decades. However, to date, the problem of their structure has not been definitely resolved, which has led to a vast diversity of terms used for their designation in the literature. Using two‐dimensional nuclear magnetic resonance, we have investigated AcDAG from the mature seeds of Euonymus maximowiczianus, from which we have been able to both identify a correlation of the methyl group in acetic acid residue with protons at the carbon atom at sn‐3 position in the glycerol residue of the AcDAG molecule and, for the first time, demonstrate that this correlation is observed exclusively with one carbon atom at the α‐position, but not with two as would have been expected in case of a racemic mixture. Moreover, results of our analysis of AcDAG isolated from the seeds of E. maximowiczianus directly confirm that diacylglycerol‐3‐acetyl‐transferase is responsible for their biosynthesis, which reveals a strict specificity not only to acetyl‐CoA as one of the substrates but also to the sn‐3‐position of the glycerol residue in sn‐1,2‐diacylglycerol during their biosynthesis.     

Chromatographic resolution of chiral diacylglycerol derivatives: Potential in the stereospecific analysis of triacyl-sn-glycerols

Diacylglycerols have been separated as their (S)-(+)-or (R)-(−)-1-(1-naphthyl)ethyl urethanes by high performance liquid chromatography (HPLC) on a column of silica gel with 0.5% 2-propanol in hexane as the mobile phase. The elution order of components derivatized with the (S)-form of the reagent was 1,3-, followed by 1,2-, and finally 2,3-diacyl-sn-glycerols. The elution order of 1,2- and 2,3-diastereomers was reversed when the (R)-form of 1-(1-naphthyl)ethyl isocyanate was used for derivatization. Single-acid 1,2- and 2,3-diastereomers were separated to the baseline with a resolution factor from 5.2–5.7, and the resolution factor between 1,3- and 1,2- or 2,3-diacyl-sn-glycerol derivatives was more than 23. Molecular species of single-acid diacylglycerol derivatives were separated in the sequence 18∶1<18∶0<18∶2<16.0. In order to assess this methodology as part of a procedure for the stereospecific analysis of triacyl-sn-glycerols, we prepared diacyl-rac-glycerols from maize oil, evening primrose oil and egg yolk triacylglycerols by partial hydrolysis with ethyl magnesium bromide. The 1,3-, 1,2- and 2,3-diacyl-sn-glycerols as (S)-(+)-1-(1-naphthyl)ethyl urethanes were isolated and their fatty acid compositions were determined. Although this only permitted an indirect determination of the compositions of positionssn-1,-2 and-3, it was sufficient to indicate the potential of the methodology because results comparable to those published earlier were achieved.     

Synthesis and mass spectrometry of 1-acyl and 3-acyl-sn-glycerol carbonates

sn-Glycerol-1,2-carbonate was prepared fromD-serine,sn-glycerol-2,3-carbonate fromL-serine, via 1-0- or 3-0-benzyl-sn-glycerol, respectively.sn-Glycerol-2,3-carbonate was also prepared fromD-mannitol orD-serine following thesn-glycerol-3-β,β,β-trichloroethylcarbonate route.sn-Glycerol-1,2-carbonate andsn-glycerol-2,3-carbonate were acylated with saturated and unsaturated fatty acid chlorides to form 3-acyl-sn-glycerol-1,2-carbonates and 1-acyl-sn-glycerol-2,3-carbonates, respectively. The mass spectra of the enantiomeric monoacyl-sn-glycerol carbonates showed molecular ions and acyl cations (RCO⁺) of high intensity. The heterocyclic dioxolan-2-one ring was remarkably stable during electron impact.