Quantitative measurement of prostaglandins E2 and E3 by selected ion monitoring

A method for the simultaneous quantative analysis of prostaglandin E₂ (PGE₂) and PGE₃ is described. The PG were analyzed by selected ion monitoring as the methyl ester-TMS ether derivatives of PGB₂ and PGB₃, respectively. The internal standard for the quantification of both species was [3,3,4,4-²H₄]PGE₂. A linear response over the range 0.6–50 ng (1.7–143 pmoles) was demonstrated for PGE₃. The chromatographic conditions used (2% SP-2330 column) afforded nearly baseline separation of the prostaglandins. New standard curves for PGE₃ must be developed each time the ion source parameters are changed. In a typical calibration run, the instrumental precision, expressed as coefficient of variation, ranged from 1.1 to 7.2% for PGE₂ (3 to 100 ng injected) and from 1.6 to 11.1% for PGE₃ (1.5–50 ng injected). The method was applied to the PG analysis of rat renomedullary tissues. The recovery of synthetic PGE₂ added to medullary homogenates was 100.5±1.7% (mean±SEM, n=9), and the recovery of PGE₃ was 91.3±1.4% (n=9).     

Simultaneous mass spectrometric measurement of prostaglandins E1 (PGE1) and PGE2 with a deuterated internal standard

The use of (3,3,4,4-²H₄)PGE₂ as internal standard for the quantitative measurement of PGE₁ by gas chromatography-mass spectrometry is reported. A method for the simultaneous analysis of PGE₁ and PGE₂ is also described. The prostaglandins were analyzed by selected ion monitoring as the methyl ester-trimethylsilyl ether (ME-TMS) derivatives of PGB₁ and PGB₂, respectively. In all cases, a linear response over the range 1 to 70 ng (2.8 to 198 pmoles) was demonstrated. The use of this procedure is exemplified by its application to the PG analysis of sheep seminal vesicles. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Gangliosides of bovine optic nerve

Gangliosides from bovine optic nerve were analyzed. The optic nerve contained 129, 98, 97, 80, 31 and 12 μg of GM₁, GD_1a, GD_1b, GT_1b, GD₃ and GQ₁ gangliosides, respectively, per g of tissue wet wt. These 6 gangliosides altogether contributed 97% of the total sialic acid. GM₃ and GM₂ gangliosides contributed the remaining 3% of total sialic acid. Stearoyl (18∶0) was the predominant acyl group (61–76%) in all gangliosides. There was a marked variation in acyl group composition between GT₁ and most of the other major gangliosides except GD₃. In comparison to the other gangliosides, GT₁ contained a lower proportion of the stearoyl group and a higher proportion of the oleoyl, nervonoyl and the long chain acyl groups. Both GT₁ and GD₃ gangliosides contained proportionately higher levels of long chain acyl groups (20∶0→24∶0) than did other gangliosides. GD₃ gangliosides showed 2 bands on thin layer chromatography, and the upper band was more distinct than the lower band.     

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu²⁻-induced inactivation of PON1 in the presence of Ca²⁺, two CLA isomers exhibited a remarkable protection (E _max, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H₂O₂ or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu²⁺-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.     

Structural Importance of the Acyl Group in Substrate Specificity of Purified Bovine Lysophospholipase D

The structural importance of the acyl group in lysophosphatidylcholine (LPC) as substrate of purified bovine lysophospholipase D (lysoPLD) was investigated. Among LPCs with saturated acyl chains, the K _m value decreased according to the length of the acyl chain (C12–C16) up to the palmitoyl group, while the V _m value showed no remarkable change. But, the extension of the acyl size to C18, as observed with 1-stearoyl LPC (K _m, 8.5 mM), rather resulted in a remarkable increase in the K _m value. Meanwhile, the introduction of one double bond in the C18 saturated acyl chain led to a remarkable reduction in the K _m value, as observed with 1-oleoyl LPC (K _m, 0.48 mM). Furthermore, 1-linoleoyl LPC (K _m, 56 μM) with two double bonds exhibited a smaller K _m value than 1-oleoyl LPC, suggesting that the unsaturation degree might be important in augmenting the binding affinity of LPCs. A similar phenomenon was also observed with 1-arachidonoly LPC (K _m, 79 μM) or 1-docosahexaenoyl LPC (K _m, 36 μM). Overall, the order of catalytic efficiency (V _m/K _m value) of those LPCs seemed to be affected by the K _m value rather than the V _m value, which differed by at most threefold among LPC derivatives. Next, the introduction of a hydroperoxide group into 1-linoleoyl-LPC or 1-arachidonoyl LPC led to a further reduction in K _m values (1-hydroperoxylinoleoyl LPC, 26 μM; 1-hydroperoxyarachidonoyl LPC, 33 μM), accompanied by a further increase in the V _m/K _m values. Additionally, phosphatidylcholines (PCs) with an oxidized acyl chain at sn-2 position were found to be efficient as 1-palmitoyl LPC as substrates of lysoPLD. Taken together, the catalytic efficiency of LPCs or oxidized PCs as substrates of lysoPLD seems to be determined by the property of the acyl chain, length of the acyl chain, unsaturation degree and oxidation status.     

Temperature-dependent deacylation of molecular species of phosphatidylcholine by microsomal phospholipase A2 of thermally acclimated rainbow trout,Salmo gairdneri 1

Using the ratios of kinetic parameters, V/Km, the deacylation of different molecular species of 1-palmitoyl,2-acyl phosphatidylcholine via microsomal phospholipase A₂ (PLA₂) was studied in liver tissue of thermally acclimated rainbow trout (Salmo gairdneri). In general, PLA₂ from fish acclimated to cold temperatures showed an order of preference for the acyl moieties of 18∶1>18∶1>18∶2>18∶0. Trout acclimated to warm temperatures generally preferred 18∶0 PC, but the actual order of preference depended on the temperature of the assays and the presence of endogenous lipids in the enzyme preparation. At 5 C, the particulate (microsomal) enzyme preferred 18∶0>18∶2>18∶1, but a lipid-free preparation of the enzyme preferred 18∶2>18∶0>18∶1. At 20 C, particulate enzyme preferred 18∶1>18∶0>18∶2 but purified enzyme preferred 18∶0>18∶2>18∶1. Thus, assay temperature and the presence of microsomal lipids had a greater effect on PLA₂ from fish acclimated to warm temperatures than fish acclimated to cold temperatures. The substrate preference of PLA₂ is discussed with reference to the previously observed changes in membrane fatty acid composition that occur with thermal acclimation in rainbow trout.     

Effect of ATP on the microsomal desaturation of unsaturated fatty acids

The effect of ATP on the microsomal desaturation of linoleic acid to γ-linolenic acid was studied in a system in vitro with the following results: (1) preincubation of rat liver microsomes with ATP alone in N₂ or in the presence of CoA and Mg⁺⁺ followed by subsequent incubation with 1-¹⁴C-linoleic acid plus NADH in O₂ resulted in enhancement of 1-¹⁴C-linoleic acid desaturation when compared with control samples in which no preincubation was performed; (2) the preincubation of the microsomes with ATP, Mg⁺⁺ and CoA in the presence of 1-¹⁴C-linoleic acid decreased the desaturation of the labeled acid to γ-linolenic acid upon subsequent incubation with NADH, as a consequence of incorporation of the acid into the microsomal lipids; (3) the increase of linoleic acid desaturation depended on the ATP concentration during preincubation and followed a sigmoidal curve. It was specific for ATP, and neither GTP, CTP, ADP nor AMP produced a similar effec. However, GTP or CTP could replace ATP as a cofactor in the microsomal desaturation of free linoleic acid to γ-linolenic, suggesting that directly or indirectly they may activate conversion of the free acid to linoleyl-CoA; (4) preincubation of microsomes with ATP activated the acylation of CoA. However, this activation showed no quantitative correlation with enhancement of the desaturation reaction; (5) addition of ATP also stimulated conversion of linoleyl-CoA to γ-linolenic acid. This enhancement was not related to inhibition of the linoleyl-CoA hydrolase; (6) however, in spite of these results, preincubation with ATP did not increase the initial velocity of linoleic acid or linoleyl-CoA desaturation; (7) preincubation of microsomes with ATP also increased the 6-desaturation of oleic acid and α-linolenic acid but did not increase the 9-desaturation of plamitic and stearic acid.     

Species variation in serum levels of prostaglandins and their precursor acids

Levels of prostaglandin (PG) precursor acids, and PGE₂, PGF_2α and TXB₂ in sera of adult animals (5/species) were determined by gas chromatography and radioimmunoassay, respectively. The level of arachidonic acid in horse serum lipids was the lowest (0.61±0.03%), and that in dog serum lipids was the highest (18.1±1.8%). Bovine serum lipids contained considerable amounts of 20∶3ω6 (precursor of monoene PG) and 20∶5ω3 (precursor of triene PG) in addition to arachidonic acid. Thromboxane B₂ was not the major species of endoperoxide metabolite synthesized by platelets from arachidonic acid in male ruminants and pig. The concentration of TXB₂ in the serum of the lactating cow was more than 50 times greater than that of ovariectomized cows or of bulls. Although TXB₂ was the major species of endoperoxide metabolite synthesized by human platelets, its serum concentration was much lower than that of nonruminant animals except the pig. These results showed that there were considerable variations in levels of PG and their precursors among various species of animals. The species variation in PG and TXB₂ concentrations was not simply attributed to the differences in platelet concentration blood.     

Highly efficient synthesis of polymer brushes with PEO and PCL as side chains via click chemistry

We reported that well-defined polymer brushes could be prepared efficiently via the grafting-onto approach and the polymer brushes grafted with poly(ethylene oxide) (PEO), poly(ε-caprolactone) (PCL) and mixed PEO/PCL have been obtained. Firstly, poly(glycidyl methacrylate)s, PG₁₀₀ and PG₉₄₀, were prepared by controlled radical polymerization and their epoxy groups were reacted with sodium azide to introduce azido groups quantitatively. Then the PG derivatives bearing azido groups, poly(3-azido-2-hydroxypropyl methacrylate)₁₀₀ (PGA₁₀₀), were reacted with the alkynyl terminated either PEO (ay-PEO₁₁₃) or PCL (ay-PCL₁₇) through copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction under mild temperature. At a molar feed ratio of ay-PEO₁₁₃:azido unit = 1:1, the coupling reaction with ay-PEO₁₁₃ proceeded nearly quantitatively. At the same feed, the conversion of grafting ay-PCL₁₇ dropped to ca. 80%. Amphiphilic polymer brushes with randomly grafted PEO and PCL branches were also obtained by feeding the mixed ay-PEO and ay-PCL during CuAAC. By coupling ay-PEO₁₁₃ onto a long backbone PGA₉₄₀, wormlike PEO brushes with an average length of 140 nm were prepared as demonstrated by AFM. This molecular worm was further labeled with Rhodamine B and then was incubated with liver cancer cells HepG2. Preliminary results indicated that the wormlike brushes could be internalized by the cells easily, suggesting the potential application as advanced vehicles for delivery.     

Oxidative activation of guanylate cyclase by prostaglandin endoperoxides and fatty acid hydroperoxides

Purified prostaglandin endoperoxides (PGG₂ and PGH₂) and hydroperoxides (15-OOH-PGE₂) as well as fatty acid hydroperoxides (12-OOH-20∶4, 15-OOH-20∶4, and 13-OOH-18∶2) were examined as effectors of soluble splenic cell guanylate cyclase activity. The procedures employed for the preparation and purification of these components circumvented the use of diethyl ether which obscured effects of lipid effectors because of contaminants presumed to be ether peroxides which were stimulatory to the cyclase. Addition of prostaglandin endoperoxides or fatty acid hydroperoxides to the reaction mixture led to a time-dependent activation of guanylate cyclase activity; 2.5-to 5-fold stimulation was seen during the first 6 min. The degree of stimulation and rate of activation were dependent on the concentration of the fatty acid effector; when initial velocities (6 min) were assessed, half maximal stimulation was achieved in the range of 2 to 3 μM. However, by extending the incubation time to 90 min, similar maximal increases in specific activity could be achieved with 3 or 10 μM PGG₂ or PGH₂. Activation of guanylate cyclase upon addition of prostaglandin endoperoxides or fatty acid hydroperoxides was prevented or reversed by the thiol reductants dithiothreitol (3 to 5 mM) or gluthathione (10 to 15 mM). Na₂S₂O₄, not known as an effective reducing agent of disulfieds, prevented but was relatively ineffective in reversing activation after it had been induced by PGG₂. Pretreatment of the enzyme preparation with increasing concentrations of N-ethyl-maleimide in the range of 0.01 to 1.0 mM prevented activation by PGG₂ without effecting basal guanylate cyclase activity. These observations indicate that fatty acid hydroperoxides and prostaglandin endoperoxides promote activation of the cyclase by oxidation of enzyme-related thiol functions. In contrast, PGE₂, PGF_2α, hydroxy fatty acids (13-OH-18∶2, 12-OH-20∶4) as well as saturated (18∶0), monoenoic (18∶1), dienoic (18∶2), and tetraenoic (20∶4) fatty acids were ineffective in promoting cyclase activation in the range of 1 to 10 μM. Studies to identify the species of the rapidly metabolized prostaglandin endoperoxides that serve as effectors of the cyclase indicated that PGG₂ but not 15-OOH-PGE₂ (the major buffer-rearrangement product of PGG₂) is most likely an activator. In the case of PGH₂ a rapidly generated (30 sec) metabolite of PGH₂ was found which contained a hydroperoxy or endoperoxy functional group and was equally as effective as PGH₂ as an apparent activator of the enzyme. The combined effects of PGG₂ and dehydroascorbic acid, another class of activator, exhibited additivity with respect to the rate at which the time-dependent activation was induced. These results suggest that activation of soluble guanylate cyclase from splenic cells can be achieved by the oxidation of sulfhydryls that may be associated with specific hydrophobic sites of the enzyme or a related regulatory component.     

Fatty acid and product selectivities of potato tuber lipid acyl hydrolase in esterification reactions with glycerol in organic media

Fatty acid [FA; butanoic (C₄); octanoic (C₈); tetradecanoic (C₁₄); and cis-9,12-octadecadienoic (C_18:2) acids] reaction selectivity and the corresponding acyl profiles in differentially accumulating acylglycerol (AG) products (mono-, di-, and triacylglycerols; MAG, DAG, TAG, respectively) were evaluated for Celite™-immobilized potato tuber lipid acyl hydrolase (LAH)-mediated esterification reactions in isooctane at 35°C and water activity of 0.19. The ordinal pattern of FA selectivities was C₈>C₁₄>C_18:2>C₄, and the AG products accumulating were α-MAG>DAG>β-MAG>TAG. A dimensionless expression for fatty acid partitioning coefficient (FAPC) was contrived to represent the partitioning patterns of specific FA into specific AG pools on the basis of an equivalent extent of FA reaction. These FAPC values indicated that preferential partitioning of FA was as follows: C₄ was preferentially partitioned into TAG, DAG, and β-MAG; C₈ was preferentially partitioned into DAG; C₁₄ was preferentially partitioned into α,β-MAG; C_18:2 was preferentially partitioned into α,β-MAG and TAG. These findings infer that the tendency for LAH-mediated esterifications to accumulate MAG is based, in part, on a constraint in reactivity of α-MAG of ≥10 acyl carbon groups to serve as acceptors for further esterification events. The general approach taken in this study may assist in identifying the discrete steps in assembling structured glycerides where different biocatalysts exhibit the greatest degree or control of reaction selectivity.     

Growth hormone and liver mitochondria: Effects on phospholipid composition and fatty acyl distribution

Effects of growth hormone on phospholipid composition and fatty acyl distribution were studied in liver mitochondria of hypophysectomized rats. After hypophysectomy, only cardiolipin showed a 25% decrease. Its fatty acyl distribution, which consisted mainly of linoleic acid (55–60%) and oleic acid (20%), was unchanged. In phosphatidylcholine and phosphatidylethanolamine fractions the contents of docosahexaenoic and arachidonic acids were decreased with a concomitant increase in linoleic acid content. These changes could be accounted for by small but significant decreases in the activities of Δ⁹-desaturase (sucrose-induced), Δ⁵-desaturase and mitochondrial elongation enzymes. The activities of Δ⁶-desaturase NADH cytochrome b₅ ferri-reductase, cytochrome b₅, NADH cytochrome c reductase and microsomal elongation enzymes remained virtually unchanged. Injection of bovine growth hormone daily for seven days restored cardiolipin and fatty acyl distribution and the enzyme activities. From these and other results, we conclude that growth hormone-dependent increase of respiratory activity of liver mitochondria may be partly mediated by the hormonal effects on membrane lipid distribution.     

Lipase-catalyzed monoesterification of 1-O-hexadecylglycerol in organic solvents

A simple method is presented to esterify 1-O-hexadecyl-rac-glycerol using lipases in different organic solvents. The following fatty acids were used: C_14∶0, C_16∶0, C_18∶0, C_18∶1, and C_18∶2. Monoesterification was achieved by using a limiting amount of the fatty acid. Both the 1-O-hexadecyl-3-O-acylglycerol and the 2-O-acylglycerol were obtained in a total yield of 75% and a ratio of 7∶1 in dichloromethane after 3 d. Chromatographic data for the monoesters, useful for the identification of the natural products, are given (gas-liquid chromatography, thin-layer chromatography, reverse-phase thin-layer chromatography). The structure was confirmed by a chemical synthesis of 1-O-hexadecyl-2-O-hexadecanoylglycerol. The 3-O-glyceride was also formed by acyl migration, as the minor component. The monoesters were separated by column chromatography and characterized by ¹H and ¹³C nuclear magnetic resonance spectra.     

Quantitative studies on methyl ketone formation in butteroil: Effect of temperature

A quantitative study of the effect of various heat treatments on methyl ketone formation in butteroil was conducted. Methyl ketone formation was found to follow first-order reaction kinetics. The energy of activation was calculated to be approximately 26.55 kcal/mole. The ketones were formed in a fairly constant molar ratio approximating to the following C₁₅,2∶C₁₃,1∶C₁₁,1∶C₉,1∶C₇,2∶C₅,1. The temperature-time conditions had little or no effect on the ratio.     

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: Regulation by a high-affinity Zn2+ binding site

Saccharomyces cerevisiae cells were demonstrated to contain lysophosphatidylcholine (lysoPtdCho) acyltransferase (E.C. 2.3.1.23) activity. The enzyme displayed K _m(app) of 69 μM for lysoPtdCho and 152 μM for oleoyl CoA. Enzyme activity was not affected by the addition of 1 mM Mg²⁺, Mn²⁺, Ca²⁻, or 200 mM EDTA. However, Zn²⁺ inhibited lysoPtdCho acyltransferase activity to 33% control values at 0.1 mM and to 7% at 1.0 mM Zn²⁺. To further explore the possibility that lysoPtdCho acyltransferase may contain a high-affinity Zn²⁺ binding site, we tested the strong Zn²⁺ chelator o-phenanthroline for its ability to inhibit enzyme activity. LysoptdCho acyltransferase activity was inhibited to 18 and 27%, respectively, those of control values in the presence of 2 and 1 mM o-phenanthroline, implying that a high-affinity Zn²⁺ binding site exists in lysoPtdCho acyltransferase or in an accessory protein that is essential for protein stability and/or activity. Saccharomyces cerevisiae lysoPtdCho acyltransferase activity displayed a broad lysoPtdCho fatty acyl chain substrate specificity utilizing lysoPtdCho molecules ranging in length from C₁₀−C₂₀ (the entire range tested). In addition, the enzyme was capable of using the ether-linked analog of lysoPtdCho, 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine, as a substrate. The ability of S. cerevisiae to incorporate radiolabeled 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine into phosphatidylcholine in vitro was exploited to demonstrate a direct precursor-product relationship between lysoPtdCho molecules and their incorportation into phosphatidylcholine in vivo. Identical labeling results were obtained in S. cerevisiae cells disrupted for their major transacylase activity, PLB1, demonstrating that the incorporation of lysolipid was via acyltransferase, and not transacylase, activity.     

3-carboxy-4-nitrophenyl-dithio-1,1′,2-trisnorsqualene: A site-directed inactivator of yeast oxidosqualene cyclase

The role and location of essential thiol groups in 2,3-oxidosqualene cyclase fromSaccharomyces cerevisiae was examined (i) by comparing inactivation properties of two known thiol reagents, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-nitro-5-thiocyanobenzoic acid (NTCB), with 3-carboxy-4-nitrophenyl-dithio-1,1′,2-trisnorsqualene (CNDT-squalene), a new thiol reagent designed as a sitedirected inactivator of oxidosqualene cyclase and (ii) by testing the ability of the substrate to protect the enzyme against inactivation by the reagents. All reagents gave a time-dependent inactivation following pseudo-first order kinetics. DTNB and CNDT-squalene showed comparable inactivation ability (K_i=0.67 and 1.21 mM), whereas NTCB was less effective (K_i=15.6 mM). Strong differences between the two most active inhibitors, DTNB and CNDT-squalene, were observed when the enzyme was saturated with substrate prior to incubation with the thiol reagent. While substrate did not protect the enzyme against the inactivation caused by DTNB, a reduction in the inactivation ability of CNDT-squalene was observed under protection conditions. The data suggest that the squalene-like inactivator modifies a thiol group located at the active site of the enzyme.     

A31P nuclear magnetic resonance investigation of acyl group transfer from phosphatidylcholine to yield lysophosphatidylcholine in human plasma

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to measure the rate of acyl transfer from phosphatidylcholine (lecithin, PC) in whole plasma and in high density lipoprotein (HDL). Spectral deconvolution was used to resolve overlapping resonances in the³¹P NMR spectra of the phospholipids. Mean values of the acyl group transfer rates from PC in plasma and HDL were 36 μmol L⁻¹h⁻¹ and 19 μmol L⁻¹h⁻¹, respectively. The reciprocal nature of the decrease in the spectral peak intensities of PC, compared to the increase in the intensities of the lysolecithin (lysoPC) peaks, suggested a substrate/product relationship consistent with the action of lecithin:cholesterol acyltransferase (LCAT), the enzyme responsible for the esterification of free cholesterol in plasma. LCAT involvement was confirmed by measuring the cholesterol esterification rate based on the¹³C NMR spectra obtained on lipid extracts from plasma that had been incubated at 37°C. Within experimental error, the rate of lysoPC formation in plasma was shown to be equal to that of cholesteryl ester formation.     

Milk fat structure of a patient with type 1 hyperlipidemia

Structural analyses were performed on milk fat samples obtained 3–10 days postpartum from a lactating patient with primary Type 1 hyperlipidemia. The milk triacylglycerols contained 3–7% C₁₀, 14–21% C₁₂, 20–30% C₁₄, 22–26% C₁₆ and 20–30% C₁₈ (largely oleic) acids. Gas liquid chromatographic (GLC) analyses of the X-1,3- and X-1,2-diacylglycerols on polar siloxane columns showed a markedly non-random association of acyl chains. Stereospecific analyses indicated that the short chain length fatty acids were confined essentially to the sn-3-position of the triacylglycerol molecule. Furthermore, these acids were largely absent from the phosphatidylcholines and the endogenous sn-1,2-diacylglycerols of the milk fat. It is concluded that the short chain fatty acids are incorporated into the milk triacylglycerols during the final stage of biosynthesis via the phosphatidic acid pathway, and that the overall fatty acid distribution is consistent with the 1-random 2-random 3-random hypothesis.     

Acyl Migration Kinetics of 2-Monoacylglycerols from Soybean Oil via <Superscript>1</Superscript>H NMR

The acyl migration kinetics of neat 2-monoacylglycerol (2-MAG) to form 1-MAG was determined using ¹H NMR spectroscopy to monitor the β-proton integration ratios of the two species over time. 2-MAG was synthesized by the Novozym 435-catalyzed alcoholysis of soybean oil and isolated by solvent extraction or molecular distillation at a mole fraction (X _2-MAG) of 0.94 relative to total MAG. The kinetics parameters of the neat 2-MAG acyl migration were investigated over the temperature range of 23–80 °C. The 2-MAG mol fraction remained unchanged at 23 °C over the course of 168 h and reached an equilibrium of X _2-MAG = 0.09 at only 80 °C. Modeling of the kinetics data revealed a 2-MAG half life (t _1/2) of 3,500 and 22.8 h at 23 and 80 °C, respectively, with an activation energy of 79.0 ± 6.5 kJ mol⁻¹. The use of ¹H NMR spectroscopy proved an expedient method for monitoring the acyl migration in 2-MAG compared to other reported methods (e.g. GC, HPLC, and ¹³C-NMR spectroscopy), requiring no sample manipulation and minimizing the deleterious effects of high temperatures and solvent exposure. Product names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may be suitable.