Purification of 2-Monoacylglycerols Using Liquid CO<Subscript>2</Subscript> Extraction

The fatty acid moiety of 2-monoacyl-sn-glycerol (2-MAG) undergoes spontaneous acyl migration to the sn-1(3) position, resulting in a thermodynamic equilibrium of approximately 1:9 of 2-MAG to 1(3)-monoacyl-sn-glycerol (1-MAG). Spontaneous acyl migration is an impediment to synthesizing and isolating specific 2-MAG for use as intermediates in the synthesis of structured triacylglycerols. 2-Monooleoyl-sn-glycerol was synthesized by the enzymatic ethanolysis of triolein and isolated by liquid CO₂ extraction. The resultant MAG, diacylglycerol, and fatty acid ethyl esters were quantified by ¹H NMR and supercritical fluid chromatography. The low polarity of the CO₂ and mild extraction temperature (25 °C) resulted in very low spontaneous acyl migration rates, allowing the MAG to be isolated in 80% yield and in a very high 2-MAG:1-MAG ratios of ≥93 mol%.     

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.     

Palmitoleic (16:1 cis-9) and cis-Vaccenic (18:1 cis-11) Acid Alter Lipogenesis in Bovine Adipocyte Cultures

Our objectives were to: (1) confirm elongation products of palmitoleic acid (16:1 cis-9) elongation in vitro using stable isotopes and (2) evaluate if exogenous supplementation of palmitoleic acid, elongation products, or both are responsible for decreased desaturation and lipogenesis rates observed with palmitoleic acid supplementation in bovine adipocytes. Stromal vascular cultures were isolated from adipose tissue of two beef carcasses, allowed to reach confluence, held for 2?days, and differentiated with a standard hormone cocktail (day?0). On day?2, secondary differentiation media containing 1 of 4 fatty acid treatments [0?μM fatty acid (control), or 150?μM palmitic (16:0), palmitoleic, or cis-vaccenic (18:1 cis-11)] was added for 4?days. On day?6, cells were incubated with [¹³C] 16:1, [¹³C] 2, or [¹³C] 18:0 to estimate elongation, lipogenic, and desaturation rates using gas chromatography-mass spectrometry. Enrichment of [¹³C] 18:1 cis-11 confirmed 18:1 cis-11 is an elongation product of 16:1. Additionally, [¹³C] label was seen in 20:1 cis-13 and cis-9, cis-11 CLA. Synthesis of [¹³C] 16:0 from [¹³C] 2 was reduced (P?<?0.05) in palmitoleic acid and cis-vaccenic acid-treated compared with control cells following 36?h incubation. By 12?h of [¹³C] 18:0 incubation, cells supplemented with palmitoleic acid had reduced (P?<?0.05) [¹³C] 18:1 cis-9 compared with all other treatments. Gene expression and fatty acid results support isotopic data for lipogenesis and desaturation. Therefore, palmitoleic acid is actively elongated in vitro and its elongation product, cis-vaccenic acid, can also reduce lipogenesis. However, inhibition of desaturation can be directly attributed to palmitoleic acid and not its elongation products, 18:1 cis-11 or 20:1 cis-13.     

Influence of Solid Supports on Acyl Migration in 2-Monoacylglycerols: Purification of 2-MAG via Flash Chromatography

Soybean oil 2-monoacylglycerol (2-MAG) was synthesized via the Novozym 435-catalyzed ethanolysis of triacylglycerols and purified by conventional liquid–liquid extraction. The 2-MAG was subjected to incubation at 20 and 40 °C in the presence of five solid commercial support materials, Lewatit, Silica Gel 60, Alumina–Neutral Brockman Activity 1, Amberlyst-15, and SBA-15, to determine their effects on acyl migration rates. Lewatit and SBA-15 did not catalyze acyl migration rates after 144 h, while silica gel slightly increased migration rates. The more polar alumina and the cationic Amberlyst 15 significantly increased migration rates. Flash chromatography purification of 2-MAG using silica gel as the immobile phase developed with an acetone/hexane binary gradient proved to be a comparable purification method to liquid–liquid extraction, resulting in 60 % 2-MAG yield, no residual triacylglycerol, diacylglycerol, or glycerol co-products, and a 95 mol% 2-MAG purity vs. 1-MAG, demonstrating that flash chromatography did not catalyze acyl migration.     

Analysis of Ceramides in Soy Sauce Oil

Soy sauce oil is a by-product of soy sauce derived from the lipids in soybeans, wheat, and koji mold (Aspergillus oryzae or A. sojae). In the present study, sphingolipids were detected in soy sauce oil for the first time. The concentration of sphingolipids as sphingoid bases was 240?±?50?mg/kg. Ceramide was the major component, and free sphingoid bases and cerebroside were minor components. The abundance ratios of the incorporated sphingoid bases were as follows: 4-hydroxysphinganine (t18:0), 13?%; 4-hydroxy-8-sphingenine (t18:1^Δ8), 29?%; sphinganine (d18:0), 4.7?%; 8-sphingenine (d18:1^Δ8), 18?%; 4,8-sphingadienine (d18:2^Δ4,Δ8), 16?%; 9-methyl-trans-4,trans-8-sphingadienine (9-Me d18:2^Δ4,Δ8), 19?%; and trans-4-sphingenine (d18:1^Δ4), 1.2?%. Two of the incorporated sphingoid bases (t18:1^Δ8 and d18:1^Δ8) were isolated and another (d18:2^Δ4,Δ8) was partly purified, while three of the ceramides were isolated and identified. The structures of the other ceramides were determined by liquid chromatography-mass spectrometry. Two groups of ceramides were observed, including one group containing t18:0, t18:1^Δ8, d18:1^Δ8, d18:2^Δ4,Δ8, and 9-Me d18:2^Δ4,Δ8 with 2-hydroxy fatty acids and the other containing t18:0 and t18:1^Δ8 with normal fatty acids. Many of these ceramides corresponded to ceramides or ceramide moieties of cerebrosides found in the soybeans, wheat, and koji mold used in soy sauce production.     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

Analysis of positional distribution of fatty acids in palm oil by13C NMR spectroscopy

The¹³C NMR spectrum of the carbonyl carbons of the acyl groups of triacylglycerols of palm oil has been shown to give the composition of saturated, oleic and linoleic acyl groups at the 1,3-positions and at the 2-position of the glycerol moiety. Except for the lack of differentiation of the saturated fatty acids, the¹³C NMR technique provides the same information as the tedious enzymatic hydrolysis cum fatty acid analysis. The carbonyl carbon of the linolenic acyl group (18∶3,[cis, cis, cis]-9, 12, 15) has a chemical shift which is only 0.005 ppm to low frequency of that of the linoleic acyl group (18∶2,[cis, cis]-9, 12), so that the two resonances may not be distinguishable (or resolved) even at a high magnetic field.     

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.     

Fatty Acid Desaturation and Elongation Reactions of <Emphasis Type="Italic">Trichoderma</Emphasis> sp. 1-OH-2-3

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16:0) mainly to stearic acid (18:0), and further to oleic acid (c9-18:1), linoleic acid (c9,c12-18:2), and α-linolenic acid (c9,c12,c15-18:3) through elongation, and Δ9, Δ12, and Δ15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9,cis-12-hexadecadienoic acid were also produced from 16:0 by the strain. This strain converted n-tridecanoic acid (13:0) to cis-9-heptadecenoic acid and further to cis-9,cis-12-heptadecadienoic acid through elongation, and Δ9 and Δ12 desaturation reactions, respectively. trans-Vaccenic acid (t11-18:1) and trans-12-octadecenoic acid (t12-18:1) were desaturated by the strain through Δ9 desaturation. The products derived from t11-18:1 were identified as the conjugated linoleic acids (CLAs) of cis-9,trans-11-octadecadienoic acid and trans-9,trans-11-octadecadienoic acid. The product derived from t12-18:1 was identified as cis-9,trans-12-octadecadienoic acid. cis-6,cis-9-Octadecadienoic acid was desaturated to cis-6,cis-9,cis-12-octadecatrienoic acid by this strain through Δ12 desaturation. The broad substrate specificity of the elongation, and Δ9 and Δ12 desaturation reactions of the strain is useful for fatty acid biotransformation.     

Effects of conjugated linoleic acid isomers on the hepatic microsomal desaturation activities in vitro

The influence of individual conjugated linoleic acid (CLA) isomers on the Δ6 desaturation of linoleic and α-linolenic acids and on the Δ9 desaturation of stearic acid was investigated in vitro, using rat liver microsomes. The Δ6 desaturation of 18∶2n−6 was decreased from 23 to 38% when the ratio of 9cis,11trans-18∶2 to 18∶2n−6 increased from 0.5 to 2. The compound 10trans,12cis-18∶2 exhibited a similar effect only at the highest concentration. The Δ6 desaturation of α-linolenic acid was slightly affected by the presence of CLA isomers. The sole isomer to induce an inhibitory effect on the Δ9 desaturation of stearic acid was 10trans,12cis-18∶2.     

Absorption and distribution of deuterium-labeledtrans- andcis-11-octadecenoic acid in human plasma and lipoprotein lipids

Triglycerides of deuterium-labeledtrans-11-,trans-11-cis-11- andcis-9-octadecenoic acid (11t-18∶1-²H, 11c-18∶1-²H) were simultaneously fed to two young adult male subjects. Plasma lipids from blood samples collected periodically for 48 hr were analyzed by gas chromatography-mass spectroscopy. The results indicate (i) the Δ11-18∶1-²H acids and 9c-18∶1-²H were equally well absorbed; (ii) relative turnover rates were higher for the Δ11-18-1-²H acids in plasma triglycerides; (iii) incorporation of the Δ11-18∶1-²H acids into plasma phosphatidylcholine was similar to 9c-18∶1-²H, but distribution at the 1-and 2-acyl positions was substantially different; (iv) esterification of cholesterol with 11t-18∶1 was extremely low; (v) chain shortening of the Δ11-18∶1-²H acids was 2–3 times greater than for 9c-18∶1-²H; (vi) no evidence for desaturation or elongation of the 18∶1-²H acids was detected; and (vii) a 40% isotopic dilution of the 18∶1-²H acids in the chylomicron triglyceride fraction indicated the presence of a substantial intestinal triglyceride pool. Based on our present knowledge, these metabolic results for Δ11-18∶1 acids present in hydrogenated oils and animal fats indicate that the Δ11 isomers are no more likely than 9c-18∶1 to contribute to dietary fat-related health problems.     

Selective uptake of fatty acids by the yeast Yarrowia lipolytica

Yarrowia lipolytica, when cultivated on mixtures of free fatty acid substrates was found to remove C12:0, C14:0, ^Δ9C18:1, ^{Δ9, 12}C18:2 and ^{Δ9, 12, 15}C18:3 at significantly higher rates than C16:0 and C18:0, regardless of fatty acid composition of the initial substrate. C12:0, C14:0 and ^{Δ9, 12, 15}C18:3 were specifically and completely removed from the substrate, while ^Δ9C18:1 and ^{Δ9, 12}C18:2 concentrations decreased by 55‐80 wt‐% in comparison with initial concentrations. In contrast, concentration of C18:0 increased 2.1—3.5 fold in the substrate. Although C18:0 was removed slowly from the substrate, this fatty acid was selectively accumulated in the storage lipid. Inversely, only low quantities of ^Δ9C18:1 and ^{Δ9, 12}C18:2 and traces of C12:0, C14:0 and ^{Δ9, 12, 15}C18:3 were accumulated in the storage lipid. During storage lipid breakdown, cellular C16:0 and ^Δ9C18:1 were taken up more rapidly than C18:0. We concluded that the capability of Yarrowia lipolytica to selectively remove several fatty acids from the substrate and accumulate others could be used in modification of the composition of selected mixtures of fatty acids and probably of common fats, to produce “new” fats with a predetermined composition.     

Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Synthesis,Purification, and Acyl Migration Kinetics of 2-Monoricinoleoylglycerol

2‐Monoricinoleoylglycerol (2‐MRG) was synthesized by the Novozym 435 catalyzed alcoholysis of castor oil in excess ethanol (1:70 mol:mol) at ambient temperature. Due to the fatty acid C12‐OH group, conventional liquid–liquid extraction methods developed for less polar, non‐hydroxylated 2‐monoacylglycerols (2‐MAG) proved inadequate for 2‐MRG purification. Alternatively, 2‐MRG was purified by normal‐phase flash chromatography (FC) on silica gel using a binary acetone‐hexane gradient mobile phase. Gram quantities of 2‐MRG were isolated in 63 % yield and contained no residual diacylglycerol (DAG), which fail to separate using liquid–liquid extraction methods. The 2‐MRG was obtained at ~90 mol% relative to 1‐MRG, proving that the FC method did not appreciably catalyze acyl migration. ¹H‐NMR spectroscopy was used to monitor the spontaneous acyl migration of isolated 2‐MRG from 20 to 80 °C. The relative energy of activation calculated from the Arrhenius relationship of the 2‐MRG acyl migration rate constants was 82.9 kJ/mol. This was ~two‐fold higher than the theoretical ΔG_298.15 calculated from molecular modeling using density functional calculations (B3LYP/6?31 + G*) of 2‐MRG, the ketal ring transition state, and 1‐monoricinoleoylglycerol (1‐MRG). The synthesis and isolation methods reported herein provide a convenient means to access useful intermediates for functionalized structured lipids.     

Separation of isomeric lysophospholipids by reverse phase HPLC

A reverse-phase high performance liquid chromatography (HPLC) method was developed which resolved isomers of lysophosphatidylcholine (LPC) differing in the location of the aliphatic chain (sn-1 orsn-2 position) and the position (Δ⁶ or Δ⁹) or geometric configuration (cis ortrans) of the olefin group in monounsaturated species. LPC isomers containing an acyl substituent at thesn-2 position eluted before their 1-acyl-sn-glycero-3-phosphocholine (1-acyl LPC) counterparts. The retention times of both thesn-1 andsn-2 isomers of monounsaturated species increased in the order Δ⁹-cis < Δ⁹-trans < Δ⁶-cis. The integrated ultraviolet absorbance (203 nm) in binary mixtures of the Δ⁹-cis and Δ⁶-cis 2-acyl lysophospholipid isomers correlated with the lipid phosphorus content of corresponding column eluates (r-0.994). Thus, the present method will facilitate synthesis of isomerically pure diradylphospholipids by providing homogeneous lysophospholipid precursors and help simplify the quantitative analysis of unsaturated lysophospholipid species.     

Distribution of (n-9) and (n-7) Isomers of Monounsaturated Fatty Acids in Indian Mustard (Brassica juncea)

The seed oils of 19 Indian mustard varieties were analyzed for fatty acid composition using GC–MS, reporting the presence of n-7 isomers of C18:1, C20:1 and C22:1 fatty acids. n-7 isomers namely cis-vaccenic (C18:1), 11, eicosenoic(C20:1) and 13-cis-docosenoic (C22:1) acids ranged from 0.61 to 1.73, 1.04 to 1.69 and 0.58 to 1.17 %, respectively. The average values of C20:1(n-7) was highest (1.36) amongst the three acids. Nervonic acid was also reported in the range of 0.69 to 2.52 %. The ratios of (n-7)/(n-9) ranged from 6.22 to 14.62, 12.38 to 27.35 and 2.01 to 3.24 % for C18:1, C20:1 and C22:1 fatty acids, respectively. The variety RLM-619 had the lowest elongation ratio (ER) as 0.44 and the highest desaturation ratio (DR) as 0.41 indicating higher efficiency of the desaturation pathway than for other varieties. The oleic desaturation ratio (ODR) values varied from 0.68 (Basanti) to 0.75 (GM-2) with a mean value of 0.72 and linoleic desaturation ratio from 0.40 (Basanti) to 0.49 (Pusa Bold) with a mean value of 0.45. Palmitoleic acid showed positive correlation with C18:1(n-7), C20:1(n-7), C20:2, C22:0, C22:1(n-7), C24:1, (n-7)/(n-9) ratio of C18:1 and C22:1 and a positive trend with ER but a significant negative correlation with C18:3, DR and ODR. The results indicated that palmitoleic acid is an important intermediate component in the synthesis of long chain (n-7) fatty acids.     

The effects oftrans fatty acids on fatty acyl Δ5 desaturation by human skin fibroblasts

The effectiveness of different fatty acids as inhibitors of fatty acyl Δ5 desaturation activity in human skin fibroblasts has been investigated. When incubated with 2.25 μM [¹⁴C] eicosatrienoate (20∶3ω6) in otherwise lipid-free medium, these cells rapidly incorporate the radiolabeled fatty acid into cellular glycerolipids and desaturate it to produce both [¹⁴C] arachidonate and [¹⁴C] docosatetraenoate. The Δ5 desaturation activity can be enhanced by prior growth of the cells without serum lipids. Elaidate (9t–18∶1) is a potent inhibitor of Δ5 desaturation whiletrans-vaccenate (11t–18∶1) is virtually without effect. Oleate and linoleate are only mildly inhibitory. Linoelaidate (9t, 12t–18∶2) is more inhibitory than linoleate but significantly less effective than elaidate. The effects of elaidate can be readily overcome by increasing the concentration of exogenous eicosatrienoate. Studies with a variety oftrans monounsaturates of differing chain lengths indicate that the ω9trans fatty acids are potent inhibitors of Δ5 desaturation, while ω7trans fatty acids are relatively ineffective. Intact human fibroblasts could thus be important in characterizing novel fatty acids as selective inhibitors of arachidonate synthesis in vivo.     

Methodology for the In Vivo Measurement of the Δ<Superscript>9</Superscript>-Desaturation of Myristic,Palmitic, and Stearic Acids in Lactating Dairy Cattle

There is limited methodology available to quantitatively assess the activity of the Δ⁹-desaturase enzyme in vivo without chemically inhibiting the enzyme or using radioactively labeled substrates. The objective of these experiments was to develop methodology to determine the incorporation and desaturation of ¹³C-labeled fatty acids into milk lipids. In a preliminary experiment, 3.7 g [1-¹³C]myristic acid ([1-¹³C]14:0), 19.5 g [1-¹³C]palmitic acid ([1-¹³C]16:0), 20.0 g [1-¹³C]stearic acid ([1-¹³C]18:0) were combined and infused into the duodenum of a cow over 24 h. In a following experiment, 5.0 g [1-¹³C]14:0, 40.0 g [1-¹³C]16:0, and 50.0 g [1-¹³C]18:0 were infused into the abomasums of separate cows as a bolus over 20 min or continuously over 24 h. Milk fat was extracted using chloroform:methanol. Fatty acids were methylated, and fatty acid methyl esters (FAME) were converted to dimethyl disulfide derivatives (DMDS). The FAME and DMDS were analyzed by gas chromatography mass spectrometry. In the preliminary experiment, ¹³C enrichment in 14:0 but not 16:0 or 18:0 was observed. When dosage amounts were increased in the following experiment, peak enrichments from the bolus infusion were observed at 8 h. Enrichments for continuous infusion peaked at 16 h for 14:0 and 18:0, and at 24 h for 16:0. The Δ⁹-desaturase products of these fatty acids were estimated to be 90% of cis-9 14:1, 50% of cis-9 16:1, and 59% of cis-9 18:1. This study demonstrates that ¹³C-labeled fatty acids may be utilized in vivo to measure the activity of the Δ⁹-desaturase enzyme.     

The influence oftrans-acids on desaturation and elongation of fatty acids in developing brain

trans-Monounsaturated acids account for up to 3% of the total octadecenoic acyl chains of human brain lipids. To investigate the influence oftrans-acids on desaturation and chain elongation of fatty acids, in vitro and in vivo experiments with rat brain were performed. In the in vitro assays of Δ9 desaturation, Δ6 desaturation and chain elongation,trans,trans-dienoic acid was inhibitory, particularly to chain elongation. Slight differences between the inhibitory effects oftrans-monoenoic acids and theircis-isomers were observed. In an in vivo model, unlabeled fatty acid (stearate, oleate, elaidate, linoleate, linoelaidate, arachidonate, ortrans-monoene from margarine) was injected simultaneously with [1-¹⁴C] linoleic acid into the brains of suckling rats. Linoelaidate and oleate inhibited desaturation and elongation of linoleate, whereas elaidate, stearate andtrans-monoene from margarine were stimulatory. While the demonstration of differences betweencis andtrans monoenic isomers required relatively high levels of the test acids, it appears thattrans-acids can influence desaturation and elongation enzymes that lead to acyl chain modification in the central nervous system. Portions of this study were presented at the meeting of the American Oil Chemists' Society and the International Society for Fat Research in New York, NY, April 1980.     

Reaction selectivity of <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> (PS-30) lipase as influenced by monoacylation of <Emphasis Type="Italic">sn</Emphasis>-glycerol

Reaction selectivities were determined in multicompetitive reactions mediated by Burkholderia cepacia lipase (Amano PS-30) at a water activity of 0.19 in hexane. Saturated FA (C4–C18 even chain) and oleic acid (C18∶1) were reacted with a single alcohol, glycerol, α-or β-MAG, containing C4, C10, C16, or C18∶1 individually as alcohol cosubstrate. Similar odrinal patterns of FA selectivity, with C8, C10, and C16 preferred over others, were generally observed for incorporation of FA into specific acylglycerol (AG) pools of the 24 specific cases evaluated. The three exceptions were enrichment of C14 and C18 in the MAG pool with α-C16-MAG, substrate, and a general suppression of >C8 incorporation into the TAG pool for reactions with α-C10- and α-C16-MAG. PS-30 lipase selectivity toward MAG was in descending order: α/β-C4-MAG>β-C10-MAG>β-C16-MAG>α/β-C18∶1-MAG>α-C10-MAG>α-C16-MAG. Selectivity in channeling CX of the original CX-MAG substrates into higher AG species was in descending order: α-C10-MAG∼α-C16-MAG>α-C18∶1-MAG>β-C10-MAG∼β-C16-MAG∼β-C18∶1-MAG >α/β-C4-MAG. Generally, MAG were better acyl donors than FA for esterification reactions leading to DAG formation. These observations are relevant to the design of biocatalytic processes intended to yield specifically structured TAG.