Metabolic aspects of positional monounsaturated fatty acids isomers

Absorption and distribution of positionalcis andtrans octadecenoic acid isomers in lipids from rat, egg and human tissues are reviewed. Selected data on enzyme, single-cell, and whole-animal studies with positional octadecenoic acid isomers are summarized and compared.     

Rapid and direct quantitative analysis of positional fatty acids in triacylglycerols using 13C NMR

There is increasing evidence that the positional fatty acid composition (FAC) of TAG is more important than total FAC with regard to nutrition values of edible oils and fats. A rapid and direct regiospecific analysis of positional fatty acids in TAG using ¹³C NMR was developed to overcome the tedious conventional methods which involve enzymatic hydrolysis, Grignard chemical degradation, and chromatography analysis. A set of NMR data acquisition parameters and processing methods had proven their excellent versatility and applicability on various types of oils and fats, with systematic error of 1.0 mol%. It was found that there are discrepancies between the regiospecific analysis results obtained by the current method and by conventional methods. Probable acyl migration occurring in the hydrolysis process in conventional methods is a noted problem. As the current ¹³C NMR method is a direct measurement and no hydrolysis of the sample is needed, acyl migration during the analysis is eliminated. As a result, the saturation level is always higher at sn‐1, 3 positions and lower at sn‐2 position in TAG structure of oils in the regiospecific data obtained from the current ¹³C NMR than that from conventional methods. In the absence of laborious chemical derivatization, this method is simple, accurate, and user‐friendly for researchers, especially for nutritionists to support their nutritional studies from the perspective of positional FAC of edible oils and fats.     

Analysis for geometrical and positional isomers of fatty acids in partially hydrogenated fats

A liquid Chromatographic procedure for frac-tionation and analysis of methyl esters from fats is described. First, esters are separated by liquid chromatography on a partially vulcanized rubber column into trienoate, dienoate, monoenoate-palmitate and stearate fractions. The monoenoate-palmitate fraction is separated by liquid chro-matography on a silver-saturated cation exchange resin into palmitate,trans monoenoate and cis monoenoate fractions. Double bond positions intrans and cis monoenoates are located by ozoniza-tion and gas chromatography of fragments. This separation procedure requires less time and uses simpler apparatus with smaller samples than a previously described method based on counter-current distribution. Data from analyses of two liquid oils and four shortenings are presented. No. Utiliz. Ees. Dev. Div., ARS, USDA.     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: II. Trans isomers

This study examined the effect of the fatty acid positional distribution and of the triacylglycerol (TG) composition on heat-induced trans isomerization of linoleic and linolenic acids. For this, we synthesized diacid TG molecules that were acylated only with linoleic acid (L) or with linolenic acid (Ln) along with palmitic acid (P). The fatty acid of interest was positioned either in the central position (PLP and PLnP, respectively) or in one of the two outer positions (PPL and PPLn, respectively). Monoacid TG, i.e., trilinolein and trilinolenin, were also synthesized and mixed with tripalmitin in a 1:2 ratio. This model TG was also compared to another TG model, which consisted of a canola oil and its randomized counterpart whose fatty acid positional distribution and TG composition were determined by means of high-performance liquid chromatography. After heating, the content of trans isomers was determined by gas-liquid chromatography with a polar capillary column. In model TG, polyunsaturated fatty acids in monoacid TG (LLL and LnLnLn) exhibited the highest degree of isomerization, compared to diacid TG, and this effect was greatest at 220°C. At this temperature, an effect of the TG structure was observed only with linolenic acid. In that situation, 18:3n-3 acylated in the central position of the TG molecule (PLnP) displayed the highest sensitivity to trans geometrical isomerization. Although to a lesser extent, the same trends as for the pure TG model were observed with the canola oil model with regard to the influence of the fatty acid positional distribution and TG molecular species.     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: II. Trans isomers

This study examined the effect of the fatty acid positional distribution and of the triacylglycerol (TG) composition on heat-induced trans isomerization of linoleic and linolenic acids. For this, we synthesized diacid TG molecules that were acylated only with linoleic acid (L) or with linolenic acid (Ln) along with palmitic acid (P). The fatty acid of interest was positioned either in the central position (PLP and PLnP, respectively) or in one of the two outer positions (PPL and PPLn, respectively). Monoacid TG, i.e., trilinolein and trilinolenin, were also synthesized and mixed with tripalmitin in a 1:2 ratio. This model TG was also compared to another TG model, which consisted of a canola oil and its randomized counterpart whose fatty acid positional distribution and TG composition were determined by means of high-performance liquid chromatography. After heating, the content of trans isomers was determined by gas-liquid chromatography with a polar capillary column. In model TG, polyunsaturated fatty acids in monoacid TG (LLL and LnLnLn) exhibited the highest degree of isomerization, compared to diacid TG, and this effect was greatest at 220°C. At this temperature, an effect of the TG structure was observed only with linolenic acid. In that situation, 18:3n-3 acylated in the central position of the TG molecule (PLnP) displayed the highest sensitivity to trans geometrical isomerization. Although to a lesser extent, the same trends as for the pure TG model were observed with the canola oil model with regard to the influence of the fatty acid positional distribution and TG molecular species.     

Desaturation of positional and geometric isomers of monoenoic fatty acids by microsomal preparations from rat liver

A range ofcis- andtrans-monoenoic fatty acids was tested as substrates for desaturation in microsomal preparations from rat liver.Trans-monoenoic acids were generally desaturated in the Δ9 position to the same extent as stearic acid. Acids with Δ7-trans- and Δ11-trans-olefinic unsaturation produced Δ7-trans,9-cis- and Δ9-cis,11-trans-conjugated dienoic acids, respectively, but the Δ8-trans- and Δ10-trans-monoenoic acids did not give Δ8,9- or Δ9,10-allenes. Of thecis-monoenoic acids examined, only those with double bonds at or beyond the Δ14 position gave any measurable Δ9 desaturation. When Δ9 desaturation of long chain saturated acids was inhibited by adding sterculic acid, these saturated acids were desaturated at the Δ5 and Δ6 positions. Many of the monoenoic acids tested were also desaturated at the Δ5 and/or Δ6 positions, although the percentage conversions were always low. Δ9-cis,11-trans-, Δ9-cis,12-trans- and Δ9-cis,13-trans-dienoic acids, produced in situ by Δ9 desaturation of the corresponding monoenoic acids, were extensively desaturated in the Δ6 position. These results are discussed in terms of: (a) the various models proposed to explain the substrate specificities of the desaturases, and (b) the metabolism of unnatural fatty acids ingested from dietary sources.     

GLC and TLC analysis of isopropylidene derivatives of isomeric polyhydroxy acids derived from positional and geometrical isomers of unsaturated fatty acids

Gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were used to investigate the isomeric positional geometrical isopropylidene derivatives of nine isomeric dihydroxy esters, four isomeric methyl 9,10-12-trihydroxystearates, and eight isomeric methyl 9,10-12,13-tetrahydroxystearates prepared from unsaturated fatty acids. The isopropylidenes derived fromcis andtrans monounsaturated fatty acids were easily separated on both polar and nonpolar columns. Positional isopropylidenes derived from positional isomers of monounsaturated fatty acids were not separated on either liquid phase but were resolved by TLC. Four of the eight isomeric isopropylidenes derived from the four geometrical isomers of linoleic acid were resolved on the polar column; the other four isomers eluted as a single peak. The four isomeric isopropylidene-trifluoroacetate derivatives derived from ricinoleic and ricinelaidic acids were also resolved on the polar column. GLC analyses were carried out with liquid phases of ethylene glycol succinate methyl silicone polymer (EGSS-X) and methyl silicone polymer (SE-30) packed columns. Isopropylidenes, in addition to their applicability for the resolution of polyhydroxy acid mixtures, are particularly useful for the determination of double bond positions and geometrical configurations of fatty acids without cleavage. Under contract with the U. S. Atomic Energy Commission.     

Determination of positional distribution of butyryl groups in milkfat triacylglycerols,triacylglycerol mixtures,and isolated positional isomers of triacylglycerols by gas chromatography and1H nuclear magnetic resonance spectroscopy

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol;X,Y=long-chain acyls) of saturated triacylglycerols (TAG) with 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%) methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz¹H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The¹H NMR spectra of three interesterified mixtures of three monoacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups atsn-1(3)- andsn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.     

Rapid separation of neutral lipids,free fatty acids and polar lipids using prepacked silica sep-Pak columns

A method is described for the separation of neutral lipid, free fatty acid and polar lipid classes using small (600 mg), prepacked silica Sep-Pak columns. Combinations of hexane and methyltertiarybutylether were used to progressively elute cholesteryl ester first then triglyceride from the column. After column acidification, fatty acids were eluted followed by cholesterol. Recoveries of these lipids were 96% or greater. Polar lipids were eluted from the column using combinations of methyltertiarybutylether, methanol and ammonium acetate. Phospholipid classes could not be separated completely from each other. Phosphatidylethanolamine and phosphatidylinositol eluted together, whereas the more polar phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were eluted as a second fraction. Recoveries of each phospholipid was greater than 98%.     

Rapid separation of gangliosides using strong anion exchanger cartridges

A method utilizing strong anion exchanger cartridges (InertSep SAX) was developed to separate gangliosides. Total lipids extracted from rat brain is able to be rapidly separated into neutral and acidic lipids rapidly. Neutral lipids were passed through the SAX cartridge while acidic lipids adsorbed onto the cartridge and were eluted by chloroform/methanol/4.0 M aqueous ammonium acetate (5:10:1, by volume). Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) were separated individually according to their characteristics by elution with increasing concentration of ammnonium acetate (0 - 4.0 M). The gangliosides yield of this procedure was higher than 95%.     

High performance liquid chromatography and glass capillary gas chromatography of geometric and positional isomers of long chain monounsaturated fatty acids

Positional and geometrical isomers of monounsaturated long chain fatty acids were analyzed by the combination of high performance liquid chromatography (HPLC) and glass capillary gas chromatography (GC). A preparative group separation ofcis andtrans isomers of the monounsaturated fatty acid methyl esters was achieved according to chain length by reversed-phase HPLC, and using a highly sensitive interference refractive index detector. After collection of the different fractions containingcis andtrans forms of the monounsaturated fatty acid methyl esters, the fractions were analyzed for their content of positional isomers using glass capillary GC with Silar-5 CP as stationary phase. The preparative step in the HPLC was also used analytically for the determination of the ratio between thecis andtrans monounsaturated fatty acids. A comparison was made between the results obtained with the HPLC technique and the results of a GLC technique with a packed OV-275 column. There was a good correlation between the 2 techniques with a tendency to highertrans values with the HPLC technique (4%). It was shown with reference substances that 18∶1ω6-cis to ω11-cis and 18∶1ω5-trans to ω12-trans, the most common monounsaturated fatty acid isomers in partially hydrogenated vegetable oils, could be almost quantitatively recovered in the HPLC step. Most of the individual positional isomers of monounsaturated fatty acids of varying chain length could be separated and determined in the glass capillary GC step with the exception of those isomers containing the double bond in a relatively high ω-position. The relative standard deviation of the technique as determined with reference substances was better than 4%. The described technique was applied to the analysis of the isomeric monounsaturated fatty acid content in partially hydrogenated vegetable and marine oils, and about 5 samples a day could be executed. Part of this work has been presented at the ISF/AOCS World Congress, New York (1980)JAOCS 58, (4), 1981, abstr. no. 184.     

Synthesis of triacylglycerol containing conjugated linoleic acid by esterification using two blended lipases

We have developed an efficient esterification for the synthesis of triacylglycerol (TAG) containing conjugated linoleic acids (CLA) using a blend of two powdered lipases. Two pairs of blended lipases promoted the esterification. Rhizomucor miehei lipase, plus Alcaligenes sp. lipase and Penicillium cammembertii MAG and DAG lipase plus Alcaligenes sp. lipase were used. At the optmal ratio of two lipases, the content of TAG containing CLA (TAG-CLA) in all glycerols reached 82–83% after 47 h using 1 wt% of lipases. With R. miehei lipase plus Alcaligenes sp. lipase, the reaction time to obtain ca. 60% of TAG-CLA was one-third of that needed with R. miehei lipase alone. The optimal ratio of two lipases differed between these two pairs. The optimal ratio was 70–80 wt% of R. miehei lipase in the last stage of the reaction, whereas it was over a wide range of 10–90 wt% for P. camembertii lipase. In the blend of R. miehei lipase plus Alcaligenes sp. lipase, activity remained very high after 10 cycles of esterification (every 47 h) and could be used in the industrial production of TAG-CLA.     

Determining the relative amounts of positional isomers in complex mixtures of triglycerides using reversed-phase high-performance liquid chromatography-tandem mass spectrometry

A reversed-phase HPLC-tandem mass spectrometry (RP-HPLC-MS-MS) method was refined for the positional analysis of complex mixtures of TAG. This method has the advantages of speed, ease of automation, and specificity over traditional digestion-based methods for the positional analysis of TAG. Collision-induced dissociation (CID) of ammoniated TAG in an ion-trap mass spectrometer produced spectra that were dependent on the FA position. Dominant DAG fragments were formed from the loss of a FA moiety from the ammoniated TAG species. The loss of FA in the outer positions was favored over their loss in the central position. The combination of RP-HPLC and CID produced spectra that were free of the isotope effects that can complicate spectral interpretation in existing methods. The combination also provided selectivity based on the chromatographic fractionation of TAG, in addition to the selectivity inherent in the CID process. Proof-of-concept experiments were performed with binary mixtures of TAG from the SOS/SSO, OSO/OOS, and the PSO/POS/SPO positional isomer systems (where S is 18∶0, stearic acid; O is 18∶1 (cis-9), oleic acid; and P is 16∶0, palmitic acid). Plots of fractional DAG fragment intensities vs. fractional composition of the binary mixtures were linear. These plots were used to determine the fractional composition of each of these isomeric systems in a variety of vegetable oils and animal fats. Current limitations, future developments, and applications of this method are discussed.     

Rapid isolation of high-affinity protein binding peptides using bacterial display

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.     

Method for separation of particles using a rotating titled liquid column (RTC). II. Closed and open-bottom column systems

A new method for selective separation of particulate materials has been developed, using a rotating tilted liquid column (RTC) with closed or open bottom. As particles move with RTC velocities determined by pseudo-circular loci which approach eccentrically to the inside wall of RTC, the most influential factor for the separation is the interaction between the particles and the column wall. To evaluate the influence of the interaction, we calculated the loci with a parameter Aω/V₀ (where A is the radius of the column, ω is the angular velocity of the column, and V₀ is the sedimentation velocity).Exploratory tests in closed column indicate that RTC velocity V(ω, θ) is represented by the following equation: V(ω, θ)/sin θ = uV₀ ? bAω/V₀, where θ is the inclination of the column to the horizontal, u = 1.103, and b = 0.16 [LT^?1] (at θ = 30 – 45°, 26 – 30 °C).In a practical application of this method, the open-bottom column was used for separation of fine particles such as starch particles.     

Comprehensive two dimensional gas chromatographic separation of fatty acids methyl esters with online reduction

The accurate analysis of complex fats and oils often requires combining data from multiple GC separations, fractionation by LC prior to GC analysis, and identifying fatty acids or confirming their identities by mass spectrometry and synthetic reference materials. Comprehensive two dimensional gas chromatography markedly increases the number of resolved fatty acid methyl esters (FAME) in a single separation experiment by combining the separations of two capillary columns. The addition of a capillary reducer in front of the second capillary column provides unique separation patterns and structural information for fatty acids based on simple elution rules.     

Separation of fatty acids by crystallization using two liquid phases

First, the phase diagrams for the water+ethanol+fatty acids system were described, and the separation method was considered. Second, the separation of lauric acid+myristic acid mixture in the ethanol aqueous solution including ethanol by liquid-liquid extraction and crystallization has been examined. The extraction of fatty acids from the organic phase to the aqueous solution has been done. Fatty acids in the aqueous solution were crystallized using the aqueous SLE surface. The solvent-free mole fraction of lauric acid in the aqueous solution was a little higher than that in the organic phase. Fine crystals made in the aqueous solution had better purity for lauric acid than the solvent-free mole fraction of lauric acid in the aqueous solution. The effects of the mole fraction of ethanol in the aqueous solution, the mole fraction of lauric acid in the organic solution, extraction temperature and crystallization temperature on the purity of the crystals were reported.     

螺旋构件强化排液的泡沫分离塔用于牛血清蛋白分离

引言 泡沫分离技术又称泡沫吸附分离技术[1],是以气泡作为分离介质,利用被分离组分在气液两相界面吸附性质的差异进行浓缩溶液中表面活性组分.在泡沫分离过程中,通过空气分布器在泡沫塔液相中产生气泡,气泡沿着轴向向上流动,被分离表面活性组分吸附在气泡的气液两相界面上,当吸附接近平衡后,气泡离开液相,在液相上方形成泡沫相.     

Reverse osmosis separation of organic acids in aqueous solutions using porous cellulose acetate membranes

Reverse osmosis data obtained using porous cellulose acetate membranes and aqueous feed solutions involving one of 22 monocarboxylic acids, seven dicarboxylic acids, and four hydroxycarboxylic acids have been analyzed. The operating pressure used was 250 psig in all cases, and the solute concentration used was ～100 ppm in most cases. The results yield the following physicochemical criteria for preferential sorption at the membrane–solution interface with respect to the un-ionized acid. At pK_a = 4.2 (for monocarboxylic acids), or Taft number (σ^*) = 0.6 or Hammett number (σ) = 0, neither the un-ionized acid nor water is preferentially sorbed at the membrane–solution interface; at pK_a < 4.2 (for monocarboxylic acids) or σ^* > 0.6 or σ > 0, the un-ionized acid is preferentially sorbed at the membrane–solution interface. For practical purposes, preferential sorption of water at the membrane–solution interface may be considered negligible in the σ^* region of 0 to 0.6. The results also show that the criterion of acidity of the molecule governing the extent of its repulsion or attraction at the membrane–solution interface is valid for both ionized and un-ionized acid. Further, when the acid molecule contains three or more straight-chain carbon atoms not associated with a ? COOH group, the nonpolar character of the molecule also affects its separation in reverse osmosis.