Mass Spectrometry of the Lithium Adducts of Diacylglycerols Containing Hydroxy FA in Castor Oil and Two Normal FA

Castor oil can be used in industry. The molecular species of triacylglycerols containing hydroxy fatty acids (FA) in castor oil have been identified. We report here the identification of twelve diacylglycerols (DAG) containing hydroxy FA in castor oil using positive ion electrospray ionization mass spectrometry of the lithium adducts. They were RR (diricinolein, R is ricinoleate), RL, RS, R‐diOH18:0, R‐diOH18:1, R‐diOH18:2, R‐triOH18:0, R‐triOH18:1, R‐triOH18:2, diOH18:0‐diOH18:1, diOH18:1‐diOH18:1 and diOH18:1‐diOH18:2. The MS² fragment ions, [M + Li ? FA]⁺ and [FA + Li]⁺, from the lithium adducts of DAG containing hydroxy FA (one or two hydroxy FA), were used for the identification. The additional fragment ions from the neutral losses of FA lithium salts [M + Li ? FALi]⁺ were used for the identification of eleven DAG containing two normal FA in a soybean oil bioconversion product. The MS² fragment ions from the neutral losses of FA lithium salts [M + Li ? FALi]⁺ were not detected from the DAG containing hydroxy FA. The DAG containing FA with more hydroxyl groups than the other FA on the same DAG molecule tended to have a prominent fragment ion [FA + Li]⁺ and an undetectable fragment ion [M + Li ? FA]⁺ while the FA was the more hydroxylated FA. Also the less hydroxylated FA of a DAG tended to have a prominent fragment ion [M + Li ? FA]⁺ and an undetectable fragment ion [FA + Li]⁺ while the FA was the less hydroxylated FA.     

Identification of TAG and DAG and their FA Constituents in Lesquerella (Physaria fendleri) Oil by HPLC and MS

Castor oil has many industrial uses because of its high content (90 %) of the hydroxy fatty acid, ricinoleic acid (OH¹²18:1⁹). Lesquerella oil containing lesquerolic acid (Ls, OH¹⁴20:1¹¹) is potentially useful in industry. Ten molecular species of diacylglycerols and 74 molecular species of triacylglycerols in lesquerella (Physaria fendleri) oil were identified by electrospray ionization mass spectrometry as lithium adducts of acylglycerols in the HPLC fractions of lesquerella oil. Among them were: LsLsO, LsLsLn, LsLsL, LsLn–OH20:2, LsO–OH20:2 and LsL–OH20:2. The structures of the four new hydroxy fatty acid constituents of acylglycerols were proposed by the MS of the lithium adducts of fatty acids as (comparing to those in castor oil): OH¹²18:2^9,14 (OH¹²18:2^9,13 in castor oil), OH¹²18:3^9,14,16 (OH18:3 not detected in castor oil), diOH^12,1318:2^9,14 (diOH^11,1218:2^9,13 in castor oil) and diOH^13,1420:1¹¹ (diOH20:1 not detected in castor oil, diOH^11,1218:1⁹ in castor oil). Trihydroxy fatty acids were not detected in lesquerella oil. The differences in the structures of these C18 hydroxy fatty acids between lesquerella and castor oils indicated that the polyhydroxy fatty acids were biosynthesized and were not the result of autoxidation products.     

Ratios of Regioisomers of the Molecular Species of Triacylglycerols in Lesquerella (Physaria fendleri) Oil Estimated by Mass Spectrometry

The ratios of regioisomers of 72 molecular species of triacylglycerols (TAG) in lesquerella oil were estimated using the electrospray ionization mass spectrometry of the lithium adducts of TAG in the HPLC fractions of lesquerella oil. The ratios of ion signal intensities (or relative abundances) of the fragment ions from the neutral losses of fatty acids (FA) as α‐lactones at the sn‐2 position (MS³) of the molecular species of TAG were used as the ratios of the regioisomers. The order of the preference of FA incorporation at the sn‐2 position of the molecular species of TAG in lesquerella was as: normal FA > OH18 (monohydroxy FA with 18 carbon atoms) > diOH18 > OH20 > diOH20, while in castor was as: normal FA > OH18 > OH20 > diOH18 > triOH18. Elongation (from C₁₈ to C₂₀) was more effective than hydroxylation in lesquerella to incorporate hydroxy FA at the sn‐1/3 positions. The block of elongation in lesquerella may be used to increase the content of hydroxy FA, e.g., ricinoleate, at the sn‐2 position of TAG and to produce triricinolein (or castor oil) for industrial uses. The content of normal FA at the sn‐2 position was about 95 %, mainly oleate (38 %), linolenate (31 %) and linoleate (23 %). This high normal FA content (95 %) at the sn‐2 position was a big space for the replacement of ricinoleate to increase the hydroxy FA content in lesquerella oil. The content of hydroxy FA at the sn‐1/3 positions was 91 % mainly lesquerolic acid (85 %) and the content of normal FA was 6.7 % at the sn‐1/3 position in lesquerella oil.     

Comparative analysis of triacylglycerols from Olea europaea L. fruits using HPLC and MALDI‐TOFMS

MALDI‐TOFMS and HPLC are two analytical methods that were used to characterize triacylglycerols (TAG) of the Meski, Sayali, and Picholine Tunisian olive varieties. The HPLC chromatograms of the oils showed the presence of 15 TAG species, among which triolein (OOO) was the most abundant (21–48%). In the Sayali cultivar, OOO was the predominant TAG species followed by POO and LOO. However, the minor TAG molecules were represented by LnLO and LnLP. MALDI mass spectra produced sodiated ([M + Na]⁺) and potassiated ([M + K]⁺) TAG molecules; only the major TAG were potassiated [OOO + K] ([OOO + K]⁺, [POO + K]⁺, and [LOO + K]⁺). In contrast to the HPLC chromatograms, the MALDI mass spectra showed 13 peaks of TAG. The major peak was detected at m/z 907, which corresponds to OOO with an Na⁺ adduct. The results from both HPLC and MALDI techniques predict the fatty acid composition and their percentages for each olive variety. Practical applications: TAG are the main components in vegetable oils. These biomolecules determine the physical, chemical, and nutritional properties of the oils. The nutritional benefits of TAG are related to DAG (moderate plasma lipid level) and esterified FA, which are intermediate biosynthetic molecules of TAG. TAG analysis is necessary to discriminate between oils of different origin, since some oils have similar FA profiles. Olive products, oils, and table olives, are the main diet sources of TAG in the Mediterranean countries. In this work, chromatographic and spectrometric methods were used for TAG analysis and characterization of Tunisian olive varieties.     

The bottom-up solution to the triacylglycerol lipidome using atmospheric pressure chemical ionization mass spectrometry

Presented here is an approach to representing the data from atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG) using a set of one, two, or three Critical Ratios. These Critical Rations may be used directly to provide structural information concerning the regioisomeric composition of the triacylglycerols (TAG), and about the degree of unsaturation in the TAG. An AAA-type, or Typel, TAG has only one Critical Ratio, the ratio of the protonated molecule, [M+H]⁺, to the DAG fragment ion, [AA]⁺. The Critical Ratio for a Type I TAG is [MH]⁺/Σ[DAG]⁺, and the mass spectrum of a Type I TAG can be reproduced from only this one ratio. An ABA/AAB/BAA, or Type II, TAG has two Critical Ratios, the [MH]⁺/Σ[DAG]⁺ ratio and the [AA]⁺/[AB]⁺ ratio. The [AA]⁺/[AB]⁺ ratio for a single TAG or TAG mixture can be compared with the [AA]⁺/[AB]⁺ ratios of pure regioisomeric standards, and the percentage of each regioisomer can be estimated. The abundance of the protonated molecule and the abundances of the two [DAG]⁺ fragment ions can be calculated from the two Critical Ratios for a Type II TAG. To calculate the abundances, the Critical Ratios are processed through the Bottom-Up Solution to the TAG lipidome. First, Critical Limits are calculated from the Critical Ratios, and then the Critical Ratios are classified into Cases by comparison with the Critical Limits. Once the Case classification is known, the equation for the abundance of each ion in the mass spectrum is given by the Bottom-Up Solution. A Type III TAG has three different FA and three Critical Ratios. The [MH]⁺/Σ/[DAG]⁺ ratio is the first Critical Ratio, the [AC]⁺/([AB]⁺+[BC]⁺) ratio is the second Critical Ratio, and the [BC]⁺/[AB]⁺ ratio is the third Critical Ratio. The second critical ratio for a Type III TAG can be compared with regioisomeric standards to provide an estimate of the percentage composition of the regioisomers. The three Critical Ratios for a Type III TAG can be processed through the Bottom-Up Solution to calculate the four ion abundances that make up the APCI-MS mass spectrum. The Critical Ratios constitute a reduced data set that provides more information in fewer values than the raw abundances.     

The Updated Bottom Up Solution Applied to Atmospheric Pressure Photoionization and Electrospray Ionization Mass Spectrometry

The Updated Bottom Up Solution (UBUS) was recently applied to atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) of triacylglycerols (TAG). This report demonstrates that the UBUS applies equally well to atmospheric pressure photoionization (APPI) MS and to electrospray ionization (ESI) MS. Critical Ratio 1 (CR1), the [MH]⁺/Σ[DAG]⁺ or [MNH₄]⁺/Σ[DAG]⁺ ratio, does not exhibit the same strongly sigmoidal shape as it does by APCI‐MS. CR1 varies more widely for APPI‐MS than by APCI‐MS, having a maximum value of 11.8, indicating a much greater effect of unsaturation on ion ratios in APPI‐MS than APCI‐MS. Critical Ratio 2, the [AA]⁺/[AB]⁺ ratio for Type II TAG or [AC]⁺/([AB]⁺+[BC]⁺) ratio for Type III TAG, allows quantification of regioisomers of TAG, and shows good agreement for APPI‐MS to regioisomer quantification determined by APCI‐MS. Critical Ratio 3, the [BC]⁺/[AB]⁺ ratio for Type III TAG, reveals new trends relating the degree of unsaturation by APPI‐MS, and shows that structural assignments made by ESI‐MS are in good agreement to APCI‐MS data. In addition to providing valuable structural information, the Critical Ratios also constitute a reduced data set that allows APPI‐MS or ESI‐MS mass spectra to be reconstructed when processed through the UBUS. Quantification by APPI‐MS of vitamin D in the gelcaps gave values of 42.90 ± 0.83 μg, or 1716 ± 33 international units, in good agreement with APCI‐MS.     

Preparation of stearidonic acid‐enriched triacylglycerols from Echium plantagineum seed oil

HPLC analysis of Echium plantagineum seed oil shows a complex triacylglycerol (TAG) profile. TAG species were separated on an analytical scale by HPLC and their fatty acid (FA) composition is reported. GLC analyses showed that some TAG fractions reached a stearidonic acid (SDA, 18:4n‐3) percentage significantly higher than that in the original oil. TAG separation on a bigger scale was also essayed, by means of a gravimetric normal‐phase chromatographic column, using silver ion‐silica gel as stationary phase. Gradient elution with solvents of increasing polarity was applied, allowing the separation of valuable TAG species containing γ‐linolenic acid (GLA, 18:3n‐6), α‐linolenic acid (ALA, 18:3n‐3) and SDA as the main constituents (more than 85% of the total FA). An enzymatic hydrolysis reaction showed the distribution of FA in the isolated species of TAG. SDA was the major FA in the sn‐2 position (more than 50% of total FA), followed by ALA (19%) and GLA (18.5%).     

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.     

Changes in olive oil composition due to microwave heating

The effects of microwave heating on some components of extra-virgin olive oil were studied. Traditional parameters, including free acidity, peroxide value and ultraviolet absorbance values at 232 and 268 nm, were determined in six extra-virgin olive oil samples before and after the microwave treatment. Significant differences (P<0.01) were detected for free acidity, peroxide, and ultraviolet absorbance at 268 nm; also, the absorbances at 232 nm showed significant differences (P<0.05) between treated and untreated samples. The glycerolic fractions, triacylglycerols (TAG), diacylglycerols (DAG), and monoacylglycerols (MAG), were isolated by thin-layer chromatography. The respective percentage fatty acid (FA) composition and percentage amount were obtained by high-resolution gas chromatography with an internal standard. For the most abundant TAG fraction, the stereospecific analysis was carried out to obtain the FA percentage compositions of the three sn-positions. Small but significant modifications were observed regarding the decrease in the TAG percentage and increases in the DAG and MAG percentage amounts. No significant changes were observed for the FA compositions of TAG, DAG, and MAG fractions before and after the treatment. Nevertheless, the results of TAG stereospecific analysis showed losses of unsaturated FA in all sn-positions. Higher percentage changes in the sn-1- than in sn-2-position of TAG were observed. Regarding the volatile fraction, different profiles were obtained after the treatment.     

Characterization of triacylglycerols in madeira laurel oil by HPLC-atmospheric pressure chemical ionization-MS

Madeira laurel oil was fractionated by liquid extraction combined with TLC, and TAGs were analyzed by HPLC coupled with atmospheric pressure chemical ionization-MS (APCI-MS). Eluted molecular species compositions of the eluted TAG in the complex natural mixture were determined by GC identification of FAME and byLC-atmospheric pressure chemical ionization (APCI)-MS analysis of the lipid. The APCI-MS spectra of most TAG exhibited [M+H]⁺ and [M−RCOO]⁺ ions, which defined the M.W. and the molecular association of fatty acyl residues, respectively. Despite the relatively high degree of saturation, with a saturated/unsaturated ratio of 0.70, no totally saturated TAG nor mixed asymmetric TAG with two saturated FA (SSM or SSU, where S is saturated, M is monounsaturated, and U is unsaturated) were found. This type of molecular structure provides a possible explanation for the relatively low m.p. (12–15°C) and also the high oxidative resistance observed.     

The regiospecific position of 18∶1 cis and trans monoenoic fatty acids in milk fat triacylglycerols

TAG of butterfat were fractionated according to the type and degree of unsaturation into six fractions by silver-ion HPLC. The fractions containing TAG with either cis-or trans-monoenoic FA were collected and fractionated further by reversed-phase HPLC to obtain fractions containing cis TAG of ACN:DB (acyl carbon number:double bonds) 48∶1, 50∶1, and 52∶1 as well as trans 48∶1, 50∶1, and 52∶1. The FA compositions of these fractions were elucidated by GC. The MW distribution of each fraction was determined by ammonia negative-ion CI-MS. Each of the [M-H]⁻ parent ions was fractionated further by collision-induced dissociation with argon, which gave information on the location of cis-and trans-FA between the primary and secondary positions of TAG. The results suggest that the sn-positions of the monoenoic cis-and trans-FA depend on the two other FA present in the molecule. With 14∶0 FA in the TAG molecule, the 18∶1 FA in the sn-2 position are mostly present as cis-isomers. When there is no 14∶0 in the TAG molecule, the trans-18∶1 isomers seem to be more common in the sn-2 position. Also when other long-chain FA are present, the trans-isomers are more likely to be located in the secondary (sn-2) position.     

Incorporation of laurate and hydroxylaurate into phosphatidylcholines and acylglycerols in castor microsomes

Because castor produces oil with a high content of hydroxyl FA (90% ricinoleate), we were interested in determining the flexibility of castor seed microsomes in incorporating other hydroxyl FA into castor oil. To this end, we incubated the [¹⁴C]-labeled 12:0 FA laurate (La), 11-hydroxylaurate, and 12-hydroxylaurate with castor microsomes that were capable of synthesizing castor oil. The molecular species of PC and acylglycerols (AG) incorporating these nonendogenous FA of castor were identified by reversed-phase C₈ and C₁₈ HPLC, respectively. [¹⁴C]Laurate was incorporated into the molecular species of PC and AG at levels of 10 and 4%, respectively, that of [¹⁴C]ricinoleate. Similar to those from the incorporation of six [¹⁴C]FA reported previously [ricinoleate (R), oleate (O), linoleate (L), linolenate (Ln), stearate (S), and palmitate (P)], the molecular species of PC incorporating [¹⁴C]laureate were LLa-PC>PLa-PC>OLa-PC>LnLa-PC>SLa-PC>RLa-PC. The molecular species of AG incorporating [¹⁴C]laurate were RRLa>LaLa>RLa>RLLa>ROLa>LOLa>LLLa>LLa>LLnLa>RSLa>OOLa. The retention times for lipids incorporating laurate were similar to those of lipids incorporating linolenate, because the equivalent carbon numbers of laurate and linolenate are the same. Relative retention times of the molecular species of PC and AG containing laurate are also reported here. The incorporation of 11-hydroxylaurate and 12-hydroxylaurate into PC and AG was not detected.     

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.     

Structural characterization of triacylglycerols using electrospray ionization-MS<Superscript>n</Superscript> ion-trap MS

Structural analysis of mixtures of TAG requires the determination of the M.W. of each compound, the characterization of the structure(s) of the individual FA substituents, and identification of the relative disposition of the acyl groups on the glycerol backbone (regiospecificity). In this study we demonstrated that ion-trap MS in combination with electrospray ionization provides an easy and detailed characterization of the TAG structure. We showed that electrospray mass spectra are characterized by intense molecular ion adducts and that the acyl group disposition on the TAG backbone can be determined on the basis of relatively well-defined quantitative differences of fragment ions produced from MS² data. Moreover, additional spectral data can be generated by MS³ experiments on moieties containing individual acyl ion fragments. When hyphenated with chromatographic separations ion-trap electrospray MS might become a routine and powerful way to analyze TAG mixtures.     

The Simulacrum System as a Construct for Mass Spectrometry of Triacylglycerols and Others

A construct called a simulacrum is defined that provides all possible solutions to a sum of two mass spectral abundances, based on values (abundances) or ratios of those values. The defined construct is applied to atmospheric pressure chemical ionization mass spectrometry (MS) of triacylglycerols (TAG). A simulacrum has precisely defined components, specifically a simulacrum sum, four Possibilities to Observe, two Cases, and eight solutions. A simulacrum with no restrictions is the First General Form of a Simulacrum. When one value is specified to be 1 (as in MS), the construct is called a Unit Simulacrum, also called the First Specified Form of a Simulacrum. When one value is 1 and no value can be greater than 1 (the two specifications dictated by mass spectrometry), the construct is called the Second Specified Form of a Simulacrum, or the Mass Spectrometry Simulacrum. Simulacra are used with three Critical Ratios calculated from raw abundances in mass spectra of TAG to provide structural information about the degree of unsaturation in TAG, the identity and quantity of regioisomers, and other structural characteristics. Three‐level‐deep nested simulacrum solutions yield the recently reported Updated Bottom Up Solution, from which the protonated molecule, [MH]⁺, and all diacylglycerol‐like fragments, [DAG]⁺, of TAG can be reproduced from the Critical Ratios. Thus, the simulacrum solutions constitute a reduced data set in which more information is provided in fewer values than raw abundances, such that the Critical Ratios constitute a compact library of mass spectra.     

Analysis of the seed oil of Heisteria silvanii (Olacaceae)—A rich source of a novel C18 acetylenic fatty acid

Besides some usual fatty acids (FA), two conjugated ene-yne acetylenic FA [trans-10-heptadecen-8-ynoic acid (pyrulic acid) (7.4%), and trans-11-octadecen-9-ynoic acid (ximenynic acid) (3.5%)], a novel ene-yne-ene acetylenic FA [cis-7, trans-11-octadecadiene-9-ynoic acid (heisteric acid) (22.6%)], and 9,10-epoxystearic acid (0.6%) could be identified in the seed oil of Heisteria silvanii (Olacaceae). Two further conjugated acetylenic FA [9,11-octadecadiynoic acid (0.1%) and 13-octadecene-9,11-diynoic acid (0.4%)] were identified tentatively by their mass spectra. The FA mixture has been analyzed by gas chromatography/mass spectrometry (GC/MS) of their methyl ester and 4,4-dimethyloxazoline derivatives. The structure of heisteric acid was elucidated after isolation via preparative silver ion thin-layer chromatography and by various spectroscopic methods [ultraviolet; infrared; ¹H, ¹³C nuclear magnetic resonance (NMR); ¹H−¹H and ¹H−¹³C correlation spectroscopy]. To determine the position of the conjugated ene-yne-ene system, the NMR spectra were also measured after addition of the lanthanide shift reagent Resolve-Al EuFOD^TM. Furthermore, the triyglyceride mixture was analyzed by high-temperature GC and high-temperature GC coupled with negative chemical ionization MS. A glass capillary column coated with a methoxy-terminated 50%-diphenyl-50%-dimethylpolysiloxane was used for the separation of the triacylglycerol (TAG) species. No evidence of decomposition of the TAG species containing conjugated ene-yne-ene FA was observed. Twenty-six species of the separated TAG were identified by means of their abundant quasi molecular ion [M−H]⁻ and their corresponding carboxylate anions [RCOO]⁻ of the fatty acids, respectively. The major molecular species of the TAG were found to be 16:0/18:1/18:1, 16:0/18:1/18:3 (heisteric acid), 17:2 (pyrulic acid)/18:1/18:1, 18:1/18:1/18:3 (heisteric acid). The TAG containing acetylenic FA showed an unexpected increase of the retention time in comparison to the TAG containing usual FA, thus making the prediction of the elution order of lipid samples containing acetylenic FA difficult.     

Production of FAME from acid oil,a by-product of vegetable oil refining

Simple alkyl FA esters have numerous uses, including serving as biodiesel, a fuel for compression ignition (diesel) engines. The use of acid-catalyzed esterification for the synthesis of FAME from acid oil, a by-product of edible vegetable oil refining that is produced from soapstock, was investigated. Soybean acid oil contained 59.3 wt% FFA, 28.0 wt% TAG, 4.4 wt% DAG, and less than 1% MAG. Maximum esterification occurred at 65°C and 26 h reaction at a molar ratio of total FA/methanol/sulfuric acid of 1∶15∶1.5. Residual unreacted species under these conditions, as a fraction of their content in unesterified acid oil, were FFA, 6.6%; TAG, 5.8%; and DAG, 2.6%. This corresponds to estimated concentrations of FFA, 3.2%; TAG, 1.3%; and DAG, 0.2%, on a mass basis, in the ester product. In an alternative approach, the acylglycerol species in soapstock were saponified prior to acidulation. High-acid (HA) acid oil made from this saponified soapstock had an FFA content of 96.2 wt% and no detectable TAG, DAG, or MAG. Optimal esterification conditions for HA acid oil at 65°C were a mole ratio of FFA/methanol/acid of 1∶1.8∶0.17, and 14 h incubation. FAME recovery under these conditions was 89% of theoretical, and the residual unesterified FFA content was approximately 20 mg/g. This was reduced to 3.5 mg/g, below the maximum FFA level allowed for biodiesel, by washing with NaCl, NaHCO₃, and Ca(OH)₂ solutions. Alternatively, by subjecting the unwashed ester layer to a second esterification, the FFA level was reduced to less than 2 mg/g. The acid value of this material exceeded the maximum allowed for biodiesel, but was reduced to an acceptable value by a brief wash with 0.5 N NaOH.     

Acylglycerols Containing Trihydroxy Fatty Acids in Castor Oil and the Regiospecific Quantification of Triacylglycerols

Ricinoleate, a monohydroxy fatty acid in castor oil, has many industrial uses. Dihydroxy and trihydroxy fatty acids can also be used in industry. We report here the identification of diacylglycerols (DAG) and triacylglycerols (TAG) containing trihydroxy fatty acids in castor oil. The C₁₈ HPLC fractions of castor oil were used for mass spectrometry of the lithium adducts of acylglycerols to identify trihydroxy fatty acids and the acylglycerols containing trihydroxy fatty acids. Two DAG identified were triOH18:1–diOH18:1 and triOH18:0–OH18:1. Four TAG identified were triOH18:1–OH18:1–OH18:1, triOH18:0–OH18:1–OH18:1, triOH18:1–OH18:1–diOH18:1 and triOH18:0–OH18:1–diOH18:1. The structures of these two newly identified trihydroxy fatty acids were proposed as 11,12,13-trihydroxy-9-octadecenoic acid and 11,12,13-trihydroxyoctadecanoic acid. The locations of these trihydroxy fatty acids on the glycerol backbone were almost 100% at the sn-1,3 positions or at trace levels at the sn-2 position. The content of these acylglycerols containing trihydroxy fatty acids was at the level of about 1% or less in castor oil.     

Effects of a Single and Short-Term Ingestion of Diacylglycerol on Fat Oxidation in Rats

This study examines the effect of diacylglycerol (DAG) oil consisting mainly of 1,3-species on fat oxidation as a possible mechanism for anti-obesity. We examined the following: (1) the long-term (23-week) effects of a DAG oil diet on the development of obesity; (2) the effect of a single ingestion of DAG oil on fat oxidation; and, (3) the short-term (2-week) effect of a DAG oil diet on fat metabolism in rats. Rats fed a DAG oil diet accumulated significantly less body fat compared to rats fed a triacylglycerol (TAG) oil diet, each oil possesses a similar fatty acid composition. More ¹⁴C-CO₂ was expired and less ¹⁴C-radioactivity was incorporated into visceral fat after administration of a tracer emulsion containing 1,3-[oleoyl-1-¹⁴C] diolein compared to [carboxyl-¹⁴C] triolein. Indirect calorimetry showed respiratory quotients were significantly lower in the DAG oil diet group than in the TAG oil diet group. More ¹⁴C-CO₂ was expired and less ¹⁴C-radioactivity was incorporated into visceral fat in the DAG oil diet group than in the TAG oil diet group after a single intragastric administration of [carboxyl-¹⁴C] triolein. These results suggest the following. (1) DAG oil has an inhibitory effect on diet-induced fat accumulation. (2) 1,3-DAG, a major component of DAG oil, is more susceptible to oxidation. (3) A short-term ingestion of DAG oil increases fat utilization at the whole body level and results in increased oxidation of dietary fat. The stimulated fat oxidation might be one explanation for the anti-obesity effect of long-term DAG oil ingestion.     

Comparison of the lymphatic transport of radiolabeled 1,3-dioleoylglycerol and trioleoylglycerol in rats

It has been reported that, compared with TAG, DAG suppresses postprandial hypertriacylglycerolemia and reduces visceral fat levels in experimental animals and humans. To clarify the mechanism responsible for these beneficial effects, we compared the lymphatic transport of 1,3-DAG, a major isomer of DAG, and TAG in rats. Male SD rats, after insertion of a cannula into the thoracic duct, were given 1,3-di[1-¹⁴C]oleoylglycerol or tri[1-¹⁴C]oleoylglycerol via a stomach tube. The 24-h receovery of the radioactivity from 1,3-di[¹⁴C]oleoylglycerol in the lymph was slightly but significantly lower than that from tri[¹⁴C]oleoylglycerol (81.3±1.0 vs. 86.5±1.2%, respectively). However, in the first 1-h interval after administration, the recovery of radioactivity from 1,3-dioleoylglycerol was almost half of that from trioleoylglycerol (17.5±2.0 vs. 31.1±1.4%). The amount of TAG and phospholipids secreted into the lymph was significantly lower 1 h after the administration of 1,3-dioleoylglycerol compared with that after the administration of trioleoylglycerol. More than 90% of the radioactivity recovered in the lymph in the first 3 h was distributed in the TAG fraction for both 1,3-dioleoylglycerol and trioleoylglycerol. These results suggest that slower lymphatic transport of 1,3-DAG compared with TAG could be a factor in the suppression of postprandial hypertriacylglycerolemia. The possibility that the slower lymphatic transport of DAG contributes to the antiobesity action observed in the feeding of 1,3-DAG cannot be excluded.