Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of Feeding Pigs Increasing Levels of C 18:1 Trans Fatty Acids on Fatty Acid Composition of Backfat and Intramuscular Fat as well as Backfat Firmness

Forty Large White pigs were fed from 30kg to 103kg body mass on diets supplemented with 6% of pure high-oleic sunflower oil (HO) or HO plus increasing amounts of partially hydrogenated rape seed oil (HR; 1.85%, 3.70%, 5.55%), containing high levels of j 6 to j 11 C 18:1 trans fatty acid isomers. Increasing dietary C 18: trans fatty acids resulted in a linear increase in C 18:1 trans fatty acids and conjugated linoleic acid (cis-9, trans-11 CLA) in backfat (BF) as well as in neutral lipids (NL) and phospholipids (PL) of M. long. dorsi. Thus, the rate of bioconversion of trans vaccenic acid (TVA) into CLA and incorporation of C 18:1 trans and CLA into pig adipose tissue was not limited up to 25g total C 18:1 trans fatty acids including 3.3g of TVA perkg feed. BF was higher in C 18:1 trans fatty acids and CLA than M. long. dorsi NL and PL. In BF and NL the sum of saturated fatty acids (SFA) increased with increasing dietary amounts of HR, while in PL SFA were reduced. Thus, according to their physical properties, C 18:1 trans fatty acids partly replaced SFA in PL. Firmness of backfat was also significantly increased (P<0.05) with increasing amounts of HR in feed.     

Thermal degradation and isomerisation kinetics of triolein studied by infrared spectrometry and GC-MS combined with chemometrics

Triolein, a triglyceride containing oleic acid as the only acid moiety in the glyceride molecules has been isothermally treated at 280, 300, and 325 °C in glass vials under nitrogen atmosphere. The products formed during the thermal treatment at each temperature have been analysed both by infrared spectrometry and GC-MS. The GC-MS analysis was performed after derivatisation of the fatty acids into their methyl esters (FAMEs).Chemometric tools were used in determining the concentrations of the main products namely triolein and trieaidin in the thermally treated mixtures. The concentration profiles of the trielaidin formed during thermal treatment at the above three temperatures were used in determining activation energy for the cis-trans isomerisation of triolein.The combined analysis reveals that the thermal treatment induces not only cis-trans isomerisation but also fission and fusion in the molecules. Furthermore, migration of the double bond in oleic and elaidic acids forming cis and trans isomers of the 18:1 acid was also observed. The heat-induced isomerisation in triolein follows a zeroth order reaction with an activation energy 41 ± 5 kcal/mol.     

Quantification of retinyl-β-glucuronides in rat urine by reversed-phase high-performance liquid chromatography with ultraviolet detection

A method is presented for the quantitation of the glucuronide conjugates of 4-oxo-all-trans-, 4-oxo-13-cis-, 13-cis-, 9-cis- and all-trans-retinoic acids in rat urine utilizing solid-phase extraction and gradient reversed-phase HPLC. The range of the R.S.D. (relative standard deviation) for both the inter- and intra-assay precision was 1.45,2–11.60%. The recovery of all retinoyl-β-glucuronides from rat urine ranged between 89 and 99%. The limit of detection was 0.01 μg/ml using 5 ml of rat urine. This method was applied to quantitate the amount of retinoyl-β-glucuronides produced in urine after the single and multiple oral administrations of 13-cis-, 9-cis- and all-trans-retinoic acids to rats.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Rapid determination of trans-fatty acids in human adipose tissue: Comparison of attenuated total reflection infrared spectroscopy and gas chromatography

A rapid attenuated total reflection (ATR) infrared (IR) spectroscopy procedure was used for quantitating the levels of total trans-fatty acid methyl ester (FAME) derivatives in neat (without solvent) test samples isolated from human adipose tissue. This procedure requires no weighing of the laboratory sample. The single-beam spectrum of the trans-containing FAMEs was ‘ratioed' against that of a reference material having only cis double bonds in order to obtain a symmetric absorption band at 966 cm⁻¹ on a horizontal background. A single-reflection ATR diamond cell that requires only about 1 μl of neat FAMEs was used. The average level of trans-fatty acids in human adipose tissue found by ATR (3.07±0.27%) was generally higher than that obtained by gas chromatography (2.59±0.20%). Reasons for such a difference are discussed.     

Interaction kinetics of liposome-incorporated unsaturated fatty acids with fatty acid-binding protein 3 by surface plasmon resonance

The role of heart-type fatty acid-binding protein (FABP3) in human physiology as an intracellular carrier of fatty acids (FAs) has been well-documented. In this study, we aimed to develop an analytical method to study real-time interaction kinetics between FABP3 immobilized on the sensor surface and unsaturated C18 FAs using surface plasmon resonance (SPR). To establish the conditions for SPR experiments, we used an FABP3-selective inhibitor 4-(2-(1-(4-bromophenyl)-5-phenyl-1H-pyrazol-3-yl)-phenoxy)-butyric acid. The affinity index thus obtained was comparable to that reported previously, further supporting the usefulness of the SPR-based approach for evaluating interactions between FABPs and hydrophobic ligands. A pseudo-first-order affinity of FABP3 to K⁺ petroselinate (C18:1 Δ6 cis), K⁺ elaidate (C18:1 Δ9 trans), and K⁺ oleate (C18:1 Δ9 cis) was characterized by the dissociation constant (K_d) near micromolar ranges, whereas K⁺ linoleate (C18:2 Δ9,12 cis/cis) and K⁺ α-linolenate (C18:3 Δ9,12,15 cis/cis/cis) showed a higher affinity to FABP3 with K_d around 1 × 10⁻⁶ M. Interactions between FAPB3 and C18 FAs incorporated in large unilamellar vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and FAs (5:1 molar ratio) were also analysed. Control DMPC liposomes without FA showed only marginal binding to FABP3 immobilized on a sensor chip while liposome-incorporated FA revealed significant responses in sensorgrams, demonstrating that the affinity of FAs to FABP3 could be evaluated by using the liposome-incorporated analytes. Significant affinity to FABP3 was observed for monounsaturated fatty acids (K_d in the range of 1 × 10⁻⁷ M). These experiments demonstrated that highly hydrophobic compounds in a liposome-incorporated form could be subjected to SPR experiments for kinetic analysis.     

Catabolism of Branched Chain Amino Acids Contributes Significantly to Synthesis of Odd-Chain and Even-Chain Fatty Acids in 3T3-L1 Adipocytes

The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel ¹³C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully ¹³C-labeled tracers, [U-¹³C]valine, [U-¹³C]leucine, [U-¹³C]isoleucine and [U-¹³C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0) and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.     

Determination of 13-cis-retinoic acid and its major metabolite, 4-oxo-13-cis-retinoic acid, in human blood by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10–2000 and 50–2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.     

Identification of enriched conjugated linoleic acid isomers in cultures of ruminal microorganisms after dosing with 1-<Superscript>13</Superscript>C-linoleic acid

Most studies of linoleic acid biohydrogenation propose that it converts to stearic acid through the production of cis-9 trans-11 CLA and trans-11 C18:1. However, several other CLA have been identified in ruminai contents, suggesting additional pathways may exist. To explore this possibility, this research investigated the linoleic acid biohydrogenation pathway to identify CLA isomers in cultures of ruminai microorganisms after dosing with a ¹³C stable isotope. The ¹³C enrichment was calculated as [(M+1/M)×100] in labeled minus unlabeled cultures. After 48 h incubation, significant ¹³C enrichment was observed in seven CLA isomers, indicating their formation from linoleic acid. All enriched CLA isomers had double bonds in either the 9,11 or 10,12 position except for trans-9 cis-11 CLA. The cis-9 trans-11 CLA exhibited the highest enrichment (30.65%), followed by enrichments from 21.06 to 23.08% for trans-10 cis-12, cis-10 trans-12, trans-9 trans-11, and trans-10 trans-12 CLA. The remaining two CLA (cis-9 cis-11 and cis-10 cis-12 CLA) exhibited enrichments of 18.38 and 19.29%, respectively. The results of this study verified the formation of cis-9 trans-11 and trans-10 cis-12 CLA isomers from linoleic acid biohydrogenation. An additional five CLA isomers also contained carbons originating from linoleic acid, indicating that pathways of linoleic acid biohydrogenation are more complex than previously described.     

Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen

Ruminal microorganisms hydrogenate polyunsaturated fatty acids (PUFA) present in forages and thereby restrict the availability of health-promoting PUFA in meat and milk. The aim of this study was to investigate PUFA metabolism and the influence of PUFA on members of the ruminal microflora. Eleven of 26 predominant species of ruminal bacteria metabolised linoleic acid (LA; cis-9,cis-12–18:2) substantially. The most common product was vaccenic acid (trans-11–18:1), produced by species related to Butyrivibrio fibrisolvens. α-Linolenic acid (LNA; cis-9,cis-12,cis-15–18:3) was metabolised mostly by the same species. The fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n − 3)) and docosahexaenoic acid (DHA; 22:6(n − 3)) were not metabolised. Cellulolytic bacteria did not grow in the presence of any PUFA at 50 μg ml⁻¹, nor did some butyrate-producing bacteria, including the stearate producer Clostridium proteoclasticum, Butyrivibrio hungatei and Eubacterium ruminantium. Toxicity to growth was ranked EPA > DHA > LNA > LA. Cell integrity, as measured using propidium iodide, was damaged by LA in all 26 bacteria, but to different extents. Correlations between its effects on growth and apparent effects on cell integrity in different bacteria were low. Combined effects of LA and sodium lactate in E. ruminantium and C. proteoclasticum indicated that LA toxicity is linked to metabolism in butyrate-producing bacteria. PUFA also inhibited the growth of the cellulolytic ruminal fungi, with Neocallimastix frontalis producing small amounts of cis-9,trans-11–18:2 (CLA) from LA. Thus, while dietary PUFA might be useful in suppressing the numbers of biohydrogenating ruminal bacteria, particularly C. proteoclasticum, care should be taken to avoid unwanted effects in suppressing cellulolysis.     

Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were −470 mV for the 13-cis configuration of the retinal Shiff base in bR and −757 mV for the all-trans configuration in H₂O, and −433 mV for the 13-cis configuration and −742 mV for the all-trans configuration in D₂O. The solvent isotope effect (ΔV=V(D₂O)−V(H₂O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13-cis configuration and −788 mV for the all-trans configuration; and for the E204Q mutant they were −491 mV for the 13-cis configuration and −769 mV for the all-trans configuration. Replacement of the Glu¹⁹⁴ or Glu²⁰⁴ residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13-cis configuration and −760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.     

Cation binding cyclic peptides composed of imino acid residues

Cyclo(L -Pro-Sar)_n (n = 2–4) with moderate flexibility and hydrophobicity of molecular structure was synthesized, and the characteristics of these cyclic peptides and their metal complexes in acetonitrile were investigated in connection with the residual properties using ¹³C-nmr measurements. The cyclic tetrapeptide cyclo(L -Pro-Sar)₂ showed a sterically hindered phenomenon in acetonitrile in which the amide backbone adopted a cis-trans-cis-trans sequence. The cyclic hexapeptide cyclo(L -Pro-Sar)₃ existed as a mixture of several conformers whose interconversion is slow on the nmr time scale, including cis-cis-trans and/or cis-trans-trans arrangement of the Sar-Pro bond. Finally, it was demonstrated that the cyclic octapeptide cyclo(L -Pro-Sar)₄ behaved as a mixture of multiple conformers which allowed for cis-trans isomerism about the Pro-Sar peptide bond, of which 20–30% had the all-cis Sar-Pro bond isomer and the remaining 70–80% had one (or more) cis Sar-Pro bond isomer. ¹³C-nmr spectra also demonstrated that cyclo(L -Pro-Sar)_n (n = 3,4) formed a 1:1 ion complex whose conformation was characterized by an all-trans peptide bond in the presence of excess metal salt. Cation binding studies, using CD measurements, established that the ion selectivity of cyclo(L -Pro-Sar)₄ in acetonitrile decreased in the order, Ba²⁺ > Ca²⁺ > Na⁺ > Mg²⁺ > Li⁺.     

Hormonal effects on fatty acid binding and physical properties of rat liver plasma membranes

Summary The fluorescent fatty acids,trans-parimaric andcis-parinaric acid, were used as analogs of saturated and unsaturated fatty acids in order to evaluate binding of fatty acids to liver plasma membranes isolated from normal fed rats. Insulin (10^–8 to 10^–6 m) decreasedtrans-parinaric acid binding 7 to 26% whilecis-parinaric acid binding was unaffected. Glucagon (10^–6 m) increasedtrans-parinaric acid binding 44%. The fluorescence polarization oftrans-parinarate,cis-parinarate and 1,6-diphenyl-1,3,5-hexatriene was used to investigate effects of triiodothyronine, insulin and glucagon on the structure of liver plasma membranes from normal fed rats or from rats treated with triiodothyronine or propylthiouracil. The fluorescence polarization oftrans-parinarate,cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene was 0.300±0.004, 0.251±0.003, and 0.302±0.003, respectively, in liver plasma membranes from control rats and 0.316±0.003, 0.276±0.003 and 0.316±0.003, respectively, in liver plasma membranes from hyperthyroid rats (p<0.025,n=5). Propylthiouracil treatment did not significantly alter the fluorescence polarization of these probe molecules in the liver plasma membranes. Thus, liver plasma membranes from hyperthyroid animals appear to be more rigid than those of control animals. The effects of triiodothyronine, insulin and glucagon addedin vitro to isolated liver plasma membrane preparations were also evaluated as follows: insulin (10^–10 m) and triiodothyronine (10^–10 m) increased fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene in liver plasma membranes while glucagon (10^–10 m) had no effects. These hormonal effects on probe fluorescence polarization in liver plasma membranes were abolished by pretreatment of the rats for 7 days with triiodothyronine. Administration of triiodothyronine (10^–10 m)in vitro increased the fluorescence polarization of trans-parinaric acid in liver plasma membranes from propylthiouracil-treated rats. Thus, hyperthyroidism appeared to abolish thein vitro increase in polarization of probe molecules in the liver plasma membranes. Temperature dependencies in Arrhenius plots of absorption-corrected fluorescence and fluorescence polarization oftrans-parinaric acid,cis-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene were noted near 25°C in liver plasma membranes from triiodothyronine-treated rats and near 18°C in liver plasma membranes from propylthiouracil-treated rats. In summary, hormones such as triiodothyronine, insulin and glucagon may at least in part exert their biological effects on metabolism by altering the structure of the liver plasma membranes.     

Transmembrane localization of cis-isomers of zeaxanthin in the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the 9-cis and 13-cis isomers of zeaxanthin on the molecular organization and dynamics of dimyristoylphosphatidylcholine (DMPC) membranes were investigated using conventional and saturation recovery EPR observations of the 1-palmitoyl-2-(14-doxylstearoyl)phosphatidylcholine (14-PC) spin label. The results were compared with the effects caused by the all-trans isomer of zeaxanthin. Effects on membrane fluidity, order, hydrophobicity, and the oxygen transport parameter were monitored at the center of the fluid phase DMPC membrane. The local diffusion-solubility product of oxygen molecules (oxygen transport parameter) in the membrane center, studied by saturation-recovery EPR, decreased by 47% and 27% by including 10 mol% 13-cis and 9-cis zeaxanthin, respectively; whereas, incorporation of all-trans zeaxanthin decreased this parameter by only 11%. At a zeaxanthin-to-DMPC mole ratio of 1:9, all investigated isomers decreased the membrane fluidity and increased the alkyl chain order in the membrane center. They also increased the hydrophobicity of the membrane interior. The effects of these isomers of zeaxanthin on the membrane properties mentioned above increase as: all-trans < 9-cis ≤ 13-cis. Obtained results suggest that the investigated cis-isomers of zeaxanthin, similar to the all-trans isomer, are located in the membrane interior, adopting transmembrane orientation with the polar terminal hydroxyl groups located in the opposite leaflets of the bilayer. However, the existence of the second pool of cis-zeaxanthin molecules located in the one leaflet and anchored by the terminal hydroxyl groups in the same polar headgroup region cannot be completely ruled out.     

Biodiesel production from rice bran oil and supercritical methanol

In this study, production of biodiesel from low cost raw materials, such as rice bran and dewaxed-degummed rice bran oil (DDRBO), under supercritical condition was carried out. Carbon dioxide (CO₂) was employed as co-solvent to decrease the supercritical temperature and pressure of methanol. The effects of different raw materials on the yield of biodiesel production were investigated. In situ transesterification of rice bran with supercritical methanol at 30 MPa and 300 °C for 5 min was not a promising way to produce biodiesel because the purity and yield of fatty acid methyl esters (FAMEs) obtained were 52.52% and 51.28%, respectively. When DDRBO was reacted, the purity and yield were 89.25% and 94.84%, respectively. Trans-FAMEs, which constituted about 16% of biodiesel, were found. They were identified as methyl elaidate [trans-9], methyl linoleaidate [trans-9, trans-12], methyl linoleaidate [cis-9, trans-12], and methyl linoleaidate [trans-9, cis-12]. Hydrocarbons, which constituted about 3% of the reaction product, were also detected.     

Effects of dietary sources of vegetable fats on performance of dairy ewes and conjugated linoleic acid (CLA) in milk

Two experiments were carried out to study the effects of supplementing the ration of lactating ewes with vegetable fats (sunflower oil, SO or hydrogenated palm oil, HPO; HIDROPALM^®) on diet digestibility, milk yield and milk composition, and on the concentration of the conjugated linoleic acid (CLA) C18:2 cis-9 trans-11 and C18:1 trans-11 (vaccenic acid, VA) and other main fatty acids in milk fat. Treatments involved a control diet, without added oil, and 2 diets supplemented with either 12 g/kg SO or 12 g/kg HPO on a dry matter (DM) basis. In the first experiment, 6 non-pregnant, non-lactating Lacaune ewes were used following a 3 × 3 replicated Latin Square design. Addition of vegetable fat supplement to the diet increased digestibility of DM, organic matter (OM) and crude protein (CP), but did not affect that of the ether extract (EE), neutral detergent fibre (NDF) or acid detergent fibre (ADF). In the second experiment, 60 Lacaune dairy ewes mid-way through lactation (120 ± 12 days in milk, 0.98 ± 0.03 kg/day average milk yield) were divided into three equal-sized groups each of which was assigned to one of the three experimental diets for 4 weeks. Compared with the control treatment, supplementation with HPO increased milk yield and energy-corrected milk. But neither vegetable fat supplement modified percentages of fat and protein in milk. Supplementation with HPO increased C14:1, C16:1 and C16:0 content and reduced C18:0 and C18:1 cis-9 content in milk fat. Supplementation with SO increased the VA content in milk fat by 36% and that of cis-9 trans-11 CLA by 29% in comparison with the control diet. Supplementation with HPO led to milk fat with 15% more cis-9 trans-11 CLA than control milk. In conclusion, adding a moderate dose of HPO or SO to the diets increased CLA concentration in milk fat. Nevertheless, supplementation with SO was more effective than HPO in increasing CLA concentration in milk fat and reducing the atherogenicity index, improving milk quality from the human health standpoint.