A whole body, in vivo, fatty A\acid balance method to quantify PUFA metabolism (desaturation, elongation and beta-oxidation)

Currently there are several contrasting methods utilized for estimating elongation and desaturation of fatty acids and their general metabolism. The majority of these methods involve an ex vivo approach, requiring expensive and sophisticated equipment, likely to result in considerable variation in enzyme activity between and within species. In the present paper we introduce a further development of the whole-body fatty acid balance method for the estimation of the elongation and desaturation of fatty acids. This method though receiving considerable attention because of its simplicity and reliability has yet to be presented in detail. Theoretically, the whole-body fatty acid balance method can potentially be applied to any organism and requires little more than a gas chromatography unit for fatty acid analysis and elementary calculations. As such in this paper we attempt to spell out in detail the theoretical basis and the methods of application drawing specific examples. Using the present method it is possible to measure the fate of individual fatty acids towards desaturation, elongation and oxidation and calculate the elongase, Δ-6 desaturase and Δ-5 desaturase activities.

Simultaneous determination of irinotecan (CPT-11) and SN-38 in tissue culture media and cancer cells by high performance liquid chromatography: Application to cellular metabolism and accumulation studies

A simple and sensitive HPLC method was developed to simultaneously determine CPT-11 and its major metabolite SN-38 in culture media and cell lysates. Camptothecin (CPT) was used as internal standard (I.S.). Compounds were eluted with acetonitrile–50 mM disodium hydrogen phosphate buffer containing 10 mM sodium 1-heptane-sulfonate, with the pH adjusted to 3.0 using 85% (w/v) orthophosphoric acid (27/73, v/v) by a Hyperclon ODS (C18) column (200 mm × 4.6 mm i.d.), with detection at excitation and emission wavelengths of 380 and 540 nm, respectively. The average extraction efficiencies were 96.9–108.3% for CPT-11 in culture media and 94.3–107.2% for CPT-11 in cell lysates; and 87.7–106.8% for SN-38 in culture media and 90.1–105.6% for SN-38 in cell lysates. Within- and between-day precision and accuracy varied from 0.1 to 10.3%. The limit of quantitation (precision and accuracy <20%) was 5.0 and 2.0 ng/ml for CPT-11 and 1.0 and 0.5 ng/ml for SN-38 in culture media and cell lysates, respectively. This method was successfully applied to quantitate the cellular accumulation and metabolism of CPT-11 and SN-38 in H4-II-E, a rat hepatoma cell line.

Effects of dietary fat and conjugated linoleic acid on plasma metabolite concentrations and metabolic responses to homeostatic signals in pigs

Sixteen female cross-bred (Large White × Landrace) pigs (initial weight 65 kg) with venous catheters were randomly allocated to four treatment groups in a 2×2 factorial design. The respective factors were dietary fat (25 or 100 g/kg) and dietary conjugated linoleic acid (CLA; 0 or 10 g CLA-55/kg). Pigs were fed every 3 h (close to ad libitum digestible energy intake) for 8 d and were bled frequently. Plasma glucose and non-esterified fatty acid (NEFA) responses to insulin and adrenaline challenges were determined on day 8. Plasma concentrations of NEFA were significantly increased (10·5 and 5·4 % for low- and high-fat diets respectively, P=0·015) throughout the experiment, suggesting that there was a possible increase in fat mobilisation. The increase in lipolysis, an indicator of ß-adrenergic stimulated lipolysis, was also evident in the NEFA response to adrenaline. However, the increase in plasma triacylglycerol (11·0 and 7·1 % for low- and high-fat diets respectively, P=0·008) indicated that CLA could have reduced fat accretion via decreased adipose tissue triacylglycerol synthesis from preformed fatty acids, possibly through reduced lipoprotein lipase activity. Plasma glucose, the primary substrate for de novo lipid synthesis, and plasma insulin levels were unaffected by dietary CLA suggesting that de novo lipid synthesis was largely unaffected (P=0·24 and P=0·30 respectively). In addition, the dietary CLA had no effect upon the ability of insulin to stimulate glucose removal.

Dietary conjugated linoleic acid differentially alters fatty acid composition and increases conjugated linoleic acid content in porcine adipose tissue

Conjugated linoleic acids (CLA) have been shown to decrease body fat content in pigs. It is possible that feeding pigs diets rich in CLA may increase carcass lipid CLA to levels that could provide health benefits when included as a part of a healthy diet. Therefore, the aim of the present study was to determine whether dietary CLA supplementation has any effect on the fatty acid composition of subcutaneous and intramuscular adipose tissue in pigs. Thirty-five female cross bred (Large White X Landrace) pigs (initial weight 57·2 kg and initial P2 back fat 11·5 mm) were used in the present study. Pigs were housed individually and randomly allocated to one of six dietary treatments (0·00, 1·25, 2·50, 5·00, 7·50 and 10·00 g CLA55 (55 g CLA isomers/100 g total fatty acids; Natural Lipids Ltd, Hovdebygda, Norway)/kg)
and fed their respective diets for 8 weeks. Twelve CLA isomers in the diet and in pig tissue lipids were separated by Agþ-HPLC. CLA was incorporated at fivefold higher levels in subcutaneous fat as compared with intramuscular fat and in a dose-dependant manner. Overall, the transfer efficiency of CLA was maximized at 5·00 g CLA55/kg. However, there was clear selectivity in the uptake or incorporation of cis,trans-9,11 isomer over the trans,cis-10,12 isomer. In general, CLA supplementation produced significant changes in skeletal muscle and adipose tissue fatty acid composition, indicating that dietary CLA had a potent affect on lipid transport and metabolism in vivo. Significant increases in myristic, palmitic and palmitoleic acids and a reduction in arachidonic acid were observed, suggesting an alteration in
activity of Δ5-, Δ6- and Δ9-desaturases in pig adipose tissue. In conclusion, feeding pigs diets supplemented with CLA increases carcass lipid CLA, but also results in changes in the fatty acid profile in pig fat that could potentially outweigh the benefits of CLA.

Dietary conjugated linoleic acid improves carcass leanness without altering meat quality in the growing pig

One constraint facing the pig industry is that ad libitum feeding can often result in high levels of body fat and technologies which can reduce the ratio of lean to fat deposition in the pig are continually being explored. Conjugated linoleic acids have been shown to decrease body fat content in pigs. Therefore, the aim of this study was to determine whether dietary conjugated linoleic acids supplementation has any effect on meat quality and carcass characteristics in finisher pigs. Sixty female crossbred (Large White × Landrace) pigs (average initial weight 56.6 ± 1.9 kg and average initial P2 backfat 11.4 ± 1.3 mm) were used in the present study. Pigs were individually housed and randomly allocated to 1 of 6 dietary treatments: 0, 0.125, 0.25, 0.50, 0.75 or 1.0% (w/w) of conjugated linoleic acids-55. The wheat-based diets were formulated to contain 14.3 MJ DE and 9.3 g available lysine per kg and were fed ad libitum for 8 weeks. Pigs were slaughtered and meat quality was determined on the longissimus thoracis using standard techniques. Dietary conjugated linoleic acids reduced subcutaneous back fat in a linear manner with effects being most pronounced in the middle back fat layer. There was also a linear (P<0.001) decrease in intramuscular fat with increasing dietary conjugated linoleic acids supplementation. However, there was no effect of conjugated linoleic acids on subjective measures of marbling of the loin. Also, loin muscle ultimate pH (P = 0.94), lightness values (P = 0.46) subjective colour scores (P = 0.79), cooking loss (P = 0.71), drip loss (P = 0.40), shear force (P = 0.61) and subjective measures of wetness/firmness (P = 0.19) were unaffected. Dietary conjugated linoleic acids did not alter oxidation, as measured by the level of TBARs at day 1 post-slaughter (P = 0.38) or after 9 days of simulated retail display (P = 0.35). These data confirm that dietary conjugated linoleic acids can improve carcass quality by decreasing back fat depths without having any detrimental effects on meat quality.

Gas chromatography-chemical ionization-mass spectrometric fatty acid analysis of a commercial supercritical carbon dioxide lipid extract from New Zealand green-lipped mussel (Perna canaliculus)

Supercritical fluid extracts of New Zealand green-lipped mussels (NZGLM) have been suggested to have therapeutic properties related to their oil components. The large number of minor FA in NZGLM extract was characterized by a GC-CIMS/MS method that excels at identification of double-bond positions in FAME. The extract contained five major lipid classes: sterol esters, TAG, FFA, sterols, and polar lipids. The total FA content of the lipid extract was 0.664 g/mL. Fifty-three unsaturated FA (UFA) were fully identified, of which 37 were PUFA, and a further 21 UFA were detected for which concentrations were too low for assignment of double-bond positions. There were 17 saturated FA, with 14∶0, 16∶0, and 18∶0 present in the greatest concentration. The 10 n−3 PUFA detected included 20∶5n−3 and 22∶6n−3, the two main n−3 FA; n−3 PUFA at low concentrations were 18∶3, 18∶4, 20∶3, 20∶4, 21∶5, 22∶5, 24∶6, and 28∶8. There were 43 UFA from the n−4, n−5, n−6, n−7, n−8, n−9, n−10, n−11 families, with 16∶2n−4, 16∶1n−5, 18∶1n−5, 18∶2n−6, 20∶4n−6, 16∶1n−7, 20∶1n−7, 16∶1n−9, 18∶1n−9, and 20∶1n−9 being the most abundant. In general, we estimated that FAME concentrations greater than 0.05% (w/w) were sufficient to assign double-bond positions. In total, 91 FA were detected in an extract of the NZGLM, whereas previous studies of fresh flesh from the NZGLM had reported identification of 42 FA. These data demonstrate a remarkable diversity of NZGLM FA.

The a-linolenic acid content of green vegetables commonly available in Australia

Green vegetable consumption has long been considered to have health benefits mainly due to the vitamins, minerals and phytonutrients (such as vitamin C, folate, antioxidants etc) contained in a vegetable-rich diet. Additionally, green vegetables are known to contain a relatively high proportion of omega-3 polyunsaturated fatty acids (PUFAs), primarily in the form of alpha-linolenic acid (18:3n-3). However, there are no data available on the fatty acid composition and concentration of green vegetables commonly consumed in Australia. The present study determined the fatty acid content of 11 green vegetables that are commonly available in Australia. The total fatty acid concentrations of the vegetables under study ranged from 44 mg/100 g wet weight in Chinese cabbage to 372 mg/100 g in watercress. There were three PUFAs in all vegetables analyzed; these were 16:3n-3, 18:2n-6, and 18:3n-3 fatty acids. Sample vegetables contained significant quantities of 16:3n-3 and 18:3n-3, ranging from 23 to 225 mg/100 g. Watercress and mint contained the highest amounts of 16:3n-3 and 18:3n-3, and parsley had the highest amount of 18:2n-6 in both percentage composition and concentration. Mint had the highest concentration of 18:3n-3 with a value of 195 mg/100 g, while watercress contained the highest concentration of 16:3n-3 at 45 mg/100 g. All 11 green vegetables contained a high proportion of PUFAs, ranging from 59 to 72% of total fatty acids. The omega-3 PUFA composition ranged from 40 to 62% of total fatty acids. Monounsaturated fatty acid composition was less than 6% of total fatty acids. The proportion of saturated fatty acids ranged from 21% in watercress and mint to 32% of total fatty acids in Brussels sprouts. No eicosapentaenoic and docosahexaenoic acids were detected in any of the samples. Consumption of green vegetables could contribute to 18:3n-3 PUFA intake, especially for vegetarian populations.

Omega-3 polyunsaturated fatty acid contents of canned meats avaialbe in Australia

Total lipid content of 20 species of canned meats available in Australia ranged from 2% in chicken (Hormel, USA) to 41% in stewed pork (Ma Ling, China). Total n-3 polyunsaturated fatty acids (PUFA) ranged from 30 in canned chicken (Hormel) to 659 mg/100 g in chicken hot dog (Tulip, Denmark). The 18:2n-6 was the predominant PUFA, ranging from 187 in corned beef (Hamper, Australia) to 2832 mg/100 g in chicken luncheon meat (Tulip). Other main PUFA, in order of concentration, were 18:3n-3, ranging from 14 in canned chicken (Hormel) to 590 mg/100 g in chicken hot dog (Tulip); conjugated 18:2n-6 (CLA) from 1 in chicken (Hormel) to 135 mg/100 g in corned mutton (Colonial, Australia); 20:4n- 6 from 11 in camp pie (Tom Piper, Australia) to 73 mg/100 g in spiced ham (Hormel); and 22:5n-3 from 5 in chicken (Hormel) and chicken luncheon (Almaraai, Jordan) to 45 mg/100 g in stewed pork (Ma Ling). Total saturated fatty acids (SFA) ranged from 598 to 14 660 mg/100 g, with 16:0 predominant followed by 18:0. Total monounsaturated fatty acid concentration ranged between 813 to 20 218 mg/100 g with 18:1 the major fatty acid. Trans 18:1 ranged from 10 to 698 mg/100 g. The canned meats contained 20 and 22-carbon long chain n-3 PUFA at levels comparable with or greater than those in fresh lean meat.

Does perinatal ω-3 polyunsaturated fatty acid deficiency increase appetite signaling?

Objective: To investigate the effect of maternal dietary ω-3 polyunsaturated fatty acid (PUFA) deficiency and repletion on food appetite signaling.
Research Methods and Procedures: Sprague-Dawley rat dams were maintained on diets either supplemented with (CON) or deficient in (DEF) ω-3 PUFA. All offspring were raised on the maternal diet until weaning. After weaning, two groups remained on the respective maternal diet (CON and DEF groups), whereas a third group, born of dams fed the DEF diet, were switched to the CON diet (REC). Experiments on food intake began when the male rats reached 16 weeks of age. Food intake was stimulated either by a period of food restriction, by blocking glucose utilization (by 2-deoxyglucose injection), or by blocking β-oxidation of fatty acids (by β-mercaptoacetate injection).
Results: DEF animals consumed more than CON animals in response to all stimuli, with the greatest difference (1.9-fold) demonstrated following administration of 2-deoxyglucose. REC animals also consumed more than CON animals in response to food restriction and 2-deoxyglucose but not to β-mercaptoacetate.
Discussion: These findings indicate that supply of ω-3 PUFA, particularly during the perinatal period, plays a role in the normal development of mechanisms controlling food intake, especially glucoprivic (i.e. reduced glucose availability) appetite signaling. Dietary repletion of ω-3 PUFA from 3 weeks of age restored intake responses to fatty acid metabolite signaling but did not reverse those in response to food restriction or glucoprivic stimuli.

Conjugated linoleic acid suppresses myogenic gene expression in a model of human muscle cell inflammation

Proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha}, contribute to muscle wasting in inflammatory disorders, where TNF{alpha} acts to regulate myogenic genes. Conjugated linoleic acid (CLA) has shown promise as an antiproliferative and antiinflammatory agent, leading to its potential as a therapeutic agent in muscle-wasting disorders. To evaluate the effect of CLA on myogenesis during inflammation, human primary muscle cells were grown in culture and exposed to varying concentrations of TNF{alpha} and the cis-9, trans-11 and trans-10, cis-12 CLA isomers. Expression of myogenic genes (Myf5, MyoD, myogenin, and myostatin) and the functional genes creatine kinase (CK) and myosin heavy chain (MHC IIx) were measured by real-time PCR. TNF{alpha} significantly downregulated MyoD and myogenin expression, whereas it increased Myf5 expression. These changes corresponded with a decrease in both CK and MHC IIx expression. Both isomers of CLA mimicked the inhibitory effect of TNF{alpha} treatment on MyoD and myogenin expression, whereas myostatin expression was diminished in the presence of both isomers of CLA either alone or in combination with TNF{alpha}. Both isomers of CLA decreased CK and MHC IIx expression. These findings demonstrate that TNF{alpha} can have specific regulatory effects on myogenic genes in primary human muscle cells. A postulated antiinflammatory role of CLA in myogenesis appears more complex, with an indication that CLA may have a negative effect on this process.

Nanostructure and hydrogen bonding in interpolyelectrolyte complexes of poly(ε-caprolactone)-block-poly(2-vinyl pyridine) and poly(acrylic acid)

Nanostructured poly(ε-caprolactone)-block-poly(2-vinyl pyridine) (PCL-b-P2VP)/poly(acrylic acid) (PAA) interpolyelectrolyte complexes (IPECs) were prepared by casting from THF/ethanol solution. The morphological behaviour of this amphiphilic block copolymer/polyelectrolyte complexes with respect to the composition was investigated in a solvent mixture. The phase behaviour, specific interactions and morphology were investigated using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, optical microscopy (OM), dynamic light scattering (DLS) and atomic force microscopy (AFM). Micelle formation occurred due to the aggregation of hydrogen bonded P2VP block and polyelectrolyte (PAA) from non-interacted PCL blocks. It was observed that the hydrodynamic diameter (D_h) of the micelles in solution decreased with increasing PAA content up to 40 wt%. After 50 wt% PAA content, D_h again increased. The micelle formation in PCL-b-P2VP/PAA IPECs was due to the strong intermolecular hydrogen bonding between PAA homopolymer units and P2VP blocks of the block copolymer. The penetration of PAA homopolymers into the shell of the PCL-b-P2VP block copolymer micelles resulted in the folding of the P2VP chains, which in turn reduced the hydrodynamic size of the micelles. After the saturation of the shell with PAA homopolymers, the size of the micelles increased due to the absorption of added PAA onto the surface of the micelles.

Bis(3-endo-camphoryl)phosphinic acid, a non-racemic helical supramolecular host with aquapores

Bis(3-endo-camphoryl)phosphinic acid (1) was prepared by the reaction of the lithium enolate of D-(+)-camphor and phosphorous trichloride followed by an oxidative work up. Compound 1 crystallizes from wet toluene as monohydrate 1·H2O, which was investigated by X-ray crystallography. Molecules of 1 are associated by strong hydrogen bonds giving rise to the formation of a supramolecular helix. The interior channel of the helix is filled by a one-dimensional (1D) string of water molecules that are also associated by hydrogen bonding. The 1D string adopts a twisted zigzag conformation. Although the hydrogen bond networks are not cross-linked both the screw of the helix and the twist of the 1D string of water molecules are left-handed (M) and controlled by the chiral camphoryl residues situated on the exterior of the helix. The overall supramolecular structure is strongly reminiscent of aquaporin-1, a significant membrane-channel protein responsible for the transport of water into the cells.

An improved method for the purification of rat liver-type fatty acid binding protein from Escherichia coli

We have developed expedient and reliable methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins. We have examined enzyme purification strategies suited to large-scale processing and present a chromatographic sequence that serves as a pilot model for industrial scale preparation of cyclosporin synthetase from cyclosporin producing fungi. A chromatographic sequence consisting of ammonium sulfate precipitation → gel filtration → hydrophobic interaction chromatography → anion exchange chromatography, yielded an electrophoretically homogeneous cyclosporin synthetase preparation (Coomassie G-250 brilliant blue staining). Furthermore, a native polyacrylamide gel electrophoresis system was developed for the isolation of active cyclosporin synthetase enzyme from crude extracts of cyclosporin producing fungi. The environmental factors affecting enzyme stability and the continuity of the in vitro cyclosporin biosynthetic reaction-temperature, pH, and substrate depletion were assessed and manageable conditions have been defined for sustainable cyclosporin biosynthesis with enzyme isolates. Cyclosporin synthetase exhibited an optimal temperature range of 24–29 °C and a pH optimum of 7.6. The native enzyme displayed a pI of 5.7, as determined by isoelectric focusing. The industrial implementation of an in vitro biosynthetic approach could potentially prove useful for the production of important therapeutic cyclosporins which occur as only minor fermentation by-products.

Isolation and characterization of polyunsaturated fatty acid producing Thraustochytrium species : screening of strains and optimization of omega-3 production

An isolation program targeting Thraustochytrids (marine fungoid protists) from 19 different Atlantic Canadian locations was performed. Sixty-eight isolates were screened for biomass, total fatty acid (TFA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) content. Analysis of fatty acid methyl ester results discerned four distinctive clusters based on fatty acid profiles, with biomass ranging from 0.1 to 2.3 g L−1, and lipid, EPA, and DHA contents ranging from 27.1 to 321.14, 2.97 to 21.25, and 5.18 to 83.63 mg g−1 biomass, respectively. ONC-T18, was subsequently chosen for further manipulations. Identified using 18S rRNA gene sequencing techniques as a Thraustochytrium sp., most closely related to Thraustochytrium striatum T91-6, ONC-T18 produced up to 28.0 g L−1 biomass, 81.7% TFA, 31.4% (w/w biomass) DHA, and 4.6 g L−1 DHA under optimal fermentation conditions. Furthermore, this strain was found to produce the carotenoids and xanthophylls astaxanthin, zeaxanthin, canthaxanthin, echinenone, and β-carotene. Given this strain’s impressive productivity when compared to commercial strains, such as Schizochytrium sp. SR21 (which has only 50% TFA), coupled with its ability to grow at economical nitrogen and very low salt concentrations (2 g L−1), ONC-T18 is seen as an ideal candidate for both scale-up and commercialization.

Transesterification of fish oil to produce fatty acid ethyl esters using ultrasonic energy

This study evaluated the production of fatty acid ethyl esters from fish oil using ultrasonic energy and alkaline catalysts dissolved in ethanol. The feasibility of fatty acid ethyl ester production was determined using an ultrasonic bath and probe, and between 0.5 and 1% KOH (added to the fish oil). Furthermore, factors such as ultrasonic device (bath and probe), catalyst (KOH and C2H5ONa), temperature (20 and 60 °C), and duration of exposure (10–90 min) were assessed. Sodium ethoxide was found to be a more efficient catalyst than KOH when transesterifying fish oil. Ultrasonic energy applied for greater than 30 min at 60 °C using 0.8% of C2H5ONa as a catalyst transesterified over 98% fish oil triglycerides to fatty acid ethyl esters. It is reasonable to conclude that the yield of fatty acid ethyl esters produced by applying ultrasonic energy to fish oil is related to the sonication time. Due to increases in the surface area contact between the reactants and the catalyst, ultrasonic energy has the potential to reduce the production time required by a conventional large-scale commercial transesterification method that uses agitation as a way of mixing.