Reginald F. Cross

Conjugated linoleic acid decreases fat accretion in pigs: evaluation by dual-energy X-ray absorptiometry

Thirty female Large White x Landrace pigs (average weight 57.2 (sd 1.9) kg) were allocated to one of six dietary treatments containing 0, 1.25, 2.5, 5.0, 7.5 or 10.0 g 55 % conjugated linoleic acids (CLA) isomers (CLA-55)/kg diet and fed for 8 weeks. Each pig was scanned at 0, 28 and 56 d and again at post slaughter using dual-energy X-ray absorptiometry (DXA) to determine the temporal pattern of body composition responses. Values determined by DXA were adjusted using regression equations generated from validation experiments between chemically and DXA-predicted values. Overall, there was a significant linear reduction in fat content with the increasing levels of CLA in the diet (P=0.007, P=0.011, P=0.008 at week 4, week 8 and for the carcass, respectively). The greatest improvement was recorded at the early stages of CLA supplementation and for the highest dose of CLA (week 4, -19.2 % compared with week 8, -13.7 %). In the first 4 weeks of feeding CLA, pigs receiving 10 g CLA-55/kg diet deposited 93 g less fat/d than pigs fed basal diets (P=0.002) compared with only 6 g less fat than control animals in the final 4 weeks. Lean content and lean deposition rate were maximised at 5 and 2.5 g CLA-55/kg diet for the first 4 weeks (P=0.016) and the final 4 weeks of treatment respectively. DXA estimates of bone mineral content and bone mineral density were not affected by CLA supplementation throughout the experiment. These data demonstrate that dietary CLA decreases body fat in a dose-dependent manner and that the response is greatest over the initial 4 weeks of treatment.

Comparison of silver-ion high-performance liquid chromatographic quantification of free and methylated conjugated linoleic acids.

Silver-ion high-performance liquid chromatography was used to fractionate a mixture of conjugated linoleic acid (CLA) isomers (as the free fatty acids, CLAFFA) in commercial CLA mixtures and biological samples. Due to the unchanged retention mechanism, it was assumed that the elution order of the isomers remained the same as that of methyl esters separated on the same column. The most abundant isomers, cis/trans 10,12-18:2 and cis/trans 9,11-18:2, were separated better as free acids on a single column than in the methyl ester form. Quantification of the CLA standard was used as the reference profile to evaluate different methylation methods commonly used to prepare CLA methyl esters for quantitation. Acid- and vuigi base-catalyzed derivatization methods resulted in CLA intraisomerization and losses in total conjugated dienes content. Acid (HCl and BF3) methylations significantly elevated the level of trans,trans isomers and significantly reduced the cis/trans isomers. Base methylation, tetramethylguanidine/methanol, resulted in loss of trans,trans isomers, and a substantial loss of total underivatized conjugated dienes. Other catalysts such as the trimethylsilyldiazomethane produced additional peaks of unidentified artifacts. The analysis of CLAFFA appears to provide more accurate quantification of CLA isomers in commercial and biological samples.

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 Ag+-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 delta5-, delta6- and delta9-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.

Mixed mode retention and the use of competing acid for the Ag+-HPLC analysis of underivatized conjugated linoleic acids.

Summary Fatty acids (FAs) and fatty acid residues are generally methylated and analyzed by GC. The reasons for this are partly historic and partly because of the sensitivity advantage of flame ionization detection over UV absorption by the carboxylic acid functionality in saturated FAs. However, for strongly absorbing unsaturated acids such as the conjugated linoleic acids (CLAs), the sensitivity advantage is greatly reduced. Hence there seems little reason to waste time and introduce errors associated with methylation. Remarkably, this appears not to have been recognized. In this paper we describe our method development for the analysis of underivatized CLAs by silver ion HPLC separation on the ChromSpher Lipids column. Using mobile phases previously optimized for the analysis of the methylated CLAs, retention is excessive and a competing acid is required. Various combinations of small concentrations of acetic acid (3.0 ‐ 2.5%) with acetonitrile (0.0 ‐ 0.025%), respectively, yield similar resolution and run times. As well as its role as a competing acid, acetic acid acts as a general strong solvent and thus can be used alone as a modifier (without acetonitrile). However, for slightly shorter run times a mobile phase of 2.5% acetic acid and 0.025% acetonitrile was chosen as the optimum mobile phase for analysis. The separation of the free CLAs is clearly superior to those previously published and obtained in this study for the methylated CLAs. The additional specific strong interactions of the underivatized CLAs seem certain to be due to hydrogen bonding between the CLA carboxylic acid functionality and the large number of residual silanols on the surface of the silica support of the stationary phase.

The separation of dihydrofolate reductase inhibitors and the determination of pKa,1 values by capillary zone electrophoresis

CZE was used as a method to separate eight dihydrofolate reductase inhibitors (DHFRI). Separation of the eight DHFRI was difficult to achieved and occurs only under very specific conditions. Baseline resolution occurred in 250 mM phosphate buffer of pH 2.1 at 13 kV. In an attempt to understand the mechanism of separation, pKa,1 values have been determined and the effective hydrodynamic radii estimated by modeling the molecules. The consequential plot of Z/r versus the measured electrophoretic mobility gave a good linear correlation, thus demonstrating the fundamental nature of the separation mechanism.

Salt effects in capillary zone electrophoresis I. Dependence of electrophoretic mobilities upon the hydrodynamic radius

An examination of the derivation of the equation relating electrophoretic mobility (μep) to charge and the hydrodynamic radius (r) shows that the 1r dependence arises from an approximation. A more generalised approach yields a 1r2 dependence and includes the known 1√I dependence as well. Nine totally ionised sulphonamides have been used to test the proposed relationship between μep and r, and at each of four buffer concentrations utilised the correlations favour 1r2. For large molecules, μep has frequently been demonstrated to be proportional to aMr23, which can be equivalent to 1r2. However, it has been suggested that there may be a transition from 1r dependence for small molecules to a 1r2 dependence for large molecules. Hence we have used literature data for a series of twenty-two peptides varying in size between 3 and 39 amino acids long to test that theory and the generality of the 1r2 dependence. The calculations indicate a consistent dependence upon 1r2.

Salt effects in capillary zone electrophoresis: II. Mechanisms of electrophoretic mobility modification due to Joule heating at high buffer concentrations

Abstract Five sulphonamides spanning a range of initial degrees of ionisation have been used as probes for the study of the mechanisms of electrophoretic mobility modification caused by increasing buffer concentrations (5–210 mM sodium phosphate) in capillary zone electrophoresis (CZE). Partition experiments at the CZE operational pH and a second pH where the analytes are uncharged permits independent determinations of the salt and temperature effects upon ionisation. At the CZE temperature, the underlying salt effects due to nonideal solute–solvent interactions are demonstrated. These are selective and indicate that the buffer concentration may be manipulated to facilitate resolution of analytes with differing degrees of ionisation. These selectivities are heightened by Joule heating. At elevated temperatures, the relative effects upon ionisation and viscosity were determined. These magnitudes were then combined and used in conjunction with the deviations in electrophoretic mobilities from the extrapolated behaviour of dilute buffers to estimate mean internal capillary temperatures.

Analysis of reduced glutathione using a reaction with 2,4'-dichloro-1-(naphthyl-4-ethoxy)-s-triazine (EDTN)

A fluorescent adduct was formed between 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine (EDTN) and reduced glutathione in a reaction at 37 °C and pH 9.2. This reaction was used as the basis of an assay for reduced glutathione. The fluorescence was examined at an excitation wavelength of 319 nm and an emission wavelength of 425 nm after extraction of residual unreacted EDTN with methylene dichloride and subsequent dilution of the aqueous phase with ethanol containing 0.01 percent Triton X-100. The reaction rate was low at pH 7 but was accelerated by addition of preparations containing the enzyme glutathione-S-transferase. The adduct gave a discrete peak using isocratic elution with HPLC on a Nova-pak C18 3 μm reverse phase column and a solvent system of methanol: 0.1 M phosphate buffer pH 6.3 (40∶60). An analytical concentration range of 24 to 240 μM reduced glutathione was obtained with an ultraviolet detection system but the concentration range was 7.5 to 75 μM when a fluorescence detection system was used. Adducts of other mercapturic acid pathway thiol compounds were not formed at 37 °C under the conditions used and hence did not interfere in the assay. They were formed by heating EDTN and the respective thiol compound at 60 °C for 30 min and they clearly separated from the reduced glutathione compound on HPLC analysis. A strong reaction was observed with digitonin while solutions of tyrosine, at 10 mM concentration, also reacted but these reactants are unlikely to interfere with reduced glutathione analysis in biological systems. When adduct formation was used to estimate reduced glutathione concentrations in some mammalian and plant tissues the reaction using 2,4′-dichloro-l-(naphthyl-4-ethoxy)-s-triazine and HPLC separation gave the same results as ano-phthaldialdehyde assay for liver and muscle but the HPLC method gave slightly lower values for other mammalian and plant tissues. The differences were attributed to other material in the tissue extracts which was fluorescing at the same wavelengths as the reduced glutathione adduct.

Salt effects in capillary zone electrophoresis: III. Systematic and selective factors in the high ionic strength separation of sulphonamides in sodium phosphate buffers

Abstract The effect of high concentrations of sodium phosphate buffer (65–210 mM) on the capillary zone electrophoretic separation of 23 sulphonamides and the neutral marker have been examined at pH 7 and 18 kV. In 65 mM phosphate, all ionised analytes (21 of the 23 sulphonamides) are resolved sufficiently for screening purposes. At higher buffer concentrations, general resolution clearly improves due to the systematic salt effect, which provides increased separation space. This effect is progressively enhanced by Joule heating via increases in the electrophoretic mobilities of the analytes migrating away from the detector. However, one pair of analytes displays unfavourable selectivity changes and comigrates at higher buffer concentrations. This has been demonstrated to be due to selective but dissimilar salt effects upon the degrees of ionisation of the two analytes that leads to a convergence of their electrophoretic mobilities. Eighteen sulphonamides were baseline-resolved between 174 and 210 mM sodium phosphate buffer.

Objective testing for the dependence of electrophoretic mobilities upon size in capillary zone electrophoresis

SummaryEighteen peptides have been modeled. From the volumetric data derived, and published mobilities, the relationship between electrophoretic mobility (μep) and the hydrodynamic radius(r) has been examined. Objective testing with respect to size has been achieved by the log-log version of generalized relationship. (1) From the gradient of the plot versus log r(2.02) there is good support for the inverse square law (μep α 1/r2). Equivalent calculations using molecular weight (Mr) and the number of amino acid residues (n) similarly lead to μep α 1/Mr2/3 and μep α 1/n2/3, respectively. However, the strength of the correlation is diminished as the precision of the representation of size is degraded. (2) An examination of the effect of size at fixed charge and a statistical analysis of the charge distribution on the peptides leads to the conclusion that deviations from the averaged behaviour arise from a charge-induced volumetric effect. Taken together, (1) and (2) indicate that whilst net charge and total size can describe average electrophoretic behaviour well, these parameters are inadequate to describe the specific mobilities of individual analytes.Objective analysis of alkylpyridine data indicates μep α 1/rx where x=2.6–2.8 (depending upon the nature of the r values utilized), but is certainly ≠1 as may have been presumed. A very small range of values may be responsible for this surprising result.