Gas chromatographic profiles of plasma total lipids as indicators of dietary history. Correlation with carbohydrate and alcohol intake based on 24-h dietary recall.

Quantitative gas chromatographic estimates of the major lipid classes and molecular species in fasting plasma were correlated with total carbohydrate, starch, fibre, sucrose and alcohol intake based on 24-h dietary recall. Spearman coefficients (rs) and tests of significance (P) were obtained for groups of 775 males and 471 females aged 20-59 years from a Toronto-McMaster Lipid Research Clinics Population Study. The most significant correlations varying from rs 0.1 to 0.2 and P 0.001 to 0.0005 (n = 400-773) were between increased intake of alcohol and increased ratios of C50/C54 triacylglycerols, C34/C36 phosphatidylcholines and phosphatidylcholine/free cholesterol (PC/FC) of plasma. Increase in total dietary carbohydrate, starch and fibre correlated with decreasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios (rs = -0.1-0.2; P less than 0.002-0.04; n = 400-773). In contrast, consumption of high levels of alcohol was associated with increasing C50/C54 triacylglycerol, C34/C36 phosphatidylcholine and PC/FC ratios. A high intake of alcohol (50-150 ml per day) distinguished itself from other simple carbohydrate-induced lipid profiles by its marked effect on increased C50/C52 triacylglycerol and PC/FC ratio.     

NUTRITIONAL AND PHYSIOLOGICAL SIGNIFICANCE OF LIPIDS

This paper reviews how intricately dietary fat regulates cholesterol metabolism. The effects of fats on plasma cholesterol are far more complex than predicted. Not only the fatty acid composition but also the triacylglycerol configuration of dietary fat influence its function. Extensive comprehension is thus required for this problem.     

EFFECT OF DIETARY LIPID ON UV LIGHT CARCINOGENESIS IN THE HAIRLESS MOUSE

Abstract— Isocaloric feeding of diets varying in lipid content to albino hairless mice has shown that their susceptibility to skin tumorigenesis induced by simulated solar UV light was not affected by the level of polyunsaturated fat, 5% or 20%. However a qualitative effect of dietary lipid was demonstrated. Mice fed 20% saturated fat were almost completely protected from UV tumorigenesis when compared with mice fed 20% polyunsaturated fat. Multiple latent tumours were detected in the saturated fat-fed mice by subsequent dietary replenishment, suggesting that a requirement for dietary unsaturated fat exists for the promotion stage of UV-induced skin carcinogenesis.     

Characterization of fatty acid and triacylglycerol composition in animal fats using silver-ion and non-aqueous reversed-phase high-performance liquid chromatography/mass spectrometry and gas chromatography/flame ionization detection

Fatty acid (FA) and triacylglycerol (TG) composition of natural oils and fats intake in the diet has a strong influence on the human health and chronic diseases. In this work, non-aqueous reversed-phase (NARP) and silver-ion high-performance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection and gas chromatography with flame-ionization detection (GC/FID) and mass spectrometry detection are used for the characterization of FA and TG composition in complex samples of animal fats from fallow deer, red deer, sheep, moufflon, wild boar, cock, duck and rabbit. The FA composition of samples is determined based on the GC/FID analysis of FA methyl esters. In total, 81 FAs of different acyl chain length, double bond (DB) number, branched/linear, cis-/trans- and DB positional isomers are identified. TGs in animal fats contain mainly monounsaturated and saturated FAs. High amounts of branched and trans-FAs are observed in the samples of ruminants. In NARP mode, individual TG species are separated including the separation of trans- and branched TGs. Silver-ion mode provides the separation of TG regioisomers, which enables the determination of their ratios. Great differences in the preference of unsaturated and saturated FAs in the sn-2 position on the glycerol skeleton are observed among individual animal fats. Unsaturated FAs are preferentially occupied in the sn-2 position in all animal samples except for wild boar with the strong preference of saturated FAs in the sn-2 position.     

Determination of total <Emphasis Type="Italic">trans</Emphasis> fats and oils by infrared spectroscopy for regulatory compliance

The mandatory requirement in many countries to declare the amount of trans fat present in food products and dietary supplements has led to a need for sensitive and accurate methodologies for the rapid quantitation of total trans fats and oils. Capillary gas chromatography (GC) and infrared spectroscopy (IR) are the two methods most commonly used to identify and quantify trans fatty acids for food labeling purposes (see the article by Delmonte and Rader in this ABC issue for a detailed presentation of GC methodology). The present article provides a comprehensive review of the IR technique and the current attenuated total reflection (ATR) Fourier-transform (FT) IR methodologies for the rapid determination of total trans fats and oils. This review also addresses potential sources of interferences and inaccuracies in FTIR determinations, particularly those done at low trans levels. Recent observations have shown that the presence of saturated fats caused interferences in the FTIR spectra observed for trans triacylglycerols. The recognition and resolution of previously unresolved quantitative issues improved the accuracy and sensitivity of the FTIR methodology. Once validated, it is anticipated that the new negative second-derivative ATR-FTIR procedure will make IR spectroscopy more suitable than ever, and a rapid alternative and/or complementary method to GC, for the rapid determination of total trans fats for regulatory compliance. Figure Infrared light bouncing inside an internal reflection crystal     

Regiospecific characterisation of the triacylglycerols in animal fats using high performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry

High performance liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI MS) was applied to the characterisation of triacylglycerols (TAGs) in animal fats. The major TAGs in four fats (beef, chicken, lamb and pork) were identified and positional isomers assigned according to their APCI mass spectra. Beef and lamb fat TAGs were confirmed as containing higher proportions of saturated fatty acids compared with those of chicken and pork. HPLC-APCI MS was also shown to be of value in providing regiospecific information for the fatty acids in individual TAG species. For example, beef and lamb fat were shown to contain both cis- and trans-isomers of the 18:1 fatty acid, whilst chicken and pork contained only the cis-isomer. When the position of fatty acid substitution was determined from the APCI spectra, whilst the cis- 18:1 was predominantly found in the 2-position of the TAG, the trans-18:1 showed a preference for the 1/3-position. Similarly, it was confirmed that although the 2-position of beef, chicken and lamb fat TAGs was dominated by unsaturated fatty acids, in pork fat, a characteristically high proportion of palmitic acid was seen in this position. The TAGs identified compared well with those reported previously. The distributions of 2-position fatty acids seen in lamb and pork fat compared favourably with those obtained by the more traditional method of lipase degradation. Although the distributions for chicken and beef showed some discrepancies, these can be attributed to weaknesses in the quantification procedure or the specificity of the lipase. Overall, the technique of HPLC-APCI MS has been shown to be very powerful for the regiospecific analysis of animal fats.     

Synthesis of enantiopure structured triacylglycerols

The synthesis of nine enantiopure structured triacylglycerols (TAGs) of the AAB type is described, all possessing the (S)-absolute configuration. Six of them possess one saturated and two identical unsaturated fatty acyl chains, whereas the remaining three possess two identical saturated fatty acids along with one unsaturated fatty acid. The former group was synthesized by a five-step chemoenzymatic route involving a highly regioselective immobilized Candida antarctica lipase, starting from enantiopure (R)-solketal. The second group was prepared by a fully chemical five-step approach based on the same chiral precursor. Such enantiopure TAGs are strongly required as standards for the enantiospecific analysis of intact TAGs in fats and oils.     

Effects of a high alpha-linolenate and high linoleate diet on hemolysis and lipid peroxidation of rat erythrocytes.

alpha-Linolenic acid (18:3n-3) is known to autoxidize several fold faster than linoleic acid (18:2n-6). Feeding a high alpha-linolenate or a high linoleate diet to rats resulted in significant changes in the n-3/n-6 ratios of 20 and 22 carbon highly unsaturated fatty acids in erythrocytes. However, the rates of hemolysis observed in N2- or O2-atmosphere were similar between the two dietary groups. No significant amounts of conjugated dienes were detected and no measurable changes in the fatty acid compositions were observed during the incubations, indicating that the hemolysis occurred without involving significant lipid peroxidation. When stimulated with a free radical initiator, [2,2'-azobis-(2-amidinopropane)dihydrochloride] (AAPH), hemolysis occurred more rapidly, conjugated dienes formed and unsaturated/saturated ratios of phospholipid fatty acids decreased. However, no statistically significant difference was observed in these parameters of the two dietary groups. These results indicate that hemolysis occurs without involving lipid peroxidation but is accelerated by free radicals through lipid peroxidation, and that the difference in autoxidizabilities of alpha-linolenate and linoleate is not reflected in the rates of hemolysis and autoxidation in rat erythrocytes.     

Evaluation of low trans fat edible oils by attenuated total reflection-Fourier transform infrared spectroscopy and gas chromatography: a comparison of analytical approaches

Current interest by the food industry in exploring reformulation options that lower the content of trans fat in edible fats and oils requires methods to accurately measure low levels of trans fat. In the present study, the quantitation of trans fat in 25 edible fat and oil samples was evaluated using two current analytical approaches, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and gas chromatography with flame ionization detection (GC-FID) according to Official Methods of the American Oil Chemists' Society. Significant differences between the ATR-FTIR and reference GC-FID quantitations were found for samples with a trans fat content <2% of total fat. These discrepancies could be explained, in part, by the presence of certain oil constituents (e.g., vitamins, carotenoids, high levels of saturated fat) that produced absorbance bands at or near 966 cm(-1) in the ATR-FTIR spectra, a region that was previously identified as being characteristic of isolated trans double bonds. Results demonstrate that the natural content of such oil constituents could result in significant overestimations of trans fat when ATR-FTIR is used to analyze edible fats and oils with a trans fat content <2% of total fat.     

Hypertension does not stimulate the development of hypercholesterolemia or fatty liver induced by a high cholesterol/cholate diet in rats

A high cholesterol/cholate diet induced hypercholesterolemia and fatty liver in both spontaneously hypertensive rats (SHR) and normotensive control rats (WKY). However, in contrast to previous concepts, the levels of cholesterol ester, triacylglycerol and phosphatidylcholine in plasma as well as triacylglycerol in liver were higher in WKY than in SHR fed a normal diet. The high cholesterol/cholate diet elevated the levels of plasma cholesterol, plasma cholesterol ester and hepatic triacylglycerol, and the extent of elevation was significantly higher in WKY than in SHR. Increases both in monoene/saturated ratios, an indication of elevated delta 9-desaturase activity, and in linoleate/arachidonate ratios, a possible indication of impaired desaturation-elongation activity, were observed in hepatic and plasma lipids of both strains fed the high cholesterol/cholate diet. The increases in monoene/saturated ratios were similar in both strains, but the increases in the linoleate/arachidonate ratios were higher for the plasma cholesterol esters of WKY than of SHR. The n-6/n-3 ratios of plasma and hepatic lipids were higher in WKY than in SHR throughout the experiments. These diet-induced changes observed in hepatic and plasma lipids were not reflected in the aortic lipids. Thus, hypertension per se does not promote the development of hyperlipemia and fatty liver induced by a high cholesterol/cholate diet. Our results also suggest that the metabolism of polyenoic fatty acids is different between SHR and WKY.     

Energetics of cholesterol transfer between lipid bilayers

It is believed that natural biological membranes contain domains of lipid ordered phase enriched in cholesterol and sphingomyelin. Although the existence of these domains, called lipid rafts, is still not firmly established for natural membranes, direct microscopic observations and phase diagrams obtained from the study of three-component mixtures containing saturated phospholipids, unsaturated phospholipids, and cholesterol demonstrate the existence of lipid rafts in synthetic membranes. The presence of the domains or rafts in these membranes is often ascribed to the preferential interactions between cholesterol and saturated phospholipids, for example, between cholesterol and sphingomyelin. In this work, we calculate, using molecular dynamics computer simulation technique, the free energy of cholesterol transfer from the bilayer containing unsaturated phosphatidylcholine lipid molecules to the bilayer containing sphingomyelin molecules and find that the affinity of cholesterol to sphingomyelin is higher. Our calculations of the free-energy components, energy and entropy, show that cholesterol transfer is exothermic and promoted by the favorable change in the lipid-lipid interactions near cholesterol and not by the favorable energy of cholesterol-sphingomyelin interaction, as assumed previously.     

Development of a DSC method for determination of certain technological parameters of margarine and mixed-fat spread

Nutritional-physiological research of near past decades had established the real nutritional value of fats and oils. In the former theory the nutritional value of fats and oils is influenced mainly by the rate of saturated/unsaturated fats. It was ruled out, and positive, inert or risk physiological effect of every single fatty acid had been established. The health-care effect of omega-3 fatty acid mainly by the favourable (<3:1) rate of omega-6/omega-3 was established, as inert was concerned the saturated C₁₆ fatty acid and the maximal amount of trans-fatty acids carrying health risk in fats was legally regulated in some countries. These nutritional-physiological requirements were mainly fulfilled by margarine producers and the elementary fats were selected in such a way that they should meet these requirements. Our method helps to the producers to quickly determine the amount of the liquid and solid fat at certain temperature and/or to adjust the technological temperature parameters. The main steps of our method are: a./ determination of cooling rate (K min^–1) of the crystallizer device; b./ determination of the rate of liquid/solid fat at 10°C temperature. This value is used for the determination of the rate of fats and oils as a function of technology and required consistency firmness (spread ability); c./ determination of the temperature from the cooling curve where the crystallization of most part of the fat has finished. This value is used for the determination of outlet temperature parameter of product coming out from the crystallizer device for margarines or mixed-fat spreads with water-in-oil system.     

Lack of effect of oral supplementation with antioxidants on cholesterol oxidation product concentration of human plasma,as revealed by an improved gas chromatography method

A gas chromatographic method was successfully applied to determine cholesterol oxidation products (COPs) in human plasma. The linearity, precision, recovery and sensitivity of the method were determined. Oral supplementation with a combination of vitamin E (800 IU), C (1 g) and β-carotene (24 mg), given for 21 days to 21 patients, did not significantly decrease plasma COP content. No correlations (n = 26) were found between initial plasma COP content and the following parameters: age, body mass index, plasma content of α-tocopherol, cholesterol, high-density lipoprotein cholesterol and triglycerides, and fat, natural antioxidant and oxidized lipid intake. Differences in plasma COP content between type 2 diabetic (n = 6) and nondiabetic (n = 20) patients were not statistically significant. The results from this study lead us to hypothesize that the nonenzymatic oxidation of cholesterol in plasma is negligible compared to COPs originating from the diet. This article also includes a comprehensive review of the drawbacks of the analytical methods of COP determination in plasma and serum.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C18 and C20) and fatty alcohols (C16 to C26) derived from wax esters. Most of these acids and alcohols were unsaturated in the omega-7 position and were accompanied by smaller amounts of the saturated and omega-5 monounsaturated analogues. glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (omega-5 and omega-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Interactions of Triton X-100 with sphingomyelin and phosphatidylcholine monolayers: influence of the cholesterol content

The presence of microdomains, called lipid rafts, in biological membranes is usually explained by lateral segregation between specific lipids and proteins. These rafts present similarities with the membrane domains isolated by their non-ionic detergent-resistance at 4 degrees C. They are enriched in sphingomyelin and cholesterol as compared with the outer leaflet of eukaryotic cell membranes. To understand the role played by the lipids enriched in rafts in their resistance to solubilization by detergents, the interactions between these lipids and the non-ionic detergent Triton X-100 were studied by using different lipid monolayers at the air-water interface. The influence of Triton X-100 on the Langmuir isotherms (i.e. surface pressure/area isotherms) of monolayers containing sphingomyelin and cholesterol at different mole ratios was analyzed and the results were compared with the influence of Triton X-100 on monolayers containing a phosphatidylcholine bearing a saturated and an unsaturated fatty acid (i.e. palmitoyloleylphosphatidylcholine) and cholesterol. This phosphatidylcholine was chosen since the phosphatidylcholines present in rafts isolated from bovine kidney could contain about 50% of saturated fatty acids. Triton X-100 induces an increase in the condensing effect observed as compared with ideal mixture of phospholipid/cholesterol. Triton X-100-induced changes in the morphology of the monolayers were visualized by Brewster angle microscopy, which confirmed the differences of behavior observed by analyzing the isotherms.     

Regulating the size and stabilization of lipid raft-like domains and using calcium ions as their probe

We apply a means to probe, stabilize, and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 10-30 nm. In vitro, mixing saturated and unsaturated lipids results in microdomains, which are unstable and coalesce. This inconsistency is puzzling. It has been hypothesized that biological line-active surfactants reduce the line tension between saturated and unsaturated lipids and stabilize small domains in vivo. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated, and a hybrid (1-saturated-2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged, and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active cosurfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line tension between them. These findings are consistent with a recent theory and provide insight into the self-organization of lipid rafts, their stabilization, and size regulation in biomembranes.     

Melting,Crystallization, and In Vitro Digestion Properties of Fats Containing Stearoyl-Rich Triacylglycerols

Fats containing the stearoyl-rich triacylglycerols (TAGs) of 1,2-distearoyl-3-oleoylglycerol (SSO) and 1,3-dioleoyl-2-stearoylglycerol (OSO) were synthesized via the lipase-catalyzed acidolysis of tristearin (SSS)-rich fat and oleic acids, followed by solvent fractionation. Their physicochemical properties and in vitro digestibilities were compared. The SSS-, SSO-, and OSO-rich fats comprised 81.6%, 52.9%, and 33.1% stearic acid, respectively, whereas oleic acid comprised 2.9%, 37.5%, and 56.2%, respectively. The SSS-, SSO-, and OSO-rich fats contained the TAGs of SaSaSa (100.00%), SaSaMo (86.98%), and MoSaMo (67.12%), respectively, and the major TAGs were SSS, SSO, and OSO, respectively. Melting and crystallization temperatures were higher and fat crystals were larger and densely packed in the descending order of SSS-, SSO and OSO-rich fats. Both in vitro multi-step digestion and pH-stat digestion were more rapid for OSO- than SSO-rich fat. Oleic acid was digested faster than stearic acid during the initial digestion, then the rate decreased, whereas that of stearic acid increased over prolonged digestion. Fats that were richer in stearoyl at the sn-1,3 position of TAG melted and crystallized at higher temperatures, had a densely packed microstructure of large fat crystals and were poorly digested. Stearic acid imparts the essential physical attributes of melting and crystallization in solid fats, and the low digestible stearoyl-rich fat would be a viable substitute for trans fatty acids in food lipid industry.     

Optimized analysis oftrans-octadecenoic acids in edible fats

Summary Trans fatty acids (TFA) in edible fats are of interest, since they are suspected to influence serum lipid levels. Thus, to estimate the atherogenic potency of edible fats a precise analytical method for the determination of TFA is required. The present study does not only aim at the quantitative analysis of total C18:1 TFA, but also at the determination of the isomeric pattern oftrans-octadecenoic acids in different fats. The combination of argentation TLC with high resolution gas chromatography allows the quantification of 10trans- and 9cis-octadecenoic acid peaks. This method can be applied to hydrogenated vegetable fats as well as to ruminant fats and tissue lipids from different species. Based on this two-dimensional chromatography the intake of single TFA isomers with food can be determined, which is of importance with respect to the hypothesis, that the negative metabolic activity originates only from certain isomers, and to the considerable variation of isomeric patterns in different edible fats. Based on the precise determination of the totaltrans-octadecenoic acid content in milk fats by TLC and GC, a formula has been derived by regression analysis, which allows the evaluation of total C18:1 TFA contents from a routine GC analysis. Though the 61 milk fats used covered a wide scope of feeding and lactation conditions, a correlation coefficient of 0.9991 was achieved.     

Influence of oleic acid on the structure of a mixture of hydrated model stratum corneum fatty acids and their soaps

The phase behaviour of a hydrated (32% (w/w) water) mixture of saturated and unsaturated fatty acids and their soaps has been examined. By progressively increasing the proportion of oleic acid, it could be shown that this unsaturated component exists partly as a separate phase within the others. The unsaturated fatty acids form an H_II phase at 25°C, whereas the saturated fatty acids form a fatty acid—soap crystal under the conditions employed. The results help to explain the role played by the fatty acids (and also cholesterol) within the lipid fraction of human stratum corneum. A feature of this study is a comparison of the results obtained by X-ray diffraction, differential scanning calorimetry and Fourier transform infrared spectroscopy.     

Hypolipidemic effects of hydrolyzed xyloglucan

Xyloglucan is found in the primary cell walls of higher plants, where it plays important biological roles. It also exists in certain seeds, for example tamarind, as a reserve polysaccharide. Some xyloglucan oligosaccharides possess plant growth promotion activity. We prepared hydrolyzed xyloglucan from the tamarind seed xyloglucan using endo-(1 → 4)- β -glucanase and examined its effects on lipid metabolism in rats fed a high fat diet. Among the plasma lipids, total lipid, cholesterol, triglyceride and β - lipoprotein were each reduced 14 - 17% by hydrolyzed xyloglucan. Hydrolyzed xyloglucan significantly reduced both adipose tissue weight and plasma GOT. Among the hepatic lipids, total lipid, cholesterol, triglyceride and phospholipid were significantly reduced by hydrolyzed xyloglucan. These hypolipidemic effects may be important for the treatment and prevention of geriatric diseases, including diabetes and cardiac disorders.