Dietary calcium impairs tomato lycopene bioavailability in healthy humans.

Lycopene (LYC) bioavailability is relatively low and highly variable, because of the influence of several factors. Recent in vitro data have suggested that dietary Ca can impair LYC micellarisation, but there is no evidence whether this can lead to decreased LYC absorption efficiency in humans. Our objective was to assess whether a nutritional dose of Ca impairs dietary LYC bioavailability and to study the mechanism(s) involved. First, in a randomised, two-way cross-over study, ten healthy adults consumed either a test meal that provided 19-mg (all-E)-LYC from tomato paste or the same meal plus 500-mg calcium carbonate as a supplement. Plasma LYC concentration was measured at regular time intervals over 7 h postprandially. In a second approach, an in vitro digestion model was used to assess the effect of increasing Ca doses on LYC micellarisation and on the size and zeta potential of the mixed micelles produced during digestion of a complex food matrix. LYC bioavailability was diminished by 83 % following the addition of Ca in the test meal. In vitro, Ca affected neither LYC micellarisation nor mixed micelle size but it decreased the absolute value of their charge by 39 %. In conclusion, a nutritional dose of Ca can impair dietary LYC bioavailability in healthy humans. This inhibition could be due to the fact that Ca diminishes the electrical charge of micelles. These results call for a thorough assessment of the effects of Ca, or other divalent minerals, on the bioavailability of other carotenoids and lipophilic micronutrients.

Plasmalogen metabolism-related enzymes in rat brain during aging: influence of n-3 fatty acid intake.

Plasmalogens (Pls) are phospholipids containing a vinyl-ether bond at the sn-1 position of the glycerol backbone. They represent between 1/2 and 2/3 of the ethanolamine phospholipids in the brain. During aging, the Pls content in human brain falls down. However, the role of Pls metabolism-related enzymes in the regulation of Pls levels remains to be determined. Dihydroxyacetone phosphate acyltransferase (DHAP-AT) is the enzyme involved in the first step of Pls biosynthesis. In the brain, a phospholipase A2, which selectively acts on Pls, has been isolated (Pls-PLA2s). In this work, we aimed to evaluate the impact of DHAP-AT (a key enzyme of Pls biosynthesis) and Pls-PLA2 (a specific Pls degradation enzyme) on the evolution of Pls content in the rat brain during aging. The influence of n-3 fatty acid intake was also evaluated. Littermates from two generations of n-3 deficient rats were fed an equilibrated diet containing either alpha-LNA alone or with two doses of DHA. After weaning, 3, 9 or 21 months of diet, rats were sacrificed. Enzymatic assays were performed, Pls levels were assessed and the sn-2 position of ethanolamine Pls was analyzed. DHAP-AT activity significantly increased between weaning and 3 months with a concomitant increase of brain Pls, which reached maximal levels after 9 months. Then, Pls levels and DHAP-AT activity significantly decreased while Pls-PLA2s activity significantly increased. Dietary n-3 fatty acids had no effect on DHAP-AT activity and on Pls levels. In conclusion, the increase of brain Pls content in the first part of the life may be related to the high increase of DHAP-AT activity, probably stimulated by DHA. In aged animals, the decrease of Pls levels may mainly be caused to an increase of their degradation by Pls-PLA2. Dietary DHA may not oppose the physiologic aging.

Acute in vivo administration of a fish oil-containing emulsion improves post-ischemic cardiac function in n-3-depleted rats.

A novel i.v. lipid preparation (MCT:FO) containing 80% medium chain-triacylglycerols and 20% fish oil was recently developed to rapidly replenish cell membrane phospholipids with omega 3 (n-3) polyunsaturated fatty acids (PUFA). In regard of this property, we investigated the effect of a single i.v. administration of MCT:FO on the recovery of cardiac function after ischemia in control and n-3-depleted rats. Results were compared with those obtained either with a control preparation, where FO was replaced by triolein (MCT:OO), or with saline. Saline (1 ml) or lipid preparation (also 1 ml) was injected as a bolus via the left saphenous vein. After 60 min the heart was removed and perfused for 20 min in normoxic conditions according to Langendorff. Thereafter, the heart was subjected to a 20 min zero-flow normothermic ischemia, followed by 40 min reperfusion. Cardiac mechanical and metabolic functions were monitored. In control rats, the previous administration of a lipid preparation (MCT:FO or MCT:OO) versus saline improved cardiac function during aerobic reperfusion post-ischemia. N-3-depleted rats showed decreased basal cardiac function and impaired recovery following ischemia. However, the bolus injection of MCT:FO opposed the deleterious effect of long-term n-3-deficiency and, in this respect, was superior to MCT:OO over the first 20 min of reperfusion. This novel approach to rapidly correct n-3 PUFA-deficiency might be clinically relevant and offer interesting perspectives in the management of acute ischemic accidents.

Preferential incorporation of dietary cis-9,cis-12,trans-15 18:3 acid into rat cardiolipins.

Cardiolipins from mitochondria of different rat organs (heart; liver and kidney) appear to be privileged targets for the incorporation of cis-9,cis-12,trans-15 18:3 acid, a compound commonly found in deodorized edible linolenic acid-containing oils. When this acid (together with other linolenic acid geometrical isomers (LAGI)) is fed at high load to rats that had been reared on a fat-free diet since weaned for a few days, it replaces the endogenously synthesized monoenoic acids that had accumulated in cardiolipin during fat deficiency. Although there is no discrimination in deposition of any LAGI in adipose tissue triacylglycerols, a high selectivity of incorporation of the cis-9,cis-12,trans-15 18:3 acid over other isomers (including the all-cis 18:3(n-3) acid) is observed either in diradylphospholipids or in cardiolipins. However, cis-9,cis-12,trans-15 18:3 acid accumulates in cardiolipins at a considerably higher level than in other phospholipids (11 times in liver, 5-7 times in heart and kidney). It reaches 22-24% of total fatty acids in cardiolipins from heart and liver, and 13-14% in kidney. The cis-9,cis-12,trans-15 18:3 acid is esterified to both the 1(1")- and 2(2")-positions of liver mitochondria cardiolipin, with a well-marked selectivity for positions 1(1"). Its 1(1")/2(2") selectivity ratio is about the same as that of 18:2(n-6) acid: 2.1 vs 2.2. It is concluded that the trans-15 ethylenic bond is probably perceived as a single bond by enzymic systems that ensure acylation of cardiolipins. The cis-9,cis-12,trans-15 isomer is able to reverse the fatty acid modifications induced in cardiolipins by a diet devoid of essential fatty acids, in a way similar to that of 18:2(n-6) acid supplementation.

Oxidative metabolism of [1-14C] mono-trans isomers of linoleic and alpha-linolenic acids in the rat.

Trans polyunsaturated fatty acids are formed during processing of vegetable oils such as deodorization and frying. The oxidative metabolism of linoleic and alpha-linolenic acids and of their mono-trans isomers (9cis,12trans-18:2, 9trans,12cis-18:2 and 9cis, 12cis,15trans-18:3, 9trans,12cis,15cis-18:3, respectively) was studied in fasting rats. A single dose of 18.5 MBq of each [1-14C] labelled fatty acid (260 microg) was orally given to the animals. The 14CO2 expired was monitored during 24 h. Radioactive countings of the CO2-trapping agent were performed at regular intervals up to 24 h after oral administration of the radiolabelled fatty acid. Radioactive countings were also performed on several tissues (liver, heart, brain, kidneys, sus-epidydimal fat, gastrocnemian muscle, gastrointestinal tract and carcass). The 14CO2 production 24 h after oral administration of the fatty acid ranged from 55.5% to 68.7% of the radioactivity administered for the C18:2 isomers and from 69.7% to 73.5% for the C18:3 fatty acids. From 6 to 24 h, 14CO2 recovery was significantly higher after oral administration of 9cis, 12trans-18:2 than after giving both other octadecadienoic isomers. 14C retention per gram of tissue in the liver and in the heart was significantly lower after feeding 9cis,12trans-18:2 than after administration of both other C18:2 isomers. 14C retention per gram of tissue in the muscle was significantly lower after administration of both trans C18:2 isomers compared to linoleic acid. Neither 14CO2 recoveries nor 14C retentions were significantly different after administration of the three octadecatrienoic acids. The difference observed in 14CO2 recovery within the dienes was probably not due to a higher specificity of the enzymes involved in the beta-oxidation sequence for the Delta12trans double bond, as previously reported. Indeed, due to the labelling of the fatty acids on the carboxyl end, 14C values recorded in the CO2-trapping agent were only due to the first cycle of beta-oxidation.

Metabolites of conjugated isomers of linoleic acid (CLA) in the rat.

Metabolism of conjugated isomers of linoleic acid (CLA) in rats was studied by feeding high quantities of CLA (180 mg/day) for six days to animals that had been reared on a fat-free diet for two weeks. After this period, animals were sacrificed and liver lipids extracted. High-performance liquid chromatography (HPLC) analyses with UV detection revealed the presence of conjugated polyunsaturated fatty acids in the total liver lipid methyl esters. After isolation by HPLC, three fatty acid metabolites were identified by gas liquid chromatography coupled with mass spectrometry as being C20:3 delta 8,12,14, C20:4 delta 5,8,12,14 and C20:4 delta 5,8,11,13. A higher quantity of C20:4 delta 5,8,12,14 compared to C20:4 delta 5,8,11,13 was observed. These must arise from the elongation and desaturation of 18:2 delta 10,12 and 18:2 delta 9,11, respectively.

Effects of aging and dietary n-3 fatty acids on rat brain phospholipids: focus on plasmalogens.

The aging brain undergoes modifications in the lipid composition of cell membranes and especially in plasmalogens. These phospholipids represent between one-half and two-thirds of the ethanolamine phospholipids in the brain. They are known to facilitate membrane fusion and act as endogenous antioxidants. During normal aging and in some pathological conditions, plasmalogen and DHA levels fall. In this context, we aimed to evaluate the influence of n-3 FA intake on plasmalogens in the brain during aging. Littermates from two generations of n-3-deficient rats were fed an n-3-deficient diet or an equilibrated diet containing either alpha-linolenic acid alone (alpha-LNA) or with two doses of DHA (0.3 or 0.6% w/w). After weaning, 9 mon of diet, or 21 mon of diet, plasmalogen levels were assessed, and the sn-2 substitutions of plasmenylethanolamines were analyzed in the cortex, striatum, and hippocampus. Our results showed that plasmalogen contents were not influenced by the diet. Plasmalogen levels were significantly decreased in aged rats compared with adults, whereas DHA levels increased in the hippocampus and remained stable in the cortex and striatum. DHA levels were significantly and similarly increased in total phospholipids and especially in plasmenylethanolamines after 9 mon of diet containing alpha-LNA alone or combined with DHA. This study showed that each structure sustained specific age-induced modifications. Dietary n-3 FA may not oppose the physiological decrease in brain plasmalogen levels during aging. Moreover, alpha-LNA appears to be equally as potent as preformed DHA at replacing DHA in the brain of our rat model.

Effects of three trans isomers of eicosapentaenoic acid on rat platelet aggregation and arachidonic acid metabolism.

Three trans isomers of eicosapentaenoic acid (EPA) were added to rat platelets stimulated with arachidonic acid (AA) in order to compare their effects on platelet aggregation and on AA oxygenation with those of EPA. The production of metabolites from radiolabelled 20:5delta 17trans was studied also. EPA induced an inhibition of platelet aggregation of 26.7 +/- 6.6% for a 20:5/20:4 ratio equal to 1. The 20:5delta 11trans and the 20:5delta 11trans,17trans were twice as antiaggregant. In contrast, the 20:5delta 17trans induced similar antiaggregant effect as its cis homologue. Each fatty acid showed a dose-dependent effect. In opposition to EPA, 20:5delta 17trans was also able to induce platelet aggregation (12 +/- 4.9% at 5 microM). With regards to the metabolism of AA, 20:5delta 11trans, 20:5delta 17trans and 20:5delta 11trans,17trans (20:5/20:4 = 1) reduced the formation of the cyclooxygenase metabolites (-63%, -37% and -68%, respectively) and enhanced that of 12-HETE (+67%, +38% and +74%, respectively) as compared to EPA. The analysis showed that radiolabelled 20:5delta 17trans was metabolized into five compounds which remained to be identified. The Rf of three of these compounds (X1, X2 and X4) were those of the metabolites of EPA. Experiments using baicalein induced an inhibition of the production of X2. This suggested that this compound was formed through the 12-lipoxygenase pathway. In the same way, using indomethacin as inhibitor, we observed that X1 and X4 were produced by the cyclooxygenase pathway. Our results suggest that the trans double bond in the delta 11 position may be responsible of the different physiological effects of the trans polyunsaturated fatty acids as compared to their cis homologue (EPA). Furthermore, 20:5delta 17trans seems to be recognised by the enzymatic system as 20:4 n-6.

No effect of dietary trans isomers of alpha-linolenic acid on platelet aggregation and haemostatic factors in european healthy men. The TRANSLinE study.

The aim of this study was to investigate the effect of trans alpha-linolenic acid on platelet aggregation and blood haemostasis. A randomized, double blind dietary intervention trial was carried out with healthy male volunteers (n=88) in three European centers. After a 6-week washout period where subjects avoided foods containing all trans fats, subjects either continued for 6 weeks with a low trans diet or a diet where trans alpha-linolenic acid provided 0.6% of energy (supplied as oil, margarine, cheese, muffins, and biscuits). At the end of the washout period the intake of trans polyunsaturated fats was 58+/-115 mg/day; this increased in patients on the high trans diet by +1344+/-328 mg/day, compared with +10+/-67 mg/day in patients on the low trans diet (p<0.01). The change in trans alpha-linolenic acid in plasma cholesteryl esters was 0.26+/-0. 20 on the high trans and 0.00+/-0.07% of fatty acids on the low trans diet (p<0.001). No effect of the high trans diet was observed on platelet aggregation: collagen EC(50) high trans 157+/-100, low trans 152+/-90 ng/mL (NS); U44619 EC(50) high trans 81+/-61, low trans 59+/-27 nM (NS). The high trans diet did not affect platelet thromboxane production, fibrinogen levels, factor VII, activated factor VIIa, or plasminogen activator inhibitor activity. There were no center-specific differences in response to the high trans diet. A relatively high amount of trans alpha-linolenic acid for 6 weeks does not increase the risk of coronary heart disease by promoting platelet aggregation and blood coagulation.

Utilization of high-performance liquid chromatography as an enrichment step for the determination of cyclic fatty acid monomers in heated fats and biological samples.

A method was developed to determine traces of cyclic fatty acid monomers (CFAM) in oils and animal tissues. This method is a combination of some techniques developed earlier but with the enrichment step being achieved by high-performance liquid chromatography (HPLC) instead of urea inclusion. After transformation of the lipids into methyl esters, the latter were hydrogenated after addition of an internal standard (methyl heptadecanoate or ethyl hexadecanoate). The mixture was enriched in CFAM by HPLC on a semi-preparative C18 reversed-phase column using acetonitrile-acetone (90:10, v/v) at 4 ml/min. The enriched fraction containing the CFAM and the internal standard was then analysed by gas chromatography on a polar column (cyanosilicone phase). This method was developed using known mixtures of CFAM isolated from both heated sunflower and linseed oils. Small amounts of CFAM (50 microg/g of sample) were determined with good reproducibility without any loss during the HPLC enrichment step and with no modification of the relative proportions of the CFAM in the mixture. This method can be applied to either heated fats and oils or biological samples (heart cell culture) that contain only traces of CFAM. Ethyl hexadecanoate (16:0 ethyl ester) can be used as an internal standard for samples containing small amounts of 17:0.

Stereoselective synthesis of (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid, and their [1-14C]-radiolabeled analogs.

To study the metabolic fate of conjugated linoleic acid isomers, we synthesized, in seven steps, from 1-heptyne, (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid, and their [1-(14)C]-analogs. In the case of (6Z,10E,12Z)-octadecatrienoic acid, a series of palladium-catalyzed cross-coupling reactions between 1-heptyne and (E)-1,2-dichloro-ethene, a coupling reaction with a Grignard reagent and cleavage of the dioxolane gave (E)-dodec-4-en-6-ynal 3. Stereoselective Wittig reaction between aldehyde 3 and triphenyl-[5-(tetrahydro-pyran-2-yloxy)-pentyl]-phosphonium provided a dienyne. Stereocontrolled reduction of the triple bond and replacement of the tetrahydropyranyl group by a bromine gave (5Z,9E,11Z)-1-bromo-heptadeca-5,9,11-triene 10. Formation of the alkenyl lithium derivative and carbonation with CO(2) furnished (6Z,10E,12Z)-octadecatrienoic acid. (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid was obtained by the same route but using triphenyl-[5-(tetrahydro-pyran-2-yloxy)-heptyl]-phosphonium iodide for the Wittig reaction. [1-(14)C]-analogs were obtained from the bromides by carbonation with (14)CO2. In all cases, chemical or radiochemical purities were found to be better than 95% after purification by flash chromatography on silica gel (>99% after additional purification by RP-HPLC). Metabolism studies in animals are in progress.

Sequential substitution of 1,2-dichloro-ethene: a convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-.

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadecadienoic acids with high radiochemical and isomeric purities (>98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO(2) gave (9Z,11E)-[1-(14)C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (<2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.

Synthesis of (6Z,9Z,11E)-octadecatrienoic and (8Z,11Z,13E)-eicosatrienoic acids and their [1-(14)C]-radiolabeled analogs.

In order to study the metabolic pathway and the physiological effects of 9c,11t-18:2 (major isomer of conjugated linoleic acid) and its C(18:3) and C(20:3) metabolites, 6c,9c,11t-18:3 and 8c,11c,13t-20:3 and their [1-(14)C]-radiolabeled analogs were prepared stereoselectively by total synthesis. The 8c,11c,13t-20:3 was obtained in 11 steps. The synthesis involves a highly stereoselective Wittig reaction between 3-(t-butyldiphenylsilyloxy)propanal and the ylide of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt which gave (3Z)-1-(t-butyldiphenylsilyloxy)-10-(2-tetrahydropyranyloxy)dec-3-ene in a first step. Then the t-butyldiphenylsilyl derivative was deprotected selectively and the resulting alcohol function was converted via a bromide into a phosphonium salt. The second stereoselective Wittig condensation between the phosphonium salt and commercial (2E)-non-2-enal under cis-olefinic conditions using Lithium hexamethyldisilazide as base afforded the (7Z,10Z,12E)-1-(2-tetrahydropyranyloxy)nonadeca-7,10,12-triene in a very good isomeric purity. The intermediate product was brominated and transformed by reaction with magnesium into Grignard reagent, which was one-carbon elongated by unlabeled or labeled carbon dioxide to obtain the 8c,11c,13t-20:3 in good isomeric purity (95%) and high radiochemical purity for its [1-(14)C]-radiolabeled analog (99%). 6c,9c,11t-18:3 was synthesized in a similar way by using 5-(2-tetrahydropyranyloxy)pentanylphosphonium salt in place of 7-(2-tetrahydropyranyloxy)heptanylphosphonium salt in a first step. Other reactions were unchanged and products were obtained in similar yields. Similar to 8c,11c,13t-20:3, the 6c,9c,11t-18:3 was obtained in a very good isomeric purity (95%) and its [1-(14)C]-radiolabeled analog in a high radiochemical purity (95%).

Large-scale preparation of (9Z,12E)-[1-(13)C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid and their [1-(13)C] all-cis isomers.

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z, 12E)-[1-(13)C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z, 15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-(13)C]-octadeca-9, 12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-(13)C]-octadeca-9,12, 15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised.

Trans mono- and polyunsaturated fatty acids in human milk.

OBJECTIVE: The aim of the present study was to identify the trans isomers of C18 fatty acids in some human milk samples. SUBJECTS: Ten human milk samples from French women were collected in a local milk bank in order to assess their trans mono and polyunsaturated fatty acids (PUFA) content. INTERVENTION: The fatty acid profile was examined using methyl and isopropyl ester derivatives. The combination of gas-liquid chromatography, high-performance liquid chromatography and silver nitrate thin-layer chromatography was needed to describe the detailed fatty acid compositions, including the trans isomers of unsaturated C18 fatty acids. RESULTS: All the samples contained trans isomers of C18:1 acid (mean level 1.9 +/- 0.2% of total fatty acids), with trans vaccenic acid being the major isomer. The samples also contained various isomers of linoleic and alpha-linolenic acid, but at lower levels. Trans isomers of PUFA are the same as those present in deodorised or deep-fried oils. One sample presented an abnormally high degree of isomerisation of alpha-linolenic acid (almost 50%). This was related to the dietary habit or consuming foods that were deep-fried in rapeseed oil. This milk sample also contained some cyclic fatty acid monomers. CONCLUSION: The human milk samples collected in this study contained some trans fatty acids, including isomers of essential fatty acids. This should be taken into account in the dietary intake of the newborn.

The effect of dietary trans alpha-linolenic acid on plasma lipids and platelet fatty acid composition: the TransLinE study.

OBJECTIVE: To collect (i) baseline data and (ii) execute a large multicentre study examining the effect of trans alpha-linolenic acid on its incorporation into plasma lipids and on risk factors for coronary heart disease. DESIGN: Male volunteers were recruited and the habitual diet assessed by a 4-d weighed record. Fatty acid composition of plasma and platelet lipids were determined by gas chromatography at baseline. After a 6 week run-in period on a trans 'free' diet, male volunteers were randomised to consume 0.6 % of energy trans alpha-linolenic acid or to continue with a diet 'low' in trans alpha-linolenic acid for 6 weeks. SETTING: Three European university research departments supported by the research and development departments of the food industry. SUBJECTS: Male volunteers (88) recruited by local advertisement. METHODS: Replacement of 30 % of the fat of the habitual diet by margarine, oil and foods. Rapeseed oil was deodorised especially to produce the trans 'free' and 'high' trans foods for this study. The incorporation and conversion of trans alpha-linolenic acid into plasma lipids and platelets was assessed by gas chromatography and dietary compliance was verified by 4-d weighed record. RESULTS: Less trans alpha-linolenic acid isomers are incorporated into human plasma lipids in French volunteers than in Dutch or Scottish volunteers consuming their habitual diets. Trans 'free' alpha-linolenic acid-rich oil can be produced by careful deodorization during refining. The 'high' trans diet provided 1410+/-42 mg/d trans isomers of alpha-linolenic acid, whilst the 'low' trans group consumed 60+/-75 mg/d. The change in plasma lipid and platelet fatty acid composition documented that trans linolenic isomers are incorporated and converted to a trans isomer of eicosapentaenoic acid. Only the 15-trans alpha-linolenic acid is incorporated into plasma cholesteryl esters. The group consuming low trans diet had a slightly higher intake of fat, especially saturated and monounsaturated fat. CONCLUSIONS: Trans 'free' rapeseed oil, rich in alpha-linolenic acid, can be produced by careful deodorization. Dietary records show good compliance. Dietary trans isomers of alpha-linolenic acid are incorporated in plasma lipids and converted to long-chain polyunsaturated fatty acids. Their effects on risk factors for coronary heart disease and their metabolism will be reported elsewhere. SPONSORSHIP: European Commission (FAIR 95-0594 grant). European Journal of Clinical Nutrition (2000) 54, 104-113

Induction of hepatic drug-metabolizing enzymes by cyclic fatty acid monomers in the rat.

The effects of cyclic monomers on the activities of several drug-metabolizing enzymes were evaluated. Female Wistar rats were fed, for 4 wk, a semi-synthetic diet containing different quantities of cyclic monomers isolated from linseed oil heated at 275 degrees C for 12 hr under nitrogen. Microsomal proteins and cytochrome c were significantly increased in rats fed a diet containing 0.1 or 1% cyclic monomers. Aminopyrine demethylation, a model reaction preferentially induced by phenobarbital, was increased by this treatment. NADPH-cytochrome P-450 reductase was also stimulated. Moreover, ethoxyresorufin deethylation, known to be greatly increased by methylcholanthrene-type inducer was only increased threefold by this treatment. The activity of p-nitrophenol UDP-glucuronosyl transferase decreased while the conjugation of bilirubin was stimulated. These results suggest that cyclic monomers isolated from heated linseed oil show some characteristics of phenobarbital-type inducers.

Cyclic fatty acid monomers from dietary heated fats affect rat liver enzyme activity.

This study was conducted to investigate the effects of dietary cyclic fatty acid monomers (CFAM), contained in heated fat from a commercial deep-fat frying operation, on rat liver enzyme activity. A partially hydrogenated soybean oil (PHSBO) used 7 d (7-DH) for frying foodstuffs, or 0.15% methylated CFAM diets was fed to male weanling rats in comparison to a control group fed a nonheated PHSBO (NH) diet in a 10-wk experiment. All diets were isocaloric with 15% fat. Animals fed either CFAM or 7-DH diets showed increased hepatic content of cytochrome (cyt.) b5 and P450 and increased activity of (E.C. 1.6.2.4) NADPH-cyt. P450 reductase in comparison to the control rats. In addition, the activities of (E.C. 2.3.1.21) carnitine palmitoyltransferase-I and (E.C. 1.1.1.42) isocitrate dehydrogenase were significantly decreased when compared to that of rats fed the NH diet. A significantly depressed activity of (E.C. 1.1.1.49) glucose 6-phosphate dehydrogenase was also observed for these animals compared to the control rats fed NH diet. Moreover, liver and microsomal proteins were significantly increased when CFAM or 7-DH diets were fed to animals in comparison to controls while liver glycogen was decreased significantly in experimental groups of rats. The results obtained in this study indicate that the CFAM in the diet from either synthetic sources or used fats increase the activity of liver enzyme systems that detoxify them.

Separation of 20:4n-6 and 20:4n-7 by capillary gas-liquid chromatography.

The use of a capillary column coated with 100% cyanopropyl polysiloxane (CP(TM)Sil 88) allows the separation of several fatty acids associated with fat deficiency. Starting with liver mitochondrial phospholipids of weanling rats fed a fat-free diet, an unusual fatty acid was isolated, along with 20:4n-6, by thin-layer chromatography on AgNO3-impregnated silica gel plates. After partial hydrazine reduction of these acids, the resulting monoenes were isolated and subjected to ozonolysis in BF3/methanol. The resulting monomethyl and dimethyl esters were identified by gas chromatography/mass spectrometry. Our data indicate that the unusual component corresponds to 20:4n-7. Based on published biochemical and analytical studies and on our own chromatographic retention data, some of the other unusual fatty acids were tentatively identified as 18:2n-7, 20:2n-7 and 20:3n-7. The CP(TM)Sil 88 column appears to be a simple and useful tool for the separation of fatty acids of the palmitoleate series.

Improved methods for the isolation and study of the C18, C20 and C22 monoethylenic fatty acid isomers of biological samples: Hg adducts, HPLC, AgNO3-TLC/FID, and ozonolysis.

The monoethylenic isomers of C18, C20 and C22 chain lengths of the depot fat of a nonhominid primate (cynomolgus monkeys,Macaca fascicularis), fed a partially hydrogenated herring oil (IV=76.0) for 30 months, were examined by 2 different approaches. The first isolation method involved preparative gas liquid chromatography and argentation thin layer chromatography (TLC). The second sequence involved a chain-length fractionation system based on the TLC of the methoxy-bromomercuri quence involved a chain-length fractionation system based on the TLC of the methoxy-bromomercuri adducts of the total methyl esters to isolate groups of acids of common degrees of unsaturation, and then high performance liquid chromatography on a reverse-phase column. In both cases, the monoethylenic isomer distribution was determined by ozonolysis in BF3/MeOH. Comparable results were obtained with the 2 methods. The second approach is recommended for small biological samples, especially for those containing a relatively high proportion of di- and other polyethylenic isomers which might interfere.