Compositional changes of fatty acids in the 1(1")-and 2(2")-positions of cardiolipin from liver, heart, and kidney mitochondria of rats fed a low-fat diet.

Cardiolipins from liver, heart and kidney mitochondria of rats fed a fat-free diet for 66 days have been analyzed for their fatty acid composition and positional distribution. The main effect was a dramatic decrease of linoleic acid which was counterbalanced by increases in the levels of palmitoleic, oleic and cis-vaccenic acids. Linoleic acid remains asymmetrically distributed between positions 1(1") and 2(2") with a positive selectivity for positions 1(1"). Its decrease is considerably faster in positions 2(2") than in positions 1(1"), which would suggest different rates of fatty acid turnover. Fat deficiency induces the appearance of 18:2(n-7) and a significant increase of 20:3(n-6) (dihomo-gamma-linolenic acid) in liver and kidney cardiolipins. In contrast, 20:3(n-6) level remains unchanged in other mitochondrial phospholipids. 18:2(n-7) and 20:3(n-6) are almost evenly distributed between both pairs of positions. Both acids have a common structural feature, that is double bonds in positions 8 and 11. 20:3(n-9) accumulates in large amounts in other mitochondrial phospholipids, but not in cardiolipins. Although surprising, 20:3(n-6) has thus to be considered as a specific marker of deficiency for cardiolipins when it is esterified to positions 1(1"). Taking into account various analytical data, it would appear that positions 1(1") of cardiolipins can only incorporate unsaturated fatty acids containing at least one cis double bond in position 8 or 9, with no other double bond between these positions and the carboxylic group.

Steady-state fluorescence polarization study of structurally defined phospholipids from liver mitochondria of rats fed elaidic acid.

In vivo-modified phospholipids from rat liver mitochondria were used to study the effect of trans-fatty acid incorporation into phospholipids on the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) embedded in phospholipid liposomes. Pure fractions of mitochondria phospholipids were prepared and analyzed for their fatty acid compositions and fatty acid positional distribution. In rats fed a diet enriched with trielaidin, elaidic acid (trans-9 18:1 acid) was extensively incorporated in position 1 of phosphatidylcholine (PC; 31% of fatty acids esterified to this position), phosphatidylethanolamine (PE; 42.5%) and phosphatidylinositol (PI; 43%). Less than 10% of the incorporated elaidic acid was esterified to position 2 of these phospholipids. More than 90% of elaidic acid esterified to position 1 displaced saturated acids. Consequently, about one-third of PC molecules and two-fifths of PE and PI molecules contained one molecule of elaidic acid instead of one saturated fatty acid molecule in their 1-position. On the other hand, cardiolipin, which is naturally practically devoid of saturated acids, was particularly resistant to elaidic acid incorporation. The fluorescence polarization of DPH incorporated in liposomes made of PC-PI and of PC-PI-PE from liver mitochondria of rats fed or not fed elaidic acid was measured. No significant differences between phospholipids containing or not containing elaidic acid could be detected. Values of DPH fluorescence polarization for all samples were comprized between 0.133 and 0.135 at 25 degrees C. We thus conclude that when elaidic acid replaces saturated fatty acids in phospholipids, even in a high proportion (one-third), the physical state of acyl chains in the hydrophobic core of membranes is not grossly modified. Thus, elaidic acid seems to behave like a saturated fatty acid, not only biochemically for the acylation of phospholipids, but also physically.

Inhibition of pancreatic lipase by mixed micelles of diethyl p-nitrophenyl phosphate and bile salts.

Solubility and Sephadex filtration assays have shown that dissolved diethyl p-nitrophenyl phosphate can be included into bile salt micelles with a partition coefficient of 32 : 1. This inclusion is probably a prerequisite for the organophosphate to inhibit lipase. The essential role played by colipase confirms that the primary step in the inhibition is an interaction of lipase with bile salt containing micelles. Therefore, it appears that the requirements of lipase towards specific substrates and inhibitors are very similar. The inhibition rate strongly depends on the total bile salt concentration and on the micellar concentration of the organophosphate. This effect may be explained, at least qualitatively, by a competition between simple and mixed micelles for the binding of colipase and lipase.

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.

On the formation of a ternary complex between lipase, colipase and micelles of amphipathic compounds.

Pancreatic lipase, colipase and 1-glyceryl octyl ether or lysolecithin micelles were shown to form a ternary complex probably resulting from the fixation of the enzyme to the binary colipase-micelle association already well documented in the case of bile salt micelles. Kinetic assays show that the colipase favours the lipase activity on tributyrin emulsions in presence of 1-glyceryl octyl ether. The UV spectrum of a derivative of lipase in which two 2-hydroxy 5-nitrobenzyl groups are attached to tryptophan, is distinctly modified by addition of 1-glyceryl octyl ether micelles in the presence of colipase.

[Effect of the addition of hog pancreatic colipase on the permeability to glucose and the phase transition of phosphatidyl choline liposomes]. / Effet de l'addition de la colipase pancréatique de porc sur la perméabilité au glucose et la transition de phase de liposomes de phosphatidyl choline.

An interaction between porcine pancreatic coli-pase and lecithin liposomes is demonstrated by gel filtration assays. The extent of the colipase penetration into the phospholipid bilayer was assessed by permeability and calorimetry studies carried out on the liposome colipase complex. The addition of colipase to liposomes induces a three fold increase in the permeability to [6-H3] glucose. This result reflects a perturbation in the bilayer which may be the consequence of the colipase interaction. The phase transition temperature is not modified by the added colipase. This observation suggests that the perturbation brought by the protein does not affect the acyl chain packing of the bulk lipid. On the other hand the enthalpy of transition (delta H) is decreased from 8.9 to 7.8 kcal/mole by the addition of colipase to the lipid. This could be explained by the interaction of the colipase with neighbouring acyl chains which do not participate in the cooperative melting of the bulk lipid. In agreement with previous spectrophotometric observations, the present results are indicative of hydrophobic interactions between colipase and bilayer hydrocarbon chains.

Effects of preterm infant formula supplementation with alpha-linolenic acid with a linoleate/alpha-linolenate ratio of 6: a multicentric study.

OBJECTIVE: To investigate the effects of a milk formula supplemented with a alpha-linolenic acid (ALA) (18:2 n-6/18:3 n-3 ratio near 6/1) on plasma and red blood cell (RBC) fatty acids (FAs) in premature infants and compare with a non supplemented formula (18:2 n-6/18:3 n-3 = 22/1). DESIGN AND SUBJECTS: Infants of mothers who elected not to breast-feed were randomly assigned to either a high alpha linolenic formula (HLF: n = 31) group or a low alpha-linolenic formula (LLF: n = 32) control group. Infants fed human milk (HM: n = 25) were enrolled concurrently as a reference group. Anthropometric and biological measurements were made after two days (D2) and 15 d (D15) of enteral feeding and at the 37th week (W37) of postconceptual age. In HLF, the 18:3 n-3 content was 1.95% of total FAs (0.77% of total energy) and the 18:2 n-6/18:3 n-3 ratio was near 6/1. In LLF, the 18:3 n-3 content was 0.55% of total FAs (0.22% of total energy) and the 18:2 n-6/18:3 n-3 ratio was 22/1. RESULTS: ALA supplementation had minimal effect on the n-6 series, did not alter the anthropometric data and confirmed the conversion of ALA into docosahexaenoic acid (DHA). Throughout the study, it maintained, the RBC membrane DHA values within the confidence interval of those obtained in the HM group. Such was not the case with LLF CONCLUSION: alpha-linolenic acid supplementation (from Rapeseed oil and in a 18:2 n-6/18:3 n-3 ratio = 6) in premature infant formula can contribute efficiently to the maintenance of the n-3 status in the premature newborns.

In vitro behavior of marine lipid-based liposomes. Influence of pH, temperature, bile salts, and phospholipase A2.

To deliver polyunsaturated fatty acids (PUFA) by the oral route, liposomes based on a natural mixture of marine lipids were prepared by filtration and characterized in media that mimic gastrointestinal fluids. First the influence of large pH variations from 1.5-2.5 (stomach) to 7.4 (intestine) at the physiological temperature (37 degrees C) was investigated. Acidification of liposome suspensions induced instantaneous vesicle aggregation, which was partially reversible when the external medium was further neutralized. Simultaneously, complex morphological bilayer rearrangements occurred, leading to the formation of small aggregates. These pH- and temperature-dependent structural changes were interpreted in terms of osmotic shock and lipid chemical alterations, i.e., oxidation and hydrolysis, especially in the first hours of storage. Besides, oxidative stability was closely related to the state of liposome aggregation and the supramolecular organization (vesicles or mixed micelles). The effects of bile salts and phospholipase A2 (PLA2) on the liposome structures were also studied. Membrane solubilization by bile salts was favored by preliminary liposome incubation in acid conditions. PLA2 showed a better activity on liposome structures than on the corresponding mixed lipid-bile salt micelles. As a whole, in spite of slight morphological modifications, vesicle structures were preserved after an acid stress and no lipid oxidation products were detected during the first 5 h of incubation. Thus, marine lipids constituted an attractive material for the development of liposomes as potential oral PUFA supplements.

Positional distribution of fatty acids in cardiolipin of mitochondria from 21-day-old rats.

Pure cardiolipins (1,3-diphosphatidylglycerol) were prepared from mitochondria of heart, liver and kidney from 21-day-old male Wistar rats and submitted to Naja naja venom phospholipase A2 (EC 3.1.1.4) action. Incubation conditions were controlled carefully, and a complete hydrolysis of cardiolipin to lysocardiolipin (di [1 (1'') acyl sn-glycero-3-phosphoryl] 1',3'-sn-glycerol) and fatty acids from positions 2(2'') was obtained in less than two hr practically without side reactions. Cardiolipins from the three organs contained low levels of saturated fatty acids; stearic acid accounted for 0.4-0.7% and palmitic acid for 1.4-3.5% of total fatty acids. These percentages apparently depended on the organ. In all three cases, linoleic acid was the major component, but its percentage varied from 62-78% of total fatty acids. Acyl chains linked to positions 1 (1'') of all three cardiolipin preparations exhibited a similar pattern: they were composed of linoleic acid for 85-89%. This fatty acid also was the main component esterified at position 2 (2''), but its percentage was much more variable: from 39.8% in heart to 51.2% in kidney and 67.8% in liver mitochondria. The remaining acids comprised octadecenoic and polyunsaturated fatty acids with more than 18 carbon atoms in different proportions. As opposed to other phospholipids, cis-vaccenic acid, and not oleic acid, was the main octadecenoic acid present in cardiolipins. Octadecenoic acids were nine- to 10-fold more concentrated at positions 2 (2'') than at positions 1 (1''). The percentage of cis-vaccenic acid was four- to five-fold higher than that of oleic acid at positions 2 (2''), whereas oleic acid dominated at positions 1 (1'').(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of alkenyl chain profile in plasmalogens of rat heart mitochondria by dietary trielaidin.

Effects of dietary trielaidin upon the alkenyl chain profile of plasmalogens were studied using heart mitochondria of rats fed a semi-purified diet containing 10% of fat supplement in which elaidic acid accounted for 69% of total fatty acids. Alkyl substituted dioxane (ASD) derivatives of the alkenyl groups of plasmalogens were prepared and analyzed by silver nitrate TLC and by GLC on different phases (BDS and OV-275). After two months of feeding the experimental diet, 40% of the ASD contained a trans-octadecenyl chain, suggesting that dietary elaidic acid was reduced in vivo to the corresponding alcohol and incorporated into plasmalogens. There was a simultaneous decrease in the percentage of ASD containing saturated chains, but the percentage of ASD substituted with cis-octadecenyl chains was not significantly affected. These observations suggested that elaidic acid may compete with saturated fatty acids, but not with cis-octadecenoic acids during the plasmalogen biosynthesis. Feeding trielaidin did not seem to have any significant influence on the relative proportions of plasmalogens, which accounted for 11-12%, on a phosphorus basis, of total heart mitochondria phospholipids.

Lymphatic absorption of nonvolatile oxidation products of heated oils in the rat.

The lymphatic absorption of nonvolatile oxidation products (NVOP) formed during heating of fats was studied. Heated colza or soybean oils or synthetic triglycerides containing a definite aromatic or alicyclic fatty acid were fed to thoracic duct-cannulated rats. Tritium-labeled triolein was added to each dietary fat, as an internal standard, in order to calculate the percentage of lymphatic absorption of the ingested NVOP. Results show that 4% of the total polymeric acids, 53% of the total oxidized monomeric acids and 96% of the total cyclic monomeric acids were recovered in the lymphatic lipids. Gas liquid and quantitative thin layer chromatography of these 3 classes indicated that, within a NVOP class, the various constituents did not present the same absorption rate. The lymphatic absorptions of individual oxidized monomers were between 25 and 93%. Concerning the polymer fraction, the lymphatic recoveries were 1% (nonpolar dimers), 6.8% (polar dimers) and 12% (polar oligomers). Aromatic acids were absorbed to a lesser degree (50-60%) than cyclohexenic acids (91-98%).

Follow-up of the delta4 to delta16 trans-18:1 isomer profile and content in French processed foods containing partially hydrogenated vegetable oils during the period 1995-1999. Analytical and nutritional implications.

A survey of the total content of trans-18:1 acids and their detailed profile in French food lipids was conducted in 1995-1996, and 1999. For this purpose, 37 food items were chosen from their label indicating the presence of partially hydrogenated vegetable oils (PHVO) in their ingredients. The content as well as the detailed profile of these isomers was established by a combination of argentation thin-layer chromatography and gas liquid chromatography (GLC) on long polar capillary columns. With regard to the mean trans-18:1 acid contents of extracted PHVO, a significant decrease was observed between the two periods, i.e., from 26.9 to 11.8% of total fatty acids. However, only minor differences were noted in the mean relative distribution profiles of individual trans-18:1 isomers with ethylenic bonds between positions delta4 and delta16 for the two periods. The predominant isomer was delta9-18:1 (elaidic) acid, in the wide range 15.2-46.1% (mean, 27.9+/-7.2%) of total trans-18:1 acids, with the delta10 isomer ranked second, with a mean of 21.3% (range, 11.6 to 27.4%). The content of the unresolved delta6 to delta8 isomer group was higher than the delta11 isomer (vaccenic acid), representing on average 17.5 and 13.3%, respectively. Other isomers delta4, delta5, delta12, delta13/delta14, delta15, and delta16, were less than 10% each: 1.0, 1.6, 7.4, 7.1, 1.8, and 1.0%, respectively. However, considering individual food items, it was noted that none of the extracted PHVO were identical to one another, indicating a considerable diversity of such fats available to the food industry. A comparison of data for French foods with similar data recently established for Germany indicates that no gross differences occur in PHVO used by food industries in both countries. Estimates for the absolute mean consumption of individual isomers from ruminant fats and PHVO are made for the French population and compared to similarly reconstructed hypothetical profiles for Germany and North America. Differences occur in the total intake of trans-18:1 acids, but most important, in individual trans-18:1 isomer intake, with a particular increase of the delta6-delta8 to delta10 isomers with increasing consumption of PHVO. It is inferred from the present and earlier data that direct GLC of fatty acids is a faulty procedure that results (i) in variable underestimates of total trans-18:1 acids, (ii) in a loss of information as regards the assessment of individual isomeric trans-18:1 acids, and (iii) in the impossibility of comparing data obtained from human tissues if the relative contribution of dietary PHVO and ruminant fats is not known.

Trans fatty acids in adipose tissue of French women in relation to their dietary sources.

This study reports the fatty acid composition of subcutaneous adipose tissue in French women with special emphasis on the content of trans fatty acids originating from two main dietary sources, ruminant fats and partially hydrogenated vegetable oils (PHVO). Adipose tissue trans fatty acid levels from 71 women, recruited between 1997 and 1998, were determined using a combination of capillary gas chromatography and silver nitrate thin-layer chromatography. Results indicate that on average cis monounsaturates accounted for 47.9% of total fatty acids, saturates for 32.2%, and linoleic acid for 14.4%. Cis n-3 polyunsaturates represented only 0.7%. Total content of trans fatty acids was 2.32 +/- 0.50%, consisting of trans 18:1 (1.97 +/- 0.49%), trans 18:2 (0.28 +/- 0.08%), and trans 16:1 (0.06 +/- 0.03%). Trans 18:3 isomers were not detectable. The level of trans fatty acids found in adipose tissue of French women was lower than those reported for Canada, the United States, and Northern European countries but higher than that determined in Spain. Therefore, trans fatty acid consumption in France appears to be intermediate between that of the United States or North Europe and that of Spain. Based on the equation of Enig et al., we estimated the mean daily trans 18:1 acid intake of French women at 1.9 g per person. The major trans 18:1 isomer in adipose tissue was delta11 trans, as in ruminant fats. Estimates of relative contribution of trans fatty acid intake were 55% from ruminant fats and 45% from PHVO. This pattern contrasts sharply with those established for Canada and the United States where PHVO is reported to be the major dietary source of trans fatty acids.

Interactions of colipase with bile salt micelles. 2. Study by dialysis and spectrophotometry.

The finding reported in the preceding paper that colipase is able to bind one sodium taurodeoxycholate micelle per molecule was confirmed by dialysis and spectrophotometry. Dialysis in the presence of labelled sodium taurodeoxycholate provided a direct qualitative proof of taurodeoxycholate binding to colipase. This binding was found to occur only above the critical micelle concentration. But, dialysis did not give any information about the composition of the associations, because equilibrium was not attained at the end of the assays. Addition of sodium taurodeoxycholate above the critical micelle concentration was also observed to induce a strong perturbation of the ultraviolet spectrum of one or several of the three tyrosines of colipase. The variation of the perturbation as a function of sodium taurodeoxycholate concentration was consistent with the binding of a single micelle to colipase. The dissociation constant calculated in "micelle molarity" was approximately 1 X 10(-4) M. The colipase-bile salt micelle association can fix one molecule of lipase to form a ternary complex which represents an interesting model of a protein-protein interaction mediated by an organized lipid structure. The ternary complex is probably also a model for lipase-substrate interactions in the presence of an amphipath.

[Implications of the incorporation of transisomers of unsaturated fatty acids into cell membranes]. / Implications de l'incorporation d'isomeres trans d'acides gras insaturés au niveau des membranes cellulaires.

The incorporation of dietary trans fatty acids is shown into intestinal brush border and heart mitochondrial membranes obtained from rats fed with either partially hydrogenated soybean oil or with a synthetic triglyceride containing elaidic or linelaidic acid. The distribution of dietary linelaidate in the major phospholipid classes from inner membranes of mitochondria is determined. Heart mitochondria from normal rats and linelaidic acid fed rats are compared with respect to their swelling rate and their oxidative phosphorylation. The influence of dietary linelaidate on the activity of some brush border-associated enzymes is also studied. On the basis of the authors' observations and those made by others with microorganism cels or models of biological membranes (liposomes and monomolecular films), the possible implications of trans fatty acids incorporation on membrane functions are reviewed.

[Time-course changes in the alkenyl and acyl chain composition of plasmalogens from the heart and kidneys of rats ingesting trielaidin]. / Evolution au cours du temps de la composition en chaînes alkényles et acyles des plasmalogènes de mitochondries de coeur et de reins chez des rats ingérant de la triélaïdine.

Weaned rats were fed for 7 or 32 days a semi-purified diet containing 10% of fat supplement in which elaidic acid accounted for 64.5% of total fatty acids. Alkenyl groups from plasmalogens of kidney and heart mitochondria were analysed as their alkyl-substituted dioxane derivatives by gas liquid chromatography (GLC). Acyl chains of total and individual phospholipids (phosphatidylcholine: PC; phosphatidylethanolamine: PE, diacyl and alkenyl-acyl forms; phosphatidylinositol: PI; cardiolipin: CL) were also analysed by GLC. Maximum level of elaidic acid incorporation was attained after only one week on the experimental diet in either total or individual phospholipids. Heart mitochondria phospholipids contained about 40% more trans-monounsaturated acids (20.9%) than kidney mitochondria phospholipids (12.5%) after one month. trans-Monounsaturated acids (t-18: 1 plus t-16: 1 acids) were incorporated in individual phospholipids from both organs in decreasing order: PI greater than PE (total) greater than PC much greater than CL. The order of incorporation of elaidic acid seemed to be related to the content of stearic acid in these phospholipids when the diet was devoid of elaidic acid (PI greater than PE greater than PC much greater than CL). The content of elaidic acid in alkenyl-acyl-glycerophosphoethanolamine (alkenyl-acyl-GPE) was the same (6% of total fatty acids) in both organs. The time-course incorporation of trans-monounsaturated alkenyl chains was different. After one week on the elaidic acid-enriched diet, they represented only 65% (kidney) or 43% (heart) of the values reached after one month. At the end of the feeding period, the trans-monounsaturated alkenyl chains were also about 40% less in total plasmalogens from kidney mitochondria (27.8%) than from heart mitochondria (46%). trans-Monounsaturated acyl chains were incorporated at a higher rate in the 2-position of alkenyl-acyl-GPE than trans-monounsaturated alkenyl chains in their 1-position. Our results confirm the unique observation of Kramer et al. (1985) who noticed that the fatty acid composition of phospholipids was much more rapidly equilibrated than the alkenyl chain composition of plasmalogens (1 week vs 2-3 weeks) from heart of rats fed diets supplemented with different oils.