Robert L. Wolff

Content and distribution oftrans-18:1 acids in ruminant milk and meat fats. Their importance in european diets and their effect on human milk

Thetrans-18:1 acid content and distribution in fats from ewe and goat milk, beef meat and tallow were determined by a combination of capillary gas-liquid chromatography and argentation thin-layer chromatography of fatty acid isopropyl esters. Thetrans isomers account for 4.5 ± 1.1% of total fatty acids in ewe milk fat (seven samples) and 2.7±0.9% in goat milk fat (eight samples). In both species, as in cow, the main isomer is vaccenic (trans-11 18:1) acid. The distribution profile oftrans-18:1 acids is similar among the three species. The contribution of ewe and goat milk fat to the daily intake oftrans-18:1 acids was estimated for people from southern countries of the European Economic Community (EEC): France, Italy, Greece, Spain, and Portugal. It is practically negligible for most of these countries, but in Greece, ewe and goat milk fat contributeca. 45% of the daily consumption oftrans-18:1 acids from all dairy products (0.63 g/person/day for a total of 1.34 g/person/day). Thetrans-18:1 acid contents of beef meat fat (ten retail cuts, lean part) and tallow (two samples) are 2.0 ± 0.9% and 4.6%, respectively, of total fatty acids (animals slaughtered in winter). Here too, the main isomer is vaccenic acid. Othertrans isomers have a distribution pattern similar to that of milk fat. Beef meat fat contributes less than one-tenth of milk fat to thetrans-18:1 acid consumed. The daily per capita intake oftrans-18:1 acids from ruminant fats is 1.3–1.8 g for people from most countries of the EEC, Spain and Portugal being exceptions (ca. 0.8 g/person/day). In France, the respective contributions of ruminant fats and margarines to the daily consumption oftrans-18:1 acids are 1.7 and 1.1 g/person/day (60 and 40% of total, respectively). These proportions, based on consumption data, were confirmed by the analysis of fat from milk of French women (ten subjects). The mean content oftrans-18:1 acids in human milk is 2.0 ± 0.6%, with vaccenic acid being the major isomer. Based on the relative levels of thetrans-16 18:1 isomer, we could confirm that milk fat is responsible for the major part of the daily intake oftrans-18:1 acids by French people. The daily individual intake oftrans-18:1 isomers from both ruminant fats and margarines for the twelve EEC countries varies from 1.5 g in Spain to 5.8 g in Denmark, showing a well-marked gradient from the southwest to the northeast of the EEC.

Evaluation of sequential methods for the determination of butterfat fatty acid composition with emphasis ontrans-18:1 acids. Application to the study of seasonal variations in french butters

The successive steps of an integrated analytical procedure aimed at the accurate determination of butterfat fatty acid composition, includingtrans-18:1 acid content and profile, have been carefully checked. This sequential procedure includes: dispersion of a portion of butter in hexane/isopropanol (2:1, vol/vol) with anhydrous Na2SO4, filtration of aliquots of the suspension through a microfiltration unit, subsequent preparation of fatty acid isopropyl esters (FAIPE) with H2SO4 as a catalyst, and analysis of total FAIPE by capillary gas-liquid chromatography (GLC). Isolation oftrans-18:1 isomers was by silver-ion thin-layer chromatography (Ag-TLC), followed by extraction from the gel of combined saturated andtrans-monoenoic acids with a biphasic solvent system. Analysis of these fractions by GLC allows the accurate quantitation oftrans-18:1 acids with saturated acids (14:0, 16:0, and 18:0) as internal standards. A partial insight in the distribution oftrans-18:1 isomers can be obtained by GLC on a CP Sil 88 capillary column (Chrompack, Middelburg, The Netherlands). All steps of the procedure are quite reproducible, part of the coefficients of variation (generally less than 3%, mainly limited to butyric and stearic acids) being associated with GLC analysis (injection, integration of peaks) and, to a lesser extent, to FAIPE preparation. FAIPE appear to be of greater practical interest than any other fatty acid esters, including fatty acid methyl esters (FAME), for the quantitation of short-chain fatty acids, because peak area percentages, calculated by the integrator coupled to the flame-ionization detector, are almost equal (theoretically and experimentally) to fatty acid weight percentages and do not require correction factors. With this set of procedures, we have followed in detail the seasonal variations of fatty acids in butterfat, with sixty commercial samples of French butter collected at five different periods of the year. Important variations occur around mid-April, when cows are shifted from forage and concentrates during winters spent in their stalls to fresh grass in pastures. At this period, there is a decrease of 4:0–14:0 acids and of 16:0 (−2 and −6%, respectively), while 18:0 andcis- plustrans-18:1 acids rise suddenly (2 and 5%, respectively). These modifications then progressively disappear until late fall or early winter. Other variations are of minor quantitative importance. Although influenced by the season, the content of 18:2n-6 acid lies in the narrow range of 1.2–1.5%.Trans-18:1 acids, quantitated by GLC after Ag-TLC fractionation, are at their highest level in May–June (4.3% of total fatty acids), and at their lowest level between January and the end of March (2.4%), with a mean annual value of 3.3%. The proportion of vaccenic (trans-11 18:1) acid, relative to totaltrans-18:1 isomers, is higher in spring than in winter, with intermediate decreasing values in summer and fall, which supports the hypothesis that the level of this isomer is linked to the feed of the cattle, and probably to the amount of grass in the feed.

trans-Polyunsaturated fatty acids in French edible rapeseed and soybean oils

The fatty acid compositions of rapeseed and soybean oils marketed in France have been determined by gas liquid chromatography on a fused-silica capillary column coated with a 100% cyanopropyl polysiloxane stationary phase. Under the operating conditions employed, methyl esters of linolenic acid geometrical isomers could be separated and quantitated easily without any other complementary technique. With only one exception, all samples under study (eight salad oils and five food samples) contain geometrical isomers of linolenic acid in measurable, although variable, amounts. Totaltrans-18:3 acids may account for up to 3% of total fatty acids. This value corresponds to a degree of isomerization (percentage oftrans isomers relative to total octadecatrienoic acids) of 30%. Examination of our data indicates that the distribution pattern of linolenic acid geometrical isomers does not depend on the degree of isomerization. The two main isomers always have thec,c,t and thet,c,c configurations. These isomers occur in the almost invariable relative proportions of 47.8±1.7% and 41.1±1.0%, respectively. The third mono-trans isomer is present in lower amounts−6.5±0.7%. The only di-trans isomer that can be quantitated with sufficient accuracy is thet,c,t isomer (4.9±1.5%). Mono-trans isomers of linoleic acid are also present in these oils. However, their maximum percentages are lower than those determined for linolenic acid geometrical isomers. In the oils showing the highest degrees of isomerization,trans isomers of linoleic acid account for 0.5% (rapeseed oils) and 1% (soybean oils) of total fatty acids. Taking into account all data, it would appear that the probability of isomerization of linolenic acid is about 13–14 times that of linoleic acid.

Improvement in the resolution of individualtrans-18:1 isomers by capillary gas-liquid chromatography: Use of a 100-m CP-Sil 88 column

Doubling the length of a CP-Sil 88 capillary column (Chrompack, Middelburg, The Netherlands) from 50 to 100 m remarkably improves the resolution of individualtrans-18:1 isomers from either ruminant fats or partially hydrogenated oils. Although the use of a 50-m column gives interesting results, it does not allow sufficient resolution of thetrans-10 andtrans-11 18:1 isomers. Moreover, thetrans-6 totrans-9 18:1 isomers emerge as a single group of peaks, whereas thetrans-12 isomer is only partly resolved from the adjacenttrans-11 andtrans-13 plustrans-14 isomers. With the 100-m column, thetrans-9,trans-10, andtrans-12 18:1 isomers are almost base-line resolved from other isomers. However, with both columns, it is not possible to separate the critical pair oftrans-13 andtrans-14 18:1 acids which co-elute under a single peak. Despite this minor drawback, the 100-m CP-Sil 88 column appears to be of great interest for the separation and the quantitation of most individualtrans-18:1 acids. Except for the use of argentation thin-layer chromatography, there is no need for complementary techniques, such as ozonolysis. This simple and powerful tool may be applied to ruminant fats, partially hydrogenated oils, and human tissue lipids.

Fatty acid composition of some pine seed oils

The fatty acid composition of seeds from seven species of the genusPinus (P. pinaster, P. griffithii, P. pinea, P. koraiensis, P. sylvestris, P. mughus, andP. nigra) was established. Pine seeds are rich in oil (31–68% by weight) and contain several unusual polymethylene-interrupted unsaturated fatty acids with acis-5 ethylenic bond. These are thecis-5,cis-9 18:2,cis-5,cis-9,cis-12 18:3,cis-5,cis-11 20:2, andcis-5,cis-11,cis-14 20:3 acids, with a trace ofcis-5,cis-9,cis-12,cis-15 18:4 acid. Their percentage relative to total fatty acids varies from a low of 3.1% (P. pinea) to a high of 30.3% (P. sylvestris), depending on the species. The majorcis-5 double bond-containing acid is generally thecis-5,cis-9,cis-12 18:3 acid (pinolenic acid). In all species, linoleic acid represents approximately one-half the total fatty acids, whereas the content of oleic acid varies in the range 14–36% inversely to the sum of fatty acids containing acis-5 ethylenic bond. The easily available seeds fromP. koraiensis appear to be a good source of pinolenic acid: their oil content isca. 65%, and pinolenic represents about 15% of total fatty acids. These values appear to be rather constant.Pinus pinaster, which is grown on several thousand acres in the southwest of France, is an interesting source ofcis-5,cis-11,cis-14 20:3 acid (7% in the oil, which isca. 35% of the dehulled seed weight), an acid sharing in common three double bonds with arachidonic acid. Apparently,P. sylvestris seed oil contains the highest level ofcis-5 double bond-containing acids among pine seed oils that have ever been analyzed.

Heat-induced geometrical isomerization of α-linolenic acid: Effect of temperature and heating time on the appearance of individual isomers

The formation of linolenic acid geometrical isomers (LAGIs) was studied in linseed oil that was heated under vacuum in sealed ampoules at different temperatures (190–260°C) for several durations (2–16 h). A temperature of about 190°C seems to be necessary to induce the formation of LAGIs. At higher temperatures, disappearance of linolenic acid follows a first-order kinetic. The formation of LAGIs increases with both heating time and temperature, degrees of isomerization of linolenic acid higher than 50–60% could easily be obtained by simply heating the oil under vacuum. Side reactions remain at a low level. The mean probabilities of isomerization of individual ethylenic bonds are similar to those determined in linolenic acid-containing oils marketed in European countries, 41.9, 4.7 and 53.3% for double bonds in positions 9, 12 and 15, respectively. The di-trans t,c,t (trans,cis,trans) isomer is formedvia the mono-trans c,c,t andt,c,c isomers by a two-step reaction. The proportions of thec,c,t andt,c,c isomers (relative to total LAGIs) decrease linearly with the heating time. The proportion of thec,t,c isomer is only slightly affected by this parameter; however, it increases with temperature. The proportion of thet,c,t isomer increases linearly with heating time at each tested temperature, at the expense of thec,c,t andt,c,c isomers. However, there is no simple relationship linking the disappearance of each of the mono-trans isomers and the formation of the di-trans isomer.

Follow-up of the Δ4 to Δ16 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 Δ4 and Δ16 for the two periods. The predominant isomer was Δ9–18∶1 (elaidic) acid, in the wide range 15.2–46.1% (mean, 27.9±7.2%) of total trans-18∶1 acids, with the Δ10 isomer ranked second, with a mean of 21.3% (range, 11.6 to 27.4%). The content of the unresolved Δ6 to Δ8 isomer group was higher than the Δ11 isomer (vaccenic acid), representing on average 17.5 and 13.3%, respectively. Other isomers Δ4, Δ5, Δ12, Δ13/Δ14, Δ15, and Δ16, 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 Δ6–Δ8 to Δ10 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.

Effects of Pinus pinaster and Pinus koraiensis seed oil supplementation on lipoprotein metabolism in the rat

The aim of the present study was to assess the effect of vegetal oils obtained from Pinus pinaster and P. koraiensis seeds on plasma lipoprotein levels and apolipoprotein (apo) gene expression in rats. These oils contain two particular fatty acids of the Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) family: all-cis-5,9,12-18:3 (pinolenic) and/or all-cis-5,11,14-20:3 (sciadonic) acids. Rats were fed for 28 d a diet containing 5% (w/w) oil supplement. Two control diets were prepared to match the fatty acid composition of P. pinaster or P. koraiensis oils with the exception of Δ5-UPIFA, which were replaced by oleic acid. Pinus pinaster seed oil decreased serum triglycerides by 30% (P<0.02), very low density lipoprotein (VLDL)-triglycerides by 40% (P<0.01), and VLDL-cholesterol by 33% (P<0.03). Pinus koraiensis seed oil decreased serum triglycerides by 16% [not statistically significant (ns)] and VLDL-triglycerides by 21% (ns). Gel permeation chromatography and nondenaturating polyacrylamide gel electrophoresis showed a tendency of high density lipoprotein to shift toward larger particles in pine seed oil-supplemented rats. Finally, P. pinaster seed oil treatment was associated with a small decrease of liver apoC-III (P<0.02) but not in apoE, apoA-I, or apoA-II mRNA levels. The levels of circulating apo were not affected by pine seed oil supplementation. In conclusion, P. pinaster seed oil has a triglyceride-lowering effect in rats, an effect that is due to a reduction in circulating VLDL.

General characteristics of Pinus spp. seed fatty acid compositions, and importance of Δ5-olefinic acids in the taxonomy and phylogeny of the genus.

The Δ5-unsaturated polymethylene-interrupted fatty acid (Δ5-UPIFA) contents and profiles of gymnosperm seeds are useful chemometric data for the taxonomy and phylogeny of that division, and these acids may also have some biomedical or nutritional applications. We recapitulate here all data available on pine (Pinus; the largest genus in the family Pinaceae) seed fatty acid (SFA) compositions, including 28 unpublished compositions. This overview encompasses 76 species, subspecies, and varieties, which is approximately one-half of all extant pines officially recognized at these taxon levels. Qualitatively, the SFA from all pine species analyzed so far are identical. The genus Pinus is coherently united—but this qualitative feature can be extended to the whole family Pinaceae—by the presence of Δ5-UPIFA with C18 [taxoleic (5,9–18∶2) and pinolenic (5,9,12–18∶3) acids] and C20 chains [5,11–20∶2, and sciadonic (5,11,14–20∶3) acids]. Not a single pine species was found so far with any of these acids missing. Linoleic acid is almost always, except in a few cases, the prominent SFA, in the range 40–60% of total fatty acids. The second habitual SFA is oleic acid, from 12 to 30%. Exceptions, however, occur, particularly in the Cembroides subsection, where oleic acid reaches ca. 45%, a value higher than that of linoleic acid. α-Linolenic acid, on the other hand, is a minor constituent of pine SFA, almost always less than 1%, but that would reach 2.7% in one species (P. merkusii). The sum of saturated acids [16∶0 (major) and 18∶0 (minor) acids principally] is most often less than 10% of total SFA, and anteiso-17∶0 acid is present in all species in amounts up to 0.3%. Regarding C18 Δ5-UPIFA, taxoleic acid reaches a maximum of 4.5% of total SFA, whereas pinolenic acid varies from 0.1 to 25.3%. The very minor coniferonic (5,9,12,15–18∶4) acid is less than 0.2% in all species. The C20 elongation product of pinolenic acid, bishomo-pinolenic (7,11,14–20∶3) acid, is a frequent though minor SFA constituent (maximum, 0.7%). When considering C20 Δ5-UPIFA, a difference is noted between the subgenera Strobus and Pinus. In the former subgenus, 5,11–20∶2 and sciadonic acids are ≤0.3 and ≤1.9%, respectively, whereas in the latter subgenus, they are most often ≥0.3 and ≥2.0%, respectively. The highest values for 5,11–20∶2 and sciadonic acids are 0.5% (many species) and 7.0% (P. pinaster). The 5,11,14,17–20∶4 (juniperonic) acid is present occasionally in trace amounts. The highest level of total Δ5-UPIFA is 30–31% (P. sylvestris), and the lowest level is 0.6% (P. monophylla). Uniting as well as discriminating features that may complement the knowledge about the taxonomy and phylogeny of pines are emphasized.

Contribution oftrans-18∶1 acids from dairy fat to european diets

Twelve commercial samples of French butter, purchased in October–November, and 12 other samples, purchased in May–June, were analyzed with particular attention to theirtrans-octadecenoic acid contents. The isomeric fatty acids were quantitated by a combination of gas-liquid chromatography (GLC) of total fatty acids as isopropyl esters on a polar capillary column (CPSil 88) and of silver nitrate-impregnated thin-layer chromatography followed by GLC of the pooled saturated (used as internal standards) andtrans-octadecenoic acid fractions. Autumn butters contained 3.22±0.44%trans-octadecenoic acids (relative to total fatty acids), whereas those collected during the spring contained 4.28±0.47% (P<0.01). Minimum and maximum values for the two sets of butters were 2.46 (autumn) and 5.10% (spring), respectively. The annual mean value for thetrans-octadecenoic acid content in all butter samples was 3.8% of total fatty acids (ca. 2% for thetrans-11 18∶1 acid). This value allows calculation of the daily individual intake oftrans-octadecenoic acids from dairy products by populations of member states of the European Economic Community (EEC). It varies from 0.57 g (Portugal) to 1.66 g (Denmark). The mean value for the twelve countries of the EEC is 1.16 g/person/d, which is close to data published for the United States. In France, the consumption oftrans octadecenoic acids from dairy fat is higher than that from margarines (ca. 1.5 vs. 1.1 g/person/d).