Fatty acid composition and nutritional relevance of most widely consumed margarines in Spain

Este estudio examina la composición de ácidos grasos de las margarinas de mayor consumo en España en el año 2000, incluyendo ácidos grasos trans. Todas las margarinas contenían al menos 20% de linoleico, siendo el contenido medio del 38%. Los ácidos grasos saturados (laúrico y mirístico) no sobrepasaron el 4% del total de ácidos grasos. La mayoría de las margarinas contenían menos del 5% de ácidos grasos trans C18:1, aunque la variabilidad era elevada entre las distintas marcas (coeficiente de variación:112%), siendo la mediana del 2.5%; los ácidos grasos trans poliinsaturados C18:2 y C18:3 no representaron más del 1%. Índices nutricionalmente importantes como el cociente ácidos grasos saturados/insaturados, índices trombogénico y aterogénico, fueron menores de 0,5. Los resultados sugieren que las margarinas españolas muestran un cambio hacia una distribución de ácidos grasos más saludable, aunque debido a la gran variabilidad en su composición, la información del etiquetado debe mejorar para evi tar per turbaciones en la trad ic ional d ie ta Mediterránea de España.


INTRODUCTION
The Mediterranean diet has been described as a useful model for providing recommended energy and nutrient levels for different population groups (Moreiras-Varela, 1989).Spain and most of the European Mediterranean countries show the lowest level of cardiovascular disease (CVD) mortality in Europe (Uemura and Pisa, 1988).Traditionally olive oil has been the main source of fat in the Mediterranean diet, although there has been a gradual increase in margarine and butter consumption during recent years.Although the direct consumption of margarine in Spain is still low (2 g/person/day), the intake of foods containing margarine in their composition (industrial bakery products, biscuits, etc.) is rising (Serra-Majem et al., 1993).
There has been controversy surrounding the safety of margarine.For example, it has been reported that margarine can safety be consumed in a prudent diet because it is a source of polyunsaturated fatty acids (PUFA), lacks cholesterol and contains a level of saturated fatty acids (SFA) lower than that of animal fat (Beare-Rogers et al., 1979).However, Willett et al. (1994) identified margarine as one of the major sources of trans fatty acids.Several epidemiological studies have shown an association between dietary trans fatty acids and CVD but these studies have been contradictory (Willet et al., 1993;Roberts et al., 1995;Fritsche et al., 1998).It is known that the nutritional quality of margarines depends on several factors: fat sources, manufacturing process, etc.To achieve the solid consistency necessary for margarines, manufacturers are permitted to blend the unmodified liquid oils with a small amount of natural solid fat or to hydrogenate some edible oils, which involves the formation of trans fatty acids (Bourr,  1985; Hernández and Boatella, 1986).Margarine industry has made great strides in making its products more heart-healthy and is leading the effort to reduce fat, saturated fat and trans fat (Michels and  Sacks, 1995; Stender et al., 1995; Ovesen et al.,  1996).However, in different countries the processes Grasas y Aceites followed to achieve these goals have modified the nutritional characteristics of the products in different ways.
For these reasons, the aim of the present study was to evaluate the fatty acid composition of margarines widely consumed in Spain in the year 2000 and their propensity to influence the incidence of coronary heart diseases.

Sources and sample preparation
Twelve brands of margarines widely consumed in Spain (O.C.U., 1999) were purchased from local suppliers in 2000.The products were from the following margarine-producing factories: Unilever food Spain S.A., Leioa, Spain; van Dijk Food Products, Lopik, Holland; Dia S.A., Getafe, Spain; Grupo Unigro S.A., Valladolid, Spain; Koninklijke Brinkers, Zeewalde, Holland.Samples of the same brand were bought and analyzed in May and again in September 2000, in order to observe differences between batches.Samples were stored under refrigeration (4 o C) until they were analyzed.Margarines were homogenized and each sample was analyzed twice.

Analysis of fatty acids
Total lipids were extracted from the margarines with chloroform/methanol (2:1, v/v) according to the method of Folch et al. (1957).Fatty acid methyl esters (FAME) were prepared with methanolic HCl (1:1) at 85 o C for 1 h and dissolved in hexane (Stoffel et al., 1959).FAME of samples from the year 2000 were analyzed by gas-liquid chromatography (GLC) using a SP-2560 fused silica capillary column (100 m x 0.25 mm inner diameter, 20 µm film thickness; Suppelco, Bellefonte, PA) in a Hewlett-Packard 5890 gas chromatograph (Hewlett-Packard, Avondale, PA).The oven temperature was programmed for 39 minutes at 175 o C and was increased to 230 o C at a rate of 3 o C per minute and held for 14 minutes.Helium was used as the carrier gas at a pressure of 290 kPa.Peaks were identified by comparison with their retention times with appropriate FAME standards (Sigma Chemical Company, Urbana, IL).Finally, the double bond position of isomers were confirmed by GLC-mass spectrometry analysis of their 2-alkenyl-4,4-dimethyloxazoline (DMOX) derivatives (Zhang et al., 1988).
For GLC-mass spectrometry analysis, a VG Analytical MS System (Model 7070EQ, VG Analytical, Manchester, England), equipped with an 11/250 data system interfaced to a Varian gas chromatograph (Model Vista 6000, Varian Associates, Palo Alto, CA) was operated at an ionization energy of 70 eV.GLC separation of the DMOX derivatives was performed on the same SP-2560 capillary column described for the FAME analysis.Helium was the carrier gas.The oven temperature was programmed from 140 o C at 1.5 o C per minute to 220 o C and held for 15 minutes.For the final determination of the composition of the C18:1 isomers in margarines, the FAME were fractionated by silver-nitrate thin-layer chromatography and further GLC, to avoid the overlap of trans C18:1 (12-trans, 13-trans, 14-trans, 15-trans octadecenoic acid) with cis C18:1 isomer peaks (Ulberth and Henninger, 1992).

Statistical analysis
The contents of the various fatty acids are given in g per 100 g total fatty acids.The coefficient of variation (CV) was calculated in order to estimate the variability of each fatty acid among the different margarines analyzed.The median was estimated in order to identify the dispersion of data.To evaluate differences between batches a Two-way analysis of variance (ANOVA) considering brand of margarine and fatty acids was assayed.This analysis was followed by LSD test when the differences were significant.

RESULTS
The samples were analyzed in May and again in September 2000 in order to observe possible differences between batches.Table I shows the distribution of identified fatty acids (mean percentage of total fatty acid weight of each brand of margarine in both batches, and mean ± SD and median of total margarines), as well as their aggregated values, SFA, MUFA, PUFA and total trans fatty acids.We did
The total trans fatty acid content varied greatly between the different brands of margarine (Table I) (CV = 129%) with M4 and M10 showing up to 30 times the levels found in M2 and M5.Because of the mean value is not very representative of a collective when the dispersion of the data is very high, we have calculated the median value for the fatty acids studied.Only trans fatty acids presented a median value (2.5%) far away from the mean value (5.1%).These total trans fatty acids were mainly trans C18:1 type, while trans C18:2 and trans C18:3 polyunsaturated acids did not exceed 1% of the total fatty acids.
The derived ratios n-6/n-3, SFA/UFA, AI, and TI of the margarines analyzed are reported in Table II.The SFA/UFA, AI and TI values were significantly low in all margarines, and below 0.5 in all cases.

DISCUSSION
Margarines have come a long way from the lard and tallow used in the late 1800 to the liquid fat blends and the introduction of hydrogenated fats in last years of the 20th century.Although some studies have pointed to a trend in the 90s to use "hard" type margarines (Annon, 1991;Ovesen et al., 1996;Ovesen et al., 1998), our study shows that the most widely consumed margarines in Spain in the present moment are semisoft and soft margarines, containing between 20-40% or more than 40% study highlight the importance of informing the consumer in order to avoid upsetting the traditional Mediterranean diet that characterizes Spain.