Dietary trans α‐linolenic acid does not inhibit Δ5‐ and Δ6‐desaturation of linoleic acid in man

Dietary trans monoenes have been associated with an increased risk of heart disease in some studies and this has caused much concern. Trans polyenes are also present in the diet, for example, trans α‐linolenic acid is formed during the deodorisation of α‐linolenic acid‐rich oils such as rapeseed oil. One would expect the intake of trans α‐linolenic acid to be on the increase since the consumption of rapeseed oil in the western diet is increasing. There are no data on trans α‐linolenic acid consumption and its effects. We therefore carried out a comprehensive study to examine whether trans isomers of this polyunsaturated fatty acid increased the risk of coronary heart disease. Since inhibition of Δ6‐desaturase had also been linked to heart disease, the effect of trans α‐linolenic acid on the conversion of [U‐¹³C]‐labelled linoleic acid to dihomo‐γ‐linolenic and arachidonic acid was studied in 7 healthy men recruited from the staff and students of the University of Edinburgh. Thirty percent of the habitual fat was replaced using a trans ‘free’‐ or ‘high’ trans α‐linolenic acid fat. After at least 6 weeks on the experimental diets, the men received 3‐oleyl, 1,2‐[U‐¹³C]‐linoleyl glycerol (15 mg twice daily for ten days). The fatty acid composition of plasma phospholipids and the incorporation of ¹³C‐label into n‐6 fatty acids were determined at day 8, 9 and 10 and after a 6‐week washout period by gas chromatography‐combustion‐isotope ratio mass spectrometry. Trans α‐linolenic acid of plasma phospholipids increased from 0.04 ? 0.01 to 0.17 ? 0.02 and cis ? ‐linolenic acid decreased from 0.42 ? 0.07 to 0.29 ? 0.08 g/100 g of fatty acids on the high trans diet. The composition of the other plasma phospholipid fatty acids did not change. The enrichment of phosphatidyl ¹³C‐linoleic acid reached a plateau at day 10 and the average of the last 3 days did not differ between the low and high trans period. Both dihomo‐γ‐linolenic and arachidonic acid in phospholipids were enriched in ¹³C, both in absolute and relative terms (with respect to ¹³C‐linoleic acid). The enrichment was slightly and significantly higher during the high trans period (P<0.05). Our data suggest that a diet rich in trans α‐linolenic acid (0.6% of energy) does not inhibit the conversion of linoleic acid to dihomo‐γ‐linolenic and arachidonic acid in healthy middle‐aged men consuming a diet rich in linoleic acid.     

Virgin hemp seed oil: An interesting niche product

Virgin hemp seed oil is not widespread on the market, although it is characterised by an interesting fatty acid composition with a high content of polyunsaturated fatty acids. Linoleic acid is the predominant fatty acid, which comes, together with α‐linolenic acid (18:3n‐3), to approximately 80% of the total fatty acids. From a nutritional point of view, up to 7% γ‐linolenic acid (18:3n‐6) and 2.5% stearidonic acid (18:4n‐3) are very interesting. The total amount of tocopherols is high between 80 and 110 mg/100 g, with γ‐tocopherol as the main tocopherol (85%). Due to the high amount of unsaturated fatty acids, hemp seed oil is very susceptible to oxidative deterioration, which results in a fast impairment of the oil during storage. In addition, the high amounts of chlorophyll in the oil due to harvesting of high amounts of immature seeds require light protection, which is often neglected because of merchandising purposes. The virgin oil is characterised by a nutty taste with a slightly bitter aftertaste. The use of virgin hemp seed oil is recommended during mild processing of food without heat.     

Lipase‐mediated hydrolysis of flax seed oil for selective enrichment of α‐linolenic acid

Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation.     

Analysis of Fatty Acid and Lignan Composition of Indian Germplasm of Sesame to Evaluate Their Nutritional Merits

An attempt was made to individually analyze a germplasm collection of 54 indigenous Indian sesame cultivars for fatty acid and lignan composition of their seed oil by gas chromatography and high performance liquid chromatography, respectively. The entries varied in their fatty acid and lignan composition. The mean percentage contents of palmitic, stearic, oleic, linoleic and α‐linolenic acids ranged between 10–22, 5–10, 38–50, 18–43 and less than 1 whereas sesamol, sesamin and sesamolin scored between 3–37, 27–67, 20–59 of the total percentage of lignan, respectively. The highest percentage of α‐linolenic acid (ALA) was obtained in Var‐9 (1.3 % of the total fatty acids). Among the lignans, high sesamin content is considered to be significant, particularly in terms of long shelf life and nutraceutical value of sesame seed oil. The study has broadened our understanding related to differential biochemical composition of the rich sesame germplasms, thereby providing us with a useful groundwork for identifying potential targets and suitable cultivars for genetic engineering approaches to be undertaken in order to improve the nutritional quality of sesame oil, which in turn would be beneficial towards human health.     

N‐3 fatty acids for human nutrition: stability considerations

Currently there is great interest in dietary n‐3 fatty acids to promote health. The food industry aims to produce food products enriched in α‐linolenic acid (Ln), eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) to reduce some of the physiological effects of linoleic acid (L), the major polyunsaturated fatty acid in our diet. However, the goal is hampered by the susceptibility of the n‐3 fatty acids to oxidation. As a result the sensory and nutritional quality of such foods deteriorates. Lipid scientists therefore have to find a way to stabilise these fatty acids. Innovative technologies to protect n‐3 polyunsaturates using antioxidants, adequate preparation, refining and packaging of the oil are needed. In this paper we review the inherent stability and the stabilisation of these nutritionally valuable polyunsaturated fatty acids.     

Current advances in sunflower oil and its applications

The fatty acid and triacylglycerol composition of a vegetable oil determine its physical, chemical and nutritional properties. The applications of a specific oil depend mainly on its fatty acid composition and the way in which fatty acids are arranged in the glycerol backbone. Minor components, e. g. tocopherols, also modify oil properties such as thermo‐oxidative resistance. Sunflower seed commodity oils predominantly contain linoleic and oleic fatty acids with lower content of palmitic and stearic acids. High‐oleic sunflower oil, which can be considered as a commodity oil, has oleic acid up to around 90%. Additionally, new sunflower varieties with different fatty acids and tocopherols compositions have been selected. Due to these modifications sunflower oils possess new properties and are better adapted for direct home consumption, for the food industry, and for non‐food applications such as biolubricants and biodiesel production.     

Effect of Agronomical Practices on the Nutritional Quality of Virgin Olive Oil at Different Ripening Stages

There is a need to verify the quality of organically produced olive oils and to compare them to conventional ones. The objective of this study was to assess possible differences in nutritional quality between agronomic practices in Picual and Hojiblanca olive oil varieties at different stages of olive ripeness. The results showed that organic versus conventional cultivation did not consistently affect acidity, peroxide index or spectrophotometric constants of the virgin olive oils considered in this study. On the contrary, phenol content, oxidative stability, tocopherol content and fatty acid composition were affected by the agronomical practices. Principal component analysis indicated that linolenic acid and β‐tocopherol were mainly responsible for discriminating Hojiblanca organic oils, whereas total phenols, palmitoleic acid and α‐tocopherol were the major contributors to differentiating Picual conventional oils. Lignoceric and stearic acids were related to oils from unripe and ripe olive fruits, respectively. Long‐term experiments are required to confirm these results.     

Canola Proteins Used as Co‐Emulsifiers with Phospholipids Influence Oil Oxidability,Enzymatic Lipolysis,and Fatty Acid Absorption in Rats

The objective of this study is to formulate and characterize oil‐in‐water emulsions with plant‐derived ingredients only, that is, proteins extracted from canola oil bodies, used as co‐emulsifiers with a canola lecithin, and to assess their suitability for food applications. Using the protein extract increases the chemical stability of rapeseed oil emulsions toward oxidation, based on the delay in conjugated diene formation under accelerated storage conditions, and favors pancreatic lipase activity. Bioaccessibility of rapeseed fatty acids is compared in lymph‐duct‐cannulated rats fed oil or emulsion. Fatty acid absorption by the intestine is increased by 78% when the oil is emulsified with canola proteins as co‐emulsifier: 28.7 mg mL^?1 versus 16.1 mg mL^?1 for oil (p < 0.05). In vitro lipolysis results are in overall agreement with fatty acid absorption in vivo. Practical Applications: Results obtained for rapeseed oil emulsified with canola proteins and phospholipids suggest that increased bioaccessibility of n‐3 polyunsaturated fatty acids could be offered in vegan food products.     

Oxidative Stability of α‐Linolenic Acid in Corn Chips Enriched with Linseed Oil Pro/Antioxidative Activity of Tocopherol

Extruded products, particularly those which are corn‐based, are widely consumed salty or sweet snacks; moreover, they very often provide a basis for breakfast for people around the world. Extruded products are characterised by a low nutritional value, and a particularly low content of polyunsaturated fatty acids of the n‐3 group. An attempt was made to enrich extruded corn crisps with α‐linolenic acid (ALA) through the addition of refined linseed oil at an amount of 5 %. Corn crisps were produced with the addition of the oil concerned so that the concentration of ALA in the finished product was at least 2 g 100 g^?1 (in a portion). With such a content of ALA, the crisps may be classified as ‘functional food’ in accordance with Commission Regulation (EU) No 432/2012 of May 2012. The following were tested: oxidative stability, and changes to the content of ALA during 6‐month storage of crisps with the addition of linseed oil and various concentrations of δ‐tocopherol and ascorbic acid. The crisps were packed in polyamide/polyethylene barrier film packages (30/70 µm), using either atmospheric air of argon for the packaging process. The study showed that with each applied concentration of δ‐tocopherol added to the linseed oil (200–800 mg 100 g^?1), it had a strong pro‐oxidant effect. Packaging in argon atmosphere play very protective role in ALA stabilisation in functional corn crisp.     

Low linolenic soybeans and beyond

Low linolenic soybean oil is the first in a series of modified oilseed products to be introduced to meet food company and consumer needs. Consumer packaged goods and foodservice companies are currently using this oil to successfully replace partially hydrogenated soybean oil, resulting in the reduction of trans fatty acids from the food supply. In addition to meeting consumer demand for healthier foods, many food processors have chosen low linolenic soybean oil based on taste, performance and cost benefits. Seed companies continue to utilize traditional breeding, marker assisted breeding and biotechnology approaches to modify oilseeds that produce oils with health and nutrition benefits. Additional modified oilseeds are at various stages of development. Soybeans with increased levels of stearic acid are being developed as an alternative to partially hydrogenated fats and high saturate fats required to provide solids and structure to food. High stability fry oils with increased levels of oleic acid, reduced levels of linolenic acid as well as a version with lower saturated fat are being developed. Soybeans are also being modified to offer more sustainable sources of omega‐3s including stearidonic acid and eicosapentaenoic acid/docosahexaenoic acid which will result in more efficacious sources of omega‐3s compared to alpha‐linolenic acid‐containing vegetable oil and improved functionality/stability compared to fish and algal oils.     

Chemical Characterization of Six Microalgae with Potential Utility for Food Application

Microalgae contain high levels of proteins, carbohydrates, and lipids, and have found a useful application in enhancing the nutritional value of foods. These organisms can also synthesize long‐chain fatty acids in the form of triacylglycerols, such as α‐linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linolenic acid (LA), γ‐linolenic acid (GLA) and arachidonic acid (AA). The aim of this study was to determine the chemical composition and measure protein, carbohydrates, fibers, lipids as well as the fatty acids composition of six microalgae species with potential application in the food industry. Two freshwater species, Chlorella vulgaris and Spirulina platensis, and four marine species, Nannochloropsis oculata, Nannochloropsis gaditana, Porphyridium cruentum, and Phaeodactylum tricornutum, were used in the experiments. Intracellular protein was the most prominent algal component (42.8–35.4 %), followed by carbohydrate + fiber (32.3–28.6 %), and lipids (15.6–5.3 %). N. gaditana is rich in saturated fatty acids, mainly palmitic acid (5.1 g/100 g), while the cells of S. platensis and C. vulgaris algae are abundant in GLA (1.9 g/100 g) and ALA (2.8 g/100 g) acids, respectively. P. cruentum differs from other algae, because it contains a large amount of AA (3.7 g/100 g). The marine microorganisms N. oculata and P. tricornutum are also a source of essential long‐chain polyunsaturated fatty acids (LC‐PUFA‐?3), mainly composed of EPA and DHA. Our results suggest that the freshwater species C. vulgaris and S. platensis are attractive nutritional supplements because of their low fiber and high protein/carbohydrate contents, while the marine species P. tricornutum and N. oculata can enrich foods with LC‐PUFA‐ω3, because of their favorable ω3/ω6 ratio.     

Triacylglycerols from viper bugloss (Echium vulgare L.) seed bio‐oil

Triacylglycerols (TAG) in viper bugloss oil were isolated from raw pressed oil by silicic acid column chromatography. The obtained blend of TAG was separated by silver ion thin‐layer chromatography (TLC Ag⁺) into nine fractions, varying in terms of unsaturation level and molecular polarity. The composition of TAG in viper bugloss oil was determined by HPLC coupled with a diode‐array detector and an evaporative light‐scattering detector. The results showed that the first three fractions were combinations of TAG containing palmitic, oleic and linoleic acids. Fractions 4 and 6 contained TAG of a similar acid composition as above, but with the addition of γ‐linolenic acid. The remaining fractions (7–9) were the most varied in acid composition. They were found to contain 26–39% palmitic acid, 12–15% oleic acid, 13–41% linoleic acid 8–24% γ‐linolenic acid, 1.5–5.5% α‐linolenic acid and 1–5% stearidonic acid. The analysis of fatty acid allocation in TAG of viper bugloss lipids revealed that linoleic acid (ranging from 2 to 100%) was the only acid found in all isolated fractions. In the investigated oil, the predominant TAG included: LnLnG (11.38%), LnLnSt (11.17%), LnGSt (7.71%), LnStSt (6.19%) and LnLnLn (5.44%). Almost 86% of the TAG contained α‐linolenic acid, while γ‐linolenic and stearidonic acids amounted to 49 and 38%, respectively.     

Geometrical isomerization of polyunsaturated fatty acids in physically refined rapeseed oil during plant‐scale deodorization

The effect of the operating temperature (between 220 and 270 °C) on the formation of trans isomers of linoleic and linolenic acids in physically refined rapeseed oil during deodorization in a plant‐scale semicontinuous tray‐type deodorizer (capacity 10 t/h) was investigated. The industrial procedures of physical refining consisted of a two‐step bleaching and deodorization process. The degree of isomerization of linoleic acid ranged from 0.33 to 4.77% and that of linolenic acid from 4.43 to 45.22% between 220 and 270 °C, respectively. A relation between the logarithm of the degree of isomerization and the deodorization temperature can be approximated by statistically highly significant linear functions for both linoleic and linolenic acids. Oleic acid was resistant to the heat‐induced geometrical isomerization. The values found for the ratio between the degrees of isomerization of linolenic and linoleic acids, slightly decreasing with increasing temperature, were equal to 13.6 and 12.9 at 230 and 240 °C, respectively. Two trans isomers of linoleic acid, exclusively with one double bond isomerized into trans configuration, and four trans isomers of linolenic acid, mostly with one double bond isomerized into trans configuration, were determined in deodorized rapeseed oils. Linolenic acid was observed to be the main source responsible for the formation of nearly all trans fatty acids in physically refined rapeseed oil. At 235 °C, a deodorization temperature considered as a reasonable technological compromise, the content of trans fatty acids in plant‐scale physically refined rapeseed oil was less than 1% of total fatty acids, which would be acceptable for further application.     

Nutritional effects of partially hydrogenated low erucic rapeseed oils

The incidence of cardiac lesions in male rats fed rapeseed oil (Brassica campestris, cultivar ‘Span’) was lower with partially hydrogenated oil (iodine value 78) than with the liquid oil which had been treated in various ways. Another rapeseed oil (Brassica napus, cultivar ‘Tower’) was similarly improved when hydrogenated to iodine value 76.6, but not at iodine value 97.1, as demonstrated in both Sprague-Dawley and Wistar rats. The improved nutritional quality of hydrogenated oil appeared not to be related to the decreased concentration of linolenic acid, because that fatty acid in linseed oil with or without erucic acid did not increase the incidence of lesions. A relatively high concentration of docosahexaenoic acid in the cardiac fatty acids was observed in adversely affected groups, but a lower concentration was found with the appropriately hydrogenated rapeseed oil. Presented in part at the AOCS Meeting, Chicago, September 1976.     

Performance of near-infrared reflectance spectroscopy (NIRS) in routine analysis of C18 unsaturated fatty acids in intact rapeseed

This study was aimed to evaluate the performance of near-infrared reflectance spectroscopy (NIRS) in the analysis of the oil composition for fatty acids like oleic (C18:1), linoleic (C18:2) and linolenic (C18:3) in zero-erucic acid rapeseed (Brassica napus L.). Intact-seed samples of 1094 lines from a breeding programme for high-oleic acid rapeseed were analyzed by both NIRS and gas chromatography (GC). Previously developed calibration equations were initially used for NIRS analyses. The accuracy of NIRS was considerably improved by including some samples of the actual breeding population into the original calibration set and developing new calibration equations. The inclusion of twenty randomly selected samples led to a reduction of the standard error of performance (SEP) from 2.6% to 1.9% for oleic, from 3.8% to 2.0% for linoleic, and from 1.1% to 0.9% for linolenic acid. The application of the new equations to the remaining population of 1074 samples resulted in coefficients of correlation between NIRS and GC values of 0.95 for oleic, 0.92 for linoleic, and 0.90 for linolenic acid. Furthermore, the effectiveness of a selection for high oleic, high linoleic, or low linolenic acid content based on NIRS data was demonstrated. The results of this study will help potential users to choose the optimal selection strategy in routine analysis of C18 unsaturated fatty acids by NIRS within a breeding programme.     

Borage and evening primrose oil

The paper gives a short overview about the production and composition of borage (Borago officinalis) and evening primrose (Oenothera biennis) oil considering special aspects of the production as cold‐pressed oil. Both oils are characterized by a remarkable amount of γ‐linolenic acid, which has some nutritional advantages. The fatty acid composition of evening primrose oil is dominated by linoleic acid with about 72% and about 13% γ‐linolenic acid, while borage oil consists of twice the amount of γ‐linolenic acid and only 38% linoleic acid. The amount of saturated fatty acids is higher in borage oil. The tocopherol composition of both oils is dominated by γ‐tocopherol, with borage oil containing twice the amount compared to evening primrose oil.     

Preparation of stearidonic acid‐enriched triacylglycerols from Echium plantagineum seed oil

HPLC analysis of Echium plantagineum seed oil shows a complex triacylglycerol (TAG) profile. TAG species were separated on an analytical scale by HPLC and their fatty acid (FA) composition is reported. GLC analyses showed that some TAG fractions reached a stearidonic acid (SDA, 18:4n‐3) percentage significantly higher than that in the original oil. TAG separation on a bigger scale was also essayed, by means of a gravimetric normal‐phase chromatographic column, using silver ion‐silica gel as stationary phase. Gradient elution with solvents of increasing polarity was applied, allowing the separation of valuable TAG species containing γ‐linolenic acid (GLA, 18:3n‐6), α‐linolenic acid (ALA, 18:3n‐3) and SDA as the main constituents (more than 85% of the total FA). An enzymatic hydrolysis reaction showed the distribution of FA in the isolated species of TAG. SDA was the major FA in the sn‐2 position (more than 50% of total FA), followed by ALA (19%) and GLA (18.5%).     

Deodorization of vegetable oils: Prediction of trans polyunsaturated fatty acid content

Kinetics of the formation of trans linoleic acid and trans linolenic acid were compared. Pilot plant-scale tests on canola oils were carried out to validate the laboratory-scale kinetic model of geometrical isomerization of polyunsaturated fatty acids described in our earlier publication. The reliability of the model was confirmed by statistical calculations. Formation of the individual trans linoleic and linolenic acids was studied, as well as the effect of the degree of isomerization on the distribution of the trans fatty acid isomers. Oil samples were deodorized at temperatures from 204 to 230°C from 2 to 86 h. Results showed an increase in the relative percentage of isomerized linolenic and linoleic acid with an increase in either the deodorization time or the temperature. The percentage of trans linoleic acid (compared to the total) after deodorization ranged from <1 to nearly 6%, whereas the percentage of trans linolenic acid ranged from <1 to >65%. Applying this model, the researchers determined the conditions required to produce a specially isomerized oil for a nutritional study. The practical applications of these trials are as follows: (i) the trans fatty acid level of refined oils can be predicted for given deodorization conditions, (ii) the conditions to meet increasingly strict consumer demands concerning the trans isomer content can be calculated, and (iii) the deodorizer design can be characterized by the deviation from the theoretical trans fatty acid content of the deodorized oil.     

Alpha‐Linolenic Acid,but Not Palmitic Acid,Negatively Impacts Survival,Asexual Reproductive Rate,and Clonal Offspring Size in Hydra oligactis

Hydra, as sit‐and‐wait predators with limited food selectivity, could serve as model organisms for the analysis of the effect of a particular dietary component on growth and reproduction. We investigated the effect of food quality and of diets enriched with palmitic (PAM) or α‐linolenic acid (ALA) on the life history traits of two hydra species: Hydra oligactis and Hydra vulgaris. We tested the hypothesis that a diet enriched with polyunsaturated fatty acids (PUFA) can stimulate growth and reproduction in simple metazoans with a sit‐and‐wait type of predatory strategy. Our results revealed that a diet based on Artemia nauplii, which are not a natural food for freshwater hydra, stimulated growth, asexual reproduction, and survival in hydra. Artemia nauplii were characterized by the highest lipid content of all used food sources. The analysis of the fatty acid content of hydra indicated the domination the n‐6 fatty acids over n‐3 (eicosapentaenoic acid [EPA], docosahexaenoic acid [DHA], and ALA). Arachidonic acid appeared to be the dominant PUFA in Hydra, irrespective of diet supplementation with palmitic acid or ALA. The dietary supplementation of ALA negatively affected the survival, asexual reproductive rate, and size of clonal offspring of H. oligactis and had no effect on the life history traits of H. vulgaris. Our results also suggest that the hydras are not able to efficiently convert ALA into other essential fatty acids, such as EPA and DHA. To our knowledge, this is the first report about the adverse effects of n‐3 fatty acid supplementation in primitive metazoans such as hydra.     

Fatty acid profile of high consumption foods by school population of the region Metropolitan of Chile, including trans fatty acid content

The Chilean school population has shown remarkable changes in their eating habits, incorporating high fat content foods to their diet, which could explain the current high obesity rates. A great proportion of these foods uses industrial fats whit high content of saturated fatty acids (SFA) and potentially contains trans fatty acids (TFA) as a result of partial oil hydrogenation or as a natural form in ruminant animals fat. The purpose of this investigation was to determine fatty acid profile, including trans fatty acids, from food consumed by the Metropolitan Region school population. A food consumption survey was applied to 203 Metropolitan Region of Chile school-children, which consisted of questions about food consumption frequency and consumer preferences. Based on the results, ten types of food products were selected. Fat content, fatty acids profile, including trans fatty acid, were determinate by GLC according to the UNE 5509 Norm. Each analysis was carried out in duplicate and three samplings were performed. It was found that a great percentage of the analyzed foods showed low TFA content (<1%). However, some of them presented remarkable saturated fatty acids content, generating a PUFA/SFA ratio with poor fat nutritional quality. The low TFA concentration found in the food may be explained by a decrease in the use of hydrogenated fats, following PAHO/WHO recommendation, which are being applied in Chile.