The EFSA Journal (2004) 81, 1-49

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1 The EFSA Journal (2004) 81, 1-49 Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to the presence of trans fatty acids in foods and the effect on human health of the consumption of trans fatty acids (Request N EFSA-Q ) (adopted on 8 July 2004) SUMMARY Occurrence in foods Trans fatty acids (TFA) are unsaturated fatty acids that have at least one double bond in the trans configuration. Some polyunsaturated TFA have a conjugated structure (e.g. conjugated linoleic acid [CLA] in milk fat), i.e. have double bonds which are not separated by a methylene group, but most have isolated (non-conjugated) double bonds. While most unsaturated fatty acids in foods have the cis configuration, TFA are also present. TFA in foods originate from three main sources: - bacterial transformation of unsaturated fatty acids in the rumen of ruminant animals; - industrial hydrogenation (used to produce semi-solid and solid fats that can be used for the production of foods such as margarines, shortenings, and biscuits) and deodorization (a necessary step in refining) of unsaturated vegetable oils (or occasionally fish oils) high in polyunsaturated fatty acids; - during heating and frying of oils at high temperatures. A large number of TFA isomers of monounsaturated and polyunsaturated fatty acids, including positional isomers of individual fatty acids, occur in foods. n fat of ruminant milk and meat products the main TFA are isomers of the monounsaturated fatty acid, oleic acid, with vaccenic acid (18:1t, n-7) predominating (about 30-50% of total trans-18:1 isomers in milk fat). However, trans isomers of other monounsaturated fatty acids (e.g. 14:1 and 16:1) as well as of polyunsaturated fatty acids (18:2 and 18:3) also occur. n foods containing partially hydrogenated vegetable oils the main TFA are also isomers of oleic acid, with elaidic acid (18:1t, n-9) accounting for typically 20-30% of total trans-18:1 isomers and vaccenic acid 10-20%. The TFA profiles of ruminant fat and partially hydrogenated vegetable oils show considerable overlap with many TFA isomers in common, although present in different proportions; proportions of TFA isomers also vary among different hydrogenated oils. Partially hydrogenated fish oils also contain trans 20:1 and 22:1 isomers. Dairy and beef fat typically contains around 3-6% TFA (wt. % of total fatty acids), while levels in lamb and mutton can be somewhat higher. The TFA content in margarines and fat spreads may vary considerably, depending on the proportion of partially hydrogenated oils used. Data from the TRANSFAR study carried out in 14 European countries during showed that soft spreads generally had TFA contents ranging from below 1% up to 17%, while hard stick margarines contained somewhat higher levels. Data from more recent analyses show that TFA levels in most edible fats are below 1-2%. n many cases this reduction has been accompanied Page 1 of 49

2 by an increased level of saturated fatty acids (SFA). The TFA content of bakery products (rusks, crackers, pies, pirogues, cookies, biscuits, wafers etc), as well as some breakfast cereals with added fat, French fries, soup powders and some sweets and snack products, may vary considerably (from below 1% up to 30% of total fatty acids) depending on the type of fat used. Vegetable oils and liquid margarines have a low proportion of TFA, usually below 1%. Dietary intake n the EU, mean daily intakes of TFA for 14 different countries estimated in the TRANSFAR study for , ranged from 1.2 to 6.7 g/d and 1.7 to 4.1 g/d among men and women, respectively, corresponding to % and % of energy, respectively. ntake was lowest in the Mediterranean countries. Mean intakes of saturated fatty acids ranged from 10.5 to 18% of total energy intake, with the lowest intakes in Southern Europe. somers of 18:1 (oleic acid) contributed 54-82% of the total TFA. Major sources of TFA were edible fats and ruminant fat, with bakery products and French fries as additional contributing foods in some countries. The contribution of TFA from ruminant fat ranged from about 30 to 80% of total TFA, corresponding to % of energy. More recent dietary surveys indicate that the intakes of TFA have decreased in a number of EU countries, mainly due to reformulation of food products, e.g. fat spreads, to reduce the TFA content. Absorption and metabolism Digestion and absorption of food TFA occurs in a similar manner to other fatty acids. After absorption, TFA follow the same metabolic routes as other fatty acids and selective accumulation in tissues does not occur. Ultimately, TFA are oxidised to provide energy. Although there is some evidence from in vitro and animal studies that conversion of essential fatty acids is inhibited by TFA, metabolism of essential fatty acids is unlikely to be impaired by TFA when intakes of essential fatty acids meet recommended levels. Health effects Evidence from many controlled human intervention studies indicates that consumption of diets containing TFA, like diets containing mixtures of SFA, consistently results in increased serum LDL cholesterol (LDL-C), compared with consumption of diets containing cismonounsaturated or cis-polyunsaturated fatty acids. The effect shows a linear dose response with serum LDL-C indicating that effects are proportional to amounts of TFA consumed. Elevated LDL-C has been causally linked to coronary heart disease; thus, higher intakes of TFA may increase risk for coronary heart disease (CHD). The available evidence does not provide a definitive answer to the question of whether TFA have an effect on LDL-C different to a mixture of SFA on a gram-for-gram basis. Evidence from controlled human intervention studies also indicates that consumption of diets containing TFA results in decreased serum HDL cholesterol (HDL-C), compared with consumption of diets containing SFA, cis-monounsaturated or cis-polyunsaturated fatty acids. The relationship shows a linear dose response. As a consequence of their effects on LDL-C and HDL-C, TFA, relative to other fatty acids, increase total cholesterol to HDL-C ratio. Lowered HDL-C levels and increased total cholesterol to HDL-C ratio have been shown to be associated with an increased risk of cardiovascular disease (CVD) in epidemiological studies. Page 2 of 49

3 Evidence from controlled human intervention studies also indicates that, relative to diets containing SFA, cis-monounsaturated or cis-polyunsaturated fatty acids, consumption of diets containing TFA results in increased concentrations of fasting triacylglycerol (TAG). The relationship shows a linear dose response. Elevated TAG is positively associated with the risk for cardiovascular disease in epidemiological studies. There is some evidence indicating that TFA increase lipoprotein(a), especially in people with elevated lipoprotein(a) concentrations, but the significance of this for cardiovascular risk is unclear. Human intervention studies do not provide evidence that TFA have any effects on blood pressure, in vitro LDL-oxidizability or haemostatic function (e.g. markers of platelet aggregation, coagulation or fibrinolysis). Human studies do not provide consistent evidence that, at current intakes in European countries, effects of fatty acids on insulin sensitivity are different for an isocaloric substitution of TFA by SFA, oleic acid or linoleic acid. n most of the human intervention studies monounsaturated TFA from hydrogenated vegetable oils were evaluated. No human intervention studies have been carried out to evaluate the effects of TFA from ruminant fat, and indeed such studies are not practicable. Thus it is not possible to determine whether there are differences between TFA from ruminant fat and TFA from hydrogenated vegetable oils in their effects on metabolic risk parameters such as LDL-C or HDL-C. Prospective epidemiological studies consistently support the findings from intervention studies for an association between higher intakes of TFA and increased risk of CHD. n the prospective cohort studies that compared the effects of TFA and SFA, the effects of TFA were stronger than those of a mixture of SFA. Epidemiological evidence for a possible relationship of TFA intake with cancer, type 2 diabetes, or allergy is weak or inconsistent. The proportion of TFA in plasma and tissue lipids in infants is inversely related with the essential fatty acid status. Only a few of studies have investigated the relationship of TFA levels in tissues with early development. While these studies have not established a causal link, effects of TFA on foetal and early infant growth and development need further research. Studies of supplemental CLA (mixtures of the trans-10,cis-12 and cis-9,trans-11 isomers) have provided evidence of potential beneficial health effects, e.g. reduction of body fat, improved immune response, improved profile of blood lipids, with no evidence of adverse effects. However, results are inconsistent and effects may differ between the CLA-isomers. There is some evidence of adverse effects on lipid and glucose metabolism and on insulin sensitivity of supplemental CLA in humans for the trans-10,cis-12 isomer. However, these effects were observed only at intake levels one or two orders of magnitude higher than those corresponding to intake from foods. Few studies have investigated the health effects in humans of naturally occurring CLA from foods and evidence is weak and inconsistent with respect to any health effects at current levels of intake (average intake of CLA from food is estimated to be about 0.3 g/day in Europe). Page 3 of 49

4 Analysis of TFA TFA may be measured in a wide range of food products by infra red spectroscopy, which estimates total non-conjugated TFA, or by gas chromatography or high pressure liquid chromatography, which can measure individual TFA with a high degree of precision. At present, there are no methods of analysis applicable to a wide range of foods that can distinguish between TFA which are naturally present in foods (e.g. in ruminant products) and those formed during the processing of fats, oils or foods. This is because of the overlap in TFA profiles of ruminant fats and hydrogenated oils and the varying proportions of TFA isomers among different hydrogenated fats. KEY WORDS Trans fatty acids, hydrogenated oil, health effects, cholesterol, cardiovascular disease. BACKGROUND n March 2003, following notification in 2002, the Danish Authorities adopted legislation which introduced, with effect from 1 June 2003, limits on the level of trans fatty acids, except naturally occurring trans fatty acids in animal fats, in oils and all processed foodstuffs containing fats and oils as ingredients. This legislation provides that eventually food products must not contain more than 2 g of trans fatty acids per 100 g of fats or oil in the product as sold to the final consumer. This restriction would not apply to naturally occurring trans fatty acids and conjugated linoleic acid (CLA). The Danish Authorities indicated that the measure was justified on public health grounds and was aiming at minimising the risk of cardiovascular disease. Following the notification by the Danish Authorities for the proposed measure some Member States made comments on the proposal and it emerged that views differed on this issue. Certain Member States considered that the level of trans fatty acids in foodstuffs should be restricted as much as possible. Other Member States did not consider there was evidence that trans fatty acids consumed in a varied diet give rise to health problems. Several Member States considered that the issue should be discussed at the European Community level. n view of the divergent opinions of the Member States and the Community interest in this matter the European Commission decided to seek the opinion of the European Food Safety Authority. TERMS OF REFERENCE n accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002, the European Commission requests the European Food Safety Authority (EFSA) to issue a scientific opinion on the presence of trans fatty acids in foods and on the effect on human health of the consumption of trans fatty acids. n this context the Authority is asked: To take into account the evidence on all trans fatty acids in foods, including the ingredients of food products, both those that are naturally present, such as in certain animal fats, and those occurring as a result of manufacturing processes, such as hydrogenation of oils; Page 4 of 49

5 To advise - whether the evidence indicates any specific effects on health of trans fatty acids - whether the effects, if any, differ according to the food source, and - how the effects, if any, compare to effects on health of other types of fatty acids; To advise, if there are effects on health, whether the effects are associated with a specific level of intake of trans fatty acids in the context of the overall diet. n addition, the Authority is asked to advise if there are any methods of analysis that can distinguish between trans fatty acids which are naturally present in fats and those formed during the processing of fats, oils or foods. The EFSA is asked to report the analytical sensitivity of any such methods. ASSESSMENT 1. NTRODUCTON Fatty acids can be classified according to their number of double bonds. Saturated fatty acids (SFA) have no double bonds, while monounsaturated fatty acids (MUFA) have one double bond and polyunsaturated fatty acids (PUFA) have two or more double bonds. The position of the double bond can vary along the carbon chain and its position can be indicated in several ways. When counted from the carboxyl-end (-COOH) of the molecule, the so-called x - nomenclature is applied, while the n-x or ωx classification is used when counting starts from the methyl-end (-CH 3 ). Thus, n-9 means that the double bond is located at the ninth carbon atom from the methyl-end. These double bonds can have either the cis or trans configuration. Cis means that the two carbon (C)-atoms (or hydrogen (H)-atoms) adjacent to the double bound point into the same direction, while in the trans configuration the two carbon atoms point into opposite directions. As an example, elaidic acid [trans-c18:1(n-9)] and oleic acid [cis-c18:1(n-9)] are shown in Figure 1. These two molecules are so-called geometrical isomers. n or ω O = H 3 C C Oleic acid Cis-C18:1(n-9) OH H 3 C Elaidic acid Trans-C18:1(n-9) C = O OH Figure 1. Structure of oleic acid and elaidic acid Most unsaturated fatty acids in the diet have the cis configuration, but trans fatty acids (TFA) are also present. These fatty acids originate from several sources and both trans monounsaturated and trans polyunsaturated fatty acids exist. Trans PUFA have at least one trans double bond and may therefore also have double bonds in the cis configuration. Page 5 of 49

6 TFA in the diet are derived from several sources. First, they are formed by bacterial transformation of unsaturated fatty acids in the first stomach (rumen) of ruminant animals. Secondly, industrial hydrogenation and deodorization of vegetable oils high in polyunsaturated fatty acids (or fish oils) may result in the formation of TFA. Finally, trans double bonds can be formed during heating and frying of oils at extreme temperatures. Hydrogenation is used to produce semi-solid and solid fats that can be used for the production of foods such as margarines, shortenings, and biscuits. When all double bonds are hydrogenated, a SFA is formed. However, the cis double bond may also isomerize into a trans double bond without net uptake of hydrogen. Furthermore, double bonds can migrate along the molecule, which results in the formation of positional isomers. Thus, through hydrogenation many different molecules can be formed, although trans MUFA are the most prevalent. Conjugated linoleic acid (CLA) refers to a mixture of positional and geometric natural isomers of linoleic acid, whose double bounds can be in either trans or cis configuration, and differing from most natural PUFA in that the double bounds are not separated by a methylene carbon, but conjugated. 2. FOOD CONTENT AND DETARY NTAKE 2.1 Occurrence of trans fatty acids in foods TFA in foods are mixtures of several monounsaturated and polyunsaturated fatty acids with different carbon chain length. n addition positional isomers of individual fatty acids usually occur. n ruminant fat trans 18:1 isomers dominate, but trans isomers of 14:1 and 16:1 as well as of PUFA (18:2 and 18:3) also occur. n milk and ruminant fat conjugated isomers of linoleic acid (CLA), which may contain one or two trans double bonds, are found (Ha et al. 1989). n partially hydrogenated vegetable oils 18:1 trans isomers dominate but small amounts of other trans monoene and polyene isomers are also found (Becker, 1998; Van Poppel, 1998; Aro et al., 1998b and c; van Erp-Baart et al., 1998). Partially hydrogenated fish oils also contain trans 20:1 and 22:1 isomers. The 18:1 trans fatty acids that occur in foods consist of mixtures of different positional isomers. More than ten 18:1 trans isomers have been identified, both in partially hydrogenated fats and in ruminant fat (Precht et al., 2001; Wolff et al., 2000; Seppänen-Laakso et al., 1996). The percentage distribution of the various isomers differs between ruminant and hydrogenated fats and also among different hydrogenated fats (Table 1). n ruminant fat trans-vaccenic acid (18:1t, n-7) is the major isomer, constituting 30-50% of the total amount of trans-18:1 isomers. n partially hydrogenated fats the position of the double bond is more evenly distributed along the carbon chain (Precht et al., 2001; Wolff et al., 2000; Seppänen-Laakso et al., 1996). Elaidic acid (18:1t, n-9) constitutes typically 20-30% of the trans-18:1 isomers and vaccenic acid 10-20%. n partially hydrogenated fish oils a range of different trans isomers of longer chain fatty acids (C20-22) also occur. Page 6 of 49

7 Table 1. Typical proportions (% of total trans 18:1 isomers) of positional 18:1 transisomers in ruminant and industrially hydrogenated fats from conventional foods Trans 18:1 isomer n-x position of double bond -position of DB Milk fat, goat Milk fat, ewe Milk fat, cow ndustrially hydrogenated fats n n n a n a n n-7 (vaccenic acid) n n-9 (elaidic acid) n-10 to n n-13 5 < 1 < 1 < 1 2 n-14 4 < 1 < 1 < 1 1 Data compiled from Precht et al., 2001; Wolff et al., 2000; and Seppänen-Laakso et al., a Sum of n-4 and n-5 isomers n the TRANSFAR study about 1300 foods from 14 European countries (Belgium, Denmark, Finland, France, Germany, Greece, taly, celand, the Netherlands, Norway, Portugal, Spain, Sweden and the United Kingdom) were analysed for fatty acids including TFA (Van Poppel et al., 1998). The analytical technique used leads to underestimation of 18:1 trans isomers of about 20%. Fat in dairy products from cow (butter, milk and cheese) typically contains around 3-6% TFA (weight % of total fatty acids). Similar levels are found in goats and sheep milk fat (Aro et al., 1998a and b). The TFA content in fat from beef is in the same order as in milk fat, while levels in lamb and mutton can be somewhat higher. The TFA content of fat from pork and poultry are generally below 1% of total fatty acids, but the content may vary, primarily depending on the TFA content of the feed (Aro et al., 1998a). The TFA content in margarines and spreads for household use may vary considerably, depending on the proportion of partially hydrogenated fats used. Data from the TRANSFAR study showed that soft spreads generally had levels ranging from below 1% up to 17%, while hard stick margarines contained somewhat higher levels (Aro et al., 1998b). The sampling was for most countries done during n some countries TFA levels in many edible fats for household use have been reduced (e.g. Sweden, Becker, 2003; Norway, Norwegian food composition table, 2001; Denmark, Hansen and Leth, 2000; Greece, Triantafillou et al., 2003). n many cases this has been accompanied by an increased level of saturated fatty acids. Vegetable oils and liquid margarines have a low proportion of TFA, usually below 1%. The TFA content of bakery products (rusks, crackers, pies, pirogues, cookies, biscuits, wafers, etc.) may vary considerably depending on the type of fat used. n the TRANSFAR study, TFA content varied from below 1% up to 30% of total fatty acids (van Erp-Baart et al., 1998). Also some breakfast cereals with added fat, French fries, soup powders and some sweets and snack products were shown to contain high TFA levels (20-40% of total fatty acids) (Aro et al., 1998c). These are generally foods with a high fat content (20-40 g/100 g edible portion). 2.2 Dietary intake n the TRANSFAR study intake of TFA was calculated from available national food consumption surveys using the analytical data produced within the project (Hulshof et al., Page 7 of 49

8 1999). Average daily intakes of TFA ranged from 1.2 to 6.7 g/day and 1.7 to 4.1 g/day among men and women, respectively, corresponding to % and % of energy (E%), respectively. n populations with the highest average TFA intake (2.1 E%), the 90 th percentile was 2.8 E%. ntake was lowest in the Mediterranean countries, but also below 1% of energy in Finland and Germany. Moderate intakes were seen in Belgium, the Netherlands, Norway and the UK and the highest intake in celand. 18:1 isomers contributed 54-82% of the total TFA. Major sources of TFA were edible fats and ruminant fat, with bakery products and French fries as additional contributing foods in some countries. The contribution of TFA from ruminant fat ranged from about 30 to 80%, corresponding to E%. The intake of SFA contributed E%, the intakes being lowest in South Europe (Hulshof et al., 1999). According to more recent dietary surveys the intake of both TFA and SFA has decreased in many of these countries (Männistö et al., 2003; Steingrimsdottir et al., 2003; Anon, 2003; Henderson et al., 2004). n Finland, average intakes in 2002 were 0.5 E% compared with 0.9 E% in ; in celand, 1.5 E% in 2002 compared with 2 E% in ; in Norway, 1 E% in compared with 1.5 E% in There are limited data on the current intake of trans fatty acids in children. A Finnish study found intakes corresponding to E% in 3-year old children (Salo et al., 2000). n the UK, mean (P97.5) intake of TFA in year old children was 1.5 (3.2) E% (Gregory et al., 1995) and in 4-18 year olds was 1.4 (2.1) E% (Gregory et al., 2000). Data from USA indicate that the average intakes of children and adolescents are similar to those of adults (Allison et al., 1999). 3. ABSORPTON, TRANSPORT, METABOLSM 3.1 Digestion, absorption and incorporation into blood and tissue lipids t has been estimated that approximately 95% of trans MUFA is absorbed, which is very similar to the rate of absorption of other fatty acids (Baer et al., 2003). Other studies did also not suggest that the position of the double bond is an important determinant of the absorption efficiency of cis and trans fatty acids with eighteen carbon atoms (Emken 1984). After absorption, trans MUFA follow the same metabolic routes as other fatty acids (Emken, 1984; Vidgren et al., 1998). TFA are not the end product of any metabolic pathway in humans. Thus, these fatty acids and their conversion products - as present in blood and tissue lipids - originate from dietary sources. t can, however, not be excluded that some TFA are derived from intermediate products of de novo fatty acid synthesis or the β-oxidation pathway. This contribution, if any, is very small, as many studies have shown a positive relation between blood and tissue levels with dietary intakes (Chen et al., 1995; Mensink and Hornstra, 1995; Mansour et al., 2001). Trans monounsaturated and polyunsaturated fatty acids have been detected in adipose tissue, blood cells, serum lipoproteins, kidney, brain, heart, liver, aorta, jejunum and human milk (Heckers et al., 1977; Chardigny et al., 1993; Chardigny et al., 1995). t has further been reported that the isomeric distribution of trans MUFA in adipose tissue was similar to that in partially hydrogenated vegetable oils (Chen et al., 1995). 1. Trans fatty acids are desaturated and elongated by the same enzymes as their cis counterparts. n vitro studies with rat liver microsomes suggest that - except for 8, 9 and 10 trans isomers - C18:1 positional isomers are good substrates for 9 desaturase Page 8 of 49

9 (Mahfouz et al., 1980). Consequently, cis-trans and trans-cis fatty acids can be formed. Some trans isomers of C18:1 are also substrates for 6 and 5 desaturases and can also be elongated into C20 and C22 fatty acids (Pollard et al., 1980). 2. Trans isomers of linoleic acid can be elongated and desaturated into trans isomers of arachidonic acid (C20:4n-6), although at a lower rate than linoleic acid (Grandgirard et al., 1989; Beyers and Emken, 1991). Trans isomers of α-linolenic acid can be converted into trans isomers of docosahexaenoic acid (DHA; C22:6n-3) (Grandgirard et al., 1989). 3. Ultimately, all trans fatty acids are oxidized. As compared to their cis counterparts, trans isomers of oleic and α-linolenic acids were oxidized to the same extent, while isomerization increased the postprandial oxidation of linoleic acid (DeLany et al., 2000; Bretillon et al., 2001) Effects on n-3 and n-6 metabolism Many studies have found a negative relationship between the proportions of trans isomers in tissue and blood lipids with those of essential fatty acids and their metabolites. These findings can be interpreted in at least two ways. First of all, TFA intake is inversely related to the intake of essential fatty acids, as especially linoleic and α-linolenic acid are converted into trans isomers during the hydrogenation process. Secondly, it has been postulated that TFA inhibit the conversion of essential fatty acids, thereby increasing the requirements for essential fatty acid intake. For rats, however, it has been demonstrated that these effects can easily be overcome when linoleic acid exceeds 2% of energy (Zevenbergen et al., 1988). n vitro and animal studies do suggest that both the cis and trans monoenoic positional isomers may inhibit 6 and 5 desaturation of linoleic and α-linolenic acids. n contrast, C18:1 9tr increased 9 desaturation of stearic acid, while C18:1 11tr and C18:2 9tr,12tr had no effects on 9 desaturase activity (Rosenthal and Whitehurst 1983; Cook and Emken 1990). t should be noted that cis fatty acids are the preferred substrates for desaturases. t is further not known if these in vitro and animal findings can be extrapolated to the human in vivo situation. n a recent human study, however, no effect of trans α-linolenic acid was found on the conversion of linoleic acid (Scrimgeour et al., 2001). Therefore, the general consensus is that in adults elongation and desaturation of essential fatty acids is unlikely to be impaired by current intakes of trans monounsaturated fatty acids, when intakes of essential fatty acids meet recommended intakes (FNB, 2002). t should be noted however that no data on the effects of TFA on the desaturation and elongation of linoleic and α-linolenic acid are available for special groups (e.g. children, pregnant women). 4. TRANS FATTY ACDS AND METABOLC RSK PARAMETERS Many human intervention studies have addressed the effects of TFA on metabolic risk parameters. n most of these studies, focus was on the effects of trans MUFA from hydrogenated vegetable oils. n most studies, no information was given on the amounts of the various positional isomers of elaidic acid, which means it is not possible to consider the importance of the position of the double bond. Effects of TFA from hydrogenated fish oil or of trans PUFA from vegetable origin have been addressed in only a few studies. No human intervention studies however have been carried out to evaluate specifically the effects of trans MUFA from dairy origin or from ruminants in general. n contrast to the amounts of TFA in Page 9 of 49

10 hydrogenated fats and oils, it is extremely difficult to manipulate the amount of TFA in dairy products to such an extent that effects on metabolic risk parameters can reliably be tested in human intervention studies. Several studies however have compared the effects of butter with those of soft and brick-type margarines. These studies though do not provide clear information on the effects of TFA from different sources. The compositions of the food products tested differed in so many aspects, that it is impossible to attribute the observed differences exclusively to the type of TFA. These studies will therefore not be discussed here. 4.1 Serum total, LDL, and HDL cholesterol One of the first controlled intervention studies that specifically examined the effects of trans MUFA from hydrogenated oils on the serum lipoprotein profile was published in 1990 (Mensink and Katan, 1990). From that study it was concluded that trans MUFA significantly raised serum total and LDL cholesterol (LDL-C) concentrations and lowered those of HDL cholesterol (HDL-C), as compared with an iso-energetic amount of oleic acid. As a result, the total to HDL cholesterol ratio was also increased. n that study, a diet rich in a mixture of SFA - mainly palmitic and stearic acids - was also included. Compared with the saturated-fat diet, the diet rich in trans MUFA did not change LDL-C, but lowered HDL-C and hence the total to HDL cholesterol ratio. Despite these clear results, this study was criticised as the amount of trans MUFA employed (11% of total energy) by far exceeded habitual intakes. Results of later well-controlled studies that tested lower intakes of trans MUFA however lead to comparable conclusions (Table 2) (Zock and Katan, 1992; Judd et al., 1994; Almendingen et al., 1995; Aro et al., 1997; Judd et al., 1998; Müller et al., 1998; Lichtenstein et al., 1999; Judd et al., 2002; Lovejoy et al., 2002). n one study, effects of TFA from partially hydrogenated fish oil were examined (Almendingen et al., 1995). t was concluded that these TFA changed lipid risk indicators for cardiovascular disease at least as unfavourably as butterfat. n another intervention study it was also found that trans MUFA decreased LDL particle size, which might be expected to increase small dense LDL (Mauger et al., 2003). These results were however not confirmed by another intervention study (Cuchel et al., 1996). Small, dense LDL is positively related with the risk for cardiovascular disease (Kraus, 2001). A recent meta-analysis has combined the results of well-controlled studies on the effects of trans monounsaturated fatty acids on serum lipoproteins (Mensink et al., 2003b). t was estimated that increasing the intake of trans MUFA with 1% of energy at the expense of carbohydrates significantly increased LDL-C concentrations with mmol/l, while HDL-C concentrations did not change. The estimated change in the total to HDL cholesterol ratio was (Table 3). n this meta-analysis, effects of other fatty acids on the serum lipoprotein profile were also estimated. From Table 3 it is clear that the LDL-C increasing effect of trans MUFA is very similar to that of palmitic acid (C16:0) and slightly less than that of lauric and myristic acids (C12:0 and C14:0). Compared with stearic acid (C18:0), cis monounsaturated and cis polyunsaturated fatty acids [mainly oleic acid, C18:1(n-9) and linoleic acid, C18:2(n-6)] trans MUFA increase LDL-C. Further, trans MUFA have no effect on HDL-C when compared with carbohydrates, while all of the fatty acids examined increase HDL-C. As a consequence, trans MUFA have, in comparison with the other fatty acids examined, the most unfavourable effect on the total to HDL cholesterol ratio, which is associated with an increased risk in cardiovascular disease in epidemiological studies (Stampfer et al., 1991). A meta-analysis found a dose-dependent linear relationship between the intake of trans monounsaturated fatty acids with concentrations of serum LDL-C and HDL-C (Katan et al., 1995; Mensink et al., 2003b) and the LDL to HDL cholesterol ratio (Ascherio et al., 1999). No evidence for a threshold effect was observed. Page 10 of 49

11 From the values in Table 3, it is also possible to calculate the predicted change on the serum lipoprotein profile when one fatty acid is exchanged for another. For example, when 1 percent of energy from trans MUFA is exchanged for cis MUFA the expected change in the total to HDL cholesterol ratio is ( 0.026) = A few cross-sectional epidemiological studies have focused on the relation between the proportion of TFA in tissues, which reflects dietary intakes, or the dietary TFA intake and the lipid profile. Relationships between the proportions of 19 geometric and positional isomers in adipose tissue with serum lipid concentrations were examined by Hudgins et al. (1991). Weak positive associations were reported between the proportion of trans-c14:1(n-9) with serum total cholesterol and VLDL-C (very low density lipoprotein cholesterol) concentrations, and between trans-c18:1(n-7) (vaccenic acid) with serum total and LDL-C concentrations. Positive associations between TFA intake with serum LDL-C, and the total to HDL cholesterol ratio were also found in another study (Troisi et al., 1992). Plasma trans-c16:1(n-7) was positively related with plasma total and LDL cholesterol, and with triacylglycerol. Negative relations were seen with plasma HDL-C concentrations, and with the total to HDL cholesterol ratio. n the TRANSFAR study, a negative association was found between trans-c18:1(n-9) with serum LDL-C concentrations. n contrast, associations with trans-c14:1(n-5) and trans-c22:1 were positive (Van de Vijver et al., 2000). n a Costa Rican population, a positive association was observed between the intake of TFA and LDL particle size. No significant relations were observed with plasma HDL-C and LDL-C levels. According to the authors, this lack of clear association may be due to the low intake of TFA, ranging from 0.5 to 1.7% of energy (Kim and Campos 2003). Altogether, relationships in cross-sectional epidemiological studies between TFA intake or biomarkers of TFA intake with the serum lipid profile are weak, but statistically significant, and in line with the outcomes of the intervention studies. 4.2 Triacylglycerol An increased concentration of fasting triacylglycerol (TAG) is positively associated with the risk for cardiovascular disease (Hokanson and Austin, 1996). n all well-controlled intervention studies that have addressed the effects of TFA on serum lipoprotein cholesterol concentrations, effects on fasting triacylglycerol concentrations have also been studied (Tables 2 and 3). From these studies, it has been estimated that under isoenergetic conditions effects of trans MUFA on fasting TAG concentrations are similar to those of a mixture of carbohydrates (Mensink et al., 2003b), while the other fatty acids lower fasting TAG concentrations. n this respect, a dose-dependent linear relationship was observed between the intake of trans MUFA with concentrations of serum TAG (Katan et al., 1995). Thus, decreasing the intake of trans MUFA at the expense of an isoenergetic amount of other fatty acid is expected to result in lower fasting TAG concentrations. 4.3 Lipoprotein(a) Lipoprotein(a) [Lp(a)] is an LDL particle with an extra glycoprotein [apoprotein (a)], attached through a disulfide link. High concentrations of Lp(a) are related with an increased risk for cardiovascular disease (Wild et al., 1997). Effects of trans MUFA on Lp(a) have been investigated in several well-controlled intervention studies (Table 4). n some of these studies Lp(a) concentrations were increased after consumption of diets rich in TFA, as compared with diets rich in SFA or cis unsaturated fatty acids (Mensink et al., 1992; Almendingen et al., 1995; Aro et al., 1997; Clevidence et al., 1997; Judd et al., 1998). Changes in Lp(a) were positively Page 11 of 49

12 related to initial concentrations (Mensink et al., 1992; Clevidence et al., 1997). Other studies however did not observe statistically significant effects (Müller et al., 1998; Lichtenstein et al., 1999). Based on epidemiological data (Wild et al., 1997), it is not expected that, for people with normal Lp(a) concentrations, the observed increases in Lp(a) would have a significant impact on cardiovascular risk. t can however not be excluded that individuals with elevated concentrations of Lp(a) would benefit from a reduction in the intake of trans MUFA. 4.4 Susceptibility to LDL oxidation Oxidation of unsaturated fatty acids in the LDL particle is a crucial step in the formation of an atherosclerotic plaque. As yet, however, there is no validated reference method that reflects the in vivo susceptibility of LDL to oxidation (Mensink et al., 2003a). Therefore, effects of increased intakes of trans MUFA have - like of other fatty acids and carbohydrates - mainly been examined using in vitro techniques. Although the relevance of these in vitro findings for the in vivo situation is uncertain (Mensink et al., 2003a), these studies did not reveal any adverse effects of TFA from hydrogenated vegetable or fish oils on in vitro LDL-oxidizability (Table 5) (Nestel et al., 1992; Cuchel et al., 1996; Halvorsen et al., 1996). 4.5 Haemostatic function A few human intervention studies have assessed, in fasting plasma, the effects of TFA on markers for platelet aggregation, coagulation and fibrinolysis, three determinants of haemostatic function (Table 6). Almendingen et al. (1996) have found that a diet enriched in partially hydrogenated soybean oil unfavourably changed concentrations of plasminogen activator inhibitor type 1 (PA-1) antigen and PA-1 activity as compared with a diet high in partially hydrogenated fish oil or butter fat. Fibrinogen levels were adversely affected by butter compared to partially hydrogenated fish oil. Mutanen and Aro (1997) however concluded that stearic and trans MUFA from partially hydrogenated vegetable oil had similar effects on markers of coagulation and fibrinolysis. Collagen-induced platelet aggregation however was positively changed by TFA, as compared to stearic acid. No effects however were found on in vitro tromboxane B 2 production, adenosine diphosphate (ADP)-induced aggregation, and production of endothelial prostacyclin (PG 2 ) (Mutanen and Aro 1997). Comparable effects of cis and trans MUFA on coagulation factors have been reported by Louheranta et al. (1999). Finally, Sanders et al. (2000) did not find any evidence that a single meal rich in trans monounsaturated fatty acids had a different impact on postprandial activation of factor V than meals enriched with other fatty acids. These intervention studies therefore do not provide evidence that TFA from hydrogenated oils have an impact on haemostatic function. 4.6 nsulin sensitivity When compared with SFA and oleic acid, consumption of 20% of energy from trans MUFA did not change fasting concentrations of glucose and insulin in obese patients with type 2 diabetes (Table 7). Postprandial insulin secretions were however increased on the diets rich in SFA and TFA (Christiansen et al., 1997). These results could not be confirmed by studies with healthy volunteers that employed lower intakes of TFA (Louheranta et al., 1999; Lovejoy et al., 2002; Lichtenstein et al., 2003). Noteworthy, Lichtenstein et al (2003) did not observe a doseresponse relationship between the intake of trans MUFA with fasting glucose or insulin concentrations or with the HOMA-index, a marker for insulin resistance. These intervention Page 12 of 49

13 studies therefore suggest that - at extreme high intakes - trans MUFA may have the same effects on postprandial insulinemia in obese subject with type 2 diabetes as SFA have. At lower intakes, TFA did not adversely affect insulin sensitivity of healthy volunteers. n a cross-sectional study with 38 subjects, of which 5 subjects had type 2 diabetes and 4 subjects had impaired glucose tolerance, no relationships were observed between TFA intake and markers of insulin resistance (Lovejoy et al., 2001). Also, no associations were observed with the proportions of trans monounsaturated or trans polyunsaturated fatty acids of serum cholesterol esters. Negative effects of self-reported intakes of total fat, and SFA and MUFA on markers of insulin resistance were however observed. n conclusion, the limited number of human studies do not provide consistent evidence that, at current intakes in European countries, the effects of fatty acids on insulin sensitivity are not different for an isocaloric substitution of TFA by SFA, oleic acid or linoleic acid. 4.7 Blood pressure Effects of trans MUFA from hydrogenated oils on blood pressure has been examined in only a few trials with normotensive, healthy subjects (Table 8). When compared with SFA, oleic acid or linoleic acid, no effects of trans MUFA on systolic or diastolic blood pressure were found (Mensink et al., 1991; Zock et al., 1993; Lichtenstein et al., 2003). 5. TRANS FATTY ACDS AND RSK FOR DSEASE 5.1 Cardiovascular disease The association between the intake of TFA with cardiovascular risk has been examined in five prospective cohort studies (Willett et al., 1993; Ascherio et al., 1996; Hu et al., 1997; Pietinen et al., 1997; Oomen et al., 2001). n all these studies, a positive association was observed between the intake of TFA and cardiovascular risk (Table 9). These relationships were more pronounced than for any other fatty acid (Ascherio et al., 1996; Hu et al., 1997; Pietinen et al., 1997). Except for one study (Ascherio at al. 1996), the p for trend reached statistical significance, indicating that the relative risk increased, the higher the intake of total TFA. Three studies looked into details between the source of TFA and the risk for cardiovascular disease. Results from two studies suggested (Willett et al., 1993; Pietinen et al., 1997) that the relation between the intake of TFA and cardiovascular risk was limited to TFA from hydrogenated sources. This was not confirmed by the third study (Oomen et al., 2001). t should be noted however that the intake of TFA from animal origin may have been too low to observe any relationships. n the study of Pietinen et al. (1997), for example, the intake of TFA from vegetable origin in the highest quintile was 5.6 g and from animal origin 2.5 g. This may have masked any association. When intakes were comparable (1.6 g from vegetable TFA versus 1.5 g from animal TFA), relative risks were similar. Based on the data of prospective cohort studies, Weggemans et al. (2004) have recently reviewed the literature and concluded that no differences in the risk of coronary heart disease is evident between total, ruminant and industrial TFA when the intake is below 2.5 g per day. The relation between the intake of TFA with the risk of cardiovascular disease has also been examined in several case-control studies. Studies published after 1990 are summarised in Table Page 13 of 49

14 10. At this point, it should be noted that case-controls studies are in general more sensitive to information bias, selection bias and confounding than prospective cohort studies. One study suggested higher total TFA intakes in cases as compared to controls. This relationship was limited to TFA from hydrogenated vegetable oils (Ascherio et al., 1994). As in prospective cohort studies, it can not be excluded that the intake of TFA from animal origin was not high enough to observe any clear relationships. n the Euramic study, the proportion of C18:1 trans fatty acids in adipose tissue was not different between cases and controls. After exclusion of two Spanish centres with far lower proportion of adipose TFA, there was however a tendency for higher proportion of TFA in the cases (Aro et al., 1995). Roberts et al. (1995) found no relation between the proportion of trans isomers of oleic or linoleic acids and sudden cardiac death. Also, the proportions of trans isomers of C16:1, C18:1 or C18:2 in plasma phospholipids were not different between patients with angiographically documented CHD and controls (Van de Vijver et al., 1996). n Costan Rican adults, the proportion of trans isomers of C16:1 and C18:2 in adipose tissue were higher in patients with a first nonfatal myocardial infarction than in controls. Proportion of trans-c18:1 however did not differ (Baylin et al., 2003). n a final study, no difference between cases and controls was found for TFA intake. The proportion in adipose tissue of trans isomers from both vegetable and animal origin was however the highest in controls (Clifton et al., 2004). n the 16 cohorts of the Seven Countries Studies, a positive relationship was observed between the intake of TFA and 25-year mortality from coronary heart disease (Kromhout et al., 1995). n summary, results from case-control studies are inconsistent. From prospective cohort studies, results are uniform: a high intake of TFA is associated with an increased risk of CHD. n the prospective cohort studies that compared the effects of TFA and SFA, the effects of TFA were more pronounced than those of SFA. 5.2 Diabetes n the Nurses Health Study a positive relationship was observed between the intake of TFA and the risk of development of type 2 diabetes. Subgroup analyses revealed that effects were primarily observed in obese women, possibly due to the fact that these women are already more insulin resistant compared with non-obese women. Negative relationships were seen for linoleic acid and marine n-3 fatty acids from fish. No relationships were observed with the intakes of SFA, MUFA, and total fat (Salmerón et al., 2001). n contrast, the owa Women s Health Study only found an inverse relationship between PUFA and vegetable fat with incidence of type 2 diabetes, but not with any type of fatty acids or with animal fat (Meyer et al., 2001). For men also, no relationships were observed between the total or fatty acid intake and the risk of type 2 diabetes in the Health Professionals Follow-up Study. Relationships with vegetable or animal fat intake were also not statistically significant (Van Dam et al., 2002). n these three prospective cohort studies (Meyer et al., 2001; Salmerón et al., 2001; Van Dam et al., 2002), associations with the source of TFA were not examined (Table 11). 5.3 Cancer Prospective cohort studies on the relationship between TFA and cancer risk are summarised in Table 12. The results from the Nurses Healthy Study suggested that TFA intake was negatively related with the risk of breast cancer. An inverse association was also found for MUFA, while a positive association was seen for fatty acids from fish (Holmes et al., 1999). From the Netherlands Cohort Study on Diet and Cancer a weak, positive, association was found between Page 14 of 49

15 the intake of total TFA and postmenopausal breast cancer incidence. This relationship was in particular evident for predominantly vaccenic acid. A weak, positive, relationship was also reported for CLA. Negative associations were observed for oleic acid and α-linolenic acid (Voorrips et al., 2002). The relationship between the intake of TFA or its content in adipose tissue with cancer risk has also been addressed in several case-control studies (Table 13). London et al. (1993) concluded that the intake of specific fatty acids, including TFA, were not related to malignant or benign breast disease. n the Euramic study, the TFA content of subcutaneous adipose tissue was positively associated with risk for breast cancer (Kohlmeier et al., 1997). A third study found a lower proportion of TFA and a higher proportion of oleic acid in women with positive lymph nodes than in women with negative lymph nodes (Petrek et al., 1997). For colon cancer, no relationship between the intake of TFA was observed in men, but a weak positive association in women. Cis unsaturated fatty acids were not related to colon cancer risk (Slattery et al., 2001). McKelvey et al. (1999) concluded that foods containing partially hydrogenated vegetable oils were not associated with colorectal adenomatous polyps. n an ecological study, significant correlations were found between TFA in adipose tissue and incidence of colon and breast cancer. No association was found with prostate cancer (Bakker et al., 1997). 5.4 Early growth and development As mentioned above, a negative relationship exists between the proportions of trans isomers in tissue and blood lipids with those of essential fatty acids and their metabolites. Similar relationships exist in in neonatal blood and cord tissue, despite dietary intake data that indicate adequate availability of essential fatty acids in the mother s diets (Al et al., 1996; Hornstra, 2000). Also, a relationship exists between the maternal TFA and essential fatty acid status with those of the infant (Elias and nnis 2001). As essential fatty acids and their longer-chain metabolites are essential for foetal growth and development, several studies have examined the relationship between the level of TFA and early growth and development (Table 14). n premature infants, Koletzko observed a negative relationship between the proportion of TFA in plasma phospholipids and cholesteryl esters with birth weight (1992). n full-term infants however such relatonships were not observed (Decsi et al., 2001). Elias and nnis however observed a negative relationship between the proportion of TFA in cholesteryl esters from infant cord arterial plasma samples with length of gestation. No statistically significant relationships were observed with birth weight or birth length. Also, the proportion of TFA in the triacylglycerol and phospholipid fractions were not related to length of gestation, birth weight or birth length (Elias and nnis, 2001). Van Houwelingen and Hornstra (1994) have also reported as preliminary findings, a negative relationship between trans-c18:1 in umbilical arterial vessel walls with birthweight and head circumference, but these associations disappeared after correction for gestational age (Hornstra, 2000). n conclusion, the proportion of TFA in plasma and tissue lipids in infants is inversely related with that of essential fatty acids and their metabolites. Only a limited number of studies have looked for relations between TFA levels in tissues and early development. While these data have not established a causal link, effects of TFA on foetal and early growth and development needs further research. Page 15 of 49

16 5.5 Asthma and allergies n the nternational Study of Asthma and Allergies in Childhood (SAAC Steering Committee, 1998), a positive relationship was observed between the prevalence of asthma, allergic rhinoconjunctivitis, and atopic eczema with the intake of TFA. These relationships were not observed for intakes of cis-monounsaturated or cis-polyunsaturated fatty acids (Weiland et al., 1999). Though results may provide a basis for further research, the observed relationships of this ecological study do as yet not provide a reason to limit the intake of TFA. 6. CONJUGATED LNOLEC ACD (CLA) CLA is naturally present in meats (e.g. beef, lamb) and dairy products (e.g. milk, cheese) derived from ruminants where they may represent 0.5 2% of fatty acids (Lin, Boylston et al., 1995). Most (70-90 %) of the CLA in these foods is the cis-9,trans-11 isomer (Angel 2004; McLeod, LeBlanc et al., 2004). Chemically produced CLA, which is commercially available and used for dietary supplementation, is usually a mixture containing about equal proportions (40% each) of two main isomers, cis-9, trans-11 CLA and trans-10, cis-12 CLA, and 20% other isomers. The estimated total CLA consumption in the USA from foods for men and women has been reported to be 212 and 151 mg/day, respectively (Ritzenthaler et al., 2001). According to these authors the main dietary sources of CLA are dairy products (60%), beef (32%), pork (3%), poultry (2%) and other foods (3%) (Ritzenthaler, McGuire et al., 2001). n the EU, although not precisely determined, an estimate from the available data indicates the average consumption of CLA to be around 300 mg per day. n some countries (e.g. Australia) consumption of CLA can be up to 1.5 g/day (Parodi, 1994). There is an increasing amount of specific scientific literature on the biological effects of CLA (for current citations to the published scientific literature on CLA, see see also Angel, 2004). The published animal studies and clinical trials indicate the possibility that CLA may have some health benefits, e.g. reduction of body fat gain, immune response, and improvement in blood lipids (see Pariza, 2004). However, conflicting effects of CLA have been reported depending on number of days of treatment, dose, animal species used, proportion of each isomer. Most of the experiments have used a mixture of different isomers (see Pariza, 2004), while it is increasingly evident that the different CLA isomers have different or even opposite biological effects (Rodriguez et al., 2002; Pariza, 2004). Several clinical studies, using concentrations of 1.7 to 6.8 g CLA per day, of supplemental CLA showed no effects on blood lipid profiles (Blankson et al., 2000; Smedman and Vessby, 2001; for other references see Pariza, 2004). A recent review (Terpstra, 2004) reported that only one study in humans showed a significant HDL-cholesterol-lowering effect of CLA; in all the other reviewed studies there were no significant effects on plasma total, LDL-, and HDLcholesterol concentrations or on plasma triacylglycerol concentrations. Thus, CLA appears to have no effect on plasma lipids (Terpstra, 2004). However, results from Riserus et al. (2002, 2004a and b) in obese people indicate that the trans-10,cis-12 CLA isomer may cause significant impairment of the peripheral insulin sensitivity as well as elevation of blood glucose and serum lipid concentrations. Page 16 of 49

17 Elias and nnis (2001) observed a negative correlation between the proportion of CLA in infant plasma cholesteryl esters and birth weight and length of gestation of term infants. A negative correlation was also seen for total TFA and gestation length. n a prospective cohort study, Voorrips et al. (2002) found weak, but significant, associations between intake of CLA and total TFA from foods and breast cancer risk in Dutch postmenopausal women. However, in a case-control study in Finnish postmenopausal women, dietary CLA, serum CLA and transvaccenic acid were significantly lower in cases than in controls (Aro et al., 2000). n conclusion, while there is some evidence of adverse effects of supplemental CLA in humans for the trans-10,cis-12 isomer, no such effects were observed for CLA supplements containing mixtures of the trans-10,cis-12 and cis-9,trans-11 isomers. Furthermore, the adverse effects of the trans-10,cis-12 isomer were observed only at intake levels one or two orders of magnitude higher than those corresponding to intake from foods. Few studies have investigated the health effects in humans of naturally occurring CLA from foods and evidence is weak and conflicting with respect any health effects at current levels of intake. 7. METHODS OF ANALYSS Methods used to analyse TFA in foods of natural origin or formed during fats and oils processing are of two types. Methods based on infrared spectroscopy only measure the total amount of TFA (non-conjugated) in a sample, while separation of the different isomers containing one, two or three double bonds may only be achieved using methods based on gas liquid (GLC) or high performance liquid chromatography (HPLC). For HPLC and GLC analyses the general approach consists of three steps: the extraction of the fat from the complex food or tissue matrix, is followed by the conversion of the total lipids into methyl esters or other derivatives which are then analyzed by GLC or HPLC or a combination of both methods. nfrared analyses may be carried out on the lipid extract or after conversion into derivatives. Generally, most lipid analysts use a mixture of chloroform Methanol (2:1 by volume) to extract lipids from animal, plant and bacterial tissue (Folch et al., 1957; Bligh and Dyer, 1959). For dairy products containing short chain fatty acids, an alternative extraction using isopropanol and hexane was developed (Wolff, 1995). n order to analyze the fatty acid components of lipids it is usually necessary to prepare nonpolar derivatives of various kinds, but mainly methyl esters. Acid catalysed transesterification has been widely used (Christie, 2002). However, the utilization of base-catalyzed methylation is recognized to be best for esterified lipids (Christie et al., 2001), especially for samples like milk fat containing conjugated fatty acid (CLA) as acid catalysis can cause isomerization of these fatty acids. 7.1 nfrared methods The determination of total TFA (non-conjugated) by the different infrared spectroscopic methods is based on the C-H out of plane deformation band observed at 966 cm-1, which is characteristic of isolated double bonds with trans configuration (Mossoba et al., 2003). The trans double bonds are found mainly in monounsaturated fatty acids either from natural origin (ruminant fat), or in hydrogenated vegetable oils but also in other minor methylene or nonhttp:// Page 17 of 49

18 methylene interrupted dienes which are mainly found in milk fat or in partially hydrogenated oils. When the trans content is low, an interference is found in foods such as milk fat which contains conjugated unsaturation. This is due to the fact that conjugated fatty acids exhibit absorption bands which are close to 966 cm-1 (990 and 950 cm-1 for the c/t, tc, and 990 cm-1 for the ditrans isomer). Samples also containing free fatty acids must be first esterified as the band near 935 cm-1 due to the O-H out of plane deformation would interfere with the band at 966 cm-1. This method (AOCS, 2004a) when using an infrared attenuated total reflection (ATR) liquid cell is applicable to the accurate determination of isolated trans bond in food samples with trans levels equal to or greater than 1.0 % of total fatty acids. f an ATR cell is not available, and an infrared transmission liquid cell (1 mm path length, NaCl windows) is used, AOCS method Cd 14-95, is recommended (AOCS, 2004b). 7.2 Gas-liquid (GLC) and high performance liquid chromatographic (HPLC) methods GLC has been the most widely used analytical method to analyze trans monounsaturated and polyunsaturated fatty acids. Cis and trans monounsaturated fatty acid isomers are now partially resolved on long capillary columns with highly polar stationary phases (for example 120 m BPX 70 column [Juanéda, 2002]). However, some overlap exists between some cis and trans geometrical isomers. This is because the retention time range for the late eluting trans 18:1 positional isomers is the same as that for the cis 18:1 positional isomers ( 6-14). n fact, the major analytical problem is that the cis fraction of milk fat or partially hydrogenated oil samples is mainly composed of the 18:1(n-9) isomer (oleic acid), while many isomers are usually found in the trans fraction. However, the 11t-18:1 isomer (vaccenic acid) is the predominant trans isomer in milk fat while the 9t, 10t, 11t and 12t-18:1 are the major trans monounsaturated isomers in partially hydrogenated oils. t is however possible to avoid this extensive overlap of the geometrical isomers using a prefractionation of the cis and trans 18:1 isomers by silver-ion thin layer chromatography (TLC) or HPLC on C18 reversed phase columns (Juanéda, 2002). After addition of an internal standard, each isolated TLC fraction is then analyzed by GLC on a highly polar column which permits quantification of most of the isomers. n any case, the 13t-18:1 and 14t-18:1 are only separated on a BPX 70 column (120 m in length) using hydrogen as the carrier gas (Juanéda, 2002). Samples only containing methylene interrupted 18:2 and/or 18:3 geometrical isomers can be readily analyzed by GLC on the same highly polar columns (Sébédio, 1991). For example, polyunsaturated fatty acid geometrical isomers of C18:3(n-3) (α-linolenic acid) which may be found in used frying oils can be separated as their methyl esters on a CP SL 88 column (Sébédio, 1991). Out of the 8 possible geometrical isomers of α-linolenic acid, only two (c,t,t, and t,c,t) are not resolved. However these two isomers can be resolved on the same column using isopropyl esters. An alternative method would be the utilization of HPLC on silver nitrate columns (Juanéda et al., 1994) which permits to separate in one run the 8 possible geometrical isomers. However, this method requires expensive equipment and would not be available to every laboratory. For complex samples containing mixtures of isomeric mono and polyunsaturated fatty acids such as milk fat, which contains cis and trans 16:1, 18:1 and 18:2 isomers including conjugated Page 18 of 49

19 fatty acids, a prefractionation step is mandatory prior to GLC or HPLC analyses. C18 reversed phase HPLC is the method of choice to fractionate such complex lipid samples as fatty acids can be separated on the basis of the number of ethylenic bonds and on the number of carbon atoms. Utilization of two C18 reversed phase columns in series also permits to separate the cis and trans 18:1 isomers (Juanéda, 2002). Each fraction collected by HPLC is then analyzed by GLC or HPLC as described previously. A particular attention should be paid to the analysis of the fraction containing CLA as these isomeric fatty acids containing two conjugated double bonds may isomerize with higher temperatures and during transesterification using acid catalyzed procedures as previously described (Christie et al., 2001). GC using polar columns is sufficient to quantify the major isomers (Christie et al., 2001). However detailed analysis of the di-trans isomers can only be obtained by AgNO 3 -HPLC (Juanéda et al., 2001). n conclusion, GLC analysis on highly polar columns is certainly the most convenient method to quantify TFA in a complex food matrix. However a prefractionation step should be used in order to avoid the overlap between some minor cis and trans 18:1 isomers. Quantification of 0.5 µg trans per mg of fat is then easily achieved. 7.3 Naturally occuring TFA vs industrially produced TFA At the present time, there is no method which permits to distinguish naturally occuring TFA from those produced industrially, as for example during the hydrogenation process. This is because of the overlap in TFA profiles of ruminant fats and hydrogenated oils and the varying proportions of TFA isomers among different hydrogenated fats. However, methods for estimation of the relative contributions of these two sources of trans 18:1 fatty acids to the total trans contents have been proposed. The first one, proposed by Wolff (1995) is based on the relative abundance of the 16-18:1 isomer in ruminant fats (~8% of the total trans 18:1 isomers) and on its almost absence in partially hygrogenated vegetable oils (~0.5% of the total trans 18:1 isomers). Knowing the quantity of the isomer (determined by GLC as previously described), and using the equation described by Wolff (1995), it is then possible to estimate the contribution of each source to the total trans content. A second method to estimate the contribution of naturally occuring TFA and of industrially produced TFA in a food matrix has been proposed by Precht and Molkentin (Precht and Molkentin, 2000) who analyzed over 2100 bovine milk samples of different origins from 14 European countries and found a good correlation between the amount of CLA and total trans C18:1 and vaccenic acid (correlation coefficients of 0.97 and 0.97 respectively). Determination by GLC as previously outlined (Christie et al., 2001) of the 9c,11t-18:1 isomer, the major conjugated fatty acid of natural origin, would allow to determine the corresponding contribution of naturally occuring trans C 18:1 to the total trans content. However, these approaches cannot be applied with confidence to a wide range of foods to distinguish between TFA which are naturally present in foods (e.g. in ruminant products) and those formed during the processing of fats, oils or foods. Page 19 of 49

20 8. CONCLUSONS TFA in foods A large number of TFA isomers of monounsaturated and polyunsaturated fatty acids, including positional isomers of individual fatty acids, occur in foods. n fat of ruminant milk and meat products the main TFA are isomers of the monounsaturated fatty acid, oleic acid, with vaccenic acid (18:1t, n-7) predominating (about 30-50% of total trans-18:1 isomers in milk fat). However, trans isomers of other MUFA (e.g. 14:1 and 16:1) as well as of PUFA (18:2 and 18:3) also occur. n foods containing partially hydrogenated vegetable oils the main TFA are also isomers of oleic acid, with elaidic acid (18:1t, n-9) accounting for typically 20-30% of total trans-18:1 isomers and vaccenic acid 10-20%. The TFA profiles of ruminant fat and partially hydrogenated vegetable oils show considerable overlap with many TFA isomers in common, although present in different proportions; proportions of TFA isomers also vary among different hydrogenated oils. Partially hydrogenated fish oils also contain trans 20:1 and 22:1 isomers. Dairy and beef fat typically contains around 3-6% TFA (weight % of total fatty acids), while levels in lamb and mutton can be somewhat higher. The TFA content in margarines and fat spreads may vary considerably, depending on the proportion of partially hydrogenated oils used. Data from the TRANSFAR study carried out in 14 European countries during showed that soft spreads generally had TFA contents ranging from below 1% up to 17%, while hard stick margarines contained somewhat higher levels. Data from more recent analyses show that TFA levels in most edible fats are below 1-2%. n many cases this reduction has been accompanied by an increased level of SFA. The TFA content of bakery products (rusks, crackers, pies, pirogues, cookies, biscuits, wafers etc), as well as some breakfast cereals with added fat, French fries, soup powders and some sweets and snack products, may vary considerably (from below 1% up to 30% of total fatty acids) depending on the type of fat used. Vegetable oils and liquid margarines have a low proportion of TFA, usually below 1%. Dietary TFA intake n the EU, mean daily intakes of TFA for 14 different countries estimated in the TRANSFAR study for , ranged from 1.2 to 6.7 g/day and 1.7 to 4.1 g/day among men and women, respectively, corresponding to % and % of energy, respectively. ntake was lowest in the Mediterranean countries. Mean intakes of SFA ranged from 10.5 to 18% of total energy intake, with the lowest intakes in Southern Europe. somers of 18:1 (oleic acid) contributed 54-82% of the total TFA. Major sources of TFA were edible fats and ruminant fat, with bakery products and French fries as additional contributing foods in some countries. The contribution of TFA from ruminant fat ranged from about 30 to 80% of total TFA, corresponding to % of energy. More recent dietary surveys indicate that the intakes of TFA have decreased in a number of EU countries, mainly due to reformulation of food products, e.g. fat spreads, to reduce the TFA content. Page 20 of 49

21 Absorption and metabolism Digestion and absorption of food TFA occurs in a similar manner to other fatty acids. After absorption, TFA follow the same metabolic routes as other fatty acids and selective accumulation in tissues does not occur. Ultimately, TFA are oxidised to provide energy. Although there is some evidence from in vitro and animal studies that conversion of essential fatty acids is inhibited by TFA, metabolism of essential fatty acids is unlikely to be impaired by TFA when intakes of essential fatty acids meet recommended levels. Health effects of TFA Evidence from many controlled human intervention studies indicates that consumption of diets containing TFA, like diets containing mixtures of SFA, consistently results in increased serum LDL-C, compared with consumption of diets containing cis-monounsaturated or cispolyunsaturated fatty acids. The effect shows a linear dose response with serum LDL-C indicating that effects are proportional to amounts of TFA consumed. Elevated LDL-C has been causally linked to coronary heart disease; thus, higher intakes of TFA may increase risk for coronary heart disease (CHD). The available evidence does not provide a definitive answer to the question of whether TFA have an effect on LDL-C different to a mixture of SFA on a gram-for-gram basis. Evidence from controlled human intervention studies also indicates that consumption of diets containing TFA results in decreased serum HDL-C, compared with consumption of diets containing SFA, cis-monounsaturated or cis-polyunsaturated fatty acids. The relationship shows a linear dose response. As a consequence of their effects on LDL-C and HDL-C, TFA, relative to other fatty acids, increase total cholesterol to HDL-C ratio. Lowered HDL-C levels and increased total cholesterol to HDL-C ratio have been shown to be associated with an increased risk of cardiovascular disease (CVD) in epidemiological studies. Evidence from controlled human intervention studies also indicates that, relative to diets containing SFA, cis-monounsaturated or cis-polyunsaturated fatty acids, consumption of diets containing TFA results in increased concentrations of fasting triacylglycerol (TAG). The relationship shows a linear dose response. Elevated TAG is positively associated with the risk for cardiovascular disease in epidemiological studies. There is some evidence indicating that TFA increase lipoprotein(a), especially in people with elevated lipoprotein(a) concentrations, but the significance of this for cardiovascular risk is unclear. Human intervention studies do not provide evidence that TFA have any effects on blood pressure, in vitro LDL-oxidizability or haemostatic function (e.g. markers of platelet aggregation, coagulation or fibrinolysis). Human studies do not provide consistent evidence that at current intakes in European countries effects of fatty acids on insulin sensitivity are different for an isocaloric substitution of TFA by SFA, oleic acid or linoleic acid. n most of the human intervention studies monounsaturated TFA from hydrogenated vegetable oils were evaluated. No human intervention studies have been carried out to evaluate the effects of TFA from ruminant fat, and indeed such studies are not practicable. Thus it is not possible to determine whether there are differences between TFA from ruminant fat and TFA from Page 21 of 49

22 hydrogenated vegetable oils in their effects on metabolic risk parameters such as LDL-C or HDL-C. Prospective epidemiological studies consistently support the findings from intervention studies for an association between higher intakes of TFA and increased risk of CHD. n the prospective cohort studies that compared the effects of TFA and SFA, the effects of TFA were stronger than those of a mixture of SFA. Epidemiological evidence for a possible relationship of TFA intake with cancer, type 2 diabetes or allergy is weak or inconsistent. The proportion of TFA in plasma and tissue lipids in infants is inversely related with the essential fatty acid status. Only a few of studies have investigated the relationship of TFA levels in tissues with early development. While these studies have not established a causal link, effects of TFA on foetal and early infant growth and development need further research. Studies of supplemental CLA (mixtures of the trans-10,cis-12 and cis-9,trans-11 isomers) have provided evidence of potential beneficial health effects, e.g. reduction of body fat, improved immune response, improved profile of blood lipids, with no evidence of adverse effects. However, results are inconsistent and effects may differ between the CLA-isomers. There is some evidence of adverse effects on lipid and glucose metabolism and on insulin sensitivity of supplemental CLA in humans for the trans-10,cis-12 isomer. However, these effects were observed only at intake levels one or two orders of magnitude higher than those corresponding to intake from foods. Few studies have investigated the health effects in humans of naturally occurring CLA from foods and evidence is weak and inconsistent with respect to any health effects at current levels of intake (average intake of CLA from food is estimated to be about 0.3 g/day in Europe). Analysis of TFA TFA may be measured in a wide range of food products by infrared spectroscopy, which estimates total non-conjugated TFA, or by gas chromatography or high pressure liquid chromatography, which can measure individual TFA with a high degree of precision. At present, there are no methods of analysis applicable to a wide range of foods that can distinguish between TFA which are naturally present in foods (e.g. in ruminant products) and those formed during the processing of fats, oils or foods. This is because of the overlap in TFA profiles of ruminant fats and hydrogenated oils and the varying proportions of TFA isomers among different hydrogenated fats. DOCUMENTATON PROVDED TO EFSA Aro A, van Amelsvoort JMM, Becker W, van Erp-Baart MA, Kafatos A, Leth T, van Poppel G (1998). Trans fatty acids in dietary fats and oils from 14 European countries: the TRANSFAR study. J Food Compos Anal 11: Hulshof KFAM, van Erp-Baart MA, Anttolainen M, Becker W, Church SM, Couet C, Hermann-Kunz E, Kesteloot H, Leth T, Martins, Moreiras O, Moschandreas J, Pizzoferrato L, Rimestad AH, Thorgeirsdottir H, van Amelsvoort JMM, Aro A, Kafatos AG, Lanzmann- Petithory D, van Poppel G (1999). ntake of fatty acids in Western Europe with emphasis on trans fatty acids: the TRANSFAR study. Eur J Clin Nutr 53: Page 22 of 49

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30 Mutanen M and Aro A (1997). Coagulation and fibrinolysis factors in healthy subjects consuming high stearic or trans fatty acid diets. Thromb Haem 77: Nestel P, Noakes M, Belling B, McArthur R, Clifton P, Janus E, Abbey M (1992). Plasma lipoprotein lipid and lp[a] changes with substitution of elaidic for oleic acid in the diet. J Lipid Res 33: Norwegian food composition table (2001). Den store matvaretabellen 2001, Statens Råd for Ernæring og Fysisk Aktivitet, Gyldendal, Oslo. Oomen CM, Ocké MC, Feskens EJM, van Erp-Baart M-AJ, Kok FJ, Kromhout D (2001). Association between trans fatty acid intake and 10-year risk of coronary heart disease in the Zutphen elderly study: a prospective population-based study. Lancet 357: Pariza MW (2004). Perspective on the safety and effectiveness of conjugated linoleic acid. Am J Clin Nutr 79 (Suppl. 6): 1132S-1136S. Parodi PW (1994). Conjugated linoleic acid: an anticarcinogenic fatty acid present in milk fat. Aust J Dairy Technol 49: Petrek JA, Hudgins LC, Ho M, Bajorunas DR, Hirsch J (1997). Fatty acid composition of adipose tissue, an indication of dietary fatty acids, and breast cancar prognosis. J Clin Oncol 15: Pietinen P, Ascherio A, Korhonen P, Hartman AM, Willett WC, Albanes D, Virtamo J (1997). ntake of fatty acids and risk of coronary heart disease in a cohort of Finnish men. The alphatocopherol, beta-carotene cancer prevention study. Am J Epidemiol 145: Pollard MR, Gunstone FD, James AT, Morris LJ (1980). Desaturation of positional and geometric isomers of monoenoic fatty acids by microsomal preparations from rat liver. Lipids 15: Precht D and Molkentin J (2000). Frequency distributions of conjugated linoleic acid and trans fatty acid contents in European bovine milk fats. Milchwissenschaft 55: Precht D, Molkentin J, Destaillats F, Wolff RL (2001). Comparative studies on individual isomeric 18:1 acids in cow, goat, and ewe milk fats by low-temperature high-resolution capillary gas-liquid chromatography. Lipids 36: Riserus U, Arner P, Brismar K, Vessby B (2002). Treatment with dietary trans10cis12 conjugated linoleic acid causes isomer-specific insulin resistance in obese men with the metabolic syndrome. Diabetes Care 25: Riserus U, Smedman A, Basu S, Vessby B (2004a). Metabolic effects of conjugated linoleic acid in humans: the Swedish experience. Am J Clin Nutr 79 (Suppl. 6): 1146S-1148S. Riserus U, Vessby B, Arner P, Zethelius B (2004b). Supplementation with trans10 cis12- conjugated linoleic acid induces hyperproinsulinaemia in obese men: close association with impaired insulin sensitivity. Diabetologia 47: Ritzenthaler KL, McGuire MK, Falen R, Shultz TD, Dasgupta N, McGuire MA (2001). Estimation of conjugated linoleic acid intake by written dietary assessment methodologies Page 30 of 49

31 underestimates actual intake evaluated by food duplicate methodology. J Nutr 131(5): Roberts TL, Wood DA, Riemersma RA, Gallagher PJ, Lampe FC (1995). Trans isomers of oleic acid and linoleic acids in adipose tissue and sudden cardiac death. Lancet 345: Rodríguez E, Ribot J, Palou A (2002). Trans-10, cis-12, but not cis-9, trans-11 CLA isomer, inhibits brown adipocyte thermogenic capacity. Am J Physiol Regul ntegr Comp Physiol 282: R Rosenthal MD and Whitehurst MC (1983). Selective effects of isomeric cis and trans fatty acids on fatty acyl D9 and D6 desaturation by human skin fibroblasts. Biochim Biophys Acta 753: Salmerón J, Hu FB, Manson JE, Stampfer MJ, Colditz GA, Rimm EB, Willett WC (2001). Dietary fat intake and risk of type 2 diabetes in women. Am J Clin Nutr 73: Salo P, Seppanen-Laakso T, Laakso, Seppanen R, Niinikoski H, Viikari J, Simell O (2000). Low-saturated fat, low-cholesterol diet in 3-year-old children: effect on intake and composition of trans fatty acids and other fatty acids in serum phospholipid fraction: The STRP study. Special Turku coronary Risk factor ntervention Project for children. J Pediatr 136: Sanders TAB, de Grassi T, Miller GJ, Morrissey JH (2000). nfluence of fatty acid chain length and cis/trans isomerization on postprandial lipemia and factor V in healthy subjects (postprandial lipids and factor V). Atherosclerosis 149: Scrimgeour CM, Macvean A, Fernie CE, Sebedio JL, Riemersma RA, investigators obott (2001). Dietary trans α-linolenic acid does not inhibit D5- and D6-desaturation of linoleic acid in man. Eur J Lipid Sci Technol 103: Sébédio JL (1991). Chromatographic techniques applied to the analyses of heated fats and oils. Chromatography and analysis: Seppänen-Laakso T, Laakso, Backlund P, Vanhanen H, Viikari J (1996). Elaidic and transvaccenic acids in plasma phospholipids as indicators of dietary intake of 18:1 trans-fatty acids. J Chromatogr B Biomed Appl 687: Siguel EN and Lerman RH (1993). Trans-fatty acid patterns in patients with angiographically documented coronary artery disease. Am J Cardiol 71: Slattery ML, Benson J, Ma KN, Schaffer D, Potter JD (2001). Trans-fatty acids and colon cancer. Nutr Cancer 39: Smedman A and Vessby B (2001). Conjugated linoleic acid supplementation in humansmetabolic effects. Lipids 36: Stampfer MJ, Sacks FM, Salvini S, Willett WC, Hennekens CH (1991). A prospective study of cholesterol, apolipoproteins, and the risk of myocardial infarction. N Engl J Med 325: Steingrimsdottir L, Thorgeirsdottir H, Olafsdottir AS. The diet of celanders. Dietary survey of the celandic Nutrition Council Main findings. Reykjavik Page 31 of 49

32 Terpstra AH (2004). Effect of conjugated linoleic acid on body composition and plasma lipids in humans: an overview of the literature. Am J Clin Nutr 79: SAAC (nternational Study of Asthma and Allergies in Childhood) Steering Committee (1998). Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema: SAAC. Lancet 351: Triantafillou D, Zografos V, Katsikas H (2003). Fatty acid content of margarines in the Greek market (including trans-fatty acids): a contribution to improving consumers information. nt J Food Sci Nutr 54: Troisi R, Willett WC, Weiss ST (1992). Trans-fatty acid intake in relation to serum lipid concentrations in adult men. Am J Clin Nutr 56: Van Dam RM, Willett WC, Rimm EB, Stampfer MJ, Hu FB (2002). Dietary fat and meat intake in relation to risk of type 2 diabetes in men. Diabetes Care 25: Van de Vijver LPL, van Poppel G, van Houwelingen A, Kruyssen ACM, Hornstra G (1996). Trans unsaturated fatty acids in plasma phospholipids and coronary heart disease: a casecontrol study. Atherosclerosis 126: Van de Vijver LPL, Kardinaal AFM, Couet C, Aro A, Kafatos A, Steingrimsdottir L, Amorim Cruz JA, Moreiras O, Becker W, van Amelsvoort JMM, Vidal-Jessel S, Salminen, Moschandreas J, Sigfússon N, Martins, Carbajal A, Ytterfors A, van Poppel G (2000). Association between trans fatty acid intake and cardiovascular risk factors in Europe: the TRANSFAR study. Eur J Clin Nutr 54: Van Erp-Baart M-A, Couet C, Cuadrado C, Kefatos A, Stanley J, Van Poppel G (1998). Trans fatty acids in bakery products from 14 European countries. J Food Comp Anal 11: Van Houwelingen AC and Hornstra G (1994). Trans fatty acids in early human development. n: Galli C, Simopoulos AP, Tremoli E (eds) Fatty acids and lipids: biological aspects. Vol 75: World Rev Nutr Diet. Karger, Basel, pp Van Poppel G (1998). Trans fatty acids in Europe. The TRANSFAR Study. J Food Comp Anal 11: Vidgren HM, Louheranta AM, Agren JJ, Schwab US, Uusitupa MJ (1998). Divergent incorporation of dietary trans fatty acids in different serum lipid fractions. Lipids 33: Voorrips LE, Brants HAM, Kardinaal AFM, Hiddink GJ, van den Brandt PA, Goldbohm RA (2002). ntake of conjugated linoleic acid, fat, and other fatty acids in relation to postmenopausal breast cancer: the Netherlands Cohort Study on Diet and Cancer. Am J Clin Nutr 76: Weggemans RM, Rudrum M, Trautwein EA (2004). ntake of ruminant versus industrial trans fatty acids and risk of coronary heart disease - what is the evidence? Eur J Lipid Sci Technol 106: Weiland SK, von Mutius E, Hüsing A, Asher M, on behalf of the SAAC Steering Committee (1999). ntake of trans fatty acids and prevalence of childhood asthma and allergies in Europe. Lancet 353: Page 32 of 49

33 Wild SH, Fortmann SP, Marcovina SM (1997). A prospective case-control study of lipoprotein(a) levels and apo(a) size and risk of coronary heart disease in Stanford Five-City Project participants. Arterioscler Thromb Vasc Biol 17: Willett WC, Stampfer MJ, Manson JE, Colditz GA, Speizer FE, Rosner BA, Sampson LA, Hennekens CH (1993). ntake of trans fatty acids and risk of coronary heart disease among women. Lancet 341: Wolff RL, Combe NA, Destaillats F, Boue C, Precht D, Molkentin J, Entressangles B (2000). 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 Analytical and nutritional implications. Lipids 35: Wolff RL (1995). Content and distribution of trans 18:1 acids in ruminant milk and meat fats. Their importance in European diets and their effect on human milk. J Am Oil Chemists Soc 72: Zevenbergen JL, Houtsmuller UMT, Gottenbos JJ (1988). Linoleic acid requirement of rats fed trans fatty acids. Lipids 23: Zock PL, Blijlevens RAMT, de Vries JHM, Katan MB (1993). Effects of stearic acid and trans fatty acids versus linoleic acid on blood pressure in normotensive women and men. Eur J Clin Nutr 47: Zock PL and Katan MB (1992). Hydrogenation alternatives: effects of trans fatty acids and stearic acid versus linoleic acid on serum lipids and lipoprotein in humans. J Lipid Res 33: PANEL MEMBERS Wulf Becker, Daniel Brasseur, Jean-Louis Bresson, Albert Flynn, Alan A. Jackson, Pagona Lagiou, Geltrude Mingrone, Bevan Moseley, Andreu Palou, Hildegard Przyrembel, Seppo Salminen, Stephan Strobel and Hendrik van Loveren. ACKNOWLEDGEMENT The Scientific Panel on Dietetic Products, Nutrition and Allergies wishes to thank Ronald P. Mensink and Jean-Louis Sébédio for their contributions to the draft opinion. Page 33 of 49

34 Table 2. Dietary trans fatty acids and serum lipid and lipoprotein concentrations: results from well-controlled intervention trials + Authors Mensink and Katan, 1990 No. of men/ women Design Days of test period 25 / 34 X 21 Zock and Katan, / 30 X 21 Judd et al., / 29 X 42 Almendingen et al / 0 X 21 Aro et al., / 49 // 35 Judd et al., / 23 X 35 Müller et al., / 27 X 17 Lichtenstein et al., / 18 X 35 Judd et al., / 0 X 35 Lovejoy et al., / 13 X 28 Test diet V C V V V V V Main source for trans fatty acids somerized high oleic acid sunflower oil Hydrogenated high oleic acid sunflower oil Not specified Partially hydrogenated soybean (diet ) or fish oil (diet ) Partially hydrogenated high oleic acid sunflower oil Not specified Partially hydrogenated soybean oil Partially hydrogenated soybean oil Not specified Not specified Total Fatty acid composition (% of daily energy intake) Lipid or lipoprotein (mmol/l, except Total : HDL) S Total : M P T TC LDL-C HDL-C C16 C18 HDL a 2.67 a 1.42 a a 3.04 a 1.25 a,b a 3.14 a 1.42 b a,b 4.89 a 4.90 b 5.26 a,b,c 5.46 a,d 5.52 b,e 5.61 c,d,e 5.32 a 5.11 a,b 5.42 b a 4.97 a 4.87 a 4.74 a 4.61 b 4.45 a,b 5.82 a 5.84 b 6.00 c 6.08 d 6.28 a,b,c 4.72 a,b,c 4.59 d,e,f,g 4.99 a,d,h 4.98 b,e,i 4.78 f,h,i 4.96 c,g 3.78 a 3.93 a 3.90 b 2.83 a,b 3.00 a 3.07 b 3.34 a,b,c 3.54 a,d 3.60 b 3.64 c,d 3.81 a 3.58 a,b 3.94 b 1.47 a,b 1.41 a 1.37 b 1.42 a,b 1.40 c 1.38 a,d 1.47 b,c,d 1.05 a 1.05 b 0.98 a,b a 1.42 a a 1.36 a Only studies with a thorough control of food intake, with dietary fatty acids being the sole variable, and designs that eliminated the effect of nonspecific drifts of the outcome variables with time, were selected. X, Cross-over or Latin square design; //, Parallel design. C, control diet. Lipid values are corrected for differences between groups when on the control diets. S: Saturated fatty acids, M: Monounsaturated fatty acids, P: Polyunsaturated fatty acids, T: Trans fatty acids, Total: Total saturated fatty acids, C16: Palmitic acid, C18: Stearic acid. TC: Total cholesterol, LDL-C: LDL-cholesterol, HDL-C: HDL-cholesterol, Total:HDL: Total to HDL cholesterol ratio, TG: Triacylglycerol. Values sharing a common superscript are significantly different (P<0.05). The total to HDL cholesterol ratio was not presented in all studies. For reasons of uniformity, values have therefore been calculated from mean total cholesterol and mean HDL cholesterol concentration. Consequently, no P-values can be given a 3.27 a 3.21 a 2.90 a 2.88 b 2.61 a,b 3.98 a,b 4.01 c 4.11 d 4.24 a 4.34 b 3.05 a,b,c 2.95 d,e,f,g 3.36 a,d,h,i 3.32 b,e,h 3.10 f 3.21 c,g,i a 1.32 a,b 1.43 b a,b,c 1.24 a,d,e,f,g 1.16 d,h 1.17 e,i,j 1.16 b,f 1.30 c,g,h,i,j 1.23 a 1.28 a,b 1.23 b TG 0.81 a,b 0.94 a 0.94 b 0.95 a 1.04 a 1.00 b 1.03 a,b 1.11 a 1.16 b,c 1.07 c a 1.09 a a 1.50 b 1.68 c 1.66 d 1.76 b 1.03 a 0.88 a,b,c,d 1.02 b 1.06 c 1.13 d,e 0.97 e Page 34 of 49

35 Table 3. Estimated effects for the change in serum lipids and lipoproteins for a group of subjects when one percent of energy in the diet from carbohydrates is replaced isocalorically by a particular fatty acid Fatty acid Trans-monounsaturates Lauric acid Myristic acid Palmitic acid Stearic acid Cis-monounsaturates Cis-polyunsaturates Change 95% C Change 95% C Change 95% C Change 95% C Change 95% C Change 95% C Change 95% C Total cholesterol (mmol/l) to to to to to to to LDL cholesterol (mmol/l) to to to to to to to HDL cholesterol (mmol/l) to to to to to to to Total to HDL cholesterol ratio to to to to to to to Triacylglycerol (mmol/l) to to to to to to to Page 35 of 49

36 Table 4. Dietary trans fatty acids and serum lipoprotein(a) concentrations: results from well-controlled intervention trials Authors No. of men/women Design Days of test period Mensink et al., / 34 X 21 Mensink et al., / 30 Almendingen et al., 1995 X / 0 X 21 Aro et al., / 50 // 35 Clevidence et al., / 29 X 42 Judd et al., / 23 X 35 Müller et al., / 27 X 17 Lichtenstein et al., 1999) 18 / 18 X 35 Test diet C V V V Main source for trans fatty acids somerized high oleic acid sunflower oil Hydrogenated high oleic acid sunflower oil Partially hydrogenated soybean (diet ) or fish oil (diet ) Partially hydrogenated high oleic acid sunflower oil Not specified Not specified Partially hydrogenated soybean oil Partially hydrogenated soybean oil Total Fatty acid composition (% of daily energy intake) S C16 C18 M P T Only studies with a thorough control of food intake, with dietary fatty acids being the sole variable, and designs that eliminated the effect of nonspecific drifts of the outcome variables with time, were selected. X, Cross-over or Latin square design; //, Parallel design. C, control diet. Lp[a] values are corrected for differences between groups when on the control diets. S: Saturated fatty acids, M: Monounsaturated fatty acids, P: Polyunsaturated fatty acids, T: Trans fatty acids, Total: Total saturated fatty acids, C16: Palmitic acid, C18: Stearic acid. Lp(a): Lipoprotein(a). Values sharing a common superscript are significantly different (P<0.05) Lp(a) (mg/l) 32 a 45 a 26 a 69 a 69 b 85 a,b 91 a,b 103 a 121 b 270 a 321 a 238 a 238 b 247 c 219 a,b,c 186 a,b 202 a 197 b Page 36 of 49

37 Table 5. Dietary trans fatty acids and LDL oxidation: results from intervention trials Authors Nestel et al., 1992 No. of men/ women Design + Days of test period 27 / 0 X 21 Test diet Main source for trans fatty acids Hardened canola/ palmolein Total Fatty acid composition (% of daily energy intake) S M P T C16 C <1.0 Results of parameters to measure in vitro LDL oxidative modification No significant effects on LDL oxidation rate, diene concentration, malondialdehyde concentration, or lag time to oxidation No significant effects on lag time to oxidation Cuchel et al., = Hydrogenated / SC corn oil Partially No statistically significant effects on hydrogenated Halvorsen et X conjugated dienes, lipid peroxides, 31 / 0 21 soybean (diet al., 1996 SC uptake of LDL by macrophages, or ) or fish oil relative electrophoretic mobility of LDL (diet ) + X, Cross-over or Latin square design; =, Sequential design. SC: Strictly controlled, which means a thorough control of food intake, with dietary fatty acids being the sole variable. S: Saturated fatty acids, M: Monounsaturated fatty acids, P: Polyunsaturated fatty acids, T: Trans fatty acids, Total: Total saturated fatty acids, C16: Palmitic acid, C18: Stearic acid. Page 37 of 49

38 Table 6. Dietary trans fatty acids and hemostatic function: results from intervention trials Authors Almendingen et al., 1996 Mutanen and Aro 1997 No. of men/women 31 / 0 31 / 49 Design + X SC // SC Days of test period Test diet C Main source for trans fatty acids Partially hydrogenated soybean (diet ) or fish oil (diet ) Partially hydrogenated high oleic acid sunflower oil Total Fatty acid composition (% of daily energy intake) S M P T C16 C Significant parameter (unit) Fibrinogen (g/l) 3.1 b 3.0 a 2.9 a,b Significant parameter (unit) PA-1 (U/mL) 8.8 b 13.5 a,b 10.7 a Significant parameter (unit) <<center in cell>> PA-1 ag (ng/ml) 16.2 b 21.5 a,b 16.5 a Nonsignificant parameters Factor Vc FPA D-dimers tpa antigen β-tg Fibrinogen Factor Vc tpa PA-1 D-dimers Fibrinogen Factor V Louheranta et / 14 X 28 Not specified al., X, Cross-over or Latin square design; //, Parallel design. SC: Strictly controlled, which means a thorough control of food intake, with dietary fatty acids being the sole variable. S: Saturated fatty acids, M: Monounsaturated fatty acids, P: Polyunsaturated fatty acids, T: Trans fatty acids, Total: Total saturated fatty acids, C16: Palmitic acid, C18: Stearic acid Values sharing a common superscript are significantly different (P<0.05). PA-1 (ag): Plasminogen activator inhibitor (antigen), Factor Vc: Factor V coagulant activity, FPA: Fibrinopeptide A, tpa: tissue plasminogen activator, β-tg: β- Thromboglobulin Page 38 of 49