Tag Archives: lipids

Bile salts: definition, functions, enterohepatic circulation, synthesis

Bile salts: contents in brief

What are bile salts

Bile salts and bile acids are polar cholesterol derivatives, and represent the major route for the elimination of the steroid from the body.
They are molecules with similar but not identical structures, and diverse physical and biological characteristics.
They are synthesized in the liver, stored in the gallbladder, secreted into the duodenum, and finally, for the most part, reabsorbed in the ileum.
Because at physiological pH these molecules are present as anions, the terms bile acid and bile salts are used herein as synonyms.

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Chemical structure of bile salts

Bile Salts
Fig. 1 – Chemical Structures of the Most Abundant Bile Acids

Bile salts have similarities and differences with cholesterol molecule.
Like the steroid, they have a nucleus composed of four fused rings: three cyclohexane rings, labeled A, B and C, and a cyclopentane ring, labeled D. This structure is the perhydrocyclopentanophenanthrene, more commonly known as steroid nucleus.
In higher vertebrates, they have 24 carbon atoms, as the side chain is three carbons shorter than the original. In lower vertebrates, bile acids have 25, 26, or 27 carbon atoms. The side chain ends with a carboxyl group, ionized at pH 7, that can be linked to the amino acid glycine or taurine (see below).
In addition to the hydroxyl group at position 3, they have hydroxyl groups at positions 7 and/or 12.
All this makes them much more polar than cholesterol.

Bile Salts
Fig. 2 – Cholic Acid Structure

Since A and B rings are fused in cis configuration, the planar structure of the steroid nucleus is curved, and it is possible to identify:

  • a concave side, which is hydrophilic because the hydroxyl groups and the carboxyl group of the side chain, with or without the linked amino acid, are oriented towards it;
  • a convex side, which is hydrophobic because the methyl groups present at position 18 and 19 are orientated towards it.

Therefore, having both polar and nonpolar groups, they are amphiphilic molecules and excellent surfactants. However, their chemical structure makes them different from many other surfactants, often composed of a polar head region and a nonpolar tail.

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Primary, conjugated and secondary bile salts

Bile Salts
Fig. 3 – Conjugated Bile Acids

Primary bile acids are those synthesized directly from cholesterol in the hepatocytes. In humans, the most important are cholic acid and chenodeoxycholic acid, which make up 80% of all bile acids. Before being secreted into the biliary tree, they are almost completely conjugated, up to 98%, with the glycine or taurine, to form glycoconjugates and tauroconjugates, respectively. In particular, approximately 75% of cholic acid and chenodeoxycholic acid are conjugated with glycine, to form glycocholic acid  and glycochenodeoxycholic acid, the remaining 25% with taurine, to form taurocholic acid and taurochenodeoxycholic.
Conjugated bile acids are molecules with more hydrophilic groups than unconjugated bile acids, therefore with a increased emulsifying capacity. In fact, conjugation decreases the pKa of bile acids, from about 6, a value typical of non-conjugated molecules, to about 4 for glycocholic acid, and about 2 for taurocholic acid. This makes that conjugated bile acids are ionized in a broader range of pH to form the corresponding salts.
The hydrophilicity of the common acid and bile salts decreases in the following order: glycine-conjugated < taurine-conjugated < lithocholic acid  < deoxycholic acid  < chenodeoxycholic acid < cholic acid <ursodeoxycholic acid.
Finally, conjugation also decreases the cytotoxicity of primary bile acids.

Secondary bile acids  are formed from primary bile acids which have not been reabsorbed from the small intestine. Once they reach the colon, they can undergo several modifications by colonic microbiota to form secondary bile acids (see below). They make up the remaining 20% of the body’s bile acid pool.

Another way of categorizing bile salts is based on their conjugation with glycine and taurine and their degree of hydroxylation. On this basis, three categories are identified.

  • Trihydroxy conjugates, such as taurocholic acid and glycocholic acid.
  • Dihydroxy conjugates, such as glycodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and taurodeoxycholic acid. They account for about 60% of bile salts present in the bile.
  • Unconjugated forms, such as cholic acid, deoxycholic acid, chenodeoxycholic acid, and lithocholic acid.

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Function of bile acids

All their physiological functions are performed in the conjugated form.

  • They are the major route for the elimination of cholesterol from human body.
    Indeed, humans do not have the enzymes to break open the cyclohexane rings or  the cyclopentane ring of the steroid nucleus, nor to oxidize cholesterol to CO2 and water.
    The other mechanism to eliminate the steroid from the body is as cholesterol per se in the bile.
  • Bile salts are strong surfactants. And in particular, di- and trihydroxy conjugates are the best surfactants among bile acids, much more effective than unconjugated counterparts, since they have more polar groups.
    Once in contact with apolar lipids in the lumen of the small intestine, the convex apolar surface interacts with the apolar lipids, such as triglycerides, cholesterol esters, and ester of fat-soluble vitamins, whereas the concave polar surface interacts with the surrounding aqueous medium. This increases the dispersion of apolar lipids in the aqueous medium, as it allows the formation of tiny lipid droplets, increasing the surface area for:

lipase activity, mainly pancreatic lipase, (bile salts also play a direct role in the activation of this enzyme);

intestinal esterase activity.

Subsequently, they facilitate the absorption of lipid digestion products, as well as of fat soluble vitamins by the intestinal mucosa thanks to the formation of mixed micelles.
Bile acids perform a similar function in the gallbladder where, forming mixed micelles with phospholipids, they prevent the precipitation of cholesterol.
Note: as a consequence of the arrangement of polar and nonpolar groups, bile acids form micelles in aqueous solution, usually made up of less than 10 monomers, as long as their concentration is above the so-called critical micellar concentration or CMC.

  • At the intestinal level, they modulate the secretion of pancreatic enzymes and cholecystokinin.
  • In the small and large intestine, they have a potent antimicrobial activity, mainly deoxycholic acid, in particular against Gram-positive bacteria. This activity may be due to oxidative DNA damage, and/or to the damage of the cell membrane. Therefore, they play an important role in the prevention of bacterial overgrowth, but also in the regulation of gut microbiota composition.
  • In the last few years, it becomes apparent their regulatory role in the control of energy metabolism, and in particular for the hepatic glucose handling.

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Enterohepatic circulation of bile salts

After fat intake, enteroendocrine cells of the duodenum secrete cholecystokinin into the blood stream. Hormone binding to receptors on smooth muscle cells of the gallbladder promotes their contraction; the hormone also causes the relaxation of the sphincter of Oddi. All this results in the secretion of the bile, and therefore of bile acids into the duodenum.
Under physiological conditions, human bile salt pool is constant, and equal to about 3-5 g. This is made possible by two processes:

  • their intestinal reabsorption;
  • their de novo synthesis (see below).

Up to 95% of the secreted bile salts is reabsorbed from the gut, not together with the products of lipid digestion, but through a process called enterohepatic circulation.
It is an extremely efficient recycling system, which seems to occur at least two times for each meal, and includes the liver, the biliary tree, the small intestine, the colon, and the portal circulation through which reabsorbed molecules return to the liver. Such recirculation is necessary since liver’s capacity to synthesize bile acids is limited and insufficient to satisfy intestinal needs if the bile salts were excreted in the feces in high amounts.
Most of the bile salts are reabsorbed into the distal ileum, the lower part of the small intestine, by a sodium-dependent transporter within the brush border of the enterocytes, called sodium-dependent bile acid transporter or ASBT, which carries out the cotransport of a molecule of bile acid and two sodium ions.
Within the enterocyte, it is thought that bile acids are transported across the cytosol to the basolateral membrane by the ileal bile acid-binding protein or IBABP. They cross the basolateral membrane by the organic solute transporter alpha-beta or OSTα/OSTβ, pass into the portal circulation, and, bound to albumin, reach the liver.
It should be noted that a small percentage of bile acids reach the liver through the hepatic artery.
A hepatic level, their extraction is very efficient, with a first-pass extraction fraction ranging from 50 to 90%, a percentage that depends on bile acid structure. The uptake of conjugated bile acids is mainly mediated by a Na+-dependent active transport system, that is, the sodium-dependent taurocholate cotransporting polypeptide or NTCP. However, a sodium-independent uptake can also occur, carried out by proteins of the family of organic anion transporting polypeptides or OATP, mainly OATP1B1 and OATP1B3.
The rate limiting step in the enterohepatic circulation is their canalicular secretion, largely mediated by the bile salt export pump or BSEP, in an ATP-dependent process. This pump carries monoanionic bile salts, which are the most abundant. Bile acids conjugated with glucuronic acid or sulfate, which are dianionic, are transported by different carriers, such as MRP2 and BCRP.

Note: serum levels of bile acids vary on the basis of the rate of their reabsorption, and therefore they are higher during meals, when the enterohepatic circulation is more active.

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Intestinal metabolism of bile acids

Bile Salts
Fig. 4 – Intestinal Bile Acid Metabolism

Bile acids which escape ileal absorption pass into the colon where they partly undergo modifications by intestinal microbiota and are converted to secondary bile acids.
The main reactions are listed below.

  • Deconjugation
    On the side chain, hydrolysis of the C24 N-acyl amide bond can occur, with release of unconjugated bile acids and glycine or taurine. This reaction is catalyzed by bacterial hydrolases present both in the small intestine and in the colon.
  • 7α-Dehydroxylation
    Quantitatively, it is the most important reaction, carried out by colonic bacterial dehydratases that remove the hydroxyl group at position 7 to form 7-deoxy bile acids. In particular, deoxycholic acid is formed from cholic acid, and lithocholic acid, a toxic secondary bile acid, from chenodeoxycholic acid.
    It should be noted that 7α-dehydroxylation, unlike oxidation and epimerization (see below), can only occur on unconjugated bile acids, and therefore, deconjugation is an essential prerequisite.
  • Oxidation and epimerization
    They are reactions involving the hydroxyl groups at positions 3, 7 and 12, catalyzed by bacterial hydroxysteroid dehydrogenases. For example, ursodeoxycholic acid derives from the epimerization of chenodeoxycholic acid.

Some of the secondary bile acids are then reabsorbed from the colon and return to the liver. In the hepatocytes, they are reconjugated, if necessary, and resecreted. Those that are not reabsorbed, are excreted in the feces.
Whereas oxidations and deconjugations are carried out by a broad spectrum of anaerobic bacteria, 7α-dehydroxylations is carried out by a limited number of colonic anaerobes.
7α-Dehydroxylations and deconjugations increase the pKa of the bile acids, and therefore their hydrophobicity, allowing a certain degree of passive absorption across the colonic wall.
The increase of hydrophobicity is also associated with an increased toxicity of these molecules. And a high concentration of secondary bile acids in the bile, blood, and feces has been associated to the pathogenesis of colon cancer.

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Soluble fibers and reabsorption of bile salts

The reabsorption of bile salts can be reduced by chelating action of soluble fibers, such as those found in fresh fruits, legumes, oats and oat bran, which bind them, decreasing their uptake. In turn, this increases bile acid de novo synthesis, up-regulating the expression of the 7α-hydroxylase and sterol 12α-hydroxylase (see below), and thereby reduces hepatocyte cholesterol concentration.
The depletion of hepatic cholesterol increases the expression of the LDL receptor, and thus reduces plasma concentration of LDL cholesterol. On the other hand, it also stimulates the synthesis of HMG-CoA reductase, the key enzyme in cholesterol biosynthesis.
Note: some anti-cholesterol drugs act by binding bile acids in the intestine, thereby preventing their reabsorption.

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Synthesis of primary bile acids

Bile Salts
Fig. 5 – Primary Bile Acid Biosynthesis

Quantitatively, bile acids are the major product of cholesterol metabolism.
As previously said, enterohepatic circulation and their de novo synthesis maintain a constant bile acid pool size. In particular, de novo synthesis allows the replacement of bile salts excreted in the faces, about 5-10% of the body pool, namely ~ 0.5 g/day.
Below, the synthesis of cholic acid and chenodeoxycholic acid, and their conjugation with the amino acids taurine and glycine, is described.
There are two main pathways for bile acid synthesis: the classical pathway and the alternative pathway. In addition, some other minor pathways will also be described.

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The classical or neutral pathway

In humans, up to 90% of bile salts are produced via the classical pathway (see fig. 5), also referred to as “neutral” pathway since intermediates are neutral molecules.
It is a metabolic pathway present only in the liver, that consists of reactions catalyzed by enzymes localized in the cytosol, endoplasmic reticulum, peroxisomes, and mitochondria, and whose end products are the conjugates of cholic acid and chenodeoxycholic acid.

  • The first reaction is the hydroxylation at position 7 of cholesterol, to form 7α-hydroxycholesterol. The reaction is catalyzed by cholesterol 7α-hydroxylase or CYP7A1 (E.C. 1.14.14.23). It is an enzyme localized in the endoplasmic reticulum, and catalyzes the rate-limiting step of the pathway.

Cholesterol + NADPH + H+ + O2 → 7α-Hydroxycholesterol + NADP+ + H2O

  • 7α-Hydroxycholesterol undergoes oxidation of the 3β-hydroxyl group and the shift of the double bond from the 5,6 position to the 4,5 position, to form 7α-hydroxy-4-cholesten-3-one. The reaction is catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase or HSD3B7 (E.C. 1.1.1.181), an enzyme localized in the endoplasmic reticulum.
  • 7α-Hydroxy-4-cholesten-3-one can follow two routes:

to enter the pathway that leads to the synthesis of cholic acid, through the reaction catalyzed by 7α-hydroxy-4-cholesten-3-one 12α-monooxygenase or sterol 12α-hydroxylase or CYP8B1 (E.C. 1.14.18.8), an enzyme localized in the endoplasmic reticulum;

to enter the pathway that leads the synthesis of chenodeoxycholic acid, through the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase or AKR1D1 (E.C. 1.3.1.3), a cytosolic enzyme.

It should be underlined that the activity of sterol 12α-hydroxylase determines the ratio of cholic acid to chenodeoxycholic acid, and, ultimately, the detergent capacity of bile acid pool. And in fact, the regulation of sterol 12α-hydroxylase gene transcription is one of the main regulatory step of the classical pathway.

Therefore, if 7α-hydroxy-4-cholesten-3-one proceeds via the reaction catalyzed by sterol 12α-hydroxylase, the following reactions will occur.

  • 7α-Hydroxy-4-cholesten-3-one is hydroxylated at position 12 by sterol 12α-hydroxylase, to form 7α,12α-dihydroxy-4-cholesten-3-one.
  • 7α,12α-Dihydroxy-4-cholesten-3-one undergoes reduction of the double bond at 4,5 position, in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase, to form 5β-cholestan-7α,12α-diol-3-one.
  • 5β-Cholestan-7α,12α-diol-3-one undergoes reduction of the hydroxyl group at position 4, in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase or AKR1C4 (EC 1.1.1.213), a cytosolic enzyme, to form 5β-cholestan-3α,7α,12α-triol.
  • 5β-Cholestan-3α,7α,12α-triol undergoes oxidation of the side chain via three reactions catalyzed by sterol 27-hydroxylase or CYP27A1 (EC 1.14.15.15). It is a mitochondrial enzyme also present in extrahepatic tissues and macrophages, which introduces a hydroxyl group at position 27. The hydroxyl group is oxidized to aldehyde, and then to carboxylic acid, to form 3α,7α,12α-trihydroxy-5β-cholestanoic acid.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoic  acid is activated to its coenzyme A ester, 3α,7α,12α-trihydroxy-5β-cholestanoyl-CoA, in the reaction catalyzed by either very long chain acyl-CoA synthetase or VLCS (EC 6.2.1.-), or bile acid CoA synthetase or BACS (EC 6.2.1.7), both localized in the endoplasmic reticulum.
  • 3α,7α,12α-Trihydroxy-5β-cholestanoyl-CoA is transported to peroxisomes where it undergoes five successive reactions, each catalyzed by a different enzyme. In the last two reactions, the side chain is shortened to four carbon atoms, and finally cholylCoA is formed.
  • In the last step, the conjugation, via amide bond, of the carboxylic acid group of the side chain with the amino acid glycine or taurine occurs. The reaction is catalyzed by bile acid-CoA:amino acid N-acyltransferase or the BAAT (EC 2.3.1.65), which is predominantly localized in peroxisomes.
    The reaction products are thus the conjugated bile acids: glycocholic acid and taurocholic acid.

If 7α-hydroxy-4-cholesten-3-one does not proceed via the reaction catalyzed by sterol 12α-hydroxylase, it enters the pathway that leads to the synthesis of chenodeoxycholic acid conjugates, through the reactions described below.

  • 7α-Hydroxy-4-cholesten-3-one is converted to 7α-hydroxy-5β-cholestan-3-one in the reaction catalyzed by 3-oxo-Δ4-steroid 5β-reductase.
  • 7α-Hydroxy-5β-cholestan-3-one is converted to 5β-cholestan-3α,7α-diol in the reaction catalyzed by 3α-hydroxysteroid dehydrogenase.

Then, the conjugated bile acids glycochenodeoxycholic acid and taurochenodeoxycholic acid are formed by modifications similar to those seen for the conjugation of cholic acid, and catalyzed mostly by the same enzymes.

Note: unconjugated bile acids formed in the intestine must reach the liver to be reconjugated.

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The alternative or acidic pathway

It is prevalent in the fetus and neonate, whereas in adults it leads to the synthesis of less than 10% of the bile salts.
This pathway  (see fig. 5) differs from the classical pathway in that:

  • the intermediate products are acidic molecules, from which the alternative name “acidic pathway”;
  • the oxidation of the side chain is followed by modifications of the steroid nucleus, and not vice versa;
  • the final products are conjugates of chenodeoxycholic acid.

The first step involves the conversion of cholesterol into 27-hydroxycholesterol in the reaction catalyzed by sterol 27-hydroxylase.
27-Hydroxycholesterol can follow two routes.

Route A

  • 27-hydroxycholesterol is converted to 3β-hydroxy-5-cholestenoic acid in a reaction catalyzed by sterol 27-hydroxylase.
  • 3β-Hydroxy-5-cholestenoic acid is hydroxylated at position 7 in the reaction catalyzed by oxysterol 7α-hydroxylase or CYP7B1 (EC 1.14.13.100), an enzyme localized in the endoplasmic reticulum, to form 3β-7α-dihydroxy-5-colestenoic acid.
  • 3β-7α-Dihydroxy-5-cholestenoic acid is converted to 3-oxo-7α-hydroxy-4-cholestenoic acid, in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase.
  • 3-Oxo-7α-hydroxy-4-cholestenoic acid, as a result of side chain modifications, forms chenodeoxycholic acid, and then its conjugates.

Route B

  • 27-Hydroxycholesterol is converted to 7α,27-dihydroxycholesterol in the reaction catalyzed by oxysterol 7α-hydroxylase and cholesterol 7α-hydroxylase.
  • 7α,27-Dihydroxycholesterol is converted to 7α,26-dihydroxy-4-cholesten-3-one in the reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid oxidoreductase;

7α, 26-Dihydroxy-4-cholesten-3-one can be transformed directly to conjugates of chenodeoxycholic acid, or can be converted to 3-oxo-7α-hydroxy-4-colestenoic acid,  and then undergo side chain modifications and other reactions that lead to the synthesis of the conjugates of chenodeoxycholic acid.

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Minor pathways

There are also minor pathways (see fig. 5) that contribute to bile salt synthesis, although to a lesser extent than classical and alternative pathways.

For example:

  • A cholesterol 25-hydroxylase (EC 1.14.99.38) is expressed in the liver.
  • A cholesterol 24-hydroxylase or CYP46A1 (EC 1.14.14.25) is expressed in the brain, and therefore, although the organ cannot export cholesterol, it exports oxysterols.
  • A nonspecific 7α-hydroxylase has also been discovered. It is  expressed in all tissues and appears to be involved in the generation of oxysterols, which may be transported to hepatocytes to be converted to chenodeoxycholic acid.

Additionally, sterol 27-hydroxylase is expressed in various tissues, and therefore its reaction products must be transported to the liver to be converted to bile salts.

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Bile salts: regulation of synthesis

Regulation of bile acid synthesis occurs via a negative feedback mechanism, particularly on the expression of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase.
When an excess of bile acids, both free and conjugated, occurs, these molecules bind to the nuclear receptor farnesoid X receptor or FRX, activating it: the most efficacious bile acid is chenodeoxycholic acid, while others, such as ursodeoxycholic acid, do not activate it.
FRX induces the expression of the transcriptional repressor small heterodimer partner or SHP, which in turn interacts with other transcription factors, such as liver receptor homolog-1 or LRH-1, and hepatocyte nuclear factor-4α or HNF-4α. These transcription factors bind to a sequence in the promoter region of 7α-hydroxylase and 12α-hydroxylase genes, region called bile acid response elements or BAREs, inhibiting their transcription.
One of the reasons why bile salt synthesis is tightly regulated is because many of their metabolites are toxic.

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References

Chiang J.Y.L. Bile acids: regulation of synthesis. J Lipid Res 2009;50(10):1955-66. doi:10.1194/jlr.R900010-JLR200

Gropper S.S., Smith J.L. Advanced nutrition and human metabolism. 6h Edition. Cengage Learning, 2012 [Google eBook]

Moghimipour E., Ameri A., and Handali S. Absorption-enhancing effects of bile salts. Molecules 2015;20(8); 14451-73. doi:10.3390/molecules200814451

Monte M.J., Marin J.J.G., Antelo A., Vazquez-Tato J. Bile acids: Chemistry, physiology, and pathophysiology. World J Gastroenterol 2009;15(7):804-16. doi:10.3748/wjg.15.804

Rawn J.D. Biochimica. Mc Graw-Hill, Neil Patterson Publishers, 1990

Rosenthal M.D., Glew R.H. Medical biochemistry – Human metabolism in health and disease. John Wiley J. & Sons, Inc., Publication, 2009

Sundaram S.S., Bove K.E., Lovell M.A. and Sokol R.J. Mechanisms of Disease: inborn errors of bile acid synthesis. Nat Clin Pract Gastroenterol Hepatol 2008;5(8):456-68. doi:10.1038/ncpgasthep1179


Trans fatty acids

Trans fatty acids: contents in brief

Definition and chemical structure of trans fatty acids

Trans fatty acids (TFA) or trans-unsaturated fatty acids or trans fats are unsaturated fatty acids with at least one a double bond in the trans or E configuration.

Trans fatty acids
Fig. 1 -Cis and Trans Isomers

Carbon-carbon double bonds show planar conformation, and so they can be considered as plains from whose opposite sides carbon chain attaches and continues. “The entry” and “the exit” of the carbon chain from the plain may occur on the same side of the plain, and in this case double bond is defined in cis or Z configuration, or on opposite side, and in that case it is defined in trans configuration.
Unsaturated fatty acids most commonly have their double bonds in cis configuration; the other, less common configuration is trans.
Cis bond causes a bend in the fatty acid chain, whereas the geometry of trans bond straightens the fatty acid chain, imparting a structure more similar to that of saturated fatty acids.

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Properties of fats rich in trans fatty acids

Below, some distinctive characteristics of the fats rich in trans fats, that make them particularly suited for the production of margarines and vegetable shortening used in home and commercial cooking, and manufacturing processes.

  • Bent molecules can’t pack together easily, but linear ones can do it.
    This means that trans fatty acids contribute, together with the geometrically similar saturated fatty acids, to the hardness of the fats in which they are, giving them a higher melting point.
    Heightening the melting point of fats means that it is possible to convert them from liquid form to semi-solids and solids at room temperature.
    Note: trans fats tend to be less solid than saturated fatty acids.
  • They have:

a melting point, consistency and “mouth feel” similar to those of butter;
a long shelf life at room temperature;
a flavor stability.

  • They are stable during frying.

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Sources of trans fatty acids

Dietary TFA come from different sources briefly reviewed below.

  • In industrialized countries, greater part of the consumed trans fatty acids, in USA about 80 percent of the total, are produced industrially, in varying amounts, during partial hydrogenation of edible oils containing unsaturated fatty acids (see below).
  • They are produced at home during frying with vegetable oils containing unsaturated fatty acids.
  • They come from bacterial transformation of unsaturated fatty acids ingested by ruminants in their rumen (see below).
  • Another natural source is represented by some plant species, such as leeks, peas, lettuce and spinach, that contain trans-3-hexadecenoic acid, and rapeseed oil, that contains brassidic acid (22:1∆13t) and gondoic acid (20:1∆11t). In these sources trans fatty acids are present in small amounts.
  • Very small amounts, less than 2 percent, are formed during deodorization of vegetable oils, a process necessary in the refining of edible oils. During this process trans fatty acids with more than one double bond are formed in small amounts. These isomers are also present in fried foods and in considerable amounts in some partially hydrogenated vegetable oils (see below).

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Industrial trans fatty acids

Hydrogenation is a chemical reaction in which hydrogen atoms react, in the presence of a catalyst, with a molecule.
The hydrogenation of unsaturated fatty acids involves the addition of hydrogen atoms to double bonds on the carbon chains of fatty acids. The reaction occurs in presence of metal catalyst and hydrogen, and is favored by heating vegetable oils containing unsaturated fatty acids.

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Partial hydrogenation of vegetable oils

The process of hydrogenation was first discovered in 1897 by French Nobel prize in Chemistry, jointly with fellow Frenchman Victor Grignard, Paul Sabatier using a nickel catalyst.
Partially hydrogenated vegetable oils were developed in 1903 by a German chemist, Wilhelm Normann, who files British patent on “Process for converting unsaturated fatty acids or their glycerides into saturated compounds”. The term trans fatty acids or trans fats appeared for the first time in the Remark column of the 5th edition of the “Standard Tables of Food Composition” in Japan.

Trans fatty acids
Fig. 2 – From Oleic Acid to Vaccenic Acid

During partial hydrogenation, an incomplete saturation of the unsaturated sites on the carbon chains of unsaturated fatty acids occurs. For example, with regard to fish oil, trans fatty acid content in non-hydrogenated oils and in highly hydrogenated oils is 0.5 and 3.6%, respectively, whereas in partially hydrogenated oils is 30%.
But, most importantly, some of the remaining cis double bonds may be moved in their positions on the carbon chain, producing geometrical and positional isomers, that is, double bonds can be modified in both conformation and position.
Below, other changes that occur during partial hydrogenation are listed.

Trans fatty acids
Fig. 3 – Vegetable Shortenings

Partially hydrogenated vegetable oils were developed for the production of vegetable fats, a cheaper alternative to animal fats. In fact, through hydrogenation, oils such as soybean, safflower and cottonseed oils, which are rich in unsaturated fatty acids, are converted into semi-solid fats (see above).
The first hydrogenated oil was cottonseed oil in USA in 1911 to produce vegetable shortening.
In the 1930’s, partial hydrogenation became popular with the development of margarine.
Currently, per year in USA, 6-8 billion tons of hydrogenated vegetable oil are produced.

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Ruminant trans fatty acids

Ruminant trans fats are produced by bacteria in the rumen of the animals, for example cows, sheep and goats, using as a substrate a proportion of the relatively small amounts of unsaturated fatty acids present in their feedstuffs, that is, feed, plants and herbs. And, considering an animal that lives at least a year, and has the opportunity to graze and/or eat hay, there is a season variability in unsaturated fatty acids intake, and trans fats produced. In fact, in summer and spring, pasture plants and herbs may contain more unsaturated fatty acids than the winter feed supply.

Trans fatty acids
Fig. 4 – The sn Positions of Triglycerides

Then, TFA are present at low levels in meat and full fat dairy products, typically <5% of total fatty acids, and are located in the sn-1 and sn-3 positions of the triacylglycerols, whereas in margarines and other industrially hydrogenated products they appear to be concentrated in the sn-2 position of the triacylglycerols.
Ruminant trans fatty acids are mainly monounsaturated fatty acids, with 16 to 18 carbon atoms, and constitute a small percentage of the trans fatty acids in the diet (see below).

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Isomers of dietary trans fatty acids

Trans fatty acids
Fig. 5 – trans-C18:1 Isomers

The most important cluster of trans fatty acids is trans-C18:1 isomers, that is, fatty acids containing 18 carbon atoms plus one double bond, whose position varies between Δ6 and Δ16 carbon atoms. In both sources, the most common isomers are those with double bonds between positions Δ9 and Δ11.
However, even if these molecules are present both in industrial and ruminant TFA, there is a considerable quantitative difference. For example, vaccenic acid (C18:1 Δ11t) represents over 60 percent of the trans-C18:1 isomers in ruminant trans fatty acids, whereas in industrial ones elaidic acid (C18:1Δ9t) comprises 15-20 percent and C18:1 Δ10t and vaccenic acid over 20 percent each others.

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Trans fatty acids: effects on human health

Ruminant trans fatty acids, in amounts actually consumed in diets, are not harmful for human health (see below).
Conversely, consumption of industrial trans fats has neither apparent benefit nor intrinsic value, above their caloric contribution, and, from human health standpoint they are only harmful, having adverse effects on:

  • serum lipid levels;
  • endothelial cells;
  • systemic inflammation;
  • other risk factors for cardiovascular disease;

Moreover, they are positively associated with the risk of coronary heart disease (CHD), and sudden death from cardiac causes and diabetes.
Note: further in the text, we will refer to industrial trans fatty acids as trans fats or trans fatty acids.

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Trans fatty acids: effects at plasmatic level

Low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) plasma levels are well-documented risk markers for the development of coronary heart disease (CHD).

  • High LDL-C levels are associated with an increased incidence of ischemic heart disease.
  • High HDL-C levels are associated with a reduced incidence of the risk.

For this reason, the ratio between total cholesterol level and HDL-C is often used as a combined risk marker for these two components in relation to the development of heart disease: the higher the ratio, the higher the risk.
TFA, as previously said, have adverse effects on serum lipids.
These effects have been evaluated in numerous controlled dietary trials by isocaloric replacement of saturated fatty acids or cisunsaturated fatty acids with trans fats. It was demonstrated that such replacement:

  • raises LDL-C levels;
  • lowers HDL-C levels, in contrast to saturated fatty acids that increase HDL-C levels when used as replacement in similar study;
  • increases the ratio of total cholesterol to HDL-C, approximately twice that for saturated fatty acids, and, on the basis of this effect alone, trans fatty acids has been estimated to cause about 6% of coronary events in the USA.

Furthermore, trans fats:

  • produce a deleterious increase in small, dense LDL-C subfractions, that is associated with a marked increased in the risk of CHD, even in the presence of relatively normal LDL-C;
  • increase the blood levels of triglycerides, and this is an independent risk factor for CHD;
  • increase levels of Lp(a)lipoprotein, another important coronary risk factor.

But on 2004 prospective studies have shown that the relation between the intake of trans fatty acids and the incidence of CHD is greater than that predicted by changes in serum lipid levels alone. This suggests that trans fats influence other risk factors for CHD, such as inflammation and endothelial-cell dysfunction.

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Trans fatty acids, inflammation and endothelial-cell dysfunction

The role of inflammation in atherosclerosis, and consequently in CHD, is burgeoned in the last decade.
Interleukin-6, C-reactive protein (CRP), and an increased activity of tumor necrosis factor (TNF) system are markers of inflammation.
In women greater intake of trans fatty acids is associated with increased activity of TNF system, and in those with a higher body mass index with increased levels of interleukin-6 and CRP. For example, the difference in CRP seen with an average intake of trans fats of 2.1% of the total daily energy intake, as compared with 0.9%, correspond to an increased risk of cardiovascular disease of 30%. Similar results have been reported in patients with established heart disease, in randomized, controlled trials, in in vitro studies, and in studies in which it has been analyzed membrane levels of trans fatty acids, a biomarker of their dietary intake.
So, trans fats promote inflammation, and their inflammatory effects may account at least in part for their effects on CHD that, as seen above, are greater than would be predicted by effects on serum lipoproteins alone.
Attention: the presence of inflammation is an independent risk factor not only for CHD but also for insulin resistance, diabetes, dyslipidemia, and heart failure.

Another site of action of TFA may be endothelial function.
Several studies have suggested the association between greater intake of trans fats and increased levels of circulating biomarkers of endothelial dysfunction, such as E-selectin, sICAM-1, and sVCAM-1.

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Other effects of trans fatty acids

In vitro studies have demonstrate that trans fats affect lipid metabolism through several pathways.

  • They alter secretion, lipid composition, and size of apolipoprotein B-100 (apo B-100).
  • They increase cellular accumulation and secretion of free cholesterol and cholesterol esters by hepatocytes.
  • They alter expression in adipocytes of genes for peroxisome proliferator-activated receptor-γ (PPAR- γ), lipoprotein lipase, and resistin, proteins having a central roles in the metabolism of fatty acids and glucose.

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Industrial trans fatty acids and CHD

Industrial trans fats are independent cardiovascular risk factor.
Since the early 1990s attention has been focused on the effect of trans fatty acids on plasma lipid and lipoprotein concentrations (see above).
Furthermore, four major prospective studies covering about 140,000 subjects, monitored for 6-14 years, have all found positive epidemiological evidence relating their levels in the diet, assessed with the aid of a detailed questionnaire on the composition of the diet, to the risk of CHD. These four studies are:

  • “The Health Professionals Follow-up study” (2005);
  • “The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study” (1997);
  • “The Nurses’ Health Study” (2005);
  • “The Zutphen Elderly Study” (2001).

These studies cover such different populations that the results very probably hold true for the populations as a whole.
A meta-analysis of these studies have shown that a 2% increase in energy intake from industrial TFA was associated with a 23% increase in the incidence of CHD. The relative risk of heart disease was 1.36 in “The Health Professionals Follow-up Study”, 1.14 in “The Alpha-Tocopherol Beta-Carotene Cancer Prevention Study”; 1.93 (1.43-2.61) in “The Nurses’ Health Study”, and 1.28 (1.01-1.61) in “The Zutphen Elderly Study”.
So,  there is a substantially increased risk even at low levels of intake: 2% of total energy intake, for a 2,000 Kcal diet is 40 Kcal or about 4-5 g of fat corresponding to a teaspoonful of fat!
Moreover, in three of the studies, the association between the intake of industrial trans fats and the risk of CHD was stronger than a corresponding association between the intake of saturated fatty acids and the risk of heart disease. In “The Zutphen Elderly Study”, this association was not investigated.
Because of the adverse effects of industrial trans fatty acids, for the same authors are unethical conducting randomized long-term trials to test their effects on the incidence of CHD.
So, avoidance of industrial trans fats, or a consumption of less 0.5% of total daily energy intake is necessary to avoid their adverse effects, far stronger on average than those of food contaminants or pesticide residues.

Further evidence
A study conducted in an Australia population with a first heart attack and no preceding history of CHD or hyperlipidemia has showed a positive association between levels of trans fatty acids in adipose tissue and the risk of nonfatal myocardial infarction.
It was shown that adipose tissue C18:1Δ7t, found in both animal and vegetable fats, was an independent predictor of a first myocardial infarction, that is, its adipose tissue level is still a predictor for heart disease after adjustment for total cholesterol. Again, it appears that only a minor part of the negative effects of trans fats occurs via plasma lipoproteins.
During the course of this study, mid-1996, TFA were eliminated from margarines sold in Australia (see below). This was a unique opportunity to investigate the temporal relationship between trans fat intake and their adipose tissue levels. It was demonstrated that trans fats disappear from adipose tissue of both case-patients and controls with a rate about 15% of total trans fats/y.
Another study conduct in Costa Rica have found a positive association between myocardial infarction and trans fatty acids.
Interestingly, in a larger, community-based case-control study, levels of trans fats in red blood cell membranes were associated, after adjustment for other risk factors, with an increase in the risk of sudden cardiac death. Moreover, the increased risk appeared to be related to trans-C18:2 levels, that were associated with a tripling of the risk, but not with cell membrane levels of trans-C18:1,  the major trans fatty acids in foods (see above).

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Trans fatty acids and diabetes (H3)

In a prospective study covering 84,204 female nurses, from “The Nurses’ Health Study”, aged 34–59 y, analyzed from the 1980 to 1996, with no cancer, diabetes, or cardiovascular disease at base line, the intake of trans fatty acids was significantly related to the risk of developing type 2 diabetes. And, after adjustment for other risk factors trans fat intake was positively associated with the incidence of diabetes with a risk up to 39% greater.
Data from controlled intervention studies showed that TFA could impair insulin sensitivity in subjects with insulin resistance and type 2 diabetes (saturated fatty acids do the analogous response, with no significant difference between TFA and them) more than unsaturated fatty acids, in particular the isomer of conjugated linoleic acid (CLA) trans-10, cis-12-CLA. Be careful because some dietary supplements contain CLA isomers and may be diabetogenic and proatherogenic in insulin-resistant subjects.

No significant effect was seen in insulin sensitivity of lean, healthy subjects.

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Ruminant trans fatty acids and the risk of CHD

Four prospective studies have evaluated the relation between the intake of ruminant trans fatty acids and the risk of CHD: no significant association was identified.
In another study published on 2008 was analyzed data from four Danish cohort studies that cover 3,686 adults enrolled between 1974 and 1993, and followed for a median of 18 years. In Denmark, consumption of dairy products is relatively high and the range of ruminant trans fat intake is relatively broad, up to 1.1% of energy. Conversely, in the other countries, ruminant trans fatty acid consumption for most people is substantially lower than 1% of energy, in USA about 0.5% of energy. After adjustment for other risk factors, no significant associations between ruminant TFA consumption and incidence of CHD were found, confirming, in a population with relatively high intake of ruminant trans fatty acids, conclusions of four previous prospective studies.
So ruminant trans fats, in amounts actually consumed in diets, do not raise CHD risk.
The absence of risk of CHD with trans fats from ruminants as compared with industrial trans fatty acids  may be due to a lower intake. In the USA, greater part of trans fats have industrial origin (see above); moreover trans fat levels in milk and meats are relatively low, 1 to 8% of total fats.
The absence of a higher risk of CHD may be due also to the presence of different isomers. Ruminant and industrial sources share many common isomers, but there are some quantitative difference (see fig. 4):

  • vaccenic acid level is higher in ruminant fats, 30-50% of trans isomers;
  • trans-C18:2 isomers, present in deodorized and fried vegetable oils, as well as in some partially hydrogenated vegetable oils, are not present in appreciable amounts in ruminants fats.

Finally other, still unknown, potentially protective factors could outweigh harmful effects of ruminant trans fats.

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Trans fatty acids: legislation regulating their content

USA
Until 1985 no adverse effects of trans fatty acids on human health was demonstrated, and in 1975 a Procter & Gamble study showed no effect of trans fats on cholesterol.
Their use in fast food preparation grew up from 1980’s, when the role of dietary saturated fats in increasing cardiac risk began clear. Then, it was led a successful campaign to get McDonald’s to switch from beef tallow to vegetable oil for frying its French fries. Meanwhile, studies began to raise concerns about their effects on health. On 1985 Food and Drug Administration (FDA) concluded that TFA and oleic acid affected serum cholesterol level similarly, but from the second half of 1985 their harmful began clear, and the final proof came from both controlled feeding trials and prospective epidemiologic studies.
On 2003 FDA ruled that food labels, for conventional foods and supplements, show their content beginning January 1, 2006. Notably, this ruling was the first substantive change to food labeling since the requirement for per-serving food labels information was added in 1990.
On 2005 the US Department of Agriculture made a minimized intake of trans fatty acids a key recommendation of the new food-pyramid guidelines.
On 2006 American Heart Association recommended to limit their intake to 1% of daily calorie consumption, and suggested food manufacturers and restaurants switch to other fats.
On 2006 New York City Board of Health announced trans fat ban in its 40,000 restaurants within July 1, 2008, followed by the state of California in 2010-2011.

Australia
After June 1996 they were eliminated from margarine sold in Australia, that before contributed about 50% of their dietary intake.

Europe
On March 11, 2003 the Danish government, after a debate started in 1994 and two new reports in 2001 and 2003, decided to phase out the use of industrial trans fats in food before the end of 2003. Two years later, however, the European Commission (EC) asked Denmark to withdraw this law, which was not accepted on the European Community level, unfortunately. However, in 2007, EC decided to closes its infringement procedure against Denmark because of increasing scientific evidence of the danger of this type of fatty acids.
The Danish example was followed by Austria and Switzerland in 2009, Iceland, Norway, and Hungary in 2011, and most recently, Estonia and Georgia in 2014. So, about 10% of the European Union population, about 500 million people, lives in countries where it is illegal to sell food high in industrial trans fats.
Governments of other European Union countries instead rely on the willingness of food producers to reduce trans fatty acid content in their products. This strategy has proved effective only for Western European countries (see below).

Canada
Canada is considering legislation to eliminate them from food supplies, and, in 2005, ruled that pre-packaged food labels show their content.

Trans fatty acids
Fig. 6 – Cookies High in Industrial Trans Fats

Therefore, with the exception of the countries where the use of trans fats in the food industry was banned, the only way to reduce their intake in the other countries is consumer’s decision to choose foods free in such fatty acids, avoiding those known containing them, and always reading nutrition facts and ingredients because they may come from margarine, vegetable oil and frying. Indeed, for example in the USA, the producers of foods that contain less than 0.5 g of industrial trans fatty acids per serving can list their content as 0 on the packaging. This content is low but if a consumer eats multiple servings, he consumes substantial amount of them.
Be careful: food labels are not obligatory in restaurants, bakeries, and many other retail food outlets.

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Trans fatty acids and food reformulation

Public health organizations, including the World Health Organization in September 2006, have recommended reducing the consumption of industrial trans fatty acids; only in USA the near elimination of these fatty acids might avoid between 72,000 and 280,000 of the 1.2 million of CHD events every year.
Food manufacturers and restaurants may reduce industrial TFA use choosing alternatives to partially hydrogenated oils.
In Denmark, their elimination (see above) from vegetable oils did not increase consumption of saturated fatty acids because they were mostly replaced with cisunsaturated fatty acids. Moreover, there were no noticeable effects for the consumer: neither increase in the cost nor reduction in availability and quality of foods.
In 2009, Stender et al. have shown that industrial trans fatty acids in food such as French fries, cookies, cakes, and microwave-oven popcorn purchased in USA, South Africa, and many European Country can be replaced, at similar prices, with a mixture of saturated, monounsaturated, and polyunsaturated fatty acids. Such substitution has even greater nutritional benefit than one-to-one substitution of industrial trans fats with saturated fatty acids alone. However, be careful because only in French fries with low industrial trans fats the percentage of saturate fatty acids remains constant, whereas in cookies and cakes is in average +33 percentage points and microwave-oven popcorn +24 percentage points: saturated fatty acids are less dangerous than industrial trans fats but more than mono- and polyunsaturated fatty acids.
The same research group, analyzing some popular foods in Europe, purchased in supermarkets, even of the same supermarket chain, and fast food, namely, McDonald’s and Kentucky Fried Chicken (KFC), from 2005 to 2014, showed that their TFA content was reduced or even absent in several Western European countries while remaining high in Eastern and Southeastern Europe.
In 2010 Mozaffarian et al. evaluated  the levels of industrial trans fats and saturated fatty acids in major brand-name U.S. supermarket and restaurant foods after reformulation to reduce industrial trans fatty acid content, in two time: from 1993 through 2006 and from 2008 through 2009. They found a generally reduction in industrial trans fat content without any substantial or equivalent increase in saturated fatty acid content.

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Foods high in trans fatty acids: examples and values

Many foods high in trans fats are popularly consumed worldwide.
In USA greater part of these fatty acids comes from partially hydrogenated vegetable oils, with an average consumption from this source that has been constant since the 1960′s.
It should be noted that the following trans fatty acid values must be interpreted with caution because, as previously said, many fast food establishments, restaurants and industries may have changed, or had to change the type of fat used for frying and cooking since the analysis were done.
The reported values, unless otherwise specified, refer to percentage in trans fatty acids/ 100 g of fatty acids.

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Margarine

Trans fatty acids
Fig. 7 – Margarine

Among foods with trans fats, stick or hard margarine had the highest percentage of them, but levels of these fatty acids have declined as improved technology allowed the production of softer margarines which have become popular. But there are difference in trans fatty acid content of margarine from different countries. Below some examples.

  • The highest content, 13-16.5%, is found in soft margarine from Iceland, Norway, and the UK.
  • Less content is found in Italy, Germany, Finland, and Greece, 5.1%, 4.8%, 3.2%, and 2.9% respectively).
  • In Portugal, The Netherlands, Belgium, Denmark, France, Spain, and Sweden margarine trans fat content is less than 2%.

USA and Canada lag behind Europe, but in the USA, with the advent of trans fat labeling of foods and the greater knowledge of the risk associated with their consumption by the buyers, change is occurring. For this reason, at now, in the USA margarine is considered to be a minor contributor to the intake of TFA, whereas the major sources are commercially baked and fast food products like cake, cookies, wafer, snack crackers, chicken nuggets, French fries or microwave-oven popcorn (see below).

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Vegetable shortenings

Trans fatty acid content of vegetable shortenings ranges from 6% to 50%, and varies in different country: in Germany, Austria and New Zealand it is less than France or USA.
However, like margarines, their trans fat content is decreasing. In Germany it decreased from 12% in 1994 to 6% in 1999, in Denmark is 7% (1996) while in New Zealand is about 6% (1997).

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Vegetable oils

At now, non-hydrogenated vegetable oils for salad and cooking contain no or only small amounts of trans fats.
Processing of these oils can produce minimal level of them, ranged from 0.05g/100 food for extra virgin oil to 2.42 g/100 g food for canola oil. So, their contribution to trans fat content of the current food supply is very little.
One exception is represented by Pakistani hydrogenated vegetable oils whose TFA content ranges from 14% to 34%.

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Prepared soups

Among foods with trans fats, prepared soups contain significant amount of them, ranging from 10% of beef bouillon to 35% of onion cream. So, they contribute great amount of such fatty acids to the diet if frequently consumed.

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Processed foods

Thanks to their properties (see above), trans fatty acids are used in many processed foods as cookies, cakes, croissants, pastries and other baked goods. And, baked goods are the greatest source of these fats in the North American diet. Of course, their trans fat content depends on the type of fat used in processing.

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Sauces

Mayonnaise, salad dressings and other sauces contain only small or no-amounts of trans fats.

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Human milk and infant foods

Trans fat content of human milk reflects the trans fat content of maternal diet in the previous day, is comprised between 1 and 7%, and is decreasing from 7.1% in 1998 to 4.6% in 2005/2006.
Infant formulas have trans fat values on average 0.1%-4.5%, with a brand up to 15.7%.
Baby foods contain greater than 5% of trans fats.

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Fast foods and restaurant’s foods

Trans fatty acids
Fig. 8 – French Fries

Vegetable shortenings high in trans fats are used as frying fats, so fast foods and many restaurant’s foods may contain relatively large amounts of them. Foods are fried pies, French fries, chicken nuggets, hamburgers, fried fish as well as fried chicken.
In articles published by Stender et al. from 2006 to 2009, it is showed that for French fries and chicken nuggets their content varies largely from nation to nation, but also within the same fast food chain in the same country, and even in the same city, because of the cooking oil used. For example, oil used in USA and Peru outlets of a famous fast food chain contained 23-24% of trans fats, whereas oil used in many European countries of the same fast food chain contained about 10%, with some countries, such as Denmark, as low as 5% and 1%.
And, considering a meal of French fries and chicken nuggets, in serving size of 171 and 160 g respectively, purchased at McDonald‘s in New York City, it contained over 10 g of TFA, while if purchased at KFC in Hungary they were almost 25 g.
Below, again from the work of Stender et al. it can see a cross-country comparison of trans fat contents of chicken nuggets and French fries purchased at McDonald ‘s or KFC.

Chicken nuggets and French fries from McDonald’s:

  • less than 1 g only if the meals were purchased in Denmark;
  • 1-5 g in Portugal, the Netherlands, Russia, Czech Republic, or Spain;
  • 5-10 g in the United States, Peru, UK, South Africa, Poland, Finland, France, Italy, Norway, Spain, Sweden, Germany, or Hungary.

Chicken and French fries from KFC:

  • less than 2 g if the meals were purchased UK (Aberdeen), Denmark, Russia, or Germany (Wiesbaden);
  • 2-5 in Germany (Hamburg), France, UK (London or Glasgow), Spain, or Portugal;
  • 5-10 in the Bahamas, South Africa, or USA;
  • 10-25 g in Hungary, Poland, Peru, or Czech Republic.

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