Category Archives: Lipids

Bile salts: definition, functions, enterohepatic circulation, synthesis

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

References

Chiang J.Y.L. Bile acids: regulation of synthesis. J Lipid Res 2009;50(10):1955-66 [PDF]

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 [Article]

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 [Article]

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 [Abstract]

Chemical composition of olive oil

Olive oil constituents

Olive Oil
Fig. 1 – EVOO

From a chemical point of view, we can identify in the olive oil two fractions, depending on the behavior in the presence of heating and strong alkaline solutions (concentrated solutions of KOH or NaOH):

  • the saponifiable fraction, which represents 98-99% of the total weight, is composed of substances that form soaps in the above conditions;
  • the unsaponifiable fraction, which represents the remaining 1-2% of the total weight, is composed of substances that fail to form soaps in the above conditions.

Saponifiable fraction of olive oil

It is composed of saturated and unsaturated fatty acids, esterified almost entirely to glycerol to form triglycerides (or triacylglycerols). To a much lesser extent, diglycerides (or diacylglycerols), monoglycerides (monoacylglycerols), and free fatty acids are also found.
Unsaturated fatty acids make up 75 to 85% of the total fatty acids. Oleic (O) and linoleic (L) acids are the most abundant ones; palmitoleic, eptadecenoic, gadoleic and alpha-linolenic (Ln) acids are present in lower/trace amounts.

Oleic Acid
Fig. 2 – IOOC and Fatty Acids

Oleic acid is the major fatty acid in olive oils. According to the rules laid down by the International Olive Oil Council (IOOC), its concentration must range from 55% to 83% of total fatty acids.
Linoleic acid is the most abundant polyunsaturated fatty acid in olive oil; its concentration must vary between 2.5% and 21% (IOOC). Because of its high degree of unsaturation, it is subject to oxidation; this means that an oil high in linoleic acid becomes rancid easily, and thus it may be stored for a shorter time.
In a Mediterranean-type diet, olive oil is the main source of fat: therefore, oleic acid, among monounsaturated fatty acids, and linoleic acid, among polyunsaturated fatty acids, are the most abundant fatty acids.
alpha-Linolenic acid must be present in very low amount, according to the IOOC standards ≤1%. It is an omega-3 polyunsaturated fatty acid, which may have health benefits. However, because of to its high degree of unsaturation (higher than that of linoleic acid), it is very susceptible to oxidation, and therefore it promotes rancidity of the olive oil that contains it.
Saturated fatty acids make up 15 to 25% of the total fatty acids.
Palmitic (P) (7.5-20%) and stearic (S) acids (0.5-5%) are the most abundant saturated fatty acids; myristic, heptadecanoic, arachidic, behenic and lignoceric acids may be present in trace amounts.

The presence of fatty acids that should be absent or present in amounts different than those found is a marker of adulteration with other vegetable oils. On this regard, particular attention is paid to myristic, arachidic, behenic, lignoceric, gadoleic and alpha-linolenic acids, whose limits are set by IOOC.

Fatty acid composition is influenced by several factors.

  • The climate.
  • The latitude.
  • The zone of production.
    Italian, Spanish and Greek olive oils are high in oleic acid and low in palmitic and linoleic acids, while Tunisian olive oils are high in palmitic and linoleic acids but lower in oleic acid. Therefore, oils can be divided into two groups:

one rich in oleic acid and low in palmitic and linoleic acids;
the other high in palmitic and linoleic acids and low in oleic acid.

  • The cultivar.
  • The degree of olive ripeness at the time of oil extraction.
    It should be noted that oleic acid is formed first in the fruit, and data seem to indicate a competitive relationship between oleic acid and palmitic, palmitoleic, and linoleic acids.

Triglycerides of olive oil

Olive Oil
Fig. 3 – The sn Positions of Triglycerides

As previously said, fatty acids in olive oil are almost entirely present as triglycerides.
In small percentage, they are also present as diglycerides, monoglycerides, and in free form.
During triglyceride biosynthesis, thanks to the presence of specific enzymes, only about 2% of glycerol binds palmitic acid in the sn-2 position (also the percentage of stearic acid in the sn-2 position is very low); for the most part, the sn-2 position is occupied by oleic acid.
On the contrary, if we consider oils that have undergone a nonenzymatic esterification, the percentage of palmitic acid in the sn-2 position increases significantly.
Note: sn = stereospecific numbering

Among triglycerides present in significant proportions in olive oil, there are:

  • OOO: 40-59%;
  • POO: 12-20%;
  • OOL: 12.5-20%;
  • POL:  5.5-7%;
  • SOO: 3- 7%.

POP, POS, OLnL, OLnO, PLL, PLnO are present in smaller amounts.
Trilinolein (LLL) is a triglyceride that contains three molecules of linoleic acid. Its low content is an indicator of an oil of good quality.
Triglycerides containing three saturated fatty acids or three molecules of alpha-linolenic acid have not been reported.

Diglycerides and monoglycerides of olive oil

Their presence is due to an incomplete synthesis and/or a partial hydrolysis of triglycerides.
The content of diglycerides in virgin olive oil ranges from 1% to 2.8%. 1,2-Diglycerides prevail in fresh olive oil, representing over 80% of the diglycerides. During oil storage, isomerization occurs with a progressive increase of the more stable 1-3 isomers, which after about 10 months become the major isomers.
Therefore, the ratio 1,2/1,3-diglycerides may be used as an indicator of the age of the oil.
Monoglycerides are present in amounts lower than diglycerides, <0.25%, with 1-monoglycerides far more abundant than 2-monoglycerides.

Unsaponifiable fractions of olive oil

It is composed of a large number of different molecules, very important from a nutritional point of view, as they contribute significantly to the health effects of olive oil.
Furthermore, they are responsible for the stability and the taste of olive oil, and are also used to detect adulteration with other vegetable oils.
This fraction includes tocopherols, sterols, polyphenols, pigments, hydrocarbons, aromatic and aliphatic alcohol, triterpene acids, waxes, and minor constituents.
Their content is influenced by factors similar to those seen for fatty acid composition, such as:

  • the cultivar;
  • the degree of ripeness of the olive;
  • the zone of production;
  • the crop year and olive harvesting practices;
  • the storage time of olives;
  • the oil extraction process;
  • the storage conditions of the oil.

It should be noted that many of these compounds are not present in refined olive oils, as they are removed during the refining processes.

Polyphenols

They make up 18 to 37% of the unsaponifiable fraction.
They are a very heterogeneous group of molecules with nutritional and organoleptic properties  (for example, oleuropein and hydroxytyrosol give oil its bitter and pungent taste).
For a more extensive discussion, see: ” Polyphenols in olive oil: variability and composition.”

Hydrocarbons

Olive Oil
Fig. 4 – Squalene

They make up 30 to 50% of the unsaponifiable fraction.
Squalene and beta-carotene are the main molecules.
Squalene, isolated for the first time from shark liver, is the major constituent of the unsaponifiable fraction, and constitutes more than 90% of the hydrocarbons. Its concentration ranges from 200 to 7500 mg/kg of olive oil.
It is an intermediate in the biosynthesis of the four-ring structure of steroids, and it seems to be responsible of several health effects of olive oil.
In the hydrocarbon fraction of virgin olive oil, n-paraffins, diterpene and triterpene hydrocarbons, isoprenoidal polyolefins are also found.
Beta-carotene acts both as antioxidant, protecting oil during storage, and as dye (see below).

Sterols

They are important lipids of olive oil, and are:

  • linked to many health benefits for consumers;
  • important to the quality of the oil;
  • widely used for checking its genuineness.
    On this regard, it is to underline that sterols are species-specific molecules; for example, the presence of high concentrations of brassicasterol, a sterol typically found in Brassicaceae (Cruciferae) family, such as rapeseed, indicates adulteration of olive oil with canola oil.

Four classes of sterols are present in olive oil: common sterols, 4-methylsterols, triterpene alcohols, and triterpene dialcohols. Their content ranges from 1000 mg/kg, the minimum value required by the IOOC standard, to 2000 mg/kg. The lowest values are found in refined oils because of the refining processes may cause losses up to 25%.

Common sterols or 4α-desmethylsterols
Olive Oil
Fig. 5 – beta-Sitosterol

Common sterols are present mainly in the free and esterified form; however they have been also found as lipoproteins and sterylglucosides.
The main molecules are beta-sitosterol, which makes up 75 to 90% of the total sterol, Δ5-avenasterol, 5 to  20%, and campesterol, 4%. Other components found in lower amounts or traces are, for example, stigmasterol, 2%, cholesterol, brassicasterol, and ergosterol.

4-methylsterols

They are intermediates in the biosynthesis of sterols, and are present both in the free and esterified form. They are present in small amounts, much lower than those of common sterols and triterpene alcohols, varying between 50 and 360 mg/kg. The main molecules are obtusifoliol, cycloeucalenol, citrostadienol, and gramisterol.

Triterpene alcohols or 4,4-dimethylsterols

They are a complex class of sterols, present both in the free and esterified form. They are found in amounts ranging from 350 to 1500 mg/kg.
The main components are beta-amyrin, 24-methylenecycloartanol, cycloartenol, and butyrospermol; other molecules present in lower/trace amounts are, for example, cyclosadol, cyclobranol, germanicol, and dammaradienol.

Triterpene dialcohols

The main triterpene dialcohols found in olive oil are erythrodiol and uvaol.
Erythrodiol is present both in the free and esterified form; in virgin olive oil, its level varies between 19 and 69 mg/kg, and the free form is generally lower than 50 mg/kg.

Tocopherols

They make up 2 to 3% of the unsaponifiable fraction, and include vitamin E.
Of the eight E-vitamers, alpha-tocopherol represents about 90% of tocopherols in virgin olive oil. It is present in the free form and in very variable amount, but on average higher than 100 mg/kg of olive oil. Thanks to its in vivo antioxidant properties, its presence is a protective factor for health. Alpha-tocopherol concentration seems to be related to the high levels of chlorophylls and to the concomitant requirement for deactivation of singlet oxygen.
Beta-tocopherol, delta-tocopherol, and gamma-tocopherol are usually present in low amounts.

Pigments

In this group we find chlorophylls and carotenoids.
In olive oil, chlorophylls are present as phaeophytins, mainly  phaeophytin a (i.e. a chlorophyll from which magnesium has been removed and substituted with two hydrogen ions), and confer the characteristic green color to olive oil. They are photosensitizer molecules that contribute to the photooxidation of olive oil itself.
Beta-carotene and lutein are the main carotenoids in olive oil. Several xanthophylls are also present, such as antheraxanthin, beta-cryptoxanthin, luteoxanthin, mutatoxanthin, neoxanthin, and violaxanthin.
Olive oil’s color is the result of the presence of chlorophylls and carotenoids and of their green and yellow hues. Their presence is closely related.

 Triterpene acids

They are important components of the olive, and are present in trace amounts in the oil.
Oleanolic and maslinic acids are the main triterpene acids in virgin olive oil: they are present in the olive husk, from which they are extracted in small amount during processing.

Aliphatic and aromatic alcohols

Fatty alcohols and diterpene alcohols are the most important ones.
Aliphatic alcohols have a number of carbon atoms between 20 and 30, and are located mostly inside the olive stones, from where they are partially extracted by milling.

Fatty alcohols

They are linear saturated alcohols with more than 16 carbon atoms.
They are found in the free and esterified form and are present, in virgin olive oil, in amount not generally higher than 250 mg/kg.
Docosanol (C22), tetracosanol (C24), hexacosanol (C26), and octacosanol (C28) are the main fatty alcohols in olive oil, with tetracosanol and hexacosanol present in larger amounts.
Waxes, which are minor constituents of olive oil, are esters of fatty alcohols with fatty acids, mainly of palmitic acid and oleic acid. They can be used as a criterion to discriminate between different types of oils; for example, they must be present in virgin and extra virgin olive oil at levels <150 mg/kg, according to the IOOC standards.

 Diterpene alcohols

Geranylgeraniol and phytol are two acyclic diterpene alcohols, present in the free and esterified form. Among esters present in the wax fraction of extra virgin olive oil, oleate, eicosenoate , eicosanoate, docosanoate, and tetracosanoate have been found, mainly as phytyl derivatives.

Volatile compounds

More than 280 volatile compounds have been identified in olive oil, such as hydrocarbons, the most abundant fraction, alcohols, aldehydes, ketones, esters, acids, ethers and many others. However, only about 70 of them are present at levels higher than the perception threshold beyond which they may contribute to the aroma of virgin olive oil.

Minor components

Phospholipids are found among the minor components of olive oil; the main ones are phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol.
In the unfiltered oils, trace amounts of proteins may be found.

 References

Gunstone F.D. Vegetable oils in food technology: composition, properties and uses. 2th. Edition. Wiley J. & Sons, Inc., Publication, 2011

Pasqualone A., Sikorska E., Gomes T. Influence of the exposure to light on extra virgin olive oil quality during storage. Eur Food Res Technol 2005;221:92-8 [Abstract]

Servili M., Sordini B., Esposto S., Urbani S., Veneziani G., Di Maio I., Selvaggini R. and Taticchi A. Biological activities of phenolic compounds of extra virgin olive oil. Antioxidants 2014;3:1-23 [Abstract]

Skin, blood pressure, rheumatoid arthritis and gamma-linolenic acid

Healthy skin and gamma-linolenic acid

gamma-Linolenic acid (GLA), an omega-6 essential fatty acid, like its precursor linoleic acid (the most abundant polyunsaturated fatty acid in human skin epidermis, where it’s involved in the maintenance of the epidermal water barrier), plays important roles in the physiology and pathophysiology of the skin.
Studies conducted on humans revealed that gamma-linolenic acid:

  • improves skin moisture, firmness, roughness;
  • decreases transepidermal water loss (one of the abnormalities of the skin in essential fatty acid deficiency animals).
Skin and gamma-Linolenic Acid
Fig. 1 – GLA

Using guinea pig skin epidermis as a model of human epidermis (they are functionally similar), it was demonstrated that supplementation of animals with gamma-linolenic acid-rich foods results in a major production of PGE1 and 15-HETrE in the skin (as previously demonstrated in in vitro experiments).
Because these molecules have both anti-inflammatory/anti-proliferative properties supplementation of diet with gamma-linolenic acid acid-rich foods may be an adjuncts to standard therapy for inflammatory/proliferative skin disorders.

Supplemental sources of GLA

The main supplemental sources of gamma-linolenic acid are oils of the seeds of:

  • borage (20%-27% of the total fatty acids);
  • black currant (from 15% to 19% of the total fatty acids);
  • evening primrose (from 7% to 14% of the total fatty acids), and

Role of gamma-linolenic acid in lowering blood pressure

The relationship between dietary fatty acid intake and blood pressure mainly comes from studies conducted on genetically modified rats that spontaneously develops hypertension (a commonly used animal model for human hypertension).
In these studies many membrane abnormalities were seen so hypertension in rat model may be related to change in polyunsaturated fatty acid metabolism at cell membrane level.
About polyunsaturated fatty acids, several research teams have reported that gamma-linolenic acid reduces blood pressure in normal and genetically modified rats (greater effect) and it was purported by interfering with Renin-Angiotensin System (that promote vascular resistance and renal retention) altering the properties of the vascular smooth muscle cell membrane and so interfering with the action of angiotensin II.
Another possible mechanism of action of gamma-linolenic acid to lower blood pressure could be by its metabolite dihomo-gamma-linolenic acid: it may be incorporated in vascular smooth muscle cell membrane phospholipids, then released by the action of phospholipase A2 and transformed by COX-1 in PGE1 that induces vascular smooth muscle relaxation.

Role gamma-linolenic acid in treatment of rheumatoid arthritis

Borago officinalis
Fig. 2 – Borage

In a study conducted by Leventhal et al. on 1993 it was demonstrated the dietary intake of higher concentration of borage oil (about 1400 mg of gamma-linolenic acid/day) for 24 weeks resulted in clinically significant reductions in signs and symptoms of rheumatoid arthritis activity.
In a subsequent study by Zurier et al. on 1996 the dietary intake of an higher dose (about 2.8 g/day gamma-linolenic acid) for 6 months reduced, in a clinically relevant manner, signs and symptoms of the disease activity; patients who remained for 1 year on the 2.8 g/day dietary gamma-linolenic acid exhibited continued improvement in symptoms (the use of gamma-linolenic acid also at the above higher dose is well tolerated, with minimal deleterious effects). These data underscore that the daily amount and the duration of gamma-linolenic acid dietary intake do correlate with the clinical efficacy.

References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Fan Y.Y. and Chapkin R.S. Importance of dietary gamma-linolenic acid in human health and nutrition. J Nutr 1998;128:1411-14 [Abstract]

Leventhal L.J., Boyce E.G. and Zurier R.B. Treatment of rheumatoid arthritis with gammalinolenic acid. Ann Intern Med 1993 119:867-73 [Abstract]

Miller C.C. and Ziboh V.A. Gammalinolenic acid-enriched diet alters cutaneous eicosanoids. Biochem Biophys Res Commun 1988 154:967-74 [Abstract]

Zurier R.B., Rossetti R.G., Jacobson E.W., DeMarco D.M., Liu N.Y., Temming J.E., White B.M. and Laposata M. Gamma-linolenic acid treatment of rheumatoid arthritis. A randomized, placebocontrolled trial. Arthritis Rheum 1996 39:1808-17 [Abstract]

Examples of foods with trans fats

Foods high in trans fats

Many foods with trans fats are popularly consumed worldwide.
In USA greater part of these trans fats come from partially hydrogenated vegetable oils (about 80%), and the average consumption of trans fats from this source has been constant since the 1960′s.

It should be noted that trans fat values must be interpreted with caution because many fast food establishments, restaurants and industries may have changed the type of fat used for frying and cooking since the analysis were done (e.g. on July 1, 2008 in New York trans fats are banned in its 40.000 restaurants).

Foods with trans fats: margarine

Foods with Trans Fats
Fig. 1 – 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 fat content of margarine from different country:

  • the greatest contents are found in soft margarine from Iceland, Norway, and the UK (13-16,5% of total fatty acids);
  • less contents are found in Italy, Germany, Finland, and Greece (5.1%, 4.8%, 3.2%, and 2.9% of total fatty acids, respectively);
  • in Portugal, The Netherlands, Belgium, Denmark, France, Spain, and Sweden margarine trans fat contents are less than 2% of the total fatty acids.

USA and Canada lag behind Europe, but with advent of trans fat labeling of foods in USA change is occurring. For this reason, at now, in USA margarine is considered to be only a minor contributor of the total trans fats, 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).

Foods with trans fats: shortenings

Trans fat content of shortening 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, trans fat content of shortenings is decreasing; i.e. in German shortenings it decreased from 12% of total fatty acids in 1994 to 6% in 1999, in Denmark is 7% (1996) while in New Zealand is about 6% (1997).

Foods with trans fats: vegetable oils

At now, nonhydrogenated 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 g food for extra virgin oil to 2,42 g/100 g food for canola oil. So their contribution to trans fat content to the current food supply is very little.
One exception is represented by Pakistani hydrogenated vegetable oils (vegetable ghee/vanaspati) whose trans fat content range from 14% to 34% of total fatty acids.

Foods with trans fats: 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 trans fats to the diet if frequently consumed.

Foods with trans fats: processed foods

Thanks to their long shelf life as their flavor stability, trans fats are used in many processed foods as cookies, cakes, croissants, pastries and other baked goods.
Baked goods are the greatest source of these fats in North American diet. Of course, their trans fat contents depend on the type of fat used in processing. In USA after 2006, when labeling laws were implemented, many processed foods have been reformulated and contain less than 0,5 g of industrial trans fats per serving; so producers can list their content as 0 on the packaging, but it‘s not 0!

Foods with trans fats: sauces

Mayonnaise, salad dressings and other sauces contribute only small or no-amounts of trans fats to the diet.

Foods with trans fats: human milk and infant foods

Trans fat content of human milk reflects the trans fatty acid content of maternal diet in the previous day. In human milk it comprise 1%-7% of the total fatty acids but 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% of the total fatty acids.
Baby foods contain greater than 5% of trans fats.

Foods with trans fats in fast foods and restaurants

Foods with Trans Fats
Fig. 2 – French Fries

Shortenings with high amounts of trans fats are used as frying fats, so fast foods and many restaurant’s foods may contain relatively large amounts of them.
Foods with trans fats are fried pies, French fries, chicken nuggets, hamburgers, fried fish as well as fried chicken.
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 because of the cooking oil used. For example oil used in USA and Peru outlets of a famous fast food chain contains 23-24% of trans fats, whereas oil used in many European countries of the same fast food chain contains about 10% trans fats, with some countries as low as 5% and 1% (Denmark).
On 2006, Stender et al. reported that a meal of French fries and chicken nuggets purchased at McDonald ‘s in New York City contained over 10 g of trans fats, while if purchased at Kentucky Fried Chicken in Hungary they are almost 25 g.
Again, from the work of Stender et all. it can see a cross-country comparison of trans fat contents of chicken nuggets and French fries purchased at McDonald ‘s or Kentucky Fried Chicken: trans fat contents vary depending on the country and even the city and often in the same city.

Chicken nuggets and French fries from McDonald’s:

  • less then 1 g in industrial trans fats 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 Kentucky Fried Chicken:

  • less than 2 g in industrial trans fats 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.
References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Eckel R.H., Borra S., Lichtenstein A.H., Yin-Piazza D.Y. Understanding the Complexity of Trans fatty acid reduction in the American diet. American Heart Association trans fat conference 2006 report of the trans fat conference planning group. Circulation 2007;115:2231-46; originally published online Apr 10, 2007 [Abstract]

Mozaffarian D., Jacobson M.F., Greenstein J.S. Food Reformulations to reduce trans fatty acids. N Eng J Med 2010;362:2037-39 [PDF]

Okie S. New York to trans fats: you’re out! N Engl J Med 2007;356:2017-21 [PDF]

Stender S., Astrup A., Dyerberg J. What went in when trans went out?. N Engl J Med 2009;361:314-16 [PDF]

Stender S., Dyerberg J. and Astrup A. Consumer protection through a legislative ban on industrially produced trans fatty acids in foods in Denmark. Scand J Food Nutr 2006;50:155-60 [Abstract]

Stender S., Dyerberg J., Astrup A. High levels of trans fat in popular fast foods. N Engl J Med 2006;354:1650-2 [PDF]

Dietary trans fatty acids: industrial and natural sources

Industrial and natural sources of dietary trans fatty acids

Dietary trans fatty acids come from different sources:

  • they can come from industrial processing, being the by-product of partial hydrogenation of unsaturated vegetable oils;
  • they can be produced naturally by plants and animals.

Dietary trans fatty acids from partial hydrogenation of vegetable oils

Dietary Trans Fatty Acids: Hydrogenation Process
Fig. 1 – Hydrogenation of Oleic Acid

In industrialized countries, greater part of the consumed trans fatty acids are produced industrially (in USA about 80%), in varying amounts, during partial hydrogenation of edible oils containing unsaturated fatty acids.
Hydrogenate means to add hydrogen atoms to unsaturated sites (that is, on a double bond) on the carbon chains of fatty acids by heating vegetable oils in presence of metal catalyst and hydrogen.
During the partial hydrogenation, an incomplete saturation of the unsaturated sites on the carbon chains of unsaturated fatty acids occurs: some double bonds remain, but they may be moved in their positions on the carbon chain, producing geometrical and positional isomers (double bonds are modified in both conformation and position).
Notably, with regard to fish oil, trans fatty acid content in non-hydrogenated oils and in highly hydrogenated oils is 0,5 and 3,6%, whereas in partially hydrogenated oils is 30%.
Hydrogenation converts vegetable oils into semisolid fats for use in:

  • margarines and shortenings;
  • commercial cooking;
  • manufacturing processes.

It should be noted that partial hydrogenation largely destroys alpha-linolenic acid, the plant-based omega-3-fatty acid.
Industrial trans fatty acids (ITFA) have adverse effects on:

  • serum lipid levels (total and LDL-cholesterol);
  • 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.

Industrial trans fatty acids are an independent cardiovascular risk factor.
Their adverse effects are seen at low level of intake: for a person consuming 2000 kcal/d, 20-60 kcal from industrial trans fatty acids, equivalent to about 2-7 g or 1-3% of the total energy intake, is enough.
So, avoidance of industrial trans fatty acids, or a consumption of less 0,5% of total daily energy intake is necessary to avoid their adverse effects (these are far stronger, on average, than those of food contaminants or pesticide residues!).

Dietary trans fatty acids from deodorization of vegetable oils

Very small amounts of trans fatty acids (less than 2 percent) are formed during deodorization of vegetable oils, a process unrelated to partial hydrogenation and necessary in the refining of edible oils. During this process trans fatty acids with more than one double bond are formed in small amounts (if the isomer contains 18 carbon atoms it is marked C18:2). These isomers are also present in fried foods and in considerable amounts in partially hydrogenated vegetable oils (e.g. soybean oil).

Dietary trans fatty acids from animals

A natural source comes from bacterial transformation of a proportion of the relatively small amounts of unsaturated fatty acids ingested by ruminants in their rumen.
They are present at low levels in meat and full fat dairy products from cows, sheep, and other ruminants (typically <5% of total fatty acids).

Dietary trans fatty acids from vegetables

Another natural source is represented by some plant species, and plant-derived foods as:

  • leeks, peas, lettuce and spinach, that contain trans-3-hexadecenoic acid;
  • rapeseed oil, that contains brassidic acid and gondoic acid.

In these sources trans fatty acids are present in small amounts.

“Homemade”dietary trans fatty acids

They are produced at home during frying with vegetable oils.

Isomers of dietary trans fatty acids

Dietary Trans Fatty Acids: ITFA
Fig. 2 – ITFA

The most important cluster of trans fatty acids both animal and industrial origin is isomers containing 18 carbon atoms  plus one double bond (C18:1) whose position varies between the Δ6 and Δ16 carbon atoms of molecule.
Even if the same trans fatty acids are largely present in industrial trans fatty acids and in trans fatty acids from ruminants, there is a considerable quantitative difference between individual molecules in the two different sources.
The most common isomers in both sources are those with double bond in position between Δ9 and Δ11, but Δ11-C18:1 or vaccenic acid represents over 60% of the trans C18:1 isomers in ruminant trans fatty acids, whereas in industrial ones  Δ9-C18:1 or elaidic acid comprises 15-20%, and Δ10-C18:1 and Δ11-C18:1 over 20% each others.

References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Lemaitre R.N., King I.B., Raghunathan T.E. et al. Cell membrane trans-fatty acids and the risk of primary cardiac arrest. Circulation 2002;105:697-01 [Abstract]

Lichtenstein A. H., Ausman L., Jalbert S.M , Schaefer E.J. Effect of different forms of dietary hydrogenated fats on serum lipoprotein cholesterol levels. N Engl J Med 1999;340:1933-40 [Abstract]

Lopez-Garcia E., Schulze M.B., Meigs J.B., Manson JA.E, Rifai N., Stampfer M.J., Willett W.C. and Hu F.B. Consumption of trans fatty acids is related to plasma biomarkers of inflammation and endothelial dysfunction. J Nutr 2005;135:562-66 [Abstract]

Mozaffarian D. Commentary: Ruminant trans fatty acids and coronary heart disease-cause for concern? Int J Epidemiol 2008;37:182-84 [Extract]

Mozaffarian D., Katan M.B., Ascherio A., Stampfer M.J., Willett W.C. Trans fatty acids and cardiovascular disease. N Engl J Med 2006;354:1601-13 [Abstract]

Oomen C.M., Ocke M.C., Feskens E.J., van Erp-Baart M.A., Kok F.J., Kromhout D. Association between trans fatty acid intake and 10-year risk of coronary heart disease in the Zutphen Elderly Study: a prospective population-based study. The Lancet 2001;357:746-51 [Abstract]

Willett W. and Mozaffarian D. Ruminant or industrial sources of trans fatty acids: public health issue or food label skirmish? Am J Clin Nutr 2008;87:515-6 [PDF]

Trans fatty acids: definition and chemistry

What are trans fatty acids?

Trans fatty acids (TFA) or partially hydrogenated fatty acids are unsaturated fatty acids, a class of lipids, with at least one a double bond in the trans configuration.
They may result from:

In industrialized countries, the greater part of the consumed trans fatty acids are produced industrially, in varying amounts, during partial hydrogenation of edible oils containing monounsaturated and polyunsaturated fatty acids.
Note: partially hydrogenated vegetable oils were developed in the United States in 1903 as a cheaper alternative to animal fats.

While ruminant trans fatty acids, in amounts actually consumed in diets, are not harmful for human health, consumption of industrial partially hydrogenated fatty acids has neither apparent benefit nor intrinsic value, above their caloric contribution: from human health standpoint they are only harmful.

Chemical structure of trans fatty acids

Fatty acids

Fatty acids are made up by carbon (from the “smallest”, formic acid, containing only one carbon atom, up to fatty acids with over 30 carbon atoms), hydrogen and oxygen atoms linked to form a carboxylic group (the “hydrophilic head” of the molecule) that continues in a more or less long chain of carbon atoms linked each other by chemical bonds that may be single or double (the “hydrophobic tail”). Hydrogen atoms are linked to carbon and oxygen atoms.
Fatty acids are defined:

  • saturated, if chemical bonds between carbon atoms of the chain are all simple;
  • monounsaturated, if a double bond is present in the chain;
  • polyunsaturated, if two or more double bonds are present between single bonds of the chain.

Monounsaturated fatty acids and polyunsaturated fatty acids can be defined as unsaturated.

Cis and trans isomers

Trans Fatty Acids
Fig. 1 – Cis and Trans Isomers

Carbon-carbon double bonds show planar conformation and so they can be considered as plans 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 plan, and in this case double bond is defined in cis 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.
Unsaturated fatty acids with at least one double bond in trans configuration are called trans fatty acids.
Cis bond causes a bend in the fatty acids chain, whereas the geometry of trans bond straightens the fatty acid chain, imparting a structure more similar to that of saturated fatty acids.
Bent molecules can’t pack together easily but linear ones can do it and this gives the fats in which they are 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; for this reason fats containing a greater part of saturated fatty acids, or the geometrically similar partially hydrogenated fatty acids, are solid at room temperature (partially hydrogenated fatty acids tend to be less solid than saturated fats).

In industrially trans fatty acids-containing fats, cyclic monomers as well as intramolecular linear dimmers are also present.

References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Ascherio A., Katan M.B., Zock P.L., Stampfer M.J., Willett W.C. Trans fatty acids and coronary heart disease. N Engl J Med 1999;340:1994-8 [Abstract]

 Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Mozaffarian D. Commentary: Ruminant trans fatty acids and coronary heart disease-cause for concern? Int J Epidemiol 2008;37:182-84 [Extract]

Mozaffarian D., Katan M.B., Ascherio A., Stampfer M.J., Willett W.C. Trans fatty acids and cardiovascular disease. N Engl J Med 2006;354:1601-13 [Abstract]

Stender S., Dyerberg J. The influence of trans fatty acids on health. Fourth edition 2003 (from Danish Nutrition Council; publ. no. 34)

Willett W. and Mozaffarian D. Ruminant or industrial sources of trans fatty acids: public health issue or food label skirmish? Am J Clin Nutr 2008;87:515-6 [PDF]

Omega-3 fatty acid supplements in the secondary prevention of CVD

Omega-3 fatty acids and prevention of CVD

Omega-3 Fatty Acid Supplements: DHA-Docosahexaenoic acid
Fig. 1 – DHA

Studies conducted on Greenland Eskimos, which consume large amount of fish or marine mammals rich in omega-3 fatty acids and have a low incidence of cardiovascular disease (CVD), have suggested a protective effects of such fatty acids against these disease. Results of other epidemiological studies, randomized trials and animal investigations, have also suggested that omega-3 fats, and in particular long-chain omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have cardiovascular effects. These studies indicate that they have anti-inflammatory, antiatherogenic, and antiarrhythmic effects, which are considered plausible mechanisms for reducing the risk of cardiovascular disease.

Omega-3 fatty acid supplements and secondary prevention of CVD

In a study published on Archives of Internal Medicine a research team, using a meta-analysis of randomized, double-blind, placebo-controlled trials, has evaluated the preventive effect of omega-3 fatty acid supplements (omega-3 fatty acid supplements for at least 1 year, with a daily dose of EPA or DHA ranged from 0.4 to 4.8 g/d, and a follow-up period ranged from 1.0 to 4.7 years) in the secondary prevention of cardiovascular disease, i.e. among patients with a history of cardiovascular disease (not in healthy individuals).
The study involved 20485 patients, male or female aged ≥18 years, 10259 randomized to a placebo group and 10226 randomized to an intervention group. Placebo groups received vegetable oils (sunflower oil, olive oil, and corn oil), mixed fatty oil, and other “inert” or ill-defined substances (aluminum hydroxide and unspecified placebo).
The meta-analysis showed insufficient evidence of a secondary preventive effect of omega-3 fatty acid supplements against overall cardiovascular events, which include peripheral vascular disease, angina and unstable angina, transient ischemic attack and stroke, fatal and nonfatal myocardial infarction, sudden cardiac death, cardiovascular death, congestive heart failure, and nonscheduled cardiovascular interventions (i.e., coronary artery bypass surgery or angioplasty).
Moreover, no significant preventive effect was observed in subgroup analyses by the following: history of cardiovascular disease, concomitant medication use (lipid lowering agents, no lipid-lowering agents, or antiplatelet agents only), country location (Western Europe, Northern Europe, United States, or Asia), inland or coastal geographic area, methodological quality of the trial, duration of treatment, type of placebo material in the trial (oil vs nonoil), dosage of EPA or DHA, or use of fish oil supplementation only as treatment.

Conclusion

The study showed insufficient evidence of a secondary preventive effect of omega-3 fatty acid supplements against overall cardiovascular events among patients with a history of cardiovascular disease.

References

Kwak S.M., Myung S-K., Lee Y.J., Seo H.G., for the Korean Meta-analysis Study Group. Efficacy of omega-3 fatty acid supplements (eicosapentaenoic acid and docosahexaenoic acid) in the secondary prevention of cardiovascular disease. A meta-analysis of randomized, double-blind, placebo-controlled trials. Arch Intern Med 2012;172(9):686-694 [Abstract]

Trans fats, omega-3 fatty acid and risk of non-Hodgkin lymphoma

Fat and protein intake and risk of non-Hodgkin lymphoma

In a clinic-based study published on Journal of Nutrition a research team evaluated the association of dietary fat and protein intake with risk of non-Hodgkin lymphoma in 603 cases (105 diffuse large B-cell lymphoma, 146 follicular lymphoma, and 218 chronic lymphocytic leukemia/small lymphocytic lymphoma) and 1007 frequency-matched controls.

non-Hodgkin Lymphoma: Trans Fat Cartoon
Fig. 1 – Trans Fat Cartoon

While omega-3 fatty acid intake was inversely associated with non-Hodgkin lymphoma risk, trans fatty acid intake was positively associated with risk and there was no association with total, animal, plant-based, or saturated fat intake.
When examining intake of specific foods, fresh fish and total seafood intakes were inversely associated with risk, whereas intakes of processed meat, milk containing any fat, and high-fat ice cream intakes were positively associated with risk.

In conclusion, the study showed that diets high in omega-3 fatty acids and total seafood were inversely associated with non-Hodgkin lymphoma risk, whereas diets high in trans fatty acids, processed meats, and higher fat dairy products were positively associated with risk.

References

Charbonneau B., O’Connor H.M., Wang A.H., Liebow M., Thompson C.A., Fredericksen Z.S., Macon W.R., Slager S.L., Call T.G., Habermann T.M., and Cerhan J.R. Trans fatty acid intake is associated with increased risk and n3 fatty acid intake with reduced risk of non-Hodgkin lymphoma. J Nutr 2013;143:672-681 [Abstract]

Artificial trans fats: businness and health

The 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” and the term trans fatty acids or trans fats (they are produced  during partial hydrogenation of edible oils containing monounsaturated and polyunsaturated fatty acids) appeared for the first time in the Remark column of the 5th edition of the “Standard Tables of Food Composition” in Japan.

Partially hydrogenated vegetable oils were developed as a cheaper alternative to animal fats.
Moreover, they:

  • contribute to the hardness of fat in which they are who can be semi-solids and solids (they are used to make margarine or shortening with a melting point, consistency and “mouth feel” similar to those of butter);
  • have a long shelf life at room temperature;
  • have flavor stability and be stable during frying.

Note: per year in USA 6-8 billion pounds of hydrogenated vegetable oil are produced.

The war on artificial trans fats

Trans Fats
Fig. 1 – Shortening

The first hydrogenated oil was cottonseed oil in USA in 1911 to produce vegetable shortening.
So, before this date, the only trans fats in human diet were those derived from ruminants.
In the 1930’s partial hydrogenation became popular with the development of margarine; through hydrogenation, oils such as soybean, safflower and cottonseed oil, which are rich in unsaturated fatty acids, are converted to margarines and vegetable shortenings.
Until 1985 no adverse effects of trans fats on human health was demonstrated and in 1975 Procter & Gamble study shows no effect of partially hydrogenated fats on cholesterol.
Their use in fast food preparation grow up from 1980’s when the role of dietary saturated fats in increasing cardiac risk began clear; 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 in USA Food and Drug Administration (FDA) concludes that trans fats and monounsaturated fat oleic acid affect serum cholesterol level similarly but from the second half of 1985 their harmful began clear and the final proof comes from both controlled feeding trials and prospective epidemiologic studies.
After June 1996 they were eliminated from margarine sold in Australia, which before contributed about 50% of the dietary intake of trans fatty acids in such country.
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 industrially produced trans fats (ITFA) in food before the end of 2003; two years later, however, the European Commission asked Denmark to withdraw this law, which was not accepted on the EU level, unfortunately.
Canada is considering legislation to eliminate industrially produced trans fats from food supplies.
On 2003 FDA ruled that food labels (for conventional foods and supplements) show trans fat content beginning January 1, 2006. Notably, this ruling is 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 fats a key recommendation of the new food-pyramid guidelines.
On 2006 American Heart Association recommends to limit their intake to 1% of daily calorie consumption and suggests food manufacturers and restaurants switch to other fats.
On 2006 New York City Board of Health announces trans fat ban in its 40.000 restaurants within July 1, 2008.

References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Ascherio A., Katan M.B., Zock P.L., Stampfer M.J., Willett W.C. Trans fatty acids and coronary heart disease. N Engl J Med 1999;340:1994-8 [Abstract]

Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Eckel R.H., Borra S., Lichtenstein A.H., Yin-Piazza D.Y. Understanding the Complexity of Trans fatty acid reduction in the American diet. American Heart Association trans fat conference 2006 report of the trans fat conference planning group. Circulation 2007;115:2231-46; originally published online Apr 10, 2007 [Abstract]

Mozaffarian D., Jacobson M.F., Greenstein J.S. Food Reformulations to reduce trans fatty acids. N Eng J Med 2010;362:2037-39 [PDF]

Okie S. New York to trans fats: you’re out! N Engl J Med 2007;356:2017-21 [PDF]

Stender S., Astrup A., Dyerberg J. What went in when trans went out?. N Engl J Med 2009;361:314-16 [PDF]

Stender S., Dyerberg J. and Astrup A. Consumer protection through a legislative ban on industrially produced trans fatty acids in foods in Denmark. Scand J Food Nutr 2006;50:155-60 [Abstract]

Stender S., Dyerberg J. The influence of trans fatty acids on health. Fourth edition 2003 (from Danish Nutrition Council; publ. no. 34)

Long chain fatty acid synthesis

Fatty acid synthesis

Fatty Acid Synthesis
Fig. 1 – Long Chain Fatty Acids

When excess calories are consumed from carbohydrates or proteins, such surplus is used to synthesize fatty acids and then triacylglycerols, while it doesn’t occur if the excess come from fats.

De novo fatty acid synthesis in plants and animals

De novo fatty acid synthesis is largely similar among plants and animals.
It occurs in chloroplasts of photosynthetic cells of higher plants, and in cytosol of animal cells by the concerted action of two enzymes: acetyl CoA carboxylase and fatty acid synthase.
Fatty acid synthase is a multienzyme complex that catalyzes a repeating four-step sequence by which the fatty acyl chain is extended by two carbons, at the carboxyl end, every each passage through the cycle; this four-step process is the same in all organisms.
In animals, the primary site for lipid metabolism is liver, not the adipose tissue.  However, adipose tissue is  a major organ system in which fatty acid synthesis occurs, though in humans it is less active than in many other animal species.

Fatty Acid Synthesis
Fig. 2 – Palmitic Acid Synthesis

Although myristic, lauric and a trace of stearic acids may also be produced, in animals and plants the main product of these reactions is palmitic acid.
It should be noted that in certain plants, such as palm and coconut, chain termination occurs earlier than palmitic acid release: up to 90% of the fatty acids produced and then present in the oils of these plants are between 8 (caprylic acid) and 14 (myristic acid) carbons long (palmitic acid: 16 carbon atoms).

Synthesis of long chain saturated and unsaturated fatty acids

Fatty Acid Synthesis
Fig. 3 – Palmitic Acid Metabolism

Palmitic acid is the commonest saturated fatty acid in plant and animal lipids, but generally it is not present in very large proportions because it may be undergo into several metabolic pathways.
In fact:

  • it is the precursor of stearic acid;
  • it may be desaturated (insertion of a double bond into fatty acid chain) to palmitoleic acid, the precursor of all fatty acids of omega-7 or n-7 family, in a reaction catalyzed by Δ9-desaturase, an ubiquitous enzyme in both plant and animal kingdoms and the most active lipid enzyme in mammalian tissues, the same enzyme that catalyzes the desaturation of stearic acid to oleic acid (see below).
    Note: Δ9- desaturase inserts double bounds in the 9-10 position of the fatty acid carbon chain, position numbered from the carboxyl end of the molecule, and:

if the substrate is palmitic acid, the double bond will appear between n-7 and n-8 position of the chain (in this case numbered from the methyl end of the molecule), so producing palmitoleic acid, the founder of omega-7 series;
numbering-atoms-palmitic-acid

if the substrate is stearic acid, the double bond will appear between n-9 and n-10 position of the chain and oleic acid will be produced.
numbering-atoms-stearic-acid

  • It may be esterified into complex lipids.

Of course, in plants and animals there are fatty acids longer and/or more unsaturated than these just seen thanks to modification systems (again desaturation and elongation) that catalyze reactions of fatty acid synthesis that are organism- tissue- and cell- specific.

Fatty Acid Synthesis
Fig. 4 – Stearic Acid Metabolism

For example, stearic acid may be:

  • elongated to arachidic, behenic and lignoceric acids, all saturated fatty acids, in reactions catalyzed by elongases.
    Again, chain elongation occurs, both in mitochondria and in the smooth endoplasmic reticulum, by the addition of two carbon atom units at a time at the carboxylic end of the fatty acid through the action of fatty acid elongation systems (particularly long and very long saturated fatty acids, from 18 to 24 carbon atoms, are synthesized only on cytosolic face of the smooth endoplasmic reticulum);
  • desaturated, as seen, to oleic acid, an omega-9 or n-9 fatty acid, in a reaction catalyzed by Δ9-desaturase. Several researchers have postulated that the reason for which stearic acid is not hypercholesterolemic is its rapid conversion to oleic acid.
Fatty Acid Synthesis
Fig. 5 – Oleic Acid Metabolism

Oleic acid is the start point for the synthesis of many other unsaturated fatty acids by reactions of elongation and/or desaturation.

In fact:

Omega-3 and omega-6 PUFA synthesis

Fatty Acid Synthesis
Fig. 6 – Omega-3 and Omega-6 Synthesis

Animal tissues can desaturate fatty acids in the 9-10 position of the chain, thanks to the presence of Δ9 desaturase; as previously seen, if the substrate of the reaction is palmitic acid, the double bond will appear between n-7 and n-8 position, with stearic acid between n-9 and n-10 position, so leading to formation respectively of palmitoleic acid and oleic acid.
Animals lack Δ12- and Δ15-desaturases, enzymes able to desaturate carbon carbon bonds beyond the 9-10 position of the chain. For these reason, they can’t produce de novo omega-3 and omega-6 PUFA (which have double bonds also beyond the 9-10 position), that are so essential fatty acids.
Δ12- and Δ15-desaturases are present in plants; though many land plants lack Δ15-desaturase, also called omega-3 desaturase, planktons and aquatic plants in colder water possess it and produce abundant amounts of the omega-3 fatty acids.

References

Akoh C.C. and Min D.B. “Food lipids: chemistry, nutrition, and biotechnology” 3th ed. 2008

Bender D.A. “Benders’ dictionary of nutrition and food technology”. 2006, 8th Edition. Woodhead Publishing. Oxford

Burr G. and Burr M. A new deficiency disease produced by the rigid exclusion of fat from the diet. J Biol Chem 1929;82:345-67 [Full Text]

Chow Ching K. “Fatty acids in foods and their health implication” 3th ed. 2008

Cozzani I. e Dainese E. “Biochimica degli alimenti e della nutrizione”. Piccin Editore, 2006

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

Stipanuk M.H.. Biochemical and physiological aspects of human nutrition. W.B. Saunders Company-An imprint of Elsevier Science, 2000