Nutrition

Carotenoids: structure, classification and function

Carotenoids are a class of fat-soluble pigments widely present in nature.
They are yellow, orange, and red organic compounds, made up of 8 isoprene units, and have many conjugated double bonds. Their hydrocarbon chain can undergo modifications which significantly influence the biological properties.
Discovered in the first half of the 1800s, carotenoids are synthesized by all photosynthetic organisms, including plants, macroalgae and microalgae, and by some non-photosynthetic organisms, such as some types of fungi, bacteria, and insects, such as pea aphids, some species of gall midges, and spider mites. In plants and microalgae they are synthesized and accumulated in plastids.
They form, with polyphenols and glucosinolates, the group of phytochemicals.
In the course of evolution, thanks to their chemical and physical properties, they have proved to be extremely versatile, as they can perform many functions in both plants and animals, among which the antioxidant activity is very important. Furthermore, in humans, some carotenoids serve as vitamin A precursor.
There are more than 750 different carotenoids, more than 100 have been found in fruits and vegetables, and about 40 are consumed in significant amounts by humans.
Thanks to their color, carotenoids are used as food additives.

CONTENTS

The discovery

Carotenoids were discovered in the first half of the nineteenth century.
The first carotenoid to be isolated was beta-carotene, in 1831, thanks to the work of the German pharmacist Heinrich Wilhelm Ferdinand Wackenroder, who called “carotene” the yellow pigment crystallized from the carrot root.
Jöns Jacob Berzelius, considered one of the fathers of modern chemistry, named the yellow pigments extracted from autumn leaves xanthophylls, from the Greek xanthos, which means yellow, and phyllon, which means leaf.
Mikhail Semyonovich Tsvet, an Italian-Russian botanist, who invented chromatography in 1906, managed to separate leaf pigments, namely, chlorophylles from carotenes and xanthophylls, thanks to column chromatography, and called the yellow-to-orange pigments carotenoids.

Chemical structure

Carotenoids are a class of lipids having as a basic structure a linear sequence of 8 isoprene units. Therefore, they are terpenes, specifically tetraterpenes, and then consist of 40 carbon atoms.
The isoprene units are joined in a 1,5 positional relationship, that is, head to tail, except in the middle of the hydrocarbon chain, where the positional relationship is 1,6, that is, tail to tail. This reverses the order making the molecule symmetrical.
Carotenoids are characterized by the presence of an extended double-bond system, 3 to 13 of which can be conjugated.

Skeletal formulas of some carotenoids
The hydrocarbon chain of carotenoids can undergo hydrogenation, dehydrogenation, introduction of oxygen atoms, cyclization of one or both ends to form an ionone rings, and may be esterification with fatty acids. Such modifications allow the formation of many different structures, and most of them are described by the chemical formula C40H56On, with 0≤n≤6.
Traditionally, carotenoids have common names related to the biological source from which they are extracted, although they can be named using IUPAC nomenclature.

Isomerism

Carotenoids have many double bonds which can exhibit cis–trans isomerism, also known as geometric isomerism. Although in theory their double bonds can be in a cis– or trans-configuration, in nature the trans-configuration prevails, as it is thermodynamically more stable than cis-configuration, thanks to the lower steric hindrance between the substituents.
The rotation around the simple bonds, or sigma bonds, allows the formation of conformational isomers, as well. When the double bonds are on the same side with respect to the sigma bond, namely, cis in reference to the single bond, the conformation is called s-cis conformation; conversely, when the double bonds are on opposite sides with respect to the sigma bond, namely, trans in reference to the single bond the conformation is called s-trans conformation. In nature, acyclic carotenoids are mainly in the s-trans conformation, as it has the least steric hindrance. When the polyene chain ends with cyclic structures, the rotation around the C6-C7 sigma bond gives rise to the formation of a 6-s-cis isomer with less steric hindrance and therefore energetically favored.
It should be noted that the steric hindrance and the dimensions of the carotenoids influence the ability to interact with enzymes and to form supramolecular structures.
Most carotenoids also have a chirality center or a chirality axis, which causes optical isomers to exist. In these cases, to give a unique and unambiguous name to the to the molecule, the RS system is used instead of the Fischer-Rosanoff convention, which is used only for carbohydrates and amino acids.

Classification

Carotenoids can be classified on the basis of the presence or absence of oxygen or cyclic structures.
Based on the presence or absence of oxygen, they are divided into xanthophylls and carotenes.
Carotenes, which are oxygen-free molecules, are made up of only carbon and hydrogen atoms, and have chemical formula C40H56. Examples of carotenes are alpha-carotene, beta-carotene, delta-carotene, zeta-carotene, phytoene, and lycopene.
Xanthophylls also contain oxygen atoms, have chemical formula C40H56On, with 0˂n≤6, and can, in turn, be divided into:

  • hydroxycarotenoids, which contain at least one hydroxyl group, such as alpha-cryptoxanthin, beta-cryptoxanthin, zeaxanthin and lutein;
  • epoxycarotenoids, which have at least one epoxy group, such as antheraxanthin, auroxanthin and luteoxanthin;
  • ketocarotenoids, which have one or more carbonyl groups, such as astaxanthin and canthaxanthin.

Depending on the presence or absence of rings in the molecule, carotenoids can be divided into cyclic and acyclic.

  • Cyclic carotenoids contain one or two cyclic structures, and, compared to acyclic carotenoids, are shorter, but with a greater steric hindrance and a greater space occupied. Examples are alpha-carotene, beta-carotene, gamma-carotene and delta-carotene.
  • Acyclic carotenoids are formed by a linear carbon chain. Examples are lycopene, zeta-carotene, phytoene, and phytofluene.

Finally, there are also uncommon or species-specific carotenoids, such as bixin, capsanthin and capsorubin.

Solubility

Carotenoids are hydrophobic molecules, therefore soluble in organic solvents, solubility which varies according to the substituents present. Given their hydrophobicity, they are found in cell membranes. In most cases, the xanthophylls have the polar groups at the ends of the polyene chain, and, when present in cell membranes, in order to minimize the energy of the system, they arrange themselves in such a way that the polar groups are in contact with the polar groups of the lipid bilayer.
Carotenoids can also have access to the aqueous environment if in association with proteins, to which they bind non-covalently. In plasma, they are transported by lipoproteins.

Color

The conjugated double bond system, acting as a chromophore, is responsible for the color of carotenoids. However, only carotenoids that have at least seven conjugated double bonds are colored; hence, phytofluene, which has five conjugated bonds, or phytoene, which has three conjugated double bonds, are colorless.
As the number of conjugated double bonds increases, the color changes from yellow to orange to red; for example, lutein, alpha-cryptoxanthin, and violaxanthin are yellow, alpha-carotene, beta-carotene, and gamma-carotene are orange, and lycopene is red.

Role in plants

Carotenoids play many roles in plants.
They contribute to cell protection against oxidative damage, increase the absorption of sunlight, are the precursors of phytohormones, such as abscisic acid, and are involved in signaling pathways. Furthermore, with chlorophylls and anthocyanins, a class of flavonoids, the major type of polyphenols, they contribute to the color of leaves, fruits, vegetables, and grains, color which, attracting animals, promotes pollination and seed dispersion. Finally, they can act as repellent agents for phytophages and pathogens, as well.

Quenching of singlet oxygen

In organisms that carry out oxygenic photosynthesis, carotenoids contribute to the protection from damage caused by excessive solar radiation. When the sunlight arriving at the photosystem II antenna complex exceeds the conversion capacity of the reaction centers, the excess energy can cause chlorophyll to remain in its excited state, which can lead to the formation of its triplet state. The excess energy can be transferred from chlorophyll to molecular oxygen causing the formation of singlet oxygen, a reactive oxygen species, capable of damaging the photosynthetic apparatus and, more generally, nucleic acids, proteins, membrane unsaturated fatty acids, as well as enzyme cofactors.
Carotenoids can prevent oxidative damage caused by singlet oxygen in two ways.
They can directly quench the singlet oxygen. In this case the energy is transferred to the carotenoid by the singlet oxygen, which decays to the triplet ground state, while the carotenoid reach the triplet state, then releasing the excess energy in the form of heat. The capacity of quench singlet oxygen increases with the number of conjugated double bonds, being maximal at nine or more conjugated double bonds. In vitro studies seem to indicate that monocyclic carotenoids are able to quench singlet oxygen more efficiently than acyclic structures, and that the presence of carbonyl groups, such as in astaxanthin, improves the antioxidant potential compared to those with hydroxyl groups, probably thanks to the extended system of conjugated double bonds. It seems that each carotenoid, before undergoing degradation reactions, is able to quench about 1000 singlet oxygen molecules.
Carotenoids are also able to prevent the formation of singlet oxygen by absorbing the energy of the excited chlorophyll, so turning into a triplet state. As the triplet state of carotenoids has an energy level lower than that of singlet oxygen, it spontaneously decays to the fundamental state.

Free radical scavenging activity

Carotenoids are able to scavenge free radicals, such as hydroxyl, peroxyl, and superoxide radicals, and nitric oxide radical. Their action can occur in four different ways.

  • Carotenoid can donate an electron to the radical cation, producing the radical cation of the carotenoid, while the radical becomes a stable molecule. The carotenoid radical cation can be regenerated to parent carotenoid by other cellular antioxidants, such as vitamin E, vitamin C and glutatione.
  • A proton can be transferred from the carotenoid to the radical species, which becomes a neutral molecule, while the carotenoid becomes a radical anion.
  • The neutralization of the free radicals can also occur as a result of addition reactions, as in the case of the neutralization of hydroxyl and peroxyl radicals.
  • An hydrogen atom can be transfer from carotenoid to the reactive oxygen species, which leads to a neutral-resonance-stabilized carotenoid radical.

Light-harvesting role

Carotenoids are present in the antenna complexes as accessory light-harvesting pigments, mainly associated with antenna proteins.
Thanks to the system of conjugated double bonds, they are able to absorb sunlight in the range of 400-500 nm. The absorbed energy is then conveyed to the reaction centers.
The absorption spectra of individual carotenoids depend on the length of the conjugation network and the type of functional groups present.
The main carotene involved in light-harvesting is beta-carotene, present in the core of photosystems I and II of all organisms that carry out an oxygenic photosynthesis, whereas lutein, violaxanthin, neoxanthin and zeaxanthin, the main xanthophylls in plants, are bound to the light-harvesting complexes.

Food sources

Humans are not able to synthesize carotenoids. They take them from food, and store them mainly in liver and adipose tissue, but also in lung, kidney, brain, and bone, where they play many functions.
Of all the carotenoids identified, about a hundred have been found in foods consumed by humans, while, if we consider the single food item, generally there are one to five main carotenoids, accompanied by others present in traces or in any case small quantities.
Fruits and vegetables are the major source of carotenoids in the human diet. Among vegetables, leafy greens are rich in beta-carotene, the most abundant carotenoid in human diet and tissues, and lutein, followed by violaxanthin and neoxanthin, carrot root is rich in beta-carotene, pumpkin in alpha-carotene, tomato in lycopene, and peppers in capsanthin and capsorubin. Carotenoids in fruits are very varied, and those in ripe fruit can be different from those in unripe fruit. Furthermore, most of the carotenoids present in ripe fruits are esterified with fatty acids. However, those of some fruits, in particular fruits that remain green when ripe, such as kiwis, undergo limited or absent esterification.
Chicken eggs, thanks to the fact that laying hens mostly eat corn, are a good source of some carotenoids, such as lutein and zeaxanthin, which are responsible for the yellow-orange color of egg yolk.
Finally, mollusks and crustaceans, which together with fish are the main sources of long-chain omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid, are the main source of carotenoids produced by microalgae and rarely found in plants, such as astaxanthin in shrimp, but also in salmon.

Food additives

Carotenoids used in food industry as additives, unlike naturally occurring carotenoids, are relatively unstable, being susceptible to the action of oxygen and sunlight, auto-oxidation, and dispersion into food ingredients which facilitates their degradation. Furthermore, at body and food storage temperatures the added carotenoids, due to their high melting point, are in crystalline form; to overcome this problem, they are used in the form of an oil-in-water emulsion.
Thanks to their coloring power, they are used as colours in foods which, due to processing or storage, lose part of their natural colour, or to standardize the colour of the products, as in the case of beverages, fruit juices or sausages, or to intensify their colour, thus improving their appearance.
Carotenoids are also precursors of compounds responsible for the flavor and aroma of some foods, hence, they can be used as flavour enhancers.
Furthermore, some carotenoids are vitamin A precursors and are used to fortify foods.
Finally, it should be emphasized that, when added to food, carotenoids are not powerful antioxidants.

Carotenoids and human health

In humans, carotenoids play important roles.
The most important role is to be precursors of vitamin A, which controls the expression of nearly 700 genes. Not all carotenoids are vitamin A precursors; only those with an unsubstituted beta-ionone ring are vitamin A precursors. The most important is beta-carotene which, having two unsubstituted beta-ionone rings, is converted into two molecules of the vitamin. Alpha-carotene and beta-cryptoxanthin, having only an unsubstituted beta-ionone ring, provide one molecule of vitamin A, hence, they have 50 percent provitamin A activity. The importance of provitamin A carotenoids is underlined by the fact that vitamin A deficiency can lead to night blindness and xerophthalmia, delay the growth and regeneration of the mucous membranes, increases the mortality rate caused by infectious diseases due to immune-related disorders, and is the single most important cause of childhood blindness in developing countries.
Carotenoids contribute to human health also by preventing the development of numerous chronic diseases through different mechanisms than provitamin A activity, such as antioxidant and anti-inflammatory actions, and photoprotection. Here are some examples. It has been observed that supplementation with lutein and zeaxanthin, two xanthophylls which accumulate in the macula lutea, is associated with an improvement in visual function and a reduction in the risk of progression of age-related macular degeneration. Lutein and zeaxanthin also seem to have a preventive effect on the development of cataracts and retinopathies, and lutein also a positive effect on cognitive functions. Among carotenes, the consumption of lycopene–rich foods has been associated with a reduction in the incidence of certain types of cancer, such as prostate and stomach cancer, and a reduction in cardiovascular risk.

Plasma carotenoids

Alpha-carotene, beta-carotene, lycopene, lutein, beta-cryptoxanthin and zeaxanthin make up more than 90 percent of the carotenoids present in plasma.
Carotenoid plasma concentrations depend on several factors including methods of cooking foods, the amount of lipids in the diet, individual variables regarding the processes of lipid digestion and lipid absorption, in which bile salts play an essential role, and vitamin A levels in the body. Considering the latter factor, a subject with low vitamin A levels may have a high conversion rates of provitamin A carotenoids, and this could be reflected in a lower plasma concentration.

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