Functional Properties of Carotenoids in Human Health

Abstract

Carotenoids are compounds of great dietery importance. Recent interest in carotenoids has been stimulated by epidemiological studies that strongly suggest that consumption of carotenoid-rich foods reduces the incidence of several diseases such as cancers, cardiovascular diseases, age-related macular degeneration, cataracts, diseases related to low immune function, and other degenerative diseases. Health benefits of carotenoids are derived from the fruits and vegetables in the diet, particularly from cooked products containing oil, or from supplements of their extracts, such as tomato sauce, dried tomatoes, or those suspended in oil. This article provides a brief overview of the chemistry of carotenoids—their absorption, transport, bioavailability, metabolism, and their action as antioxidants, and in the prevention of a number of common diseases.

Keywords:

• Carotenoids
• Human health
• Toxicity
• Bioavailability
• Nutrition
• Absorption and transport

INTRODUCTION

Carotenoids are often considered to be plant pigments, and they occur universally in the chloroplasts of all higher plants and algae. They are most obvious, however, when they occur in non-photosynthetic tissues, where they are responsible for the yellow, orange, and red colours of many fruits, flowers, and roots. Carotenoids are also pigments found in microbes, not only in photosynthetic membranes of photobacteria and cyanobacteria, but also in non-phototrophic bacteria and fungi. They are also important pigments in animals, especially birds (yellow and red feathers and egg yolk), fish and crustaceans (orange-red skin and flesh), and invertebrate animals, where in the free form or as complexes with proteins, they are responsible for a wide variety of colours. These animal carotenoids are, however, of plant origin, being assimilated from the diet.[1]

Early explorations of carotenoids stemmed from curiosity of the appealing yellow, orange, and red colours in fruits and vegetables. But recent interest in carotenoids has been stimulated by epidemiological studies that strongly suggest that consumption of carotenoid-rich foods reduces the incidence of cancers,[2,3] cardiovascular diseases,[4,5] age-related macular degeneration (AMD),[6] cataracts,[5,7] diseases related to low immune function[4,8,9] and other degenerative diseases.

To be able to have any beneficial action, the carotenoids must be absorbed, transported, and deposited in certain tissues. All carotenes and many xanthophylls, could be absorbed by humans. Although about 90% of the lycopene in dietary sources is found in the linear, all-trans conformation, human tissues contain mainly cis-isomers.[10] Several research groups have suggested that cis-isomers of lycopene are better absorbed than the all-trans form because of the shorter length of the cis-isomer, the greater solubility of cis-isomers in mixed micelles, and/or as a result of the lower tendency of cis-isomers to aggregate.[11,12]

It seems that the epoxyxanthophylls such as violaxanthin and neoxanthin, which are abundant in plants particularly in green tissues, cannot be incorporated into and used by the human body. This brings up the important aspect of the bioavailability of dietary carotenoids, which depends on other dietary factors, especially the contribution of dietary fats. Carotenoids may be absorbed more efficiently from cooked than from uncooked foods, probably because cooking and processing liberates the carotenoids from their usual molecular environment, making them more accessible for solubilization.[13,14,15]

Research in the last 70 years has shown that the carotenoid pigments present in fruits and vegetables are the main dietary source of Vitamin A for most people, especially those in poorer countries, where Vitamin A deficiency is a serious problem. Beta–carotene is the main compound with provitamin A activity.[16] When incorporated in the diet, it may be broken down into two molecules of retinol (Vitamin A) by the action of the enzyme β-carotene-15, 15′-dioxygenase in the intestine. However, β–carotene is not the only carotenoid with provitamin A activity and any carotenoid with at least one unsubstituted β-ring can undergo similar cleavages. Current attention is centered on the action of β-carotene as an antioxidant, as it may interfere in free radical oxidation (such as the peroxidation of lipids), typical of many degenerative diseases.

In short, carotenoids are compounds of great dietery importance not only as precursors of Vitamin A, but also as molecules that take part in cell protection and consumer attraction due to the visual colour they provide to food. This article provides a brief overview of the chemistry of carotenoid, their absorption, transport, bioavailability, metabolism, and their action as antioxidants, as well as in the prevention of a number of common diseases.

CAROTENOIDS — CHEMISTRY AND OCCURRENCE

Carotenoids are tetra-terpenoid C₄₀ compounds because they are formed by eight isoprenoid (IP) units. The IP units are joined in a head-to-tail pattern, but the order is inverted at the molecular center. The two central methyl side chains are thus in a 1, 6-positional relationship and the remaining non-terminal methyl groups are in a 1, 5-positional relationship. All carotenoids (i.e., carotenes and xanthophylls) may be derived from the acyclic (C₄₀H₅₆) lycopene by (a) hydrogenation, (b) dehydrogenation, (c) cyclization, (d) oxygen insertion, (e) double bond migration, (f) methyl migration, (g) chain elongation, and (h) chain shortening.

Carotenoids are traditionally grouped into (a) carotenes and (b) xanthophylls. Carotenes (e.g., β-carotene from carrots and lycopene from tomatoes) are strictly hydrocarbons. Xanthophylls are oxygenated carotenoids, containing alcohol, carbonyl, or other functional groups. The xanthohylls, lutein, and capsanthin are widely used as colourants and nutrients in the food and animal feed industries. These carotenoids are typically derived from marigold (Tagetes erecta L.) flowers and paprika (Capsicum annuum L.), respectively. Epoxy carotenoids (e.g., violxanthin and antheraxanthin) comprise a major group of xanthophylls, but the physiological significance of which is unclear. Those carotenoids lack an end group and with the carbon skeleton shortened are called apocarotenoids.

Structurally, the carotenoids may be acyclic (e.g., lycopene) or contain a ring of six carbons at one or both ends of the molecule (e.g., β-carotene). Traditionally, trivial names have been given to carotenoids after discovery, which in most cases refers to the natural sources from which they have been isolated, for example, zeaxanthin from maize (Zea mays) and lycopene from tomato (Lycopersicon esculentum L.). Because this system does not include any structural information, a semi-systematic nomenclature was defined that gives structural information and additional reference to the parent carotene. This system was approved by the International Union of Pure and Applied Chemists (IUPAC), which has published in a compendium of rules for the nomenclature of carotenoids.[17,18] Greek letters are used to designate the end groups that may be present in the carotenoid molecule.

The presence of a large number of conjugated double bonds in the carotenoid molecule makes it possible to have numerous geometric isomers. In practice, however, most carotenoids are naturally present in the trans form (E). A few are present naturally in cis form (Z) such as bixin in annatto seeds (Bixa orellana) or pro-lycopene (a carotenoid with several double bonds and Z-configuration) present in certain varieties of tomatoes.

Carotenoids are widespread among living organisms, including both plants and animals, but they are found in greater concentration and variety in plants, as well as certain bacteria, which are the main organisms that are able to synthesize them.

In tomatoes, peppers, and citrus fruits, carotenogenesis is closely associated with the ripening process. During the process of ripening, there are both qualitative and quantitative changes in pigment synthesis. It should be noted that a number of changes takes place as plants mature: (a) destruction of chlorophyll, (b) unmasking of other pigments and (c) denovo synthesis of carotenoids and anthocyanins.

Carotenoids are abundant in green plant tissues, but because of the presence of the green cholorophyll, their colour is mostly masked. The predominant carotenoids are the β‐carotene, lutein, violaxanthin and neoxanthin. Zeaxanthin, γ-carotene, β-cryptoxanthin, and antheraxanthin are also found in small amounts.

In fruits, xanthophylls are normally found in greater amounts than carotenoids. For instance, the predominant pigments in maize are lutein and zeaxanthin, while in mango (Mangifera indica) and persinmmon (Diospyros kaki), β-cryptoxanthin and zeaxanthin predominate. An exception is tomato in which, the major pigment is a carotenoid (lycopene). Certain carotenoids are limited to a single plant species. Capsanthin and capsorubin are found almost exclusively in ripe fruits of the genus Capsicum and are responsible for their attractive red colours.[19,20] The orange (Citrus sinensis) contains varying amounts of β‐citraurin and β-citranaxanthin (both apo-carotenoids), together with β-cryptoxanthin, lutein, antheraxanthin, violaxanthin and traces of their carotene precursors.[21]

Xanthophylls are found in the free form in green plant tissues of the vegetables in our diet. However, in non-dietary green plant tissues, due to leaf senescence and ripening, coinciding with the transformation of chloroplasts into chromoplasts, the carotenoids undergo esterification with different fatty acids. Esterification does not alter chromatic characteristics of the carotenoids. Moreover, esterification is related to the capacity of the plant (in particular fruits and flowers) to overproduce and accumulate carotenoids. The change in esterification profile of the xanthophylls in fruits of red pepper has been proposed recently as a ripening index.[22]

The carotenoids are lipophilic substances and thus insoluble in aqueous medium, except in certain cases where highly polar functional groups are present, as in norbixin, a carotenoid with dicarbonyl acid structure.

The structural difference between bicyclic β-carotene and acyclic lycopene resides in the fact that β-carotene can be metabolized into a series of shortened molecules, culminating in the formation of retinoic acid by an excentric cleavage pathway or to retinal by a central cleavage pathway.

The presence of the long, extensive system of conjugated double bonds (or polyene chain) is responsible for one of the most distinctive characteristics of the carotenoids, which is light absorption. A chromophore with seven or more double bonds gives the capacity of absorbing light in the visible range and consequently, the observation of colours spanning from yellow to red via a great variety of orange tones. Moreover, the polyene chain makes the carotenoid molecule extremely susceptible to isomerizing and oxidizing conditions like light, heat or acids.

The properties of carotenoid pigments in vitro may be different to those in vivo because of the interaction with the physiochemical environment (mainly lipids and proteins) surrounding the pigments. This can be particularly critical regarding the functionality and action of the carotenoids in vivo.

ABSORPTION AND TRANSPORT OF CAROTENOIDS

Recently, it has been shown that the absorption of certain carotenoids is not passive — as believed for a long time — but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1).[23,24] A number of genes involved in vitamin A transport and metabolism have been recently identified.[25,26] The rate limiting steps in the lymphatic absorption of vitamin A involve intracellular processing of vitamin A within the enterocyte. The key steps appear to be related to chylomicron formation and secretion and are closely coupled with fat absorption.[27]

Borel, Drai, Faure, Fayol, Galabert, Laromiguière, and Le Moël[28] reviewed our current knowledge about intestinal absorption and cleavage of carotenoids. New facts about carotenoid absorption have emerged, while some controversies about cleavage are close to an end. Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, — such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject.[24]

The precise mechanism of beta-carotene cleavage by β-carotene 15,15′ monooxygenase (EC 1.14.99.36) [formerly called β-carotene 15,15′ dioxygenase (EC 1.13.11.21)] has been discovered recently, and a second enzyme, which cleaves the β-carotene molecule asymmetrically also has been found.[29,30] β-carotene 15,15′ monooxygenase only acts on the 15,15′ bond, thus forming two molecules of retinal from one molecule of β-carotene by central cleavage. A study has shown that this enzyme can act on all carotenoids.[29] Researchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the ββ-carotene 15,15′ monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However, the conversion of provitamin A carotenoids into vitamin A is only partial, and requires a satisfactory protein status.[28]

To date, there are more than 600 naturally occurring carotenoids, of which 50 to 60 have been found in foods commonly consumed by man.[31,32] The most common carotenoids found so far in man include, lutein, zeaxanthin, β-cryptoxanthine, lycopene, α‐and β-carotene, and their geometric isomers.[33,34]

Since humans and animals are unable to synthesize carotenoids in their bodies, they obtain them exclusively from the diet. Dietary carotenoids in food exist in 2 major forms: (a) as true solutions in oil (i.e., lipid droplets) or (b) as parts of matrices within the fruit or vegetable (i.e., pigment-protein complexes in a variety of plant cell structures).[16]

Due to their highly lipophilic property, carotenoids follow the same absorptive pathway as other lipids. Absorption is defined as movement of dietary carotenoids or metabolites of carotenoids to the lymphatic or portal circulation.[35]

The extraction of carotenoids from fruits and vegetable matrices begins in the stomach, where it transfers to the lipid fraction.[36] The efficiency of transfer depends on the characteristics of the matrix, lipophilic properties of the carotenids, pH of the medium,[37] and characteristics of the lipids (long or short-chain, saturated or unsaturated).

Carotenoid absorption involves: (a) digestion of food matrix, (b) dispersion in lipid emulsion particles, (c) formation of bile salt micelles, (d) transport across the unstirred water layer (UWL) to intestinal epithelial cells, (e) repackaging of carotenoids into chylomicrons within the epithelial cells, and (f) plasma transport of carotenoids and/or their metabolites.

Digestion of Food Materials

The stomach is important in initiating lipid digestion. Aproximately 20% of ingested triacylglycerols (TAGs) are hydrolyzed in the stomach by gastric lipase. In addition, gastric muscle contractions, gastric acidity and pepsin, mash food particles, and release dietary carotenoids from their matrix and protein interactions.

Dispersion of Carotenoids in Liquid Emulsion Particles

Once the carotenoids are released, the lipophilic carotenoids would dissolve in the oily phase of lipid droplets. Mixing and shearing forces from normal digestive-tract-movement bring about the formation of a fine lipid emulsion and the contents of the stomach pass into the duodenum.

The emulsion has a TAG core surrounded by a mono-molecular layer of partially digested proteins, polysaccharides and lipids, especially phospholipids and partially ionized fatty acids.[38,39]

The solubility and location of the polar carotenoids (xanthophylls) and the non-polar carotenoids (carotenes) in the emulsion differ.[40] Carotenes are thought to be incorporated almost exclusively in the TAG core of the emulsion, whereas the more polar xanthophylls distribute preferentially at the emulsion surface.[40] Other lipid-soluble nutrients with polar groups such as α-tocopherol and trans-retinoic acid are also thought to locate at the droplet surface.[38] The significance of the location in an emulsion is that the surface components can spontaneously transfer from lipid droplets to micelles, whereas components associated with the emulsion core require digestion of TAG before transfer.[40]

The enzyme best suited to hydrolyze TAGs in emulsions is pancreatic colipase-dependent lipase,[39] which is one reason why pancreatic insufficiency decreases plasma carotenoid concentration.[41]

Formation of Bile Salt Micelles

A major difference between absorption of other dietary lipids and carotenoids is that the carotenoids seem to have an absolute requirement for bile salt micelles.[42,43] However, the fatty acids can be absorbed in the absence of micelles.[38]

In the duodenum, the emulsion particles are further stabilized by the addition of bile salts and phospholipid lecithin, both of which, is important for the emulsification of fats and carotenoids. However, the transfer of lipophilic compounds from emulsion particles to bile salt micelles is not fully understood.

The polar portions of the bile salts and lecithin molecules are highly soluble in water, whereas most of the remaining portions of their molecules are highly soluble in fat. Therefore, the fat-soluble portions of these bile salts and lecithin molecules dissolve in the surface layer of the fat globules, with the polar portions projecting outwards. The water-soluble bile salt micelles thus act as a transport medium to carry the carotenoids and other intraluminal lipids, which would otherwise be relatively insoluble, to the brush borders of the intestinal epithelial cells.[43]

The polar parts being soluble in the surrounding aqueous fluids, greatly decrease the interfacial tension of the fat. Under these conditions, the globular lipid particles can be broken up into many minute particles easily in the small intestine. Each time the diameters of the fat globules are significantly decreased as a result of agitation in the small intestine, the total surface area of the fat increases many times.[43]

The solubility of carotenoids in bile salt micelles is limited and varies with intraluminal concentration of carotenoids. Canfield, Fritz, and Tarara[44] studied the incorporation of β-carotene into bile salt micelles designed to resemble those seen in the lumen of small intestine. While the solubility of carotenoids differ in emulsions, the overall solubility of polar and non-polar carotenoids are similar in bile salt micelles.[40]

Depending on their polarity, carotenoids may be solubilized independently into different regions of the bile salt micelles. El-Gorab and Underwood[45] found that β-carotene was soluble in the hydrophobic core of the micelle, whereas retinol was incorporated into the surface. Rather than competing for solubility into bile salt micelles, retinol expanded the micelle and enhanced β-carotene solubility into the internal core of the micelle. Additional research is needed to determine if the polar xanthophylls and non-polar carotenes solubilize independently into bile salt micelles, and how they influence each other's incorporation into bile salt micelles.

Transport Across the Unstirred Water Layer to Intestinal Epithelial Cells

Bile salt micelles must pass through a 40 μm deep unstirred water layer (UWL) at the surface of intestinal epithelium to deliver their contents to the apical portion of the enterocytes.

It was demonstrated by Westergaard and Dietschy,[46] that movement through the UWL was rate-limiting for absorption of lipids, whereas passage through the microvilli-membrane was very rapid. The bile salt micelles serve as a reservoir for carotenoids and other lipids, which then move across the UWL as monomers down a concentration gradient to the brush border membrane. This model makes it easy to visualize the polar xanthophylls diffusing through the final aqueous phase to the microvillus surface but difficult to envision the movement of the very hydrophobic nonpolar carotenes, although it might be similar to the movement of lipids.

Although the mechanism of carotenoid transfer from the bile salt micelles through the microvilli membrane is unclear, there is general agreement that the rate of transfer is dependent on intramicellar concentration of carotenoids and that the carotenoids are taken up unchanged by the mucosal cell.[28]

Repackaging of Carotenoids into Chylomicrons within the Epithelial Cells

Once the carotenoid is inside the enterocyte, its fate depends on its structure. Still unsolved is how the flow of hydrophobic carotenoids within the enterocyte is controlled. However, it was found that dioxygenase and celular retinol binding protein [CRBP (II)] are regulated by the same mechanism involving long-chain fatty acids and their metabolites.[47]

Humans are somewhat unique in that they can cleave provitamin A active carotenoids to Vitamin A within the intestinal mucosal cells or they can absorb a whole variety of carotenoids intact. Most species do not absorb carotenoids intact. The ferret and the pre-ruminant calf absorb carotenoids intact and are appropriate animal models to study transport and metabolism of carotenoids.[48,49] If a carotenoid contains an unsubstituted β‐ionone ring with a polyene side chain of at least 11 carbon atoms, it can be cleaved enzymatically to Vitamin A.[50] It is clear from a number of animal studies that as dietary carotene levels increase, the efficiency of their conversion to Vitamin A is substantially decreased. This suggests that regulatory mechanism(s) are in place to limit mucosal cell uptake, transport, or conversion of vitamin A when dietary carotenes are high or when Vitamin A status is adequate.[35]

Most carotenoids and retinyl esters are incorporated into water-soluble, lipoprotein particles called chylomicrons (CM) within the epithelial cells.[51] Cellular events that regulate or facilitate the incorporation of carotenoids into CM are not yet understood. However, some carotenoids and/or their metabolites are directly absorbed from the small intestine into the portal circulation. There is also a possibility of enterohepatic circulation of carotenoids, as biliary secretion of carotenoids was reported in humans.[41]

Plasma Transport of Carotenoids and/or Their Metabolites

Newly absorbed carotenoids, together with retinyl esters and small amount of retinols are transported on CM from the intestinal mucosa via lymphatic system into the general circulation.[51]

Chylomicrons containing exogenous TAGs and cholesterol adhere to binding sites on the inner surface (endothelium) of the capillaries in skeletal muscle and adipose tissue. The TAG component of CM is hydrolysed by extracellular lipo-protein lipase. The tissues then take up the liberated fats. But it is unclear whether there is a transfer of carotenoids from CMs to extrahepatic tissues prior to uptake of CM remnants by the liver.[35]

The CMs shrink in size as their component TAGs are progressively hydrolysed until they are reduced to cholesterol-enriched remnants.[50] The CM remnants retaining apolipoproteins B48 and E on their surface will interact with receptors on hepatocytes and be taken up by the liver after dissociating from the capillary endothelium. A small amount of the CM remnants is also takenup by other tissues. The hepatocytes incorporate much of the remnants’ internalized carotenoids into lipo-protein particles.[16]

Krinsky, Cornwell, and Oncley[52] were among the first to determine the distribution of carotenoids among the various lipoprotein classes. Their results were also supported by later studies.[53,54,55] Hydrocarbon carotenoids predominate in the core of very low-density lipo-proteins (VLDLs) and low-density lipo-proteins (LDLs). Whereas xanthophylls are distributed approximately equally between high density lipo-proteins (HDLs) and LDLs in human serum. Parker[56] suggests that the actual content of β-carotene per unit lipid may be greater in HDLs than in LDLs. He also mentioned that the surface area of LDLs is almost twice that of HDLs but the content of xanthophylls (lutein and zeaxanthin) is greater in HDLs than in LDLs. This apparent favour of xanthophylls for HDLs over LDLs has yet to be explained.

The kind of distribution accords with the hydrophobicity of the carotenoids and lipo-proteins and was not affected by high dietary carotenoid or supplement intakes or prolonged dietary depletion.[57] This might be due to the small fraction of carotenoid content in the mass of the lipoprotein particle.[50]

In-vitro studies have shown that carotenoids do not transfer from one lipoprotein class to another.[58,50] Carotenoids are thus involved in a complex and probably cyclic metabolic pathway involving the intestinal chylomicrons, the liver, plasma lipoproteins, and peripheral tissues.

PLASMA AND TISSUE CAROTENOID CONTENT

Willstaedt and Lindqvist[59] studied the carotenoid content and relative concentrations in human blood as early as 1936. It should be emphasized that carotenoids are non-covalently bound to lipoproteins and are not homeostatically controlled. Their plasma concentrations thus, depend mainly on amounts in the diet. When subjects were fed a diet deficient in carotenoids, plasma carotenoid levels decreased.[60] Other factors affecting plasma carotenoid concentrations include intestinal absorption efficiencies, tissue uptake, release of carotenoids from the tissues into the plasma, and their catabolic rates.

Lutein, lycopene, zeaxanthin, β-cryptoxanthin, β-carotene, and α-carotene comprise 60 to 70% of the total plasma carotenoid content.[32,61] Although the distribution and amount of carotenoids in individuals differ much, each person maintains a fairly constant pattern for at least a month, reflecting a fairly uniform diet during that period, abetted by the presumed buffering effect of tissue carotenoid concentrations.

Carotenoids are found in all tissues in the adult human body, mainly in fatty tissues, liver, and the plasma.[62] Human skin is known to accumulate β-carotene, lutein, lycopene, and canthaxanthin.[63] High carotenoid intake, particularly β-carotene canthaxanthin may result in carotenodermia.[64]

Around 90% of the carotenoids in the body are found in the tissues and around 10% in the plasma. In nearly all tissues studied, the concentrations of lycopene and β-carotene were the highest, lutein/zeaxanthin were intermediate, while cryptoxanthin and α-carotene were the lowest.[11,65,66,67]

Tissue concentration of carotenoids in most instances showed the same distribution pattern as in plasma from the same population group. Some exceptions are the preferential acculmulation of β-carotene in the pineal gland,[68] the corpus luteum and lutein/zeaxanthin in the macula of the eye.

CAROTENOID BIOAVAILABILITY IN HUMAN SUBJECTS

The molar equivalency of retinol, the active form of vitamin A, of small amounts of β-carotene in oil is approximately 0.5,[69] whereas that of carotenoids in rapidly stir-fried vegetables is very poor (< 0.05).[70] Carotenoids in fruits seem to be better utilized than those from vegetables. This problem in defining a general factor for converting provitamin A carotenoids to Vitamin A is not new. The FAO/WHO[71] suggested that 6 μg of all-trans-β-carotene or 12 μg of all-trans-provitamin A carotenoids in food is equivalent to 1 μg of all-trans-retinal. Most national and international committees have used the same values due to absence of precise information about this problem. (See section 6.4 for recent update on these figures).

To date, it is still not possible to assign actual Vitamin A values to specific plant foods consumed within a meal context nor is it possible to assign specific values to the absorption efficiency of a specific carotenoid from a specific food or preparation.[72]

In many cases, the debate has been confused by the various meanings of the term “bioavailabilty.” This is because it can be addressed in various ways, depending on whether the issue is the Vitamin A value of the carotenoid or the carotenoid per se. In this article, the term “bioavailabilty” will refer to the absorption of carotenoid per se, i.e., proportion of carotenoids ingested, which becomes available to the body for metabolic processes. Data generated from a promising cell culture model for studying intestinal absorption of carotenoids suggest the participation of a specific epithelial transporter.[73] The identification of such transporters in humans represents an exciting challenge in carotenoid research to understand fully the intestinal absorption and transport process of those compounds.

FACTORS AFFECTING BIOAVAILABILTY OF CAROTENOIDS

In addition to knowing the amount of individual carotenoids present in food or formulations it is important to know the bioavailabilty of carotenoids with respect to the person or experimental animals in question. Several environmental, dietary, physiological, and matrix factors influence the bioavailability of carotenoids from food or formulations have been summarized by de Pee and West.[70] These factors are discussed below. Furthermore, a quantification of these factors in human subjects would enable better prediction of carotenoid bioavailabilty from certain foods under specified circumstances.

Species of Carotenoids

The bioavailability of hydrocarbon carotenes such as β-carotene is relatively lower than that of the oxygenated xanthophylls such as lutein and zeaxanthin. Due to their polar nature, the xanthophylls are more easily incorporated into the outer portions of bile salt micelles within the gastrointestinal tract and can be easily taken up by enterocyte membranes and eventually chylomicrons, thus increasing their bioavailability. This is supported by the work of van het Hof, Brouwer, West, Haddeman, Steegers-Theunissen, van Dusseldorp, Westrate, Eskes, and Hautvast,[14] who showed that the absorbability of lutein from vegetables was 5 times than that of β-carotene. The high relative absorbability of lutein compared with that of β-carotene is also in line with the findings of Johnson, Hammond, Yeum, Qin, and Wang.[74]

Isomeric Forms

Several geometric isomers of carotenoids exist in food and human tissues. The major β-carotene isomer in human plasma circulation is the all-trans- β-carotene, with smaller amounts of 13-cis and 9-cis β-carotene.[75] However, circulating levels of cis β‐carotene are not responsive to increased consumption of that isomer.[11] Recent studies on the serum response to a single large oral dose of either all-trans-β-carotene or 9-cis β‐carotene in men indicated that the all-trans isomer attains a far greater postprandial concentration than the cis isomer.[76] You, Parker, and Goodman,[77] using stable isotopes, showed that substantial proportions of oral doses of 9-cis β-carotene can undergo isomerization to all-trans-β-carotene between ingestion and appearance in plasma.

Many factors like heat (cooking), light and pH can cause isomerization of the trans form to the cis. The bioavalilability of isomers of other carotenoids and their importance to human health and disease remains to be explored.

Carotenoid Esters

Many orange-coloured fruits contain beta-cryptoxanthin in its non-esterified, as well as its esterified form. β-cryptoxanthin are of particular interest, since they are a major source of Vitamin A, often only secondary to β-carotene. Cryptoxanthin and other carotenoids like zeaxanthin and lutein are found in considerable amounts in human plasma and tissues but they occur only in their free form and not in the esterified form. In a recent study, Breithaupt, Weller, Wolters, and Hahn[78] found that subjects fed native beta-cryptoxanthin esters from papaya (Carica papaya L.) or non-esterified beta-cryptoxanthin in equal total amounts had the same increased levels of plasma beta cryptoxanthin concentration after 6 to 12 hours. Their study indicates comparable bioavailability of both non-esterified beta-cryptoxanthin and mixtures of beta-cryptoxanthin esters. The results support the existence of an effective enzymatic cleavage system accepting various beta-cryptoxanthin esters.

Lutein is a yellow carotenoid widely distributed in plants and in animal tissues. The lutein molecule is often found in nature esterified with fatty acids. Tests of the bioavailability in healthy humans of supplements of lutein (as crystals in an oil suspension) and lutein esters (as powder) indicated that both forms are equally well absorbed at intakes of about 20 mg per day.[79,80] The solubility properties of the formulations were found to be important factors.[81] The optimal absorption of lutein esters may require more fat in a meal than is needed for carotenes or vitamin E.

Amount of Carotenoids

The efficiency of β-carotene absorption from the diet is higher at low dose levels than at high dose levels.[35] This is also true for lycopene.[11] Absorption of carotenoids appears to be linear at dosages up to 20 – 30 mg but becomes limiting at higher levels because of factors like solubility.[40] The retinol equivalency for β-carotene and other provitamin A carotenoids differ much and are recently summarized in Table 1.

Table 1 The retinol equivalency for β-carotene and other provitamin A carotenoids differ much and were recently summarized in Higdon.[82]

Retinol Activity Equivalency (RAE) for β-carotene and other provitamin A carotenoids
Quantity consumed	Retinol equivalency	RAE ratio
1 mcg of dietary or supplemental vitamin A	1 mcg of retinol∗	1:1
2 mcg of supplemental beta-carotene	1 mcg of retinol	2:1
12 mcg of dietary beta-carotene	1 mcg of retinol	12:1
24 mcg of dietary alpha-carotene	1 mcg of retinol	24:1
24 mcg of dietary beta-cryptoxanthin	1 mcg of retinol	24:1
∗One International Unit (IU) is equivalent to 0.3 mcg of retinol, and one mcg of retinol is equivalent to 3.33 IU of retinal (Adapted from Higdon[82]).

Food Matrix

The matrix in which carotenoids are embedded in a food source is important in determining its bioavailabilty. Release from the physical matrix and the timely solubilization in the lipid droplets during the early stages of digestion are initial steps to carotenoid absorption. It is widely accepted that (a) carotenoids from commercial preparations such as, from oil or emulsion preparations are more bioavailable than those found in fruit and vegetable matrix (b) carotenoids are generally less bioavailable from raw than from processed fruits and vegetables.[51]

In some plant food materials, carotenoids occur in crystalline form, such as α- and β-carotene in carrots and lycopene in tomatoes, while in orange and yellow fruits, carotenoids are dissolved in oil droplets. In dark green vegetables, such as spinach, carotenoids are present in protein complexes.[14,83] These carotenoproteins have inhibitory effect upon carotenoid digestion and absorption.[51]

It was shown by Riedl, Linseisen, Hoffmann, and Wolfram,[84] that some dietary fibres reduce the absorption of carotenoids in healthy young women.

Food Processing

Cooking and mechanical homogenization increases the bioavailability of carotenoids, the mechanism by which this occurs is most likely due to the release of the carotenoid from the food matrix and from protein complexes.[85,86] Dietz, Sri Kantha, and Erdman[87] reported an increase in extractability of carotenoids in carrots after steaming. However, prolonged exposure to high temperatures (boiling) reduced the bioavailability of carotenoids by increasing the oxidation and production of more isomers. Therefore, though destruction of matrix can reduce matrix effect, excessive processing could also destroy carotenoid molecules. An optimum balance must be reached between maximal destruction of matrix and minimal destruction of carotenoid molecules.

The improved effect on bioavailability by processing may not occur for xanthophylls.[88] In contrast, van het Hof et al.[14] showed significantly higher plasma responses of lutein after ingestion of chopped spinach than after ingestion of whole-leaf spinach for 4 days.

CAROTENOID METABOLISM

There is very little information on the metabolic fate of non-provitamin A carotenoids that are not metabolised to retinol. It is generally assumed that they undergo oxidation (photobleaching in the skin), cleavage and polyene chain shortening by a process analogous to β-oxidation of fats and that the unmetabolized remnants are detoxified in the liver by the addition of sugar residues and are eliminated in the feces and urine.[38] The observation that large intakes of carotenoids can result in yellowing of the skin might suggest that the skin is a significant excretory mechanism.[64] Many aspects of the metabolism of retinoids in vertebrates remain controversial and poorly understood. Because few chemical reactions are possible for this group of compounds, furthering our knowledge of isoprenoid transformation in plants could be beneficial to our understanding of how retinoids and carotenoids are transformed in vertebrates.[89]

Metabolic Transformation of β-Carotene

That β-carotene is converted biologically into Vitamin A in mammals was first shown in 1930.[90] For many years, the pathways for its conversion were unclear, largely because the conversion rate is relatively slow and cell-free preparations of tissues were inactive, β-carotene was rapidly oxidized chemically to various derivatives and the resolving power and/or sensitivity of available methods was limited.

It was known that retinal was the sole metabolic product of oxidation of β-carotene and the enzyme responsible was carotenoid 15, 15′-dioxygenase (EC 1.13.11.21). Retinals can undergo modifications involving oxido-reduction, dehydratation rearrangement, and glycosylation.[91] Thus the central cleavage was deemed to be the major pathway in oxidation of β-carotene in mammals.[27,92]

Metabolic Transformation of Lutein and Zeaxanthin

Metabolic pathways leading to the formation of lutein and zeaxanthin metabolites detected in human serum were summarized by, Khachik, Englert, Beecher, and Smith Jr.[93] The driving force for the metabolic conversion is the oxidation of the allylic hydroxyl group of lutein to form 3-hydroxy-β,∊-caroten-3′-one which in turn undergoes reduction either with retention of configuration to revert to lutein or with epimerization at C-3′ to form 3′-epilutein. The results from 2 human trials have confirmed that although ingestion of purified lutein results in an increase in the plasma zeaxanthin concentration, the concentration level of 3′-epilutein remained constant.[94] This suggested that after 3′-epilutein formation, the compound undergoes either double bond migration giving zeaxanthin or allylic oxidation forming 3-hydroxy- β,∊-caroten-3′-one. The absolute configuration of the metabolites of lutein and zeaxanthin (mono-and diketo-carotenoids), which can determine the most probable pathway for these oxidation reactions in humans is still not known. The possible metabolic pathway leading to lutein dehydration products in humans were previously described by Khachik et al.[93] The presence of the direct oxidation product of lutein and 3′-epilutein (metabolite of lutein and zeaxanthin) in human retina suggests that lutein and zeaxanthin may act as antioxidants to protect the macula against short-wavelength visible light.[95]

Metabolism of Other Common Carotenoids

α-carotene is cleaved to retinal and α-retinal, presumably by carotenoid 15–15′-dioxygenase.[96] Lycopene, although absorbed well from oily solutions and taken up by the liver and other organs, is metabolized in poorly understood pathways.[97] Many isomers of lycopene are present in human plasma and tissues, of which the major ones are all-trans and 5-cis lycopene.[97] Canthaxanthin, is not metabolized to detectable products in rats and squirrel monkeys or humans.[98] In chickens, however, a portion is reduced to the mono- and dihydroxy derivatives, which in turn are acylated.[99] Capsanthin a major and exclusive red ketocarotenoid in paprika is well-absorbed when orally administered to men. It is associated equally with HDL and LDL in plasma and is cleared rapidly from circulation[100] but no metabolites of capsanthin have yet been identified. Recently, Perez-Galvez, Martin, Sies, and Stahl[101] showed that the bioavailability of the pepper-specific carotenoids capsanthin and capsorubin from paprika oleoresin was very low. However, oleoresin from paprika is a suitable source for the provitamin A carotenoids beta-carotene and beta-cryptoxanthin and the macular pigment zeaxanthin.

9′-cis-bixin, a methyl ester of the dicarboxylic acid 9′-cis-norbixin and its congeners are found in annatto seeds. Extracts of annatto are commonly used as food colorants. When 9′-cis-bixin is ingested by human volunteers, it is also well absorbed and rapidly cleared from plasma.[102] 9′-cis-bixin is demethylated to dicarboxylic acid norbixin and isomerized to all-trans bixin in vivo.[102] Geometric and optical isomers of astaxanthin have been reported in the plasma of middle-aged men.[76] Epoxide carotenoids - lutein epoxide and violaxanthin, were predominant in fatty tissues of both malignant and benign tumors. Epoxide carotenoids-mutatoxanthin and lutein epoxide and other carotenoids such as zeaxanthin, canthaxanthin, lutein, and neoxanthin were predominant in neoplastic material. β-carotene and lutein epoxide were found in all tissues, alpha carotene was found in 50% of them. Antheraxanthin was present in fatty tissue only.[103] Other carotenoids such as neoxanthin and violaxathin, though found in green vegetables, have not been detected in human plasma.[31]

CAROTENOIDS AS ANTIOXIDANTS

The sun provides energy necessary for life on earth and its electromagnetic spectrum covers γ- and x-ray, ultraviolet (UV) and visible light, as well as infrared (IR) and microwave radiation. UV and visible radiation play a particularly important role as far as photo-oxidative stress and diseases related to sun exposure are concerned. This includes the spectrum from 280–750 nm that comprises UVB (280–315nm), UVA (315–380 nm), and visible light (380–750 nm). Most of the UV light is absorbed by the ozone layer of the atmosphere and less than 4% of the UVB intensity reaches the earth's surface. However, the thickness of the shielding ozone layer is decreasing; therefore, exposure to UV light increases. UV light is directly damaging to living organisms by inducing chemical reactions with relevant biomolecules.[104]

Visible light drives photosynthesis that is essential for converting radiation energy. However, visible light also causes damage resulting from physico-chemical processes, resulting in reactive oxygen species being formed in light exposed tissues. The modification of biologically important molecules in photo-oxidative reactions has been associated with pathological processes in the development of several diseases of light-exposed tissues, including cataract, age-related macular degeneration (AMD), skin cancer, skin aging, or skin erythema formation.

Biological oxidants arise from environmental sources, for example, in the skin from exposure to ultraviolet light. They are also produced endogenously. Two major sources are mitochondria and phagocytic cells. Mitochondria generate superoxide during ordinary oxidative metabolism, probably at the ubiquinone-cytochrome b level of the mitochondrial electron transport chain.[105] The superoxide radical can be damaging in itself, but also has the potential to produce the extremely destructive hydroxyl radical.[106]

Superoxide can dismutate to produce hydrogen peroxide. Hydrogen peroxide is subject to Fenton chemistry in the presence of transition metal catalysts to produce hydroxyl radical. Phagocytic cells also produce superoxide and hydrogen peroxide during their oxidative burst and in addition, they produce nitric oxide and hypochlorous ions. They may be an important source of oxidants, especially in chronic infections.[107] Hydroxyl radicals react with vulnerable sites on lipids, proteins and DNA at a rate, which is essentially diffusion-limited.[108]

Carotenoids are one of a variety of biological antioxidant compounds. They are singlet oxygen scavengers and many of them are interlinked in cycles of regeneration and recycling.[109] In the human organism, carotenoids are part of the antioxidant defense system. They interact synergistically with other antioxidants; mixtures of carotenoids are more effective than single compounds.[110]

Carotenoids and Singlet Oxygen

Transitions between energy states are associated with changes in the electron configuration of the molecule, which may influence its reactivity with other species. In the singlet oxygen state (¹O₂), all valence electrons are paired and have opposite spins. Therefore, electrostatic repulsion will be great, resulting in an excited state of ground molecular oxygen, which has a relatively long excited state lifetime; long enough to react with other moieties of high electron density such as C = C bonds. The resulting hydroperoxides can then cleave to initiate a conventional free radical chain reaction.

Singlet oxygen can act as a DNA damaging agent.[111,112,113] This oxidant can react with deoxyguanosine and with guanine in DNA leading to induction of at least four different DNA base modifications constituting premutational lesions. The induction of single-stranded breaks in the oxidized DNA in aging of human Retinal Pigment Epithelial (RPE) cells has been established recently.[114] DNA strand breaks but not oxidized DNA base damages, blocked transcription by RNA polymerase II.[115] Antioxidants that reduce gene damage also reduce cell death.[116]

Carotenoids scavenge singlet molecular oxygen by physical or chemical quenching.[117,118] Singlet oxygen quenching is dependent on the carotenoid incorporated into the tissues; xanthophylls exhibit a marked reduction in efficiency compared to the hydrocarbon carotenoids. Lycopene and β-carotene exhibit the fastest singlet oxygen quenching rate constants with lutein the least efficient.[119]

Interaction of Carotenoids with Free Radicals

As well as the ability to quench excited states, carotenoids can also react with free radicals. However, unlike the quenching of singlet oxygen, which mainly leads to energy dissipation as heat, the reactions of a carotenoid with a free radical will lead to electron transfer or possibly addition reactions. The major fact to note is that the “odd” electron, which characterizes a free radical is not lost. Thus for a free radical (R∗) reacting with a carotenoid (CAR), reactions shown below are expected, depending on the redox potentials of the species involved.

and

Furthermore, carbon-centered radicals are known to react readily (by addition), with oxygen, giving peroxyl radicals.

Thus, it is to be expected that antioxidant properties of carotenoids and other antioxidants, may well depend on the oxygen concentrations present.

Because of their structures, vitamin A and carotenoids can autoxidize when O₂ tension increases, and thus are most effective antioxidants at low oxygen tensions that are typical of physiological levels found in tissues.[120]

Herein lies the difference in the chain breaking antioxidant properties of carotenoids and tocopherol; the tocopheroxy radical is much less reactive with oxygen than the carotene radical. The greater the oxygen tension, the more likely it is that carotenoids will participate in lipid peroxidation. However, carotenoids are weaker antioxidants compared to Vitamin E.[121]

CAROTENOIDS AND CANCER

Carotenoids such as capsanthin and related carotenoids isolated from the fruits of red paprika Capsicum annuum L. have been reported to possess anti-tumor promoting activity.[122] Carcinogenesis is considered a multistage process involving initiation, promotion and progression. The mechanisms involved in the conversion of a normal cell to one with a malignant phenotype are not fully understood yet.

Tumor initiation is caused by spontaneous or carcinogen-induced genetic (DNA) damage. Some important initiators are mutagens, genotoxic pro-oxidants and radiation. At this stage, β-carotene may exert its protective efforts by detoxifying, quenching or trapping the active intermediates, which react with the DNA. Pregnane X Receptor (PXR)-mediated upregulation of certain genes such as CYP3A4/CYP3A7 and CYP3A5 as well as multi drug resistance proteins, MDR1 and MRP2 by carotenoids points to a potential interference on the metabolism of xenobiotic and endogenous carcenogenic compounds.[123] In patients with Alzheimer disease, a significant inverse relationship between lymphocyte DNA 8-OHdG content (indicative of DNA damage) and plasma levels of lycopene, lutein, alpha-carotene, and beta-carotene, respectively, was observed.[124] In a recent study of 160 breast cancer patients and 226 healthy women, Kim, Ahn, and Lee‐Kim[125] found that high blood levels of several antioxidants were strongly associated with a low risk of breast cancer. In premenopausal women, high levels of beta-carotene, lutein/zeaxanthin and vitamin A were associated with 67, 87, and 85% reductions in breast cancer risk, respectively. In postmenopausal women, high levels of vitamin A, beta-carotene, alpha-carotene, lycopene, and lutein/zeaxanthin were associated with 92, 72, 80, 75, and 88% reductions in adjusted breast cancer risk, respectively. In a study by De Flora, Bagnasco, and Vainio,[126] carotenoids and vitamin A appear to work via multiple mechanisms, which would support a potential protective role in cancer initiation and in the pathogenesis of other mutation-related diseases.

Tumor promotion means growing and proliferation of the initiated abnormal cells. Promotion may be reversible for a long time. For example, prostrate cancer in humans may have a latent period of up to 40 years.[127] The potency of many promoters (including granulocytes activated by inflammation) to produce active oxygen species provides a rationale for the use of β -carotene, a known singlet oxygen quencher.

Tumor progression: Carotenoids in general, and lycopene in particular, may be effective anticarcinogenic agents by inhibiting cell growth through apoptosis induction.[128] Furthermore, β-carotene has been shown to enhance gap-junction communications, irrespective of its provitamin A activity.[129] Gap junctions of cell membranes are considered to be part of cell-to-cell communication, and it is believed that increased communication between normal and initiated cells may restrict the rate of initiated cells expressing the neoplastic phenotype. The up-regulation of gap junctions by β-carotene and lycopene is considered to be caused by the increased expression of connexin 43, a junctional protein.[130,131]

When the cells become more autonomous, a conversion occurs to a malignant tumor, by invading healthy tissue and developing metastatic foci at various sites of the body, known as clinical cancer manifestation. Animal studies indicate that β-carotene may also protect against tumor progression. It was reported that β-carotene protected 45% of the cases against skin cancer in mice, when administered after irradiation but was without any effect when given before irradiation.[132]

Synergy Between Carotenoids and Phytonutrients

The epidemiological and laboratory studies reviewed suggest a cancer-preventive activity for carotenoids and led to collaborative large intervention studies with synthetic β-carotene. However, the use of a single plant-derived compound in human cancer prevention studies has not been successful, revealing either no beneficial effect[133] or even a negative effect.[134,135] These results led to the hypothesis that a single micronutrient cannot replace the power of the concerted action of multiple compounds derived from a diet rich in fruits and vegetables when examining the possible effects on the effects of cancer.[131]

To support the hypothesis that a concerted action of several micronutrients is responsible for the anticancer activity of diet enriched with fruits and vegetables, it has to be shown that plant-derived constituents, such as carotenoids, have the ability to synergize with other phyto-nutrients.[136]

Carotenoids and Transcription

Carotenoids modulate the basic mechanisms of cell proliferation, growth factor signaling, and gap-junction intercellular communication to produce changes in the expression of many proteins participating in these processes, for example, connexins, cyclins, cyclin-dependent kinases, and their inhibitors.[137] Therefore, the question that arises is by what mechanisms do carotenoids affect so many diverse cellular pathways? The changes in the expression of multiple proteins suggest that the initial effect of carotenoids involves modulation of transcription. This may be due to either direct interaction of the carotenoid molecules or their derivatives with transcription factors, e.g., with ligand-activated nuclear receptors or indirected modification of transcriptional activity, e.g., via changes in status of cellular redox, which affects redox-sensitive transcription systems such as AP-1, NFκB and antioxidant response element (ARE).[138]

Emerging evidence now suggests that derivatives of retinoids or the carotenoids themselves may also modulate the activity of transcription factors. For example, the synergistic inihibition of cancer cell proliferation by lycopene in combination with 1,25(OH)₂D₃ or retinoic acid,[136] the ligands of two members of the nuclear receptor super-family, suggests that lycopene or one of its derivatives may also interact with members of this family of receptors.

Induction of phase II enzymes, which conjugate reactive electrophiles and act as indirect antioxidants, appears to be an effective means for achieving protection against a variety of carcinogens in animals and humans. Transcriptional control of the expression of these enzymes is mediated, at least in part, through ARE found in the regulatory regions of their genes. The transcription factor Nrf2, which binds to ARE, appears to be essential for the induction of phase II enzymes, such as glutathione S-transferases (GSTs), NAD(P)H: quinine oxidoreductase (NQO1) (139) as well as the thiol-containing reducing factor, thioredoxin.[125] Constitutive hepatic and gastric activities of GST and NQO1 were decreased by 50–80% in Nrf2-deficient mice compared with normal mice.[139]

Some carotenoids are capable of inducing phase II metabolizing enzymes, p-nitrophenol-UDP-glucuronosyl transferase and NQO1 in rats and in transiently transfected mammary cancer and hepatocarcinoma cells.[140] These results suggest that the lycopene-induced increase in the levels of glutathione (GSH) and the phase II enzyme glutathione S-transferase, inactivate carcinogens by forming conjugates that are less toxic and readily excreted.

Modulation of Apoptotic Signalling by Carotenoids in Cancer Cells

Little is known regarding the mechanisms of action at molecular levels by which carotenoids modulate cancer process. It appears that carotenoids can bring about changes in gene expression and protein activity in the cells. There is some evidence to support the theory that carotenoid molecules may interfere in cancer related molecular pathways and change the expression of many proteins involved in: a) cell proliferation, differentiation, apoptosis and angiogenesis; b) carcinogen detoxification; c) DNA damage and repair; and d) immunosurveillance.[141] Carotenoids seem to affect gene expression either directly by interference with the control apparatus of the gene expression machinery or by virtue of metabolites or metabolic conditions induced (hormonal status, cellular redox status, etc.) that, in turn, alter cell functions implicated in the cancer process. These observations raise issues about possible dosage levels for carotenoid administration, possible synergy, as well as antagonistic interactions between carotenoids and other dietary components as discussed earlier.

Effects of Carotenoids on Caspase Cascade

β-carotene-induced apoptotic pathway requires activation of caspase-3, which has been defined as a key executioner involved in apoptosis induced by many stimuli and is also necessary for the nuclear change associated with apoptosis, such as chromatin condensation. Carotenoids can initiate caspase-3 cascade mainly by interacting with a signal complex on cell membranes, which induces caspase-8 activation by changes in conformation of some membrane-associated “death” receptors and then by operating through a non-receptor signaling pathway within cytoplasm, which induces caspase-9 activation.[142]

Effects of Carotenoids on Mitochondria

The involvement of mitochondria in the pro-apoptotic effects of carotenoids has been clearly demonstrated by the fact that β-carotene induced the release of cytochrome c from mitochondria and altered the mitochondrial membrane potential (Δψ_m) in different tumor cells.[142] Most of the current data are compatible with the notion that the loss of Δψ_m constitutes an irreversible event in the proapoptotic process and that anticancer agents, such as retinoids, induce apoptosis by provoking a disruption of Δψ_m.[143] Interestingly, it has been demonstrated that the synthetic retinoid CD437 was able to alter Δψ_m and consequently, to induce apoptosis only in respiration-efficient squamous carcinoma cells. When an inhibitor of mitochondrial DNA synthesis, such as ethidium bromide, was added to the cells, the pro-apoptotic action of this retinoid was diminished, illustrating that mitochondrial respiration was required for the effect on apoptosis.[144]

Effects of Carotenoids on Genes and/or Proteins Involved in Apoptosis

β-carotene has been reported to induce an oxidative stress in tumor oral cells, resulting in the expression of stress proteins, such as heat-shock protein (hsp)70 and/or hsp90, which are nuclear binding proteins involved in apoptosis.[145] Both 9-cis and all-trans-β-carotene are able to induce an intracellular accumulation of hsp70 in cervical dysplasia-derived cells and the treated cells showed morphological changes indicative of apoptosis.[145]

Two isoforms of cyclo-oxygenase (COX) participate in regulating cell growth; COX-1 is constitutively expressed in most cells and COX-2 is an inducible enzyme in response to cellular stimulai.[146] They reported that an increase in COX-2 expression is deeply related to carcinogenesis and tumor promotion. One of the possible mechanisms by which COX-2 can induce tumorigenesis is through its ability to act as an anti-apoptotic gene. It has been shown that β-carotene is able to down regulate the expression of COX-2 in colon cancer cells and such an effect was accompanied by apoptosis induction. This observation is particularly interesting in view of the fact that COX-2 expression is regulated by peroxisome proliferators-activated receptor (PPAR)-γ and PPARs are suggested to be modulated by carotenoids.[140]

ROLE OF CAROTENOIDS AGAINST CANCERS

In vitro cell culture experiments have shown that carotenoids inhibit cell proliferation, transformation and micronucleus formation as well as modulating expression of certain genes. These properties are consistent with a protective effect against carcinogenesis.[147] One of the earliest discoveries in this respect was that certain carotenoids, in vitro, increased gap junctional communication in a dose-dependent manner.[148] Since this early study, there has been a wealth of investigations on individual carotenoids and their effect of cancers.

Lung Cancer

Observational epidemiological studies in the 1960s and 1970s showed a consistent inverse association between fruit and vegetable intake and the risk of lung cancer and other cancers.[149] Further studies suggested that β-carotene may be the compound responsible for this association, although they did not provide direct evidence that this was the case. Therefore, several β-carotene intervention trials were started in the 1980s and 1990s to test this hypothesis.[150,151] These trials showed that β-carotene did not reduce the risk of lung cancer and in the trial using Finnish male heavy smokers there was an increase in lung cancer perhaps because β-carotene was acting as a pro-oxidant rather than an antioxidant in these circumstances.[152,135] Recently, Wright, Mayne, Swanson, Sinha, and Alavcanja[153] have reported that the consumption of a wide variety of vegetables has a greater bearing on lung cancer than intake of any specific carotenoid or total carotenoids.

Breast Cancer

Clavel-Chapelon, Niravong, and Joseph[154] reviewed epidemiologic literacture on breast cancer and found that β-carotene was considered in 16 case-controlled studies. Higher β-carotene consumption was associated with a lower risk of breast cancer in 11 studies, with significant results in four and the remaining five showing no association. A review by Cooper, Eldridge, and Peters[155] showed a mixed picture, although high lycopene levels were associated with decreased risk in one study.[156] The mechanism of action of lycopene in the inhibition of breast cancer is associated with inhibition of cell cycle progression at the G1 phase.[157] In contrast, a large-scale study of 39,876 women in the US showed no association of plasma lycopene levels with a reduced risk of breast cancer in middle-aged and older women.[158]

Prostrate cancer

The first interest in the hypothesis that dietary lycopene protects against prostrate cancer was in 1995, when Giovannucci, Rimm, Liu, Stampfer, and Willet[159] reported that high-lycopene foods were associated with a reduced risk of prostrate cancer, in the Health Professionals Follow-Up Study. Intake of lycopene in a lipid diet, e.g. tomato sauce or pizza was particularly effective.[159] It is known that supplementation of the diet with tomato sauce leads to a substantial increase in total lycopene in serum and prostrate.[160] There have been several updates since the first report in 1995 and Giovannucci has reviewed some 72 such studies. There have been about 57 reported inverse associations between tomato intake or lycopene blood level and risk of cancer at a defined anatomic site, with 35 being statistically significant. At the present time, the accumulated data suggest that intake of tomato or tomato products may be associated with a lower prostrate cancer[161] but it is not certain that lycopene is the only compound in the tomato that contributes to this effect. Other carotenoids and phytochemicals in the fruit may contribute also.[162,160] Interestingly, it has also been shown that a high tomato diet can reduce leukocyte oxidative DNA damage and prostrate tissue oxidative damage in patients already diagnosed with prostrate cancer,[163] thus suggesting that such food can be used in treatment of prostrate cancer as well as its prevention.

Colorectal Cancer

β-carotene intake has been inconsistently associated with colorectal cancer,[164] but the effects of other carotenoids have only been studied recently. Slattery, Benson, Curtin, Ma, Schaeffer, and Potter[165] used dietary data to show that lutein was inversely associated with colon cancer in both men and women, whilst other carotenoids such as lycopene, zeaxanthin and β-cryptoxanthin were unremarkable. In contrast, a more recent study concluded that there was no association between dietary intake of carotenoids and colorectal cancer risk.[166]

Skin Protection

Recent studies support the hypothesis that oral sun protectants may probably be more efficient than topical ones.[167] The protective effects are thought to be related to the antioxidant properties of the carotenoids. During ultraviolet (UV) irradiation, skin is exposed to photo-oxidative damage induced by the formation of ROS. Photo-oxidative damage affects cellular lipids, proteins, and DNA and is considered to be involved in the formation of erythema, premature aging of the skin, photodermatoses, and skin cancer.

Carotenoids are efficient scavengers of ROS. Several animal studies and in vitro experiments provided evidence that carotenoids and tocopherols prevent UV light–induced skin lesions and protect against skin cancer.[168] It is known that plasma and skin carotenoid concentrations decrease on exposure to UV irradiation.[169]

CAROTENOIDS AND AGE-RELATED MACULAR DEGENERATION (AMD)

AMD is one of the most commonly occurring conditions among older adults in the Western countries. The two major types of AMD are neovascular/exudative (N/E) and atrophic or nonexudative macular degeneration. All patients appear to begin with the atrophic form of the disease, with a proportion progressing to the N/E form. Although an estimated 80% of all AMD patients have the atrophic form, the N/E form may be responsible for approximately 90% of the severe visual loss (20/200 or worse) from AMD.[170] Each of the AMD manifestations is associated with age and decreased central visual acuity. The atrophic form is characterized by drusen and pigment epithelial atrophy, the N/E form is characterized by retinal pigment epithelial detachments, choroidal neovascular membranes, and disciform scars.[171]

There is much interest on the role of nutritional antioxidants (Vitamin C, E, lutein, zeaxanthin and zinc) because of the evidence that they help to prevent the oxidative damage involved in the pathogenesis of AMD and cataract. Using intervention studies involving humans, lutein supplementation was shown to result in increased macular pigment and improved vision in patients with AMD and other ocular diseases. In this regard, it is of interest to note that animal toxicology studies invovling lutein have led to the classification of purified crystalline lutein as generally recognized as safe (GRAS).[15]

CORONARY HEART DISEASE

Coronary heart disease (CHD) is one of the primary causes of death in the Western world. The emphasis of research so far has been on the relationship between serum cholesterol levels and the risk of CHD. More recently, oxidative stress induced by reactive oxygen species (ROS) is also considered to play an important part in the etiology of this disease. Dietary lycopene and β-carotene have been shown to prevent the formation of oxidized LDL, a key player in the pathogenesis of atherosclerosis and CHD.[172] The most impressive population-based evidence comes from a multi-center case-control study in which subjects from 10 European countries were evaluated for a relationship between their antioxidant status and acute myocardial infarctions. After adjusting for a range of dietary variables, only lycopene levels, not β-carotene levels, were found to be protective.[173,174] Serum lycopene concentration may play a role in the early stages of atherosclerosis. Recently a prospective, nested, case-control study was conducted by Harvard University researchers on 39,876 women. The study showed that higher plasma lycopene concentrations are associated with a lower risk of cardiovascular disease in middle-aged and elderly women.[175] These findings suggest that dietary lycopene or other carotenoids consumed as oil-based or oil-containing products confer cardiovascular benefits.

CAROTENOIDS AND IMMUNE FUNCTION

Beta-carotene has been shown to enhance immune functions in humans. A recent study on whether vegetables rich in carotenoids, such as beta-carotene or lycopene, modulate immune functions in healthy humans showed that increased plasma carotenoid concentrations after vegetable juice consumption are accompanied by a time-delayed modulation of immune functions in healthy men consuming a low-carotenoid diet.[176] Early studies demonstrating the ability of dietary carotenes to prevent infections have left open the possibility that the action of these carotenoids may be through their prior conversion to vitamin A. Subsequent studies to demonstrate the specific action of dietary carotenoids have used carotenoids without provitamin A activity such as lutein, canthaxanthin, lycopene and astaxanthin. In fact, these nonprovitamin A carotenoids were as active, and at times more active, than beta-carotene in enhancing cell-mediated and humoral immune response in animals and humans.[177] In recent years, new physiological functions of vitamin A have been identified, including its role in immune defense. The antioxidant potential of carotenoids is thought to account for their health benefits. The inverse correlations between C-reactive potein (CRP) and beta-carotene or retinal, indicate either decreased synthesis or increased utilization of these antioxidants in children with acute infections.[178] Recent studies have also indicated that elderly people with a high plasma beta-carotene concentration may have a lower occurrence of acute respiratory infections.[179] Animal studies have shown that Vitamin A and retinoid content in a diet influences the cytokine response in non-sensitized and also ovalbumin-sensitized mice. Therefore these molecules may serve as active modulators of cytokine production in vivo that are responsible for the induction and persistence of atopic diseases.[180]

CONCLUDING REMARKS

The scientific research to date has demonstrated an array of health benefits clearly associated with carotenoids in the diet. Health benefits are derived from the fruits and vegetables in the diet, particularly from cooked products containing oil, or from supplements of their extracts, such as tomato sauce, dried tomatoes, or those suspended in oil. These carotenoids are almost always present as mixtures and work in synergy to bring about their beneficial effects.