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The central retina, known as the macula, is responsible for central and colour vision. The macula is characterised by a yellow colour, attributable to the presence of macular pigment (MP).1 MP is composed of lutein (L) and zeaxanthin (Z), two hydroxycarotenoids, which are entirely of dietary origin, and the retinal metabolite of L: meso-zeaxanthin (meso-Z).2,3 Age-related macular degeneration (AMD) is the advanced form of age-related maculopathy (ARM), which results in loss of central vision, and is the leading cause of blindness in the Western World.4
Although the pathogenesis of ARM is incompletely understood, there is a growing body of evidence to suggest that cumulative blue light damage and/or oxidative stress plays a role.5 At the macula, MP filters out blue light at a pre-receptoral level1 and quenches free radicals.6 These actions are consistent with the hypothesis that MP protects against AMD.
This article reviews the dietary sources of the macular pigment constituents, and the factors affecting carotenoid digestion and absorption into the blood stream. Supplemental L and Z studies are also discussed.
The macular carotenoids
Carotenoids are part of a vast spectrum of natural pigments that are synthesised de novo by all plants, where they play a critical role in the photosynthetic process. Carotenoids are also found in non-photosynthetic microorganisms (bacteria, yeast, and moulds), where they are known to protect against the detrimental effect of excess light and oxygen.7
Carotenoids share many common properties: they are insoluble in water but dissolve in organic solvents they are bleached upon exposure to light or atmospheric oxygen and they possess an absorption band near the blue to violet end of the visible spectrum.7
Carotenoids have a basic C40 structure, which can be further modified by the addition of C5 units to give C45 and C50 carotenoids, or by oxidation, which results in carotenoids containing less than 40 carbon atoms. The central carbon chain, of alternating single and double bonds, carry cyclic or acyclic end groups. The extended system of conjugated double bonds contributes to their major biochemical functions, and is responsible for their colour.
Carotenoids are generally subdivided into two groups: the carotenes composed only of carbon and hydrogen atoms and the xanthophylls, which carry at least one oxygen atom ß-carotene, a carotene, and lycopene are prominent members of the carotene group, while lutein (L), zeaxanthin (Z), alpha and ß-cryptoxanthin and astaxanthin are important xanthophylls.8
The xanthophylls, L and Z, have two hydroxyl groups, one on each side of the molecule. Structurally, L and Z are isomers of one another (Figure 1), and differ only in the position of the double bond in the six-carbon ring located on the right side of the carbon chain.9
Isomerisation in carotenoids is commonplace, and both these isomers can show further cis/trans isomerisation by changes in the geometrical configuration about the double bonds located in the hydrocarbon backbone. In general, the all-trans form is thermodynamically most stable and predominant in nature, but several cis isomers of carotenoids are present in blood and tissues.2,10
Foods rich in L and Z
Over 600 carotenoids have been described in nature however, humans, who obtain their carotenoids solely through metabolism of carotenoid-rich foods, typically ingest about 40. Thirty-four of these carotenoids have been identified in human serum including 13 carotenoids, 13 geometrical isomers and eight metabolites.11
Fruits and vegetables are the most important source of carotenoids in the human diet. Using high performance liquid chromatography, Sommerberg and coworkers,12 provided comprehensive information on the content of L and Z, in a wide variety of fruits and vegetables, the results of which are seen in Table 1. It was found that the highest amount of L and Z among all the fruits and vegetables used in this study were in egg yolk and maize corn. The highest mol per cent of L was found in maize corn, followed by egg yolk, kiwi fruit, seedless red grapes, zucchini squash and pumpkin. Z on the other hand was the major carotenoid in orange pepper. Green leafy vegetables such as spinach, celery, brussels sprouts and broccoli, which have previously been the recommended sources for carotenoids, were relatively high in L (47 - 22 mol per cent), but very low in Z concentration (0-3 mol per cent).
The source yielding the highest concentration of L and Z in combination was egg yolk. In fact, a study comparing L enriched eggs to pure L supplement, found that the eggs, with their lipid matrix, were more bioavailable for L absorption into the blood stream.13 Cholesterol is a risk factor for coronary artery disease and due to their high cholesterol content, a restricted intake of eggs has been recommended for many years. However, studies have shown that a higher intake of cholesterol through the addition of eggs in the diet, not only increases total cholesterol but also HDL cholesterol.14,15 Since HDL cholesterol is protective against atherosclerosis, egg consumption should have no severe adverse effects on cardiac risk factors. However, a more recent study investigating the effectiveness of supplementation with chicken egg yolks (1.3 egg yolk/day containing 380µg L and 280µg Z), found a significant increase in serum L and Z, but also an increase of plasma LDL-cholesterol by 8-11 per cent (p<0.05).16 Therefore, the effect of egg yolk supplementation on lipoprotein concentrations and profiles will have to be further investigated before recommending eggs as a primary dietary source of L and Z.
Lycium barbarum L is a small red berry known as Fructus lycii and wolfberry in the West, and Kei Tze and Gou Qui Zi in Asia. Wolfberry contains high concentrations of Z dipalmitate (up to 5mg/100g) and is valued in Chinese culture for being food for vision.17,18 Although concentrations of Z in wolfberry are high, the bioavailability is relatively low, and food-based products with enhanced bioavailability are being developed to utilise this, one of the richest natural sources of Z.19
L and Z supplements are available in both esterified and non-esterified forms, alone or in combination with other vitamins and micronutrients. A large number of manufacturers offer such preparations, with increasing numbers of multivitamin tablets now including L and/or Z in their formulations. Esterified L and/or Z supplements have been demonstrated to be more bioavailable than free L and/or Z.20,21
The average daily intake of L and Z in the US is reported to be 2.0-2.3mg daily for men and 1.7-2.0mg daily for women,22 while nutritional supplements containing L deliver from 0.25 mg to 20 mg daily.
The optimal dosage for ocular health is, as yet, unknown23 however, scientific literature has demonstrated that a daily intake of up to 40 mg of L and Z over a period of two months causes no noticeable toxicity or adverse reactions.24 Furthermore, Fijians consume an average of 25 mg L daily throughout a lifetime without any reported toxic effects.25
Digestion, absorption and transport of L and Z
Absorption may be defined as the movement of a substance to the lymphatic and/or portal circulation from the gastrointestinal system. Several processes are required for optimal absorption of carotenoids. These include: adequate digestion of the food matrix in order to release the carotenoids formation of lipid micelles in the small intestine uptake of carotenoids by intestinal mucosal cells transport of carotenoids to the lymphatic and/or portal circulation. Carotenoids enter the circulation as a component of chylomicrons.26 Following uptake by the liver, carotenoids are re-secreted on lipoproteins, which are believed to deliver carotenoids to various tissues.
Lipoproteins are classified into the following five groups: chylomicrons very low density lipoproteins (VLDL) intermediate density lipoproteins (IDL) low density lipoproteins (LDL) high density lipoproteins (HDL).27 Chylomicrons are synthesised by the intestine and deliver dietary triglycerides to muscle and adipose tissue, and dietary cholesterol to the liver.
The majority of plasma carotenoids are transported on LDL, with 55 per cent of total carotenoids associated with this lipoprotein, whereas HDL is associated with 33 per cent, and VLDL is associated with 10-19 per cent, of the total carotenoids.28 However, xanthophylls, including L and Z, are relatively equally distributed between LDL and HDL,26,29 with a progressive decrease in the content of L and Z from light to dense LDL.29 This finding has prompted the suggestion that an individual's lipoprotein profile may influence the transport and delivery of these carotenoids to the retina, with a consequential impact on MP.
Bioavailability of carotenoids
Bioavailability is defined as the fraction of an ingested nutrient that is available to the body for utilisation in normal physiological functions, or for storage. The mnemonic 'SLAMENGHI' includes all the factors that may influence the rate of absorption of carotenoids and the overall bioavailability of the carotenoids ingested: Species of carotenoids, Linkages at the molecular level, Amount of carotenoid, Matrix, Effectors, Nutrient status, Genetics, Host-related factors, and Interactions among these variables.30-32 The two most important factors influencing carotenoid bioavailability from fruit and vegetable food sources are Matrix and Effectors, which are discussed here.
A. Bioavailability of carotenoids from different food matrices
Disruption of the food matrix and release of the constituent carotenoids is the first step in absorption. Carotenoids occur in food plants either as part of the chloroplasts of green leafy vegetables, as part of the chromoplasts of fruits, or as semi-crystalline membrane-bound solids, found in carrot and tomato.33 ß-carotene from fruits has been found to be 2.6 to six times more effectively absorbed into plasma, compared to that from green leafy vegetables, leading to speculation that chloroplasts may not be as effectively disrupted as chromoplasts in the intestinal tract.34
Studies investigating the effect of food matrix on carotenoid bioavailability compare plasma response to supplementation with vegetables or fruits, to that of pure carotenoid supplements, giving a 'relative carotenoid bioavailability'.
The relative bioavailability of ß-carotene from vegetables compared with purified ß-carotene, ranges from between 3 to 6 per cent for green leafy vegetables, 19-34 per cent for carrots and 22-24 per cent for broccoli.35-40 Van het Hof and co-workers carried out a study to assess the bioavailability of ß-carotene and L from a mixed vegetable diet.41 This four-week study involved 54 healthy volunteers, 22 of whom consumed a high vegetable diet (490g/d), a further 22 of whom consumed a low vegetable diet (130g/d), and 10 of whom consumed a low vegetable diet, supplemented with pure ß-carotene (6mg/d) and L (9mg/d). The responses of plasma ß-carotene and L to the high vegetable diet were 14 per cent and 67 per cent, respectively. Similarly, a study investigating the relative bioavailability of ß-carotene and L from spinach, found L (45 per cent) was more bioavailable than ß-carotene (5.1 per cent).39 A study investigating the bioavailability of L, ß-carotene, alpha and gamma tocopherol from broccoli found only L had significant serum increases in both men and women, whereas gamma tocopherol showed significant changes in women only.42 L, which is a dihydroxycarotenoid, is approximately 0.1 per cent as lipophilic as ß-carotene, which would enable its release into an aqueous environment, and could, in part, explain its increased bioavailability.
Wolfberry is rich in Z dipalmitate, and a single-blinded, placebo-controlled human intervention trial, investigating the effects of supplementation with whole wolfberries demonstrated a 2.5-fold increase in fasting plasma Z concentrations, indicating that Z in wolfberries is readily bioavailable.22 Furthermore, results from a randomised, single-blind cross-over study, examining absorption of non-esterified and native esterified Z (Z palmitate from wolfberries), demonstrated an enhanced bioavailability of Z dipalmitate compared with the non-esterified form.25
Disruption of the food matrix
The intactness of the cellular matrix is also considered a determinant of the bioavailability of carotenoids from fruits and vegetables and, therefore, varying food forms in which the carotenoids are consumed (ie vegetable juice versus raw or cooked vegetables), can result in differences in the plasma carotenoid response.
However, the disruption of the matrix affects the bioavailability of different carotenoids differently and the literature is somewhat conflicting.
As early as 1948, Van Zeben and Hendriks reported that homogenisation improved the bioavailability of ß-carotene from carrots in humans.43 A study investigating the bioavailability of ß-carotene and L from spinach, found the plasma response of L was significantly increased (by 14 per cent) when the spinach was consumed as chopped spinach instead of whole leaf spinach, whereas the plasma response of ß-carotene was not affected.40
In a similar study, the relative bioavailability of ß-carotene and L from whole leaf, minced and liquefied spinach, compared to carotenoid supplement, was 5.1 per cent, 6.4 per cent and 9.5 per cent, respectively, for ß-carotene, and for L it was 45 per cent,52 per cent and 55 per cent, respectively.39 Therefore, this study reports a much lower increase in the plasma response of L when spinach was chopped and, conversely, shows a larger relative increase in ß-carotene plasma response to the spinach chopping.
With respect to juicing, a 70 per cent difference was found in the bioavailability of ß-carotene from raw carrots versus carrot juice consumed by well-nourished adult females over a six-week period, although the difference was not significant.38 A study examining the serum response to consumption of vegetable juice versus raw or cooked vegetables, found ß-carotene and L were significantly higher in the vegetable juice group than the raw or cooked vegetable group (p<0.05 and p=0.05, respectively). However, no significant differences were found with ß-carotene, ß-cryptoxanthin and lycopene.44
Cooking enhances the carotenoid content measured in vegetables, which is possibly due to the increased extractability of the carotenoids as a result of the heating of the vegetable matrix, and possibly loosening their binding to proteins.45-47 Mechanical homogenisation and heat treatment is used in the processing of tomatoes into tomato paste and, interestingly, plasma lycopene responses are far greater (22-380 per cent) after consumption of tomato paste than that for the same amount of lycopene consumed from fresh tomatoes.48,49 Furthermore, consumption of tomato juice heated with oil was observed to increase serum lycopene concentration compared to unheated tomato juice alone. The heating of the juice is believed to release lycopene from protein complexes and hence improve its absorption.50
The effect of varying the preparation of wolfberry on Z bioavailability was investigated using three formulations: berries homogenised in hot (80°C) water, warm (40°C) skimmed milk and hot (80°C) skimmed milk.23 Z peaked at six hours post-ingestion for all formulations however, the hot skimmed milk preparation had a three-fold enhanced bioavailability of Z compared with both the classical hot water and warm skimmed milk treatment of wolfberries. This study indicates that the bioavailability of Z from this rich source can be increased and there is a growing interest in developing food-based products with enhanced bioavailability.
B Effectors of absorption
- Dietary lipids
Dietary lipids, consumed along with carotenoids, provide a hydrophobic phase where the carotenoids can be solubilised, and hence facilitate their release from their food matrices. Incorporation of the released carotenoids into mixed micelles is an important step in the absorption process. Among other factors, formation of these micelles is dependent on the presence of fat in the intestine. Therefore, ingestion of fat along with carotenoids is thought to be crucial to their absorption.51
Several investigators have studied the importance of dietary fat for the absorption of ß-carotene and from their findings it appears that 5g of fat in a meal is sufficient to ensure carotenoid uptake.52-54 Enhancement of L and L ester bioavailability by lipids has also been demonstrated.55,56 - Dietary fibre
The bioavailability of carotenoids from vegetables and fruits may be affected by the fibre content. It has been suggested that fibre interferes with the micelle formation, but the results to date are contradictory.39,57,58 Rock and Swendseid57 reported that adding 12g dietary citrus pectin to controlled meals with 25mg synthetic ß-carotene (0.48g pectin/mg ß-carotene) reduced the increase in plasma ß-carotene. However, Castenmiller and co-workers carried out a similar study with half the concentration of dietary fibre (0.23g pectin/mg ß-carotene) and were unable to demonstrate any effect of dietary fibre on carotenoid absorption.39 A study investigating L bioavailability, showed that meals enriched with various fibres (0.15g/Kg weight), had an inhibitory effect on L absorption.59 - Inhibition of lipid absorption
Anti-obesity drugs, aimed at diminishing fat absorption, can also impair the absorption of fat-soluble carotenoids. Sucrose polyester (eg Olestra), used as a fat substitute, decreases plasma carotenoid levels by 20 to 120 per cent. The more lipophilic the carotenoid the more pronounced the negative effect. It appears that the carotenoids were incorporated with the non-absorbable sucrose polyester, rather than into micelles formed from the dietary fat.60-62 Phytosterols, plant sterols used to lower cholesterol absorption, have also been shown to reduce carotene and xanthophyll absorption.63,64 - Interaction between carotenoids
Carotenoids are ingested with other nutrients and micronutrients and, therefore, interactions at the intestinal level are inevitable. Competition for absorption may occur at the level of micellar incorporation, intestinal uptake, lymphatic transport or at more than one level.51 Antagonism can be demonstrated when carotenoids are concurrently given. ß-carotene has been shown to interfere with L65,66 and canthaxanthin67,68 absorption. Again, literature on carotenoid competition for absorption is conflicting, for example, some studies show a reduction in lycopene concentration during supplementation with ß-carotene,54,69 whereas others found no effect,70,71 or even an enhancing effect.72,73
Long-term ß-carotene supplementation has been reported as having limited or no effect on plasma concentrations of other carotenoids. However, the supplements may have been ingested at times of the day other than those at which foods rich in carotenoids were consumed.74 Furthermore, a study on vegetable-borne ß-carotene, L and lycopene demonstrated competition for incorporation into chylomicrons, but the medium term (three weeks) carotenoid plasma concentrations showed no adverse effects.75
Supplementation studies of L and Z in humans
There have been several studies investigating plasma response to supplemental L and/or Z in humans.76-83 Bone and co-workers,76 investigated the effect of a range of L doses (2.4-30mg/day) as well as a high Z dose (30mg/day) on the serum L and Z concentration. Serum L concentrations for each subject reached a plateau that was correlated with the dose (r=0.58, p<0.001). The plateau concentrations ranged from 0.28µM to 2.7µM. The supplemental 30 mg/day of Z was less well absorbed with a plateau of 0.5µM. However, the Z used in this study was crystalline, unesterified and incorporated in gelatin/starch beadlets, which exhibited little or no tendency to dissolve in vegetable oil, therefore lowering the bioavailability of this supplemental Z. While the supplemental L was esterified and prepared as an oleoresin that was easily solubilised in vegetable oil, rendering the supplemental L more bioavailable.
Previous studies from this group, reported a 10-fold increase in serum L concentration with 30mg/day L77 and supplementation with 2.4 mg/day L led to a 100 per cent increase in serum L.78 While Berendschot reported a five-fold increase in serum L in response to a 10mg/day L supplementation.79
In these studies, the serum L and Z declines to pre-supplementation levels following discontinuation of the supplements. Serum carotenoid depletion has been shown to follow first order kinetics, with the half life for L being 76 days and that for Z being 36 days.80
A more recent study reported an average three-fold increase in serum L in subjects taking 5mg/day L for six months.81 In another six month study, subjects were supplemented with 10mg L, 10mg Z, or 10mg of L combined with 10mg Z.82 The average serum L increase was seven-fold for the L group, the average serum Z was 27-fold for the Z group, and the average serum L and Z increases were four-fold and 14-fold respectively for the group receiving both L and Z. The lower response to supplementation of L and Z together could be due to the lower relative bioavailability when administering both carotenoids together, or possibly due to competition for uptake into the plasma due to their high chemical similarity.
Interestingly, Bone and co-workers carried out a study where they included the non-dietary meso-Z with supplemental L and Z.83 The supplement consisted of unesterified carotenoids: 14.9mg of meso-Z 5.5mg L and 1.4mg of Z. The presence of serum meso-Z, in addition to L and Z, was confirmed, demonstrating that meso-Z, although not normally present in the diet, can be absorbed.
Conclusion
Observational studies analysing the relationship between dietary intake of L and Z and the serum concentration of these carotenoids have demonstrated a positive and significant relationship. Although dietary sources of L and Z are well documented, the bioavailability, and factors influencing their bioavailability from various food sources, need to be further investigated.
Supplementation with L and Z augments serum levels of these carotenoids. The optimal dosage for ocular health is, as yet, unknown however, both esterified L and Z appear to be more bioavailable than non-esterified forms, and high dosages (up to 40 mg/day) have not shown any noticeable toxicity or adverse reactions. In the majority of individuals, increased serum concentrations of the xanthophyll carotenoids are reflected in an increase in macular pigment levels, which in turn provides protection against AMD. Until the recommended daily allowances of the macular carotenoids are calculated, it is prudent for eye care professionals to advise their patients to maintain a diet rich in the relevant fruits and vegetables and, if supplementation is advocated, one should follow the manufacturer's guidelines carefully.
? Dr Orla O'Donovan, Dr Edward Loane and Stephen Beatty are based at the Macular Pigment Research Group, Waterford Institute of Technology