Carotenoids

418 George Britton, Synnøve Liaaen-Jensen and Hanspeter Pfander D. How Might the Effects be Mediated? 1. Via antioxidant action Harmful reactive oxygen species and free radicals are continuously produced in the body during normal cellular functioning and are introduced from exogenous sources. Damage caused by these is associated with aging and with the incidence and progression of serious diseases including cancer, CHD, age-related macular degeneration and neurodegenerative conditions. Although the body has a battery of defences against this (enzymes and endogenous antioxidants), it is widely believed that dietary supplementation with antioxidants can be a part of a protective strategy to minimize the oxidative damage, especially in the elderly and other vulnerable populations. But can carotenoids be counted among the effective antioxidants in vivo? Since the concept was first raised in 1984, much of the research on carotenoids and human health has been driven by the prospect that carotenoids may be among the group of antioxidants in fruits and vegetables that help to prevent damage caused by oxidizing free radicals. It is enlightening to compare the conclusions given in that paper [5] with the many other interpretations published by other reviewers and commentators. Carotenoids have been shown to have antioxidant activity in vitro at physiological oxygen tensions. Most work has been done with model systems with oxidizing free radicals generated artificially from azo-initiators. Assays have usually addressed parameters of lipid peroxidation, most commonly the TBARS reaction, the reliability of which in the presence of carotenoids has been questioned; carotenoid oxidation products can give a positive reaction. Experimental design and control must be rigorous. Purity of the carotenoid tested is essential; if it is not free from peroxides, completely different results can be obtained. Comparison between different studies and different systems is, therefore, fraught with difficulty. Model and non-biological studies of antioxidant effects are usually undertaken under conditions that do not resemble those in vivo. To demonstrate antioxidant activity in a natural system in vivo is extremely difficult; the system is so complex, conditions are difficult to control and are not uniform within the system, natural carotenoid concentrations are low and many interactions with other substances are possible. There is, however, evidence that a synergistic/cooperative interaction between carotenoids and antioxidants such as tocopherols, ascorbic acid, and flavonoids, may play a role in the biological antioxidant network. This does not prove that carotenoids are antioxidants in vivo, however. It may be that the antioxidants protect the carotenoid and prevent the formation of pro-oxidant carotenoid peroxides and peroxy radicals, or regulate the formation of oxidative breakdown products that could have biological actions?

Editors’ Assessment 419 2. Via metabolites A question that frequently arises is whether any effect attributed to a carotenoid is in fact due to a metabolite/breakdown product rather than to the intact carotenoid itself (Chapter 18). For any carotenoid, there are many products, as complex mixtures. Any one, or combination, could have some biological activity. The roles of vitamin A and retinoic acid are well known but evidence is accumulating to suggest that other non-retinoid breakdown products may be biologically active. Some possibilities that have been suggested are: (i) β-Carotene cleavage products of different chain length or cleavage products of other carotenoids, bearing structural features (end group or chain) in common with retinoids, could act as retinoid agonists or antagonists. (ii) Many carotenoid oxidation products have reactive α,β-unsaturated aldehyde structures. By binding to side-chain amino groups or, for dialdehydes, cross-linking, these could modify properties and activity of enzymes or other proteins. (iii) The biological activity of many compounds is based on size, shape and position of functional groups. Although otherwise structurally unrelated, a carotenoid breakdown product could have the right topography to mimic some other molecule, e.g. vitamin D, hormones, and act as an agonist or antagonist. 3. Via the immune system Carotenoid status is associated with immunocompetence (Chapter 17). Effects of several different carotenoids on various parameters of the human immune response system have been demonstrated, mostly in experiments in vitro. It is known that vitamin A strongly influences the immune system. When this is extended to provitamin A carotenoids, it is always possible that any action is via vitamin A. Effects are also seen, however, with some non-provitamin A carotenoids, especially lutein and astaxanthin. The question of whether antioxidant effects, or action via non-retinoid carotenoid metabolites may be involved in any such effects has been raised. This potentially interesting topic merits further careful investigation. E. Reports of Other Health Effects Possibilities of other effects of carotenoids on other aspects of health have been proposed. Most have not been pursued, but some do look interesting, though much more work is needed to substantiate any of these suggestions. The work is so far not extensive enough to warrant specific coverage in this Volume, but the topics are worth further consideration.

420 George Britton, Synnøve Liaaen-Jensen and Hanspeter Pfander 1. Water-soluble carotenoids Carotenoids are usually not soluble in water and are therefore located in a hydrophobic environment in vivo. Carotenoid dicarboxylic acids, however, have appreciable solubility in water, allowing them to remain in an aqueous environment in cells or fluids. Effects of the carotenoid dicarboxylic acid crocetin (538) and its disodium salt on haemorrhagic shock and wound trauma have been reported and attributed to increasing pulmonary oxygen flow to tissues [6]. It is proposed that the dipolar structure and its associated water shell impart special properties to the molecule. It would be interesting to know if similar effects could be found with carotenoid dicarboxylic acids of other chain lengths. HOOC COOH crocetin (538) . 2. Bone health Bone health is the result of two opposing processes, bone formation and bone resorption. These are regulated by various factors, notably hormones and vitamin D. β-Carotene, lycopene and other carotenoids have been associated with a role in supporting bone formation via increasing levels of bone-forming proteins [7]. This would constitute a benefit of carotenoids against brittle bones in the elderly. In contrast, high intake of vitamin A is associated with loss of bone mineral density and risk of osteoporosis (Chapter 9). 3. Metabolism and mitochondria In recent years there are increasing suggestions that carotenoids (most experimental work has been with astaxanthin) may help to offset the imbalance known as ‘metabolic syndrome’ by increasing mitochondrial efficiency, energy metabolism and especially fat metabolism, thereby helping to reduce obesity and enhance athletic performance [8]. In intense and prolonged exercise, the level of metabolism in mitochondria in the muscles is high and oxidative stress greatly increases. Also, according to the ‘mitochondrial theory of aging’, oxidative damage, to DNA, protein and lipids, accumulates in the mitochondria over the lifetime of the organism. Mitochondrial dysfunction leads to many consequences, including pro-oxidative changes in redox homeostasis and efflux of mitochondrial components, notably cytochrome c. This has been linked to effects on signalling mechanisms, impairment of the immune response and neurodegenerative conditions (Chapters 11 and 17). The indications are interesting enough to merit further rigorous study, which should be extended to other carotenoids.

Editors’ Assessment 421 F. Final Comments: The Big Questions (i) “Apart from their clear function as dietary precursors of vitamin A, do carotenoids have other functions or actions that are beneficial to human health?” A number of associations have been reported between higher concentrations of carotenoids in the blood and reduced risk of serious diseases such as cancer and CHD. Also actions of carotenoids on various cellular and molecular processes have been demonstrated in cells in culture. Intervention trials to try to show a direct relationship between administered pure carotenoid intake and disease risk have been less informative; conflicting results are common. (ii) “Are carotenoids important antioxidants in vivo?” Although it is clear that carotenoids can serve as antioxidants in vitro, there is no unequivocal evidence for their functioning in this way in vivo. Indeed, carotenoid concentrations are low compared to those of recognized antioxidants such as vitamins C and E, so activity as a general antioxidant seems unlikely, but some specialized action in a particular sub-cellular environment such as a membrane, or in particular tissues, cannot be ruled out. All aspects must be considered. It is suggested, for example, that protection of LDL by lycopene as an antioxidant may be a factor in reducing risk of CHD. If this is looked at carefully, calculations show that, on average, there is only about one carotenoid molecule per four LDL particles. This does not seem compatible with a major antioxidant role. (iii) “Do carotenoids help to provide protection and reduce risks of serious degenerative diseases?” The honest answer to this and to most of the questions about the roles of carotenoids in human health is that we simply do not know. After many years of extensive and painstaking research, definite answers and unequivocal evidence are hard to find, though there is much circumstantial and indicative evidence. Much time and many resources have been spent chasing one popular idea to the exclusion of others or simply repeating experiments with one carotenoid after another. Would we now be better informed and have a better understanding, for example, if some of the massive research effort devoted to elucidating the proposed role of carotenoids as antioxidants had been channelled in other directions? (iv) “Might some other carotenoids be important for health?” Few of the hundreds of natural carotenoids have been studied for effects on human health. It is always possible that some, even ones that are not components of a normal diet, could have biological activity, though the question of safety and toxicity must be observed. One interesting structure is 3,3’-dihydroxyisorenieratene (161) which combines structural elements of carotenoids and tocopherol.

422 George Britton, Synnøve Liaaen-Jensen and Hanspeter Pfander HO OH 3,3'-dihydroxyisorenieratene (161) (v) “Is it good for health to take carotenoid supplements?” The bulk of evidence and the consensus of opinion supports the conclusion that intake of carotenoids at the levels obtained in a normal balanced diet is safe and may be beneficial, but the safety of larger intake, especially the high doses often taken as supplements is, at best, not proven. The reader must judge from the evidence. There is much uncertainty and it would not be wise or responsible of us to advocate the routine use of carotenoid supplements at levels above the normal dietary level of 5 mg/day. We make no recommendation either way; personally, however, we are not convinced to take supplements but do try to maintain a good supply of carotenoids from our normal diet. (vi) Finally, have we any advice for the researcher taking up the exciting challenges in this field? Evaluate the literature carefully and critically. Look at experimental design and make your own interpretation. Don’t rely only on interpretation by the author or by other reviewers or commentators. Make sure that any ideas and conclusions are compatible with the properties of the carotenoid as it exists in its natural state and surroundings. So enjoy it and: Above all, be open-minded and expect the unexpected!! References [1] R. Peto, R. Doll, J. D. Buckley and M. B. Sporn, Nature, 290, 201 (1981). [2] J. A. Olson, in Modern Nutrition in Health and Disease (ed. M. E. Shils, J. A. Olson, A. C. Ross and M. Shike), p. 521, Saunders, Philadelphia (1998). [3] R. S. Dueker, Y. Lin, A. B. Buchholz, P. D. Schneider, M. W. Lamé, H. J. Segall, J. S. Vogel and A. J. Clifford, J. Lipid Res., 41, 1790 (2000). [4] F. Tourniaire, W. Leung, C. Méplan, A.-M. Minihane, S. Hessel, J. von Lintig, J. Flint, H. Gilbert, J. Hesketh and G. Lietz, Carotenoid Sci., 12, 57 (2008). [5] G. W. Burton and K. U. Ingold, Science, 224, 569 (1984). [6] L. J. Giassi, A. K. Poynter and J. L. Gainer, Shock, 18, 585 (2002). [7] S. Sahni, M. T. Hannan, J. Blumberg, L. A. Cupples, D. P. Kiel and K. L. Tucker, Am. J. Clin. Nutr., 89, 416 (2009). [8] M. Ishikara, Carotenoid Sci., 12, 3 (2008).

423 Index A Accelerator mass spectrometry 153, 388, 411 Acceptable daily intake of carotenoids 79 Acute myocardial infarction 125, 289–295 Age-related macular degeneration (AMD) Chapter 15 AIDS 180, 363, 375, 376 Alzheimer’s disease 373, 374 Angina pectoris 287, 290 Antioxidant/pro-oxidant properties of carotenoids Chapter 12 factors that determine antioxidant or pro-oxidant behaviour 251 relevance in vivo 259 Antioxidant response element 224, 225, 378, 396 Apoptosis Chapter 11, 252, 256, 270, 357, 366, 376, 385–398 AREDS trial 327, 328 ATBC trial 79, 251, 295, 296, 392, 415, 416 Athletic performance 420 Azo-initiators 238, 242, 258, 418 B Beadlets 75–79, 88, 136, 229, 345 Beaver Dam Eye Study 326 Bioavailability Chapter 7 absorption, transport, and deposition in tissues 116-131 definition 115 influential factors 133–138, 166, 167 Biofortification 101, 184–186 Biomarkers 10, 43, 128, 197–207, 229, 230, 241, 247, 248, 261, 270, 277, 294, 297, 414 Biopsy 202, 203, 328, 349 Bitot’s spots 178 Bone health 177, 395, 420 Breast-feeding 155, 186

424 C C30 RP-HPLC column 19, 25, 26 Cancer Chapter 13 Canthaxanthin crystals in the eye 72, 307, 416 Capsules 74–77, 90 CARET trial 251, 295, 415 Carotenodermia 57, 77, 341 Carotenoid aggregates 3, 58, 62, 230, 253, 416 Carotenoid analysis Chapter 2 non-invasive methods 43, 143, 202, 314, 338, 414 quality control 201 Carotenoid metabolites Chapter 18 summary of beneficial and harmful effects 402 Carotenoid purity, importance 418 Carotenoid supplements Chapter 4 legislation and claims 70, 71 pharmacological doses 139, 176, 182, 186, 222, 224, 248, 415 regulatory approval 95 safety 324 Carotenoids in food Chapter 3 factors that affect composition and content 57–60 good sources 55 Carotenoids or carotenoid-rich food 207, 371, 413 Cataract 71, 77, 80, 150, 191, 260, Chapter 15 Cell cycle Chapter 11 Chinese wolfberry 72, 320, 325 Chylomicrons 115–142, 155, 156, 175, 176 CIELAB 65 Cis/trans isomers see Geometrical isomers Comet assay 243, 249, 252, 399 Connexins 213, 229, 401 Conversion factors 150–169, 174 methods to determine 153–163 summary of experimental values 163, 164 Coronary heart disease Chapter 14 Cosmetics 68, 91, 100, 342, 350 Cyclins 214–218, 377 Cystic fibrosis 318 Cytochrome c 219, 377 Cytochrome P450 227, 228, 253, 392, 397, 402

425 D Dazzle, glare and haze 312 Delayed type hypersensitivity 350, 366–379 Delivery to cell cultures 229, 342 Dendritic cells 228, 365, 370 Detoxication 224, 227, 396, 398, 415, 421 Diarrhoea 140, 179–183, 371 Dietary fat 135, 136, 167 Dietary fibre 135–137, 166, 167, 205, 413 E Epidemiology Chapter 10 Erythrocytes 249, 420 Erythropoietic protoporphyria 80, 317, 345, 415 Exercise-induced oxidative damage 372 Eye Chapter 15 E/Z Isomers see Geometrical isomers F Food composition tables 32, 55, 63–65, 153, 154, 187, 198, 271, 411 analytical precision 64, 411 Food frequency questionnaires 140, 191, 198, 199 design and limitations 198 Food matrix 100, 115–117, 132, 142, 161, 165, 166, 379, 411 Food preparation 161, 166 Food processing 115, 133 Food quality and safety 99 Formulations 4, 73–78, 86–90, 95, 187, 255, 320, 411 Fortification 4, 70, 182–187 Functional foods 1, 45, 99, 188 Functional genomics 230 Functions, actions and associations 409 G Gap junction communication 213, 229, 243, 391, 395, 401, 402 Geometrical isomers and isomerization 2, 3, 9, 13, 15, 16, 21, 25, 32, 39, 49, 58–63, 73, 75, 78, 85–88, 118, 119, 122, 123, 134, 135, 386, 388, 389, 416 Gastrointestinal tract 131–142, 150, 195, 259

426 Genetic modification 3, 50–53, 89, 93, 96, Chapter 6, 184–187, 413 acceptability of GM products 112 examples of GM crops with altered carotenoid compositions 102 prospects for GM microorganisms 95, 96 Golden rice 4, 52, 101, 111, 112, 165, 169, 185 Growth spurt in children 182 H Haemorrhagic shock 420 Haem oxygenase-1 224, 225, 255, 352, 396 Hazard ratio 273 HDL 121, 142, 317 Hepatic stellate cells 169, 175 Heterochromatic flicker photometry 314–320, 328 HIV 150, 180, 249, 376, 377 HPLC Chapter 2, 48, 63, 64, 76, 134, 158, 160, 201–203, 305, 314, 325, 338, 342, 343, 416 internal standards 28 multiple peaks, injection artefacts 21 Human genome 139, 144, 187, 412 Human papilloma virus 271, 280, 281 Human tissue samples – analysis 9, 39 Hypervitaminosis A 176 I IARC 79 Immune response system 290, Chapter 17, 409, 419 and cancer 374 introduction to the immune system 364–366 role of reactive oxygen species 366, 367 Infections 115, 167, 169, 177–180, 183, 186 Inflammatory response 212, 223, 226, 228, 337, 350, Chapter 17, 391 Intrinsically labelled food 161–163, 169 Ischaemic shock 291–296 Isotopic labelling 153, 157, 159, 161, 169, 183, 187, 411 In vitro digestion model 140, 142 K Keratomalacia 177, 178 Keratosis 342

427 L Leukaemia 219, 221 LDL 116–123, 142, 242, 247, 257, 260, 287, 317, 421 Lipase 10, 121, 123, 183 Lipofuscin 316, 317, 323, 326 Lipoproteins Chapter 7, 156, 226, 240, 247 Liposomes 230, 246, 257, 258, 342, 351, 352 Lipoxygenase 58, 61 Lycopenodermia 341 M Macula lutea Chapter 15 Malaria 140 Matrix metalloproteases 223, 254, 336, 337, 349 Measles 150, 179–183 Melanoma 282, 346 Metabolic syndrome 420 Metabolomics 108, 357 Micelles 78, Chapter 7, 230, 246, 257, 258 Microarrays 5, 6, 217, 230, 357 Mitochondria 125, 219, 235, 256, 367, 368, 372, 373, 376, 392, 402, 420 Mitochondrial DNA 354, 355 Mitochondrial theory of aging 373, 420 N Nanoparticles 255, 342, 352 Natural carotenoid production Chapters 4–6 environmental consequences 417 Natural killer cells 289, 290, 364 Natural or synthetic carotenoids? 417 Neurodegenerative diseases 257, 367, 373, 374, 377, 418, 420 NHANES survey 154, 193, 326 Night blindness 150, 173, 177 Non-responders 122, 144, 158, 187, 260, 319, 414 O Obesity 420 Odds ratio 273 Oncogenes 215, 226, 346

428 Oral tanning agent 77, 307, 416 Osteoporosis 177, 420 P Parasite infection 139, 140, 167, 169, 182, 365 Passive diffusion 119, 120, 144 Phase I enzymes 228, 397 Phase II enzymes 224, 225, 378, 396–402 Photoaging 335, 348–350 Physicians’ Health Study 291 Plant breeding Chapter 6, 184, 187 Polymorphisms 187, 230, 412 Polyunsaturated fatty acids (PUFA) 236, 241, 257, 323, 327 Postprandial chylomicron response 140, 156, 157, 166 PPAR 226–229, 377, 390 Prostaglandins 219, 226, 241, 378 Prostate-specific antigen 207, 261 Protein malnutrition 167, 175, 179, 181 Proteomics 357, 366 Pulmonary oxygen flow 420 R Reactive oxygen species Chapter 12 Red palm oil 53, 72, 79, 85, 127, 132, 157, 183, 185, 187, 198, 417 Redox-sensitive proteins 218 Relative risk 273 Resonance Raman spectroscopy 43, 132, 143, 202, 203, 317, 326, 341, 342, 356, 414 validation of method 202, 203 Respiratory burst 367, 371 Respiratory infection 150, 178–180, 371, 373 Responders 122, 158, 187, 411, 412 Retinal pigment epithelium (RPE) 226, 249, 256, 259, 304, 307, 316, 322, 324 Retinal responders/non-responders 328 Retinoid receptors (RAR, RXR) 222, 390 Retinol activity equivalent (RAE) 176 Retinol binding protein (RBP) 126, 140, 175, 176 Retinol equivalent (RE) 154, 162, 176 Rheumatoid arthritis 374

429 S Seafood 54, 150 Signalling mechanisms 5, 181, Chapter 11, 212, 213, 219, 336 Skin Chapter 16 carotenoid concentrations with/without supplementation 339 structure and UV penetration 336, 337 SLAMENGHI 131 Smoke condensate (TAR) 218, 219, 225 SR-B1 116, 119–124, 130, 144 Stable isotopes 143, 157, 159, 161, 169 Sunburn 77, 335, 351 Sunscreen 77, 347–349, 359 Sun protection factor 347, 348 T TBARS 237, 241, 242, 247, 248, 254, 351, 418 and β-carotene oxidation products 245 Total antioxidant capacity 237, 240, 247 Toxicity 88, 95, 175, 176, 183–186, 230, 325 Transgenic tomatoes 51, 106–109, 413 U UVA, UVB Chapter 16 V Variability between individuals, 135, 141, 187, 318, 319, 411 VLDL 116, 121, 142, 156 Visual acuity 125, 302, 312, 328 Vitamin A deficiency Chapter 9 W WHEL study 275 Wound trauma 420 Wrinkling 37, 348

430 X Xanthophyll acyl esters 11, 18, 26, 27, 30, 36, 39, 51, 60, 78, 80, 134, 319, 340 Xanthophyll-binding protein 124, 125, 309, 317, 318 Xenobiotics 224, 227 Xerophthalmia 150, Chapter 9 stages and definitions 177, 178 Y Yellow carrots 122, 138, 413

431 Postscript This Volume brings to an end our work on this Carotenoids series, in which we have tried to tell the whole carotenoid story. It has been a demanding task that has taken over our lives for many years. But it has also been a ‘labour of love’. We have learned a lot and hope this passes on to the reader. When the time comes for a new ‘Carotenoids’ project, it will be in new hands, seen with fresh eyes and bringing fresh ideas. Whoever undertakes this task, we wish them well. In devoting ourselves to this project we have inevitably deprived others of our time and attention, and we thank our friends and colleagues and especially our families for their understanding and support during these long years. And we hope all who read and use these books will come to enjoy the challenges and beauty of carotenoids as much as we have. George Britton Synnøve Liaaen-Jensen Hanspeter Pfander