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Albert J. Augustin Karlsruhe
20 figures, 5 in color, and 17 tables, 2005
Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney
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Library of Congress CataloginginPublication Data
Nutrition and the eye / volume editor, Albert J. Augustin.
p. ; cm. – (Developments in ophthalmology, ISSN 02503751 ; v. 38) Includes bibliographical references and indexes. ISBN 3805578385 (hard cover : alk. paper)
1. Eye–Diseases–Prevention. 2. Eye–Diseases–Nutritional aspects. 3. Dietary supplements–Therapeutic use.
[DNLM: 1. Dietary Supplements. 2. Eye Diseases–prevention & control. 3. Drug Evaluation. 4. Nutrition. WW 140 N9756 2005] I. Augustin, Albert J.
II. Series. RE48.N88 2005 617.7�061–dc22
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
© Copyright 2005 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acidfree paper by Reinhardt Druck, Basel ISSN 0250–3751 ISBN 3–8055–7838–5
VI List of Contributors VII Preface
Augustin, A.J. (Karlsruhe)
Schreier, P. (Würzburg)
Janisch, K.M.; Milde, J.; Schempp, H.; Elstner, E.F. (Freising)
Stahl, W. (Düsseldorf)
89 Selenium, Selenoproteins and Vision
Flohé, L. (Magdeburg)
103 Nutritional Supplementation to Prevent Cataract Formation
Meyer, C.H.; Sekundo, W. (Marburg)
E.F. Elstner, Prof. Dr.
TUM Weihenstephan, Center of Life and Food Sciences Lehrstuhl für Phytopathologie Am Hochanger 2 DE85350 Freising (Germany)
MOLISA GmbH Universitätsplatz 2 DE39106 Magdeburg (Germany) Tel. �49 331 7480950 EMail email@example.com
K. M. Janisch, Dr.
TUM Weihenstephan, Center of Life and Food Sciences Lehrstuhl für Phytopathologie Am Hochanger 2 DE85350 Freising (Germany)
Department of Ophthalmology Philipps University of Marburg RobertKochStrasse 4 DE35037 Marburg (Germany) Tel. �49 6421 2862616 Fax �49 6421 2865678 EMail firstname.lastname@example.org
J. Milde, Dr.
TUM Weihenstephan, Center of Life and Food Sciences Lehrstuhl für Phytopathologie Am Hochanger 2 DE85350 Freising (Germany)
H. Schempp, Dr.
Ursula Schmidt-Erfurth, Prof. Dr. med.
Universitätsklinik der Augenheilkunde und Optometrie Währingergürtel 1820 AT1090 Vienna (Austria) Tel. �43 140 4007930 Fax �43 140 4007932 EMail email@example.com
Peter Schreier, Prof. Dr.
Lehrstuhl für Lebensmittelchemie Universität Würzburg Am Hubland DE97074 Würzburg (Germany) EMail firstname.lastname@example.org
Walter Sekundo, Priv.-Doz. Dr. med.
Department of Ophthalmology Philipps University of Marburg RobertKochStrasse 4 DE35037 Marburg (Germany) Tel. �49 6421 2862643 Fax �49 6421 2865678 EMail email@example.com
W. Stahl, Prof. Dr.
Heinrich Heine University Düsseldorf Institute of Biochemistry and Molecular Biology I PO Box 101007 DE40001 Düsseldorf (Germany) Tel. �49 211 8112711 Fax �49 211 8113029 EMail firstname.lastname@example.org
In the last 10 years, there has been growing interest in antioxidants and food supplements. Many diseases of the human body, especially those of the eye, are initiated by oxidative metabolites leading to oxidative tissue damage. One of the most important agents contributing to oxidative damage of eye tissue is the physiological stimulus – light. These effects were studied extensively in the early 1980s. However, successful procedures for lens removal and replacement by intraocular lenses have reduced the enthusiasm for further research. New approaches for the treatment of agerelated macular degeneration (AMD) have resulted in an overwhelming increase in the interest to continue with research in the field of AMD and related diseases. This was further enhanced by the finding that the lens can protect the macula by filtering the highenergy portion of the visible spectrum of light. In addition, we have learned that numerous diseases of the retina are mediated by oxidative tissue damage. This damage can be initiated and propagated by light and/or inflammatorymediated mechanisms as in AMD or by the result of oxidative metabolites of another origin. The generation of advanced glycation end products and the consecutive propagation of oxidative processes play an important role in the pathogenesis of proliferative diabetic retinopathy. Interestingly, there is an association between the generation of oxidative metabolites and inflammatory reaction and expression of growth factors such as VEGF, with cause and effect being taken into consideration.
In the aging organism, several antioxidative protective mechanisms are reduced in both their function and concentration. Consequentially, the pharmaceutical industry has put an overwhelming amount of food supplements on the market, which is confusing for physician and consumer alike. In addition, we know that the application of antioxidative agents can lead to a prooxidative or counterreaction of those substances and disturb the balance of the antioxidative network. Fortunately, basic research findings have enhanced our knowledge of free radical mediated processes. These findings are an important contribution when making recommendations for which of antioxidative substances should be given.
This textbook is divided into two major sections: (1) basic research focusing on the major compounds of nutrition and food supplements, and (2) clinical research providing the latest information on the results of important clinical studies. The first section gives further insight into the mechanisms of action of major substances relevant to antioxidants and food supplements in relation to eye diseases. Recommendations for maximum consumption of the respective substances are given. The consequence and relevance of one of the most important trace elements – selenium – is discussed in a separate section. This is also true for vitamins E and C as well as for lutein and zeaxanthin, the physiological macular pigment. The second section focuses on both anterior and posterior segment diseases which might be influenced by food supplementation and/or antioxidants. The latest relevant studies for daily clinical work are discussed. In addition, the oxidative pathomechanisms of the most important disease processes are explained.
It is therefore hoped that this textbook, intended for clinicians and basic vision scientists, will enhance the interest in oxidative processes in eye diseases and increase research activity, especially in eye diseases leading to blindness such as diabetic retinopathy and AMD.
Albert J. Augustin
Lehrstuhl für Lebensmittelchemie, Universität Würzburg, Würzburg, Germany
The actual knowledge about food sources, functions and nutrientnutrient interactions, deficiencies as well as chemopreventive properties including risk/benefit considerations of vitamins C and E, selected carotenoids and flavonoids, as well as copper, zinc and selenium, and �lipoic is reviewed.
Copyright © 2005 S. Karger AG, Basel
Evidence from both epidemiological and experimental observations has shown that the high consumption of fruits and vegetables may help to prevent diseases in humans. Because of their welldocumented properties, several vitamins, polyphenols and flavonoids, as well as trace elements have found particular attention as potential chemopreventive agents in our diet.
In the following, the actual knowledge about sources, functions and interactions, deficiencies as well as chemopreventive properties of vitamins C and E, carotenoids, flavonoids, copper, zinc and selenium, as well as �lipoic acid is summarized.
Vitamin C, Lascorbic acid (R5[(S)1,2dihydroxymethyl]3,4dihydroxy5Hfuran2one), is a watersoluble vitamin. Unlike most mammals, humans do not have the ability to make their own vitamin C. Therefore, we must obtain vitamin C through our diet.
As shown in table 1, different fruits and vegetables vary in their vitamin C content. In several foods, such as cabbage, Lascorbic acid is found in form of
Table 1. Vitamin C in selected foods
Food (raw) Vitamin C, mg/100 g
Beef, pork, fish 0–2 Liver, kidney 10–40
Cow 1–2 Human 3–6
Brussels sprouts 90–150 Carrot 5–10 Oat, rye, wheat 0 Kale 120–180 Potato 10–30 Rhubarb 10 Spinach 50–90 Tomato 20–35
Acerola 1,300 Apple 10–30 Banana 10 Citrus fruits 40–50 Guava 300 Sea buckthorn 160–800 Strawberry 40–90
ascorbigen A (B) that is split into Lascorbic acid in the course of cooking. However, thermal treatment usually destroys a considerable part of vitamin C.
There are many functions related to vitamin C. It is required for the synthesis of collagen and also plays an important role in the syntheses of norepinephrine and carnitine . Recent research also suggests that vitamin C is involved in the metabolism of cholesterol to bile acids .
Vitamin C is a highly effective antioxidant and can protect proteins, lipids, carbohydrates, and nucleic acids from damage by free radicals and reactive oxygen species that can be generated during normal metabolism as well as through exposure to toxins and pollutants. Vitamin C may also be able to regenerate other antioxidants, such as vitamin E .
However, vitamin C can interact in vitro with some free metal ions to produce potentially damaging free radicals. Although free metal ions are not generally found under physiological conditions, the idea that high doses of vitamin C might be able to promote oxidative damage in vivo has received a great deal of attention. A recent review found no sufficient scientific evidence that vitamin C promotes oxidative damage under physiological conditions or in humans .
Severe vitamin C deficiency has been known for many centuries as the disease scurvy. By the late 1700s the British navy was aware that scurvy could be cured by eating oranges or lemons, even though vitamin C would not be isolated until the early 1930s. At present, scurvy is rare in developed countries because it can be prevented by as little as 10mg of vitamin C daily.
In the USA, the recommended dietary allowance (RDA) for vitamin C was revised upward from 60mg daily for men and women (table 2). The recommended intake for smokers is 35 mg/day higher than for nonsmokers, because smokers are under increased oxidative stress and generally have lower blood levels of vitamin C .
Much of the information regarding vitamin C and the prevention of chronic disease is based on prospective studies in which vitamin C intake is assessed in large numbers of people who are followed over time to determine whether they develop specific chronic diseases.
Until recently, the results of most studies indicated that low or deficient intakes of vitamin C were associated with an increased risk of cardiovascular
Table 2. RDA for vitamin C in the USA
|Life stage||Age||Males, mg/day||Females, mg/day|
|Adults||19 years and older||9075|
|Smokers||19 years and older||125||110|
|Pregnancy||18 years and younger||–||80|
|Pregnancy||19 years and older||–||85|
|Breastfeeding||18 years and younger||–||115|
|Breastfeeding||19 years and older||–||120|
diseases and that modest dietary intake of about 100mg/day was sufficient to reduce the risk among nonsmoking men and women . Several studies failed to find significant reductions in the risk of coronary heart disease (CHD) among vitamin C supplement users [5, 6]. The First National Health and Nutrition Examination Study (NHANES I)  found that the risk of death from cardiovascular diseases was 42% lower in men and 25% lower in women who consumed
�50mg/day of dietary vitamin C . Recent results from the Nurses’ Health Study based on the followup of more than 85,000 women over 16 years also suggest that higher vitamin C intakes may be cardioprotective . In this study, vitamin C intakes of �300 mg/day from diet plus supplements or supplements were associated with a 27–28% reduction in CHD risk. However, in those women who did not take vitamin C supplements, dietary vitamin C intake was not significantly associated with CHD risk. This finding is inconsistent with data from numerous other prospective cohort studies that found inverse associations between dietary vitamin C intake of vitamin C plasma levels and CHD risk [1, 10]. Data from the National Institutes of Health (NIH) indicated that plasma and circulating cells in healthy, young subjects became fully saturated with vitamin C at a dose of 400 mg/day . The results of the NHANES I study and the Nurses’ Health Study suggest that maximum reduction of cardiovascular disease risk may require vitamin C intakes high enough to saturate plasma and circulating cells, and thus the vitamin C body pool .
From a number of studies it has been concluded that increased consumption of fresh fruits and vegetables is associated with a reduced risk of several types of cancer . Such studies are the basis for dietary guidelines which recommend at least 5 servings of fruits and vegetables per day.
In several casecontrol studies the role of vitamin C in cancer prevention has been investigated. Most have shown that higher intakes of vitamin C are associated with a decreased incidence of several cancers. In general, prospective studies in which the lowest intake group consumed �86 mg of vitamin C daily have not found differences in cancer risk, while studies finding significant cancer risk reductions found them in people consuming at least 80–110 mg of vitamin C daily .
Although most large prospective studies found no association between breast cancer and vitamin C intake, two recent studies described dietary vitamin C intake to be inversely associated with breast cancer risk in certain subgroups . In the Swedish Mammography Cohort, women who consumed an average of 110mg/day of vitamin C had a 39% lower risk of breast cancer compared to women who consumed an average of 31 mg/day . A number of observational studies have found increased dietary vitamin C intake to be associated with decreased risk of stomach cancer, and laboratory experiments indicate that vitamin C inhibits the formation of carcinogenic compounds in stomach. Infection with Heliobacter pylori is known to increase the risk of stomach cancer and also appears to lower the vitamin C content of stomach secretions. Although two intervention studies did not find a decrease in the occurrence of stomach cancer with vitamin C supplementation , more recent research suggests that vitamin C supplementation may be a useful addition to standard H. pylori eradication therapy in reducing the risk of gastric cancer .
Decreased vitamin C levels in the lens of the eye have been associated with increased severity of cataracts in humans. Some, but not all studies have observed increased dietary vitamin C intake  and increased blood levels of vitamin C  to be associated with decreased risk of cataracts. Those studies that have found a relationship suggest that vitamin C intake may have to be �300 mg/day for a number of years before a protective effect can be detected . Recently, a 7year controlled intervention trial of a daily antioxidant supplement containing 500mg of vitamin C, 400IU of vitamin E, and 15mg of �carotene in 4,629 men and women found no difference between the antioxidant combination and a placebo on the development and progression of agerelated cataracts . Therefore, the relationship between vitamin C intake and the development of cataracts requires further clarification.
Table 3. Tocopherols and tocotrienols
|�T.||H||CH3||CH3||2R,4�R,8�R||[�]25456 � 2.4�|
A number of possible problems with very large doses of vitamin C have been suggested, mainly based on in vitro experiments or isolated case reports, including: genetic mutations, birth defects, cancer, atherosclerosis, kidney stones, ‘rebound’ scurvy, increased oxidative stress, excess iron absorption, and vitamin B12 deficiency. However, none of these adverse health effects have been confirmed, and there is no reliable scientific evidence that large amounts of vitamin C are toxic or detrimental to health. With the latest RDA published in 2000, a tolerable upper intake level (UL) of 2 g daily was recommended .
The term vitamin E comprises a family of several antioxidants, i.e. tocopherols and tocotrienols (cf. formula and table 3). �Tocopherol is the only form of vitamin E found in the largest quantities in the blood and tissue . As �tocopherol is the form of vitamin E that appears to have the greatest nutritional significance, it will be the primary topic of the following discussion.
Major sources of �tocopherol in the diet include vegetable oils (olive, sunflower, safflower oils) nuts, whole grains and green leafy vegetables. All forms of vitamin E occur naturally in foods, but in varying amounts. Selected examples are given in table 4.
The main function of �tocopherol in humans appears to be that of an antioxidant. The fatsoluble vitamin is suited to intercepting free radicals and preventing a chain reaction of lipid destruction. Aside from maintaining the integrity of cell membranes throughout the body, �tocopherol also protects the lipids in low density lipoproteins (LDL) from oxidation.
Several other functions of �tocopherol have been described. It is known to inhibit the activity of protein kinase C, an important cell signaling molecule, as well as to affect the expression and activity of immune and inflammatory
Table 4. Amounts of vitamin E in selected foods (mg/100 g)
�Tocopherol �Tocopherol �Tocopherol Others
Milk, cow 0.08 – – –
pasteurized Milk, human 0.5 – – – Egg 0.7 – 0.4– Butter 2.2 – – – Margarine 14.0– – – Olive oil 11.9 0.1 0.6 – Maize oil 25.0055.8
.65 2.5 Wheat germ oil 192.0 50.8 30.4 6.8 Spinach 1.6 – 0.1 0.8 Tomato 0.8 – 0.1 – Wheat, whole 1.00–
.4 2.9 grain
cells. In addition, �tocopherol has been shown to inhibit platelet aggregation and to enhance vasodilation [21, 22].
The function of �tocopherol in humans is still unclear. Its blood levels are generally 10 times lower than those of �tocopherol. Limited in vitro and animal tests indicate that �tocopherol or its metabolites may play a role in the protection of the body from damage of free radicals [23, 24], however these effects have not been demonstrated convincingly in humans.
In one recent prospective study increased plasma �tocopherol levels were associated with a significantly reduced risk of developing prostate cancer, while significant protective associations for increased levels of plasma �tocopherol were found only when �tocopherol levels were also high . These limited findings, in addition to the fact that taking �tocopherol supplements may lower
�tocopherol levels in blood, have activated the interest for additional research on the effects of dietary and supplemental �tocopherol on health .
Vitamin E deficiency has been observed in individuals with malnutrition, genetic defects affecting the �tocopherol transfer protein and fat malabsorption syndromes. Severe vitamin E deficiency results mainly in neurological symptoms (ataxia and peripheral neuropathy), myopathy, and pigmented retinopathy. For this reason, people who develop peripheral neuropathy, ataxia or retinitis pigmentosa should be screened for vitamin E deficiency .
Table 5. RDA for (RRR)�tocopherol in the USA
|Adults||19 years and older||15||15|
Although true vitamin E deficiency is rare, suboptimal intake of vitamin E is quite common. The National Health and Nutrition Examination Survey III (NHANES III) investigated the dietary intake and blood levels of �tocopherol in 16,295 multiethnic adults. 27% of white participants, 41% of AfricanAmericans, 28% of MexicanAmericans and 32% of the other participants were found to have blood levels of �tocopherol �20 �mol/l, a value chosen because the literature suggests an increased risk for cardiovascular disease below this level .
The RDA for vitamin E was previously 8 mg/day for women and 10mg/day for men, but it was revised in 2000  (table 5).
The results of large observational studies suggest that increased vitamin E consumption is associated with decreased risk of myocardial infarction or death from heart disease in both men and women. Each study was a prospective study which measured vitamin E consumption in presumably healthy people and followed them for number of years to determine how many of them were diagnosed with, or died as a result of heart disease. In two of the studies, those individuals who consumed �7 mg of �tocopherol in food were only approximately 35% as likely to die from heart disease as those who consumed �3–5 mg of
�tocopherol [29, 30]. Two other large studies found a significant reduction in the risk of heart disease only in those women and men who consumed �tocopherol supplements of at least 67 mg of (RRR)�tocopherol daily [31, 32]. Recently, several studies have observed plasma or red blood cell levels of �tocopherol to be inversely associated with the presence or severity supplements in patients with heart disease have not shown vitamin E to be effective in preventing heart attacks or death [33, 34].
Several large prospective studies have failed to find significant associations between �tocopherol intake and the incidence of lung cancer or breast cancer . A placebocontrolled intervention study designed to look at the effect of �tocopherol supplementation on lung cancer in smokers found a 34% reduction in the incidence of prostate cancer in smokers given supplements of 50mg of synthetic �tocopherol (equivalent to 25 mg of (RRR)�tocopherol) daily .
To date, ten observational studies have examined the association between vitamin E consumption and the incidence and severity of cataracts. Of these studies, five found increased vitamin E intake to be associated with protection from cataracts, while five reported no association [36, 37]. A recent intervention trial of a daily antioxidant supplement containing 500 mg of vitamin C, 400 IU of vitamin E, and 15 mg of �carotene in 4,629 men and women found that the antioxidant supplement was not different than a placebo in its effects on the development and progression of agerelated cataracts over a 7year period . Another invention trial found that a daily supplement of 50mg of synthetic �tocopherol daily (equivalent to 25 mg (RRR)�tocopherol) did not alter the incidence of cataract surgery in male smokers . Thus, the relationship between vitamin E intake and the development of cataracts requires further clarification.
�Tocopherol has been shown to enhance specific aspects of the immune response. For instance, 200 mg of synthetic �tocopherol (equivalent to 100 mg (RRR)�tocopherol) daily for several months increased the formation of antibodies in response to hepatitis B vaccine and tetanus vaccine in elderly adults . Whether �tocopherolassociated enhancements in the immune response actually translate to increased resistance to infections in older adults remains to be determined .
Few side effects have been noted in adults taking supplements of �2,000 mg of �tocopherol daily ([RRR] or racemic �tocopherol). However, results of longterm �tocopherol supplementation have not been studied. In addition
Table 6. Tolerable UL for �tocopherol in the USA
Age group UL, mg/day (IU/day) d�tocopherol
Infants 0–12 months Not possible to establish Children 1–3 years 200 (300) Children 4–8 years 300 (450) Children 9–13 years 600 (900) Adolescents 14–18 years 800 (1,200) Adults 19 years and older 1,000 (1,500)
to setting the new RDA for �tocopherol in 2000, an upper limit (UL) for �tocopherol was given (table 6). An UL of 1,000 mg daily of �tocopherol of any form would be the highest dose unlikely to result in potential hemorrhage in adults .
Copper (Cu) is an essential trace element for animals and humans. In the body, copper shifts between the cuprous (Cu1�) and the cupric (Cu2�) forms, though the majority of the body’s copper is in the Cu2� form. The ability of copper to easily accept and donate electrons explains its important role in oxidoreductions and scavenging of free radicals .
Copper is found in a wide variety of foods and is most plentiful in organ meats, shellfish, nuts, and seeds. Wheat bran cereals and whole grain products are also good sources of copper. According to national surveys, the average dietary intake of copper in the USA is approximately 1.0–1.1 mg/day for adult women and 1.2–1.6 mg/day for adult men.
Copper is a critical functional component of a number of essential enzymes. Some of the physiological functions known to be copperdependent are discussed below.
Cytochrome c oxidase plays an essential role in cellular energy production. By catalyzing the reduction of molecular oxygen to water, an electrical gradient is generated used by the mitochondria to create ATP .
Another cuproenzyme, lysyl oxidase, is required for the crosslinking of collagen and elastin. The action of lysyl oxidase helps to maintain the integrity of connective tissue in the heart and blood vessels and plays a role in bone formation .
Ceruloplasmin (ferroxidase I) and ferroxidase II have the capacity to oxidize ferrous iron (Fe2�) to ferric iron (Fe3�), the form of iron that can be loaded onto the protein transferring for transport to the site of red blood cell formation. Although the ferroxidase activity of these two cuproenzymes has not yet been proven to be physiologically significant, the fact that iron mobilization from storage sites is impaired in copper deficiency supports their role in iron metabolism .
A number of reactions essential to normal function of the brain and nervous system are catalyzed by cuproenzymes. Monoamine oxidase (MAO) plays a role in the metabolism of norepinephrine, epinephrine, and dopamine. MAO also functions in the degradation of serotonin which is the basis for the use of MAO inhibitors as antidepressants . The myelin sheath is made of phospholipids whose synthesis depends on cytochrome c oxidase activity .
In addition, tyrosinase is required for the formation of the pigment melanin. Melanin is formed in the melanocytes and plays a role in the pigmentation of the hair, skin and eyes .
Furthermore, copper has several antioxidant functions. Superoxide dismutase (SOD) is operative as an antioxidant by catalyzing the conversion of superoxide radicals to hydrogen peroxide which can subsequently be reduced to water enzymatically . Two forms of SOD contain copper, i.e. (i) copper/zinc SOD is found within most cells of the body, including red blood cells, and
(ii) extracellular SOD is a coppercontaining enzyme found in high levels in the lung and low levels in blood plasma .
Finally, copperdependent transcription factors regulate transcription of specific genes. Thus, cellular copper levels may affect the synthesis of proteins by enhancing or inhibiting the transcription of specific genes. They include genes for Cu/Zn SOD, catalase, and proteins related to the cellular storage of copper .
An adequate copper nutritional status appears to be necessary for normal iron metabolism and red blood cell formation. Iron has been found to accumulate in the livers of copperdeficient animals, indicating that copper (probably in the form of ceruloplasmin) is required for iron transport to the bone marrow for red blood cell formation . Infants fed a high iron formula absorbed less copper than infants fed a low iron formula, suggesting that high iron intakes may interfere with copper absorption in infants .
High supplemental zinc intakes of �50mg/day for extended periods of time may result in copper deficiency. High dietary zinc increases the synthesis of metallothionein, which binds certain metals and prevents their absorption by trapping them in intestinal cells. Metallothionein has a stronger affinity for copper than zinc, so, high levels of metallothionein induced by excess zinc cause a decrease in intestinal copper absorption. High copper intakes have not been found to affect zinc nutritional status [42, 47].
Although vitamin C supplements have produced copper deficiency in laboratory animals, the effect of vitamin C supplements on copper nutritional status in humans is less clear. In one human study, vitamin C supplementation of 1,500 mg/day for 2 months resulted in a significant decline in ceruloplasmin oxidase activity . In another one, supplements of 605 mg of vitamin C/day for 3 weeks also resulted in decreased ceruloplasmin oxidase activity, although copper absorption did not decline. Neither of these studies found vitamin C supplementation to adversely affect copper nutritional status.
Clinically evident copper deficiency is uncommon. Serum copper and ceruloplasmin levels may fall to 30% of normal in cases of severe copper deficiency. One of the most common clinical signs of copper deficiency is an anemia that is unresponsive to iron therapy but corrected by copper supplementation. The anemia is thought to result from defective iron mobilization due to decreased ceruloplasmin activity. Copper deficiency may also result in neutropenia, a condition that may be accompanied by increased susceptibility to infection. Abnormalities of bone development related to copper deficiency are most common in copperdeficient lowbirthweight infants and young children. Less common features of copper deficiency may include loss of pigmentation, neurological symptoms, and impaired growth [43, 44].
Individuals at Risk of Deficiency
Highrisk individuals include premature infants, especially those with low birth weight, infants with prolonged diarrhea, infants and children recovering from malnutrition, individuals with malabsorption syndromes, including celiac disease, sprue and short bowel syndrome due to surgical removal of a large portion of the intestine. Individuals receiving intravenous total parental nutrition or other restricted diets may also require supplementation with copper and other trace elements [43, 44]. Recent research indicates that cystic fibrosis patients may also be at increased risk of copper insufficiency .
Table 7. RDA for copper in the USA
|Adults||19 years and older||900||900|
A variety of indicators were used to establish to RDA for copper, including plasma copper concentration, serum ceruloplasmin activity, SOD activity in red blood cells, and platelet copper concentration . The RDA for copper reflects the results of depletionrepletion studies and is based on the prevention of deficiency (table 7).
While severe copper deficiency results in cardiomyopathy in some animal species, the pathology differs from atherosclerotic cardiovascular diseases prevalent in humans. Studies in humans have produced inconsistent results and their interpretation is hindered by the lack of a reliable marker of copper nutritional status. Outside the body, free copper is known to be a prooxidant and is frequently used to produce LDL oxidation. Recently, ceruloplasmin has been found to stimulate LDL oxidation in vitro , however, there is little evidence that copper or ceruloplasmin promotes LDL oxidation in vivo. In addition, SOD and ceruloplasmin are known to have antioxidant properties leading to assume that copper deficiency rather than excess copper increases the risk of cardiovascular diseases .
Several epidemiologic studies have found increased serum copper levels to be associated with increased risk of cardiovascular disease. A recent prospective study in the USA examined serum copper levels in more than 4,400 men and women at the age of 30 years and older . During the following 16 years, 151 participants died from CHD. After adjusting for other risk factors of heart disease, those with serum copper levels in the two highest quartiles had a significantly greater risk of dying from CHD. Three other casecontrol studies conducted in Europe had similar findings. Serum copper largely reflects serum ceruloplasmin and is not a sensitive indicator of copper nutritional status. Serum ceruloplasmin levels are known to increase by 50% or more under certain conditions of physical stress, such as trauma, inflammation, or disease. Because over 90% of serum copper is carried in ceruloplasmin, elevated serum copper may simply be a marker of the inflammation that accompanies atherosclerosis.
In contrast to the serum copper findings, two autopsy studies found copper levels in heart muscle to be lower in patients who died of CHD than those who died of other causes . Additionally, the copper content of white blood cells has been positively correlated with the degree of patency of coronary arteries in CHD patients [54, 55] and patients with a history of myocardial infarction had lower concentrations of extracellular SOD than those without such a history .
While in small human studies adverse changes in blood cholesterol levels, including increased total and LDLcholesterol levels and decreased HDLcholesterol levels  were found, other studies have not confirmed those results . Copper supplementation of 2–3 mg/day for 4–6 weeks did not result in clinically significant changes in cholesterol levels [51, 59]. Recent research has also failed to find evidence that increased copper intake increases oxidative stress. In a multicenter placebocontrolled study, copper supplementation of 3 and 6 mg/day for 6 weeks did not result in increased susceptibility of LDL ex vivo oxidation . Moreover, in vitro the oxidizability of red blood cells decreased , indicating that relatively high intakes of copper do not increase the susceptibility of LDL or red blood cells to oxidation.
Osteoporosis has been observed in infants and adults with severe copper deficiency, but it is not clear whether marginal copper deficiency contributes to osteoporosis. Research regarding the role of copper nutritional status in agerelated osteoporosis is limited. Serum copper levels of 46 elderly patients with hip fractures were reported to be significantly lower than matched controls . A small study in premenopausal women who consumed an average of 1 mg of dietary copper daily, reported a decreased loss of bone mineral density from the lumbar spine after copper supplementation of 3 mg/day for 2 years . Marginal copper intake of 0. 7mg/day for 6 weeks significantly increased a measurement of bone resorption (breakdown) in healthy adult males . However, copper supplementation of 3–6 mg/day for 6 weeks had no effect on biochemical markers of bone resorption or bone formation in a study of healthy adult men and women .
The exact mechanism of the action of copper on the immune system function is not yet known. Adverse effects in insufficient copper appear most
Table 8. Tolerable UL for copper in the USA
Age group UL, mg/day
Infants 0–12 months Not possible to establish Children 1–3 years 1 Children 4–8 years 3 Children 9–13 years 5 Adolescents 14–18 years 8 Adults 19 years and older 10
pronounced in infants. Infants with Menkes disease, a genetic disorder that results in severe copper deficiency, suffer from frequent and severe infections [66, 67]. In a study of 11 malnourished infants with evidence of copper deficiency, the ability of certain white blood cells to engulf pathogens increased significantly after 1 month of copper supplementation . More recently, 11 men on a lowcopper diet (0.66 mg copper/day for 24 days and 0.38 mg/day for another 40 days) showed a decreased proliferation response when mononuclear cells were presented with an immune challenge in cell culture . While severe copper deficiency has adverse effects on immune function, the effects of marginal copper insufficiency in humans are not clear.
Copper toxicity is rare in the general population. Acute copper poisoning has occurred through the contamination of beverages by storage in coppercontaining containers as well as from contaminated water supplies . In the USA, the healthbased guideline for a maximum water copper concentration of 1.3 mg/l is enforced by the Environmental Protection Agency (EPA) . Symptoms of acute copper toxicity include abdominal pain, nausea, vomiting, and diarrhea which help to prevent additional ingestion and absorption of copper. More serious signs of acute copper toxicity include severe liver damage, kidney failure, coma, and death.
As to healthy individuals, doses of up to 10mg daily have not resulted in liver damage. For this reason, the UL for copper was set at 10mg/day from food and supplements  (table 8).
Selenium is a trace element that is essential in small amounts but can be toxic in larger quantities. Humans and animals require selenium for the function of a number of selenoproteins. During their synthesis, selenocysteine is incorporated into a very specific location in the amino acid sequence in order to form a functional protein. Unlike animals, plants do not appear to require selenium. However, when selenium is present in the soil, plants incorporate it nonspecifically into compounds that usually contain sulfur [72, 73].
The richest food sources of selenium are organ meats and seafood, followed by muscle meats. In general, there is a wide variation in the selenium content of plants and grains, as the incorporation of selenium into plant proteins is dependent only on soil selenium content. Brazil nuts grown in areas of Brazil with seleniumrich soil may provide �100 �g of selenium in one nut, while those grown in seleniumpoor soil may contain 10 times less . In the USA, grains are a good source of selenium, but fruits and vegetables tend to be relatively poor sources. In general, drinkingwater is not a significant source of selenium. The average dietary intake of adults in the USA has been found to range from about 80 to 110 �g/day.
At least 11 selenoproteins have been characterized and there is evidence that additional selenoproteins exist.
Glutathione Peroxidases. Four seleniumcontaining glutathione peroxidases (GPx) have been identified, i.e. (i) cellular or classical GPx, (ii) plasma or extracellular GPx, (iii) phospholipid hydroperoxide GPx, and (iv) gastrointestinal GPx . Although each GPx is a distinct selenoprotein, they are all antioxidant enzymes that reduce potentially damaging reactive oxygen species, such as hydrogen peroxide and lipid hydroperoxides to harmless products like water and alcohols by coupling their reduction with the oxidation of glutathione.
Sperm mitochondrial capsule selenoprotein was once thought to be a distinct selenoprotein, but now appears to be phospholipid hydroperoxide GPx .
Chemopreventive Compounds in the Diet
Thioredoxin Reductase. Thioredoxin reductase participates in the regeneration of several antioxidant systems, possibly including vitamin C. Maintenance of thioredoxin in a reduced form by thioredoxin reductase is important for regulating cell growth and viability [75, 77].
Iodothyronine Deiodinases. The thyroid gland releases very small amounts of biologically active thyroid hormone (triiodothyronine, T3) and larger amounts of an inactive form of thyroid hormone (thyroxine, T4) into the circulation. Most of the biologically active T3 in the circulation and inside cells is created by the removal of one iodine atom from T4 in a reaction catalyzed by seleniumdependent iodothyronine deiodinase enzymes. Through their actions on T3, T4 and other thyroid hormone metabolites, three different seleniumdependent iodothyronine deiodinases (types I, II, and III) can both activate and inactivate thyroid hormone, making selenium an essential element for normal development, growth, and metabolism through the regulation of thyroid hormones [75, 78].
Selenoprotein P and W.
Selenoprotein P is found in plasma and is also associated with vascular endothelial cells. Although the function of selenoprotein P has not been clarified to date, it has been suggested to function as a transport protein as well as an antioxidant capable of protecting endothelial cells from peroxynitrite damage [75, 79]. Selenoprotein W is found in the muscle. Although its function is presently unknown, it is thought to play a role in muscle metabolism .
Incorporation of selenocysteine into selenoproteins is directed by the genetic code and requires the enzyme selenophosphate synthetase. A selenoprotein itself, selenophosphate synthetase catalyzes the synthesis of monoselenium phosphate, a precursor of selenocysteine which is required for the synthesis of selenoproteins [73, 75].
As an integral part of the GPx and thioredoxin reductase, selenium probably interacts with every nutrient that affects the prooxidant/antioxidant balance of the cell. Other minerals that are critical components of antioxidant enzymes include copper, zinc, and iron. Selenium as GPx also appears to support the activity of �tocopherol in limiting the oxidation of lipids. Animal studies indicate that selenium and �tocopherol tend to spare one another and that selenium can prevent some of the damage resulting from �tocopherol deficiency in models of oxidative stress. Thioredoxin reductase also maintains the antioxidant function of vitamin C by catalyzing its regeneration .
Selenium deficiency may impair the effects of iodine deficiency. Iodine is essential for the synthesis of thyroid hormone, but the iodothyronine deiodonases are also required for the conversion of T4 to the biologically active thyroid hormone T3. Selenium supplementation in a small group of elderly individuals decreased plasma T4, indicating increased deiodinase activity with increased conversion to T3 .
Insufficient selenium intake results in decreased activity of the GPx. Even when severe, isolated selenium deficiency does not usually result in obvious clinical illness.
Individuals at Risk of Deficiency
Clinical selenium deficiency has been observed in chronically ill patients who were receiving total parenteral nutrition (TPN) without added selenium for prolonged periods of time. Muscular weakness, muscle wasting, and cardiomyopathy have been observed. TPN solutions are now supplemented with selenium to prevent such problems. People who have had a large portion of the small intestine surgically removed or those with severe gastrointestinal problems, such as Crohn’s disease, are also at risk for selenium deficiency due to impaired absorption. Specialized medical diets used to treat metabolic disorders, such as phenylketonuria, are often low in selenium. Specialized diets that will be used exclusively over long periods of time should have their selenium content assessed to determine the need for selenium supplementation .
Keshan disease is a cardiomyopathy that affects young women and children in a seleniumdeficient region of China. The incidence of Keshan disease is closely associated with very low dietary intakes of selenium and poor selenium nutritional status. Despite the strong evidence that selenium deficiency is a fundamental factor in the etiology of Keshan’s disease, the seasonal and annual variation in its occurrence suggests that an infectious agent is also involved. Coxsackie virus is one of the viruses that has been isolated from Keshan patients and this virus has been found to be capable of causing an inflammation of the heart called myocarditis in seleniumdeficient mice. Studies in mice indicate that oxidative stress induced by selenium deficiency results in changes in the viral genome capable of converting a relatively harmless viral strain to a myocarditiscausing strain [80, 81]. Though not proven in Keshan disease, selenium deficiency may result in a more virulent strain of virus with the potential to invade and damage the heart muscle.
is characterized by osteoarthritis and is associated with poor selenium status in areas of northern China, North Korea, and eastern Siberia. The disease affects children between the ages of 5 and 13 years. Unlike Keshan disease, there is little evidence that improving selenium nutritional status prevents KashinBeck disease. Thus, the role of selenium deficiency in the
Table 9. RDA for selenium in the USA
|Adults||19 years and older||55||55|
etiology of KashinBeck disease is less certain. A number of other causative factors have been suggested for KashinBeck disease, including fungal toxins in grain, iodine deficiency, and contaminated drinkingwater .
The most recent RDA is based on the amount of dietary selenium required to maximize the activity of the antioxidant enzyme glutathione peroxidase in blood plasma  (table 9).
From the theory, optimizing selenoenzyme activity could decrease the risk of cardiovascular diseases by reducing lipid peroxidation and influencing the metabolism of cell signaling molecules known as prostaglandins. However, prospective studies in humans have not demonstrated strong support for the cardioprotective effects of selenium. While one study found a significant increase in illness and death from cardiovascular disease in individuals with serum selenium levels �45 �g/l compared to matched pairs �45 �g/l , another study using the same cutoff points for serum selenium found a significant difference only in deaths from stroke . A study of middleaged and elderly Danish men found an increased risk of cardiovascular disease in men with serum selenium levels �79 �g/l , but several other studies found no clear inverse association between selenium nutritional status and cardiovascular disease risk . In a multicenter study in Europe, toenail selenium levels and risk of myocardial infarction (hear attack) were only associated in the center where selenium levels were the lowest . While some epidemiologic evidence suggests that low levels of selenium may increase the risk of cardiovascular diseases, definitive evidence regarding the role of selenium in preventing cardiovascular diseases will require defined clinical trials.
There is good evidence that selenium supplementation at high levels reduced the incidence of cancer in animals. More than twothirds of over 100 published studies in 20 different animal models of spontaneous, viral, and chemically induced cancers found that selenium supplementation significantly reduced tumor incidence . The evidence indicates that the methylated forms of selenium are the active species against tumors, and these methylated selenium compounds are produced at the greatest amounts with excess selenium intakes. Selenium efficiency does not appear to make animals more susceptible to develop cancerous tumors .
A prospective study of more than 60,000 female nurses in the USA found no association between toenail selenium levels and total cancer risk . In a study of Taiwanese men with chronic viral hepatitis B or C infection decreased plasma selenium concentrations were associated with an even greater risk of liver cancer . A casecontrol study within a prospective study of over 9,000 Finnish men and women examined serum selenium levels in 95 individuals subsequently diagnosed with lung cancer and 190 matched controls . Lower serum selenium levels were associated with an increased risk of lung cancer and the association was more pronounced in smokers. In this Finnish population, selenium levels were only about 60% of that common in generally observed other Western countries.
Another casecontrol study within a prospective study of over 5,000 male
health professionals in the USA found a significant inverse relationship between toenail selenium content and the risk of prostate cancer in 181 men diagnosed with advance prostate cancer and 181 matched controls . In individuals whose toenail selenium content was consistent with an average intake of 159 �g/day, the risk of advance prostate cancer was only 35% of those individuals with toenail selenium content consistent with an intake of 86 �g/day. Within a prospective study of more than 9,000 JapaneseAmerican men, a casecontrol study that examined 249 confirmed cases of prostate cancer and 249 matched controls found the risk of developing prostate cancer to be 50% less in men with serum selenium levels in the highest quartile compared to those in the lowest quartile , while another casecontrol study found that men with prediagnostic plasma selenium levels in the lowest quartile were 4–5 times more likely to develop prostate cancer than those in the highest quartile . In contrast, one of the largest casecontrol studies to date found a significant inverse association between toenail selenium and the risk of colon cancer, but no associations between toenail selenium and the risk of breast cancer or prostate cancer .
Human Intervention Trials. (1) Undernourished populations: An intervention trial undertaken among a general population of 130,471 individuals in five townships of Qidong, China, a highrisk area for viral hepatitis B infection and liver cancer, provided table salt enriched with sodium selenite to the population of one township (20,847 people) using the other four townships as controls. During an 8year followup period, the average incidence of liver cancer was reduced by 35% in the seleniumenriched population while no reduction was found in the control populations. In a clinical trial in the same region, 226 individuals with evidence of chronic hepatitis B infection were supplemented with either 200 �g of selenium in the form of a seleniumenriched yeast tablet or a placebo yeast tablet daily. During the 4year followup period, 7 out of 113 individuals supplemented with the placebo developed primary liver cancer while none of the 113 subjects supplemented with selenium developed liver cancer . (2) Wellnourished populations: In the USA, a doubleblind, placebocontrolled study of more than 13,000 older adults with a history of nonmelanoma skin cancer found that supplementation with 200 �g/day of seleniumenriched yeast for an average of 7.4 years was associated with a 51% decrease in prostate cancer incidence in men . The protective effect of selenium supplementation was the greatest in those men with lower baseline plasma selenium and prostatespecific antigen (PSA) levels. Surprisingly, recent results from this study indicate that selenium supplementation increased the risk of one type of skin cancer (squamous cell carcinoma) by 25% . Although selenium supplementation shows promise for the prevention of prostate cancer, its effects on the risk for other types of cancer is unclear. In response to the need to confirm these findings, several large placebocontrolled trials designed to further investigate the role of selenium supplementation in prostate cancer prevention are presently under way [100, 101].
Several mechanisms have been proposed for the cancer prevention effects of selenium, (i) maximizing the activity of selenoenzymes and improving antioxidant status, (ii) improving immune system function, (iii) affecting the metabolism of carcinogens, and (iv) increasing the levels of selenium metabolites that inhibit tumor cell growth. A twostage mode has been proposed to explain the different anticarcinogenic activities of selenium at different doses. At nutritional or physiologic doses (�40–100 �g/day in adults) selenium maximizes antioxidant selenoenzyme activity and probably enhances immune system function and carcinogen metabolism. At supranutritional or pharmacologic levels (�200–300 �g/day in adults) the formation of selenium metabolites, especially methylated forms of selenium, may also exert anticarcinogenic effects.
Selenium deficiency has been associated with impaired function of the immune system. Moreover, selenium supplementation in individuals who are not seleniumdeficient appears to stimulate the immune response. In two small studies, healthy [102, 103] and immunosuppressed individuals  supplemented with 200 �g/day of selenium as sodium selenite for 8 weeks showed an enhanced immune cell response to foreign antigens compared with those taking a placebo. A considerable amount of basic research also indicates that selenium plays a role in regulating the expression of cytokines .
Selenium deficiency appears to enhance the virulence or progression of some viral infections. The increased oxidative stress resulting from selenium deficiency may induce mutations or changes in the expression of some viral genes. Selenium deficiency results in decreased activity of GPx. Coxsackie virus has been isolated from the blood of some sufferers of Keshan disease, suggesting that it may be a cofactor in the development of this cardiomyopathy associated with selenium deficiency in humans .
Although selenium is required for health, high doses can be toxic. Acute and fatal toxicities have occurred with accidental or suicidal ingestion of gram quantities of selenium. Clinically significant selenium toxicity was reported in 13 individuals taking supplements that contained 27.3 mg per tablet due to a manufacturing error. Chronic selenium toxicity, selenosis, may occur with smaller doses of selenium over long periods of time. The most frequently reported symptoms of selenosis are hair and nail brittleness and loss. Other symptoms may include gastrointestinal disturbances, skin rashes, a garlic breath odor, fatigue, irritability, and nervous system abnormalities. In an area of China with a high prevalence of selenosis, toxic effects occurred with increasing frequency when blood selenium concentrations reached a level corresponding to an intake of 850 �g/day in adults based on the prevention of hair and nail brittleness and loss and early signs of chronic selenium toxicity . The UL of 400 �g/day for adults (see table 10) includes selenium obtained from food which averages about 100 �g/day for adults in the USA as well as selenium from supplements.
Zinc is an essential trace element for all forms of life. Clinical zinc deficiency in humans was first described in 1961, when the consumption of diets
Table 10. Tolerable UL for selenium Age group UL, �g/day in the USA Infants 0–6 months
45 Infants 6–12 months
60 Children 1–3 years
90 Children 4–8 years
150 Children 9–13 years
280 Adolescents 14–18 years 400 Adults 19 years and older 400
with low zinc bioavailability (due to high phytic acid) content was associated with ‘adolescent nutritional dwarfism’ in the Middle East . Since then, zinc insufficiency has been recognized by a number of experts as an important public health issue, especially in developing countries .
Shellfish, beef, and other red meats are rich sources of zinc. Nuts and legumes are good plant sources. Zinc bioavailability is relatively high in meat, eggs, and seafood because of the relative absence of compounds that inhibit zinc absorption. The zinc in whole grain products and plant proteins is less bioavailable due to their relatively high content of phytic acid, a compound that inhibits zinc absorption . The enzymatic action of yeast reduces the level of phytic acid in foods. Recently, national dietary surveys in the USA estimated that the average dietary zinc intake was 9 mg/day for adult women and 13 mg/day for adult men .
Numerous aspects of cellular metabolism are zincdependent. Zinc plays important roles in growth and development, the immune response, neurological function, and reproduction. On the cellular level, the function of zinc can be divided into three categories: (i) catalytic, (ii) structural, and (iii) regulatory .
membranes increases their susceptibility to oxidative damage and impairs their function .
(iii) Zinc finger proteins have been found to regulate gene expression by acting as transcription factors. Zinc also plays a role in cell signaling and has been found to influence hormone release and nerve impulse transmission. Recently, zinc has been found to play a role in apoptosis .
Taking large quantities of zinc (�50mg/day) over a period of weeks can interfere with copper bioavailability. High intake of zinc induces the intestinal synthesis of metallothionein. It traps copper within intestinal cells and prevents its systemic absorption. More typical intakes of zinc do not affect copper absorption and high copper intakes do not affect zinc absorption .
Iron. Supplemental (38–65 mg/day of elemental iron) but not dietary levels of iron may decrease zinc absorption. This interaction is important in the management of iron supplementation during pregnancy and lactation and has led to the recommendation of zinc supplementation for pregnant and lactating women taking �60mg/day of elemental iron [114, 115].
Calcium. High levels of dietary calcium impair zinc absorption in animals, but it is uncertain whether this occurs in humans. Increasing the calcium intake by 890mg/day in the form of milk or calcium phosphate (total calcium intake 1,360mg/day) reduced zinc absorption and zinc balance in postmenopausal women , but increasing the calcium intake of adolescent girls by 1,000 mg/ day in the form of calcium citrate malate (total calcium intake 1,667 mg/day) did not affect zinc absorption or balance . Calcium in combination with phytic acid reduces zinc absorption.
The bioavailability of dietary folate is increased by the action of zincdependent enzyme, suggesting a possible interaction between zinc and folic acid. In the past, some studies found low zinc intake to decrease folate absorption while other studies described folic acid supplementation to impair zinc utilization in individuals with marginal zinc status [109, 110]. However, a more recent study found that supplementation with a high dose of folic acid (800 �g/day) for 25 days did not alter zinc status in a group of students being fed with low zinc diets (3.5 mg/day) nor did zinc intake impair folate utilization .
Information about zinc deficiency was mostly derived from the study of individuals born with acrodermatitis enteropathica, a genetic disorder resulting from the impaired uptake and transport of zinc. The symptoms of severe zinc deficiency include the slowing or cessation of growth and development, delayed sexual maturation, characteristic skin rashes, chronic and severe diarrhea, immune system deficiencies, impaired wound healing, diminished appetite, impaired taste sensation, night blindness, swelling and clouding of the corneas, and behavioral disturbances. Before the cause of acrodermatitis enteropathica was known, patients typically died in infancy. Oral zinc therapy results in the complete remission of symptoms, though it must be maintained indefinitely in individuals with the genetic disorder . Although dietary zinc deficiency is unlikely to cause severe zinc deficiency in individuals without a genetic disorder, zinc malabsorption or conditions of increased zinc loss, such as severe burns or prolonged diarrhea, may also result in severe zinc deficiency.
|Adults||19 years and older||11||9|
|Pregnancy||18 years and younger||–||12|
|Pregnancy||19 years and older||–||11|
|Breastfeeding||18 years and younger||–||13|
|Breastfeeding||19 years and older||–||12|
More recently, is has become apparent that milder zinc deficiency contributes to a number of health problems, especially common in children who live in developing countries. The lack of a sensitive indicator of mild zinc deficiency hinders the scientific study of its health implications. However, controlled trials of moderate zinc supplementation have demonstrated that mild zinc deficiency contributes to impaired physical and neuropsychological development, and increased susceptibility to lifethreatening infections in young children .
The RDA for zinc is listed for all age groups because infants, children, and pregnant and lactating women are at increased risk of zinc deficiency. Since a sensitive indicator of zinc nutritional status is not readily available, the RDA for zinc was based on a number of different indicators of zinc nutritional status and represents the daily intake likely to prevent deficiency in nearly all individuals in a specific age and gender group  (table 11).
Growth and Development
In the 1970s and 1980s, several randomized placebocontrolled studies of zinc supplementation in young children with significant growth delays were conducted in Denver, Colorado. Modest zinc supplementation (5.7 mg/ day) resulted in increased growth rates compared to placebo . More recently, a number of larger studies in developing countries observed similar results with modest zinc supplementation. A metaanalysis of growth data from zinc intervention trials recently confirmed the widespread occurrence of growthlimiting zinc deficiency in young children, especially in developing countries . Although the exact mechanism for the growthlimiting effects of zinc deficiency are not known, recent research indicates that zinc availability affects cellsignaling systems that coordinate the response to growthregulating hormone, insulinlike growth factor1 (IGF1) .
Low maternal zinc nutritional status has been associated with diminished attention in the newborn infant and poorer motor function at 6 months of age. Zinc supplementation has been associated with improved motor development in very lowbirthweight infants, more vigorous activity in Indian infants and toddlers, and more function activity in Guatemalan infants and toddlers . Additionally, zinc supplementation was associated with better neuropsychologic functioning (e.g., attention) in Chinese students, but only when zinc was provided with other micronutrients . Two other studies failed to find an association between zinc supplementation and measures of attention in children diagnosed with growth retardation. Although initial studies suggest that zinc deficiency may depress cognitive development in young children, more controlled research is required to determine the nature of the effect and whether zinc supplementation is beneficial .
Adequate zinc intake is essential in maintaining the integrity of the immune system  and zincdeficient individuals are known to experience increased susceptibility to a variety of infectious agents . Agerelated declines in immune function are similar to those associated with zinc deficiency, and certain aspects of immune function in the elderly have been found to improve with zinc supplementation . For instance, a randomized placebocontrolled study in men and women over 65 years of age found that a zinc supplement of 25 mg/day for 3 months increased levels of CD4 T cells and cytotoxic T lymphocytes compared to placebo . However, other studies have not found zinc supplementation to improve parameters of immune function, indicating that more research is required before any recommendations regarding zinc and immune system response in the elderly can be made.
It has been estimated that 82% of pregnant women worldwide are likely to have inadequate zinc intakes. Poor maternal zinc status has been associated with a number of adverse outcomes of pregnancy, including low birth weight, premature delivery, and labor and delivery complications. However, the results of maternal zinc supplementation trials in the USA and developing countries have been mixed . Although some studies have found maternal zinc supplementation to increase birth weight and decrease the likelihood of premature delivery, two recent studies in Peruvian and Bangladeshi women found no difference between zinc supplementation and placebo in the incidence of low birth weight or premature delivery [130, 131]. Supplementation studies designed to examine the effect of zinc supplementation on labor and delivery complications have also generated mixed results, though few have been conducted in zincdeficient populations .
AgeRelated Macular Degeneration
A leading cause of blindness in people over the age of 65 is a degenerative disease of the macula known as agerelated macular degeneration (AMD). Observational studies have not demonstrated clear associations between dietary zinc intake and the incidence of AMD [132–134]. A randomized controlled trial attracted the interest as it was found that 200 mg/day of zinc sulfate (91 mg/day of elemental zinc) over 2 years reduced the loss of vision in patients with AMD . However, a later trial using the same dose and duration found no beneficial effect in patients with a more advance form of AMD in one eye . A large randomized controlled trial of daily antioxidant (500 mg of vitamin C, 400 IU of vitamin E, and 15 mg of
�carotene) and highdose zinc (80mg of zinc and 2 mg of copper) supplementation found that the antioxidant combination plus highdose zinc and highdose zinc alone significantly reduced the risk of advanced macular degeneration compared to placebo in individuals with signs of moderate to severe macular degeneration in at least one eye . At present, there is little evidence that zinc supplementation would be beneficial to people with
Table 12. Tolerable UL for zinc in the Age group UL, mg/day USA Infants 0–6 months
4 Infants 7–12 months
5 Children 1–3 years
7 Children 4–8 years
12 Children 9–13 years
23 Adolescents 14–18 years
34 Adults 19 years and older
early signs of macular degeneration but further randomized controlled trials are warranted .
Isolated outbreaks of acute zinc toxicity have occurred as a result of consumption of food or beverages contaminated with zinc released from galvanized containers. Signs of acute zinc toxicity are abdominal pain, diarrhea, nausea, and vomiting. Single doses of 225–450mg of zinc usually induce vomiting. Milder gastrointestinal distress has been reported at doses of 50–150 mg/day of supplemented zinc. Metal fume fever has been reported after the inhalation of zinc oxide fumes. Profuse sweating, weakness, and rapid breathing may develop within 8 h of zinc oxide inhalation and persist 12–24 h after exposure is terminated [109, 110].
The major consequence of longterm consumption of excessive zinc is copper deficiency. Total zinc intakes of 60mg/day (50mg supplemental and 10mg dietary zinc) have been found to result in sign of copper deficiency. In order to prevent copper deficiency, the US UL of intake was set for adults at 40mg/day, including dietary and supplemental zinc  (table 12).
Carotenoids: �-Carotene, �-Carotene, �-Cryptoxanthin, Lycopene, Lutein and Zeaxanthin
Carotenoids are a class of more than 600 naturally occurring pigments synthesized by plants, algae, and photosynthetic bacteria. These colored molecules are the sources of the yellow, orange, and red colors of many plants . Fruits and vegetables provide most of the carotenoids in the human diet; � and �carotene, �cryptoxanthin, lutein, zeaxanthin, and lycopene are the most common carotenoids in the diet.
Carotenoids in foods are mainly in the alltrans form although cooking may result in the formation of other isomers. Average intakes for the major carotenoids in the US diet are :
� and �Carotene are provitamin A carotenoids, i.e. they can be converted by the body to vitamin A. The vitamin A activity of �carotene in foods is 1/12 that of retinol. Thus, it would take 12 �g of �carotene from foods to provide the equivalent of 1 �g of retinol. The vitamin A activity of �carotene from foods is 1/24 that of retinol, so it would take 24 �g of �carotene from foods to provide the equivalent of 1 �g of retinol. Orange and yellow vegetables like carrots and winter squash are rich sources of � and �carotene. Spinach is also a rich source of �carotene.
Like � and �carotene, �cryptoxanthin is a provitamin A carotenoid. The vitamin A activity of �cryptoxanthin from foods is 1/24 that of retinol. Orange and red fruits as well as vegetables like sweet red peppers and oranges are particularly rich sources of �cryptoxanthin.
Lycopene gives tomatoes, pink grapefruit, watermelon, and guava their red color. It has been estimated that 80% of the lycopene in the diet comes from tomatoes and tomato products such as tomato sauce, tomato paste, and ketchup . Lycopene is not a provitamin A carotenoid, i.e. the body cannot convert lycopene to vitamin A.
Lutein and zeaxanthin are both from the class of carotenoids known as xanthophylls. They are not provitamin A carotenoids. Some methods used to quantify lutein and zeaxanthin in foods do not separate the two compounds, so they are mostly reported as lutein � zeaxanthin. Lutein and zeaxanthin are present in a variety of fruits and vegetables. Dark green leafy vegetables, like spinach and kale, are particularly rich sources of lutein and zeaxanthin.
Vitamin A Activity. �Carotene, �carotene, and �cryptoxanthin are provitamin A carotenoids, their essential function recognized in humans is to serve as a source of vitamin A .
Antioxidant Activity. In plants, carotenoids have the important antioxidant function of quenching singlet oxygen . Among them, lycopene is one of the most effective quenchers of singlet oxygen . Although important for plants, the relevance of singlet oxygen quenching to human health is less clear. Carotenoids can also inhibit lipid peroxidation, but their actions in humans appear to be more complex . At present, it is unclear whether the biological effects of carotenoids in humans are a result of their antioxidant activity or other nonantioxidant mechanisms.
The long system of alternating double and single bonds common to all carotenoids allows them to absorb light in the visible range of the spectrum. This feature has particular relevance to the eye, where lutein and zeaxanthin efficiently absorb blue light. Reducing the amount of blue light that reaches the structures of the eye that are critical to vision may protect them from lightinduced oxidative damage .
Intercellular Communication. Carotenoids can facilitate communication between neighboring cells grown in culture by stimulating the synthesis of connexion proteins . Carotenoids increase the expression of the gene encoding a connexion protein, an effect that appears unrelated to the vitamin A or antioxidant activities of various carotenoids .
Immune System Activity. As vitamin A is essential for normal immune system function, it is difficult to determine whether the effects of provitamin A carotenoids are related to their vitamin A activity or other activities of carotenoids. Although some clinical trials have found that �carotene supplementation improves several biomarkers of immune function [149–151], increasing intakes of lycopene and lutein did not result in similar improvements in immune function biomarkers .
Although consumption of provitamin A carotenoids can prevent vitamin A deficiency, no deficiency symptoms have been identified in people consuming lowcarotenoid diets if they consume adequate vitamin A . After reviewing the published scientific research, the Food and Nutrition Board of the Institute of Medicine concluded that the existing evidence in 2000 was insufficient to establish a RDA or adequate intake for carotenoids.
Evidence that LDL oxidation plays a role in the development of atherosclerosis led to investigations of the role of antioxidant compounds such as carotenoids in the prevention of cardiovascular diseases . A number of casecontrol and crosssectional studies have found higher blood levels of carotenoids to be associated with significantly lower measures of carotid arteryintimamedia thickness [154–159]. Higher plasma carotenoids at baseline have been associated with significant reductions in cardiovascular disease risk in some prospective studies [160–162], but not in others [163–165]. While the results of several prospective studies indicate that people with higher intakes of carotenoidrich fruits and vegetables are at lower risk of cardiovascular disease [166–168], it is not yet clear whether this effect is a result of carotenoids or other factors associated with diets high in carotenoidrich fruits and vegetables.
In contrast to the results of epidemiologic studies suggesting that high dietary intakes of carotenoidrich fruits and vegetables may decrease cardiovascular disease risk, four randomized controlled studies found no evidence that �carotene supplements in doses ranging from 20 to 50 mg/day were effective in preventing cardiovascular diseases [169–172]. Based on these results, it has been concluded that there was good evidence that �carotene supplements provided to benefit in the prevention of cardiovascular disease in middleaged and older adults [173, 174]. Although diets rich in �carotene have generally been associated with reduced cardiovascular disease risk in observational studies, there is no evidence that �carotene supplementation reduces cardiovascular disease risk.
AgeRelated Macular Degeneration
In Western countries, degeneration of the macula is the leading cause of blindness in older adults. The only carotenoids found in the retina are lutein and zeaxanthin. By preventing a substantial amount of the blue light entering the eye from reaching underlying structures involved in vision, lutein and zeaxanthin may protect them from lightinduced oxidative damage which is thought to play a role in the pathology of AMD . It is also possible, though not proven, that lutein and zeaxanthin act directly to neutralize oxidants formed in the retina. Epidemiologic studies provide some evidence that higher intakes of lutein and zeaxanthin are associated with lower risk of AMD . However, the relationship is not clear. While crosssectional and retrospective casecontrol studies found that higher levels of lutein and zeaxanthin in the diet [176–182], blood, and retina were associated with lower incidence of AMD, two prospective cohort studies found no relationship between baseline dietary intakes or serum levels of lutein and zeaxanthin and the risk of developing AMD over time [183–185]. Thus, it seems to be premature to recommend supplements without data from randomized controlled trials . The available scientific evidence suggests that consuming at least 6 mg/day of lutein and zeaxanthin from fruits and vegetables may decrease the risk of AMD [176–178].
The only published randomized controlled trial designed to examine the effect of a carotenoid supplement on the risk AMD used �carotene in combination with vitamin C, vitamin E, and zinc because lutein and zeaxanthin were not commercially available as supplements at the time the trial began . Although the combination of antioxidants and zinc lowered the risk of developing advanced macular degeneration in individuals with signs of moderate to severe macular degeneration in at least one eye, it is unlikely that the benefit was related to �carotene since it is not present in the retina. Supplementation of male smokers in Finland with 20mg/day of �carotene for 6 years did not decrease the risk of AMD compared to placebo .
Ultraviolet light and oxidants can damage proteins in the eye’s lens causing structural changes that result in the formation of opacities known as cataracts. As people grow old, cumulative damage to lens proteins often results in cataracts that are large enough to interfere with vision .
The potential for increased intakes of lutein and zeaxanthin to prevent or slow the progression of cataracts has been pointed out . Three large prospective cohort studies found that men and women with the highest intakes of foods rich in lutein and zeaxanthin, i.e. spinach, kale, and broccoli, were 20–50% less likely to require cataract extraction [189, 190] or develop cataracts . Additional research is required to determine whether these findings are related specifically to lutein and zeaxanthin intake or to other factors associated with diets high in luteinrich foods.
In addition it has to be mentioned that �carotene supplementation (20mg/day) for more than 6 years did not affect the prevalence of cataracts or the frequency of cataract surgery in male smokers living in Finland . In contrast, a 12year study of male physicians in the USA found that �carotene supplementation (50mg every other day) decreased the risk of cataracts in smokers but not in nonsmokers . Two randomized controlled trials examined the effect of an antioxidant combination that included �carotene, vitamin C, and vitamin E on the progression of cataracts. While one study found no benefit after more than 6 years of supplementation , the other study found a small decrease in the progression of cataracts after 3 years of supplementation . Altogether, the results of randomized controlled trials suggest that the benefit of �carotene in slowing the progression of agerelated cataracts does not outweigh the potential risks.
Lung Cancer. The results of early observational studies suggested that an inverse relationship between lung cancer risk and �carotene intake was often assessed by measuring blood levels of �carotene [195, 196]. More recently, the development of databases for other carotenoids in food has allowed to estimate dietary intakes of total and individual dietary carotenoids more accurately. In contrast to early retrospective studies, recent prospective cohort studies have not consistently found inverse associations between �carotene intake and lung cancer risk. Analysis of dietary carotenoid intake and lung cancer risk in two large prospective cohort studies in the USA that followed more than 120,000 men and women for at least 10 years revealed no significant association between dietary �carotene intake and lung cancer risk . However, men and women with the highest intakes of total carotenoids, �carotene, and lycopene were at significantly lower risk of developing lung cancer than those with the lowest intakes. Dietary intakes of total carotenoids, lycopene, �cryptoxanthin, lutein, and zeaxanthin, but not �carotene, were associated with significant reductions in lung cancer in a 14year study of more than 27,000 Finnish male smokers , whereas only dietary intakes of �cryptoxanthin and lutein, and zeaxanthin were inversely associated with lung cancer risk in a 6year study of more than 58,000 Dutch men . A recent analysis of the pooled results of six prospective cohort studies in North America and Europe also found no relationship between dietary �carotene intake and lung cancer risk, although those with the highest �cryptoxanthin intakes had a risk of lung cancer that was 24% lower than those with the lowest intakes . While smoking remains the strongest risk factor for lung cancer, results of recent prospective studies using accurate estimates of dietary carotenoid content suggest that diets rich in a number of carotenoids – not only �carotene – may be associated with reduced lung cancer risk.
In addition, the effect of �carotene supplementation on the risk of developing lung cancer has been examined in three large randomized placebocontrolled trials. In Finland, the �Tocopherol �Carotene (ATBC) cancer prevention trial evaluated the effects of 20mg/day of �carotene and/or 50mg/day of �tocopherol on more than 29,000 male smokers , and in the USA, the �Carotene and Retinol Efficacy Trial (CARET) evaluated the effects of a combination of 30mg/day of �carotene and 25,000 IU/day of retinol (vitamin A) in more than 18,000 men and women who were smokers, former smokers, or had a history of occupational asbestos exposure . Unexpectedly, the risk of lung cancer in the groups taking �carotene supplements was increased by 16% after 6 years in the ATBC participants and increased by 28% after 4 years in the CARET participants. The Physicians Health Study (PHS) examined the effect of �carotene supplementation (50 mg every other day) on cancer risk in more than 22,000 male physicians in the USA, of whom only 11% were current smokers . In that lower risk population, �carotene supplementation for more than 12 years was not associated with an increased risk of lung cancer. Although the reasons for the increase in lung cancer risk are not yet clear, experts feel that the risks of highdose �carotene supplementation outweigh any potential benefits for cancer prevention, especially in smokers or other highrisk populations [173, 202].
Prostate Cancer. The results of several prospective cohort studies suggest that lycopenerich diets are associated with significant reduction in the risk of prostate cancer . In a study of more than 47,000 health professionals followed for 8 years, those with the highest lycopene intake had a risk of prostate cancer that was 21% lower than those with the lowest lycopene intake . Those with the highest intakes of tomatoes and tomato products (accounting for 82% of total lycopene intake) had a risk of prostate cancer that was 35% lower and a risk of aggressive prostate cancer that was 53% lower than those with the lowest intakes. Similarly, a prospective study of Seventh Day Adventist men found that those who reported the highest tomato intakes were at significantly lower risk of prostate cancer  and that those with the highest plasma lycopene levels were at significantly lower risk of developing aggressive prostate cancer . However, dietary lycopene intake was not related to prostate cancer risk in a prospective study of more than 58,000 Dutch men . Thus, it is not clear whether the prostate cancer risk reduction observed in some epidemiologic studies is related to lycopene itself, other compounds in tomatoes, or other factors associated with lycopenerich diets.
Bioavailability of Carotenoids
The bioavailability of carotenoids is influenced by a number of factors. In general, purified carotenoids in oil (supplements) are more bioavailable than carotenoids in foods . In particular, the bioavailability of �carotene from supplements is much higher than from foods. One study found that the bioavailability of �carotene from spinach was only 14% of that of purified �carotene in oil . In contrast, the bioavailability of lutein from spinach was 67% of that of purified lutein in oil.
The relatively low bioavailability of carotenoids from foods is partly due to the fact that they are associated with proteins in the plant matrix. Carotenoids are associated with chloroplasts in green leafy vegetables and chromoplasts in fruit. Chopping, homogenizing, and cooking disrupt the plant matrix, increasing the bioavailability of carotenoids . The bioavailability of lycopene from tomatoes is substantially improved by heating tomatoes in oil [210, 211].
High doses of �carotene supplements (�30mg/day) and the consumption of large amounts of carotenerich foods have resulted in carotenodermia and, analogously, high intakes of lycopenerich foods or supplements may result in lyopenodermia. Adverse effects of lutein and zeaxanthin have not been reported.
Unlike vitamin A, high doses of �carotene taken by pregnant women have not been associated with increased risk of birth defects. However, the safety of highdose �carotene supplements in pregnancy and lactation has not been well studied. Although there is no reason to limit dietary �carotene intake, pregnant and breastfeeding women should avoid consuming �3 mg/day (5,000 IU/day) of �carotene from supplements unless they are prescribed under medical supervision .
Interactions among Drugs and Carotenoids
The cholesterollowering agents colestyramine and colestipol can reduce absorption of fatsoluble vitamins and carotenoids as can mineral oil and orlistat, a drug used to treat obesity. Colchicine, a drug used to treat gout, can cause intestinal malabsorption. However, longterm use of 1–2 mg/day of colchicine did not affect serum �carotene levels . Increasing gastric pH through the use of proton pump inhibitors decreased the absorption of a single dose of a �carotene supplement but it is not known if the absorption of dietary carotenoids is affected [214–216].
The results of metabolic studies suggest that high doses of �carotene compete with lutein and lycopene for absorption when consumed at the same time. However, the consumption of highdose �carotene supplements did not adversely affect serum carotenoid levels in longterm clinical trials [217–220].
Phenylchromane derivatives having a 2phenylchromane (� flavane) skeleton are called flavonoids. They represent the single, most widely occurring group of phenolic phytochemicals. Flavonoids are classified according their oxidation level of their central C ring. These variations define the families of flavones, flavanols, flavanones, flavonols, flavandiols (leukoanthocyanidins) and flavylium salts (anthocyanidins). Structural differences between the various members of these families are mainly caused by the hydroxylations in different positions, methylations of individual hydroxyl groups, and glycosidation by various sugars. The most common sugars are Dglucose, Lrhamnose, Dgalactose, Dglucuronic acid, Dgalacturonic acid, Larabinose, and Dxylose. �Glycosidation prevails in the Dseries, while Lsugars are �glycosidically bound. In total, several thousand different flavonoids have been described .
In the human diet, chlorogenic acid (e.g. from coffee, carrots), ferulic acid
(e.g. from cereals), flavonols (e.g. from onions, vegetables, tea, apples), catechin and other flavan3ols (e.g. from apples, grapes, chocolate), isoflavones (e.g. from soybeans) as well as lignans (e.g. from cereals) constitute the major classes.
The lack of reliable compositional data and the fact that intake of any single plant phenol will be highly dependent on the types of food plants consumed, means that it is not possible to provide definitive values of intake in human populations. In the 1970s, it was generally assumed that the average intake of dietary flavonoids is in the range of 1g/day. This figure has been questioned later. Related studies of the flavonoid content of common human beverages indicate consumption rates in the lower milligram ranges, in which tea and onions are major contributors.
The presence of a phenolic group in a natural flavonoid would be expected to provide antimicrobial activity and the addition of further phenolic groups might be expected to enhance this activity. In fact, one of the undisputed functions of flavonoids and related polyphenols is their role in protecting plants against microbial invasion. This not only involves their presence in plants as constitutive agents but also their accumulation as phytoalexins in response to microbial attack .
Yet one further property of flavonoids that has been researched recently has had antiviral activity, most notably against the human immune deficiency virus (HIV). Some flavonoids appear to have direct inhibitory activity on the virus. That is apparently true for baicalin (5,6,7trihydroxyflavone7glucoronide) from Scutellaria baicalensis . Other flavonoids are inhibitory to enzymes required for viral replication.
In addition, it is now generally accepted that flavonoids, along with other plant polyphenols, play a role in protecting plants against both insect and herbivorous mammals, i.e. they are active in plantanimal interactions. In recent years, attention has mainly been focused on simple phenolic constituents or on the polymeric proanthocyanidins, but further research has also been concerned with lowmolecularweight flavones, flavonols, and isoflavones .
In the last decade, the medicinal properties of flavonoids came increasingly into the center of research. The most important effects will briefly be discussed in the following.
Antioxidant Properties and Enzyme Inhibition
Flavonoids have been shown to act as scavengers of various oxidizing species, such as superoxide anion, hydroxyl radical or singlet oxygen. Flavonoids do not react specifically with a single species and so a number of different evaluation methods have been developed which makes comparison of the various studies very difficult. The structural conditions for the antioxidation activity have been reviewed recently .
A possible contributory mechanism to the antioxidant activity of flavonoids is their ability to stabilize membranes by decreasing membrane fluidity. A series of representative flavonoids partition into the hydrophobic core of the membrane, causing a dramatic decrease in lipid fluidity in this region of the membrane .
Flavonoids are known to inhibit key enzymes in mitochondrial respiration . Some flavonoids also inhibit the enzyme xanthine oxidase which catalyzes the oxidation of xanthine and hypoxanthine to uric acid. During the reoxidation of xanthine oxidase, both superoxide radicals and hydrogen peroxide are produced. Obviously, flavons show higher inhibitory effect than flavonols and hydroxyl groups at both C3 and C3� are essential for high superoxide scavenging activity .
The in vitro antioxidative activities have been recognized for decades, but it is still not clear whether there are in vivo beneficial effects.
Flavonoids may inhibit the cyclooxygenase and/or the 5lipoxygenase pathways of arachidonate metabolism. The various structuralrelated effects have been reviewed comprehensively .
Flavonoids may act in a number of different ways on the various components of blood, such as platelets, monocytes, LDL, and smooth muscles. Platelets are key participants in atherogenesis and proinflammatory indicators, such as thromboxane A2, PAF and serotonin are produced from them. Flavonoids may inhibit platelet adhesion, aggregation and secretion .
In a survey of 65 flavonoids for procoagulant activity, 18 were found to inhibit the interleukin1induced expression of tissue factor on human monocytes . Flavonols, such as kaempferol, quercetin and myricetin have been shown to inhibit adenosine deaminase activity in the endothelial cells of the aorta whereas flavones were found to be inactive .
Flavonoids have also been shown to be potent inhibitors of the oxidative modification of LDL by macrophages. They also inhibit the cellfree oxidation of LDL mediated by copper sulfate .
Coronary Heart Disease
In most countries a high intake of saturated fats is strongly correlated with high mortality from CHD, but this is not the case in some regions of France (socalled ‘French paradox’). This anomaly has been attributed to the regular intake of red wine in the diet . Epicatechin and quercetin might be more important than resveratrol in reducing CHD. It is suggested that the combination of antioxidant phenolics in wine may protect against atherogenesis with regular longterm consumption. There are several other studies considering the effect of flavonoids on CHD and the role of dietary antioxidant flavonoids protecting against CHD has been more widely reviewed .
Cytotoxic Antitumor Activity
There have been many bioassayguided searches for cytotoxic antitumor agents in plants, especially those known to be used in folk medicine for this purpose. This has led to the isolation and identification of numerous active constituents from all the different flavonoid classes. However, the choice and number of all lines used in these bioassays has been very variable and it is difficult to draw general conclusions from them. The literature has been reviewed comprehensively .
The main group of flavonoids that is well known to possess estrogenic activities are the isoflavones, such as genistein. In a normal human diet the presence of such active flavonoids is generally considered to be harmless because no single phytoestrogen is present in sufficient quantity to have physiological consequences. However, this may not be the case for vegetarians, especially those who eat a large percentage of legumes in their diet which has a high isoflavonoid content, such as soya and pulses.
Other Biological Activities
It is well known that some flavonoids can act as antispasmolytic agents by relaxing smooth muscles in various parts of the mammalian body. An impressive example for a flavone with anxiolytic and anticonvulsive activity is the most recently studied hispidulin . Flavonoids may also exhibit antibacterial , antifungal , and antimalaria  activities.
Risk/benefit evaluations are under investigation worldwide, however, the lack of sufficient data does not allow to draw final conclusions. Nonetheless, the recently published statements on safety aspects of functional food including flavonoids show that scientists clearly stress the importance to substantially evaluate the safety to health, functionality and claims as well as recommend observations after the market introduction .
Also known as thioctic acid, �lipoic acid is a naturally occurring compound that is synthesized by plants and animals, including humans. �Lipoic acid contains two sulfur molecules that can be oxidized or reduced. This feature allows �lipoic acid to function as a cofactor for several important enzymes as well as a potent antioxidant. Only the Risomer of �lipoic acid is synthesized naturally. Conventional chemical synthesis of �lipoic acid results in a 50/50
R:S (racemic) mixture of the two optical isomers .
(R) Lipoic acid (R) Dihydro lipoic acid
Most �lipoic acid in food is derived from lipoamidecontaining enzymes and is bound to lysine (lipoyllysine). Animal tissues that are rich in lipoyllysine include kidney, heart, and liver, whereas lipoyllysinerich plant sources comprise spinach, broccoli, and tomatoes. Somewhat lower amounts of lipoyllysine have been measured in peas, Brussels sprouts, and rice bran.
Digestive enzymes do not break the bond between �lipoic acid and lysine effectively. Thus, it has been hypothesized that most dietary �lipoic acid is absorbed as lipoyllysine, and free �lipoic acid has not been detected in the circulation of humans who are not taking �lipoic acid supplements. Although
�lipoic acid is found in a wide variety of foods from plant and animal sources, quantitative information on the �lipoic acid content of food is limited.
In its proteinbound form, R�lipoic acid is a required cofactor for several multienzyme complexes inside the mitochondria. The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetylCoA; the �ketoglutarate dehydrogenase complex catalyzes the metabolism of three amino acids, leucine, isoleucine, and valine. The glycine cleavage system is a multienzyme complex that catalyzes the formation of 5,10methylene tetrahydrofolate, an important cofactor in nucleic acid synthesis.
When large amounts of free �lipoic acid are available, e.g., under supplementation, �lipoic acid is also able to function as an antioxidant . Free �lipoic acid is rapidly taken up by cells and reduced intracellularly to
�dihydrolipoic acid (DHLA). DHLA is the only form that functions directly as an antioxidant . Because DHLA is rapidly eliminated from cells, the extent to which its antioxidant effects can be sustained remains unclear. DHLA is a potent reducing agent and has the capacity to regenerate a number of oxidized forms, i.e. of vitamin C, glutathione, and coenzyme Q10 which are able to regenerate oxidized
�tocopherol, forming an antioxidant network. DHLA can be regenerated from
�lipoic acid through the activity of enzymes present in cells .
Certain free metal ions like iron and copper can induce oxidative damage by catalyzing reactions that generate highly reactive free radicals. Both �lipoic acid and DHLA may chelate or bind metal ions in a way that prevents them from generating free radicals . At present, this property has only been demonstrated in vitro.
Glutathione is an important watersoluble antioxidant that is synthesized from the sulfurcontaining amino acid cysteine. The availability of cysteine inside a cell determines its rate of glutathione synthesis. Although increases in intracellular DHLA are shortlived, DHLA may also improve intracellular antioxidant capacity by inducing glutathione synthesis .
The protein �1antiprotease is an inhibitor of the enzyme elastase. Oxidation inactivates �1antiprotease, leading to increased activity of elastase and degradation of elastin in the lungs, a process that has been implicated in chronic obstructive pulmonary disease (COPD). In vitro, DHLA can act as a reducing factor for the enzyme, peptide methionine sulfoxide reductase (PMSR) which can reduce and reactive oxidized �1antiprotease . Whether �lipoic acid contributes to the repair of oxidized proteins in living organisms remains to be determined.
Nuclear factor�B (NF�B) is known as a transcription factor, as it is able to bind to DNA and affect the rate of transcription of certain genes that have NF�B binding sites. NF�B plays an important role in regulating genes related to inflammation and the pathology of a number of diseases, including atherosclerosis, cancer and diabetes . Physiologically relevant concentrations of �lipoic acid have been found to inhibit the activation of NF�B when added to cells in culture .
AP1 is another transcription factor that can be affected by both reactive oxygen species (ROS) and certain antioxidants within cells. Treating cells in culture with DHLA has been found to inhibit the activity of AP1 by decreasing the expression of the gene for cfos, one of the proteins that makes up the functional AP1 complex .
�Lipoic acid deficiency has not been described, suggesting that humans are able to synthesize enough to meet their needs of enzyme cofactors. Increased destruction of the cofactor form of �lipoic acid may underlie the pathology of some diseases. In arsenic toxicity, arsenic can form a complex with �lipoic acid in dehydrogenase enzymes, leaving it inactive . Circulating antibodies to lipoamidecontaining enzyme subunits have been isolated in patients with primary biliary cirrhosis .
In aging rats, shortterm dietary supplementation with R�lipoic acid has been found to decrease mitochondrial ROS production and improve mitochrondrial function [246, 247]. A series of studies in aged rats found that combined dietary supplementation of R�lipoic acid and acetylLcarnitine improved mitochondrial energy metabolism, decreased oxidative stress, increased physical activity, and improved measures of shortterm memory [248, 249]. As these findings are very encouraging, the researchers caution that these studies used relatively high doses of the compounds only for 1 month. It is not yet known whether taking relatively high doses of R�lipoic acid and acetylLcarnitine will be for the benefit of aging rats in the long term or will have similar effects in humans.
Pharmacologic doses of �lipoic acid, i.e. many times higher than the amount a person could synthesize or obtain from foods, have been prescribed to treat diabetic patients in Germany since the late 1960s . Data from animal studies suggest that the Risomer may be more effective in improving insulin sensitivity than the Sisomer [251, 252], but this possibility has not been tested in any published human trials.
Oxidative Stress. A number of studies in individuals with diabetes (types 1 and 2) indicate that they are under increased oxidative stress, a condition that is believed to contribute to the vascular and neurological complications of diabetes. Although �lipoic acid supplementation has been found to reduce measures of oxidative stress in animal models of diabetes, evidence that
�lipoic acid reduced oxidative stress in humans with diabetes is limited. In a nonrandomized crosssectional study, 33 patients with type 1 or type 2 diabetes who had taken 600 mg/day of �lipoic acid orally for at least 3 months, had lower levels of plasma lipid peroxidation than did 74 diabetics who did not take �lipoic acid . An intervention trial in 10 diabetic patients found that plasma lipid peroxides were significantly lower after taking 600 mg/day of �lipoic acid orally for 60 days compared to baseline . Oral �lipoic acid supplementation (600 mg/day) has been found to decrease NF�B activation in the white blood cells of type 1 diabetics  and patients with diabetic nephropathy (kidney damage). The formation of advanced glycation end products (AGEP) also leads to glucosemediated damage in diabetes. �Lipoic acid has been found to prevent the formation of AGEP in vitro .
Diabetic Peripheral Neuropathy.
Over onethird of diabetics develop peripheral neuropathy. In addition to the pain and disability caused by diabetic neuropathy, it is a leading cause of lower limb amputation in diabetics . The results of several large randomized controlled trials indicate that maintaining blood glucose at nearly normal levels is the most important step in decreasing the risk of diabetic neuropathy. However, such intensive blood glucose control may not be achievable in all diabetic patients.
Oxidative stress has been implicated in the pathology of diabetic neuropathy, and �lipoic acid is approved for the treatment of diabetic neuropathy in Germany. At least 15 clinical trials have examined the effect of �lipoic acid treatment on symptoms of diabetic neuropathy with mixed results, especially in smaller studies. Modest benefits have been observed in several large multicenter trials. More than 300 type 2 diabetics with symptomatic peripheral neuropathy were randomly assigned to intravenous treatment with 100, 600 or 1,200 mg/day of �lipoic acid or placebo for 3 weeks . Symptom scores were significantly improved in those that received intravenous infusions of at least 600 mg/day of
�lipoic acid compared to placebo. A subsequent multicenter trial randomly assigned 509 type 2 diabetics with symptomatic peripheral neuropathy to one of different treatments . Although symptom scores did not differ significantly from baseline in any of the groups, assessments of sensory and motor deficits by trained physicians were significantly improved after 3 weeks of intravenous �lipoic acid therapy and nonsignificantly improved at the end of 6 months of oral �lipoic acid therapy. A smaller randomized controlled trial examined the effect of longterm oral �lipoic acid supplementation on the results of electrophysiological nerve conduction studies in 65 diabetic patients with symptomatic peripheral neuropathy . Those who took �lipoic acid showed significant improvements in 3 out of 4 nerve conduction assessments compared to those who took placebo.
Overall, the available research suggests that oral doses of at least 600 mg/day of �lipoic acid may offer some benefit in the alleviation of neuropathy symptoms and deficits, especially when used in conjunction with effective treatment aimed at normalizing blood glucose levels.
Vascular Complications. Endothelial function in individuals with diabetes (types 1 and 2) is often impaired and diabetics are at increased risk for vascular disease. Several small preliminary studies in humans have examined the effect of �lipoic acid administration on endothelial function. In one study, intraarterial infusions of �lipoic acid improved endotheliumdependent vasodilation in 39 diabetic patients, but not in 11 healthy controls . Oral supplementation of 1,200 mg/day of �lipoic acid for 6 weeks improved a measure of capillary perfusion in the fingers of 8 diabetic patients with peripheral neuropathy . In an uncontrolled, nonrandomized study of 84 diabetic patients, plasma thrombomodulin levels, a marker of compromised endothelial function, decreased significantly in the 35 diabetics that took 600 mg/day of
There is evidence that the enantiomers of �lipoic acid have different biological activities. Within the mitochondria, R�lipoic acid is reduced to DHLA, the more potent antioxidant, 28 times faster than S�lipoic acid. However, in the cytosol S�lipoic acid is reduced to DHLA twice as fast as R�lipoic acid. One study in humans found R�lipoic acid to be more bioavailable than S�lipoic acid when taken orally . Rlipoic acid was more effective than Slipoic acid in enhancing insulinstimulated glucose transport and metabolism insulinresistant rat skeletal muscle , and R�lipoic acid was more effective than racemic �lipoic acid and S�lipoic acid in preventing cataracts in rats . Almost all studies of �lipoic acid supplementation in humans have been performed using racemic �lipoic acid. At present, it is not known whether R�lipoic acid is more effective as an antioxidant than racemic lipoic acid when taken by humans in pharmacologic doses.
In general, �lipoic acid doses of 600 mg/day have been well tolerated. Doses as high as 1,200 mg/day (600 mg, twice a day) for 2 years and 1,800 mg/day (600 mg, 3 times a day) for 3 weeks did not result in adverse effects when given to patients with diabetic neuropathy under medical supervision. There are no reports of toxicity from �lipoic acid overdose in humans. In individuals with diabetes and/or impaired glucose tolerance, �lipoic acid supplementation may lower blood glucose levels. Individuals on diabetic medications should monitor blood glucose levels. Diabetic medication doses may need to be adjusted to avoid hypoglycemia. As controlled safety studies in pregnant and lactating women are not available, the use of �lipoic acid supplements by pregnant or breastfeeding women is not recommended .
The support kindly provided by FRUIT, International Fruit Foundation, Heidelberg, is greatly acknowledged.
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Peter Schreier Lehrstuhl für Lebensmittelchemie, Universität Würzburg Am Hubland, DE–97074 Würzburg (Germany) EMail email@example.com
K.M. Janisch, J. Milde, H. Schempp, E.F. Elstner
TUM Weihenstephan, Center of Life and Food Sciences, Lehrstuhl für Phytopathologie, Freising, Germany
All inflammatory processes include oxygenactivating processes where reactive oxygen species are produced. Intrinsic radical scavenging systems or compounds administered with food warrant metabolic control within certain limits. Antioxidants, which in many cases are free radical scavengers or quenchers of activated states, comprise a vast number of classes of organic molecules including most prominently the phenolics. In this report, mechanisms of protection from oxidative damage by the antioxidants vitamin C and E and flavonoids, as present in most plant extracts used as natural drugs, are summarized. For this purpose the principle of oxygen activation during representative disease processes and the protective actions of antioxidants are outlined in short.
Copyright © 2005 S. Karger AG, Basel
Reactive oxygen species (ROS) occur in the healthy metabolism as byproducts of oxidative processes such as the respiratory chain in mitochondria. Their detoxification is maintained with antioxidant enzymes (e.g. superoxide dismutase, catalase, glutathione peroxidase) and antioxidants (e.g. ascorbic acid, glutathione, tocopherol) and marks only small problems for a healthy individual. Certain incidences, such as ageing, inflammation, ischemia, and smoking, cause a rise in the oxidative stress. Thus the metabolism has to cope with ROS and ROS metabolism plays a crucial role in pathogenesis [1–3].
ROS include superoxide radicals (O2 ), hydrogen peroxide (H2O2), hydroxyl radicals (OH), singlet oxygen (1O2), peroxynitrite (ONNOH) and hypochloric acid (HOCl). A oneelectron transfer onto oxygen generates superoxide radicals. They dismutate spontaneously to hydrogen peroxide and oxygen at neutral to slight acidic pH; whereas superoxide dismutases (SOD) catalyze
Fig. 1. Structure of ascorbic acid.
the dismutation independently from the pH. Hydrogen peroxide can also be generated via a twoelectron transfer onto molecular oxygen, a reaction carried out by certain oxidases. Catalases detoxify hydrogen peroxide resulting in water and oxygen. A further oneelectron transfer onto hydrogen peroxide produces the most reactive ROS, hydroxyl radicals. They react quickly with organic molecules in their surroundings generating further radicals such as alkyl, peroxyl radicals damaging the tissue with these interactions [1–4]. Singlet oxygen originates physically from the transfer of light energy onto molecular (triplet) oxygen through photosensitizers. It impairs organic molecules in the ground state [1, 4, 5].
ROS are known to be involved in many diseases and the process of ageing. In the eye, diseases such as agerelated macular degeneration (AMD) and cataract are related to this. In both diseases, the high consumption of oxygen during normal metabolism and the exposure to light evolve oxidative stress in the tissue of the retina and lens . In the course of AMD, phagocytic processes of the retinal pigment epithelial cells (RPE) and accumulation of lipofuscin, an agerelated protein debris of the metabolism with photosensitizer abilities, enhance the oxidative strain in the retina additionally [5–8]. The degeneration of the lens fibers occurring in cataract is furthermore linked with smoking, cardiovascular diseases and diabetes , events already associated with the involvement of ROS. The protective mechanisms of the antioxidants vitamin C and E and flavonoids as well as their involvement in eye diseases are summarized in the following. Carotenoids are not considered here as they are discussed in detail in another chapter of this book.
The natural occurring vitamin ascorbic acid (vitamin C) is the most important hydrophilic antioxidant for human metabolism to maintain health. It is a dibasic acid with an enediol structure within the heterocyclic furanolactone ring (fig. 1) . Ascorbic acid is able to chelate transition metals as a bidentate ligand but also possesses reducing abilities. It scavenges ROS (e.g. superoxide radical, hydroxyl radical, hydrogen peroxide, singlet oxygen) and is oxidized by a variety of other oxidants such as halogens, quinones, and phenoxyl radicals [9–12]. The oxidation of ascorbate to dehydroascorbate is a twoelectron redox process. The loss of the two hydrogen ions and one electron forms the ascorbyl radical which is an acid and relatively stable compared to other radical ROS [9, 10]. The ascorbyl radicals disproportionate to ascorbic acid and dehydroascorbic acid. A further electron loss leads from the ascorbyl radical to dehydroascorbate. Dehydroascorbate still has low reducing abilities and can be oxidized further; it is reduced enzymatically to regenerate ascorbic acid. Therefore, ascorbic acid and its oxidation product dehydroascorbic acid form a reversible redox couple with the ascorbate radical as important intermediate contributing to the antioxidant function [9, 10, 13, 14]. Due to the reducing properties, ascorbic acid serves as one primary defense in the aqueous milieu. This scavenging ability is important in the eye where radiation and oxidative stress demand higher protection. Ascorbic acid interacts with glutathione and
tocopherol as an antioxidant defense line [9, 11]. Ascorbate cannot directly scavenge lipophilic radicals occurring in membranes but it reduces the tocopheroxyl radicals bound in the membrane in the lipidaqueous phase transition. In aqueous milieus, ascorbate protects glutathione, another watersoluble antioxidant of the cell, on its own expense and vice versa [9, 11, 13, 14]. The interaction of ascorbic acid with tocopherol and glutathione depicts a potent defense line in ocular tissue, protecting lens proteins and retina tissue of photooxidative damage.
Humans are unable to synthesize ascorbic acid and it is therefore actively absorbed from the diet by sodiumdependent transport systems in the intestine. The ascorbic acid concentrations of single tissues is tightly controlled and achieved with active cellular transporters for ascorbic acid and dehydroascorbic acid. The normal concentration of ascorbic acid in plasma is about 8–14 mg/l. As ocular tissue accumulates ascorbic acid, the concentrations are 100 times higher in the retina, 20fold increased in the aqueous humor and in the lens the level is 10fold the level of plasma [8, 9, 15]. Supplementation with ascorbic acid leads to a rise in the ascorbate levels in ocular tissue [8, 9]. The influence of ascorbic acid on the prevention of cataract and AMD is not yet confirmed. Epidemiological studies give inconsistent results. There are several studies concerned with nutritional supplement and cataract development, but due to their retrospective character they are prone to be biased prior to the diagnosis of cataract [16–18]. There are eight studies considering ascorbate intake and cataract prevalence. Four of these studies found a decreased prevalence for cataract when plasma ascorbic acid levels were high, some of these findings were not significant after adjustment for sex and age [8, 16, 18]. The other three studies found no association between ascorbic acid intake and plasma concentrations and prevalence of cataract. The last study showed an increased
CH3 CH3 CH3
CH3 CH3 CH3
Homologue R1 R2 R3
Fig. 2. Structures of tocopherol (a) and tocotrienol (b) and pattern of substitution of the homologues (c).
risk for cataract and high ascorbate plasma levels [16, 18]. There are reports indicating correlation between ascorbic acid intake and retinal levels, but for the progression of AMD increasing levels of carotenoids are of more importance than of ascorbate which had no influence at all on the progression of AMD [8, 16].
Vitamin E is the most important lipidsoluble antioxidant in humans. It is a scavenger of peroxyl radicals and therefore inhibits the chain reactions in lipid peroxidation. The generic term ‘vitamin E’ is primarily a nutritional term and describes the eight tocopherol (, , , ; fig. 2a) and tocotrienol (, , ,
; fig. 2b) homologues which exhibit vitamin E activity. Tocopherols are derivatives of the 2methyl6chromanol with a phytyl side chain attached at position C2; the , , and forms differing in the numbers and positions of their methyl groups at the ring (fig. 2c). The tocotrienols vary from the corresponding tocopherols in the isoprenoid side chain which is unsaturated at C3, C7 and C11. As tocopherol is the most abundant and active form in vivo, the term vitamin E refers now to tocopherol in the literature [3, 19, 20]. The methyl groups at the aromatic ring are crucial for the biological activity, the order of activity is: . Furthermore, the hydroxyl group is important for the antioxidant activity of the tocopherols as it donates a hydrogen atom to free radicals . The chainbraking properties are the result of the much faster reaction of tocopherols and tocotrienols with lipid peroxyl radicals than the reaction of these radicals with adjacent fatty acids and/or membrane proteins. In addition, tocopherols also react with singlet oxygen and superoxide radicals which contribute to their protective properties . The arising tocopherol radicals can undergo different fates. They can react either with other tocopheryl radicals to give dimers or with other peroxyl radicals to give stable products [3, 19]. It is known that tocopheryl radicals are oxidized to
tocopherylquinone which is metabolized and excreted in the urine . The tocopheryl radicals can be recycled by reductants such as ubiquinol, polyphenolic compounds or ascorbic acid. The regeneration of the tocopherols with ascorbic acid is the major pathway for tocopheryl radicals in vivo and explains the synergistic antioxidant effects of these two antioxidants observed in vitro [3, 19, 20].
Vitamin E is absorbed by passive diffusion in the small intestine after its solubilization in micelles formed from fatty acids and bile acids. There are no differences in the absorption of the diverse homologues . Vitamin E is transported in the blood within the lipoproteins and the normal plasma concentration in human ranges from 15 to 40 M . Supplementation of vitamin E results in an increase of the plasma level of about 2 to 3fold . The concentrations of tocopherol in the lens are about 1,573–2,550 ng/g wet weight and 257–501 ng/g wet weight for tocopherol [22, 23]. It maintains the reduced glutathione levels in the lens and aqueous humor by enhancing the glutathione recycling [24, 25]. Supplementation of vitamin E does not enhance the concentration in the lens . The rod outer segments and RPE contain high quantities of vitamin E. These tissues are sensitive to changing plasma levels of vitamin E . Three studies correlated significantly high plasma tocopherol levels (20 M) with a lesser prevalence of cataract, this could not be confirmed by a fourth study [8, 18, 24, 25]. High dietary intake of vitamin E was not correlated with decreasing risks for cataract but dietary intake of vitamin E is hard to estimate as the use of readymade foods and diverse brands of oil containing varying concentrations and compositions of tocopherols [5, 24]. There is no clear conclusion about pure vitamin E supplements; only one study found a significant lower prevalence for cataract whereas several other studies reported either nonsignificant inverse associations or no effects at all [18, 24, 25]. Combined supplements of multiple vitamins and/or minerals reduced the risk for cataract in several studies [8, 25]. As the retinal tissue is more sensitive to changes of vitamin E levels than the lens, in vitro studies suggest that
Fig. 3. Structure of flavan.
dietary intake of vitamin E or supplements have more influence in AMD than in cataract [5, 26]. There are only two studies (BDES and EDCC study) on this subject, the BDES study reporting a significant reduced risk for large macular drusen with high vitamin E intake. This significance was lost after adjustment for total vitamin E intake (dietary and supplement) [5, 8]. The other study found no significant association between dietary intake of vitamin E, with or without supplements, and AMD [5, 8]. High plasma levels of tocopherol were correlated significantly positive with a reduction of AMD in three studies, whereas two other studies found no significant associations [5, 8].
Flavonoids belong to the secondary metabolites in plants stored as glucosides in the vacuole. The basic structure of flavonoids is the flavan structure (fig. 3). Precursors for the flavonoid synthesis are three malonylCoA (ring A and C) and one 4cumaroylCoA (ring B) . Flavonoids exhibit several positive health aspects: they are anticancerogenic, antimutagenic, antiviral, antioxidant, immunestimulating and estrogenactive; they inhibit lipid peroxidation, lowdensitiy lipoprotein (LDL) oxidation and chelate transition metals [27–29].
The maximal radical scavenging and/or antioxidant properties are given by [30, 31]: (a) the dihydroxyl groups at position 3 and 4 in ring B; (b) the 2,3double bond in combination with the 4oxo group in ring C; (c) the hydroxyl group at position 3 in ring C, and (d) the hydroxyl groups at position 5 and 7 in ring A.
After the reaction with radicals, the arising aroxyl radicals are stable enough not to undergo further chain reactions. The aroxyl radicals disproportionate on the one hand back to the parental flavonoid and otherwise to a quinoid structure . Protective redox systems involving ascorbate and vitamin E can be extended with flavonoids interacting in a ‘cascade’ thus including lipophilic systems within this reaction. This was reported to work either synergistically or additive or showing a vitamin E or vitamin E and C sparing effect, respectively [33–36].
The knowledge about the bioavailability, uptake and metabolism is important to judge their pharmacological significance. Flavonoids occur in the diet as aglycones and glucosides. It is known that the aglycones as well as the glucosides pass the acidic conditions in the stomach unaltered . They are either absorbed passively via diffusion as aglycones or actively via the sodiumdependent glucose/galactose transporters in the small intestine. Lactase/phlorizin hydrolase and cytosolic glucosidase in the brush border of the small intestine are able to hydrolyze the glucosides and enhance the concentration of the aglycones for free diffusion. The absorbed flavonoids reach the liver to undergo further metabolism. Glucosides are hydrolyzed from the liver and all existing aglycones are converted by enzymes of the detoxification metabolism into glucuronides, sulfates and/or methylates which are the circulating forms in blood/plasma besides ring fission products derived from colon metabolism [37–40]. Detected physiological concentrations of quercetin are 0.5 and 0.1 M for isorhamnetin . Numerous reports describe effects on eye diseases on the basis of plant extracts, mainly containing flavonoids: Extracts from Primula macrophylla containing 3,4dihydroxychalcone and 3methoxyflavone are in use for several eye diseases in Pakistan and Afghanistan ; flavone glucosides (tetrahydroxytrimethoxyflavone oligoglucosides) from the heart wood of Pongamina pinnata from India are used for healing eye diseases ; anticataractic properties on the basis of the inhibition of aldose reductase as an initiating enzyme of photooxidative and degenerative reactions in the lens are reported for flavonoids (acacetin, apigenin, luteolin, linarin) from ‘Buddleja Flos’ or Chrysanthemum boreale [43, 44]. Furthermore, aqueous flavonoids from Propolis, the ‘bee glue’ used from bees to coat their hives, cure eye infections due to antiinflammatory and antiviral effects . An overview on natural therapies on ocular disorder is presented by Head . The conclusion is that increased circulation to the optic nerve and antioxidant functions help to prevent and potentially to cure cataracts and glaucoma. Unfortunately, there are no reports or studies on the possible concentrations of flavonoids in the various tissues of the eye after either supplementation or treatment.
The abovementioned regeneration cycles for tocopherol, ascorbate and flavonoids were intensively investigated in our laboratory. At diene conjugation in the copperinduced LDL oxidation, a widely accepted assay for studies concerning lipid peroxidation occurring in vivo in LDL particles at the progress of atherogenesis, cooperative effects of tocopherol and ubiquinol were determined. Both compounds are consumed during diene conjugation in a clear pecking order: tocopherol disappears as soon as approximately 85% of ubiquinol is consumed and diene conjugation sets on with the complete consumption of
Lag phase prolongation (min)
500 400 300 200 100 0
Ascorbate 2.5M 403min
|Calculated lag phase prolongation: 160min|
Fig. 4. Synergistic effects of ascorbic acid and rutin in copperinduced LDL oxidation.
tocopherol . Synergistic implications of ascorbic acid and rutin, a naturally occurring flavonoid, were determined in LDL oxidation (fig. 4). Rutin is able to prolong the onset of diene conjugation ( lag phase prolongation), whereas ascorbic acid has either prooxidative (1 M ) or a slight protective effect (2.5 M ), respectively (fig. 4). Addition of rutin and ascorbic acid led to a synergistic lag time prolongation, indicating interactions between these two antioxidants which enhances their single antioxidant properties and prevents prooxidative effects . LDL particles can be loaded with lipophilic antioxidants to investigate their impact on LDL oxidation and to study interactions between the loaded lipophilic compound and hydrophilic samples added to the assay. In the case of lycopene or luteinloaded LDL, no significant prolongation was determined (fig. 5), but the addition of rutin led to a synergistic prolongation of the lag time (fig. 5), indicating interactions between the lipophilic antioxidant in the LDL particle and the hydrophilic compound in the surrounding aqueous milieu of the assay . These results support the findings of several eye studies on AMD and cataract prevention which supplemented with multivitamin preparations. The results of the studies indicate that a reasonable composition of multivitamins possibly enriched with minerals provides a reduction for the incident of cataract and AMD [8, 16, 18, 25, 26].
The presented data show clearly that the antioxidant status of the eye plays a crucial role in pathogenesis of AMD and cataract, but other factors such as genetics, way of life or environmental influences need to be considered as well.
Fig. 5. Synergistic effects of lycopene or luteinloaded LDL with rutin in copperinduced LDL oxidation.
The influence of flavonoids has to be studied intensively to create a valid data basis. Furthermore, the data for vitamin C and E are inconsistent at the moment and need onward investigations. At least the combination of single antioxidants such as vitamin C and E and flavonoids are a possible field for future studies, but carotenoids need to be included as they contribute extensively to the antioxidant status in the eye.
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K.M. Janisch TUM Weihenstephan, Center of Life and Food Sciences Lehrstuhl für Phytopathologie Am Hochanger 2, DE–85350 Freising (Germany) EMail firstname.lastname@example.org–muenchen.de
Augustin A (ed): Nutrition and the Eye. Dev Ophthalmol. Basel, Karger, 2005, vol 38, pp 70–88
Heinrich Heine University Düsseldorf, Institute of Biochemistry and Molecular Biology I, Düsseldorf, Germany
The yellow color of the macula lutea is due to the presence of the carotenoid pigments lutein and zeaxanthin. In contrast to human blood and tissues, no other major carotenoids including �carotene or lycopene are found in this tissue. The macular carotenoids are suggested to play a role in the protection of the retina against lightinduced damage. Epidemiological studies provide some evidence that an increased consumption of lutein and zeaxanthin with the diet is associated with a lowered risk for agerelated macular degeneration, a disease with increasing incidence in the elderly. Protecting ocular tissue against photooxidative damage carotenoids may act in two ways: first as filters for damaging blue light, and second as antioxidants quenching excited triplet state molecules or singlet molecular oxygen and scavenge further reactive oxygen species like lipid peroxides or the superoxide radical anion.
Copyright © 2005 S. Karger AG, Basel
Carotenoids comprise a class of natural lipophilic pigments which are found in plants, algae, bacteria, yeasts, and molds . They are responsible for many of the yellow, orange and red hues of fruits and flowers. Chlorophyll masks the carotenoids in green leaves but in autumn, as the chlorophyll levels decline, the color of the carotenoids becomes visible and produces the yellows and reds of autumn foliage. As accessory pigments, carotenoids participate in photosynthetic processes and are involved in mechanisms of photoprotection in higher plants, dissipating excess light energy through the xanthophyll cycle, with the formation of zeaxanthin from violaxanthin . Carotenoids can also be found in many animal species, and are important colorants in birds, insects, fish, and crustacean, although animals are not capable of synthesizing carotenoids de novo and depend on dietary supply. About 600 different carotenoids have been characterized and new ones continue to be identified . Among the huge variety of structurally different carotenoids about 50 occur in the human diet with �carotene being the most prominent [4, 5]. Epidemiological studies clearly show that the consumption of a diet rich in fruit and vegetables is correlated with a lower risk for a number of diseases including some types of cancer, as well as cardiovascular, neurodegenerative and ophthalmological disorders . Among the dietary components, micronutrients have been suggested to be involved in the protection against such agerelated diseases . Carotenoids are dietary constituents, provided in high amounts by fruit and vegetables, and they likely play a role in disease prevention .
�Carotene and some other members of the carotenoid family are socalled provitamin A compounds. After absorption, they are cleaved by specific
enzymes and significantly contribute to human vitamin A supply which is one of the most important biological features of carotenoids. Vitamin A is essential for vision, growth and development. However, carotenoids also reveal other biological properties apparently contributing to health and to the prevention of diseases [9, 10]. Most of the carotenoids, including the major dietary nonprovitamin A compounds such as lutein, zeaxanthin, and lycopene, are very efficient antioxidants, provide photoprotection, and trigger cellular communication.
Within the last decade possible health effects of the carotenoids lutein and zeaxanthin have attracted attention, and levels of adequate supply were discussed with respect to beneficial effects on ocular health [11, 12]. There is increasing evidence from epidemiological studies that an increased intake of the macular pigments lutein and zeaxanthin is inversely associated with the risk for agerelated macular degeneration (AMD), a disease which affects the elderly and is a major cause of irreversible blindness in Western countries. Although effects of lutein and zeaxanthin in the prevention of AMD remain to be proven in intervention studies, they exhibit physicochemical and biochemical properties which make them suitable compounds for photoprotection of the retina.
It should be noted that various synthetic carotenoids and extracts from carotenoidrich plants are also used as food colorants, additives to animal feeds, nutritional supplements and for cosmetical and pharmaceutical purposes. In many multivitamin formulas, single carotenoids or carotenoid mixtures are included.
The chemical structures of lutein, zeaxanthin and �carotene are presented in figure 1. All members of the carotenoid family are tetraterpenoids composed of a central carbon chain with conjugated double bonds carrying different linear
Fig. 1. Structures of selected carotenoids.
or cyclic substituents. Based on their composition, carotenoids are divided into two subgroups: the oxygen free carotenes and the xanthophylls (oxocarotenoids) which contain at least one oxygen atom in their structure. Lutein and zeaxanthin are substituted with two hydroxyl groups at the 3 and 3�position of the ionone rings and can be formally assigned as dihydroxy derivatives of �and �carotene, respectively. Due to the presence of hydroxyl groups they are more polar than carotenes. In both compounds, nine carboncarbon double bonds of the polyene backbone are fully conjugated, whereas the double bonds in the ring are only partially in conjugation. The pattern of conjugated double bonds determines the lightabsorbing properties and influences the antioxidant activity of carotenoids. The absorption maximum of lutein is around 445 nm, the one of zeaxanthin 450nm; the molar extinction coefficients (�mol) at these wavelengths are in the range of 140,000–145,000 cm�1mol�1. Thus, both carotenoids are efficiently absorbing blue light . Because of the presence of chiralic centers, lutein and zeaxanthin may occur in several stereoisomeric forms, three in the case of zeaxanthin and eight for lutein. In plants only one major stereoisomer of lutein, [(3R,3�R,6�R)�,�carotene3,3�diol], and one of zeaxanthin, [(3R,3�R)�, �carotene3,3�diol] is found because of stereospecific biosynthesis. Consequently, these isomers are also dominant in our diet and the ones preferentially provided to the organism. According to the number of double bonds, several cis/trans (E/Z) configurations are possible for a given molecule. In homogenous solutions, carotenoids tend to isomerize and form a mixture of mono and polycis isomers in addition to the alltrans form. When incorporated into the food matrix the compounds are more resistant towards isomerization. Generally, the alltrans form is predominant in nature but several cis isomers of carotenoids are present in blood and tissues [10, 14]. Absorption spectra of carotenoid cis
isomers exhibit an additional absorption band at a characteristic position about 120nm below the wavelength of the absorption maximum . The intensity of the socalled ‘cis peak’ depends on the position of the cis bond and is most intensive when the central carboncarbon double bond of the molecule is in cis configuration.
The key molecule in carotenoid biosynthesis is isopentenyl diphosphate used to build up ‘step by step’ the carotenoid phytoene . Several enzymatic dehydrogenation reactions lead to the acyclic carotenoid lycopene. �Carotene,
�carotene, lutein, zeaxanthin and an array of other carotenoids are synthesized via subsequent cyclization, dehydrogenation, and oxidation reactions. A great number of enzymes involved in carotenoid biosynthesis have been identified and their DNA sequence has been determined. Methods of modern gene technology provide the tools to modulate carotenoid biosynthesis in plants and microorganisms and to change their carotenoid pattern . Single carotenoids or groups of carotenoids may be enriched by directed synthesis and genetically modified plants, algae or microorganisms may be used as suitable sources for selected carotenoids.
Lutein and zeaxanthin are among the most abundant carotenoids in our diet and mainly provided by fruit and vegetables. Carotenoid analyses have improved in the last decade, however, before the introduction of suitable stationary phases it was difficult to separate and quantify lutein and zeaxanthin with standard chromatographic methods. Therefore, their levels in carotenoid databases for foods are often reported as the sum of both compounds (table 1). In the US National Health and Education Survey, the intake provided with a Western diet has been estimated to be 1.3–3.0mg/day of lutein and zeaxanthin combined . In the German National Food Consumption Survey an average lutein intake of 1.9 mg/day was calculated, slightly increasing with age . Lutein contributed about 35% to the total carotenoid consumption. Similar data have been reported for other countries. The Food Habits of Canadians Study provided intake data for lutein (adults aged 18–65) as being 1.4 mg/day .
Table 1. Dietary sources of lutein and zeaxanthin
|Dietary source||Lutein � zeaxanthin|
|�g/100 g edible portion|
However, the studies also show that there is a broad range of intakes which apparently depends on individual preferences for specific foods. In the German study, green leafy salads and spinach have been identified as the major sources of lutein and zeaxanthin providing about 50% of the total supply. Lettuce, spinach, corn, broccoli and oranges were identified as the foods contributing most of the lutein to the Canadian diet. Further important sources are egg yolks, other green vegetables and fruits such as green beans, green peas, brussels sprouts, cabbage, kale, or peaches and tangerines. Also, fruit juices contribute to the intake of xanthophylls. In most plants the content of lutein by far exceeds that of zeaxanthin and ratios of 7:1 to 4:1 have been reported. Corn contains quite high amounts of zeaxanthin and it has been suggested that it is an important source for this specific carotenoid. Also, some potato varieties contain considerable amounts of lutein and zeaxanthin with the latter dominating in some of the strains .
Lutein and zeaxanthin are further found in algae, other microorganisms and the petals of many yellow flowers which are used as natural sources of these carotenoids. The hydroxy groups of both compounds can be esterified with various naturally occurring fatty acids [20, 21]. Thus, mono and diesters of lutein and zeaxanthin also occur in plants. Lutein and zeaxanthin dipalmitates,
Table 2. Carotenoid serum or plasma levels (nmol/ml)
|Ref.||Subjects (n)||Lutein � zeaxanthin||�Cryptoxanthin||Lycopene||�Carotene||�Carotene||Sum of carotenoids|
|84 111 85 33 86 3,480 29 54 87 57 88 98 22 220 22 180 Mean � SD||0.26 0.35 0.36 0.18 0.49 0.46 0.27 0.25 0.33 � 0.11||0.32 0.17 0.22 0.25 0.24 0.17 0.17 0.12 0.21 � 0.06||0.39 0.26 0.40 0.22 1.06 0.58 0.43 0.43 0.47� 0.26||0.05 0.08 0.08 nd 0.09 0.11 0.08 0.06 0.08 � 0.02||0.22 0.25 0.34 0.38 0.58 0.34 0.32 0.21 0.33 � 0.12||1.24 1.11 1.40 1.03 2.46 1.66 1.27 1.07 1.41 � 0.47|
|nd � Not determined.|
dimyristates, several mixed esters as well as monomyristates and further monoesters have been identified in the petals of the marigold flower (Tagetes erecta). Depending on sources and processing, nutritional supplements may contain lutein esters, with much smaller amounts of zeaxanthin esters, and/or free lutein and zeaxanthin.
Generally, the carotenoid pattern in human plasma is determined by the variety of fruit and vegetables ingested within the diet . Table 2 lists the blood levels of the major carotenoids reported in several studies. In the different studies, average lutein plus zeaxanthin concentrations were in the range of 0.18–0.49 nmol/ml. The ratio between lutein and zeaxanthin is usually between
3:1 and 5:1 . Both compounds are also found, together with other members of the carotenoid family, in human tissues including liver, kidney, lung, and skin [24, 25]. In skin, small amounts of carotenol fatty acid esters were identified, among them are lineolate, palmitate, oleate, myristate, and stearate monoand diesters of lutein, zeaxanthin, 2�,3�anhydrolutein, �cryptoxanthin and
�cryptoxanthin . It has been suggested that carotenol esters in human skin may be formed by reesterification of xanthophylls following absorption.
Carotenoid uptake from the diet follows the pathway of lipophilic nutrients. After digestion of food, carotenoids are incorporated into micelles formed from dietary lipids and bile acids, which facilitate absorption into the intestinal mucosal cell. Further, carotenoids are incorporated into chylomicrons which are released into the lymphatic system. In the blood, carotenoids appear initially in the chylomicron and VLDL fraction, whereas the levels in other lipoproteins such as LDL and HDL rise at later time points with peak levels at 24–48 h. The major vehicle of hydrocarbon carotenoids such as lycopene and �carotene is the LDL, whereas the polar xanthophylls are more equally distributed between LDL and HDL.
The bioavailability of carotenoids is influenced by several factors and may
vary within a wide range. Dietary fat, consumed together with the carotenoids, improves the absorption. Lutein plasma responses were higher when lutein esters were consumed together with a highfat spread compared to coingestion with a lowfat meal . On the contrary, bioavailability is decreased when certain types of dietary fiber are coingested. Depending on the type of fiber, plasma levels of lutein and other carotenoids, calculated as area under the plasma response curve (AUC0–24 h), were diminished by 40–75% . When carotenoids are consumed together with nonabsorbable fat replacers like sucrose polyesters, absorption is impaired ; xanthophylls are less affected than carotenes.
Processing of carotenoidcontaining products usually improves bioavailability. Disruption of cellular structures and release of the compounds from the food matrix as well as improved accessibility to lipophilic subcellular compartments have been suggested to play a role . When carotenoids were supplied with a diet rich in vegetables, the bioavailability of lutein was higher than that of �carotene . After ingestion of a supplement rich in �carotene which contained small amounts of lutein and zeaxanthin a preferential increase of the xanthophylls in the chylomicron fraction of lipoproteins was observed .
In human and animal studies, interactions between carotenoids during absorption have been described . Lutein exhibited an inhibitory effect on
�carotene uptake which was most pronounced when lutein was the predominant carotenoid in the source. �Carotene cleavage, however, was not affected . Competition between single carotenoids for incorporation into micelles and exchange of the compounds between lipoproteins apparently play a role, and may explain the different blood responses.
In dietary supplements nowadays on the market and containing lutein, the carotenoid may be present in two different forms – either esterified with different fatty acids or unesterified as socalled ‘free’ lutein. There is an ongoing debate regarding possible differences in the bioavailability of lutein from both forms. However, present data do not provide evidence that lutein from fatty acid esters is less bioavailable than ‘free’ lutein . Other factors such as dissolution of the formulation appear to be more important and should be considered for the evaluation of xanthophyll bioavailability. After ingestion of a single dose of zeaxanthin dipalmitate from wolfberry or nonesterified zeaxanthin (equal amounts), somewhat higher AUC values were determined in the group supplemented with the ester . No differences in plasma responses were found after application of �cryptoxanthin esters from papaya and nonesterified �cryptoxanthin . Based on the present data it is suggested that ester hydrolysis is not a limiting step in lutein and zeaxanthin absorption and that the esters exhibit at least an equivalent bioavailability compared to the ‘free’ compounds.
Esters of carotenoids are usually not detectable in human blood. However, when esterified lutein was supplied for a longer period of time, free lutein concentrations increased significantly, and in addition small amounts of lutein esters were detectable in the serum of some of the volunteers . However, the contribution of lutein esters to total lutein was less than 3%.
Present knowledge on lutein and zeaxanthin metabolism is scarce, but metabolism and conversion play a role in the formation of the specific carotenoid pattern of the macula lutea (see below). In studies supplementing lutein and zeaxanthin, metabolites of the compounds have been detected in serum but have been characterized only partially . Several carotenoids appearing in human serum are suggested to be derived from both xanthophylls, either by dehydration or oxidation of the hydroxyl group, the latter yielding mono and diketo derivatives of the parent carotenoids .
The identity of lutein and zeaxanthin as major carotenoids in the macular pigment of humans and other primates has been well established [39–43]. Both carotenoids have been identified by UVVis spectroscopy, mass spectrometry and by chromatographic methods comparing elution profiles with authentical standards. They are also dominating the carotenoid pattern of the entire retina but the concentration in the macula lutea is about 100 times higher than in the peripheral retina. It has been calculated that the levels of the macular pigments range from 0.3 to 1.3 mM , which is much higher than carotenoid concentrations found in any other tissue.
Limited information is available on the effect of carotenoid supplementation and macula pigment density. However, first studies show that upon supplementation with lutein the density of the macula pigment increases .
In addition to lutein and zeaxanthin, an oxidation product of lutein (3hydroxy�,�carotene3�one) was identified; some other oxocarotenoids were also found but in minor amounts . Several geometrical isomers of lutein and zeaxanthin, including 9cislutein, 9�cislutein, 13cislutein, 13�cislutein, 9ciszeaxanthin, and 13ciszeaxanthin were determined in human and monkey retinas. It is unknown if the cis isomers have specific roles compared the alltrans forms of the macular carotenoids.
Although only one stereoisomeric form of lutein, (3R,3�R,6�R)�, �carotene3,3�diol, and of zeaxanthin, (3R,3�R)�,�carotene3,3�diol, is provided with the diet, considerable amounts of a second stereoisomer of zeaxanthin, (3R,3�S)�,�carotene3,3�diol, were identified in the central region of macula lutea applying chiral column chromatography . The latter isomer has been assigned as mesozeaxanthin and differs from ‘natural’ zeaxanthin by the configuration at the 3�carbon atom. The UVVis spectra of zeaxanthin and mesozeaxanthin are identical. Mesozeaxanthin is not detectable in considerable amounts in other human tissues or blood [46, 47]. Thus, it has been suggested that the isomer is specifically formed in ocular tissues. Two different mechanisms have been discussed both assuming that mesozeaxanthin is generated from the lutein stereoisomer (3R,3�R,6�R)�,�carotene3,3�diol. One pathway proposes the intermediate formation of an oxidation product involving the allylic 3�hydroxy group of lutein, the other suggests that the isomerization results from a direct migration of the isolated double bond in the �ring of lutein, preserving the configuration at the 3�carbon atom [16, 42, 47].
Interestingly, lutein, zeaxanthin and mesozeaxanthin levels and ratios vary in different regions of the macula lutea [16, 48]. Moving from excentric positions of the retina towards the fovea (center) carotenoid concentrations increase and the lutein to zeaxanthin ratio changes in favor of zeaxanthin. Lutein is the dominating carotenoid in the outer segments of the retina (lutein:zeaxanthin about 2:1), whereas zeaxanthin is the major carotenoid in the center and the ratio is reversed. In accordance with dietary supply, the amount of lutein in human serum exceeds that of zeaxanthin by the factor 3–4. Applying chiral chromatography, the distribution pattern of zeaxanthin isomers (3R,3�R)�,�carotene3,3�diol, and (3R,3�S)�,�carotene3,3�diol (mesozeaxanthin) was analyzed. Both isomers are found in about equal amounts (ratio 1:1) in the center of the macula lutea, whereas the contribution of mesozeaxanthin to total zeaxanthin is gradually decreasing moving to peripheral areas. The reasons for the enrichment of mesozeaxanthin in the central area and the spatial distribution pattern of lutein and zeaxanthin are unknown.
It is generally accepted that xanthophyllbinding proteins with high affinity for the hydroxylated ionone rings are involved in the directed transport of macular carotenoids. However, detailed knowledge on the structures and properties of these proteins is lacking. It is also unknown whether the spatial and structural distribution of carotenoids within the retina is related to specific functions and, if so, what biochemical or physicochemical properties may underlie such functional aspects. There is some evidence that the orientation of xanthophylls in cellular membranes might play a crucial role.
Analysis of transverse retina sections shows that high levels of the carotenoids are present in the photoreceptor axons. Tubulin is found in abundance in the receptor axon layer of the fovea. There is evidence for specific xanthophylltubulin interactions and it has been suggested that the protein serves as a locus for the deposition of macular carotenoids . Substantial amounts of lutein and zeaxanthin were also determined in rod outer segments . Based on the localization of the carotenoids and their chemical properties it has been suggested that they protect photoreceptors by filtering blue light and provide additional protection by scavenging reactive oxygen species.
The development and existence of an organism in the presence of oxygen is associated with the generation of reactive oxygen species (ROS), even under physiological conditions. ROS are responsible for the oxidative damage which may affect all types of biological molecules, including DNA, lipids, protein and carbohydrates [51–53]. Such damage is discussed as pathobiochemical mechanism playing a crucial role in the development of various diseases [54, 55]. Some of the most relevant ROS are: singlet oxygen (1O2), peroxyl radicals (ROO�), the superoxide anion radical (O2��), the nitric oxide radical (NO�), peroxynitrite (ONOO�), and hydrogen peroxide (H2O2). ROS are either radicals (molecules that contain at least one unpaired electron) or reactive nonradical compounds such as excited state molecules like 1O2. These reactive intermediates are often summarized by the term oxidants or prooxidants.
The prooxidant load is counteracted by a diversity of antioxidant defense systems operative in biological systems which include antioxidant enzymes, lowmolecularweight antioxidants, trace elements and specific proteins. An antioxidant has been defined as ‘any substance that, when present in low concentrations compared to that of an oxidizable substrate, significantly delays or inhibits the oxidation of that substrate’ [51–53].
The major enzymes directly involved in the detoxification of ROS are superoxide dismutase, which is scavenging O2��, as well as catalase and glutathione peroxidases reducing hydrogen peroxide and organic hydroperoxides, respectively.
Several endogenous lowmolecularweight compounds are also involved in antioxidant defense, of which glutathione is the most prominent. Other endogenous compounds such as ubiquinol10, urate, or bilirubin also contribute to antioxidant defense.
With the human diet, an array of different compounds possessing antioxidant activities are provided to the organism. The most prominent representatives of dietary antioxidants are carotenoids, ascorbate (vitamin C), tocopherols (vitamin E), and polyphenols. Most groups of dietary antioxidants comprise a number of structurally different compounds . Taken together, the organism is protected against oxidative damage by a network of defense systems, which may act synergistically and fulfill specific tasks depending on their reactivity and distribution in tissues and at the subcellular level.
When ROS are produced in excessive amounts and not sufficiently detoxified, the steadystate balance between the prooxidant load and the antioxidant network may be disrupted. A disbalance in favor of the prooxidants potentially leading to damage has been defined as ‘oxidative stress’ .
Upon light exposure, a cascade of photoinduced reactions takes place in the exposed tissues [57, 58]. As primary event, light interacts with a suitable chromophore which mediates desired biological responses but may also initiate damaging reaction sequences. Photoinduced damage may directly affect the chromophore, e.g. formation of pyrimidine dimers in the DNA. However, the chromophor can also act as photosensitizer initiating subsequent photochemical reactions. Porphyrins, flavins, DNA bases, amino acids or lipofuscin have been shown to act as photosensitizers. Upon illumination, the photosensitizer is excited from the ground state to the first excited singlet state, followed by conversion to the triplet state via intersystem crossing. According to the postexcitational chemistry the excited state can react in two ways – either by type I or type II photooxidation reactions [58–60]. A type I mechanism involves hydrogenatom abstraction or electrontransfer reactions between the excited state of the photosensitizer and a cellular substrate, yielding free radicals and radical ions. Such intermediates undergo further reaction sequences, leading to the deterioration of proteins, lipids or DNA and finally damage important cellular structures. As the primary reaction product of a type II photooxidation reaction, singlet molecular oxygen (1O2) is generated via energy transfer from the excited photosensitizer to ground state oxygen. 1�g O2, the major form of electronically excited, singlet oxygen, is a dienophilic intermediate which reacts with polyunsaturated fatty acids, DNA bases, histidine, tyrosine or tryptophan residues forming cyclic or acyclic peroxides. Peroxides are unstable compounds and undergo further rearrangement, elimination or Fentontype reactions, yielding modified biomolecules or free radicals. Again, radicalinitiated reaction chains take place and impart damage to biological and cellular structures.
Lipofuscin, a heterogenous conglomerate of biomolecules and breakdown products, has been assigned as the ‘aging pigment’ and is found in various postmitotic cells. In the retinal pigment epithelium, lipofuscin is present as micrometersized spherical particles and an accumulation has been observed in the process of macular degeneration. The pigment is characterized by its yellow autofluorescence upon exposure to blue light and it has been suggested that fluorescent components of lipofuscin granules are at least in part responsible for phototoxic reactions . The composition of lipofuscin is poorly defined and the fluorophores have only been partially characterized. However, it has been demonstrated that retinal lipofuscin is a photoinducible generator of ROS, and illumination of lipofuscin with visible light leads to extragranular lipid peroxidation, enzyme inactivation, and protein oxidation .
Carotenoids, including lutein and zeaxanthin, efficiently scavenge ROS which is considered to be an important biological task of these compounds. According to their unique structure they are efficient scavengers of singlet molecular oxygen and of peroxyl radicals. Further, they are effective deactivators of electronically excited sensitizer molecules which are involved in the generation of radicals and singlet oxygen [63, 64].
The interaction of carotenoids with 1O2 depends largely on physical quenching which involves direct energy transfer between the molecules. The energy of singlet molecular oxygen is transferred to the carotenoid resulting in the formation of ground state oxygen and a triplet excited carotenoid. Dissipating its energy by interaction with the surrounding solvent, the carotenoid returns to its ground state and no further reactions take place. Because the carotenoids remain intact during physical quenching of 1O2 or excited sensitizers, they can be reused severalfold in quenching cycles.
The efficacy of carotenoids for physical quenching is related to the number of conjugated double bonds present in the molecule which determines their lowest triplet energy level. Lutein, zeaxanthin, �carotene and structurally related carotenoids have triplet energy levels close to that of 1O2 thus, enabling efficient energy transfer. They are the most efficient naturally occurring quenchers for 1O2 with quenching rate constants around 5–12 � 109 M�1s�1. However, the singlet oxygen quenching activity of carotenoids is dependent on the environment . When incorporated into a model membrane, xanthophylls were less active than hydrocarbon carotenoids. In this system, lycopene and �carotene exhibited the fastest singlet oxygen quenching rate constants whereas lutein and zeaxanthin were less efficient.
In contrast to physical quenching, chemical reactions between the excited oxygen and carotenoids are of minor importance, contributing less than 0.05%
to the total quenching rate. However, this process finally leads to the decomposition of the carotenoid, known as photobleaching. In vitro experiments have demonstrated that lutein and zeaxanthin are more stable than �carotene and lycopene under photooxidative conditions. In a mixture of the four carotenoids the loss of lutein and zeaxanthin was less pronounced when the solution was irradiated with UV light in the presence of the sensitizer Rose Bengal . The macular carotenoids were also more resistant towards irradiation with sunlight.
Among the various other ROS which are formed in the organism, carotenoids most efficiently scavenge peroxyl radicals. These are generated in the process of lipid peroxidation, and scavenging of this species interrupts the reaction sequence which would otherwise finally lead to damage of lipophilic compartments. Due to their lipophilicity and specific property to interact with peroxyl radicals, carotenoids are thought to play an important role in the protection of cellular membranes against oxidative damage . The antioxidant activity of carotenoids regarding the deactivation of peroxyl radicals likely depends on the formation of radical adducts forming a resonancestabilized carboncentered radical.
A variety of products have been detected subsequent to oxidation of carotenoids, including carotenoid epoxides and apocarotenoids of different chain length . It should be noted that these compounds might possess biological activities and interfere with signaling pathways when present in unphysiologically high amounts .
The antioxidant activity of carotenoids depends on the oxygen tension present in the system [70, 71]. At low partial pressures of oxygen such as those found in most tissues under physiological conditions, �carotene was found to inhibit oxidation. In contrast, prooxidant activities of carotenoids have been demonstrated in several in vitro experiments at high oxygen tension. However, it is still not known if prooxidant properties of carotenoids play a role in vivo.
A number of in vitro studies were carried out in order to compare the antioxidant activities of structurally different carotenoids. The results vary very much depending on the system used for investigation. The mechanism and rate of scavenging is strongly dependent on the nature of the oxidizing radical species and less dependent on the carotenoid structure [72, 73].
When the antioxidant activity of carotenoids was assayed in multilamellar liposomes measuring formation of thiobarbituric acidreactive substances (TBARS) after challenge with 2,2�azobis(2,4dimethylvaleronitrile) (AMVN) the following ranking was determined: lycopene ��carotene ��cryptoxanthin �zeaxanthin ��carotene �lutein . In the TEAC assay, which investigates the potency to scavenge the ABTS radical, lutein and zeaxanthin exhibited comparable activities and were somewhat less efficient than �carotene and lycopene .
In a model system using egg yolk lecithin liposomal membranes, UVinduced lipid oxidation was also slowed down by lutein and zeaxanthin. In this system, zeaxanthin appeared to be a better photoprotector during prolonged UV exposure. It was suggested that the differences in the protective efficacy of lutein and zeaxanthin in lipid membranes are attributable to a different organization of zeaxanthinlipid and luteinlipid membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane whereas the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane was thought to play a role in photoprotection [76, 77].
There is evidence that the antioxidant effects of carotenoids depend on the concentration in the system with an optimal concentration for each compound. When human skin fibroblasts (loaded with single carotenoids) were exposed to UVB light, lycopene, �carotene, and lutein were capable of decreasing UVinduced formation of TBARS, an indicator for lipid oxidation. The amounts of carotenoid needed for optimal protection were divergent: 0.05, 0.40, and
0.30 nmol/mg protein for lycopene, �carotene, and lutein, respectively. At levels below the optimum, less protection was found whereas at higher levels prooxidant effects were observed .
Carotenoids are part of a complex antioxidant network, and it has been suggested that interactions between structurally different compounds with variable antioxidant activity provide additional protection against increased oxidative stress. For example, vitamin C, the most powerful watersoluble antioxidant in human blood and tissues, acts as a regenerator for vitamin E in lipid systems . Synergistic interactions against UVAinduced photooxidative stress have been observed in cultured human fibroblasts when combinations of antioxidants were applied with �carotene as the main component . The antioxidant activity of carotenoid mixtures was assayed in multilamellar liposomes, measuring the inhibition of the formation of TBARS . Mixtures were more effective than single compounds, and the synergistic effect was most pronounced when lycopene or lutein were present. The superior protection of mixtures may also be related to specific positioning of different carotenoids in membranes.
The photoreceptors in the retina are susceptible to damage by light, particularly blue light . As already mentioned, pigments like lipofuscin may act as photosensitizers and have been considered to be involved in pathways leading to photooxidative damage. Upon irradiation with blue light, lipofuscin fluorophores mediate cellular damage and induce apoptosis .
Fig. 2. Mechanisms of protection against photooxidative damage.
Based on the spectral properties of carotenoids, it has been postulated that one of the important tasks of lutein and zeaxanthin in the macula is filtering of blue light. However, all major dietary carotenoids including �carotene and lycopene are efficient blue light filters in homogenous solution. Thus, it remains unclear why lutein and zeaxanthin should be preferably used as filtering compounds in the retina. It is known that spectral properties as well as antioxidant activities change with the environment. Therefore, the filtering effects of lutein and zeaxanthin in comparison to those of lycopene and
�carotene were investigated in membrane model using unilamellar liposomes . Liposomes were loaded in the hydrophilic core space with a fluorescent dye, excitable by blue light, and various carotenoids were incorporated into the lipophilic membrane. The fluorescence emission in carotenoidcontaining liposomes was lower than in controls when exposed to blue light, indicating a filter effect. At low concentrations, all carotenoids exhibited similar activities. However, the xanthophylls could be incorporated in higher amounts into the membrane and showed a better filtering efficacy than �carotene or lycopene.
Evidence from epidemiology, animal studies, and in vitro experiments supports the hypothesis that the major macular pigments, lutein and zeaxanthin, protect the central retina against degenerative processes. The unique distribution, localization and the high levels of both carotenoids within the macula lutea add further evidence for a specific protective function in this tissue. Due to the physicochemical properties of carotenoids it is likely that the major task of the macula carotenoids is related to the protection of the central retina from photooxidative damage. Filtering blue light and scavenging ROS are most likely the mechanisms of protection (fig. 2). Upon supplementation, levels of lutein increase in the macula and high levels of intake are apparently related to a lower risk for AMD. One of the major tasks for the future will be to provide unequivocal evidence that an increased consumption of macular carotenoids helps to prevent AMD.
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Prof. Dr. W. Stahl Heinrich Heine University Düsseldorf Institute of Biochemistry and Molecular Biology I PO Box 101007, DE–40001 Düsseldorf (Germany) Tel. �49 211 8112711, Fax �49 211 8113029, EMail email@example.com
Augustin A (ed): Nutrition and the Eye. Dev Ophthalmol. Basel, Karger, 2005, vol 38, pp 89–102
MOLISA GmbH, Magdeburg, Germany
Selenium biochemistry is reviewed in respect to its presumed relevance to agerelated ocular diseases. Selenium is an essential trace element that exerts its physiological role as selenocysteine residue in at least 25 distinct selenoenzymes in mammals. Lack of GPx1 due to alimentary selenium deprivation has been inferred to induce cataract in rats and was demonstrated to cause cataracts in mice by targeted gene disruption. The role of other selenoproteins in the eye remains to be worked out. Selenium in excess of the tiny amounts required for selenoprotein synthesis is toxic in general and causes cataracts in experimental animals. Clinical evidence for a protective role of selenium in the development of cataract, macula degeneration, retinitis pigmentosa or any other ocular disease is not available, likely because suboptimum selenium intake, as it may result from unbalanced diet, does not cause any pathologically relevant selenium deficiency in the eye. At present, there is neither theoretical nor an empirical basis to expect beneficial effects of selenium supplementation beyond the dietary reference intakes of 55 �g/day in the context of ocular diseases.
Copyright © 2005 S. Karger AG, Basel
In the context of ocular diseases, selenium is most frequently quoted as an agent that causes cataracts in experimental rodents. However, deficient alimentary supply of the essential trace element is also implicated in the development of cataracts [1, 2]. The latter effect is commonly discussed to be related to an antioxidant action of selenium. Since oxidative damage is also believed to contribute to agerelated macula degeneration and retinitis pigmentosa, the presumed antioxidant selenium might equally be relevant to these diseases. Supportive experimental or clinical data, however, are scarce, and the seemingly conflicting findings demand a critical reevaluation that is based on solid knowledge of the biological roles of selenium.
This article will therefore briefly summarize relevant aspects of selenium biochemistry in mammals, compile the knowledge on selenoproteins with special emphasis on those present in the eye, try to explain experimental or clinical data by established molecular events and finally line out what should reasonably be considered to become clinically important.
Unspecific Selenium Effects versus Enzymatic
Selenium exerts its beneficial biological role as constituent of an estimated total of 25 distinct proteins (table 1) . They comprise five thioldependent peroxidases, commonly called glutathione peroxidases (GPx), three deiodinases (DI), which are involved in the synthesis and degradation of the thyroid hormones, three thioredoxin reductases (TR), the selenium transport protein (SelPP), the selenophosphate synthetase that is required for the synthesis of all other selenoproteins, and a variety of further proteins known by deduced amino acid sequence, the biological role of which is still poorly defined .
In these proteins, selenium is present as one or more selenocysteine residues that are integrated into the amino acid chains at specific positions. The specific incorporation of selenocysteine into the proteins is determined by a complex coding mechanism, wherein the stop codon TGA is recoded by means of a secondary mRNA structure called SECIS (for selenocysteine incorporation sequence) and the pertinent translation factors, SBP2 and mSelB. The former recognizes the SECIS, the latter a specific selenocysteylloaded tRNA(ser)sec. Interestingly, charging of tRNA(ser)sec differs from the common pathway; the tRNA has first to be loaded with serine. The seryl residue is then transformed into a selenocysteyl residue by selenocysteine synthase with selenophosphate as substrate. If the charged selenocysteyltRNA(ser)sec is not sufficiently available, the stop codon nature of TGA becomes dominant again despite the presence of the SECIS in the particular mRNA, that means the ‘selenoprotein’ is truncated at the position where selenocysteine was to be inserted [reviewed in 4].
An important phenomenon to understand the biological consequences of selenium shortage is the ‘hierarchy of selenoproteins’. The term describes the observation that the individual selenoproteins respond differently to selenium restriction. Out of the wellinvestigated selenoproteins, the classical glutathione peroxidase, GPx1, and GPx3, the extracellular variant, decline most readily in selenium deficiency and recover slowly upon resupplementation. In contrast, GPx2, the gastrointestinal form, and phospholipid hydroperoxide GPx (GPx4) remain reasonably high even in moderate to severe selenium deficiency, the remaining selenoproteins ranking in between. The underlying molecular mechanism is not completely understood. One of the reasons of the fast decline and slow recovery of GPx1 and GPx3 is a degradation of the pertinent mRNAs in
Table 1. Selenoproteins 2003 
|Cytosolic or classical GPx||cGPx, GPx1|
|Phospholipid hydroperoxide GPx||PHGPx, GPx4|
|Plasma GPx||pGPx, GPx3|
|Gastrointestinal GPx||GIGPx, GPx2|
|5�deiodinase, type 1||5�DI1|
|5�deiodinase, type 2||5�DI2|
|5deiodinase, type 3||5DI3|
|Mitochondrial thioredoxin reductase||SelZf1|
|Thioredoxin reductase homologs||SelZf2|
|15kDa selenoprotein (T cells)|
|Selenoprotein R (methionine sulfoxide||MrsB|
|Selenoprotein N (knockout causes||SelN|
|muscular dystrophy with|
|spinal rigidity and restrictive|
response to selenium deprivation. A seleniumdependent affinity shift of RNAbinding proteins likely contributes to the differential mRNA stabilities. Out of the proteins of the selenoprotein machinery only SBP2 might function as the required selenium sensor, as it only binds tRNA(ser)sec if this is charged with selenocysteine. However, since SBP2 does not directly interact with the mRNA, it would have to cooperate with an RNAbinding protein such as mSelB to signal the selenium status to the mRNA/protein complex, which is also called the selenosome [reviewed in 4, 5].
Equally important is the differential delivery of selenium to particular tissues. Privileged organs that retain their selenium status in pronounced selenium deficiency are thyroid, brain and testis. The molecular basis of this up to recently mysterious phenomenon is now emerging. Foodderived selenium is incorporated in the liver into SelPP, a protein with up to 17 selenocysteine residues (10 in humans). SelPP is secreted into the circulation apparently to deliver its selenium to sites of particular demand. The present hypothesis is that SelPP binds to a receptor in the privileged tissues, is internalized there and degraded to provide selenium for de novo synthesis of selenoproteins. This view is corroborated by a dramatic drop of selenium content in privileged tissues associated with elevated selenium levels in livers of SelPP knockout mice . Unfortunately, it has so far not been investigated whether ocular tissues are similarly supplied with selenium, as is the brain.
It remains to be discussed what happens to adsorbed selenium beyond the amount required for the synthesis of selenoproteins. To some extent this depends on the chemical nature of the particular seleno compound. Selenomethionine, for instance, is stochastically incorporated into proteins instead of methionine, the consequences thereof being unclear. As a rule, however, bioavailable selenium, be it selenite from drinking water or selenoamino acids from meat or fish protein, are transformed to the same intermediate, selenide. In case of selenite, the reduction can be achieved directly by any of the thioredoxin reductases or by reaction with GSH to selenaglutathione trisulfide and reduction thereof by glutathione reductase or thioredoxin reductases. The selenoamino acids are transformed by the transulfuration pathway and, ultimately, H2Se is released from selenocysteine by (seleno)cysteine lyase . H2Se serves as the precursor of selenophosphate, which is used for selenoprotein synthesis, as outlined above. Any excess beyond the tiny genetically determined demand needs to be disposed immediately, because seleno compounds are highly reactive and accordingly toxic and no storage mechanism, apart from the limited capacity of SelPP synthesis, is known in mammals. The first line of defense against excess selenium is SAMdependent methylation of H2Se. It yields the volatile monoand dimethylselenium derivatives that are exhaled and account for the smell of rotten horseradish or garlic that is typical of acute selenium poisoning. These volatile metabolites easily penetrate the bloodbrain barrier and are the main culprits of selenium’s neurotoxicity. Further methylation yields the trimethylselenonium ion that is excreted with the urine. Once the methylation capacity is exhausted, which usually results from chronic overexposure to selenium, the element starts to disclose it socalled antioxidant potential – with disastrous consequences [more details reviewed in 2, 4, 7].
For sake of clarity it has to be stressed that selenium is not an antioxidant, either in the chemical meaning of this term or in a biological sense. As will be discussed below, selenium plays an important role in the metabolism of H2O2 and other hydroperoxides by being a functional heteroatom in peroxidases and thereby contributes to the prevention of free radical formation and related tissues damage. But this does not justify its mislabeling as an ‘antioxidant’. In chemical terminology, an antioxidant is a compound that reacts with free radicals, thereby becomes transformed into a less reactive radical itself, and thus slows down or terminates free radical chain reactions. None of the selenium compounds contained in food or metabolites thereof meets these characteristics. More importantly, the mass law implies that the efficacy of an antioxidant increases with its concentration. What instead happens in ‘supranutritional dosing’ of selenium is easily predicted and has been amply verified experimentally : The excess selenium ends up in a pool of selenium compounds of the oxidation state �2, i.e. selenides or selenols. Being strong reductants, such compounds react with the most abundant oxidant, i.e. molecular dioxygen (O2), with formation of superoxide anion radicals and hydrogen peroxide (H2O2). The seleno compounds thereby oxidized are enzymatically reduced as outlined above, and undergo autoxidation again. In short, any selenium surpassing the biological needs and the very limited storage capacity starts redox cycling, which is the most efficient way to cause oxidative damage in biological systems.
These basic principles of selenium biochemistry disclose why the therapeutic window of any selenium compound is inevitably small. They further reveal why the symptoms of chronic selenium intoxication, which is associated with the oxidative stress markers typical of redox cyclers, often resemble those of selenium deficiency, which results in impaired detoxification of hydroperoxides.
The majority of the mammalian selenoproteins were discovered in the past decade. Accordingly, understanding of their biology is mostly limited, and their presence in ocular tissues has been verified in exceptional cases only.
It must be inferred that the eye contains cytosolic (TR1) and mitochondrial (TR3) thioredoxin reductases that are indispensable for ribonucleotide reduction via thioredoxin and also determine other functions of the pleiotropic redox mediators of the thioredoxin family. Similarly, each cell has to be equipped with selenophosphate synthetase to synthesize pivotal selenoproteins such as the thioredoxin reductases. Selenoprotein W prevails in muscle and nervous tissue but its presence in the eye remains to be established. Virtually nothing is
Fig. 1. The glutathione peroxidase reaction. a The catalytic triad that is strictly conserved by the whole GPx superfamily with the only exception that selenocysteine may be replaced by cysteine, which is associated with low peroxidases activity. Residue numbers of bovine GPx1 are given as example. b The catalytic cycle of GPx that is essentially characterized by redox shuttling of its selenium moiety. In case of GPx1, GSH serves as reducing substrate, other isoenzymes accept different thiols (RSH).
known about iodothyronine deiodinases (DI) in the eye. They regulate local thyroid hormone activity by generating the active 3,5,3�triiodothyronine from thyroxin (DI1 and DI2) or degrading thyroxin and 3,5,3�triiodothyronine to reverse T3 (3,3�,5�triiodothyronine) or other inactive compounds, respectively (DI1 and DI3), and their potential relevance to ocular affections in thyroid disturbances such as Graves’ disease would certainly be of interest.
The only two selenoproteins that have unambiguously been shown to be present in the eye are GPx1 and GPx3. As early as 1965, i.e. long before the selenoprotein nature of any mammalian enzyme was recognized , the ‘classic’ cytosolic glutathione peroxidase (GPx1) was isolated from lens by the pioneer of eye biochemistry, Antoinette Pirie . The extracellular form GPx3, which is primarily derived from the kidney, was found to be also synthesized in the ciliary body  and to be released into the aqueous humor . Like all other members of the GPx family, these two enzymes reduce H2O2, organic hydroperoxides and peroxynitrite at the expense of thiols with high efficiency. Depending on the nature of the hydroperoxide, the bimolecular rate constants for the reaction of reduced enzyme with ROOH ranges between some 106 and 108 M�1s�1. These extreme rate constants depend on the selenium moiety, which forms a catalytic triad consisting of a selenocysteine, a glutamine and a tryptophan residue wherein the selenol function of the selenocysteine is dissociated and polarized for nucleophilic attack on peroxo groups  (fig. 1). In case of GPx1, the reducing substrate is glutathione (GSH), whereas GPx3 also accepts thioredoxin and glutaredoxin as reductants. The metabolic context of GPx1 is
Fig. 2. Main metabolic context of GPx1. The selenoprotein is typically supplied with reduction equivalents (NADPH) from the pentose phosphate shunt. Deficiencies in this pathway may impair regeneration of GSH and, in consequence, hydroperoxide (ROOH) detoxification via GPx1. Accumulating hydroperoxide, by decomposing into alkoxyl (RO�) and hydroxyl radical (�OH), may then initiate free radical chain reactions.
straightforward. The reducing substrate GSH is regenerated by glutathione reductase, which predominantly receives its reduction equivalents as NADPH from the pentosephosphate shunt (fig. 2). Instead, GPx3 depends on the tiny amounts of thiols that are released into the extracellular space and has therefore been addressed as ‘orphan enzyme’ . The lack of any known thiolregenerating system in the extracellular space limits the capacity of GPx3 to cope with an extensive hydroperoxide challenge. The different localization of these otherwise similar enzymes thus points to distinct biological roles: While GPx1 has been established as the most important device of hydroperoxide detoxification in general, GPx3 may regulate the extracellular peroxide tone that is implicated in the biosynthesis of inflammatory lipid mediators by lipoxygenases and may affect other signaling cascades [reviewed in 5, 7].
The interest of ophthalmologists in the relationship of the glutathione system and cataract development dates back to the 1950s of the last century and was extensively reviewed by Kinoshita  already in 1964. The observations were: (i) the lens has been reported to have a GSH content that, with about 10mM, surpasses that of any other tissue ; (ii) GSH of the lens drops with age ; (iii) it is even more decreased in cataractous lenses , where
(iv) glutathionylated proteins increase .
The first to recognize the link of these findings to H2O2 detoxification was evidently Antoinette Pirie, who not only identified GPx1 in the lens but simultaneously presented a source of H2O2 that attacks the lens from the aqueous humor, where it is formed by autoxidation of another ‘antioxidant’, ascorbate .
The role of H2O2 in inducing cataract was then corroborated by Srivastava and Beutler  by incubating rabbit lenses with tyrosine and tyrosinase, which produces superoxide and/or H2O2 as byproduct. Preceding cataract development GSH became oxidized and released to the medium as GSSG. Interestingly, a similar loss of GSH in the lens and export of GSSG to the aqueous humor was observed upon naphthalene feeding to rabbits , which likely is mediated by a metabolite of naphthalene, 1,2naphthoquinone. The latter is a known redox cycler which oxidizes GSH by continuously generating H2O2 but aggravates the loss of GSH by reacting to covalent adducts . Analogous reactions of 1,2naphthoquinone with SH groups of �and �crystallin, that are favored at low GSH concentrations, lead to insoluble colored proteins that contribute to cataract manifestations.
The latter findings reveal that H2O2 itself is not necessarily the agent that induces the cataractogenic protein modification. H2O2derived free radicals may be the main culprits, and oxygencentered radicals may be directly formed in the eye, e.g. by UV irradiation. To mimic such radical damage, rats were poisoned with the herbicide diquate. This compound is reduced univalently by NADPH and induces cataract via a radical that does not cause any significant loss of GSH . Further evidence for GSHindependent cataracts is provided by genetics. A dominant cataract mutant (Nop/�) did not display any abnormalities in the enzymes related to the glutathione redox balance. In particular the activities of GPx, glutathione reductase and glucose6phosphate dehydrogenase were not affected . Similarly, mutations in the Huntingtin interacting protein were found to be associated with cataracts .
On the other hand, inverse genetics finally corroborate the early hypotheses on hydroperoxides being key players in cataract development and the GSH system being protective: GPx1�/� mice spontaneously develop complete lens opacification at an age of 15 months. This is preceded by progressive nuclear light scattering, distortion of fiber membranes and distension of interfiber space starting at 3 weeks of age and lamellar cataracts between 6 and 10 months .
This finding is of particular interest, since it represents the so far only phenotype observed in unstressed GPx1�/� mice. These animals grow normally if not exposed to hydroperoxides, bacterial toxins that trigger an oxidative burst in phagocytes, redoxcycling herbicides or viral infections [reviewed in 7]. It has therefore to be concluded that the lens, in contrast to other tissues, is physiologically exposed to a certain oxidative stress that needs continuous protection by the selenoprotein GPx1. In short, the GSHdependent hydroperoxide detoxification may thus be rated as one of the genetically validated systems that protect against agerelated cataract formation. The knockout experiments also provide a rational to explain the early observations of cataract development in seleniumdeprived rodents [23, 24], which thus is likely the consequence of GPx1 deficiency.
How then does the proven protection by selenium against cataract formation comply with the cataractogenic potential of the very same element? Cataracts can consistently be induced in young rats, rabbits and guinea pigs by selenite at dosages below the threshold causing acute systemic toxicity. The narrow time window of susceptibility and the comparatively low dosages suggested specific effects of selenite on the lens, and indeed a variety of phenomena have been observed in this cataract model that are not easily explained by unspecific selenium toxicity, e.g., a dramatic increase in calcium and phosphate in the lens, binding of radioactive selenium to lens proteins [compiled in 2], impairment of protein tyrosine phosphorylation and phosphatidylinositol3kinase activity , and calciuminduced proteolysis of �crystallin . However, whatever the seemingly specific mechanism of lens toxicity may be, all these effects are observed at dosages that surpass the ones required for optimum production of selenoproteins by more than three orders of magnitude: several milligrams/kilogram instead of 1 �g/kg. Under these conditions, selenium has clearly changed its face from an essential micronutrient to a prooxidative redox cycler. Accordingly, the total reducing capacity of the lens is decreased, as is evident from a decrease in GSH, NADPH and total protein sulfhydryls, and markers of oxidative damage such as malondialdehyde are substantially increased . It therefore may be doubted if the seemingly specific effects of selenium reflect more than sites of the young lenses that are particularly prone to oxidative damage. In line with this view, seleniteinduced cataract could be inhibited in vitro and in vivo with antioxidant extracts of green tea (Camellia sinensis)  or other antioxidants . In conclusion, as an essential trace element up to daily dosages of 1 �g/kg body weight, selenium prevents cataract formation by guaranteeing optimum H2O2 detoxification via GPx1. If given in marked excess, it induces cataracts by causing oxidative damage to the lens.
The unequivocal demonstration of the indispensability of GPx1 in the eye of mice justifies discussing the potential impact of the selenoenzyme in respect to ophthalmic diseases believed to be caused or aggravated by oxidative stress. Since GPx1 belongs to the group of selenoproteins that declines rapidly in selenium deficiency, the problem addressed is intimately related to the question if selenium supplementation can ameliorate such diseases. To state it right away, supportive clinical evidence is surprisingly scarce.
To our knowledge there is not a single report that relates a genetic deficiency of GPx1 or of any other selenoprotein to ocular diseases. Congenital cataract was reported in 1 out of 14 cases of heredited deficiency of GSH biosynthesis . The evidence for a role of GPx1 in the human eye becomes slightly more persuasive if the GSHregenerating system GPx1 depends on is considered. Glucose6phosphate dehydrogenase (G6PD) deficiency has for long been implicated as a risk factor for cataract formation , but the attempts to statistically verify an association of G6PD deficiency and cataract incidence remained largely disappointing [30, 31]. The results varied between geographical regions and, likely, between different types of G6PD mutations. Nevertheless, a trend towards higher cataract incidence was observed at least in younger or presenile patients [30, 32–34]. Likely the genetic deficiencies in GSH regeneration remain silent as long as the subjects are not challenged by prooxidant agents, as they do in respect to associated hematological disorders. A statistically verified association of cataract incidence and selenium deficiency is equally missing and has not even been reported for countries where selenium deficiency syndromes such as Keshan disease or KashinBeck disease were endemic. At first glance, this surprises in view of the experimental background. It may be revealing, however, that cataract induction in rats required secondgeneration selenium deprivation. This implies that either the selenium deficiency must be extreme and has to be sustained for years to mimic the GPx1 status of GPx1�/� mice or that the lenses of rats and man, respectively, are less prone to oxidative damage. Evidently a critically low selenium status of the eye is hardly achieved even at low, and not at all at suboptimum supply, because the eye is likely as privileged in selenium supply as the nervous tissue in general.
The second ophthalmic disease believed to result from, or to be aggravated by, oxidative damage, is retinitis pigmentosa (RP). The pigmented deposits reminding of lipofuscin are considered to result from cooxidation of unsaturated lipid and proteins. The most convincing clinical support of the oxidative damage hypothesis of RP is the almost consistent association of the disease with untreated genetic deficiency of the �tocopherol transfer protein that leads to a dramatic decrease of vitamin E in nervous tissue, ataxia and mental retardation . The preventive efficacy of megadoses of vitamin E (up to 2g/day)
is commonly attributed to its antioxidant capacity that essentially consists in scavenging peroxyl radicals of lipids. The products of this reaction, hydroperoxides of fatty acids or complex lipids are substrates of GPx’s, in particular of GPx4. This biochemical link is often quoted to explain the synergism of selenium and vitamin E in preventing experimental deficiency syndromes [2, 7]. However, GPx4, which is the isoenzyme specialized for the prevention of lipid peroxidation, ranks high in the hierarchy of selenoproteins, will not markedly decrease, unless selenium deficiency is extreme. The few clinical studies addressing this problem did not suggest any link between RP and selenium deficiency. Unexpectedly, GPx1, which readily declines in selenium deficiency, was found to be elevated in RP [36, 37], a finding that, in principle, corroborates the oxidative stress hypothesis for RP, but rules out the contribution of selenium deficiency as a common etiological factor.
For agerelated macular degeneration (ARMD) finally, the assumption of a radiationtriggered oxidative damage is theoretically most appealing. The invoked oxidative processes, however, are initiated by singlet oxygen, which is more readily quenched by the carotenoids of the macula lutea than by any other nonenzymatic or enzymatic process. Accordingly, the macula lutea protects itself against potential damage by the focused light by accumulation of carotenoids, specifically lutein and zeaxanthin, which are responsible for its typical color . The protective role of other antioxidants or enzymatic mechanism is less clear. A study on 18 ARMD patients showed a lower blood glutathione reductase activity but comparable GPx activity when compared to agematched controls . More recently, a genome scan for ARMDrelated markers identified a region on chromosome 5, where GPx3 is located . Altogether, however, the evidence for an impact of the selenium status to ARMD is weak.
Unfortunately, clinical trials with patient numbers that promise statistical power have preferentially been performed with complex mixtures of minerals and vitamins that are believed to act primarily as antioxidants. Despite the high number of patients involved, e.g., in the Linxian cataract study or the ARED study, beneficial effects, if evident at all, can hardly be attributed to any of the individual components of the supplement mixtures. The kind of study design may be relevant to evaluate the sense of current trends to expect miracles from supranutritional dosages of micronutrients, be they antioxidants or not. The dosedependent switch in mechanism of action, which here has been exemplified for selenium and could easily be extended to other micronutrients, makes the outcome of such studies hard to interpret. The problem whether ocular diseases are aggravated by oxidative stress and which presumed antioxidant might retard disease manifestation could not be solved this way.
Selenium has an established role in ocular physiology. As an integral part of glutathione peroxidase type 1, it prevents oxidative damage and, in consequence, cataract formation in the eye lens of rodents, as is demonstrated by alimentary selenium deprivation and genetic disruption of the GPx1 gene. The roles of other selenoproteins in the eye remain to be established.
Excess selenium that is not incorporated specifically into selenoproteins causes cataracts in postnatal animals presumably via redox cycling.
There is no theoretical basis, nor any experimental evidence for the hope that any selenium supplementation that exceeds the dietary reference intakes of 55 �g/day has a beneficial effect on the eye. Moreover, animal experimentation reveals that only severe and sustained selenium deficiency affects eye physiology. Accordingly, any reliable clinical data revealing a beneficial effect of selenium on ocular diseases are missing.
Taking into account the documented difficulties to induce any pathologically relevant selenium deficiency in the eyes of experimental animals and the known hazards of excess selenium, supranutritional selenium supplementation to prevent agerelated ocular diseases can at present not be recommend.
In view of the established function of selenium in the eye, it nevertheless appears advisable to screen oxidative stressrelated ocular diseases for disturbances of selenium biochemistry that, in exceptional cases, might be a cause or complicating factor of the disease.
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Prof. Dr. Leopold Flohé MOLISA GmbH, Universitätsplatz 2 DE–39106 Magdeburg (Germany) Tel. �49 331 7480950, EMail firstname.lastname@example.org
Carsten H. Meyer, Walter Sekundo
Department of Ophthalmology, Philipps University of Marburg, Marburg, Germany
Age-related cataract (ARC) is the leading cause of blindness in the world, particularly in developing countries. In contrast, cataract surgery has become the most frequent surgical procedure in people aged 65 years or older in the Western world, causing a considerable financial burden to the health care system. The development of cataracts is mainly an age-related phenomenon, although socioeconomic and lifestyle factors appear to influence their development,
e.g. smoking has been found to directly influence ARC. A key role in the pathomechanism of the crystalline lens alteration is played by glucose metabolism and associated effected redox potential, which may induce oxidative damages. Aldose reductase blockers were able to prevent the development of diabetic cataracts in experimental studies, however clinical trials were interrupted due to unclear side effects. Other drugs with radical scavenging properties were effective in in vitro and in vivo experiments, but could not be proven to be efficient and safe in preclinical human trials. A number of epidemiological studies showed an increased risk of nuclear or cortical cataract in people with low blood levels of vitamin E. It is also known that the measured levels of ascorbic acid decline with increasing age in the lens. �-Carotin and other non-polar carotenoids seem to be missing and may therefore only play a minor role. Polarized carotenoid lutein and zeaxanthin are available in low concentrations and may therefore have some direct effects. The results of the present interventional studies are still controversial. While the Linxian studies indicated that the prevalence for nuclear cataract was reduced by the supplementation with retinol/zinc or vitamin C/molybdenum, the AREDS trial showed no effect of the antioxidant formulation on the development or progression of ARC. Again, while the REACT study demonstrated a statistically significant positive treatment effect 2 years after treatment for the US patients and for both subgroups (US � UK) after 3 years, no effect was observed in UK patients alone. In another US study, the Physician Health Study, no positive or negative effect of �-carotin was observed. Taken together, these studies suggest that any effect of antioxidants on cataract development is likely to be very small and probably is of no clinical or public health significance, thus removing a major rationale for ‘anticataract’ vitamin supplementation among health-conscious individuals.
Copyright © 2005 S. Karger AG, Basel
Age-related cataract (ARC) is the leading cause of blindness (40%) in the world [56, 71, 78]. The estimated number of currently 20 million people blinded by cataract will double by the year 2020 [3, 57, 58, 66, 69, 83]. First, this article starts with a survey about the physiology and concentration of vitamins and other substances in the lens. Subsequently, epidemiological studies on nourishment and cataract are discussed. Finally, we present several clinical interventional studies on the use of antioxidative agents and discuss their findings.
The development of cataracts is mainly an age-related phenomenon, although socioeconomic and lifestyle factors may also influence their development. Smoking has been found to directly influence ARC in many cross-sectional [3, 21, 26, 32, 38, 39, 42, 87] and longitudinal studies , while caffeine and drinking alone had no effect on ARC [26, 57]. In addition, both low income and educational level are also related to increased cataract rate as well as morbidity and mortality in general [15, 40, 43]. The causative pathology of these associations with increased cataracts is not clear, but may be influenced by health care, noxious environments, and high-risk behaviors [27, 46, 51, 58, 124]. Associations between cataract and risk of death were recently observed in an African-descent population [40, 70, 80].
Severe cataract is a critical problem especially in developing countries, although it also affects the more developed countries as well. Cataract surgery has become the most frequent surgical procedure in people aged �65 years in the USA, with an estimated annual cost of USD 3.4 billion . Despite the fact that surgical treatment is relatively simple, it is known that dietary contingent aspects may play an important role in the decline of its risk, and therefore prevent or postpone costly cataract surgery. In this regard there is an ongoing discussion on the preventive effects of antioxidative and microsupplementary agents, because many oxidative, especially photooxidative processes are known to be important etiologic factors in cataract formation [10, 62, 107, 120].
The primary function of the transparent crystalline lens is to refract and focus light onto the retina . As the lens does not have its own blood supply, all essential nutrients need to diffuse from the aqueous through the surrounding capsular bag and each cellular membrane into the cell. The entry metabolite transport into the epithelial cells and fibers of the lens is processed by a variety of mechanisms, including active transportation as well as diffusion processes. The maintenance of constant water hydration (approximately 69%) is also energy-consuming and depends to a great extent on metabolic activity. Significant alterations in metabolic processes result in loss of transparency and cataract formation. Most metabolic energy (90%) derives from oxidation of glucose to lactic acid by a process called anaerobic glucolysis. In addition, the lens also contains a rather unusual pathway of glucose known as the sorbitol pathway, which involves the direct utilization of non-phosphorylated sugars .
As the lens ages, there is a progressive increase in the level of albuminoid proteins, which render more of the lens fibers. The structural proteins (�-, �-, and
�-crystallines) constitute most of the dry weight of the lens. During aging the concentration of various lens proteins increases, especially albuminoid fraction �-crystallines are the largest (800 kDa, compared to 20 kDA for the �-crystalline) [59, 60, 81, 82].
The general pathogenesis of human cataracts is believed to be the result of multiple factors acting over many years. Mechanisms of syn- and co-cataractogenesis [16, 26, 33] explain cataract formation due to an accumulation of several cataract risk factors. Syn-cataractogenesis represents the combination of two (or more) damaging factors that lead in combination to lens opacities. In co-cataractogenesis the direct cataractogenic effect of a substance is promoted
when it is in combination with a subliminal factor that, on its own, has no effect. The term agerelated cataract includes three different forms of lens opacity: cortical, nuclear, and posterior subcapsular (PSC) (fig. 1–3). Each of these has its own distinctive pathogenic changes and distinctive risk factors. While ultraviolet light exposure [100, 111, 122] and steroid administration have been linked to the cortical cataract, cigarette smoking has been consistently associated with the nuclear cataract [65, 67, 110]. A variety of biochemical, animal, and human studies suggested that oxidative changes of the lens proteins may cause lenses to become opaque [7, 9, 11, 14, 17, 119, 126].
Early investigations in the 1930s on the pathophysiology of cataract formation led to considerations that the metabolisms can be influenced by medical treatment . H.K. Müller, Chairman at the Department of Ophthalmology at the University of Bonn, pursued a conservative cataract therapy and established the Clinical Institute for Experimental Ophthalmology [85, 86, 88] some 50 years ago. Major investigations during the 1950s to 1970s on lens metabolism, aging processes of the lens proteins and their impact on lens transparency were the key results to consider a therapy for ‘senile cataract’ [3, 44, 45, 50, 89].
A key role in the pathomechanism of the crystalline lens alteration is played by the glucose metabolism and associated effected redox potential, which may induce oxidative damages [48, 89, 93]. Therefore, aging is not the primary cause, but rather a background of the cataractogenesis as some interactions occur during life. Multiple cross-reactions are responsible for the multifactorial cataractogenesis [100, 111, 122]. With increasing light absorption,
Fig. 1. An anterior cortical cataract is seen as a diffuse whitish opacity virtually under the anterior lens capsule. It can be located centrally (as in this photograph), close to the equator of the lens or ‘spread’ from the periphery toward the optical center.
Fig. 2. a A typical nuclear cataract (2� nuclear sclerosis) is characterized by a yellowish appearance of the nucleus when viewed at the slitlamp. b In advanced stages of nuclear sclerosis the lens turns brown: an entity known as ‘brunescent cataract’.
the crystalline lens builds additional disulfide bridges and glycosylations at the protein level exaggerating to large biochemical complexes and networks. It is noteworthy to stress that diabetes may also trigger certain cataractogenic interactions. Two additional mechanisms are also briefly mentioned, namely
(a) the damage of epithelial cells before or during their differentiation may induce a false differentiation or persisting cellular damage, leading to PSC cataract, and (b) the entire metabolism of the lens may be disturbed by toxic events. All three conditions mentioned cause oxidative damages and persistent impairment. The above-named results led to a variety of experimental and
Fig. 3. A PSC cataract usually presents as a large white plaque behind the nucleus and is best appreciated using retroillumination.
therapeutical attempts and trials on cataract prophylaxis, which can be divided essentially into two groups.
Group A: Substances and substance mixtures aimed at slowing down the aging process of the lens through an optimization of the lenses provision.
These preparations mainly target the diabetic metabolism (type 1 diabetes mellitus), its effect on the activity of aldose reductase and the resulting accumulation of sorbitol. Efficient blockers of aldose reductase were developed in the 1960s and 1970s .
Aldose reductase blockers such as Sorbinil® (Pfizer) were able to prevent the development of diabetic cataracts in experimental studies. Although systemically or locally given, they could prevent cataract in animal experiments ; clinical trials using Sorbinil® were interrupted due to unclear side effects. In clinical trials other products demonstrated an extreme long persistence in the lens, therefore no suitable dosage could be found and the clinical trials were stopped. Pyruvate was also used to prevent oxidative damage of the crystalline lens . Although promising attempts to stimulate the metabolism of glutathione were published in experimental studies [9, 18, 23], there are no clinical studies investigating initial drugs (prodrug) products.
Finally, Tempol® was developed to protect lens epithelial cells from H2O2 radical damages