Validation of markers for their relevance to health and disease.

Validation of markers requires two different steps. The first one is the analytical validation including analytical procedures, definition of standard reference, and round-tests analysis. The second one is the assessment of the biological relevance of the selected marker. This biological relevance may be in the field of physiology to measure a physiological function, or in the area of pathology to measure a disease. Validation of markers is a time-consuming task that needs cooperation between laboratories. On the other hand a validated marker is a keystone for scientists to share their results and to explore more efficiently a given biological system.

Introduction: markers of oxidative damage and antioxidant modulation.

The question of the function of antioxidants in human health can only be resolved when validated markers of oxidative damage are available. Markers allow determination of the effect of antioxidants on damage to DNA, proteins, and lipids. The final goal is the execution of human intervention studies based on markers that are clearly linked to physiological function. These data will be critical in determining the effectiveness of nutrients in promoting health and wellbeing and reducing disease risk.

Do iron and vitamin C co-supplementation influence platelet function or LDL oxidizability in healthy volunteers?

OBJECTIVE: To examine the effect of co-supplementation with iron and vitamin C on antioxidant status, platelet function and low density lipoprotein oxidation in normal healthy volunteers. DESIGN: The study was carried out with two groups of 20 subjects each acting as their own control, comparing presupplemention with postsupplemention. One group was supplemented with iron and the RDA level of vitamin C and the second group with iron and 260 mg/d vitamin C. SETTING: The International Antioxidant Research Centre, The Guy's, King's College and St Thomas's School of Biomedical Science, Guy's Campus, London. SUBJECTS: Forty normal healthy volunteers, recruited from the staff of the Medical School and Hospital in which two volunteers withdrew during the study. INTERVENTIONS: Subjects in both studies were randomly assigned to one of two groups (5 males and 5 females group) and received supplements containing iron (14 mg/d) and either 60 mg/d (Group A) or 260 mg/d (Group B) vitamin C for 12 wk. Blood samples were taken at 6 wk and 12 wk, and prior to supplementation and analysed for iron and antioxidant status (transferrin bound iron, vitamin C and E, and beta-carotene levels) in both studies. Samples from the first study were analysed for the susceptibility of LDL isolated from plasma to Cu2+-induced oxidation and samples from the second for platelet function. RESULTS: Transferrin-bound iron was significantly increased (P < 0.05) at 12 wk, in Group A subjects (from 14.9+/-5.3 micromol/1 to 19.5+/-2.3 micromol/l; mean+/-s.d.; n=19), whereas those in Group B showed a significant increase (P < 0.05) after 6 wk (from 15.8+/-4.5 micromol/l to 20.4+/-6.6 micromol/l; n = 19) which decreased at 12 wk (16.3+/-5.0 micromol/l). Plasma total ascorbate significantly increased from an initial level of 59.3+/-21.3 micromol/l to 87.6+/-29.0 micromol/l after 6 wk and 81.7+/-11.4 micromol/l after 12 wk following the Group B supplementation, but only after 12 wk in Group A (from 64.0+/-24.8 micromol/l to 77.2+/-13.2 micromol/l). Plasma alpha-tocopherol concentrations were significantly increased after 6 wk and 12 wk with both levels of supplementation (from 24.2+/-5.71 micromol/l Group A and 23.4+/-5.3 micromol/l Group B to 26.3+/-5.5 micromol/l and 25.71+/-4.7 micromol/1 respectively at 12wk). The mean lag phase to oxidation of low density lipoprotein (LDL) was significantly increased in subjects in Group B after 12 wk ingestion of iron and 260 mg vitamin C (from 80.0+/-14.8 min to 97.2+/-16.9 min; n = 9). Platelet sensitivity to ADP-induced aggregation was significantly decreased (P < 0.05) by 12 wk in Group A (from EC50 2.3 < or = 1.3 microM to 3.7+/-2.2 microM; n = 10), whereas those receiving higher vitamin C showed a significant decrease (P < 0.05; from EC50 1.9+/-0.6 microM to 3.1+/-1.8 microM) after 6wk which subsequently increased towards presupplemental levels (2.6+/-1.6 microM). Platelets from the latter subjects showed a significant reduction in ADP-induced ATP secretion at both 6wk and 12 wk. CONCLUSION: The results show modest beneficial effects on LDL oxidation and platelet function following supplementation with iron and vitamin C. No evidence for pro-oxidant effects was observed.

Functional food science and defence against reactive oxidative species.

This paper assesses critically the science base that underpins the argument that oxidative damage is a significant causative factor in the development of human diseases and that antioxidants are capable of preventing or ameliorating these disease processes. The assessment has been carried out under a number of headings, and some recommendations for future research are made based on the present day knowledge base. The knowledge database (1) Consideration of the basic science that underlies understanding of the role of free radicals in causing cellular pathologies, and the role of antioxidants in preventing this, shows that an imbalance of reactive oxygen species and antioxidant defence systems may lead to chemical modifications of biologically relevant macromolecules. This imbalance provides a logical pathobiochemical mechanism for the initiation and development of several disease states. Experimental data obtained in vivo provide evidence that antioxidants function in systems that scavenge reactive oxygen species and that these are relevant to what occurs in vivo. The relevance in vivo of these observations depends inter alia on knowledge of the uptake and distribution of the antioxidant within the human body, and on what tissue levels of the antioxidant may be expected in relation to dietary levels. (2) There is some way to go until validated precise methods are available for measuring biomarkers of oxidative damage in human subjects in vivo under minimally invasive conditions. With respect to oxidative damage in DNa, HPLC and GC-mass spectrophotometry methods have both merits and limitations. Lipid oxidation products in plasma are best measured as isoprostanes or as lipid hydroperoxides using specific HPLC techniques. Development of isoprostane measurement will advance specificity and precision. The measurement of oxidative damage to proteins has some potential but such methods have not been effectively exploited. (3) Epidemiological studies support the hypothesis that the major antioxidant nutrients vitamin E and vitamin C, and beta-carotene (which may or may not be acting as an antioxidant in vivo), may play a beneficial role in prevention of several chronic disorders. More research is needed on the impact of other non-nutrient compounds, such as other carotenoids and flavonoids, on human health. In general, human intervention studies using hard end-points are the gold standard. Trials are restricted mainly to the major antioxidants and do not allow firm conclusions because of inconsistent findings, an insufficient number of studies and the use of varying doses. There is evidence that large doses of beta-carotene may be deleterious to the health of certain subgroups of the population such as heavy habitual smokers. (4) With respect to the safety of administration of supplementary vitamins, vitamin C is safe at levels of supplementation up to 600 mg/d, and higher levels, up to 2000 mg/d, are without risk. Vitamin E has a very low human toxicity and an intake of 1000 mg/d is without risk; 3200 mg/d has been shown to be without any consistent risk. Large intakes of beta-carotene must be viewed with caution because they have been shown to confer detriment to a population at high risk of lung cancer when administered after many years of high risk (smoking) behaviour. Until further work clarifies the situation in heavy smokers with respect to taking supplements, larger doses should be avoided by such individuals. There is little reliable information about the human toxicology of flavonoids and related non-nutrient antioxidant constituents of the diet. (5) The food industry has long experience in the control of oxidative damage in foods and this experience can be used to advantage for the protection of food antioxidants which are beneficial. Some of these, such as vitamins C and E and beta-carotene, are well known, and strategies for their protection in foods are already exploited by food technologies. (ABSTRACT TRUNCATED)

The effects of iron and vitamin C co-supplementation on oxidative damage to DNA in healthy volunteers.

The effects of co-supplementing healthy volunteers with iron (14 mg/day ferrous sulphate) and vitamin C (either 60 mg/day or 260 mg/day as ascorbic acid) on levels of oxidative DNA damage in white blood cells were studied. The subjects were divided into two groups: one group of 20 volunteers with a higher mean initial level of plasma vitamin C (71.9 +/- 14.0 mumol/l) and a second group of 18 volunteers with a lower mean level (50.4 +/- 25.8 mumol/l). In the first group there was a significant rise in several oxidative DNA base damage products and in total oxidative DNA damage in DNA extracted from white blood cells, but not in 8-hydroxyguanine, after 6 weeks of supplementation. However, after 12 weeks levels returned approximately to normal. In the group with the lower initial level of plasma ascorbate, presupplemental levels of oxidative DNA damage were higher and decreased on supplementation with iron and ascorbate. Since oxidative DNA damage has been suggested as a risk factor for the development of cancer, the implications of increased levels in well-nourished subjects after iron/ascorbate supplementation are disturbing in view of the frequent use of dietary supplements containing both iron salts and ascorbate.

Defense against reactive oxygen species.

As part of the European Commission Concerted Action on Functional Food which was managed by the International Life Sciences Institute (Europe) a series of Theme Papers was produced which examined the 'state of the art' with respect to the subject matter and made recommendations for research. This paper is a summary of the paper concerned with Defence Against Reactive Oxygen species. Having reviewed the scientific literature the authors concluded that certain stringent criteria, which they identified, would need to be satisfied in order to be able to conclude that free radical events are involved in certain human diseases, and that antioxidants are capable of modulating these events and thus reducing the risk of disease. Although there is some evidence that would lead to this conclusion the authors demonstrated that there is at present insufficient evidence available on which to base a firm conclusion that antioxidants are capable of reducing risk of disease, and very little evidence that addresses the important question as to how much of the nutrients concerned are required in the diet to achieve the objective of reducing risk. Research priorities address the need in particular for the development and validation of cellular markers of oxidative damage which are required before there can be new human studies that address the question. There is also a need for more information as to the pharmacokinetics of uptake from diet, distribution and cellular concentration of the antioxidants.

Reference values for alpha-tocopherol and beta-carotene in the Whitehall II Study.

Plasma alpha-tocopherol, beta-carotene, serum lipids and their derived ratios were determined in British Civil Servants (n = 7177) at the second medical examination of the Whitehall II Study, a longitudinal study of cardiovascular disease. For plasma alpha-tocopherol the non-parametric 95% reference interval (90% confidence limits) for the total population was: 11.1 (10.9-11.3)-51.5 (50.6-52.7) mumol/l. For plasma beta-carotene the non-parametric reference interval for the total population was: 0.05 (0.05-0.05)-2.14 (2.08-2.21) mumol/l. The latter interval was wider than those previously published with a higher mean (0.61 mumol/l) and median (0.75 mumol/l). Plasma beta-carotene concentrations were higher in women than men with age-adjusted means of 0.70 and 0.57 mumol/l respectively (p < 0.001). This may reflect differences in diet, lifestyle and metabolism between the sexes. The alpha-tocopherol/cholesterol ratio, as in other surveys, did not vary with age. Among men, current- and ex-smokers had a higher alpha-tocopherol/cholesterol ratio than never-smokers with age-adjusted means of 4.18, 4.19 mumol/mmol and 4.05 mumol/mmol respectively. This difference is as yet unexplained. Follow-up of these subjects will help to clarify the role of antioxidant nutrients as protective factors for cardiovascular disease and cancer.

Effects of co-supplementation of iron with ascorbic acid on antioxidant--pro-oxidant balance in the guinea pig.

The relationship between intake of iron with ascorbic acid and their uptake into the plasma and liver of guinea pigs was studied. The influence on the antioxidant/pro-oxidant balance of liver microsomes was also determined. Animals were fed a standard pelleted diet low in iron and ascorbic acid for 35 days. The pellet diet was supplemented by oral dosing with a solution containing either maintenance dietary levels of ascorbic acid and iron, or one of three regimens that increased the dosage of these substances ten fold. There were no significant differences in animal growth rate or food intake between these regimens. Liver and plasma total ascorbate levels were significantly increased (p < 0.05) in animals receiving either ascorbic acid alone (liver 126 +/- 36 micrograms/g tissue wet wt. and plasma 51.7 +/- 17.0 microM; n = 9) or ascorbic acid and iron (105 +/- 18 micrograms/g and 40.3 +/- 15.3.0 microM; n = 8) compared to controls (84 +/- 36 micrograms/g and 15.3 +/- 8.5 microM; n = 11). Total iron levels in the liver (76.7 +/- 7.3 micrograms/g; control; n = 6) and plasma (2.4 +/- 0.03 mg/l; control) were not significantly raised in animals under these conditions of iron or ascorbate intake. Liver microsomes isolated from animals receiving iron had a greater susceptibility to oxidative stress in terms of malondialdehyde production during auto-oxidation compared to those from control animals under the same conditions. This effect was eliminated on combining ascorbic acid with the iron supplementation, suggesting that oral administration of vitamin C has a protective rather than a pro-oxidant effect under these circumstances.

Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease?

Recent research about the role of free radical derivatives of oxygen and nitrogen in biological systems has highlighted the possibility that antioxidants, such as vitamin E, that prevent these processes in vitro may be capable of carrying out a similar function in living organisms in vivo. There is increasing evidence that free radical reactions are involved in the early stages, or sometimes later on, in the development of human diseases, and it is therefore of particular interest to inquire whether vitamin E and other antioxidants, which are found in the human diets, may be capable of lowering the incidence of these diseases. Put simply, the proposition is that by improving human diets by increasing the quantity in them of antioxidants, it might be possible to reduce the incidence of a number of degenerative diseases. Of particular significance to these considerations is the likely role of the primary fat-soluble dietary antioxidant vitamin E in the prevention of degenerative diseases such as arteriosclerosis, which is frequently the cause of consequent heart attacks or stroke, and prevention of certain forms of cancer, as well as several other diseases. Substantial evidence for this proposition now exists, and this review is an attempt to give a brief account of the present position. Two kinds of evidence exist; on the one hand there is very substantial basic science evidence which indicates an involvement of free radical events, and a preventive role for vitamin E, in the development of human disease processes. On the other hand, there is also a large body of human epidemiological evidence which suggests that incidence of these diseases is lowered in populations having a high level of antioxidants, such as vitamin E, in their diet, or who have taken steps to enhance their level of intake of the vitamin by taking dietary supplements. There is also some evidence which suggests that intervention with dietary supplements of vitamin E can result in a lowered risk of disease, in particular of cardiovascular disease, which is a major killer disease among the developed nations of the world. The intense interest in this subject recently has as its objective the possibility that, by making some simple alterations to dietary lifestyle, or by enhancing the intake of vitamin E by fortification of foods, or by dietary supplements, it may be possible to reduce substantially the risk of a large amount of common, highly disabling human disease. By this simple means, therefore it may be possible to improve substantially the quality of human life, in particular for people of advancing years.

Selenium status in pregnant women of a rural population (Zaire) in relationship to iodine deficiency.

Endemic myxoedematous cretinism has been associated with combined selenium and iodine deficiency in several areas of Zaire. To determine selenium and iodine status across the country, serum selenium and thyroid function parameters including urinary iodide were determined at prenatal clinics in 30 health centres of rural villages distributed over the whole country. Only in Bas-Zaire was the mean serum selenium level similar to that in non-deficient areas (80-120 ng/ml); in the regions of Bandunda and Kasai levels were marginally decreased (55-80 ng/ml), and in Kivu, Haut-Zaire, Equateur and Shaba they were marginally or moderately decreased (< 55 ng/ml). The frequency of abnormally low urinary iodide (< 5 micrograms/dl) varied from 20% in the region of Bas-Zaire to 50% in Kasai (P < 0.001), and to still higher percentages in the 5 other regions of Zaire (Bandundu, 57%; Kivu, 63%; Equateur, 72%; Shaba, 76%; Haut-Zaire, 84%). With the exception of Bas-Zaire, biochemical maternal hypothyroidism (serum TSH > 5mU/l) was present in every region, with a frequency ranging from 3% in Kivu to 12% in Equateur. Iodine deficiency affects most of the Zairean population and requires public health measures on a larger scale than previously estimated. Combined iodine and selenium deficiency affects Equateur, Haut-Zaire and Kivu, where endemic myxoedematous cretinism occurs, but also Shaba, where it was not previously described. Besides combined iodine and selenium deficiency which is permissive, another factor (thiocyanate?) must be taken into account to explain the peculiarly elevated prevalence of endemic myxoedematous cretinism in Central Africa.

Will the 'good fairies' please prove to us that vitamin E lessens human degenerative disease?

Recent research about the role of free radical derivatives of oxygen and nitrogen in biological systems has highlighted the possibility that antioxidants, such as vitamin E, that prevent these processes in vitro may be capable of carrying out a similar function in living organisms in vivo. There is increasing evidence that free radical reactions are involved in the early stages, or sometimes later on, in the development of human diseases, and it is therefore of particular interest to inquire whether vitamin E and other antioxidants, which are found in the human diets, may be capable of lowering the incidence of these diseases. Put simply, the proposition is that by improving human diets by increasing the quantity in them of antioxidants, it might be possible to reduce the incidence of a number of degenerative diseases. Of particular significance to these considerations is the likely role of the primary fat-soluble dietary antioxidant vitamin E in the prevention of degenerative diseases such as arteriosclerosis, which is frequently the cause of consequent heart attacks or stroke, and prevention of certain forms of cancer, as well as several other diseases. Substantial evidence for this proposition now exists, and this review is an attempt to give a brief account of the present position. Two kinds of evidence exist; on the one hand there is very substantial basic science evidence which indicates an involvement of free radical events, and a preventive role for vitamin E, in the development of human disease processes. On the other hand, there is also a large body of human epidemiological evidence which suggests that incidence of these diseases is lowered in populations having a high level of antioxidants, such as vitamin E, in their diet, or who have taken steps to enhance their level of intake of the vitamin by taking dietary supplements. There is also some evidence which suggests that intervention with dietary supplements of vitamin E can result in a lowered risk of disease, in particular of cardiovascular disease, which is a major killer disease among the developed nations of the world. The intense interest in this subject recently has as its objective the possibility that, by making some simple alterations to dietary lifestyle, or by enhancing the intake of vitamin E by fortification of foods, or by dietary supplements, it may be possible to reduce substantially the risk of a large amount of common, highly disabling human disease. By this simple means, therefore it may be possible to improve substantially the quality of human life, in particular for people of advancing years.

Practical approaches to low density lipoprotein oxidation: whys, wherefores and pitfalls.

The purpose of this review is to bring together the different approaches for studying the oxidation of low density lipoproteins and try to identify some critical factors which will permit greater comparability between laboratories. These issues are discussed both in terms of the variety of exogenous mediators of oxidation applied (transition metal ions, haem proteins, azo initiators, peroxynitrite, cells etc.) and their raisons d'être, as well as the methodologies (formation of conjugated dienes, hydroperoxides, decomposition products of lipid peroxidation, altered surface charge, macrophage uptake) applicable to the different stages of the oxidation and the factors underlying their accurate execution and interpretation.

The effects of ascorbic acid and iron co-supplementation on the proliferation of 3T3 fibroblasts.

Exposure of 3T3 fibroblasts to FeII reveals a concentration-dependent inhibition of cell proliferation compared to control cells, the apparent threshold for this iron-mediated effect being 5 microM FeII. The inhibition of cell proliferation was accompanied by an enhancement of total malondialdehyde (MDA) levels (as detected directly by hplc) in the cells at higher iron concentrations. The co-supplementation of FeII with varying concentrations of ascorbic acid over the range 5 microM to 240 microM had no significant effect on the threshold for iron toxicity or lipid peroxidation. These results show that there is neither a significant exacerbation of the pro-oxidant effect of FeII nor any protective effect of ascorbate when cultures of 3T3 mouse fibroblasts are exposed to co-supplementation regimes of iron with ascorbic acid.

Safety of antioxidant vitamins and beta-carotene.

Epidemiologic evidence links high antioxidant status with low risk of degenerative disease. Optimal intakes of antioxidants may not be achievable by diet alone; supplements may be taken, particularly in subgroups of the population at high risk. It is thus necessary to ensure that antioxidant supplements are safe and free from side effects. The toxicity of vitamin E is low; no mutagenic, teratogenic, or carcinogenic effects are known and in double-blind studies in which large amounts of vitamin E were used in humans, no side effects occurred. High concentrations are contraindicated in subjects with vitamin K-associated blood coagulation disorders, and the toxicity in normal subjects ingesting large amounts of vitamin E over long periods requires additional investigation. Toxicity of beta-carotene also is low. Evidence from human toxicity trials is not available but there is much circumstantial evidence that 15-50 mg/d is without side effects except for hypercarotenemia in some subjects at high intakes. The findings of more lung cancer in subjects who smoked and who were given 20 mg beta-carotene/d than in those given a placebo could be influenced by the cancer being well advanced before beta-carotene administration. Massive anecdotal evidence exists that vitamin C (at > or = 1 g/d) is safe. Exhaustive literature searches have failed to reveal a controlled study of vitamin C toxicity in human subjects. Anxiety exists about oxalate stone formation, uricosuria, vitamin B-12 destruction, mutagenicity, and iron overload, but the consensus is that adverse effects do not occur in healthy subjects ingesting large amounts of vitamin C.

Levels of antioxidant nutrients in plasma and low density lipoproteins: a human volunteer supplementation study.

A human supplementation study was undertaken in order to investigate the correlation between the intake of individual daily dosages of vitamin E (300 mg), vitamin C (250 mg), or beta-carotene (15 mg) of eight week duration and their uptake in vivo in plasma and LDL. The effects of a combined supplement of vitamin E, vitamin C and beta-carotene (Redoxon protector-75 mg, 150 mg, 15 mg respectively) were also investigated. The results show that on supplementation with the individual antioxidants the increases in plasma alpha-tocopherol:cholesterol levels lie in the 1.5-2 fold range and the beta-carotene:cholesterol ratios give a mean 3.5 fold enhancement. The combined supplement containing the same level of beta-carotene as the single dosage achieved comparative levels of uptake in plasma. The level of plasma vitamin C appears to be maximal at about 100 microM regardless of the pre-supplementation level.

How 'safe' are antioxidant vitamins?

Interest in a putative disease-preventive role for the so-called antioxidant nutrients derives from a large body of evidence suggesting that oxidative damage is a contributing cause of many life-shortening diseases. Since their use is an otherwise healthy population, it is important that such agents be virtually free of toxicity. The agents of most interest are alpha-tocopherol (vitamin E), ascorbic acid (vitamin C) and beta-carotene. When used for disease prevention, the doses given are several-fold greater the Recommended Dietary Allowance (RDA), the latter being based on amounts necessary for the prevention of classic deficiency conditions recognised decades ago. alpha-Tocopherol, ascorbic acid and beta-carotene are remarkably well tolerated and free from toxicity. Consequently, they are well suited for testing as preventive agents, since their use does not require any toxicity monitoring except in unusual circumstances. An example of the latter would be in patients who are vitamin K deficient, perhaps through anticoagulation with drugs such as warfarin, in which case use of high doses of alpha-tocopherol may increase the bleeding tendency.

Is there a significant role for lipid peroxidation in the causation of malignancy and for antioxidants in cancer prevention?

alpha-Tocopherol (alpha-T) uptake and its relationship to cell proliferation and lipid peroxidation was studied in a baby hamster kidney cell line (BHK-21/C13) and its polyoma virus-transformed malignant counterpart (BHK-21/PyY cells). The principal findings were as follows. (a) The level of lipid peroxidation judged by malondialdehyde (MDA) measurement by HPLC, was higher in the transformed cells than in the nontransformed cells. Oxidative stress by 374 mM Fe3+/10 mM ADP caused a significant increase in the level of MDA of a similar magnitude in both cell types. Addition of 7, 14, and 21 mM alpha-T caused no diminution of the MDA level in the unstressed cells and abolished the increase in MDA seen in the stressed cells. (b) The endogenous level of alpha-T in the transformed cells was lower than in the nontransformed cells and all of the measurable alpha-T in these cells was destroyed by the oxidative stress. Supplementation of the cells with alpha-T caused an increase in the level of alpha-T that was proportional to the level of inclusion of alpha-T in the medium. (c) Growth was stimulated by 7 and 14 mM alpha-T but not by the higher levels of inclusion in the medium. The growth stimulation was much larger in the transformed cells (163% of growth in the unsupplemented medium) than in the nontransformed cells (120%). (d) These results demonstrate that, in this cell system, the growth-stimulating ability of alpha-T is unrelated to the ability of alpha-T to control lipid peroxidation and that the level of peroxidation is increased in the malignant state. The difference between the findings reported here and earlier work showing increased levels of alpha-T and decreased levels of peroxidation in transformed malignant cells is discussed and possible explanations for it are advanced.