Iris F. F. Benzie

FERRIC REDUCING/ANTIOXIDANT POWER ASSAY: DIRECT MEASURE OF TOTAL ANTIOXIDANT ACTIVITY OF BIOLOGICAL FLUIDS AND MODIFIED VERSION FOR SIMULTANEOUS MEASUREMENT OF TOTAL ANTIOXIDANT POWER AND ASCORBIC ACID CONCENTRATION

Publisher Summary This chapter discusses ferric reducing/antioxidant power (FRAP) assay. The ferric reducing/antioxidant power (FRAP) assay is a recently developed, direct test of “total antioxidant power.” The FRAP assay is robust, sensitive, simple, and speedy and facilitates experimental and clinical studies investigating the relationship among antioxidant status, dietary habits, and risk of disease. Measurement of the total antioxidant power of fresh biological fluids—such as blood plasma—can be measured directly; the antioxidant content of various dietary agents can be measured objectively and reproducibly and their potential for improving the antioxidant status of the body investigated and compared. The FRAP assay is also sensitive and analytically precise enough to be used in assessing the bioavailability of antioxidants in dietary agents to help monitor longitudinal changes in antioxidant status associated with an increased intake of dietary antioxidants and to investigate the effects of disease on antioxidant status.

Lipid peroxidation: a review of causes, consequences, measurement and dietary influences

In this review the process of lipid peroxidation and the atherogenicity of peroxidied lipids are discussed. Recent findings with regard to the effect of selected dietary factors on susceptibility of lipids to oxidative stress and on antioxidant defences are analysed with particular reference to their potential use in the prevention and treatment of atherogenesis and, by extension, coronary heart disease. Laboratory methods of assessing antioxidant defences, lipid peroxidation and the effects of lipid peroxidation are also reviewed and discussed with particular reference to their ability to assess in vivo oxidative stress and lipid peroxidation status. A range of oxidative stress indices are presented and their limitations discussed, but the main focus is on the most commonly used laboratory test for lipid peroxidation, the thiobarbituric acid reacting substances (TBARS) test. Finally, the influence of selected dietary factors on measured peroxidation status is discussed, with particular reference to the antioxidant vitamins C (ascorbic acid) and E (alpha tocopherol) and the type of fatty acids (mono- and poly-unsaturated) in the diet.

Evolution of dietary antioxidants

Oxygen is vital for most organisms but, paradoxically, damages key biological sites. Oxygenic threat is met by antioxidants that evolved in parallel with our oxygenic atmosphere. Plants employ antioxidants to defend their structures against reactive oxygen species (ROS; oxidants) produced during photosynthesis. The human body is exposed to these same oxidants, and we have also evolved an effective antioxidant system. However, this is not infallible. ROS breach defences, oxidative damage ensues, accumulates with age, and causes a variety of pathological changes. Plant-based, antioxidant-rich foods traditionally formed the major part of the human diet, and plant-based dietary antioxidants are hypothesized to have an important role in maintaining human health. This hypothesis is logical in evolutionary terms, especially when we consider the relatively hypoxic environment in which humans may have evolved. In this paper, the human diet is discussed briefly in terms of its evolutionary development, different strategies of antioxidant defence are outlined, and evolution of dietary antioxidants is discussed from the perspectives of plant need and our current dietary requirements. Finally, possibilities in regard to dietary antioxidants, evolution, and human health are presented, and an evolutionary cost-benefit analysis is presented in relation to why we lost the ability to make ascorbic acid (vitamin C) although we retained an absolute requirement for it.

Consumption of green tea causes rapid increase in plasma antioxidant power in humans

Green tea contains polyphenolic antioxidants that have shown anticarcinogenic properties in animal and in vitro experimental studies. Current data regarding absorption and bioavailability of tea antioxidants in humans, however, are conflicting. In this study, plasma and urine antioxidant power after ingestion of green tea was measured using the ferric reducing/antioxidant power (FRAP) assay (US patent pending) to assess absorption, systemic distribution, and renal excretion of green tea antioxidants in healthy adults. Results showed that absorption of green tea antioxidants was rapid, with peak increase in plasma FRAP of around 4% at 40 minutes after ingestion: mean increase was 44 +/- 9 (SE) mumol/l. Excretion of polyphenolic antioxidants was also fast, peaking at 60-90 minutes, with significant correlation between urinary FRAP values and urinary total phenolic concentrations (r = 0.845, p < 0.001). In control studies, no increase in plasma or urine FRAP values was seen after intake of water. Although the amount of antioxidants absorbed was relatively small and the increase in plasma antioxidant power was of short duration, results demonstrate that some potentially anticarcinogenic polyphenolic antioxidants in green tea enter the systemic circulation soon after ingestion and cause a significant increase in plasma antioxidant status. This increase may, in turn, lower oxidative damage to DNA and so decrease risk of cancer.

Interactions between vitamins C and E in human subjects.

Despite convincing in vitro evidence, a vitamin C-E interaction has not been confirmed in vivo. This study was designed to examine the effects of supplementation with either vitamin C or E on their respective plasma concentrations, other antioxidants, lipids and some haemostatic variables. Fasting blood was collected before and after intervention from thirty healthy adults in a double-blinded crossover study. Baselines for measured variables were established after 2 weeks of placebo supplementation, followed by daily supplementation with 73.5 mg RRR-alpha-tocopherol acetate or 500 mg ascorbic acid, and placebo, for 6 weeks. A 2 month washout preceded supplement crossover. Mean values showed that plasma lipid standardised alpha-tocopherol increased with ascorbic acid supplementation: from 4.09 (sem 0.51) to 4.53 (sem 0.66) micromol/mmol total cholesterol plus triacylglycerol (P < 0.05), and plasma ascorbic acid increased from 62.8 (sem 14.9) to 101.3 (sem 22. 2) micromol/l (P < 0.005). Supplementation with (RRR)-alpha-tocopherol acetate increased plasma alpha-tocopherol from 26.8 (sem 3.9) to 32.2 (sem 3.8) micromol/l (P < 0.05), and lipid-standardised alpha-tocopherol from 4.12 (sem 0.48) to 5.38 (sem 0.52) micromol/mmol (P < 0.001). Mean plasma ascorbic acid also increased with vitamin E supplementation, from 64.4 (sem 13.3) to 76. 4 (sem 18.4) micromol/l (P < 0.05). Plasma ferric reducing (antioxidant) power and glutathione peroxidase (U/g haemoglobin) increased in both groups, while urate, total cholesterol and triacylglycerol levels decreased (P < 0.05 throughout). Results are supportive of an in vivo interaction between vitamins C and E.

Acute hyperglycemia and oxidative stress: direct cause and effect?

Oxidative stress is increased in Type 2 diabetes and this appears to underlie the development of diabetic complications. Increased oxidative stress is claimed to be triggered directly by acute (sudden-onset) hyperglycemia, but published data do not clearly support a direct cause and effect relationship. In this article, published evidence of a direct prooxidant effect of acute hyperglycemia is presented and discussed in some detail, and conflicts, controversies, and problems are highlighted. Evidence for glucose variability as a possible important trigger of oxidative stress in diabetes is reviewed, with some speculation as to how the field would be advanced if there were more widespread recognition about the role that wide fluctuations in glucose concentration play in diabetic complications. Possible direct or indirect antioxidative effects of various drugs used in the treatment of diabetic subjects are discussed because these may have influenced current understanding of the link between hyperglycemia and oxidative stress. The aims are to reveal the divergence between the available evidence and the accepted view that acute hyperglycemia is a direct trigger of oxidative stress and to suggest areas of research that will help resolve current controversies in this important and challenging area.

Ganoderma lucidum (‘Lingzhi’), a Chinese medicinal mushroom : biomarker responses in a controlled human supplementation study

Lingzhi (Ganoderma lucidum) is a woody mushroom highly regarded in traditional medicine and is widely consumed in the belief that it promotes health and longevity, lowers the risk of cancer and heart disease and boosts the immune system. However, objective scientific validation of the putative health benefits of Lingzhi in human subjects is lacking, and issues of possible toxicity must be addressed. The present double-blinded, placebo-controlled, cross-over intervention study investigated the effects of 4 weeks Lingzhi supplementation on a range of biomarkers for antioxidant status, CHD risk, DNA damage, immune status, and inflammation, as well as markers of liver and renal toxicity. It was performed as a follow-up to a study that showed that antioxidant power in plasma increased after Lingzhi ingestion, and that 10 d supplementation was associated with a trend towards an improved CHD biomarker profile. In the present study, fasting blood and urine from healthy, consenting adults (n 18; aged 22-52 years) was collected before and after 4 weeks supplementation with a commercially available encapsulated Lingzhi preparation (1.44 g Lingzhi/d; equivalent to 13.2 g fresh mushroom/d) or placebo. No significant change in any of the variables was found, although a slight trend toward lower lipids was again seen, and antioxidant capacity in urine increased. The results showed no evidence of liver, renal or DNA toxicity with Lingzhi intake, and this is reassuring. The present study of the effects in healthy, well-nourished subjects provides useful, new scientific data that will support controlled intervention trials using at-risk subjects in order to assess the therapeutic effect of Lingzhi in the promotion of healthy ageing.

The comet assay as a tool for human biomonitoring studies: The ComNet Project

The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. Studies often involve small numbers of subjects, and design may be sub-optimal in other respects. In addition, comet assay protocols in use in different laboratories vary significantly. In spite of these difficulties, it is appropriate to carry out a pooled analysis of all available comet assay biomonitoring data, in order to establish baseline parameters of DNA damage, and to investigate associations between comet assay measurements and factors such as sex, age, smoking status, nutrition, lifestyle, etc. With this as its major objective, the ComNet project has recruited almost 100 research groups willing to share datasets. Here we provide a background to this project, discussing the history of the comet assay and practical issues that can critically affect its performance. We survey its diverse applications in biomonitoring studies, including environmental and occupational exposure to genotoxic agents, genoprotection by dietary and other factors, DNA damage associated with various diseases, and intrinsic factors that affect DNA damage levels in humans. We examine in depth the quality of data from a random selection of studies, from an epidemiological and statistical point of view.

Effects of Dietary Antioxidants on Human DNA Ex Vivo

The protective effect of fruits and vegetables against cancer is well established. It is believed that this effect is mediated by antioxidants and decreased oxidative damage to DNA. However, the identity of the antioxidant(s) responsible is not clear. Moreover, a potentially damaging pro-oxidant effect of some antioxidants has been reported. In this study the ex vivo effects of several dietary antioxidants, including quercetin, various catechins, ascorbic acid and f -tocopherol, were investigated, at concentrations up to 200 u w M, using the single cell gel electrophoresis (comet) assay for DNA damage. Lymphocytes from three healthy subjects were pre-incubated with these antioxidants, and the comet assay was performed on treated, untreated, challenged and unchallenged cells in parallel, oxidant challenge being induced by 5 u min exposure to hydrogen peroxide (final concentrations H 2 O 2 : 30, 45, or 60 u w M). Results using this ex vivo cellular assay showed protection by some antioxidants (quercetin, caffeic acid), no effect by some (catechin, epicatechin, catechin gallate, epicatechin gallate) and an apparently damaging effect by others (epigallocatechin, epigallocatechin gallate). Damage may have been caused by production of H 2 O 2 from these polyphenolics. Neither ascorbic acid nor f -tocopherol protected or damaged DNA. Further study of the role of quercetin and caffeic acid in DNA protection is needed.

Plasma ascorbic acid: measurement, stability and clinical utility revisited.

AIMS To compare plasma ascorbic acid results by the colorimetric FRASC (Ferric Reducing/Antioxidant and Ascorbic Acid) assay and a reference HPLC method; to re-examine plasma ascorbic acid stability, and anticoagulant effect. DESIGN AND METHODS For method comparison, 31 plasma samples were tested by both methods. For stability, matching EDTA, heparin, citrate and fluoride/oxalate plasma, stored under different conditions of time and temperature, was measured. RESULTS FRASC is an acceptable alternative to HPLC for plasma ascorbic acid: precision, limit of detection and recovery were similar, and results by the two methods were indistinguishable: mean (95% CI) difference:1.8 (-1.1-4.6; n = 31) micromol/L. Ascorbic acid was most stable in heparinized plasma. Marked loss (p < 0.05) in EDTA plasma occurred within 30 min of blood collection. CONCLUSIONS FRASC offers a speedy and reliable alternative to HPLC for plasma ascorbic acid. Heparin is proposed as the anticoagulant of choice; loss of ascorbic acid is rapid in EDTA plasma ex vivo.