Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1997-07-11
2001-01-23
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S028000, C436S063000, C436S164000
Reexamination Certificate
active
06177260
ABSTRACT:
FIELD OF THE INVENTION
This invention concerns a method of measuring the antioxidant (reducing) power and antioxidant concentration of a sample, in particular various biological fluids, pharmaceuticals, beverages, herbs, herbal remedies and foodstuffs.
BACKGROUND OF THE INVENTION
In the invention described, the terms “antioxidant (or reducing or reductive) capacity, antioxidant (or reducing or reductive) power, antioxidant (or reducing or reductive) ability, antioxidant (or reductive or reducing)” are held to be synonymous and interchangeable. Oxidising species, or reactive oxidant species, are known to contribute to the cause of a number human diseases. In situations or conditions where antioxidant defence is inadequate, there may be oxidative changes to DNA, protein and lipids, which may lead to various degenerative diseases, such as coronary hear disease, cancer, diabetes, arthritis and cataracts (Gey K. F., Br. Med. Bull., (1993), 49, 679-99; Steinberg D., Circulation, (1992), 85, 2338-44; Cutteridge J. M. C., (1994) 91, 133-40; Gey K. F. et al, Am. J. Clin. Nutr., (1993), 57, 787S-797S).
Measurement of antioxidant status of biological fluids and tissues may be useful in the study of pro-oxidant:antioxidant balance and the risk of, or defence against, diseases associated with oxidative damage. The relative contributions to this by individual antioxidants, and assessment of the potential for ingestion or treatment with foodstuffs, pharmaceuticals, vitamin preparations, herbs etc., to improve antioxidant status may be useful in helping to plan risk reduction/health promoting strategies.
The term “antioxidant,” can be employed as a generic term describing a group of compounds which may prevent the generation of oxidising species, remove such species or inactivate them, thus preventing, delaying or minimising oxidative changes to important biomolecules. Members of this group are diverse in structure and action, and include metal chelating compounds and enzymes such as catalase and superoxide dismutase. However, one important antioxidant sub-group comprises electron donating (i.e. reducing) antioxidant, such as (but not limited to) ascorbic acid (vitamin C), alpha, beta, gamma and delta tocopherols and tocotrienols (collectively known as “vitamin E”), uric acid, bilirubin, phenolic group compounds such as flavonoids, and thiol group-containing compounds such as protein and glutathione. Electron donating antioxidants, also known as scavenging and chain breaking antioxidants, destroy or inactivate oxidising species by means of electron transfer from “antioxidant” to the oxidant. In this application, the term “antioxidant” is used hereafter to describe such electron donating antioxidants.
Up until now, most tests used for measuring the antioxidant power of a sample have measured the ability of the sample to withstand the oxidative effects of reactive species purposely generated in the reaction mixture. For example, depletion of antioxidants denoted by a change in signal, such as the rate of oxygen utilisation (Wayner, D. D. M. et al, Biochim. Biophys. Acta, (1987), 924, 408-419, or chemiluminescence (GB 2245062; Popov, I. N., Free Radicals Biol. Med., (1994), 17, 267-71; Whitehead, T. P., et al, anal. Chim, Acta., (1992), 266, 265-277; Lissi, E., Free Radicals Biol. Med., (1995), 16, 581-90). However, these methods require specialised equipment and can be time consuming. Therefore, these methods are difficult to sue and limited in their applications. As a result of the lack of an inexpensive, simple, rapid and efficient method of measuring individual antioxidant and/or total antioxidant (reducing) power of biological samples, there is insufficient data on the clinical utility of assessing antioxidant status in the various disorders thought to be associated with oxidative changes/oxidative stress.
Specific antioxidants, such as ascorbic acid, are known to play a major role in antioxidant defence. However, most methods of measuring the concentration of ascorbic acid in samples are non-specific, redox linked colourimetric methods (Pachla L.A. et al, J. Assoc. Anal. Chem., (1985), 68 1-12). A more specific method has been disclosed by Liu L. S., et al, J. Chrom., (1993), 612, 63-70, which requires use of HPLC. However, this method is both time consuming and expensive. Lewin G., et al, J. Biochem. Biophys. Meth., (1994), 28, 277-82, has recently disclosed a sensitive method of measuring ascorbic acid using a photochemiluminescence method, however this method requires specialised equipment. Moreover, owing to the time consuming nature of most methods used to date, pre-treatment of samples is usually needed in order to stabilise the ascorbic acid content.
Furthermore, it is time consuming, expensive and difficult to the point of impracticality to attempt to measure all possible constituent antioxidants within a heterogeneous sample, such as blood plasma, urine, plant material, foodstuffs etc. Therefore, a method suitable for the speedy, specific and sensitive measurement of individual antioxidant(s) and/or net, overall or total antioxidant power of a wide range of sample types would have considerable advantages over currently available methods. This would especially be true if such a method could be performed on samples with no or minimal pre-treatment. Such a method would facilitate observational, clinical and nutritional studies into antioxidant defence and the role of oxidative stress in the aetiology and severity of chronic disease.
SUMMARY OF THE INVENTION
The term FRAP in the description of the invention is the ferric reducing (antioxidant) power of a sample. Also, the term FRASC is the ferric reducing antioxidant power (FRAP) and the ascorbic acid concentration of the sample, as measured by a modification of the FRAP assay in which both of these indices of antioxidant defence are measured by a single test.
It is an object of the invention to provide a simple and efficient method for measuring the antioxidant power of a sample.
It is a further, and more specific, object of the invention to provide a simple and efficient method for measuring the antioxidant power of a sample and the amount of antioxidants in a sample.
It is still a further, and more specific, object of the invention to provide a simple and efficient method for measuring the antioxidant power of a sample and the amount of a specific antioxidant in a sample.
It is still a further, and more specific, object of the invention to provide a simple and efficient method for measuring the antioxidant power of a specific antioxidant in a sample.
It is still a further, and more specific, object of the invention to provide a simple and efficient method for measuring the antioxidant power of a specific antioxidant in a sample and the amount of the specific antioxidant in a sample.
Other objects and advantages of the invention will become apparent as the description proceeds.
Redox indicators are known to undergo physiochemical change, for example a colour change, when reduced by antioxidants in a sample. This physicochemical change can be easily monitored by known methods, for example by monitoring a change in absorption, or emission, of electromagnetic radiation in a sample or by other methods that are known to monitor redox reactions. The method of the invention utilises the ability of antioxidants to reduce metal complexes to their lower oxidation state or to take part in any electron transfer reaction (i.e. a redox reaction) which causes a concomitant, direct physicochemical change in the redox partner taking the role of oxidant. For example, Fe
III
-TPTZ is reduced by antioxidants to its Fe
II
-TPTZ form, which is blue in colour, and hence results in an increase in absorbance at 593 nm.
the redox reaction described above is only intended to be representative of the many suitable redox reactions that may be known to those in the art. Many other known redox reactions or variations therefore are likely to be found useful for the present purposes and can be investigated by sample experimentation. The term redox indicator is sue
Benzie Iris F. F.
Strain John Joseph
Leydig , Voit & Mayer, Ltd.
The Hong Kong Polytechnic University
Weber Jon P.
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