WO2005052575A1

WO2005052575A1 - Molecular markers of oxidative stress

Info

Publication number: WO2005052575A1
Application number: PCT/IB2004/003748
Authority: WO
Inventors: Claude Charuel; Thomas Andrew Clayton; Jeremy Ramsey Everett; John Christopher Lindon; Jeremy Kirk Nicholson
Original assignee: Pfizer Corp Belgium; Pfizer Ltd Great Britain; Pfizer Corp SRL
Current assignee: Pfizer Corp Belgium; Pfizer Ltd Great Britain; Pfizer Corp SRL
Priority date: 2003-11-28
Filing date: 2004-11-15
Publication date: 2005-06-09
Anticipated expiration: 2006-05-28

Abstract

The invention relates to a method of detecting molecular markers that are indicative of oxidative stress and the molecular markers thus detected. The invention also relates to a method of identifying oxidative stress in a living organism, and further methods for determining whether compounds induce or alleviate oxidative stress, and further relates to methods for reducing or preventing oxidative stress in an organism.

Description

MOLECULAR MARKERS OF OXIDATIVE STRESS

This invention relates to a method of detecting molecular markers that are indicative of oxidative stress and the molecular markers thus detected. The invention also relates to a method of identifying oxidative stress in a living organism, and further methods for determining whether compounds induce or alleviate oxidative stress, and further relates to methods for reducing or preventing oxidative stress in an organism

Background to the Invention Oxidative stress in biological systems has been defined as 'a disturbance of the pro-oxidant/anti-oxidant balance in favour of the former leading to potential damage' (Sies, H. (1991) Oxidative Stress II. Oxidants and Antioxidants. Academic Press, London). The term refers to the situation where there is a serious excess of reactive oxygen and/or nitrogen species in relation to the capacity of the anti-oxidant defences of an individual. Reactive oxygen and nitrogen species that are potentially damaging to biological systems include, amongst others, the superoxide and hydroxyl radicals, hydrogen peroxide, hypochlorous acid, nitric oxide and peroxynitrite. Such reactive species may arise from normal biological processes or in response to an applied stimulus. Biological systems exist to combat these oxidative challenges and these anti-oxidant defences include the destruction of hydrogen peroxide under the action of the enzyme catalase or through the combined action of glutathione and glutathione peroxidase. Oxidative stress is believed to be an important factor in the damage caused by various toxins and to have an important role in several human diseases and in the ageing process (Miccadei, S., Kyle, M. E., Gilfor, D. and Farber J. L. (1988) Toxic consequences of the abrupt depletion of glutathione in cultured rat hepatocytes. Arch. Biochem. Biophys., 265, 311-320; Halliwell, B. and Gutteridge, J. M. C. (1999) Free radicals in biology and medicine, 3^r Edition. Oxford University Press, Oxford.). Oxidative stress contributes to many diseases including autoimmune diseases, cancer, neurodegenerative diseases, heart attack and stroke. Oxidative stress is known to have a role in asthma, in mechanisms of neurodegeneration, in impaired mitochondrial function and in redox regulation. Oxidative stress is a common cause of damage to the kidney and kidney disease. Oxidative stress is also involved in impairment of glucose transport and in neutrophil oxidation and plays a role in inflammation.

Oxidative stress is a potentially damaging condition. Hence it is desirable to provide biological tests or indicators as to whether an individual has a disturbance of the pro-oxidant/anti-oxidant balance. It is known that the detection of significant changes in the oxidative or anti-oxidative status of an individual is possible using enzyme assays to monitor the altered activity of various antioxidant defense enzymes such as superoxide dismutase, catalase and glutathione peroxidase (Sodhi et al (1997) Study of oxidative stress in rifampicin-induced hepatic injury in young rats with and without protein-energy malnutrition. Human Exp. Toxicol, 16, 315-321). Oxidative stress is known to lead to lipid peroxidation hence methods have been developed to test for this and for other breakdown products as a measure of oxidative stress levels in a biological system. For example the measurement of the concentration of thiobarbituric acid reactive substances is used as an indicator of lipid peroxidation (Ohkawa et al (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem, 95, 351 - 358). Likewise, malondialdehyde (MDA), a breakdown product of lipid peroxidation can be measured (Yeo HC, Helbock HJ, Chyu DW, Ames BN. Assay of malondialdehyde in biological fluids by gas chromatography-mass spectrometry. Anal Biochem. 1994;220:391-396). Arachidonic acid peroxidation products called isoprostanes are also used as markers for oxidative stress in vivo ( Wang Z, Ciabattoni G, Creminon C, Lawson JA, Fitzgerald GA, Patrono C, Maclouf J. Immunological characterization of urinary 8- epi-prostaglandin F2 alpha excretion in man. J Pharmacol Exp Ther. 1995;275:94- 100). Oxidative status can also be evaluated by measuring serum hydroperoxides using the chromogenic reaction with N,N-diethyl- 7-phenylenediamine (Alberti A, Bolognini L, Macciantelli D, Carratelli M. The radical cation of Ν,Ν-diethyl-para- phenylenediamine, a possible indicator of oxidative stress in biological samples, Res Chem Intermed, 2000, 26, 253 - 267). Methods are known for detecting reactive oxidizing species in a sample which can cause oxidative stress for example assays for hydrogen peroxide by the phenol red method (Pick et al (1980) A simple colourimetric method for the measurement of hydrogen peroxide produced by cells in culture. J. Immunol. Methods., 38, 161 - 170). Other indicators of oxidative status include thiol content (Sedlak et al, 1968, Estimation of total protein bound and non-protein bound sulfhydrl groups in tissues with Ellman's reagent. Anal biochem, 25, 192 - 205) and glutathione loss or the increase in oxidized glutathione (Varma et al, Prevention of Intracellular Oxidative Stress to Lens by Pyruvate and its Ester, Free Rad Res, 28, 131 - 135). Hence a number of markers of oxidative stress are known and the level of such markers can be determined using a variety of methods. However, there are several problems and limitations associated with these methods. Many of the methods detect damage associated with the prior presence of oxidative stress rather than oxidative stress itself. Assays of enzyme activity require specialised techniques and skills. Additionally, many of the existing molecular markers of oxidative stress are measured indirectly, for example by using multi step assay procedures or chemical derivatisation protocols which require the addition of reagents to the biological sample and are both time consuming and subject to inaccuracy and artefact of measurement. Hence it is advantageous to provide molecular markers of oxidative status which are closely associated with oxidative stress and which can be more directly detected in a biological sample both rapidly and with certainty and without the absolute requirement of adding reagents to the sample itself. Accordingly and in view of the potentially damaging effect of oxidative stress it is desirable to provide a method for identifying new molecular markers indicative of oxidative stress, and to provide a new method of identifying oxidative stress or oxidative status in an animal subject employing the quantity measurement of such molecular markers in test samples. It would also be desirable for such a method to be effective in the direct and rapid identification and quantity measurement of the relevant molecular markers in the test samples. Furthermore it is desirable to provide a method for determining whether a subject suffering from oxidative stress or a disturbance of the pro-oxidant/anti-oxidant balance in favour of the former, with the possibility of potential biological damage, will benefit from the administration of an antioxidant therapy, and additionally to provide a method, involving the administration of antioxidant therapy, for treating individuals suffering, or potentially suffering, from oxidative stress. It is also desirable to provide a method of screening a compound for its ability to induce oxidative stress or to reduce oxidative stress in an animal or in cell cultures in- vitro and for quantifying such effects, thus allowing the detection and quantification of potentially damaging or beneficial effects of compounds, in relation to oxidative stress, in animal subjects or in cell cultures in-vitro.

Brief Description Of Drawings

Figure 1. Hypersuccinic aciduria, hyperacetic aciduria, hypo2-oxoglutaric aciduria and hypohippuric aciduria after dosing rifampicin (1000 mg/kg). 'a' shows a selected region of the 1H NMR spectrum of the 24-17 hours pre-dose urine sample from rat X. 'b' shows the same region of the 1H NMR spectrum of the 0-7 hours post-dose urine sample from rat X. The two spectra are scaled to constant intensity for the creatinine CH peak.

Brief Description of the Invention This invention makes available a method of detecting molecular markers that are indicative of oxidative stress and a method for identifying oxidative stress in an animal. The invention further provides a method for screening a compound for its ability to induce oxidative stress or for its ability to reduce oxidative stress in an animal or in cell cultures in-vitro and for quantifying the potency of a compound in inducing or reducing oxidative stress. Additionally the invention provides a therapeutic method for treating an animal subject diagnosed with a condition of oxidative stress. We have identified particular molecular components of biological samples, for example body fluids, body tissues and cell cultures, whose quantities may or do vary in conditions of oxidative stress and have identified these molecular components as molecular markers of oxidative stress. We have also shown that some of these molecular entities are well suited to being directly measured in urine samples from animal subjects with the minimum of intervention into the sample measured. Thus, such analyses have the potential to provide a rapid and non-invasive assessment of whether an animal subject is in an abnormal oxidative state. Similar analyses should be possible with cell cultures. Using such analyses in cell cultures or animal subjects, test compounds could potentially be evaluated for their ability to induce or alleviate oxidative stress.

Detailed Description of the Invention

Thus the present invention comprises several aspects, designated (I) to (VIII) below.

(I) This aspect provides a method of detecting molecular markers indicative of oxidative stress comprising;

a) providing a first and second sample set of the same type of sample of body fluid or body tissue obtained by the same sampling protocol from members of a single animal species, such that the first and second sample sets contain a plurality of molecular components,

b) the first sample set being derived from a first animal population not having oxidative stress,

c) the second sample set being derived from a second animal population which has, or has been arranged to have oxidative stress,

d) measuring a first and second signal or signals derived from the molecular components of the samples of the first and second sample sets respectively, the signal or signals being indicative of the quantity of the molecular components in the measured sample, and based on the measurements deriving for each of the first and second sample sets a first and second quantity data set describing the quantities of the molecular components of the sample sets,

e) comparing the first and second quantity data sets and thereby identifying one or more molecular components which are higher or lower in quantity in the latter relative to the former data set,

f) selecting one or more of the molecular components identified in step (e), g) determining the molecular identity of the selected molecular component(s) and identifying the molecular component(s) as molecular marker(s) of oxidative stress;

h) repeating steps a) to e) a number of times, wherein the animals are arranged to have oxidative stress has been provided by a different means at each repeat, and determining if there is a consistent change in the potential marker on each occasion in order to more fully confirm an selected marker as a marker of oxidative stress or by finding a rational chemical or biochemical explanation for the usefulness of the selected marker as a marker of oxidative stress.

The molecular components typically include the combination of chemical and/or biochemical species that comprise the sample. This includes the molecules found in living organisms and may for example comprise fats, proteins, nucleic acids, carbohydrates, and their constituent subunits, minerals, vitamins, hormones, metabolic substrates, intermediates or products, cofactors, coenzymes and prosthetic groups.

The sampling protocol preferably involves using a first animal and second animal population that are phenotypically homogeneous apart from the fact that the second population has been arranged to have oxidative stress or alternatively has been selected for the reason that it is already subject to a condition of oxidative stress; the first and second populations should otherwise be as similar as possible and lacking any other disease, infection or inborn errors in metabolism. Preferably the first animal and second animal population are subject to the same environmental and nutritional states. More preferably the two populations are regulated to receive the same diet, since diet can affect the composition of body fluids and body tissues. The first and second sample set are preferably of the same sample type, that is the same body fluid type, for example both urine or both blood plasma or the same body tissue type, for example both liver or both kidney. More preferably samples to be compared should be treated as near as possible identically as part of the sampling protocol prior to taking measurements, for example with regard to homogenisation, dialysis, lysis, sedimentation, precipitation, centrifugation, clarification or filtration of a sample; and/or with regard to temperature of collection, storage on ice, snap freezing, slow freezing, and/or with regard to the time period and temperature at which the sample is stored; and/or with regard to dilution with a solvent for example water, addition of buffers and salts, additionally with regard to the addition of preserving agents such as azide, glycerol, anticlotting factors and/or with regard to pH.

Preferably the signal or signals are any measurable signal or signals or pattern of signals which are characteristic of the presence, absence or quantity of the molecular components in the sample of body fluid or body tissue. Preferably the signal or signals or patterns of signals result from the output of measurements taken by techniques such as nuclear magnetic resonance (NMR) spectroscopy and/or any other chemical analysis techniques such as mass spectroscopy (MS), infrared (IR) spectroscopy, RAMAN spectoscopy, ultra violet (UV) or visible spectroscopy, gas chromatography (GC) and high performance liquid chromatography (HPLC), liquid chromatography (LC) or by using any combination of such techniques e.g. GC-MS or HPLC-MS or HPLC-NMR or HPLC-NMR-MS. Preferably the chosen analytical method is one that provides quantitative multi-component analysis. Most preferably the signal or signals or patterns of signals result from the output of measurements taken by nuclear magnetic resonance (NMR) spectroscopy for example 1H nuclear magnetic resonance (NMR) spectroscopy. Preferably, the first and second sample sets are analysed in exactly the same way. More preferably the first and second sample sets will be analysed using the same piece(s) of analytical equipment, without any change in operating conditions. Most preferably the first and second sample sets will be analysed on the same day using the same equipment, preferably with samples from the first and second sample sets being run alternately or in some randomised order to avoid or minimise any time-related effects.

The quantity of a molecular component in a sample of body fluid or body tissue is preferably determined from measurements taken from an NMR, infrared (IR), RAMAN, ultra violet (UV), fluorescent, visible or mass spectrum of the sample of body fluid or body tissue. Preferably spectral peak heights and/or peak areas or absorbance values or ion counts or other electrical measurements are used to determine the quantity of a molecular component giving rise to an associated spectral peak. In particular the quantity may be determined by comparing such a measurement to a corresponding measurement for a reference compound, which is already present or is added at a known quantity to the sample of body fluid or body tissue as an internal reference prior to measurement of the sample. Alternatively the quantity may be determined from the spectral peak by reference to an extinction coefficient of the relevant molecular component and application of the Beer Lambert Law. Alternatively the quantity of a molecular component in a sample of body fluid or body tissue may be determined from measurements taken from a gas chromatography (GC) chromatogram, a high performance liquid chromatography (HPLC) chromatogram or a liquid chromatography (LC) chromatogram, the quantity normally being proportional to the area of the peak corresponding to the eluted molecular component. In such cases, the quantity of a molecular component is preferably determined by reference to a peak area of a reference compound, which is already present or is added in known quantity to the sample of body fluid or body tissue as an internal reference prior to measurement of the sample, or is determined with respect to a suitable calibration curve for the component being quantified. Alternatively the quantity of a molecular component in a sample of body fluid or body tissue is determined from measurements taken by using any combination of chromatographic and spectroscopic techniques e.g. GC-MS, the quantity being determined using a combination of the above spectral and chromatographic measures.

Most preferably the quantity of a molecular component in a sample of body fluid or body tissue may be determined from measurements taken from an NMR spectrum such as an ^!H NMR spectrum of the sample of body fluid or body tissue. NMR spectroscopy can be successfully used for the examination of small (ca. 10-20 mg) samples of solid tissue (e.g. Moka et al. (1997), Magic angle spinning proton nuclear magnetic resonance spectroscopic analysis of intact kidney tissue samples, Analytical Communications, 34, 107-109). However, this requires a special technique known as Magic Angle Spinning (MAS). Preferably, any 'solid' tissue samples would be 'snap' frozen in liquid nitrogen immediately after collection and subsequently stored at -80C pending analysis. Collection and storage vessels should be selected which will not contaminate the samples by leakage of plasticisers or other substances. Each NMR spectrum may be normalised, or scaled, to give the same total integration as every other NMR spectrum in a data set to which the spectrum is to be compared. Additionally it is preferable to scale the 1H NMR data from a body fluid or body tissue sample to a constant integration for an internal reference peak. An endogenous creatinine or allantoin peak may be used as an internal reference for determining a relative quantity of a molecular component in a urine sample and scaling urinary data to a constant creatinine peak helps to eliminate differences in excretion that are related to body mass. 1H NMR peak heights and/or peak areas related to molecular components in the measured sample may be measured since both measures are indicative of the quantity of the correlated molecular components, but peak areas are the more reliable measures. Most preferably peak height ratios or peak area ratios are calculated relative to an internal reference peak, to give a quantity of the molecular component relative to the reference peak. With plasma samples it has proven useful to use glucose as an internal reference for the 1H NMR spectra in conjunction with separate glucose determinations on each sample.

Preferably the quantity data sets comprise signal-derived quantity data correlated to one or more molecular components of a test sample, allowing comparison of particular molecular component quantity values between data sets derived from different test samples and subsequent identification of any quantity variations between data sets.

Where the quantities of the molecular components in a sample of body fluid or body tissue are determined using chromatography the molecular identity of the selected molecular component is preferably obtained by isolation of the selected molecular component from the elution flow and analysis by Mass Spectroscopy, NMR or other molecular or chemical assay. Alternatively the molecular identity of the selected molecular component may be obtained by reference to internal standards of known chemical or biochemical entities introduced into the test sample prior to chromatography.

Where the quantities of the molecular components in a sample of body fluid or body tissue are determined using spectroscopic measurements the molecular identity of a selected molecular component is preferably obtained by reference to a library or database of spectra associated with the relevant spectroscopy technique. Alternatively the molecular identity of a selected molecular component may be obtained by adding to the test sample a quantity of an essentially pure known biochemical or chemical entity to judge if the spectral peak(s) related to the selected component is/are increased thus identifying the selected molecular component as the same as the added entity. Alternatively reference spectra of essentially pure chemical or biochemical entities may be recorded and compared to the spectrum of the test sample to identify the molecular identity of the selected molecular component. Alternatively where the spectroscopy technique is NMR or Mass Spectroscopy or another suitable technique, the molecular identity of the selected molecular component may be deduced directly from the spectrum using de-novo structure solution methods. More preferably the quantity of the molecular components in a sample of body fluid or body tissue are determined from measurements taken from an NMR spectrum and the molecular identity of a selected molecular component is obtained by adding to the test sample a quantity of an essentially pure known biochemical or chemical entity to judge if the NMR spectral peak(s) related to the selected component is/are increased thus identifying the selected molecular component as the same as the added entity. Alternatively reference NMR spectra of essentially pure chemical or biochemical entities may be recorded and compared to the spectrum of the test sample to identify the molecular identity of the selected molecular component. Most preferably the quantity of the molecular components in a sample of body fluid or body tissue are determined from measurements taken from an NMR spectrum and the molecular identity of the selected molecular component is obtained by reference to a data library or other publication correlating peak data from NMR spectra with particular chemical or biochemical entities, examples of such sources are; 1) Pouchert, C. J. and Behnke, J. (1993). The Aldrich Library of 13C and IH FT NMR Spectra. Edition I. Published by Aldrich Chemical Co., Inc. 2) Fan, T. W.-M. (1996) Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear

Magnetic Resonance Spectroscopy, 28, 161 - 219. 3) Nicholson, J. K., Foxall, P. J. D., Spraul, M., Farrant, R. D. and Lindon, J. C. (1995) 750 MHz IH and 1H-13C NMR Spectroscopy of Human Blood Plasma. Analytical Chemistry, 67, 793 - 811.

The animal population in either of steps (b) or (c) of the method of aspect (I) of the invention may consist of a single animal; or more preferably of two or more animals. Usually the animals in steps (b) or (c) will be of the same species. Usually the animals will be all of the same sex, alternatively mixed sex populations may be used in steps (b) and (c).

Furthermore in a particular embodiment the second animal population is the same as the first animal population and the first and second sample sets are provided at different time points separated by the induction of oxidative stress in the animal population the induction of oxidative stress in the animal follows the animal population being arranged to have oxidative stress.

The second animal population may be arranged to have oxidative stress by administration of a compound causing oxidative stress such as; rifampicin or antimycin A or des-acetylrifampicin or 3-formylrifampicin or hydrogen peroxide or paraquat or menadione or nitrogen dioxide or carbon tetrachloride or methyl chloride or allyl formate or allyl alcohol or acrolein or alloxan or dialuric acid, preferably rifampicin. The compound may also be a free radical or may be a compound that is metabolised to generate free radicals. The compound may alternatively be a compound, or a compound that is metabolised to a compound which interferes with antioxidant defences, for example by depleting glutathione. The compound may alternatively be a compound, or a compound that is metabolised to a compound, which produces oxidative stress through redox cycling.

(II) This aspect of the invention provides an assay for the determination of the degree of oxidative stress in a living organism or group of living organisms, wherein the change in the quantity of a molecular marker in a test sample from the organism(s) determined relative to the quantity of a molecular marker in a reference sample or samples from a reference organism or group of reference organisms is taken as indicative of the degree of oxidative stress, wherein the molecular marker is selected from the group comprising; 2-oxoglutarate, 2-oxoglutaric acid, succinate, succinic acid, glyoxylate, glyoxylic acid, formate, formic acid, oxaloacetate, oxaloacetic acid, malonate, malonic acid, 2-oxoadipate, 2-oxoadipic acid, glutarate, glutaric acid, 2- oxoheptanedioate, 2-oxoheptanedioic acid, adipate, adipic acid, acetate, acetic acid, hippurate, hippuric acid, glycine, ammonia, ammonium ion, L-glutamic acid and L- glutamate.

The living organism(s) may be any unit of life that can exist independently and includes cells which compose biological tissues and which may exist independently in culture as well as more advanced living organisms such as members of the of the animal kingdom. Preferably the organism is selected from the phylum of chordates including mammals, fish, amphibians, reptiles and birds. Mammal are preferred for example a human, a mouse, a rat, a pig, a cow, a bull, a sheep, a horse, a dog or a rabbit or any farmed animal or any animal, for example an animal used for the purpose of breeding. Preferably the animal is a rat, most preferably a human.

Preferably the reference organism or group of reference organisms are derived from an organism population not having oxidative stress or not being arranged to have oxidative stress.

(Ill) This aspect of the invention provides a method for identifying oxidative stress in an animal or group of animals of the same species using the assay as described in aspect (II), said method comprising;

a) determining the quantity of a molecular marker in a test sample or set of samples of body fluid or body tissue taken from the animal(s), the marker being associated with oxidative stress in that animal species,

b) comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress,

c) the presence of oxidative stress in the animal or group of animals being identified by a higher or lower value of the former quantity, for the test sample, relative to that in the latter, for the reference sample,

wherein the molecular marker is selected from the group comprising; 2-oxoglutarate, 2-oxoglutaric acid, succinate, succinic acid, glyoxylate, glyoxylic acid, formate, formic acid, oxaloacetate, oxaloacetic acid, malonate, malonic acid, 2-oxoadipate, 2- oxoadipic acid, glutarate, glutaric acid, 2-oxoheptanedioate, 2-oxoheptanedioic acid, adipate, adipic acid, acetate, acetic acid, hippurate, hippuric acid, glycine, ammonia, ammonium ion, L-glutamate and L-glutamic acid.

Preferably the presence of oxidative stress in an animal or group of animals is identified by a higher, or increased, quantity of any one of; formate, formic acid, acetate, acetic acid, malonate, malonic acid, succinate, succinnic acid, glutarate, glutaric acid, adipate, adipic acid, ammonia and ammonium ion; or by a lower, or decreased, quantity of any one of; glyoxylate, glyoxylic acid, oxaloacetate, oxaloacetic acid, 2-oxoglutarate, 2-oxoglutaric acid, 2-oxoadipate, 2-oxoadipic acid, 2-oxoheptanedioic acid, 2-oxoheptanedioate, hippurate, hippuric acid, glycine, L- glutamate and L-glutamic acid.

In this aspect of the invention a quantity of more than one molecular marker may be determined. In a modification of the method of (III) in step (a) a quantity ratio of two molecular markers is determined for the test sample, and in step (b) that ratio is compared to a predetermined reference sample quantity ratio or range of quantity ratio of two molecular markers selected from the group consisting of the group set forth as in (III) and pyruvate and pyruvic acid. In this aspect of the invention the two molecular markers of the quantity ratio may be selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid , formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2-oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2-oxoheptanedioate or 2-oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L-glutamate or L-glutamic acid and ammonia or ammonium ion;

The population of the animal species not suffering from oxidative stress as referred to in step (b) may comprise one or more animals of the species.

The predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress, as referred to in step (b) of aspect III, may be obtained by determining the quantity of a molecular marker in a reference sample or set of samples of body fluid or body tissue taken from the latter animal(s) not suffering from oxidative stress, the marker being associated with oxidative stress in that animal species. This may be done in the manner of a control or reference process at the time when the method provided in aspect III is performed. (IV) In this aspect of the invention there is provided a method for screening a compound for its ability to induce oxidative stress in an animal or group of animals using the assay as described in aspect (II), said method comprising,

a) providing a first reference sample or set of samples of body fluid or body tissue taken from an animal or group of animals prior to dosing with a test compound,

b) dosing the animal or group of animals with the test compound,

c) providing a corresponding test sample or set of samples of body fluid or body tissue taken from the animal or group of animals post dosing,

d) determining the quantity of a molecular marker in the reference sample(s) and comparing it with the quantity of the molecular marker in the test sample(s), a higher or lower quantity of the marker(s) in the latter relative to the former being indicative of the ability of the compound to cause oxidative stress in the animal or group of animals and wherein the molecular marker is selected from the group consisting of the group set forth as in (III). Preferably, the reference sample(s) and the corresponding test sample(s) will all be of the same type of body fluid or body tissue. In this aspect of the invention a quantity of more than one molecular marker may be determined. In this aspect of the invention a plurality of animals of the same type may be provided at step (a) and the effect of the test compound assessed in relation to the group as a whole, and a progressively increasing quantity of the test compound may be dosed to a plurality of animals at step (b), with different animals or groups of animals receiving different doses, the comparison of the quantity of molecular marker(s) at step (d) being made for each of the several animals or groups of animals and the higher or lower quantity of the molecular marker(s) in each of the several animals are compared to each other in order to estimate the quantity of the test compound that produces a given percentage change in molecular marker quantity in the animals. Preferably the plurality of animals are all members of the same species.

In a modification of the above method (TV) or in step (d) a quantity ratio of two molecular markers may be determined in the reference sample(s) and the ratio(s) obtained compared with the corresponding quantity ratio(s) in the test sample(s) wherein the two molecular markers are selected from the group consisting of the group set forth as in (III) and pyruvate and pyruvic acid.

(V) In this aspect of the invention there is provided a method of screening a compound for its ability to induce oxidative stress in an animal or group of animals using the assay as described in aspect (II), said method comprising,

a) dosing the animal or group of animals with a test compound

b) providing a test sample of body fluid or body tissue taken from the animal(s) post dosing,

c) determining the quantity of a molecular marker in the test sample(s), the marker being associated with oxidative stress in that animal species, and comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress, a higher or lower quantity of the molecular marker in the former quantity for the test sample relative to the latter quantity for the reference sample being indicative of the ability of the compound to cause oxidative stress in the animal or group of animals and wherein the molecular marker is selected from the group consisting of the group set forth as in (III).

In this aspect of the invention a quantity of more than one molecular marker may be determined

The population of the animal species not suffering from oxidative stress as referred to in step (c) of aspect V may comprise one or more animals of the species.

The predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress, as referred to in step (c) of aspect V, may be obtained by determining the quantity of a molecular marker in a reference sample or set of samples of body fluid or body tissue taken from the latter animal(s) not suffering from oxidative stress, the marker being associated with oxidative stress in that animal species. This may be done in the manner of a control or reference process at the time when the method provided in aspect III is performed.

In this aspect of the invention a plurality of animals of the same type will normally be provided and the effect of the test compound assessed in relation to the whole group. Furthermore, a progressively increasing quantity of the test compound may be dosed to a plurality of animals at step (a), with different animals or groups of animals receiving different doses, the comparison of the quantity of molecular marker(s) at step (c) being made for each of the several animals or groups of animals in order to estimate the quantity of the test compound which produces a given effect in terms of a given percentage change in molecular marker quantity in the animals. Preferably the plurality of animals are all members of the same species.

In a modification of aspect (V) of the invention at step (c) a quantity ratio of two molecular markers may be determined for the test sample, and that ratio may be compared to a predetermined reference sample quantity ratio or range of quantity ratio of two molecular markers selected from the group consisting of the group set forth as in (III) and pyruvate and pyruvic acid;

As in aspect (III) of the invention, where the methods of aspects (III) and (V) involve comparison of a ratio of two molecular markers they may be selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid, formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2-oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2-oxoheptanedioate or 2-oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L-glutamate or L-glutamic acid and ammonia or ammonium ion;

(VI) In this aspect of the invention there is provided a method for treating an animal or group of animals diagnosed with a condition of oxidative stress comprising the use of the assay as described in aspect (11) or (III) to identify the presence of oxidative stress in that animal or group of animals, then; administering antioxidant therapy to the animal or group of animals if the presence of oxidative stress is diagnosed, i.e. if the quantity of the molecular marker (in the test sample) from the animal or group of animals is higher or lower relative to the quantity of the molecular marker (in the reference sample) from a reference animal or group of animals and wherein the molecular marker is selected from the group consisting of the group as in (III); the reference animal or group of reference animals are preferably derived from an animal population not having oxidative stress.

In this aspect of the invention the method may comprise the steps of; providing a test sample of body fluid or body tissue taken from the animal, determining the quantity of a molecular marker in the test sample and comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress. In this aspect of the invention a quantity of more than one molecular marker may be determined. Preferably an animal subject diagnosed with a condition of oxidative stress is an animal suffering from diseases linked to oxidative stress and shows indications of oxidative stress when tested using known tests for oxidative stress. For example the animal may demonstrate any the following physiological states; inflammation, autoimmune diseases, cancer, neurodegenerative diseases, heart attack and stroke, asthma, impaired mitochondrial function and redox regulation; damage to the kidney, kidney disease. The animal may demonstrate indications of oxidative stress when tested using known tests for oxidative stress for example by measurement of any of the following using known assay methods, arachidonic acid byproducts such as isoprostanes, serum hydroperoxides using the chromogenic reaction withN,N-diethyl-p-phenylenediamine, the radical cation of Ν,Ν-diethyl-para- phenylenediamine, hydrogen peroxide, total protein bound and non-protein bound sulfhydryls (thiols), glutathione loss or the increase in oxidized or lipid peroxidation using known tests for thiobarbituric acid reactive substances malondialdehyde (MDA. Alternatively, the animals should be found to show indications of oxidative stress when tested using known enzyme assays to monitor the altered activity of various antioxidant defense enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Again in this aspect of the invention in step (b) a quantity ratio of two molecular markers may be determined (for the test sample), and that ratio compared to a predetermined reference sample quantity ratio or range of quantity ratios of two molecular markers selected from the group consisting of the group set forth as in (III) and pyruvate and pyruvic acid and the two molecular markers of the quantity ratio may be selected from the paired groups comprising; succinate or succinic acid and 2- oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid, formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2-oxoadipate or 2- oxoadipic acid, adipate or adipic acid and 2-oxoheptanedioate or 2-oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L-glutamate or L- glutamic acid and ammonia or ammonium ion;

In this aspect of the invention the method may comprise the steps of;

a) providing a test sample of body fluid or body tissue taken from the subject post administration of the antioxidant therapy,

b) determining the quantity of a molecular marker(s) in the test sample and comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker(s) characteristic of a population of that animal species not suffering from oxidative stress to determine if the condition of oxidative stress has improved wherein the molecular marker(s) is selected from the group consisting of the group set forth as in (III);

Preferably the condition of oxidative stress in the animal is determined to have improved if the comparison in (b) demonstrates a lower, or decreased, quantity of any one of; formate, formic acid, acetate, acetic acid, malonate, malonic acid, succinate, succinnic acid, glutarate, glutaric acid, adipate, adipic acid, ammonia and ammonium ion; or a higher, or increased, quantity of any one of; glyoxylate, glyoxylic acid, oxaloacetate, oxaloacetic acid, 2-oxoglutarate, 2-oxoglutaric acid, 2-oxoadipate, 2- oxoadipic acid, 2-oxoheptanedioic acid, 2-oxohepatanedioate, hippurate, hippuric acid, glycine, L-glutamate and L-glutamic acid In any of the methods of treatment of the invention the antioxidant therapy may comprise the administration of an alpha-oxo acid or its corresponding anion; for example selected from the group comprising; glyoxylate, glyoxylic acid, oxaloacetate, oxaloacetic acid, 2-oxoglutaric acid, 2-oxoglutarate; alternatively the antioxidant therapy may comprise the administration of L-glutamate or L-glutamic acid or glycine.

(VII) This aspect of the invention provides a method of screening a compound for its ability to reduce oxidative stress in an animal or group of animals using the assay as described in aspect (II) or (111) said method comprising,

a) providing a reference sample, or set of test samples, of body fluid or body tissue taken from an animal(s) suffering from oxidative stress,

b) dosing the animal(s) with a test compound,

c) providing a test sample, or set of test samples, of body fluid or body tissue taken from the animal(s) post dosing with the test compound,

d) determining the quantity of a molecular marker in the reference samρle(s) and comparing it with the quantity of the molecular marker in the corresponding test sample(s), a higher or lower quantity of the marker in the latter test sample(s) relative to the former reference sample(s) being indicative of the ability of the compound to reduce oxidative stress in the animal(s) and wherein the molecular marker is selected from the group consisting of the group set forth in as in (III);

In an alternative embodiment the animal or animals in step (b) which are dosed with the test compound, and from which in step (c) the test sample is provided, may comprise an equivalent but different animal or set of animals from that in step (a), i.e. equivalent in that they are suffering from oxidative stress and are of the same type for example, the same species and preferably same sex and age. Thus in this embodiment a comparison is made between a control or reference group of animals, i.e. which remains undosed, and a test group of animals which is dosed with a test compound. In this aspect of the invention a plurality of animals of the same type will normally be provided at step (a) and the effect of the test compound assessed in relation to the whole group. Furthermore, a progressively increasing quantity of the test compound may be dosed to a plurality of animals at step (b), with different animals or groups of animals receiving different doses, the comparison of the quantity of molecular marker(s) at step (d) being made for each of the several animals or groups of animals in order to estimate the quantity of the test compound that produces a given effect for example by comparison of the quantity of the molecular marker(s) in each of the several animals to each other to estimate the quantity of the test compound which produces a given percentage change in molecular marker quantity in the animals. Preferably the plurality of animals are all members of the same species.

Again in step (d) a quantity ratio of two molecular markers may be determined in the reference sample and that ratio may be compared with a quantity ratio of two molecular markers in the test sample wherein the two molecular markers are selected from the group consisting of the group set forth as in (III) and pyruvate and pyruvic acid.

hi any preceding aspect of the invention the sample(s) may be selected from the group of; urine, saliva, blood serum, blood plasma, blood, sweat, tears, faeces, bile, semen, vaginal secretions, cerebrospinal fluid, aqueous humour, vitreous humour, synovial fluid, peritoneal fluid and pericardial fluid, pleural fluid, amniotic fluid, maternal milk, breath and breath condensate, body tissue such as liver tissue or kidney tissue or tissue of any body organ, a tissue homogenate, a tissue extract, a tissue cell extract and a tissue cell lysate.

(VIII) This aspect of the invention provides a method of screening a test compound for its ability to alter the level of oxidative stress in a test culture of cells using the assay as described in (II) said method comprising, a) obtaining one or more reference samples from a reference cell culture,

b) introducing a quantity of the test compound into the test culture of cells,

c) obtaining one ore more test samples from the test culture of cells,

d) determining the quantity of a molecular marker in the one or more test samples and comparing it with the quantity of the molecular marker in the one or more reference samples, a higher or lower quantity of the molecular marker in the latter relative to the former being indicative of the ability of the compound to alter the level of oxidative stress in the culture of cells and wherein the molecular marker is selected from the group consisting of the group set forth in (III).

In this aspect of the invention the reference culture may be the same as the test culture. The reference cell culture and/or test cell culture may be under oxidative stress alternatively the reference cell culture and/or test cell culture may not be under oxidative stress. The test and reference cell cultures may be cultures of cells of the same species. The test and reference cell cultures may be cultures of prokaryotic or eukaryotic cells, preferably eukaryotic. The cell cultures may be of cells comprising any structural unit of a living organism, for example heart cells or skin cells or lung cells or liver cells or kidney cells or brain cells or neural cells, preferably liver cells or kidney cells, most preferably liver cells. It is preferable to culture both reference and test cell cultures under similar conditions, particularly temperature of growth and medium from which the cells are grown also the level of oxygenation of the cultures. hi this aspect of the invention a quantity of more than one molecular marker may be determined.

In a modification of this aspect of the invention a quantity ratio of two molecular markers may be determined for the test sample(s) and may be compared to a quantity ratio or range of quantity ratio of two molecular markers determined for the reference sample(s) wherein the two molecular markers are selected from the group consisting of the group set forth (III) and pyruvate and pyruvic acid. As in aspect (III) of the present invention, where the methods of aspect (III) and aspects (VIII) involve a comparison of a ratio of two molecular markers they may be selected from the paired groups comprising; succinate or succinic acid and 2- oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid, formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2-oxoadipate or 2- oxoadipic acid, adipate or adipic acid and 2-oxoheptanedioate or 2-oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L-glutamate or L- glutamic acid and ammonia or ammonium ion.

In any of the aspects of the invention the quantity of a molecular marker may be determined using nuclear magnetic resonance (NMR) spectroscopy and/or any other chemical analysis techniques such as mass spectroscopy (MS), infrared (IR) spectoscopy, gas chromatography (GC) and high performance liquid chromatography (HPLC) or by using any combination of such techniques e.g. GC-MS. Furthermore a test sample or subject may be treated prior to analysis with one or more chemical reagents so as to produce derivative(s) of one or more molecular components, so as to enhance data recovery or to improve sample stability.

In any aspect of the invention where a molecular marker is used it may be a chemical or biochemical entity in the sample of body fluid or body tissue, which is identified to be indicative of the presence, absence or level of oxidative stress. Preferably the molecular marker is a metabolic substrate, intennediate or product, structural protein, nucleic acid, transport or receptor protein, lipid, carbohydrate, vitamin, amino acid, peptide, hormone, immunological protein, protein associated with metabolic or genetic control, catalytic protein, enzyme or their associated cofactors. Most preferably the molecular marker is a metabolic substrate, intermediate or product.

In any aspect of the present invention wherein a sample or a body fluid sample is used it may be preferably selected from the group comprising, saliva, blood serum, blood plasma, blood, sweat, tears, faeces, bile, semen, vaginal secretions, cerebrospinal fluid, synovial fluid, peritoneal fluid and pericardial fluid, pleural fluid, amniotic fluid, maternal milk, aqueous humour, vitreous humour, breath, breath condensate. The body fluid is more preferably blood or urine, and is most preferably urine. In any aspect of the present invention wherein a sample or body tissue sample is used it may be preferably selected from the group comprising; any tissue forming a structural unit in a living organism for example a tissue of any body organ such as liver tissue, kidney tissue, brain tissue, heart tissue, lung tissue, skin tissue or may be a tissue homogenate or a tissue extract or a tissue cell extract or a tissue cell lysate of such a body tissue. Preferably the body tissue is a tissue of a body organ. More preferably the body tissue is of the kidney and most preferably it is of the liver.

In any aspect of the present invention the animal may be any living organism of the animal kingdom but is more preferably selected from the phylum of chordates including mammals, fish, amphibians, reptiles and birds. Preferably the animal is a mammal, for example a human, a mouse, a rat, a pig, a cow, a bull, a sheep, a horse, a dog or a rabbit but may be any living animal including any farmed animal or any animal used for the purpose of breeding. Preferably the animal is a rat, most preferably a human.

In any aspect of the present invention, wherein animals not suffering from oxidative stress are used (for example as reference animals) they are preferably normal healthy animals free from diseases linked to oxidative stress and not showing indications of oxidative stress when tested using known tests for oxidative stress. More preferably the animals should not be found to show indications of oxidative stress when tested using known tests for oxidative stress for example by measurement of any of the following using known assay methods, arachidonic acid byproducts such as isoprostanes, serum hydroperoxides using the chromogenic reaction with N,N-diethyl- -phenylenediamine, the radical cation of Ν,Ν-diethyl-para-phenylenediamine, hydrogen peroxide, total protein bound and non-protein bound sulfhydrl , glutathione loss or the increase in oxidized or lipidperoxidation using known tests for thiobarbituric acid reactive substances malondialdehyde (MDA. Alternatively, the animals should not be found to show indications of oxidative stress when tested using known enzyme assays to monitor the altered activity of various antioxidant defense enzymes such as superoxide dismutase, catalase and glutathione peroxidase. In any aspect of the invention where a determination is made as to whether or not a quantity is higher or lower in quantity than another quantity or quantity range that detemiination is preferably made using a statistical procedure. For example multivariate statistical procedures such as pattern recognition methods for example partial least squares or partial least squares discriminant analysis may be used to compare quantity data sets and thereby identify quantity values which are higher or lower in quantity between the data sets. Thus molecular markers of oxidative stress may be identified from multivariate data by the application of procedures such as pattern recognition methods for example partial least squares or partial least squares discriminant analysis. Preferably a statistical procedure is employed that enables the determination whether observed differences in measured quantities or quantity values of a molecular component are statiscally significant in that they are statistically different from that quantity or range of quantity expected or measured in the absence, or possibly in the presence, of oxidative stress. The procedure is preferably any standard statistical procedure for assessment of statistical significance, more preferably procedures such as; tests of hypotheses, tests of significance, rules of decision, or decision rules. Typically the level of significance, or significance level, of the selected statistical procedure, often denoted by P, is pre-specified, in practice, preferably a significance level of 0.05 or 0.01 or 0.001 is used, although other values may be used. Critical values corresponding to P = 0.05, 0.01, and 0.001 are tabulated for many commonly used statistics, such as those for the *t-test, -test and *chi- squared test, and may be used in the assessment of judging significance. Preferably a 0.001 to 0.05 significance level is used. Most preferably a 0.05 significance level is used. Where a quantity value is compared to a range of quantity values then significance is preferably judged by determination of the standard deviation of the quantity value from the mean of the distribution of the range of quantity values, typically a value of 3 standard deviations from the mean is taken as being significant, normalisation of the distribution may be necessary using standard procedures prior to calculation of the standard deviation. Preferably 1, 2 and 3 standard deviations from the mean is taken as being significant, most preferably 3 standard deviations from the mean is taken as being significant. In any aspect of the present invention wherein the quantity of a molecular component of a sample of body fluid or body tissue is determined, that determination is preferably made from measurements taken from an NMR, infrared (IR), RAMAN, ultra violet (UV), fluorescent, visible or mass spectrum of the sample. Preferably spectral peak heights, peak areas or ion counts are used to quantify the molecular component(s) giving rise to the associated spectral peak(s). Further preferably the quantity is determined by comparison to a spectral peak height or area or ion count of a reference compound, which is already present or is added at a known quantity to the sample of body fluid or body tissue as an internal reference prior to measurement of the sample. Alternatively the quantity is determined from the spectral peak by reference to an extinction coefficient of the relevant molecular component and application of the Beer Lambert Law. Alternatively the quantity of a molecular component in a sample of body fluid or body tissue is determined from measurements taken from a gas chromatography (GC), a high performance liquid chromatography (HPLC) or a liquid chromatography (LC) chromatogram, the quantity normally being proportional to the area of the peak corresponding to the eluted molecular component. Preferably the quantity is determined with reference to a peak area of a reference compound, which is added at a known quantity to the sample of body fluid or body tissue as an internal reference prior to measurement of the sample, or is determined with respect to a suitable calibration curve for the component being quantified. Alternatively the quantity of a molecular component in a sample of body fluid or body tissue is determined from measurements taken by using any combination of such techniques e.g. GC-MS the quantity being determined using a combination of the above spectral and chromatographic measures.

Most preferably the quantity of a molecular component in a sample of body fluid or body tissue is determined from measurements taken from an NMR spectrum of the sample of body fluid or body tissue. Preferably each NMR spectrum is normalised, or scaled, to give the same total integration as every other NMR spectrum in a data set to which the spectrum is to be compared. Additionally and also preferable is to scale the 1H NMR data from the body fluid or body tissue sample to a constant integration for the internal reference peak. More preferably the endogenous creatinine or allantoin peak is used as the internal reference for determining the quantity of a molecular component in a urine sample. Scaling urinary data to constant creatinine or allantoin helps to eliminate differences in excretion that are related to body mass. Further preferably a peak height or a peak area related to a molecular component in the measured sample may be measured since both measures are indicative of the quantity of the correlated molecular component, although peak areas are more reliable quantity measures than peak heights. Most preferably peak height ratios or peak area ratios are calculated relative to an internal reference peak, to give a quantity of the molecular component relative to the reference peak. For NMR spectra of blood plasma samples glucose is preferably used as the internal reference in conjunction with separate glucose determinations on each sample. In any aspect of the present invention where an animal is dosed, that, dose can be delivered by any known or standard method. Preferably the dose is delivered orally, intravenously, injected parenterally, injected intramuscularly, injected subcutaneously, by inhalation, by suppository, pessary or topically. Preferably the dose of the compound is in the range from 0.01 to 1000 mg/kg body weight of the subject to be treated, preferably 0.1 to 20 mg/kg. Alternatively the dose may be delivered by intravenous infusion, preferably at a dose which is of the range from 0.001-100 mg/kg/hr. Typically, the actual dosage, which will be most suitable for an individual animal will depend on the age, weight, sex and response of the particular animal. The above dosages are exemplary of the average case. In any aspect of the present invention, wherein an estimate is made of the quantity of the test compound that produces a given percentage change in molecular marker quantity in the animals, that estimate is preferably made by correlating the quantity of the test compound against the relevant molecular marker quantity, preferably including the quantity value of the relevant molecular marker in the absence of any test compound, in order to derive the mathematical relationship between the two variable sets from which the estimate can be derived and the quantity of the test compound which produces a given percentage change in molecular marker quantity in the animals can be estimated. Preferably the mathematical relationship between the two variable sets is derived by graphical methods, more preferably using curve fitting procedures. Alternatively the mathematical relationship between the two variable sets is derived by parametric methods and/or statistical methods.

As used herein the term oxidative stress is typically understood to refer to a disturbance of the pro-oxidant/anti-oxidant balance in favour of the former with the potential of leading to potential biological damage. The term refers to the situation where there is a serious excess of reactive oxygen and/or nitrogen species in relation to the capacity of the anti-oxidant defenses of an individual. Reactive oxygen and nitrogen species which are potentially damaging to a biological system include, amongst others, the superoxide and hydroxyl radicals, hydrogen peroxide, hypochlorous acid, nitric oxide and peroxynitrite. Such reactive species may arise from normal biological processes or in response to an applied toxicological stimulus. Oxidative stress is believed to be an important factor in the damage caused by various toxins and to have an important role in several human diseases and in the ageing process and is known to lead to lipid damage and lipid peroxidation. Oxidative stress also contributes to many diseases including inflammation, autoimmune diseases, cancer, neurodegenerative diseases, heart attack and stroke. Oxidative stress is known to have a role in asthma, neurodegeneration, impaired mitochondrial function and redox regulation; oxidative damage is a common cause of damage to the kidney and kidney disease; impairment of glucose transport; neutrophil oxidation and a plays a role in inflammation.

As used herein the term body fluid is typically understood to include extracellular fluids of the animal body for example; saliva, blood serum, blood plasma, blood, sweat, tears, faeces, bile, semen, vaginal secretions, cerebrospinal fluid, synovial fluid, peritoneal fluid and pericardial fluid, pleural fluid, vitreous humour, aqueous humour, amniotic fluid, maternal milk, breath, breath condensate. It is to be appreciated that the biochemical composition of intracellular fluids are reflected in the extracellular tissue fluid and consequently in the circulating blood of organisms which contacts that tissue. Alterations in blood composition may, in turn, be reflected in altered urinary composition and other body fluids. Thus, disease states, biological conditions which cause disruption of normal metabolic processes and consequent disease or toxic assaults, due to an unusual challenge such as a dose of a particular chemical compound e.g. a drug substance, which effect metabolic processes of the body are likely to be reflected in altered compositions of the body fluids. Body fluids thus provide important sample sources for testing for a disease state, metabolic state or oxidative state of the body as represented in the body fluid sample. Major alterations in such body fluids are frequently caused when toxins, for example liver or kidney toxins, are administered and inherent factors such as major enzyme deficiencies can also be identified from the body fluids. In the context of toxicology studies, changes in endogenous body fluid components that are induced by dosed substances may be used to assess toxic effects, associated levels of toxicity of compounds and also to identify relevant defensive processes or to monitor the progress of a therapeutic treatment on an organism.

As used herein the term animal is to be understood to include any living organism of the animal kingdom. Of particular relevance are the vertebrates including mammals, fish, amphibians, reptiles and birds. The term animal is particularly to be understood to include mammals such as a human, a mouse, a rat and other rodents, a pig, a cow, a bull, a sheep, a horse, a dog or a rabbit or any farmed animal or any animal, for example an animal used for the purpose of breeding.

As used herein the term molecular components as used herein in reference to samples is typically understood to include the combination of molecular chemical and/or biochemical species which comprise a biological sample such as for example a sample of body fluid or body tissue. The term includes the molecules found in living organisms and may comprise fats, proteins, nucleic acids, carbohydrates, minerals, vitamins, hormones, metabolic substrates, intermediates or products, cofactors, coenzymes and prosthetic groups.

As used herein the term molecular marker is typically understood to refer to a chemical or biochemical entity or quantity of that entity in the sample of body fluid or body tissue or a statistically associated combinations of entities, for example a ratio of quantities that is indicative of a state of oxidative stress, disease or toxicity associated with oxidative stress and may also be associated with a clinical outcome due to oxidative stress. Examples of such molecular markers include chemical and biological molecules, for example metabolic substrates, intermediates or products, structural proteins, nucleic acids, transport and receptor proteins, immunological proteins, proteins associated with metabolic or genetic control, catalytic proteins, enzymes and their associated cofactors, lipids, phospholipids, fats, carbohydrates, minerals, vitamins, hormones, cofactors, coenzymes and prosthetic groups Additionally the term molecular marker is also understood to include measurable signal or signals or function, including levels of activity of biological processes for example gene and protein expression and levels of activity of cellular signalling pathways, associated with such chemical or biochemical entities. Furthermore the term molecular marker is also understood to include the quantity of the chemical or biochemical entity in the sample of body fluid or body tissue or a statistically quantity associated with a combinations of entities, for example a ratio of quantities that is indicative of a state of oxidative stress, disease or toxicity associated with oxidative stress and may also be associated with a clinical outcome due to oxidative stress.

The following examples illustrate the embodiments and principles of the invention.

Examples

Introduction

Rifampicin (also known as rifampin) is a semi-synthetic antibiotic having antibacterial and tuberculostatic properties. Rifampicin is hepatotoxic leading to jaundice and can cause the elevation of plasma transaminases (Scheuer et al., 1974). Other features of rifampicin hepatotoxicity include impaired cholesterol synthesis (Zitkowa et al., 1982) and interference with bilirubin transport and conjugation, leading to hyperbilirubinaemia (Timbrell, 1991). Sodhi et al. (1997) reported that growing Wistar rats treated with rifampicin at 50 mg/kg/day for two weeks showed marked changes in the oxidative/anti-oxidative 'profile' of the liver; the treatment produced a significant increase in hepatic lipid peroxidation and decreased hepatic activities of superoxide dismutase, catalase and glutathione peroxidase. The latter two changes were the most substantial and would be likely to lead to an intracellular excess of hydrogen peroxide and Sodhi et al. speculated that an altered oxidative/anti- oxidative profile is closely associated with rifampicin-induced hepatic injury. There are various ways by which rifampicin might cause oxidative stress. Most obviously, rifampicin is a hydroquinone and it seems likely that rifampicin or one or more of its metabolites would behave similarly to the structurally analogous rifamycin SV and would undergo redox cycling leading to the production of superoxide and the loss of chemical reducing power in the form of NADPH. Excess hydrogen peroxide would be generated by the action of superoxide dismutase on the superoxide. The loss of NADPH would tend to inhibit the re-conversion of oxidized glutathione (GSSG) to glutathione (GSH) diminishing the capacity of the rat to combat excess hydrogen peroxide and free radicals (Halliwell and Gutteridge, 1999; Timbrell, 1991).

Outline of Study The study was conducted in accordance with the requirements of the relevant national legislation and local guidelines. Fifteen male Sprague-Dawley rats (ca. 7 weeks old at the start of the study and ca. 250g in mass at the time of dosing) were obtained from Charles River (France). They were kept in individual metabolic cages and given free access to water and a standard rodent diet. Rifampicin was administered as a single dose at two dose levels (400 and 1000 mg/kg) selected on the basis of literature information. The rifampicin was dosed orally as a suspension in an aqueous solution of methyl cellulose and Tween. The control rats were dosed orally with the same volume (lOml/kg) of the solution of methyl cellulose and Tween. Of the total of fifteen rats, five received the high dose of rifampicin, five received the low dose of rifampicin and five received the control treatment. Individual pre- and post-dose urine samples were collected, into ice-cooled vessels containing sodium azide, for seven hours daily. The rats were euthanased (using CO₂) at ca. 168 hours post- dosing.

NMR spectroscopic analysis of urine samples

Urine samples were prepared for NMR analysis by mixing 400 μl of urine with 200 μl of phosphate buffer (an 81:19 (v/v) mixture of 0.2 M Na₂HPO₄ and 0.2 M NaH₂PO_4; pH 7.4); if insufficient urine was available the shortfall was made up with purified water with a minimum of 200 μl of urine being used. The urine-buffer mixture was left to stand for 10 minutes at room temperature and then centrifuged at 13,000 rpm for a further 10 minutes to remove suspended particulates. 500 μl of 'clear' buffered urine was transferred to an NMR tube and 50 μl of a TSP/D₂O solution added. TSP (sodium 3-trimethylsilyl-[2, 2, 3, 3- HUj-1-propionate) is a chemical shift reference compound (δ 0) used in the NMR experiment and the D O provided a field/frequency lock for the NMR spectrometer. The concentration of the TSP/D₂O solution was such as to give a final TSP concentration of 0.1 mM in the NMR tube. The NMR analyses were carried out at 303K on a Bruker AMX 600 MHz NMR spectrometer with the standard NOESYPRESAT pulse sequence used to reduce the size of the water signal Quantitation of metabolites from NMR spectra

The urine samples selected for metabolite quantitation were those collected from 24- 17 hours pre-dose and from 0-7, 24-31, 48-55 and 144-151 hours post-dose. These samples were referred to as the day -1, day 1, day 2, day 3 and day 7 samples respectively, with dosing being performed at the start of day 1. Endogenous creatinine was used as the internal reference for the quantitation. This method of quantitation was chosen in preference to a measurement of metabolite excretion rates because analysis indicates that the latter can be badly affected by incomplete voiding of the bladder during the 7-hour urine collection periods with such errors being especially likely if the dosed compound causes a reduction in water drinking. Creatinine is an accepted internal reference for the quantitation of urinary components with the amount of creatinine excreted over a set period being proportional to muscle mass under nonnal circumstances. The NMR signals quantified were the acetate singlet at ca. δ 1.92, the succinate singlet at ca. δ 2.41, the central peak of the 2- oxoglutarate triplet at ca. δ 3.02, the creatinine singlet at ca. δ 4.05, the hippurate doublet at ca. δ 7.84 and the formate singlet at ca. δ 8.46. For ease of analysis, peak heights were measured instead of peak areas. Peak height ratios were then calculated relative to the height of the creatinine singlet at ca. δ 4.1.

Statistical analysis of data

All statistical analyses were carried out to compare dosed groups with same-study controls at the same time point. The method used was based on an analysis of variance performed on rank-transformed data, this being a method that does not assume a normal distribution of results. Pair-wise comparisons were then made as appropriate. The minimum acceptable level of significance for each test was P = 0.05.

Results The reported changes are in comparison to same-study controls sampled at the same time-point. Selected urinary excretion data are summarised in Tables 1 - 4. Both doses of rifampicin caused significant day 1 increases in the urinary excretion of acetate.

Both doses of rifampicin caused major day 1 and day 2 decreases in the urinary excretion of 2-oxoglutarate but by day 7 the levels of 2-oxoglutarate were approximately normal. Rifampicin dosing clearly increased day 1 succinate excretion in certain animals but overall the dosed groups showed no statistically significant difference from controls.

Both doses of rifampicin caused substantial day 2, day 3 and day 7 increases in formate excretion with a noticeably smaller increase on day 3 than on days 2 and 7.

Both doses of rifampicin caused extraordinary and persistent decreases in the urinary excretion of hippurate, which appeared to be loosely associated with the formate increases.

Discussion

The urinary findings are generally supportive of the idea that H₂O₂-induced oxidative effects are occurring in the rifampicin-dosed rats and the liver would be a likely site for such reactions. From the results it is possible that H O -induced oxidative decarboxylation of pyruvate might be producing acetate and urinary acetate increases which were found after rifampicin dosing. Pyruvate is not present in sufficient quantity to be readily NMR-visible in normal rat urine and it was therefore difficult to observe pyruvate changes. Decreases in the urinary level of 2-oxoglutarate are very commonly found with hepatotoxins (Clayton, 2001) but the decreases found after dosing rifampicin were particularly dramatic and this might possibly be due to H₂O₂- induced oxidative decarboxylation, which could produce succinate. Increases in the urinary level of succinate are not normally found after dosing hepatotoxins (Clayton, 2001) and the rifampicin-induced succinate increases are, therefore, especially supportive of the link between H₂O oxidative effects and production of succinate. It is worth noting that the conversion of 2-oxoglutarate to succinate would be particularly effective in producing a 1H NMR-visible increase in the product of the decarboxylation. The reason is that, whilst the relevant methylenes are not chemically equivalent in 2-oxoglutarate, they become chemically and magnetically equivalent in succinate; thus, 2-oxoglutarate' s pair of triplets is converted to a strong singlet in succinate. h the case of oxalacetate and malonate both these compounds the methylenic protons are in a very acidic environment, being situated between two adjacent carbonyls, and might be invisible to 1H NMR because of fast exchange with water protons. The observed increases in urinary formate might have arisen through hydrogen peroxide-induced oxidative decarboxylation of glyoxylic acid, which is normally produced in large quantity by the mammalian liver (Holmes and Assimos, 1998).

It should be noted that microbiological contamination of urine can possibly lead to increased levels of both formate and acetate (Sweatman et al. 1993) but, with the precautions taken in the present study, such microbiological effects are assumed to be absent.

The extraordinary and persistent decreases in urinary hippurate might be a consequence of the rifampicin-induced destruction of the gut microflora, since the gut micro flora are believed to have a role in the metabolic fate of benzoic acid (Phipps et ah, 1998). Another possible cause of the observed hippurate decrease might be glycine depletion in the liver, where hippurate is normally synthesized from glycine and benzoic acid. Glycine depletion might result from glycine oxidation or could possibly arise through decreased glycine synthesis from glyoxylate. Decreased glycine synthesis from glyoxylate is a possible explanation because glyoxylate might be converted to formate through oxidative decarboxylation. Furthermore, the apparent correlation in timing between the urinary formate increases and the urinary hippurate decreases suggests that there is less synthesis of hippurate because of the oxidative decarboxylation of glyoxylic acid. However another possible cause of the loss of urinary hippurate could be because the benzoic acid precursor has been attacked by the hydroxyl radical. Both decarboxylation and hydroxylation reactions are possible when aromatic carboxylic acids are attacked by the hydroxyl radical and the products of such reactions have been used to measure the amount of hydroxyl radical in biological systems (Halliwell and Gutteridge, 1999). In fact, sodium benzoate is used as a food/drink preservative and could be considered as an antioxidant. Hence, there are reasons to suggest that a decrease in urinary hippurate could be a useful marker of oxidative stress.

Tables

Table 1. The urinary excretion of formate measured relative to creatinine. Group Day - 1 Day + 2 Day + 3 Day + 7 Controls 0.083 0.056 0.058 0.044 Rifampicin 400 mg/kg 0.069 0.21** 0.15** 0.46* Rifampicin 1000 mg/kg 0.071 0.31*** 0.18** 0.61** Formate excretion is expressed relative to creatinine as internal reference with the quoted values being (height of formate peak at δ 8.46)/(height of creatinine methylene peak at δ 4.05). Each of the quoted values is the average for the relevant group, with n = 5 usually. The means marked with one or more asterisks (*, ** or ***) are those where the values for the treated group show a statistically significant difference (P = 0.05) from the values for the relevant control group. * designates a significant difference at P = 0.05; ** designates a significant difference at P = 0.01; *** designates a significant difference at P = 0.001.

Table 2. The urinary excretion of formate expressed relative to same day controls.

Group Day - 1 Day + 2 Day + 3 Day + 7 Controls 1.0 1.0 1.0 1.0 Rifampicin 400 mg/kg 0.8 3.7 2.5 11 Rifampicin 1000 mg kg 0.9 5.5 3.1 14 The values quoted are derived from the data shown in Table 1 by taking ratios with respect to the relevant control value. The values are quoted to a maximum of 2 s.f. and to a maximum of 1 d.p.. Values equal to or greater than 2 are given in bold. Table 3. The urinary excretion of 2-oxoglutarate, succinate and acetate.

Day - 1 (0-7 hours) Day + 1 (0-7 hours) Group 2-OG Succinate Acetate 2-OG Succinate Acetate

Controls 1.94 1.04 0.129 1.99 0.98 0.151 Rifampicin 400 mg/kg 2.16 1.02 0.123 0.05*** 1.38 0.392* Rifampicin 1000 mg/kg 2.39 1.14 0.116 0.19** 1.94 0.545** 2-OG denotes 2-oxoglutarate. The values quoted are the averages of the peak height ratios measured relative to the creatinine singlet at δ 4.05. These values are quoted to a maximum of 3 s.f. The means marked with one or more asterisks (*, ** or ***) are those where the values for the treated group show a statistically significant difference (P = 0.05) from the values for the relevant control group. * designates a significant difference at P = 0.05; ** designates a significant difference at P = 0.01; *** designates a significant difference at P = 0.001.

Table 4. The urinary excretion of hippurate expressed relative to same day controls*.

Group Day +2 Day + 7 Controls 1.0 1.0 Rifampicin 400 mg/kg 0.063 0.081 Rifampicin 1000 mg/kg 0.065 0.042 * These figures are derived from the individual values for the ratio (height of hippurate doublet at δ 7.84/height of creatinine peak at δ 4.05). The values quoted are group averages expressed as a multiple of the average level in the controls sampled at the same time-point. The values are quoted to 2 significant figures. No statistical analysis is provided but the decreases in urinary hippurate excretion caused by rifampicin were extremely clear.

References

Halliwell, B. and Gutteridge, J. M. C. (1999) Free radicals in biology and medicine, 3 ,rd Edition. Oxford University Press, Oxford. Scheuer et al. (1974) Rifampicin hepatitis: A clinical and histological study.

Lancet, 1, 421-425.

Timbrell J. A. (1991) Principles of Biochemical Toxicology, 2nd Edition. Taylor and Francis, London, Bristol PA. Zitkowa et al (1982) Causes for rifampicin hepatotoxicity, An experimental study. Czechosolvak. Med., 5, 210-217.

Sodhi et al (1997) Study of oxidative stress in rifampicin-induced hepatic injury in young rats with and without protein-energy malnutrition. Human Exp. Toxicol, 16, 315-321. Clayton T. A. (2001). PhD thesis. University of London.

Sweatman et al (1993) NMR of biofluids: detection of ²H-acetate and ²H-formate in urine as an indicator of microbiological contamination. J. Pharmaceut. Biomed. Anal., 11, 169-172.

Holmes and Assimos (1998). Glyoxylate synthesis, and its modulation and influence on oxalate synthesis. J. Urol., 160, 1617-1624.

Phipps et al (1998) Effect of diet on the urinary excretion of hippuric acid and of dietary-derived aromatics in the rat. A complex interaction between diet, gut microflora and substrate specificity. Xenobiotica, 28, 527-537.

Claims

1) A method of detecting molecular markers indicative of oxidative stress comprising;

c) the second sample set being derived from a second animal population which has been arranged to have, or has, oxidative stress,

f) selecting one or more of the molecular components identified in step (e),

g) determining the molecular identity of the molecular component(s) and identifying the molecular component(s) as a molecular marker(s) of oxidative stress. 2) The method according to claim 1 wherein the animal population in either of steps (b) or (c) consists of a single animal.

3) The method according to claim 1 wherein the animal population in either of steps (b) or (c) consists of two or more animals.

4) The method according to any one of claims 1 to 3 wherein the second animal population is the same as the first animal population and where the first and second sample sets are provided at different timepoints separated by the induction of oxidative stress in the animal population.

5) The method according to any one of claims 1 to 4 wherein the second animal population is arranged to have oxidative stress by administration of a compound causing oxidative stress.

6) The method according to any one of claims 1 to 5 wherein the compound causing oxidative stress is selected from the group comprising; rifampicin, hydrogen peroxide, paraquat and menadione, nitrogen dioxide, carbon tetrachloride, methyl chloride, allyl formate, allyl alcohol, acrolein, alloxan and dialuric acid.

7) An assay for the determination of the degree of oxidative stress in a living organism, wherein the change in the quantity of a molecular marker in a test sample from the organism determined relative to the quantity of a molecular marker in a reference sample from reference organism is taken as indicative of the degree of oxidative stress, wherein the molecular marker is selected from the group comprising; 2-oxoglutarate, 2-oxoglutaric acid, succinate, succinic acid, glyoxylate, glyoxylic acid, formate, formic acid, oxaloacetate, oxaloacetic acid, malonate, malonic acid, 2- oxoadipate, 2-oxoadipic acid, glutarate, glutaric acid, 2-oxohepatanedioate, 2- oxoheptanedioic acid, adipate, adipic acid, acetate, acetic acid, hippurate, hippuric acid, glycine, ammonia, ammonium ion, L-glutamic acid and L-glutamate.

8) A method of identifying oxidative stress in an animal using the assay as claimed in claim 7, said method comprising; a) determining the quantity of a molecular marker in a test sample of body fluid or body tissue taken from the animal, the marker being associated with oxidative stress in that animal species,

c) the presence of oxidative stress in the animal being identified by a higher or lower value of the former quantity, for the test sample, relative to the latter, for the reference sample,

wherein the molecular marker is selected from the group comprising; 2-oxoglutarate, 2-oxoglutaric acid, succinate, succinic acid, glyoxylate, glyoxylic acid, formate, formic acid, oxaloacetate, oxaloacetic acid, malonate, malonic acid, 2-oxoadipate, 2- oxoadipic acid, glutarate, glutaric acid, 2-oxohepatanedioate, 2-oxoheptanedioic acid, adipate, adipic acid, acetate, acetic acid, hippurate, hippuric acid, glycine, ammonia, ammonium ion, L-glutamic acid and L-glutamate.

9) The method according to claim 8 wherein the quantity of more than one molecular marker is determined.

10) A modification of the method according either of claims 8 or 9 wherein in step (a) a quantity ratio of two molecular markers is determined for the test sample, and in step (b) that ratio is compared to a predetermined reference sample quantity ratio or range of quantity ratio of two molecular markers selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid.

11) The method according to claim 10 wherein the two molecular markers of the quantity ratio are selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid , formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2- oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2-oxohepatanedioate or 2- oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L- glutamate or L-glutamic acid and ammonia or ammonium ion.

12) A method of screening a compound for its ability to induce oxidative stress in an animal using the assay as claimed in claim 7, said method comprising;

a) providing a reference sample of body fluid or body tissue taken from an animal prior to dosing with a test compound,

b) dosing the animal with the test compound,

c) providing a corresponding test sample of body fluid or body tissue taken from the animal post dosing,

d) determining the quantity of a molecular marker in the reference sample and comparing it with the quantity of the molecular marker in the test sample, a higher or lower quantity of the marker in the latter relative to the former being indicative of the ability of the compound to cause oxidative stress in the animal and wherein the molecular marker is selected from the group consisting of the group set forth in claim 8.

13) The method according to claim 12 wherein the quantity of more than one molecular marker is determined.

14) A method according to either of claims 12 or 13 wherein a plurality of animals are provided at step (a) and a progressively increasing quantity of the test compound is dosed at step (b) to the several animals, the comparison of the quantity of molecular marker(s) at step (d) being made for each of the several animals and the higher or lower quantity of the molecular marker(s) in each of the several animals are compared to each other to estimate the quantity of the test compound which produces a given percentage change in molecular marker quantity in the animals.

15) A modification of the method according to any one of claims 12 to 14 wherein in step (d) a quantity ratio of two molecular markers is determined for the reference sample and that ratio is compared with a quantity ratio of two molecular markers in the test sample wherein the two molecular markers are selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid.

16) A method of screening a compound for its ability to induce oxidative stress in an animal using the assay as claimed in claim 7 said method comprising,

a) dosing the animal with a test compound

b) providing a test sample of body fluid or body tissue taken from the animal post dosing,

c) determining the quantity of a molecular marker in the test sample and comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker characteristic of a population of that animal species not suffering from oxidative stress, a higher or lower quantity of the molecular marker in the former relative to the latter being indicative of the ability of the compound to cause oxidative stress in the animal and wherein the molecular marker is selected from the group consisting of the group set forth in claim 8.

17) The method according to claim 16 wherein the quantity of more than one molecular marker is determined.

18) A method according to either one of claims 16 or 17 wherein a plurality of animals are provided at step (a) and a progressively increasing quantity of the test compound is dosed to the several animals, the comparison of the quantity of molecular marker(s) at step (c) being made for each of the several animals and the higher or lower quantity of the molecular marker(s) in each of the several animals are compared to each other to estimate the quantity of the test compound which produces a given percentage change in molecular marker quantity in the animals.

19) A modification of the method according to any one of claims 16 to 18 wherein at step (c) a quantity ratio of two molecular markers is determined for the test sample, and that ratio is compared to a predetermined reference sample quantity ratio or range of quantity ratio of two molecular markers selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid.

20) The method according to either of claims 15 or 19 wherein the two molecular markers of the quantity ratio are selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid , formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2-oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2- oxohepatanedioate or 2-oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L-glutamate or L-glutamic acid and ammonia or ammonium ion

21) A method of treating an animal diagnosed with a condition of oxidative stress comprising;

a) use of the assay as claimed in either of claims 7 or 8 to identify the presence of oxidative stress in that animal then,

b) administering antioxidant therapy to the animal if the quantity of the molecular marker in the reference sample is higher or lower relative to the test sample and wherein the molecular marker is selected from the group consisting of the group set forth in claim 8.

22) The method according to claim 21 wherein the quantity of more than one molecular marker is determined.

23) A modification of the method according to either of claims 21 or 22 wherein in step (b) a quantity ratio of two molecular markers is determined for the test sample, and that ratio is compared to a predetermined reference sample quantity ratio or range of quantity ratio of two molecular markers selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid. 24) The method according to claim 23 wherein the two molecular markers of the quantity ratio are selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid , formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2- oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2-oxohepatanedioate or 2- oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L- glutamate or L-glutamic acid and ammonia or ammonium ion

25) The method according to any one of claims 21 to 24, further comprising the steps of;

b) determining the quantity of a molecular marker(s) in the test sample and comparing that quantity with a predetermined reference sample quantity or quantity range of the molecular marker(s) characteristic of a population of that animal species not suffering from oxidative stress to determine if the condition of oxidative stress has improved wherein the molecular marker(s) is selected from the group consisting of the group set forth in claim 8.

26) The method according to any one of claims 21 to 25 wherein the antioxidant therapy comprises the administration of an alpha-oxo acid or its corresponding anion.

27) The method according to any one of claims 21 to 25 wherein the antioxidant therapy comprises the administration of L-glutamate or L-glutamic acid or glycine.

28) The method according to claim 26 wherein the alpha-oxo acid or its corresponding anion selected from the group comprising; glyoxylate, glyoxylic acid, oxaloacetate, oxaloacetic acid, 2-oxoglutaric acid, 2-oxoglutarate. 29) A method of screening a compound for its ability to reduce oxidative stress in an animal using the assay as claimed in claim 7 or 8, said method comprising,

a) providing a reference sample of body fluid or body tissue taken from an animal suffering from oxidative stress,

b) dosing the animal with a test compound,

c) providing a test sample of body fluid or body tissue taken from the animal post dosing,

d) determining the quantity of a molecular marker in the reference sample and comparing it with the quantity of the molecular marker in the test sample, a higher or lower quantity of the marker in the latter test sample relative to the former reference sample being indicative of the ability of the compound to reduce oxidative stress in the animal and wherein the molecular marker is selected from the group consisting of the group set forth in claim 8.

30) The method according to claim 29 wherein the quantity of more than one molecular marker is determined.

31) A method according to either one of claims 29 or 30 wherein a plurality of animals are provided at step (a) and a progressively increasing quantity of the test compound is dosed to the several animals, the comparison of the quantity of molecular marker(s) at step (d) being made for each of the several animals and the higher or lower quantity of the molecular marker(s) in each of the several animals are compared to each other to estimate the quantity of the test compound which produces a given percentage change in molecular marker quantity in the animals.

32) A modification of the method according to any one of claims 29 to 31 wherein in step (d) a quantity ratio of two molecular markers is determined for the reference sample and that ratio is compared with a quantity ratio of two molecular markers in the test sample wherein the two molecular markers are selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid.

33) A method of screening a test compound for its ability to alter the level of oxidative stress in a test culture of cells using the assay as claimed in claim 7 said method comprising,

a) obtaining one or more reference samples from a reference cell culture,

b) introducing a quantity of the test compound into the test culture of cells,

c) obtaining one or more test samples from the test culture of cells,

d) determining the quantity of a molecular marker in the one or more test samples and comparing it with the quantity of the molecular marker in the one or more reference samples, a higher or lower quantity of the molecular marker in the latter relative to the former being indicative of the ability of the compound to alter the level of oxidative sfress in the culture of cells and wherein the molecular marker is selected from the group consisting of the group set forth in claim 8.

34) The method according to claim 33 wherein the reference culture is the same as the test culture.

35) The method according to either of claims 33 or 34, wherein the reference cell culture or test cell culture are under oxidative stress

36) The method according to either of claims 33 or 34, wherein the reference cell culture or test cell culture are not under oxidative stress

37) The method according to any one of claims 33 to 36 wherein the quantity of more than one molecular marker is determined. 38) A modification of the method according to any one of claims 33 to 36 wherein a quantity ratio of two molecular markers is determined for the test sample(s) and is compared to a quantity ratio or range of quantity ratio of two molecular markers determined for the reference sample(s) wherein the two molecular markers are selected from the group consisting of the group set forth in claim 8 and pyruvate and pyruvic acid.

39) The method according to claim 38 wherein the two molecular markers of the quantity ratio are selected from the paired groups comprising; succinate or succinic acid and 2-oxoglutarate or 2-oxoglutaric acid, acetate or acetic acid and pyruvate or pyruvic acid , formate or formic acid and glyoxylate or glyoxylic acid, malonate or malonic acid and oxaloacetate or oxaloacetic acid, glutarate or glutaric acid and 2- oxoadipate or 2-oxoadipic acid, adipate or adipic acid and 2-oxohepatanedioate or 2- oxoheptanedioic acid, hippurate or hippuric acid and formate or formic acid, L- glutamate or L-glutamic acid and ammonia or ammonium ion

40) A method according to any one of claims 1 to 32, wherein the sample is selected from the group of; urine, saliva, blood serum, blood plasma, blood, sweat, tears, faeces, bile, semen, vaginal secretions, aqueous humour, vitreous humour, cerebrospinal fluid, synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid, amniotic fluid, breath, breath condensate, liver tissue and kidney tissue.

41) A method according to any one of claims 1 to 40 wherein quantity of the molecular marker(s) is determined using nuclear magnetic resonance (NMR) spectroscopy and/or any other chemical analysis techniques such as mass spectroscopy (MS), infrared (IR) spectoscopy, gas chromatography (GC) and high performance liquid chromatography (HPLC) or by using any combination of such techniques e.g. GC-MS.

42) A method according to claim 1 to 41 wherein the test sample or samples are treated prior to analysis with one or more chemical reagents so as to produce derivative(s) of one or more molecular components, so as to enhance data recovery or to improve sample stability.