+

WO2005052575A1 - Marqueurs moleculaires du stress oxydatif - Google Patents

Marqueurs moleculaires du stress oxydatif Download PDF

Info

Publication number
WO2005052575A1
WO2005052575A1 PCT/IB2004/003748 IB2004003748W WO2005052575A1 WO 2005052575 A1 WO2005052575 A1 WO 2005052575A1 IB 2004003748 W IB2004003748 W IB 2004003748W WO 2005052575 A1 WO2005052575 A1 WO 2005052575A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
oxidative stress
molecular
animal
sample
Prior art date
Application number
PCT/IB2004/003748
Other languages
English (en)
Inventor
Claude Charuel
Thomas Andrew Clayton
Jeremy Ramsey Everett
John Christopher Lindon
Jeremy Kirk Nicholson
Original Assignee
Pfizer Limited
Pfizer Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0327743A external-priority patent/GB0327743D0/en
Application filed by Pfizer Limited, Pfizer Inc. filed Critical Pfizer Limited
Publication of WO2005052575A1 publication Critical patent/WO2005052575A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • 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
  • 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.
  • 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.
  • 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.
  • 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).
  • MDA malondialdehyde
  • 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 ⁇ , ⁇ -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.
  • FIG. 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.
  • 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.
  • the invention provides a therapeutic method for treating an animal subject diagnosed with a condition of oxidative stress.
  • This aspect provides a method of detecting molecular markers indicative of oxidative stress comprising;
  • the first sample set being derived from a first animal population not having oxidative stress
  • the second sample set being derived from a second animal population which has, or has been arranged to have oxidative stress
  • step (e) 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • NMR nuclear magnetic resonance
  • MS mass spectroscopy
  • IR infrared
  • RAMAN spectoscopy
  • UV ultra violet
  • UV gas chromatography
  • HPLC high performance liquid chromatography
  • LC liquid chromatography
  • the chosen analytical method is one that provides quantitative multi-component analysis.
  • 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.
  • NMR nuclear magnetic resonance
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • LC liquid chromatography
  • 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.
  • 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.
  • 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).
  • MAS Magic Angle Spinning
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the animals in steps (b) or (c) will be of the same species.
  • the animals will be all of the same sex, alternatively mixed sex populations may be used in steps (b) and (c).
  • 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.
  • 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,
  • 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.
  • 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.
  • the animal is a rat, most preferably a human.
  • 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.
  • 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;
  • 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.
  • 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.
  • a quantity of more than one molecular marker may be determined.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the plurality of animals are all members of the same species.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • the plurality of animals are all members of the same species.
  • 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;
  • 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;
  • the reference animal or group of reference animals are preferably derived from an animal population not having oxidative stress.
  • 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.
  • a quantity of more than one molecular marker may be determined.
  • 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.
  • 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 ⁇ , ⁇ -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.
  • MDA thiobarbituric acid reactive substances malondialdehyde
  • 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 oxal
  • the method may comprise the steps of;
  • 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
  • the antioxidant therapy may comprise the administration of an
  • 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,
  • test compound 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the plurality of animals are all members of the same species.
  • a quantity of more than one molecular marker may be determined
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • NMR nuclear magnetic resonance
  • IR infrared
  • GC gas chromatography
  • HPLC high performance liquid chromatography
  • 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.
  • a molecular marker 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.
  • 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.
  • the molecular marker is a metabolic substrate, intermediate or product.
  • a sample or a body fluid sample 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.
  • a sample or body tissue sample 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the animal is a rat, most preferably a human.
  • 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.
  • 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 ⁇ , ⁇ -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.
  • MDA malondialdehyde
  • 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.
  • 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.
  • 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.
  • 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.
  • procedures such as pattern recognition methods for example partial least squares or partial least squares discriminant analysis.
  • 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.
  • the level of significance, or significance level, of the selected statistical procedure 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.
  • a 0.001 to 0.05 significance level is used.
  • Most a 0.05 significance level is used.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • glucose is preferably used as the internal reference in conjunction with separate glucose determinations on each sample.
  • the dose is delivered orally, intravenously, injected parenterally, injected intramuscularly, injected subcutaneously, by inhalation, by suppository, pessary or topically.
  • 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.
  • the dose may be delivered by intravenous infusion, preferably at a dose which is of the range from 0.001-100 mg/kg/hr.
  • 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.
  • 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.
  • the mathematical relationship between the two variable sets is derived by graphical methods, more preferably using curve fitting procedures.
  • the mathematical relationship between the two variable sets is derived by parametric methods and/or statistical methods.
  • 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.
  • 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.
  • 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 vitreous humour
  • aqueous humour aqueous humour
  • 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.
  • toxins for example liver or kidney toxins
  • 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.
  • 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.
  • 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.
  • 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.
  • molecular markers examples 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.
  • 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.
  • Rifampicin also known as rifampin
  • 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.
  • 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).
  • 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 CO 2 ) at ca. 168 hours post- dosing.
  • Urine samples were prepared for NMR analysis by mixing 400 ⁇ l of urine with 200 ⁇ l of phosphate buffer (an 81:19 (v/v) mixture of 0.2 M Na 2 HPO 4 and 0.2 M NaH 2 PO 4; pH 7.4); if insufficient urine was available the shortfall was made up with purified water with a minimum of 200 ⁇ l 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 ⁇ l of 'clear' buffered urine was transferred to an NMR tube and 50 ⁇ l of a TSP/D 2 O solution added.
  • TSP sodium 3-trimethylsilyl-[2, 2, 3, 3- HUj-1-propionate
  • ⁇ 0 chemical shift reference compound
  • the concentration of the TSP/D 2 O 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. ⁇ 1.92, the succinate singlet at ca. ⁇ 2.41, the central peak of the 2- oxoglutarate triplet at ca. ⁇ 3.02, the creatinine singlet at ca. ⁇ 4.05, the hippurate doublet at ca. ⁇ 7.84 and the formate singlet at ca. ⁇ 8.46.
  • peak heights were measured instead of peak areas. Peak height ratios were then calculated relative to the height of the creatinine singlet at ca. ⁇ 4.1.
  • 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.
  • 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.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne une méthode de détection de marqueurs moléculaires révélateurs du stress oxydatif et les marqueurs moléculaires ainsi détectés. Elle concerne également une méthode d'identification du stress oxydatif dans un organisme vivant, et d'autres méthodes permettant de déterminer si des composés induisent ou atténuent le stress oxydatif, ainsi que des méthodes permettant de réduire ou de prévenir le stress oxydatif dans un organisme.
PCT/IB2004/003748 2003-11-28 2004-11-15 Marqueurs moleculaires du stress oxydatif WO2005052575A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB0327743.1 2003-11-28
GB0327743A GB0327743D0 (en) 2003-11-28 2003-11-28 Molecular markers of oxidative stress
US55893704P 2004-04-02 2004-04-02
US60/558,937 2004-04-02

Publications (1)

Publication Number Publication Date
WO2005052575A1 true WO2005052575A1 (fr) 2005-06-09

Family

ID=34635448

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2004/003748 WO2005052575A1 (fr) 2003-11-28 2004-11-15 Marqueurs moleculaires du stress oxydatif

Country Status (1)

Country Link
WO (1) WO2005052575A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007083632A1 (fr) 2006-01-20 2007-07-26 Keio University Méthode pour déterminer un stress oxydatif
JP2010527624A (ja) * 2007-05-31 2010-08-19 バイオクレイツ ライフ サイエンス エージー 酸化ストレスレベルを測定するためのバイオマーカーおよび方法
WO2011010103A1 (fr) * 2009-07-22 2011-01-27 Imperial Innovations Limited Méthodes et utilisations
WO2011010104A1 (fr) * 2009-07-22 2011-01-27 Imperial Innovations Limited Procédés
US7981399B2 (en) 2006-01-09 2011-07-19 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
US20130149388A1 (en) * 2010-01-04 2013-06-13 Joseph Louie MCCLAY Metabolomics-based biomarkers for lung function
WO2016081534A1 (fr) * 2014-11-19 2016-05-26 Metabolon, Inc. Biomarqueurs pour la stéatose hépatique et leurs procédés d'utilisation

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6096556A (en) * 1998-02-09 2000-08-01 Washington University Method for the determination of oxidative stress
WO2001009118A2 (fr) * 1999-07-29 2001-02-08 Patrick T Prendergast Composes dithiolthione pour le traitement de troubles neurologiques et pour renforcer la memoire
US20030044878A1 (en) * 2001-05-09 2003-03-06 Christiaan Leeuwenburgh Method and system to test a substance for inflammatory or oxidant properties
US20030211622A1 (en) * 1998-06-29 2003-11-13 Roberts L. Jackson Methods and compositions to assess oxidative brain injury

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6096556A (en) * 1998-02-09 2000-08-01 Washington University Method for the determination of oxidative stress
US20030211622A1 (en) * 1998-06-29 2003-11-13 Roberts L. Jackson Methods and compositions to assess oxidative brain injury
WO2001009118A2 (fr) * 1999-07-29 2001-02-08 Patrick T Prendergast Composes dithiolthione pour le traitement de troubles neurologiques et pour renforcer la memoire
US20030044878A1 (en) * 2001-05-09 2003-03-06 Christiaan Leeuwenburgh Method and system to test a substance for inflammatory or oxidant properties

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM), BETHESDA, MD, US; January 1998 (1998-01-01), MADEKIN A S ET AL: "[Antioxidant therapy of vision organ disorders in the Chernobyl accident liquidators]", XP002320997, Database accession no. NLM9584637 *
KOWLURU R A ET AL: "Retinal glutamate in diabetes and effect of antioxidants.", NEUROCHEMISTRY INTERNATIONAL. APR 2001, vol. 38, no. 5, April 2001 (2001-04-01), pages 385 - 390, XP002321001, ISSN: 0197-0186 *
SODHI C P ET AL: "Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats", DRUG AND CHEMICAL TOXICOLOGY AN INTERNATIONAL JOURNAL FOR RAPID COMMUNICATION, vol. 20, no. 3, 1997, pages 255 - 269, XP009045089, ISSN: 0148-0545 *
VESTNIK OFTALMOLOGII. 1998 JAN-FEB, vol. 114, no. 1, January 1998 (1998-01-01), pages 31 - 34, ISSN: 0042-465X *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7981399B2 (en) 2006-01-09 2011-07-19 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
US8486690B2 (en) 2006-01-09 2013-07-16 Mcgill University Method to determine state of a cell exchanging metabolites with a fluid medium by analyzing the metabolites in the fluid medium
EP1975621A1 (fr) * 2006-01-20 2008-10-01 Keio University Methode pour determiner un stress oxydatif
EP1975621A4 (fr) * 2006-01-20 2009-02-11 Univ Keio Methode pour determiner un stress oxydatif
WO2007083632A1 (fr) 2006-01-20 2007-07-26 Keio University Méthode pour déterminer un stress oxydatif
US8309056B2 (en) 2006-01-20 2012-11-13 Keio University Method for determination of oxidative stress
US7964177B2 (en) 2006-01-20 2011-06-21 Keio University Method for determination of oxidative stress
JP2010527624A (ja) * 2007-05-31 2010-08-19 バイオクレイツ ライフ サイエンス エージー 酸化ストレスレベルを測定するためのバイオマーカーおよび方法
WO2011010104A1 (fr) * 2009-07-22 2011-01-27 Imperial Innovations Limited Procédés
WO2011010103A1 (fr) * 2009-07-22 2011-01-27 Imperial Innovations Limited Méthodes et utilisations
US20130149388A1 (en) * 2010-01-04 2013-06-13 Joseph Louie MCCLAY Metabolomics-based biomarkers for lung function
US9823204B2 (en) * 2010-01-04 2017-11-21 Lineagen, Inc. Metabolomics-based biomarkers for lung function
WO2016081534A1 (fr) * 2014-11-19 2016-05-26 Metabolon, Inc. Biomarqueurs pour la stéatose hépatique et leurs procédés d'utilisation

Similar Documents

Publication Publication Date Title
EP1540560B1 (fr) Phenotypage metabolique
Griffin et al. NMR spectroscopy based metabonomic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat
Berger et al. Relationship between dietary intake and tissue levels of reduced and total vitamin C in the nonscorbutic guinea pig
Hollinger et al. Case management and plasma half-life in a case of brodifacoum poisoning
Basoglu et al. Nuclear magnetic resonance (NMR)-based metabolome profile evaluation in dairy cows with and without displaced abomasum
Bertram et al. NMR-based metabonomic studies reveal changes in the biochemical profile of plasma and urine from pigs fed high-fibre rye bread
Chamorro-García et al. Transgenerational metabolomic fingerprints in mice ancestrally exposed to the obesogen TBT
Trexel et al. Macrophage-mediated clofazimine sequestration is accompanied by a shift in host energy metabolism
Mills et al. Sample processing alters glutathione and cysteine values in blood
Guo et al. Rosmarinic acid alleviates ethanol-induced lipid accumulation by repressing fatty acid biosynthesis
Lee et al. Associations between local acidosis induced by renal LDHA and renal fibrosis and mitochondrial abnormalities in patients with diabetic kidney disease
Stratton et al. Urinary excretion of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans
Ito et al. A single oral supplementation of nicotinamide within the daily tolerable upper level increases blood NAD+ levels in healthy subjects
Li et al. Metabolomics profiling reveals the mechanism of caffeic acid in extending lifespan in Drosophila melanogaster
WO2005052575A1 (fr) Marqueurs moleculaires du stress oxydatif
Vendramini et al. Serum metabolomics analysis reveals that weight loss in obese dogs results in a similar metabolic profile to dogs in ideal body condition
Tian et al. The hematinic effect of Colla corii asini (Ejiao) using 1 H-NMR metabolomics coupled with correlation analysis in APH-induced anemic rats
KR20120022849A (ko) 파브리병을 위한 마커 및 치료제로서의 테트라하이드로바이오프테린의 용도
Yang et al. Metabolomic studies on the systemic responses of mice with oxidative stress induced by short-term oxidized tyrosine administration
US6133039A (en) In vivo method for determination of oxidative stress
Shaghaghi et al. Glutamine restores mitochondrial respiration in bleomycin-injured epithelial cells
Aramaki et al. Urinary excretion of 2‐methylacetoacetate, 2‐methyl‐3‐hydroxybutyrate and tiglylglycine after isoleucine loading in the diagnosis of 2‐methylacetoacetyl‐CoA thiolase deficiency
Salek et al. A metabolomic study of brain tissues from aged mice with low expression of the vesicular monoamine transporter 2 (VMAT2) gene
Tripathi et al. Aluminum‐mediated metabolic changes in rat serum and urine: A proton nuclear magnetic resonance study
US20040191913A1 (en) Method of evaluation of antioxidation ability by measuring redox balance in vivo

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DPEN Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载