Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2001-05-31
2004-04-27
Gitomer, Ralph (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S007100
Reexamination Certificate
active
06727075
ABSTRACT:
BACKGROUND OF THE INVENTION
Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by a progressive decline in cognitive function, as well as by numerous amyloid plaques, neurofibrillary tangles (NFTs) and extensive neuronal loss in the brains of AD patients (Morrison-Bogorad et al., 1997, In
The Molecular and Genetic Basis of Neurological Disease
, Second edition, Butterworth-Heinemann, eds., pp 581-600). Although epidemiolgic studies have failed to identify a single cause of AD, genetic studies have implicated several mutations in three separate genes on different chromosomes that encode the amyloid-(A) precursor proteins (APP), presenilin-1 (PS-1), and presenilin-2 (PS-2) as the cause of autosomal dominantly inherited AD in a subset of kindreds with familial AD (FAD) (Van Duijn, 1996, J. Neurol. Neurosurg. Psychiatry 60:478-488; Goedert et al., 1997, In:
The Molecular and Genetic Basis of Neurological Disease
, Second edition, Butterworth-Heinemann, eds. pp. 613-628; Selkoe, 1997, Science 275:630-631). In addition, the 4 allele of the apolipoprotein E (APOE) gene has been shown to be a genetic risk factor for AD (Selkoe, 1997, Science 275:630-631). However, all of the known FAD mutations account for less than 5% of affected patients, since the majority of AD cases are sporadic and there is only modest evidence in support of familial clustering (Hardy, 1997, Proc. Natl. Acad. Sci USA 94:2095-2097).
Despite this heterogeneity, common factors may be involved in the pathogenesis of both hereditary and sporadic AD. These factors may promote the formation of A deposits and NFTs, as well as the massive degeneration of neurons in selected regions of all AD brains (Morrison-Bogorad et al., 1997 In:
The Molecular and Genetic Basis of Neurological Disease
, Second edition, Butterworth-Heinemann, eds. pp. 581-600). It has been suggested that the abnormal processing or production of A and plaque formation are pivotal events in the pathogenesis of the disease (Scheuner et al., 1996, Nature Med. 2:864-870; Mattson et al., 1992, Neurosc. 12:376-389). Furthermore, aggregated, but not monomeric species of A are hypothesized to induce the dysfunction and death of neurons in vitro by a range of mechanisms (Busciglio et al., 1995, Neuron. 14:879-888; Thomas et al., 1996, Nature 380:168-171; Behl et al., 1994, Cell 77:817-827). It has been hypothesized that AD brain regions which have accumulations of numerous A-rich senile plaques (SPs) are loci of elevated oxidative stress, perhaps reflective of an inflammatory reaction (Hensley et al, 1994, Proc. Natl. Acad. Sci. USA 91:3270-3274). Furthermore, it has been suggested that oxidant stress may be of functional importance in the pathogenesis of AD and that the production of reactive oxygen species (ROS) in the brain leads to lipid peroxidation and neuronal degeneration in AD (Gotz et al., 1994, Proc. Natl. Acad. Sci. USA 91:3270-3274).
Although there has been much speculation that ROS may play an important role in AD, there have been few data in support of this hypothesis. Efforts to elucidate the role of lipid peroxidation and oxidant stress in vivo have been hampered by the paucity of reliable quantitative molecular markers. Currently available molecular markers have been of limited value due to their chemical instability or their lack of sensitivity or specificity (Gutteridge and Halliwell, 1990, Trends Biochem. Sci. 15:129-1365).
The few studies which have been reported thus far of lipid peroxidation in the AD brain have provided evidence for increased lipid peroxidation by measuring levels of thiobarbituric acid reactive substances (TBARS) (Subbarao et al., 1990, J. Neurochem. 55:342-345; Palmer and Burns, 1994, Brain Res. 645:338-342; Lovell et al., 1995, Neurology 45:1594-1601; Balazs and Leon, 1994, Neuroch. Res. 19:1131-1137). However, the validity of this method is limited because it measures other aldehydes conjugated to TBARS, as well as non-lipid related chromogens. Recently, two separate groups of investigators have reported no difference in the level of TBARS and lipid hydroperoxides in AD versus control brains (Lyras et al., 1997, J. Neurochem. 68:2061-2069; Hayn et al., 1996, Life Sci. 59:537-544). Immunohistochemical data suggest the presence in AD brain of stable by-products of lipid peroxidation (Montine et al., 1997, J. Neuropath. Exper. Neurol. 56:866-871; Sayre et al., 1997, J. Neurochem. 68:2092-2097). While increased levels of 4-hydroxynonenal in post-mortem CSF of AD patient has been reported, no such quantitative data are available for this compound in AD brains (Lovell et al., 1997, Neurobiol. Aging 18:457-461).
Thus, there is an unmet need in the art for compositions and methods relating to molecular markers of oxidant stress or lipid peroxidation in a mammal for use in the diagnosis, treatment and development of therapeutics for diseases which manifest oxidant stress, such as Alzheimer's disease. The present invention meets these needs.
BRIEF SUMMARY OF THE INVENTION
The invention relates to a method of measuring the level of lipid peroxidation in a mammal suspected of having an oxidant stress syndrome or disease. The method comprises a) obtaining a first sample of a tissue or body fluid from the mammal; b) assessing the level of an isoprostane molecular marker for lipid peroxidation present in the first sample; and, c) comparing the level of the isoprostane molecular marker present in the first sample with the level of the isoprostane molecular marker present in a second sample of a tissue or body fluid obtained from an otherwise identical mammal which is not afflicted with an oxidant stress syndrome or disease, wherein an elevated level of the isoprostane molecular marker in the first sample relative to the level of the isoprostane molecular marker in the second sample, is indicative of an elevated level of lipid peroxidation in the mammal, thereby indicating the presence of an oxidant stress syndrome or disease in the mammal.
In one aspect, the method further comprises after a) and prior to b) isolating from the first sample the isoprostane molecular marker.
In another aspect, the elevated level of lipid peroxidation comprises an elevated level of a reactive oxygen species (ROS).
In yet another aspect, the elevated level of lipid peroxidation comprises an elevated level of inflammation.
In one embodiment, the elevated level of inflammation comprises elevated cyclooxygenase (COX) activity.
In yet a further aspect, the oxidant stress disease is Alzheimer's disease.
In another aspect, the isoprostane molecular marker is selected from the group consisting of iPF
2&agr;
-III, iPF
2&agr;
-VI and 8,12-iso-iPF
2&agr;
-VI.
In an additional aspect, the tissue is brain tissue.
In one embodiment, the brain tissue is selected from the group consisting of brain frontal pole tissue and brain temporal pole tissue.
In another embodiment, the body fluid is selected from the group consisting of cerebrospinal fluid (CSF), plasma and urine.
The invention also relates to a method of diagnosing an oxidant stress syndrome or disease in a mammal. The method comprises a) obtaining a first sample of a tissue or body fluid from the mammal; b) assessing the level of the isoprostane molecular marker present in the first sample; and, c) comparing the level of the isoprostane molecular marker present in the first sample with the level of the isoprostane molecular marker present in a second sample of a tissue or body fluid obtained from an otherwise identical mammal which is not afflicted with the oxidant stress syndrome or disease, wherein an elevated level of the isoprostane molecular marker in the first sample relative to the level of the isoprostane molecular marker in the second sample, is indicative of an elevated level of lipid peroxidation in the mammal, whereby the oxidant stress syndrome or disease is diagnosed in the mammal.
In one aspect, the method further comprises after a) and before b) isolating from the first sample the isoprostane molecular marker.
Also included in the invention is a method of m
Fitzgerald Garret A.
Pratico Domenico
Rokach Joshua
Trojanowski John Q.
Gitomer Ralph
Morgan & Lewis & Bockius, LLP
The Trustees of the University of Pennsylvania
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