Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
1999-03-04
2001-02-06
Leary, Louise N (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S004000, C435S975000
Reexamination Certificate
active
06183981
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of pathology and diagnostics. More particularly, it concerns the early diagnosis of late-onset sporadic Alzheimer's Disease (AD). More specifically, the invention relates to detecting changes in the level of mitochondrial cytochrome oxidase for diagnosing and monitoring AD.
2. Description of Related Art
Alzheimer's Disease (AD) is characterized by initial memory loss, followed by progressive loss of neurons leading to dementia and loss of all nervous functions, and eventually death. AD is now the fourth-largest killer of adults 65 and older, and this disease impacts one of every three families in the United States (Gonzalez-Lima and Gonzalez-Lima, 1987), and affects over 13 million people world-wide. As the population trends lead to an increase in the number of older people, this figure will only increase.
AD may be categorized in one of two forms; familial AD or sporadic AD Familial AD accounts for only about 5% to 10% of all Alzheimer's cases and has an unusually early onset, generally before the age of fifty. Familial AD is inherited and follows conventional patterns of Mendelian inheritance. This form of AD has been linked to nuclear chromosomal abnormalities. In contrast, the second form of AD, sporadic AD, is a late-onset disease which is not inherited or caused by nuclear chromosomal abnormalities. This late-onset form of the disease is the more common type of AD and is believed to account for approximately 90 to 95% of all Alzheimer's cases. The cause of sporadic AD is not known.
Mitochondrial DNA defects are thought to be involved in a number of degenerative diseases, including Leber's hereditary optic neuropathy and myoclonic epilepsy lactic acidosis and stroke (MELAS). Such defects also are thought to involved in the “sporadic” (non-Mendelian) occurrence of neurodegenerative diseases such as Parkinson's and AD. Further, considering that the mitochondrion is the site of the electron transport pathway, it is not surprising that aberrant electron function has been described in both Parkinson's disease and AD. Cytochrome oxidase (CO, also known as ferrocytochrome c:oxygen oxidoreductase, cytochrome aa3, EC 1.9.3.1) is a key component of the mitochondrial electron transport chain, where it is responsible for the activation of oxygen for aerobic energy metabolism in all eukaryotic cells. CO is the terminal rate-limiting enzyme for cellular respiration. Since the brain relies almost exclusively on the aerobic metabolism of glucose for its energy, CO function is essential for normal brain function.
In recent years it has been reported that defects in cytochrome oxidase, may be involved in AD. Parker et al. (1990) showed that patients with AD have reduced cytochrome oxidase activity. Further, it has also been shown that when rats are infused with sodium azide, a specific inhibitor of cytochrome oxidase, the rats suffer from a form of behavior dysfunction characterized by impaired memory and learning (Bennett et al., 1992). These rats mimic the effect of AD in humans. In addition, the sodium azide-treated rats failed to display long-term potentiation, demonstrating loss of neuronal plasticity. Therefore, a chronic defect in CO activity in AD leads to oxidative damage to mitochondrial DNA and preferential cell death in the brain (Mecocci et. al., 1994).
The diagnosis of AD is by clinical observation and definitive diagnosis is only accomplished by pathological examination at autopsy. There have been many attempts to diagnose AD by identifying differences in biological markers such as protease nexin II and apolipoprotein E alleles, as well as monitoring cathepsin D in the patient's cerebrospinal fluid (U.S. Pat. No. 5,686,269), monitoring amyloid precursor protein (U.S. Pat. No. 5,705,401), and monitoring concentration of neuronal membrane phospholipids and phospholipid metabolites (U.S. Pat. No. 5,545,566). However, these approaches generally have a low rate of success or involve invasive techniques that are at best uncomfortable and often unreliable.
Additionally, measurement of increased levels of neuronal thread protein in cerebrospinal fluid of Alzheimer's patients has been proposed as a diagnostic test (de la Monte et al., 1992). Recently, this test has been available commercially as AD7C™ from NYMOX. It involves a lumbar puncture performed by a physician to obtain the cerebrospinal fluid. But AD7C™ has the disadvantage that it cannot be used as an early diagnostic test effectively because it relays on the increase in a protein resulting from neuronal degeneration. Therefore, significantly elevated levels of neuronal thread protein in cerebrospinal fluid can only be detected reliably after considerable irreversible neuronal death has occurred.
Because AD is progressive in nature, the efficiency of a cure could critically depend upon early detection. Additionally, the value of any new therapy could be better assessed if a rapid, safe and effective diagnostic procedure were available to monitor the progress of AD in patients following treatment. Therefore, there is a distinct need for a reliable, non-invasive, diagnostic laboratory test of AD so that this debilitating disorder may be detected at its earliest stages for efficient and effective intervention and treatment. The present invention is designed to address these needs.
SUMMARY OF THE INVENTION
In order to overcome deficiencies in the art, the present invention provides a quantitative assay for early diagnosis of late-onset sporadic Alzheimer's Disease (AD) in living individuals using peripheral tissue biopsy. The assay also may be used for postmortem diagnosis.
Thus, the present invention provides a method of diagnosing late-onset sporadic AD comprising the steps of obtaining a sample from a human subject; assessing cytochrome oxidase activity in nucleated cells of the sample; and comparing the cytochrome oxidase activity in nucleated cells of the sample with the cytochrome oxidase activity of a standard, wherein a decrease in cytochrome oxidase activity in nucleated cells of the sample, with respect to the standard, indicates that the subject has AD.
In preferred embodiments, the standard is cytochrome oxidase activity of like cells from an individual not afflicted with AD. In other preferred embodiments, the method further comprises assessing cytochrome oxidase activity from the like cells. In particularly preferred embodiments, the sample is a superficial tissue biopsy. In more defined embodiments, the biopsy is from a tissue selected from the group consisting of muscle, dermis, epidermis, bone marrow, peripheral ganglion or nerve. In certain preferred embodiments, the sample may be frozen and sectioned.
In particular aspects of the present invention, the assessing comprises providing diaminobenzidine (DAB) to cells of the sample and measuring oxidation of DAB to an indamine polymer. It is contemplated that measuring oxidation of DAB to an indamine polymer, when calibrated with spectrophotometry provides quantitative cytochemistry.
In particular embodiments, the cytochrome oxidase activity is quantified. In defined embodiments, the diagnostic cytochrome oxidase activity level of cells of the sample is significantly lower than the mean cytochrome oxidase activity level of the like cells. The term “significantly lower” as used herein is a cytochrome oxidase activity in the sample that is lower than the cytochrome oxidase in like cells. In preferred embodiments, the cytochrome oxidase activity is 10% or more below 15% or more below; 20% or more below; 25% or more below; 30% or more below; 35% or more below; 40% or more below; 45% or more below; 50% or more below; 55% or more below; 60% or more below; 65% or more below; 70% or more below; 75% or more below; 80% or more below; 85% or more below or 90% or more below the mean cytochrome oxidase activity level of the like cells.
Also contemplated by the present invention is a method for monitoring a treatment for l
Board of Regents , The University of Texas System
Fulbright & Jaworski
Leary Louise N
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