Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2000-08-03
2002-05-07
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C435S007400, C435S004000
Reexamination Certificate
active
06383771
ABSTRACT:
FIELD OF THE INVENTION
This invention relates in general to methods to monitor the energy enhancing effect of the reduced form of species such as nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADPH), Coenzyme Q10, reduced form of Coenzyme Q10, adenosine triphosphate (ATP) and physiologically acceptable salts thereof, through determination of the relative activity of the energy-producing enzyme NADH cytochrome C reductase in whole blood.
BACKGROUND OF THE INVENTION
Every living cell needs energy to survive. This energy is produced, according to a process known as oxidative phosphorylation, in form of the chemical entity adenosine triphosphate (ATP).
The key enzyme in the production of ATP is NADH cytochrome C reductase, also known as Complex I-III. This enzyme reduces cytochrome C by using the reducing agent NADH, the reduced form of nicotinamide adenine dinucleotide. The reduced cytochrome C is then oxidized by the enzyme cytochrome C oxidase (Complex IV) to form water. In other words, the reduced form of NADH, also called Coenzyme I, uses ubiquitous oxygen in the cell to form water and 3 ATP molecules in accordance with the following general reaction scheme:
NADH+H
+
+½O
2
+3P
i
+3ADP→NAD
+
+3ATP+4H
2
O.
Thus, with one NADH molecule, three ATP molecules are obtained having an energy of approximately 21 kilocalories. This process is set forth schematically in
FIG. 1
, where FeS is Reiske iron sulfur protein; ADP=adenosine diphosphate; and b
562
, b
566
, c
1
, a and a
3
are cytochromes. The enzymes depicted in
FIG. 1
are referred to as Complex I (NADH:ubiquinone oxidoreductase); Complex II (succinate dehydrogenase); Complex III (ubiquinone:cytochrome C oxidoreductase); Complex IV (cytochrome C oxidase); and Complex V (ATP synthase). These enzymes, whose energy-related functions occur in the mitochondria of the cell, are assembled from 13 polypeptides coded by the mitochondrial DNA (mtDNA) and approximately 50 polypeptides coded by the nuclear DNA (nDNA). This system of five complexes also constitutes what is referred to as the electron transport chain (ETC), the common pathway for cellular energy metabolism, through which enzyme-catalyzed redox processes achieve electron transfer among critical substrate species.
NADH cytochrome C reductase is the first and key enzyme of this energy producing process. The greater the activity of NADH cytochrome C reductase, the higher the cellular output of energy. Illustratively, the more energy a cell needs, the more NADH it contains. For example, heart cells have 90 &mgr;g/g tissue; brain and muscle cells contain 50 &mgr;g/g tissue; liver cells contain 40 &mgr;g/g; and red blood cells contain 3 &mgr;g/g tissue. Thus, the activity of NADH cytochrome C reductase, directly linked to the amount of NADH present in the cell, reflects the energy producing capacity of a cell. Alberts, B., Bray, D., Lewis, J., Raff, H., Roberts, K., and Watson, J. D., “Energy Conversion: Mitochondria and Chloroplasts,” in
Molecular Biology of the Cell
, 3rd Ed., Garland Publishing Inc., pp. 653-720, 1994; Lehninger, A. L., “Vitamins and Coenzymes,” in
Biochemistry
, 2nd Ed., The John Hopkins University School of Medicine, Worth Publishers, Inc., pp. 337-342, 1975.
It has been shown in a variety of diseases (the so-called mitochondrial diseases) that energy production, in particular the activity of NADH cytochrome C reductase (Complex I-III), is decreased. This has been demonstrated not only in brain and muscle tissue, but also in platelets. Cooper, J. M., Mann, V. N., Krige, D., and Schapira, A. H. V., “Human mitochondrial complex I dysfunction,”
Biochemica et Biophysica Acta
1101, 198-203 (1992); Mizuno, Y., et al., “Deficiencies in Complex I Subunits of the Respiratory Chain in Parkinson's Disease,”
Biochemical and Biophysical Research Communication
163, 1450-1455 (1989); Shoffner, J. M., Wafts, R. L., Juncos, J. L., Torroni, A., and Wallace, D. C., “Mitochondrial Oxidative Phosphorylation Defects in Parkinson's Disease,”
Ann. Neurol
. 30, 332-339 (1991). This has been found both in patients with Parkinson's disease (PD) and in patients with Alzheimer's disease. A further demonstration of the link between cytochrome C reductase activity and disease conditions involves the Parkinson inducing toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that irreversibly inhibits and destroys NADH cytochrome C reductase in certain brain areas causing Parkinsonian-like symptoms. Benecke, R., Strumper, P., and Weiss, H.,
Brain
1993, Vol. 116, Part 6, pp. 1451-1463. These and other similar findings have significant implications for investigations into the etiology of conditions such as Alzheimer's and Parkinson's diseases.
Another known enzyme toxin is azidothymidine (AZT) which is used in the treatment of AIDS patients. This toxin damages NADH cytochrome C reductase, causing a reduction of energy production in the cell. Dalakas, M. C., IIIa, I., Pezeshkpour, G. H., Laukaftis, J. P., Cohen, B, and Griffin, J. L. “Mitochondrial myopathy caused by long-term zidovudine therapy,”
New Engl. J.Med
. 322, 1098-1105 (1990). By measuring the activity of NADH cytochrome C reductase in muscle tissue biopsy, it was demonstrated that AZT destroys the enzyme's activity, consequently blocking the energy production of the cells leading to muscle atrophy.
In addition to substances known to have an inhibitory effect on the activity of key enzymes related to cellular processes for the production of energy, there are also substances, such as the reduced form of nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), either endogenous or introduced exogenously, that are able to enhance NADH cytochrome C reductase activity and, consequently, cellular energy production. These two co-enzymes, and their pharmaceutically acceptable salts, have been shown to be useful in the treatment of Parkinson's Disease (PD). The effectiveness of these agents for this purpose is disclosed in U.S. Pat. Nos. 4,970,200, 5,019,516, and 5,332,727, the disclosures of which are incorporated herein by reference. In addition, these substances are effective in the treatment of Alzheimer's disease, as disclosed in U.S. Pat. No. 5,444,053, the disclosure of which is also incorporated herein by reference. These substances have also been demonstrated to be effective in supplying additional energy to healthy individuals as disclosed in EP 0 496 479 131.
Assay methods for the determination of NADH cytochrome C reductase activity have been described for many tissues, in particular muscle, liver, brain and heart cells. See, for example, Hatefi, Y. and Stiggall, D. L. (1978b), “Preparation and properties of NADH:cytochrome C oxidoreductase (Complex II),” in
Methods in Enzymology
, 53, Fleischer, S. and Packer, L., eds, pp 5-10, Academic Press, New York, 1978; Trounce, I., Byrne, E. and Marzuki, S., “Decline in skeletal muscle mitochondrial respiratory chain function: Possible factors in ageing,”
Lancet
1989, 637-639 (assay of muscle tissue); Yen, T.-C., et al., “Liver mitochondrial respiratory functions decline with age,”
Biochem. Biophys. Res. Comm
. 165, 994-1003 (1989) (assay of liver tissue); Nakagawa-Hattori, Y., et al., “Is Parkinson's disease a mitochondrial disorder?”
J. Neurol. Sci
. 107, 29-33 (1992) assay of muscle tissue obtained post-mortem); Mizuno, Y., et al., “Effects of 1-methyl-1-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenylpyridinium ion activities of the enzymes in the electron transport system in mouse brain,”
J. Neurochem
. 48, 1787-1793 (1987) (assay of mouse brain tissue); Reichman, H. et al., “Respiratory chain and mitochondrial deoxyribonucleic acid n blood cells from patients with focal and generalized dystonia,”
Movement Disorders
9, 597-600 (1994) (assays of platelet homogenate).
Indeed, cofactors in oxidative phosphorylation processes have seen widespread use i
Birkmayer Pharmaceuticals
Kenyon & Kenyon
Witz Jean C.
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