Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-03-22
2001-10-23
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06306609
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for the detection of nitric oxide in fluid media, e.g., mammalian body fluids, as well as articles and reagents useful therefor. In one aspect, the present invention relates to non-invasive methods for the detection of nitric oxide in body fluids. In another aspect, the present invention relates to articles useful for collection and detection of nitric oxide in a variety of fluid media. In yet another aspect, the present invention relates to methods to monitor patients for underproduction or overproduction of nitric oxide.
BACKGROUND OF THE INVENTION
Nitric oxide (NO), a gaseous free radical, was once considered mainly as an environmental pollutant from car exhausts and in city smog and cigarette smoke. This view toward nitric oxide was changed in 1987, the year in which NO was discovered to be produced in humans (see, for example, Ignarro et al., in
Proc. Natl. Acad. Sci., USA
, 84:9265-69 (1987) and Palmer et al., in
Nature
, 327:524-26 (1987)). First identified as an endothelium-derived relaxation factor, NO is now recognized as a new kind of cell signaling molecule that regulates the functions of many mammalian cells and tissues.
NO is generated by the enzymatic cleavage of L-arginine, catalyzed by the nitric oxide synthase enzyme (NOS; see, for example, Rodeberg et al., in
Am. J. Surg
., 170:292-303 (1995), and Bredt and Snyder in
Ann. Rev. Biochem
., 63:175-95 (1994)). Three different isoforms of NOS have been isolated, cloned, sequenced and expressed, i.e., endothelial cell derived nitric oxide synthase (eNOS), neuronal cell derived nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS). The eNOS and nNOS isoforms are expressed constitutively, and both enzymes require an increase in intracellular calcium for activation. Under physiological conditions, a low output of NO is continuously produced by the eNOS isoform, which is present in numerous cells, including endothelium and neurons. This low level of nitric oxide is involved in a variety of regulatory processes, e.g., blood vessel homeostasis, neuronal communication and immune system functions.
On the other hand, under pathophysiological conditions, a high output of NO is produced by the inducible, calcium-independent NOS isoform (iNOS), which is expressed in numerous cell types, including endothelial cells, smooth muscle cells and macrophages, upon activation with cytokines or endotoxin. These high levels of nitric oxide production (by cytokine-activated macrophages) contribute to, in part, the ability of these cells to kill bacteria or tumor cells and hence play an important role in host defense. However, while excessive NO helps destroy invading microbes, the systemic overproduction of NO by numerous types of cytokine-activated cells, including endothelial cells, smooth muscle cells, macrophages and hepatocytes, may trigger a cascade of events that can result in a variety of inflammatory and infectious diseases and conditions, such as tissue injury, shock, multiple organ failure and death. In fact, abnormally elevated levels of nitric oxide have recently been associated with many inflammatory and infectious diseases or conditions, such as, for example, septic shock, over expression of cytokines, diabetes, allograft rejection, inflammatory bowel disease, rheumatoid arthritis, stroke, multiple sclerosis, and the like.
Similarly, production of an insufficient amount of nitric oxide is also capable of causing a variety of disease states, such as, for example, neonatal persistent pulmonary hypertension, pre-eclampsia, adult respiratory distress syndrome, post-angioplasty restenosis, impotence, and the like.
Nitric oxide is a potent vasodilator (see, for example, Palmer in
Arch. Surg
., 128:396-401 (1993) and Radomski & Moncada in
Thromb. Haemos
., 70:36-41 (1993)). For example, in blood, NO produced by the endothelium diffuses isotropically in all directions into adjacent tissues. As NO diffuses into the vascular smooth muscle, it binds to guanylate cyclase enzyme, which catalyzes the production of cGMP, inducing vasodilation (see, for example, Ignarro, L. J.,
Ann. Rev. Toxicol
. 30:535-560 (1990); Moncada, S.,
Acta Physiol. Scand
., 145:201-227 (1992); and Lowenstein and Snyder,
Cell
, 70:705-707 (1992)).
The overproduction of nitric oxide causes an extreme drop in blood pressure, resulting in insufficient tissue perfusion and organ failure, syndromes that are associated with many diseases and/or conditions (e.g., septic shock, stroke, over expression of cytokines, allograft rejection, and the like). The overproduction of nitric oxide is triggered by a number of stimuli, such as the overproduction of inflammatory cytokines (e.g., the overproduction of interleukin-1, interferons, endotoxin, and the like). Additionally, the overproduction of NO has been found to be one of the major side-effects of cytokine therapy (see, for example, Miles et al., in
Eur. J. Clin. Invest
., 24:287-290 (1994) and Hibbs et al., in
J. Clin. Invest
., 89:867-877 (1992)). Thus, abnormally elevated nitric oxide levels have been associated with many inflammatory and infectious diseases.
The half-life of NO in vivo is only 3-5 seconds, a short lifetime that makes it very difficult to detect and quantify. Several biophysical techniques have been developed for the measurement of NO levels in aqueous solution. These include chemiluminescence assay (see, for example, Downes et al.,
Analyst
, 101:742-748 (1976)), oxyhemoglobin assay (see, for example, Kelm and Schrader,
Cir. Res
., 66:1561-1575 (1990)), GC-MS detection (see, for example, Palmer et al.,
Nature
(London), 327:524-526 (1987)), and nitrosyl-hemoglobin formation detected by electron paramagnetic resonance (EPR) spectroscopy at liquid nitrogen temperature (see, for example, Lancaster et al.,
Proc. Natl. Acad. Sci. USA
, 87:1223:1227 (1990)).
Production of NO can also be indirectly detected by measuring its end products, NO
2
−
/NO
3
−
(see, for example, Palmer et al., supra). None of these techniques in their present form, however, can be used for in vivo detection of NO production. Recently, an invasive electrochemical microsensor to detect NO levels in blood vessels of healthy human volunteers has been described (see, for example, Vallance et al.,
Lancet
, 346:153-154 (1995)).
Dithiocarbamates are a class of low molecular-weight sulphur-containing compounds that are effective chelators (see, for example, Shinobu et al.,
Acta Pharmacol
et
Toxicol
., 54:189-194 (1984)). For example, diethyldithiocarbamate (DETC) is used clinically for the treatment of nickel poisoning. Recently, it was found that N-methyl-D-glucamine dithiocarbamate (MGD) chelates with ferrous iron as a two-to-one [(MGD)
2
-Fe] complex, which in turn interacts strongly with NO, forming a stable and water-soluble complex in aqueous solution, i.e., [(MGD)
2
-Fe—NO] (see, for example, Lai & Komarov,
FEBS Lett
., 345:120-124 (1994)). The latter complex gives rise to a sharp three-line spectrum with g
iso
=2.04, characteristic of a nitrosyl-Fe-dithiocarbamate complex which can readily be detected by EPR spectroscopy at ambient temperatures. This method of detecting NO in body fluids in real time has recently been described by Lai in U.S. Pat. No. 5,358,703.
There is, however, still a need in the art for more rapid, preferably non-invasive methods for the detection of nitric oxide in fluid media such as mammalian body fluids.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention, non-invasive methods have been developed for the measurement of NO levels in a variety of fluid media, e.g., in mammalian body fluids. The present invention embraces the use of a semi-permeable membrane bag containing a nitric oxide reacting substance (e.g., [(MGD)
2
-Fe]) to trap NO diffusing into the bag, and a simple physical or chemical detection method to measure the levels of [(MGD)
2
-Fe—NO] or other end products in the bag.
Since NO is a neutral gas molecule, it is
Foley & Lardner
Le Long V.
Medinox Inc.
Padmanabhan Kartic
Reiter Stephen E.
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