Patent Publication Number: US-5891735-A

Title: Method for measuring nitric oxide in nitrosyl (FeII)-hemoglobin and S-nitrosohemoglobin

Description:
RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Application Number 60/003,801 filed on Sept. 15, 1995. 
     BACKGROUND OF THE INVENTION 
     Endothelium-derived relaxing factor (EDRF), is a product of the normal endothelial cell, and has both vasodilatory and antiplatelet properties (Furchgott, R. F. et al., Nature, 288:373-376 (1980); Moncada, S. et al., Biochem. Pharmacol, 38:1709-1713 (1989); Azuma, H. et al., Brit. J. Pharmacol. 88:411-415 (1986) and Radomski, M. W. et al., Brit. J. Pharmacol. 92:639-642 (1987)). Pharmacologic studies suggest that disease states as varied as septic shock, hyper-homocysteinemia, atherosclerosis, and hypoxia-induced pulmonary hypertension may be associated with abnormal concentrations of EDRF in the vascular milieu (Westernberger, U. et al., Free Rad. Res. Comm. 11:167-168 (1990); Yamamoto, H. et al., J. Clin. Invest. 81:1752-1758 (1988); Dinh-Xuan, A. T. et al., Engl. J. Med. 324:1539-1547 (1991)). This bioactive substance is believed to be equivalent to nitric oxide, or a chemical congener or adduct thereof (Palmer, R. M. G. et al., Nature 327:524-525 (1987); Ignarro, L. J. et al., Proc. Natl. Acad. Sci. 84:9265-9269 (1987)). Among the species of importance as biological adducts of nitric oxide are S-nitrosothiols, which are adducts with the sulfhydryl groups of amino acids, peptides, and proteins. 
     It has been demonstrated that nitric oxide and authentic EDRF react with free thiol groups of proteins under physiologic conditions in vitro, to form S-nitroso-proteins. These nitric oxide adducts have bioactivities which are comparable to nitric oxide, but exhibit half-lives on the order of hours, significantly longer than that of EDRF (Stamler, J. S. et al., Proc. Natl. Acad. Sci. 89:444-448 (1992)). 
     Under normal circumstances, the concentration of nitric oxide in blood or plasma is believed to be quite low (in the 1 nM range) and its half-life of the order of 0.1 second. Its high degree of reactivity toward oxygen and redox metals, in conjunction with its extremely short half-life, have made the routine measurement of blood levels in both normal and disease states most difficult by standard methods, such as chemiluminescence spectroscopy, electron paramagnetic resonance spectroscopy, or differential absorbance spectroscopy of hemoglobin (Martin, W. et al., J. Pharmacol. Exp. Therap. 237:529-538 (1986); Downes, M. J. et al., Analyst 101:742-748 (1976); Kelm, M. et al., Circ. Res. 66:1561-1575 (1990); Arroyo, C. M. et al., Free Rad. Res. Comm. 14:145-155 (1991) and Goretsky, J. et al., J. Biol. Chem. 263:2316-2323 (1988)). In fact, it is generally assumed in the field that such measurements are not feasible by currently used methods. 
     Nitrosonium (NO + ) is a short lived species which is too unstable to exist freely in biological systems, and felt to be non-detectable by chemiluminescence. Nitric oxide exists in the S-nitrosothiol adduct, not as nitric oxide but rather as a nitrosonium equivalent. Thus, it behaves chemically in a manner which more closely resembles NO +   than NO -   (nitric oxide). 
     Hemoglobin (Hb) is a tetramer comprised of two alpha and two beta subunits. In human Hb, each subunit contains one heme, while the beta (β) subunits also contain highly reactive thiol (SH) groups (cysβ93) (Olson, J. S., Meth. of Enzym., 76:631-651 (1981; Antonini &amp; Brunori, In Hemoglobin and Myoglobin in Their Reactions with Ligands, American Elsevier Publishing Co., Inc., New York, pp.29-31 (1971)). These cysteine residues are highly conserved among species. Nitric oxide (NO) interacts with hemoglobin at its metal centers, whereas S-nitrosothiols (RSNOs) can donate the NO group to β93 cysteine residues. 
     Nitric oxide is known to bind tightly to hemoglobin, forming nitrosyl(FeII)-hemoglobin. Interactions of NO with ahemoglobin are believed to be a major route of NO metabolism in biological systems. It follows that levels of NO-hemoglobin in blood should be an excellent indication of endogenous NO production. However, methods have not been developed that are sufficiently sensitive to make this determination in vivo (Beckman, J. S. et al., Methods in Nitric Oxide Research, Feelisch and Stainler, J. S. eds, Wiley, Chichester, U.K. (1996)). Specifically, electron paramagnetic resonance (EPR) has been used previously to measure nitric oxide bound to the Fe of the heme. However, under normal physiological conditions, circulating levels of NO-hemoglobin in blood are below the detection limit. The insensitivity of EPR makes this method impossible to use to monitor all but a gross change in NO-hemoglobin from normal levels. Only in pathophysiological states such as sepsis and pregnancy, which are characterized by NO overproduction, can EPR be used to detect a measurable level for NO in blood. 
     EPR measurements also suffer from being cumbersome and expensive. An alternative method of measuring NO by assaying nitrite/nitrate in body fluids also suffers from insensitivity. 
     SUMMARY OF THE INVENTION 
     The invention is a method for measuring nitrosyl(FeII)-hemoglobin in blood using a series of steps in which a protein fraction is prepared from blood cells and the protein fraction is assayed for the formation of nitric oxide by photolysis of the protein fraction followed by measuring a chemiluminescence signal generated by a chemical reaction between nitric oxide and ozone. cl DETAILED DESCRIPTION 
     The invention relates to a method for determining the concentration of nitrosyl(FeII)-hemoglobin in a blood sample, thereby serving as a measure of the level of NO in the animal or human from which the blood sample has been taken. The method is related to one used previously for the measurement of S-nitrosoproteins and smaller molecular weight S-nitrosothiols in plasma (See U.S- Ser. No. 5,459,076; Oct. 17, 1995. The contents of this patent are hereby incorporated by reference in their entirety.) However, the primary focus of the present invention is on assaying for nitrosyl(FeII)-hemoglobin rather than S-nitrosothiols. 
     In contrast to the previous method, in which the red blood cells were removed and discarded from the sample to be analyzed, the subject invention method uses the red blood cells. The method measures NO which has reacted with the thiol groups of hemoglobin in the form of S-nitroso-hemoglobin (SNO-Hb) as well as NO bound to the Fe of the heme (nitrosyl(FeII)-hemoglobin or Hb(FeII)NO). As shown in the table, the level of S-nitroso-hemoglobin in venous blood is negligible compared to the level of Hb(FeII)NO. Therefore, to specifically measure the level of Hb(FeII)NO in venous blood, it is unnecessary to include steps in which Hb samples are divided into two aliquots which are then either treated or not treated with a 10-fold excess of HgCl 2  over the protein concentration. Reaction of Hb with HgCl 2  removes NO from thiol groups selectively, without disturbing NO bound at the heme. Values for NO obtained from the HgCl 2  reaction, if significant, should be subtracted from the total NO obtained for the measurements without the HgCl 2  reaction, to obtain an accurate value for Hb(FeII)NO. 
     In one embodiment of the invention, a blood sample is taken from a mammal, such as a human, and the solid parts including cells are isolated away from the remaining fluid. The cells are then lysed by standard methods, and a protein fraction is prepared. Before quantitating nitric oxide adducts (nitrosonium adducts, which include low molecular weight S-nitrosothiols such as S-nitrosoglutathione and high molecular weight S-nitrosothiols such as S-nitroso-proteins), it is preferable to first remove low molecular weight S-nitrosothiols endogenous to the red blood cells, which would also contribute to the NO value, by a step which separates low molecular weight molecules away from the red blood cell proteins (referred to as desalting). This step can include, for example, dialysis or column chromatography based on separation by size of the molecules. A further step is to subject the protein fraction to photolysis, as in a photolysis cell, where it is irradiated with light of the appropriate wavelength to liberate NO from the various forms of hemoglobin. The resulting NO is detected by reaction with ozone. 
     One embodiment of the invention utilizes a chemiluminescence apparatus in which a photolysis cell is linked directly to the reaction chamber and detector portion, thereby bypassing the pyrolyzer. A sample of the blood protein fraction is injected into the photolysis cell, either directly, or as chromatographic effluent from a high-performance liquid or gas chromatography system which is connected to the photolysis cell. 
     The sample is then irradiated with a mercury vapor lamp, and directed through a series of cold traps, where liquid and gaseous fractions which are less volatile than nitric oxide (such as nitrite and nitrate) are eliminated, leaving only free nitric oxide remaining in the cell. The nitric oxide is then transported by a gaseous stream, preferably helium, into the chemiluminescence spectrometer. In the alternative, other inert gases may be used. 
     Once present in the chemiluminescence spectrometer, the free nitric oxide is detected by its chemical reaction with ozone, resulting in the generation of signals that are recorded on a digital integrator. If desired, flow rates and illumination levels in the photolysis cell can be adjusted to cause complete photolysis of the S-nitric oxide bond of the S-nitrosothiol compounds. Flow rates and illumination levels may be adjusted by routine methods available in the art, in order to achieve optimal cleavage of the bond between the particular adduct and nitric oxide, nitrosonium or nitroxyl, whichever is bound. 
     In a variation, the invention relates to a method for detecting S-nitrosothiols, including primarily S-nitroso-hemoglobin (SNO-Hb) in a blood sample. This method comprises inactivating the chemiluminescence, signal-generating capability of any nitric oxide which is associated with a thiol, in the protein fraction derived from the blood sample, and determining the amount of thiol-bound nitric oxide by measuring the quantitative difference between total nitric oxide and nitric oxide remaining after inactivation. 
     A particular embodiment of this variation relates to a method in which the protein fraction derived from the blood sample is treated with a source of mercurous ion, followed by air incubation, which oxidizes the nitric oxide and nitrosonium and renders them undetectable. Compounds suitable for pretreatment include Hg 2  Cl 2  and other mercurous ion salts and organic mercurials. The treated sample is then injected into the photolysis cell, where NO +   is converted to NO -   (nitric oxide) and the nitric oxide is detected by the chemiluminescence method described above. The amount of nitric oxide which is specifically derived from S-nitrosothiols is determined by comparing the chemiluminescence signal generated by the mercurous ion-treated sample, with a chemiluminescence signal generated by a sample of the equivalent biological fluid which is not treated with mercurous ion prior to injection into the photolysis cell. 
     In a further embodiment of the claimed invention, the methods described herein may be utilized to determine the presence of a disease state which involves abnormal levels of nitric oxide and its biologically active equivalents, by monitoring Hb(FeII)NO and SNO-Hb levels in blood, and more particularly, Hb(FeII)NO in venous blood from a patient. The ability to specifically assay for Hb(FeII)NO in venous blood distinguishes this assay over previously known methods. Nitric oxide adducts represent a pool of bioactive nitric oxide in physiological systems. Therefore, in disease states in which the pathogenesis derives from the effects of abnormal levels of nitric oxide, these methods provide a means for the clinician to determine the presence of, and monitor the extent of, the disease state. Such information enables the clinician to determine the appropriate pharmacological intervention necessary to treat the disease state. Such disease states and medical disorders include, but are not limited to, septic shock, cardiogenic shock, hypovolemic shock, atherosclerosis, hyperhomocysteinemia, venous thrombosis, arterial thrombosis, coronary occlusion, pulmonary embolism, cerebrovascular accidents, vascular fibrosis, ectopia lentis, osteoporosis, mental retardation, skeletal deformities, pulmonary hypertension, malignancy, infections, inflammation, asthma, tolerance to narcotics and central nervous system disorders. Furthermore, the use of these methods is not limited to these diseases. This method can be of use in assaying biologically active nitric oxide equivalents in any disease state or medical disorder in which nitric oxide is implicated. 
    
    
     EXAMPLES 
     Example 1 
     Methods and Control Experiments 
     Initially, to modify the chemiluminescence apparatus for use in the detection method, a photolysis cell, consisting of a borosilicate glass coil (3 m×0.64 cm o.d.×1 mm i.d., turned to a diameter of 6 cm and a width of 12 cm), with a purge stream of helium (5 L/min), was linked directly to the reaction chamber and detector portion of a chemiluminescence apparatus (Model 543 Thermal Energy Analyzer, Termedix, Inc., Woburn, Mass.), thereby bypassing the pyrolyzer. Then, a 5-100 μl volume sample was either introduced directly, or as a chromatographic effluent from an attached high-performance liquid or gas chromatography system into the photolysis cell, and irradiated with a 200-watt mercury vapor lamp (vertically mounted in the center of the photolysis coil on TEFLON towers). The effluent from the photolysis coil was directed to a series of cold traps where liquid and gaseous fractions less volatile than nitric oxide (such as nitrite and nitrate), were removed. 
     The nitric oxide was then transported by a helium stream into the chemiluminescence spectrometer, in which free nitric oxide was detected by reaction with ozone. Signals were recorded on a digital integrator (Model 3393A, Hewlett Packard, Andover, Mass., USA). Flow rates and illumination levels in the photolysis cell were designed to result in complete photolysis of the S-nitric oxide bond, as confirmed by analysis of effluent from the photolysis cell, according to standard methods (Saville, B., Analyst 83:670-672 (1958)). 
     To determine the fraction of nitric oxide which was derived from S-nitrosothiols, several additional steps were included in the method described above. Prior to its injection into the photolysis cell, an aliquot of the same sample was treated with an 8 to 10-fold excess of Hg over protein concentration followed by air incubation, according to standard methods (Saville, B., Analyst 83:670-672 (1958)), in order to oxidize the nitric oxide displaced as nitrosonium and render it undetectable. Nitric oxide concentrations from samples alternatively subjected to or not subjected to, pretreatment with HgCl 2 , were compared, to determine how much of the nitric oxide detected was derived specitically from S-nitrosothiols. Similarly, as an added measure of confirmation, S-nitrosothiols and free nitric oxide were compared using nitric oxide concentrations in samples alternatively exposed to, or not exposed to, photolysing illumination. 
     S-nitroso-L-cysteine, S-nitroso-glutathione, S-nitroso-N-acetyl-L-cysteine and S-nitroso-bovine serum albumin were synthesized by exposing the respective thiols to acidified NaNO 2 , according to routine methods, and standard curves were generated (Stamler, J. S. et al., Proc. Natl. Acad. Sci. 89:444-448 (1992)). Similarly, standard curves were derived for nitric oxide generated from acidified NaNO 2 , or from a saturated solution of nitric oxide gas serially diluted immediately before measurement in airtight syringes. Concentration-response curves were linear with correlation coefficients of ≧0.98 in all cases. Limits of sensitivity were approximately 0.1 pM, and intraassay variability was ±3%. 
     The response of this system to pure S-nitrosothiols was first examined using S-nitroso-L-cysteine as a standard. The concentration of the stock solution from which serial dilutions were made was determined by standard methods and confirmed by the optical density at 340 nm (Saville, B., Analyst 83:670-672 (1958); and Stamler, J. S. et al., Proc. Natl. Acad. Sci. 89:444-448 (1992)). Serial dilutions were prepared with concentrations ranging from 100 μM to 0.1 pM. The chemiluminescence signal was linear over this concentration range (correlation coefficient of ≧0.98). 
     As additional controls, S-nitroso-glutathione, and S-nitroso-N-acetyl-L-cysteine were also synthesized and responses measured. In the absence of photolysis, the nitric oxide signal was below the limits of detectability. Similarly, HgCl 2  pretreatment of the samples, followed by incubation in air to oxidize (and render undetectable) the liberated nitric oxide led to a loss of &gt;99% of the chemiluminescence signal in all cases. 
     Example 2 
     Endogenous Levels of S-nitrosohemoglobin and Nitrosyl(FeII)-Hemoglobin in Blood 
     To determine if SNO-Hb is naturally occuring in the blood, and if so, its relationship to the O 2  transport capacity and nitrosylated-heme content of red cells, we developed an analytical approach to assay the S-nitrosothiol and nitrosyl-heme content of erythrocytes. Arterial blood was obtained from the left ventricle of anesthetized rats by direct puncture and venous blood was obtained from the jugular vein and inferior vena cava. Hb was then purified from red cells and assayed for RSNO and (FeII)NO content. Arterial blood contained significant levels of SNO-Hb, whereas levels were virtually undetectable in venous blood. (See table.) Measurements made 45 minutes after infusion of the NO synthase inhibitor N.sup.ω -monomethyl-L-arginine (L-NMMA) (50 mg/kg), showed a depletion of SNO-Hb as well as total Hb-NO (82 and 50±18%, respectively; n=3-5; p&lt;0.05). These data establish the endogenous origin of SNO-Hb, although some environmental contribution is not excluded. The arterial-venous distribution seen for SNO-Hb was reversed in the case of Hb(FeII)NO, which was detected in higher concentrations in partially deoxygenated (venous) erythrocytes. Accordingly, the proportion of nitrosylated protein thiol and heme appears to depend on the oxygenation state of the blood. Consistent with these findings, Wennmalm and coworkers have shown that Hb(FeII)NO forms mainly in venous (partially deoxygenated) blood (Wennmalm, A., et al., Br. J. Pharmacol., 106(3):507-508 (1992)). However, levels of Hb(FeII)NO in vivo are typically too low to be detected (by EPR) and SNO-Hb is EPR-silent (i.e. it is not paramagnetic). Thus, photolysis-chemiluminesence represents an important technological advance, as it is the first methodology capable of making quantitative and functional assessments of NO binding to Hb under normal physiological conditions. 
     Blood was obtained from the left ventricle (arterial) and jugular vein (venous) of anesthetized Sprague-Dawley rats. Comparable venous values were obtained in blood from the inferior vena cava. Red blood cells were isolated by centrifugation at 800 g, washed three times in phosphate buffered saline at 4° C., lysed by the addition of 4-fold excess volume of deionized water containing 0.5 mM EDTA, and desalted rapidly across G-25 columns according to the method of Penefsky at 4° C. In 24 rats, Hb samples were divided in two aliquots which were then treated or not treated with 10-fold excess HgCl 2  over protein concentration as measured by the method of Bradford. Determinations of SNO-Hb and Hb(FeII)NO were made by photolysis-chemiluminescence as described below. In 12 additional rats, further verification of the presence of SNO-Hb was made by assaying for nitrite after HgCl 2  treatment. Specifically, samples (with and without HgCl 2 ) were separated across Amicon-3 (Centricon filters, m.w. cut off 3,000) at 40° C. for 1 h, and the low molecular weight fractions collected in airtight syringes containing 1 μM glutathione in 0.5N HCl. Under these conditions, any nitrite present was converted to S-nitrosoglutathione, which was then measured by photolysis-chemiluminescence (detection limit ˜1 nM). SNO-Hb was present in all arterial samples, and levels determined by this method (286 ±±33 nM) were virtually identical to and not statistically different from those shown in the table. In venous blood, SNO-Hb was undetectable (0.00±25 nM); levels were not statistically different from those given above. 
     Method of Assay on Rat Blood 
     As a part of the assay, a highly sensitive photolysis-chemiluminescence methodology was employed, which had been used for measuring RSNOs (S-nitrosothiols) in biological systems (Gaston, B., et al., (1993); Stamler, J. S., et al., (1992)). The method involves photolytic liberation of NO from the thiol, which is then detected in a chemiluminesence spectrometer by reaction with ozone. The same principle of operation can be used to cleave (and measure) NO from nitrosyl-metal compounds (Antonini, E. Brunori, M. In Hemoglobin and Myoglobin in Their Reactions with Ligands, American Elsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). With adjustment of flow rates in the photolysis cell, complete photolysis of the NO ligand of Hb(FeII)NO could be achieved. Standard curves derived from synthetic preparations of SNO-Hb, Hb(FeII)NO, and S-nitrosoglutathione were linear (R&gt;0.99), virtually . superimposable, and revealing of sensitivity limits of approximately 1 nM. Two analytical criteria were then found to reliably distinguish SNO-Hb from Hb(FeII)NO: 1) signals from SNO-Hb were eliminated by pretreatment of samples with 10-fold excess HgCl 2 , while Hb(FeII)NO was resistant to mercury challenge; and 2) treatment of SNO-Hb with HgCl 2  produced nitrite (by standard Griess reactions) in quantitative yields, whereas similar treatment of Hb(FeII)NO did not. UV/VIS spectroscopy confirmed that NO remained attached to heme in the presence of excess HgCl 2 . 
     
                       TABLE
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Endogenous levels of S-nitrosohemoglobin and
nitrosyl(FeII)-hemoglobin in blood
Site       SNO-Hb (nM)
                      Hb(FeII) NO (nM)
______________________________________
Arterial   311 ± 55*
                      536 ± 99†
Venous     32 ± 14 894 ± 126
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 *P &lt; 0.05 vs venous;
 †P &lt; 0.05 for paired samples vs venous
 
    
     Equivalents 
     Those skilled in the art will know, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.