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Nitric oxide measurements during endotoxemia.

Endotoxemia is defined as an alteration in cellular oxygen metabolism initiated by infection. Excessive production of NO has been implicated in the pathogenesis of endotoxemia (1). Inducible NO synthase (iNOS)[3] activity stimulated at the site of infection is primarily responsible for the sustained release of NO (2). The increase in NO leads to myocardial depression (3) and vascular dysfunction associated with vasorelaxation and hypotension. Decreased skin perfusion, reduced mental alertness, and increased serum lactate concentrations are also characteristics of endotoxemia (4). Endotoxemia remains the main cause of death in intensive care units. It has a mortality rate in the US of 50-60% (5). The release of multiple endogenous mediators plays a causative role in initiating an uncontrolled inflammatory response (6). One of those mediators has been identified as NO.

NO is generated by two constitutive NOS (cNOS) isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), which control relaxation of smooth muscles and modify central and peripheral nervous system function. Dysfunction of cNOS is associated with reduced production of NO, which leads to severe ischemia/reperfusion injury, hypertension, and hypercholesterolemia (7). In contrast, excessive and continuous production of NO by iNOS over prolonged periods contributes significantly to circulatory failure and hypotension as observed during endotoxemia (8). Several endotoxins activate the transcription and translation of iNOS, the third NOS isoform (9). This induction of iNOS occurs mainly in leukocytes, macrophages, hepatocytes, cardiac myocytes, and, to a lesser degree, in vascular smooth muscle (8). Induction of iNOS at the site of infection is responsible for the massive and uncontrolled generation of NO, which occurs during endotoxemia (2, 6).

Cytotoxicity is often directly attributed to NO. However, NO is only moderately reactive and shows low cytotoxicity at physiologic concentrations (10). The major decay route for NO in mammalian systems is by its extremely rapid reaction with superoxide ([O.sub.2.sup.-]) to form peroxynitrite (OONO[sup.-]; Fig. 1). It is one of the fastest reactions in mammalian physiology (K = 6.7 X [10.sup.9] L x [mol.sup.-1] x [s.sup.-1]) (11). The final product (peroxynitrite) is usually harmless. However, after protonation (p[K.sub.a] = 6.8), peroxynitric acid is formed, which, at high concentrations, is very cytotoxic. Peroxynitric acid undergoes either homolytic cleavage to form the hydroxyl free radical (OH*) and the nitrogen dioxide free radical ([N[O.sub.2)]*) or heterolytic cleavage to form the nitronium cation (N[O.sub.2.sup.+)] and the hydroxide anion (O[H.sup.-]) (12). These three extremely reactive moieties (OH*, N[O.sub.2;] and N[O.sup.2.sup.+]) are most likely the source of the observed cytotoxicity erroneously attributed to NO (13).

The total concentration of NO during endotoxemia has been estimated by the determination of the concentration of NO decay products (N[O.sub.2.sup.-] + N[O.sub.3.sup.-]). This indirect approach has demonstrated a significant increase in the concentration of N[O.sub.2.sup.-] + N[O.sub.3.sup.-] during endotoxemia as measured in a variety of body fluids (9,14,15). However, these measurements provide no information concerning diffusible active NO, which is capable of activating smooth muscle relaxation. The diffusible NO plays a crucial role in hypotension during endotoxemia. Therefore, the indirect measurement of NO degradation products (N[O.sub.2.sup.-] + N[O.sub.3.sup.-]) may provide very misleading information concerning the degree of vasorelaxation within the cardiovascular system.

This report describes both direct electrochemical measurements of the diffusible concentration of NO (active NO concentration) and indirect measurements of the NO final decay products (total amount of NO produced is expressed as a sum of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] concentration) during endotoxemia in an animal model. These results were correlated with chemiluminescence measurements of [O.sub.2.sup.-] and peroxynitrite, which are generated simultaneously with NO during endotoxemia.


Materials and Methods


Normotensive Wistar-Kyoto male rats (280-320 g) were maintained at 22 [+ or -] 1 [degrees]C with a 12-h light/ 12-h dark cycle and were allowed water and standard rat chow ad libitum.


Rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. They were intubated and ventilated with room air using a Harvard small animal ventilator set at a tidal volume of 2.5 mL and a breathing rate of 100 breaths/min. The porphyrinic NO sensor was placed in a pulmonary artery wall during the surgery, and its exact localization was determined during postmortem examination. Polyurethane catheters were inserted into the left jugular and left femoral veins for drug administration. The endotoxin lipopolysaccharide [(LPS); Escherichia coli serotype 0127: B8; Sigma] was dissolved in saline and subsequently administered as an intravenous infusion (2 mg x [kg.sup.-1] x [min.sup.-1] for 10 min; total dose, 20 mg/kg). [N.sup.G]-Monomethyl-L-arginine (L-NMMA), a nonspecific cNOS and iNOS inhibitor, was purchased from Sigma Chemical, and L-N6(1-iminoethyl)-lysine (L-NIL), a selective iNOS inhibitor, was from LC Laboratories Co. Each of the inhibitors was dissolved in 0.1 mol/L phosphate buffer (pH 7.4) and then injected as an intravenous bolus (20 mg/kg) 45 min before the start of the LPS infusion. Another catheter was placed in the right carotid artery for intermittent blood sampling.


The isolated pulmonary artery was placed in modified Hank's balanced salt solution (HBSS; Sigma) at 4 [degrees]C and pH 7.4. The composition of HBSS is as follows: NaCl (137 mmol/L), Tris-HCl (10 mmol/L), Mg[Cl.sub.2] (1 mmol/L), KCl (5 mmol/L), Ca[Cl.sub.2] (1.8 mmol/L), MgS[O.sub.4] (0.8 mmol/L), K[H.sub.2]P[O.sub.4] (0.44 mmol/L), [Na.sub.2[HP[O.sub.4] (0.33 mmol/L), and L-arginine (0.1 mmol/L). Under the dissection microscope (Wild M3G; Leica), the pulmonary artery was cleared of adjacent tissue and cut into small segments (2-3 mm). When polyethylene glycol-superoxide dismutase (PEGSOD; Sigma) was used, it was added to HBSS at a dose of 100 kU/L of medium 10 min before injection of the calcium ionophore (A23187).


Two types of NO sensors were used for the in vivo and in vitro experiments. Their design was based on previously developed and well-characterized, chemically modified carbon-fiber technology (16-18). The porphyrinic sensor is free of interference from all reagents used in these experiments and from substances that may have been found in mammalian blood to concentrations at least two orders of magnitude greater than their expected concentrations. The NO sensor is also insensitive to blood pressure changes (19) and has a small thermal coefficient (1.8% of signal/[degrees]C). Additionally, the sensor is free of piezoelectric noise and is not affected by induced currents generated by contracting muscles of the cardiac systems (16, 20). The catheter-protected porphyrinic NO sensor for in vivo measurements was mounted on the tip of a truncated needle. Briefly, the needle from an intravenous catheter unit, 24 gauge and 25 mm long (Angiocath; Becton Dickinson), was roughened along the shaft and then truncated and polished flat, so that it was 3 mm shorter than its 24-gauge, protective catheter. A bare copper wire with a diameter of 0.1 mm and coated with conducting epoxy and a single carbon fiber (6 [micro]m in diameter; protruding 3 mm) were mounted inside the hollow needle on the protruding carbon-fiber tip. A highly conductive polymeric porphyrin was deposited on the protruding carbon-fiber tip by the use of 0.50 mmol/L nickel(II) tetrakis(3-methoxy-4-hydroxyphenyl)porphyrin (TMHPPNi) in 0.1 mol/L NaOH and the cyclic voltammetry deposition technique. After the deposition process, the carbon fiber tip was immersed three times for 6-7 s in a 10 g/L alcohol solution of Nafion (Aldrich).

For in vivo measurements, smooth muscle tissue was pierced with the catheter needle and positioned at the desired place in the pulmonary arterial wall. When the position of the catheter was secure, the needle was removed and replaced with the porphyrinic sensor. The auxiliary electrodes, platinum and silver/silver chloride (SSCE) electrodes, were placed in contact with the adjacent tissue. For in vitro measurements, an open arterial strip was placed in a thermostated organ chamber and two auxiliary electrodes of the three-electrode system (the platinum as a counter electrode and the silver/silver chloride as a reference electrode) were positioned in the organ chamber near the arterial strip. The porphyrinic sensor without catheter protection was lowered near the surface (5 [+ or -] 2 [micro]m) of the previously positioned opened arterial ring with the aid of the dissecting microscope and micromanipulator. Chronoamperometry was used for fast (0.1-s response time) and continuous measurement of the change of NO concentration with time. The data were collected using a PAR model 273 voltammetric analyzer (EG&G Princeton Applied Research) interfaced with an IBM Pentium computer with data acquisition and control software (Research Electrochemistry Software, Ver. 4.30; EG&G Princeton Applied Research). Before and after in vivo and in vitro measurements, each sensor was calibrated using NO calibration solutions at NO concentrations of 2 X 10-9 to 2 X 10-5 mol/L prepared as described previously (21).

N[O.sub.2.sup.-] AND N[O.sub.3.sup.-] MEASUREMENTS

At the same time as the electrochemical measurements of active NO in the tissue during endotoxemia, indirect measurements of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] (final products of NO decay) were performed using the Griess method (22). The Griess reagent is specific for N[O.sub.2.sup.-]; therefore, N[O.sub.2.sup.-] was reduced to NO,- by metallic cadmium. NOZ- and N03 content was determined as N[O.sub.2.sup.-] from absorbance measurements at 540 run using a calibration curve.


The concentration of [O.sub.2.sup.-] was determined by a chemiluminescence method (23). [O.sub.2.sup.-] produced chemiluminescence from lucigenin (bis-N-methylaridinium N[O.sub.3.sup.-]; Sigma), which was detected by a scintillation counter (Beckmann 6000 LS with a single-photon monitor). Each tissue sample (0.8-1.5 mg) was immersed in 2 mL of HBSS adjusted to pH 7.4 at 25 [degrees]C. Lucigenin was added to the solution to a final concentration of 0.25 mmol/L. All measurements of [O.sub.2.sup.-] were made in a similar manner after a 2-min incubation in HBSS. For the nonbasal determination of [O.sub.2.sup.-], the 2-min incubation period was followed by the injection of 20 [micro]L of 1 mmol/L A23187 calcium ionophore. A23187 is a receptor-independent cNOS agonist and is needed to measure maximal cNOS production of NO. Calibration curves were constructed on the basis of the photons emitted by the [O.sub.2.sup.-] generated after treatment of xanthine with xanthine oxidase.


A chemiluminescence method was also used to determine the concentration of peroxynitrite (22). Photon counts were recorded with a scintillation counter (Beckman 6000LS, with a single-photon monitor). Each tissue sample (8-10 mg) was immersed in 0.5 mL of HBSS adjusted to pH 7.4 at 25 [degrees]C, and then calcium ionophore A23187 solution was added to a final concentration of 20 [micro]mol/L. Alkaline sodium bicarbonate luminol solution (1.0 mL) was added after 3 s to achieve a final concentration of 400 [micro]mol/L luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and 50 mmol/L bicarbonate at pH 10.0. The basal concentration of peroxynitrite was obtained in the same manner in the absence of tissue.


Values were expressed as means [+ or -] SE, with P <0.05 considered statistically significant. Statistical evaluation was done by ANOVA followed by unpaired Student Mest. All analyses were performed with the statistical software Microcal Origin (Microcal Software, Inc.).



The total concentration of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] in blood plasma increases during endotoxemia induced by administration of LPS (Fig. 2). After 3 h of endotoxemia, a statistically significant increase in the total concentration of N[O.sub.2.sup.-] plus N[O.sub.3.sup.-] was observed, which reached a plateau of 10 [+ or -] 1.8 [micro]mol/L after 5 h. The plateau concentration was approximately fourfold higher than the basal concentration.

However, the direct, in vivo electrochemical measurement of NO using the porphyrinic sensor implanted into the rat pulmonary artery clearly showed two phases of NO release during endotoxemia (Fig. 3a). In the early acute phase, after administration of 20 mg/kg LPS, a sharp increase in the production of NO above its basal concentration of 52 [+ or -] 10 nmol/L was observed. The rate of increase for NO was 0.19 nmol x [L.sup.-1] x [s.sup.-1]. After 540 [+ or -] 25 s, the NO concentration reached a plateau of 145 [+ or -] 18 nmol/L above the basal concentration that persisted for 4 [+ or -] 0.6 min. The plateau was followed by a decrease in the concentration of NO at the rate of -0.07 nmol x [L.sup.-1] x [s.sup.-1]. After 45 min, a second increase in the concentration of NO was observed, but at a much slower rate of <0.02 nmol x [L.sup.-1] x [s.sup.-1]. A second plateau of NO concentration was established 90 [+ or -] 10 min after administration of LPS. The selective inhibitor of iNOS, L-NIL, at the dose of 20 mg/kg decreased the NO concentration only during the second phase of endotoxemia (~5 min after LPS administration; Fig. 3B). Administration of the L-NMMA (20 mg/kg) nonselective inhibitor of eNOS, nNOS, and iNOS decreased the concentration of NO during both the acute phase and the second phase of endotoxemia (Fig. 3C).

To estimate the potential maximum concentration of NO produced by eNOS during endotoxemia, in vitro changes in endothelial NO concentration were measured in freshly excised pulmonary artery after stimulation with 10 [micro]mol/L calcium ionophore A23187 (Fig. 4). The single carbon-fiber porphyrinic sensor was placed close (5 [+ or -] 2 [micro]m) to the surface of the endothelial cells in an excised segment of pulmonary artery, and NO concentrations were recorded at different times after the administration of LPS. To estimate the influence of [O.sub.2.sup.-] on calcium-stimulated endothelial NO release, NO was measured after endotoxemia in the presence or absence of 100 kU/L PEG-SOD (membrane-permeable SOD). The basal concentration of NO was 45 [+ or -] 5 and 27 [+ or -] 8 nmol/L in control and endotoxic tissue, respectively. In the presence of PEG-SOD, the basal concentration significantly increased in endotoxic (40 [+ or -] 3 nmol/L), but not in the control (48 [+ or -] 5 nmol/L). NO release stimulated by calcium ionophore in the controls was increased by 15% from 437 [+ or -] 6 nmol/L to 504 [+ or -] 7 nmol/L in the presence of PEG-SOD. However, in endotoxic artery, the concentration of NO increased by -50% in the presence of PEG-SOD (increase from 262 [+ or -] 3 nmol/L to 547 [+ or -] 5 nmol/L; P <0.001; n = 6).




Endothelial NO production was substantially decreased during the first 3 h of endotoxemia (Figs. 4 and 5) because of the increased generation of [O.sub.2.sup.-]. The increase of [O.sub.2.sup.-] was confirmed indirectly with an experiment performed in the presence of PEG-SOD. In the presence of PEG-SOD, the NO concentration was restored to the concentration observed before endotoxemia. The maximal production of NO by endothelium during different phases of endotoxemia was determined by activating cNOS with the receptor-independent agent A23187 (Fig. 5; n = 6).


The previous experiments using PEG-SOD suggest that the increase in the production of [O.sub.2.sup.-] during endotoxemia is responsible for the decrease in the concentration of NO (Figs. 4 and 5). During the time period (60 min) of endotoxemia, there was no significant decrease in the eNOS protein as determined by Western blot. Therefore, the decrease of the production of NO is attributable solely to an increase in the generation of [O.sub.2.sup.-]. To confirm this hypothesis, a chemiluminescence method was used to directly measure the concentration of [O.sub.2.sup.-] during endotoxemia in freshly isolated tissue. The mean basal concentration (before endotoxemia) of [O.sub.2.sup.-] in the pulmonary artery was 16 [+ or -] 2 nmol/L (Fig. 6). The change in the total concentration of [O.sub.2.sup.-] with time after LPS administration was measured after the maximal stimulation of NO release by A23187 (Fig. 6). One hour after LPS administration, the concentration of [O.sub.2.sup.-] increased to 30 [+ or -] 3 nmol/L, reaching a plateau of 41 [+ or -] 4 nmol/L after 6 h (Fig. 6; n = 6). In the presence of L-NMMA, the concentration of OZ- decreased ~60%. The decrease in OZ-concentration is similar to the decrease in the NO concentration in the presence of L-NMMA.




Peroxynitrite release was measured in vitro during the maximal stimulation of the release of NO by A23187 (Fig. 7), and it paralleled the release of [O.sub.2.sup.-] (Fig. 6) throughout endotoxemia. After 1 h, the concentration of peroxynitrite increased approximately twofold compared with the control. A plateau was established after 3 h of endotoxemia. The maximum concentrations of NO, [O.sub.2.sup.-], and peroxynitrite before and 3 h after LPS administration are shown in Fig. 7.



The direct and continuous in vivo measurement of diffusible NO in the pulmonary artery during endotoxemia demonstrated two distinct increases in the concentration of NO with time (Fig. 3A). The first increase in the concentration of NO occurred ~10 min after the administration of LPS and was attributable to the activation of eNOS. A similar pattern of NO release is observed after stimulation of endothelial cells with typical agonists of NO, such as bradykinin or acetylcholine. The second increase in the diffusible concentration of NO was observed -1 h after the onset of endotoxemia and was a result of the activation of iNOS. These direct electrochemical measurements of NO differ markedly from the indirect estimation of total NO by spectroscopic measurements of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] concentrations. This indirect measurement of degradation products revealed a steady increase in the concentration of NO with time during endotoxemia (Fig. 2). Therefore, the distinct biphasic temporal fluctuations detected in the concentration of biologically active NO by the porphyrinic microsensor were not detected with the less sensitive and more indirect method of measuring NO degradation products. The in vivo measurement of NO may be performed with the use of a modified porphyrinic microsensor placed in a catheter or needle, which can be located in tissue or in circulating blood for the direct determination of NO (17). Although technically more demanding, this method provides a precise measurement of fluctuations of NO concentrations with time.

The measurement of NO and its metabolites in septic patients poses a challenge to the clinical laboratory. The indirect spectroscopic measurement of N[O.sub.2.sup.-] and N[O.sub.3.sup.-], indicators of NOS activity and the total production of NO, is based on the detection of the degradation products of NO in plasma or urine (13,22). However, experimental evidence supporting the validity of this assumption is limited. In a recent study, mice lacking the eNOS gene had 40% less plasma N[O.sub.2.sup.-] and N[O.sub.3.sup.-] compared with wild-type mice (24). Thus, the absence of eNOS leads to a reduction in plasma N[O.sub.2.sup.-] and N[O.sub.3.sup.-] not compensated by other NOS isoforms. However, this measurement still fails to account for temporal variations in concentrations of NO in plasma and specific organs (2).

The evaluation of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] in urine also has important limitations. The rate of excretion of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] in the urine is directly related to renal function (25). NO is generated from the following reaction: L-arginine + NADPH + [O.sub.2] [right arrow] [N.sup.G]-hydroxyl-L-arginine [right arrow] citrulline + NO + NAD[P.sup.+] (26). The cellular elements in infected urine contain an increased amount of NO generated by iNOS compared with uninfected control specimens. The iNOS represents an endogenous source of NOZ- production in the urine specimen (27, 28). Under appropriate physiological conditions, NO and, therefore, its degradation products, N[O.sub.2.sup.-] and N[O.sub.3.sup.-], can be produced nonenzymatically (29-31). Hydrogen peroxide generates NO from n- or L-arginine by a nonenzymatic pathway (30). The [O.sub.2.sup.-] anion oxidizes the intermediate, [N.sup.G]-hydroxy-Larginine, to NO (30, 31). Therefore, inhibition of cNOS and/or iNOS activity, increased in septic shock (32), will not completely stop NO production because the nonenzymatic synthetic pathways would not be altered. In acidic urine, some N[O.sub.2.sup.-] is converted to NO (33). A variety of disease states lead to increased or decreased total production of NO and, therefore, alterations in N02excretion in the urine (22, 34-36). Consequently, the concentration of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] in urine is not only an indicator of a urinary tract infection, but also a monitor of the total body production of NO (13, 22, 33-36).

The first phase of NO release during endotoxemia is catalyzed by calcium-dependent eNOS, which is activated by an increase in unbound cytosolic free calcium. Inhibition of calcium flux into the cell inhibits NO release (1, 7). LPS activates intracellular calcium gradients, which stimulate eNOS to produce NO, water, and L-citrulline from L-arginine and OZ (8) (Fig. 1). To estimate maximal cNOS activity at different times after the induction of endotoxemia, we used the calcium ionophore A23187 to increase intracellular calcium influx (Figs. 4-7). Concurrently, during this first phase of NO release, the concentration of [O.sub.2.sup.-] also increased (Fig. 6). This LPS-stimulated production of [O.sub.2.sup.-] is inhibited by the presence of the nonspecific NOS inhibitor L-NMMA. The generation of [O.sub.2.sup.-] occurs during endotoxemia after there is a local depletion of L-arginine of <[10.sup.-4] to [10.sup.-3] mol/L. At this low concentration of L-arginine, there is an insufficient concentration gradient for this substrate to bind to the active site of cNOS (37). In the absence of L-arginine or tetrahydrobiopterin, cNOS function expands to the catalysis of two reactions: the one-electron reduction of [O.sub.2] to [O.sub.2.sup.-] and the five-electron oxidation of L-arginine to NO. When these two reactions take place in close proximity, the extremely rapid reaction of [O.sub.2.sup.-] with NO produces peroxynitrite, which, after protonation, rearranges to form the stable product N[O.sub.3.sup.-](9). Using isolated enzymes, Klatt et al. (38) found that both eNOS and nNOS, but not iNOS, can produce [O.sub.2.sup.-] in L-arginine- or tetrahydrobiopterin-limiting environments. Therefore, the decrease in the production of NO observed throughout endotoxemia (Fig. 5) can be attributed to the increased production of [O.sub.2.sup.-]. eNOS appears to be a major source of [O.sub.2.sup.-]. However, in endothelial cells, there are several other sources of [O.sub.2.sup.-] generation, including prostaglandin metabolism, cytochrome P450, and processes stimulated by protein kinase C or xanthine oxidase. Therefore, the net concentration of NO measured by the porphyrinic sensor depends not only on NO generated by cNOS, but also on the concentration of [O.sub.2.sup.-]. The increasing concentration of [O.sub.2.sup.-] reacts with NO to produce peroxynitrite, which leads to a reduction in bioactive NO (Fig. 5), not a steady increase in NO as suggested by the determination of N[O.sub.2.sup.-] and N[O.sub.3.sup.-] (Fig. 2). In conclusion, the indirect measurements of NO using the concentration of NOZ- and NO,- during endotoxemia fails to provide an accurate release of bioactive NO. Because of the fast consumption of NO by OZ-, the actual concentration of active NO in the cardiovascular system is relatively low, and this concentration can be monitored only by using direct, real-time measurement.

This work was supported by a grant from the William Beaumont Hospital Research Institute and Public Health Grant (HL 60900). We thank T. Kosaski for technical assistance in the preparation of the manuscript.


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[1] Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701. [2] Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, M1 48073.

[3] Nonstandard abbreviations: i-, c-, e-, and nNOS, constitutive, inducible, endothelial, and neuronal NO synthase; LPS, lipopolysaccharide; L-NMMA, [N.sup.G]-monomethyl-L-arginine; L-NIL, L-[N.sup.G]-(1-iminoethyl)-lysine; HBSS, Hank's balanced salt solution; and PEG-SOD, polyethylene glycol-superoxide dismutase.

* On sabbatical leave from: Department of Biochemistry, Medical University of Gdansk, and Laboratory of Cellular and Molecular Nephrology, Medical Research Center of the Polish Academy of Science, 80-210 Gdansk, Poland. [dagger] Address correspondence to this author at: Department of Chemistry and Biochemistry, Ohio University, Clippinger Laboratories, Athens, OH 45701. Fax 740-593-9641; e-mail

Received January 19, 2001; accepted March 15, 2001.
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Title Annotation:Endocrinology and Metabolism
Author:Brovkovych, Viktor; Dobrucki, Lawrence W.; Brovkovych, Svitlana; Dobrucki, Iwona; Kalinowski, Leszek
Publication:Clinical Chemistry
Date:Jun 1, 2001
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