Plasma Nitrosothiols Contribute to the Systemic Vasodilator Effects of Intravenously Applied NO
Experimental and Clinical Study on the Fate of NO in Human Blood
Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 μmol/L. The presence of red blood cells reduced the formation of plasma RSNOs. Intravenous infusion of 30 μmol/min NO in healthy volunteers increased plasma levels of RSNOs and induced systemic hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the brachial artery and forearm microvasculature. Intravenous application of S-nitrosoglutathione, a potential carrier of bioactive NO, mimicked the vascular effects of NO, whereas nitrite and nitrate were inactive. Changes in plasma nitrosothiols were correlated with vasodilator effects after intravenous application of S-nitrosoglutathione and NO. These findings demonstrate that in humans the pharmacological delivery of NO solutions results in the transport and delivery of NO as RSNOs along the vascular tree.
The continuous production and release of endothelial NO plays an important role in vascular homeostasis1 and cardiac function.2,3⇓ The supposedly rapid conversion of NO to biologically inactive metabolites in human blood formed the rationale for inhalation NO therapy, because the short half-life of NO should confine its effect to the pulmonary circulation.4 However, recent evidence suggests that higher doses of inhaled NO may exert side effects beyond the pulmonary circulation.5,6⇓ Red blood cells (RBCs) are believed to be a major sink for NO by virtue of the rapid co-oxidation reaction of NO with oxyhemoglobin to form methemoglobin (metHb) and nitrate. Alternatively, NO may react with hemoglobin (Hb) to form either nitrosylhemoglobin (NOHb) or S-nitrosohemoglobin (SNOHb).6,7⇓ In addition to its reaction with RBCs, NO has to interact at some stage with plasma constituents, especially in view of the existence of an RBC-free zone close to the vessel wall.8
A better knowledge of the fate of NO in plasma is an important prerequisite for a proper understanding of its physiology in blood and in the human circulation in general. Recently, we provided evidence that intra-arterially applied NO can be transported in a bioactive form over significant distances along the forearm circulation.9 To date, no data have been reported on the systemic dilator effects of intravenously applied NO in the human peripheral vasculature, presumably because of its supposedly rapid clearance from blood. Our data challenge this current dogma. In the present study, we demonstrate that the intravenous infusion of NO solution results in the transport and delivery of NO as S-nitrosothiols (RSNOs), which are accompanied by systemic hemodynamic effects and vasodilation in conduit and resistance vessels.
Materials and Methods
In Vitro Studies on the Metabolism of NO in Plasma
To estimate the decay of NO in different media, NO was added to either blood, plasma, a solution containing physiological levels of human serum albumin (0.6 mmol/L), or PBS in a closed reaction chamber (Figure 1). Changes in NO concentration were determined with a commercial NO-sensitive Clark-type electrode. Simultaneous measurements of NO and oxygen were performed in an acrylic chamber maintained at 37.5±0.05°C under stirring (200 rpm) after purging of the different media with argon for 10 minutes. This treatment resulted in dissolved O2 levels equivalent to concentrations in the arterial circulation (120 to 150 μmol/L). After 20 minutes of equilibration, NO was added to the reaction chamber to achieve concentrations of 1, 5, and 10 μmol/L NO in PBS. Because an appropriate signal was obtained at a final concentration of 5 μmol/L NO, subsequent experiments in albumin-containing solution, plasma, and blood were conducted using this concentration.
Aliquots of saline stock solutions of NO10 were added to plasma and whole blood to further study the quantitative and kinetic aspects of oxidative and nitrosative chemistry. The formation of plasma RSNOs was measured after NO incubation in the absence and presence of RBCs from undiluted whole blood samples. Venous blood was collected into tubes containing heparin (10 U/mL) and centrifuged for 20 minutes at 800g and 37°C. Plasma was checked for hemolysis by spectroscopy. Stock solutions of S-nitrosoalbumin (SNOAlb) were prepared freshly.11
Determination of Nitrite, Nitrate, and RSNOs
For the determination of plasma nitrite and RSNOs, plasma was diluted 1:5 in ice-cold 0.9% saline containing N-ethylmaleimide (5 mmol/L) and EDTA (2 mmol/L)9 and measured by a chemiluminescence analyzer (Sievers 280 NOA).11 NO adducts transported in whole blood (RBCs) were measured using identical procedures. Nitrate concentrations in plasma was measured by high-performance liquid chromatography with direct UV detection.12
In Vivo Studies and Vascular Study Protocols
Healthy nonsmoking volunteers who underwent regular physical examinations and routine chemical analyses and who had no present or past evidence of arterial hypertension, hypercholesterolemia, chronic heart failure, or diabetes mellitus were studied supine in a quiet air-conditioned room (21°C) in the morning. The study protocol was approved by the ethics committee of Heinrich-Heine-University. Changes in forearm blood flow (FBF) were measured at 10-second intervals using standard techniques of mercury-in-rubber strain-gauge plethysmography.13 The diameter of the brachial artery was measured with a 15-MHz linear array transducer proximal above the antecubital fossa at end diastole by an automated analysis system.14 All high-resolution ultrasound tracings and plethysmography data were analyzed in a blinded fashion.
To test the hypothesis that NO exerts systemic effects in vivo, four sets of investigations were carried out. In the first set, to assess systemically effective doses without the risk of eliciting serious side effects, 6 doses of 5 μmol were applied at 3-minute intervals to achieve a cumulative dose of 30 μmol NO after completion of a preliminary dose-finding study. To achieve exact quantification of changes in FBF, the area under the curve of the signal recorded over 3-minute intervals was analyzed after each NO application. In a second set of studies, the dilator responses in conduit and resistance vessels were compared after intravenous application of the cumulative bolus dose (30 μmol, corresponding to 0.4 to 0.6 μmol NO/kg body wt). Volume-matched saline control solutions or NO solutions were infused in a double-blinded fashion and in random order. In a third set of experiments, the in vivo increase of plasma RSNOs and their hemodynamic effects were investigated. NO was infused (30 μmol/min) into the dorsal hand vein, and samples drawn from the cubital veins of the ipsilateral and contralateral forearm were analyzed (see Figure 5). In a fourth set of experiments, potential vasodilator effects of the NO metabolites nitrite, nitrate (n=3), and S-nitrosoglutathione (GSNO, 1 μmol [n=6] and 4 μmol [n=1]) were assessed. The kinetics of changes in plasma RSNO and hemodynamics and the concomitant formation of NO adducts in RBCs were determined after application of NO (n=6) and GSNO (n=4).
Data are reported as mean±SEM, and values of P<0.05 were considered significant. Changes in FBF, blood pressure, heart rate, and brachial artery diameter were analyzed by the nonparametric Wilcoxon test. Differences in plasma RSNO levels between ipsilateral and contralateral blood samples were analyzed by the Mann-Whitney test.
Decomposition of NO in Human Plasma and Blood
In agreement with the literature,15 NO decay in PBS was more rapid at increasing NO concentrations (data not shown). The decay of NO (5 μmol/L) in various media estimated as the time interval from maximal to half-maximal response occurred according to the following rank order: PBS (182±15 seconds), albumin-containing solution (132±21 seconds), and plasma (68±12 seconds). The addition of NO to the blood did not result in any detectable signal of free NO, indicating that its half-life is shorter than the reaction time of the electrode (<5 seconds) under these conditions.
Metabolic Conversion of NO
Human plasma from fasted healthy volunteers contained the following concentrations of NO-related metabolites: 147±68 nmol/L nitrite, 16.4±3.4 μmol/L nitrate, and 15±6 nmol/L RSNOs (n=5). To gain insight into the concentration dependence of oxidative and nitrosative NO metabolism in plasma, increasing concentrations of NO (6 to 200 μmol/L) were added. The majority of the applied NO was rapidly converted to nitrite, and this was paralleled by the formation of RSNOs, whereas the increase in nitrate was nonsignificant (Figures 2A through 2C). Even with supraphysiological concentrations of NO, the pool of nitrosatable sites in human plasma appeared to be nonsaturable. Using lower concentrations of NO (0.2 to 2.0 μmol/L), we also measured a significant increase in plasma nitrosothiols, which was less in the presence of blood cells (Figure 2D
Dynamics of NO Metabolism in Plasma
To obtain information on the dynamics of NO conversion, a time-course study on the formation of its metabolites was carried out. By incubating plasma with 40 μmol/L NO over a period of 10 minutes, the formation of nitrite occurred almost instantaneously (Figure 3). The majority of RSNOs were formed immediately after the addition of NO to the plasma, but maximum levels were not achieved before 5 minutes of incubation. Again, the increase in nitrate was nonsignifican
Site and Mechanisms of Plasma Nitrosation
Freshly obtained human plasma was separated into a 20-kDa ultrafiltrate (low molecular weight [LMW]) and a high molecular weight (HMW) fraction. Then, aqueous NO solution was added to aliquots of native plasma and the two plasma fractions, and the respective formation of RSNOs was measured after 5 minutes, the incubation time at which RSNO levels are maximal (Figure 3). The amount of RSNOs detected in the HMW plasma fraction (which is composed largely of SNOAlb) corresponded to 95% of that in whole plasma. Only small amounts of RSNOs were determined in the LMW plasma fraction, and the total nitroso content in the two individual fractions corresponded to 96±8% of the level in whole plasma. Importantly, when both fractions were recombined before the addition of NO, RSNO levels were almost identical to those in native plasma, indicating the absence of any conceivable cooperative catalytic effect regarding albumin nitrosation (native plasma 848±62 nmol/L, HMW 807±70 nmol/L, LMW 10±4 nmol/L, and HMW/LMW recombined 811±78 nmol/L), corroborating published data.16 Using chemiluminescence, we found that SNOAlb released NO spontaneously (1.4±0.2 pmol NO/min from 10 nmol SNOAlb, pH 7.4, room temperature, n=3), and this was further potentiated in a temperature-, light-, ascorbate-, and thiol-dependent manner (data not shown), confirming earlier reports.17
Intravenous Application of NO Solution Into the Human Systemic Circulation
The in vivo studies were performed in a study population of 31 healthy individuals (26 men and 5 women) with a mean age of 29±1 years, a body weight of 68±2 kg, a body length of 176±2 cm, a heart rate of 67±2 bpm, and 118±3 mm Hg systolic and 72±2 mm Hg diastolic blood pressure. Baseline FBF amounted to 2.5±0.3 mL/min per 100 mL tissue. A slight increase in FBF (and thus evidence for a systemic effect of NO) was observed shortly after intravenous application of 5 μmol NO. Repeated applications of the same dose of intravenously applied NO resulted in a sustained vasodilation, as evidenced by a further increase in FBF and a consecutive decrease in blood pressure (Figure 4), which was accompanied by a minor increase in heart rate (from 68±2 to 70±1 bpm, P=NS). On repeated NO application, these circulatory responses appeared to become subject to counterregulation. In a further preliminary set, we demonstrated that intravenous application of a single NO bolus of 30 μmol could be applied and that it dilated not only forearm resistance vessels but also the brachial artery (4.24±0.26 to 4.43±0.24 mm, P=0.1, n=4
In a third set of studies, infusion of NO (30 μmol over 1 minute) into the dorsal hand vein significantly increased (from 38±6 to 283±110 nmol/L) RSNO levels in the cubital vein proximal to the infusion site (Figure 5A). RSNO levels in the vein of the opposite arm peaked at a later time point (59±8 nmol/L), consistent with the circulatory transit time (Figure 5B). NO infusion significantly increased brachial artery diameter from 4.42±0.12 to 4.61±0.12 mm (Figure 5C) and increased FBF from 1.58±0.16 to 2.18±0.24 mL/100 mL tissue per minute (Figure 5D). Maximal contralateral RSNO concentrations preceded maximal dilator responses in the forearm microvasculature and macrovasculature, consistent with the involvement of RSNOs in the circulatory response. Mean arterial blood pressure dropped significantly (from 93±3 to minimally 88±1 mm Hg), whereas heart rate remained unaffected (66±5 versus 67±5 bpm). All parameters reached baseline values within 45 to 60 minutes. Volume-matched saline controls had no significant effect on baseline values.
Bolus applications of nitrite or nitrate (30 μmol each) affected neither vascular tone nor plasma RSNO levels (n=3). To investigate whether the application of RSNOs into the human circulation would elicit systemic effects similar to those of NO, GSNO was administered. Injection of a 1 μmol bolus of GSNO increased plasma RSNOs, brachial artery diameter, and FBF (Figures 6A through 6C). This was accompanied by a decrease in blood pressure and an increase in heart rate (mean blood pressure 82±4 to 72±4 mm Hg, heart rate 62±4 to 81±4 bpm; for both, P=0.028). Changes in all three parameters were dose dependent, inasmuch as all responses were potentiated by a 4-fold increase in the GSNO dose (n=1) (in Figure 6, compare panels A through C with panels D through F
Concomitant changes in NO adducts in RBCs on the application of NO and GSNO are depicted in Figure 7. The increase of venous NO adducts in RBCs was almost 6-fold higher on application of NO compared with GSNO. In Figure 8, the mean values of the original data from Figures 5 through 7 are depicted to relate the changes in vascular responses and biochemical signals on intravenous application of NO and GSNO. With both agents, the changes in plasma RSNOs were uniformly correlated with the vasodilation at the level of conduit and resistance arteries (Figures 8C and 8D), whereas the correlations for the RBC-associated NO adducts were different for GSNO and NO (Figures 8A and 8B
The key findings of the present study are as follows: (1) In vitro, supraphysiological concentrations of NO added to plasma are subject not only to oxidative decomposition but also to nitrosative chemistry, forming HMW RSNOs in a nonsaturable manner. In the presence of RBCs, the increase of plasma RSNO concentration was reduced. (2) In vivo, pharmacological delivery of NO elicited a significant formation of RSNOs and a long-lasting dilation of human conduit and resistance arteries, supporting the notion that in vivo plasma RSNOs contribute to the transport and delivery of NO in the human systemic circulation. (3) The oxidative decomposition products of NO, nitrite, and nitrate appear to be biologically inactive under these conditions.
Oxidative Metabolism of NO and Nitrosative Chemistry in Human Plasma
In the present study, the major product of the oxidative breakdown of NO in plasma was found to be nitrite, with only a minor portion being converted to nitrate, which is in line with previous reports.12,18⇓ NO decomposition in aqueous buffer solution and plasma was dependent on the concentration of both oxygen and NO. The given concentration of NO was removed more rapidly from plasma and albumin-containing solution compared with PBS. Our findings are consistent with the recent demonstration that the diffusional field and life span of NO is critically determined by the presence of proteins and lipids forming hydrophobic environments favoring protein nitros(yl)ation.19–21⇓⇓ The formation of plasma RSNOs from NO has been demonstrated earlier, 22 with reported levels being in the micromolar range. In humans, basal RSNO levels reported in the literature range from 24±9 nmol/L6 to 9.2±1.6 μmol/L.23 This discrepancy may be explained either by marked species differences or by the different analytical approaches. In agreement with several recent reports,24,25⇓ we have detected basal RSNO levels in the nanomolar range and demonstrate in the present study that basal levels of plasma RSNOs can be increased severalfold by exposure to NO. With supraphysiological concentrations of 200 μmol/L NO, protein binding sites for NO were still not saturated. Most important, plasma RSNOs were formed after adding NO at lower concentrations (0.2 to 2 μmol/L), equivalent to those recently determined at the surface of endothelial cells.26 RBCs, which represent a potential sink for added NO, considerably reduced the formation of plasma RSNOs. The finding that a small amount of plasma RSNOs could still be detected in the presence of RBCs may be explained by the possibility that a portion of this NO escaped the reaction with RBCs and may have reacted with plasma species. Alternatively, RSNOs recovered in plasma may originate from RBCs, which dominated the uptake of NO and have also been shown to export nitrosothiols.27 These findings underscore the potential of plasma as a physiological NO carrier. Furthermore, our in vitro data support the notion that RSNOs have the potential to carry NO, inasmuch as it is stable for a limited period of time, and its decomposition is accompanied by the release of NO.
Biological Implications of Nitrosation Chemistry in Human Plasma
The significance of plasma RSNOs as a potential transport and delivery system for NO in the human circulation has not been established. Our in vitro data suggest that the addition of NO in the form of aqueous solution to plasma leads to the formation of RSNOs. However, whether such an increase is sufficient to elicit a biological response and whether these findings can be extrapolated to the in vivo situation in blood with RBCs being present as a potentially important intravascular sink for NO were unclear. The rapid intraerythrocytic conversion of NO to either NOHb, SNOHb, or metHb and nitrate also raises the question of whether NO can ever elicit a systemic vasodilator effect via plasma RSNOs at all. In the present study, we demonstrate for the first time that repeated intravenous application of NO exerts systemic hemodynamic effects, as judged by plethysmographic measurement of a significant increase in FBF in the contralateral arm and a transient and modest decrease in arterial blood pressure. In a separate set of experiments, we extended these findings by demonstrating that intravenous NO application does not elicit a vasodilator effect on resistance vessels alone but also causes dilation of conduit arteries.
To further support the notion that plasma RSNOs are involved in NO transport, we aimed to provide evidence for the formation of such species in vivo. Infusion of the same dose of NO into the dorsal hand vein increased plasma RSNOs in blood sampled from the proximal antecubital vein. Most important, these RSNOs were recovered within a time period equivalent to circulatory transit in the opposite arm. This was followed by dilator responses in the brachial artery and forearm microvasculature. The onset of brachial artery dilation occurred simultaneously to that in the downstream microvasculature, indicating that this effect was secondary to the applied NO itself and not a result of shear-related endothelial NO formation due to flow increases in the microvasculature. To the best of our knowledge, these data indicate for the first time that exogenously applied NO is transported in the form of plasma nitroso species, exerting systemic vasodilator effects. Taken together, these results provide unequivocal evidence for the occurrence and hemodynamic consequences of nitrosation chemistry in the human circulation.
To further identify the nature of carriers for bioactive NO in human plasma, we intravenously applied the NO metabolites found in our in vitro experiments. Nitrite and nitrate at comparable doses were vasodilatory inactive, ruling out the possibility that these products of oxidative NO decomposition could have functioned as NO carriers under the conditions of the present study. In support of the conclusion that RSNOs are involved in the systemic vascular effects of intravenously applied aqueous NO solution, its hemodynamic response was mimicked by intravenous application of a LMW nitrosothiol. GSNO was chosen over SNOAlb because GSNO is likely to have a lower risk for potential immunological side effects and because SNOAlb has been described as undergoing rapid transnitrosation to form LMW nitrosothiols in vivo.28 The peak in plasma RSNOs either coincided with or preceded the changes in brachial artery diameter, and FBF and was dose dependent (Figure 6). Most important, circulating plasma RSNO levels were in the same range after intravenous application of either GSNO or NO. Thus, with both interventions, the amount of plasma RSNOs necessary to elicit a given dilation at the level of both the conduit and resistance vessels was comparable (Figures 8C and 8D). Given the similarity in the onset of dilation between NO and GSNO, transnitrosation reactions do not appear to be rate-limiting in vivo, which is in agreement with recent reports.21
Consumption of plasma NO by RBCs is reduced by increased flow, an unstirred plasma layer surrounding the RBCs, the cell-free zone near the vascular wall, and a reduced diffusion rate over the cellular membrane.8,29,30⇓⇓ However, in the present study, the formation of RSNO and the dilatory response lasted several orders of magnitude longer than the previously suggested half-life of NO in vivo, thus ruling out the possibility that the transport of free NO contributes significantly to the sustained dilation seen in both conduit and resistance vessels.
Besides oxidative and nitrosative reactions in plasma, NO may undergo alternative metabolic routes (such as reactions with reactive oxygen species), diffuse into vascular tissues and blood cells, and react with Hb within RBCs. NO and oxyhemoglobin may rapidly react to form nitrate and metHb. Depending on the prevailing oxygenation state of Hb, NO can react with Hb to form either NOHb31,32⇓ or SNOHb.7,33⇓ In line with previous reports, we found a transient increase in these NO adducts, the sum of which is depicted in Figure 7. The concentration of NO adducts in RBCs increased >6-fold on NO application compared with GSNO application, although both were applied at doses eliciting comparable hemodynamic responses. Intravenous application of NO at the chosen dose might favor formation of NO adducts in RBCs because of the facilitated access to RBCs, whereas the NO delivered by GSNO may be selectively transferred to plasma constituents or released to the vascular wall.
Interestingly, the ratio of the biochemical signal (change in plasma and RBC adducts of NO) and the observed biological response (dilation of conduit and resistive vessels) was different on infusion of GSNO and NO. For blood cells, which mainly consist of RBCs, this relationship was diverging (Figures 8A and 8B). The identical degree of vasodilation was associated with a significant higher formation of NO adducts on application of NO compared with GSNO. Thus, at least at these supraphysiological NO doses, the measured intracellular NO adducts might represent only a spillover with minor importance for the observed vasodilatory responses. It further argues against a one-to-one shift of formed NO adducts from RBC to plasma at these pharmacological doses of NO. In contrast, in plasma, on NO and GSNO infusion a given increase in RSNO was uniformly associated with a significant vasodilation (Figures 8C and 8D), supporting our notion that plasma RSNOs are involved in the observed vasodilator activity of NO solutions. In addition, NO originated from RBCs might contribute to this effect.
Continuous intravenous infusion of appropriate doses of NO in the form of an aqueous solution may be useful to influence platelet function and vascular tone by means of modulating endogenous blood-borne NO carriers and may represent a conceptually attractive new approach for the treatment of cardiovascular disorders, which are associated with a reduced bioavailability of NO. However, it has to be considered that doses of applied NO in the present study were in the pharmacological rather than the physiological range. Cautious advice has to be made about therapeutic application of such doses to patients either for short- or long-term use. In addition, to find the appropriate doses, the most suitable route of NO administration has to be determined. Inhalation, in contrast to intravenous application, might elicit different NO bioactivity. We applied NO intravenously using either repetitive boluses of 5 μmol or bolus infusion at 30 μmol/min. In vitro, it has been demonstrated that bolus application of NO favors artificial nitrosation.34 Whether in vivo bolus administration compared with infusions would alter the ratio for oxidative and nitrosative chemistry cannot be clarified by our experiments. It has to be considered that using pharmacological doses of NO may mask physiological reaction mechanisms. The mode of reaction of in vivo formation of plasma RSNOs on NO application should be investigated further.
This study was supported by the Biomedizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, by grants from the Deutsche Forschungsgemeinschaft (DFG) (Ke 405/4, Pr 643 2/1, and Sonderforschungsbereich 612), and by funds from NIH grant HL-69029-01 (to Dr Feelisch). Dr Rassaf is a research fellow funded by the DFG (Ra 969/1-1). The technical assistance of S. Matern, G. Dömer, and C. Ferfers is gratefully acknowledged.
↵*Both authors contributed equally to this study.
Original received March 18, 2002; revision received July 24, 2002; accepted August 14, 2002.
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