Evidence for in vivo transport of bioactive nitric oxide in human plasma (original) (raw)

In the present study we report on the kinetics and vasodilator potency of authentic NO in the human forearm circulation and demonstrate that sterile aqueous NO solutions can be administered intra-arterially in humans without complications or unwanted side effects. NO in saline solution dilates conduit and resistance arteries at doses lower than those currently used in NO inhalation therapy. The observed biological effects lasted considerably longer than ex vivo studies on its half-life in blood had predicted. Intravascular application of authentic NO was found to lead to the in vivo formation of plasma nitrite, nitrate, and _S_-nitrosothiols, and only the latter, together with free NO, appears to be involved in the flow response elicited.

Dilator effects of authentic NO. Effects of intra-arterially applied NO on blood flow were unexpected and, to our knowledge, had not been reported before. NO appears to have a uniform effect along the vascular tree, in that it dilated conduit and resistance arteries, which are regulated in a mutually independent manner. The observation that dilation of the radial artery occurred prior to the dilator action on resistance vessels rules out that initial radial artery dilation was mediated by an endothelium-dependent, flow-mediated mechanism. Nevertheless, an additional flow-mediated component secondary to the NO-mediated increase in flow in the downstream microvasculature may contribute to the prolonged dilation of the radial artery. Alternatively, counter-regulatory vascular signaling mechanisms may reverse the NO-induced dilation in resistance arteries, which are also critically controlled by the tissue supply/demand ratio for nutrients. Conduit arteries are less sensitive to such opposing mechanisms. The duration of flow increase, and thus the biological half-life of NO, was much longer than expected from the half-life of NO estimated from ex vivo studies in blood (5, 6, 29). This may be due to the longer-lasting activation of vascular downstream signaling cascades, formation of intermediate NO adducts (see below), or a reduced NO consumption under flow conditions.

In the absence of RBCs, the half-life of NO in aerated buffer solutions or plasma has been reported to be in the range of 1.5–6 minutes (5, 30). In a static system, RBCs act as an effective sink for NO, reducing its half-life in plasma to something on the order of 1.8 milliseconds (5). However, the consumption of NO by RBCs has been suggested to be reduced due to the presence of an unstirred layer of plasma around RBCs, resulting in an effective diffusional resistance (6). Therefore, intraerythrocytic hemoglobin reacts with NO up to 1000 times more slowly than does free hemoglobin. Consumption of plasma NO by RBCs is further hampered due to flow-related effects. Using an isolated microvessel preparation, Liao et al. (31) have demonstrated that consumption of NO by RBCs progressively decreases with increasing flow, suggesting that under these conditions hemoglobin contained in RBCs is not acting as an effective NO scavenger. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which has been estimated to reach up to 25% of the luminal diameter in thickness (32).

Taken together, these experimental data suggest that the lifespan of NO in plasma in vivo is in the range of seconds to minutes and that NO may thus be transported as such at considerable distances along the vascular bed. Our data are in agreement with this interpretation, since the onset of flow increases after intra-arterial application of aqueous NO solutions occurred within seconds and was almost identical to the response kinetics following stimulation of endogenous NO synthesis in the same vascular bed (Figure 3). This suggests that at least the initial part of the vascular effects observed in the present study was mediated by NO itself.

Metabolism of NO and nitrosation chemistry in vivo. Intra-arterial NO application resulted in an increase in plasma levels of nitrite, nitrate, and RSNOs, with nitrate representing the highest and RSNOs the smallest percentage of the recovered N-oxides. About 30% of the applied NO dose was not recovered. One possible explanation for this is that a part of the applied NO may have been converted to metabolites other than the ones measured by us or entered blood cells and tissue and thus gone undetected. Alternatively, since this portion consisted mainly of NO, it might have escaped into ambient air before becoming oxidized to nitrite or converted to nitrate, although this possibility is admittedly speculative.

In a previous study performed in the same vascular bed, we have demonstrated that plasma nitrite rather than nitrate levels reflect regional changes in eNOS activity (10). These findings suggested that NO, which is released from the endothelium into the RBC-free plasma zone lining the vascular wall, is subject to oxidative decomposition in a manner independent of hemoglobin. In line with this notion, we now demonstrate that, after application of aqueous NO solution into the human forearm vasculature, significant amounts of NO can be recovered as nitrite in the draining antecubital vein. However, as plasma nitrite accounted for only 5% of the amount of NO infused, alternative metabolic routes have to be considered. The net increase in plasma nitrate levels (>90 μmol/l) was more than tenfold higher than that of nitrite (>8 μmol/l; Figure 4). Several potential routes of nitrate formation have to be considered: Conversion of nitrite to nitrate within the erythrocyte is considered unlikely to contribute much to the increase in plasma nitrate levels, because both nitrite uptake by RBCs and nitrite oxidation by hemoglobin are relatively slow processes (33, 34) that far exceed total passage time through the forearm circulation, which is in the range of 4–10 seconds (28). It appears rather unlikely that such a conversion took place during blood sampling or sample processing (in an unstirred blood sample), since samples were drawn within seconds, almost instantly cooled down using chilled vials, and diluted. However, if our hypothesis was correct and NO transported in plasma as such, the possibility that part of the applied NO might have entered RBCs and been converted to nitrate during sample processing cannot be excluded. Whatever the mechanism of nitrate formation, the complete lack of vasodilator activity of intra-arterially infused nitrite and nitrate rules out any role for these metabolites in NO delivery under normoxic conditions.

Earlier studies have demonstrated that the interaction of NO with albumin results in the formation of SNOAlb and that the latter represents the principal nitrosothiol in plasma (13, 14). We therefore made no further attempt to dissect the minor fraction of low–molecular weight RSNOs from the total plasma RSNO pool. In the present investigation, we determined basal RSNO levels to be in the low nanomolar range, which is in agreement with most other recent studies in human plasma (14). Whether nitrosation in vivo takes place exclusively at the level of plasma constituents or includes circulating blood cells as well remains to be investigated. Despite a net increase of only 30 nM (Figure 4), the formation of RSNOs appears to account, at least in part, for the dilator effects of NO on conduit and resistance arteries. On first sight, the increases in plasma RSNOs observed during NO infusion might appear disappointingly small. However, it is important to note that comparable concentrations of GSNO (5 nmol/min infused to a flow of ∼200 ml/min in the brachial artery corresponds to 25 nM) closely matched the vasodilator profile of authentic NO. While the onset of vasodilation seen with GSNO was delayed compared with that of the NO infusion, the sustained vasodilation observed with NO (Figure 3) may be mediated by the in vivo formation of plasma RSNOs. GSNO was chosen over SNOAlb for reasons of simplicity of application (no need for separation and nitrosation of autologous albumin) and to avoid possible unwanted immunological reactions due to increased amounts of protein-bound SNO epitopes. The possibility that part of the NO-induced dilation in our experiments was mediated by SNOHb (23) or NOHb (20) cannot be excluded at present, but it appears rather slight, because intraerythrocytic hemoglobin reacts with NO up to 1000 times more slowly than does free hemoglobin, and consumption of plasma NO by RBCs is further hampered due to flow-related effects (31). However, these pathways have not been specifically addressed in our study, and further investigations are warranted to elucidate the potential role of hemoglobin in the transport of NO under physiological conditions.

Therapeutic implications. Collectively, we have demonstrated that authentic NO, when applied as aqueous solution into the human artery, exerts local vasodilator effects at significant distances from the site of application. Part of these effects appears to be mediated via free NO, another part via nitrosation of plasma constituents. Systemic application of authentic NO may compensate for the NO deficiency seen in various cardiovascular diseases associated with endothelial dysfunction. The route of administration and the chosen dose may critically determine NO transport and biological effects. The latter depend on uptake, transport, and liberation of NO along the vascular tree by physiological blood-borne carriers, the intercellular contact of blood cells during passage through the microvasculature, and the interaction of plasma NO stores with the endothelium and vascular smooth muscle. The metabolism and distribution of NO and thus its range of action may exhibit significant spatial heterogeneity in vivo, depending on vessel size, hematocrit, flow velocity, and shear rate (35, 36) as well as oxygen saturation and tissue pO2. These variables are expected to affect NO consumption in the RBC-free plasma zone near the vascular wall, and within RBCs (31, 37). Matters are further complicated by the fact that the RBC membrane represents a heterogenous sink (5, 38), which may critically determine the effects of NO on the microcirculation (39). These denominators might be affected differently by intra-arterial, intravenous, or inhaled NO substitution therapy. The dose range required for NO-induced vasodilation in humans was hitherto unknown. The doses of NO applied in the present study are considerably lower than those in current use for inhalation therapy of critically ill patients (40). Those patients typically receive 25–35 parts per million NO, resulting in a net uptake of 5.5–7.7 μmol NO per minute of inhalation. In a recent study aimed to assess the formation of NOHb and potential peripheral vasodilator effects (22), NO was inhaled at 80 ppm for 1 hour, resulting in a net uptake of 1080 μmol NO, a dose more than 30-fold higher than the highest dose applied in the present study. Yet the magnitude of the peripheral dilator effects of NO was less than those described in the present study. Whether the application of aqueous NO solution may offer therapeutic advantages over existing NO-related therapies and furthermore be suitable to exert systemic biological effects in vivo remains to be investigated.