The reaction of nitrite with oxyHb can involve multiple steps with complex concentration dependencies and many intermediate products. The reaction kinetics then are bimodal, with a slow phase (initiation), during which oxyHb is oxidized to metHb, that precedes a rapid propagation phase. The process is autocatalytic, as the propagation species is generated in multiple copies during one reaction cycle. Earlier attempts to explain the mechanism have been reviewed [35
]. Our recent kinetic reexamination of this reaction suggests that the most crucial intermediates, H2
radical act exclusively in the initiation and the propagation phases, respectively [18
]. In this study we have attempted to examine this reaction in the context of nitrite supplementation in hemolytic disorders, and to ascertain if the oxidative intermediates generated during the progression of this reaction are likely to be of concern. For this reason we have focused on relevant concentration ranges of hemoglobin (up to 30 µM in plasma [16
]) and on plasma components that may alter the reaction characteristics. As shown in this study, common plasma antioxidants (AA, UA) regulate the reaction by limiting the quantity of intermediate reactive products (as H2
radical, ferrylHb species).
Our EPR and spectrophotometric results for equimolar nitrite/oxyHb mixtures indicate that no free radicals are detected during the whole time course of the reaction. When 10-fold excess of nitrite over oxyHb was used, the situation in plasma and PBS vis a vis free radical formation differed dramatically; with an total absence of free radicals in plasma and clear transient presence of protein-based radical in PBS that coincided with the observed fast phase of the reaction (). The observed monotonous kinetics of the reaction up to ~4-fold nitrite excess over oxyHb suggests that a burst of oxidizing reactive intermediates does not occur.
Natural levels of nitrite in plasma are in the nanomolar range of 200–500 nM [2
]. Nitrite/oxyHb molar ratios of 1/1 and 10/1 in our study were chosen with two possible nitrite supplementation routes in mind. Supplementing nitrite by dietary sources with green leaf vegetables as a preferred source, 100 g
of spinach would lead to total nitrite dose in range of 1 µmol/kg [1
], or around 1 µM nitrite in blood. Therefore, concentrations of nitrite in plasma would unlikely to exceed its natural levels much more than 5-fold by dietary changes only. In this case, with extracellular oxyHb in plasma of ~1.4 µM, diet-modified nitrite/oxyHb molar ratio is ~1/1 without hemolysis and drops to ~1/30 during the hemolysis, so free radical formation in plasma is not expected. However, in case of acute NO· deficiency, more direct route to deliver nitrite is needed. In nitrite infusion-based therapies much higher levels of nitrite in plasma are possible, which could potentially lead to the possibility of free radical formation in the vasculature, such as ferrylHb species [13
]. However, vasodilatory effects of nitrite occur even under a low pharmacologic concentration regime which is unlikely to generate a burst of oxidizing species. Infusion of nitrite into human forearm brachial artery to its final concentration in blood of 2 µM caused 30% increase of blood flow [4
]. When 100-fold higher concentration of nitrite was used (200 µM), much higher increase of blood flow was observed (175%), but one could argue that achieving such a large increase in blood flow could be unnecessary or even dangerous in some cases. Also, intravenous infusion instead of intra-arterial could take advantage of the higher deoxyHb content of venous blood to make it possible to lower the necessary dose.
Reactive free radical species can be scavenged by antioxidants present in plasma. The two main antioxidants in plasma are ascorbic acid and uric acid with physiological concentration range of 20–60 µM and 300–400 µM, respectively [36
]. The ability of both to destroy or at least significantly limit free radicals formed in case of 10-fold excess of nitrite over oxyHb in PBS suggests that the risk of free radical formation in nitrite-based therapies can be lowered by concurrent administration of antioxidants as previously shown [39
]. FerrylHb and free radicals which are potential cause of oxidative stress in the vasculature, seem to be formed only at very non-physiological conditions of nitrite, such as may occur during nitrite poisoning [40
] and should not play a major role in most processes involving cell-free plasma Hb in vivo
Interestingly, at conditions of equimolar nitrite and oxyHb, the rate of metHb formation was significantly higher than in PBS (). At normal conditions, cell-free hemoglobin and heme are rapidly bound to haptoglobin (Hp) and hemopexin, respectively [24
] and cleared out of the blood. We hypothesized that the increased rate of metHb formation observed in plasma might be caused by the presence of Hp. Earlier it had been reported that heme environment in Hb in Hp–Hb complex is slightly altered when compared to free Hb [41
]. Also, Hp binds primary to the Hb dimer.
It is known that the Hb–Hp complex retains NO· scavenging activity [17
], but to our best knowledge no report had been made on how Hb-binding to Hp affects Hb reaction with nitrite ion. Several studies also report that Hp–Hb complex formation prevents oxidative damages caused by free Hb in vasculature [32
Based on observed accelerated metHb formation from cell-free Hb in plasma () we further examine the effect of Hp presence on Hb-nitrite reaction kinetics. When the Hp–Hb complex was incubated with nitrite, we observed biphasic decay with a ferrylHb intermediate clearly present in MLR deconvolution (). This observation is in agreement with published data [43
], where formation of ferrylHb from oxyHb as well as from metHb was not inhibited, but increased by Hp–Hb complex formation. However, according to the proposed reaction mechanism for oxyHb with nitrite [18
], as well as the observed acceleration of metHb formation in plasma, we would expect to see some traces of protein-based free radicals in plasma, at least in the case of 10-fold nitrite excess, which we did not observe. The main difference between PBS and plasma in this regard is likely to be the presence of plasma antioxidants. To examine this hypothesis we used antioxidants added to the reaction mixture of nitrite/Hp–Hb in PBS at the beginning of the reaction. Indeed, after addition of 50 µM ascorbic acid and 100 µM uric acid only slow monotonic oxyHb decay, but not autocatalysis was observed.
Catalase added at the beginning of the reaction also prevented the autocatalytic burst both in presence and in absence of Hp, showing that Hp did not open an alternative reaction path leading to autocatalysis. We assume that formation of Hp–Hb complex with Hb dimer conformation in R state is responsible for enhancing the reaction kinetics.
In this study we provide insight into reaction of cell-free Hb with nitrite at low oxyHb and nitrite regimes as expected in plasma at physiological and pharmacological ranges and we also examine possible interactions with other plasma components. We found no autocatalytic burst of oxidant formation under these conditions, unlike what has been observed at high nitrite:Hb ratios and/or high Hb concentrations [19
]. The presence of ferrylHb-based radicals was confirmed in case of 4-fold and higher excess of nitrite over oxyHb, and at low nitrite:oxyHb ratios when Hp–Hb complex was formed prior to the reaction. Addition of ascorbic and uric acids at concentrations normally found in plasma abolished radical formation, and added catalase scavenged H2
. We conclude that effective pharmacological doses of nitrite, especially when complemented with antioxidant effects of plasma, are not likely to be limited vis a vis
free radical or ferrylHb formation by this reaction in vivo
during nitrite-based therapies in diseases accompanied by hemolysis or otherwise due to NO· deficiency [49
]. Our study concentrated on cell-free hemoglobin, however, the major part of hemoglobin found in blood is of course encapsulated in red blood cells. One can ask about the consequences of nitrite infusion on this portion of hemoglobin. Based on the overwhelming ratio of hemoglobin over nitrite and the presence of antioxidants and metHb reductase system inside the cell we would predict that therapeutically desirable increases of nitrite concentration in plasma would have only a small effect on redox processes inside the red blood cell. Based on the results from our in vitro
study, we believe that for the case of nitrite infusion therapy used for diseases accompanied by hemolysis, the determining factor vis a vis
radical formation in the vasculature would be the cell-free hemoglobin. The risk of free radical formation in blood in direct nitrite infusion-based therapies can be further lowered by co-administration of sufficient doses of naturally occurring antioxidants, such as ascorbic acid (which itself could potentially participate in the reduction of nitrite to NO). However, this study concerns itself only with the reactions of nitrite in the plasma and therefore can not completely rule out any potentially detrimental effects of nitrite that may result from alternative routes of administration. Our results also suggest that pharmacological doses should be relatively low, below the nitrite/free Hb molar ratio of 4; however, to give recommendations for doses, a clinical trial is necessary.