Red Blood Cells (RBCs) need to deform and squeeze through narrow capillaries. Decreased deformability of RBCs is, therefore, one of the factors that can contribute to the elimination of aged or damaged RBCs from the circulation. This process can also cause impaired oxygen delivery, which contributes to the pathology of a number of diseases. Studies from our laboratory have shown that oxidative stress plays a significant role in damaging the RBC membrane and impairing its deformability. RBCs are continuously exposed to both endogenous and exogenous sources of reactive oxygen species (ROS) like superoxide and hydrogen peroxide (H2O2). The bulk of the ROS are neutralized by the RBC antioxidant system consisting of both non-enzymatic and enzymatic antioxidants including catalase, glutathione peroxidase and peroxiredoxin-2. However, the autoxidation of hemoglobin (Hb) bound to the membrane is relatively inaccessible to the predominantly cytosolic RBC antioxidant system. This inaccessibility becomes more pronounced under hypoxic conditions when Hb is partially oxygenated, resulting in an increased rate of autoxidation and increased affinity for the RBC membrane. We have shown that a fraction of peroxyredoxin-2 present on the RBC membrane may play a major role in neutralizing these ROS. H2O2 that is not neutralized by the RBC antioxidant system can react with the heme producing fluorescent heme degradation products (HDPs). We have used the level of these HDP as a measure of RBC oxidative Stress. Increased levels of HDP are detected during cellular aging and various diseases. The negative correlation (p < 0.0001) between the level of HDP and RBC deformability establishes a contribution of RBC oxidative stress to impaired deformability and cellular stiffness. While decreased deformability contributes to the removal of RBCs from the circulation, oxidative stress also contributes to the uptake of RBCs by macrophages, which plays a major role in the removal of RBCs from circulation. The contribution of oxidative stress to the removal of RBCs by macrophages involves caspase-3 activation, which requires oxidative stress. RBC oxidative stress, therefore, plays a significant role in inducing RBC aging.

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.Nitric oxide (NO) 1 synthesized by endothelial nitric-oxide synthase diffuses both to the smooth muscle cells as well as the lumen (1, 2). The reaction of NO with smooth muscle cell guanylate cyclase plays a crucial role in the regulation of vascular tone and blood flow (3). In the lumen NO is either oxidized by oxygen resulting in the formation of nitrite (4) or is taken up by red blood cells (5). In red blood cells, NO rapidly reacts with oxyhemoglobin to form methemoglobin (Hb(III)) and nitrate and with deoxyhemoglobin (deoxyHb) to form heme-nitrosylated hemoglobin (Hb(II)NO) (6, 7). Earlier studies, therefore, assumed that the only effect of red blood cells on the bioactivity of nitric oxide was to scavenge and inactivate NO limiting its availability for vasodilation. This perspective was challenged by studies of Stamler and co-workers (8 -13) who proposed an active role for red blood cells in the delivery of NO. Their model is based on the potential binding of NO released from arterial endothelial cells to the few vacant deoxyhemes to form Hb(II)NO and to the ␤-93 cysteine to form S-nitrosohemoglobin (SNOHb) (11). The relative affinity for these two hemoglobin sites was assumed to depend on the hemoglobin quaternary conformation with heme-bound NO favored by the T-state present in deoxygenated hemoglobin transferred to the cysteine in ...

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.

Heme proteins play a major role in various biological functions, such as oxygen sensing, electron transport, signal transduction, and antioxidant defense enzymes. Most of these reactions are carried out by redox reactions of heme iron. As the heme is not recycled, most cells containing heme proteins have the microsomal mixed function oxygenase, heme oxygenase, which enzymatically degrades heme to biliverdin, carbon monoxide, and iron. However, the red cell with the largest pool of heme protein, hemoglobin, contains no heme oxygenase, and enzymatic degradation of the red cell heme occurs only after the senescent red cells are removed by the reticuloendothelial system. Therefore, only nonenzymatic heme degradation initiated when the heme iron undergoes redox reactions in the presence of oxygen-producing reactive oxygen species takes place in the red cell. Unlike enzymatic degradation, which specifically attacks the alpha-methene bridge, reactive oxygen species randomly attack all the carbon methene bridges of the tetrapyrrole rings, producing various pyrrole products in addition to releasing iron. This review focuses on the literature related to nonenzymatic heme degradation with special emphasis on hemoglobin, the dominant red cell heme protein.