Effect of incubation with crystalloid solutions or medications on packed red blood cells.

BACKGROUND: American Association of Blood Banks (AABB) guidelines suggest that packed red blood cells (PRBCs) be administered through a dedicated intravenous (IV) catheter. Literature supporting this broad-scope declaration are scarce. Obtaining additional IV access is painful, costly, and an infectious risk. We evaluated the effect of co-incubating PRBCs with crystalloids and medications on PRBC hemolysis, membrane deformability, and aggregation, as well as medication concentration. METHODS: PRBCs were co-incubated 5 minutes with plasma, normal saline (NS), 5% dextrose in water (D5W), Plasmalyte, epinephrine (epi), norepinephrine (norepi), dopamine (dopa), or Propofol (prop). Samples were then assessed for hemolysis (free hemoglobin, serum potassium), membrane deformability (elongation index [EI]), aggregation (smear, critical shear stress [mPa]) and drug concentration (High Performance Liquid Chromatography/Tandem Mass Spectrometry [LCMS-MS]). Significance (p ≤ 0.05) was determined by Wilcoxon-paired comparisons or Wilcoxon/Kruskall Willis with post-hoc Dunn's test. RESULTS: Compared to co-incubation with plasma: 1) co-incubation resulted in significantly increased hemolysis only when D5W as used (free hemoglobin, increased potassium); 2) EI trended lower when co-incubated with D5W and trended toward higher when co-incubated with prop; 3) aggregation was significantly lower when PRBCs co-incubated with NS, D5W, or Plasmalyte, and trended lower when co-incubated with epi, norepi, or dopa. Medication concentrations were between those predicted by distribution only in plasma and distribution through the entire intra- and extracellular space. CONCLUSION: Our data suggest that 5 minutes of PRBC incubation with isotonic crystalloids or catecholamines does not deleteriously alter PRBC hemolysis, membrane deformability, or aggregation. Co-incubation with D5W likely increases hemolysis. Propofol may promote hemolysis.

Red blood cells stored 35 days or more are associated with adverse outcomes in high-risk patients.

BACKGROUND: Clinical trials have shown that longer red blood cell (RBC) storage duration does not worsen outcomes; however, these studies included few RBCs near the end of the 42-day storage limit. We tested the hypothesis that these "oldest" RBCs are associated with adverse outcomes. STUDY DESIGN AND METHODS: In a retrospective study, 28,247 transfused patients given 129,483 RBC units were assessed. Morbidity, mortality, and length of stay (LOS) were compared in patients transfused exclusively with RBCs stored not more than 21 days versus patients transfused exclusively with RBCs stored 28 days or more and patients transfused exclusively with RBCs stored 35 days or more. RESULTS: After risk adjustment, ≥35-day RBCs were associated with increased morbidity (adjusted odds ratio [adjOR], 1.19; 95% confidence interval [CI], 1.07-1.32; p = 0.002), but ≥28-day RBCs were not (adjOR, 1.06; 95% CI, 0.97-1.15; p = 0.2). Neither ≥35-day nor ≥28-day RBCs were associated with increased mortality. In critically ill patients, ≥35-day RBCs were associated with increased morbidity (adjOR, 1.25; 95% CI, 1.08-1.44; p = 0.002) and mortality (adjOR, 1.38; 95% CI, 1.08-1.74; p = 0.009), but ≥28-day RBCs were associated with neither. In older patients, ≥35-day RBCs were associated with increased morbidity (adjOR, 1.22; 95% CI, 1.04-1.42; p = 0.01), but not mortality (adjOR, 1.28; 95% CI, 0.96-1.71; p = 0.1), and ≥28-day RBCs were associated with neither. LOS was increased for both ≥28- and ≥35-day RBCs for all patients and the critically ill and older subgroups. CONCLUSIONS: RBCs transfused in the last 7 days of their 42-day storage limit may be associated with adverse clinical outcomes in high-risk patients.

Oxidative stress and rheologic properties of stored red blood cells before and after transfusion to surgical patients.

BACKGROUND: The loss of structural and functional integrity of red blood cells (RBCs) during storage, collectively referred to as "storage lesion," has been implicated in reduced oxygen delivery after transfusion. RBCs are highly susceptible to oxidative damage from generation of reactive oxygen species by autoxidation of hemoglobin. Therefore, we examined whether increased oxidative stress (OS) in stored RBCs is associated with impaired cell membrane deformability before or after transfusion. STUDY DESIGN AND METHODS: Thirty-four patients undergoing multilevel spine fusion surgery were enrolled. OS in RBCs was assessed by the presence of fluorescent heme degradation products and methemoglobin, which were measured with fluorimetric and spectrophotometric methods, respectively. Deformability and aggregation were determined by ektacytometry in stored RBCs, autologous salvaged RBCs, and posttransfusion blood samples. RESULTS: OS in stored RBCs was significantly increased with longer storage (R = 0.54, p = 0.032) and significantly higher than that in fresh RBCs (9.1 ± 1.3 fluorescent arbitrary units vs. 7.7 ± 0.9 fluorescent arbitrary units, p < 0.001). Deformability decreased (R = -0.60, p = 0.009) with increasing storage duration. OS was elevated (p < 0.05) and deformability was decreased (p < 0.05) in postoperative blood from patients who had undergone moderate (≥4 RBC units) but not minimal or no transfusion. Neither the decrease in deformability of RBCs nor the aggregation changes were correlated with OS. CONCLUSIONS: Although stored RBCs show signs of increased OS and loss of cell membrane deformability, these changes were not directly correlated and were only evident after moderate but not lower dose transfusion in postoperative surgical patients. These findings suggest that factors other than OS may contribute to impaired rheology with stored RBCs in the clinical setting.

The Impact of Surgery and Stored Red Blood Cell Transfusions on Nitric Oxide Homeostasis.

BACKGROUND: Cell-free hemoglobin (Hb) forms in stored red blood cells (RBCs) as a result of hemolysis. Studies suggest that this cell-free Hb may decrease nitric oxide (NO) bioavailability, potentially leading to endothelial dysfunction, vascular injury, and multiorgan dysfunction after transfusion. We tested the hypothesis that moderate doses of stored RBC transfusions increase cell-free Hb and decrease NO availability in postoperative surgical patients. METHODS: Twenty-six patients undergoing multilevel spine fusion surgery were studied. We compared those who received no stored RBCs (n = 9) with those who received moderate amounts (6.1 ± 3.0 units) of stored RBCs over 3 perioperative days (n = 17). Percent hemolysis (cell-free Hb), RBC-NO (heme-NO), and plasma nitrite and nitrate were measured in samples from the stored RBC bags and from patients' blood, before and after surgery. RESULTS: Posttransfusion hemolysis was increased approximately 3.5-fold over preoperative levels (P = 0.0002) in blood samples collected immediately after surgery but not on postoperative days 1 to 3. Decreases in both heme-NO (by approximately 50%) and plasma nitrite (by approximately 40%) occurred postoperatively, both in nontransfused patients (P = 0.036 and P = 0.026, respectively) and transfused patients (P = 0.0068 and P = 0.003, respectively) and returned to preoperative baseline levels by postoperative day 2 or 3. Postoperative plasma nitrite and nitrate were decreased significantly in both groups, and this change was slower to return to baseline in the transfused patients, suggesting that blood loss and hemodilution from crystalloid administration contribute to this finding. CONCLUSIONS: The decrease in NO metabolites occurred irrespective of stored RBC transfusions, suggesting this decrease may be related to blood loss during surgery and hemodilution rather than to scavenging of NO or inhibition of NO synthesis by stored RBC transfusions.

2,3-Diphosphoglycerate Concentrations in Autologous Salvaged Versus Stored Red Blood Cells and in Surgical Patients After Transfusion.

BACKGROUND: Stored red blood cells (RBCs) are deficient in 2,3-diphosphoglycerate (2,3-DPG), but it is unclear how autologous salvaged blood (ASB) compares with stored blood and how rapidly 2,3-DPG levels return to normal after transfusion. Therefore, we compared levels of 2,3-DPG in stored versus ASB RBCs and in patients' blood after transfusion. METHODS: Twenty-four patients undergoing multilevel spine fusion surgery were enrolled. We measured 2,3-DPG and the oxyhemoglobin dissociation curve (P50) in samples taken from the ASB and stored blood bags before transfusion and in blood samples drawn from patients before and after transfusion. RESULTS: The mean storage duration for stored RBCs was 24 ± 8 days. Compared with fresh RBCs, stored RBCs had decreased 2,3-DPG levels (by approximately 90%; P < 0.0001) and a decreased P50 (by approximately 30%; P < 0.0001). However, ASB RBCs did not exhibit these changes. The mean 2,3-DPG concentration decreased by approximately 20% (P < 0.05) in postoperative blood sampled from patients who received 1 to 3 stored RBC units and by approximately 30% (P < 0.01) in those who received ≥4 stored RBC units. 2,3-DPG was unchanged in patients who received no stored blood or ASB alone. After surgery, 2,3-DPG levels recovered gradually over 3 postoperative days in patients who received stored RBCs. CONCLUSIONS: Stored RBCs, but not ASB RBCs, have decreased levels of 2,3-DPG and a left-shift in the oxyhemoglobin dissociation curve. Postoperatively, 2,3-DPG levels remain below preoperative baseline levels for up to 3 postoperative days in patients who receive stored RBCs but are unchanged in those who receive only ASB RBCs.

Red blood cell membrane-facilitated release of nitrite-derived nitric oxide bioactivity.

The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO(+) ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.

New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease.

Sickle cell disease (SCD) is associated with increase in oxidative stress and irreversible membrane changes that originates from the instability and polymerization of deoxygenated hemoglobin S (HbS). The relationship between erythrocyte membrane changes as assessed by a decrease in deformability and oxidative stress as assessed by an increase in heme degradation was investigated. The erythrocyte deformability and heme degradation for 27 subjects with SCD and 7 with sickle trait were compared with normal healthy adults. Changes in both deformability and heme degradation increased in the order of control to trait to non-crisis SCD to crisis SCD resulting in a very significantly negative correlation between deformability and heme degradation. However, a quantitative analysis of the changes in deformability and heme degradation for these different groups of subjects indicated that sickle trait had a much smaller effect on deformability than on heme degradation, while crisis affects deformability to a greater extent than heme degradation. These findings provide insights into the relative contributions of erythrocyte oxidative stress and membrane damage during the progression of SCD providing a better understanding of the pathophysiology of SCD.

The effect of intermittent pneumatic compression of legs on the levels of nitric oxide related species in blood and on arterial function in the arm.

BACKGROUND: Intermittent pneumatic compression (IPC) of legs exerts beneficial local vascular effects, possibly through local release of nitric oxide (NO). However, studies demonstrating systemic transport of nitrogen oxide species and release of NO prompt the question of whether IPC could also exert nonlocal effects. We tested whether IPC (1) affects systemic levels of nitrite, S-nitrosothiols and red blood cell (RBC) NO, and (2) exerts vasoactive effects in the brachial artery (BA), although this hypothesis-generating pilot study did not investigate cause and effect relationship between (1) and (2). METHODS: In 10 healthy subjects, ages 24-39 years, we measured plasma nitrite, plasma S-nitrosothiols and RBC-NO from venous blood samples drawn before and after IPC treatment. We also measured BA responses to 5 min of upper arm occlusion at rest and during 1 h of leg IPC. RESULTS: There was a significant decrease in plasma nitrite (112±26 nM to 90±15 nM, p=0.0008) and RBC-NO (129±72 nM to 102±41 nM, p=0.02). Plasma S-nitrosothiols were unchanged (5.79±4.81 nM to 6.27±5.79 nM, p=0.3). BA occlusion-mediated constriction (OMC) was significantly attenuated with IPC treatment (-43±13% to -33±12%, p=0.003). High-flow mediated BA dilation was unchanged (13.3±9.4% to 11.5±7.2%, p=0.2). CONCLUSION: Plasma nitrite, RBC-NO, and BA OMC decreased with leg IPC. We hypothesize that this decrease in circulatory pool of plasma nitrite and RBC-NO may result from the transfer of their NO-bioactivity from blood to the hypoxic arm tissue, to be stored and released under hypoxic stress and oppose OMC. Future studies should investigate whether IPC-induced decreases in brachial OMC are caused by the changes in systemic NO activity, and whether leg IPC could benefit distant arterial function in systemic cardiovascular disease.

The quaternary hemoglobin conformation regulates the formation of the nitrite-induced bioactive intermediate and the dissociation of nitric oxide from this intermediate.

Deoxyhemoglobin reduces nitrite to nitric oxide (NO). In order to study the effect of the hemoglobin quaternary conformation on the nitrite reaction, we compared T-state deoxyhemoglobin with R-state deoxyhemoglobin produced by reacting hemoglobin with carboxypeptidase-A prior to deoxygenation. The nitrite reaction with deoxyhemoglobin was followed by chemiluminescence, electron paramagnetic resonance and visible spectroscopy. The initial steps in this reaction involve the binding of nitrite to deoxyhemoglobin followed by the formation of an electron delocalized metastable intermediate that retains potential NO bioactivity. This reaction is shown by visible spectroscopy to occur 5.6 times faster in the R-state than in the T-state. However, the dissociation of NO from the delocalized intermediate is shown to be facilitated by the T-quaternary conformation with a 9.6 fold increase in the rate constant. The preferred NO-release in the T-state, which has a higher affinity for the membrane, can result in the NO diffusing out of the RBC and being released to the vasculature at low partial pressures of oxygen.

Red blood cells induce hypoxic lung inflammation.

Hypoxia, which commonly associates with respiratory and cardiovascular diseases, provokes an acute inflammatory response. However, underlying mechanisms are not well understood. Here we report that red blood cells (RBCs) induce hypoxic inflammation by producing reactive oxygen species (ROS) that diffuse to endothelial cells of adjoining blood vessels. Real-time fluorescence imaging of rat and mouse lungs revealed that in the presence of RBC-containing vascular perfusion, hypoxia increased microvascular ROS, and cytosolic Ca(2+), leading to P-selectin-dependent leukocyte recruitment. However, in the presence of RBC-free perfusion, all hypoxia-induced responses were completely inhibited. Because hemoglobin (Hb) autoxidation causes RBC superoxide formation that readily dismutates to H(2)O(2), hypoxia-induced responses were lost when we inhibited Hb autoxidation with CO or nitrite, or when the H(2)O(2) inhibitor, catalase was added to the infusion to neutralize the RBC-derived ROS. By contrast, perfusion with RBCs from BERK-trait mice that are more susceptible to Hb autoxidation and to hypoxia-induced superoxide production enhanced the hypoxia-induced responses. We conclude that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H(2)O(2) that diffuses to the lung microvascular endothelium, thereby initiating Ca(2+)-dependent leukocyte recruitment. These findings are the first evidence that RBCs contribute to hypoxia-induced inflammation.

Nitrite enhances RBC hypoxic ATP synthesis and the release of ATP into the vasculature: a new mechanism for nitrite-induced vasodilation.

A role for nitric oxide (NO) produced during the reduction of nitrite by deoxygenated red blood cells (RBCs) in regulating vascular dilation has been proposed. It has not, however, been satisfactorily explained how this NO is released from the RBC without first reacting with the large pools of oxyhemoglobin and deoxyhemoglobin in the cell. In this study, we have delineated a mechanism for nitrite-induced RBC vasodilation that does not require that NO be released from the cell. Instead, we show that nitrite enhances the ATP release from RBCs, which is known to produce vasodilation by several different methods including the interaction with purinergic receptors on the endothelium that stimulate the synthesis of NO by endothelial NO synthase. This mechanism was established in vivo by measuring the decrease in blood pressure when injecting nitrite-reacted RBCs into rats. The observed decrease in blood pressure was not observed if endothelial NO synthase was inhibited by N(omega)-nitro-L-arginine methyl ester (L-NAME) or when any released ATP was degraded by apyrase. The nitrite-enhanced ATP release was shown to involve an increased binding of nitrite-modified hemoglobin to the RBC membrane that displaces glycolytic enzymes from the membrane, resulting in the formation of a pool of ATP that is released from the RBC. These results thus provide a new mechanism to explain nitrite-induced vasodilation.

Nitrite-induced improved blood circulation associated with an increase in a pool of RBC-NO with no bioactivity.

The reduction of nitrite by RBCs producing NO can play a role in regulating vascular tone. This hypothesis was investigated in rats by measuring the effect of nitrite infusion on mean arterial blood pressure (MAP), cerebral blood flow (CBF) and cerebrovascular resistance (CVR) in conjunction with the accumulation of RBC-NO. The nitrite infusion reversed the increase in MAP and decrease in CBF produced by L-NAME inhibition of e-NOS. At the same time there was a dramatic increase in RBC-NO. Correlations of RBC-NO for individual rats support a role for the regulation of vascular tone by this pool of NO. Furthermore, data obtained prior to treatment with L-NAME or nitrite are consistent with a contribution of RBC reduced nitrite in regulating vascular tone even under normal conditions. The role of the RBC in delivering NO to the vasculature was explained by the accumulation of a pool of bioactive NO in the RBC when nitrite is reduced by deoxygenated hemoglobin chains. A comparison of R and T state hemoglobin demonstrated a potential mechanism for the release of this NO in the T-state present at reduced oxygen pressures when blood enters the microcirculation. Coupled with enhanced hemoglobin binding to the membrane under these conditions the NO can be released to the vasculature.

Heme degradation and oxidative stress in murine models for hemoglobinopathies: thalassemia, sickle cell disease and hemoglobin C disease.

Red blood cells with abnormal hemoglobins (Hb) are frequently associated with increased hemoglobin autoxidation, accumulation of iron in membranes, increased membrane damage and a shorter red cell life span. The mechanisms for many of these changes have not been elucidated. We have shown in our previous studies that hydrogen peroxide formed in association with hemoglobin autoxidation reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent emission bands and the release of iron. Heme degradation was assessed by measuring the fluorescent band at ex 321 nm. A 5.6 fold increase in fluorescence was found in red cells from sickle transgenic mice that expressed exclusively human globins when compared to red cells from control mice. When sickle transgenic mice co-express the gamma M transgene, that expresses HbF and inhibits polymerization, heme degradation is decreased. Mice expressing exclusively hemoglobin C had a 6.9 fold increase in fluorescence compared to control. Heme degradation was also increased 3.5 fold in beta-thalassemic mice generated by deletion of murine beta(major). Membrane bound IgG and red cell metHb were highly correlated with the intensity of the fluorescent heme degradation band. These results suggest that degradation of the heme moiety in intact hemoglobin and/or degradation of free heme by peroxides are higher in pathological RBCs. Concomitant release of iron appears to be responsible for the membrane damage that leads to IgG binding and the removal of red cells from circulation.

Potential Modulation of Vascular Function by Nitric Oxide and Reactive Oxygen Species Released From Erythrocytes.

The primary role for erythrocytes is oxygen transport that requires the reversible binding of oxygen to hemoglobin. There are, however, secondary reactions whereby the erythrocyte can generate reactive oxygen species (ROS) and nitric oxide (NO). ROS such as superoxide anion and hydrogen peroxide are generated by the autoxidation of hemoglobin. NO can be generated when nitrite reacts with hemoglobin forming an HbNO+ intermediate. Both of these reactions are dramatically enhanced under hypoxic conditions. Within the erythrocyte, interactions of NO with hemoglobin and enzymatic reactions that neutralize ROS are expected to prevent the release of any generated NO or ROS. We have, however, demonstrated that partially oxygenated hemoglobin has a distinct conformation that enhances hemoglobin-membrane interactions involving Band 3 protein. Autoxidation of the membrane bound partially oxygenated hemoglobin facilitates the release of ROS from the erythrocyte. NO release is made possible when HbNO+, the hemoglobin nitrite-reduced intermediate, which is not neutralized by hemoglobin, is bound to the membrane and releases NO. Some of the released ROS has been shown to be transferred to the vasculature suggesting that some of the released NO may also be transferred to the vasculature. NO is known to have a major effect on the vasculature regulating vascular dilatation. Erythrocyte generated NO may be important when NO production by the vasculature is impaired. Furthermore, the erythrocyte NO released, may play an important role in regulating vascular function under hypoxic conditions when endothelial eNOS is less active. ROS can react with NO and, can thereby modulate the vascular effects of NO. We have also demonstrated an inflammatory response due to erythrocyte ROS. This reflects the ability of ROS to react with various cellular components affecting cellular function.

Measurement of plasma nitrite by chemiluminescence without interference of S-, N-nitroso and nitrated species.

Recent studies have demonstrated that plasma nitrite (NO2-) reflects endothelial nitric oxide synthase activity and it has been proposed as a prognostic marker for cardiovascular disease. In addition, NO2- itself has been shown to have biological activities thought to be triggered by reduction back to NO in blood and tissues. The development of sensitive and reproducible methods for the quantitative determination of plasma NO2- is, therefore, of great importance. Ozone-based chemiluminescence assays have been shown to be highly sensitive for the determination of nanomolar quantities of NO and NO-related species in biological fluids. We report here an improved direct chemiluminescence method for the determination of plasma NO2- without interference of other nitric oxide-related species such as nitrate, S-nitrosothiols, N-nitrosamines, nitrated proteins, and nitrated lipids. The method involves a reaction system consisting of glacial acetic acid and ascorbic acid in the purge vessel of the NO analyzer. Under these acidic conditions NO2- is stoichiometrically reduced to NO by ascorbic acid. Fasting human plasma NO2- values were found in the range of 56-210 nM (mean=110+/-36 nM). This method has high sensitivity with an accuracy of 97% and high precision (CV<10%) for determination of plasma nitrite. The present method is simple and highly specific for plasma NO2-. It is particularly suited for evaluating vasculature endothelial NO production that predicts the risks for cardiovascular disease.

Nitric oxide redox reactions and red cell biology.

Three hypotheses explain a role for red blood cells (RBCs) in delivering NO to the vasculature: (a) "the SNOHb hypothesis" involves the uptake of NO by RBCs with NO transferred from the heme to the beta-93 thiol in the R quaternary conformation, followed by the release to membrane thiols in the T quaternary conformation; and (b and c) "the nitrite hypotheses" bypass the intrinsic difficulties of transporting the highly reactive NO, by reutilizing the nitrite formed when NO reacts with oxygen. Deoxyhemoglobin reduces this nitrite back to NO. The distinction between the two nitrite mechanisms depends on the importance of intermediate species formed during nitrite reduction. Without bioactive intermediates, the NO must be immediately released to avoid binding to deoxyhemoglobin. The "nitrite intermediate hypothesis" enables the RBCs to store a pool of potentially bioactive NO until it is released from the cell. In this review, we critically compare these different proposals for the transport/delivery of NO by RBCs. We also compare the redox properties in the RBCs associated with NO with the redox properties associated with oxygen.

Ferriheme catalyzes nitric oxide reaction with glutathione to form S-nitrosoglutathione: A novel mechanism for formation of S-nitrosothiols.

S-nitrosothiols (SNO) perform many important functions in biological systems, but the mechanism by which they are generated in vivo remains a contentious issue. Nitric oxide (NO) reacts with thiols to form SNO only in the presence of a molecule that will accept an electron from either NO or the thiol. In this study, we present evidence that ferriheme accepts an electron from NO or glutathione (GSH) to generate S-nitrosoglutathione (GSNO) in vitro under anaerobic or hypoxic (2% O2) conditions. Ferriheme formed charge transfer-stable complexes with NO to form ferriheme-NO (heme-Fe(II)-NO+) and with GSH to form ferriheme-GS (heme-Fe(II)-GS•) under anaerobic conditions. The reaction between GSH and the heme-Fe(II)-NO+ complex or between NO and the heme-Fe(II)-GS• complex resulted in simultaneous reductive ferriheme nitrosylation (heme-Fe(II)NO) and the generation of GSNO. Thus, ferriheme is readily reduced to ferroheme in the presence of NO and GSH together, but not with either individually. The reaction between NO and the heme-Fe(II)-GS• complex to generate GSNO occurred more rapidly than NO was consumed by endothelial cells, but not red blood cells. In addition, pretreatment of endothelial cells with ferriheme or the ferriheme-GS complex generated SNO upon addition of NO under hypoxic conditions. The results of this study raise the possibility that in vivo, ferriheme can complex with GSH to form ferriheme-GS complex (heme-Fe(II)-GS•), which rapidly reacts with NO to generate GSNO under intracellular oxygen levels. The GSNO formation by this mechanism is more efficient than any other in vitro mechanism(s) reported so far.

Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase.

Catalase and glutathione peroxidase (GSHPX) react with red cell hydrogen peroxide. A number of recent studies indicate that catalase is the primary enzyme responsible for protecting the red cell from hydrogen peroxide. We have used flow cytometry in intact cells as a sensitive measure of the hydrogen-peroxide-induced formation of fluorescent heme degradation products. Using this method, we have been able to delineate a unique role for GSHPX in protecting the red cell from hydrogen peroxide. For extracellular hydrogen peroxide, catalase completely protected the cells, while the ability of GSHPX to protect the cells was limited by the availability of glutathione. The effect of endogenously generated hydrogen peroxide in conjunction with hemoglobin autoxidation was investigated by in vitro incubation studies. These studies indicate that fluorescent products are not formed during incubation unless the glutathione is reduced to at least 40% of its initial value as a result of incubation or by reacting the glutathione with iodoacetamide. Reactive catalase only slows down the depletion of glutathione, but does not directly prevent the formation of these fluorescent products. The unique role of GSHPX is attributed to its ability to react with hydrogen peroxide generated in close proximity to the red cell membrane in conjunction with the autoxidation of membrane-bound hemoglobin.

Red cell perturbations by amyloid beta-protein.

Amyloid beta-protein (A beta) accumulation in brain is thought to be important in causing the neuropathology of Alzheimer's disease (AD). A beta interactions with both neurons and microglial cells play key roles in AD. Since vascular deposition of A beta is also implicated in AD, the interaction of red cells with these toxic aggregates gains importance. However, the effects of A beta interactions with red blood cells are less well understood. Synthetic amyloid beta-protein (1-40) was labeled with biotin and preincubated at 37 degrees C for 4, 14 and 72 h to produce fibrils. Flow cytometry was used to study the binding of these fibrils to red cells. The amyloid fibrils had a high affinity for the red cell with increased binding for the larger fibrils produced by longer preincubation. Bovine serum albumin (BSA) did not reverse the binding, but actually resulted in a more efficient binding of the A beta fibrils to the red cells. The interaction of A beta with red cells increased the mean cell volume and caused the cells to become more spherical. This effect was greater for the longer fibrils. At the same time the interaction of A beta with red cells produced an increase in their fluorescence measured after 16-h incubation at 37 degrees C. This increase in fluorescence is attributed to the formation of fluorescent heme degradation products. The effect of prior hemoglobin oxidation, catalase inhibition and glutathione peroxidase inhibition indicated that the amyloid-induced oxidative damage to the red cell involved hydrogen peroxide-induced heme degradation. These results suggest that amyloid interactions with the red cell may contribute to the pathology of AD.

Heme degradation by reactive oxygen species.

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.