EDITORIAL COMMENT
Hemodialysis HyperhemolysisA Novel Mechanism of Endothelial Dysfunction and Cardiovascular Risk?*
Chenell L. Donadee, MD and
Mark T. Gladwin, MD*
Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; and the Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania
* Reprint requests and correspondence: Dr. Mark T. Gladwin, Pulmonary, Allergy, and Critical Care Medicine, NW 628 Montefiore Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213 (Email: gladwinmt{at}upmc.edu).
Key Words: nitric oxide • nitrite • red blood cells • hemolysis • hemodialysis • flow-mediated vasodilation • hemoglobin • arginase
In this issue of the Journal, Meyer et al. (1) investigated the role of hemodialysis-induced intravascular hemolysis on nitric oxide (NO) bioavailability and vascular dysfunction in patients with end-stage renal disease (ESRD). They showed an increase in hemolysis and the levels of cell-free plasma hemoglobin (Hb) during hemodialysis that was associated with NO scavenging and impaired flow-mediated dilation, an index of endothelial NO generation during shear stress. This study is important because it is the first study of this phenomenon in an iatrogenic setting and it adds to a growing body of literature suggesting that even trace levels of cell-free plasma Hb impair endothelial NO signaling. This process has been termed hemolysis-associated endothelial dysfunction.
Nitric oxide is a potent endogenous vasodilator produced tonically by the endothelium. It is now well accepted that cell-free oxyhemoglobin in the plasma functions as an NO scavenger, producing methemoglobin and nitrate (NO3
–) in a reaction that is fast and irreversible.
 | ([Equation 1]) |
Normally, in the absence of hemolysis, the amount of NO scavenged by Hb is greatly limited by the Hb sequestration within the red blood cell plasma membrane. This sequestration produces major bulk diffusional barriers to NO, which include an unstirred layer around the red blood cell, an intrinsic membrane permeability barrier to NO, and a cell-free zone along the endothelium in laminar flowing blood (2,3). These combined barriers reduce the reaction rate of NO with intracellular Hb by approximately 1,000-fold. Thus, when states of hemolysis disrupt the red blood cell plasma membrane and release Hb into the plasma, NO scavenging reaction rates increase by 1,000. This effect is compounded by the extravasation of the Hb tetramer and dimer into the interstitial space. Remarkably, kinetic models suggest that levels of plasma Hb as low as 1 µM have the potential to impair endothelial NO signaling, and heme concentrations as low as 6 µM have been found to impair NO-dependant vasodilation in vivo in patients with sickle cell disease (2,4). In the current study, Meyer et al. (1) found 16.8 µM heme in patients undergoing hemodialysis, nearly 3 times that amount. Hemolysis also releases arginase-1, an intracellular enzyme that when released into the plasma reacts with L-arginine to form ornithine, effectively reducing L-arginine availability for conversion to NO by endothelial NO synthase (3,5–7).
The effects of cell-free plasma Hb on NO consumption have also been shown in clinical trials of Hb-based blood substitutes, in which various cell-free Hb preparations given to humans and animals showed dose-dependent effects on vasoconstriction, pulmonary hypertension, and systemic hypertension (3,8,9). Induced intravascular hemolysis in a dog model produced NO consumption and subsequent vasoconstriction and renal dysfunction (10). This phenomenon has also been noted in patients with sickle cell disease, a chronic hemolytic state (2). In these studies, the vasoconstrictive effects of NO depletion caused by hemolysis could be counteracted by inhaled NO, which reacts directly with the cell-free Hb in the pulmonary circulation, oxidizing it to methemoglobin, which cannot then scavenge the NO systemically (2,10). A recent study published in the Journal of Clinical Investigation by Boretti et al. (11) reports that cell-free plasma Hb has potent oxidative properties that are associated with hypertensive effects. They also found that increasing the levels of haptoglobin in the circulation reduced this oxidative stress and limited both the hypertensive effects and the renal insufficiency associated with plasma hemoglobinemia.
The current proof-of-concept study by Meyer et al. (1) is the first to demonstrate NO consumption by cell-free Hb in the iatrogenic setting of hemodialysis. They hypothesized that hemodialysis produces an increase in cell-free Hb that would then scavenge NO, causing measurable vascular effects. They measured flow-mediated dilation of the brachial artery before and after hemodialysis sessions in 14 patients with ESRD of varied etiology. Flow-mediated dilation is a method that measures the vasodilation response of the brachial artery to the shear stress of blood flow. In this method, a blood pressure cuff produces hand ischemia for 5 min, and after release of the cuff there is an acute increase in hyperemic blood flow to the arm. This increased blood flow produces upstream increased blood flow in the large conduit arteries (brachial and radial arteries). This produces secondary shear stress, which activates the endothelial NO synthase in the endothelium, causing a subsequent delayed vasodilation after 30 to 60 s. This vasodilation can be measured by assessing the brachial or radial artery diameter using high-fidelity Doppler ultrasound. The shear-dependent vasodilation is largely determined by endothelial NO synthase activity. Meyer et al. (1) found that flow-mediated dilation was decreased after hemodialysis and inversely correlated to the amount of cell-free Hb (p = 0.041).
This finding was supported by assays that directly showed increased NO consumption by the plasma collected after hemodialysis. The NO consumption by cell-free Hb was further substantiated by adding ferricyanide (FeCN6) to the post-hemodialysis plasma. The FeCN6 oxidizes heme from the ferrous Fe+2 to ferric Fe+3 state, which has very low affinity for NO. When FeCN6 was added to the post-hemodialysis plasma, it significantly decreased the affinity of cell-free Hb for NO, and NO consumption returned to pre-hemodialysis levels. Increased levels of cell-free Hb and arginase-1 were found in the post-hemodialysis samples, presumably caused by hemolysis from the hemodialysis treatment and mechanical hemolysis of red blood cells. However, other markers of hemolysis, such as increased lactate dehydrogenase, bilirubin, haptoglobin, and change in amount of red blood cells, were not detected as would be expected with red blood cell breakdown (1). This is surprising, but may be caused by very small changes in their levels that were not detectable by the quantifying methodologies used.
Hemodialysis is a routine procedure for a large and growing population of patients with ESRD, a disorder associated with an increased prevalence of cardiovascular diseases such as hypertension, coronary artery disease, and cerebrovascular disease (12). This study provides evidence for a novel mechanism of iatrogenically induced vascular dysfunction in this at-risk population. The vascular changes detected also raise concern about other clinical procedures that may produce similar hemolysis because of mechanical shearing forces such as extracorporeal membrane oxygenation, cardiopulmonary bypass, ventricular assist devices, and intraoperative blood salvage machines. We hypothesize that this phenomenon may be operative in red blood cell transfusion, especially in cases of older blood or massive transfusions in which there is a reduced red cell life span after infusion. Further investigation into cell-free plasma Hb-mediated NO consumption in these clinical settings is clearly warranted.
Appreciation of a role for iatrogenic hemolysis and hemolysis-associated endothelial dysfunction leads to potential novel approaches to therapy. Initial studies indicate that this may be in the form of NO donors or exogenous haptoglobin, which could possibly be administered with HD or other inciting procedures to counteract these vascular effects (2,10,11). Another development in our understanding of Hb–NO biology is the appreciation that Hb possesses a nitrite reductase and anhydrase activity that can convert nitrite to NO and N2O3, respectively, offsetting the Hb-dependent NO scavenging by vasodilation. Nitrite reacts with deoxygenated Hb to form methemoglobin and NO (13–15).
 | ([Equation 2]) |
We have shown that this reaction is under allosteric control, so that it is most efficient near the Hb P50 (where the hemes are half-saturated with oxygen) and contributes to the control of blood flow (13,16–18). Recently, we have discovered that export of NO activity may involve the intermediacy of N2O3 through anhydrase activity (19), where this reaction occurs via a fast reaction of NO and HbFe3+ bound to nitrite.
 | ([Equation 3]) |
Consistent with this theory, 2 recent studies have examined the addition of nitrite to Hb-based oxygen carriers and found that low concentrations of nitrite reverse the vasoconstrictive effects (20,21). According to this hypothesis, low doses of nitrite could be given before hemodialysis, cardiopulmonary bypass, or transfusion of aged blood to limit the cardiovascular toxicity of NO scavenging.
In summary, confirmation of the current studies would suggest that hemodialysis-associated endothelial dysfunction caused by NO scavenging by cell-free plasma Hb may affect a very large population and open the door to novel therapies that could limit this pathobiology.
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Footnotes
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Dr. Gladwin has received research grant support in the form of a Collaborative Research and Development Agreement between the U.S. government and IKARIA, and is listed as a coinventor on a U.S. government patent for the use of nitrate salts for cardiovascular indications.
* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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