Hemoglobin is perhaps the most intensively studied of the biologically important molecules. Much is known of its structure, its function, and its regulation. In addition to well-characterized processes of oxygen, carbon dioxide, and carbon monoxide transport, new data suggest a key role of hemoglobin as a carrier of nitric oxide. In this review, we describe the basis of this interaction, as well as its clinical relevance to such problems as acute respiratory distress syndrome, percutaneous transluminal coronary angioplasty, and transplant allograft survival.Hemoglobin is essential to life. Recent observations relating hemoglobin-bound nitric oxide (NO) to vascular dynamics and oxygen delivery have emphasized the central role of this multipurpose carrier molecule in health and disease. The purposes of this review are (1) to examine the essential features of hemoglobin function, (2) to delineate the novel relationship between hemoglobin and NO, and (3) to focus on these NO-hemoglobin functions as they relate to pathological states encountered in surgical practice.STRUCTURE AND FUNCTION OF HEMOGLOBINAdult hemoglobin is a tetramer of 2 α and 2 β subunits. Each subunit of the hemoglobin tetramer contains a pocket in which the iron-containing ring (heme) is suspended. The central iron (Fe2+) is covalently attached to nitrogen groups in the heme, and pulled slightly above the ring by a histidine of the hemoglobin subunit. Without the Fe2+in heme, the protein portion of hemoglobin could not bind or transport oxygen. Similarly, the heme portion alone, without the globin protein, would rapidly react with oxygen to form water and ferric heme (methemoglobin, Fe3+).The complementary nature of the heme moiety to globin protein interaction is illustrated by carbon monoxide (CO) poisoning. The potentially paralyzing effect of CO on cellular electron transport is prevented by virtue of the structure of the globin molecule. Free heme binds CO with an affinity 25,000 times that of oxygen. Steric hindrance by the globin protein reduces the essentially irreversible CO-heme interaction to a hemoglobin affinity for CO of only 200 times that of oxygen.In this fashion, hemoglobin protects cells from lethal CO exposure while permitting CO release from hemoglobin in the lung, albeit slowly.All of the available hemoglobin sites for oxygen binding are saturated in the lungs. When a single oxygen molecule binds the hemoglobin tetramer, the oxygen affinity of the hemoglobin increases, making it more likely that second, third, and fourth oxygen molecules will also bind. Conversely, in the peripheral tissue, where oxygen tension is lower, the release of 1 oxygen molecule enhances the release of the other oxygen molecules. Cooperative, noncovalent interactions between the globin subunits result in this familiar sigmoidal relationship of hemoglobin oxygen saturation and oxygen pressure. Oxygen binding pulls the iron, normally suspended above the porphyrin ring, into the heme plane. This motion of the iron and the attached histidine triggers a conformational change in the entire hemoglobin tetramer that increases its affinity for oxygen.The affinity for oxygen is also influenced by the microenvironment of tissues. As respiring cells produce carbon dioxide, carbamate is formed at the N terminus of the hemoglobin molecule. When the N terminus is no longer positively charged and salt bridges form between the α and β subunits, oxyhemoglobin affinity decreases. This is the basis of the Bohr effect that facilitates hemoglobin uptake of oxygen in the lungs and release of oxygen in the tissues.LIFE AND DEATH OF NONitric oxide is generated from multiple endogenous and exogenous sources and has diverse physiologic functions.Vascular endothelial cell–derived NO acts directly on adjacent vascular smooth muscle via a cyclic guanosine monophosphate mechanism to cause vasorelaxation, with an increase in regional blood flow and oxygen delivery.Macrophages use NO in the destruction of phagocytosed material, while the central nervous system uses NO as a neurotransmitter. Platelet aggregation and activation is known to be inhibited by NO, while bronchoconstriction is attenuated.Other cell types, including hepatocytes, and myocardial cells, are known to possess different forms of NO-producing enzymes.The differential regulation of these enzyme systems by various cytokines and other cell signals is crucial to the biological effect of NO.While NO plays a role in physiologically appropriate diastolic relaxation, NO also mediates the aberrant myocardial depression during the inflammatory response.In the liver, NO plays a protective role during oxidative stress, during inflammatory injury, and is able to inhibit cytokine-induced apoptosis.Nitroglycerin, nitroprusside, and amyl nitrite serve as clinically accessible exogenous NO donors available to the surgeon.Nitric oxide reacts with a number of biologically important molecules. When NO combines with free oxygen, nitrite species are formed.Covalent interaction with thiol groups leads to formation of S-nitrosylated proteins, thus both modulating their function, and creating a reservoir of NO.Superoxide oxygen radicals interact with NO to form cytotoxic peroxynitrite (ONOO−).When NO combines directly with the heme of oxyhemoglobin, ferrous iron (Fe2+) is reduced to form ferric methemoglobin (Hb[Fe3+]). Conversely, NO combines with the heme site of deoxyhemoglobin to form Hb[Fe2+]NO.This scavenging of NO by hemoglobin results in vasoconstriction(Figure 1).Figure 1.Nitric oxide (NO) is derived from exogenous and endogenous sources. While S-nitrosohemoglobin increases blood flow by delivery of NO to vascular smooth muscle, the heme portion of hemoglobin scavenges NO, resulting in vasoconstriction. Free radicals react with NO, forming cytotoxic peroxynitrite.HEMOGLOBIN IS AN NO DONORBy comparing the structure of hemoglobin across many species, a surprisingly limited number of amino acid sequences are constant. The most phylogenetically constant amino acids (and, by inference, the most crucial to function) include those surrounding the heme pocket, as well as a cysteine at position 93 on the surface of the β subunit (cys93β).This cysteine, a thiol, is capable of binding NO and forming S-nitrosohemoglobin (SNO-Hb). While heme rapidly scavenges local endothelial cell–generated NO, inducing vasoconstriction when hemoglobin is perfused through isolated aortic segments, SNO-Hb exhibits vasodilatory properties when perfused through the same in vitro system.Furthermore, the deletion of cys93β of the perfused hemoglobin results in vasoconstriction. Jia et aland Stamler et alhave proposed that the role for this highly conserved cysteine is to bind NO in the lungs, and to deliver NO to peripheral tissues as a fundamental mechanism of blood flow and blood pressure control.In a physiologically predictable fashion, the deoxygenated form of SNO-Hb is more apt to release NO and cause vasodilation than the oxygenated form.When SNO-Hb is infused into animals breathing room air, they exhibit an increase in cerebral blood flow. In contrast, there is no alteration in cerebral blood flow with infusion of SNO-Hb in animals under hyperoxic conditions.Furthermore, endogenous SNO-Hb is detected in the arterial blood alone, while Hb[Fe2+]NO is present in venous blood in animals breathing room air; SNO-Hb predominates in both sides of the circulation under hyperoxic conditions. These data imply formation of SNO-Hb in the lung, by a yet unidentified mechanism, with subsequent release of NO from cys93β in the tissue arterioles to cause vasodilation, counteracting the decreased blood flow from NO scavenging by heme. This process occurs only when peripheral oxygen tension is low, such as with breathing room air, ensuring a balance between oxygen delivery and demand(Figure 2).Figure 2.Oxygenated S-nitrosohemoglobin (SNO-HbO2) is formed in the lungs. In the peripheral vasculature, both nitric oxide (NO) and oxygen (O2) are released. Nitric oxide delivery causes vasodilation and counteracts scavenging of NO by hemoglobin (HbFe2+-NO) to ensure oxygen delivery matches oxygen demand.Nitric oxide released from hemoglobin does not simply diffuse out of red blood cells and into vascular smooth muscle to promote vasodilatation. Plasma NO is bound to albumin and other cysteine containing low molecular weight proteins (thiols). Data suggest NO is sequentially transferred from hemoglobin to plasma protein thiols, and to vascular smooth muscle cells.The specific mechanism of the NO transfer remains to be elucidated.CLINICAL IMPLICATIONS OF SNO-HbAcute Respiratory Distress SyndromeInhaled NO is an attractive potential treatment for patients with acute respiratory distress syndrome (ARDS). Nitric oxide should not only direct pulmonary blood flow toward ventilated alveoli, enhancing oxygen uptake in the lung, but also vasodilate peripherally, via SNO-Hb, promoting systemic oxygen delivery. Indeed, NO is purported to lower pulmonary hypertension, a central feature of ARDS.Improvement in peripheral tissue oxygen delivery, as well as enhanced mitochondrial utilization of oxygen in the periphery, has been reported with inhaled NO therapy in patients with ARDS.Surprisingly, recent randomized clinical trials on the use of inhaled NO in patients with ARDS conclude no effect on mortality or duration of mechanical ventilation.Furthermore, any positive effect on the lung's ability to oxygenate blood appears transient, lasting hours to days.This therapeutic failure is consistent with the generation of the unstable, and highly toxic peroxynitrite, formed by the interaction of NO and superoxide radicals.The enhanced oxygen delivery afforded by inhaled NO could impair mitochondrial electron transport through these toxic by-products.Ultimately, the therapeutic potential of exogenous NO will depend on striking a balance between NO-enhanced oxygen delivery and the production of destructive NO radicals.AngioplastyPercutaneous transluminal coronary angioplasty for the treatment of coronary artery disease is limited by the high incidence of restenosis. Restenosis is the result of endothelial cell injury, release of cytokines, and resultant proliferation and migration of smooth muscle cells underlying the damaged endothelium.Interestingly, this post–percutaneous transluminal coronary angioplasty vascular remodeling does not lead to clinically relevant blockage in all patients.Nitric oxide may modulate this process of intimal hyperplasia and restenosis. Addition of an NO donor reduces the degree of intimal thickening, and enhances the regeneration of the endothelial cells, reforming the surface of the injured blood vessel.Similar attenuation of restenosis has been reported following administration of an NO-albumin conjugate, and with gene transfection of the NO-generating enzyme, NO synthase, into endothelial cells.Furthermore, prolonged inhalation of NO has been shown to attenuate intimal proliferation after angioplasty in the rat.This latter beneficial effect of augmenting NO delivery to the injured blood vessel may be mediated by SNO-Hb. However, the specific role of SNO-Hb in modulating vessel injury remains undefined.Cardiac TransplantationA diffuse coronary arterial intimal hyperplasia affects 40% of transplanted hearts and is the dominant limitation to donor organ survival. Endothelial injury appears to be the inciting event of posttransplantation neointimal hyperplasia. Studies of human heart recipients 1 to 3 years after cardiac transplantation reveal defects in endothelium dependent, NO-mediated vasodilation.Theories invoking reaction to major histocompatibility complex antigens on the endothelial cells of the donor organ, unanswered variable expression of adhesion molecules on the endothelium, or bystander damage to the endothelium from reaction to donor white blood cells have been proposed to explain this diffuse allograft vasculopathy. Surprisingly, work in inbred animals indicates that the transplant vasculopathy progresses even if the donor organ is protected from the recipient immune system by donor organ replacement back into the donor strain.Conversely, vasculopathy is prevented if the allograft is transplanted back into the donor strain within 4 days (but not later) of the original transplant. This time dependence suggests that early injury to the endothelium of the transplanted organ is responsible for the diffuse vasculopathy. Indeed, cardiac endothelial dysfunction (decreased NO activity) at 2 weeks after human heart transplantation predicts the development of cardiac vasculopathy at 1 year.Thus, decreased NO activity is not only a marker for endothelial dysfunction, but it may play a role in the pathogenesis of allograft vasculopathy. Inhibition of NO synthase in animals exacerbates renal, cardiac, and aortic allograft rejection, while transfection of NO synthase suppresses vasculopathy.Thus, an NO delivery system used early in the posttransplantation period is a promising strategy to attenuate allograft vasculopathy.Hemoglobin-Based Blood SubstitutesThe development of stroma-free hemoglobin (SFH) as a blood substitute has been limited by the prominent side effect of systemic hypertension. Stroma-free hemoglobin becomes an unregulated scavenger of NO in the vasculature. Although this hypertension may be mediated partially by α2-adrenergic receptors, the hypertensive effects of SFH involve an interaction at the heme site of artificial hemoglobin with NO.Intraluminal scavenging of NO alone may be insufficient to bring about all of the hypertensive effects of SFH.Gould et alpostulate that extravasation of SFH across the endothelial cell barrier with binding of NO at its vascular smooth muscle target is the cause of hypertensive effects. Thus, polymerized forms of SFH that prevent extravasation should promote normotensive oxygen delivery. A preliminary clinical trial supports this hypothesis.Sickle Cell AnemiaIn addition to vascular dynamics, NO may influence the oxygen affinity of some altered forms of hemoglobin. 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JAMA Surgery – American Medical Association
Published: Apr 1, 1999