American Journal of Hematology 82:134–144 (2007) Methemoglobin—It’s Not Just Blue: A Concise Review Jay Umbreit* PPD Inc., Wilmington, North Carolina Hemoglobin has functions besides carrying oxygen to the tissues, and regulates vascular tone and inflammation via a redox couple with methemoglobin. Hemoglobin has iron in the reduced valance Fe(II) and methemoglobin has iron in the oxidized valance Fe (III), with a free energy capable of producing water from oxygen. In generating methemoglobin the couple functions as a nitrite reductase. The degree of oxidation of hemoglobin senses the oxygen level in the blood and uses its ability to produce nitric oxide from nitrite to control vascular tone, increasing blood flood when the proportion of oxygenated hemoglobin falls. Additional cardiovascular damage is produced by methemoglobin mediated oxidation of light density lipo- proteins, accelerating arteriosclerosis. In addition, the release of heme from methemoglobin is an important factor in inflammation. These physiologic functions are paralleled by the well-described role in the oxidation of various drugs resulting in methemoglobinemia. Am. J. Hematol. 82:134–144, 2007. VC 2006 Wiley-Liss, Inc. Key words: inflammation; atherosclerosis; methylene blue INTRODUCTION The auto-oxygenation generates Hgb(Fe(III)), called methemoglobin, and superoxide. At least in vitro Hemoglobin’s function in oxygen carriage is so the superoxide undergoes dismutation to hydrogen overwhelmingly important that it has obscured some peroxide and oxygen. The hydrogen peroxide is rap- of the other functions hemoglobin plays in physiol- idly decomposed by catalase. The 6th coordinate of ogy. The heme iron is carried in an (approximately) the methemoglobin is occupied with water. If not im- ferrous state (Fe(II)), the reduced form that can be mediately destroyed the hydrogen peroxide would oxidized to the ferric Fe(III) form (methemoglobin), react with Hgb(Fe(II))O to produce ferrylhemo- analogous to the cytochrome system. It is coupled to 2 globin, Hgb(Fe(IV))¼¼O with a rhombic heme that redox cycles in the cell, and is recycled itself. This reacts with further hydrogen peroxide to produce allows for the generation of two types of cyclic path- free Fe(III) and porphyrin degradation products [1]. ways. In the first, driven by the NAD-cytochrome b5 reductase, hemoglobin and methemoglobin are cycled (Fig. 1). In the second, a cell redox cycle system is driven by the oxidation of hemoglobin, with metha- HgbðFeðIIÞÞO2 ! HgbðFeðIIIÞÞ þ O2 moglobin as the product (Fig. 2). These complicated þ systems have important roles in inflammation and 2O2 þ 2H ! O2 þ H2O2 vascular regulation. H2O2 þ HgbðFeðIIÞÞO2 ! HgbðFeðIVÞÞ¼¼O HgbðFeðIVÞÞ¼¼O ! FeðIIIÞþporphyrin breakdown: REACTION OF HEMOGLOBIN WITH OXYGEN The transport of oxygen requires oxygen reversibly *Correspondence to: Jay Umbreit, PPD Inc., 3151 S. 17th Street, bound to ferrous hemoglobin, HgbFe(II). The oxy- Wilmington, NC 28412. E-mail: [email protected] genated hemoglobin Hgb(Fe(II))O2 is a very stable molecule but does slowly auto-oxidize at a rate Received for publication 27 December 2005; Accepted21 June 2006 of about 3%/day. This rate is accelerated at lower Published online 19 September 2006 in Wiley InterScience (www. oxygen tensions if the hemoglobin is partially oxy- interscience.wiley.com). genated. The chemistry is actually quite complex. DOI: 10.1002/ajh.20738 VC 2006 Wiley-Liss, Inc. Concise Review: Methemoglobin—It’s Not Just Blue 135 Fig. 1. The hemoglobin/methemoglobin reaction results in the production of defined products. The methemoglobin is Fig. 2. A redox couple in the cell can result in the produc- recycled by a reductase. [Color figure can be viewed in the tion of methemoglobin. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley. online issue, which is available at www.interscience.wiley. com.] com.] It has been difficult to detect ferrylhemoglobin, Hgb codon. The membrane form of the reductase also \ (Fe(IV)) in vivo, likely because of a comproportio- comes in two forms, localized in the ER or the mito- " nation between ferrylhemoglobin and oxyhemoglo- chondrion inner membrane. In the ER the enzyme par- bin, Hgb(Fe(II))O2, to produce Hgb(Fe(III))O2 [2]. ticipates in lipid metabolism (including biosynthesis of cholesterol and in P450 mediated drug metabolism), ð ð ÞÞ¼¼ þ ð ð ÞÞ ! Hgb Fe IV O Hgb Fe II O2 but in the mitochondrion membrane it mediates the HgbðFeðIIIÞÞO2 regeneration of ascorbate from the ascorbate radical. In the absence of myristolation the N-terminus acts as The ferrylhemoglobin is very oxidative, and the a signal recognition peptide and targets the protein to hydroxgen peroxide breakdown products accumulate be inserted into the ER. Myristolation lowers the abil- in vivo and may initiate further oxidative damage [3]. ity to interact with the signal recognition particle and These are not thought to be the major pathway for makes the protein available for post-translational tar- hemoglobin or heme degradation under most physio- geting to the mitochondrial membrane. logic conditions. Those cases of congenital methemoglobinemia due to an enzyme defect in the reductase have been classi- When the hemoglobin, Hgb(Fe(II))O2, is auto-oxi- dized to methemoglobin, Hgb(Fe(III)), the methemo- fied into 4 types [6]. Type 1 is a deficiency in cyto- globin, is recycled back to hemoglobin Hgb(Fe(II)) chrome b5 reductase limited to erythrocytes [7,8]. Type so that in the steady state the amount of intracellular 1 has few symptoms other than visible cyanosis, such methemoglobin is <1%. The methemoglobin is reduced as occasional complaints of headache, fatigue, and by the NADH-cytochrome b5-metHgb reductase. In exertional dyspnea. The methemoglobin levels exceed addition, reduction can be done by several alternative 25% of the total hemoglobin. Type 2 is more pervasive pathways such as NADPH-dependent MetHgb reduc- and is associated with a generalized systemic deficiency tase and direct reduction by intracellular ascorbate due to the alterations in lipid metabolism affecting a and glutathione [4]. multitude of tissues, particularly the central nervous system [9,10]. There is an unremitting, progressive neu- rological deterioration with mental retardation, micro- NADH-CYTOCHROME b5 REDUCTASE cephaly, opisthotonus, athetoid movements, and gen- There are two forms of the NADH-cytochorme b5 eralized hypertonia. Type 3 is no longer recognized as reductase (diaphorase 1, DIA1) in humans. A soluble, a separate entity since it was shown [11] to be identical erythrocyte restricted form, which is active in methe- to type 1. Type 4 was described in a single case, and is moglobin reduction, and a ubiquitous membrane asso- manifested by an attenuated concentration of cyto- ciated form involved in lipid metabolism [5]. In the rat chrome b5 [12]. the two forms are generated from alternative tran- To date, with new mutations being described often, scripts differing in the first exon. In the rat exon I has there are 33 different mutations known in unrelated an in-frame initiation codon, which is used inefficiently patients of different ethnicity with recessive congenital and allows the use of a downstream AUG in the sec- methemoglobinemia [13]. For the 28 exon mutations, ond exon, generating the soluble form. Likewise, in 17 have been associated with Type 1, 15 with Type 2, humans there are two transcripts, M and S. The S tran- and one mutation was common to both 1 and 2. The script is not detectable, except in erthrocytes. The posi- Type 1 typically consists of missense mutations re- tion of the first AUG is shifted in man, but analogous lated to enzyme stability. Type 2 are typically dele- to the rat there seems to be an internal initiation tions or premature stop codons resulting in truncated American Journal of Hematology DOI 10.1002/ajh 136 Concise Review: Umbreit Fig. 3. Oxygenated hemoglobin uses the oxygen to pro- duce nitrate, and in the process methemoglobin. The meth- emoglobin is recycled by the reductase. or unstable proteins, and those associated with FAD binding residues [14,15]. Fig. 4. Mechanism for oxidation of nitrite. While the mech- Recently another factor controlling methemoglobin anism is still unclear, one electron is taken from Fe(II) to was reported, an antioxidant protein AOP2 that pro- form Fe(III) and one from the substrate and the two elec- tects against methemoglobin formation. It is associated trons added to oxygen to eventually reduce oxygen to with the hemoglobin complex and may be involved in water. the normal protection of the heme pocket [16]. trations of NO to form to permit some vaso-dilation [28,29]. This allows a portion of the NO to escape REACTION WITH NITRIC OXIDE, both the diffusion into the vascular smooth muscle NITRITE, AND NITRATE and the reaction with hemoglobin in the RBC. This Nitric oxide is a gas that is continuously produced NO is able to react with oxygen to form nitrite, or to by nitric oxide synthetase in the endothelial cells using nitrosylate free thiols, amines, and other unidentified L-arginine as the substrate. The constitutive enzyme components [30]. requires calcium and calmodulin. NO is responsible for control of the systemic [17] and coronary artery 2NO þ O2 ! 2NO2 vascular [18,19] tone, acting as an endothelial relaxing À þ ð ð ÞÞ À þ þ ! ð ð ÞÞ factor [20,21]. The effects of NO are however limited to NO2 Hgb Fe II O2 2H Hgb Fe III þ À þ the local area surrounding the cells producing the gas. NO3 H2O NO rapidly diffuses from the endothelial cell into the vessel lumen and enters the red blood cell. It While the mechanism for this is complex and contro- reacts differently with oxyhemoglobin and deoxyhe- versial, overall the reaction takes one electron from moglobin.