General Concepts The potential for normal RBCs to undergo auto-oxidative destruction is great because the cell is loaded with 20-mM hemoglobin, most of which is bonded to oxygen at the iron(Fe++) atom in heme. The bond that allows the reversible association and dissociation of oxy gen from the heme moiety of hemoglobin involves the partial trans fer of an electron from iron(Fe++) to oxygen. Consequently, oxygen then has an extra electron, making it a superoxide radical. Ordinarily, when oxygen leaves hemoglobin, it returns the electron. If it does not, a highly reactive superoxide ion is released, leaving behind it an iron(Fe+++) moiety called methemoglobin.
HbFe2+O2 → HbFe3+ + O2−1
Methemoglobin cannot reversibly bind oxygen. Methemoglobin in itself is not harmful to RBCs, but if the oxidative assault persists, methemoglobin is converted to hemichromes, which are variably denatured hemoglobin intermediates in which the distal histidine unit binds to the oxidized heme. This step is associated with conversion from a high to a low spin state, as measured by electron spin resonance. Continued oxidation leads to the irreversibility of hemichrome oxidation, precipitation, and eventually the formation of Heinz bodies. Hemichromes and Heinz bodies can destroy membrane function directly or by causing the oxidation of membrane proteins and lip ids. Approximately 3% of hemoglobin is converted to methemoglobin each day, but the finding that only 1% of hemoglobin normally is in the form of methemoglobin indicates that mechanisms preventing oxidation in RBCs are in effect. These mechanisms are limited because RBCs lack the ability to either efficiently generate ATP or synthesize enzymes. The primary means for preventing or addressing oxidant injury are the generation of the reduced form of nicotinamide adenine dinucleotide (NADH) via the Embden–Meyerhof glycolytic pathway and the generation of nicotinamide adenine dinucleotide phosphate (NADPH) via the hexose monophosphate shunt. NADH is used to reduce methemoglobin by cytochrome b5 reductase, and NADPH is used to reduce glutathione and for catalase activity. Defects in this defense system against oxidation lead to an enhanced tendency to oxidative hemolysis. Examples are G6PD deficiency states.26 G6PD catalyzes the initial rate-limiting step in the hexose monophosphate shunt. Deficiencies lead to a reduced ability to generate NADPH in response to oxidant stress. Any agent or event that interferes with the smooth offloading of oxygen enhances the generation of O2−1 and methemoglobin, as indicated in the equation. If the reducing power of the RBC is inadequate, hemichromes and Heinz bodies are generated. Many agents appear to cause oxidative hemolysis by interfering with the smooth functioning of the heme cleft.
Pathophysiology
After the oxidative attack has been initiated, the sequence pro ceeds along a recognizable track. The oxidative attack is directed at hemoglobin and the RBC membrane. However, these structures are not clearly separable because the precipitated hemichrome and Heinz bodies come to lie against the cytosolic face of the membrane. Methemoglobin may be detectably elevated, with levels as high as 50% to 60% of total hemoglobin. The hemichromes, by themselves or with their iron portions acting as a Fenton reagent, mediate the generation of hydroxyl free radicals, which add their effect to that of superoxide and hydrogen peroxide. Lipid peroxidation may take place, leading to membrane blebbing and cell lysis, as well as the loss of asymmetry of the phospholipid membrane bilayer. Movement of phosphatidylserine and phosphatidylethanolamine to the outer bilayer of the membrane results in increased recognition by macro phages in the reticuloendothelial system. Membrane proteins may be crosslinked, with binding of denatured, oxidized hemoglobin to the membrane cytoskeleton, which may increase splenic macrophage recognition. In addition, the RBCs are rigid and susceptible to trapping in sinusoidal structures, whether or not they have Heinz bodies lying against the membrane. In vitro evidence suggests that oxidized RBCs are increasingly susceptible to phagocytosis by macrophages. These features may account for extravascular destruction. The oxidative lesions can be severe enough to cause intravascular destruction as well, producing hemoglobinemia and hemoglobinuria.
The smear may show bite cells, which look as if a macrophage had taken a bite, removing a Heinz body-containing segment of the membrane. RBC rigidity may result in irregularly shaped cells because these undeformable cells are unable to undergo elastic recoil after fighting their way through the sinus wall. Recurrent loss of membrane material may produce spherocytes. Severe hemolysis may produce the kind of circulating ghost or hemighost called a blister cell or bite cell. These RBCs have an empty veil of membrane on one side and puddled hemoglobin on the other. A Heinz body preparation may be positive. However, the absence of bite cells does not rule out the diagnosis.
The clinical picture is determined by the specific agent used. Screening for G6PD deficiency or a related disorder using an enzyme assay or the ascorbate cyanide test may be useful. Although any defect in the antioxidant defense mechanisms, such as G6PD deficiency, considerably increases the susceptibility to hemolysis, many agents can produce oxidant hemolysis even in persons with normal defense mechanisms (see the box on Agents That Cause Oxidative Hemolysis).
Paraquat ingestion has occurred inadvertently and in suicide attempts. Profound cyanosis with methemoglobinemia can occur within hours, with levels of 120% or higher. The condition may be succeeded by hemolysis, with Heinz bodies seen in appropriate preparations of RBCs.
Toxic ingestion or inhalation of nitrites may occur in suicide attempts from industrial exposures; via diets high in pickled or smoked foods; through intentional recreational use; or in infants from formulas prepared using well water high in nitrates, which are reduced to nitrites in the infant gut. Nitrites bind to hemoglobin, producing methemoglobinemia, which may be so profound as to produce coma. If methylene blue infusion does not quickly turn the chocolate color of blood back to normal, the physician must consider the possibility that the patient is G6PD deficient and therefore unable to generate adequate amounts of NADPH (dis cussed earlier in the section Drug-Induced Oxidative Hemolysis: General Concepts). In that case, exchange transfusion may be life saving. Benzocaine topical anesthesia in the form of a spray or cream can cause severe methemoglobinemia, with cyanosis and dyspnea requiring methylene blue treatment.
In a case of almost fatal oxidative hemolysis, hydrogen peroxide was injected directly into the Hickman catheter of a patient with AIDS because some persons infected with HIV had circulated a pamphlet suggesting that hydrogen peroxide could be used therapeutically to control HIV infection. We now are seeing AIDS patients with dapsone-induced methemoglobinemia and hemolytic anemia. Methemoglobinemia, if severe, is treated as described in the preceding paragraph and in Chapter 44.
Pyridium (phenazopyridine) can cause oxidative hemolysis even in the absence of renal disease. This agent is commonly used for the treatment of bladder irritation. The Physician’s Desk Reference recommends maximum therapy of 2 days. However, patients not uncommonly are given a prescription for 1 to 4 weeks of therapy.
It has been recognized for more than 130 years that therapy with dapsone causes oxidative hemolysis. In the past, dapsone was used primarily to treat leprosy and dermatitis herpetiformis and was not often encountered as a cause of oxidative hemolysis. Dapsone has come into more widespread use in some communities as a very effective prophylactic agent against Pneumocystis carinii pneumonia in patients with AIDS. The reduced levels of glutathione reported in patients with AIDS may enhance dapsone toxicity. Dapsone is also used in the treatment of malaria where it regularly causes hemolytic anemia in G6PD-deficient patients. Some clinics screen potential recipients for G6PD deficiency and, if results are negative, proceed with dapsone therapy. However, dapsone can cause oxidative attack on normal RBCs, leading sequentially to methemoglobinemia, Heinz bodies, and hemolysis, all occurring at generally accepted standard doses. Dapsone is metabolized to a hydroxylamine derivative that is directly toxic to RBCs.
