Williams Hematology



Definition and History
Etiology and Pathogenesis

Toxic Methemoglobinemia

Nadh-Diaphorase Deficiency

Hemoglobins M

Low-Oxygen Affinity Hemoglobins


Mode of Inheritance
Clinical Features
Laboratory Features

Toxic Methemoglobinemia

Nadh-Diaphorase Deficiency

Abnormal Hemoglobins

Differential Diagnosis
Therapy, Course, and Prognosis

Toxic Methemoglobinemia

Hereditary Methemoglobinemia

Chapter References

Cyanosis, blue discoloration of the skin and mucous membranes, is usually due to a change in the color of hemoglobin. Commonly this is due to a high concentration of deoxyhemoglobin because of cardiorespiratory failure or right-to-left shunting. Cyanosis also may indicate that an abnormal hemoglobin is present or that there is an increased concentration of a normally present hemoglobin derivative. Methemoglobin is a reversible oxidation product of hemoglobin and can be present in excess amounts either because of rapid oxidation of hemoglobin by drugs or toxic chemicals or because of a hereditary defect in the methemoglobin-reducing system. Hemoglobins M are mutant hemoglobins that cannot be adequately reduced by the enzymatic systems of the red cell. Sulfhemoglobins are irreversible denaturation products of hemoglobin which can produce cyanosis even when present in relatively low and harmless quantities.
Toxic methemoglobinemia is effectively treated by intravenous infusion of methylene blue, which links the highly efficient NADPH-reducing system to methemoglobin.

Acronyms and abbreviations that appear in this chapter include: G-6-PD, glucose-6-phosphate dehydrogenase.
A bluish discoloration of the skin and mucous membranes, designated cyanosis, has been recognized since antiquity as a manifestation of lung or heart disease. Cyanosis resulting from drug administration has also been recognized since before 1890.1 Toxic methemoglobinemia occurs when various drugs or toxic substances either oxidize hemoglobin directly in the circulation or facilitate its oxidation by molecular oxygen.
In 1912 Sloss and Wybauw reported a case of a patient with idiopathic methemoglobinemia.2 Later Hitzenberger3 suggested that a hereditary form of methemoglobinemia might exist, and subsequently numerous such cases were reported.4 In 1948 Hörlein and Weber5 described a family in which eight members over four generations manifested cyanosis. The absorption spectrum of methemoglobin was abnormal. They demonstrated that the defect must reside in the globin portion of the molecule. Subsequently Singer suggested that such abnormal hemoglobins be given the designation hemoglobin M.6
The existence of abnormal hemoglobins that cause cyanosis through quite another mechanism was first recognized in 1968 with the description of hemoglobin Kansas.7 Here the cyanosis was not due to methemoglobin, as occurs in hemoglobin M, but rather to an abnormally low oxygen affinity of the mutant hemoglobin. Thus, at normal oxygen tensions a large amount of deoxygenated hemoglobin is present in the blood.
The cause of still another form of methemoglobinemia that occurs independently of drug administration and without the existence of any abnormality of the globin portion of hemoglobin was first explained by Gibson,8 who clearly pointed to the site of the enzyme defect, NADH diaphorase.
Sulfhemoglobinemia refers to the presence in the blood of hemoglobin derivatives that are defined by their characteristic absorption of light at 620 nm even in the presence of cyanide.
Methemoglobinemia decreases the oxygen-carrying capacity of blood, because the oxidized iron cannot reversibly bind oxygen. Moreover, when one or more iron atoms have been oxidized, the conformation of hemoglobin is changed so as to increase the oxygen affinity of the remaining ferrous heme groups. In this way methemoglobinemia exerts a dual effect in impairing the supply of oxygen to tissues.9
Hemoglobin is continuously oxidized in vivo from the ferrous to the ferric state. The rate of such oxidation is accelerated by many drugs and toxic chemicals, including sulfonamides, lidocaine and other aniline derivatives, and nitrites. A vast number of chemical substances may cause methemoglobinemia.10,11 and 12 Some of the agents that are responsible for clinically significant methemoglobinemia in current clinical practice are listed in Table 49-1.


NADH diaphorase catalyzes a step in the major pathway for methemoglobin reduction. This enzyme reduces cytochrome b5, using NADH as a hydrogen donor. The reduced cytochrome b5 reduces, in turn, methemoglobin to hemoglobin (see Chap. 26). A steady-state methemoglobin level is achieved when the rate of methemoglobin formation equals the rate of methemoglobin reduction, either through the NADH-diaphorase system or through the relatively minor auxiliary mechanisms such as direct chemical reduction by ascorbate and reduced glutathione. A NADPH-linked enzyme, NADPH diaphorase, does not play a role in methemoglobin reduction except when a linking dye such as methylene blue is supplied (see “Treatment,” below). A marked diminution in the activity of NADH diaphorase will result in the accumulation of the brown pigment in circulating erythrocytes.
Accordingly, hereditary deficiency of the enzyme that reduces cytochrome b5, NADH diaphorase (sometimes designated cytochrome b5 reductase), is one of the causes of methemoglobinemia. A number of mutations of NADH diaphorase have been identified at the nucleotide level.41,42,43,44,45,46,47,48 and 49
Most of the patients with this disorder merely have methemoglobinemia, and these have been classified as having type I disease. In type II disease, deficiency also exists in nonerythroid cells, such as fibroblasts and lymphocytes.50 Patients with this form of disease are afflicted, in addition to methemoglobinemia, with a progressive encephalopathy and with mental retardation. The finding that fatty acid elongation is defective in the platelets and leukocytes of such patients51 may provide a clue to the type of defect that could occur in the central nervous system, where fatty acid elongation plays an important role in myelination. Occasionally patients with deficiency of NADH diaphorase in nonerythroid cells do not suffer any neurologic disorder, and it has been suggested that they be designated as having type III disease.42,52
A combination of both increased hemoglobin oxidation and decreased methemoglobin reduction also may occur. Since the activity of NADH diaphorase is normally low in newborn infants,53 they are particularly susceptible to the development of methemoglobinemia. Thus, serious degrees of methemoglobinemia have been observed in infants as a result of toxic materials, such as aniline dyes used on diapers,54 and the ingestion of nitrate-contaminated water.55 Bacterial action in the intestinal tract may reduce nitrates to nitrites, which in turn cause methemoglobinemia. In rural areas, fatal methemoglobinuria in infants due to wells contaminated with nitrates still occurs.56
Heterozygotes for NADH-diaphorase deficiency are not usually clinically methemoglobinemic. However, under the stress of administration of drugs which normally induce only slight, clinically unimportant, methemoglobinemia such persons may become severely cyanotic because of methemoglobinemia.57
An animal model of NADH-diaphorase deficiency has been described in the cat.58,59 and 60
The molecular mechanisms by which hemoglobin binds oxygen and releases it are discussed in detail in Chapter 28. Heme is held in a hydrophobic “heme pocket” between the E and F a-helices of each of the four globin chains. The iron atom in the heme forms four bonds with the pyrrole nitrogen atoms of the porphyrin ring and a fifth covalent bond with the imidazole nitrogen of a histidine residue in the nearby F a-helix (Fig. 49-1).61 This histidine, residue 87 in the a chain and 92 in the b chain, is designated as the proximal histidine. On the opposite side of the porphyrin ring the iron atom lies adjacent to another histidine residue to which, however, it is not covalently bonded. This distal histidine occupies position 58 in the a chain and position 63 in the b chain. Under normal circumstances oxygen is occasionally discharged from the heme pocket as a superoxide anion, removing an electron from the iron and leaving it in the ferric state. The enzymatic machinery of the red cell efficiently reduces the iron to the divalent form, converting the methemoglobin to hemoglobin (see Chap. 26).

FIGURE 49-1 Diagrammatic representation of the heme group inserted into the heme pocket. A, proximal histidine; B, distal histidine. (a) In the deoxygenated form the larger ferrous atom lies out of the plane of the porphyrin ring. (b) In the oxygenated form the now smaller “ferric-like” atom can slip into the plane of the porphyrin ring. As a result, the proximal histidine and the helix F into which it is incorporated are displaced. (Lehmann and Huntsman,61 with permission.)

In most of the hemoglobins M, tyrosine has been substituted for either the proximal or the distal histidine. Tyrosine can form an iron-phenolate complex that resists reduction to the divalent state by the normal metabolic systems of the erythrocyte. Four hemoglobins M are a consequence of substitution of tyrosine for histidine in the proximal and distal sites of the a and b chains. As shown in Table 49-2, these four hemoglobins M have been designated by the geographic names Boston, Saskatoon, Iwate, and Hyde Park. Analogous His®Tyr substitutions in the a chain of fetal hemoglobin have also been documented and have been designated hemoglobins FM-Osaka62 and FM-Fort Ripley.63


Another hemoglobin M, Hb MMilwaukee, is formed by substitution of glutamic acid for valine in the sixty-seventh residue of the b chain rather than substitution of tyrosine for histidine. The glutamic acid side chain points toward the heme group, and its g-carboxyl group interacts with the iron atom, stabilizing it in the ferric state.
It is rare for methemoglobinemia to occur as result of hemoglobinopathies other than hemoglobin M, but hemoglobinChile (b28 Leu®Met) is such a hemoglobin. Producing hemolysis only with drug administration, this unstable hemoglobin is characterized clinically by chronic methemoglobinemia.70
In some hemoglobin variants the deoxy conformation of the hemoglobin molecule is favored because the angle of the heme is altered from that found normally in deoxyhemoglobin. Such changes occur in HbHammersmith, HbBucuresti, HbTorino, and HbPeterborough. In other instances the quaternary conformation is changed by mutations involving the a1b2 contact (HbKansas, HbTitusville, and HbYoshizuka). Properties of abnormal hemoglobins associated with low oxygen affinity are summarized in Table 49-3.


In response to the improved tissue oxygen supply brought about by a right-shifted oxygen dissociation curve, the “oxygen sensor”73 of the body decreases the output of erythropoietin.71 As a result, the steady state level of hemoglobin is diminished; mild anemia is characteristic of patients with hemoglobins with a decreased oxygen affinity.
Sulfhemoglobin derives its name from the fact that it can be produced in vitro from the action of hydrogen sulfide on hemoglobin74 and that the feeding of elemental sulfur to dogs has been associated with sulfhemoglobinemia.75 Sulfhemoglobin may contain one excess sulfur atom.76,77 and 78 Sulfhemoglobinemia has been associated with the ingestionof various drugs, particularly sulfonamides, phenacetin, acetanilid, and phenazopyridine.79,80 It also occurs independent of drug use and has been thought to be related to chronic constipation or to purging.81 Some patients with sulfhemoglobinemia or a past history of this disorder appear to have increased levels of red blood cell GSH.82 The reason for this increase and its relationship to sulfhemoglobinemia is not clearly understood, but it may be of significance that some of the types of drugs which have been associated with sulfhemoglobinemia cause an elevation of red cell GSH levels,83 probably by activating the enzyme glutathione synthetase83 or by increasing intracellular glutamate levels.84
Cyanosis due to abnormal hemoglobins is inherited as an autosomal dominant disorder. In contrast, hereditary methemoglobinemia due to NADH-diaphorase deficiency is inherited in an autosomal recessive fashion. Evidence for the occurrence of hereditary sulfhemoglobinemia85 is not convincing, and it is likely that the single family reported represents a hemoglobin M hemoglobinopathy.
Methemoglobinemia may be chronic or acute. Severe acute methemoglobinemia, usually the consequence of drug ingestion or toxic exposure, can produce symptoms of anemia, since methemoglobin lacks the capacity to transport oxygen. Acutely developing levels of methemoglobin exceeding 60 to 70 percent of the total pigment may be associated with vascular collapse, coma, and death,24,29 although recovery was documented in one patient with a level as high as 81.5% of the total pigment.86
Chronic methemoglobinemia, whether due to exposure to drugs or toxins or to hereditary causes, is usually asymptomatic. In instances when the methemoglobin levels are very high (>20% of the total pigment) mild erythrocytosis is occasionally noted. Patients with hemoglobins M or with low oxygen affinity hemoglobin also manifest cyanosis. In the case of a-chain variants, the dusky color of the infants will be noted at birth, but the clinical manifestations of b-chain variants become apparent only after b-chains have largely replaced the fetal a-chains at 6 to 9 months of age. In spite of the impaired hemoglobin function, no cardiopulmonary symptoms are observed and there is no clubbing. In the case of Hb MSaskatoon and Hb MHyde Park hemolytic anemia with jaundice may be present. The hemolytic state may be exacerbated by administration of sulfonamides.87
Toxic Methemoglobinemia
In toxic methemoglobinemia an elevated level of methemoglobin is found, but the activity of NADH diaphorase is normal.
In hereditary methemoglobinemia due to NADH-diaphorase deficiency, between 8 and 40 percent of the hemoglobin is in the oxidized (methemoglobin) form. The blood may have a chocolate-brown color. NADH-diaphorase activity is best measured using ferricyanide as a receptor, measuring the rate of oxidation of NADH.88,89 The residual level of enzyme activity is usually less than 20 percent of normal in patients with methemoglobinemia due to deficiency of this enzyme. An immunoassay has been described,90 but such an assay would not detect mutants in which enzyme molecules with impaired catalytic activity are present. For unknown reasons, glutathione reductase activity is usually also diminished.91 Cytochrome b5 assays92 may be useful if diaphorase activity is normal.
The spectrum of normal methemoglobin A at pH 7.0 is illustrated in Fig. 49-2.93 Hemoglobins M may be differentiated from methemoglobin formed from hemoglobin A by their absorption spectra in the range of 450 to 750 nm. Since only some 20 to 35 percent of the total hemoglobin will ordinarily be hemoglobin M, the mixed spectra of methemoglobin A and hemoglobin M may be difficult to interpret. Therefore, it is preferable to perform these spectral studies on purified hemoglobin M isolated by electrophoretic or chromatographic means.61

FIGURE 49-2 Absorption spectra at pH 7.0. A; B, methemoglobin A; B, methemoglobin MBoston; C, methemoglobin MSaskatoon; D, methemoglobin A fluoride complex. For purposes of comparison all the optical densities have been made equal to 0.61 at 500 nm. (Gerald and George,93 with permission of the American Association for the Advancement of Science.)

All hemoglobin M samples should be converted to methemoglobin so that any difference found in electrophoresis will be due to the amino acid substitution and not to the different charge of the iron atom. Electrophoresis at pH 7.1 is most useful for separation of hemoglobins M since the imidazole groups of histidine have a net positive charge at this pH, while at higher pH levels the histidines and the substituting tyrosines are both neutral.
The hemoglobins M differ in their reactivity to cyanide and to azide ions.94
Sulfhemoglobin is detected in the lysate of blood treated with ferricyanide, cyanide, and ammonia by comparing the optical density at 620 nm with that at 540 nm.95,96
Cyanosis due to methemoglobinemia or sulfhemoglobinemia should be differentiated from cyanosis due to cardiac or pulmonary disease particularly when right-to-left shunting is present. In the latter instances the arterial oxygen tension will be low, while in methemoglobinemia and sulfhemoglobinemia it should be normal. One should be certain, however, that the oxygen tension was measured directly and not deduced from the percent saturation of hemoglobin. Blood from a patient with cyanosis due to arterial oxygen desaturation promptly becomes bright red upon being shaken with air. In addition, these causes of cyanosis are readily differentiated by carrying out quantitative blood methemoglobin and sulfhemoglobin levels. Because of the potential lethal nature of high levels of methemoglobin, and because prompt treatment may be life saving, a high index of suspicion is important. A patient with cyanosis whose arterial blood is brown with a that is found to be normal on blood gas examination is likely to have methemoglobinemia. One should not rely on the readings of a pulse oximeter, since false readings may be obtained in the presence of methemoglobin. Rapid examination of a blood sample using an automatic analyzer such as a CoOximeter is the first step in confirming the diagnosis. However, as pointed out under “Laboratory Features” above, although treatment should not be delayed, direct spectrophotometric analysis should be carried out on the pretreatment sample as soon as possible to distinguish between methemoglobinemia and sulfhemoglobinemia.
A family history is usually helpful in differentiating hereditary methemoglobinemia due to NADH-diaphorase deficiency from hemoglobin M disease. The former has a recessive mode of inheritance, the latter a dominant mode. Thus, cyanosis in successive generations suggests the presence of hemoglobin M; normal parents but possibly affected sibs implies the presence of NADH-diaphorase deficiency. Consanguinity is more common in NADH-diaphorase deficiency. In NADH-diaphorase deficiency incubation of the blood with small amounts of methylene blue will result in rapid reduction of the methemoglobin; in hemoglobin M disease such reduction does not take place. The absorption spectra of methemoglobin and its derivatives are normal in NADH-diaphorase deficiency; they are abnormal in hemoglobin M disease. In the case of toxic methemoglobinemia cyanosis is generally of relatively recent origin, and a history of exposure to drug or toxin may usually be obtained; in hereditary methemoglobinemia a history of lifelong cyanosis may usually be elicited.
Toxic Methemoglobinemia
Acute toxic methemoglobinemia may represent a serious medical emergency. Because of the loss of oxygen-carrying capacity of the blood and because of the left shift in the oxygen dissociation curve that occurs when methemoglobin is present in high concentration,97 acute methemoglobinemia may be life threatening when the level of the pigment exceeds half of the total circulating hemoglobin.
Methylene blue is an effective treatment for patients with methemoglobinemia because NADPH formed in the hexose monophosphate pathway can rapidly reduce this dye to leukomethylene blue in a reaction catalyzed by NADPH diaphorase. Leukomethylene blue, in turn, nonenzymatically reduces methemoglobin to hemoglobin.98 An exception to the efficacy of this treatment exists in those patients who are G-6-PD deficient (see Chap. 45). In these subjects methylene blue would not only fail to give the desired effect on methemoglobin levels but might compound the patient’s difficulty by inducing an acute hemolytic episode.99 In patients with acute toxic methemoglobinemia who are symptomatic or whose methemoglobin level is rising rapidly, the intravenous administration of 1 or 2 mg methylene blue per kg body weight over a period of 5 min is the preferred treatment because of its very rapid action.20 Use of excessive amounts of methylene blue should be avoided: the administration of repeated doses of 2 mg methylene blue/kg body weight has produced acute hemolysis even in patients with normal G-6-PD levels.21 The response to treatment is so rapid, with marked lowering or normalization of methemoglobin levels within an hour or two, that no other treatment is usually needed, but the patient should be observed carefully because continued absorption of a toxic substance from the gastrointestinal tract may cause recurrence of the methemoglobinemia. In patients who are in shock blood transfusion may be helpful. Cimetidine, used as a selective inhibitor of N-hydroxylation, may decrease the methemoglobinemia produced by dapsone in patients with dermatitis herpetiformis.100
The course of hereditary methemoglobinemia is benign, but patients with this disorder should be shielded from exposure to aniline derivatives, nitrites, and other agents which may, even in normal persons, induce methemoglobinemia. Hereditary methemoglobinemia due to NADH-diaphorase deficiency is readily treated by the administration of ascorbic acid, 300 to 600 mg orally daily divided into three or four doses. While intravenously administered methylene blue is very effective in correcting this type of methemoglobinemia, it is not suitable for the long-term therapy that needs to be given if the state is to be treated at all.
The iron phenolate complex which exists in the hemoglobins M prevents the reduction of ferric to ferrous iron. For these reasons the methemoglobinemia does not respond to administration of ascorbic acid or of methylene blue. No effective treatment exists for the cyanosis that is present in patients with abnormal hemoglobins with reduced oxygen affinity.
Sulfhemoglobinemia is almost always a benign disorder. Unlike methemoglobin, sulfhemoglobin does not produce a left shift in the oxygen dissociation curve but rather decreases the affinity of hemoglobin for oxygen.79 The disorder tends to recur repeatedly in the same persons after exposure to drugs but does not generally appear to affect their overall health. Unlike methemoglobin, sulfhemoglobin cannot be converted to hemoglobin. Thus, once sulfhemoglobinemia occurs it will persist until the erythrocytes carrying the abnormal pigment reach the end of their life span.

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Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology



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  2. If anyone can help please email ptcruiser1@optusnet.com.au
    My 22year old daughter has had central cyanosis for two years and no cause can be found! All heart and breathing normal, suspected hemoglobinopathy, no improvement when given oxygen and normal pulseox, it never has completely resolved but does vary in severity and causes a feeling of dsypnea. Also she has constant anemia for ten years, doctors very perplexed, negative test for methemoglobinemia which they were sure it was, and it has persisted way longer than blood turnover
    ,any ideas welcome, thanks, jo

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