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792 Part VI: The Erythrocyte Chapter 50: Methemoglobinemia and Other Dyshemoglobinemias 793

CLINICAL FEATURES methemoglobin. The Rad-57 uses eight wavelengths of light instead of
Drug Ingestion the usual two and is thereby able to measure more than two species of
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human hemoglobin. In addition to the usual SpO value, the Rad-57
2
Methemoglobinemia may be chronic or acute and acquired or con- displays SpCO and SpMet, which are the pulse oximeter’s estimates of
genital. Acquired severe acute methemoglobinemia, usually the conse- COHb and methemoglobin concentrations, respectively. In a study on
quence of drug ingestion or toxic exposure, can produce symptoms of healthy human volunteers in whom controlled levels of methemoglobin
anemia, since methemoglobin lacks the capacity to transport oxygen. and COHb were induced, the Rad-57 measured COHb with an uncer-
Symptoms may include shortness of breath, palpitations, and vascu- tainty of ±2 percent within the range of 0 to 15 percent and measured
lar collapse. Chemicals that induce methemoglobinemia are often also methemoglobin with an uncertainty of 0.5 percent within the range of 0
capable of causing hemolysis, and a combination of hemolytic anemia to 12 percent, the usefulness of this instrument has been verified also
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and methemoglobinemia may occur. Chronic methemoglobinemia, by other studies. 70,71
whether a result of exposure to drugs or toxins or of hereditary causes,
is usually asymptomatic. Cyanosis, even if present, may not be discern-
able in persons with very dark skin coloration. In instances when the Cytochrome b5 Reductase Deficiency
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methemoglobin levels are chronically very high (>20 percent of the total In hereditary methemoglobinemia resulting from cytochrome b5 reduc-
pigment), mild erythrocytosis may be noted (Chap. 57). tase deficiency, between 8 and 40 percent of the hemoglobin is in the
form of methemoglobin. The blood may have a chocolate-brown color
and cyanosis is present. Cytochrome b5 reductase activity is best mea-
M Hemoglobins sured using ferricyanide as a receptor, measuring the rate of oxidation
Patients with hemoglobin M also manifest cyanosis. In the case of of NADH. 72,73 The residual level of enzyme activity is usually less than
α-globin variants, the dusky color of the infants will be noted at birth, 20 percent of normal in patients with methemoglobinemia resulting
but the clinical manifestations of β-globin variants become apparent from deficiency of this enzyme. An immunoassay has been described,
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only after β chains have largely replaced the fetal γ chains at 6 to 9 but such an assay would not detect mutants in which enzyme mole-
months of age. In spite of the impaired hemoglobin function, no cardio- cules with impaired catalytic activity are present. For unknown reasons,
pulmonary symptoms are observed and there is no clubbing. In the case glutathione reductase activity (Chap. 47) is usually also diminished. 75

of HbM Saskatoon and HbM Hyde Park , hemolytic anemia with jaundice may be
present. The hemolytic state may be exacerbated by administration of
sulfonamides. 61 Cytochrome b5 Deficiency
Cytochrome b5 assays may be useful if cytochrome b5 reductase activity
is normal, and the presence of hemoglobin M is ruled out. 76
Cytochrome b5 Reductase Deficiency
Hereditary methemoglobinemia resulting from cytochrome b5 reduc-
tase deficiency may, as noted above, be associated with mental retar- M Hemoglobins
dation, failure to thrive and early death. In one case, skeletal anomalies Optical Spectrum Figure 50-2 illustrates the spectrum of normal
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were documented as well. 62 methemoglobin A at pH 7.0. Hemoglobins M may be differentiated
from methemoglobin formed from hemoglobin A by its absorption
LABORATORY FEATURES spectrum in the range of 450 to 750 nm. Because only some 20 to
35 percent of the total hemoglobin will ordinarily be the hemoglobin M,
Toxic Methemoglobinemia the mixed spectra of methemoglobin A and the hemoglobin M may be
In toxic methemoglobinemia, an elevated level of methemoglobin is difficult to interpret. Therefore, it is preferable to perform these spectral
found, but the activity of cytochrome b5 reductase is normal. Methe- studies on purified hemoglobin M isolated by electrophoretic or chro-
moglobin levels are best measured using the change of absorbance of matographic means. 56
methemoglobin at 630 nm that occurs when cyanide is added, convert- Electrophoresis All hemoglobin M samples should be converted
ing the methemoglobin to cyanmethemoglobin, a principle used in the to methemoglobin so that any difference found in electrophoresis will
Evelyn–Malloy method. 63,64 Errors in diagnosis are frequently made be the result of the amino acid substitution and not the different charge
when automated instruments designed to estimate levels of reduced of the iron atom. Electrophoresis at pH 7.1 is most useful for separation
hemoglobin, oxygenated hemoglobin, methemoglobin, and carboxyhe- of hemoglobins M because the imidazole groups of histidine have a net
moglobin (COHb) are used. Most automated instruments do not prop- positive charge at this pH, while at higher pH levels the histidines and
erly make this distinction. 65,66 the substituting tyrosines are both neutral.
The clinical incidence of methemoglobinemia can be overesti- Other Biochemical Methods The hemoglobins M differ in their
mated by cooximeter measurements compared to the more specific reactivity to cyanide and to azide ions. This property may help to iden-
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Evelyn–Malloy method. Evelyn-Malloy method involves direct spec- tify the subunit affected, as the iron-phenolate bonds are stronger in the
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trophotometric analysis and should be used when methemoglobinemia α-chain variants than in the β-chain variants. However, definitive iden-
is suspected. This is achieved by lysing the blood in a slightly acid buffer tification of the variant requires peptide or DNA analysis. Hemoglo-
and measuring the optical density at 630 nm before and after adding a bins that cause cyanosis because of a diminished oxygen affinity may be
small amount of neutralized cyanide. The absorption of methemoglobin detected by determining the oxygen dissociation curve of blood, being
at this wavelength disappears when it is converted to cyanmethemoglo- certain that the 2,3-bisphosphoglycerate (2,3-BPG) level is normal,
bin. Although this method was described in 1938, it remains the most or by estimating the oxygen dissociation curve of hemoglobin, which
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accurate technique for the estimation of methemoglobin in the blood. has been stripped of 2,3-BPG by extensive dialysis against an appro-
Details of its performance can be found in an earlier edition of this text priate buffer. Many of the hemoglobins with decreased oxygen affinity
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and elsewhere. 61 are unstable (Chap. 49) and will precipitate in the isopropanol stabil-
An eight-wavelength pulse oximeter, Masimo Rad-57 (Rainbow-SET ity test. In many laboratories, it may be easier to analyze the coding
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Rad-57 Pulse CO-Oximeter, Masimo Inc, Irvine, CA), has been sequence of the globin chains at the DNA level than to attempt to deter-
approved by the FDA for the measurement of both COHb and mine the properties of the hemoglobin. 79

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