Postscript

Description

Methemoglobin refers to the oxidation of ferrous iron (Fe²⁺) to ferric iron (Fe3⁺) within the hemoglobin molecule.¹
In red blood cells RBCs), an equilibrium normally exists between hemoglobin and methemoglobin. In adults, about l% of the hemoglobin is in the met-form; 99% remains in the normal state. Methemoglobinemia occurs when the concentration of methemoglobin in RBCs is >5%.
Note that while hemoglobinemia refers to the presence of free hemoglobin in the plasma, methemoglobinemia refers to increased levels of methemoglobin within circulating RBCs.
Methemoglobin:
- Is formed from unoxygenated hemoglobin.
- Is not capable of carrying oxygen or carbon dioxide.
- Decreases the available oxygen supply.
- Shifts the oxygen-hemoglobin dissociation curve to the left (increased affinity of hemoglobin for oxygen, reduced offloading of oxygen to tissues).

Classification

Congenital:
- Rare
- Includes:
  - Enzymopathy – autosomal recessive variants in the CYB5R3 gene
  - Hemoglobinopathy (Hemoglobin M) – autosomal dominant variants in the globin genes
Acquired:
- More common
- Mainly due to the exposure to substances that cause oxidation of Hb both directly or indirectly.

Pathophysiology

Oxidation and reduction:
- Oxidation involves the extraction of electrons from a substrate.
- Reduction involves the transfer of electrons to a substrate.
- A substance has been:
  - Oxidized if it loses an electron
  - Reduced if it gains an electron
- Oxidation/reduction reactions are termed “redox” reactions because they always occur together (i.e., for one substance to be reduced, another must be oxidized).
- When oxidation occurs within the heme moiety of hemoglobin, methemoglobinemia (MHb) is formed.²
- Many agents that cause methemoglobinemia have other effects on the RBC, e.g., on the membrane, causing some degree of hemolysis.
Susceptibility of hemoglobin to oxidation:
- Hemoglobin molecules contain iron within a porphyrin heme structure:
  - The iron in hemoglobin is normally found in the Fe²⁺state.
  - The iron moiety of hemoglobin can be oxidized to the Fe³⁺state to form methemoglobin.
- Because RBCs are exposed to high levels of oxygen, a small degree of physiologic MHb formation occurs continuously (called auto-oxidation):
  - Formation of methemoglobin and conversion back to the normal ferrous state (by reduction [addition of an electron]) occurs at low levels during normal RBC metabolism.
  - Normally, the formation and reduction of methemoglobin are balanced, and the steady-state level of methemoglobin is approximately 1% of total hemoglobin.
- Deoxyhemoglobin has a tense (T) conformation and is about tenfold more susceptible to oxidation than oxyhemoglobin, which has a relaxed (R) conformation.
- Low pH and organic phosphates accelerate hemoglobin oxidation rates by the shift of the T –> R conformational equilibrium towards the T conformation.
Clinically significant methemoglobinemia occurs when there is an imbalance between two processes, increased production of methemoglobin or decreased reduction.
Defense mechanisms against oxidation of hemoglobin:
- Oxidation is prevented by a variety of non-enzymatic and enzymatic systems in RBCs.
- Since RBCs are unable to synthesize new protein, antioxidant enzymes degrade with time. As a result, older cells are more susceptible to oxidation.
- The red cell has a significant reducing capacity by:
  - Reducing oxidant compounds such as H₂0₂ before they react with hemoglobin to form methemoglobin:
    - Reduced glutathione (GSH):
      - In the hexose monophosphate shunt, the enzymatic conversion of glucose-6-phosphate (G-6-P) to 6-phosphogluconate (6-PG) by glucose-6-phosphate dehydrogenase (G6PD) results in production of NADPH.
      - NADPH, in turn, can be used to reduce oxidized glutathione (GSSG) to reduced glutathione (GSH).
      - In the presence of the enzyme glutathione peroxidase, GSH can combine with oxidant compounds capable of changing hemoglobin to the met-form, especially H₂0₂.
      - This pathway plays a minor role in regulating levels of methemoglobinemia; G6PD deficiency is not normally associated with methemoglobinemia.
    - Catalase – reduces H₂0₂ to H₂0 and 0₂.
    - Ascorbic acid
    - Glutathione (without glutathione peroxidase)
    - Sulfhydryl compounds
    - Flavin
    - Tetrahydrobiopterin
    - Cysteamine
    - Reduced cysteine on protein molecules
  - Reducing methemoglobin back to normal hemoglobin as soon as it is formed:
    - Reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (Cyb5R):
      - Methemoglobin levels are normally kept low (approximately 1 percent) by the RBC enzyme cytochrome b5 reductase (Cyb5R),³ which reduces (adds an electron to) the heme in hemoglobin, converting it back to the ferrous (Fe²⁺) state.⁴
      - Glycolytic intermediates that produce reduced nicotinamide adenine dinucleotide (NADH) serve as the original electron donors.
      - Electrons travel from glycolytic intermediates to NADH to cytochrome-b5 reductase to cytochrome b5 and finally to MHb.
      - Cyb5R is a cytosolic cytochrome, and the only physiologically important pathway for methemoglobin reduction. Accounts for approximately 99% of daily MHb reduction.
      - Congenital deficiency of Cyb5R is the principal form of inherited methemoglobinemia.
      - The gene localized to chromosome 22q13qter, and a number of mutations have been identified.
      - Certain acute/toxic exposures can overwhelm the ability of Cyb5R to reduce methemoglobin. Heterozygotes for pathogenic variants in Cyb5R may be at increased risk for methemoglobinemia following these exposures.

- - - NADPH-dependent methemoglobin reductase (NADPH-MetHb reductase):⁵
      - In this pathway, electrons are derived from NADPH that is generated by glucose-6-phosphate dehydrogenase (G6PD) in the hexose monophosphate (pentose phosphate) shunt.
      - However, there is normally no electron acceptor present in RBCs to interact with NADPH.
      - As a result, the pathway to convert NADPH to NADP is only activated by extrinsic electron acceptors such as methylene blue (MB), ascorbic acid and riboflavin.⁶
      - This is the mechanism by which MB therapy reverses methemoglobinemia in severely affected individuals.⁷
      - The requirement of G6PD for generating NADPH explains why MB therapy is ineffective in individuals with G6PD deficiency.
      - The primary function of this reductase is probably to metabolize oxidant xenobiotics and not MHb.

Formation of methemoglobin has two consequences on oxygen binding/delivery:
- The ferric iron in methemoglobin does not bind oxygen. Therefore, methemoglobin does cannot transport oxygen.
- The ferric heme in the hemoglobin tetramer also causes the remaining normal ferrous hemes within the same tetrameric hemoglobin molecule to have increased O₂ affinity. resulting in shift of the oxygen dissociation curve to the left.
As a result of these two changes (inability to bind oxygen and left-shifting of the oxygen-hemoglobin curve), methemoglobin causes functional anemia.

Causes

Hereditary:
- Enzyme deficiency:
  - Hereditary methemoglobinemia is a rare recessively inherited disorder due to deficiency of reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (Cyb5R) due to pathogenic variants in the CYB5R3 gene.
  - Homozygotes:
    - Methemoglobin levels of more than 1.5 g/dl (about 10%).
    - Present with cyanosis.
  - Heterozygotes:
    - Have approximately 50% enzyme activity but without cyanosis.
    - In response to oxidant drugs, severe cyanosis may develop because of methemoglobinemia.
  - Cyb5R deficiency has been classified into:
    - Type I (erythrocyte)
      - Most common type.
      - Reductase deficiency restricted to RBCs.
      - Caused by variants in RBC-specific isoform CYB5R3 that affect the stability of soluble form, which is easily degraded and cannot be readily replenished because mature RBCs lack the ability to synthesize new proteins.
      - Most of these variants affect the stability of the Cyb5R enzyme, not its catalytic activity.
      - Accounts for approximately 90 percent of CYB5R3 disease variants.
      - Usually present with no clinical manifestations other than cyanosis.
    - Type II (generalized)
      - Rare, severe form of the disease.⁸
      - Causes methemoglobinemia as well as developmental delay and neurologic manifestations related to loss of Cyb5R function in non-erythroid cell types.
      - This accounts for approximately 10 percent of CYB5R3 disease variants.
      - These variants affect the catalytic activity or decrease the transcription of the Cyb5R enzyme in RBCs (soluble form) and other cells (membrane-bound form).
- Hemoglobin M:
  - Hemoglobin with an amino acid substitution in the alpha or beta chains resulting in a complex in which the iron is held in the methemoglobin form.⁹
  - This mutation allows for the stabilization of iron in the ferric [Fe3+] state.
  - Transmission is autosomal dominant (homozygous state would presumably be incompatible with life).
  - At least 12 Hb M disease variants have been described, including:
    - Boston
    - Fort Ripley
    - Hyde Park
    - Iwate
    - Kankakee
    - Osaka
    - Saskatoon
  - Individuals with Hb M disease have chronic methemoglobinemia and may have cyanosis but are usually otherwise asymptomatic.
  - Methemoglobin concentrations usually do not exceed 25% to 30%.
  - Methylene blue and ascorbic acid have no effect on the reduction of methemoglobin in this disease.

Acquired:¹⁰
- Exposure to an oxidizing chemical:
  - Most common cause of methemoglobinemia.
  - Common agents include:
    - Aniline
    - Benzocaine
    - Dapsone
    - Phenazopyridine (pyridium)
    - Nitrites
    - Nitrates
    - Naphthalene
    - Chlorates
  - Some agents directly oxidize hemoglobin, while others exert an indirect effect.
  - Many drugs that produce MHb are not themselves the causative agents. Instead these drugs are metabolized to an oxidative free radical.

ANNALS OF EMERGENCY MEDICINE 34:5 NOVEMBER 1999

- - Systematic review of 148 publications representing 159 cases of acquired MHb:
    - Cocaine-based anesthetics and dapsone accounted for 45/87 (52 %)

Transfusion and Apheresis Science 61 (2022) 103299

- Idiopathic:
  - Second most common cause of MHb.
  - Often related to systemic acidosis
- Dietary
  - Dietary nitrites in well water.
  - Occurs in very young infants.

Clinical Presentation

The diagnosis of methemoglobinemia should be suspected in case of unexplained cyanosis and hypoxemia that does not resolve with supplemental oxygen. Concern for the diagnosis of methemoglobinemia is further increased by any of the following:¹¹
- Prior history of methemoglobinemia
- Positive family history for methemoglobinemia
- Positive genetic testing for a disease variant in one of the methemoglobinemia genes
- Known exposure to methemoglobinemia-inducing substance
Because methemoglobin cannot carry oxygen and because the oxygen dissociation curve is shifted to the left, most symptoms (except for cyanosis) related to the blood’s decreased oxygen-carrying capacity.
Most patients with type I congenital methemoglobinemia are asymptomatic except for cyanosis (only of cosmetic significance), but some forms may have serious morbidity (some patients might experience headache, exertional dyspnea, and fatigue).
Children with type II CYB5R deficiency are at risk for microcephaly and experience developmental delay, other neurologic abnormalities such as seizures, and failure to thrive.
Acquired methemoglobinemia can be severe or even fatal, depending on the proportion of methemoglobin.
Severity of presentation depends on:
- Percentage of MetHb
- Rate of increase in MetHb levels
- The intrinsic ability of the patient to prevent or reduce MetHb
- The patient’s underlying functional status
Symptoms depend on the level of MHb and its rapidity of formation, and include:¹²
- Dyspnea
- Anxiety
- Headache
- Fatigue
- Weakness
- CNS depression
- Dizziness
- Cyanosis
- Seizures
Signs include:
- Cyanosis:
  - As little as 1.5 g methemoglobin in 100 ml blood (1.5 g%) can produce clinically recognizable cyanosis.
  - In patients without anemia, cyanosis first appears at methemoglobin concentrations of about 15%.
  - As the cyanosis increases, the lips and mucous membranes develop a brownish cast unlike the bluish discoloration seen in oxygen desaturation.¹³
  - Cyanosis is not specific for methemoglobinemia; it can be caused by high levels of deoxygenated hemoglobin (>4 g/dL), sulfhemoglobin (>0.5 g/dL), or methemoglobin (>1.5 g/dL).
- Seizures
- Coma
The lethal human concentration of MetHb is in the range of 70% to 80%.

ANNALS OF EMERGENCY MEDICINE 34:5 NOVEMBER 1999

Diagnosis

Overview:
- Methemoglobin is expressed as a concentration or a percentage and this is further described under evaluation. Percentage of methemoglobin is calculated by dividing the concentration of methemoglobin by the concentration of total hemoglobin.
- The normal level of methemoglobin is 1.0–1.5%.
- The diagnosis is confirmed when the MetHb level is higher than 5%.
- The finding of an SaO₂ of approximately 85% and failure of the SaO₂ to improve with administration of supplemental oxygen (refractory hypoxemia) are clues that may raise the suspicion for methemoglobinemia.¹⁴
- Patients typically have normal amounts of dissolved O2 in their blood (pO₂) despite the low O2 saturation on pulse oximetry.
Pulse oximetry:
- Standard pulse oximeters measure oxygen saturation by passing 2 wavelengths of light through the tissues into a detector. By measuring the changes in absorbance at each wavelength, they can identify the absorbance caused by hemoglobin bound to molecular oxygen in the pulsing arterial blood.
- Routine pulse oximetry cannot detect methemoglobin.¹⁵
- Methemoglobin has a peak absorbance at 631 nm. At this wavelength, the absorption of oxyhemoglobin is negligible.
- The pulse oximeter measures light absorbance at only 2 wavelengths:
  - 660 nm
  - 940 nm
- Both oxyhemoglobin and deoxyhemoglobin absorb light at 660 and 940 nm; it is the ratio of the absorbance at the 2 wavelengths from which a pulse oximeter determines oxygen saturation:
  - A ratio of absorbance (660 nm/940 nm) of 0.43 corresponds to 100% oxygen saturation.
  - A ratio of 3.4 corresponds to 0% oxygen saturation.
  - In the absence of a dyshemoglobin, an absorbance ratio of 1.0 corresponds to an oxygen saturation of approximately 85%.
- MHb absorbs light almost equally at both 660 and 940 nm:
  - In the presence of 100% methemoglobin, therefore, the absorbance ratio of light at 660 nm over 940 nm is about 1.0 and the pulse oximeter reading is approximately 85% oxygen saturation.
  - At lower levels of MHb, oxygen saturation measured by pulse oximetry is slightly lower. However, when MHb levels reach 30% to 35%, the light absorbance ratio reaches a plateau, and the pulse oximeter reading becomes stable in the 82% to 86% range independent of actual MHb levels.¹⁶
  - The pulse oximeter detects significant levels of MHb as mild to moderate oxygen desaturation; unfortunately, it cannot be used to determine the actual percentage of MHb in the blood.
Noninvasive co-oximetry:¹⁷
- Co-oximeter is also a simplified spectrophotometer, but unlike a pulse oximeter, it measures light absorbance at 4 different wavelengths (multiple wavelength oximetry.
- A specialized modification of standard pulse oximetry in which absorbance is also measured at a fixed wavelength of 630 nm.
- These wavelengths correspond to specific absorbance characteristics of:
  - Deoxyhemoglobin
  - Oxyhemoglobin
  - Carboxyhemoglobin
  - Methemoglobin
- A peak absorbance of light at 630 nm is used to characterize MHb.
Arterial blood gas testing:
- When CO-oximetry is not available, elevated MetHb concentration determined with arterial blood gas testing.
- The vast majority of blood gas analyzers in use in the United States are able to detect methemoglobin by its absorbance spectrum at 631 nm.
- The method of assaying methemoglobin on a blood gas is measurement of the absorption spectrum using co-oximetry.
- An arterial or venous sample can be used.
- Generally, the result is expressed as a percentage of methemoglobin.
- Some instruments interpret all readings in the 630 nm range as methemoglobin; thus, false positives may occur in the presence of other pigments, including sulfhemoglobin, MB, and certain drugs.
- Because patients with methemoglobinemia have normal amounts of dissolved O₂ in their blood, they have normal PaO₂ despite the low O₂ saturation on pulse oximetry, and the calculated O₂ saturation of Hb seems to be falsely elevated.
- SaO₂ calculations are falsely normal due to the assumption that all hemoglobin is either oxyhemoglobin or deoxyhemoglobin. The difference between the depressed SpO₂ measurement and the falsely normal SaO₂ calculation is known as the “saturation gap.”
Direct measurement of MetHb:
- Methemoglobin can be quantified using a reaction with cyanide (the Evelyn-Malloy method).
- Cyanide binds to the positively charged methemoglobin, eliminating its peak absorbance at 630 to 635 nm.
- The subsequent addition of ferricyanide converts the entire specimen to cyanmethemoglobin for measurement of the total hemoglobin concentration.
Chocolate-colored blood:
- The primary diagnostic consideration in a patient with cyanosis is to differentiate deoxyhemoglobin from MHb. Blood containing high concentrations of MHb appears chocolate brown as opposed to the dark red/violet of deoxygenated blood. Venous blood samples in methemoglobinemia are brown, typically at levels of 10% or higher.
Other assays:
- CBC, peripheral smear, Heinz body preparation and hemolytic indices:
  - Hemolytic anemia may follow drug-induced methemoglobinemia, especially with exposure to dapsone, sulfasalazine, or phenacetin.
  - The anemia is characterized by Heinz bodies (precipitated hemoglobin or globin subunits due to denaturation of hemoglobin in erythrocytes) and fragmented red blood cells.
- Hemoglobin electrophoresis and DNA sequencing of the globin chain gene can be used to identify hemoglobin M.
- Specific enzyme assays (nicotinamide adenine dinucleotide [NADH]–dependent reductase, cytochrome b5 reductase) may be determined to diagnose inherited cases.

Treatment

Acquired methemoglobinemia with methemoglobin levels above 30 percent (or lower if symptomatic from hypoxia) is a medical emergency that requires prompt clinical suspicion and rapid evaluation and treatment.
Remove the inciting oxidant stressor/precipitating agent.
Provide supportive care by stabilizing the airway, breathing, and circulation.
If MetHb level is at least 30% in asymptomatic patients and at least 20% in symptomatic patients, consider infusion of either:
- Methylene blue (MB):
  - Treatment of choice for acute toxic methemoglobinemia with methemoglobin levels >30 percent or or those who are symptomatic with methemoglobin levels between 20 and 30 percent
  - Acts faster than ascorbic acid
  - Effectiveness of MB is also better established
  - Should not be used in patients with G6PD deficiency
    - MB is not effective for treatment of methemoglobinemia in these individuals
    - MB can precipitate hemolysis in individuals with G6PD deficiency
- Ascorbic acid:
  - in individuals with G6PD deficiency (in whom MB can precipitate hemolysis)
  - has reducing potential
  - an be used to treat severe or symptomatic methemoglobinemia when MB is unavailable or contraindicated
Exchange transfusion and hyperbaric oxygen have been reported to be beneficial in severe disease according to case reports, but there are no controlled trials of these approaches

Dapsone-associated methemoglobinemia

Dapsone is a sulfone antibiotic that decreases folate synthesis by inhibiting the enzyme dihydropteroate synthetase.
Commonly used in:
- Prevention of Pneumocystis jiroveci pneumonitis (PJP) in both cancer and HIV patients (considered the best alternative treatment for PCP prophylaxis in those who cannot tolerate trimethoprim-sulfamethoxazole (TMP-SMX).
- Treatment of leprosy and dermatitis herpetiformis.
Dapsone reaches peak concentration 2–6 hours after ingestion, with a half-life of 20–30 hours.
In the liver, it undergoes:
- Reversible acetylation by N-acetyltransferase (NAT2) to monoacetyl dapsone.
- Cytochrome P450-mediated N-hydroxylation to the toxic metabolite dapsone hydroxylamine:
  - The hydroxylamine metabolites are retained in the circulation for a long period as they undergo
    enterohepatic recirculation.
  - Quickly taken up by the erythrocytes where they (in particular, dapsone monohydroxylamine) are primarily responsible for the hematologic adverse effects of methemoglobinemia and hemolysis.
Hemolytic anemia or methemoglobinemia develops in about 4%–13% of patients who are given dapsone.
1 study involving 16 patients who underwent solid organ transplant and were receiving prophylactic dapsone
reported that methemoglobinemia presented at a median 48 (range 7–809) days after treatment began.
A common cause of acquired methemoglobinemia. In a series of 138 cases of methemoglobinemia, dapsone accounted for 42 percent, with a mean methemoglobin level of 7.6 percent (range 2 to 34 percent).
In a series of 167 children with hematologic malignancy or aplastic anemia receiving dapsone for Pneumocystis (PCP) prophylaxis, 32 (19 percent) developed methemoglobinemia (median level: 9 percent, range 3.5 to 22 percent). The child with 22 percent methemoglobin also had G6PD deficiency.
Symptoms have occurred at MetHb levels ranging from 1.9% to 26.8%, mostly at the 100-mg dose.
Cimetidine, an H2 receptor antagonist, is an alternative treatment for dapsone-induced methemoglobinemia. Cimetidine competes with dapsone for cytochrome P450 enzymes with decreased production of the toxic hydroxylamine metabolite, which decreases methemoglobin.

Rasburicase-associated methemoglobinemia

Rasburicase is a recombinant urate oxidase enzyme that:
- Lowers uric acid levels by converting it into allantoin, a water-soluble compound that can be excreted in the urine.
- Results in production of hydrogen peroxide (H₂O₂), a potent oxidant that can also generate methemoglobin.
Approval/indications
- Rasburicase was approved by the Food and Drug Administration (FDA):
  - in 2002 for the treatment of hyperuricemia in pediatric patients at risk of developing tumor lysis syndrome (TLS).
  - in 2009 for adult patients with TLS.
- Recommended by the American Society of Clinical Oncology in the Guidelines for the management of pediatric and adult tumor lysis syndrome since 2008.
FDA-approved dose is 0.2 mg/kg for up to 5 days.
Glucose-6-phosphate dehydrogenase (G6PD) deficiency:
- G6PD-deficient individuals are more susceptible to methemoglobinemia from rasburicase.
- Rasburicase is contraindicated in G6PD-deficient patients to prevent hemolysis and methemoglobinemia.
- The package insert recommends screening high-risk patients, such as those of African or Mediterranean ancestry, for G6PD deficiency before administering the medication.
Pre-marketing studies revealed an incidence of less than 1% of hemolysis and methemoglobinemia among 703 patients.¹⁸
In a multicenter clinical trial involving 1069 patients (682 children and 387 adults), four cases of hemolytic anemia were reported (subsequently, G6PD deficiency was diagnosed in one patient), and three patients were reported to have methemoglobinemia.
Systematic review of reports of rasburicase‑induced hemolytic anemia and methemoglobinemia:
- 40 studies identified reporting 43 cases of methemoglobinemia and hemolytic anemia induced by rasburicase.
- Median age of the patients was 50 years old.
- Males represented 81.4%.
- Black race 71.4%.
- 60.5% of the patients received rasburicase for TLS treatment. 30.2% for TLS prevention.
- The median time to symptom onset was 24 h (2–72 h).
- The median methemoglobinemia peak was 10.0% (range 4.7–23.1%).
- The median time to methemoglobinemia peak was 24 h (6–72 h).
- The median baseline hemoglobin (prior to receiving rasburicase) was 11.6 g/dL (7–16.3).
- The median hemoglobin nadir was 6.10 g/dL.
- The median lowest oxygen saturation was 80% (50–90%).
- G6PD status:
  - Deficient in 72.1%
  - Normal in 9.3%
  - Not tested in 18.6%
- Treatment included:
  - Packed RBC transfusion
  - Methylene blue in 27.9% of patients
  - Ascorbic acid n 20.9% of patients.

1 / 0