A patient with both methemoglobinemia and G6PD deficiency: A therapeutic conundrum
Authors: N. Scott Reading, José A. Ruiz‐Bonilla, Robert D. Christensen, William Cáceres‐Perkins, Josef T. Prchal
Journal: American Journal of Hematology (2017)

A 42-year-old Puerto Rican male was referred to the VA hospital with a medical history of hypertension, hyperlipidemia, post-traumatic stress disorder, depression, intermittent cyanosis, shortness of breath, headaches, myalgia, and a recent diagnosis of methemoglobinemia. Our initial evaluation showed a well-nourished non-cyanotic male with stable vital signs and normal physical exam. His laboratory blood results showed a methemoglobin level of 8.0% (reference range <0.8%), cytochrome B5 reductase (CYB5R) enzyme activity level of 3.1 IU/gHb (reference range 8.2-19.2), hemoglobin (Hb) concentration of 16.8 g/dL, WBC count of 6600/µL, platelet count of 231,000/µL; arterial blood gases with 97% oxygen saturation, pH 7.39, PO2 91.7, PCO2 44.9, HCO3 26.6. Hemoglobin electrophoresis revealed no abnormalities. His glucose-6-phosphate dehydrogenase (G6PD) enzyme activity level was low at 0.9 IU/gHb (reference range 4.6-13.5). His prescription profile was lisinopril, clonazepam, simvastatin, and niacin and no recent exposure to dapsone, fava beans, antimalarial drugs, local anesthetics, or sulfa drugs. The patient was born to non-consanguineous parents and was a non-smoker, non-alcoholic, and non-illicit drug user. Methemoglobinemia is a rare disorder and its initial symptoms can be vague and particularly when methemoglobin levels are low the condition can easily be misdiagnosed or go unrecognized. In this case the diagnosis of methemoglobinemia remained unrecognized for 4 years. A review of his medical records reflects the patient's journey in receiving the current diagnosis. Initial symptoms of shortness of breath and fatigue upon exertion arose while the patient was deployed in military service as a fire fighter in the Middle East. Patient was taking candesartan for hypertension. Patient reported exposure to insect repellant (DEET), insecticides, JP8 fuels, dust, and smoke from burning trash and feces during this period. An initial diagnosis of hypoxemia was made by pulse oxymetry at a level 2 expeditionary (military) medical support facility and supplemental oxygen was administered but later discontinued due to lack of effective response. Patient was medically released from active duty (REFRAD) due to symptoms attributed to anxiety disorder. The patient self-reported good health aside from hypertension prior to entering active military service. Although chest pain, dizziness, and shortness of breath are reported side effects of candesartan the patient reported that it was well tolerated prior to the deployment. As clinical symptoms began shortly after arriving in country and due to his responsibilities in the military an environmental aspect to his symptoms cannot be ruled out. Anti-malarial prophylactics were routinely provided to military personnel. However, we were unable to document whether or not it was administered to the patient. Living conditions while deployed are particularly harsh (meager housing, separation from family, poor nutrition, long work hours, hazardous environment) and may have contributed to increased levels of stress and anxiety. A combination of medication and environmental elements may have aggravated an underlying, unrecognized condition in the patient. Hypoxia, properly diagnosed through pulse oximetry, is a common symptom of a variety of disorders often manifested by cyanosis if deoxygenated hemoglobin levels exceed 5 g/dL. In such an austere environment anxiety manifested by shortness of breath and fatigue upon exertion would not be uncommon. There is always a potential for complications when multiple medications are taken by patients, particularly in a less structured environment found in a wartime theater. Although G6PD deficiency testing is conducted prior to administration of anti-malarial prophylactics, it is possible that positive screening (no such positive or negative documentation could be obtained) might have prevented the use of these drugs. The intermittent nature of his recurring symptoms and the complexity of his medicinal regime after returning home (treatment for anxiety, PTSD, general myalgia, etc.) may have increased the complications of his diagnosis. Cardiopulmonary disorders are a common underlying cause of cyanosis/hypoxemia and accompanying general malaise.1 Thus, studies of blood oxidation and arterial-venous shunting should be undertaken. Patient's symptoms had progressed to include headaches and generalized myalgia. Upon return to the United States his case was evaluated by his primary care physician, and later at pain and PTSD clinics without major improvement. Patient's prescription regime was altered to include clonazepam, simvastatin, niacin and lisinopril. Cardiopulmonary evaluation was conducted by chest CT scan, PFT, echocardiography which eliminated pulmonary etiology and shunt anomalies as the cause of his symptoms. However, a right bundle branch block was discovered during the course of these investigations but it was deemed noncontributory to his symptoms. The absence of cardiopulmonary disorders and continued hypoxemia required investigation into less common causes of hypoxemia. Environmental and rare effectors of hypoxia are low total oxygen pressure, carbon monoxide poisoning, hemoglobinapathies (low oxygen affinity hemoglobin variants), and inactivation of hemoglobin by genetic or exogenous means (congenital or acute toxic methemoglobinemia, sulfhemoglobinemia). The laboratory work up included normal blood counts and red cell indexes, vitamin B12 and folate levels, hepatitis serology, hemoglobin electrophoresis, and heavy metal analysis (Pb, Hg, As). Most tests were returned with normal values. However, an observation of dark-brown colored blood during arterial blood gas analysis led to the discovery of elevated methemoglobin levels (12%). The patient at this point was referred to VA hospital. Our initial analysis confirmed the diagnosis and red blood cell (RBC) enzyme assessments revealed decreased CYB5R and G6PD enzyme activity, see first paragraph. Molecular analysis of G6PD and CYB5R3 revealed a hemizygous double mutation c.376A > G/c.968T > C (G6PD Betica/A-) in the G6PD gene and a homozygous c.775C > T (p.R259Y) mutation of CYB5R3 gene. A family study revealed his father had a normal G6PD activity level of 8.5 IU/gHb and decreased CYB5R activity level of 4.8 IU/g; his mother had a decreased G6PD activity level of 1.5 IU/gHb and a normal CYB5R enzyme level of 9.1 IU/gHb; and both daughters were heterozygous for both CYB5R and G6PD enzyme deficiencies. The diagnostic workup prior to referral to the VA system resulted in the discovery of methemoglobinemia and subsequent evaluation revealed a patient with sustained high levels of methemoglobin. Methemoglobin is formed by the oxidation of ferrous (Fe2+) iron to the ferric (Fe3+) iron in hemoglobin. This results in blood that has a brownish/blue color that does not revert to red on exposure to oxygen.2 Although hemoglobin is oxidized in vivo, it is continuously reduced back to the ferrous state by the physiological CYB5R enzyme system. Under normal circumstances, methemoglobin levels are maintained at 0.8% or less.3 The most common form of congenital methemoglobinemia is an autosomal recessive disorder due to CYB5R deficiency, resulting in chronic cyanosis in most affected individuals. Cyanosis is present when total methemoglobin level exceeds 1.5 g/dL (i.e., 10% or greater when hemoglobin concentration is in the normal range 15 gm%) but cyanosis may not be apparent when the patient is anemic or in those individuals with rare CYB5R mutations whose methemoglobin level is <10%, such as was present in the propositus. There are two distinct CYB5R deficiency phenotypes. In type I deficiency, gene mutations result in an unstable protein that only affects erythrocyte enzyme levels, wherein most patients are asymptomatic with chronic cyanosis, but some report non-specific symptoms such as fatigue, dyspnea, and headache. Type I deficiency is common in several populations; Athabaskan Alaskans,4 Navajo Indians,5 Yakut population in Siberia,6, 7 and some Chinese populations7, 8 but has also been identified as an endemic disorder in Puerto Rico.9 Mutations causing type II deficiency severely decrease enzyme activity in all tissues resulting in a severe clinical phenotype with early mortality.1, 10 Although a genetic cause for methemoglobinemia was discovered, individuals with low CYB5R enzyme levels are still subject to exogenous factors overwhelming RBC CYB5R enzyme causing acute toxic methemobloinemia which can be life threatening. As many medications can give rise to acute toxic methemoglobinemia we reviewed his prescription history. Traditional treatment of methemoglobinemia is the administration of methylene blue to activate the dormant NADPH reduction pathway to reduce methemoglobin to ferrous hemoglobin. However, due to the presence of G6PD deficiency administration of methylene blue is contraindicated because it can act as both a reductant and an oxidant, causing acute hemolysis in G6PD deficient individuals. The alternative treatment using high levels of vitamin C and B2 utilizes an indirect reductive pathway to return methemoglobin back to ferrous hemoglobin. Review of medication history revealed multiple concurrent prescriptions such as lisinopril, clonazepam, simvastatin, and niacin. Two of the medications, niacin and lisinopril, were identified as possible negative effectors in methemoglobinemia or G6PD deficiency, although neither of them has been conclusively proven to have a negative interactions. As a precaution, niacin was discontinued but methemoglobin levels were not materially changed. Lisinopril, on the other, had had been tolerated by the patient for many years and with no documented hemolytic episode. As methylene blue was contraindicated, therapy with 250 mg bid ascorbic acid and 120 mg riboflavin was started. He continued to have less frequent dyspnea and headaches without cyanosis, and repeated studies showed methemoglobin levels that were decreased and maintained between 1.3% and 8.5%. The initial “hypoxic” cyanotic episode while in active service with poor response to oxygen supplementation could have been secondary to methemoglobinemia, that is, acute toxic methemoglobinemia. Treatment goals were established including an exercise program, weaning from oxygen therapy, and continuation of ascorbic acid and riboflavin. He has been educated to avoid medications and food that provoke hemolytic and/or methemoglobinemia exacerbation episodes for patients with CYB5R deficiency and G6PD deficiency. We report a Puerto Rican patient with autosomal recessive type I CYB5R deficiency and previously undiscovered G6PD deficiency, who presented with a transient cyanosis while in military service. Only after a rigorous biochemical and genetic workup were the underlying causes of his symptoms uncovered and proper management initiated. The patient was homozygous for a CYB5R p.259Arg > Trp mutation in addition to carrying a G6PD mutation (G6PD Betica/A-). Because of the concomitant G6PD and CYB5R deficiencies he could not be treated with methylene blue and an alternative approach was required. A regimen of ascorbic acid and riboflavin was prescribed, which ameliorated his sporadic cyanosis and symptoms. Our patient illustrates the diagnostic dilemma faced when considering methylene blue therapy for methemoglobinemia. As typical for subjects with most G6PD deficient variants, he had no previous history of hemolysis but was not clearly exposed to usual hemolysis initiating agents or methylene blue. Unlike most patients who carry two mutated CYB5R alleles, he did not have chronic cyanosis and thus his CYB5R deficiency was not diagnosed until his midlife. Fortunately, in this instance, we were aware of both G6PD and CYB5R deficiencies and thus initiated an alternative therapy for CYB5R deficiency other than methylene blue. However, for individuals with acute toxic methemoglobinemia, in whom previous testing for G6PD deficiency has not been performed, methylene blue administration would be detrimental. There is a paucity of information on epidemiology and phenotype of CYB5R deficiency in Puerto Rico; however, this and previous reports9, 10 suggest that CYB5R deficiency may be relatively moderate and thus present without chronic cyanosis increasing the likelihood of the condition not being diagnosed. Nevertheless, the patient would be expected to be prone to acute symptomatic exacerbation upon exposure of certain drugs and chemicals. Further, coexistent G6PD deficiency, both due to African and Mediterranean variants, are frequent in Puerto Rico. Thus, if proper diagnoses of his conditions during his first episode of cyanosis had been made and methylene blue administered it could have been associated with potentially serious consequences. Unfortunately, validated laboratory diagnosis of G6PD deficiency takes too long to be practical in deciding about methylene blue therapy in acute and emergency settings. To address this challenge, there have been attempts to develop commercially available rapid screening tests for G6PD deficiency. Recent studies have identified there are a number of short-comings associated with these tests: low sensitivity (<85%) for detecting individuals with moderate and severe deficiency, a high degree of false G6PD normal results in males and heterozygous females, and difficulty interpreting the readout signal.11, 12 We suggest a newly developed simple approach for detecting acute hemolysis, which could be utilized in urgent situations and in particular in areas where both these genetic variants coexist as in Puerto Rico. Hemolysis can be rapidly detected and monitored by the end-tidal breath carbon monoxide (ETCO) levels. ETCO levels quantitatively correlates with catabolism of heme and thus rapidly assesses significant hemolysis.13 A newly-developed instrument, CoSense® Monitor (Capnia Inc., Redwood City, CA), has been FDA approved for rapid detection of ETCO. The device is noninvasive, safe, compact and portable, provides results in minutes, and utilizes a single-use nasal cannula (Precision Sampling Set) for quantifying ETCO levels. The device has been used successfully for evaluation of neonatal hemolysis.14 In the event of acute toxic methemoglobinemia in a patient with an unknown G6PD enzyme status the use of this instrument with gradual dose escalation would assess the tolerance of a patient to methylene blue therapy. Importantly, it would be prudent to establish the minimal dose of methylene blue that can be administered to invoke a modest reduction response and not clinically challenging hemolysis. In an analogous clinical scenario, structured studies have been proposed15 and performed16 for assessing hemolytic potential of antimalarial agents. We suggest such a study should be designed in Puerto Rico prospectively testing the administration of small doses of methylene blue to patients with acute symptomatic cyanotic methemoglobinemia to determine the optimal dosage required to affect reduction of methemoglobin and prevent hemolysis in individuals with concomitant G6PD deficiency.