L-arginine, a nitric oxide precursor, reduces dapsone-induced methemoglobinemia in rats

Dapsone use is frequently associated to hematological side effects such as methemoglobinemia and hemolytic anemia, which are related to N-hydroxylation mediated by the P450 enzyme system. The aim of the present study was to evaluate the influence of L-arginine supplementation, a precursor for the synthesis of nitric oxide, as single or multiple dose regimens on dapsone-induced methemoglobinemia. Male Wistar rats were treated with L-arginine at 5, 15, 30, 60 and 180 mg/kg doses (p.o., gavage) in single or multiple dose regimens 2 hours prior to dapsone administration (40 mg/kg, i.p.). The effect of the nitric oxide synthase inhibitor L-NAME was investigated by treatment with multiple doses of 30 mg/kg (p.o., gavage) 2 hours before dapsone administration. Blood samples were collected 2 hours after dapsone administration. Erythrocytic methemoglobin levels were assayed by spectrophotometry. The results showed that multiple dose supplementations with 5 and 15 mg/kg L-arginine reduced dapsone-induced methemoglobin levels. This effect is mediated by nitric oxide formation, since the reduction in methemoglobin levels by L-arginine is blocked by simultaneous administration with L-NAME, a nitric oxide synthase inhibitor.

O uso da dapsona é frequentemente associado a efeitos adversos hematológicos, como a metemoglobinemia e anemia hemolítica, ambos relacionados com a N-hidroxilação mediada pelo sistema P450. O objetivo do estudo foi avaliar a influência da suplementação de L-arginina, um precursor da síntese de óxido nítrico, administrado em regime de dose única ou múltipla na metemoglobinemia induzida pela dapsona. Ratos machos Wistar foram tratados com L-arginina (po, gavagem) em dose única ou múltipla de 5, 15, 30, 60 e 180 mg/kg 2 horas antes da administração de dapsona (40 mg/kg, ip). O efeito do L-NAME, um inibidor de óxido nítrico sintase (NOS), foi avaliado através do tratamento com doses múltiplas de 30 mg/kg. Amostras de sangue foram coletadas duas horas após a administração de dapsona. A concentração de metemoglobina eritrocitária foi analisada por espectrofotometria. Os resultados mostraram que a suplementação em dose múltipla de 5 e 15 mg/kg de L-arginina reduziu os níveis de metemoglobina induzida pela dapsona. Este efeito é mediado pela formação de óxido nítrico, uma vez que a redução nos níveis de metemoglobina pela L-arginina é bloqueada pela administração simultânea de L-NAME, um inibidor da óxido nítrico sintase.

Methemoglobinemia: from diagnosis to treatment.

BACKGROUND AND OBJECTIVES: Methemoglobin is the oxidized form of hemoglobin, which does not bind oxygen and increases the affinity of oxygen for the partially oxidized portion of hemoglobin. Increased levels of methemoglobin in the blood are secondary to congenital changes and exposure to several chemical agents, resulting in a disorder with several differential diagnoses, which it can lead to death if it is not treated. The objective of this report was to review this subject, emphasizing relevant information for the clinical management of patients with methemoglobinemia. CONTENTS: When the concentration of methemoglobin in the blood is above 1.5%, the patient develops cyanosis, the main characteristic of this disorder. The color of the arterial blood changes to dark brown with normal PaO2. One should suspect the diagnosis in patients with cyanosis and low saturation (SpO2) without significant cardiopulmonary dysfunction. Co-oximetry is the gold standard and defines the diagnosis. Treatment should be based on whether the syndrome is acute or chronic (etiology) and on the severity of symptoms. Blood levels of methemoglobin are important, especially in acute cases. Basic treatment includes removal of the agent responsible for the disorder, administration of oxygen, and observation. Severe cases should be treated with the specific antidote, methylene blue, which is not effective in some situations. CONCLUSIONS: Methemoglobinemia is a potentially severe disorder, whose diagnosis depends on a high degree of suspicion. In general, anesthesiologists are the first to detect the problem in the preoperative period and should lead the treatment.

Heterogeneity of the molecular biology of methemoglobinemia: a study of eight consecutive patients.

Congenital methemoglobinemia can be caused by mutations involving five different genes. We studied the etiology and molecular biology of eight consecutive patients with methemoglobinemia. Four had b5R mutations; two were novel. A novel intronic mutation caused markedly reduced mRNA resulting in type II methemoglobinemia. Three patients had acquired methemoglobinemia without any b5R mutations.

Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001.

Seven new mutations in the nicotinamide adenine dinucleotide reduced-cytochrome b(5) reductase gene leading to methemoglobinemia type I.

Cytochrome b(5) reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.

A compound heterozygote in the NADH-cytochrome b5 reductase gene from a Chinese patient with hereditary methemoglobinemia type I.

To elucidate the mutation in the nicotinamide adenine dinucleotide-cytochrome b5 reductase (b5R) gene from a Chinese patient with hereditary methemoglobinemia type I, we analyzed the coding sequences of b5R cDNA from the patient and from normal subjects by direct sequencing the reverse transcriptase-polymerase chain reaction (RT-PCR) products. The PCR-amplified genomic DNA fragments of the b5R gene from the patient, his mother, and normal controls were analyzed by restriction enzymes MspI and RsaI. A compound heterozygote Arg57Gln (CGG-->CAG)/Cys203Tyr (TGC-->TAC) was found in the b5R gene from the patient, and a CGG-->CAG mutant allele occurred in a chromosome inherited from his mother, while TGC-->TAC occurred in a chromosome inherited from his father. In this report, we discuss a compound heterozygote first observed in the b5R gene from a patient with hereditary methemoglobinemia type I.

Dapsone toxicity: some current perspectives.

1. Dapsone is a potent anti-inflammatory and anti-parasitic compound, which is metabolised by cytochrome P-450 to hydroxylamines, which in turn cause methaemoglobinaemia and haemolysis. However, during the process of methaemoglobin formation, erythrocytes are capable of detoxifying the hydroxylamine to the parent drug, which may either reach the tissues to exert a therapeutic effect or return to the liver and be re-oxidised in a form of systemic cycling. This glutathione-dependent effect, combined with the un-ionised state of the drug at physiological pH, may contribute to its efficacy. 2. Paradoxically, other aspects of the glutathione-dependent cycling of the hydroxylamine metabolite may contribute to the major adverse reaction of the drug, agranulocytosis. Erythrocytes exposed to the metabolite and repeatedly washed may still release the hydroxylamine in sufficient concentration to kill mononuclear leucocytes in vitro. Thus, erythrocytes may be a conduit for the hydroxylamine to reach the bone marrow to covalently bind to granulocyte precursors, which may trigger an immune response in certain individuals and may lead to the potentially fatal eradication of granulocytes from the circulation. 3. Attempts to increase patient tolerance to dapsone have been most successful using a metabolic inhibitor to reduce hepatic oxidation of the drug to the hydroxylamine. Methaemoglobin formation in the presence of cimetidine was maintained at 30% below control levels for almost 3 mo, and patients' reported side effects such as headache and lethargy were significantly reduced. 4. As clinical application of new and safer dapsone analogues is years away, the use of cimetidine provides an immediate route to increasing patient compliance during dapsone therapy, especially in those maintained on dapsone dosages in excess of 200 mg/day.

Four new mutations in the NADH-cytochrome b5 reductase gene from patients with recessive congenital methemoglobinemia type II.

Recessive congenital methemoglobinemia (RCM) due to NADH-cytochrome b5 reductase (cytb5r) deficiency leads to two different types of diseases. In the type I form, cyanosis is the only symptom, and the soluble enzyme is defective in red blood cells. In the type II form, cyanosis is associated with severe mental retardation and neurologic impairment; the enzymatic defect is systemic, involving both soluble and membrane-bound isoforms. We characterized mutations responsible for cytb5r deficiency in three unrelated patients with severe RCM type II. The first patient presented a homozygous exon 5 skipping. The only mutation detected was a homozygous G to C transversion at position +8, downstream from the 5' splice site of exon 5. We suggest that this unusual mutation might be responsible for the abnormal splicing of the primary transcripts, resulting in frameshift with premature STOP codon. The second mutation found corresponds to a homozygous C to T transition changing the Arg-218 codon to a premature STOP codon in exon 8. The third case was a compound heterozygote, carrying two different mutant alleles in the cyb5r gene. One allele presented a missense mutation with replacement of Cys-203 (TGC) by Arg (CGC) in exon 7. The second allele carried a 3-bp deletion (TGA) of nucleotides 815 to 817, modifying two contiguous codons in exon 9 of the cDNA with loss of Met-272. These results confirm the genetic polymorphism of cytb5r gene mutations identified in RCM type II, as observed for the mutations described in the RCM type I, and shed light on the molecular bases of the two different diseases associated with cytb5r deficiency.

Concise review: methemoglobinemia.

The ferrous iron of hemoglobin is exposed continuously to high concentrations of oxygen and, thereby, is oxidized slowly to methemoglobin, a protein unable to carry oxygen. To restore hemoglobin function, methemoglobin (ferrihemoglobin) must be reduced to hemoglobin (ferrohemoglobin). Under physiological conditions, methemoglobin reduction is accomplished mainly by red cell NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) so efficiently that there is insignificant amounts of methemoglobin in the circulating blood. However, should methemoglobin formation be increased--e.g., due to the presence of oxidant drugs, or an abnormal methemoglobin not amenable to reduction (hemoglobin M), or a deficiency in red cell cytochrome b5 reductase--methemoglobinemia will result. Most methemoglobinemias have no adverse clinical consequences and need not be treated. Under certain conditions, such as exposure to large amounts of oxidant or in young infants, rapid treatment is necessary. In hereditary cytochrome b5 deficiency, treatment is often directed at improving the poor cosmetic effect of persistent cyanosis with the minimum amount of drugs to give satisfactory clinical results.

[Acute poisoning caused by methemoglobinemia-inducing substances. 6 cases]. / Intossicazione acuta da sostanze metaemoglobinizzanti. Sei casi.

Six patients were admitted to intensive care in Lodi Hospital with acute poisoning by substances causing methemoglobinaemia. They were treated with antidotes, purifying substances, blood exchange and symptomatic intensive care. The patients were observed 1-3 hours after ingestion of the poison. Three patients (50%) died. This is in line with reports in the literature given the severity of the poisoning. Three patients recovered completely and without sequelae. The importance of early diagnosis with particular attention to anamnestic data and the peculiar clinical symptoms of this syndrome is emphasised as essential for the prompt treatment required. A new classification for the causes of methemoglobinaemia based on pathogenetic criteria is proposed.

Enzymopenic hereditary methemoglobinemia: a clinical/biochemical classification.

Recessively inherited NADH-cytochrome B5 reductase deficiency, when present in the homozygous or doubly heterozygous form, is manifested by two different clinical presentations, depending on the nature and cellular distribution of the mutant enzyme. The observations supporting a clinical and biochemical classification of enzymopenic hereditary methemoglobinemia are summarized. Type I, with deficiency demonstrable only in the erythrocytes, presents as uncomplicated, benign methemoglobinemia. Type II, generalized cytochrome B5 deficiency demonstrable in all of the tissues that have been examined, is accompanied by severe, lethal, progressive neurological disability, in addition to methemoglobinemia. Type III deficiency is limited to hematopoietic cells and resembles Type I clinically. Type IV, also clinically like Type I, is associated with deficiency of the cofactor, cytochrome B5. Except for Type IV, the different types appear to be the result of mutations in paired alleles of a gene on chromosome 22 that affect the catalytic activity or stability of the cytochrome B5 reductase.