The objective of this work was to determine the methemoglobinemia and correlate with dapsone levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by high performance liquid chromatography and methemoglobinemia by spectrophotometry. The mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean methemoglobin levels in male was 3.09 µg/mL; 191%, and in female was 2.84 ± 1.67%. No correlations were seen between dapsone levels and methemoglobin in male and female patients. Our results demonstrated that the dosage of dapsone in leprosy treatment does not promote a significant methemoglobinemia

MARTINS, Ana de Nazaré Silva

RIVERA, Juan Gonzalo Bardález

SALGADO, Claudio Guedes

SILVA, Jorge Pereira da

VIEIRA, José Luiz Fernandes

The Brazilian Journal of Infectious Diseases

AbstractThe objective of this work was to determine the methemoglobinemia and correlate with dapsone levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by high performance liquid chromatography and methemoglobinemia by spectrophotometry. The mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean methemoglobin levels in male was 3.09 μg/mL; 191%, and in female was 2.84 ± 1.67%. No correlations were seen between dapsone levels and methemoglobin in male and female patients. Our results demonstrated that the dosage of dapsone in leprosy treatment does not promote a significant methemoglobinemia

Fernandes Vieira, Jose Luiz

Bardarez Riveira, Juan Gonzalo

Silva Martins, Ana de Nazaré

da Silva, Jorge Pereira

Salgado, Claudio Guedes

Elsevier - Publisher Connector 

Methemoglobinemia and dapsone levels in patients with leprosy 

319
BR
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Methemoglobinemia and dapsone levels 
in patients with leprosy
Authors
Jose Luiz Fernandes 
Vieira1
Juan Gonzalo Bardarez 
Riveira2
Ana de Nazaré Silva 
Martins2
Jorge Pereira da Silva1
Claudio Guedes Salgado1
1Toxicology Professor of 
Universidade Federal do 
Pará Pharmacy School, 
Belém, PA, Brazil.
2Undergraduated students 
of Universidade Federal do 
Pará, Belém, PA, Brazil.
Submitted on: 06/25/2009
Approved on: 10/02/2009
Correspondence to:
Jose Luiz Fernandes 
Vieira
Laboratório de 
Toxicologia,
Universidade Federal 
do Pará
Rua Augusto Corrêa, 1
Belém – PA – Brazil
Phone: +55-91-32017733
E-mail: jvieira@ufpa.br 
We declare no conﬂ ict of 
interest.
ABSTRACT
The objective of this work was to determine the methemoglobinemia and correlate with dapsone 
levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with 
laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by 
high performance liquid chromatography and methemoglobinemia by spectrophotometry. The 
mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean 
methemoglobin levels in male was 3.09 µg/mL; 191%, and in female was 2.84 ± 1.67%. No cor-
relations were seen between dapsone levels and methemoglobin in male and female patients. Our 
results demonstrated that the dosage of dapsone in leprosy treatment does not promote a signifi -
cant methemoglobinemia.
Keywords: methahemoglobinemia, dapsone, leprosy. 
[Braz J Infect Dis 2010;14(3):319-321]©Elsevier Editora Ltda.
Dapsone (4-4´-diaminodiphenylsulfone, DDS) 
is a chemical analogue of sulfapyridine, syn-
thesized in 1908. It is a part of the multidrug 
regimen recommended for the treatment of 
leprosy, but it is also used against a number 
of noninfectious inflammatory diseases.1 
DDS acts in the same way as sulfonamides, 
inhibiting the synthesis of dihydrofolic acid 
through competition with para-aminoben-
zoate for the active site of dihydropteroate 
synthetase. The anti-inflammatory action 
of DDS is associated with the interference 
in neutrophil chemotactic migration, β2 in-
tegrin (CD11b/ CD18)-mediated adherence 
of human neutrophils in vitro and with the 
activation or function of the G-protein (Gi 
type) that initiates the signal transduction 
cascade common to chemotactic stimuli.1,2
DDS is absorbed readily from the gas-
trointestinal tract with bioavailability of 
more than 86%. The peak plasma concen-
tration after 100 mg of oral DDS is attained 
between 2 to 8 hours. The drug shows lin-
ear pharmacokinetics within the therapeutic 
range and the time-course after oral admin-
istration fits a 2-compartment model. DDS 
is distributed for all organs including skin, 
liver, kidneys and erythrocytes. It is metab-
olized via acetylation or N-hydroxylation. 
The latter reaction yields the hydroxylamine, 
a potentially toxic metabolite produced by 
cytochrome P-450 enzymes. About 85% of 
DDS is excreted in the urine, mainly as glu-
curonide and 10% is excreted in the bile.3
Adverse effects associated with DDS in-
clude dose-related hemolysis, methemoglo-
binemia (MeHb), peripheral neuropathy, 
agranulocytosis, aplastic anemia, and sulfone 
syndrome (fever, malaise, exfoliative dermati-
tis or morbilliform rash, hepatic dysfunction, 
lymphadenopathy, MeHb, and hemolytic 
anemia).2 MeHb is the most common side 
effect of dapsone and is formed by hydroxy-
lamine metabolite, which is capable of being 
co-oxidized with hemoglobin in the red blood 
cell. MeHb can occur either in congenital or 
acquired forms. The fi rst is present at bird and 
manifests in two distinct forms. Type I is an 
erythrocyte form with a defi ciency of NAD-
Hcytochrome b5 reductase gene and Type II 
is a generalized form that is characterized by 
a b5 reductase defi ciency in all tissues. Ac-
quired forms are usually pharmacokinetically 
induced responses that result in an increase in 
rate of oxidation of hemoglobin to methemo-
globin and, a number of chemicals have been 
implicated. The equilibrium between haemo-
globin and methaemoglobin is maintained by 
a particular mechanism. Methaemoglobin is 
reduced to haemoglobin by the NADHcyto-
Este é um artigo Open Access sob a licença de CC BY-NC-ND
320
chrome b5 reductase enzyme establishing a steadystate 
level of about 1% of total haemoglobin.4-7 
The objective of this work was to determine the 
MeHb levels and correlate with dapsone plasma levels 
in multibacillary leprosy patients under leprosy multi-
drug therapy.
Were enrolled 15 adult male and 15 adult female pa-
tients with laboratory and clinical confirmation of multi-
bacillary leprosy from the State Reference Unit for Lepro-
sy Treatment Dr. Marcello Candia, Marituba, PA, Brazil. 
Exclusion criteria included incapacitating erythema no-
dosum leprosum, severe neuritis, SIDA, tuberculosis, and 
malaria. Their characteristics were as follows (means ± 
SDs): age, 28 ± 13.1 years (age range; 18 to 37 years); body 
weight, 64.21 ± 12.1 kg, erythrocyte count, 4.21 ± 0.52 X 
106/µL; white blood count, 11,300 ± 4,700/µL. Informed 
consent was obtained from all subjects. This study was 
approved by the ethics committee of the Tropical Medi-
cine Center of Universidade Federal do Pará.
Each patient received the standard multibacillary lepro-
sy multi-drug therapy of rifampin (600 mg) and clofazimine 
(300 mg) monthly, supervised, and dapsone (100 mg) 
and clofazimine (50 mg) daily, unsupervised. At the 
time of the study, all patients had received dapsone for 
at least one month. 
Blood samples were taken in the steady-state; i.e., 
three days after the administration of supervised dose. 
All samples were taken before dapsone intake; i.e., trough 
levels were measured. MeHb was determined according 
Heggesh et al. (1970).8 Dapsone was analyzed by high 
performance liquid chromatography with ultraviolet 
detection (Pro Star – Varian, Walnut, CA-USA), as de-
scribed previously.9 The column was an ODS C18 4.6 X 
250 mm (Supelco Inc. Bellefonte PA, USA). The method 
involved liquid-liquid extraction of drug from plasma 
samples with diethyl-ether. The mobile phase consisted 
of 20% acetonitrile v/v. Phenacetin (100 µg/mL) was used 
as internal standard. The analytical procedure validated 
in our laboratory demonstrate that within-day and day-
to-day coefficients of variation were 10.7 and 14.1%, re-
spectively. Mean extraction recovery of dapsone was 90%. 
The stability of blank plasma spiked with dapsone was 60 
days. Rifampin, clofazimine, prednisone and thalidomide 
do not interfered in the detections of dapsone. 
Data are presented as mean ± SD. The concentrations 
of dapsone between patients were compared by Student’s 
t, with p-values of < 0.05 considered to indicate signifi-
cant differences, and Pearson coefficient to estimate the 
correlation between the variables. Statistical evaluations 
were conducted using the statistical computer package 
STATISTICA (Statsoft, Tulsa, Okla., USA).
The mean concentration of dapsone in male plasma 
samples in the steady state was 1.42 ± 1.65 µg/mL, rang-
ing from 0.22 to 6.9 µg/mL, and in the female samples 
was 2.42 ± 2.28 µg/mL, ranging from 0.24 to 8.0 µg/
mL. These results are consistent with previous work in 
healthy volunteers after 100 mg of oral dapsone, which 
show dapsone levels ranging from 1.10 to 2.33 µg/mL, 
and demonstrated that the bioavailability of dapsone is 
similar between healthy volunteers and leprosy patients. 
No difference was observed between male and female 
dapsone plasma levels.10
It has been demonstrated that the compliance with 
prolonged leprosy therapy was enhanced when dapsone 
was associated with others drugs and, in this study, 90% 
of patients presented therapeutic levels of dapsone for 
leprosy multi-drug therapy of 0.5 to 5.0 µg/mL, and 
correlated with previous report of adherence in leprosy 
multi-drug therapy studies where the compliance was 
above of 85%.1
The MeHb levels of male patient was 3.09 ± 1.91 %, 
ranged from 1.14% to 8.33%, and in female patients was 
2.84 ± 1.67%, ranged from 0.28% to 5.89%. No differ-
ence was observed between male and female patients. 
73% of patients presented MeHb levels above the val-
ues of unexposed population, but no signs or symptoms 
of MeHb were observed.2,4 It has been demonstrate that 
the symptoms of MeHb generally correlate with MetHb 
levels. At levels above 10%, cyanosis becomes clinically 
apparent. Exertion dyspnea, tachycardia, dizziness, chest 
pain, and headache occur with levels up 20%. At levels 
> 50%, arrhythmias, seizures, and depressed conscious-
ness may be seen, levels above 85% are life threatening. 
Our results are consistent with previous work that show 
the risk of dapsone-dependent side effects is very low if 
plasma concentration is below 5 mg/L.2,4-7
No significances were seen in Pearson coefficients 
between dapsone concentrations and MeHb levels in 
both groups. In male patients was -0.3419 and 0.355 in 
female patients. This finding does not support the evi-
dence of a good relationship between these variables in 
the therapeutic use of dapsone. Our results provide evi-
dence suggesting that the dosage of dapsone in leprosy 
treatment does not promote an important MeHb.
 
Methemoglobinemia and dapsone levels in leprosy
321Braz J Infect Dis 2010; 14(3):319-321
Vieira, Riveira, Martins et al.
REFERENCES
1. Zhu IY, Stiller MJ. Dapsone and sulfones in dermatology: over-
view and update. J Am Acad Dermatol. 2001; 45:420-34.
2. Coleman MD. Dapsone toxicity: some current perspective. Gen 
Pharmacol. 1995; 26:1461-7.
3. Zuidema J, Hilbers-Modderman ESM, Merkus FWHM. Clini-
cal pharmacokinetics of dapsone. Clin Pharmacokinet 1986; 
11:299-315.
4. Halim NKD, Ogbeide E. Haematological alterations in leprosy 
patients treated with dapsone. East Afr Med J. 2002; 79:100-2.
5. Kaur I, Metha M, Agnihotri N et al. Dapsone-induced meth-
emoglobinemia in leprosy patients. Int J Lepr 2001; 69:247-9.
6. Vage C, Saab N, Woster PM, Svensson CK. Dapsone-induced 
hematologic toxicity: comparison of the methemoglobin-form-
ing ability of hydroxylamine metabolities of dapsone in rat an 
human blood. Toxicol Appl Pharmacol 1994; 129:309-16.
7. Reilly TR, Woster PM, Svensson CK. MMethemoglobin 
formation by hydroxylamine metabolites of sulfamethox-
azole and dapsone: implications for differences in adverse 
Drug Reactions. J Pharmacol Exp Ther 1999; 288: 951-9.
8. Hegesh E, Gruener RN, Bockwosky R, Shuvat HJ. A sensi-
tive micromethod for the determination of methemoglob-
in in blood. Clin Chim Acta 1970; 30: 679-82. 
9. Kwadijk S, Torano JS. High-performance liquid chroma-
tographic method with ultraviolet detection for the deter-
mination of dapsone and its hydroxylated metabolite in 
human plasma. Biomed Chromatogr 2002; 16:203-8.
10. Ab G, Brocavich JM, Etzel JV et al. Evaluation of effects of 
altered gastric pH on absorption of dapsone in healthy vol-
unteers. Antimicrob Agents Chemoter 1994; 38:2227-9.


Repositório Institucional da Universidade Federal do Pará

319BRIEF COMMUNICATION Methemoglobinemia and dapsone levels in patients with leprosyAuthorsJose Luiz Fernandes Vieira1Juan Gonzalo Bardarez Riveira2Ana de Nazaré Silva Martins2Jorge Pereira da Silva1Claudio Guedes Salgado11Toxicology Professor of Universidade Federal do Pará Pharmacy School, Belém, PA, Brazil.2Undergraduated students of Universidade Federal do Pará, Belém, PA, Brazil.Submitted on: 06/25/2009Approved on: 10/02/2009Correspondence to:Jose Luiz Fernandes VieiraLaboratório de Toxicologia,Universidade Federal do ParáRua Augusto Corrêa, 1Belém – PA – BrazilPhone: +55-91-32017733E-mail: jvieira@ufpa.br We declare no conﬂ ict of interest.ABSTRACTThe objective of this work was to determine the methemoglobinemia and correlate with dapsone levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by high performance liquid chromatography and methemoglobinemia by spectrophotometry. The mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean methemoglobin levels in male was 3.09 µg/mL; 191%, and in female was 2.84 ± 1.67%. No cor-relations were seen between dapsone levels and methemoglobin in male and female patients. Our results demonstrated that the dosage of dapsone in leprosy treatment does not promote a signifi -cant methemoglobinemia.Keywords: methahemoglobinemia, dapsone, leprosy. [Braz J Infect Dis 2010;14(3):319-321]©Elsevier Editora Ltda.Dapsone (4-4´-diaminodiphenylsulfone, DDS) is a chemical analogue of sulfapyridine, syn-thesized in 1908. It is a part of the multidrug regimen recommended for the treatment of leprosy, but it is also used against a number of noninfectious inflammatory diseases.1 DDS acts in the same way as sulfonamides, inhibiting the synthesis of dihydrofolic acid through competition with para-aminoben-zoate for the active site of dihydropteroate synthetase. The anti-inflammatory action of DDS is associated with the interference in neutrophil chemotactic migration, β2 in-tegrin (CD11b/ CD18)-mediated adherence of human neutrophils in vitro and with the activation or function of the G-protein (Gi type) that initiates the signal transduction cascade common to chemotactic stimuli.1,2DDS is absorbed readily from the gas-trointestinal tract with bioavailability of more than 86%. The peak plasma concen-tration after 100 mg of oral DDS is attained between 2 to 8 hours. The drug shows lin-ear pharmacokinetics within the therapeutic range and the time-course after oral admin-istration fits a 2-compartment model. DDS is distributed for all organs including skin, liver, kidneys and erythrocytes. It is metab-olized via acetylation or N-hydroxylation. The latter reaction yields the hydroxylamine, a potentially toxic metabolite produced by cytochrome P-450 enzymes. About 85% of DDS is excreted in the urine, mainly as glu-curonide and 10% is excreted in the bile.3Adverse effects associated with DDS in-clude dose-related hemolysis, methemoglo-binemia (MeHb), peripheral neuropathy, agranulocytosis, aplastic anemia, and sulfone syndrome (fever, malaise, exfoliative dermati-tis or morbilliform rash, hepatic dysfunction, lymphadenopathy, MeHb, and hemolytic anemia).2 MeHb is the most common side effect of dapsone and is formed by hydroxy-lamine metabolite, which is capable of being co-oxidized with hemoglobin in the red blood cell. MeHb can occur either in congenital or acquired forms. The fi rst is present at bird and manifests in two distinct forms. Type I is an erythrocyte form with a defi ciency of NAD-Hcytochrome b5 reductase gene and Type II is a generalized form that is characterized by a b5 reductase defi ciency in all tissues. Ac-quired forms are usually pharmacokinetically induced responses that result in an increase in rate of oxidation of hemoglobin to methemo-globin and, a number of chemicals have been implicated. The equilibrium between haemo-globin and methaemoglobin is maintained by a particular mechanism. Methaemoglobin is reduced to haemoglobin by the NADHcyto-JUN-2010.indd   319 28/7/2010   10:35:34320chrome b5 reductase enzyme establishing a steadystate level of about 1% of total haemoglobin.4-7 The objective of this work was to determine the MeHb levels and correlate with dapsone plasma levels in multibacillary leprosy patients under leprosy multi-drug therapy.Were enrolled 15 adult male and 15 adult female pa-tients with laboratory and clinical confirmation of multi-bacillary leprosy from the State Reference Unit for Lepro-sy Treatment Dr. Marcello Candia, Marituba, PA, Brazil. Exclusion criteria included incapacitating erythema no-dosum leprosum, severe neuritis, SIDA, tuberculosis, and malaria. Their characteristics were as follows (means ± SDs): age, 28 ± 13.1 years (age range; 18 to 37 years); body weight, 64.21 ± 12.1 kg, erythrocyte count, 4.21 ± 0.52 X 106/µL; white blood count, 11,300 ± 4,700/µL. Informed consent was obtained from all subjects. This study was approved by the ethics committee of the Tropical Medi-cine Center of Universidade Federal do Pará.Each patient received the standard multibacillary lepro-sy multi-drug therapy of rifampin (600 mg) and clofazimine (300 mg) monthly, supervised, and dapsone (100 mg) and clofazimine (50 mg) daily, unsupervised. At the time of the study, all patients had received dapsone for at least one month. Blood samples were taken in the steady-state; i.e., three days after the administration of supervised dose. All samples were taken before dapsone intake; i.e., trough levels were measured. MeHb was determined according Heggesh et al. (1970).8 Dapsone was analyzed by high performance liquid chromatography with ultraviolet detection (Pro Star – Varian, Walnut, CA-USA), as de-scribed previously.9 The column was an ODS C18 4.6 X 250 mm (Supelco Inc. Bellefonte PA, USA). The method involved liquid-liquid extraction of drug from plasma samples with diethyl-ether. The mobile phase consisted of 20% acetonitrile v/v. Phenacetin (100 µg/mL) was used as internal standard. The analytical procedure validated in our laboratory demonstrate that within-day and day-to-day coefficients of variation were 10.7 and 14.1%, re-spectively. Mean extraction recovery of dapsone was 90%. The stability of blank plasma spiked with dapsone was 60 days. Rifampin, clofazimine, prednisone and thalidomide do not interfered in the detections of dapsone. Data are presented as mean ± SD. The concentrations of dapsone between patients were compared by Student’s t, with p-values of < 0.05 considered to indicate signifi-cant differences, and Pearson coefficient to estimate the correlation between the variables. Statistical evaluations were conducted using the statistical computer package STATISTICA (Statsoft, Tulsa, Okla., USA).The mean concentration of dapsone in male plasma samples in the steady state was 1.42 ± 1.65 µg/mL, rang-ing from 0.22 to 6.9 µg/mL, and in the female samples was 2.42 ± 2.28 µg/mL, ranging from 0.24 to 8.0 µg/mL. These results are consistent with previous work in healthy volunteers after 100 mg of oral dapsone, which show dapsone levels ranging from 1.10 to 2.33 µg/mL, and demonstrated that the bioavailability of dapsone is similar between healthy volunteers and leprosy patients. No difference was observed between male and female dapsone plasma levels.10It has been demonstrated that the compliance with prolonged leprosy therapy was enhanced when dapsone was associated with others drugs and, in this study, 90% of patients presented therapeutic levels of dapsone for leprosy multi-drug therapy of 0.5 to 5.0 µg/mL, and correlated with previous report of adherence in leprosy multi-drug therapy studies where the compliance was above of 85%.1The MeHb levels of male patient was 3.09 ± 1.91 %, ranged from 1.14% to 8.33%, and in female patients was 2.84 ± 1.67%, ranged from 0.28% to 5.89%. No differ-ence was observed between male and female patients. 73% of patients presented MeHb levels above the val-ues of unexposed population, but no signs or symptoms of MeHb were observed.2,4 It has been demonstrate that the symptoms of MeHb generally correlate with MetHb levels. At levels above 10%, cyanosis becomes clinically apparent. Exertion dyspnea, tachycardia, dizziness, chest pain, and headache occur with levels up 20%. At levels > 50%, arrhythmias, seizures, and depressed conscious-ness may be seen, levels above 85% are life threatening. Our results are consistent with previous work that show the risk of dapsone-dependent side effects is very low if plasma concentration is below 5 mg/L.2,4-7No significances were seen in Pearson coefficients between dapsone concentrations and MeHb levels in both groups. In male patients was -0.3419 and 0.355 in female patients. This finding does not support the evi-dence of a good relationship between these variables in the therapeutic use of dapsone. Our results provide evi-dence suggesting that the dosage of dapsone in leprosy treatment does not promote an important MeHb. Methemoglobinemia and dapsone levels in leprosyJUN-2010.indd   320 28/7/2010   10:35:34321Braz J Infect Dis 2010; 14(3):319-321Vieira, Riveira, Martins et al.REFERENCES1. Zhu IY, Stiller MJ. Dapsone and sulfones in dermatology: over-view and update. J Am Acad Dermatol. 2001; 45:420-34.2. Coleman MD. Dapsone toxicity: some current perspective. Gen Pharmacol. 1995; 26:1461-7.3. Zuidema J, Hilbers-Modderman ESM, Merkus FWHM. Clini-cal pharmacokinetics of dapsone. Clin Pharmacokinet 1986; 11:299-315.4. Halim NKD, Ogbeide E. Haematological alterations in leprosy patients treated with dapsone. East Afr Med J. 2002; 79:100-2.5. Kaur I, Metha M, Agnihotri N et al. Dapsone-induced meth-emoglobinemia in leprosy patients. Int J Lepr 2001; 69:247-9.6. Vage C, Saab N, Woster PM, Svensson CK. Dapsone-induced hematologic toxicity: comparison of the methemoglobin-form-ing ability of hydroxylamine metabolities of dapsone in rat an human blood. Toxicol Appl Pharmacol 1994; 129:309-16.7. Reilly TR, Woster PM, Svensson CK. MMethemoglobin formation by hydroxylamine metabolites of sulfamethox-azole and dapsone: implications for differences in adverse Drug Reactions. J Pharmacol Exp Ther 1999; 288: 951-9.8. Hegesh E, Gruener RN, Bockwosky R, Shuvat HJ. A sensi-tive micromethod for the determination of methemoglob-in in blood. Clin Chim Acta 1970; 30: 679-82. 9. Kwadijk S, Torano JS. High-performance liquid chroma-tographic method with ultraviolet detection for the deter-mination of dapsone and its hydroxylated metabolite in human plasma. Biomed Chromatogr 2002; 16:203-8.10. Ab G, Brocavich JM, Etzel JV et al. Evaluation of effects of altered gastric pH on absorption of dapsone in healthy vol-unteers. Antimicrob Agents Chemoter 1994; 38:2227-9.JUN-2010.indd   321 28/7/2010   10:35:34

Methemoglobinemia and dapsone levels in patients with leprosy

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Methemoglobinemia and dapsone levels in patients with leprosy

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Repositório Institucional da Universidade Federal do Pará