R ßv isiü d ü S oc ied ade B rasileira de M ed icina Tropical 24(2): 111-114, abr-jun, 1991
ISONIAZID ACETYLATING PHENOTYPE IN PATIENTS
WITH PARACOCCIDIOIDOMYCOSIS A N D ITS
RELATIONSHIP WITH SERUM SULFADO XIN LEVELS,
GLUCOSE-6-PHOSPHATE DEHYDROGENASE A N D
GLUTATHIONE REDUCTASE ACTIVITIES.
Benedit o Barraviera, Paulo Câmara Marques Pereira, Jussar a Marcondes Machado, Maria Julia de Souza,
Car los Robert o G. Lima, Paulo Robert o Curi, Rinaldo Poncio Mendes and Dom ingos Alves Meira.
T h e a u t h o r s e v a l u a t e d th e i s o n i a z i d a c e t y l a t i n g p h e n o t y p e a n d m e a s u r e d h e m a to c r it, h e m o g lo b i n , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e a n d g l u t a t h i o n e r e d u c t a s e a c t i v i t i e s p l u s s e r u m s u l-f a d o x i n l e v e l s in 3 9 p a t i e n t s w ith p a r a c o c c i d i o i d o m y c o s i s ( 3 3 m a l e s a n d 6 l-f e m a l e s ) a g e d 1 7 to 5 8 y e a r s . T w e n ty o n e ( 5 3 . 8 4 % ) o f th e p a t i e n t s p r e s e n t e d a s l o w a c e t y l a t i n g p h e n o t y p e a n d 1 8 ( 4 6 . 1 6 % ) a f a s t a c e t y l a t i n g p h e n o t y p e . G lu c o s e - 6 - p h o s p h a t e - d e h y d r o g e n a s e ( G 6 P D ) a c t i v i t y w a s d e c r e a s e d in 5( 2 3 . 8 0 % ) s l o w a c e t y l a t o r s a n d in 4 ( 2 2 . 2 2 % ) f a s t a c e t y l a t o r s . G l u t a t h i o n e r e d u c t a s e a c t i v i t y w a s d e c r e a s e d in 1 4 ( 6 6 . 6 6 % ) s l o w a c e t y l a t o r s a n d in 1 2 ( 6 6 . 6 6 % ) f a s t a c e t y l a t o r s . S e r u m l e v e l s o f f r e e a n d t o t a l s u l f a d o x i n W ere h i g h e r in s l o w a c e t y l a t o r ( p < 0 . 0 2 ) . A n a l y s i s o f t h e r e s u lts < p e r m itte d u s t o c o n c lu d e t h a t s e r u m s u lf a d o x i n le v e l s a r e r e l a te d to th e a c e t y l a t o r p h e n o t y p e . F u r th e r m o r e , s u l f a d o x i n l e v e l s w e r e a l w a y s a b o v e 5 0 jig/ml, a value considered therapeutic. Glutathione reductase deficiency observed in 66% of patients may be related to the intestinal malabsorption of
n u tr ie n ts , a m o n g th e m r ib o f la v in , a F A D p r e c u r s o r v ita m in , i n p a t i e n t s w i th p a r a c o c e i d io i d o m y c o s i s .
K e y w o r d s : I s o n i a z i d a c e t y l a t i n g p h e n o t y p e . P a r a c o c c i d i o i d o m y c o s i s . G l u c o s e 6 p h o s -p h a t e d e h y d r o g e n a s e ( G 6 P D ) . G l u t a t h i o n e r e d u c ta s e . S u l f a d o x i n .
The therapeutic conduct adopted by us thus far® 18 for paracoccidioidomycosis is divided into two stages, i.e. acute and maintenance treatment Several drugs have been employed for acute treatment, among them sulfa drugs alone17 or in combination with trimethoprim^ 7 18. For maintenance, the use of slow- excretion sulfa drugs is recommended for a period of at least two years 18. These drugs, because of their oxidizing action, may cause serious side effects in patients with genetic red blood cell defects1 3 4 6 9 14 21. Among such defects are glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase deficiency3 14. Glutathione reductase deficiency also may be an acquired defect due to deficiency of riboflavin, a FAD precursor vitamin^ 14 These
Department of Infectious and Parasitologic Diseases, and Section of Statistics, School of Medicine of Botucatu, Uni versity Paulist State, São Paulo, SP, Brazil.
Research Supported by F U N D U N E SP, Grant 007/89 and CNPq, Grant 300398/88-3.
Correspondence address: Dr. Benedito Barraviera, Faculda de de Medicina de Botucatu/UNESP, CP: 576, 18610 Botucatu, SP, Brasil.
Recebido para publicação em 2 8 /1 2 /9 0 .
defects may cause attacks of hemolytic anemia in patients treated for paracoccidioidomycosis, with con sequent severe worsening of clinical signs and progno sis.
In addition, the literature is controversial with respect to the effect of isoniazid acetylating phenotype on serum levels of sulfones, sulfonamides and isonia zid. Some investigators8 1° 27 believe that the acetyla ting phenotype is intimately related to the serum levels of these drugs, whereas others24 disagree.
In view of the above considerations, the objecti ves of the present study were to determine the isoniazid acetylating phenotype and to evaluate correlation of it with G6PD and glutathione reductase activity and with serum levels of free and total sulfadoxin in patients with paracoccidioidomycosis.
MATERIAL AND METHODS
The present study was conducted at the Para- cocciodioidomycosis Outpatient Clinic of the Division of Infectious and Parasitologic Diseases of the School of Medicine of Botucatu/UNESP, São Paulo, Brazil, from 1987 to 1989. Thirty-nine patients (6 females and 33 males, aged 19 to 64 years) with a confirmed diagnosis of paracoccidioidomycosis were studied. The clinical form of the diseasel3 was chronic in 29 and subacute progressive in 10 patients. All patients
B arraviera B , Pereira P C M , M a c ha d o J M , So u za M J, L im a C RG , C u riP R , M e nde s R P, M eira D A . Iso n ia zid acetylating p he no typ e inp atien ts w ith paracoccidioidom ycosis a n d its relationship w ith serum sulfado xin levels,glucose-6-phosphate dehydrogenase a n d g lu tathione reductase activities. R e v is ta da S oc ied ad e B rasileira de M ed icin a T ropical 2 4 :1 1 1 -1 1 4 , a h r ju n , 1991
were under maintenance treatment with one tablet (500 mg) of sulfadoxin twice a week. As recommended by Mendes1®, the medication was taken on Mondays and Thursdays and blood samples were collected on Wednesdays at about 2:00 p.m.. All patients had been under maintenance treatment for at least one month and followed medical prescriptions rigorously. They were tested for isoniazid acetylating phenotype and G6PD and glutathione reductase activities and sub mitted to measurement of hematocrit, hemoglobin and serum levels of free and total sulfadoxin.
Isoniazid acetylating phenotype was determi ned before the beginning of treatment by the colorime tric test of Eidus et aU2 and Hodgkin et al1^ and the patients were classified as fast acetylators when acetylated isoniazid was more than 65% and as slow acetylators when acetylated isoniazid was less than 65%, according these authors *2 15
G6PD and glutathione reductase activities were determined by the technique standardized by Barra viera et aP. The normal values were 221.10±21.06 ^.g/min for G6PD activity, and 81.27±9.54 fxg/min for glutathione reductase. Hematocrit ievels were determined by the microhematocrit method and hemo globin levels by the cyanomethemoglobin method20.
Serum sulfadoxin levels were measured by the techique of Bratton & Marshall11 which is used to evaluate free and total sulfadoxin levels.
Two study groups were proposed for evaluation of the results: Group 1: slow acetylators; and Group 2: fast acetylators.
Data were analyzed statistically using the pai red “t” test2^. To determine the interrelationship of the variables, the linear correlation coefficient betwe en pairs of variables was calculated.
RESULTS
Analysis of the results presented in Table 1 showed that 21(53.84%) patients were slow acetyla- tor and 18(46.16%) fast acetylators.
Hematocrit and hemoglobin levels were redu ced only among male patients, 5 of whom (27.77%) were fast acetylators and 6(28.57%) slow acetylators.
G6PD activity was decreased in 5(23.80%) slow acetylators and 4(22.22%) fast acetylators. Glutathione reductase activity was decreased in 14(66.66%) slow acetylators and in 12(66.66%) fast acetylators.
Serum levels of free and total sulfadoxin were significatively higher among slow acetylators (p < 0,05 and p < 0,02, respectively). There was a negative correlation between slow acetylating pleno- type and serum level of free sulfa (ro,ol = -0,45).
DISCUSSION
Isoniazid acetylating phenotype and G6PD and glutathione reductase activities are genetically deter mined traits^ 6 8 10 12 15 The first is determined by major autosomal genes and is related to the acetylating capacity of hepatic acetyl transferase. One of the functions of this enzyme is to inactivate sulfones, sulfonamides and isoniazid10 1215. Population stu dies'9 26 have demonstrated two types of acetylators, i.e., fast and slow, the frequency of each varying from population to population. American Indians, Japanese and Eskimos most often present a fast acetylating phenotype2^. In Brazil, Beiguelman et al10 evaluated the acetylating phenotype of Caucasian and Black patients with tuberculosis and detected 57% slow acetylators among Caucasians and 50% among Blacks. In the present study, the prevalence of acetyla ting phenotype was quite similar to that found by these investigators1®.
G6PD activity is determined by genes located on the X chromosome14 21. Thus, only females can manifest homozygous or heterozygous trait. In the present study, 3 patients heterozygous for G6PD deficiency were detected. Among the six G6PD- deficient male patients, only two developed anemia. It should be pointed out that reduced number of patients
Table 1 - D i s t r i b u t i o n o f a c e t y l a t i n g p h e n o t y p e , h e m a to c r i t , h e m o g lo b in , g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e ( G 6 P D ) a n d g l u t h a t i o n e r e d u c t a s e ( G R ) a c t i v i t i e s a n d s e r u m l e v e l s o f t o t a l a n d f r e e s u lf a d o x in .
Acetylating
phenotype Hematocrit
(%) X + s
Hemoglobin (g%) X ± s
G6PD (jug/min)
X ± s
GR Og/min)
X ±s
Total Sulfadoxin
(Mg/ml) X + s
Free Sulfadoxin
(jug/ml) X ± s
Slow 49.61 + 14.46± 208.42± 62.73± 190.24+* 154.04±**
n=21) 7.54 2.21 38.46 21.08 54.10 41.54
Fast 48.88± 13.76+ 214.03+ 64.26+ 152.98+ 125.04±
(n=18) 7.01 1.95 31.01 16.24 37.91 32.39
Statistical analysis: * t=2.45; p<0.02.
** t=2.40; p<0.05; differences significatives in respect to the fast acetylating group.
B ar ru v ie ra B , Pereira P C M , M ac ha do J M , S o u za M J, L im a C RG , C ur iP R , M e nd es R P, M e ir a D A . Is o n ia zid acetyla tin gp he notype in pa tie n ts w ith paraco ccid ioido m y co sis a n d its relationship with serum sulfad oxin levels,glucose-6-phosphate dehydrogenase a n d glu tathio ne reductase
activities. R ev is ta da Soc ieda de B rasileira de M ed icin a T ropical 24:111-114, abr-jun, 1991
w ith anem ia detected am ong d eficien t individuals under the effect o f oxid izing drugs such as sulfadoxin
can b e explained by the type o f d eficien cy predom i nating in B razilian populations1 4 T h e deficien cy
encountered here is p o ssib ly o f the A frican type, w ith a consequent tolerance o f red cells to oxid izing drugs3 4 6 14 21
From a genetical view point, glutathione re-ductas deficien cy follow s the autosom al recessive m odel, the detection o f d eficient individuals bein g a
very infrequent occurrence1 14. F o r perfect functio ning, this enzym e n eed s N A D P H generated through
the p en to se pathw ay3 1421 and F A D , a phosphoryla-ted riboflavin derivative2 9. Barraviera e t al2, in a
study o f individuals from the Brazilian A m a z o n region, d etected d ecreased glutathione reductase activity due
to riboflavin d eficien cy cau sed b y the peculiar dietary habits o f that region. In addition, the elevated rate o f helm inth infestation d etected am ong A m a z o n popula tion s m a y strongly contribute to the consum ption o f
this vitam in16. T h e elev a ted prevalence o f glutathione reductase observed in the present study m ay h ave b een related to the intestinal m alabsorption o f nutrients,
am ong them am ino acids and vitam ins, observed am ong patients w ith p rogressive paracoccidioidom y
c o s is 7 17.
O n the other hand, individuals chronically using
sulfam ide derivatives are know n to suffer stim ulation o f glutathione synthetase w hich cau ses increased synth esis o f reduced glutathione2 3 . T h ese individuals
eventually show an in crease in glutathione reductase activity. T h e present findings sh ow ed ex a c tly the
contrary, i.e., a decrease in glutathione reductase activity, strengthening the hypothesis that the nutri tio nal d eficien cy o f riboflavin m ay b e the lim iting
factor in glutathione reductase activity. It should be
pointed ou t that all anem ic patients presented decrea sed glutathione reductase activity. It has b een repor-ted 1 6 9 14 21 that the sam e drugs that ca u se anem ia
am ong G 6 P D -d efic ien t individuals can ca u se anem ia am ong glutathione reductase-d eficient subjects. In this ca s e , the nutritional d eficien cy o n the on e hand, the
chronic effect o f sulfadoxin on the other, m ay have strongly contributed to the on set o f anem ia.
T he relationship b etw een acetylating phenotype and serum lev els o f fast-excretion sulfam ide deriva tives are controversial24 27. V ree et al27 and
Barra-viera et a l8, w hen evaluating the serum sulfadiazin e le v e ls o f patients using th ese drugs and their
relationship w ith the acetyla ting phenotype,
found m ore elevated drug le v els and d ecreased
renal function am ong slow acetylators. T h e present
results agree w ith th ose reported in these studies8 27 and clearly dem onstrate the difference in serum sulfa d oxin lev els b etw een slo w and fast acetylators. O n the
other hand, serum lev els o f free sulfadoxin w ere
always above 50 jag/ml in all patients studied, levels considered therapeutic by Padilha-Gonçalves22. Thus, the posology used was appropriat since it maintained satisfactory serum levels of the drug even among fast acetylators.
Finally, determination of acetylator phenotype and of serum sulfadoxin level as well as their rela tionships with G 6 P D and glutathione reductase acti vities represents an important and very useful para meter since it permits better clinical follow-up of the patients, with early observation of the possible onset of anemia and of therapeutic faults due to insufficient circulating levels of the drug.
R E S U M O
O s a u t o r e s a v a l i a r a m o f e n ó t i p o a c e t i l a d o r d a is o n i a z i d a , h e m a tó c r i t o , h e m o g lo b i n a , a t i v i d a d e d a g l i c o s e 6 f o s f a t o d e s i d r o g e n a s e , g l u t a t i o n a r e d u t a s e e o s n í v e i s s é r i c o s d e s u l f a d o x i n a d e 3 9 d o e n t e s c o m p a r a c o c c i d í o i d o m i -c o se , s e n ã o 3 3 d o s e x o m a s -c u l in o e 6 d o f e m i n i n o , -c o m i d a d e s c o m p r e e n d i d a s e n tr e 1 7 e 5 8 a n o s . V in te e u m ( 5 3 , 8 4 % ) d o e n t e s a p r e s e n t a r a m f e n ó t i p o a c e t i l a d o r le n to e 1 8 ( 4 6 , 1 6 % ) r á p i d o . A a t i v i d a d e d a g l ic o s e - 6 - f o s f a t o d e s i d r o g e n a s e ( G 6 P D ) e s t e v e d i m i n u í d a e m 5 ( 2 3 , 8 0 % ) a c e -t i l a d o r e s l e n -t o s e 4 ( 2 2 , 2 2 % ) r á p i d o s . A a -t i v i d a d e d a g l u t a t i o n a r e d u t a s e e s t e v e d i m i n u í d a e m 1 4 ( 6 6 , 6 6 % ) a c e t i l a d o r e s l e n to s e 1 2 ( 6 6 , 6 6 % ) r á p i d o s . O s n í v e i s s é r i c o s d e s u l f a d o x i n a liv r e e t o t a l f o r a m m a i o r e s n o s a c e t i l a d o r e s l e n to s ( p < 0 , 0 2 ) . A a n á l i s e d o s r e s u l t a d o s p e r m i t e c o n c l u i r q u e o s n í v e i s s é r i c o s d e s u l f a d o x i n a r e l a c i o n a - s e c o m o f e n ó t i p o a c e t i la d o r . A l é m d i s s o , o s n í v e i s e s t iv e r a m s e m p r e a c i m a d e 5 0 f i g / m l , n í v e i s e s t e s c o n s i d e r a d o s te r a p ê u tic o s . P o r o u t r o la d o , a d e f ic iê n c i a d e g l u t a t i o n a r e d u t a s e p o d e e s t a r r e l a c i o n a d a c o m a m á a b s o r ç ã o i n t e s t i n a l d e n u tr i e n te s , e n tr e e le s r i b o f l a v i n a , v i t a m i n a p r e c u r s o r a d e F A D .
P a la v r a s - c h a v e s : F e n ó t i p o a c e t i l a d o r d a i s o n i a z i d a . P a r a c o c c i d i o i d o m i c o s e . G l i c o s e - 6 - f o s f a t o d e s i d r o g e n a s e ( G 6 P D ) . G l u t a t i o n a r e d u ta s e . S u l f a d o x i n a .
ACKNOWLEDGMENTS
The authors are grateful to FUNDUNESP and CNPq for support
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