1 s2.0 0041387975900537 main

Prévia do material em texto

Tuber& ( 1975), 56,203 
DETERMINATION OF THE ACETYLATOR PHENOTYPE USING MATRIX 
ISONIAZID 
G. A. ELLARD and PATRICIA T. GAMMON 
MRC Unit .for Laboratory Studies of Tuberculosis, Royal Postgraduate Medical School. Durane Road. 
London, WI2 OHS 
HILKKA TIITINEN* 
Nufjield Unit of Medical Genetics. Deparlnrent of Medicine. University of LiverpooI, Crown Street, Liverpool, L6Y J&X 
SUMMARY 
The development of a simple method for classifying subjects as slow or rapid acetylators 
is reported. The method is based on determining the ratio of acetylisoniazid to acid- 
labile isoniazid in the urine 23-24 hours after an oral dose of a slow-release isoniazid 
formulation. The results obtained when this method was applied to phenotype over 200 
East African tuberculosis patients are described. It is suggested that the method would 
be particularly suitable for classifying tuberculosis out-patients. 
Les auteurs rapportent la mise au point d’une methode simple pour classer les sujets en 
ipactivateurs lents et rapides. La mCthode est baste SW le rapport de l’adtylisoniazide 
sur l’isoniazide g fonction acide libre dans les urines 23 d 24 heures apr&s une dose orale 
d’isoniazide retard. Les r&ltats obtenus lorsque cette mCthode a Ctd utilisCe pour la 
dktermination du phtnotype de 200 malades tuberculeux africains, sont don&. Les auteurs 
suggkrent que la mtthode serait particuli6rement adaptCe & la classification des malades 
ambulatoires. 
RESUMEN 
Se comunica el empleo de un mCtodo simple para clasificar a las personas coma acetila- 
doras rapidas o lentas. El m&odo est6 basado en la determinacibn de la relaci6n entre 
acetilisoniazida e isoniazida gcido-labil en la orina, 23 a 24 horas despuCs de la ingesti6n 
de una dosis de isoniazida de liberaci6n lenta. Se describen 10s resultados obtenidos 
con el m&odo aplicado al fenotipo en 200 pacientes tuberculosos de Africa de1 Este. 
Se sugiere que este m&odo sen’a muy apropiado para clasificar 10s tuberculosos no 
hospitalizados. 
ZUSAMMENFASSUNG 
Berichtet wird iiber eine einfache Methode, mit der zwischen schnellen und langsamen 
* Present address: Department of Pulmonary Diseases, University Central Hospital. Helsinki, Finland. 
204 I.l,l.AKl~ 4k.I) 0.1 HL-.KS 
Azetylierern unterschieden werden kann. Bestimmt wird das Verhgltnis zwischen Azetyl- 
isoniazid und saurelabilem Isoniazid im Urin 23 - 24 Stunden nach oraler Gabe eines 
lsoniazid - Retardpraparats. Die mit dieser Methode bei 200 Afrikanern gewonnenen 
Resultate werden mitgeteilt. Die Methode diirfte sich besonders zur Klassifierung von 
ambulant behandelten Patienten eignen. 
Introduction 
When tuberculosis patients are treated with once-weekly isoniazid-containing regimens, rapid 
acetylators of isoniazid fare significantly worse than do slow acetylators (see Ellard, 1975). Recently 
several methods have been described for classifying subjects as slow or rapid acetylators of isoniazid 
based on determinations of the ratio of acetylisoniazid to acid-labile isoniazid excreted in the urine 
after giving intramuscular or intravenous doses of isoniazid (Eidus, Harnanansingh & Jessamine. 
1971; Venkataraman and others, 1972; Ellard, Gammon & Tiitinen, 1973). A procedure has also 
been described based on determining the ratio of these compounds in the urine 6-8 hours after 
oral dosage with 10 mg/kg isoniazid (Eidus and others, 1974). In this paper we describe the results 
obtained with a method that should be particularly suitable for phenotyping tuberculosis out- 
patients. In this method urine samples are obtained 23-24 hours after giving an oral dose of 600 
mg isoniazid in a slow-release formulation (‘matrix isoniazid’, Smith and Nephew HS 82; Ellard 
and others, 1973), and subjects phenotyped according to the ratio of acetylisoniazid to acid-labile 
isoniazid determined using simple calorimetric methods. 
Methods 
Collectian of’ samples 
First study - Urine collections (23-24 hour) and plasma samples (24 hour) were obtained from 
49 healthy British student volunteers after they had swallowed oral doses of 600 mg matrix isoniazid. 
Second study - Urine collections were obtained 5-6 hours after oral dosage with 40 mg/kg o.i 
sulphadimidine (Evans, 1969), and 23-24 hours after oral dosage with 600 mg matrix isoniazid. 
from 57 East African tuberculosis patients prior to treatment with regimens consisting of 4 or 8 
weeks daily streptomycin plus isoniazid plus thiacetazone followed by twice-weekly isoniazid plus 
thiacetazone (East African/British Medical Research Council, 1974~). The matrix isoniazid doses 
were given at least 24 hours after the sulphadimidine doses. 
Third study - Urine collections were obtained 23-24 hours after oral dosage with 600 mg matrix 
isoniazid from 204 East African tuberculosis patients prior to treatment with a regimen consisting 
of 2 months daily streptomycin plus isoniazid plus rifampicin plus pyrazinamide followed by 4 
months twice-weekly streptomycin plus isoniazid plus pyrazinamide (East African/British Medical 
Research Council, 1974~). 
Urine samples were also obtained over a period of 36 hours after giving 600 mg matrix isoniazid 
to a slow and a rapid acetylator. All test doses of matrix isoniazid and sulphadimidine were taken 
on an empty stomach. All samples were stored at -20°C prior to analysis in London. Because of the 
instability of acetylisoniazid in plasma (Ellard, Gammon & Wallace, 1972), plasma samples were 
analysed within a week of their collection. 
Analytical methods 
Plasma concentrations of isoniazid and acetylisoniazid were determined fluorimetrically (Ellard, 
Gammon & Wallace, 1972). The concentrations of acid-labile isoniazid in the urine were deter- 
mined calorimetrically with trinitrobenzene sulphonic acid using a modification (Ellard and others, 
ACETYLATOR PHENOTYPE 205 
. 
. . . . ??
? ?
? ?
? ?
. 
I 
s Rapid acetylators 
- Slow acetylators 
LoglO ratio of acetylisoniazid to 
isoniazid in plasma 
FIG. 1 
Distribution of volunteers according to the ratios of acetylisoniazid to isoniazid 
in the urine and plasma. (First study). 
1973) of the Dymond and Russell (1970) procedure. Urinary concentrations of acetylisoniazid were 
determined calorimetrically using a modification of the method described by Venkataraman, 
Eidus and Tripathy (1968). In this modification acid-labile hydrazones of isoniazid were first 
broken down by treating aliquots of urine (0.4 ml) with 0.05 ml 2N HCl and standing at room 
temperature for 15 min. After adding 0.05 ml 2N NaOH, the samples were reacted by the addition 
of 0.2 ml 0.5M pH 6.0 phosphate buffer, O-2 ml 20 % potassium cyanide and 0.8 ml 12.5 % chlora- 
mine-T. After standing for 2-3 min. 1 ml acetone was added and the acetylisoniazid concentrations 
calculated from the absorption of the reaction product at 550 nm. In the first study urinary con- 
centrations of acid-labile isoniazid and acetylisoniazid were also determined fluorimetrically (Ellard, 
Gammon and Wallace, 1972). Concentrations of free sulphadimidine and acid-hydrolysable 
(free plus acetylated) sulphadimidine were determined by the Bratton and Marshall procedure 
(Varley, 1962). 
Results and Discussion 
The results obtained in the first study are summarised in Table I and illustrated in Fig. 1. The 
slow acetylators (30) could readily be distinguished from the rapid acetylators (19) on the basis 
of their higher isoniazid plasma concentrations 24 hours after giving 600 mg matrix isoniazid. 
Alternatively the two phenotypes could be characterised from the ratios of acetylisoniazid to 
isoniazid present in the plasma at this time or the ratios of the excretion of acetylisoniazid to acid- 
labile isoniazid in the urine. Whichever method was used each volunteer was unambiguously 
classified in the same way. Although each of these parameters efficientlydistinguished the 2 acety- 
lator phenotypes, the most convenient method was clearly to determine the ratio of acetylisoniazid 
to acid-labile isoniazid in the urine at 23-24 hours using the simpler calorimetric methods. 
The acetylisoniazid concentrations determined in the urine samples using the calorimetric and 
C 
206 ELLARD AND OTHERS 
TABLE 1. - CHARACTERISAT~ON OF VOLUNTEERS AS SLOW AND RAPID ACETYLATORS ( 1st study) 
Concentrations @g/ml) or ratios in the plasma or urine 24 hr after an oral dose of 600 mg matrix isoniazid. 
Fluid Compounds estimated Methods** 
Plasma 
Plasma 
Urine 
Urine 
Isoniazid 
Acetylisoniazid 
isoniazid 
Acetylisoniazid 
Acid-labile isoniazid 
Acetylisoniazid 
Acid-labile isoniazid 
F 
F 
F 
c 
*Number of subjects. SGeometric means. **Methods: F, fluorimetric; C, calorimetric. 
I- 
Slow (30)* 
---- -_--_. _ 
Mean $ Range 
0.55 0.23-1.17 
1.05 0.28-2.24 
Meun 
0.06 
IO.8 
Range 
-0.02-0.14 
4.0-67.4 
2.68 I .69-6.7 I 22.8 12.9-48.0 
2.18 14.5 8.2-24.9 
tTwo-tailed t-tests between slow and rapid inactivators using 
variances. P < 0001 in every case. 
7- 
Rapid ( 19) 7_ 
! 
_- 
I -- 
/t 
--- 
14.6 
14.1 
20. I 
18.8 
logarithmically transformed data to give similar 
fluorimetric methods were closely similar. However the concentrations of acid-labile isoniazid 
determined using the calorimetric method were significantly higher than those estimated by the 
much more specific and sensitive fluorimetric method. The greatest discrepancy between the results 
obtained by the 2 methods was seen in those samples from the rapid acetylators that contained the 
lowest isoniazid concentrations. Nevertheless, whichever method was employed, the lowest urinary 
ratio of acetylisoniazid to acid-labile isoniazid of the 19 rapid acetylators was approximately 
double that of the highest of the 30 slow acetylators. It was therefore concluded that using the more 
convenient calorimetric methods rather than the more specific fluorimetric methods would be most 
unlikely to result in appreciable misclassification of the 2 phenotypes. 
The results obtained in the second study are illustrated in Fig. 2 and summarised in Table II. 
Using either method the 57 patients were unambiguously classified as 31 slow and 26 rapid acety- 
lators. In this study the sulphadimidine method appeared to give slightly better discrimination 
between the 2 phenotypes than the new method. Among the slow acetylators a direct correlation 
Method Ratio estimated 
TABLE 11. - COMPARISON OF RESULTS OBTAINED WITH NEW AND SULPHADIMIDINE METHODS (2nd study) 
New 
Sulphadimidine 
Acetylisoniazid 
Acid-labile isoniazid 
Acetylsulphadimidine 
Sulphadimidine 
-__-- -_~ ~~~~~ ~ ~~~ 
Slow (3 I)* Rapid (26) 
~___ ____- tt 
Mean** Range Mean Range 
~- - -_____ 
2.52 1.14-6.80 14.8 7.1-53.0 16.0 
0.90 0.41-I .92 4.46 2.58-8.62 18.0 
*Number of subjects. **Geometric means. 
tTwo-talled t-tests between slow and rapid inactivators using logarithmically transformed data lo give similar 
variances. Pi 0901 in every case. 
ACETYLATOR PHENOTYPE 207 
I ??
-0.5 
Loglo ratiX of acetylsulphLiTmidine 
PO 
to sulphadimine in urine 
FIG. 2 
Distribution of patients according to the ratios of acetylated to free isoniazid 
and sulphadimidine in the urine. (Second study). 
could be demonstrated between the ratios of acetylisoniazid to acid-labile isoniazid 24 hours after 
giving matrix isoniazid and of acetylsulphadimidine to sulphadimidine 5-6 hours after giving 
sulphadimidine (P<O~OOl). This finding confirms the conclusion of White & Evans (1968) that 
there are significant differences between individuals within the same phenotype, and provides 
further evidence for the close parallelism between the acetylation of isoniazid and sulphadimidine 
in man. The inaccuracy of the calorimetric isoniazid method at the concentrations encountered 
in the samples from the rapid acetylators (Ellard and Gammon, in preparation) probably prevented 
a similar correlation being demonstrated among the rapid acetylators. 
The ratios of calorimetrically-determined acetylisoniazid/acid-labile isoniazid in the urine samples 
obtained from the 204 patients investigated in the third study indicated that there were 112 slow 
acetylators (ratios < 7.0) and 92 rapid acetylators (ratios > 7.0). Since there were no significant 
differences between the three studies in the calorimetrically determined acetylisoniazid/acid-labile 
isoniazid ratios, the results are illustrated in an amalgamated form in Fig. 3. The bimodal distribu- 
tion of the results is clearly apparent and a ratio of 7 was chosen as the most suitable value for 
distinguishing between the 2 phenotypes. Only 12 (4%) of the 310 urine samples had ratios of bet- 
ween 6 and 8. The investigation of the change with time in the magnitude of the ratio of acetyli- 
soniazid/acid-labile isoniazid excreted in the urine showed that 24 hours after giving 600 mg 
matrix isoniazid it was increasing by about 4 % per hour in the slow acetylator and 8 % per hour in 
the rapid acetylator. It was therefore concluded that misclassification was unlikely to occur on 
I 
O-62 l-25 2.5 5-O 10.0 20-o 40.0 >50 
Ratio of acetylisoniazid to acid-labile isoniazid 
FIG. 3 
Distribution of subjects according to the ratios of acetylisoniazid to acid-labile 
isoniazid in the urine. (All three studies). 
ELLARD AND OTHERS 
account of patients not providing urine samples at precisely 23-24 hours. Furthermore slight 
delays in the absorption of isoniazid that could result if the test dose were taken shortly after a meal 
instead of on an empty stomach would also be unlikely to result in misclassification. The method 
would therefore appear to have the additional practical advantage of being relatively unaffected by 
slight lapses on the part of the subject to fully comply with the instructions. 
It is suggested that this method would be particularly convenient for phenotyping tuberculosis 
out-patients who could take the test dose of matrix isoniazid one day and return 23 hours later to 
provide the required urine sample. This would avoid the patient having to wait the 6-8 hours 
required in the isoniazid method of Eidus and others (1974), or in the sulphadimidine method 
(Evans, 1969; Viznerova, Slavikova and Ellard, 1973). These latter methods would however be 
more suitable for population surveys of the relative prevalence of the slow and rapid acetylator 
phenotypes. The results obtained in the second study, and a comparison of the results obtained in the 
third study with those obtained in a’large study using sulphadimidine (Viznerova, Slavikova and 
Ellard, 1973) suggest that in its present form the isoniazid matrix method may not discriminate 
between the 2 phenotypes as well as the sulphadimidine method. However further studies using 
higher doses of matrix isoniazid (30 mg/kg) and determining the urinary ratios of acetylisoniazid/ 
acid-labile isoniazid fluorimetrically, indicate that when the method is modified in this way, its 
discrimination is superior to that of the sulphadimidine method (Ellard and Gammon, in prepara- 
tion). 
Identical proportions of slow acetylators (55 ‘A) were found among the 204 East African patients 
ACETYLATOR PHENOTYPE 209 
investigated in the second and third studies, and this proportion is similar to those found in other 
populations of Negro descent (Evans, Manley & McKusick, 1960; Evans, 1962; Dufour, Knight & 
Harris, 1964; Fawcett 8c Gammon, 1975). The proportion of slow acetylators found among the 
much smaller number of British students investigated in the first study (61%) is also similar to that 
found among other populations of Caucasian descent (see Ellard, 1975). In the controlled EastAfrican clinical trials no significant differences in response to treatment could be demonstrated 
between rapid and slow acetylators, whether they were being treated for 12 months with 4 or 8 
weeks of daily streptomycin, isoniazid and thiacetazone followed by twice-weekly isoniazid plus 
thiacetazone or for 6 months with 8 weeks of daily treatment with streptomycin, isoniazid, rifam- 
picin and pyrazinamide followed by streptomycin plus isoniazid plus pyrazinamide twice-weekly 
(East African/British Medical Research Council Study, 1974~, B, respectively). 
We should like to thank the British Council and Finnish Anti-Tuberculosis Association for financially supporting 
this investigation, and Prof. D. A. P. Evans for his help and advice. 
REFERENCES 
DYMOND, L. C. & RUSSELL, D. W. (1970). Rapid determination of isonicotinic acid hydrazide in whole blood with 
2, 4, 6-trinitrobenzene sulphonic acid. Clinica Chimica Acta, 27, 513. 
DUFOUR. A. P., KNIGHT, R. A. & HARRIS, H. W. (1964). Genetics of isoniazid metabolism in Caucasian. Negro and 
Japanese populations. Science, 145, 391. 
EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL INTERMITTENT THIACETAZONE INVESTIGATION (1974~). A pilot 
study of two regimens of intermittent thiacetazone plus isoniazid in the treatment of pulmonary tuberculosis in 
East Africa. Tuber&, 55, 211. 
EAST AFRICAN/BRITISH MEDICAL RESEARCH COUNCIL (1974~). Controlled clinical trial of four short-course (Qmonth) 
regimens of chemotherapy for treatment of pulmonary tuberculosis. Second study. Lancet, 2, 1100. 
EIDU~, L., HARNANANSINGH, A. M. T. & JESSAMINE, A. G. (1971). Urine test for phenotyping isoniazid inactivators. 
American Review of Respiratory Disease, 104, 587. 
EIDU~, L., HODGKIN, M. M., SHAEFER, 0. & JESSAMINE, A. G. (1974). Distribution ofisoniazid inactivators in Eskimos 
and Canadian college students by a urine test. Revue canadienne de biologic, 33, 117. 
ELLAI~~, G. A. (1975). Variations between individuals and populations in the acetylation of isoniazid and its signifi- 
cance for the treatment of pulmonary tuberculosis. Paper presented at the 3rd Deer Lodge Symposium of 
~Clinical Pharmacology 23/9/74, to be published in Clinical Pharmacology and Therapeutics. 
ELLARD, G. A., GAMMON, P. T., POLANSKY, F., V~ZNEROVA, A., HAVL~K, I., & Fox, W. (1973). Further studies on the 
pharmacology of a slow-release matrix preparation of isoniazid (Smith and Nephew HS 82) of potential use 
in the intermittent treatment of tuberculosis. Tubercle, 54, 57. 
ELLARD, G. A.. GAMMON, P. T. & TIITINEN. H. (1973). Determination of the acetylator phenotype from the ratio 
of the urinary excretion of acetylisoniazid to acid-labile isoniazid: A study in Finnish Lapland. Tubercle, 54,201. 
ELLARD, G. A., GAMMON, P. T. & WALLACE, S. M. (1972). The determination of isoniazid and its metabolites acety- 
Iisoniazid, monoacetyl-hydrazine. diacetyl-hydrazine, isonicotinic acid and isonicotinylglycine in serum and urine. 
Biochemical Journal, 126, 449. 
EVANS, D. A. P. (1962). Pharmacogenetique. Medicine et Hygiene, 20, 905. 
EVANS, D. A. P. (1969). An improved and simplified method of detecting the acetylator phenotype. Journal ofMedical 
Genetics, 6, 405. 
EVANS. D. A. P., MANLEY, K. A. & MCKUSICK, V. A. (1960). Genetic control of isoniazid metabolism in man. British 
Medical Journal, 2, 485. 
FAWCETT. I. W., & GAMMON, P. T. (1975). The determination of the acetylator phenotype in a Northern Nigerian 
population. Tubercle, 56, 199. 
VARLEY, H. (1962). Practical Chnical Biochemistry, 3rd edition, p. 634, Heinemann, London. 
VENKATARAMAN, P., EIDUS, L., & TRIPATHY, S. P. (1968). Method for the estimation of acetylisoniazid in urine. 
Tubercle, 49, 210. 
VENKATARAMAN, P., MENON, N. K., NAIR, N. G. K., RADHAKRISHNA, S., Ross, C. & TRIPATHY, S. P. (1972). Classifi- 
cation of subjects as slow or rapid inactivators of isoniazid, based on the ratio of the urinary excretion of acety- 
lisoniazid to isoniazid. Tubercle, 53, 84. 
VIZNEROVA, A., SLAvftcovA, Z. & ELLARD, G. A. (1973). The determination of the acetylator phenotype of tuber- 
culosis patients in Czechoslovakia using sulphadimidine. Tuber&e, 54, 67. 
WHITE. T. A. & EVANS, D. A. P. (1968). The acetylation of sulfamethazine and sulfamethoxypyridazine by human 
subjects. Clinical Pharmacology and Therapeutics, 9,80.