Following an intramuscular injection of isoniazid 3 mg/kg body-weight, the urinary

excretion of isoniazid and acetylisoniazid during the periods 0-1, 1-2, 2-3 and 3-4 h was

determined for 124 patients with pulmonary tuberculosis. On the same occasion, the

serum isoniazid concentration at 4½ h was determined by microbiologic assay. The ratios

of acetylisoniazid to isoniazid in the urine collections at 2, 3 and 4 h. were bimodally

distributed. Rules were derived from these. ratios for classifying subjects as slow or

rapid inactivators of isoniazid. There was 100% agreement between the classification

based on each of these ratios and that based on the serum isoniazid concentration at

4½ h

Menon, N K

Nair, N G K

Radhakrishna, S

Ross, Chandra

Tripathy, S P

Venkataraman, P

NIRT Institutional Repository

Indian Med Res 60, 5 May 1972 pp 685-693Classification Of Subjects As Slow Or RapidInactivators Of Isoniazid, Based On TheRatio Of The Urinary Excretion OfAcetylisoniazid To Isoniazid*P. Venkataraman N.K. Menon N.G.K. Nair S. RadhakrishnaChandra Ross and S.P. TripathyTuberculosis Chemotherapy Centre † Madras-31Received for publication January 1 1972Following an intramuscular injection of isoniazid 3 mg/kg body-weight, the urinaryexcretion of isoniazid and acetylisoniazid during the periods 0-1, 1-2, 2-3 and 3-4 h wasdetermined for 124 patients with pulmonary tuberculosis. On the same occasion, theserum isoniazid concentration at 4½ h was determined by microbiologic assay. The ratiosof acetylisoniazid to isoniazid in the urine collections at 2, 3 and 4 h. were bimodallydistributed. Rules were derived from these. ratios for classifying subjects as slow orrapid inactivators of isoniazid.There was 100% agreement between the classificationbased on each of these ratios and that based on the serum isoniazid concentration at4½ h.IntroductionWith the advent of intermittent regimens in the chemotherapy of tuberculosis,the rate of inactivation of isoniazid has attained considerable importance (TuberculosisChemotherapy Centre, Madras 1970). This rate is usually determined by estimatingthe concentration of isoniazid in plasma or serum after oral, intramuscular or intra-venous administration of the drug.The present paper describes a simpler methodrequiring the collection of a single urine specimen after an intramuscular injection ofisoniazid. The method is based on the finding that slow inactivators of isoniazidexcrete a larger proportion of the dose in the free form and a smaller proportion asacetylisoniazid (and isonicotinic acid) in the urine, than do rapid inactivators (Arm-strong and Peart 1960, Mukoyama et al 1963, Peters et al 1965, Short 1962, Tiitinen1969). The efficiency of this (urine) method was assessed in parallel with that of astandard method based on microbiological assay of isoniazid in serum.Material and MethodsPatients : In all, 124 patients with newly-diagnosed pulmonary tuberculosis, the latterpart of an intake of 241 to a chemotherapy study, were included in this investigation.* This article is also being published in the Tubercle (Edin)† The Centre is under the joint auspices of the Indian Council of Medical Research, the Tamil Nadu(Madras State) Government and the World Health Organization in collaboration with the MedicalResearch Council of Great BritainI2 Urine Test for Determining Isoniazid Inactivation RateCollection of specimens :On the day of the test, a specimen of urine was collectedfrom the patient and tested for acetylisoniazid by the method of Eidus and Hamilton(1964). If the result was negative, the patient was given an intramuscular injection ofisoniazid, 3 mg/kg body-weight. Urine collections were made for the periods 0-l,1-2, 2-3 and 3-4 h (and the volumes noted), and blood taken at 4½ h. Urine andserum specimens were then stored at –20°C for periods not exceeding one week.Estimation of isoniazid in serum :The concentration of isoniazid in serum was esti-mated microbiologically, using a vertical diffusion method similar to that describedby Lloyd and Mitchison (1964) (The lower limit for the sensitivity of this method is0.2 µg/ml).It took 2 weeks for the result to become available.Estimation of isoniazid aud acetylisoniazid in urine : Estimations of isoniazid andacetylisoniazid were undertaken on each urine collection.All the urine collections ineach week were examined at a single session, after coding and rearranging them in arandom order.Isoniazid : Isoniazid was extracted by the method of Eidus and Little (1962) and wasestimated by the method of Maher et al (1957) with minor modifications.In brief,3 ml of urine was first mixed with 1 ml of 0.1 N sodium hydroxide solution and 3.2 g ofpowdered ammonium sulphate, and then shaken together with 30 ml of a solvent mixturecontaining chloroform and n- butanol (7 : 3) for 30 min in a rotary shaker. Next,20 ml of the solvent extract was shaken for 15 min with 4 ml of 0.1N sulphuric acid.A 3 ml aliquot of the resulting acid extract was then treated with 0.3 ml of a 2 % vanillinsolution in 25 % ethanol, and the colour intensity read at 380 mµ in a Unicam SP 600spectrophotometer.The concentrations of isoniazid were read from a standardgraph prepared with known concentrations of isoniazid.Acetylisoniazid : Acetylisoniazid was estimated by the method of Venkataraman et l(1968), without treatment with potassium permanganate, and expressed as equivalentof isoniazid.ResultsSerum isoniazid concentrations :The distribution of patients according to the serumisoniazid concentration at 4½ h after an intramuscular dose of 3 mg/kg is set out inGraph 1. On the basis of this histogram, patients with a concentration of 0.90 µg/mlor more were classified as slow inactivators of isoniazid, and those with a concentra-tion of 0.89 µg/ml or less as rapid inactivators.Thus, 67 (54 %) of the 124 patients inthe present investigation were slow inactivators and 57 (46 %) were rapid inactivators.Ratio of urinary excretion of acetylisoniazid to isoniazid : The ratios of the urinaryexcretion of acetylisoniazid to isoniazid at various intervals after an intramuscularinjection of isoniazid 3 mg/kg are illustrated on a logarithmic scale in Graphs 2 and 3.The logarithmic transformation was employed, with success, to equalize the errorvariance (that is, the variance between duplicate estimations of the ratio) in the rapidinactivators and the slow inactivators.P. Venkataraman et alGraph 1Distribution of 241 patients according to the serum isoniazid concentration,4½ h after an intramuscular dose of 3 mg/kg body-weight3Isoniazid concentration (µg/ml)The concentration was < 0.20 µg/ml in 16 patientsThe ratio in the first hour collection failed to separate the patients into two dis-tinct groups (Graph 2) and is therefore not considered further in this report.Con-sidering the ratios for the periods 1-2, 2-3 and 3-4 h (Graph 2), and for O-2 h(Graph 3), the distributions clearly indicate the presence of two distinct groups ofpatients – namely, the slow inactivators (left side) and the rapid inactivators (rightside). The geometric mean ratio and the range for the two groups of patients are pre-sented in Table I, together with the criterion for classifying a patient as slow inactivator.It will be seen that the critical ratio increases with increasing period after the test dose.The classification based on each of the urinary ratios (1-2, 2-3, 3-4, 0-2 h) and thatbased on the standard microbiologic method employing serum were identical for everypatient.The last column of the Table provides an index of the efficiency with which eachof the urinary ratios discriminates between slow and rapid inactivators of isoniazid–the larger the index, the more efficient is the discrimination. The indices for theratios at 2-3 and 3-4 h are similar, and were of the same order as the index (untabula-ted) obtained with the standard microbiologic method on the same set of patients.4 Urine Test for Determining Isoniazid Inactivation RateGraph 2Distributions of patients according to the ratio of acetylisoniazid to isoniazid inurinefor the periods 0-l, 1-2, 2-3 and 3-4 hLog. 10 x Ratio of acetylisoniazid to isoniazidGraph 3Distribution of patients according to the ratio of acetylisoniazid toisoniazid in urine for the period 0-2 h!Log. 10 x Ratio of acetylisoniazid to isoniazidP. Venkataraman et al 5It will also be noticed that the indices for the ratios at 1-2 and 0-2 h are slightlysmaller than the indices for the ratios at 2-3 and 3-4 h.Table I. Ratio of urinary excretion of acetylisonlazid to isoniazid after an intramuscular doseof isoniazid 3 mg/kg body-weightPeriod (hours)after test doseSlow inactivators Rapid inactivatorsMean Range Mean RangeCriterionfor slowinactivatorIndex ofdiscrimination*l - 2 0.74 0.44–1.11 2.19 1.36–5.45 <1.26 24.12 - 3 0.89 0.47–1.40 3.16 1.82–6.32 <1.58 27.43 - 4 1.02 0.43–1.56 4.47 2.48–10.76 <2.00 27.80 - 2 0.71 0.45–1.09 1.78 1.30–2.94 < 1.12 24.2*The difference in means between slow and rapid inactivators, divided by the standard error of thedifferenceHeterogeneity among rapid inactivators :Graph 4 correlates the serum isoniazidconcentration at 4½ h with the ratio of the urinary excretion of acetylisoniazid toisoniazid for the period 3-4 h.T e scattergram demonstrates a clear distinctionbetween the slow inactivators and the rapid inactivators. Also, within the rapidinactivators, it is possible to identify 8 patients (marked as x), all of whom had verylow serum isoniazid concentrations (<0.2 µg/ml) and very high urinary ratios (> 6.3).All eight patients are included in the group of 9 patients (also marked as x) who can beidentified from Graph 5 as having a high ratio for the period 2-3 h (>4.0) and for theperiod 3-4 h (>6.3).Finally, the ratios for the period 1-2 h for these eight patientswere all higher than the corresponding ratios for the remaining patients. Thesefindings demonstrate that there was heterogeneity among the rapid inactivators,and that there were probably 8 or 9 homozygous rapid inactivators in the populationexamined in the present study.Stability of isoniazid and acetylisoniazid in urine :From each of 27 patients (14 slowinactivators, 13 rapid inactivators), collections were made of the total urine excretedduring the period 24-26 h after an oral dose of a slow-release preparation of iso-niazid (35 mg/kg) or soluble isoniazid (15 mg/kg), and estimations of isoniazid andacetylisoniazid were undertaken.After adding a crystal of thymol as a preservative,aliquots of the 27 specimens were stored at 37°C and the estimations repeated at2, 4, 7, 10 and 14 days.The mean isoniazid content for the 27 patients showed a signi-ficant linear decline (P< 10-5), the reduction being 8 % by 7 days and 15 % by 14 days(Table II). In contrast, the mean acetylisoniazid content did not appear to be affectedby the storage. Further analyses, not tabulated here, showed that the classificationas slow or rapid inactivator was not altered for any patient as a result of the loss inisoniazid content from storage.6 Urine Test for Determining Isoniazid Inactivation RateGraph 4Correlation between serum isoniazid concentration at 4½ h and ratio of acetylisoniazidto isoniazid in urine for the period 3-4 hRAPIDLog. 10 x Ratio of acetylisoniazid to isoniazid in urineTable II. Effect of storage at 37°C on isoniazid and acetylisoniazid in urine*Duration of storage(days)Isoniazid(µg/ml)Acetylisoniazid(µg/ml)0 22.8 119.12 23.2 116.34 22.5 109.97 21.0 109.910 19.8 117.714 19.4 116.1*Mean of the values for 27 samplesP. Venkataraman et alGraph 5Correlation between the value-s of the ratio of acetylisoniazid to isoniazidin urine for the periods 2-3 and 3-4 hDiscussionThe methods commonly used for determining the rate of inactivation of isoniazidconsist in measuring the plasma/serum concentrations of the drug 6 h after oraldosage (Evans et al 1960, 1961, Mukoyama et l 1963, Sunahara et al 1961), the serumealf-life of the drug after intravenous or intramuscular injection (Jenne 1960, Scottet al 1969, Tiitinen 1969), or the serum isoniazid concentration 4½ h after intra-muscular injection (Gangadharam et l 1961, Tuberculosis Chemotherapy Centre,Madras 1970).The isoniazid inactivation rate can also be determined by measuringthe percentage of acetylated sulphadimidine in plasma/blood or urine 6-8 h after anoral dose of sulphadimidine (Evans 1969, Rao et al 1970).This paper describes a method for classifying subjects as slow or rapid inactivatorsby measuring the ratio of the urinary excretion of acetylisoniazid to isoniazid for theperiods 0-2, 1-2, 2-3 or 3-4 h after an intramuscular dose of isoniazid 3 mg/kg body-weight. There was 100 % agreement between the classification based on each of the8 Urine Test for Determining Isoniazid Inactivation Rateabove ratios and that based on a standard microbiologic method employing serum.Afurther study of the efficiency of each of these ratios in discriminating between slowand rapid inactivators (Table I) suggested that acollec ion of urine over the period0-2 h would be very satisfactory ;also, that no major extra benefit would be derived(a) by asking the subject to empty his bladder at 1 h and collecting only the urineexcreted during 1-2 h, or (b) by collecting the urine excreted during 2-3 or 3-4 h.The above method of classification, based on the ratio of acetylisoniazid to iso-niazid in a 0-2 h collection of urine, has several advantages.First, it does not re-quire the collection of blood, unlike many of the earlier methods. Secondly, theperiod of waiting is reduced to 2 h for the subject, and the test result can be madeavailable the same day. Lastly, since the classification is based on a ratio, the ac-curacy of the method is unlikely to be affected by the failure to obtain a complete urinecollection. A disadvantage of the method, however, is that the test dose has to beadministered by intramuscular injection.Another limitation, especially under fieldconditions, is the instability of isoniazid in urine.In the present study, storage at 37°Creduced the isoniazid content by 8 % in 7 days and by 15 % in 14 days, despite theaddition of a crystal of thymol as a preservative. However, this did not alter the classi-fication (as slow or rapid) in any instance.It is difficult to differentiate clearly between homozygous and heterozygousrapid inactivators of isoniazid on the basis of the isoniazid half-life after an oral orintramuscular dose of the drug (Scott et al 1969). In the present study, scattergramsrelating the ratio of acetylisoniazid to isoniazid in urine for the period 3-4 h withthe corresponding ratio for the period 2-3 h and with the serum isoniazid concentrationat 4½ h suggested the presence of a group of 8 or 9 homozygous rapid inactivators.Itis of interest that, according to the Hardy-Weinberg law, the expected number ofhomozygous rapid inactivators in the population under study is 8.Since the completion of this study, a method has been described by Russell (1970)for classifying subjects as slow or rapid inactivators by measuring, semi-quantitatively,the ratio of acetylisoniazid to isoniazid in a specimen of morning urine obtained afteroral dosage with 300 mg of isoniazid in 3 divided doses (after meals) on the previousday. Although such a method might be technically convenient, it is unlikely to be verypracticable under field conditions.More recently, Eidus et al (1971) have describeda method similar to ours, but collecting the urine over the period 6-8 h after anintramuscular test dose of isoniazid 8 mg/kg.AcknowledgmentWe are grateful to the nursing staff for organizing the collection of blood and urinespecimens, to our colleague Miss S. Joseph for the microbiologic assays, and toDr. G.A. Ellard and Prof. D.A. Mitchison for valuable suggestions in the preparationof the report.P. Venkataraman et al 9ReferencesArmstrong, A.R. and Peart, H.E.1960. A comparison between the behaviour of Eskimos and non-Eskimos to the administration of isoniazid.Am Rev Resp Dis81, 588.Eidus, L. and Hamilton,. E.J. 1964. A new method for the determination of N-acetylisoniazid inurine of ambulatory patients. Am Rev Resp Dis 89, 587.Eidus, L. Hamanansingh, A.M.T. and Jessamine, A.G.1971. Urine test for phenotyping isoniazidinactivators. Am Rev Resp Dis 104, 587.Eidus, L. and Little, E. 1962.Methods of assaying antituberculous drugs in biological material. II.The chemical assay of isoniazid.Am Rev Resp Dis 85, 278.Evans, D.A.P. Manley, K.A. and McKusick, V.A.1960. Genetic control of isoniazid metabolism inman. Br Med J ii, 485.Evans, D A.P. Storey, P.B. and McKusick, V.A. 1961. Further observations on the determinationof the isoniazid inactivator phenotype.Bull John Hopkins Hosp 108, 60.Evans. D.A.P. 1969. An improved and simplified method of detecting the acetylation phenotype. JMed Genetics 6, 405.Gangadharam, P.R.J. Bhatia, A.L. Radhakrishna, S. and Selkon, J.B. 1961. Rate of inactivation ofisoniazid in South Indian patients with pulmonary tuberculosis. I : Microbiological assay ofisoniazid in serum following a standard intramuscular dose.Bull WHO 25, 765.Jenne, J.W. 1960. Studies of human patterns of isoniazid metabolism using an intravenous fall-offtechnique with a chemical method. Am Rev Resp Dis 81, .Lloyd, J. and Mitchison, D.A. 1964.isoniazid. J Clin Path 17, 622.A vertical diffusion method for the microbiological assay ofMaher, J.R. Whitney, J.M. Chambers, J.S. and Stanonis, D.J.tion of isoniazid and para-aminosalicylic acid in body fluids.1957. The quantitative determina-Am Rev Tub Pul Dis 76, 852.Mukoyama, H. Sunahara, M. Ogawa, M. and Kawai, K.1963. Urinary excretion of metabolites ofisoniazid and inactivation of sulfonamide in rapid, intermediate and slow inactivators of isoniazid.Kekkaku 38, 1.Peters J.H. Miller, K.S. and Brown, P.1965. Studies on the metabolic basis for the genetically deter-mined capacities for isoniazid inactivation in man.J Pharm Exp Therap 150, 298.Rao, K.V.N. Mitchison, D.A. Nair, N.G.K. Prema, K. and Tripathy, S.P. 1970. Sulphadimidineacetylation test for classification of patients as slow or rapid inactivators of isoniazid.Br Med Jiii, 495.Russell, D.W. 1970. Simple method for determining isoniazid acetylator phenotype. Br Med J iii,4 9 5 .Scott, E.M. Wright, R.C. and Weaver,. D.W.1969. The discrimination of phenotypes for rate odisappearance of isonicotinyl hydrazide from serum. J Clin Invest 48, 1173.Short, E.I. 1962. Studies on the inactivation of isonicotinyl acid hydrazide in normal subjects andtuberculous patients. Tubercle (Edin) 43, 3.Sunahara, S. Urano, M. and Ogawa, M.tion. Science 134, 1530.1961. Genetical and geographic studies on isoniazid inactiva-Tiitinen, H. 1969. Isoniazid and ethionamide serum levels and inactivation in Finnish subjects. ScandJ Resp Dis 50, 110.Tuberculosis Chemotherapy Centre, Madras 1970. A controlled comparison of a twice-weekly andthree once-weekly regimens in the initial treatment of pulmonary tuberculosis. Bull WHO 43,143.Venkataraman, P. Eidus, L. and Tripathy, S.P.. 1968.urine. Tubercle (Edin) 49, 210.Method for the estimation of acetylisoniazid in

Inactivators of Isoniazid, Based on The Ratio of The Urinary Excretion of Acetylisoniazid To Isoniazid

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