Fluorescence test for the detection of ethionamide metabolites in urine

Tt~hercle, Lond.. (1967). 48. 291

FLUORESCENCE

TEST FOR THE DETECTION METABOL1TES IN URINE

OF ETHIONAMIDE

By P. VENKATARAMAN,L. EIDUS, S. P. TRip/crltY and S. Vlil~u Ji'om the Tttherct,losis Chemotherapy Centre*. Madras 31. hulia

SUMMARY

A test for the detection ofmetabolites ofethionamide in urine is described. I1 is based on the observatl.on that these metabolites fluoresce under the eli~ct of ultra-violet iight. in stutlies on volunteers and patients, the test showed po:;itive results in 86'.,'.,~ of specimens collected at !-3 hours after administering 0.5 g. ofethionamide, and in 100",., of those collected at 4-18 hours following administration of the drug. "llle specimens collected at 24 and 48 hours showed positive results in 85':., and 38',';i. respectively. The test is simple, specilic and reproducible.

R~SUM~ L'auteur d6crit un test pour la d6tection des m~tabolites de l'6thionamide dans i'urinc. !1 est bas6 sur i'observation que ces m6tabolites deviennent fluorescents sous l'elt'et de la lumibre ultra-violette. Au cours d'dtudes sur des volontaires et des malades, le test donnait des r6sultats positifs dans 86 o./, des spdcimens collect6s entre i fi 3 heures ape, s administration de,O,5 g d'6thionamide, et dans 100')i, de ceux collect6s 4 "3 18 heures ape,s administration de la drogue. Les sp6cimens collect6s apr~s 24 et 48 heurcs ont donn6 des r6sultats positifs dang 85 et 38';:; des cas respectivement. Le test est simple, sp6cifique et reprodt, ctible.

RESUMEN Se describe un test para detectar los metabolitos de la ethionamida en la orina. Se basa en .la observaci6n que estos metabolitos fluorescen ba jo el efec to de la luz ultravioleta. En estudios efectuados en voluntarios y e n cnfermos el test arroj6 un resultado positivo en el 86% de muestras obtenidas despues de 1 a 3 horas de administrar 0'50 g. de ethionamida, y e n el 100% de las muestras obtenidas 4 a 18 horas despues de administrar la droga, t.as muestras obtenidas a las 24 y 48 horas arrojaron resultados positivos en el 85",,/, y 38 ~',i respectivamente. El test es simple, especifico y reproducible.

Inlroduction After the introduction of cthionamide as a chemotherapeutic agent in the treatment of tuberculosis some workers (Veran and others. 1959) developed microbiological methods, and others * Under the joint auspices of the Indian Council of Medical Research. the Madras State Government. the World Health Organization and the Medical Research Council of Great Britain.

292

l u ~:Rct.t:

(Biede'r. Gerbail & Mazeau, 1961; Eidus, Greenberg & Little, 1961) chemical melthods tbr determining the concentration of this drug in biological fluids. Applying these techniques, Rist, Grumbach & Libermann (1959), Gray, Hamilton &.Eidus (1962) and Greenberg & Eidus (.1962) found that only 1-2~,~ of the ethionamide administered was excreted in an unaltered" form ~ Attempts to check the regularity of self-administration of ethionamide by testing the urine for the unaltered drug were. therefore, unsuccessful. Yamamoto and others (1962) had shown that, unlike ethionamide, some urinary metabolites o f this compound produced a purple fluorescence on exposure to ultra-violet light. These fluorescent compounds, presumably pyridones, ct-~uld be extract,".d from the urine by organic solvents. Based on these observations tile present authors attempted to develop a urine test for checkintt the self-administration of ethionamide. Prcliminary investigations indicated that an organic extract of the metabolites in the urine of a volunteer admir:istered a single dose of. 0.5 g. of elhionamidc would show detinite, though weak, fluorescence under the effect of ultra-violet light in the specimen~ collected after as long a period as 72 l'tours after administration of the drug. It was considered that this property of the urinary metabolites could be made use of in clinical practice, where ethionamide is administered on a daily basis, if the fluorescence could be made more intense ~lnd its duration shortened. These objectives were achieved by treating the chloroform-extract of the urine with alkaline potassium ferricyanide before exposure to ultra-violet light. The details of this investigation are described below.

Reagents

Materials and Melhods

1. 4M potassium phosphate bufl'er, pH7. 2. Chloroform, reagent gradc. 3. 2% aqueous solution of potassium ferricyanidc, freshly prepared. 4. N/10 sodium hydroxide solution. 5." Glacial acetic acid. All the reagents were checked initially for tile presence of any fluorescent impurity. Test Procedure 5 m]. of urine and 0.5 ml. of phosphate buffer were taken in a 15 ~,~ 150 ram. stoppered test-tube, 5 mi. of chloroform were then added and the test-tube manually shaken vigorously. The tube was then centrifuged for 5 minutes at 2090 r.p.m., the chloroform layer was transferred into another tube and 1 mi. of tile potassium ferricyanide solution followed by I tall. of N/I0 sodium hydroxide were added; the tube was again thoroughly shaken. After removing the aqueous phase by means of a Pasteur pipette, 1 ml. of glacial acetic acid was added to the chloroform extract. Tile solution was mixed well and the tube was examined in a dark room under ultra-violet light (350 nl~J.Hanovia Arc tube)with a brown paper as background. The light source was directed away fi-om the reader towards the tube. A positive result was indicated by a bluish-purple fluorescence. A faint bluish-purple fluorescence was reported as a trace positive reaction; a definite bluish-purple fluorescence was reported as positive and graded as 1 + , 2 + or 3 + according to the intensity. A very faint and uncertain fluorescence without the characteristic bluish-purple colour was recorded as 'doubtful' and interpreted as negative.

Plan .and Conduct of the Study The sensitivity, specificity and reproducibility of the test were investigated by (a) collecting urine specimens from patients and volunteers at known intervals after a single oral dose of 0.5 g. of ethionamide, (b) incorporating control specimens of urine obtained from patients and volunteers who were either receiving no drugs or'receiving drugs other than ethionamide, and

IT]tI(JNAMII)I! IS UR1NE

293

(C) performing duplicatc tests on a]iquots of urine obtained from volunteers after ~dministr;tlion of 0.5 g. eth.ionamide. All Ihe investigations wcre conducted "blind'. that is. the person undertaking the tesi wa~ not aware either of tile source or of the time of collection of any a'trine specimen. All the tests were iSerformed and interpreled by one I~erson (PVR). Sensftiv,:ty Volunteers--Each of 11 volunteers was administered 0-5 g. of elhionamide under supervision. :ffter emptying tile bladder. Specimens of urine were collected hourly up to 5 hours and at 8, 12. 24.28. 36. 48. 52 and 72 hours after taking the drug. Four specimens were not col!coted so fllat tile study is based on 139 ;peeimens. Patients-Sixty-nine patients who were receiving treatment at this Centre with drugs other than eth.ionamide and vitamin B-complex were admlnislered, under supervls~on. 0-5 g. of ethionamide, either alone or in combination with other antitubercuf0sis drugs, alter they had emptied Iheir bladders. Spccimcns o f urine were collected [t~ ~, to 72 hours (see ]'able 1) after Ihe administration. Itl a~1~220 specimens were collected. Spec~[~citr--A tot:l[ of 476 urine specimens were collected for studying the speeilicity of the test. Of these.~99 were obtained from 39 vohmteers and 60patients who did not take any drug. A further 237 were collected from 28 vohmtee~w at 1-72 hours (the majority within 24 hours) alter the adminislration of the following drugs : No oI' No. oi" Drugs atut dosage t'o[t#lte(,r.v .~l~e<'ittlells (a) Isoniazid 400 rag. (v, ith 6 rag. of pyridoxine) 5 45 (b) Pyrazinamide 2 g. 4 39 (c) Cyeloserine 0.5 g, 3 26 (d) Pyrazinamide 2 g. 4 cyeloserine 0"5 g. 2 19 (e) Nicotinamide 150 rag. I 6 (f) Nicotinamide 300 rag. 3 27 (g) Pyridoxine 280 nag. 3 27 (h) Vitamin B-complex, 4 tablets* 4 21 (i) Vitamin B-complex, 6 lablets* 3 27 One hundred and forly speeinlens were obtained from 40 patients whn received one or more of the following drugs: (a) streptomyeln (b) thioaeetazone (e) PAS (d) isoniazid (e) pyrazinamide (f) eyeloserine (g) vitamin l~-eomplex. Reproducibility This was studied on two a[iquols from each of 133 specimens ofurine obtained t~romnine volunteers wlto received 0.5 g. of ethionamide. The aliquots were allotted code numbers and tested together 'blind' by the examiner. Results

Sensitivity A large proportion of the urine specimens dolleeted as early as ohe hour after administration of 0"5 g. of eth.ionamide yielded positive results, namely 9 (82 ~,~) of f I from volunteers and 23 (88 ~.',]) of 26 from patients (Table 1). Subsequently. the test result was positi,,e in a// specimens eollecled between 3-24 hours from volunteers and between 4-18 hours from pa3ients. At 48 hours, four of the I 1 specimens from volunteers and six of 15 specimens from patients'yielded a positive result, while at 52 and at 72 hours al[ speeiraens, whether from volunteers or patiertt.% yielded negative results. * Each tablel conlained thiamine 10 rag,, ribofiavine 5 rag,, pyridoxine I illg., nlcotlnamide 10 rag.. eallcillmpanlothen,ate 3 rag. ~l13dcy~nocobal~mine I ~g.

294

"l'UBI=.RC LE TABL~ I.~PERCENIAGEOF VOLUNTEERSAND PATIENTSXVI~HPOSITIVETESI" RI'SUt.TSAFTI'R SUPERVISEDADMINISTRATIONOF0"5 (i. OF EI'IIIONAMIDE Horn" of coilecthm after administration of etltionan.ide

1 2 3 4 5

769 10~12 13~15 16---18 19~21 22~24 25--30 31~36 48 52 72

Vohmteers ... Nmnber tested

Path,nts

Positive test restdts No. ~,'o

9 9 ..

(82) (82)

9i

Itl --I Ill !1 ~"

(too)

-I I I.i '

8 5 4 o o

(73) (45) (36) (o) (o)

Number tested

13 26 17 7 3 3 3 12 7 5 8 14 43 25 8 15

Positive test restdts No. % 7

23 14 6 3~

(54)* 88 (82.) (86)

31

31 lZ ~

(100)

7i Sl 8) 12

35 !6 3 6 0 0

(86) 81 64 (38) (40) (0) (o)

* The brackets indicate percentages based on less than 25 observations. Specificity

Of 476 urine specimens from volunteers and patients who received no drugs or received drugs other than ethioaamide, 3 ('0.6~,,~) showed positive reactions. Of these, two were trace positives, one in a four-hour specimen from a volunteer who took 300 rag. of'nicotinamide and the other in an eight-hour specimen from a patient who took six tablets of vitamin-B-complex; specimens obtained at 1, 2, 8, 12 and 24 hours on the same occasion from the former yielded negative results, as did those collected at l, 2, 4, 12, 24, 30, 36 and 48 hours from the latter. T h e third positive result was a one-plus reaction in an eight-hour specimen from a volunteer who had taken four tablets of vitamin B-complex; however, all the remaining urine specimens collected from this volunteer, at 1, 2, 4 and 24 hours, yielded only negativ,, test results. All three positive reactions occurred in 137 specimens collected from volunteers (.108 specimens) and patients (29 specimens) who had been administered the B-complex vitamins, compared with none of 339 specimens from volunteers and patients who had either not received any drug or had received one or more antituberculosis drug but not the B-complex vitamins. Reproducibility

Table lI correlales the results of duplicate tests on aliquots of 133 specimens of urine collected from volunteers between 1 and 72 hours after administration of ethionamide. Identical results were obtained in 113 (85 ,~ of the specimens, lzurthermore, 56 specimens were negative in both tests and 70 specimens were positive in both tests; in all, therefore, agreement in the interpretation of the duplicate test result was found in 126 (95%) specimens. A negative result on one aliquot and a positive result on its duplicate was obtained in 7 (5%) specimens~these were all late-hour samples, one collectdd at 24 hours, and the remaining six between 30-48, hours after administration of the drug.

I{'IIIlONAMIDI-;

IN

URINI:;

295

TAiILi-: II.--.RI~sUt.TS Oi: DUPLICATE TESTS ON 133 SPi:(.Ti~.li-:NS Oi" l.JRINi!

Result o f lest on afiquot 1

Negative

Negative

56

2

0

0

0 :

58

3

18

I

0

0 ,

22

0

27

Trace

Result o f test on aliquot 2 .. Total 7)'ace

! ~-

2- ~.

3 4.

1+

2

3

17

5

2 ~3

0 0

0 0

2 0

20 1

I ' 23 2 ~, ! , 3

Total

61

23

20

26

3 '

133

Discussion

In tile chemotherapy of tuberculosis as well as other chronic diseases requiring long-term medication it is important to know whether the patient takes the prescribed medicaments regularly for a long period. Therefore an important procedure in the management of tuberculous patients is the testing of the urille for the prescribed drugs (Fox, 1958, 1962" Poole & Stradling, 1960). There are already a number of tests for PAS and isoniazid (see Hobby, 1964; Venkatara'man and others, 1965) but there are very few available for second-line antituberculosis drugs. Recently a simple tes! was introduced for the detection of pyrazinamide (Pines & Richardson, 1964) and a sensitive and reliable method was developed in this Centre for detection of pyrazirmmide al~d cycloserine (Rao and others, 1965)but there was no suitable test for the detection ofethionamide or its metabolites in urine. The method of Rao and others (1965), which involves the ~jse of alkaline sodium nitroprusside as reagent, may detect the presence of ethionamide, however, unpublished results indicate that the reaction obtained in urine specimens of volunteers taking ethionamide is weak and erratic. Yamamoto and others (1962) used Grote's reagent which also contains sodium nitroprusside as the main ingredient. Colour reactions were produced with solutions containing ethionamide in concentrations as low as 5 p.g./ml, but no positive result was obtained with urine specimens collected from persons taking this drug due to the low content o f f r e e ethionamide. The unsatisfactory results with methods based on f r e e ethionamide induced us to design a test for the detection of the metabolites of ethionamide. Bie~ler & Mazeau (1962, 1964) studied the urinary metabolites of ethionamide by ch.romatography. Attempts were made by Yamamoto and others (1962, 1963) to investigate in particular the fluorescent r,.etabolites of the above compound. These metabolites, extracted from the urine with chloroform or other organic solvents, exhibited a purple fluorescence under ultra-violet light. However, such an extract would not be suitable for a test in clinical practice as the test remains positive for too long a period, so that many specimens might be positive despite considerable irregularity in taking the medicament. By introduction of an oxidation procedure the fluorescence of the chloroform extract became more intense in specimens collected up to 10-12 hours following the administration of'the drug, the intensity declining thereafter, and the test yielding only negative reactions beyond 48 hours. The test was positive in ~all specimens collected'between 3-24 hours in volunteers and 4-18 hours in patients after the super+ised administration of 0-5 g. ofethionamide" thereafter the proportion of positive results gradually decreased, in both. groups. All specimens collected at 52 and at 72 hours were negative. From these results it can be inferred that, in patients ~eceiving 0.5 g. ofethionamide daily, a negative result clearly signifies that no drug had been taken during ~.tle preceding 4-i 8 hours" a positive result on the other hand indicates that ethionamide had been taken at some time during the previous 48 hours. Positive reactions occurred in none of the 339 specimens collected from

296

TUt3ERcLE

v o l u n t e e r s a n d patients w h o had either not received a n y d r u g or had received a n t i t u b e r c u l o s i s d r u g s o t h e r th;~n e t h i o n a m i d e (isoniazid, p y r a z i n a m i d e , cycloserine, s t r e p t o m y c i n or t h i o a c e t a zone) a n d in 3 (2-2~ o f 137 specimens from v o l u n t e e r s a n d patients w h o received v i t a m i n s o f the B-complex group. It is k n o w n t h a t m e t a b o l i t e s o f B-complex v i t a m i n s m a y p r o d u c e , on e x p o s u r e to ultra-violet light, f l u o r e s c e n c e - - y e l l o w w i t h riboflavine a n d bluish with derivatives o f nicotinic acid ( N a j j a r & W o o d , 1940, H u f f & Perizweig, 1943), but t h e s e c o u l d usually be distinguished from the c h a r a c t e r i s t i c b l u i s h - p u r p l e fluorescence o f e t h i o n a m i d e metabolites. It m a y be n o t e d that9 these posi:ive reactions o c c u r r e d in e a c h case otlly in one single specimen o u t o f several collected from the same volunteer. A l t h o u g h the incidence of such positive reactions was low in this study, the possibility o f occasional positive reactions due to high doses o f n i c o t i n a m i d e or v i t a m i n B-complex should be b o r n e in mind. T h e i n t e r p r e t a t i o n o f the test result as positive or negative was f o u n d to be r e p r o d u c i b l e " thus, of 133 specimens tested in duplicate, 56 were negative a n d 70 positive on both tests, there being only 7 (5',~',',) discrepancies. T h e s e discrepancies could be a t t r i b u t e d to the presence o f trace a m o u n t s of the m e t a b o l i t e s in specimens o f urine collected at the t e r m i n a l phase o f the excretion p e r i o d - - a p h e n o m e n c , n c o m m o n l y e n c o u n t e r e d in urine tests. in conclusion, the fluorescence test was f o u n d to be sensitive, specific and reproducible. Since the test is simple it c a n be p e r f o r m e d even in small laboratories. We are grateful to the volunteers at the Tuberculosis Chemotherapy Centre for their co-operation, and to the nursing staff fi~r tile collection of specimens of urine. REFERENCES BI~DEa, A.. GERaatL, D.. & MAZVAU. L. (1961). Dosage de F6thionamide (1314 Th) dans le serurn, ie liquid cepl'talorachidien et l'urine. ,4mds. pharm, fr., 19. 200. Blr;Dra, A. & MAZEAU.L. (1962). Etude du metabolisme de l'6thionamide chez I'homme. !. Separation de m6tabolites par chromatographic. Annls. pharm, fr., 20, 211. BIt.:l)t:.r, A. & MAZEAU, [.. ~1964). Recherches sur le m6tabolisme de i'6thionamide chcz I't'tomme. Separation el identification de certains n't6tabolites pat" ct',romatographie sur touche mince. Thdrapie, 19. 897. Elous. L., GREI:NUERG, L., & Ln'tLr~, E. (1961). Methods of assaying antituberculosis drugs in biological material. I. The chemical assay of alpha-ethyl thioisonicotinamide (Th 1314). Am. Rev. resp. Dis.. 84, 98. Fox, W. (1958). The problem ot" self-administration of drugs with particular reference to pulmonary tuberculosis. Tubercle. Lond., 39. 269. Fox, W. (1962). Self.administration of medicaments. A review of published work and a study of the problems. Bull. int. Un. Tubcr 32, 307. GRay, D. G., H,~,.MILTON. E. J., & EIDUS, L. (1962). Ciinico-laboratory studies of alpha-ethylthioisonicotinamide (Th 13 !4). Canad. reed. Assoc. J., 86, 317. Grtgt:NBEgc;, L. & En~u.s, L. (1962). Antituberculosis drugs (Th 1314 and INH) in the host organism. Br. J. Dis. Che.,t.. 56, 124.

HoBnY. G. L. (1964). Practical methods of detecting major antituberculosis drugs in the urine. Adv. Tuberc. Res.. ! 3.98. HuFI:, J. W. &. PERI.ZWEIG, W. A. (1943). N'methylnicotinamide, a metabolite of nicotv:~ic acid in the urine..i, biol. C/tent., 151), 395. NAJJAR, V. A. & WOOD, R. W. (1940). Presence of a hitherto unrecognized nicotinamide deriwltive in human urine. Proc. Sot'. exp. Biol. Med.. 44, 386. PINr:S. A. & RicHArDSON. R. J. (1964). A.simple tablet test foe the detection of pyrazinamide in the urine. Tubercle. Lond., 45. 166. POOLe, G. & STrADtJNC;, P. (1960). Chemotherapeutic pitfalls in the treatment of tuberculosis. Br. reed. J., I, 161. R^o, K. V. N., EIDus, L., Jacou, C. V., & T~n,,trttY, S. P. (1965). A simple test for the detection of pyrazinamide and 9cycloserine in urine. Tubercle, Lond., 46, 199. Rtsr, N., GgUMnaCH, F., & LmeRMaNN, D. (1959). Experiments on the antituberculosis activity of aipha-ethylthioisonicotinamide. Am. Rev. Tuberc., 79,1. VENK^'I',aRAMAN,P., EIDUS, L., RAMACHANDRaN,K., & TrlpA'rH't', S. P. (1965). A comparison of various methods for the detection of isoniazid and its metabolites in urine. Tubercle, Lond., 46, 262. \q/raN, P., Rlst, N., TRICHEREAtJ.R., MOIGNETEAU,C., TOStlVINT, Y., GU~r,rEGO, A., & BLal,",t.C. (1959). Un nouvez~u medicament antituberculeux. Le thioamide de l'acide alpha-ethylisonicotinique ou 1314Th darts le traitemet:: de la tuberculose pulmonaire. Presse Indd., 67,1597. Y^MaMOtO, M., Y^MaGUCHI, W., KUMAZaW^, Y., & T,aK,a,rlasHJ, Y. (1962). Fate of 2-ethyl-thioisonicotinamide (1314 Th) in human beings. Report 1. J. Chest Dis., 6,1036. (In Japanese). Y^MamO'rO, M.. YaMaGHUCI-~, W., & KUMaZAW.~, Y. (1963). Fate of 2-ethyl-thioisonicotinamide (1314 Th) in human beings, Report I 1. J. Chest Dis., 7, 1325. (In Japanese).