Measurement of the concentration of miracil in biological fluids

133 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL ~/[EDICINE AND HYGIENE. Vol. 41. No. 1. S e p t e m b e r , 1947.

MEASUREMENT

OF THE CONCENTRATION IN BIOLOGICAL FLUIDS*

OF MIRACIL

BY A. L. L A T N E R ,

R. V. COXON AND E. J. K I N G .

British Postgraduate Medical School, London.

Miracil D (1-diethylaminoethylamino-4-methyl-thioxanthone) has been used in Germany for the treatment of experimental schistosomiasis ( L a n c e t , 1947). Further trials of the drug are proceeding in this country and the work here described was undertaken in order to, assist the further investigation of its pharmacological behaviour. On first considering the problem of how to, measure the concentration of miracil in body fluids our attention was directed towards fluorimetric procedures of the type extensively used in the study o f mepacrine during the War. However, miracil does not fluoresce in ultra-violet ligh~ and, though it can be caused toJ do so by treatmen~ with various reagents, none of these proved satisfactory in practice, owing to. the presence of high "blank values " in normal blood. In the second place, methods depending on a dyelaking reaction, such as those described for cinchonidine (BRoDm and UDENFRmND, 1945) and for paludrine (KING, WOOTTON and GILCHRIST, t946), were tried. These were' attended with some success, but our most satisfactory results were achieved by a simple colorimetric procedure, based on measurement of the yellow colour of the miracil extracted from the blood and suitably concentrated. DYE-LAKING METHOD. This method (which we demonstrated to. the Biochemical Society in January, 1947) has not been deveI0ped beyond its preliminary stages, but it is described here since, though we have ourselves abandoned it for the present, it may be capable of further refinement and prove usefuI under special conditions, for example in climates where it is desirable to. use solvents having a relatively high boiling-point. * T h i s investigation was u n d e r t a k e n at t h e r e q u e s t of the Medical R e s e a r c h 'Council, to w h o m grateful a c k n o w l e d g e m e n t is m a d e for a g r a n t to defray expenses.

134

MIRACIL

PRINCIPLE. Miracil is extracted f r o m laked blood in the presence of sodium phosphate into ethylene dichloride. T o this solution in ethylene dichloride is added b r o m o - t h y m o l blue (buffered at p H 7) and the m i x t u r e is shaken, with the f o r m a t i o n of a c o m p o u n d of miracil and b r o m o - t h y m o l blue which is soluble in ethylene dichloride. Since the uncoupled dye is insoluble in the organic solvent, a partition of the dye takes place, and the a m o u n t of dye thus removed f r o m the aqueous phase is measured. TECHNIQUE.

Bromo-thymol blue reagent : ~ T h i s is prepared in two stages as follows : First a stock solution is made by dissolving 40 mg. of solid dye in 100 ml. of 1 per cent. alcohol ; secondly, 4 ml. of this solution are diluted to 100 ml. with buffer containing 3.63 grammes of KI-I2PO4 and 14.35 grammes of Na~HPO4.12H~O to the litre. To 5 ml. of fresh whole blood, laked with 10 ml. of water, are added 3 ml. of 0.2 M-Na~HPO4, and then 30 ml. of ethylene dichloride. The mixture is shaken vigorously for 5 minutes in a mechanical shaker, and then centrifuged for 15 minutes at 2,500 revolutions per minute. The upper aqueous layer is aspirated away, leaving the organic layer covered with a pellicle of protein material. The ethylene dichloride is transferred to another bottle, the peHicle being left behind, and is then washed once with 60 ml. of 10 per cent. NaOH and twice withthe same volume of water. At each stage the mixtures are mechanically shaken and later separated by centrifuging as in the original extraction. 25 ml. of the ethylene dichloride layer are removed by means of a teated pipette, and placed in a small stoppered measuring cylinder ; 2.5 ml. of bromothymol blue reagent are then added and the mixture is shaken mechanically for 15 minutes to bring about coupling between miracil and the dye, and is then centrifuged. One ml. of the upper aqueous layer is removed and treated with a micro-drop of l0 N-NaOH. The resulting blue colour is read in a Spekker absorptiometer, using rn~crocups, and the original miracil content is determined from a graph previously prepared. The graph is drawn from the readings obtained, after treating by the above method a series of samples of normal fresh blood to which miracil has been added in known amounts. A graph derived in this way is shown in Fig. 1 and it will be seen that the amount of dye removed from the aqueous solution (as indicated by diminution in intensity of its colour) is proportional to the concentration of miracil present. YELLOW COLOUR METHOD. Miracil exhibits a strong yellow c o l o u r when dissolved in hydrochloric acid and it was f o u n d that this colour varied with the concentration of acid present, showing an analogous t h o u g h opposite tendency to that of the fluorescence of mepacrine (BRoDIE and UDENFRIEND, 1943). This variation in the casd~of miracil is shown in Fig. 2. By transferring the miracil f r o m 5 ml. of bloed into 1 ml. of 0"04 N-HC1 and reading the colour of the latter in a Spekker absorptiometer using microcups, blood concentrations of the order of 10 to: 100 ~g./100 ml. could be handled.

A.

L.

LATNER,

R.

V.

COXON

AND

E.

J.

ICING

135

TECHNIQUE.

Calibration of Instrument. A stock standard solution containing 100 'mg. miracil hydrochloride in 1 litre 0'1 ~T-HC1 is prepared. This is stable for at least 3 months, if kept in the dark. Two ml. of this solution are diluted to 50 ml. with distilled water. Each 0"5 ml. of this dilute solution, when added to 5 ml. of fresh oxalated blood, produces a miracil content eqivalent to 40 ~g./100 ml. A series of 5 ml. blood samples can, therefore, easily be prepared containing total amounts of miracil hydrochloride equivalent to concentrations of 40, 80, 120, 160 and 200 f~g./lO0 ml. respectively. : 0-1~ o-~

0"1,: z 0-25

~ o,~

o

o-zo o

3

o-zo

Oq5 o-to

o

I

mo

~oo

~

~

I

4oo

I

soo

I

600

Fro. 1.--Results of dyelaking procedure using samples of blood containing known concentrations of drug.

Spehker absorptiometer, Hford spectrum red filter,

~0°12

S

i o-it ~o.8

go., o-z

-os

I

~L

I

I

I

I

Fzo. 2.--Relation of intensity of yellow colour of miracil solutions to the concentrafion of HC1 present.

Spekker absorptiometer, mercury light, Ilford spectrum violet filter.

40 80 mrac,~ ~Eo

IZO 16o 200 TO ~LOOD ( . ' ~ / ' O O ~ t )

FIG. 3. - - Yellow colour method for miracil. Results obtained from samples of blood containingknown concentrations of miracil. Npekker absorptiometer, mercury light, Ilford spectrum violet filter.

Each sample is haemolyzed with I0 ml. distilled water, and then made alkaline with 2'5 ml. N-NaOH solution. It is best to carry out the whole of this procedure, from the preparation of the 5 ml. blood sample onwards, in a 500 ml. separating funnel. The clear brown solution thus formed is treated with 0"I ml. caprylic alcohol, and thoroughly extracted by shaking with 25 ml. of ether. It is essential to shake the separating funnel at least 200 times. The emulsion thus formed is as a rule easily broken by the addition of 2 ml. of acetone and is allowed to stand for at least ½ hour until the two layers are well defined. If the emulsion proves especially obstinate, a few drops of absolute alcohol may be added as a further aid to inducing adequate separation. After the lower layer has been run off, the ether layer is collected and set aside in a stoppered bottle while the lower layer is returned to the funnel and again extracted with a further 25 ml. e t h e r . Separation at this stage

136

MIRACIL

usually takes place quite easily. Occasionally, however, it may be necessary m add a few drops of absolute alcohol. The ethereal phase from the second shaking is added to that obtained from the first, the residual aqueous fluid being discarded. The separating funnel is now washed out with 20 ml. 0'5 N-NaOH solution to remove any scum adhering to inner surface, and the combined ether extracts are returned to the cleaned funnel, and shaken well with a further 20 ml. 0'5 N-NaOH. The alkali layer is run of[, and the ether extract washed twice by shaking well with distilled water whose reaction has been adjusted to, p H 7'2. Each of these procedures is liable to be followed by some degree of emulsion formation, but standing for 10 minutes is usually sufficient to bring about an adequate degree of separation. It has been demonstrated that the small amount of ether contained in the emulsions, after this period of time, is usually negligible, and its loss does not materially affect the result. The ether extract, after the final washing, is freed as far as possible from the lower aqueous layer, and transferred to a clean dry 100 ml. separating funnel. This transfer is carried out by pouring the ether through the neck of the larger funnel, and not by running it off through the stem, so as to avoid carrying over any traces of emulsion which might dilute the acid to be added. 1 ml. of 0"04 N-HC1 is now introduced and the small funnel thorougMy shaken by hand for several minutes. There must be at least 600 to-and-fro movements. After being allowed to, settle, the clear lower layer, which is now, as a rule, perceptibly yellow in colonr, is run off into a micro-cup (volume 0'5 ml.) and its coloration measured in a Spekker photoelectric absorptiometer, using an ultra-violet light source, and spectral violet filters (Ilford 601). A typical calibration curve is shown ir~ Fig. 3. It can be seen that there is a satisfactory straight line relationship between the final extinction values, and the known concentrations of miracil hydrochloride. The results have been found to. be consistently reproducible, but it is advisable to check the curve at intervals to allow for possible variations in the instrument. DETERMINATION OF I~V~IRACILCONCENTRATIONIN BLOOD. 5 ml. of oxalated blood are treated as Mreadv described, and the miracil content determined (in terms of miracil hvdrochloride, I~g./100 ml.), from the calibration curve. SOLUTIONS REQUIRED.

Stock standard rniracil hvdrocModde solution : 100 mg. miracil hydrochloride are dissolved in 0"1 N-HCl and made up to 1,000 ml. by the addition of more 0'l N-HC1. This keeps for 3 months. Diluted miracll standard : 2 m|. of the above stock standard are made up to 50 ml. with distilled water.

A.

L.

LATNER,

1R. V ,

E O X O N AND

E.

J.

137

KING

0"04 N-HCI: is prepared by diluting 2 ml. N-HC1 to 50 ml. with distilled water, which has been exactly adjusted to pH 7"2 by the addition ~of dilute NaOH or HC1. It is of extreme importance that the two final washings of the ether extract be carried out with water, which has been adjusted exactly to pH 7"2This adjustment was carried out in our experiments with a Lovibond comparator using phenol red as indicator, and adding dilute HC1 or N a O H as required. For values in the neighbourhood of 20 ~g. or less ot~ miracil solidus t00 ml. blood, it is preferable to use I0 ml. of oxalated blood instead of 5 ml. RESULTS.

(a) Recoveries of added miracil. It was found that, using a calibration curve derived in the manner described above, and then checking the results against given amounts of miracil added to a series of blood samples, satisfactory recoveries could be demonstrated. The results of a set of such trials are shown in Table I.

(b). With blood from animals after dosage with miracil. Our experiments with animals were limited in scope, being confined to demonstrating that our chemical procedure would be applicable to the range TABLE I. RECOVERIES OF MIRACIL ADDED TO BLOOD I N T H E LABORATORY.

added 5 ml. blood in /~g.

.~rp. o u n t to

Equivalent level /,g. per 100 ml.

Amount recovered in /zg. Figures in brackets are percentage recoveries. First experiment.

Second ~xperiment.

Third experiment.

2

40

1-8 (90)

1"6 (80)

2"2 (110)

4

80

3'5 (87)

4.1 (102)

3"5 (87)

6

120

5-8 (96)

6.6 (110)

6.6 (Ii0)

8

160

10

200

7.6

7.3

7.6

(95)

(91)

(95)

9"7 (97)

10"0 (100)

10"3 (103)

138

MInA(JlL

of concentrations which might reasonably be expected in animals receiving therapeutic doses of miracil. It was first shown that blood from untreated rats, rabbits and monkeys (as well as human subjects) gave a negligible reading when subjected to analysis. Results obtained from rabbits a n d rats receiving miracil by parenteral injections are set out in Tables II and III. TABLE II. VALUES FOR. RABBIT BLOOD AFTER INTRAMUSCULAR DOSAGE.

Dose.

T i m e after dose in hours.

50 mg. per kg. body weight.

Blood miraeil in /~g. per 100 ml.

1

125

2

125

3

115

Died in 8 hours 25 rag. per kg. body weight

2

45

20

25

45

15

TABLE I l I . VALUES FOR RAT BLOOD FOLLOWING INTRAPERITONEAL DOSAGE (A and B are different animals).

Dose.

T i m e after dose in hours.

Blood miracil /~g. p e r 100 ml. A

25 rag. per kg. body weight.

B

40 50 20

40 30

Btood w~/s obtained from the rabbits by venipuncture and from the rats by ventricular puncture. COLORIMETRIC M E T H O D FOR URINE. A procedure based on the same principles as that described for blood can be applied equally well to urine. Since the absolute amount of miracil

A. L.

LATNE1R, 1:{. V.

COXON AND E. J. K I N G

139

:that can be conveniently recovered from this source is much greater than is the case with blood samples, a stronger solution of hydrochloric acid can be used for the final reading of the colour and direct comparison with a standard :solution of miracil can be employed in lieu of reference to a calibration curve. The technique which we have found satisfactory for the analysis of urine samples is as follows : To 50 ml. of urine in a separating funnel is added a sufficient volume (2 to 3 ml.) of 0'3 N-NaOH to render the mixture alkaline to. litmus and also 50 ml. of diethyl ether ; the funnel is vigorously shaken, After settling, the lower layer is collected in a flask and the upper layer transferred to a stoppered bottle. The lower layer (which is likely to contain some emulsion) is now returned to the funnel and shaken with a further 50 ml. of ether. On separation, the upper layer is added to the ether extract obtained from the first operation, while the lower is again returned to the funnel and mixed with about 100 ml. of water. This tends to break up any remaining emulsion -enabling a further amount of ether extract to be recovered. The whole of the ether extract is now washed with 10 ml. of 0"3 N-NaOH, any emulsification being counteracted with a few drops of absolute alcohol. The ether layer, after a further washing with water, is then shaken with exactly 10 ml. of N-HC1. The acid layer is withdrawn and its colour compared with standards made up by dissolving 5 and 25 mg. respectively of miracil in 1 litre of N-HC1. If the reading indicates a urinary content of less than 0"5 mg./1, of miracil, it is preferable to repeat the procedure using 100 ml., or even 200 ml., of urine. RESULTS.

When miracil was added artificially to normal urine in the laboratory the amount recovered over a range corresponding to 1 to 5 mg./litre did not differ from that added by more than l0 per cent. In the case of rabbits receiving a parenteral dose of 25 mg. miracil per kg. body-weight, the urine may be expected to contain about 2 mg. per litre. DISCUSSION. The colorimetric method described has the virtue of great simplicity. By running a series of estimations concurrently, manipulating one while another is standing, a large number of estimations can be carried out in a relatively short time. It is important that the larger separating funnel used for the initial separation should be scrupulously clean, since any adherent particles on its wall seem to encourage the formation of troublesome emulsions. Using fresh samples of blood we have never found a blank value greater than the equivalent of 5 ~g. per 100 ml., but if blood is kept at room temperature for longer than 2 hours, readings corresponding to 15 ~g. m a y be reached. The question of the volatility of the solvents is one which merits some mention

140

MIRACIL

since the drug under consideration is intended for combating a tropical disease, so that field trials are likeIy to be made in countries where the atmospheric temperatures are much higher than in the United Kingdom. It seems. to us that since the initial experimental work with miracil is likely to be done in a temperate climate and that there is a prospect of air-conditioning plants being installed on an increasing scale in research laboratories in tropical countries, it was justifiable to introduce a procedure involving an ether-extraction. Trials with the substitution of high-boiling petroleum ether were not successful, nor were attempts to utilize amyl alcohol and amyl acetate. On the other hand, it was found that extraction with ethylene dichloride, as in the dye-laking method, followed by re-extraction of the ethylene dichloride with 85 per cent. lactic acid offered a possible, though more tedious, alternative colorimetric procedure. Regarding the figures in Tables II and III, it should be mentioned that the animals whose blood we tested had received only single doses of the drug ; it is likely that during therapeutic r~gimes invoiving repeated doses the levels encounted will approximate to the earlier and higher ones in our Tables. So far there are not sufficient data available to enable any definite statement to, be made as to the specificity of either of the methods described, but we consider that the colorimetric is probably the more specific as well as the more sensitive. Support for this opinion is found in its negJigible blank value and also in the fact (first noticed when using the method on rabbits',urine) that some metabolic derivative of miracil is produced by animals receiving the drug which differs! from the drug itself in its behaviour towards the extractants. Thus, although this derivative is extracted from alkalinized urine b y ether, it does not pass from the ether into hydrochloric acid, and therefore does not enter into the final colorimetric measurement. Presumably a similar substance may exist in blood, but again it would be prevented from interferin~ with the colorimetric reading by being eliminated dfirin~ the extraction process. SUMMARY. 1. A method of determining the concentration of miracil in blood b y extracting it and measuring its yellow colour is described. 2. A preliminary account of a dye-laking method is also given. 3. The application of the colorimetric procedure to urine is described. 4. Some results obtained with these procedures are recorded. •t 0

Br~oDI~, B. B. & UDEN~IEND, S. & . (1945). Ibid., KING, E. J., WOOTTON, I. D. P. • Lancet. (1947). Leading artlele,

REFERENCES. (1943). J. biol. Chem., 151, 299. 158, 705. GILCI-IRIST,M. (1946). Lancet. 1,886. 1, 414.