FROM T H E DEPARTMENT OF O R A L S U R G E R Y , HEAD: PROF. JOHN HERTZ, R O Y A L SCHOOLS *OF DENTISTRY, STOCKHOLM AND UMEA, AND THE

DEPARTMENT OF PATHOLOGY, HEAD: PROF. CARL-MARTIN FAJERS, MEDICAL SGHOeOL, UMEA, SWEDEN.

DISAPPEARANCE RATE OF TRYPAN BLUE IN RAT PLASMA AFTER

INTRAPERITONEAL INJECTION

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HOLGER THILANDER Received 13.vi.62.

In order to ensure intense vital staining of the tissues repeated in- jections are recommended, and intervals ranging from 2 to 5 days haw been suggested (Cappell 1929; Romeis 1948). Since whatever the mode of administration the dye is transported from the site of injection to the various parts of the body via the circulatory apparatus, it is irn- portant to know the speed with which trypan blue disappears from the plasma after injection, for this will determine the minimum interval between the different injections.

When using a method in which repeated intraperitoneal injections of trypan blue are given a t fairly short intervals, i t is of interest to follow the variation in concentration of the dye in the plasma in the intervals between injections. Any variation in this respect between different intervals would be attributable to a change in the resorption and excretion of the dye.

The results of studies on this relationship after intravenous injection which have been performed by Gregersen & Rawson ( 1943) and Auskaps & Shaw (1955) are probably not valid for intraperitoneal injection sincc the concentration of the plasma is also affected by the speed with which the dye is taken up from the peritoneum.

In order to examine this problem the following study was performed.

M A T E R I A L A N D M E T H O D

Two experiments were carried out. In the first 3 white rats (age about 15 months) were given one intraperitoneal injection of one per cent aqueous solution of trypan blue amounting to one millilitre per 100 g body weight. Blood samples were thcn taken from the tip of the tail, the first after 15 minutes, and subsequent ones after 3/, l%, 21/4, 3, 3%, 4%, 51/+, 6, 7, 9, 12, 15, 20, and 24 hours, and then once a day for 11 days. Each time about 40 h1 of blood were drawn into a heparinized capillary tube, one end of which was then sealed. The specimen was centrifuged, the tube broken at the plasma-haematocrit border and 10 pl of plasma were diluted with 3 ml of distilled water. The absorption of the solution was determined in a Beckman B spectrophotometer at a wavelength of 590 millimicrons. The tests were performed

57

with sample cells having a light path of 5 cm. The zero was adjusted against di- stilled water. On adding 10 .u1 of unstained plasma to 3 ml of distilled water no measurable absorption values could be observed.

From the results of this experiment the optimum interval between intraperitoneal injections was taken as 24 hours. In the second experiment one injection was given daily for 6 days in succession to each of 4 rats. Following the first and last injections samples were taken during the next 24 hours according to the schedule of the pre- vious experiment. On each of the 4 intervening days one sample was taken imme- diately prior to the injection. In other respects the method was as for the previous experiment.

. . r..

.o - I I:

5 10 15 i0 2424487296120120 125 130 135 140 145 150 f t t t t hours t

r.. .o - I I:

5 10 15 20 2424487296120120 125 130 135 140 145 150 f t t t t hours t Fig. 1.

Relationship between time and extinction for plasma in 4 rats receiving one intra- peritoneal injection of one per cent aqueous trypan blue on 6 days in succession.

The arrows mark the various times of injection.

R E S U L T S

The results obtained during the first 24 hours of the second experi- ment, which are reproduced graphically in Fig. 1, were similar to the corresponding results in the first. In all of the animals the concentra- tion of trypan blue in the plasma reached its maximum after about 2 hours. The concentration fell sharply during the following two hours, and after 10 hours it had reached a value which thereafter diminished very slowly. After 12 days there was still a detectable amount of trypan blue in the plasma.

On repeated injections at 24-hour intervals the trypan blue in the plasma increased steadily. Comparison of the extinction curves shows that the increasc in concentration after the last injection was less than the corresponding increase after the first injection, and that after reaching its maximum value the concentration fell slowly and marc steadily.

The 4 animals suffered no ill effects from the administration of trypan blue and, at the end of the experiment, they were deep blue in col ou r.

D I S C U S S I O N

The resorption of dye from the sites of deposit evidently takc place quite rapidly. The curve for a single injection after the maximum con-

centration had been reached is similar to the one after intravenous injection reported by Gregersen & Rawson (1943) and Auskaps & Shaw (1955) , which, however, fell a little more rapidly. The marked drop in the amount of trypan blue in the plasma during the first few hours after the maximum concentration has been reached has been ascribed to the type of bond between the dye and the plasma protein (Rawson 1843; Gregersen & Rawson 1943), whereby the trypan blue is linked primarily to the plasma albumin and only two molecules of the dye arc linked to each albumin molecule. Any excess of trypan blue will then be bound to the plasma globulins. Since the capacity of the plasma protein to combine with trypan blue is limited, if the excess of dye is large part of it will remain free in the plasma and disappear rapidly from the circulation into the tissues and the urine. I t is this penetration into the tissues that accounts for the good staining properties of trypan blue.

Because of the low toxicity of trypan blue and the fact that the plasma concentration falls slowly after 10 hours, the interval between injections could be reduced from the proposed 2-5 days to, say, 24 hours. The continuous increase in plasma concentration in the 24-hour tests appears to be due to an increase in the amount of free trypan blue in the plasma, established by paper electrophoresis examination of plasma taken 30 hours after the last injection.

The results thus show that if an intense vital staining is required for fairly short experimental periods a suitable interval between the intra- peritoneal injections is 24 hours. Longer periods involve a risk of toxic action due to accumulation of free trypan blue in the plasma.

S U M M A R Y

In these experiments a programme for intraperitoneal injection of trypan blue was worked out by examining spectrophotometrically the disappearance rate of trypan blue in the plasma of the white rat.

The material consisted of 7 rats. It was found that injection of one niillilitre of one per cent aqueous solution of trypan blue per 100 mg body weight a t 24 hours intervals for 6 days produced no evident signs of toxicity. There was, however, an accumulation of the stain in the plasma, and electrophoresis tests showed much of it to be in thc free state.

R E F E R E N C E S

Auskaps, A . M . & Shnw, J . H . : Vital staining of calcifying bone and dentin with try-

C a p p r l l , I ) . F . : Intravitam and supravital staining. J. Path. Bact., 32: 595, 1929. Gregersrn, M . I . & Rawson, R . ‘4.: The disappearance nf T-1824 and structurally rc-

lated dyes from the blood stream. Amer. J. Physiol., 138: 698, 1943. Rawson, R . A , : The binding of T-1824 and structurally related diazo dyes by the

plasma proteins. Amer. J. Physiol., 138: 708, 1943. Rorneis, B.: Mikroskopische Technik. 16th Ed. Leibniz Verlag, Munich, 1948.

pan blue. J. Dent. Res., 3 4 : 452, 1955.