Forensic Science International 41 (1989161- 65 Elsevier Scientific Publishers Ireland Ltd.

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A COMPARATIVE PHARMACOKINETIC STUDY OF ETHANOL IN THE BLOOD, VITREOUS HUMOUR AND AQUEOUS HUMOUR OF RABBITS

P. FERNANDEZ, M. LOPEZ-RIVADULLA, J.M. LIRARES, F. TAT0 and A.M. BERMEJO

Department of Legal Medicine, Service of Forensic Toxicology, Faculty of Medicine, University of Santiago de Compostela ISpainJ

(Received May 23rd, 19881 (Revision received July 20th, 19881 (Accepted August 8th, 19881

Summary

The pharmacohinetics of ethanol in the blood, vitreous humour and aqueous humour of the rabbit after intravenous administration of single dose have been compared. Ethanol levels were determined by gas-liquid chromatography. Certain parallelism between ethanol levels in these body fluids were observed after 80 min.

Key words: Pharmacohinetics; Ethanol; Blood; Aqueous humour; Vitreous humour

Introduction

The possibility of determining ethanol in the vitreous humour and correlating the results with its blood levels was first broached by Sturner and Coumbis [l] in 1966. Since then, this hypothesis has been further developed [2--51, and some authors [6,7] have also referred to vitreous humour/blood correlation in relation to barbiturates and salicylates, although the results obtained were not as good as those for ethanol.

In this paper we compare the pharmacokinetics of ethanol in the blood, vitreous humour and aqueous humour of the rabbit, whose volume of ocular fluids is sufficient for toxicological analysis. Given the absence from the literature of any reports on the pharmacokinetics of ethanol in these ocular fluids, the data we present may be useful in interpreting results when the aqueous humour or vitreous humour constitute the sole reference sample.

Materials and Methods

A Perkin Elmer Model Sigma 3 Gas Chromatograph equipped with flame ionization detector was used. The column used was a 2 m x 6 mm glass col- umn packed with 60180 Porapak Q. The instrument parameters used were:

0379-0738/891$03.50 0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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injector and detector temperature 190 OC; column temperature 160 OC; carrier gas (nitrogen) flow rate 80 mllmin. The retention times were estimated as 102 s for the ethanol and 195 s for the internal standard (isopropanol).

Procedure The area ratio of ethanol to isopropanol was calculated according to the

method of Curry et al. [S] and represented against the ethanol concentration in whole blood and water (0.2, 0.5, 1, 2, 2.5 and 3 g/l). The calibration curves obtained were: Water: Y = 0.490 X - 0.040, T = 0.9990; Whole blood: Y = 0.475 X - 0.003, r = 0.9980.

Experimental model Eighteen white rabbits, weighing between 1600 g and 2600 g were used in

the experiment. The animals were anaesthetized with sodium pentobarbitone (40 mglkg), injected slowly into the marginal vein of the ear with constant monitoring of cornea1 reflex and respiratory rate; in some cases a booster (10 mg/kg) was necessary. Tracheotomy was then performed, and the animals connected to a respirator (Braun Melsungen. “Beat Mungspumpe nach Schu- ler DBP”) throughout the rest of the experiment. The femoral vein was drained and an Abbocath T-20 inserted and connected to a syringe contain- ing a solution of heparine in order to prevent clotting of the blood.

Ethanol dissolved in physiological serum was then injected into the mar- ginal vein of the ear in a dose of 0.4 mg/kg for 1 min approximately. Blood samples were extracted through the Abbocath inserted in the femoral vein.

The aqueous humour was removed by penetrating the anterior chamber of the eye taking care not to pierce the iris, which would cause blood to pass into the anterior chamber.

The vitreous humour was extracted through the cornea and the crystal- line, after emptying the anterior chamber of the eye in order to avoid contaminating the sample.

Samples were collected every 10 min starting 10 min after injection of alcohol. Last series of samples were collected 120 min after injection.

Results and Discussion

The average concentration/time curves for ethanol in blood, aqueous humour and vitreous humour are shown in Fig. 1.

The results show that the pharmacokinetic behaviour of ethanol from 10 to 120 min is governed by a monoexponential equation in the blood and a biexponential equation in the aqueous humour and vitreous humour, when the method of residuals was applied [9]:

Blood: C = 0.65 e-O.022 t

Aqueous humour: C = 0.95 e-0.020 t - 0.77 e-0.06’ t

Vitreous humour: C = 1.53 e-0.021 t - 1.72 e+.o45 t

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(LogAEthanolb <g/b

Time (min.>

Fig. 1. Ethanol concentration/time in: (a) blood, (b) aqueous humour and (c) vitreous humour. Each point is the average of three determinations.

The blood kinetics suggest that small doses of ethanol, which is elimi- nated by a process of biotransformation, might be insufficient to produce saturation of the enzymes involved in the process. This may explain the different pharmacokinetic parameters obtained in humans, after the administration of higher doses [lO,ll].

Figure 1 shows that the elimination half-lives of ethanol in the blood, aqueous humour and vitreous humour are similar (32 min, 35 min and 33 min, respectively). The apparent volume of distribution is estimated as 1.57 likg. Both parameters are similar to those obtained in humans [lO,ll] after the administration of analogous doses.

The rapid transport of ethanol from plasma to the ocular fluids, is prob- ably due to its low molecular weight and its chemical affinity for specific components of these fluids (high water solubility of alcohol). The levels of

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TABLE 1

RELATIONSHIPS BETWEEN ETHANOL LEVELS (g/l, IN WHOLE BLOOD (B.1, AQUEOUS HUMOUR (A.H.1 AND VITREOUS HUMOUR (V.H.1

At each time, three samples were studied.

Time hid B./A. H. B./K H. A.H./KH.

10 1.53 3.71 2.42 20 1.03 1.21 1.18 30 0.89 0.97 1.09 40 0.79 0.77 0.97 50 0.71 0.71 1.00 60 0.69 0.60 0.87 70 0.71 0.54 0.75 80 0.66 0.44 0.69 90 0.62 0.41 0.68

100 0.66 0.44 0.67 110 0.62 0.41 0.67 120 0.63 0.42 0.67

Average ratios (80 to 120 min) C.V. (80 to 120 minp

0.64 0.42 0.68 3.22% 3.76% 1.47%

‘C.V., coefficient of variation (average of 15 determinations).

ethanol in the aqueous humour and vitreous humour exceed blood ethanol levels after 30 min.

Table I shows a comparison of ethanol levels in blood, aqueous humour and vitreous humour, all of which show a certain parallelism after 80 min. The coefficients of variation for the last five times studied (15 determina- tions) do not exceed 5%. This is not the case in the previous phase, between 10 and 80 min. during which time there is much greater variation. In the 18 rabbits studied, the average ratio of blood/aqueous humour was 0.80 f 0.15 with a range of 0.62- 1.53; the average ratio of blood/vitreous humour was 0.90 f 0.38 with a range of 0.41-3.71 and the average ratio of aqueous humourlvitreous humour was 0.97 f 0.23 with a range of 0.67 - 2.42.

In 35 human autopsy cases carried out in our laboratory the average ratio of blood/vitreous humour ethanol was 0.91 f 0.14 with a range of 0.72-1.20. The average blood/vitreous humour ratios in rabbits and human cadavers were comparable. However, it is difficult to predict within what ranges of blood ethanol levels this similarity is complied with, because only one alcohol dose was studied and blood ethanol concentrations varied between 0.52 and 0.047 g/l.

In this way, one may conclude that the average ratio blood/vitreous humour obtained in this pharmacokinetic study in rabbits confirm the results found in human cadavers even though a wider range of ratios was observed at the different times studied in the rabbits.

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