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Distribution of tetracaine and its metabolite in rabbits after high versus normal spinal anesthesia
Forensic Science International 124 (2001) 130–136
Distribution of tetracaine and its metabolite in rabbits after high versus normal spinal anesthesia Yukiko Hinoa, Hidefumi Inoueb, Keiko Kudoa, Naoki Nishidaa, Noriaki Ikedaa,* a
Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan b Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Received 18 May 2001; received in revised form 15 August 2001; accepted 20 August 2001
Abstract High spinal anesthesia is one cause of sudden death associated with the spinal anesthesia. We did animal experiments to verify high spinal anesthesia by analyzing tetracaine and its metabolite, p-butylaminobenzoic acid in tissue samples. Tetracaine (0.25% in 10% glucose solution) 0.21–0.28 mg/kg was administered to two groups of rabbits to induce high and normal spinal anesthesia. Tetracaine and the metabolite in rabbit tissues were analyzed by gas chromatography–mass spectrometry, as a free base for tetracaine and as tert-butyldimethylsilyl derivative for the metabolite. In the group given high spinal anesthesia, levels of the metabolite in the brain stem were higher than in the cerebrum, cerebellum and whole blood. On the other hand, in the group given normal spinal anesthesia, the opposite results were obtained. Therefore, high spinal anesthesia induced by tetracaine can be diagnosed by comparing the concentrations of metabolite in whole blood, cerebrum, cerebellum and brain stem. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forensic toxicology; Spinal anesthesia; Tetracaine; p-Butylaminobenzoic acid; Tissue distribution
1. Introduction Tetracaine, also known as amethocaine, is one of the ester-type local anesthetic drugs and is commonly used to induce spinal anesthesia. Local anesthetic drugs used for spinal anesthesia occasionally spread to high regions of cerebrospinal nerves, and can lead to sudden death due to the direct action on the central nervous system [1–11]. In such cases, it is of important to clarify the cause of death in view of forensic toxicology. There are reports on the determination of local anesthetics in human tissues in cases of high spinal anesthesia [9,11]. However, the diagnosis of high spinal anesthesia was not made. We find no documentation of such studies. To design a method to diagnose high spinal anesthesia, we prepared animal models of high and normal spinal anesthesia by giving tetracaine in rabbits and we compared the distribution
* Corresponding author. Tel.: þ81-92-642-6124; fax: þ81-92-642-6126. E-mail address: [email protected] (N. Ikeda).
of tetracaine and one of its metabolites, p-butylaminobenzoic acid, in tissue samples.
2. Experimental 2.1. Animal experiment This experiment was reviewed by the Committee of the Ethics on Animal Experiment in Graduate School of Medical Sciences, Kyushu University and carried out under the control of the Guideline for Animal Experiment in Faculty of Medicine, Kyushu University and The Law (No. 105) and Notification (No. 6) of the Japanese Government. The rabbit model established by Langerman et al. [12] was used with some modifications. Tetracaine solution was made as follows. Twenty milligram of tetracaine hydrochloride was dissolved in 8 ml of 10% glucose solution for injection to make up a hyperbaric solution of 0.25% tetracaine hydrochloride. Male Japanese white rabbits, weighing 2.68–3.19 kg, were anesthetized with inhalation of diethyl ether, and
0379-0738/01/$ – see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 1 ) 0 0 5 8 5 - 0
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
supplementary doses were intermittently given to maintain the surgical level of anesthesia. The 10 rabbits were fixed on an operating table in the prone position and divided into two groups of five. For one group, the table was placed in the head-up position to induce normal spinal anesthesia, and on the other group, the table was placed in the head-down position to induce high spinal anesthesia. In each group, surgical fields in the lumbar areas were anesthetized with lidocaine. The skin and subcutaneous fascia were opened by a straight midline incision, between the second and fourth lumbar areas. Muscles from both sides of the third lumbar spinous process were separated by blunt dissection. The process, ligament flavum, and extradural fat were sequentially removed and the dural sac was exposed. Lumber puncture was carefully performed using a 25-gauge and 40 mm needle for nerve blockade, and 200–300 ml (0.21– 0.28 mg/kg) of the solution was slowly injected into the subarachnoid space. The dosage was determined based on the dose administered to patients for orthopedic surgery [13]. The block level of spinal anesthesia was assessed by the response to pain stimulus using pin-prick. In each group, 200–300 ml saline solution was injected into the subarachnoid space of one rabbit as control. To determine the distribution pattern of tetracaine and its metabolite following the intravenous administration of tetracaine, tetracaine (1.08 mg/kg) was injected into the marginal ear vein of another rabbit. In the rabbits given normal spinal anesthesia and intravenous injection, these rabbits were sacrificed by an overdose of thiopental. In the group given high spinal anesthesia, the rabbits died only under influence of high spinal anesthesia. The cerebrum, cerebellum, brainstem, some parts of the spinal cord and whole blood were collected from each dead rabbit then stored at 20 8C until analysis. 2.2. Reagents Tetracaine hydrochloride,dibucaine hydrochloride, p-butylaminobenzoic acid, and p-dimethylaminobenzoic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan), Teikoku Chemical Industries (Osaka, Japan), Aldrich Chemical (Milwaukee, WI, USA), and Ishizu Seiyaku (Osaka, Japan), respectively. N-(tert-butyldimethylsilyl)-Nmethyltrifluoroacetamide (MTBSTFA) used for tert-butyldimethylsilyl (t-BDS) derivatization was purchased from GL Sciences (Tokyo, Japan). Acetonitrile and tert-butyl methyl ether were of analytical grade and were purified by distillation. Other chemicals used were of analytical grade. 2.3. Analysis of tissue samples The concentrations of tetracaine and p-butylaminobenzoic acid in tissue samples were determined using our methods [13,14] but with some modifications. Extraction of tetracaine in brain and spinal cord: a potion (0.5 g) of each sample was homogenized in 3.0 ml distilled
131
water and 1.0 ml 0.2 M sodium acetate–acetic acid buffer (pH3.8) containing 0.15 mmolneostigmine (esteraseinhibitor) and 2 ml of internal standard solution (IS1: 1000 ng dibucaine, IS2: 200 ng p-dimethylaminobenzoic acid) in a 30-ml centrifuge tube. To the mixture, 10 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min then centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10-ml centrifuge tube, adjusted to pH 9.5 with 100 ml of 20% sodium carbonate. To the mixture, 2 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min, then centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 20 ml of tert-butyl methyl ether, and a 2-ml aliquot of the solution was injected onto gas chromatography–mass spectrometry (GC–MS). Extraction of p-butylaminobenzoic acid in brain and spinal cord. The organic layer from the initial extraction was transferred into a 30-ml centrifuge tube containing 2 ml of 0.5 M sodium hydroxide solution and 2 ml of 0.05 M sodium hydrogen carbonate/0.1 M sodium hydroxide buffer (pH 10.5). The mixture was then shaken for 10 min and centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10-ml centrifuge tube and neutralized by adding 4 M hydrogen chloride solution. To the solution was added 1 ml of 0.2 M sodium acetate/acetic buffer (pH 3.8) and 2 ml of tert-butyl methyl ether, and the preparation was shaken and centrifuged. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 ml of acetonitrile, and 10 ml of MTBSTFA was added to the solution for t-BDS derivatization. The solvent was kept at 60 8C for 1 h and a 2-ml aliquot of the solution was injected onto a GC–MS apparatus for p-butylaminobenzoic acid analysis. Extraction of tetracaine and p-butylaminobenzoic acid in whole blood. Concentrations of tetracaine in whole blood were determined using the above-mentioned method. Concentrations of p-butylaminobenzoic acid in whole blood were determined by single extraction with methylene chloride to completely precipitate protein, and we followed the same procedure described above [13]. Determination of tetracaine and p-butylaminobenzoic acid by GC–MS. Two extracts each containing tetracaine or the metabolite were separately introduced to GC–MS, and the same conditions of GC–MS were used for analyses of tetracaine and the metabolite. The apparatus used was a HewlettPackard 5989A GC–MS system. A fused-silica capillary column, HP-1 (12 m 0:2 mm i.d., 0.33 mm film thickness), coated with 100% dimethylpolysiloxane stationary phase was used. Splitless injection mode was selected with a valve off time of 2 min. The GC–MS conditions were as follows: the initial temperature of 100 8C was held for 2 min, then the temperature was programmed to 300 8C at a rate of 20 8C/min and maintained for 1 min. Temperatures of the injection port and transfer line were each maintained at 280 8C. Helium was used as the carrier gas with a flow rate of 1 ml/min.
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Selected ion monitoring (SIM) mode was used. For determination of tetracaine and its metabolite, ions of m/z 176, 116, 250 and 178 were selected for tetracaine, dibucaine (IS1), p-butylaminobenzoic acid and p-dimethylaminobenzoic acid (IS2), respectively.
problem was overcome by adding 0.05 M sodium hydrogen carbonate/0.1 M sodium hydroxide buffer (pH 10.5) to the aqueous layer. Lipid in the spinal cord was removed from the aqueous layer with this buffer and clean extracts were obtained. Typical chromatograms of tetracaine and its metabolite obtained from the brain and whole blood are shown in Figs. 1 and 2.
3. Results and discussion 3.1. Application of spinal anesthesia Tetracaine solution was injected into the subarachnoid space directly through a special needle used for nerve blockade. The rabbits tolerated the direct spinal anesthesia by carefully puncturing the dural sac and slowly injecting tetracaine. They showed no signs of excitation after the injection. Nerve injury of the spinal cord or leakage of the solution was never observed at the site of injection, in any rabbit. In the group given normal spinal anesthesia, the rabbits were awakened after the injection of tetracaine, and loss of response to pain stimulus was observed only below the waist. In the control rabbit given only saline solution, no change was observed. These rabbits died immediately after the injection of thiopental. In the rabbit given an intravenous injection of tetracaine, the rabbit was awakened after the injection, and analgesic skin level by pain stimulus was not observed. Moreover, the rabbit given tetracaine in a four times higher dosage (1.08 mg/kg) than that used for spinal anesthesia did not die and it was sacrificed by giving an overdose of thiopental. In the group of high spinal anesthesia, the rabbits began dozing soon after the injection of tetracaine, and the respiratory rate gradually decreased. Total paralysis of the body was apparent, and pain stimulus was not observed at the region of the neck or ear. All five rabbits fell into respiratory and cardiac arrest in the end. It took a few minutes in three of them and more than 10 min in the remaining two. This difference of survival time was probably due to slight differences of the rate and force of injection, the direction of injection, the anatomical configuration of the spinal column and positioning of the rabbit. In the control rabbit given only saline solution, no change was observed. This rabbit died immediately after the injection of thiopental. 3.2. Analysis of tetracaine and p-butylaminobenzoic acid in brain and spinal cord Our method developed for the analysis of tetracaine [13,14] in whole blood and tissues was examined to determine levels of tetracaine in brain and spinal cord. Tetracaine could be determined and there were no interfering peaks. With respect to analysis of p-butylaminobenzoic acid in the spinal cord, emulsions occurred in the aqueous layer at the step of back extraction by 0.5 M sodium hydroxide, presumably related to lipids present in the spinal cord. This
3.3. Concentrations of tetracaine and its metabolite in tissue samples The concentrations of tetracaine and its metabolite in tissue samples are shown in Tables 1–3. Tetracaine and its metabolite were not detected from control rabbits. As shown in Table 1, tetracaine was not detected in brain tissues and cervical spinal cord in the group given normal spinal anesthesia. On the other hand, tetracaine was detected in brain tissues, spinal cord and whole blood in some rabbits given high spinal anesthesia (Table 2). In the brain stem, tetracaine was detected in four of five rabbits. In addition, as shown in Table 3, tetracaine was not detected in spinal cord and brain stem in rabbit given intravenous administration. Therefore, the detection of tetracaine in brain stem was considered to be useful to diagnose high spinal anesthesia in rabbits. However, tetracaine was not detected in human tissue samples, including brain, whole blood, liver, skeletal muscle and adipose tissue in our case [14]. This was probably due to the higher cholinesterase activity in human than that in rabbit tissues [15,16]. Moreover, according to our data, tetracaine added to human blood is rapidly hydrolyzed to the metabolite. Therefore, tetracaine was not considered to be a proper target compound to determine when forensic diagnosis of high spinal anesthesia is required for human cases. As shown in Tables 1–3, the metabolite was detected in every sample. In the group given normal spinal anesthesia, the levels of the metabolite in brain stem were lower than in the cerebrum, cerebellum and whole blood, and levels of the metabolite in cervical spinal cord were the lowest in every sample (Table 1). In the rabbit given an intravenous administration, levels of metabolite in the brain stem were lower than in the cerebrum and cerebellum, as shown in Table 3. The distribution was exactly the same as that in the group given normal spinal anesthesia. Therefore, the metabolite found in the brain was derived from the circulating blood in the group given normal spinal anesthesia and an intravenous administration. In the group given high spinal anesthesia, there were differences on the concentrations of metabolite in each rabbit (Table 2). These differences were probably due to individual differences in survival time and a degree of distribution of tetracaine in the cerebrospinal fluid, etc. However, despite these differences, levels of the metabolite in the brain stem were always higher than in the cerebrum, cerebellum and whole blood. These results suggest that the metabolite found in the brain stem was mainly derived from
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
Fig. 1. SIM chromatograms of tetracaine extracted from brain and whole blood of Nos. 2 and 9 rabbits. 133
134 Y. Hino et al. / Forensic Science International 124 (2001) 130–136
Fig. 2. SIM chromatograms of metabolite extracted from brain and whole blood of Nos. 1 and 6 rabbits.
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
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Table 1 Concentrations of tetracaine and its metabolite in rabbit tissues in cases of normal spinal anesthesia (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
1 2.75 0.27 30
2 2.68 0.28 31
3 2.92 0.21 41
4 3.02 0.25 30
5 2.72 0.28 24
Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite
Cerebrum Cerebellum Brain stem
NDa ND ND
148.1 109.3 28.8
ND ND ND
105.2 65.6 61.4
ND ND ND
105.7 79.7 68.0
ND ND ND
170.7 193.6 110.7
ND ND ND
293.4 348.9 182.8
Spinal cord Cervical Thoracic Lumbar
ND 139.2 60874.3
20.2 254.5 6012.2
ND 228.8 50057.3
59.6 121.5 9456.3
ND 367.8 54586.0
60.6 123.5 2470.3
ND 1906.3 122697.8
109.4 647.4 4216.9
ND 814.6 197130.7
137.3 835.4 7705.5
Heart blood Peripheral blood
ND 4.9
170.4 104.6
126.9 141.1
79.1 104.3
ND ND
205.8 204.4
64.4 85.4
207.8 220.5
151.6 228.3
334.1 373.8
a
Not detected.
Cerebrospinal fluid is usually collected for toxicological examinations in autopsy cases of death following spinal anesthesia. However, detection of local anesthetics in cerebrospinal fluid at several sites of the spinal cord can only prove the existence of local anesthetics in the subarachnoid space and a reliable diagnosis of high spinal anesthesia is not feasible. To our knowledge, this paper is the first describing how to diagnose high spinal anesthesia. Moreover, in the case of high spinal anesthesia induced by other local anesthetics, a similar distribution pattern of drugs in brain and whole blood will be likely. Therefore, our results of animal experiments will aid in diagnosing the cause of death following spinal anesthesia induced by various kinds of local anesthetics. This method will be useful when examining human samples for forensic studies.
the cerebrospinal fluid, and it was considered that the tetracaine solution had spread to the high region of the spinal cord and brain stem, where this drug was hydrolyzed to the metabolite. Hence, tetracaine acted directly on the central nervous system, and led to death of the rabbits. Therefore, we concluded that high spinal anesthesia induced by tetracaine can be diagnosed by comparing the concentrations of metabolite in whole blood, cerebrum, cerebellum and brain stem. In the brain stem, the concentration of metabolite in medulla oblongata is most important, because this site is in close proximity to the cervical spinal cord, and the center of the respiratory system locates at this site. With regard to the spinal cord, the same distribution pattern of the metabolite was observed in both groups of rabbits. Therefore, the spinal cord is not suitable tissue for use for diagnosing high spinal anesthesia.
Table 2 Concentrations of tetracaine and its metabolite in rabbit tissues in cases of high spinal anesthesia (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
6 2.68 0.28 3
7 2.81 0.27 2
8 3.19 0.24 15
9 2.83 0.27 13
10 2.80 0.27 5
Tetracaine Metabolite Tetracaine Metabolite Tetracaine Metaholite Tetracaine Metabolite Tetracaine Metabolite Cerebrum Cerebellum Brain stem
NDa ND 69.9
79.2 70.8 303.7
ND ND 93.8
128.4 130.6 419.7
ND ND ND
537.0 512.0 845.1
ND 102.9 307.1
486.5 723.9 2825.9
307.0 3490.6 12952.6
417.4 1788.4 6960.0
Spinal cord Cervical Thoracic Lumbar
385.7 63453.4 170639.4
650.4 6074.4 6718.8
3326.5 79864.6 269978.8
648.8 1157.4 3230.5
3227.8 145632.6 142665.7
978.1 4604.5 3392.9
3659.4 96455.2 130721.3
1890.0 3940.0 5582.2
19208.2 90281.3 207471.4
2263.4 2523.8 2831.8
Heart blood Peripheral blood
372.7 551.8
251.4 171.6
88.0 122.0
168.2 87.6
102.2 59.3
151.1 123.0
51.0 87.0
461.1 368.1
55.5 60.0
173.3 141.8
a
Not detected.
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Table 3 Concentrations of tetracaine and its metabolite in rabbit tissues following intravenous administration (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
11 3.02 1.08 26 Tetracaine
Metabolite
Cerebrum Cerebellum Brain stem
268.3 199.5 NDa
4597.5 2973.1 800.3
Spinal cord Cervical Thoracic Lumbar
ND ND ND
411.0 332.4 341.8
Heart blood Peripheral blood
148.8 403.9
521.4 498.4
a
Not detected.
Acknowledgements We express our special gratitude to R. Esaki and Dr. K. Yamashita for technical assistance and M. Ohara for language assistance. Part of this work was presented at the 38th International Meeting of the International Association of Forensic Toxicologists held in Helsinki, Finland, August 2000.
References [1] U. Aromaa, M. Lahdensuu, D.A. Cozantis, Severe complications associated with epidural and spinal anaesthesias in Finland 1987–1993. A study based on patient insurance claims, Acta Anaesthesiol. Scand. 41 (1997) 445–452. [2] R.L. Carpenter, R.A. Caplan, D.L. Brown, C. Stephenson, R. Wu, Incidence and risk factors for side effects of spinal anesthesia, Anesthesiology 76 (1992) 906–916.
[3] G.L. Zeitlin, Possible decrease in mortality associated with anaesthesia. A comparison of two time periods in Massachusetts, USA, Anaesthesia 44 (1989) 432–433. [4] R.A. Caplan, R.J. Ward, K. Posner, F.W. Cheney, Unexpected cardiac arrest during spinal anesthesia: a closed claims analysis of predisposing factors, Anesthesiology 68 (1988) 5–11. [5] J. Abramowitz, Cardiac arrest during spinal anesthesia. I, Anesthesiology 68 (1988) 970. [6] M.H. Zornow, M.S. Scheller, Cardiac arrest during spinal anesthesia. II, Anesthesiology 68 (1988) 970–971. [7] D.L. Brown, R.L. Carpenter, D.C. Moore, L.D. Bridenbaugh, S.M. Rupp, D.H. Ramsey, G.E. Thompson, M.F. Mulroy, Cardiac arrest during spinal anesthesia. III, Anesthesiology 68 (1988) 971–972. [8] J.R. Jones, Cardiac arrest during spinal anesthesia. IV, Anesthesiology 68 (1988) 973. [9] K. Sakata, S. Ishigaki, M. Sakata, Distribution of lidocaine and disopyramide in human blood and tissue, a case report of death caused by spinal anesthesia, Forensic Sci. Int. 37 (1988) 1–10. [10] D.A. Davis, An analysis of anesthetic mishaps from medical liability claims, Int. Anesthesiol. Clin. 22 (1984) 31–42. [11] M. Fukui, Medicolegal study of spinal anesthesia, Jpn. J. Legal Med. 24 (1970) 136–158. [12] L. Langerman, G. Chaimsky, E. Golomb, M. Tverskoy, A.I. Kook, S. Benita, A rabbit model for evaluation of spinal anesthesia: chronic cannulation of the subarachnoid space, Anesth. Analg. 71 (1990) 529–535. [13] K. Kudo, Y. Hino, N. Ikeda, H. Inoue, S. Takahashi, Blood concentrations of tetracaine and its metabolite following spinal anesthesia, Forensic Sci. Int. 116 (2001) 9–14. [14] Y. Hino, N. Ikeda, K. Kudo, A. Tsuji, Sensitive and selective determination of tetracaine and its metabolite in human samples by gas chromatography–mass spectrometry, J. Anal. Toxicol. 24 (2000) 165–169. [15] J.R. Kambam, B. Horton, W.C.V. Parris, S.A. Hyman, M.L. Berman, B.V.R. Sastry, Pseudocholinesterase activity in human cerebrospinal fluid, Anesth. Analg. 68 (1989) 486–488. [16] D.R. Winters, B.G. Harper, I.G. Resnick, Automated procedure for estimation of blood cholinesterase activities in rabbits, Lab. Anim. Sci. 47 (1997) 407–410.
Distribution of tetracaine and its metabolite in rabbits after high versus normal spinal anesthesia Yukiko Hinoa, Hidefumi Inoueb, Keiko Kudoa, Naoki Nishidaa, Noriaki Ikedaa,* a
Department of Forensic Pathology and Sciences, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan b Department of Anesthesiology and Critical Care Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Received 18 May 2001; received in revised form 15 August 2001; accepted 20 August 2001
Abstract High spinal anesthesia is one cause of sudden death associated with the spinal anesthesia. We did animal experiments to verify high spinal anesthesia by analyzing tetracaine and its metabolite, p-butylaminobenzoic acid in tissue samples. Tetracaine (0.25% in 10% glucose solution) 0.21–0.28 mg/kg was administered to two groups of rabbits to induce high and normal spinal anesthesia. Tetracaine and the metabolite in rabbit tissues were analyzed by gas chromatography–mass spectrometry, as a free base for tetracaine and as tert-butyldimethylsilyl derivative for the metabolite. In the group given high spinal anesthesia, levels of the metabolite in the brain stem were higher than in the cerebrum, cerebellum and whole blood. On the other hand, in the group given normal spinal anesthesia, the opposite results were obtained. Therefore, high spinal anesthesia induced by tetracaine can be diagnosed by comparing the concentrations of metabolite in whole blood, cerebrum, cerebellum and brain stem. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Forensic toxicology; Spinal anesthesia; Tetracaine; p-Butylaminobenzoic acid; Tissue distribution
1. Introduction Tetracaine, also known as amethocaine, is one of the ester-type local anesthetic drugs and is commonly used to induce spinal anesthesia. Local anesthetic drugs used for spinal anesthesia occasionally spread to high regions of cerebrospinal nerves, and can lead to sudden death due to the direct action on the central nervous system [1–11]. In such cases, it is of important to clarify the cause of death in view of forensic toxicology. There are reports on the determination of local anesthetics in human tissues in cases of high spinal anesthesia [9,11]. However, the diagnosis of high spinal anesthesia was not made. We find no documentation of such studies. To design a method to diagnose high spinal anesthesia, we prepared animal models of high and normal spinal anesthesia by giving tetracaine in rabbits and we compared the distribution
* Corresponding author. Tel.: þ81-92-642-6124; fax: þ81-92-642-6126. E-mail address: [email protected] (N. Ikeda).
of tetracaine and one of its metabolites, p-butylaminobenzoic acid, in tissue samples.
2. Experimental 2.1. Animal experiment This experiment was reviewed by the Committee of the Ethics on Animal Experiment in Graduate School of Medical Sciences, Kyushu University and carried out under the control of the Guideline for Animal Experiment in Faculty of Medicine, Kyushu University and The Law (No. 105) and Notification (No. 6) of the Japanese Government. The rabbit model established by Langerman et al. [12] was used with some modifications. Tetracaine solution was made as follows. Twenty milligram of tetracaine hydrochloride was dissolved in 8 ml of 10% glucose solution for injection to make up a hyperbaric solution of 0.25% tetracaine hydrochloride. Male Japanese white rabbits, weighing 2.68–3.19 kg, were anesthetized with inhalation of diethyl ether, and
0379-0738/01/$ – see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 9 - 0 7 3 8 ( 0 1 ) 0 0 5 8 5 - 0
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
supplementary doses were intermittently given to maintain the surgical level of anesthesia. The 10 rabbits were fixed on an operating table in the prone position and divided into two groups of five. For one group, the table was placed in the head-up position to induce normal spinal anesthesia, and on the other group, the table was placed in the head-down position to induce high spinal anesthesia. In each group, surgical fields in the lumbar areas were anesthetized with lidocaine. The skin and subcutaneous fascia were opened by a straight midline incision, between the second and fourth lumbar areas. Muscles from both sides of the third lumbar spinous process were separated by blunt dissection. The process, ligament flavum, and extradural fat were sequentially removed and the dural sac was exposed. Lumber puncture was carefully performed using a 25-gauge and 40 mm needle for nerve blockade, and 200–300 ml (0.21– 0.28 mg/kg) of the solution was slowly injected into the subarachnoid space. The dosage was determined based on the dose administered to patients for orthopedic surgery [13]. The block level of spinal anesthesia was assessed by the response to pain stimulus using pin-prick. In each group, 200–300 ml saline solution was injected into the subarachnoid space of one rabbit as control. To determine the distribution pattern of tetracaine and its metabolite following the intravenous administration of tetracaine, tetracaine (1.08 mg/kg) was injected into the marginal ear vein of another rabbit. In the rabbits given normal spinal anesthesia and intravenous injection, these rabbits were sacrificed by an overdose of thiopental. In the group given high spinal anesthesia, the rabbits died only under influence of high spinal anesthesia. The cerebrum, cerebellum, brainstem, some parts of the spinal cord and whole blood were collected from each dead rabbit then stored at 20 8C until analysis. 2.2. Reagents Tetracaine hydrochloride,dibucaine hydrochloride, p-butylaminobenzoic acid, and p-dimethylaminobenzoic acid were purchased from Wako Pure Chemical Industries (Osaka, Japan), Teikoku Chemical Industries (Osaka, Japan), Aldrich Chemical (Milwaukee, WI, USA), and Ishizu Seiyaku (Osaka, Japan), respectively. N-(tert-butyldimethylsilyl)-Nmethyltrifluoroacetamide (MTBSTFA) used for tert-butyldimethylsilyl (t-BDS) derivatization was purchased from GL Sciences (Tokyo, Japan). Acetonitrile and tert-butyl methyl ether were of analytical grade and were purified by distillation. Other chemicals used were of analytical grade. 2.3. Analysis of tissue samples The concentrations of tetracaine and p-butylaminobenzoic acid in tissue samples were determined using our methods [13,14] but with some modifications. Extraction of tetracaine in brain and spinal cord: a potion (0.5 g) of each sample was homogenized in 3.0 ml distilled
131
water and 1.0 ml 0.2 M sodium acetate–acetic acid buffer (pH3.8) containing 0.15 mmolneostigmine (esteraseinhibitor) and 2 ml of internal standard solution (IS1: 1000 ng dibucaine, IS2: 200 ng p-dimethylaminobenzoic acid) in a 30-ml centrifuge tube. To the mixture, 10 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min then centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10-ml centrifuge tube, adjusted to pH 9.5 with 100 ml of 20% sodium carbonate. To the mixture, 2 ml of tert-butyl methyl ether was added and the preparation was shaken for 10 min, then centrifuged at 850 g for 15 min. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 20 ml of tert-butyl methyl ether, and a 2-ml aliquot of the solution was injected onto gas chromatography–mass spectrometry (GC–MS). Extraction of p-butylaminobenzoic acid in brain and spinal cord. The organic layer from the initial extraction was transferred into a 30-ml centrifuge tube containing 2 ml of 0.5 M sodium hydroxide solution and 2 ml of 0.05 M sodium hydrogen carbonate/0.1 M sodium hydroxide buffer (pH 10.5). The mixture was then shaken for 10 min and centrifuged at 850 g for 15 min. The aqueous layer was transferred to a 10-ml centrifuge tube and neutralized by adding 4 M hydrogen chloride solution. To the solution was added 1 ml of 0.2 M sodium acetate/acetic buffer (pH 3.8) and 2 ml of tert-butyl methyl ether, and the preparation was shaken and centrifuged. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 50 ml of acetonitrile, and 10 ml of MTBSTFA was added to the solution for t-BDS derivatization. The solvent was kept at 60 8C for 1 h and a 2-ml aliquot of the solution was injected onto a GC–MS apparatus for p-butylaminobenzoic acid analysis. Extraction of tetracaine and p-butylaminobenzoic acid in whole blood. Concentrations of tetracaine in whole blood were determined using the above-mentioned method. Concentrations of p-butylaminobenzoic acid in whole blood were determined by single extraction with methylene chloride to completely precipitate protein, and we followed the same procedure described above [13]. Determination of tetracaine and p-butylaminobenzoic acid by GC–MS. Two extracts each containing tetracaine or the metabolite were separately introduced to GC–MS, and the same conditions of GC–MS were used for analyses of tetracaine and the metabolite. The apparatus used was a HewlettPackard 5989A GC–MS system. A fused-silica capillary column, HP-1 (12 m 0:2 mm i.d., 0.33 mm film thickness), coated with 100% dimethylpolysiloxane stationary phase was used. Splitless injection mode was selected with a valve off time of 2 min. The GC–MS conditions were as follows: the initial temperature of 100 8C was held for 2 min, then the temperature was programmed to 300 8C at a rate of 20 8C/min and maintained for 1 min. Temperatures of the injection port and transfer line were each maintained at 280 8C. Helium was used as the carrier gas with a flow rate of 1 ml/min.
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Selected ion monitoring (SIM) mode was used. For determination of tetracaine and its metabolite, ions of m/z 176, 116, 250 and 178 were selected for tetracaine, dibucaine (IS1), p-butylaminobenzoic acid and p-dimethylaminobenzoic acid (IS2), respectively.
problem was overcome by adding 0.05 M sodium hydrogen carbonate/0.1 M sodium hydroxide buffer (pH 10.5) to the aqueous layer. Lipid in the spinal cord was removed from the aqueous layer with this buffer and clean extracts were obtained. Typical chromatograms of tetracaine and its metabolite obtained from the brain and whole blood are shown in Figs. 1 and 2.
3. Results and discussion 3.1. Application of spinal anesthesia Tetracaine solution was injected into the subarachnoid space directly through a special needle used for nerve blockade. The rabbits tolerated the direct spinal anesthesia by carefully puncturing the dural sac and slowly injecting tetracaine. They showed no signs of excitation after the injection. Nerve injury of the spinal cord or leakage of the solution was never observed at the site of injection, in any rabbit. In the group given normal spinal anesthesia, the rabbits were awakened after the injection of tetracaine, and loss of response to pain stimulus was observed only below the waist. In the control rabbit given only saline solution, no change was observed. These rabbits died immediately after the injection of thiopental. In the rabbit given an intravenous injection of tetracaine, the rabbit was awakened after the injection, and analgesic skin level by pain stimulus was not observed. Moreover, the rabbit given tetracaine in a four times higher dosage (1.08 mg/kg) than that used for spinal anesthesia did not die and it was sacrificed by giving an overdose of thiopental. In the group of high spinal anesthesia, the rabbits began dozing soon after the injection of tetracaine, and the respiratory rate gradually decreased. Total paralysis of the body was apparent, and pain stimulus was not observed at the region of the neck or ear. All five rabbits fell into respiratory and cardiac arrest in the end. It took a few minutes in three of them and more than 10 min in the remaining two. This difference of survival time was probably due to slight differences of the rate and force of injection, the direction of injection, the anatomical configuration of the spinal column and positioning of the rabbit. In the control rabbit given only saline solution, no change was observed. This rabbit died immediately after the injection of thiopental. 3.2. Analysis of tetracaine and p-butylaminobenzoic acid in brain and spinal cord Our method developed for the analysis of tetracaine [13,14] in whole blood and tissues was examined to determine levels of tetracaine in brain and spinal cord. Tetracaine could be determined and there were no interfering peaks. With respect to analysis of p-butylaminobenzoic acid in the spinal cord, emulsions occurred in the aqueous layer at the step of back extraction by 0.5 M sodium hydroxide, presumably related to lipids present in the spinal cord. This
3.3. Concentrations of tetracaine and its metabolite in tissue samples The concentrations of tetracaine and its metabolite in tissue samples are shown in Tables 1–3. Tetracaine and its metabolite were not detected from control rabbits. As shown in Table 1, tetracaine was not detected in brain tissues and cervical spinal cord in the group given normal spinal anesthesia. On the other hand, tetracaine was detected in brain tissues, spinal cord and whole blood in some rabbits given high spinal anesthesia (Table 2). In the brain stem, tetracaine was detected in four of five rabbits. In addition, as shown in Table 3, tetracaine was not detected in spinal cord and brain stem in rabbit given intravenous administration. Therefore, the detection of tetracaine in brain stem was considered to be useful to diagnose high spinal anesthesia in rabbits. However, tetracaine was not detected in human tissue samples, including brain, whole blood, liver, skeletal muscle and adipose tissue in our case [14]. This was probably due to the higher cholinesterase activity in human than that in rabbit tissues [15,16]. Moreover, according to our data, tetracaine added to human blood is rapidly hydrolyzed to the metabolite. Therefore, tetracaine was not considered to be a proper target compound to determine when forensic diagnosis of high spinal anesthesia is required for human cases. As shown in Tables 1–3, the metabolite was detected in every sample. In the group given normal spinal anesthesia, the levels of the metabolite in brain stem were lower than in the cerebrum, cerebellum and whole blood, and levels of the metabolite in cervical spinal cord were the lowest in every sample (Table 1). In the rabbit given an intravenous administration, levels of metabolite in the brain stem were lower than in the cerebrum and cerebellum, as shown in Table 3. The distribution was exactly the same as that in the group given normal spinal anesthesia. Therefore, the metabolite found in the brain was derived from the circulating blood in the group given normal spinal anesthesia and an intravenous administration. In the group given high spinal anesthesia, there were differences on the concentrations of metabolite in each rabbit (Table 2). These differences were probably due to individual differences in survival time and a degree of distribution of tetracaine in the cerebrospinal fluid, etc. However, despite these differences, levels of the metabolite in the brain stem were always higher than in the cerebrum, cerebellum and whole blood. These results suggest that the metabolite found in the brain stem was mainly derived from
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
Fig. 1. SIM chromatograms of tetracaine extracted from brain and whole blood of Nos. 2 and 9 rabbits. 133
134 Y. Hino et al. / Forensic Science International 124 (2001) 130–136
Fig. 2. SIM chromatograms of metabolite extracted from brain and whole blood of Nos. 1 and 6 rabbits.
Y. Hino et al. / Forensic Science International 124 (2001) 130–136
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Table 1 Concentrations of tetracaine and its metabolite in rabbit tissues in cases of normal spinal anesthesia (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
1 2.75 0.27 30
2 2.68 0.28 31
3 2.92 0.21 41
4 3.02 0.25 30
5 2.72 0.28 24
Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite Tetracaine
Metabolite
Cerebrum Cerebellum Brain stem
NDa ND ND
148.1 109.3 28.8
ND ND ND
105.2 65.6 61.4
ND ND ND
105.7 79.7 68.0
ND ND ND
170.7 193.6 110.7
ND ND ND
293.4 348.9 182.8
Spinal cord Cervical Thoracic Lumbar
ND 139.2 60874.3
20.2 254.5 6012.2
ND 228.8 50057.3
59.6 121.5 9456.3
ND 367.8 54586.0
60.6 123.5 2470.3
ND 1906.3 122697.8
109.4 647.4 4216.9
ND 814.6 197130.7
137.3 835.4 7705.5
Heart blood Peripheral blood
ND 4.9
170.4 104.6
126.9 141.1
79.1 104.3
ND ND
205.8 204.4
64.4 85.4
207.8 220.5
151.6 228.3
334.1 373.8
a
Not detected.
Cerebrospinal fluid is usually collected for toxicological examinations in autopsy cases of death following spinal anesthesia. However, detection of local anesthetics in cerebrospinal fluid at several sites of the spinal cord can only prove the existence of local anesthetics in the subarachnoid space and a reliable diagnosis of high spinal anesthesia is not feasible. To our knowledge, this paper is the first describing how to diagnose high spinal anesthesia. Moreover, in the case of high spinal anesthesia induced by other local anesthetics, a similar distribution pattern of drugs in brain and whole blood will be likely. Therefore, our results of animal experiments will aid in diagnosing the cause of death following spinal anesthesia induced by various kinds of local anesthetics. This method will be useful when examining human samples for forensic studies.
the cerebrospinal fluid, and it was considered that the tetracaine solution had spread to the high region of the spinal cord and brain stem, where this drug was hydrolyzed to the metabolite. Hence, tetracaine acted directly on the central nervous system, and led to death of the rabbits. Therefore, we concluded that high spinal anesthesia induced by tetracaine can be diagnosed by comparing the concentrations of metabolite in whole blood, cerebrum, cerebellum and brain stem. In the brain stem, the concentration of metabolite in medulla oblongata is most important, because this site is in close proximity to the cervical spinal cord, and the center of the respiratory system locates at this site. With regard to the spinal cord, the same distribution pattern of the metabolite was observed in both groups of rabbits. Therefore, the spinal cord is not suitable tissue for use for diagnosing high spinal anesthesia.
Table 2 Concentrations of tetracaine and its metabolite in rabbit tissues in cases of high spinal anesthesia (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
6 2.68 0.28 3
7 2.81 0.27 2
8 3.19 0.24 15
9 2.83 0.27 13
10 2.80 0.27 5
Tetracaine Metabolite Tetracaine Metabolite Tetracaine Metaholite Tetracaine Metabolite Tetracaine Metabolite Cerebrum Cerebellum Brain stem
NDa ND 69.9
79.2 70.8 303.7
ND ND 93.8
128.4 130.6 419.7
ND ND ND
537.0 512.0 845.1
ND 102.9 307.1
486.5 723.9 2825.9
307.0 3490.6 12952.6
417.4 1788.4 6960.0
Spinal cord Cervical Thoracic Lumbar
385.7 63453.4 170639.4
650.4 6074.4 6718.8
3326.5 79864.6 269978.8
648.8 1157.4 3230.5
3227.8 145632.6 142665.7
978.1 4604.5 3392.9
3659.4 96455.2 130721.3
1890.0 3940.0 5582.2
19208.2 90281.3 207471.4
2263.4 2523.8 2831.8
Heart blood Peripheral blood
372.7 551.8
251.4 171.6
88.0 122.0
168.2 87.6
102.2 59.3
151.1 123.0
51.0 87.0
461.1 368.1
55.5 60.0
173.3 141.8
a
Not detected.
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Table 3 Concentrations of tetracaine and its metabolite in rabbit tissues following intravenous administration (ng/g) Rabbit number Weight (kg) Dose (mg/kg) Survival time after injection (min)
11 3.02 1.08 26 Tetracaine
Metabolite
Cerebrum Cerebellum Brain stem
268.3 199.5 NDa
4597.5 2973.1 800.3
Spinal cord Cervical Thoracic Lumbar
ND ND ND
411.0 332.4 341.8
Heart blood Peripheral blood
148.8 403.9
521.4 498.4
a
Not detected.
Acknowledgements We express our special gratitude to R. Esaki and Dr. K. Yamashita for technical assistance and M. Ohara for language assistance. Part of this work was presented at the 38th International Meeting of the International Association of Forensic Toxicologists held in Helsinki, Finland, August 2000.
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