Study On Forensic Toxicokinetics Of Isoniazid In Animals

Objective:1. To establish a qualitative and quantitative analysis of INH in biological material by a TLCS and high-performance liquid chromatography after derivatization.2. To develop a toxicokinetics, dynamic distribution, postmortem distribution and postmortem redistribution and toxic decomposition kinetics model of isoniazid in rabbits and rats.3. Study the toxicokinetics, dynamic distribution, postmortem distribution, postmortem redistribution and toxic decomposition kinetics of isoniazid in rabbits and rats, and provide a scientific evidence for the forensic identification of isoniazid poisoning death.Methods1. TCLS and HPLC. The biological samples were treated with salicylaldehyde for derivatization. After being extracted with chloroform: methanol (9:1), derivate was analysed qualitatively and quantitatively by a TLCS and a high-performance liquid chromatography.2.Toxicokinetics of isoniazid in rabbits: Six rabbits were given an intragastric administration of INH a LD50 dose of isoniazid(280mg/kg)by stomach, the blood 1ml were taken from carotid artery at 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 4h, 6h after administration. The biological samples were treated by salicylaldehyde derivatization, the derivate were analyzed by TLCS for qualitative and high-performance liquid chromatography for quantitative analysis.3. Study on postmortem distribution. Six rabbits were given an intragastric administration of INH with a dose of 2 LD50 (560mg/kg). As soon as the blood pressure, respiration and ECG disappeared, the rabbits were dissected, and the specimens such as heart blood, heart liver, spleen, lung, kidney, brain, stomach, were sampled immediately, in which the concentration of INH was determined qualitatively and quantitatively by a TLCS and a high-performance liquid chromatography after derivatization.4. Study on dynamic distribution. After being given an intragastric administration of INH with a dose of LD50 (280mg/kg), three of fifteen rabbits were respectively killed at 45min, 1h, 2h, 4h, 6h, and quickly dissected, the specimens such as heart blood, heart liver, spleen, lung, kidney, brain, stomach, were sampled immediately, in which the concentration of INH was determined qualitatively and quantitatively by a TLCS and a high-performance liquid chromatography after derivatization.5. Study on postmortem redistribution of isoniazid in rats. After being given an intragastric administration of INH with a dose of 1/2 LD50(0.33g/kg) for 1h, fifty-four male rats were executed and preserved at 20℃. Six of them were dissected at 0h, 4h, 8h, 12,24 h, 48h, 72h, 96h after the death. Specimens such as heart blood, heart liver, spleen, lung, kidney, brain, stomach, were sampled immediately, in which the concentration of INH was determined qualitatively and quantitatively by a TLCS and a high-performance liquid chromatography after derivatization.6. Toxic decomposition kinetics.①Study on toxic decomposition kinetics of INH in preserved biological samples. After being given an intragastric administration of INH with a dose of 4LD50 (0.88g/kg) for 1h, 6 dog were executed, and the heart and liver of every dog were sampled and divided into four parts. Three of them were preserved at -20℃,-4℃, 20℃, the last one of blood was added with NAF to 1%, and the last part of liver was fixed in 4% formaldehyde. Both of 1% NAF added blood and 4% formaldehyde fixed liver was stored at 20℃. At 0h, 4h, 8h, 12, 24h, 48h, 72h, 96h, 3weeks, 4weeks, 2month, 3month, 4month, 5month, 6month, 7month, 8month after the death, the concentration of INH in them was determined qualitatively and quantitatively by a TLCS and a high-performance liquid chromatography after derivatization.②Study on toxic decomposition kineticsof isoniazid added in blank human blood. INH was added in blank human blood (10mg/100ml), which was divided into three parts and placed at 20℃. At 0h, 4h, 8h, 12, 24 h, 48h, 72h, 96h, 3weeks, 4weeks, 2 month, 3month, 4month after the storage, the concentration of INH in stored blood was determined qualitatively and quantitatively by a TLCS and a HPLC after derivatization.7. Statistical Methods: The data was collected by software SPSS11.5 and expressed by , drug concentration- time data was processed with WinNonlin pharmacokinetic software.Results1. Symptoms. After being given an intragastric administration of INH with a dose of LD50 for 30min, the rabbits showed a respiratory depression, coma, tonic convulsions, convulsion as grand mal epilepsy. The symptoms were alleviated at 1.5-2 h after the administration. 2 LD50 poisoned rabbits showed a respiratory depression, coma, generalized paroxysmal tonic spasm like grand mal, and died in 2h±30min.2. Toxicokinetics. Toxicokinetics of isoniazid in rabbits met one-compartment model with a first kinetics. Tmax = 0.847h, Cmax = 52.3538μg/ml, K01 = 5.828h-1, K10= 0.5702h-1, AUC = 118.138, t1/2k10=1.2156h, V=4156.55L/kg, AUC=118.138, Cmax=52.3538μg/ml.3. Dynamic distribution. At 0.75h,1h,2h,4h,6 h after being given an intragastric administration of LD50 INH. the order of INH concentration detected in rabbits were stomach, kidney, liver, heart, lung, brain, spleen; the liver, stomach, kidney, lung, brain, spleen, heart; stomach, liver, kidney, lung, brain, heart, spleen; stomach, kidney, liver, heart, brain, spleen, lung; stomach, kidney, liver ,heart, lung, brain and spleen. The toxicokinetics in each organs met one-compartment open model with a first order kinetics,Tmax was 0.961-1.416 h. T1/2 of heart, liver, spleen, lung, kidney, brain and stomach were 2.323h, 2.052h, 0.359h, 2.093h, 4.887h, 0.955h, 3.465h.4. Postmortem distribution. The order of INH detected in poisoning death rabbits after a 2 LD50 dose was stomach (131.59±15.3μg / g) > kidney (52.98±8.6μg / g)> blood (45.4±12.2μg / g)> vitreous humor (40.8±16.3μg/ml)>heart (31.224±16.15μg / g) > brain (28.2±8.5μg/ g)> spleen (25.62±1.37μg / g)> lung (20.89±5.18μg / g)> muscle (20.3±7.5μg / g)> liver (18.5±12.61μg / g) .5. Postmortem redistribution. The content of isoniazid in brain of dead rats stored at 20℃significantly increased at 2 h after death(P<0.05); the content of isoniazid in heart blood and the lungs was significantly increased(P<0.05)at 4 h after death ; the contents of isoniazid in myocardium, spleen and kidney were significantly increased at 8 h after death(P<0.05). The content of isoniazid in heart blood, heart, brain reached a peak at 12 h after death; the content of isoniazid in liver, spleen, and kidney reached a peak at 48 h after death; the content of isoniazid in lung reached the peak at 8 h after death. The contents of isoniazid in stomach wall and muscle showed no significant change. The content of isoniazid in lung, spleen, kidney, heart, heart blood, liver changed more significantly than in brain, muscle and stomach wall.6. Toxic decomposition kinetics. The toxic decomposition kinetics of isoniazid in blood and liver meets the first-order kinetics, could be expressed by Ct=Ae-αt+Be-βt. The contents of isoniazid in livers of poisoned dogs, which were stored at 20℃, 0℃, -20℃and in 4% formaldehyde(20℃), decreased significantly at 12, 12, 24 and 48 h (P <0.05), and the decomposition half-life were 6.07 d, 10.50d, 35.69d and 162.56 d; The contents of isoniazid in blood of poisoned dogs, which were stored at 20℃, 0℃, -20℃and 20℃containing 1% NaF, decreased significantly at 12, 12, 24 and 48 h (P <0.05), and the decomposition half-life were 5.27d, 21.61d, 39.13d and 103.34d. The toxic decomposition kinetics of isoniazid aeeed in blank human blood met the first-order kinetics, could be expressed by Ct=14.233e-0.0176t+71.512e-0.00026t .The decomposition half-life was 289.9d.Conclusion1. A TLCS and HPLC after derivatization for the qualitative and quantitative analysis of INH in biological material has been established in this paper, which can be applied to forensic identification of INH poisoning death.2. A toxicokinetics, dynamic distribution, postmortem distribution and postmortem redistribution and toxic decomposition kinetics model of isoniazid in rabbits and rats have been developed, which can be applied to the study on forensic toxicokinetics of INH. 3. Toxicokinetics of isoniazid in rabbits met one-compartment model with a first kinetics. Tmax = 0.847h, t1/2k10=1.2156h. Content of isoniazid in organs reached a peak at 0.961-1.416h after adminatration, the elimination half time in organs were 0.359h- 4.887h.4. The dynamics distribution of isoniazid in rabbits after an intragastric administration showes different trends, which can be used to determine the death phas in INH poisoning death.5. The postmortem distribution of INH in poisoning death rabbits after a 2 LD50 dose (stomach> kidney> blood> vitreous humor> heart> brain> spleen> lung> muscle > liver) is different from after a LD50 dose. So, besides blood, heart, lung and spleen, vitreous humor, kidney should also be sampled for the qualitative and quantitative analysis of INH in the forensic identification of INH poisoning death, and the dose and death time shoulb be taken into consideration.6. There is a postmortem redistribution of INH in poisoned rat. The content of isoniazid in kidney, blood, humor, heart, brain, spleen, lung of dead rats significantly increased, but not significantly increased in stomach and muscle. It suggests that postmortem redistribution should be taken into consideration in the forensic identification of INH poisoning death, anatomy should be carried out within 4-8 h, peripheral blood and muscle should be sampled for analysis.7. Isoniazid in the stored blood and the liver can be decomposed, the low temperature, formalin fixatione or addition of 1 % NaF can step down the decomposition. Decomposition of INH added in human blood is slower. It suggests that there is a species differences in decomposition of INH. So, samples for determination of INH should be submitted within 12-48h and analysis should be carried out as soon as possible, otherwise, samples should be frozen, added to 1%NaF, fixed with formalin, submitted and analysed as soon as possible. If the sample has stored for a long time, the decomposition kinetic parameters of INH in human blood can be used for the content of INH at the time of death.