Studies of enzyme-catalyzed modification of proteins. I. Tyrosinase-catalyzed modification of asparaginase.

Asparaginase [EC 3.5.1.1.] of Escherichia coli, an anti-tumor enzyme, was inactivated in a time-dependent fashion by mushroom tyrosinase [EC1.14.18.1.]. The inactivation did not proceed, however, when heat-inactivated tyrosinase was used. Exculusion of the atmospheric oxygen or addition of diethyldithiocarbamate, a copper selective chelating agent, prevented the inactivation. The difference absorption spectrum of tyrosinase-inactivated asparaginase versus intact asparaginase exhibited the appearance of marked absorption peaks at 300 and 350 nm. These results indicate that the tyrosyl residue(s) of asparaginase, which is essential for the activity is enzymatically modified by tyrosianes.

Postmortem diffusion of drugs from the bladder into femoral venous blood.

We describe significantly elevated drug concentrations in the femoral venous blood due probably to postmortem diffusion from the bladder. A 16-year-old deceased male was found in a shallow ditch in winter. The estimated postmortem interval was 9 days and putrefaction was not advanced. The cardiac chambers contained fluid and coagulated blood and a small amount of buffy coat clots. Diffused hemorrhages were found in the gastric mucosa. The bladder contained approximately 600 ml of clear urine. Gas chromatographic-mass spectrometric analysis of the urine disclosed allylisopropylacetylurea (a fatty acid ureide sedative), diphenhydramine, chlorpheniramine and dihydrocodeine. The cause of death was considered to be drowning due to a drug overdose and cold exposure. The concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine in the femoral venous blood (1.89, 3.27 and 3.30 microg/ml, respectively) were much higher than those in blood from the right cardiac chambers (0.294, 0.237 and 0.240 microg/ml, respectively). Urine concentrations of diphenhydramine, free dihydrocodeine and total dihydrocodeine were 22.6, 37.3 and 43.1 microg/ml, respectively. The stomach contained negligible amounts of diphenhydramine, free dihydrocodeine and total dihydrocodeine (0.029, 0.018 and 0.024 mg, respectively); concentrations of these drugs in the femoral muscle were 0.270, 0.246 and 0.314 microg/g, respectively. These results indicate that postmortem diffusion of diphenhydramine and dihydrocodeine from the bladder resulted in the elevated concentrations of these drugs in the femoral venous blood. Not only high urinary drug concentrations but also a large volume of urine in the bladder might accelerate the postmortem diffusion.

Effects of cocaine administration route on the formation of cocaethylene in drinkers: an experiment using rats.

The effects of cocaine administration route on the formation of cocaethylene, an active metabolite of cocaine produced in the presence of ethanol, were investigated using rats. When 20 mg/kg cocaine was administered into the stomach together with 2 g/kg ethanol, maximum liver concentrations of cocaine and cocaethylene, 8410 +/- 3600 and 1680 +/- 520 ng/g, respectively, were observed at 15 min. In other tissues the maximum levels of both the substances were attained in 30 min, but were much lower than those in the liver. Intramuscular administration of 20 mg/kg cocaine with 2 g/kg oral ethanol gave levels of liver cocaine as low as 103 +/- 29 to 150 +/- 35 ng/g, resulting in no detection of liver cocaethylene over the entire 180-min study period, although gradual accumulation of cocaethylene was observed in other tissues. The accumulation patterns of cocaine and cocaethylene in blood of alcohol-intoxicated rats after the i.m. administration of cocaine were similar to those in blood of drinkers after nasal insufflation of cocaine. Despite i.v. administration of 1 mg/kg cocaine with 2 g/kg oral ethanol, no detectable amounts of cocaethylene appeared in any tissues over the entire 60-min study period. The present findings are considered to be of importance in the fields of forensic and clinical toxicology for clarifying (1) the rate of in vivo cocaethylene formation and (2) the distribution of cocaine and cocaethylene in blood and tissues.

Spectra interference between diquat and paraquat by second derivative spectrophotometry.

A rapid and accurate method, combining solid-phase extraction and second-order derivative spectrophotomety approaches, is developed for the simultaneous determination of diquat (DQ) and paraquat (PQ) in blood, tissue and urine samples. Supernatant resulting from the precipitation of protein (with trichloroacetic acid) in plasma and tissue or Amberlite IRA-401 resin treated urine are passed through a mini-column packed with Wakogel gel (Silica gel). Analytes are then eluted with a non-organic solvent, 0.2mol/l HCl solution containing 2mol/l NH(4)Cl. UV spectrum of the eluent in 220-350nm range provides effective screen to detect the presence of DQ and/or PQ. In the presence of DQ or PQ alone, the analyte present is quantitated by conventional zero- or second-order derivative spectrophotometry. The calibration curve in the 0.1-5.0mg/l range for either analyte obeys Beer's law. When both DQ and PQ are present, their concentrations are determined by the peak amplitudes of their respective second-derivative spectra after the addition of alkaline dithionite reagent. Interference is negligible when the DQ/PQ concentration ratio is within the 5.0-0.2 range. Using a 2-ml of sample size, the detection limits for DQ and PQ in plasma are 0.02 and 0.005mg/l. The corresponding detection limits for urine samples (10ml sample size) are 0.004 and 0.001mg/l. Recoveries of DQ and PQ in triplicate plasma and urine samples spiked with 0.5mg/l of analytes are 93 and 85%. The precision of the proposed method resulting from triplicate study of spiked urine samples varies from 3.2 to 4.6% at 0.5mg/l of DQ and PQ, respectively.

Pitfalls when determining tissue distributions of organophosphorus chemicals: sodium fluoride accelerates chemical degradation.

This paper describes the tissue distributions of dichlorvos, an organophosphate, and chlorpyrifos-methyl, an organophosphorothioate, in a male individual who died after ingesting an insecticidal preparation containing these chemicals and the results of an in vitro stability study on dichlorvos and chlorpyrifos-methyl in blood and buffers. Tiny amounts of dichlorvos, 0.067 and 0.027 mg/L, were detected in the vitreous humor and cerebrospinal fluid, respectively. Although dichlorvos (0.082-8.99 mg/L or mg/kg) was detected in the thoracic aortic blood, thoracic inferior vena caval blood, pericardial fluid, bile, and spleen, it was strongly suggested that it had diffused postmortem from the stomach, which contained 879 mg, because no dichlorvos was detected in the other blood samples and tissues tested. Substantial amounts (0.615-4.15 mg/L) of chlorpyrifos-methyl were detected in all blood samples, and the order of its concentrations was as follows: pulmonary vessel blood > thoracic inferior vena caval blood > blood in the right cardiac chambers > blood in the left cardiac chambers approximately thoracic aortic blood > right femoral venous blood. The total amount of chlorpyrifos-methyl in the stomach was 612 mg. However, it was strongly suggested that virtually no chlorpyrifos-methyl diffused from the stomach into surrounding fluids and tissues postmortem because no chlorpyrifos-methyl was detected in the bile and little was found in the pericardial fluids. Neither compound was detected in the urine. In vitro experiments showed that dichlorvos (10 mg/L) almost disappeared from fresh (pH 7.4) and acidified (pH 6.2) blood samples within 24 and 72 h, respectively. However, 53 and 77% of the original amount of dichlorvos in 0.05M phosphate buffers at pH 7.4 and 6.2 were detected 72 h later. Chlorpyrifos-methyl (1 mg/L) was very stable in blood samples, regardless of the pH, during the 72-h study period, but in the pH 7.4 and 6.2 phosphate buffers, approximately 80% of the original amount had degraded after 72 h. These results indicate that organophosphates are degraded more rapidly by esterase activities than by chemical mechanisms and that organophosphorothioates are hydrolyzed chemically in aqueous solutions but are very stable in biological specimens and not metabolized by esterases. When sodium fluoride was added to blood samples, dichlorvos degraded completely within 15 min, and chlorpyrifos-methyl became very unstable. Thus, when analyzing samples to detect organophosphorus chemicals, this common preservative should not be added to fluid specimens.

Testing for drugs of abuse in meconium of newborn infants.

A reliable and sensitive screening procedure has been developed for drugs of abuse (amphetamines, cocaine metabolites, opiates, and phencyclidine [PCP]) in meconium from infants. The substances in meconium were extracted with chloroform-isopropanol (3:1) and screened by enzyme multiplied immunoassay technique (EMIT). The lower detection limits of the EMIT for benzoylecgonine, d-methamphetamine, morphine, and PCP were 250 ng/g, 730 ng/g, 110 ng/g, and 100 ng/g, respectively. This method was applied to meconium from 50 infants born to mothers suspected of using the drugs of abuse during pregnancy. Of the 50, 12 were positive for benzoylecgonine, seven for opiates, and one for PCP. The presence of benzoylecgonine and PCP in meconium was confirmed by gas chromatography/mass spectrometry and that of opiates by thin-layer chromatography. The routine analysis of meconium for drugs of abuse is recommended in cases where (A) urine can not be obtained or (B) urinalysis is negative for the substances despite a strong suspicion of maternal use of the substances during pregnancy.

Estimating the time between drinking and death from tissue distribution patterns of ethanol.

To establish a method for estimating the time between the last consumption of alcohol and death, we examined the ethanol levels in body fluids and tissues of rats that had been orally administered 1 g/kg ethanol. We observed the following relationships between ethanol levels in the cardiac blood (blood in the heart itself), vitreous humor, and urine: cardiac blood > vitreous humor > urine at 10 min (early absorption stage); vitreous humor > cardiac blood > urine from 20 to 50 min (late absorption stage); vitreous humor > urine > cardiac blood from 60 to 120 min (distribution stage); and urine > vitreous humor > cardiac blood at 180 min (excretion stage). It was also observed that, in cases of death immediately following drinking, ethanol levels in the stomach contents are very high, and the following ratios of ethanol levels were observed: skeletal muscle to cardiac blood--less than 1; liver to cardiac blood--around 1. buccal mucosa to cardiac blood-greater than 1. These ratios at equilibrium after drinking were around 1, lower than 1 and around 1, respectively. We also measured alcohol levels in the cardiac blood, urine, vitreous humor and stomach contents of nine cadavers who had consumed alcohol prior to death. The relationships between the time since last consumption of alcohol and relative ethanol levels in these specimens were in good accordance with the results of the animal experiments.

Effects of antineoplastics, antibiotics and antidiabetics on acetaldehyde metabolism after alcohol ingestion.

The main purpose of this study was to evaluate the inhibitory effects of 5-fluorouracil antineoplastics, cephem antibiotics containing the methyltetrazolylthiol (MTT) group and antidiabetics on aldehyde dehydrogenase (ALDH) activity in vivo and in vitro. In in vivo experiments, rats were given a 100 mg/kg dose of drugs (10 mg/kg for glibenclamide) orally or intraperitoneally. When each drug was administered singly immediately after an oral administration of 1.5 g/kg ethanol, only carmofur, an antineoplastic, produced marked increases in blood acetaldehyde concentrations. This action was also noted when ethanol was ingested 15 h after administration. The remaining drugs did not increase blood acetaldehyde concentrations. When rats were treated with carmofur at 12 h intervals for 3 consecutive days and were given 1.5 g/kg ethanol after the final treatment, blood acetaldehyde concentrations were elevated more significantly than with a single administration of carmofur. Furthermore, daily administration of cephem antibiotics containing the MTT group, latamoxef, cefamandole, cefoperazone and cefbuperazone, significantly increased blood acetaldehyde concentrations. Daily administration of sulfonylurea antidiabetics, chlorpropamide and acetohexamide, slightly increased blood acetaldehyde concentrations. Drugs causing increases in blood acetaldehyde concentrations when administration was combined with ethanol ingestion also inhibited ALDH activity in vitro. The results of the in vitro experiments roughly correlated with those of the in vivo experiments. The inhibitory effects of drugs on ALDH activity were in the following order: carmofur >> cephem antibiotics containing the MTT group > sulfonylurea antidiabetics.

Concentrations of monoethylglycinexylidide in body fluids of deceased patients after use of lidocaine for endotracheal intubation.

The objective of this study was to determine whether the postmortem concentrations in body fluids of monoethylglycinexylidide (MEGX), a major active metabolite of lidocaine, reflect the circulatory state during cardiopulmonary resuscitation following endotracheal intubation using lidocaine. The concentrations of lidocaine and MEGX in blood, pericardial fluid, bile and/or urine were measured for sixteen patients who had received endotracheal intubation using Xylocaine jelly, a 2% w/v lidocaine hydrochloride preparation. Lidocaine was detected in all of the sixteen cases. Of six patients who had survived 3 h to 10 d following endotracheal intubation, four were MEGX-positive and two were negative. No MEGX was detected in the other ten patients whose hearts had not resumed beating despite attempts at cardiopulmonary resuscitation. MEGX can be an indicator of the vital state of a patient during cardiopulmonary resuscitation; it shows the antemortem use of lidocaine under normal hepatic conditions.

Criteria for judging whether postmortem blood drug concentrations can be used for toxicologic evaluation.

Postmortem blood drug concentrations tend to be regional-dependent. The aim of this study was to establish preliminary criteria, based on analytical findings of drugs in body fluids, for objectively judging whether drug concentrations determined in postmortem blood are usable for toxicologic evaluation. The study involved 11 autopsy cases in which no obvious putrefaction was observed. Twelve drugs were assayed; the cumulative frequency of detection was 16. The drug concentrations in cerebrospinal fluid and pericardial fluid were averaged, and the ratio between this average concentration and the concentration in femoral venous blood was determined. The mean ratio was close to 1, with a small standard deviation (0.94 +/- 0.20). From these results, the following criteria were drawn: 1) when the ratio of average cerebrospinal fluid/pericardial fluid drug concentration to blood drug concentration is within a range of 0.6-1.4, the postmortem blood drug concentration is usable for toxicologic evaluation; and 2) when the ratio is outside this range, the average cerebrospinal fluid/pericardial fluid concentration should be used as an alternative to drug concentration is postmortem blood.

Pericardial fluid as an alternative specimen to blood for postmortem toxicological analyses.

In actual forensic cases, we occasionally encounter victims with their blood being completely lost. In this study, pericardial fluid has been proposed as a specimen for toxicological analysis, and its utility has been evaluated. Fifteen autopsy cases with little putrefaction were selected. Fairly good correlations were observed between blood and pericardial fluid for all drugs, neutral and basic drugs and acidic drugs with regression equations of y=1.09x - 0.086 (r=0.989, n=21), y=0.969x - 0.072 (r=0.993, n=16) and y=1.01x + 0.355 (r=0.970, n=5), respectively. The correlations of drug concentrations between blood and cerebrospinal fluid/femoral muscle were not as good as those between blood and pericardial fluid. No correlations were observed between blood and urine/bile. The ratios of pesticide concentrations in each specimen to those in blood showed a large variation. Although our study was limited to a small number of cases, we have concluded that pericardial fluid is a good sample for quantitative confirmation of analyses performed on blood samples or a quantitative alternative to blood in exsanguinated victims. Cerebrospinal fluid, urine, bile and the skeletal muscle were found to be suitable only for qualitative analyses.

Concentrations of morphine and codeine in urine of heroin abusers.

We have measured concentrations of morphine, codeine and 6-monoacetylmorphine in urine of people admitted to the Los Angeles County + University of Southern California Medical Center. Of 60 patients positive for morphine and/or codeine, 10 were judged to be heroin abusers based on positive results for 6-monoacetylmorphine, a specific metabolite of heroin. In nine of these ten patients, 0.028-39.4 microg/ml free codeine and 0.070-307 microg/ml total codeine were detected along with 1.74-218 microg/ml of free morphine and 11.2-2870 microg/ml of total morphine; the morphine-to-codeine ratios were 3.65-228 and 2.27-207 for the free forms and total amounts of these opiates, respectively. In the one patient who was negative for codeine, the concentrations of free and total morphine were 0.114 and 2.22 microg/ml, respectively. Based on our data and literature data available, the following criteria are proposed for judging heroin use from the results of urinalysis, especially when no 6-monoacetylmorphine is detected: (1) a detectable amount of free morphine exists and the concentration of total morphine is higher than 10 microg/ml; (2) a detectable amount of codeine exists; and (3) the morphine-to-codeine ratio is higher than 2 for both the free forms and total amounts of these opiates.

Can microorganisms produce alcohol in body cavities of a living person?: a case report.

Unusual endogenous ethanol production in intraabdominal bloody fluid of an individual who was stabbed in the abdomen and who developed peritonitis after a peritoneotomy is discussed. In the intraabdominal bloody fluid, 2.45 mg/g ethanol and 0.079 mg/g n-propanol were detected. The level of ethanol in the heart blood was about 1 mg/g. The level of n-propanol indicates that a large quantity of ethanol was produced endogenously in the intraabdominal bloody fluid. In an animal experiment in which rats were injected with 20 mL of 10% glucose mixed 5:1 with a presumed volume of rat blood into the abdominal cavity after injury of the small intestine to allow enterobacteria to spread into the cavity, a significant quantity of ethanol was produced in the administered fluid while the animals were alive. The antemortem ethanol production in the intraabdominal bloody fluid of the victim might have been caused by the microorganisms responsible for the peritonitis after the operation.

Distribution of free and conjugated morphine in body fluids and tissues in a fatal heroin overdose: is conjugated morphine stable in postmortem specimens?

The tissue distribution of free and conjugated morphine in a male individual who died after self-injection of heroin and methamphetamine was investigated, and the postmortem stability of morphine in the blood, liver and urine, and that of 6-monoacetylmorphine in the urine was determined. Confirmation and quantitation of morphine, 6-monoacetylmorphine and methamphetamine were performed by gas chromatography/mass spectrometry and gas chromatography, respectively. Blood levels of free and total morphine were very site-dependent with ranges of 462-1350 and 534-1570 ng/mL, respectively. Large amounts of total morphine, 5220, 4200, and 2270 ng/g, had accumulated in the stomach contents, liver, and lung, respectively. The concentration of free morphine in the cerebrospinal fluid was correlated very closely with that in the cerebrum. The proportion of free morphine in various fluids and tissues ranged from 23.0% to 98.8% of total morphine: less than 30% in the stomach contents and urine; 30-60% in the liver, cerebrospinal fluid, lung, and pericardial sac fluid; 61-90% in the spleen, right femoral muscle, myocardium, blood in the left and right ventricles of the heart, and right femoral vein blood; more than 91% in the right kidney and cerebrum. Detectable amounts of 6-monoacetylmorphine, 417 ng/mL and 78 ng/g, existed in the urine and stomach contents, respectively, indicating that this individual might have died within several hours after heroin injection. Methamphetamine concentrations in the blood were also site-dependent within the range 551-1730 ng/mL. In an in vitro experiment, free and conjugated morphine were stable in the blood and urine at 4, 18-22, and 37 degrees C for a 10-day study period. In the liver, however, conjugated morphine had been converted almost completely to free morphine at 18-22 and 37 degrees C by the end of the experiment, although it was stable at 4 degrees C. Urine 6-monoacetylmorphine, although degraded slightly at 37 degrees C, was stable at 4 and 18-22 degrees C during the experiment. Thus it appears that non-specific hydrolysis of conjugated morphine to free morphine would not occur in corpses at least for a few days after death. Femoral muscle may be a specimen of choice for roughly predicting the ratio of free to total morphine in blood even when blood specimens are not available, because the femoral muscle is relatively spared of both postmortem diffusion of drugs and bacterial invasion.

The effect of postmortem interval on the concentrations of cocaine and cocaethylene in blood and tissues: an experiment using rats.

Cocaine and cocaethylene concentrations in blood and tissues at early stages postmortem (0-6 h) were investigated using alcohol-treated rats. Gas chromatography/mass spectrometry following a liquid/liquid extraction procedure was employed to detect these drugs. Calibration curves showed good linearity in the range of 0 to 2,500 ng/mL with correlation coefficients of 0.9999 and 0.9998 for cocaine and cocaethylene, respectively. In a group treated with cocaine and ethanol orally, the liver lost over 25% of the cocaine present at death after 1 h. Conversely, the hepatic cocaethylene concentrations at this time reached more than twice those at death. Thereafter, the hepatic concentrations of cocaine and cocaethylene were maintained at a constant level until 6 h postmortem. Similar results were obtained with rats given cocaine intramuscularly. No changes in the cocaine and cocaethylene concentrations in any other tissues during the 6-h of postmortem period were observed. The forensic pathologist and toxicologist should be aware of these phenomena when selecting postmortem specimens for the analysis of cocaine and cocaethylene and take them into account when interpreting the results.

Determining the state of the deceased during cardiopulmonary resuscitation from tissue distribution patterns of intubation-related lidocaine.

The objective of this study was to determine whether the concentrations of lidocaine, used for endotracheal intubation, in body fluids and tissues reflect the state of the circulation of the deceased during cardiopulmonary resuscitation. The tissue distribution of lidocaine was investigated in seven individuals (Cases 1-7) who underwent medical treatment with endotracheal intubation using Xylocaine jelly (a 2% lidocaine hydrochloride preparation), before being pronounced dead. Six patients (Cases 1-6) had cardiopulmonary arrest on arrival at hospital. In Cases 1-4, there was no restoration of heartbeat during cardiopulmonary resuscitation. However, systemic distribution of intubation-related lidocaine was observed and the kidney-to-liver ratios of lidocaine were less than 1. In Cases 5 and 6, the heartbeat resumed temporarily with cardiac massage, and a kidney-to-liver lidocaine ratio greater than 1 was observed. In Case 7, where the patient was comatose upon admission to hospital, the kidney-to-liver ratio of lidocaine was also greater than 1. These phenomena were substantiated in animal experiments. Our results indicate that the absorption of tracheal lidocaine during the artificial circulation resulting from cardiopulmonary resuscitation results in a kidney to liver ratio of less than 1, whereas absorption during natural circulation gives a ratio greater than 1. The kidney-to-liver ratio of intubation-related lidocaine may give useful information on the state of a patient during cardiopulmonary resuscitation.

Comparative studies on tissue distributions of organophosphorus, carbamate and organochlorine pesticides in decedents intoxicated with these chemicals.

This paper describes the tissue distributions of dichlorvos, an organophosphate, chlorpyrifos-methyl, an organophosphorothioate, methomyl, a carbamate, and endrin, an organochlorine, in three individuals (Cases 1-3) who died after ingesting insecticidal preparations containing these chemicals. In Case 1 involving dichlorvos and chlorpyrifos-methyl, no dichlorvos was detected in most of the blood and tissue samples. Tiny amounts of dichlorvos (0.067 mg/L and 0.027 mg/L) were detected in the vitreous humor and cerebrospinal fluid, respectively. The chlorpyrifos-methyl concentrations in the blood samples were very site-dependent with a range of 0.615-2.24 mg/L. The tissue concentrations of chlorpyrifos-methyl were within the range 0.379-8.60 mg/kg. The total amounts of dichlorvos and chlorpyrifos-methyl in the stomach were 879 and 612 mg, respectively. The serum cholinesterase activity was 3 IU/L/37 degrees C. In Case 2 involving methomyl, the methomyl concentrations in the blood samples were very site-dependent with a range of 0.56-4.75 mg/L. The tissue concentrations of methomyl were 2.61 mg/kg or less, no methomyl being detected in the spleen, liver and kidney. The methomyl concentrations in the cerebrospinal fluid and vitreous humor were 5.37 and 4.75 mg/L, respectively. The stomach contained 85 mg methomyl. The serum cholinesterase activity was 73 IU/L/37 degrees C. In Case 3 involving endrin, the victim underwent medical treatment for 7 h after ingesting an endrin preparation. The differences in the endrin concentrations among the blood samples were small, with a range of 0.353-0.615 mg/L. The tissue concentrations of endrin were within the range 0.467-13.3 mg/kg. The endrin in the stomach (66 mg) was adsorbed almost completely on the activated charcoal that was administered for medical treatment.

Redistribution of basic drugs into cardiac blood from surrounding tissues during early-stages postmortem.

The objective of this study was to elucidate the mechanism(s) responsible for increases in the concentrations of basic drugs in cardiac blood of bodies in a supine position during early-stages postmortem. The concentrations of basic drugs in cardiac blood and other fluids and tissues of three individuals who had used one or more basic drugs were examined. The results were compared with those obtained in experiments using rabbits. In the first case, autopsy of whom was performed approximately 12 h after death, methamphetamine was detected and its concentrations were in the order: lung >> pulmonary venous blood > blood in the left cardiac chambers (left cardiac blood) >> pulmonary arterial blood > blood in the right cardiac chambers (right cardiac blood). In the second case, autopsy of whom was performed approximately 9 h after death, methamphetamine and morphine were detected and their concentrations in the left cardiac blood were roughly twice those in the right cardiac blood. The methamphetamine and morphine concentrations in the lung were 2 to 4 times higher than those in cardiac blood samples. In the third case, autopsy of whom was performed approximately 2.5 days after death, the pulmonary veins and arteries were filled with chicken fat clots. Toxicological examination revealed the presence of four basic drugs: methamphetamine, amitriptyline, nortriptyline and promethazine. Their concentrations in the lung were 5 to 300 times higher than those in cardiac blood, but postmortem increases in the concentrations of these drugs in the cardiac blood were not observed. In the animal experiments, rabbits were given 5 mg/kg methamphetamine intravenously or 20 mg/kg amitriptyline subcutaneously and sacrificed 20 min or 1 h later, respectively. The carcasses were left in a supine position at the ambient temperature for 6 h after or without ligation of the large vessels around the heart. For the groups with ligated vessels, the mean ratios of the drug concentrations in both left and right cardiac blood samples 6 to 0 h postmortem were about 1, whereas in those without ligated vessels, these ratios were about 2 and 1, respectively. The order of the methamphetamine and amitriptyline concentrations in blood and tissue samples were roughly: lungs > myocardium and pulmonary venous blood > cardiac blood, inferior vena caval blood and liver. Our results demonstrate that when bodies are in a supine position, (1) basic drugs in the lungs diffuse rapidly postmortem into the left cardiac chambers via the pulmonary venous blood rather than simply diffusing across concentration gradients, and (2) basic drugs in the myocardium contribute little to the increases in their concentrations in cardiac blood during the early postmortem period.

Medicolegal implications of drugs and chemicals detected in intracranial hematomas.

The purpose of this study was to determine how drug findings in intracranial hematomas should be assessed in forensic autopsy cases. Six cases in which intracranial hematomas containing drugs and chemicals were detected were examined in this study. Of the six cases, five were positive for drugs and chemicals that had been self-administered by the victims prior to injury. Post-traumatic time interval from injury to death was in the range 10 to 65 h. In two individuals who were positive for norephedrine or toluene, the concentrations of these substances were much higher in the intracranial hematomas than in heart blood. In an individual who was positive for phenobarbital, its concentration was only a little higher in the intracranial hematoma than in heart blood. In the remaining two cases, substantial quantities of ethanol were detected in the intracranial hematomas, but little ethanol was detected in heart blood. In three cases, some drugs were administered at hospital after the injuries. The time interval from the initial drug administration to death was 19 to 60 h. In two individuals given phenytoin and/or lidocaine intravenously, substantial amounts of these drugs were detected in the intracranial hematomas. In an individual given diazepam intravenously, a substantial quantity of diazepam was detected in heart blood, but not in the intracranial hematoma. Toxicological analysis of intracranial hematomas may be useful not only for determining whether individuals were under the influence of ethanol at the time they were injured, but also for detecting pre-traumatic usage of other drugs and chemicals. However, the medical record should be reviewed thoroughly from a toxicological view point if victims underwent medical treatment prior to death because drugs administered for the purpose of medical treatment can disseminate into preexisting intracranial hematomas, depending on the size of the hematomas.

Absorption of intubation-related lidocaine from the trachea during prolonged cardiopulmonary resuscitation.

The purpose of this study was to determine whether lidocaine is absorbed from the trachea during the artificial circulation of cardiopulmonary resuscitation. The tissue distribution of lidocaine was investigated in eight individuals (Cases 1-8) who underwent cardiopulmonary resuscitation before being pronounced dead. In Cases 1-4, there was no restoration of heart beat during cardiopulmonary resuscitation. Heart massage had been continued for 5 min in Cases 1 and 2, and for 60 min in Cases 3 and 4. Relatively high concentrations of lidocaine (more than 0.1 mg/L) were detected in the blood left in the heart and/or in the large thoracic vessels in the four cases. In Cases 1-3, a large proportion of the lidocaine detected in these blood samples may have diffused from the trachea after cessation of cardiopulmonary resuscitation since no lidocaine was detected in the cerebrospinal fluid, cerebrum, liver, right kidney, and/or right femoral muscle. In Case 4, however, tracheal lidocaine was thought to have been absorbed during cardiopulmonary resuscitation because 0.167-0.340 mg/L or mg/kg lidocaine was detected in the cerebrospinal fluid, liver, right kidney, and right femoral muscle. This was substantiated in experiments performed in rabbit carcasses given 50 microL/kg Xylocaine jelly (a 2% lidocaine hydrochloride preparation) intratracheally, followed by rhythmical thoracic compressions (100-150 times per minute) for 60 min. A possible reason for lack of absorption of lidocaine from the trachea of Case 3 during a 60-min cardiopulmonary resuscitation procedure may have been that effective blood circulation was not obtained during cardiopulmonary resuscitation because of bleeding and pulmonary collapse. Cases 5-8 survived for 3 h to 10 days after successful cardiopulmonary resuscitation; it was obvious that lidocaine was distributed to the tissues under the influence of the natural circulation. The kidney to liver lidocaine ratio in Case 4 (0.8) was much lower than that in Cases 5-8 (1.3-4.6), although the lidocaine ratio in the blood in the left ventricle when compared to blood in the right ventricle was similar in the five cases. The kidney to liver lidocaine ratio may be helpful in judging whether the lidocaine detected was absorbed during the artificial circulation of cardiopulmonary resuscitation or naturally. Additionally, postmortem diffusion of tracheal lidocaine into the blood in the left ventricle was much greater than into the blood in the right ventricle due to their anatomical location during a supine position. The pattern of tissue distribution of lidocaine gives useful information on the state of decedents during cardiopulmonary resuscitation.