Yoshiaki Hashimoto

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.

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.

Potential for Error when Assessing Blood Cyanide Concentrations in Fire Victims

The present study explores toxicologic significance of blood cyanide concentrations in fire victims. Headspace gas chromatography was used for cyanide detection. Analysis of blood samples from ten fire victims (postmortem interval = 8 h to 3 to 5 d) detected zero to 11.9 mg/L of cyanide and a large difference in cyanide concentrations among victims. Carboxyhemoglobin (COHb) saturation was in the range of 24.9 to 84.2%. To examine the effects of methemoglobinemia and postmortem interval on blood cyanide concentrations in fire victims, an experiment was carried out using rabbits as the animal model. The rabbits were sacrificed by intramuscular injection of 1 mL/kg 2% potassium cyanide 5 min after intravenous injection of 0.33 mL/kg of 3% sodium nitrite (Group A, n = 3) or physiological saline (Group B, n = 6). Average methemoglobin contents immediately before potassium cyanide administration were 6.9 and 0.8% in Groups A and B, respectively. Average cyanide concentrations in cardiac blood at the time of death were 47.4 and 3.56 mg/L, respectively. When blood-containing hearts of the rabbits (n = 3 for Group B) were left at 46 degrees C for the first 1 h, at 20 to 25 degrees C for the next 23 h and then at 4 degrees C for 48 h, approximately 85 and 46% of the original amounts of blood cyanide disappeared within 24 h in Groups A and B, respectively. After the 72-h storage period, 37 and 10%, respectively, of the original amounts of cyanide remained in the blood. When the other three hearts in Group B were left at 20 to 25 degrees C for the last 48 h without refrigeration, cyanide had disappeared almost completely by the end of the experiment. The present results and those published in the literature demonstrate that the toxic effects of cyanide on fire victims should not be evaluated based solely on the concentration in blood.

Postmortem Stability of Cocaine and Cocaethylene in Blood and Tissues of Humans and Rabbits

A study was conducted to examine the postmortem stability of cocaine and cocaethylene in rabbit blood and tissues, and to determine whether cocaethylene is produced in decomposed human specimens containing cocaine and endogenous ethanol. Heart blood, liver, brain and femoral muscle taken from rabbits 20 min after oral administration of 20 mg/kg cocaine together with 2 g/kg ethanol were kept at 20-25 degrees C for 5 days. Cocaine and cocaethylene concentrations were in the order brain > liver > muscle > blood, and showed very large intersubject variations at the time of death. Cocaine was degraded rapidly in the blood and liver. However, 12.0 +/- 8.5% and 26.2% +/- 19.4% of the original cocaine was still detectable in the brain and muscle, respectively. Cocaethylene was degraded more slowly than cocaine in all of the specimens. The pH of the blood remained around 7.4 during a 5-day period; all the other specimens showed pH values of 6.2-6.7 on and after the first day postmortem. When 10,000 ng/g cocaine was incubated with decomposed human blood, liver, brain and muscle homogenates containing 0.29-0.60 mg/g endogenous ethanol at 20-25 degrees C and 37 degrees C, no change in cocaine concentration was observed during the study period of 24 h, and no cocaethylene was detected. The pH values of the homogenates were within the range 4.2 to 5.2 at the beginning of the experiment. It was found that: 1) cocaethylene was more stable in postmortem specimens than cocaine; 2) muscle as well as brain was specimen of choice for detecting cocaine and cocaethylene postmortem; 3) cocaine was resistant to decomposition under acidic conditions; and 4) putrefactive bacteria had no ability to produce cocaethylene even in the presence of cocaine and endogenous ethanol.

Medicolegal studies on alcohol detected in dead bodies — alcohol levels in skeletal muscle

An experiment was carried out on rats to determine whether or not a skeletal muscle sample was suitable for the determination of ethanol concentration in a carcass. Gas chromatography was used to estimate the ethanol and n-propanol concentrations in the femoral muscle and intracardial blood. The ethanol concentration of each sample was corrected according to the moisture ratio of circulating blood, viz., 78.5%. The ethanol concentration ratio of blood to muscle was 1.03 two hours after ethanol administration. When the carcasses of rats pre-treated with ethanol were stored at 15 degrees C and 25 degrees C, respectively, the ethanol concentrations in muscle and blood increased with time. At all times the concentration was higher in blood than in muscle, and also higher in samples collected from the carcass stored at 25 degrees C than at 15 degrees C. When the control carcass was stored in the same manner, the postmortem production of ethanol was noticed in both blood and muscle. As in the experimental rats, the control rats exhibited a higher blood ethanol than muscle ethanol level. Again, the ethanol concentration was higher in samples collected from the carcass stored at 25 degrees C than at 15 degrees C. The ratio of ethanol to n-propanol was less than 20:1 in blood and less than 10:1 in muscle. These results suggest that skeletal muscle may be a suitable tissue for the postmortem detection of ethanol.

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.

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 Beers 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.

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.

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.

POSTMORTEM DIFFUSION OF TRACHEAL LIDOCAINE INTO HEART BLOOD FOLLOWING INTUBATION FOR CARDIOPULMONARY RESUSCITATION

This study investigated the postmortem diffusion of tracheal lidocaine into the blood after intubation in three individuals whose heart beat was not restored by cardiopulmonary resuscitation. The results are compared with those obtained in animal experiments using rabbits. The first human subject was a 3.5-month-old female baby who died of sudden infant death syndrome. She was autopsied approximately 20 h after death. A toxicological examination revealed the presence of 0.349 mg/L and 0.102 mg/L of lidocaine in the blood in the left and right ventricles of the heart, respectively. No lidocaine was detected in the cerebrum, liver, or right femoral muscle. The second subject was a 44-year-old man who died of brain swelling due to head injuries, and was autopsied approximately 20 h after death. Lidocaine concentrations in the hili of the left and right lungs were 10.9 mg/kg and 2.65 mg/kg, respectively, and 1.02 mg/L and 0.209 mg/L in the blood in the left and right ventricles of the heart, respectively. The right femoral vein blood contained only a trace amount of lidocaine; no lidocaine was detected in the cerebrum, liver, or right femoral muscle of this subject. The third subject was a 38-year-old man who died of bleeding due to a stab wound to the left thigh, and was autopsied approximately 20 h after death. Lidocaine concentrations were 1.41 mg/kg and 1.37 mg/kg in the hili of the left and right lungs, respectively, and 0.642 mg/L and 0.746 mg/L in the blood in the thoracic aorta and superior vena cava, respectively. No lidocaine was detected in the right femoral vein blood, cerebrum, liver or right femoral muscle. In the animal experiments, rabbits carcasses were left in the supine position at an ambient temperature following application of 1 mg/kg lidocaine hydrochloride into the trachea just above the bifurcation. Lidocaine concentrations of 0.550-4.03 mg/L and 3.05-7.30 mg/L were detected in the heart blood, one and three days after the lidocaine treatment, respectively; neither the cerebrum nor right femoral muscle contained detectable amounts of lidocaine. The pH values of body fluids and tissues of the human and animal corpses were below 7.0. This study has demonstrated that following intubation, tracheal lidocaine diffuses into surrounding fluids and tissues, and that this is attributable to postmortem acidosis. We suggest that, in subjects who underwent cardiopulmonary resuscitation with intra-tracheal intubation, heart blood and femoral vein blood should be analyzed for lidocaine. In addition, the pattern of distribution of lidocaine in the surrounding tissues may provide some information on the state of victims during cardiopulmonary resuscitation.