Stability of blood carbon monoxide and hemoglobins during heating

The effects of heating on hemoglobin (Hb) and carbon monoxide (CO) levels in human blood were investigated by in vitro experiments. Head-space gas chromatography (HS-GC) using a molecular sieve 5A stationary phase and thermal conductivity detection was adopted for the measurement of CO gas, and spectrophotometric methods were used for the measurement of various Hb forms, protein and heme contents. Deteriorated absorbance spectra were observed for heat-treated blood samples, and double wavelength spectrophotometry was proven to give wrong percent saturation of carboxyhemoglobin content (% CO-Hb). The blood sample taken from one fatal fire casualty gave significantly higher % CO-Hb measured spectrophotometrically, compared to that by HS-GC. Control blood or purified Hb solution, which was saturated with CO in designated extent, was heated in a sealed vial. Under the incubation below 54 degrees C, all Hb forms were stable, except for oxyhemoglobin (Hb-O(2)), which was partially oxidized to met-hemoglobin (Met-Hb). In contrast, under the incubation at 65 degrees C, Met-Hb was denatured completely to be insoluble, and Hb-O(2) was partially denatured via Met-Hb formation. CO-Hb was resistant against heating. The difference of heat susceptibility and precipitability among Hb forms resulted in artificial increase of % CO-Hb. During heating, spontaneous CO was produced from blood.     

Quantitative evaluation of volatile hydrocarbons in post-mortem blood in forensic autopsy cases of fire-related deaths

Volatile hydrocarbons in post-mortem blood from victims of fires were analyzed quantitatively by headspace gas chromatography mass spectrometry. The benzene and styrene concentrations in the blood were positively correlated with the carboxyhemoglobin (CO-Hb) concentration, which is evidence that the deceased inhaled the hydrocarbons and carbon monoxide simultaneously. By contrast, the concentrations of toluene and CO-Hb in the blood were not significantly correlated. This lack of correlation could be explained by two different sources of toluene, with low blood concentrations of toluene arising when the deceased inhaled smoke and high blood concentrations of toluene arising when the deceased inhaled petroleum vapor or other unknown vapors. The quantity of soot deposited in the respiratory tract was classified into four grades (-, 1+, 2+, 3+). The mean CO-Hb concentration in the 1+ soot group was significantly lower than those in the 2+ (p<0.05) and 3+ (p<0.01) soot groups. The blood CO-Hb concentrations in the 1+ soot group were all below 30%. Those indicated that the deceased aspirated smoke that contained both soot and carbon monoxide. The wide variation in CO-Hb concentrations for each soot classification could be caused by the different types of smoke produced by different materials. For example, petroleum combustion with a limited supply of oxygen, like in a compartment fire, may produce a large volume of dense black smoke that contains a large quantity of soot. Soot deposits in the airways and the blood CO-Hb concentration are basic and essential autopsy findings that are used to investigate fire-related deaths. The quantitative GC-MS analysis of blood volatile hydrocarbons can provide additional useful information on the cause of the fire and the circumstances surrounding the death. In combination, these three findings are useful for the reconstruction of cases.     

Simple determination of carboxyhemoglobin by double wavelength spectrophotometry of absorbance difference and the comparison with gas chromatographic method

This paper describes the spectrophotometric determination of carboxyhemoglobin (CO-Hb) in blood on the basis of double wavelength spectrophotometry of absorbance difference. Absorbance measurements are made in the 500–600 nm region at a blood dilution of 100–200-fold. Blood is diluted with a solution containing Na₂S₂O₄ to provide two components of CO-Hb and deoxyhemoglobin (deoxy-Hb). Absorbance difference at the two wavelengths at which deoxy-Hb has the same absorbance reflects only the CO-Hb component because the opposite component is nulled out of the mixture. After measurement of the absorbance difference, the measuring solution is saturated with CO gas to make all Hb derivative CO-Hb and remeasured at the same wavelengths. The percent of CO-Hb is considered the absorbance difference ratio. Results obtained by the present method was in satisfactory agreement with gas chromatographic data in blood not containing methemoglobin (Met-Hb). Comparative experiments using the gas chromatographic method and the present method were performed with samples containing Met-Hb. However, while there is a deficiency in the gas chromatographic method when the samples contain Met-Hb, the results of the present method were in close agreement with theoretical values when samples are mixed with CO-Hb, O₂-Hb and Met-Hb. Advantages of this method are that it is simple and accurate, standard curve or equation for calculation and accurate dilution are not necessary.     

Comparison of ethanol concentrations in venous blood and end-expired breath during a controlled drinking study

Concentration-time profiles of ethanol were determined for venous whole blood and end-expired breath during a controlled drinking experiment in which healthy men (n=9) and women (n=9) drank 0.40-0.65 g ethanol per kg body weight in 20-30 min. Specimens of blood and breath were obtained for analysis of ethanol starting at 50-60 min post-dosing and then every 30-60 min for 3-6 h. This protocol furnished 130 blood-breath pairs for statistical evaluation. Blood-ethanol concentration (BAC, mg/g) was determined by headspace gas chromatography and breath-ethanol concentration (BrAC, mg/2l) was determined with a quantitative infrared analyzer (Intoxilyzer 5000S), which is the instrument currently used in Sweden for legal purposes. In 18 instances the Intoxilyzer 5000S gave readings of 0.00 mg/2l whereas the actual BAC was 0.08 mg/g on average (range 0.04-0.15 mg/g). The remaining 112 blood- and breath-alcohol measurements were highly correlated (r=0.97) and the regression relationship was BAC=0.10+0.91BrAC and the residual standard deviation (S.D.) was 0.042 mg/g (8.4%). The slope (0.91+/-0.0217) differed significantly from unity being 9% low and the intercept (0.10+/-0.0101) deviated from zero (t=10.2, P<0.001), indicating the presence of both proportional and constant bias, respectively. The mean bias (BAC - BrAC) was 0.068 mg/g and the 95% limits of agreement were -0.021 and 0.156 mg/g. The average BAC/BrAC ratio was 2448+/-540 (+/-S.D.) with a median of 2351 and 2.5th and 97.5th percentiles of 1836 and 4082. We found no significant gender-related differences in BAC/BrAC ratios, being 2553+/-576 for men and 2417+/-494 for women (t=1.34, P>0.05). The mean rate of ethanol disappearance from blood was 0.157+/-0.021 mg/(g per hour), which was very close to the elimination rate from breath of 0.161+/-0.021 mg/(2l per hour) (P>0.05). Breath-test results obtained with Intoxilyzer 5000S (mg/2l) were generally less than the coexisting concentrations of ethanol in venous blood (mg/g), which gives an advantage to the suspect who provides breath compared with blood in cases close to a threshold alcohol limit.     

Forensic and chemical determination of methane in cadaveric samples

Conditions were elaborated for the determination of methane, through gas chromatography, in blood and the lung by using a 2% methanol solution as an internal standard and with the below gas-chromatography column being applied: dimensions--200 x 0.3 cm, 15% di-2-ethylhexyl sebacate on Dinochrome II (0.16 x 0.21 mm) at 110 0 degree C. The detector is of the flame-ionization type. The normal content of methane was determined for blood and for the lung--0.122 +/- 0.0074 microgram/ml and 0.20 +/- 0.04 microgram/g, respectively. Biological samples from cadavers of other persons who died due to trauma were suggested for use as controls. The method was used to examine expertise objects, blood and the lung from the cadavers of peoples who died in fire.     

A study on the combined action of CO and HCN in terms of concentration-time products

Acute toxicity at single and combined exposures of CO and HCN was studied on rats in terms of concentration-time product (ppm . min) necessary to kill animals (lethal CT). The animal was exposed individually to test gas in an animal chamber made of transparent plastics, and test gas was made in gas chamber connected to the animal chamber by a wide and short piece of plastic tube. HCN was produced by addition of NaCN solution to H2SO4 and in case of CO exposure, various amounts of pure CO were introduced. During exposure, gas samples were frequently taken. After exposure, blood sample was withdrawn from the right side of the heart. CO concentrations in the gas and blood were determined gas chromatographically. HCN in the gas sample was measured spectrophotometrically, after being absorbed into NaOH solution in a glass vessel devised by our laboratory. At single exposures, mean lethal CT for CO was 78,000 +/- 22,000 and for HCN was 4,700 +/- 940. In combined exposure, various combinations of CO and HCN were used. A fractional CT, defined as a ratio of CT to lethal CT, multiplied by 100, was calculated for each gas. A linear relationship between fractional CTs of HCN and CO was considered to show a simple additive action between the two gases. The sum of both fractional CTs averaged 100 +/- 26. On the other hand, linear relation was not observed between blood levels of the two toxicants at death.     

Breath alcohol concentration determined with a new analyzer using free exhalation predicts almost precisely the arterial blood alcohol concentration

A new breath alcohol (ethanol) analyzer has been developed, which allows free exhalation, standardizes measured exhaled alcohol concentration to fully saturated water vapor at a body temperature of 37 degrees C (43.95 mg/L) and includes a built-in self-calibration system. We evaluated the performance of this instrument by comparing standardized alcohol concentration in freely expired breath (BrAC) with arterial (ABAC) and venous (VBAC) blood alcohol concentrations in fifteen healthy volunteers who drank 0.6 g of alcohol per kg body weight. The precision (coefficient of variation, CV) of the analyzer based on in vivo duplicate measurements in all phases of the alcohol metabolism was 1.7%. The ABAC/BrAC ratio was 2251+/-46 (mean+/-S.D.) in the post-absorptive phase and the mean bias between ABAC and BrAC x 2251 was 0.0035 g/L with 95% limits of agreement of 0.033 and -0.026. The ABAC and BrAC x 2251 were highly correlated (r=0.998, p<0.001) and the regression relationship was ABAC = 0.00045 + 1.0069 x (BrAC x 2251) indicating excellent agreement and no fixed or proportional bias. In the absorption phase, ABAC exceeded BrAC x 2251 by at most 0.04+/-0.03 g/L when tests were made at 10 min post-dosing (p<0.05). The VBAC/BrAC ratio never stabilized and varied continuously between 1834 and 3259. There was a proportional bias between VBAC and BrAC x 2251 (ABAC) in the post-absorptive phase (p<0.001). The pharmacokinetic analysis of the elimination rates of alcohol and times to zero BAC confirmed that BrAC x 2251 and ABAC agreed very well with each other, but not with VBAC (p<0.001). We conclude that this new breath analyzer using free exhalation has a high precision for in vivo testing. The BrAC reflects very accurately ABAC in the post-absorption phase and substantially well in the absorption phase and thereby reflects the concentration of alcohol reaching the brain. Our findings highlight the magnitude of arterio-venous differences in alcohol concentration and support the use of breath alcohol analyzers as a stand-alone test for medical and legal purposes.     

The relationship between bedding and face-down death in infancy: mathematical analysis of a respiratory simulation system using an infant mannequin to assess gas diffusibility in bedding

Rebreathing is a model for the relationship between a prone sleeping position and sudden infant death syndrome. This study used a mechanical simulation model to establish the relationship between types of bedding and rebreathing potential for an infant placed prone (face down) at different postnatal ages. The infant mannequin was connected to a respirator set to deliver physiologically appropriate combinations of tidal volume (V(T)) and respiratory rates (RR) across a range of postnatal ages (0-18 months). Before measurements were made, CO(2) flow was regulated to 5+/-0.1% of end-tidal PCO(2) (EtCO(2)). After the model was placed in a prone position, any increase in the fractional concentration of inspired CO(2) (FiCO(2)) was measured. FiCO(2) increased immediately and rapidly, and reached a maximum value within a few minutes. The maximum FiCO(2) ranged from under 2% to over 10%, depending on the bedding. FiCO(2) was also affected by V(T) and RR. This model is not applicable to actual infants because of the large tissue stores of CO(2) in infants; however, it is useful for evaluation of gas diffusibility of bedding and will simplify the investigation of sleeping environments when a baby is found dead with its face covered by soft bedding. In general, the higher the FiCO(2), the greater the rebreathing potential. Theoretically, considering the paucity of body stores of O(2), changes in FiO(2) would be affected not by changes in FiCO(2), but by CO(2) production and gas movement around the infant's face. The rapid decrease of FiO(2) is approximated at the inverse of the FiCO(2) timecourse, suggesting the significance of not only CO(2) accumulation but also O(2) deprivation in the potential space around the baby's face.     

Kinetics of ethanol in biological sections

Ethanol concentration in alveolocapillary blood (ACB), venous blood (VB), capillary blood (CB), saliva and urine was measured in healthy men and women aged 19-45 years 20, 40, 60, 90, 120, 180, 240 and 300 min after a single intake of 20% ethanol solution in soda water in a dose 0.8 g/kg body mass. Two types of kinetic curves were established. Calculations with Vidmark equation for different biomedia were made. Ethanol levels in all BM studied coincided in the resorption phase. In the elimination phase, ethanol concentration forms a sequence: ACB < saliva < VB < urine. Correlations and correlation coefficients of ethanol concentrations in different BM were estimated. The ethanol concentration correlation urine/ACB 1.71 +/- 0.15 and VB/ACB 1.45 +/- 0.07 is proposed for use in tests for alcohol intoxication.     

A microbiological test for the diagnosis of death by drowning

The present study was aimed at demonstrating the diffusion of sea water or freshwater into the bloodstream as a consequence of water aspiration due to drowning. The study was carried out on 42 study group subjects who died by drowning in salt water (20 cases) and freshwater (22 cases) and 30 control group subjects who died from causes other than drowning. For 25 cases we obtained water samples from the aquatic locations where the victims were found. The blood samples of study and control groups were analyzed to search for faecal coliforms (FC) and faecal streptococci (FS) bacteria. The presence of FC and FS was showed by the development of blue and red colonies, respectively. From left ventricular (LV) and right ventricular (RV) blood cultures of the 20 sea drowning victims we always isolated FS and FC, whereas 19 (95%) femoral arterial (FA) and 18 (90%) femoral venous (FV) blood cultures were positive for both faecal bacteria. Related to freshwater victims, LV blood cultures showed FS presence for all the 22 cases studied (100%) and FC presence for 20 cases (90.91%). Blood cultures from RV, FV, and FA showed various patterns of faecal bacteria presence. The analysis of 25 water samples from the aquatic locations where the victims were found showed the presence of FC and FS bacteria. Blood cultures from the 30 control subjects uniformly proved the absence of faecal bacteria.     

Failure to detect elevated levels of carboxyhemoglobin in infants dying from SIDS

Carboxyhemoglobin (COHb) levels were determined in stored blood samples from 91 infants diagnosed to have died from the sudden infant death syndrome (SIDS) (0.59+/-0.41%, excluding one outlying value of 10.83%); 48 age-matched controls (0.53+/-0.38%); and three individuals who died from fire related causes (41+/-20%). No statistical differences in COHb levels were detected between blood from SIDS and control infants (p = 0.43).     

大鼠闭合性脑损伤后血清髓鞘碱性蛋白研究

用ＥＬＩＳＡ双抗体夹心方法研究了大鼠闭合性弥漫性脑损伤后,血清髓鞘碱性蛋白（ＭＢＰ）含量的时序变化。正常大鼠血清中ＭＢＰ为６．１６３３±１．５３０１ｎｇ／ｍｌ（Ｘ±Ｓ）。伤后立即死亡组大鼠的血清ＭＢＰ为１１．３８１８±２．６５７４ｎｇ／ｍｌ,伤后１５ｍｉｎ,为１０．８３１９±２．３１３５ｎｇ／ｍｌ,此血清高ＭＢＰ水平一直持续到伤后３天。伤后第４天和第５天,血清ＭＢＰ基本恢复到正常水平。立即死亡组、伤后１５ｍｉｎ组和伤后３天之内各组的ＭＢＰ水平与正常组和伤后第４天和第５天的比较,Ｐ＜０．０１。认为可进一步探讨将血清ＭＢＰ含量的检测,作为脑震荡性脑损伤的辅助生化诊断指标之一     

Site-dependent production of gamma-hydroxybutyric acid in the early postmortem period

This study compared endogenous gamma-hydroxybutyric acid (GHB) concentrations in various postmortem fluid samples of 25 autopsy cases. All bodies were stored between 10-20 degrees C until autopsy, and the intervals between death and autopsy were less than 2 days (6-48 h). GHB concentrations were measured by headspace gas chromatography after GHB was converted to gamma-butyrolactone. Endogenous GHB concentrations were significantly higher in femoral venous blood (4.6+/-3.4 microg/ml, n=23) than in cerebrospinal fluid (1.8+/-1.5 microg/ml, n=9), vitreous humor (0.9+/-1.7 microg/ml, n=8), bile (1.0+/-1.1 microg/ml, n=9) and urine (0.6+/-1.2 microg/ml, n=12). GHB concentrations were similar in blood samples taken from different sites. Cut-off limits of 30 and 10 microg/ml are proposed for blood and urine, respectively, to discriminate between exogenous and endogenous GHB in decedents showing no or little putrefaction (postmortem intervals usually 48 h or less). The criterion established for endogenous GHB in postmortem urine may also be applicable to analytical results in cerebrospinal fluid, vitreous humor and bile from deceased persons.     

急性心肌缺血再灌FOS免疫组织化学实验研究

探寻FOS蛋白在心肌早期缺血再灌流损伤中变化规律,为心性猝死的诊断提供新方法。利用SD大鼠建立心肌缺血再灌流损伤模型,设立正常、缺血对照组与缺血再灌组。心脏标本经HE染色及免疫级化观察。结果发现,在冷冻切片上缺血20min再灌流30min,再灌流区心肌细胞核呈阳性着色。但在石蜡切片上,缺血30min再灌流30min后,再灌流区才有心肌细胞核（37．76％±9．66％）呈弱阳性着色,再灌流60min后核呈棕褐色阳性染色,120min后开始减弱（35.36％±3.16％）。正常和单纯缺血组心肌细胞核未见有阳性反应。HE染色无明显病理改变。结果提示,SABC－FOS免疫组织化学方法最早可揭示心肌缺血20min再灌流30min的损伤,FOS蛋白在再灌流后60－120min之间可能有一个高峰表达。此法对显示实验性心肌早期缺血再灌流损伤有重要的价值。有望用于心性猝死的诊断。     

Dermal absorption of kerosene components in rats and the influence of its amount and area of exposure

The influences of amount and area of dermal exposure to kerosene upon the levels of kerosene components in biological samples were examined in vivo and in vitro. Thirty-two rats were randomly divided into four groups and exposed to kerosene through the abdominal skin for 2h. The amounts (soaked in cotton) and area of kerosene exposed were 1 ml/4 cm(2) in Group I, 4 ml/4 cm(2) in Group II, 4 ml/16 cm(2) in Group III and 16 ml/64 cm(2) in Group IV. Before, then 5, 10, 20, 30, 45, 60, 90 and 120 min after exposure, 0.5 ml of blood was collected. Solid tissue samples, including the exposed skin area, were harvested at 120 min. Kerosene components were analyzed by gas chromatography/mass spectrometry. Trimethylbenzens (TMBs) that are easily absorbed kerosene components, appeared at 5-20 min. The time course changes in TMB levels in blood were significantly different between Groups I and II or Groups I and III, and almost identical between Groups II and III. Similar trends were observed in tissue samples at 120 min. High concentrations of aliphatic hydrocarbons (AHCs) were detected in the exposed skin and the AHC levels were dependent on the amount of kerosene exposed per unit area. These results suggest that (1) dermal absorption of kerosene occurs soon after dermal exposure started, (2) absorption of TMBs is influenced by the total amount of kerosene rather than area of exposure, and (3) AHCs remaining in the skin at significant levels are influenced by the amount of kerosene per unit area exposed.     

Study of 5-hydroxytryptamine (serotonin) in pericardial fluid in different causes of death (II.). Experimental study of 5-HT levels in two types of shocks (hemorrhagic and septic) in dogs

In the present work we studied the levels of 5-HT in the pericardial fluid of 160 cadavers according to cause of death; such as myocardial infarction, violent asphyxia, pulmonary embolism, infections, bronchopulmonary diseases, traumatic and hemorrhagic diseases in the CNS, and multiple traumatism. We did not find significant differences in the various causes of death. A complementary study of 20 dead dogs having suffered induced shock, either hemorrhagic or septic, was made. In both series we studied the serum levels of 5-HT and the following parameters: systolic and diastolic arterial pressures, central venous pressure, hematocrit value, pH value, total proteins, albumin, PO2, and PCO2. We found the significant correlation (r = 0.828, P less than 0.01) only between 5-HT serum levels and the systolic arterial pressure in the hemorrhagic shock.     

The role of environmental factors in the causation of sudden death in infants: two cases of sudden unexpected death in two unrelated infants who were cared for by the same babysitter

We report two cases of sudden unexpected death in two unrelated African American female infants, 2 months and 4 months old. Both infants were attended to by the same babysitter in the same apartment and died 39 days apart in the same bed and in the same bedroom. The autopsy of the first infant revealed sudden unexplained death in an infant. Toxicologic analysis for carbon monoxide (CO) was not performed because it was not suspected. When the second infant died, investigation into the ambient air quality within the apartment revealed high levels of CO emanating from a poorly ventilated and defective hot water heater, which was located across a hallway from the bedroom where the two babies died. CO saturation levels in the postmortem blood samples of the two babies were elevated and were similar (13% and 14%). Nicotine and cotinine were not detected in the blood sample of the two infants. Cherry-red livor mortis was absent. Acute CO intoxication was determined to be the underlying cause of these two unexpected deaths. These two cases underscore the need to integrate ambient air analysis and postmortem CO analysis as routine components of the comprehensive death investigation of infants who die suddenly and unexpectedly.     

95例尸体血中HbCO％的分析

血中碳氧血红蛋白饱和度(简称HbCO％,下同)含量是判断一氧化碳中毒,推断火灾中尸体生前状态的依据。本文证明:血中HbCO％与其年龄、性别及不同情况下CO中毒有关。一氧化碳中毒的尸体含量较高(平均大于60％);火灾事故中遇难的尸体含量中等(平均在30～50％);而被杀后投入火场或服毒后自焚则较低(平均小于20％)。     

Tissue distribution of nitrazepam and 7-aminonitrazepam in a case of nitrazepam intoxication

We report a case of nitrazepam poisoning in which the distribution of nitrazepam and 7-aminonitrazepam was determined in body fluids and tissues. A 52-year-old woman was found dead in a shallow ditch (approximately 5 cm in depth), in the winter. Ambient temperature was 2-8 degrees C. The postmortem interval was estimated to be approximately 1 day and no putrefaction was observed. The cause of death was thought to be drowning due to nitrazepam overdose and cold exposure. Blood concentrations of nitrazepam and 7-aminonitrazepam were very site dependent (0.400-0.973 microg/ml and 0.418-1.82 microg/ml). In addition, the concentration of the same analytes in the bile were 4.08 and 1.67 microg/ml, respectively, and in the urine: 0.580 and 1.09 microg/ml, respectively. A high accumulation of both substances was observed in various types of brain tissue (2.17-6.22 microg/g and 2.49-5.11 microg/g). Only small amounts of nitrazepam and 7-aminonitrazepam were detected in the liver (0.059 and 0.113 microg/g, respectively). Large differences in the observed concentrations of nitrazepam and 7-aminonitrazepam among arterial and venous blood samples were thought to be mainly due to dilution of arterial blood by water entering the circulation through lungs at the time of death. Bacterial metabolism of nitrazepam may also have contributed to the observed differences.     

Postmortem urine immunoassay showing false-positive phencyclidine reactivity in a case of fatal tramadol overdose

This is a report of postmortem false-positive reactivity using an enzyme-multiplied urine phencyclidine (PCP) immunoassay (EMIT II+) due to a single-agent fatal tramadol overdose. An autopsy of a 42-year-old male who died alone at home revealed no identifiable lethal anatomic abnormalities, thus leading to toxicologic analysis. Femoral blood was obtained for drug testing by high-performance liquid chromatography (HPLC) and showed a tramadol level of 14.0 mg/L, 2 orders of magnitude greater than the therapeutic range (0.1 to 0.3 mg/L). Urine was also obtained and EMIT II+ immunoassay revealed positivity for PCP at 88 mAU/min. However, confirmatory testing by HPLC failed to identify PCP in either the urine or serum. To verify the suspicion that this was a false-positive PCP result, stock solutions of tramadol and its major metabolite (O-desmethyltramadol) at concentrations of 100 mg/L in 10% methanol/H2O were compared with a blank solution (10% methanol/H2O) for EMIT II+ PCP reactivity and demonstrated reactivities of 44 mAU/min and 27 mAU/min, respectively. While these individual results were below the cutoff reactivity for a positive EMIT II+ PCP result (ca. 85 mAU/min), they were much more reactive than the blank calibrator (set at 0 mAU/min). Therefore, we conclude that the immunoreactivity of tramadol and its metabolites in aggregate is responsible for the PCP immunoassay interference and false-positive result.