References for determining the time of death by potassium in vitreous humor

The different statements concerning the slope and intercept of the regression line and the 95% limits of confidence are the reason that potassium in vitreous humor is not used (at least in Germany) as an aid in estimating the time of death. The relationship between the concentration of potassium and the time of death is mainly influenced by antemortem electrolyte imbalances caused by disease and/or duration of terminal episode. The influence of terminal episode is best identified by its duration (Adelson et al., J. Forensic Sci., 8 (1963) 503–514). In order to have a method suitable for every case and to be as precise as possible we looked therefore for parameters in vitreous humor which were stable postmortem and indicating antemortem electrolyte imbalance. Urea is such a parameter, being stable postmortem (Coe, Am. J. Clin. Pathol., 51 (1969) 741–750) and useful as a marker of antemortem electrolyte imbalance. Our investigations on potassium in vitreous humor, including sudden and hospital deaths after chronic lingering disease, revealed 95% limits of confidence of ±34 h up to 120 h postmortem. Reviewing only cases with urea less than 100 mg/dl the 95% limits of confidence could be reduced to ±22 h. Considering the duration of terminal episode (<6 h) the precision was ±20 h. In this way our modified procedure is suitable for every case with the resulting precision of estimation being determined only by the duration of the terminal episode and urea concentration.     

Postmortem increase in potassium in the vitreous humor. Which parameters are suitable as indicators of antemortem agonal electrolyte imbalance?

The relationship between a rise in the potassium concentration in the vitreous humor and the time of death has been established by several authors. Sturner (1963) and Adelson (1963) have been able to demonstrate a more precise relationship between potassium and the time of death in cases of sudden death and death occurring within 6 h as opposed to death after a chronic, lingering disease. However, the duration of the terminal episode cannot always be assessed in forensic medicine. Therefore, it would be valuable if other parameters could also be established that would indicate an agonal dysregulation of electrolytes. Our study revealed that sodium, urea, chloride, and calcium are of great value in indicating antemortem electrolyte imbalance because of their stability in the vitreous humor after death. The first results established that urea may be a suitable internal standard.     

A new biochemical method for estimation of postmortem time.

Hypoxanthine (Hx) is formed by hypoxic degradation of adenosine monophosphate (AMP) and might be elevated due to antemortem hypoxia. However, it also increases after cessation of the life processes. Until now measurements of potassium in corpus vitreous humor have been used by forensic pathologists to determine postmortem time. In this study the influence of postmortem time and temperature on vitreous humor Hx and potassium levels were compared. Repeated sampling of vitreous humor was performed in 87 subjects with known time of death and diagnosis. The bodies were kept at either 5 degrees C, 10 degrees C, 15 degrees C or 23 degrees C. Hx was measured by means of HPLC and potassium by flame photometry. In 19 subjects from whom samples were obtained within 1.5 h after death, the normal level of Hx could be estimated to be 7.6 mumol/l and that of potassium to be 5.8 mmol/l. The spread of the potassium levels measured shortly after death was much greater than for the corresponding Hx levels. In the four temperature groups the Hx level increased 4.2, 5.1, 6.2 and 8.8 mumol/l per h, respectively, whereas the corresponding figures for potassium were 0.17, 0.20, 0.25 and 0.30 mmol/l per h. The vitreous humor concentration of both Hx and potassium increases fairly linearly after death. The slopes are steeper with increasing temperature. Since the scatter of the levels is greater for potassium than for Hx, the latter parameter seems to be better suited for the determination of time of death in cases without antemortem hypoxia, especially during the first 24 h.     

家兔急性有机磷中毒死后玻璃体液化学成分的变化趋势

目的 探讨家兔急性有机磷中毒死后不同时间眼玻璃体液化学成分的变化。方法用5％敌百虫制作家兔急性有机磷中毒动物模型,在死后0～72h内不同时间点,采用留置微量重复取样法吸取眼玻璃体液,以全自动生化分析仪测量眼玻璃体液中钾、镁、磷、钙、钠、氯浓度的变化,统计数据进行二项式回归方程拟合分析。结果家兔急性有机磷中毒死后72h眼玻璃体液中钾、镁、磷随PMI延长而逐渐升高,舻分别为0．9818、0．9700、0．9458,与PMI相关性好,其中30h内镁的相关性更好;钙、钠随PMI延长呈降低趋势,砰分别为0．7124、0．7355,相关性较差;氯表现不稳定掣仅为0．0869。结论家兔急性有机磷中毒死后玻璃体液中钾、镁、磷、钙、钠等离子浓度随PMI延长而改变,72h内总体衡量,钾与PMI最具有相关关系。     

Avian vitreous humor concentrations of inosine, hypoxanthine, xanthine, uric acid, uracil and uridine as influenced by age and sex: their relevance as indicators of ante-mortem hypoxia

An investigation was made to determine the effects of age and sex on postmortem concentrations (mumol/l) of inosine, hypoxanthine, xanthine, uric acid, uracil and uridine in the vitreous humor of chickens (Gallus domesticus). Five male and 5 female chickens were sampled each week from 0-10 weeks of age. Samples were collected at 0 and 24 h postmortem and analysed by HPLC. Hypoxanthine, uric acid and uridine were detected at both 0 and 24 h postmortem whereas inosine, xanthine and uracil were detected only at 24 h postmortem. Neither sex nor the sex X age interaction influenced the concentrations of the compounds investigated (P greater than 0.01). Age had a significant influence on the concentrations of all compounds at 24 h postmortem (P less than 0.0001). At 0 h postmortem, age significantly affected the levels of hypoxanthine and uridine (P less than 0.0001) but not uric acid (P = 0.014). All compounds occurred at higher concentrations at 24 than 0 h postmortem with the exception of uridine at 0 and 1 week of age. Previous studies using data confounded by age and postmortem sampling time have concluded that postmortem vitreous humor hypoxanthine concentration is a useful indicator of antemortem hypoxia. The results of this study cast doubt on that conclusion. Age and postmortem sampling time should be rigorously controlled when conducting quantitative analyses of these compounds in vitreous humor.     

Vitreous humor carbohydrate-deficient transferrin concentrations in the postmortem diagnosis of alcoholism

Deaths from the effects of alcohol intoxication are encountered routinely in forensic practice. In an important number of cases difficulty may arise in interpreting the significance of results obtained in the autopsy. In clinical practice biochemical markers, particularly serum gamma-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), aspartate transaminase (AST), carbohydrate-deficient transferrin (CDT), and erythrocyte mean corpuscular volume are used to diagnose heavy alcohol consumption. CDT is used as a reliable and specific marker. In postmortem diagnosis, because of the difficulty in interpreting blood alcohol levels and relatively non-specific pathological features, biochemical compounds have been studied for use as possible markers. The aim of this study was to evaluate the usefulness of the postmortem determination of CDT in vitreous humor as a confirmation of antemortem alcoholism. CDT levels were studied in 66 male cadavers with a mean age of 55.9 years (S.D. 17.0, range 22-87 years) with a mean postmortem interval of 17.9 h (S.D. 11.4, range 4-72 h). Cases were assigned to two diagnostic groups according to the antemortem diagnosis of alcoholism. Statistically significant differences were found for CDT and ALT concentrations between the two diagnostic groups. The highest vitreous humor levels of CDT and ALT were obtained in the group of cases with a previous diagnosis of alcoholism. Our results suggest that vitreous humor CDT levels are useful in cases where the postmortem diagnosis of alcoholism is hindered by the non-specificity of data.     

Postmortem biochemical examination of synovial fluid--a preliminary study

Vitreous humor chemistry is used for postmortem analysis since serum values of many components are thought to be reflected in vitreous humor and to be stable for a prolonged postmortem interval (pmi). A similar isolated compartment to vitreous humor is synovial fluid which up to now was hardly used for postmortem chemistry.The aim of the present examination was to compare the values of various analytes in both fluid compartments to get first hints for the reliability of the examination of synovial fluid. Therefore, in 74 cases of sudden death both fluids were taken and analysed for natrium, potassium, calcium, chloride, urea, creatinine, and glucose. The results show that the examination of synovial fluid as compared to vitreous humor is a reliable method to get hints on the premortal metabolic status since most analytes are stable postmortem. The range of all values is comparable in both fluids. The time course of glucose concentrations and, much more important, potassium concentrations is nearly similar. If no vitreous humor is available synovial fluid may be used with all precautions in diagnosis known from vitreous humor.     

The extent of postmortem drug redistribution in a rat model.

The aim of this study was to investigate the postmortem redistribution of several drugs in a rat model and to examine if any of the pharmacological properties was related to the extent of this phenomenon. One of the following drugs: phenobarbital (phenobarbitone), acetaminophen (paracetamol), carbamazepine, codeine, verapamil, amphetamine, mianserin, trimeprazine (alimemazine) or chloroquine was administered together with nortriptyline orally to rats 90 min prior to sacrifice. Heart blood was sampled immediately before sacrifice and after 2 h postmortem, as it has previously been shown that this is sufficient time for postmortem concentration changes to occur in heart blood. Blood was also sampled from the clamped abdominal inferior vena cava (representing peripheral blood) and tissue samples were taken from lungs, myocardium, liver, kidney, thigh muscle, forebrain, and vitreous humor together with a specimen from the minced carcass. Drugs were analyzed by high performance liquid or gas chromatography. For phenobarbital, acetaminophen and carbamazepine the postmortem to antemortem blood drug concentration ratios were close to 1.0 and tissue concentrations were low. The postmortem to antemortem heart blood drug concentration ratio for chloroquine (6.9 +/- 1.5) was higher than for nortriptyline (3.5 +/- 0.3), and the remaining drugs (codeine, verapamil, amphetamine, mianserin, and trimeprazine) showed ratios of the same magnitude as nortriptyline. The postmortem to antemortem blood drug concentration ratios for both heart blood and blood from the vena cava and also the lung to antemortem blood drug concentration ratio were closely related to the apparent volume of distribution for the drugs studied (p < 0.001). Accordingly, an apparent volume of distribution of more than 3-4 L/kg is a good predictor that a drug is liable to undergo postmortem redistribution with significant increments in blood levels. The postmortem drug concentration in blood from vena cava was closely related to the antemortem blood level, confirming that among the postmortem samples, the peripheral blood sample was the most representative for the antemortem blood concentration.     

Vitreous humor chemistry: the use of potassium concentration for the prediction of the postmortem interval

Upon review of the literature, extensive disagreement was found as to the usefulness of vitreous humor potassium concentration as a predictor of the postmortem interval (PMI). A pilot study of 1427 cases was performed to address this problem. The requisite statistical analysis for the prediction of PMI is inverse prediction. The 95% inverse prediction interval was found to be approximately +/- 20 h. The linear regression equation for the data was y = 0.238 chi + 6.342, with a coefficient of determination (r2) of 0.374. This r2 value means that 62.6% of the variation of potassium is unaccounted for by the variation in PMI. Further studies are required to attribute this unaccounted variation to quantifiable factors. This would narrow the inverse prediction interval and enable vitreous potassium to be a useful aid in the prediction of PMI.     

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.     

Postmortem vitreous humor beta-hydroxybutyrate: its utility for the postmortem interpretation of diabetes mellitus

Ketoacidotic coma is one of the most serious complications arising from diabetes mellitus, especially type I, and may be the cause of sudden death especially in diabetes type I. Since beta-hydroxybutyrate (beta-OHB) serum concentrations might provide more information on the severity of ketoacidosis, the aim of this study was to evaluate the concentrations of beta-OHB in vitreous humor and its correlation with other biochemical parameters during postmortem examination. We intended to ascertain the sensitivity and the specificity of these markers for diagnosing diabetes mellitus and the presence of ketoacidosis. This study involved 453 cadavers with a mean age of 57.6 years (S.D. 20.7) and a mean postmortem interval of 17.8 h (S.D. 9.6, range 2-61 h). Cases were assigned to two diagnostic groups according to the antemortem diagnosis of diabetes mellitus, based on the patients' medical records. In vitreous humor statistically significant differences were found in biochemical marker concentrations between the two diagnostic groups, the highest values being obtained in the group of subjects with a previous diagnosis of diabetes mellitus. The measurement of beta-OHB in vitreous humor may be a useful alternative to using blood during postmortem analysis. The presence of high levels of beta-OHB may help interpret the cause of death in diabetics when the autopsy result is negative.     

Vitreous Humor: Biochemical Constituents in Estimation of Postmortem Interval*,†

Abstract: Analysis of biochemical constituents of the vitreous humor can be useful in determining the postmortem interval as there is proportionate postmortem rise of potassium and fall in sodium concentration. We studied 120 autopsy cases to determine the utility of potassium, sodium, calcium, and chloride levels, and sodium/potassium ratio in estimating the postmortem interval. There was a linear relationship between vitreous potassium concentration and postmortem interval, whereas an inverse relationship between vitreous sodium/potassium ratio and postmortem interval was noted. Other factors like age, sex, cause of death, season of death, and refrigeration of sample did not influence the vitreous humor potassium values. Using the statistical tools, a new formula was derived to determine the postmortem interval based on the potassium concentration and a review of previous literature is presented. Hence, the findings of this study supported a central role of vitreous humor biochemistry in many postmortem forensic and pathological evaluations.     

Vitreous humor biochemical constituents: evaluation of between-eye differences

The present study was conducted to investigate the differences in the vitreous humor biochemical concentrations for vitreous electrolytes and calcium in the same pair of eyes at identical postmortem interval (PMI). The vitreous humor samples were collected independently in both eyes from 48 autopsies (PMI range, 4.5-84.3 hours) with documented time of death. The samples were analyzed for potassium, sodium, chloride, and calcium using a Beckman Coulter LX20 Automated Analyzer based on ion-selective electrode methodology. There were no statistically significant between-eye differences at identical postmortem interval. A significantly high correlation was observed between paired potassium concentrations of both the eyes. A highly significant linear correlation was observed between the individual eye and mean potassium concentrations of both the eyes with postmortem interval. The observed differences were not significantly correlated with postmortem interval. The results demonstrated that the between-eye differences for vitreous electrolytes and calcium are insignificant. Therefore, the utility of vitreous biochemistry, particularly potassium in postmortem interval estimation and other forensic applications, cannot be questioned solely on the basis of these differences.     

Variations in vitreous humor chemical values as a result of instrumentation

Urea nitrogen, glucose, sodium, potassium, and chloride were measured in common vitreous humor samples using a variety of instruments. There was found to be variation in values obtained by the different procedures for each of these constituents. The variation in electrolyte values between the different procedures can pose real problems in attempting to determine the presence of an antemortem dehydration or low salt condition. Possible reasons for these variations are discussed, and the normal range of values of both sodium and chloride for the different instrumentalities is provided. However, variations in values for both urea nitrogen and glucose would not pose any problems of interpretation for forensic science evaluations.     

玻璃体液化学成分规律性改变与死亡时间推断的研究进展

死亡时间（postmortem interval．PMI）的准确推断是法医尸体检验工作中的难题之一,PMI推断对刑事案件的侦察和审判具有重要的意义。人死亡后不同时间眼玻璃体液化学成分的规律性变化,如钾、镁、氨、尿素氮、肌酐和尿酸、次黄嘌呤、乳酸的含量逐渐升高,而钙、钠、酶类、葡萄糖、维生素C的含量逐渐降低,有助于比较准确地进行PMI推断。本文对其在法医学领域的研究进展进行综述。     

The use of vitreous humor as an alternative to whole blood for the analysis of benzodiazepines

In postmortem drug analysis, the most commonly used sample matrix is whole blood. However, postmortem changes can denature this matrix, resulting in a loss or degradation of drugs, thus biasing analytical findings. Vitreous humor is thought to be less affected by these changes and should, therefore, have the potential to provide a more reliable estimation of antemortem drug concentrations. To assess the usefulness of vitreous humor for the analysis of benzodiazepine drugs, vitreous humor and whole blood were obtained postmortem in 27 cases. Three benzodiazepine drugs were investigated-temazepam, diazepam, and desmethyldiazepam. For temazepam and diazepam, some correlation was found between the matrices (R2 = 0.789 and 0.724, respectively). However, for desmethyldiazepam, no correlation was observed (R2 = 0.068). Regression analysis on plots of vitreous humor versus blood concentrations produced gradients of less than 1.0 showing that, in general, levels in blood are higher than the corresponding levels in vitreous humor.     

Measurement of Postmortem 1,5‐anhydroglucitol in Vitreous Humor for Forensic Diagnosis

In forensic diagnosis, postmortem blood glucose is known to be susceptible to change after death. However, the 1,5‐anhydroglucitol (1,5‐AG) concentrations in plasma and cerebrospinal fluid (CSF) reflect the mean blood glucose level for a short period of time. In this study, we compared the postmortem 1,5‐AG concentrations in vitreous humor and CSF in 47 subjects to evaluate the utility of this concentration in the vitreous humor for forensic diagnosis. The postmortem 1,5‐AG concentrations in vitreous humor (mean±SD: 20.2 ± 8.7 μg/mL) and CSF (16.8 ± 8.7 μg/mL) did not differ significantly and showed a strong correlation (r² = 0.87, p < 0.01). These results suggest that the vitreous humor 1,5‐AG concentration provides useful information on the antemortem blood glucose level, in addition to the HbA1c value and the CSF 1,5‐AG concentration.     

Measurement of chemical analytes in vitreous humor: stability and precision studies

The analytic accuracy and precision for measurement of chemical analytes in vitreous humor (VH) are critical if results are to be used in forensic pathology. The purpose of our study was to demonstrate the stability and the reproducibility of VH sodium, potassium, chloride, glucose, urea nitrogen, acetone, and beta-hydroxybutyrate in specimens obtained from both eyes in medical examiner cases. We also compared with calculated VH osmolalities. Small but significant increases were observed in VH electrolyte concentrations in specimens refrigerated 6-12 months: sodium pre 144 mmol/L, post 151 mmol/L; potassium pre 12.0 mmol/L, post 12.8 mmol/L; chloride pre 121 mmol/L, post 123 mmol/L. No differences were observed between eyes, and within-day precision for all electrolyte measurements were excellent, (<1%). Frozen specimens showed significantly higher measured (439 mOsmol/kg) as compared with calculated osmolality (305 mOsmol/kg), with 1% within-day precision and no significance between eye variation for glucose and urea nitrogen. In 20 of 24 medical cases selected for possible ketoacidosis, measurement of beta-hydroxybutyrate concentrations appears to be a promising diagnostic biomarker for confirming suspected ketosis in medical examiner cases by means of VH analysis.     

Correlation of antemortem and postmortem digoxin levels.

Postmortem serum digoxin levels from any source routinely exceed antemortem values. Variation resulting from site of sampling gave a mean postmortem to antemortem ratio of 1.96 for heart, 1.63 for subclavian vein, and 1.42 for femoral vein samples. No correlation could be made between the postmortem interval and the increase in post-mortem serum values, irrespective of the site of sampling. A combination of femoral venous serum and vitreous humor values gave the best information for determining possible antemortem digoxin toxicity.     

家兔玻璃体液间隔多次微量取样方法研究

目的探讨间隔多次微量取样玻璃体液是否可用于死亡时间推断。方法99只家兔分为2大组(I)大组45只,再分为A、B、C、D、E五个小组(每组9只),右眼(实验眼)于死后间隔12h多次取样直至24h(A组)、48h(B组)、72h(C组)、96h(D组)和120h(E组);各小组左眼(对照眼)仅分别于死后24h、48h、72h、96h和120h取样一次。检测Ca2+、Cl-、K+、Na+和P含量,同一取样时间的双眼做配对t检验。(II)大组54只,再分为2小组F组9只,每隔12h双眼交替多次取样直至120h;G组45只,每隔12h一次取样10眼(动物左右眼均编号后随机抽取每次10眼),直至120h;检测Ca2+、K+和Na+,两组差值做t检验及PMI回归方程比较。结果E组K+有差异(P<0.05),其余A~E组双眼物质含量配对t检验均无差异(P>0.05)。F与G组物质含量差值t检验无差异(P>0.05);K+、Na+的PMI线性回归方程的斜率、截距无差异(P>0.05),Ca2+与PMI无相关性。结论控制间隔时间和取样次数,多次微量取样对死后玻璃体液元素含量的规律性变化无显著影响,可以替代传统方法进行死亡时间研究。