Postmortem biochemistry

Postmortem biochemistry may provide significant information in determining the cause of death. Due to the rapid postmortem breakdown of metabolism and active membrane transport only analytes which are stable in blood can be determined on this fluid compartment, other parameters have to be analysed on other fluid compartments like vitreous humor (VH). However, using another fluid compartment as a mirror of blood at the moment of death involves severe methodical problems. The conceptual problems of reference values in vitreous humor as a mirror of blood are addressed. Additionally, the necessary steps to be taken before calculating the discriminating values between "normal" and "diseased" are described. For all chemical determinations, a clear definition of the site of sample acquisition is necessary. Up to now chemical determinations on alternative fluids have mainly been carried out using instruments calibrated for urine or serum. Developing calibrated methods for analysis of alternative fluids is a further task for the future.     

Hyaluronidase as a liquefying agent for chemical analysis of vitreous fluid

Vitreous humor is a suitable specimen for postmortem clinical chemistry because the analytes remain relatively stable after death and they closely reflect blood levels immediately prior to death. The viscous nature of vitreous fluid, however, presents analytical problems including imprecision and inaccuracy. Various preanalytical treatments, such as boiling, high speed centrifugation, microfiltration and dilution have been used. These techniques are labor intensive and add to imprecision and inaccuracy. Because glycosaminoglycans contribute significantly to the viscosity of vitreous humor, we used hyaluronidase as a liquefying agent. We compared the results of analyses in 33 vitreous humor specimens after hyaluronidase treatment with the results after either no treatment or specimen dilution. Seventeen of the 33 specimens could not be analyzed without dilution. Even after dilution, several analytes still could not be measured. Hyaluronidase treatment negated the need for sample dilution and had no significant effect on the analyses.     

Aqueous Fluid as a Viable Substitute for Vitreous Fluid in Postmortem Chemistry Analysis

Vitreous fluid sampling for postmortem chemistry analysis is discouraged in pediatric forensic cases involving head trauma due to the risk of introducing retinal artifacts. Aqueous fluid is physically separated from the posterior chamber of the eye, and therefore, unlikely to produce vitreal artifact when sampled. Analysis of aqueous fluid is therefore proposed as a substitute for vitreous. Vitreous and aqueous fluid was sampled concurrently from 28 pediatric and 55 adult decedents, and sodium (Na), potassium (K), chloride (Cl), urea nitrogen (UN), creatinine (Cr), and glucose (Glc) concentrations were compared. Significant correlation existed between all analytes regardless of age or postmortem interval, and linear regression equations were derived. Aqueous concentrations were generally higher than vitreous for Na, K, and Cr and were marginally lower for Cl, UN, and Glc. Assuming vitreous fluid as a standard for correlating postmortem chemistry to antemortem serum values, aqueous may be a viable substitute for vitreous when expected differences are considered.     

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.     

Comparative and Correlation Studies of Biochemical Substances in Vitreous Humor and Synovial Fluid

Vitreous humor (VH) and synovial fluid (SF) become optional postmortem samples for biochemistry investigation. However, few studies have explored SF as a potential source, and there is still controversy whether the biochemical concentrations in bilateral VH are the same. We explored the concentrations of biochemical substances in both VH and SF, and compared between both sides and between the fluids. We also correlated the biochemical concentrations in both fluids. All samples were centrifuged after collection and the supernatant fluids were used for the analysis. The results indicated no significant difference in the constituent concentrations between the fluids from both sides (p > 0.05). Sodium, potassium, chloride, and magnesium concentrations were significantly higher in the VH, but in vice versa for glucose, uric acid, and creatinine concentrations (p < 0.001). Sodium, potassium, glucose, lactate, urea, uric acid, and creatinine concentrations between both fluids showed significant correlation (p < 0.05), but not chloride and magnesium concentrations.     

Postmortem Distribution of 3‐Beta‐Hydroxybutyrate

The concentrations of 3‐beta‐hydroxybutyrate (3HB) in femoral blood, urine, vitreous humor as well as pericardial and cerebrospinal fluids were retrospectively examined in a series of medico‐legal autopsies, which included cases of diabetic ketoacidosis, hypothermia fatalities without ethanol in blood, bodies presenting mild decompositional changes, and sudden deaths in chronic alcoholics. Similar increases in 3HB concentrations were observed in blood, vitreous, and pericardial fluid, irrespective of the cause of death, suggesting that pericardial fluid and vitreous can both be used as alternatives to blood for postmortem 3HB determination. Urine 3HB levels were higher than blood values in most cases. Cerebrospinal fluid 3HB levels were generally lower than concentrations in blood and proved to be diagnostic of underlying metabolic disturbances only when significant increases occurred.     

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。     

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.     

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.     

The effect of chilling, freezing, and rewarming on the postmortem chemistry of vitreous humor

The effect of chilling at the time of death on the postmortem chemistry of the vitreous humor was studied by using sheep heads obtained immediately following decapitation. One group of heads was kept at room temperature, while the remainder were chilled on ice or in ice water, then refrigerated or frozen. Vitreous humor specimens were taken at intervals over a 48-h period. Chilling inhibited the fall in the glucose concentration and the total carbon dioxide content and lessened the increase in lactic acid, compared to the room temperature group. Rapid glycolysis resumed when the heads rewarmed to room temperature starting at 6-h postmortem, but did not resume at later points. The rate of rise of the potassium and magnesium concentrations was also diminished in the chilled eyes. Freezing and thawing caused an abrupt increase in the potassium and magnesium levels, but other solutes were unaffected.     

Glucose and lactate in vitreous humor compared with the determination of fructosamine for the postmortem diagnosis of diabetes mellitus

Diabetes mellitus is a chronic metabolic illness responsible for a great number of deaths. In postmortem diagnosis, because of the difficulty involved in interpreting blood glucose levels and relatively nonspecific pathologic features, biochemical markers in vitreous humor are useful. The aim of this study was to compare the results obtained for the combined determination of lactate and glucose with fructosamine levels recorded in the vitreous humor of two diagnostic groups (one diabetic and the other nondiabetic). The authors intended to ascertain the capacity of different markers measured in vitreous humor to diagnose diabetes mellitus. Fifty-one cadavers (mean age, 58.7 years; standard deviation, 17.09) were studied. The mean postmortem interval was 16.4 hours (standard deviation, 9.05). Cases were assigned to two diagnostic groups according to whether they were previously diagnosed as either diabetic or nondiabetic. Statistically significant differences for glucose, fructosamine, and the sum values of glucose and lactate were found between the two diagnostic groups. The highest levels were obtained in the group of cases with a previous diagnosis of diabetes mellitus. After the comparison of receiver operating characteristic curves, the sum values of glucose and lactate in vitreous humor is a better predictor of antemortem diabetes mellitus than the fructosamine.     

Research Progress on Postmortem Interval Estimation by Vitreous HumorCSCD

Postmortem interval (PMI)estimation is one of the most challenging problems in the field of forensic science. Vitreous humor is a hotspot which has been used for PMI estimation and postmortem chemical analysis in forensic pathology. In order to provide novel perspectives for the future research of PMI estimation using vitreous humor, the comparison between vitreous humor with other common body fluids, the effect of temperature on vitreous humor, vitreous humor detection method and data fitting method have been reviewed in this paper. © 2018 by the Editorial Department of Journal of Forensic Medicine.     

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 (less than 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.     

Urea,Uric Acid,and Creatinine in Postmortem Blood,Vitreous Humor,and Synovial Fluid: A Comparative and Correlation Study,

Urea, uric acid, and creatinine have been demonstrated to be relatively stable in postmortem blood (BL), thus being useful for diagnostic purposes. However, no studies have explored their levels in BL, vitreous humor (VH), and synovial fluid (SF) concurrently. Therefore, we measured and compared their levels in these fluids. We also determined the effects of various factors on their levels. The results indicated that BL urea, uric acid, and creatinine levels were significantly higher than VH and SF levels. VH and SF urea levels and SF creatinine levels had a strong correlation with BL urea and creatinine levels, respectively. BL creatinine levels were higher in men than in women. BL and SF creatinine levels were negatively correlated with age. SF uric acid and BL, VH, and SF creatinine levels exhibited a positive correlation with weight. Only VH creatinine levels were positively correlated with body mass index. None of urea, uric acid, and creatinine levels were correlated with postmortem interval.     

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.     

Variations in vitreous humor chemical values as a result of pre-analytical treatment

Vitreous humor (VH) is used for postmortem diagnosis of metabolic diseases and to clarify the postmortem interval. Because of its viscous nature, this fluid has to be liquefied prior to analysis; however variations in measured concentrations of the analytes are ascribed to different pre-analytical treatment methods with regard to. The aim of this study was to compare different pre-analytical methods. Centrifugation, heat treatment, enzymatic digestion and liquefying by ultrasound were compared using a collection of 120 samples obtained from 2003 to 2007. The determined parameters were: sodium, potassium, chloride, calcium, glucose, creatinine, urea and lactate. Analyses were performed either photometrically or by using ion-selective electrodes. Heat and hyaluronidase treatment generate slightly higher and lower values in the measurement of electrolytes and glucose. However, in the determination of calcium concentration, both methods (heat especially) are associated with extreme low or high values. Only differences between ultrasound and centrifugation treatment show comparatively small variations and are close to instrument accuracy. Therefore, we recommend centrifugation, combined with mixing, as the best and easiest method in which to prepare frozen samples for analysis. Additionally, the measurement of lactate shows that analytical methods, calibrated for serum and urine, cannot be easily applied for VH.     

Is there recent progress in the estimation of the postmortem interval by means of thanatochemistry?

Numerous methods have been proposed in the last 60 years for the determination of the time since death by chemical means. Many of them were reviewed by Schleyer in his monograph on the determination of the time since death by means of thanatochemistry about 40 years ago and none of these early methods has gained any practical value since they do not meet the demands in practice (being precise, reliable, giving an immediate result). While the earlier studies were mainly carried out on blood and cerebrospinal fluid (CSF) since the late 60s most investigations have been performed on vitreous humor (VH). This is mainly due to the fact that vitreous humor is topographically isolated and well protected, and thus, autolytic changes proceed slower compared to blood and CSF. The most studied parameter in VH is potassium and even nowadays reports on the postmortem rise of vitreous potassium are published, proposing new analytical methods or statistical evaluations. Chemical parameters studied for the determination of the time since death have to be differentiated according to the underlying process (catabolism, metabolic processes, pure autolysis and diffusion, putrefactive changes). In the present paper, recent studies on thanatochemistry are discussed regarding the underlying process, the analytical methods (for instance H magnetic resonance spectroscopy (1H MR spectroscopy), immunohistochemistry), the studied fluid compartment, the statistical evaluation and the precision of death time estimation. The value of chemical methods for the determination of the time since death is up to now very limited. This is supported by the fact that field studies on the reliability and precision of death time estimation by chemical means are still scarce in the literature.     

Comparison of chemical methods for determining postmortem interval

Accurate determination of postmortem interval (PMI) is a problem for the forensic thanatologist, especially in unwitnessed deaths. A number of objective chemical methods for determining PMI have been developed, the most widely used being accumulation of potassium in the vitreous humor. The authors previously have reported a chemical method for determining PMI from the predictable accumulation or clearance of the dopaminergic metabolite 3-methoxytyramine (3-MT) in the putamen of the brain. They have extended their previous study to compare directly the accuracy of determining PMI from the level of 3-MT in putamen with the level of potassium in vitreous humor. The data indicate that 3-MT is at least as accurate as, if not more accurate than, potassium accumulation in vitreous humor, although 3-MT levels can be affected by the cause of death and drugs present at the time of death. Nevertheless, determination of both the 3-MT and potassium levels can afford the most accurate method of determining PMI; preliminary nomograms for determining PMI from both variables are presented.     

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.     

Chemical estimation of fresh water immersion intervals

Postmortem vitreous humor chemistry studies were reviewed in 25 cases in which death was followed by submersion in fresh water for known lengths of time. The potassium concentrations in cold temperature cases initially increased in a linear manner, but appeared to level off or decline after about 1 week. In hot weather cases, potassium levels rose rapidly during the first few days postmortem. The sodium and chloride levels decreased in a fairly regular fashion, with less temperature variation. Increasing variation in values began to occur at about 7 days. The data show that dilution of the contents of the eye by fresh water begins at about that time in cases of cold water submersion. A formula employing the weighted average of the potassium and chloride concentrations can be used to determine the duration of submersion in cold water over the range of 2-10 days.