Biochemical investigation of vitreus: applications in forensic medicine, especially in relation to alcohol

Vitreus biochemistry is useful in subjects dying with uraemia, dehydration, hyponatraemia; and in the presence of osmotically active substances such as alcohol and drugs. Useful tests include sodium, potassium, chloride, urea, creatinine, osmolality, osmolar gap estimation and ethanol. The significance of calcium, magnesium, zinc, copper, pH, phosphate and urate is still not clear. Transaminases and lactate dehydrogenase are found to be significantly raised; these may be associated with sudden release of hyalocyte contents at death.     

Comprehensive evaluation of pericardial biochemical markers in death investigation

Pericardial fluid (PCF) is a well-preserved cadaveric material in cases without structural damage. The present study investigated fundamental serum components of PCF, including total proteins (TP), albumin (Alb), urea nitrogen (UN), creatinine (Cr), uric acid (UA), glucose (Glu), sodium (Na), potassium (K), chloride (Cl), calcium (Ca) and magnesium (Mg) in PCF with regard to the postmortem and survival periods, and cause of death in serial medicolegal autopsy cases (n = 288) with intact pericardial and cardiac structures within 48 h postmortem. The amount of PCF (mostly 5–25 ml) showed no survival or postmortem time dependence, or difference among the causes of death. For all cases, there were moderate postmortem decreases of Na and Cl, and increases of K and Mg, which were insufficient for application to estimate the time since death; however, characteristic findings with regard to the cause of death were detected in cases of hours-long survival, especially in ‘functional causes of death’: higher Alb, UN, Cr, UA, K and Mg, and lower Na, Cl and Ca in intoxication; lower TP, Alb, Cr, K and Mg, and higher Na and Cl in hypothermia (cold exposure); higher Alb, UN, Cr, UA and Mg, and lower Glu and Ca in hyperthermia (heatstroke). These observations suggest the usefulness of pericardial biomarkers for investigating the cause and process of death to reinforce pathological and toxicological findings.     

A Case of Fatal Dehydration During Police Custody

In the forensic literature, fatal dehydration predominantly concerns young children or incapacitated elderly persons. The postmortem diagnosis of fatal dehydration can be challenging to confirm, especially if the preceding circumstances are unknown. Here presented is a case of a 23‐year‐old man who died while held in an isolation cell during police custody for 18 days. Autopsy findings were unspecific, but vitreous fluid analysis showed 192 mmol/L sodium, 179 mmol/L chloride, 16 mmol/L potassium, 352 μmol/L (3.98 mg/dL) creatinine, and 81 mmol/L (226.9 mg/dL) urea nitrogen. Based on the findings and circumstances, the cause of death was determined as severe dehydration and manner of death accident. This case illustrates the importance of performing postmortem biochemistry.     

Postmortem interval estimation by biochemical determination in birds muscle

Experimental postmortem interval estimation was assayed by various biochemical components of hens pectoral muscle. Among them, only percentage of non-protein nitrogen on total soluble protein, asparatic amino transferase activity and creatinine concentration showed significant correlation with time after death. Asparatic amino transferase was negatively correlated and non-protein nitrogen percentage and creatinine were positively correlated to postmortem interval. The stronger correlation (0.925) was found for creatinine.     

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.     

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.     

Serum biochemical markers in drowning: diagnostic efficacy of Strontium and other trace elements

The aim of our study was to analyse several serum biochemical markers in order to evaluate the discriminant capacity of trace elements individually and jointly in drowning (seawater drowning and freshwater drowning) and their correlation with age, sex, postmortem interval, time in water and concentrations of the trace elements in the drowning medium. Sixty-seven cases of drowning (53 seawater drownings (SWD); 14 freshwater drownings (FWD) and 73 control cases (other asphyxias, n=44, and other causes of death, n=29) were selected according to the scene, cause and circumstances of death, together with autopsy findings. Serum strontium (Sr), magnesium (Mg), sodium (Na), chloride (Cl), calcium (Ca), iron (Fe), urea, creatinine (Cr) and cardiac Troponine T (cTn-T) were measured in the left ventricle (Lv), right ventricle (Rv) and peripheral blood. Lv-Rv differences for each marker and Sr, Mg, Na, Cl, Ca and Fe concentrations in the drowning medium were determinated. Mean concentrations of Sr, Cl and Mg in both ventricles and peripheral serum and Lv-Rv differences and Ca Lv and Na Rv were significantly higher in cases of drowning than for other causes of death. In SWD, Sr, Mg, Ca, Na and Cl were significantly higher in Lv than in Rv as a result of aspirating water. In contrast, haemodilution is evident from the significantly higher levels of Fe and urea in Rv than in Lv in cases of SWD, and from the higher Mg and Cr levels in Rv in FWD. In the case of SWD, serum levels of Sr are confirmed as the best parameter for diagnosis, although other trace elements may also be useful, such as the serum concentrations of Mg and Cl. In the case of FWD, the joint determination of Sr and other biochemical markers, especially Fe, may increase correct diagnosis.     

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.     

Diagnosis of sudden death in infants due to acute dehydration

When infants die suddenly of acute dehydration, clinical signs and autopsy findings may be equivocal or absent, and microbiologic cultures often are not helpful. Vitreous humor electrolyte and urea nitrogen concentrations were measured in 53 infants dying of gastrointestinal infections, sudden infant death syndrome (SIDS), other infectious diseases, and miscellaneous causes to determine whether these parameters would assist in the recognition and confirmation of deaths resulting from dehydration. Significant differences were found when comparing the mean sodium and urea nitrogen levels of infants dying of gastrointestinal infections with those succumbing to SIDS or other causes. We recommend that these determinations be routinely performed whenever the gross autopsy findings are insufficient to explain the death.     

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.     

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。     

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.     

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.     

Effect of 1,2-indanedione on PCR-STR typing of fingerprints deposited on thermal and carbonless paper

The determination of the date of death from bone remains is of scientific interest but also has important legal implications. The establishment of the postmortem interval (PMI) is a very complex problem because of the great number of intrinsic factors that may alter the normal course of postmortem change, such as the age, sex, constitution and previous physiological and pathological states of the subject, and external factors. In order to evaluate the utility of X-ray diffraction and the measurement of some components in dating bone remains, a total of 69 long bones from 69 different cadavers (41 males, 28 females) with a mean age of 68 years (S.D.=17.6, range 12-97) were used. The bones were removed from cement tombs of Murcia Cemetery, where they had lain for documented times of between 7 and 54 years (S.D.=11.6, mean time 17.6 years). We have studied potassium, sulphur, nitrogen, urea, total protein, phosphorus, and some X-ray diffraction (XRD) parameters related to the degree of crystallinity of the mineral component in medullar and cortical bone zones to establish which of the two provides the most useful information for calculating the PMI. In the overall analysis of our data, we believe that the use of both XRD and biochemical analyses (especially of urea, potassium and sulphur) particularly in the cortical zone of the bone could be an alternative method for dating osseous remains.     

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.     

Post-mortem biochemical investigations of vitreous humor

For several decades vitreous humor has been used for post-mortem biochemical investigations with the objective of a post-mortem diagnosis of pre-existing diseases and the clarification of forensic issues, in particular the determination of the post-mortem interval. For the determination of measured concentrations in vitreous humor pre-analytic factors as well as analytical and instrumental variations have to be taken into consideration. The aim of this study was a methodical investigation of two methods of sample pre-treatment as influencing variables. The compared methods were centrifugation and treatment in the ultrasonic bath. The determined parameters were sodium, potassium, chloride, calcium, lactate, urea, glucose and creatinine. Analyses were performed photometrically or by an ion-selective electrode. For some of the analytes a dilution was necessary before analysing. Regarding to the two pre-treatment methods, significant differences in the measured concentrations were not found. The precision proved to be mostly unsatisfying and was clearly better in diluted samples than in undiluted aliquots. A comparison of the vitreous humor of the two ocular bulbs did not lead to significant differences.     

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.     

The eye as a chemical indicator of environmental temperature at the time of death

Vitreous humor chemistry profiles were reviewed on 133 autopsied cases in which death occurred outdoors during a six-year period, to determine whether environmental temperature at the time of death influenced chemistry values obtained at autopsy. The glucose concentration and total carbon dioxide content varied inversely with temperature. Values were significantly higher in the winter than the summer months. The mean glucose level was higher in deaths caused by cold exposure than in other deaths occurring in the cold, but individual cases could not be distinguished on the basis of chemistry values. Potassium levels tended to be slightly lower in winter. It was noted that chemistry studies could be used to help determine whether a body found outdoors in winter actually died in a different, warmer environment.     

Postmortem serum uric acid and creatinine levels in relation to the causes of death

Serum uric acid (UA) and creatinine (Cr) mainly derive from skeletal muscle tissues. Although, remarkable postmortem stability of the serum levels has been reported, there appears to be very poor knowledge of the diagnostic value in investigation of death, except for uremia. The aim of the present study was to evaluate postmortem serum UA and Cr levels using 395 forensic autopsy cases, in comparison with blood urea nitrogen (BUN), for investigation of the pathophysiology of death with special regard to the causes of death involving possible skeletal muscle damage, e.g. due to hypoxia, heat or agonal convulsions. Cr and BUN showed relatively good topographic stability in the cadaveric blood, whereas, UA was often much higher in the right heart blood than in the left heart and peripheral blood, independent of postmortem intervals. Moderate to marked elevation of Cr and BUN accompanied with hyperuricemia was observed in delayed death. In the acute death cases (survival time <30 min), UA, especially in the right heart blood, showed a considerable elevation in mechanical asphyxiation and drowning. The Cr level in fire victims with a lower carboxyhemoglobin (COHb) level (<60%) was significantly higher than in those with the possible fatal level (>60%). A similar elevation of Cr was observed in fatalities from heat stroke and methamphetamine (MA) poisoning. The observations suggested that hyperuricemia in acute death may be indicative of advanced hypoxia and that elevated Cr level may reflect the skeletal muscle damage, especially due to thermal influence.