Postmortem Vitreous Humor Research Articles

Post-mortem vitreous humour as potential specimen for detection of insulin analogues by LC–MS/MS

Differentiation of insulin analogues is required in forensic and clinical toxicology as well as in sports doping control. Immunoassay results provide only weak evidence for exogenous administration of insulin, as concentrations cannot be reliably interpreted and specific information on the insulin species remains unknown. In post-mortem blood, insulin degrades rapidly. In this study, improved methodology consisting of precipitation of proteins, immunoaffinity purification and liquid chromatography coupled to high resolution/high accuracy mass spectrometry were applied to post-mortem vitreous humour. Ten successive cases with a post-mortem interval from four to ten days were investigated for insulin analogues. The cause of death in these cases was connected with diabetes and its complications, as well as with chronic cardiovascular disease, alcoholism and cancer. In all cases, the manner of death was natural (disease). Insulin was positively detected in post-mortem vitreous humour in three cases out of ten by mass spectrometry. In two cases, the method revealed the long-acting insulin glargine (Lantus) metabolite M2 (DesB31-32 Lantus), and human insulin was detected in one case. The findings were in agreement with the documented history of insulin medication. No other obvious reason could be found for the failure of detecting insulins in the other cases than insulin degradation during the lengthy post-mortem interval. Vitreous humour is still a most prospective specimen for detection of insulin analogues post-mortem.

Interpretation of drug concentrations in an alternative matrix: the case of meprobamate in vitreous humor

The use of vitreous humor (VH) as an alternative matrix to blood in the field of forensic toxicology has been described for numerous drugs. Interpretation of drug concentrations measured in VH, as in other matrices, requires statistical analysis of a data set obtained on a significant series. In the present study, two diagnostic tests interpreting postmortem VH concentrations of meprobamate in 117 sets of autopsy data are reported. (1) A VH meprobamate concentration threshold of 28mg/l was statistically equivalent to that of blood meprobamate concentration threshold of 50mg/l distinguishing overdose from therapeutic use in blood. The intrinsic qualities of the test were good, with sensitivity of 0.95 and absolute specificity of 1. (2) A novel interpretation tool is proposed, allowing blood concentration range to be evaluated, with a known probability, from VH concentration.

Comparison of Oxycodone in Vitreous Humor and Blood Using EMIT(R) Screening and Gas Chromatographic-Mass Spectrometric Quantitation

Vitreous humor may serve as a useful alternative specimen for oxycodone analysis in death investigations where blood samples are not available or are of poor quality or limited quantity. The purpose of this study was to investigate the relationship between immunoassay results and gas chromatography-mass spectrometry (GC-MS) quantitation of oxycodone in postmortem vitreous humor and blood. When used with vitreous humor calibrators, the Microgenics DRI Oxycodone (EMIT) Assay was found to be linear from 25 to 500 ng/mL with an limit of detection of 25 ng/mL. Vitreous humor and postmortem blood precipitate immunoassay responses in 57 oxycodone-positive cases were found to be correlated (r(2) = 0.69, p < 0.01). Confirmation and quantitation of oxycodone in vitreous humor by GC-MS was linear from 50 to 1000 ng/mL with a limit of detection of 10 ng/mL and a limit of quantitation of 50 ng/mL. In 30 cases, oxycodone vitreous humor concentrations ranged from less than 50 to 945 ng/mL, and blood concentrations ranged from 103 to 768 ng/mL. The average vitreous humor/blood ratio was 1.16 and ranged from 0.12 to 3.26. Disparities between vitreous fluid and blood oxycodone concentrations were seen in a few cases.

Distribution of 11-Nor-9-Carboxy-Δ9-tetrahydrocannabinol in Traffic Fatality Cases

11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THC-COOH) distributions in postmortem specimens are rarely reported. Fifty New Jersey State Medical Examiner's cases in which automobile accident deaths suspected of involving marijuana intake were studied for the distributions of THC-COOH in postmortem urine, blood, vitreous humor, and bile specimens. Cases were selected based on immunoassay (TDx) urine test results. If the preliminary urine test indicated the presence of THC-COOH (apparent THC-COOH concentration > or = 20 ng/mL), urine, heart blood, vitreous humor, and bile specimens from the case were analyzed for THC-COOH concentrations by gas chromatography-mass spectrometry. The mean, standard deviation, and range of THC-COOH in heart blood, urine, and bile found in these 50 cases were 0.081, 0.082, and 0.016-0.33 microg/mL; 0.314, 0.415, and 0.044-2.33 microg/mL; and 12.9, 11.4, and 1.03-43.7 microg/mL, respectively. THC-COOH was absent (detection limit, 1 ng/mL) or at low concentration (< 10 ng/mL) in vitreous humor specimens. The mean, standard deviation, and range of the bile-to-blood and urine-to-blood ratios were 242, 196, and 17.2-888 and 4.70, 4.05, and 1.14-19.2, respectively. The highest concentrations of THC-COOH were found in bile and the lowest in vitreous humor. These findings are consistent with the high hydrophobicity nature of THC-COOH and further suggest that bile is the specimen of choice for detecting low level of THC-COOH in postmortem cases.

Postmortem vitreous humor β-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 (β-OHB) serum concentrations might provide more information on the severity of ketoacidosis, the aim of this study was to evaluate the concentrations of β-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 β-OHB in vitreous humor may be a useful alternative to using blood during postmortem analysis. The presence of high levels of β-OHB may help interpret the cause of death in diabetics when the autopsy result is negative.

Is vitreous humour useful for the interpretation of 3,4-methylenedioxymethamphetamine (MDMA) blood levels? Experimental approach with rabbits.

As drug instability and redistribution are factors known to affect the interpretation of post-mortem blood levels, we questioned whether post-mortem vitreous humour concentrations could be useful as predictors for the MDMA load at the time of death. In a first series of in vivo experiments using rabbits, 3,4-methylenedioxymethamphetamine (MDMA) concentrations in plasma, blood and vitreous humour were studied as a function of time after intravenous (i.v.) administration of MDMA. Equilibration between the vascular compartment and vitreous humour was attained about 1 h after i.v. MDMA administration. In a second series of experiments, the post-mortem stability of MDMA in vitreous humour in relation to ambient temperature was investigated. Post-mortem MDMA concentrations in vitreous humour were closer to the ante-mortem blood levels when compared to cardiac blood samples. These preliminary investigations in the rabbit model indicate that measurements of vitreous humour concentrations could also be of interest for predicting the blood concentration at the time of death in humans.

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 (μmol/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 × age interaction influenced the concentrations of the compounds investigated ( P > 0.01). Age had a significant influence on the concentrations of all compounds at 24 h postmortem ( P < 0.0001). At 0 h postmortem, age significantly affected the levels of hypoxanthine and uridine ( P < 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.

Postmortem time and storage temperature affect the concentrations of hypoxanthine, other purines, pyrimidines, and nucleosides in avian and porcine vitreous humor.

An HPLC method was used to determine whether postmortem time and storage temperature affect the concentrations of purines, pyrimidines, and nucleosides in avian and porcine vitreous humor. Inosine, hypoxanthine, xanthine, uric acid, uracil, uridine, and thymine were identified in the vitreous humor of chickens (Gallus domesticus). Time from death to sample collection (0-192 h) influenced the concentrations of all seven compounds (p less than 0.01 to less than 0.0001). The storage temperature of chicken carcasses before sampling (6 or 20 degrees C) had a significant influence on the concentrations of inosine, hypoxanthine, xanthine, uric acid, uracil, and thymine (p less than 0.05 to less than 0.0001). The interaction of postmortem time with temperature was significant for all seven compounds (p less than 0.01), with the rate of increase in concentration over time being higher at 20 than 6 degrees C. At 0 h postmortem, the vitreous humor of pigs (Sus scrofa) contained detectable levels of hypoxanthine, uracil, and uridine. Inosine, hypoxanthine, xanthine, guanosine, guanine, uracil, and uridine were detected after storage of pig eyes for 24 h at 20 degrees C. Between 0 and 24 h postmortem, there was a 22-fold increase in hypoxanthine concentration and a 13-fold increase in uracil concentration (p less than 0.0001). It was concluded that postmortem time and temperature are factors that must be taken into consideration when making comparisons of purines, pyrimidines, and nucleosides between groups and in relating postmortem vitreous humor hypoxanthine concentrations to antemortem hypoxia.

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.

Magnesium, potassium, sodium and calcium in post-mortem vitreous humour from humans

Levels of magnesium, potassium, sodium and calcium in post-mortem vitreous humour from human controls, fire fatalities and drowning victims have been determined. The effects of time-related internal changes, external environmental parameters and different causes of death are evaluated. Despite the positive correlation and marked increase of potassium and, to a lesser extent, of magnesium and calcium with the length of the post-mortem interval, individual biological variability severely limits the usefulness of predictions of post-mortem interval based on electrolyte metal data. At best, there is only a 2 3 chance of a prediction being within 12 h of the true value. Vitreous humour metal concentrations are affected by external influences, such as the elevated temperatures of fires which increase the rate of release of intracellular magnesium and potassium. In cases where drowning is suspected, establishment or exclusion of this cause of death is not possible on the basis of vitreous humour electrolyte metal data because of possible post-immersion diffusion across the permeable membrane of the eyeball. It appears, however, that magnesium in salt-water cases and sodium in freshwater cases are related, albeit erratically, to the length of the immersion period.

Comparative Study of Postmortem Barbiturates, Methadone, and Morphine in Vitreous Humor, Blood, and Tissue

With the introduction of radioimmunoassay (RIA) techniques, it has become toxicologically possible to determine drug concentrations in postmortem vitreous humor. This study demonstrates and confirms this toxicological feasibility. In 49 medical examiner's drug related cases, postmortem tissue levels of morphine, barbiturates, and methadone were compared to the vitreous humor.

Postmortem Vitreous Humor in Fatal Acute Iron Poisoning

The postmortem level of iron in the vitreous humor was assayed in a case of treated fatal iron poisoning. An antemortem serum iron level was 9060 micrograms/dL and postmortem blood level was 2920 micrograms/dL. A postmortem vitreous humor specimen collected by a standard technique had an iron level of 80 micrograms/dL whereas 23 controls had a mean of 12.8 +/- 4.2 micrograms/dL (range, 6 to 21 micrograms/dL). Although this elevated value could reflect some serum contamination, it may be a reflection of the toxic state.

Postmortem Biochemical Changes in Canine Vitreous Humor

Time and temperature effects on postmortem vitreous humor from 60 adult mongrel dogs were studied. After death the dogs were held at 4, 20, or 37 degrees C for intervals of 3, 6, 12, 24, or 48 h. Antemortem and postmortem vitreous was analyzed for sodium, chloride, potassium, urea nitrogen, glucose, and creatinine. Potassium levels rose with increases in temperature and time. Sodium, chloride, and urea nitrogen values were stable at 4 degrees C for 48 h; they were less stable at higher temperatures. Glucose dropped to less than half within 3 h at all temperatures. Creatinine values were inconsistent. In that postmortem glucose and sodium levels remained below antemortem levels, diagnosis of hyperglycemia and hypernatremia should be possible. Consequently, a diagnosis of hypoglycemia could not be supported; the diagnosis of hyponatremia could be made in the early postmortem period.

Postmortem Vitreous Humor Chemistry in Sudden Infant Death Syndrome and in Other Causes of Death in Childhood

Postmortem vitreous humor concentrations reflect antemortem serum chemical values. The authors measured the postmortem vitreous humor concentrations of Na+, K+, Cl-, Ca+2, Mg+2, urea nitrogen, creatinine, and total protein of 127 children who died from sudden infant death syndrome (SIDS) and other causes. Forty-seven children, 1 hour to 13 years old, had died of medical or surgical causes; 21 children, 7 weeks to 11 years old, had died following acute trauma; 59 children, 8 days to 1 year old, had died of SIDS. There was no significant difference between mean postmortem vitreous humor concentrations of those who died of medical and surgical causes and those who died after acute trauma (non-SIDS). in both groups, the mean postmortem vitreous humor concentrations of Mg+2 and Ca+2 were significantly higher in premature infants. There was direct correlation of postmortem vitreous humor concentration and postmortem interval for K+, but the variation, +/- 26 hours, was too large to be of practical importance in estimating time of death in individual cases. The three patient groups were compared and the mean postmortem vitreous humor concentrations of all eight constituents in the SIDS and non-SIDS groups were in the same ranges. In SIDS, the mean postmortem vitreous humor concentrations of Mg+2, Cl-, and urea nitrogen were significantly different from values of the non-SIDS cases, but not enough to indicate SIDS or to be informative about the etiology of SIDS. Measurement of the concentrations of postmortem vitreous humor constituents may not aid in the diagnosis of SIDS, but may aid in discovering unsuspected antemortem serum chemical abnormalities.