Meptazinol and ethanol: a fatal intoxication

Meptazinol (Meptid(?)) is an analgesic drug that is used to treat mild to moderate pain including postoperative pain, obstetrical pain, and the pain of renal colic. This case reports a death due to the combined effects of meptazinol and alcohol in a man with significant heart disease and alcoholic liver disease. A 57-year-old male was found unresponsive in his bed at home with empty blister packets of meptazinol around him. A general drug screen detected the presence of meptazinol, and caffeine and metabolites, in cardiac blood. Analysis, both quantitative (HPLC-DAD) and qualitative (HPLC-DAD, LC-MS), of meptazinol was carried out. Meptazinol was found at the following concentrations: 15.5 mg/L in unpreserved femoral blood; 18.6 mg/L in preserved (fluoride-oxalate) femoral blood; 52.1 mg/L in unpreserved cardiac blood; 16.8 mg/L in preserved vitreous; 61.7 mg/L in unpreserved urine; and 9.8 g/L in stomach contents. Ethanol, analyzed by headspace GC-FID, was present in preserved (fluoride-oxalate) femoral venous blood, urine, and vitreous at concentrations of 232 mg/100 mL, 297 mg/100 mL, and 192 mg/100 mL, respectively. Death was attributed to meptazinol and ethanol toxicity, with atherosclerotic coronary artery disease as a contributing factor.     

Post‐mortem determination of insulin using chemiluminescence enzyme immunoassay: preliminary results

Insulin determination in blood sampled during post‐mortem investigation has been repeatedly asserted as being of little diagnostic value due to the rapid occurrence of decompositional changes and blood haemolysis. In this study, we assessed the feasibility of insulin determination in post‐mortem serum, vitreous humour, bile, and cerebrospinal and pericardial fluids in one case of fatal insulin self‐administration and a series of 40 control cases (diabetics and non‐diabetics) using a chemiluminescence enzyme immunoassay. In the case of suicide by insulin self‐administration, insulin concentrations in pericardial fluid and bile were higher than blood clinical reference values, though lower than post‐mortem serum concentration. Insulin concentrations in vitreous (11.50 mU/L) and cerebrospinal fluid (17.30 mU/L) were lower than blood clinical reference values. Vitreous insulin concentrations in non‐diabetic control cases were lower than the estimated detection limit of the method. These preliminary results tend to confirm the usefulness of insulin determination in vitreous humour in situations of suspected fatal insulin administration. Additional findings pertaining to insulin determination in bile, pericardial, and cerebrospinal fluid would suggest that analysis performed in post‐mortem serum and injection sites could be complemented, in individual cases, by investigations carried out in alternative biological fluids. Lastly, these results would indicate that analysis with chemiluminescence enzyme immunoassay may provide suitable data, similar to analysis with liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and immunoradiometric assay, to support the hypothesis of insulin overdose. Copyright © 2015 John Wiley & Sons, Ltd.     

Drug concentrations in post‐mortem specimens

A meta‐analysis of drug concentrations in post‐mortem specimens is presented. The analysis involved 50 commonly used drugs and their concentrations in femoral blood, other blood (such as cardiac blood), vitreous humor, muscle, liver, kidney, brain, heart, lung, spleen, and bile. A total of 10 993 analytical results from 5375 post‐mortem cases in 388 studies were gathered and the ratios of drug concentrations in tissue material to median femoral blood concentrations were calculated. Analytical results from the laboratory's own database (years 2000–2018) were also included. The results show that the variation of ratios between post‐mortem specimens and femoral blood is highly compound dependent. This database can be utilized in interpretation of toxicological results in cases where femoral blood is not available. The specimens with similar concentrations as in femoral blood were vitreous humor, muscle, and other blood, such as cardiac blood, and the highest concentrations were generally measured from liver and bile. For these reasons we suggest the following order for biological specimens to be used for a quantitative toxicological analysis in cases where femoral blood is not available: 1. other blood, 2. muscle, 3. vitreous humor, 4. brain, 5. heart, 6. spleen, 7. kidney, 8. liver, and 9. bile.     

Summary statistics for drug concentrations in post‐mortem femoral blood representing all causes of death

Concentration distributions for 183 drugs and metabolites frequently found in post‐mortem (PM) femoral venous blood were statistically characterized based on an extensive database of 122 234 autopsy cases investigated during an 18‐year period in a centralized laboratory. The cases represented all causes of death, with fatal drug poisonings accounting for 8%. The proportion of males was 74% with a median age of 58 years compared with 26% females with a median age of 64 years. In 36% of these cases, blood alcohol concentration was higher than or equal to 0.2‰, the median being 1.6‰. The mean, median, and upper percentile (90th, 95th, 97.5th) drug concentrations were established, as the median PM concentrations give an idea of the “normal” PM concentration level, and the upper percentile concentrations indicate possible overdose levels. A correspondence was found between subsets of the present and the previously published PM drug concentrations from another laboratory that grouped cases according to the cause of death. Our results add to the knowledge for evidence‐based interpretation of drug‐related deaths.     

Determination of a selection of anti‐epileptic drugs and two active metabolites in whole blood by reversed phase UPLC‐MS/MS and some examples of application of the method in forensic toxicology cases

Quantitative determination of anti‐epileptic drug concentrations is of great importance in forensic toxicology cases. Although the drugs are not usually abused, they are important post‐mortem cases where the question of both lack of compliance and accidental or deliberate poisoning might be raised. In addition these drugs can be relevant for driving under the influence cases. A reversed phase ultra‐performance liquid chromatography‐tandem mass spectrometry method has been developed for the quantitative analysis of the anti‐epileptic compounds carbamazepine, carbamazepine‐10,11‐epoxide, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, 10‐OH‐carbazepine, phenobarbital, phenytoin, pregabalin, and topiramate in whole blood, using 0.1 mL sample volume with methaqualone as internal standard. Sample preparation was a simple protein precipitation with acetonitrile and methanol. The diluted supernatant was directly injected into the chromatographic system. Separation was performed on an Acquity UPLC® BEH Phenyl column with gradient elution and a mildly alkaline mobile phase. The mass spectrometric detection was performed in positive ion mode, except for phenobarbital, and multiple reaction monitoring was used for drug quantification. The limits of quantification for the different anti‐epileptic drugs varied from 0.064 to 1.26 mg/L in blood, within‐day and day‐to‐day relative standard deviations from 2.2 to 14.7% except for phenobarbital. Between‐day variation for phenobarbital was 20.4% at the concentration level of 3.5 mg/L. The biases for all compounds were within ±17.5%. The recoveries ranged between 85 and 120%. The corrected matrix effects were 88–106% and 84–110% in ante‐mortem and post‐mortem whole blood samples, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Drug concentrations in post‐mortem femoral blood compared with therapeutic concentrations in plasma

Therapeutic drug concentrations measured in plasma are of limited value as reference intervals for interpretation in post‐mortem (PM) toxicology. In this study, drug concentration distributions were studied in PM femoral venous blood from 57 903 Finnish autopsy cases representing all causes of death during an 11‐year period. Cause‐of‐death information was obtained from death certificates issued by forensic pathologists. Median, mean, and upper percentile (90th, 95th, 97.5th) concentrations were calculated for 129 drugs. To illustrate how PM median concentrations relate to established therapeutic ranges in plasma, a PM blood/plasma relationship was calculated for each drug. Males represented 75% of the subjects and showed a lower median age (55 yrs) than females (59 yrs). In 43% of these cases, blood alcohol concentration was higher than 0.2‰, and the median was 1.8‰. Sixty‐one (47%) of the 129 drugs showed a PM blood/plasma relationship of 1. For 22 drugs (17%), the relationship was <1, and for 46 drugs (35%), the relationship was >1. No marked correlation was found between the PM blood/plasma relationship and the volume of distribution (V_d). For 36 drugs, more than 10% of cases were fatal poisonings attributed to this drug as the main finding. These drug concentration distributions based on a large database provide a helpful reference not only to forensic toxicologists and pathologists but also to clinical pharmacologists in charge of interpreting drug concentrations in PM cases. © 2013 The Authors. Drug Testing and Analysis published by John Wiley & Sons, Ltd.     

An evaluation of the DRI‐ETG EIA method for the determination of ethyl glucuronide concentrations in clinical and post‐mortem urine

A commercial enzyme immunoassay for the qualitative and semi‐quantitative measurement of ethyl glucuronide (EtG) in urine was evaluated. Post‐mortem (n=800), and clinical urine (n=200) samples were assayed using a Hitachi 902 analyzer. The determined concentrations were compared with those obtained using a previously published liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the quantification of EtG and ethyl sulfate. Using a cut‐off of 0.5 µg/ml and LC‐MS/MS limit of reporting of 0.1 µg/ml, there was a sensitivity of 60.8% and a specificity of 100% for clinical samples. For post‐mortem samples, sensitivity and specificity were 82.4% and 97.1%, respectively. When reducing the cut‐off to 0.1 µg/ml, the sensitivity and specificity were 83.3% and 100% for clinical samples whereas for post‐mortem samples the sensitivity and specificity were 90.3 % and 88.3 %, respectively. The best trade‐offs between sensitivity and specificity for LC‐MS/MS limits of reporting of 0.5 and 0.1 µg/ml were achieved when using immunoassay cut‐offs of 0.3 and 0.092 µg/ml, respectively. There was good correlation between quantitative results obtained by both methods but analysis of samples by LC‐MS/MS gave higher concentrations than by enzyme immunoassay (EIA), with a statistically significant proportional bias (P<0.0001, Deming regression) for both sample types. The immunoassay is reliable for the qualitative and semi‐quantitative presumptive detection of ethyl glucuronide in urine. Copyright © 2012 John Wiley & Sons, Ltd.     

Investigation of cardiac glycoside levels in human post mortem blood and tissues determined by a special radioimmunoassay procedure.

Even after the introduction of radioimmunological methods the question of a cardiac glycoside causing or contributing to the death of a patient can not be answered satisfactorily. By means of a special radioimmunoassay procedure for digoxin as well as for the structurally related methyl- and acetylderivatives we measured the concentrations in human blood and post mortem tissues. We investigated the glycoside contents in the blood of intravenously digitalised (Novodigal) al) patients before and after death. At autopsy blood specimens were taken from the heart and the femoral vein. We found an increase of the glycoside level up to a highly toxic range (7--15 ng/ml) especially in the heart blood. Thus post mortem blood levels of digoxin and its derivatives are not suitable for a final decision in alleged cases of fatal poisonings. Measuring various concentrations in tussues and body fluids of the above cardiac glycosides mentioned revealed the kidney concentration to be of high value in confirming a digitalis poisoning. This organ and the heart show the highest tissue concentrations. Interpretations of fatal digitalis poisonings should be based on the additional knowlege of these concentrations. Individual cardiac glycosides may be analyzed by a combination of thin layer chromatography and radioimmunoassay.     

Further evidence for the presence of GHB in postmortem biological fluid: implications for the interpretation of findings

Analysis and interpretation of the findings for the drug of abuse gamma hydroxybutyric acid (GHB) in fatalities has become very problematic. This is primarily because of variable data in postmortem biological fluids resulting from the endogenous nature of the compound, possible postmortem production, and varying methods of detection. Preliminary studies support the use of plasma standards in determining urinary GHB concentrations and indicate measurement of GHB in postmortem biological fluids may be dependent on the method of analysis. In order to assist interpretation of postmortem data based on gas chromatography-mass spectrometry (GC-MS) analysis using GHB-d6 internal standard, the results of GHB concentrations measured routinely in postmortem blood and urine specimens in 40 fatalities received during a three-month period are shown. In all cases, GHB was not implicated in the cause of death; there was no apparent correlation between manner of death and resultant GHB concentrations. Mean concentrations of GHB determined in postmortem blood were found to be 12.3 mg/L (range = 2-29 mg/L, n = 38) and 12.6 mg/L (range = 4-25 mg/L, n = 17) (unpreserved and sodium fluoride-preserved samples, respectively) and 5.5 mg/L in unpreserved urine (range 0-18 mg/L, n = 39) and 4.8 mg/L in sodium fluoride-preserved (range 0-10 mg/L, n = 15) urine samples. Vitreous humor was available in two of the cases analyzed (GHB = 1 and 3 mg/L). The data support the potential use of sodium fluoride-preserved samples for interpretation of GHB concentrations, particularly if there has been an extended postmortem interval. In addition, interpretative cut-offs can be proposed for both postmortem blood and urine, based on the specific GC-MS method used. At blood concentrations less than 30 mg/L and at urine concentrations less than 20 mg/L, it is possible that any GHB detected could represent only endogenous GHB production.     

The effect of antioxidants on the long‐term stability of THC and related cannabinoids in sampled whole blood

The stability of cannabinoids is complex and crucial for the assessment of impaired driving caused by cannabis. Therefore, the effect of antioxidants on the long‐term stability of Δ⁹‐tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD), 11‐hydroxy‐Δ⁹‐tetrahydrocannabinol (THC‐OH), and 11‐nor‐9‐carboxy‐Δ⁹‐tetrahydrocannabinol (THC‐COOH) in whole blood samples preserved with fluoride citrate (FC) and fluoride oxalate (FX) mixtures was investigated at different temperatures. The measured concentrations of the cannabinoids in authentic whole blood preserved solely with FC or FX mixtures decreased significantly during prolonged storage at ?20°C. On average, less than 5% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage interrupted by 5 thawing/freezing cycles. The rate of decrease was greatest in FC‐preserved blood. The repeated thawing/freezing of the samples accelerated the instability progression. At 5°C approximately 60% of the initial concentrations of THC and CBD were recovered after 19 weeks of storage. No significant decrease was observed in samples stored at ?80°C during the test period of 5 months. The instability at ?20°C was to a great extend avoided by adding 30 mM ascorbic acid (ASC) to the samples before storage. Samples preserved with a combination of the FX mixture and ASC showed no significant decrease in the recovered concentrations during a 5‐month storage period interrupted by 6 thawing/freezing cycles. Samples preserved with a combination of the FC mixture and ASC showed almost similar improvements in cannabinoid stability. Other reducing agents such as sodium metabisulfite and glutathione also improved the stability in FX‐preserved blood stored at ?20°C.     

Postmortem distribution of tapentadol and N-desmethyltapentadol

Postmortem distribution concentrations of the pain medication tapentadol and its metabolite N-desmethyltapentadol are reported. Tapentadol (Nucynta?) is a synthetic mu-opioid receptor agonist that also has norepinephrine reuptake inhibitor action. The laboratory received two cases. Case 1: a 19-year-old, morbidly obese male with sudden unexpected death. Toxicology results revealed tapentadol (femoral blood: 0.77 mg/L, liver: 1.65 mg/kg), N-desmethyltapentadol (femoral blood: 0.07 mg/L, liver: 0.19 mg/kg), diazepam (femoral blood: 0.04 mg/L), nordiazepam (femoral blood: 0.06 mg/L) and amiodarone (femoral blood: 5.30 mg/L). Case 2: a 60-year-old female who died from complications following hip replacement. Only tapentadol (femoral blood: 0.26 mg/L, liver: 0.52 mg/kg) was found in the toxicology results. Quantitative results of tapentadol/N-desmethyltapentadol were achieved using liquid chromatography-tandem mass spectrometry in multiple reactions monitoring mode. This is the first known distribution study of tapentadol and N-desmethyltapentadol values in postmortem cases.     

A simple extraction and LC‐MS/MS approach for the screening and identification of over 100 analytes in eight different matrices

In the present study, a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) multi‐analyte approach using one single work‐up approach in whole blood, plasma, serum, post‐mortem blood, liver tissue, gastric content, hair, and urine was developed for fast target screening and reliable identification of 130 analytes often requested in clinical and forensic toxicology. Samples (500 μL each) of whole blood, plasma, serum, post‐mortem blood, tissue (homogenized 1 + 4 with water), as well as 3 g of distilled gastric contents, 1 mL of urine, or 20 mg of pulverized hair were extracted at different pH values with an diethyl ether‐ethyl acetate mixture (1:1). Separation and identification were performed using LC‐QTRAP with electrospray ionization in positive mode. For identification 1 scheduled multi‐reaction‐mode (sMRM) method with 390 transitions was developed covering benzodiazepines, Z‐drugs, antidepressants, neuroleptics, opioids, new synthetic drugs, and phosphodiesterase type 5 inhibitors. For positive sMRM transitions with intensities exceeding 5000 cps, dependent scans (EPI scan collision energy, 35 eV, collision energy spread, 15 eV) were performed for library search using our in‐house library. The method was developed with respect to selectivity, matrix effects, recovery, process efficiency, limit of detection, and applicability. The simple work‐up procedure was suitable for all biosamples with exception of urine in respect to low concentrated analytes, which showed median recovery values of 59%. The method was selective for 130 analytes in all 8 biosamples. For 106 analytes, the limit of detection in whole blood, plasma, and serum was lower than the lowest therapeutic concentration listed in blood level lists. Copyright © 2014 John Wiley & Sons, Ltd.     

Targeted analysis of 116 drugs in hair by UHPLC‐MS/MS and its application to forensic cases

A multi‐target method that can detect a broad range of drugs in human hair, such as hypnotics, anxiolytics, analgesics, benzodiazepines, antihistamines, antidepressants, antipsychotics, and anticonvulsants, was developed based on ultra‐high‐performance liquid chromatography–tandem mass spectrometry (UHPLC‐MS/MS). The drugs were extracted from 10 mg of washed hair by incubation for 18 h in a 25:25:50 (v/v/v ) mixture of methanol/acetonitrile/2 mM ammonium formate (8% acetonitrile, pH 5.3). For 51% of the basic drugs, the lower limits of quantification (LLOQs) were in the range of 0.05–0.5 pg/mg, and the majority (98%) were ≤ 5 pg/mg. Linearity ranged from LLOQs to 100–500 pg/mg for all the basic drugs. For acid and neutral drugs, the LLOQs ranged from 0.4 to 500 pg/mg, and linearity ranged from LLOQs to 80–40 000 pg/mg. According to published reports on concentrations attained in single dose control studies, the present method is sensitive enough to detect single‐dose drug exposure for many of the drugs. The accuracy was within 75–125% for the majority of drugs. Good precision was observed (relative standard deviations [RSD%] < 25%) for most of the compounds, including the prepared quality control (QC) hair samples. The method was applied to forensic cases and concentrations of rarely reported drugs in hair in 25 post‐mortem forensic cases were presented. Hair concentrations of amisulpride, gabapentin, mianserin, mepyramine, orphenadrine, and xylometazoline have not been previously reported. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro and in vivo metabolism and detection of 3‐HO‐PCP,a synthetic phencyclidine,in human samples and pooled human hepatocytes using high resolution mass spectrometry

The new psychoactive substance (NPS) 3‐HO‐PCP, a phencyclidine (PCP) analog, was detected in a law enforcement seizure and in forensic samples in Denmark. Compared with PCP, 3‐HO‐PCP is known to be a more potent dissociative NPS, but no toxicokinetic investigations of 3‐HO‐PCP are yet available. Therefore, 3‐HO‐PCP was quantified in in vivo samples, and the following were investigated: plasma protein binding, in vitro and in vivo metabolites, and metabolic targets. All samples were separated by liquid chromatography and analyzed by mass spectrometry. The unbound fraction in plasma was determined as 0.72 ± 0.09. After in vitro incubation with pooled human hepatocytes, four metabolites were identified: a piperidine‐hydroxyl‐and piperidine ring opened N‐dealkyl‐COOH metabolite, and O‐glucuronidated‐ and O‐sulfate‐conjugated metabolites. In vivo, depending on the sample and sample preparation, fewer metabolites were detected, as the O‐sulfate‐conjugated metabolite was not detected. The N‐dealkylated‐COOH metabolite was the main metabolite in the deconjugated urine sample. in vivo analytical targets in blood and brain samples were 3‐HO‐PCP and the O‐glucuronidated metabolite, with 3‐HO‐PCP having the highest relative signal intensity. The drug levels of 3‐HO‐PCP quantified in blood were 0.013 and 0.095 mg/kg in a living and a deceased subject, respectively. The 3‐HO‐PCP concentrations in deconjugated urine in a sample from a living subject and in post‐mortem brain were 7.8 and 0.16 mg/kg, respectively. The post mortem results showed a 1.5‐fold higher concentration of 3‐HO‐PCP in the brain tissue than in the post mortem blood sample.     

Importance of vacutainer selection in forensic toxicological analysis of drugs of abuse

The enzymatic degradation of cocaine in blood samples, even during transport to a forensic laboratory, is a common problem in toxicological analysis. This can be avoided by the use of blood-sampling devices such as gray-top Vacutainers containing the cholinesterase inhibitor sodium fluoride. In the present study, which included 147 authentic cases, blood samples were collected into two different tubes, one containing fluoride/oxalate and one without stabilizing agents. In all cases, both samples were analyzed for drugs of abuse using Abbott FPIA immunoassays after precipitation and gas chromatography-mass spectrometry (GC-MS) for quantitative analysis. The cannabinoid immunoassay showed markedly lower values in the fluoride-containing samples; this was investigated further and could be explained by hemolysis of these samples. In addition, the concentrations of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) were lower in these samples. A stability study with the THCCOOH acyl glucuronide showed that it is unstable in unpreserved serum, which could explain our observation. GC-MS quantitative data for amphetamine and derivatives, opiates, delta9-tetrahydrocannabinol, and 11-hydroxy-delta9-tetrahydrocannabinol were essentially identical; however, they also differed substantially for cocaine, cocaethylene, ecgonine methylester, and benzoylecgonine. Unexpectedly, the concentrations of benzoylecgonine in unpreserved serum were almost half as high as in the fluoride-containing samples.     

Effects of the Fas/Fas‐L pathway on fluoride‐induced apoptosis in SH‐SY5Y cells

The mechanisms underlying fluoride‐induced apoptosis in neurons still remain unknown. To investigate apoptosis, caspase‐3 activity, and mRNA expression of Fas, Fas‐L, and caspases (‐3 and ‐8) induced by fluoride, human neuroblastoma (SH‐SY5Y) cells were incubated with 0, 20, 40, and 80 mg/L sodium fluoride (NaF) for 24 h in vitro. The data show that cell viability in the 40 and 80 mg/L fluoride groups were significantly lower than that of the control group. The percentages of apoptosis in the 40 and 80 mg/L fluoride groups were markedly higher than those in the control group, and they increased with the increase in fluoride concentration. The activity of caspase‐3 and mRNA expression levels for Fas, Fas‐L, and caspases (‐3 and ‐8) in the 40 and 80 mg/L fluoride groups were significantly higher than those in the control group. An agonistic anti‐Fas monoclonal antibody (CH‐11) significantly augmented apoptosis induction by fluoride, showing a synergistic effect, while a Fas‐blocking antibody (ZB4) partly inhibited fluoride‐induced apoptosis of SH‐SY5Y cells. The results indicate that fluoride exposure could induce apoptosis in SH‐SY5Y cells, and the Fas/Fas‐L signaling pathway may play an important role in the process. © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 86–92, 2011.     

Investigation of cardiac glycoside levels in human post mortem blood and tissues determined by a special radioimmunoassay procedure

Even after the introduction of radioimmunological methods the question of a cardiac glycoside causing or contributing to the death of a patient can not be answered satisfactorily. By means of a special radioimmunoassay procedure for digoxin as well as for the structurally related methyl- and acetylderivatives we measured the concentrations in human blood and post mortem tissues.We investigated the glycoside contents in the blood of intravenously digitalised (Novodigal^®) patients before and after death. At autopsy blood specimens were taken from the heart and the femoral vein. We found an increase of the glycoside level up to a highly toxic range (7–15 ng/ml) especially in the heart blood. Thus post mortem blood levels of digoxin and its derivatives are not suitable for a final decision in alleged cases of fatal poisonings.Measuring various concentrations in tissues and body fluids of the above cardiac glycosides mentioned revealed the kidney concentration to be of high value in confirming a digitalis poisoning. This organ and the heart show the highest tissue concentrations. Interpretations of fatal digitalis poisonings should be based on the additional knowledge of these concentrations. Individual cardiac glycosides may be analyzed by a combination of thin layer chromatography and radioimmunoassay.Presented as short communication at the V^th meeting of the international academy of legal medicine, Caracas/Venezuela 4.–10. 12. 1977     

Evaluation of Post‐Mortem Effects on Global Brain DNA Methylation and Hydroxymethylation

The number of epigenetic studies on brain functions and diseases are dramatically increasing, but little is known about the impact of post‐mortem intervals and post‐sampling effects on DNA modifications such as 5‐methylcytosine (5mC) and 5‐hydroxymethylcytosine (5hmC). Here, we examined post‐mortem‐induced changes in global brain 5mC and 5hmC levels at post‐mortem intervals up to 540 min., and studied effects of tissue heat stabilization, using LUMA and ELISA. The global 5mC and 5hmC levels were generally higher in the cerebellum of adult rats than neonates. When measured by ELISA, the global 5mC content in adults, but not neonates, decreased with the post‐mortem interval reaching a significantly lower level in cerebellum tissue at the post‐mortem interval 540 min. (2.9 ± 0.7%; mean ± S.E.M.) compared to control (3.7 ± 0.6%). The global 5hmC levels increased with post‐mortem interval reaching a significantly higher level at 540 min. (0.29 ± 0.06%) compared to control (0.19 ± 0.03%). This suggests that the post‐mortem interval may confound 5mC and 5hmC analysis in human brain tissues as the post‐mortem handling could vary substantially. The reactive oxygen species (ROS) level in cerebellum also increased over time, in particular in adults, and may be part of the mechanism that causes the observed post‐mortem changes in 5mC and 5hmC. The global 5mC and 5hmC states were unaffected by heat stabilization, allowing analysis of tissues that are stabilized to preserve more labile analytes. Further studies in human samples are needed to confirm post‐mortem effects on DNA methylation/hydroxymethylation and elucidate details of the underlying mechanisms.