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Original Research European Journal of Forensic Sciences www.ejfs.co.uk DOI: 10.5455/ejfs.224357 Analytical data in support of the liver and peripheral blood concentration ratio as a marker of postmortem redistribution Iain M McIntyre ABSTRACT Forensic Toxicology Laboratory Manager, County of San Diego, Department of the Medical Examiner Address for correspondence: County of San Diego, Department of the Medical Examiner, 5570 Overland Ave., Suite 101, San Diego, CA 92123, U.S.A. Email: Iain. [email protected] Received: April 06, 2016 Accepted: May 18, 2016 Published: June 28, 2016 Objective: Postmortem redistribution (PMR) refers to the changes that can occur in drug concentrations after death. Consequently, postmortem blood concentrations may not always reflect the antemortem drug levels. A recent literature review has postulated a model describing drugs with a liver (L) to peripheral blood (P) concentration ratio less than 5 L/kg as being prone to little or no PMR, while drugs with an L/P ratio greater than 20-30 L/kg exhibit propensity for substantial PMR. Antidepressants including tricyclic antidepressants and some selective serotonin re-uptake inhibitors, for example, were obviously distinguished from drugs confirmed to be free from, or demonstrate little PMR. Methods: This current paper presents analytical L/P data from 867 postmortem cases yielding a ranking of 44 different drugs’ propensity for (and degree of) PMR. Results: The results are recorded on a continuum of propensity for PMR ranging from the lowest to the greatest. Drugs previously suspected to exhibit substantial PMR were noticeably differentiated from those thought not to demonstrate such postmortem changes–the higher the L/P, the greater the propensity for PMR. Moreover, drugs with intermediate propensity for PMR were discriminated from others with greater (or lesser) predisposition. Conclusions: The resulting classification of drugs’ propensity for–and anticipated degree of–PMR, should assist with a rational interpretation of postmortem drug concentrations for forensic experts. KEY WORDS: Forensic sciences, forensic toxicology, autopsy, liver, peripheral blood, postmortem redistribution INTRODUCTION A potentially significant issue complicating interpretation of postmortem drug concentrations results from the phenomenon referred to as postmortem redistribution (PMR). Postmortem drug concentrations in blood may not always reflect antemortem drug concentrations due to the movement of the drugs after death [1]. Accordingly, authors have argued a cautious approach in interpreting postmortem blood concentrations [2]. The mechanisms involved in PMR are both complicated and poorly understood. However, postmortem drug concentrations in blood have been thought to follow some commonly accepted trends that may aid with interpretation. In general, the characteristics of the drug itself have been used to estimate if a drug is subject to PMR. Substantial changes in blood drug concentrations have been predicted for basic, lipophilic drugs with a high volume of distribution [3]. When PMR occurs, blood specimens drawn from the central body cavity and heart generally exhibit higher drug concentrations postmortem than specimens drawn from peripheral areas, most commonly the femoral region. Diffusion of drugs from organ tissues into the blood may explain the observed phenomenon [1,4]. 178 The collection, analysis, and comparison of antemortem blood specimens would obviously be appropriate in assisting with the interpretation of postmortem blood drug concentrations, but relevant specimens are only rarely available. In a set of case studies of six drugs, concentrations in the postmortem femoral blood specimens exceeded the antemortem concentrations in five of the drugs studied, suggesting that even peripheral blood exhibited some redistribution [5]. The study reporting principally cases of overdose did not, however, convey the postmortem interval been death and autopsy. This interval (or postmortem delay) has been suggested to influence PMR [6]. The potential for redistribution of other drugs in postmortem peripheral blood has also been documented more recently [7]. In an early attempt to assess and demonstrate PMR, postmortem blood specimens were collected from two areas of the body at autopsy: A peripheral area and central area (often the heart/ cardiac) so that a comparison could be made. This approach appeared to provide a partial answer to the difficulties associated with the interpretation of postmortem drug concentrations. Prouty and Anderson [6] first detailed information about blood drug concentrations attained from the different sites for over Eur J Forensic Sci ● 2016 ● Vol 3 ● Issue 4 McIntyre: L/P ratio as a marker of postmortem redistribution 50 drugs. Then, Dalpe-Scott et al. [8] presented a tabular list of the drug concentrations from both cardiac and peripheral blood samples expressed as a ratio of cardiac to peripheral blood (C/P) for over 100 drugs. The C/P ratio became the accepted benchmark with the accepted guideline that ratios >1.0 were associated with redistribution, and high ratios indicated potential for substantial PMR [8]. This guideline was repeated in a review a few years later where many C/P ratios were republished [9]. Despite some apparent value, limitations of the C/P model, however, have been documented. While drug properties such as the volume of distribution, protein binding, and pKa were thought to contribute to PMR, a relationship between C/P and drug properties has not been established [10]. In addition, there has been little agreement as to what ratio actually defines a compound as one that is prone to substantial or minimal PMR [11]. Furthermore, reports of a C/P ratio >1.0 have been published for salicylate [5] and carisoprodol [11] which are not prone to redistribution. Arteriovenous differences, anatomic variability within individuals, and statistical chance may result in a C/P ratio >1.0 in drugs that do not redistribute. In addition, resuscitation attempts may result in a C/P ratio <1.0 [12]. Inaccurate ratios may also be obtained as an artifact of sampling on depletion of the cardiac blood volume by the collection of blood from connected blood vessels, or in cases of an acute overdose where the drug has not undergone complete absorption and/or distribution. Consequently, the traditional C/P ratios can be inconclusive and even misleading with respect to the interpretation of PMR [13]. Alternately, the liver to peripheral blood (L/P) ratio has been proposed as a marker for PMR. Ratios <5 L/kg were advocated to indicate little to no propensity toward PMR, and ratios exceeding 20-30 L/kg indicative of a propensity for substantial PMR [11]. A number of reports and a literature review elaborating on, and supporting, this model have now been published by this author [13-18]. The current paper describing analytical postmortem data from 867 cases is presented in support of the L/P ratio theory. Results were then applied to create a ranking 44 different drug’s propensity for (and degree of) PMR. METHODS Autopsy and Postmortem Specimen Collection In all cases, a full autopsy was conducted at the Medical Examiner’s Department, San Diego County. Autopsies were performed within 48 h of the recorded time of death. In cases in which the decedent was found dead, the time of death was recorded as the time found. This was not the exact time of death, which may have occurred several hours earlier. No attempt was made to establish the exact time of death for these cases. No cases showed obvious signs of decomposition; cases were not included if decomposition was noted by the medical examiner investigator or observed during the autopsy procedure. Eur J Forensic Sci ● 2016 ● Vol 3 ● Issue 4 Autopsies on the 867 cases examined in this investigation were performed over several years (2007-2015) by a staff of 10 board certified forensic pathologists. Although individual pathologists had slightly different approaches to details within the autopsy procedure, the general technique and specimen collection were consistent. The autopsy was started with a usual Y incision to allow viewing of the chest and abdominal organs. Following an initial inspection, each organ was removed for a more detailed examination. During the examination of the liver, sections of the right lobe of the liver (approximately 100 g) were collected and stored in an opaque plastic 4 ounce container without preservative. There was minimal chance of contamination from gastric contents or other sources with this section and collection technique. Upon removal of the intestines, the common iliac vein was visualized and punctured or cut and the peripheral blood specimens (generally 10-20 ml) collected and stored in standard glass tubes containing sodium fluoride (100 mg) and potassium oxalate (20 mg). Using this technique (visual identification of the iliac vein in the pelvis), the pathologist was able to ensure collection of blood returning from the leg. However, as the upper section of iliac vein was not usually clamped, there was potential for a small volume of blood to accumulate from more proximal regions in some cases. Despite this relatively minor exception, there was minimal opportunity for substantial contamination of the blood, especially from other sources. All samples were stored at 4°C until analyzed. Toxicological Analyses In general, the toxicological screening regimen consisted of the analysis of postmortem blood for alcohol and simple volatile compounds (GC-FID headspace), drugs of abuse by ELISA (at a minimum: Cocaine metabolite, opiates, methamphetamine, benzodiazepines, cannabinoids, fentanyl) (Immunalysis Inc., CA), and an alkaline drug screen by gas chromatography-mass spectrometry (GC-MS) following solid phase extraction. An acid/neutral drug screen with high-performance liquid chromatographic (HPLC)-photodiode array detection following specimen precipitation with acetonitrile was performed as required (often dictated by medications found at the scene). Positive results were confirmed and quantified by subsequent and specific techniques. Most of the drugs studied were quantified by an alkaline liquid-liquid extraction followed by GC-nitrogen-phosphorus detector detection which has been previously described [17]. Drugs determined by other procedures were carisoprodol and meprobamate by GCMS [11]; amphetamine and methamphetamine determined by GC-MS [18]; fentanyl by GC-MS [19]; lamotrigine and quetiapine, which were determined by the HPLC method [20]; acetaminophen by HPLC [21]. RESULTS AND DISCUSSION Table 1 presents the mean, standard deviation, and median L/P ratio data for 867 cases where 44 drugs were examined. The L/P values represent data determined from autopsied cases at the Department of the Medical Examiner, San Diego County. Some drugs investigated clearly exhibited a significant L/P 179 McIntyre: L/P ratio as a marker of postmortem redistribution ratio variability. This was particularly evident for a number of drugs previously considered to display substantial potential for PMR. Amitriptyline, for example, revealed a mean value of 30.4 L/kg with a standard deviation of 39.0 (for 57 cases). This substantial variability exemplifies the interpretative complexity of postmortem forensic toxicology. There are several potential explanations including individual variability, recent compared to long-term drug use, and possibly overdose cases – with potentially an inadequate time for complete distribution of the drug before sudden death. This concept has been described previously [8]. Consequently, the median value of 16.4 L/kg was projected to be a more representative value of the intrinsic L/P ratio for both this drug (amitriptyline) and most of the other drugs as well (the median is the most resistant statistic and having a breakdown point of 50%: So long as no more than half the data are contaminated, the median Table 1: L/P blood ratio data: Alphabetical listing for 44 drugs Drug Acetaminophen Amiodarone Amitriptyline Amlodipine Amphetamine Bupropion Carisoprodol Chlorpheniramine Citalopram Clomipramine Clozapine Cyclobenzaprine Desipramine Dextromethorphan Diltiazem Diphenhydramine Doxepin Doxylamine Fentanyl Fluoxetine Gabapentin Guaifenesin Hydrocodone Hydroxyzine Imipramine Lamotrigine Meprobamate Methadone Methamphetamine Metoprolol Mirtazapine Naproxen Olanzapine Paroxetine Promethazine Propoxyphene Propranolol Quetiapine Sertraline Tramadol Trazodone Venlafaxine Zolpidem Ethanol L/P: Liver/peripheral 180 Number of cases L/P (L/kg) (mean) SD L/P (L/kg) (median) 15 5 57 9 15 11 11 3 37 5 12 13 6 4 8 54 20 3 16 48 28 5 38 10 6 3 8 94 18 6 5 20 20 19 9 35 6 65 9 36 19 42 14 1.2 61.5 30.4 45.5 8.0 1.3 2.8 9.5 10.1 45.4 7.4 27.5 43.8 8.7 23.8 10.0 22.1 2.8 6.9 42.2 0.67 1.4 3.4 13.8 30.8 8.0 1.2 5.5 5.7 4.0 14.3 1.1 14.0 33.3 14.0 14.2 13.5 18.0 97.0 2.5 3.5 4.3 6.2 0.91 0.41 42.4 39.0 34.6 4.0 0.6 1.5 1.9 7.9 27.9 2.4 27.4 14.3 5.5 24.2 8.9 14.0 0.4 4.5 30.1 0.26 1.3 1.7 6.2 15.3 2.5 0.7 3.0 2.3 1.7 10.4 0.65 9.6 21.2 16.1 12.1 10.9 21.6 40.0 1.4 2.0 3.3 9.6 1.1 41.9 16.4 27.8 7.0 1.0 2.0 9.5 7.5 61.0 6.7 19.6 44.6 8.6 17.2 6.7 19.4 2.9 5.9 30.0 0.65 0.90 3.0 12.3 28.8 8.5 0.90 4.8 6.2 3.5 12.0 1.0 12.0 29.2 9.1 9.0 10.9 11.2 76.0 2.3 2.8 3.3 2.4 will not give an arbitrarily large result. The median can be used generally as a measure when a distribution is skewed, or when one requires reduced importance to be attached to outliers. Despite the fact many of the drugs did not demonstrate such large variability; the median value was applied to evaluate all drugs for purposes of consistency. Data from these 44 drugs corroborated earlier published reports supporting a relationship between the L/P ratio and susceptibility for drugs to exhibit potential for PMR [11,13,22]. Drugs known to be susceptible to significant PMR, such as the tricyclic antidepressants and some selective serotonin re-uptake inhibitors, were clearly distinguished from ethanol, gabapentin, carisoprodol, zolpidem, and other compounds not thought to exhibit substantial PMR. Several of these data have been independently published: Carisoprodol/meprobamate [11], sertraline [14], fentanyl [16], hydroxyzine [17], and methamphetamine/ amphetamine [18,22]. The data for ethanol were taken from the previous publication [23]. Nevertheless, the resulting L/P ratio (and consequent interpretation of potential for PMR) may not be upheld in all casework; especially, on occasion of substantially longer postmortem delay (>48 h), particularly for those compounds displaying extensive PMR potential. In such cases, and certainly in the event of decomposition, the possibility of considerable physical and chemical changes may cause additional and inconsistent drug redistribution, thereby increasing interpretative complexity. Furthermore, in cases of overdose, where the incomplete distribution of a drug can result in variable concentrations throughout the body’s organs and tissues, the current approach may not always be suitable. Such cases, therefore, should be interpreted even more cautiously. Collection procedures employed for postmortem blood and liver tissue samplings are also important matters for consideration. Consistency in the collection technique of postmortem blood is critical. Concentrations of many drugs have been shown to have substantial site-dependence [24]. Concentrations attained from drug analyses performed on heart (or central) blood, pericardial blood, chest blood, and perhaps subclavian blood, where drug concentrations can be erroneously elevated, may not be applicable to this particular model. Likewise, the site/location of collection of the liver sample is important. Liver concentrations may differ if collected near the lower left quadrant where contamination from gastric content can occur [25]. Nonetheless, if sample collections were consistent and contamination prevented (or at least marginalized) assumptions and calculations analogs to those made in this paper can be practicable. The results were then recorded on a continuum of propensity for PMR ranging from the lowest to the greatest [Table 2]. Again, as recognized earlier, drugs previously suspected to exhibit substantial PMR were noticeably differentiated from those thought not to demonstrate such postmortem changes – The higher the L/P, the greater the propensity for PMR. Moreover, drugs with intermediate propensity for PMR were discriminated from others with greater (or lesser) predisposition. Citalopram Eur J Forensic Sci ● 2016 ● Vol 3 ● Issue 4 McIntyre: L/P ratio as a marker of postmortem redistribution Table 2: L/P ratio: Listed in increasing propensity for PMR Drug Gabapentin Guaifenesin Meprobamate Ethanol Naproxen Bupropion Acetaminophen Carisoprodol Tramadol Zolpidem Trazodone Doxylamine Hydrocodone Venlafaxine Metoprolol Methadone Fentanyl Methamphetamine Clozapine Diphenhydramine Amphetamine Citalopram Lamotrigine Dextromethorphan Propoxyphene Promethazine Chlorpheniramine Propranolol Quetiapine Mirtazapine Olanzapine Hydroxyzine Amitriptyline Diltiazem Doxepin Cyclobenzaprine Amlodipine Imipramine Paroxetine Fluoxetine Amiodarone Desipramine Clomipramine Sertraline L/P (L/kg) 0.65 0.90 0.90 0.91 1.0 1.0 1.1 2.0 2.3 2.4 2.8 2.9 3.0 3.3 3.5 4.8 5.9 6.2 6.7 6.7 7.0 7.5 8.5 8.6 9.0 9.1 9.5 10.9 11.2 12.0 12.0 12.3 16.4 17.2 19.4 19.6 27.8 28.8 29.2 30.0 41.9 44.6 61.0 76.0 PMR: Postmortem redistribution, L/P: Liver/peripheral (L/P = 7.5 L/kg), for example, can be identified as an example of a drug which possesses propensity for PMR somewhat greater than that of venlafaxine (L/P = 3.3 mg/L), but substantially less than that of the traditional tricyclic antidepressants and some of the other SSRIs such as fluoxetine (L/P = 30.0). Furthermore, this ranking of drugs’ potential for PMR addressed the apparent incongruity observed in the interpretation of some postmortem drug concentrations. Sertraline, for example, a compound with a large volume of distribution (20-50 L/kg) has been reported with a relatively insignificant C/P ratio of <1.3 [14], and consequently, inconsistent with a drug exhibiting a substantial degree of PMR. However, postmortem blood concentrations (considered therapeutic to 1.0 or 1.5 mg/L) are commonly reported to be greater than expected in consideration of clinical investigations - studies report plasma sertraline concentrations averaging up to 0.20 mg/L, even with chronic high dosing of 300 mg daily [26]. This new analysis (displaying Eur J Forensic Sci ● 2016 ● Vol 3 ● Issue 4 L/P = 76.0 L/kg) confirmed the substantial potential for sertraline PMR and presented a more characteristic explanation of observed postmortem blood concentrations. This conclusion is also more consistent with expectation resulting from consideration of the particularly large volume of distribution. In addition, the list (showing drug ranking of potential for PMR) resolved interpretive confusion concerning other drugs such as metoprolol. Reports of inconsistent C/P ratio data have made assessment of potential PMR difficult – One report listed the C/P ratio data as l.0 or less [8] and another as 3.8 [6]. The current model confirmed the minimal potential for metoprolol PMR, with an L/P = 3.5 L/kg. One of the principal goals of these investigations was to attempt to provide a cataloging of drugs’ propensity for and, subsequently, their potential extent of PMR. Until now, most efforts in interpretation have simply described PMR by an aphorism ranging from “the drug has not been found to exhibit PMR” to “the drug is subject to PMR.” Such descriptions have never been particularly useful in the interpretation of postmortem drug concentrations, especially in relation to deducing what the drug concentration may have been at the time of death. Development of the L/P ratio model and the resulting estimation of propensity for PMR will hopefully provide a more definitive, and ultimately, a more authoritative numerical interpretation of PMR for many drugs and poisons. Although the approach described in this paper was capable of providing an accurate explanation and interpretation for data collected at this Medical Examiner’s Office, it can be anticipated (or even expected) that this will not be possible in all casework. As discussed earlier, the site of specimen collection and collection technique, excessive postmortem delay, cases of overdose, or contamination may cause susceptibility to significant changes in drug concentrations, producing additional interpretation difficulties, and rendering the current model unsuitable, or ineffective. Nevertheless, a technique to estimate potential for PMR, together with a reference list for 44 of some of the most commonly encountered drugs in postmortem forensic toxicology, is now attainable. The resulting classification of drugs’ propensity for – and anticipated degree of PMR, should assist with a rational interpretation of postmortem drug concentrations for forensic experts. ACKNOWLEDGMENTS The author would like to thank the San Diego County Chief Medical Examiner, Dr. Glenn Wagner, for making available case details described in this manuscript and Christina Meyer Escott, for collating much of the drug data. REFERENCES 1. 2. 3. Pounder DJ, Jones GR. 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Clin Chem 1994;40:498-9. Source of Support: Nil, Conflict of Interest: None declared. Eur J Forensic Sci ● 2016 ● Vol 3 ● Issue 4