6‐Monoacetylmorphine‐antibody distribution in tissues from heroin‐related death cases: An experimental study to investigate the distributive response

Abstract Heroin, a semisynthetic opioid drug synthesized from morphine, is the 3,6‐diacetyl ester of morphine (diacetylmorphine). The post‐mortem diagnosis of heroin‐related death could be an issue and usually rely on a combination of investigations, including the autopsy, histological and toxicological analysis. We conducted the present study to evaluate the correlation between the heroin concentration in biological fluids (peripheral blood, bile and urine) and the post‐mortem anti‐6‐MAM antibody expression in various tissues (brain, heart, lung, liver and kidney) using immunohistochemical staining. A quantitative analysis of the immunohistochemical reaction was carried out. 45 cases of heroin‐related death investigated at the Forensic Pathology Institutes of the University of Rome, Foggia and Pisa were included. The control group was composed of 15 cases of death due to other causes, without brain lesions and negative toxicological analysis for drugs. We found a positive immunohistochemical reaction in different organs and it was related to the timing of heroin metabolization. No reaction was found in the control group. Our findings show that immunohistochemistry can be a valuable tool for the post‐mortem diagnosis of acute heroin abuse. A better understanding of the timing of heroin's metabolism can be useful in the forensic field and for future therapeutic applications.

deaths caused by overdose. 3 In Europe, in 2019, the average retail purity of heroin ranged from 11% to 51%, with half of the countries reporting an average purity between 18% and 31%. Indexed trends show that the average purity of heroin increased by 23% between 2009 and 2019, while its price decreased by 17%. National prevalence estimates range from less than one to more than seven highrisk opioid users per 1.000 inhabitants aged 15-64 years. Overall, this translates into about 0.35% of the European population or 1 million high-risk opioid users in 2019. Heroin was the third most common drug reported by Euro-DEN Plus hospitals in 2019, present in 16% of hospital admissions for acute drug-related toxicity. Opiates were found in 10 of the 26 hospital deaths reported, usually in combination with other drugs. 4 The peak concentrations in the blood are generally at 1-5 min from an intravenous injection and 5 min after snorting or intramuscular administration. Heroin is 2-3 times more potent than morphine and the estimated minimum lethal dose is 100-200 mg, but addicts may be able to tolerate up to 10 times as much. However, fatalities have occurred after doses of 10 mg.
Compared with morphine, heroin is a more lipophilic compound and crosses the blood-brain barrier within 15-20 s and achieves relatively high brain levels; 68% of an intravenous dose is absorbed into the brain. 5 Heroin (diacetylmorphine) is rapidly transformed into its active metabolites (i.e., 6-monoacetylmorphine , morphine, morphine-3-glucuronide [M3G] and morphine-6-glucuronide [M6G]), primarily in peripheral blood and to some extent in the liver, kidney and brain 6 ; with studies showing that the narcotic effects of heroin occur primarily via its major metabolite, 6-MAM. 7 Heroin and 6-MAM are highly lipophilic, easily crossing the blood-brain barrier (BBB), yet they are rapidly metabolized to opiate agonists (i.e., morphine and M6G) and the likely neurotoxic M3G. 8 In addition, the maximal brain concentrations (T max ) of 6-MAM were achieved at 15 min after heroin administration, similar to the reported T max of naloxone (NLX). 9 The heroin-related death is a remarkable issue that encompasses relevant health, judicial and forensic consequences.
Frequently, the final diagnosis is based upon a combination of scene investigation, physical examination of the body, the autopsy, as well as histological and toxicological findings. 10 So, post-mortem diagnosis of heroin-related death, could be an enigma. 11 Toxicology data is certainly of greater importance for the diagnosis of heroin-related death. Several implications for research arise from the literature on deaths attributed to heroin overdose because blood morphine alone often cannot explain the fatal event. 12 A true heroin overdose, in the absence of poly-drugs abuse, represents a minority of cases, and a more complex mechanism of action, of an inflammatory or immunological nature, has been repeatedly considered. 13 Lack of tolerance, the synergistic effect of other toxic substances, and even repeated allergic stimuli to adulterants, or to heroin itself, to the extent of inducing anaphylactoid reactions, are theories to be taken seriously. 14 At present, illicit fentanyl, and other synthetic opioids represent the third wave of the so-called opioid overdose epidemic. 15 Research is looking for how vaccines for the treatment of opioid use disorders and reduction of opioid-induced fatal overdoses fit within the current medication-assisted treatment portfolio. 16 The aim of this study was to clarify the correlation between heroin administration and the distributive response in heroin-related death, as well as to investigate the correlation between heroin blood concentration and the immunohistochemical features of various tissues through an anti-6-MAM antibody.

| MATERIAL AND ME THODS
The processing of the data reported in this paper is covered by the general authorization to process personal data for scientific research purposes granted by the Italian Data Protection Authority (1 March 2012 as published in Italy's Official Journal no. 72 dated 26 March 2012) since the data do not entail any significant personalized impact on data subjects. Our study does not involve the application of experimental protocols; therefore, it does not require approval by an institutional and/or licensing committee nor informed consent. In all cases, local prosecutors opened an investigation, ordering that an autopsy be performed to clarify the exact cause of death.

| Samples selection
The toxicological data and the autopsy records of the 204 autopsies of drug-related death performed at the Departments of Forensic Pathology of the University of Rome, Foggia and Pisa over the period 2014-2021 were evaluated, and 45 cases of heroin-related death were selected (42 men, three women, mean age 44.4 years).
The autopsy was performed within 36 h after death. Only cases with toxicological data positive for heroin and negative for any TA B L E 1 Mean free-morphine, total-morphine, and 6-monoacetylmorphine concentrations in peripheral blood, bile and urine in the 45 cases of heroin-related death selected from the databases  Table 1.
All cases were HIV-1 negative. The control group was composed of 15 cases (five women, ten men, mean age 43.9 years) that died from car accidents (n = 3) and sudden cardiac deaths (n = 2) with no clinical history, without brain lesions and negative toxicological analysis for drugs. Those deaths were characterized by their rapidity.

| Histological and immunohistochemical analysis
The study was carried out on samples of the brain (superior frontal gyrus and hypothalamus), heart, lung, liver and kidney. In the litera- The immunohistochemical findings and the gradation of the immunohistochemical reaction have been described with an ordinary scale and the median value has been reported. For the quantitative analysis, in each immunohistochemical section, we made 20 observations TA B L E 2 Semi-quantitative evaluation and statistical analysis [statistically significant (p < 0.05)] of the immunohistochemical findings and gradation of the immunohistochemical reaction in the brain, heart, lung, liver, kidney, intravascular samples in the heroin-related deaths and in the control group    Table 3 shows the comparison between the toxicological data and immunohistochemical reactions, as well as the PMI, in our cases.

| DISCUSS ION
We used an experimental model and immunohistochemistry to in- Additionally, our findings on lung samples showed positivity in the cytoplasm of macrophages and intra-alveolar spaces can be correlated with acute respiratory depression heroin-related death. 22 Macroscopic and microscopic pathological findings in case of heroinrelated death are nonspecific and may be inadequate to render a definitive diagnosis for forensic purposes. 23 Autopsies in such cases typically show severe pulmonary congestion and oedema weighing >1000 g per lung with a dilated right ventricle. 24 In cases of chronic and, in particular, intravenous drug consumption, histopathological findings are described, nonspecific, in all organs. One of the causes of death in heroin addiction is respiratory failure, often accompanied by pulmonary complications, especially oedema. The death is generally due to severe acute poisoning, regarding the degree of opioid tolerance possessed by the subjects at the time of the lethal dose.
As Büttner stated, up to 90% of all cases of heroin-related death show brain oedema with prominent tonsillar herniation and uncal grooving at autopsy. 25 However, rapid death after heroin intake has no, or poor, morphological evidence of cell injury. Heroin-related death can occur from the following: absolute acute poisoning; intake of a quantity of heroin superior, in an absolute sense, to any tolerance; acute intoxication related to tolerance: intake of a quantity of heroin greater than the degree of tolerance of the subject; first injection death: taking a lethal dose in a non-drug addict. 26 Katz subjects with many years of intravenous drug consumption, deposits of immunoglobulin and complement detected in the pulmonary interstitium. 28,29 Acute bacterial and nonbacterial endocarditis in intravenous drug-related deaths can be observed in the myocardium. 30,31 In the kidneys, related microscopic changes to the use of heroin are defined as 'heroin-associated nephropathy'. 32 The spectrum of heroin-associated kidney diseases includes primarily acute kidney failure, glomerulopathies, such as focal segmental glomerulosclerosis, membranoproliferative glomerulonephritis, and less often with immune complex glomerulonephritis. 33 A further histological report is rhabdomyolysis after heroin consumption. 34 On the central nervous system, heroin has various effects including hypoxic-ischemic brain damage from respiratory depressing effects and neuroinflammatory response. 25,35 In an immunohistochemical study, Neri et al. investigated the expression of brain numerous markers. 36 This study demonstrated that morphine induces inflammatory response and some cytokines were overexpressed (oxygen-regulated protein 150, cyclooxygenase-2, heat

TA B L E 3 (Continued)
shock protein 70, IL-6 and IL-15). In another immunohistochemical study, Fineschi et al. confirmed that elevated concentrations of serum tryptase are associated with many heroin-related deaths. 37 In several other studies, the morphological and histopathological organs' changes of chronic heroin abuse are described. 38 One limitation of our study is the relatively small sample size.
Precise criteria, however, were used for selecting subjects. Also,

ACK N OWLED G EM ENTS
This research received no external funding.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.