From: Richard Lake ] Date: Fri, 03 Jul 1998 21:44:41 -0400 Newshawk: Richard Lake
Source: The Lancet (UK)
Contact: lancet.editorial@elsevier.co.uk
Website: http://www.thelancet.com/
Pubdate: 27 June 1998

Authors: Franco Tagliaro, Zeno De Battisti, Frederick P Smith, Mario Marigo Note: Institute of Forensic Medicine, University of Verona, Policlinico, 37134 Verona, Italy (F Tagliaro MD, Z De Battisti MD, M Marigo MD); Department of Justice Sciences, University of Alabama at Birmingham, Birmingham, AL, USA (F P Smith PhD).

Correspondence to: Dr Franco Tagliaro DEATH FROM HEROIN OVERDOSE: FINDINGS FROM HAIR ANALYSIS

Summary Background Morphine analysis of hair is used in forensic toxicology to study the addiction history of heroin addicts. To clarify the features underlying fatal heroin intake, we measured hair morphine content in a group of deceased heroin addicts, to verify a possible correlation between fatal heroin overdoses and the addiction behaviour of these individuals before death. Methods 91 deaths were attributed to heroin overdose in Verona, Italy, in 1993-96.
We analysed the hair of 37 of these individuals, and of 37 active heroin addicts, 37 former heroin users abstinent from the drug for several months, and 20 individuals with no evidence of exposure to opioids. From each individual, a hair sample of about 150 mg was analysed by RIA and high-performance liquid chromatography, to measure the morphine content.

Findings The mean morphine content in the hair of the addicts who had died was 1·15 ng/mg (SD 2·35 ng/mg; range 0-12·25 ng/mg) compared with 6·07 ng/mg (4·29; 1·15-17·0) in the active heroin addicts, 0·74 ng/mg (0·93; 0·10-3·32) in the abstinent former addicts, and values below the detection limit in the non-exposed group. Hair morphine content among those who had died was significantly lower than that in active heroin consumers (p(0·0001), but not significantly different from that in the former addicts (p0·978). Interpretation Although our findings may be subject to selection bias, since suitable hair samples were available for only 37 of the 91 addicts who had died, these findings support the theory of high susceptibility to opioid overdose after periods of intentional or unintentional abstinence, due to loss of tolerance.

Medical staff running detoxification programmes should be aware of the risk inherent in relapse to heroin after a period of abstinence. Moreover, occasional heroin use without a build-up of tolerance could also give a high risk of overdose.

Lancet 1998; 351: 1923-25 Introduction In Italy, according to official epidemiological data, heroin overdoses account for about 1000 deaths per year.1 Despite the efforts of forensic pathologists, clinical pathologists, and toxicologists, the mechanisms by which heroin overdose leads to death are not yet clear. A major reason for the lack of clarification is that blood samples taken from people who have died from heroin overdose show great variation in the amounts of biologically active metabolites of heroin present. Even in cases of acute overdose, observed blood concentrations of morphine, the main active metabolite of heroin, have ranged from 10 ng/mL to 4000 ng/mL.2,3 This range hampers the definition of a clear threshold of lethal heroin intake. The range can be partly ascribed to variable survival times after heroin injection,4-6 which in most cases are unknown, and to the rapid disappearance of heroin and its active metabolites from the blood (half-life heroin, 9 min; 6-acetylmorphine, 38 min; morphine, 80 min2). Thus, even though the blood concentrations fall, morphine bound to receptors in the central nervous system may lead to death by respiratory failure.7 Respiratory depression caused by opioids is the main physiological explanation of fatal heroin overdoses, but other explanations include metabolic variation in heroin tolerance,8,9 the toxicity of adulterants,10 pharmacological interactions with alcohol,11 and even allergic reaction to components of heroin preparations.12 However, the relevance of these factors in explaining the majority of deaths has not been statistically proved. Moreover, investigation of the mechanisms of fatal heroin overdose is hindered by gaps in individual case histories.

Any information given by relatives and friends concerning the medical history of the victim is likely to be unreliable because of the addict's lifestyle and environment. To address this issue, toxicological analysis of hair can be used in the retrospective investigation of drug use and addiction. Head hair grows at approximately 0·8-1·3 cm per month.13 Drugs can be detected in hair tissue weeks or months after intake. Exogenous compounds are incorporated into hair tissue at the root. They reach the growing hair matrix from capillary blood surrounding the hair germination centre, from skin-gland secretions,14 and, in some cases, from the external environment.15 The low metabolic activity of the hair shaft, and the protection exerted by the hair matrix components, contribute to the stability of the embedded compounds. Although contamination of the hair by drugs present in the environment,16 by hair bleaching, and by hair dyeing17 may affect the accumulation of chemicals in the hair matrix, there is consensus about the usefulness of hair analysis in the study of prevalence of drugs misuse.18 On these grounds, we used hair analysis, in addition to the usual forensic tests of biological fluids, to study heroin-linked deaths in the province of Verona, Italy. We aimed to verify a possible correlation between these deaths, and the drug use of the individual in the months before death. Methods >From among 91 heroin-related deaths between 1993 and 1996, we selected 37 individuals (29 men, eight women, aged 18-34) for hair analysis (group D). Criteria for selection were availability of hair, state of decomposition, lack of contamination of hair (blood, vomit, etc), lack of cosmetic treatments, availability of devices to sample hair during necropsy, and collaboration of the necropsy technicians.

All the cases underwent our routine pathological and toxicological analysis. Urine was screened for opioids, benzoylecgonine (cocaine metabolite), amphetamines, benzodiazepines, methadone, barbiturates, cannabinoids, and alcohol. Blood was tested for free (unconjugated) morphine, cocaine, and alcohol. Threshold positive concentrations of toxic agents in urine were as suggested by the US Department of Health and Human Services' Division of Workplace Drug Testing (300 ng/mL morphine or codeine/mL for opioids). A "positive" control group of 37 heroin addicts was also studied (group A1). The group consisted of 30 men and seven women, aged 18-32, who had just entered a detoxification programme at a local medical centre for drug addictions. Admission to the programme was based on clinical and toxicological investigations, including a positive test for opioids in urine. A second control group of 37 former chronic users of heroin, who had allegedly been abstinent from the drug for several months, was also investigated (group A2). Members of group A2 had applied for obtaining, or reobtaining, a driving licence, and according to Italian law (D Les 285, April 30, 1992) they had to be checked by physical examination and toxicological screening of their urine, to verify abstinence. They had undergone serial urine toxicological screening for opioids, benzoylecgonine, amphetamines, benzodiazepines, methadone, barbiturates, cannabinoids, and alcohol for about 40 days. Their hair was tested for morphine and cocaine. The people in group A2 showed negative results in the urine tests, but still had traces of morphine in their hair, suggesting either persisting occasional use of heroin, short abstinence from the drug, or exposure to opioids in the environment. As a "negative" control group, we chose 20 employees of the Institute of Forensic Medicine, 15 men and five women aged 27-44, with no evidence of exposure to opioids (group N). A hair sample of about 150 mg (3-5 cm long) was collected from the vertex posterior of each individual's head, by cutting with scissors as close to the scalp as possible. Hair samples were collected with the informed consent of the people in the control groups, and stored at room temperature in paper envelopes until analysis. The sample collection and pretreatment has been described in detail elsewhere.19 Hair specimens were washed with 50 mL ethyl ether and 50 mL 0·01 mol/L hydrochloric acid on a porous glass filter, then dried, cut with scissors into small fragments, and weighed. 100 mg of hair was incubated overnight in a water bath containing 2 mL 0·25 mol/L hydrochloric acid at 45°C. The incubation mixture was then neutralised with equimolar amounts of 1 mol/L sodium hydroxide, and twice extracted into organic phase (18% dichloromethane, 18% dichloroethane, 64% heptane, by volume) from a carbonate buffer pH 9, by use of a proprietary liquid-liquid extraction system. After shaking for 15 min, and phase separation by centrifugation at 500 g for 10 min, the organic phases from the two extraction steps were collected, pooled, and evaporated under an air stream. The dried extract was reconstituted with 1 mL 0·05 mol/L phosphate buffer pH 5. Qualitative analysis was done with RIA, and quantitative analysis was done with high-performance liquid chromatography (HPLC). The threshold positive level of morphine was 0·1 ng per mg of hair. The RIA was done with a commercial kit (Coat-a-Count, Diagnostic Products Corporation, Los Angeles, CA, USA). An iodine-125 labelled tracer was used, and apparatus tubes were coated with antibody highly specific for free morphine, which gave less than 2% cross-reactivity with morphine glucuronide and other major opioids.19 All results were then confirmed by HPLC, based on reverse-phase separation on a polymeric column (PLRP-S 5 µm, Polymer Labs, Church Stretton, UK), and amperometric detection at a glassy carbon electrode (+350 mV vs a silver/silver chloride reference), according to a routine procedure used in our institute.20 Quantitative blood analysis for morphine and cocaine was carried out by HPLC, with amperometric detection of morphine and fluorimetric detection of cocaine.21,22 The above methods have been used for years for routine testing of hair and blood samples.

Since 1990 the Institute of Forensic Medicine has participated in an inter-laboratory study for the validation of hair analysis techniques, promoted by the US National Institute of Standards and Technology, MD, USA. For the statistical analysis of our results we used one-way ANOVA, Student's t test for unpaired data, and the non-parametric Wilcoxon signed-rank test. Statistical tests used StatView SE+ Graphics (version 1·03). Results All urine samples from group A1 were positive for opioids. The urine samples from group A2 tested negative for all substances. All members of group D tested positive for opioids in urine. Benzodiazepines, alcohol, cannabinoids, amphetamines, and methadone were also found in the urine samples from several members of group D, but there was no specific pattern. Group N was not tested by urinalysis, because this information was not relevant for our purposes, and because toxicological urinalyses of employees are prohibited by law in Italy. The mean blood concentration of free morphine in group D was 273·3 ng/mL (SD 188·63 ng/mL, range 60-894 ng/mL). Blood morphine concentrations in group D were in all cases above established levels of toxicity.13 All members of group D were known to the police as heroin addicts. Based on this information, on the necropsy data, and on the findings at the death scene, all the deaths were attributed to acute overdose of heroin. In most cases, recent injection marks were found on the body, suggesting heroin intake by intravenous injection. The measurement of morphine in the hair of the deceased (group D) gave a mean value of 1·15 ng/mg (SD 2·35 ng/mg; range 0-12·25 ng/mg). There was no morphine in the hair of group N members. The mean morphine content of the hair of the active heroin addicts of group A1 was 6·07 ng/mg (SD 4·29; range 1·15-17·00 ng/mg). The incompletely abstinent individuals of group A2 had a mean value of 0·74 ng/mg (SD 0·93; range 0·10-3·32; figure). Mean hair morphine content (ng/mg) Horizontal barsmeans. A1active heroin addicts. Ddeceased following heroin overdose. A2incompletely abstinent individuals. One-way ANOVA showed a highly significant difference between the mean hair morphine contents found in groups A1, A2, and D (p(0·0001). The statistical comparison of the group data found that hair morphine contents were significantly lower in group D than in the active heroin consumers of group A1 (Wilcoxon test p(0·0001). However, hair morphine content in group D did not differ significantly from those of the incompletely abstinent subjects of group A2 (Wilcoxon test p0·978). As expected, the active addicts of group A1 showed higher mean morphine content in hair than the incompletely abstinent subjects of group A2 (p(0·0001). By the least square method, no linear (R20·017), exponential (R20·007), or logarithmic (R20·170) correlation was found between the amounts of morphine in the hair and in the blood of the people who died from heroin overdose.

Discussion The link between drug use and drug accumulation in hair has been examined in several earlier reports.23-26 To our knowledge, however, hair analysis has not been used to investigate the recent addiction histories of people who have died from heroin overdose. Our study was limited to the province of Verona, and may have been subject to selection bias since suitable hair samples were available for only 37 of the 91 addicts who died. However, we have shown that most fatal heroin overdoses occurred in heroin users with a much lower hair morphine content than that found in the hair of active, chronic consumers of the narcotic. On the assumption of a rough positive correlation between mean heroin intake and morphine concentrations in hair, and a hair growth rate of 1 cm/month, our findings suggest that most individuals who died from heroin overdose had virtually abstained from heroin during the 4 months preceding death. Thus, the results of this hair analysis support a theory of high susceptibility to opioid overdose after periods of intentional or unintentional abstinence. This theory has been used to explain the high number of deaths among addicts recently released from jail or on completion of a detoxification programme.

9,27 The reasons for increased susceptibility to overdose remain unclear, but it is likely that a lower heroin tolerance after a period of abstinence, or a low tolerance owing to light or irregular heroin use, leads to a corresponding decrease in the size of a fatal dose. The difference in hair opioid content between groups A1 and A2 in our study supports the continued use of hair testing in forensic analysis in cases of heroin overdose. The results of our study should indicate to the medical staff of detoxification programmes that there are risks inherent in relapse to heroin intake following abstinence from the drug. In particular, we point out the potential risk of "opioid free" detoxification programmes for individuals at risk of relapse. Moreover, occasional or recreational heroin use (eg, at weekends), an increasing heroin addiction pattern that is not characterised by dependence and tolerance, could lead to more cases of heroin overdose than is generally thought.

Contributors Franco Tagliaro was mainly responsible for writing the article, and for developing, and validating the analytical methods. Zeno De Battisti coordinated medical and pathological investigations, sample collection, and analysis. Frederick P Smith contributed to study design, and to statistical data analysis. Mario Marigo discussed, revised, and approved all aspects of the study, and the resulting paper. All researchers contributed to writing the paper. Acknowledgments We thank Carla Neri for routine urinalyses and Giovanna Carli for help.

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