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Drugs from Unusual Matrices: Using Bone Tissue as a Forensic Toxicology Specimen

Wed, 07/03/2013 - 8:03am
Lata Gautam, Claire Newland, and Michael D. Cole

Forensic toxicologists are often called upon to determine whether or not a controlled substance has been ingested by or administered to a victim of accident or crime. The availability of many tissue types is limited in decomposed or skeletonized remains, and therefore bone tissue becomes an important specimen.Forensic toxicologists are often called upon to determine whether or not a controlled substance has been ingested by or administered to a victim of accident or crime. In order to make such assessments they routinely utilize specimens including blood, other body fluids, hair, and the organs associated with drug metabolism and excretion. However, the availability of such tissue is limited in decomposed or skeletonized remains, and therefore bone tissue becomes an important specimen. There are 206 bones in the adult human body and it constitutes on average 12–15% of the total body mass. Given the biochemistry of bone growth, development, and tissue turnover, it is reasonable to assume that bones could potentially represent a significant “sink” for drugs and their metabolites. Under such circumstances this would allow determination both of recent and historical drug use. However, at present there is limited research in drug detection in bone tissue, and the implications of results obtained are not fully understood.

Although a wide range of drugs have been detected in bones or bone tissue using animal model study using rats, pigs, and mice there are limited number of publications in which human bone samples have been studied. This paper addresses two of the most significant questions to be faced by toxicologists who might use bone samples:

  1. Is there variation in drug concentration within the same skeleton?
  2. Does burial affect drug detection in skeletal tissues ?

Early studies have proved drugs can be found in bone. These drugs include ketamine, morphine, diazepam, fentanyl, olanzapine, and amitriptyline etc. Different analytical techniques have been utilized to undertake the analysis including ELISA (Enzyme-linked immunosorbent assay) and liquid and gas chromatography coupled with mass spectrometry and other detectors. In vitro studies have shown that most of the drugs that were present in the blood were also detected in the bone samples and in some cases there were drugs detected only in the bones. However the difference in concentration has been reported that could be dependent on sample preparation method. A case study published in 2005 highlights the lack of research on the concentration of drugs in the bone. Blood, different tissues, and bone samples were analyzed from a psychiatric patient, who was found dead from intoxication of olanzapine and citalopram in a home for mentally ill adults.1 These drugs were extracted by incubating 1) 1 g bone 2) cutting into thin slivers prior to incubation in water and methanol for 24 h. Higher concentrations were found while slivers were incubated in methanol and the least effective method was incubating whole bone in water. Therefore, this highlights the need for a standardized sample preparation method to avoid bias in the interpretation of the results. Moreover, different extraction methods (solvent extraction, solid phase extraction, and microwave assisted digestion) have been used. Currently there is no data available on the recovery of drugs from bones which indicates that full method validation is required. Further there is little information on the cross reactivity of the drugs extracted from bone in the ELISA screening system.

 

Can there be any variation of drug detection within the same skeleton?
In vitro studies using animal models have been conducted to investigate whether toxicological findings in one skeletal tissue would match elsewhere within the same skeleton.2-4 Different drugs such as ketamine (a phencyclidine like stimulant), phenobarbital (anticonvulsant, barbiturate), nordazepam (a metabolite of diazepam, benzodiazepine) and diazepam (benzodiazepines), morphine (opiate, benzylisoquinoline), amitriptyline (tricyclic antidepressant), and citalopram (antidepressant) were investigated. Most of the drugs studied in all bone types. Ketamine was not detected in the ulnar whereas olazapine was not detected at all in the bone. In addition, there were variations between bone type and concentration found. For example concentration of ketamine and metabolites showed up to 23 fold variation. In a study of amitryptline and its metabolite nor-tryptline the drug metabolite ratio varied only by 2–6 fold with bone type. Some authors have reported 20–40 fold site dependent variability with the higher drug levels being found in the bones in the trunk of the body.2,3 In a rat model acute an exposure study of fentanyl from fresh and decomposed skeletal remains using ELISA as the detection method gave an instrument response in the following order marrow > vertebrae > pelvi > ephiphyseal bone > diaphyseal bone, again highlighting the site dependent variability. Higher level of drugs in femoral epiphyses could be due to the presence of trabecular (spongy) bone. This could also be due to the decomposition process where drugs enter the porous bone from neighboring liquefied tissues and the circulation process where bones containing red bone marrow (in direct contact with blood) exhibit higher levels than bones with yellow bone marrow.

 

Does burial affect drug detection in skeletal tissues?
Limited data is available on this area. The only human study in the authors’ knowledge was related to the detection of morphine from the bone sample (fresh and buried for one year) of a fatally poisoned heroin addict.5 The morphine concentration was 155 ng/g , 54.4% less in the buried bone sample which weighed 17% less when compared to the fresh sample (340 ng/g). However, 6-monoacetylmorphine, the major metabolite of diamorphine was not detected and so it was not possible to determine whether the morphine was from a pharmaceutical source or derived from the ingestion of heroin by the individual.

ELISA has been used to investigate whether a benzodiazepine detected in bone tissue were actually from the bone itself or from exogenous tissue6 using a mouse model. The drug (midazolam) was detected in the bone sample however absent in the bone marrow indicating that the drugs are detected in bone tissue itself and not a result of contamination by the drug in exogenous tissue.

 

Research Areas for the Future
If we are to interpret the detection of drugs in bones in a meaningful way, there are a number of questions which need to be answered. These include:

  • Are different classes of drug sequestered at different rates in bone of a given type or differentially in bones from different parts of the skeleton?
  • How does this relate to chemistry of the drug – pKa, ability to bond with inorganic components of bone?
  • What is the concentration vs. time curve for different drug classes in buried remains?
  • In buried remains are the drugs leached and if so what environmental factors affect this (temperature, water content of the substrate, pH, microrgansims)?
  • Does post-mortem concentration in bone correlate with antemortem levels in body fluids?


Conclusion
Bones are useful in decomposed or skeletonized cases where it is not possible to obtain body fluids to determine the antemortem blood levels. Research on the detection of drugs in bone is only in its early stages and interpretation of the toxicological analysis of bone is complicated. This is partly due to the heterogeneous anatomic structure of the bone and also due to the complex heterogenous nature of bone chemistry. Therefore drugs are not completely distributed evenly throughout. As a result of substantial site dependent variation, different bones within the same skeleton can yield a wide range of drug and metabolite levels.

Due to the limited research on this sample matrix, the detection of drugs is best interpreted as evidence of exposure only. Further work is required to determine the importance of drug concentration in bone with respect to method of administration, time since drug exposure and death, and drug-drug interactions.

 

References

  1. E.L. Horak, A. J. Jenkins, Post-mortem Tissue Distribution of Olazapine and Citalopram in a Drug Intoxication, Journal of Forensic Science, 50 (3) (2005), 1-3.
  2. N.A. Desrosiers, J.H. Watterson,D. Dean, J.F. Wyman, Detection of Amitryptyline, Citalopram, and Metabolites in Porcine Bones Following Extended Outdoor Decomposition, Journal of Forensic Science, 57(2) (2012) 544-549.
  3. J.H. Watterson,N.A. Desrosiers,C.C. Betit,D. Dean, J.F. Wyman, Relative distribution of drugs in decomposed skeletal tissue, Journal of Analytical Toxicology, 34 (2010) 510-515.
  4. J.H. Watterson, J.P. Donohue,Relative distribution of ketamine and norketamine in skeletal tissues following various periods of decomposition, Journal of Analytical Toxicology, 35 (2011) 452-458.
  5. N. Raikos, H. Tsoukali, S.N. Njau, Determination of opiates in post-mortem bone and bone marrow, Forensic Science International, 123 (2001) 140-141.
  6. L.Y. Gorczynski, F.J. Melbye, Detection of Benzodiazepines in different tissues, including bone, using a quantitative ELISA assay, Journal of Forensic Science, 46(4) (2001) 916-918.

Lata Gautam holds a B.Sc. (Hons.) and M.Sc. from Tribhuvan university in Nepal and a Ph.D. in Forensic Science from Anglia Ruskin University in the U.K. She is currently a Senior Lecturer in Forensic Science at Anglia Ruskin University. Her research interests include Forensic Toxicology and the analysis of drugs from biological matrices.

Mike Cole holds a B.A. (Hons.) in Natural Sciences from the University of Cambridge, U.K. and a Ph.D. in Natural Product Chemistry from the university of London. He is currently Professor of Forensic Science and Faculty Director of Research, Knowledge Transfer, and Scholarship in the Faculty of Science and Technology at Anglia Ruskin University. His research interests include development of analytical methods for street drugs and methods for drug comparison.

Claire Newland holds a BSc (Hons) in Biological Chemistry from the University of Leicester and a BSc in the Science of Dentistry from the University of Bristol. She is currently completing her MSc in Forensic Science at Anglia Ruskin University, Cambridge. Her research interest include analysis and identification of sized drug samples.

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