Hargrove, Veronica M. PhD, FTS-ABFT; Molina, D. Kimberley MD
From the Bexar County Medical Examiner’s Office, San Antonio, TX.
Manuscript received May 16, 2013; accepted June 26, 2013.
Reprints: Veronica M. Hargrove, MS, FTS-ABFT, Bexar County Medical Examiner’s Office, 7337 Louis Pasteur Dr, San Antonio, TX 78229. E-mail: firstname.lastname@example.org.
The authors report no conflicts of interest.
Skeletal muscle constitutes a large percentage of the total body volume, making it a potentially widely available specimen for drug quantitation when blood is not available for toxicological testing. Morphine is a commonly encountered opiate in postmortem toxicology known to have stable blood concentrations in peripheral vessels. Morphine concentrations were measured in both femoral blood and skeletal muscle to assess the stability and predictability of skeletal muscle concentrations as compared with femoral concentrations. Analysis showed skeletal muscle was a sensitive matrix for the detection of morphine; however, there is significant disparity between the skeletal muscle and blood concentrations with a lack of predictability. The authors conclude that thigh skeletal muscle may be used for qualitative identification of morphine; however, interpretation of quantitative results should not be made as there does not seem to be a clear correlation between femoral blood and skeletal muscle concentrations for morphine.
In forensic toxicology, the most commonly used specimen to interpret drug concentrations is blood. However, medical examiners and forensic toxicologists are sometimes faced with cases where the proper specimens for toxicological analysis are not available. Blood may not be the most suitable specimen, or even available, in cases where there has been decomposition of the body, massive blood loss, or significant therapeutic blood transfusion. Skeletal muscle tissue, specifically thigh muscle, has been described as the preferred alternative specimen for analysis because its concentrations closely mimic those seen in peripheral blood.1
Postmortem redistribution is a commonly encountered and described phenomenon in forensic toxicology. Redistribution from tissue reservoirs is a major theory for how postmortem redistribution occurs. It is known that visceral organs, such as the lungs, heart, and liver, can serve as drug reservoirs affecting the central vasculature drug concentrations postmortem. After death, drugs stored in these reservoirs redistribute into the central vasculature, leading to a decrease in drug concentration in these tissues and an increase in the central blood concentrations over time.2 It has been shown that organs such as the lungs, liver, and cardiac muscle have higher drug concentrations and are therefore thought to allow for redistribution of drugs from these tissues into the surrounding vessels.2–5
Because skeletal muscle is present in large quantities and is affected by decomposition later than visceral organs, and it can be obtained from peripheral sites away from known tissue reservoirs, it has been argued that it should be the preferred tissue specimen for toxicological testing when blood is not available.1 However, knowing that visceral organs are known to be tissue reservoirs, it can be postulated that skeletal muscle could serve in the same capacity for the peripheral vessels. Thus, the present study was undertaken to assess whether morphine concentrations in skeletal muscle tissue from the thigh area are comparable to morphine concentrations in the adjacent femoral vein.
MATERIALS AND METHODS
Cases examined at the Bexar County Medical Examiner’s Office between February 2011 and February 2012 that had a history of known prescribed morphine use were selected for this study. Cases involving children and homicides and cases with extensive medical intervention and/or significant blood loss or trauma were excluded. Before obtaining the femoral blood sample, the femoral vein was clamped, and less than 3 mL of blood was drawn along with less than 5 g of muscular tissue. Blood samples were collected and stored in gray Vacutainer tubes (sodium fluoride preservative), and muscle samples were stored in heat sealed plastic bags. All samples were placed in a −20°C freezer until assayed. Bodies were kept refrigerated at 4°C upon arrival at the morgue. Morphine analysis was accomplished using a solid-phase extraction procedure, and samples were analyzed using liquid chromatography–tandem mass spectrometry.
A total of 18 cases met inclusion criteria for this study. The average age of the decedents was 80 years, with a range from 52 to 96 years. The majority of the cases were male (55.6%), with females accounting for 44.4%. On average, all sample collections were done within 24 hours after death (range, 3–24 hours).
There was 100% concordance between blood and muscle for qualitative morphine detection, meaning that when morphine was detected in the blood, it was also detected in the muscle. The average morphine concentration in the muscular tissue was 0.130 mg/kg (median, 0.079 mg/kg), with ranges between 0.011 and 0.501 mg/kg. The average morphine concentration in femoral blood was 0.155 mg/L (median, 0.097 mg/L), with ranges between 0.020 to 0.532 mg/L. The blood: muscle ratios averaged 2.15 (median, 0.92), with a range of 0.42 to 9.5. Table 1 shows the femoral vein blood concentrations, skeletal muscle concentration, and the ratio of blood to muscle concentrations. In the majority of the cases (75%), the blood-to-muscle ratio was less than 2.
In addition, regression analysis was performed to attempt to further delineate the relationship between blood and muscle concentrations. Polynomial analysis revealed the best fit; however, it is difficult to use and only achieved an r2 of 0.54 (Fig. 1). Linear analysis was also applied to all cases but only showed an r2 of 0.1; however, when this analysis was applied to only those cases where the blood morphine concentration was less than 0.3 mg/L, the r2 improved significantly to 0.65 (Figs. 2 and 3). These results seem to suggest that the relationship between morphine blood and muscle concentrations is predictable for the lower concentrations of morphine becoming more spurious at higher concentrations.
Given the potential utility of skeletal muscle as a valuable toxicologic sample, several studies have addressed it’s use. Previous studies examining various drugs have found skeletal muscle to be a good qualitative sample to assess for the presence or absence of drugs,4,6 consistent with what the current study found for morphine. In fact, two previous studies investigating the possibility of using muscle as an alternative specimen have concluded that most drugs can be detected in both blood and muscle7 and that muscle should be included as standard autopsy material to improve toxicological conclusions.8 Interestingly, Langford et al4 also found that central skeletal muscle (specifically the diaphragm) had higher drug concentrations than peripheral muscles, suggesting that muscle is susceptible to postmortem redistribution, similar to the changes found in the blood. However, studies have also found considerable variations in skeletal muscle concentrations leading to the conclusion that interpretation of muscle concentrations could be unreliable.4,6 In general, our results agree with these conclusions because muscle morphine concentrations did not consistently correlate with femoral blood concentrations.
Regarding specifically morphine, previous studies have shown ratios of blood to skeletal muscle concentrations to range between 0.67 and 15.5.8,9 However, the majority of these cases were overdose cases with high morphine concentrations. The present study has shown that the relationship between skeletal muscle and blood concentrations becomes more spurious at higher concentrations. This may be, in part, due to a possible effect of chronic use versus acute exposure; further research would need to be performed to address this and other possible variables.
The authors conclude that although interpretation of muscle concentrations can be difficult given the low predictability with blood concentrations, muscle does show high concordance for the qualitative assessment of the presence or absence of morphine and may be used as a screening sample, allowing for quantitation to occur on any blood available for analysis.
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