This study provides a unique resource for studying the existence of pathologies detected at forensic autopsy with particular emphasis upon the role of recent and chronic alcohol drinking in a country with a high level of hazardous drinking. These first results have shown that several of the novel alcohol biomarkers appear to perform well in post-mortem samples, and could be more widely used in other autopsy studies particularly in the absence of proxy-reported alcohol consumption patterns.
A major strength of this study is the systematic collection of information about the presence/absence of other pathologies of interest, regardless of whether or not they were considered to have contributed to the death. This goes considerably beyond what would be recorded routinely at forensic autopsy. A further strength is the systematic collection of proxy information about the deceased. However, the most unique feature of the study is the information on a wide range of informative alcohol biomarkers, several of which have not been used previously in this sort of post-mortem research study. The main weakness of the study is that it is restricted to deaths subject to forensic autopsy. These are a highly selected subset of all deaths, and thus cannot be used as a basis for estimating the overall burden of alcohol-related mortality. Furthermore, it is necessary to be aware of the potential for bias (such as recall and social acceptability) in the proxy interviews. Nevertheless, this sort of study can provide insights into the coexistence of multiple pathologies, and their relationship to patterns and levels of alcohol consumption.
One of the striking substantive findings is that over half of the deaths for whom proxy interviews were conducted were of men of non-Estonian (mainly Russian) ethnicity. This is a far higher proportion than seen in the national mortality data for men of working age, although it may partially reflect the fact that they are more likely to live in towns. However, we do know that non-Estonians have a higher mortality than Estonians for almost all causes of death in Estonia [24
], and especially alcohol-related causes [25
], consistent with known differences in binge [4
] and surrogate drinking [7
How do the results reported here compare to those from other autopsy studies? In a series of consecutive routine medico-legal autopsies in Norway 1973-1992 [27
], 47.6% of autopsies had a BAC ≥ 0.5 mg/g. In our study, 55% of autopsies had a BAC ≥ 02 mg/g, which is the legal limit for driving a vehicle in Estonia [28
This study substantially extends our understanding of the potential use of alcohol-related biomarkers at autopsy. We already know that liver enzymes, used widely to detect liver damage in living subjects, are of limited value post mortem. AST and ALT are increased by haemolysis and hypoxia and so are subject to differences in the process of dying. In contrast, GGT, as a marker of liver fibrosis and cirrhosis, is less affected by these processes.
Notably, PEth in blood, and EtG and EtS in urine all had zero or very low levels among the never/almost never drinking category. This indicates a high specificity of the novel alcohol biomarkers even in an autopsy setting. Nevertheless, the risk of post-mortem elevation needs to be carefully considered when interpreting values of PEth in post-mortem toxicology, given that some extremely high results were observed. Artefactual elevation of PEth levels may be due to ethanol produced by post-mortem fermentation in those who have not been drinking prior to death, and/or during storage in samples where the blood alcohol concentration at death was high [18
EtG and EtS are conjugated ethanol metabolites. We have demonstrated [29
] that EtG is sensitive to bacterial degradation (e.g. in the presence of E. coli) but also that EtG may be formed due to bacterial activity when ethanol is present. In contrast, EtS is not susceptible to bacterial degradation/synthesis making it the preferable biomarker for recent drinking in post-mortem toxicology. In routine clinical use, both metabolites are therefore measured as was done in this study. However, calculation of the EtG/EtS molar ratio, as an index of EtG disappearance/formation showed similar, albeit significantly higher values than in a Swedish clinical reference material (mean/median in the Estonian post-mortem sample were 2.57/2.39 mg/L; in the Karolinska reference sample 2.34/1.70 mg/L; p
= 0.0064). There was only one sample that showed a markedly different ratio. However, this indicates that bacterial interference between time of death and autopsy was not a major problem in this study. Taken together, these data suggest that these novel biomarkers perform well as quantitative markers of the frequency of recent alcohol intake (i.e. within ~1-3 days) in aetiological epidemiological research. Despite this, their use in forensic legal contexts still requires caution [31
In summary, this paper describes a unique resource for investigating the contribution of alcohol to premature mortality. The data are being further exploited to look at a number of key areas: 1) a more detailed exploration of the association between morphological pathologies, biomarkers of alcohol consumption and proxy reports of drinking behaviour; 2) assessment of the association of autopsy evidence of alcohol-induced damage and proxy reports and biomarker indicators of high levels of alcohol consumption; 3) investigating the association of proxy reports and biomarker indicators of alcohol consumption with mortality from cardiovascular conditions.
We hope that the approach that we have described will be applied by other research groups in other settings, thus helping to improve our still surprisingly limited knowledge of the post-mortem findings associated with problematic and hazardous alcohol use.