Department of Forensic Medicine, Göteborg University, Göteborg,
1 Department of Medicine, Sahlgrenska University Hospital/Östra, Göteborg University, Göteborg and
2 Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden
Received 17 November 1999; in revised form 16 November 2000; accepted 12 December 2000
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ABSTRACT |
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INTRODUCTION |
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In the clinical setting, elevated serum concentration of carbohydrate-deficient transferrin (CDT) is used as a specific marker of potentially harmful alcohol consumption (Stibler, 1991). An increased serum CDT concentration corresponds to continuous ingestion of large amounts of alcohol (Salmela et al., 1994
). Due to the long half-life of CDT in serum it is possible to detect high serum concentrations several days after an alcohol debauch (Stibler, 1991
). The CDT method thus seems to be well-suited to indicate pre-mortal alcohol misuse in an alcoholic. Thus, an elevated serum CDT concentration in combination with negative blood and/or urine ethanol concentrations would support a withdrawal-related death. However, in post-mortem analysis, clinically used cut-off levels do not seem to be applicable to serum CDT (Sadler et al., 1996
). High values of CDT may thus be false due to carbohydrate-chain dissociation from the transferrin molecule by enzymatic or bacterial interactions in blood after death (Sadler et al., 1996
).
Our hypothesis was that some of these methodological problems could be overcome if a fenced body fluid such as vitreous humour was used for post-mortem CDT analysis. Consequently, we aimed to investigate whether in the present study reliable post-mortem analysis of vitreous humour CDT concentration (VH-CDT) could be achieved by use of a commercially available assay designed for serum analysis (CDTectTM). Moreover, we intended to evaluate the usefulness of VH-CDT as a marker of alcohol misuse and possible withdrawal-related death in forensic medicine.
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MATERIALS AND METHODS |
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For each subject, the time-points were noted regarding when the person had been seen alive for the last time and when the body was found (see Table 1). Cause of death was established based on clinical and biochemical investigations according to the routines at the forensic department (Table 1
). The study was approved by the Research Ethics Committee of Göteborg University.
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Biochemical assay
Carbohydrate-deficient transferrin concentration in vitreous humour (VH-CDT).
One tube of vitreous humour from the eye from each subject was thawed, well mixed and tested in double duplicate in the same assay run using CDTectTM radioimmunoassay (Pharmacia & Upjohn Diagnostics AB, Uppsala, Sweden, nowadays Axis-Shield PLC, Oslo, Norway) according to the manufacturer's directions as detailed by Stibler et al. (1991). Briefly, after iron saturation of the samples, separation of the transferrin isoforms with pI >5.7 was accomplished by anion-exchange chromatography on microcolumns. Thereafter, these carbohydrate-deficient fractions of transferrin were quantified by a double-antibody radioimmunoassay with a standard curve comprising values from 5 to 300 U/l. The mean value was reported and used for all calculations. The intra-assay and inter-assay coefficients of variation for CDT analyses were <5 and 16.8%, respectively.
Cut-off level for VH-CDT.
Since the transferrin content in vitreous humour is <10% of that in serum (Devgun and Dunbar, 1986), it was unknown at the planning of the study whether CDT could be detected in vitreous humour. It thus seemed appropriate to use the detection limit of the commercially available test, which is 5 U/l, as the highest acceptable cut-off level of VH-CDT for recent alcohol abuse.
Ethanol concentrations
. Ethanol concentrations were measured by headspace gas chromatography (Jones and Schuberth, 1989) at the Department of Toxicology, National Institute of Forensic Medicine (Linköping, Sweden).
Statistics
Due to the small sample size, Fisher's exact test was used to compare categorical data between alcoholics and controls. P < 0.05 was considered as statistically significant.
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RESULTS |
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DISCUSSION |
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The choice of CDTectTM for analysis of VH-CDT in our study turned out well because, by chance, it seemed to be possible to use the detection limit of this test in a semiquantitative way to discriminate between alcoholics and non-alcoholics. The highly significant result in the Fisher's exact test was somewhat surprising, because it could hardly be expected that all of the alcoholics would have been in a drinking phase. However, the outcome supports that most of the alcoholics had been drinking heavily prior to death, because a raised VH-CDT level is related to alcohol consumption and not to alcoholism per se.
Two of the controls had detectable levels of VH-CDT. This may be due to selection failure; that is, they may in fact have been high-consumers of alcohol. However, since these two controls had a much longer post-mortem interval before the vitreous humour specimens were collected, time-dependent changes in CDT concentrations may have occurred also in vitreous humour, giving a false positive test. Theoretically, this hypothesis could easily be investigated by taking repeated samples of vitreous humour from some subjects at various time points for CDT analyses. However, this procedure was not considered appropriate, because it must be assumed that the barrier surrounding the vitreous humour in this way is destroyed and thus the VH-CDT will be influenced in the same manner as is noted in post-mortal serum specimens. Another cause of a false positive result could be certain eye disorders in which the protein content may increase and the protein composition may change as a consequence of the breakdown of the bloodretinal barrier (Bresgen et al., 1991).
Until more data are available on this subject, it seems reasonable to restrict the post-mortem interval for the use of VH-CDT analysis. Nevertheless, when an arbitrary, but in forensic medicine practical, post-mortem interval of 72 h for VH-CDT analysis was applied, the outcome was not considerably changed.
The conclusions from the present preliminary study, with a rather small sample, must be drawn with some caution. There is a need for more studies to compare the various CDT methods (Keating et al., 1998; Viitala et al., 1998
). If the findings of this study can be reproduced and established, analysis of CDT in vitreous humour can be useful in forensic medicine with at least two applications. First, it may make it possible to detect heavy alcohol consumption before death in forensic cases in general. Such information may explain certain confusing autopsy findings, and may also be helpful in the process of establishing cause of death in obscure cases. Secondly, in persons with known alcohol-dependence, analysis of CDT in vitreous humour, in combination with blood and urine ethanol tests, may offer an opportunity to define an alcoholic state during which an individual has died (Table 3
). This approach would make it possible to link various causes of death to certain phases of alcohol misuse and thus provide a tool to identify withdrawal-related death in alcoholics.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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