Hypoxanthine in vitrous humour
Hypoxanthine accumulates when cells are deprived of oxygen. ATP (which can be described as the cell’s “fuel”) is enzymatically broken down into hypoxanthine, which is excreted by the cells. Normally there is no (~0.5 µmol/l) hypoxanthine present in vitreous humour in living individuals.
Background
The study of the postmortem rise of hypoxanthine and potassium in vitreous humour was first published in 1991 by Rognum et al. (Forens Sci Int 1991; 51:139). Since then, the finding has been confirmed by several research groups and improvements have been made. The latest paper on this method was published by Rognum et al in 2016 (Forensic Sci. Int. 262, 160–165).
The Hypoxanthine method has been used regularly at the Department of Forensic Pathology in Oslo for 6 years. In 26 cases the time of death could be established very precisely by tactical police investigation (e.g. electronic traces). In 23 of these cases the results of the Hypoxanthine method fitted with the tactical finding within the method's confidence interval (Rognum et al. Scand J Forens Sci; 2009;1:22).
Method
Eye fluid (vitreous humour) is a clear gel that fills the space between the lens and the retina of the eye. It is an easily available and less complex fluid (contains less interfering components) than for example blood. This makes it a good object of analysis.
The vitreous humour is collected with 5 ml vacutainers. The lids should be retracted and the needle introduced near the outer canthus, so that the hole will be covered when the lids are released. Point the needle to the middle of the eye. When correctly placed you can see the tip of the needle magnified by the lens and transparent fluid slowly dripping into the vacutainer.
At the Department of Forensic Pathology, we quantify hypoxanthine with capillary electrophoresis and potassium with an ion-selective electrode. HPLC may also be used for quantification of hypoxanthine. To our knowledge, simpler methods (such as enzyme assays) are not reliable.
Accuracy and use
Both the hypoxanthine and the potassium method may be used for a longer period of time after death (4 days) than methods based on temperature drop (1 day). Moreover the hypoxanthine method is substantially more precise than the classical methods currently used.
The preliminary results from research conducted at the Department of Forensic Pathology in Oslo, show that up until 6 hours after death the confidence interval is ± 36 minutes. From 6 to 12 hours after death, the preliminary results show a confidence interval of 1.3 hours.
Precautions
The sample is of no use if the vitreous humour contains blood.
Some diseases induces hypoxia and hence there is increased levels of hypoxanthin in vitreous humour even before death. Therefore this method is not suitable in cases where the death is caused by intoxication, or other conditions which lead to hypoxia or increased hypoxanthine leves due to metabolic disorders. such as e.g gout.
Also when using this method it is of importance to consider shift in ambient temperature. For the time being this is regarded as an experimental method.