IJUP08 - Universidade do Porto

Determination of hypoxanthine in vitreous humor by a sequential
injection analysis system and its application in the estimation of
postmortem interval
Ana M. Santos 1 , Ana I. Pereira 1 , Marieta L. C. Passos 1 , S. Frazão 2 , Agostinho J.C.
Santos 2 , M. Lúcia M. F. S. Saraiva 1 and José L. F. C. Lima 1
1 REQUIMTE, Serviço de Química-Física, Faculdade de Farmácia, Universidade do Porto,
Portugal.
2 Serviço de Medicina Legal, Faculdade de Medicina, Universidade do Porto, Portugal. Serviço de
Patologia Forense, Instituto Nacional de Medicina Legal, I.P. – Delegação Norte.
The estimation of the postmortem interval (PMI), i. e., the estimation of the time since
death, is crucial to the practice of certain forensic cases. Many methods have been used to
estimate the PMI, and the determination of hypoxanthine is one of them. Hypoxanthine is a
degradation product of adenosine. Increased concentrations were reported in cases of
hypoxia as well as with prolonged PMI. Hypoxanthine is recommended as an indicator of
prolonged (cerebral) hypoxia, for example in victims of sudden infant death as well as a
new biochemical method for estimation of postmortem time [1].
In this work it is proposed to estimate the PMI on the basis of postmortem hypoxanthine
concentrations in vitreous humor. The hypoxanthine concentration is determined by the
enzymatic oxidation of the hypoxanthine to uric acid. The enzyme xanthine oxidase
catalyzes the oxidation of hypoxanthine in two steps producing xanthine in the first and
uric acid in the second, which is detected spectrophotometrically at 290 nm.
This reaction is applied in a sequential injection analysis (SIA) system [2] where it was
incorporated a column with the xanthine oxidase immobilized in activated glass beads.
Physical and chemical parameters such as pH, concentration of reagents, temperature, and
sample volume were studied in order to attain the best analytical performance and the
lower reagent consumption.
The specificity of this reaction associated with the use of an automatic SIA system
constitutes a robust, versatile, and simple alternative, adapted to the actual demands of
sample and reagents economy and reduced residue production.
References:
[1] Madea, B., Kaferstein, H., Hermann, N. and Stricht, G. (1994), Hypoxanthine in vitreous-humor
and cerebrospinal-fluid – a maker of postmortem interval and prolonged (vital) hypoxia – remarks
also on hypoxanthine in sidsl, Forensic Sci Int., 65 (1), 19-31.
[2] Ruzicka, J. and Marshal G. D. (1990), Sequential injection: a new concept for chemical
sensors, process analysis and laboratory assays, Analytica Chimica Acta, 237, 329-343.
Acknowledgements: Marieta L. C. Passos thanks Fundação para a Ciência e a Tecnologia and FSE
(III Quadro Comunitário) for the Ph.D. grant (SFRH / BD / 22752 / 2005). Authors thanks Reitoria
da Universidade do Porto the Project Investigação Científica na Pré-Graduação (2007).
100