Detection of the sarin hydrolysis product in formalin-fixed brain tissues of victims of the Tokyo subway terrorist attack

Abstract

One of the hydrolysis products of sarin (isopropyl methylphosphonofluoridate) was detected in formalin-fixed brain tissues of victims poisoned in the Tokyo subway terrorist attack. Part of this procedure, used for the detection of sarin hydrolysis products in erythrocytes of sarin victims, has been described previously. The test materials were four individual cerebellums, which had been stored in formalin fixative for about 2 years. Sarin-bound acetylcholinesterase (AChE) was solubilized from these cerebellums, purified by immunoaffinity chromatography, and digested with trypsin. Then the sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion, subjected to trimethylsilyl derivatization (TMS), and detected by gas chromatography-mass spectrometry. Peaks at m/z 225 and m/z 240, which are indicative of TMS-methylphosphonic acid, were observed within the retention time range of authentic methylphosphonic acid. However, no isopropyl methylphosphonic acid was detected in the formalin-fixed cerebellums of these 4 sarin victims, probably because the isopropoxy group of isopropyl methylphosphonic acid underwent chemical hydrolysis during storage. This procedure will be useful for the forensic diagnosis of poisoning by protein-bound, highly toxic agents, such as sarin, which are easily hydrolysed. This appears to be the first time that intoxication by a nerve agent has been demonstrated by analyzing formalin-fixed brains obtained at autopsy.