Blood-brain barrier penetration of novel pyridinealdoxime methiodide (PAM)-type oximes examined by brain microdialysis with LC-MS/MS.

To develop a new reactivator of inhibited acetylcholinesterase (AChE) that can easily penetrate the blood-brain barrier (BBB), BBB penetration of 6 known and novel pyridinealdoxime methiodide (PAM)-type oximes (alkylPAMs) with relatively high reactivation activities was examined by in vivo rat brain microdialysis with liquid chromatography-mass spectrometry (LC-MS/MS). The 50% lethal dose (LD(50)) of alkylPAMs was intravenously determined for Wistar rats, then the limit of detection, quantification range and linearity of the calibration curve of the alkylPAMs in dialysate and blood were determined by LC-MS/MS. Following 10% LD(50) intravenous administration of the alkylPAMs, 4-[(hydroxyimino) methyl]-1-(2-phenylethyl) pyridinium bromide (4-PAPE) and 4-[(hydroxyimino) methyl]-1-octylpyridinium bromide (4-PAO) appeared in the dialysate. Striatal extracellular fluid/blood concentration ratios were 0.039+/-0.018 and 0.301+/-0.183 (mean+/-SEM), respectively, 1 h after treatment. This is the first report of BBB penetration of 4-PAPE, and the concentration ratio was smaller than that of 2-PAM. The mean BBB penetration of 4-PAO was approximately 30%, indicating that intravenous administration of 4-PAO may be effective for the reactivation of blocked cholinesterase in the brain. However, the toxicity of 4-PAO (LD(50); 8.89 mg/kg) was greater than that of 2-PAM. Further investigation is required to determine the effects of these alkylPAMs in organophosphate poisoning.

New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes.

INTRODUCTION: A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). RESULTS AND DISCUSSION: This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. CONCLUSION: Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.

Hydrolysis of an acetylthiocholine by pralidoxime iodide (2-PAM).

Pralidoxime iodide (2-PAM), an antidote approved for the reactivation of inhibited acetylcholinesterase (AChE) in organophosphate poisoning, dose-dependently hydrolyzed an acetylthiocholine iodide (ASCh). The AChE (0.3 U) activity inhibited by VX analog (ENMP, 0.1 microM) increased to approximately 200% of normal levels after a dosage of 5 mM 2-PAM (control 0.132+/-0.012 U/ml, 5 mM 0.253+/-0.026 U/ml). This result indicates that 2-PAM produced a thiocholine from the ASCh by hydrolysis. High-performance liquid chromatography (HPLC) analysis was then performed to further clarify the hydrolysis of ASCh with 2-PAM. It was clear that 2-PAM was converted to acetylated 2-PAM with acetic acid produced from ASCh by hydrolysis. Next, we tried to compare this esterase-like activity of 2-PAM with that of obidoxime, which is known as a strong reactivator of inhibited AChE, and with diacetylmonoxime, known as a weak reactivator. All of these oximes showed esterase-like activity, and their strengths were consistent with those of known reactivators of inhibited AChE. These results indicate that a great deal of the data obtained previously with ASCh relating to the effects of oximes must be rechecked. It is clear that oximes easily hydrolyze ASCh. We therefore strongly caution that the method of determining AChE activity with ASCh is not suitable for examining the effects of oximes.

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues.

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.

Development of forensic diagnosis of acute sarin poisoning.

On March 20, 1995, the Tokyo subway system was subjected to a horrifying terrorist attack with sarin gas (isopropyl methylphosphonofluoridate) that left 12 persons dead and over 5000 injured. In order to diagnose the definite cause of death of the victims, a new method was developed to detect sarin hydrolysis products in the erythrocytes and formalin-fixed cerebella from four victims of sarin poisoning. Sarin-bound acetylcholinesterase (AChE) was solubilized from the specimens of sarin victims and digested with trypsin. The sarin hydrolysis products bound to AChE were released by alkaline phosphatase digestion. The digested sarin hydrolysis products were subjected to trimethylsilyl derivatization and detected by gas chromatography-mass spectrometry. Sarin hydrolysis products were detected in all sarin poisoning victims.

Pralidoxime iodide (2-pAM) penetrates across the blood-brain barrier.

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 +/- 0.053 (mean +/- SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

The BCG scar after percutaneous multiple puncture vaccination may help establish the nationalities of unidentified cadavers.

BCG vaccination using the multiple puncture device (the Heaf gun) has been used in Japan and in the United Kingdom. The appearance of the BCG scars therefore differs from that caused by the conventional intradermal BCG vaccination, which is used throughout the world. The aim of this study was to investigate the usefulness of examining BCG scars to identify the nationalities of unidentified cadavers. We investigated the BCG vaccination program not only in Japan, but also in other countries, along with the relation between the BCG scar and nationality. The results showed that the countries which domestically make and use the Heaf gun are Japan (where it is the only method that has been in use since 1968), the United Kingdom (where it has been used, in part, since 1982), and South Africa (where it has been used, in part, since 1972). In addition, for the past 10 years, the Japanese Heaf gun method has been partially applied in the Republic of Korea and in Brazil. The Heaf gun scar can be clearly distinguished from the intradermal scar, and is visible throughout a person's life when good technique is used to administer the vaccination. If the Heaf gun scar is found on the left upper arm of an unidentified Asian cadaver, it is sure to be that of a Japanese. The findings of this present study indicate that the Heaf gun scar can be examined to identify the nationalities of unidentified cadavers.

Production of gamma-hydroxybutyric acid in postmortem liver increases with time after death.

Gamma-hydroxybutyric (GHB) acid, which is becoming popular as a drug of abuse, was shown by means of gas chromatography-mass spectrometry (GC-MS) to increase in mouse liver with time after death. The amount detected was 0.8 +/- 1.0 microg/g at 3 h after death, 4.7 +/- 1.5 microg/g at 24 h, and 8.8 +/- 0.8 microg/g at 72 h. Furthermore, GHB was detected in samples from deceased persons, at concentrations of 2.6-12.0 microg/g in liver, 0.4-7.3 microg/ml in blood, and 0-2.6 microg/ml in urine, but was not detected in the blood and urine of living persons. Although 1,4-butanediol has been suggested to be a precursor of GHB produced after death, 1,4-butanediol was not detected in any of our samples. Additionally, succinate semialdehyde arising from gamma-aminobutyric acid (GABA) transamination to GHB was also barely detectable in any of our samples. This study supports previous reports that GHB is a product of postmortem decomposition. Production of GHB increases with time after death in postmortem liver; since we were unable to identify endogenous 1,4-butanediol and succinate semialdehyde in our samples, the pathway of GHB production after death remains unclear.