Methods for differential and quantitative analyses of brain neurosteroid levels by LC/MS/MS with ESI-enhancing and isotope-coded derivatization.

The analysis of changes in the brain neurosteroid (NS) levels due to various stimuli can contribute to the elucidation of their physiological roles, and the discovery and development of new antipsychotic agents targeting neurosteroidogenesis. We developed methods for the differential and quantitative analyses of the brain levels of allopregnanolene (AP) and its precursor, pregnenolone (PREG), using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using 2-hydrazino-1-methylpyridine (HMP) and its isotope-coded analogue, (2)H3-HMP (d-HMP). For the differential analysis, the brain sample of an untreated rat was derivatized with HMP, while the brain sample of a treated (stressed or drug-administered) rat was derivatized with d-HMP. The two derivatives were mixed and then subjected to LC/ESI-MS/MS. The stress- and drug (clozapine and fluoxetine)-evoked increases in the brain AP and PREG levels were accurately analyzed by the developed method. It was also possible to determine the absolute concentrations of the brain steroids when a deuterium-coded moiety was introduced to the standard steroids of known amounts by the derivatization and the resulting derivatives were used as internal standards. The HMP-derivatization enabled the highly sensitive detection and the use of d-HMP significantly improved the assay precision [the intra- (n=5) and inter-assay (n=5) relative standard deviations did not exceed 13.7%] and accuracy (analytical recovery ranged from 98.7 to 106.7%).

Characterization of erythrovirus B19 genomes isolated in liver tissues from patients with fulminant hepatitis and biliary atresia who underwent liver transplantation.

BACKGROUND: Fulminant hepatitis and biliary atresia are serious problems and their causes have not been explained well. We investigated whether or not erythrovirus B19 is a candidate etiologic agent in such liver disease patients who had undergone liver transplantation. METHODS: Liver tissues from 47 patients consisted of 28 fulminant hepatitis and 19 biliary atresia were examined to detect B19 genes by PCR and further analyzed their genomic characterization. RESULTS: B19 DNA was detected by nested PCR in 10 of 28 cases (35.7%) livers in the fulminant hepatitis group and 7 of 19 (36.8%) livers in the biliary atresia group, respectively (statistically not significant). Importantly, among the 8 hepatic B19 DNA-positive patients who had paired samples of liver and serum, the serum B19 genome was detectable in only one case. B19 mRNA was identified in all of 10 fulminant hepatitis cases with hepatic B19 DNA, but only 1 out of 7 (14.3%) cases in biliary atresia tested. Furthermore, we obtained ten isolates having the B19 genome with nearly full-length sequences. Interestingly, phylogenetic analysis based on the NS1 gene revealed three different clusters: two for isolates from fulminant hepatitis and the other for isolates from biliary atresia. CONCLUSIONS: Our results presented here suggested that B19 may be an etiologic agent of fulminant hepatitis.

Complete nucleotide sequence and phylogenetic analyses of hepatitis B virus isolated from two pileated gibbons.

We analyzed full-length sequence of hepatitis B virus (HBV) recovered from two pileated gibbons (Hylobates pileatus) originally born in East Asia. Two animals possessed a viral genome of 3182 nt in length with a 33 nt deletion in the pre-S1 region, and designated HBV PG-Makiko and HBV PG-Yohko, respectively. Both sequences had 65-90% similarity to type A-G of human HBV isolates. Phylogenetic analysis demonstrated that both isolates were distinct from the human and other nonhuman primate HBV isolates, but could be classified into gibbon isolates that were previously reported by others. Small spherical and tubular particles and large particles with outer envelopes were observed in the serum under immunoelectron microscopic examination. By immunohistochemical staining, HBsAg and HBcAg were detected in the cytoplasm and nuclei of hepatocytes, respectively. Our results suggested that HBV found in these animals is indigenous to their respective hosts and not recent acquisitions from human.

Simian TT virus (s-TTV) infection in patients with liver diseases.

Recently, we identified TTV isolates from nonhuman primates and named them simian TTV (s-TTV). To investigate the prevalence of s-TTV in humans, we examined sera from healthy individuals and patients with liver diseases in Japan for the presence of s-TTV DNA by PCR assay. s-TTV DNA was determined by nested PCR using s-TTV-specific primers designed from untranslated region of s-TTV genome. s-TTV DNA sequence was detected in three of 200 (1.5%) healthy adults but none of 48 infants without liver disease. On the other hand, s-TTV DNA was detected in 30 of 287 (10.5%) Japanese patients with liver disease. s-TTV coinfection with hepatitis B virus and hepatitis C virus were present in 16.7 and 30% of these patients, respectively, while 53.3% of patients were positive for s-TTV alone. Nucleotide sequence analyses in 20 patients confirmed that these PCR products were derived from s-TTV genome sequences and nearly 85% identical to those of s-TTV prototypes from chimpanzees. Phylogenetic analysis demonstrated that all s-TTV isolates from humans were distinguished clearly from the human TTV isolates. Furthermore, s-TTV in humans was classified into two different genotypes as well as simians. Our results indicate that generally 10.5% of Japanese patients with liver diseases were infected with s-TTV. The routes of s-TTV transmission from animal to human require clarification.