Development of an HPLC method using relative molar sensitivity for the measurement of blood concentrations of nine pharmaceutical compounds.

We developed a reliable high-performance liquid chromatographic analysis method using a relative molar sensitivity (RMS) technique that does not require an authentic, identical reference analyte material to quantify blood serum carbamazepine, phenytoin, voriconazole, lamotrigine, meropenem, mycophenolic acid, linezolid, vancomycin, and caffeine levels for routine blood concentration measurements. Carbamazepine and caffeine were also used as non-analyte reference materials to calculate the RMS of each analyte. The RMS was calculated from the ratio of the slope of the calibration equation (analyte/non-analyte reference material), then used to quantify analytes in control serum samples spiked with carbamazepine, phenytoin, voriconazole, meropenem, mycophenolic acid, linezolid or vancomycin. In addition, the concentrations of these six drugs in control serum samples determined by the proposed RMS method agreed well with that obtained using a conventional method. The proposed RMS method is a promising tool for the clinical determination of nine drugs, given the accuracy, precision, and efficiency of quantifying these analytes.

[Determination of Plasma Concentrations of Imatinib by a Novel Automated Analyzer Based on High-Performance Liquid Chromatography].

LM1010 HPLC is an emerging automated method designed for use in clinical settings. The aim of this study was to compare the analytical performance of LM1010 with the performance of traditional HPLC and LC-MS/MS in the measurement of plasma concentrations of imatinib. Seventy-eight plasma samples from 20 patients (14 men and 6 women) were collected. Plasma concentrations of imatinib in samples from the same patient were analyzed simultaneously using LM1010, HPLC and LC-MS/MS (LSI Medience Corporation). Strong correlations were seen in pairwise comparisons of results from the LM1010 and HPLC methods, the LM1010 and LC-MS/MS methods, and the LC-MS/MS and HPLC methods (Spearman's r=0.936, 0.906, and 0.953, respectively); however, the results from the LC-MS/MS method showed a positive proportional bias in comparison with the results from the LM1010 and HPLC methods, according to Deming analyses (slope=1.064 and 1.105, respectively). In Bland-Altman analyses, the LC-MS/MS method showed a positive mean bias of 98.6 and 112 ng/mL in comparison with the LM1010 and HPLC methods, respectively. Notably, results obtained using the LM1010 method were comparable to those using the HPLC method (positive mean bias=13.6 ng/mL; 95% confidence interval, -7.9-35.1 ng/mL). Biochemical parameters or drugs taken concomitantly with imatinib were not found to affect the bias of the LM1010 method. The LM1010 method can be applied to routine therapeutic drug monitoring of imatinib.

[Evaluation of Plasma Concentrations of Mycophenolic Acid in Renal Transplant Patients Using LM1010 High-performance Liquid Chromatography].

Plasma concentrations of mycophenolic acid (MPA), an immunosuppressive agent, have been measured in clinical settings using immunoassay methods or HPLC. However, immunoassay methods show cross-reactivity with metabolites of MPA glucuronide. Recently, the LM1010 high-performance liquid chromatography instrument was approved as a new general medical device. In this study, we compared the results of MPA plasma concentrations analyzed using the LM1010 method and the previously described HPLC method. Plasma samples obtained from 100 renal transplant patients (32 women and 68 men) were evaluated using both HPLC instruments. Deming regression analyses showed a very high correlation between the two instruments, with a slope of 0.9892 and an intercept of 0.0235 µg/mL (r2=0.982). Bland-Altman analysis showed an average of -0.0012 µg/mL between the LM1010 method and the previously described HPLC method. For the LM1010 method, the total run time for MPA analysis was 7 min, and the analytical time was short; however, the extraction recovery when using a spin column was extremely low for frozen plasma samples stored at -20°C for 1 month, and the volume required for the assay (150 µL) could not be collected. Thus, for the LM1010 method, analysis using fresh plasma samples was optimal. Overall, our findings showed that the LM1010 method was a rapid, accurate HPLC assay for MPA analysis and could be used in clinical practice for routine monitoring of MPA in fresh plasma samples.

Brain stem infarction in a 6-year-old boy with Down syndrome.

Infarct locations in children with arterial ischemic stroke have primarily been reported to be lobar or in the basal ganglia, and those in patients with Down syndrome (DS) and antiphospholipid syndrome (APS) are typically wide and multiple. No solitary brain stem infarctions have ever been reported in children with DS until now. Here, we report a case of brain stem infarction in a 6-year-old boy with DS who had no cardiac, renal, or intestinal complications. He exhibited ataxic gait and medial longitudinal fasciculus (MLF) symptoms at first presentation. Neuroimaging revealed a localized and isolated lesion in the midbrain. Although he did not satisfy the diagnostic criteria of APS, he showed persistently elevated levels of anticardiolipin antibody (21 U/mL; normal value <10 U/mL). Although he had the risks of a multiple vascular systems disorder, DS, and persistently elevated levels of antiphospholipid antibodies, his lesion was not similar to any of the previously reported cerebral infarctions in DS or in APS. To our knowledge, this is the first report of limited solitary brain stem infarction in a child with DS.

Autophosphorylation of heme-regulated eukaryotic initiation factor 2α kinase and the role of the modification in catalysis.

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI), functions in response to heme shortage in reticulocytes and aids in the maintenance of a heme:globin ratio of 1:1. Under normal conditions, heme binds to HRI and blocks its function. However, during heme shortage, heme dissociates from the protein and autophosphorylation subsequently occurs. Autophosphorylation comprises a preliminary critical step before the execution of the intrinsic function of HRI; specifically, phosphorylation of Ser-51 of eIF2α to inhibit translation of the globin protein. The present study indicates that dephosphorylated mouse HRI exhibits strong intramolecular interactions (between the N-terminal and C-terminal domains) compared to phosphorylated HRI. It is therefore suggested that autophosphorylation reduces the intramolecular interaction, which induces irreversible catalytic flow to the intrinsic eIF2α kinase activity after heme dissociates from the protein. With the aid of MS, we identified 33 phosphorylated sites in mouse HRI overexpressed in Escherichia coli. Phosphorylated sites at Ser, Thr and Tyr were predominantly localized within the kinase insertion region (16 sites) and kinase domain (12 sites), whereas the N-terminal domain contained five sites. We further generated 30 enzymes with mutations at the phosphorylated residues and examined their catalytic activities. The activities of Y193F, T485A and T490A mutants were significantly lower than that of wild-type protein, whereas the other mutant proteins displayed essentially similar activity. Accordingly, we suggest that Tyr193, Thr485 and Thr490 are essential residues in the catalysis.

HBx protein is indispensable for development of viraemia in human hepatocyte chimeric mice.

The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patient's serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log(10) copies ml(-1) and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

G to A hypermutation of TT virus.

APOBEC3 proteins function as part of innate antiviral immunity and induce G to A hypermutation in retroviruses and hepatitis B virus (HBV) genomes. Whether APOBEC3 proteins affect viruses that replicate without a reverse transcription step is unknown. TT virus (TTV), known to present in serum of healthy individuals and HBV carriers, has a single-stranded circular DNA genome and replicates without reverse transcription. In this study, we examined 67 blood samples obtained from healthy individuals and HBV carriers and observed G to A hypermutation of genomes of TTV in both healthy individuals and HBV carriers. During ALT flare-up in HBV carriers, G to A hypermutation of HBV increased, but TTV genomes significantly decreased in number and hypermutated TTV genomes became undetectable. Our results show that hypermutated TTV exist in healthy individuals and HBV carriers and that TTV genomes were susceptible to immune reaction directed to HBV by interacting with APOBEC3 proteins.