Performance of an automated system for quantitation of hepatitis C virus core antigen.

The performance of an automated system for the HCV core antigen assay using the Lumispot LS-2000 automated analyzer was evaluated against that of the COBAS AMPLICOR HCV MONITOR Test, version 2.0 (COBAS HCM-2) for the testing of sera from 155 chronic hepatitis C patients. The within-run coefficient of variations (CVs) and the between-day CVs were <9.6 and <8.4%, respectively. The analytical detection limit of the HCV core antigen assay was 5.0 fmol/l and it was linear up to at least 45000 fmol/l. No blood elements interfered with the assay. HCV core antigen levels were significantly correlated with HCV RNA levels in both serogroup 1 and serogroup 2 (r=0.829, P<0.001). It is estimated that 100 fmol/l of HCV core antigen level was equivalent to approximately 30000 IU/ml of HCV RNA level. Three sera had HCV core antigen levels below the detection limit of the assay and their HCV RNA levels as determined by COBAS HCM-2 assay were very low at 1000, 1100 and 1700 IU/ml, respectively. In six IFN responders among seven patients, HCV core antigen levels were in parallel with HCV RNA levels. In conclusion, since this assay demonstrated good reproducibility, a favorable dynamic range and adequate sensitivity, it may be useful as an alternative direct marker of viral level monitoring in patients with hepatitis C.

[Quantification of urinary hemoglobin by an immunological method].

The quantification method of urinary hemoglobin has not been established. We examined whether a reagent (Eiken, Tokyo) used to immunologically assay fecal hemoglobin could be utilized to quantify urinary hemoglobin. The coefficients of variation were 2.2-3.0% in the reproducibility of one-run assays using urine and 4.3-5.7% in that of multiple-run assays using the standard sample of hemoglobin. Urinary hemoglobin was unstable and decreased in a time- and temperature-dependent manner. However, addition of the hemoglobin stabilization buffer (50 mM phosphate buffer, pH 6.4) (Eiken) to urine made urinary hemoglobin stable. Urinary hemoglobin levels did not change significantly when stored at 4 degrees C for 7 days or at -80 degrees C for 30 days. The hemoglobin concentration (mean +/- SD) of urine showing 1+, 2+ and 3+ with a test strip was 385 +/- 165(n = 30), 1070 +/- 499(n = 40) and 4130 +/- 2770 ng/ml (n = 20), respectively. Urinary hemoglobin did not correlate with urinary albumin, transferrin, immunoglobulin G, N-acetyl-beta-D-glucosaminidase nor alpha 1 microglobulin. These results suggest that this immunological method using the hemoglobin stabilization buffer can be utilized for quantification of urinary hemoglobin, which may provide clinically important information.

Stereospecific analysis of omeprazole in human plasma as a probe for CYP2C19 phenotype.

Omeprazole is a class referred to as proton pump inhibitor; it acts to regulate acid production in the stomach and is used to treat various acid-related gastrointestinal disorders. In the liver, it is metabolized to varying degrees by several cytochrome P-450 (CYP) isoenzymes which are further categorized into subfamilies of related polymorphic gene products. The metabolism of omeprazole is to a large extent dependent on CYP3A4 and CYP2C19. Omeprazole is metabolized to two major metabolites, 5-hydroxyomeprazole (CYP2C19) and omeprazole sulfone (CYP3A4). Minor mutations in CYP2C19 affect its activity in the liver and, in turn, the metabolic and pharmacokinetic profiles of omeprazole. The frequency of CYP2C19 poor metabolizers in population of Asian descent has been reported to range from 10 to 20%. Accordingly, results from population studies indicate that omeprazole can be used as a probe drug for phenotyping CYP2C19. The optical isomers of omeprazole show a clear difference in their metabolism by human liver microsomes. This study demonstrates the stereospecific analysis of omeprazole in human plasma as a probe drug of CYP2C19 phenotyping. The chiral separation of omeprazole was achieved on a chiral column with circular dichroism (CD) detection and LC/MS. A good resolution of enantiomers was obtained. The column used for chiral separation was CHIRALPAK AD-RH column (4.6 x 150 mm) using phosphate buffer and (or ammonium acetate) acetonitrile as an eluent. After a single oral dose of omeprazole (20 mg), the plasma concentrations of the separate enantiomers of omeprazole were determined for 3.5 h after drug intake. The present study is useful because of the part polymorphism plays in the therapeutic effectiveness of proton pump inhibitors during the treatment of acid-related diseases.

Apolipoprotein A-I deficiency with accumulated risk for CHD but no symptoms of CHD.

We evaluated a 69-year-old Japanese woman with apolipoprotein (apo) A-I deficiency, high levels of low-density lipoprotein (LDL)-cholesterol, hypertension and impaired glucose tolerance. The patient had corneal opacity, but neither xanthomas, xanthelasma, nor tonsillar hypertrophy. She was not symptomatic for coronary heart disease (CHD), and had normal electrocardiograms at rest and exercise using a cycle ergometer. She had severely reduced levels of high-density lipoprotein (HDL)-cholesterol (0.10-0.18 mmol/l) and no apo A-I (<0.6 mg/dl). LDL-cholesterol and apo B as well as apo E were increased even under treatment with 10 mg pravastatin per day. Gel filtration chromatography revealed that in addition to VLDL and LDL fractions, she had apo A-II rich and apo E rich fractions, which were present in the HDL fraction separated by ultracentrifugation. A cytosine deletion was identified by genomic DNA sequencing of the apo A-I gene of the patient at the third base of codon 184 in the fourth exon, which led to a frame shift mutation and early termination at codon 200. This patient is the oldest among those with apo A-I deficiency reported in the literature, and she had no symptoms of CHD despite the accumulated risk for the disease.

Determination of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry.

Omeprazole is a benzimidazole compound that acts as a proton-pump inhibitor. Because the metabolism of omeprazole is mainly catalyzed by cytochrome P-450 (CYP) 3A4 and CYP2C19. the genetic polymorphism of CYP2C19 could be of clinical concern in the treatment of acid-related diseases with omeprazole. Therefore, a reliable method for omeprazole phenotyping is desirable in clinical situations. This study has demonstrated the determination of omeprazole and its metabolites in human plasma by liquid chromatography-three-dimensional quadrupole mass spectrometry with a sonic spray ionization interface. The analytical column was YMC-Pack Pro C18(50x2.0 mm I.D.) using acetonitrile-50 mM ammonium acetate (pH 7.25) (1:4) at a flow-rate of 0.2 ml/min. The drift voltage was 30 V. The sampling aperture was heated at 110 degrees C and Shield temperature was 230 degrees C. In the mass spectrum, the molecular ions of omeprazole, hydroxyomeprazole and omeprazole sulfone were clearly observed as base peaks. This method is sufficiently sensitive and accurate for pharmacokinetic studies of omeprazol.