A highly sensitive sandwich-type chemiluminescent enzyme immunoassay (CLEIA) for endothelin-1 (ET-1) in plasma which does not involve any extraction steps has been developed using two populations of polyclonal antibodies. One antibody is specific to the C-ter-minal of the endothelin family of peptides and is used as an immobilized antibody. The other antibody is a Fab fragment composed of rabbit antibodies against the N-terminal core region of ET-1 and is coupled with horseradish peroxidase (HRP). Labeled HRP activity was measured using the enhanced chemiluminescent reaction of Luminol/hydrogen peroxide devised by the authors. The assay was sensitive enough to detect 0.05 pg/well (20 amol/well) of ET-1 and had no significant cross-reactivities with other related peptides, including endothelin-3 and the endothelin precursor peptide, big ET-1. Intra-assay CVs were in the range of 8.4%-24.8% for relatively low concentrations of plasma ET-1 (2pg/mI) and 4.8% - 8.1% for higher concentrations of plasma ET-1 (10pg/mI). Inter-assay CVs were 22.6% and 11.1%, respectively. No significant interference caused by bilirubin, neutral fat, hemoglobin and selected anticoagulants was observed. Preliminary investigations indicated that the basal level of ET-1 in normal humanplasma (n=37) was 0.65pg/mI.

View full abstract

To investigate the usefulness of lactate dehydrogenase isoenzyme-1 (LD-1) and LD-1/LD ratio for diagnostic assessment of acute myocardial infarction (AMI) in the subacute stage, we studied for one month 20 patients with AMI and another 20 patients with abnormal score of LD-1 and LD-1/LD ratio randomly chosen at our outpatient clinic. Serum levels of cardiac enzymes revealed that LD-1 and LD-1/LD ratio were in the abnormal range from 48 to 168 hours after the onset of AMI. Their positiverate at 360 hours after the onset was 58% and 64%, respectively. Other cardiac enzymes, such as AST, ALT, CK and CK-MB were quickly normalized over 48 hours after onset. The 20 patients with abnormal score of LD-1 and LD-1/LD ratio in the laboratory examination had few cardiac symptoms and normal levels of other cardiac enzymes. The 20 patients, 5 had AMI, 2 angina pectoris, 2 congestive heart failure, 2 pulmonary embolism, 3 abnormal electrocardiogram, 3 malignant tumor, and 3 trauma. One or two weeks had already passed after the onset in the 5 AMI cases. LD-1 and LD-1/LD ratio might provide us with useful information for diagnosis of asymptomatic or subacute phase AMI.

View full abstract

A selective, sensitive, column-swiching-high-performance liquid chromatography employing direct injection of microserum has been developed for vancomycin quantitation in human serum and applied to clinical situations. The columns consist of a separation column filled with reversed ODS-C18 and a pretreatment column filled with a macroporous hydrophilic copolymer. A mixture of potassium phosphate buffer and acetonitrile is used as the mobile phase, and vancomycin is directly detected at a UV wavelength. The chromatogram showed a high separation factor; with no interference from other endogenous substances or combined drugs in surum. Good linearity was also obtained (0-69μmol/l). The coefficient of variation for within- day reproducibility was 1.24-2.12%, with a recovery rate of 97-106%. The minimum limit of detection was 0.03μmol/l (S/N=3). The correlation coefficient between the fluorescence polarization immunoassay and this method was 0.975. The serum concentrations in patients treated with vancomycin were monitored at regular intervals. This fast, accurate, and in expensive method, can be applied to routine clinical laboratory testing.

View full abstract

To understand the mechanism of suppression of active oxygen species production in neutrophils in diabetics, we examined the polyol pathway by using the aldose reductase inhibitor (ONO-2235) in relation to superoxide production in neutrophils.
Sorbitol levels were increased in erythrocytes from diabetic patients. Sorbitol accumulation in erythrocytes during incubation with glucose was suppressed by the addition of the inhibitor.
The superoxide production in neutrophils was increased by the incubation with glucose (25 and 50 mmol/l, p<0.01) and suppressed by the addition of the inhibitor (2.5 and 25 μ mol/l, p< 0.05-0.01).
The augmented polyol pathway in hyperglycemia may suppress the production of active oxygen species in diabetic neutrophils.

View full abstract

We determined the apoE genotypes of 85 healthy donor samples by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Two hundred ninety-two base pair fragments of the exon 4 of the apoE gene were amplified by PCR from genomic DNA extracted from peripheral blood leukocytes, and the PCR products were digested by a restriction endonuclease ; Hha I. The genotype was determined by electrophoresis on a 13 % polyacrylamide gel. These genotypes were compared with the corresponding phenotypes assessed by isoelectric focusing (IEF) and western blotting. The genotypic frequencies were as follows:ε 4/ ε 4, 3.5% ; ε4/ ε 3, 21.2%; ε 4/ ε 2, 2.4%; ε 3/ ε 3, 65.9%; ε 3/ ε 2, 7.1% and ε 2/ ε 2, 0%. The apoE genotypes were completely consistent with the phenotypes obtained by the IEF method. ApoE genotyping by the PCR-RFLP method may not only substitute for phenotyping but may also be useful to detect the variant form of apoE. Therefore, it is a reliable, useful and simple method for analysis of apoE phenotypes and rare polymorphisms.

View full abstract

This standard specifies minimum agreement for the container, namely ‘ Sample Rack, ’ in which test tubes are supported and run on a laboratory transportation system. The standard addresses the uniform characteristics of the rack considering the necessary design, size and equipment used in the process. They should include ; maximum capacity of five tubes, height of 60-100 mm (60 mm is the most desirable), length of 100 mm, width of 25 mm, and pitch of 20 mm.
The distance between the centers of any two successive tubes.

View full abstract

The JSCC terminology in connection with standards has been established to promote the standardization activity in the field of clinical chemistry. This document is based on a provisional recommendation (Kuwa, Ver. 1.0, 1992), which was accepted by the members of the JSCC in December 1995, after revision taking into account the comments received.
This document offers a protocol of the terms and definitions concerning standardization, measurement systems, methods, reference materials, measured values, calibrations, quality control materials and reference intervals.

View full abstract

The certified enzyme reference material (ERM) for the standardization of catalytic concentration measurement of enzymes was applied to the recommended methods proposed by the Japan Society of Clinical Chemistry (JSCC) for the measurement of the catalytic concentration of six serum enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase (ALP), lactate dehydrogenase (LD) and gamma-glutamyltransferase (γ -GT). This ERM should mainly be used for the establishment of transferability in the enzyme measurement system, may also be applied to the development and assessment of a measurement methods and assignment of values of enzyme concentration for enzyme calibrators and material for quality assurance systems.

View full abstract