In-vitro tests using isolated receptors for the binding of ligands, that is receptor assays, are powerful tools for determining of physiological/ pharmacological activities of biologically active substances. In this article, receptor assays for benzodiazepine drugs are described, including radioreceptor assays and non-isotopic receptor assays. Competitive binding assays using synthetic receptors are also described as a newly developed technique, in which the synthetic receptors are prepared by a molecular imprinting technique. Typical data obtained by these three receptor assays are shown and the characteristics and usefulness of each assay are reviewed.

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Recently, several methods for measuring the amylase activity have been developed using new synthetic substrates. These methods can detect released chromophores such as 4-nitrophenol (PNP), 2-chloro-4-nitrophenol (CNP), and NADH. We compared ten such methods with the blue dye-linked starch polymer (Blue Starch) method. The intra-run precision (CV) of each method, 0.4-3.3%, was acceptable. The influence from coexisting materials was negligible in each method, and the correlation among the methods was good. There was no problem with the stability of any reagents after preparation. Comparing the reactivities of pancreatic and salivary amylases as a ratio (P/S), the differences among methods became obvious.

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Antioxidant activities of bilirubin, human serum albumin (HSA) and α-tocopherol (TOH: vitamin E) were examined in vitro. A mixture of bilirubin and retinol [5.6μmol/l albuminfree unconjugated bilirubin (Bu), 5.6μmol/l albumin-bound Bu with 33μmol/l HSA, 5.6μmol/l delta bilirubin (Bd) containing 33μmol/l HSA, 33μmol/l HSA alone, 116μmol/l TOH, and/or 3.8 μmol/l retinol] was oxidized by H₂0₂ in the presence of horseradish peroxidase, and the retinol remaining after oxidation was assayed by reversed-phase HPLC. The retinol concentration in the reaction mixture was decreased to 0.21±0.04μmol/l (5.6% of the original value) after oxidation. Albumin-bound and free Bu and Bd protect retinol against oxidation. Antioxidant activities of these types of bilirubin (with equivalent levels of bilirubin and HSA) as expressed in percent of original retinol concentration are, in order of effectiveness: Bd (44.9%) >albumin-bound Bu (27.5%) >albumin-free Bu (11.9%). Albumin-bound Bu protects retinol more than HSA (9.2%) and TOH (8.1%). We investigated antioxidant activities of serum bilirubin of healthy volunteers, Gilbert's disease, hemolytic jaundice and type I Crigler-Najjar syndrome. Retinol remaining after oxidation was 103.2±11.1% (mean±SD) in healthy control sera, but was 98.9±6.6% in the latter unconjugated hyperbilirubinemic sera samples. Accordingly, bilirubin moiety showed an intrinsic antioxidant ability in vitro, but it might contribute less to antioxidation in patient's serum. Moreover, albumin-free Bu and Bd may contribute less to antioxidation in normal serum, since their serum concentrations are too low to exert antioxidant activity.

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We developed a new automated method for determining serum total and direct bilirubin using nitrite as a unique oxidizing agent. Bilirubin is quickly oxidized by nitrite in a pH3.7 buffer solution with a decrease in the absorbance at 450nm. We used n-tetradecyltrimethyl ammonium bromide as the reaction accelerator for total bilirubin measurement, and polyoxyethylene (n-alkyl or iso-alkyl) ether and polyvinylpyrrolidone as the inhibitor of oxidation with free bilirubin for direct bilirubin measurement. Our new method showed good reproducibility, no interference from coexisting substances in serum, good correlation with various commercial kits such as enzymatic and chemical methods, and reasonable cost. These results indicated the usefulness of our new method.

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The Lineweaver-Burk plot or Hanes-Woolf plot is usually used to determine enzyme kinetics parameters, such as K_m, in one-substrate reaction. However, that is often applied to twosubstrate reactions as a conventional or simplified method, because of the complexity of the nonlinear least-squares method and the difficulty in obtaining the appropriate computer program. The reliability of the enzyme kinetics parameters in two-substrate reactions determined by the simplified method was evaluated using the optimized reaction rates equation calculated by a computer program “ASNOP”. The reaction rates of aspartate aminotransferase (AST) and γ-glutamyltransferase (γ-GT) were determined throughout a wide range of substrate concentrations. The rate of AST estimated by the simplified method corresponded well with that calculated by the optimized method under the analytical condition recommended by JSCC. However, a discrepancy in the rate was observed between the two methods at high concentrations of 2-oxoglutarate, because substrate inhibition is neglected in the simplified method. The reaction rate of γ-GT as the complex Ping- Pong bibi mechanism is unreliable when estimated by the simplified method. Furthermore, when the contour lines were drawn by the response surface method (RSM), a large error was observed at regions deviating from a defined midpoint.

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The conventional bromcresol purple (BCP) method of quantitatively measuring serum albumin shows false negative results when there is interference from the sulfhydryl (SH) group in serum. To eliminate this interference, we developed a two-reagent system to measure serum albumin. Serum is pretreated with the first reagent which contains sodium dodecyl sulfate and 5, 5'-dithiobis (2-nitrobenzoic acid). Then, BCP in the second reagent develops color depending on the concentration of serum albumin. Using this new system, the SH group in serum was oxidized during the pretreatment with the first reagent and the interference was eliminated. Furthermore, the high specificity, which is one of features of BCP methods, was maintained. Good precision, good linearity, and good correlation with immunological methods were observed. This new method is more accurate than the currently used BCP methods, and thus, useful for the measurement of serum albumin.

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α₂-Macroglobulin (α₂-MG), a plasma protease inhibitor, has been proposed to be involved in glucose and lipoprotein metabolism. In this study, the relationship between α₂-MG and glucose or specific lipoproteins was examined. After glucose loading, α₂-MG levels were significantly lowered while glucose levels increased. After fat loading, α₂-MG levels were significantly elevated as well as triglyceride and remnantlike particle cholesterol (RLP-C) levels. α₂-MG values of diabetic patients were significantly higher than those of healthy subjects (222.3± 20.1 vs. 175.4±7.6mg/dl p<0.05) but decreased significantly from 220.9±17.1 to 209.4±16.6mg/dl following strict glycemic control. These data suggest that α₂-MG levels are related to the state of glucose and lipoprotein metabolism and that α₂-MG may serve as a marker of diabetes mellitus.

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