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.
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.