Effect of Protein

1872 CLINICAL CHEMISTRY, Vol. 29, No. 10, 1983 we measure serum total bile acids by the 3HSD-diaphorase method, using the “Sterognost 3a Flu” kit (Nyeg#{228}rd, Oslo, Norway), 50 L of serum + 1200 L of Tris HCI buffer (0.1 moUL, pH 9.0) + 250 L of Sterognost solution. Table 1 shows the effect of protein with and without pre-heating the sample. The “calculated cholic acid concentration” refers to the concentration calculated with the protein-free solution of cholic acid as calibrator. The protein effect withoutpro-heating corresponds to the results of Hanson and Freier. With sample pro-heating, the decrease in calculated cholic acid concentration in the range 0-80 g of BSA per liter in the sample is only about 6%, hardly of any clinical significance. At high protein concentrations (e.g., 12 g/L) in the assay mixture, the bile acid determination is also strongly influenced by protein after pro-heating. The slightly higher fluorescence units recorded after pro-heating suggest some evaporation of the sample dilutions (covered with Parafllm). To explore whether the protein influence was at the first or second enzymatic reaction, bile acid was determined with the 3HSD enzyme with fluorometric detection of generated NADH, by use of the kit “Sterognost 3a PHO” (performed as an endpoint determination). Protein had no influence on the fluorescence value. In contrast, the diaphorase step, which we investigated by adding NADH to the “Sterognost 3a Flu” reagent blank solution, which does not contain 3HSD, was inhibited by protein, as also noted by Hanson and Freier. To investigate whether the effect was an interference with the reaction or ascribable to quenching of the fluorescence of resorufin, we also added albumin after terminating the reaction. This had no effect on the generated fluorescence, suggesting that the protein inhibition is due to interference with the diaphorase reaction. The decrease in the fluorescence of the blanks in Table 1, however, does suggest that the fluorescence of resazurin is to some extent quenched by albumin.