National Registry in Clinical Chemistry

148 CliNICAL CHEMISTRY. Vol. 23, No. 1, 1977 aniline the regression equations obtained were as follows: y = 0.988x + 0.695, r = 1.00, t = 0.358,P <0.8>0.7, n = 12; y = 0.976x+ 0.799, r = 1.00, t = 1.71,P <0.2 > 0.1, n = 20; and y = 0.978x+ 0.901, r = 1.00, t = 1.71,P < 0.1 >0.05,n32. In the presence of 4-nitroaniline, valueswere y = 0.986x + 2.50,r = 1.00, t = -2.0, P = <0.1 > 0.05, n = 12; y = 0.993x + 1.90, r = 1.00, t = -2.75, P < 0.02> 0.01,n = 20; and y = 0.991x + 2.07,r = 1.00, t = -3.45, P <0.005> 0.001,n32. These results indicatethat liberation of even 100 ,smolof4-nitroaniline p r liter during the y-glutamyltransferase assay would yield at most an average inhibitionof 3% (cf.between-runprecisionof2.05%)ina monitoringperiod of 5-10 mm after the start of the reaction and that this inhibition would be absent in the 10-15 mm monitoring period. In view of the possibility that the (very minor) differences in the effect of 4-nitroaniline in the two 5-mm monitoring periods might result from inhibition of the initial reaction rate by 4nitroaniline, with a temporary lag-phase production,18 of the above sera were re-examined with the instrument in the kinetic mode. In this mode, the absorbance change2 mm after serum addition is printed over three 15-s intervals. With each serum a linear print-out was obtained. A plot of the absorbance change over the total 45-s monitoring period in the presenceof 4-nitroaniline (y) vs. that obtained in its absence (x) yielded the following regression equation: y = 0.961x + 3.01, r = 1.00, t = 2.01, P <0.1 > 0.05. From the above data it is obvious that 4-nitroaniline, even at a concentration of 100 jmol/liter, produces minimal effect, even early in the course of the reaction, and that this is no longer detectable at 10 mm. With this 4-nitroaniline concentration, the substrate solution would have an absorbance of approximately 1.5 at 405 nm, compared with the usual absorbance of 0.5 for fresh substrate. Such a substrate would be visibly highly colored and normally would be rejected. For a 100 ,smol/liter concentration of 4-nitroaniline to be releasd in the assay described in the second paragraph above would require serum with an activity of about 100 U/liter to act for 10 mm. With the Abbott analyzer and a 51-fold serum dilution, it would require a serum of activity 1000 U/liter acting over a 5-mm period. Because Martin et al. (2)have stated that 4-nitroaniline liberation even below 125 jsmol/liter would lead to significant error with fixed-point assays, this point was re-investigated with such an assay (4), though I had originally investigated thispossibility when developingthe assay in 1968 and had found the reaction toobey zero-order kinetics fora 30-min period with (three) sera of activity up to 200 U/literat 37 #{176}C. This limit of linearity was clearly stated in the published method description. Reaction progress curves were again plotted, with Tris buffer and reagent concentrations as originally specified (4) but with a buffer pH of 8.3, and also with the identical technique but with AMPD buffer and reactant concentrations as above. The measuring instrument was the Vitatron photometer previously used in the kinetic studies. I again confirmed with two sera that the reaction progress curve was linear for a 30-mm incubation period with activities up to 200 U/liter. With this assay, absorbance readings are made on a sixfoldaciddilutionofthe assay mixture, and 30-mm incubation of a serum of activity 200 U/liter liberates 4-nitroaniline at a fmal concentration of 600 smol/liter of assay mixture.