A comparison of trihydroxyindole and HPLC/electrochemical methods for catecholamine measurement in adrenal chromaffin cells

Abstract

Three commonly used methods for the determination of epinephrine and norepinephrine levels in adrenal medullary tissue were compared. Two variations of the trihydroxyindole procedure, which utilized oxidation at room temperature or 0°C, underestimated levels of total catecholamines in certain standard solutions and were unable to determine correctly their norepinephrine:epinephrine ratios. However, both the variability and the underestimation of the trihydroxyindole procedure carried out at room temperature were more pronounced than that of the trihydroxyindole assay at 0°C. In addition, we tested an isocratic HPLC method utilizing electrochemical detection which separates epinephrine from norepinephrine. The ability of this method to measure correctly total and individual catecholamine levels was superior to either trihydroxyindole procedure, as was its variability. When the three assay methods were used to measure total and individual catecholamine levels in cultured adrenal bovine chromaffin cells, both the trihydroxyindole (0°C) method and the HPLC method yielded values in agreement with those in the literature. However, the HPLC method produced data with lower error estimates. The trihydroxyindole (room temperature) assay was unable to reliably measure levels of epinephrine and norepinephrine in chromaffin cells. These comparisons of catecholamine assays demonstrated that there are circumstances under which the use of each is appropriate. In experiments where the epinephrine:norephinephrine ratio may be changing, the more accurate and precise HPLC assay may be essential, since the trihydroxyindole assays underestimate total catecholamines to varying degrees depending on this ratio. However, the HPLC method suffers from a requirement for technical sophistication for routine use. Therefore, in some laboratories and for repetitive measurement of many samples, the trihydroxyindole assay has a distinct advantage due to its easy utilization and ubiquitous materials. However, the superior results obtained with the trihydroxyindole (0°C) assay over the trihydroxyindole (room temperature) assay emphasizes the need to evaluate the trihydroxyindole procedure for the required purpose, especially if differential oxidation is used for estimation of individual catecholamine levels.