Effect of Urine pH, Storage Time, and Temperature on Stability of Catecholamines, Cortisol, and Creatinine

Abstract

In studies of stress in humans and animals, urinary excretion of catecholamines and cortisol is widely used as a stress index (1)(2). In most studies that have measured urinary catecholamines, hydrochloric acid or some antioxidant has been added to the urine samples immediately after voiding. This is probably because classic methodology demands preservation of urine specimens to prevent catecholamine degradation (3)(4)(5). On the other hand, urine is not usually acidified when glucocorticoids are to be analyzed (2)(6). Because of the difference in urine treatment required for studies of catecholamines and glucocorticoids, it has been necessary to prepare two kinds of urine samples in parallel, i.e., acidified and unpreserved. Clearly it would be preferable if a single urine treatment could be used for analysis of both stress-related hormones. However, to our knowledge, little is known about the stability of these compounds in urine kept under different conditions. In the present study, we examined changes in catecholamine and cortisol concentrations in human urine samples stored at various pH values for different periods, using HPLC and fluorometry. The stability of urinary creatinine was also investigated, because creatinine excretion is commonly used to estimate the exact timing of urine collection. In the present experiments, urinary free catecholamines were assayed using a modification of a method described previously (7). Briefly, 0.5 or 1 mL of urine was adjusted to pH 8.4–8.6 with 1 mol/L NaOH after addition of 100 pmol of isoproterenol as an internal standard and 1 mL of 0.1 mol/L Na2EDTA (pH 8.6); the urine was then adsorbed to 150 mg of alumina packed in a glass column (6 mm i.d.). The alumina was washed twice with 5 mL of water and eluted with 1 mL of 0.25 mol/L acetic acid. The eluate …