High selectivity and sensitivity is reported in the measurements of xanthine in urine by fast scan cyclic voltammetry (FSV) with a nanostructured carbon fiber sensor of 3.5 +/- 0.4 mum radius. Fabrication of the sensors for the measurements is described. Fabrication of the nanostructure at the carbon fiber sensor surface exposes surface pores. SEM images confirm the formation of the nanostructure. The results indicate that the nanostructure improves the sensitivity and limit of detection (LOD) in the measurements of xanthine and uric acid. The sensors allow rapid direct measurements of xanthine in 2000-fold diluted xanthinuric urine and of uric acid in 2000-fold diluted normal urine. The sensitivity and the LOD of xanthine is 0.40 +/- 0.02 nA microM(-1) (0.995) and 1 microM, respectively, and 0.99 +/- 0.01 nA microM(-1) (0.998) and 500 nM for uric acid. The concentration of xanthine in 2000-fold diluted xanthinuric urine is 1.6 +/- 0.2 muM from FSV and from HPLC. The concentration of xanthine and uric acid in urine can be determined by pre- or post-calibration of the sensor in buffer or by the method of standard addition.

2,8-Dihydroxyadenine (2,8-DHA) was identified by high-performance liquid chromatography with ultraviolet detection as a major metabolite in the supernatant of endothelial cells of the pulmonary artery (PAECs) and aorta (AECs), in addition to hypoxanthine, xanthine, uric acid, and uracil. Under normoxic, hypoxic, and hyperoxic conditions, the concentrations of all the identified metabolites change with time, marking the response of endothelial cells to stress, as a result of changes in cellular metabolism. Thus, the metabolites can serve as stress markers, and their concentrations can indicate the type and the level of cell stress. The results verify that PAECs adapt to survive oxidative stress of hyperoxia. However, AECs can adapt to hypoxia only for a short time and do not survive prolonged hypoxia. The role of the polyamine synthesis pathway in the formation of the unsalvaged adenine, as a possible source of 2,8-DHA, is discussed.

Fast scan cyclic voltammetry (FSV) with a nanostructured carbon fiber sensor (N-CFS) was developed for direct measurements of the purine metabolite 2,8-dihydroxyadenine (2,8-DHA; i.e. 6-amino-1H-purine-2,8-dione) in endothelial cell supernatants as a marker of cell stress. The 2,8-DHA was measured in the supernatant of aortic (AECs) and pulmonary artery endothelial cells (PAECs), which were maintained in Hank's Balanced Salt solution (HBSS) and exposed to physiological oxygen pressures as well as to oxidative stress, hypoxia (specifically 3% O(2) for AECs) and hyperoxia (20% O(2) for PAECs). Dilution of the supernatants with phosphate buffer in the ratio of 1 : 5 allowed the optimization of FSV measurements with the N-CFS in cell supernatants.The LOD for 2,8-DHA was 1 microM and the LDR was 2-15 microM with the sensitivity of (0.34 +/- 0.01) nA microM(-1) (R(2) = 0.99). The changes in 2,8-DHA concentration when the cells were exposed to stress confirm that PAECs can adapt to stress. However, the results also show that the tolerance of AECs to hypoxia is low. Cellular pathways involved in the response of PAECs and AECs to oxidative stress are outlined.