Evidence of in vivo peroxynitrite formation in patients with colorectal carcinoma, higher plasma nitrate/nitrite levels, and lower protection against oxygen free radicals

Endogenously formed nitrogen and oxygen free radicals are believed to be involved in human cancer etiology. Plasma nitrate/nitrite originates from endogenous nitric oxide production in fasting humans, decrease in superoxide scavenger activity (SSA), and free sulfhydryl groups (SH) reflects the amount of superoxide anion generated, and nitrotyrosine is believed to be formed by the interaction of tyrosine and peroxynitrite in vivo. The aim of the current study was to measure plasma nitrate/nitrite, SSA, and SH in 69 patients (mean age +/- standard deviation, 66 +/- 11 years) with colorectal carcinoma. Nitrotyrosine was measured from both the plasma and tumor tissues in 32 patients. All patients had adenocarcinoma of the colon or rectum. Twenty-five patients were classified as stage B according to Dukes: classification as modified by Astler-Coller, 13 were classified as stage C, and 31 patients were classified as stage D. To determine whether the changes are specific for colorectal cancer, 20 patients with active inflammatory bowel disease (IBD; mean age, 52 +/- 18 years) and 30 healthy volunteers, who sen ed as control subjects (mean age, 48 +/- 11 years), were studied. Plasma nitrate/nitrite was measured by the modified Griess method, SSA was measured by an electron/spin resonance spin trapping method, free SH was measured by Ellman's method, and the presence of nitrotyrosine in the plasma and tumor tissue was detected by high performance liquid chromatography (HPLC) using C-18-derivatized silica (5 mu m) column (C18S, Crestpaque, New York, NY, USA) and at a wavelength of 274 nm. Patients with colorectal carcinoma and with active IBD had a significantly higher plasma nitrate/nitrite level (51.2 +/-: 26.2 mu m and 56.0 +/- 14.6 mu m versus. 29.6 +/- 6.3 mu m; p < 0.01), and a lower SSA level (39 +/- 11.5 U/g protein and 52.0 +/- 18.9 U/g protein versus. 88 +/- 25.1 U/g protein; p < 0.05) and SH level (7.7 +/- 3.89 mu m protein and 6.4 +/- 2.1 mu m protein versus. 10.9 +/- 4.14 mu m/mg protein; p < 0.01) than control subjects. A nitrotyrosine peak could be detected in 7 of 32 specimens of cancer tissue but not in biopsy samples obtained from patients with IBD, nor in the plasma. There was no difference in nitrate/nitrite, SSA, or SH levels between subgroups of cancer patients when sex, disease duration, tumor location, or therapy were considered. The elevated plasma nitrate/nitrite with lower plasma SSA and SH levels suggest a concomitant increase in nitrogen and oxygen free radical formation in cancer patients. This change is likely not specific for tumor formation because it can also be observed in other inflammatory conditions such as IBD. The presence of nitrotyrosine in the tumor tissue is the first evidence for the in vivo formation of peroxynitrite in colorectal carcinoma.