We suggest that the estimation of the peritoneal cytokine levels might be an additional diagnostic tool that can support the early recognition of peritonitic complications in colorectal surgery.

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

No abstract

The generation of peroxynitrite (ONOO−) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO− and a potential protector against ONOO−-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO−. An ONOO−–luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO−. The nitration of tyrosine residues on proteins was measured on ONOO−-treated bovine serum albumin (BSA) in the presence or absence of 0–24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO− to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO−. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO− (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; p < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO− and αKG concentrations (y = −2 × 105 ln(x) + 1 × 106; r2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p < 0.001) and 12,658 ± 1928 cps (p < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; p < 0.001).

Exposure to cigarette smoke increase the formation of oxidatively modified proteins, lipids, DNA/RNA and carbohydrates in humans. These toxic substances are involved in several pathologies such as, e.g., cancer. Antioxidative supplementation is known to decrease the formation of oxidatively modified proteins, namely, carbonyl proteins. A newly developed combination of 2-oxoglutarate/ascorbic acid/5-hydroxy-methyl-furfur-aldehyde/carnosine was estimated as to the protection of generated carbonyl proteins in vivo and in vitro.Methods: Carbonyl protein content in human plasma of 15 smokers and 15 non-smokers were estimated with the Carbonylprotein ELISA, and in erythrocyte proteins with the spectro-photometically method. 15 smokers were randomly assigned to drink the combination of 2-oxoglutarate/ascorbic acid/5-hydroxy-methylfurfur-aldehyde over 4 weeks. Plasma carbonyl proteins were measured before and after 2 and 4 weeks of supplementation.In vitro studies of cigarette smoke modified proteins in absence or presence of the combination of 2-oxoglutarate/ascorbic acid/5-hydroxy-methyl-furfur-aldehyde/ carnosine or the separate substances 2-oxoglutarate, ascorbic acid, and 5-hydroxy-methyl-furfur-aldehyde were measured with the carbonyl protein spectrophotometically method before, after 2, 15 and 30 minutes.Results: Carbonyl protein in plasma was significantly higher in smokers (448±223 pmol/mg) as compared to non-smokers (191±40 pmol/mg; p<0.02), highly significant in erythrocyte proteins (26.8±12.9 nmol/mg vs. 12.1±0.3 nmol/mg; p<0.001). After 4 weeks the supplemented group showed significant lower carbonyl proteins (228±11 pmol/mg) as compared to the non-supplemented (325±45 pmol/mg; p<0.001).In vitro, smoking of BSA solution showed a significant reduction of generated carbonyl proteins in the presence of the combined supplements after 2 min. (2.18±0.34 nmol/mg vs. 5.94 nmol/mg±0.43 nmol/mg; p<0.001), 15 (2.81±0.31 nmol/mg vs. 7.96±0.79 nmol/mg; p<0.001), 30 min. (3.39±0.32 nmol/mg vs. 9.89 nmol/ mg±0.07 nmol/mg; p<0.001) and 60 min. (3.85±0.63 nmol/mg vs. 11.51 nmol/mg±0.94 nmol/mg; p<0.001) compared to BSA solution without supplements. Using seperate substances at concentrations of 0.106 mM 2-oxo-glutarate showed a significant reduction of carbonyl proteins after 2 min. (48.5%), 15 min. (52.3%), 30 min. (54.6%) and 60 min. (54.7%) incubation with cigarette smoke, vitamin C: 2 min. (41,4%), 15 min. (41.8%), 30 min. (42.7%) and 60 min. (45.0%). 5-HMF: 2 (13.8%), 15 (12.3%), 30 (7.8%) and 60 min. (10.9%). Conclusion:In vivo study in smokers and in vitro studies showed a significant reduction of oxidatively modified proteins in the presence of a combined supplement mixture containing 2-oxoglutarate/ascorbic acid/5-hydroxy-methyl-furfur-aldehyde/carnosine.