Alpha-Ketoglutarate: A Potential Inner Mitochondrial and Cytosolic Protector against Peroxynitrite and Peroxynitrite-Induced Nitration?

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).

Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells

“…5-HMF does have a likewise structure and antioxidative activity as vitamin C and therefore also is able to produce in media peroxides. Therefore, the protection of aKG against media-generated peroxides, like hydrogen peroxide and peroxynitrite, is suitable to stabilize 5-HMF [ 23 , 24 ] as we have seen that the carbonylated protein content of cell lysate was decreased in HF-SAR but also in Jurkat cells during incubation with aKG and 5-HMF.…”

“…Glutaminolysis is directly involved in maintaining ROS homeostasis, which is crucial for cell growth and survival [ 26 ]. aKG itself showed an anti-oxidative potential by reducing peroxides, e.g., hydrogen peroxide and peroxynitrite, to tricarboxylic acid (TCA) cycle substances such as succinate and, as side products, nitrate and NO* [ 23 ].…”

“…The longer the incubation with Jurkat cells was the better the outcome of the reduction of mitochondrial activity was; it even reached down to 10% with the highest aKG+ 5-HMF concentration. The IC50% was assessed after 72 h of incubation in Jurkat cells with a lower aKG + 5-HMF concentration of 200 µg/mL aKG + 66.7 µg/mL 5-HMF, suggesting a synergistic effect of both substances in reducing peroxides [ 23 ] and activating antioxidative acting enzymes [ 9 , 18 ].…”

Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways

“…Furthermore, the oxoglutarate dehydrogenase complex (OGDC) or α-ketoglutarate dehydrogenase complex is an enzyme complex that plays a major role in the citric acid cycle. aKG is a potent scavenger in eliminating peroxynitrite [ 1 ], a physiological nitrating agent derived from nitrosyl radial (NO • ) and superoxide anion radical (O 2 * − ; [ 2 ]), to succinate and nitrite, which can be easily converted back to NO • .…”

“…aKG is required as a potent oxidant for the reductive carboxylation bidirectional in cancer cells with mitochondrial defects to operate IDH inversely and SDH directly [ 8 ] Under intracellular acidic conditions, which does take place intracellular in cancer cells, the nonenzymatic decarboxylation of aKG in the presence of H 2 O 2 and ONOO − cannot occur. Thus, nonenzymatically generated succinate decreases, which itself is able to stabilize HIFα [ 1 ].…”