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

• Main Authors: Joachim Greilberger (Author), Michaela Greilberger (Author), Reinhold Wintersteiger (Author), Klaus Zangger (Author), Ralf Herwig (Author)
• Format: Book
• Published: MDPI AG, 2021-09-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

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; <i>p</i> < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO⁻ and αKG concentrations (<i>y</i> = −2 × 10⁵ ln(<i>x</i>) + 1 × 10⁶; <i>r</i>² = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, <i>p</i> < 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 (<i>p</i> < 0.001) and 12,658 ± 1928 cps (<i>p</i> < 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 (<i>r</i>² = 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; <i>p</i> < 0.001).

Item Description

10.3390/antiox10091501
2076-3921