One of the main reasons behind the study of free radical processes is an extremely important and contradictory role they play in living systems. Lipid peroxidation is the most well-studied process of damaging biologically important substances, since the resultant product of this process – polyunsaturated fatty acid residues – are transformed with the formation of toxic oxidation products and oxidative degradation 18910.

In our work we used a steady-state radiolysis technique to generate free radicals . It is free from the artifacts characteristic of the reagent-based methods. When exposed to radiation, water undergoes a breakdown sequence into hydroxyl radicals and other radical and molecular products (Figure 2).

Hydroxyl radicals (^·OH) are highly reactive and consequently short-lived (in vivo their half-life is approximately 10⁻⁹ seconds 23). They can also occasionally appear as byproducts of the immune response in living organisms (by macrophages and microglia) or in the mitochondria 324. Hydroxyl radicals can damage virtually all types of macromolecules: carbohydrates, nucleic acids, lipids and amino acids, which makes them very dangerous for the organism. Unlike superoxide, which can be detoxified by superoxide dismutase, hydroxyl radicals cannot be eliminated by an enzymatic reaction.

Our previous studies 252627 proved that the fragmentation of hydroxyl-containing organic compounds procceding in parallel with (proceeding vis-à-vis) the formation of carbon-centered radicals is possible under hypoxic conditions (Figure 3).

Oxygen- and nitrogen-centered radicals formation can also trigger the destruction of the biomolecules’ carbon skeleton 112728.

The data presented in Table 1 show the radiation-chemical yields of the major radiolysis products formed from Glc, its metabolites (Fru-1,6-P₂, G-3-P and Glc-6-P), as well as the reference compounds (Fru, Glu, Glc-1-P) in aqueous solutions.

As is well-known, the excessive formation of lipohydroperoxides, which in the Fenton-type reaction forms alkoxyl radicals (LO^·), is associated with the concept of ferroptosis 22. In the present study we used t-BuOOH/Fe²⁺ model system for alkoxyl radicals generation (Figure 4).

The data presented in Figure 5A show the hydroperoxide-induced yields (Y, molecule/100 initiator molecules) of the major products formed in Fru-1,6-P₂ aqueous 0.01 M deaerated solutions in the presence of t-BuOOH/Fe²⁺.

As we can see from the data presented in Table 1, the main product of free radical transformations of Fru-1,6-P₂, G-3-P, Glc-1-P and Glc-6-P initiated by ^·OH is an inorganic phosphate. The same can be stated for t-BuO^·-induced transformations of Fru-1,6-P₂ (Figure 5A). Among other products of the transformations induced by hydroxyl and tert-butoxyl radicals dicarbonyl substances such as glyoxal and methylglyoxal (in the case of Fru derivatives and G-3-P) were found. It should be mentioned that in free radical transformations of Fru-1,6-P₂ initiated by Fe²⁺/t-BuOOH the ratio between the amount of methylglyoxal and that of inorganic phosphate appeared to be by ~ 30 times higher than in the case of g-irradiation of the appropriate solutions (Figure 5A and Figure 5B).

The resultant products obtained in the experiment can arise from the generation of carbon- and oxygen-centered radicals in the initial compounds which appeared after the interaction of the initial molecules with ^•OH and t-BuO^•. High yields of inorganic phosphate (Table 1, Figure 5A) allow us to suggest that the mechanisms of free radical transformation of Fru-1,6-P₂ include two consecutive reactions of dephosphorylation of the initial molecule, as it is shown in Figure 6A. The formation of inorganic phosphate and methylglyoxal can also result from the sequence of reactions illustrated in Figure 6B.

In systems containing oxygen the radiation-chemical yield of inorganic phosphate from Fru-1,6-P₂ appearedto decrease by approximately 40% as compared to the argon-saturated systems (Table 1); while in oxygen-saturated systems glyoxal and methylglyoxal did not accumulate at all (Table 1). These experimental data can be account for the ability of molecular oxygen 29 to oxidize carbon-centered radicals at the α-position to hydroxyl groups, thereby blocking their further transformations. Figure 6A shows a similar reaction 1 between oxygen and a free radical intermediate of Fru-1,6-P₂ degradation.

According to the pertinent literature on the subject, α-oxoaldehyde such as methylglyoxal plays an important role in the non-enzymatic and enzymatic production of free radicals in biological systems 51720303132. For example, the free radical derivatives can be formed in the reaction of methylglyoxal-induced protein glycation 18. Since lysine elements are known to be one of the major targets for reactive carbonyl compounds in proteins 10; we studied the interaction of this amino acid with methylglyoxal.

The application of chemiluminescence revealed that the superoxide anion radical gets formed in the mixture of methylglyoxal with L-lysine at pH 7.8 (Figure 7). Lucigenin-dependent chemiluminescence was measured in the reaction mixture that contained the following elements: K,Na-phosphate buffer (0.1 M, pH 7.8), 20 μM lucigenin, 0.015 M methylglyoxal, 0.015 M L-lysine (upper curve); the same + SOD 120 units (lower curve). In this figure the insert stands for the kinetics of formazane formation during the reaction of methylglyoxal with L-lysine. The reaction showed by the mixture ran as follows: carbonate buffer (0.1 M, pH 9.5), 10 mM methylglyoxal and 10 mM L-lysine (upper curve); the same + SOD 120 units (lower curve).

SOD under these conditions almost completely inhibits the chemiluminescence of lucigenin, which proves some dependence between this process and the presence of О₂^·- (Figure 7, lower curve). We also assessed the superoxide anion production through the accumulation of formazan on NBT reduction in carbonate buffer, pH 9.5. (Inset, Figure 7). The accumulation of formazan under these conditions might not depend on О₂^·-, since NBT may probably be reduced by other intermediates of methylglyoxal reaction with L-lysine. Nevertheless, judging by the ability of SOD to inhibit significantly (more than 4 times) the formation of formazan under the above-mentioned conditions, we may conclude that the better part of NBT is reduced owing to the action of О₂^·- (Inset, Figure 7).

Thus, the functioning of superoxide dismutase (SOD) effect has been revealed. Superoxide-depended chemiluminescence of lucigenin in the reaction mixture contained: K,Na-phosphate buffer (0.1 M, pH 7.8), 20 μM lucigenin, 0.015 M methylglyoxal, 0.015 M L-lysine (upper curve); the same + SOD 120 units (lower curve). Inset is the kinetics of formazan formation during the reaction of methylglyoxal with L-lysine. The reaction showed by the mixture ran as follows: carbonate buffer (0.1 M, pH 9.5), 10 mM methylglyoxal and 10 mM L-lysine (upper curve); the same + SOD 120 units (lower curve).

The effect of SOD can account for the fact that this enzyme removes superoxide formed in the tested model system. Indeed, the data obtained in the experiment 17 is the evidence that О₂^·- is formed by a single-electron oxygen reduction by methylglyoxal anion radical (MGO^·-) in accordance with the reaction:

MGO^·- + О₂ → MGO + О₂^·- ......(2).

(Figure 8) shows the results of the EPR spectroscopic study of the products obtained in L-lysine reactions with methylglyoxal (Figure 8, spectra B and E). The data presented in this figure demonstrate that free radical intermediates are formed under anaerobic conditions in the reaction between L-lysine and methylglyoxal. EPR spectra were recorded 4 min after the components had been mixed under aeration (spectrum A and D) or under nitrogen purging (spectrum B and E); several samples have been purged with nitrogen for 10 min (hypoxic conditions), which was followed by another 4 min of incubation after the switchover to aerobic conditions (spectrum C and F). In samples D-F SOD (400 units/ml) was added.

The EPR spectrum recorded in the reaction of L-lysine with methylglyoxal has a multicomponent hyperfine structure. Previously, such EPR spectrum has been recorded in the reaction mixture containing L-alanine and methylglyoxal 17. In the present work, using C¹³- and N¹⁵-substituted and deuterated L-alanine derivatives it has been shown that the EPR spectrum is a superposition of signals of a methylglyoxal anion radical and a cation radical of Schiff base cross-linking (dialkylimine) that appears in the interaction between methylglyoxal and the amino acid. This statement allowed us to suggest that the EPR spectrum observed in our experiments can be also a superposition of MGO^·- signals and the cation-radical of methylglyoxal dialkylimine with lysine. Noteworthy as well, only trace quantities of free radical intermediates were registered under aeration of the reaction mixture (Figure 8, spectra A and D).

In samples D-F added SOD (400 units/ml). EPR spectra were recorded 4 min after the components had been mixed under aeration (spectrum A and D) or under nitrogen purging (spectrum B and E); several samples were purged with nitrogen for 10 min (hypoxic conditions) and followed by another 4 min of incubation after the switchover to aerobic conditions (spectrum C and F). The simulation of EPR spectra of methylglyoxal anion radical (G).

The decrease in the concentration of organic free radicals recorded by EPR in aerated reaction medium is probably not associated with the inhibition of their formation. Indeed, with nitrogen purging the content of the organic free radicals, the reaction reached its maximum in 8-9 min after the reaction components had been mixed; but after gas was replaced with air the level of these free radicals in the medium dropped dramatically (Figure 8 and Figure 9).

Under these experimental conditions SOD most probably would reduce the rate of the intensity of EPR signal decreasing during aeration (Figure 9). The EPR spectrum containing five components of hyperfine structure and a g-factor equal to 2.0045 were recorded during aeration of the reaction medium in the presence of SOD (Figure 8, spectrum F).

According to the data presented in the literature, the characteristics of the EPR spectrum presented in Figure 8 (spectrum F) correspond to the signal of cis-form of methylglyoxal anion radical (MGO^·-) 172033. This fact confirms the above-mentioned assumption that free radical intermediates of L-lysine reaction with methylglyoxal are MGO^·- and the cation radical of dialkylimine. Thus, molecular oxygen seems to interact directly with the free radical derivatives of methylglyoxal and dialkylimine; the products formed in this reaction are not registered by EPR (Figure 8 and Figure 9). However, SOD protects the methylglyoxal anion radical under aerobic conditions, which proves the possibility of MGO^·- elimination under the effect of superoxide. Indeed, it has been found out that in aqueous media О₂^·- reduces some organic radicals 3435. In particular, superoxide is an electron donor for the reduction of the protonated nitrosobenzene anion radical 36. By analogy, it can be suggested that О₂^·- interacts with the methylglyoxal anion radical, reducing it to non-radical products in accordance with the reaction:

MGO^·- + H⁺+ О₂^·- → non-radical product + О₂ .....(3)

The state-of-the-arts analysis 171830 revealed that a sequence of reactions normally result in the formation of free radicals in the interaction of amino acids with carbonyl compounds (Figure 10). The presented scheme shows that dialkylimine is a Schiff base, which is a product of interaction of methylglyoxal carbonyl groups with two L-lysine molecules. As a result of the reaction of dialkylimine with another molecule of α-oxoaldehyde, the Schiff base cation radical and MGO^·- are formed, respectively (Figure 10). Such a peculiarity of the chemical structure seems to reduce the ability of dialkylimines to participate in reactions of single-electron oxidation/reduction. Since SOD less effectively prevents the dissipation of the dialkylimine cation radical than that of methylglyoxal anion radical (Figure 8 and Figure 9), it can be suggested that a free radical derivative of dialkylimineis is mainly used in the reactions with molecular oxygen. Carbon-centered free radicals of amino acids, lipids and sugars are known to interact with molecular oxygen with the formation of peroxyl radicals, which are further transformed into non-radical products 1291213. However, in contrast to the carbon-centered allyl radicals, a free electron in the dialkylimine cation radical is localized in the system of coupled double bonds between the Schiff base nitrogen atoms and methylglyoxal carbons (Figure 10). Meanwhile, such a system of coupled double bonds is present in the molecule of paraquat (methyl viologen). The reaction of a single-electron oxygen reduction by the paraquat cation radical normally forms a paraquat dication and a superoxide anion 121. The analogous reaction between oxygen and the dialkylimine cation radical may probably occur under the conditions of our experiments.

The obtained results allow us to extend and expand the previously proposed mechanism for generating the superoxide radical during the glycation reaction of amino acids by methylglyoxal 17. The functioning of the mechanism presupposes that the reactions presented in Figure 10 result in the formation of MGO^·-, which further reduces oxygen to О₂^·- (reaction 3). We assume that О₂^·- is not only formed in the reactions involving MGO^·- but also seems to participate in them (Figure 10).

These assumptions explain the effect of oxygen on the kinetics of accumulation and dissipation of free radicals having emerged in the mixture of L-lysine and methylglyoxal. The paper 17 has shown that accumulation of MGO^·-and Schiff base cross-linked cation radical depends on the oxygen concentration only insignificantly. According to our data, this fact may be associated with the application of L-alanine as an amino acid in the cited work and by a high pH value of the reaction medium (carbonate buffer, pH 9.5). It should be pointed out that the model system proposed in the paper (phosphate buffer, pH 7.8) is much closer to physiological conditions. Thus, it is most likely that oxidative modification of proteins and other biomolecules might be the influence a local generation of superoxide produces on the interaction between the residues of L-lysine (and probably other amino acids) and α-oxoaldehydes. This phenomenon of non-fermentative superoxide generation might be an element of autocatalytic intensification of pathophysiological action of carbonyl stress.

Glycation of biopolymers by reactive carbonyl compounds (Maillard reaction) leads to the formation of the advanced glycation end products – the biomarkers of pathophysiological processes associated with diabetes and various other metabolically grounded diseases 5710. Using different regulatory pathways, these products can provoke NADPH oxidase and mitochondria to form a superoxide radical 32437. Oxidative stress in diabetes mellitus can also be triggered by inhibition of antioxidant enzymes, which, in its turn, was caused by reactive dicarbonyl compounds 1538. At the same time, as it has been noted above, the non-enzymatic reactions may get involved in the formation of О₂^·- under the carbonyl stress.

Recent studies have shown that glucose oxidation by a hydroxyl radical leads to the formation of a-oxoaldehyde as 3-deoxyglucosone 39, that agrees with the proposed mechanism of non-enzymatic synthesis of methylglyoxal. The data obtained in our study also highlight the important role the free radical reactions play in non-enzymatic transformations of sugars and other carbonyl compounds. It should be also noted that the synthesis of methylglyoxal from hexose phosphates in the presence of reactive oxygen and organic free radicals and provokes the consequent formation of О₂^·- in modifications of amino acids by methylglyoxal. Thus, under the conditions of hyperglycemia the oxidative stress can cause non-enzymatic accumulation of methylglyoxal, which, in its turn, can further stimulate oxidative stress as well. Therefore, the concepts of ‘oxidative stress’ and ‘carbonyl stress’ should be considered rather controversial, since various stages of these processes typical of these stresses not infrequently merge and converge into each other. These data may allow for proposing that molecular mechanisms provoking the diseases associated with oxidative and carbonyl stress (such as atherosclerosis and diabetes) should be more complicated than they used to be thought of and are to be studies with close attention to the interplay of reactive carbonyl compounds and ROS.