Miklos Ghyczy

Electrophilic methyl groups present in the diet ameliorate pathological states induced by reductive and oxidative stress: a hypothesis.

Reductive stress, characterised by an increased NADH:NAD+ ratio, may be as common and as important a consequence of redox imbalance as oxidative stress. It may also be an important predisposing cause of the generation of reactive oxygen species. Considerable experimental and indirect clinical evidence suggests that protection against reductive stress depends on biomolecules with electrophilic methyl groups (EMG) such as S-adenosylmethionine, betaine, carnitine and phosphatidylcholine. Pathological processes leading to reductive stress and their relief by such protective agents is reviewed and the proposed molecular mechanism is outlined. These and other EMG-containing biomolecules are part of the daily diet and may represent an important control system for redox balance.

Evidence in support of a concept of reductive stress - Reply by Ghyczy & Boros

It is generally believed that free radical damage to biological systems is exerted by the oxidative action of hydroxyl radicals. Some researchers have, however, observed that many degenerative diseases are associated with a hypoxic state that results in an increased NADH: NAD ratio, leading to a reductive cytosolic environment (Ido et al. 1997). Yet a number of natural and synthetic substances shown to be beneficial in such pathological states have been paradoxically termed `antioxidants despite the fact that they exist in oxidized form, e.g. lipoic acid. It has been recently hypothesized (Ghyczy & Boros, 2001) that biomolecules with a positively charged N or S atom and a bound methyl group, termed electrophilic methyl group, react with NADH thus ameliorating reductive stress. In this letter, preliminary data are presented in support of the contention of Ghyczy & Boros (2001) that reductive rather than oxidative stress is of clinical importance. An increasing interest in oxidative stress in diabetes mellitus (Lipinski, 2001) led to a search for a diagnostically useful method to measure the antioxidative potential of plasma. To generate hydroxyl radicals, ascorbic acid and cupric chloride were used. Unexpectedly, when these two substances were added in equimolar proportion to normal human plasma, a precipitate was formed containing mostly aggregated fibrinogen. To further investigate this phenomenon, a solution of purified fibrinogen was used and was found to form insoluble precipitate with ascorbic acid± cupric chloride mixture, however, at a concentration 10fold lower than needed in plasma. The factor responsible for the inhibition of fibrinogen aggregation in plasma was then identified as human serum albumin. Although human serum albumin has been suggested to be an antioxidant due to the presence of one -SH group, all other thirty-four cysteines exist in the oxidized form as disulfides. Hence, this protein is rather unlikely to act as an effective antioxidant agent. To elucidate a mechanism by which human serum albumin inhibits hydroxyl radical-induced fibrinogen aggregation, a spectrophotometric experiment was done in which ascorbic acid±cupric chloride was added to a solution of NAD and optical density recorded at 340 nm. A rapid increase in optical density proved that the hydroxyl radicals generated in this system have a reducing potential that was inhibited by the addition of human serum albumin. Molar proportions of the reagents used in the present experiment revealed that one molecule of human serum albumin neutralized fifteen hydroxyl radicals, indicating that about one-half of disulfide bonds in this protein underwent reduction. It was previously observed that hydroxyl radical modification of human serum albumin led to the formation of higher molecular mass complexes (Davies & Delsignore, 1987), and that limited reduction with dithiothreitol caused its aggregation and coprecipitation with other plasma proteins (Lipinski & Egyud, 1992). Consequently, human serum albumin can be considered as a sacrificial antireductive protein which when modified by hydroxyl radicals gives a signal to proteolytic degradation and elimination from the circulation. In conclusion, in view of the findings reported by Ghyczy & Boros (2001) and the results presented in this present letter, the concept of oxidative stress and antioxidants needs be revised. Not only electrophilic methyl group biomolecules, but many other substances containing reducible groups may fall into a category of antireductants rather than antioxidants. For example, highly unsaturated eicosapentanoic acid also inhibited hydroxyl radical-induced fibrinogen aggregation. In addition, reductive addition of hydroxyl groups to double bonds of pyrimidines, pyrazines, and other aromatic rings may explain beneficial action of water soluble vitamins in free radical-induced stress (Moorthy & Hayon, 1976). Finally, similar reductive mechanisms may be involved in the formation of hydroxy derivatives of nucleic acids, e.g. 8hydroxy-2 0-deoxyguanosine, generally albeit speculatively, believed to be a result of oxidative stress (Park & Floyd, 1994).