Guido R.M.M. Haenen

OXIDANTS AND ANTIOXIDANTS: STATE OF THE ART

Reactive oxygen species are regarded as merely pernicious. This is incorrect for they play a pivotal role in many physiologic reactions, such as cytochrome P450-mediated oxidations, regulation of the tone of smooth muscle, and killing of microorganisms. An imbalance in oxidant-antioxidant activity is involved in many free radical-mediated pathologies, e.g., ischemia-reperfusion and asthma. In an attempt to alleviate these pathologies with antioxidants, it should be noted that these compounds are neither specific nor mere antioxidants. Associated with antioxidant activity is a pro-oxidant action. In the development of new antioxidant therapies, the important question of how these drugs are incorporated in or commensurate with existing integrated physiologic radical-defense systems should be addressed.

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.

Transcription factor NF-κB as a potential biomarker for oxidative stress

There is increasing interest in the involvement of transcription factors, such as of the transcription factor NF-κB (nuclear factor-κB), in the pathogenesis of various diseases. NF-κB is involved in the control of the transcription of a variety of cellular genes that regulate the inflammatory response by the production of cytokines, chemokines, cell adhesion molecules and acute phase proteins. The involvement of NF-κB is especially of interest as it is activated by oxidative stress and its activation can be modulated by antioxidant compounds. The activation of NF-κB can be determined by the electromobility shift assay (EMSA) with a NF-κB binding-site-specific probe. EMSA can also be used on human mononuclear cells isolated from peripheral blood, which could make the assay applicable for clinical trials. The critical steps of the EMSA are discussed, addressing some pitfalls of the assay. The procedure that can be used to express NF-κB activity in human subjects is evaluated. This offers the possibility to use NF-κB as a functional biomarker of oxidative stress as illustrated by several examples of in vitro and in vivo studies. Chemicals/CAS: Biological Markers; NF-kappa B

Bioprocessing of Wheat Bran in Whole Wheat Bread Increases the Bioavailability of Phenolic Acids in Men and Exerts Antiinflammatory Effects ex Vivo

Whole grain consumption has been linked to a lower risk of metabolic syndrome, which is normally associated with a low-grade chronic inflammation. The benefits of whole grain are in part related to the inclusion of the bran, rich in phenolic acids and fiber. However, the phenols are poorly bioaccessible from the cereal matrix. The aim of the present study was to investigate the effect of bioprocessing of the bran in whole wheat bread on the bioavailability of phenolic acids, the postprandial plasma antioxidant capacity, and ex vivo antiinflammatory properties. After consumption of a low phenolic acid diet for 3 d and overnight fasting, 8 healthy men consumed 300 g of whole wheat bread containing native bran (control bread) or bioprocessed bran (bioprocessed bread) in a cross-over design. Urine and blood samples were collected for 24 h to analyze the phenolic acids and metabolites. Trolox equivalent antioxidant capacity was measured in plasma. Cytokines were measured in blood after ex vivo stimulation with LPS. The bioavailabilities of ferulic acid, vanillic acid, sinapic acid, and 3,4-dimethoxybenzoic acid from the bioprocessed bread were 2- to 3-fold those from the control bread. Phenylpropionic acid and 3-hydroxyphenylpropionic acid were the main colonic metabolites of the nonbioaccessible phenols. The ratios of pro-:antiinflammatory cytokines were significantly lower in LPS-stimulated blood after the consumption of the bioprocessed bread. In conclusion, bioprocessing can remarkably increase the bioavailability of phenolic acids and their circulating metabolites, compounds which have immunomodulatory effects ex vivo.

Pitfalls in a method for assessment of total antioxidant capacity.

A relatively simple and widely applied method for quantitating the total antioxidant capacity of body fluids and drug solutions based on the absorbance of the ABTS radical cation was evaluated. In this assay, the end-point is an antioxidant-induced decrease in absorbance at a fixed time. This decrease is used as an index of total antioxidant capacity. It is shown that Trolox, potassium cyanide and quercetin all decrease the absorbance of ABTS radical cations at a fixed time, but by different mechanisms. Trolox scavenges the ABTS radical, potassium cyanide inhibits radical formation, while quercetin acts by both mechanisms. Using this method antioxidant capacity may be overestimated, due to both a scavenger effect and an effect on the rate of ABTS oxidation. To distinguish between these effects, a post-addition assay was used in which the sample is added when the formation of radicals is stable. Using post- addition assay conditions enables discrimination between effects on radical scavenging and on the radical formation, two major mechanisms for antioxidant action. In extrapolating the results to an in vivo situation it should be questioned: (i) whether the peroxidase process does indeed mimic the process of radical formation in vivo, and (ii) whether the ABTS radicals do resemble the radical species involved in an in vivo situation. Results obtained in the ABTS radical-based methods should therefore be reviewed critically before the antioxidant capacity can be assessed.

Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.).

Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fractions of wheat grain. It was found that the aleurone content of these fractions was highly correlated with the antioxidant capacity of the fractions (r = 0.96, p < 0.0001). Ferulic acid appeared to be the major contributor to the antioxidant capacity in fractions with higher antioxidant capacity. The contribution of protein was rather limited. It was concluded that the antioxidant potency of wheat grain fractions is predominantly determined by aleurone content, which can be attributed to the presence of relatively large amounts of phenolic compounds, primarily ferulic acid.

CIMETIDINE AND OTHER H2 RECEPTOR ANTAGONISTS AS POWERFUL HYDROXYL RADICAL SCAVENGERS

Hydroxyl radical scavengers are able to compete with deoxyribose for the hydroxyl radicals generated in a reaction mixture. We found that the H2 receptor antagonists like cimetidine, burimamide, ranitidine, famotidine and tiotidine except for being good inhibitors in histamine-stimulated gastric acid secretion, were also very powerful hydroxyl radical scavengers. Rate constants for reaction of these drugs with hydroxyl radicals ranged from 7.7 x 10(9) Ms-1 to 14.8 x 10(9) M-1 s-1. These rate constants are much higher than for the well-known hydroxyl radical scavenger mannitol (1.7 x 10(9) M-1 s-1). In this study we investigated which part of the cimetidine molecule might be responsible for its potent hydroxyl radical scavenging activity. Testing fragments of the cimetidine molecule revealed that the guanidine moiety of cimetidine had little hydroxyl radical scavenging activity. However the other part of the molecule, the methylated imidazole with a sulfur and amino group containing side chain appeared to be a powerful hydroxyl radical scavenger.

4-Hydroxy-2,3-trans-nonenal stimulates microsomal lipid peroxidation by reducing the glutathione-dependent protection

Glutathione (GSH) protects liver microsomes against lipid peroxidation. This is probably due to the reduction of vitamin E radicals by GSH, a reaction catalyzed by a membrane-bound protein. Pretreatment of liver microsomes with 0.1 or 1mM 4-hydroxy-2,3-trans-nonenal (HNE), a major product of lipid peroxidation, reduces the GSH-dependent protection. GSH and vitamin E concentrations are not affected by this pretreatment. Pretreatment with 0.1 mM N-ethyl maleimide (NEM), a synthetic sulfhydryl reagent, resulted in a reduction similar to that with HNE of the GSH-dependent protection against lipid peroxidation. The reduction of the GSH-dependent protection by HNE and NEM is probably the result of inactivation of the membrane-bound protein by covalent binding to an essential SH group on the protein. If the GSH-dependent protection would proceed via the microsomal GSH transferase, pretreatment with NEM, which activates the microsomal GSH transferase, should enhance the GSH-dependent protection. Actually a decrease in the GSH-dependent protection is found. Apparently the GSH-dependent protection does not proceed via the microsomal GSH transferase. Also the microsomal phospholipase A2 is not involved, since addition of 0.1 mM mepacrine, an inhibitor of phospholipase A2, did not preclude the GSH-dependent protection. Once the process of lipid peroxidation, either in vivo or in vitro, has started, the protection of liver microsomes by GSH is less effective. This might be the result of formed HNE. In this way an endproduct of lipid peroxidation stimulates the process that generates this product.

Activation of the microsomal glutathione-s-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid peroxidation, and on the microsomal GSH-S-transferase (GSH-tr) in the rat was determined. In vitro incubation of liver microsomes with 5 mM acrolein for 30 sec resulted in a 2-fold activation of the GSH-tr. This activation probably proceeds via alkylation of the thiol group of the GSH-tr. In vivo administration of 1.1 mmol allyl alcohol/kg to rats did also result in a 2-fold stimulation of the GSH-tr activity. Administration of 375 mg pyrazole/kg, an inhibitor of the alcohol dehydrogenase, thus reducing the acrolein formation, prevented the in vivo stimulation of GSH-tr by allyl alcohol. This indicates that the activation of GSH-tr in vivo by allyl alcohol probably also proceeds via alkylation of the thiol group of the GSH-tr by acrolein. GSH protects liver microsomes against lipid peroxidation, probably via a free radical reductase that reduces vitamin E radicals at the expense of GSH. Incubating liver microsomes for 30 min with 0.1 mM acrolein reduced the GSH dependent protection against lipid peroxidation, probably because an essential thiol group(s) on the free radical reductase is alkylated. In vivo administration of allyl alcohol did not reduce the GSH dependent protection of the microsomes. Probably the thiol group(s) located on the free radical reductase is less accessible or less reactive than the thiol group on the GSH-tr. After administration of allyl alcohol we found no evidence for in vivo lipid peroxidation. Therefore we could not evaluate the importance of the GSH dependent protection against lipid peroxidation in vivo.

Reduction of lipoic acid by lipoamide dehydrogenase.

Racemic lipoic acid is therapeutically applied in pathologies in which free radicals are involved. The in vivo reduction of lipoic acid may play an essential role in its antioxidant effect. It was found that mitochondrial lipoamide dehydrogenase (LipDH, EC 1.8.1.4.) reduces the R-enantiomer 28 times faster than the S-enantiomer of lipoic acid. Moreover, it was observed that the metabolites of lipoic acid, bisnor-, tetranor-, and beta-lipoic acid are poor substrates of LipDH. S-lipoic acid inhibits the reduction of the R enantiomer only at relatively high concentrations. The reduction of R-lipoic acid by mitochondria-rich tissues may proceed smoothly, even if the racemic mixture is applied. This is of importance in elucidating the molecular mechanism of the pharmacotherapeutic effect of lipoic acid.