Abstract
Oxidative stress is the consequence of an imbalance between pro-oxidant and antioxidant processes. Antioxidants that counteract reactive oxygen species do not all work the same way. Both resveratrol and the more powerful 4-hydroxytyrosol are excellent reducing agents. Polyphenol treatment (red wine polyphenols, resveratrol and catechin) is associated with a significant increase in anion permeability for chloride compared with control and 2.2′-azobis-2 amidinopropan dihydrochloride affected cells. Treatment with polyphenols was associated with a significant reduction in mean ± standard error of the mean membrane lipid peroxidation compared with control and 2.2′-azobis-2 amidinopropan dihydrochloride treatment. Hemolysis data are also obtained in the previously described conditions. 4-hydroxytyrosol is shown to significantly protect red blood cells from oxidative damage by 4-hydroxynonenal. But there are paradoxical effects like uric acid and creatinine. The obtained data evidence that both creatinine and uric acid levels have influence on the ratio of both malondialdehyde/protein and 4-hydroxynonenal/protein content on red blood cell ghosts, demonstrating their possible protective role against oxidative stress at low concentrations in blood and oxidizing power at higher concentrations. Finally, polyunsaturated fatty acids do not have all this reducing power.
Keywords
- reactive oxygen species
- resveratrol
- 4-hydroxytyrosol
- uric acid
- creatinine
- polyunsaturated fatty acids
1. Introduction
Chemical stress induced by the presence, in a living organism, of an excess of reactive chemical species, generally centered on oxygen (reactive oxygen species), secondary to an increased production of the same and / or to a reduced efficiency of the physiological systems of antioxidant defense. There are many antioxidants, among these there is resveratrol. The history of resveratrol (the
2. Study on powerful antioxidants
2.1 Study on resveratrol
The reducing agent treatment efficacy was observed by evaluation of anion permeability for chloride, lipid peroxidation and hemolysis in RBCs. Anion permeability for chloride is an indicator of membrane protein damage and is evaluated in RBCs by the specific absorption of methemoglobin (CM) at 590 and 635 nm after treatment of heparinized blood with NaNO2. The measurement of the membrane lipid degradation is obtained by the determination of MDA. The lipid peroxidation susceptibility is observed after the oxidative stress induced by AAPH. The hemolysis assays are conducted on blood samples in phosphate buffer saline. To evaluate in human RBC the
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F1.png)
Figure 1.
RBC membrane anion permeability for chloride. Data are mean ± standard error of the mean on 10 determinations. *P < 0.05 according to one-way analysis of variance and Bonferroni
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F2.png)
Figure 2.
Effects of lipid peroxidation and induced by 2.2′-azobis-2 amidinopropan dihydrochloride (60 mM) (
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F3.png)
Figure 3.
Effect of 2.2′-azobis-2 amidinopropan dihydrochloride-induced hemolysis in sportive human RBCs at several times. Data are mean ± standard error of the mean on 10 determinations. Column labels are according to incubation times a = (60 min); b = (120 min); c = (180 min); d = (240 min). *
2.2 Study on 4-hydroxytyrosol
The assay (ALDetectTM
So we have TOT = MDA + HNE and then TOT-MDA = HNE. The assay is based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole (R1) with MDA and 4-hydroxyalkenals to yield a stable chromophore with maximal absorbance at 586 nm at 45°C. One molecule of either MDA or 4-hydroxyalkenal reacts with
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F4.png)
Figure 4.
Human RBC membrane concentration of MDA and HNE (μM absolute concentration in membrane samples) from controls and hypertriglyceridemic patients (A) and the same samples treated with 4-hydroxytyrosol (80 μM) (B). It is evident that for the two dosed substances each grouped data are significantly different for
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F5.png)
Figure 5.
Ratio of oxidized lipids (MDA + HNE) and membrane proteins. It is evident that all comparisons for the two dosed substances, each group of data is significantly different for
According to our data in RBC, ghosts are present both HNE and MDA products [13]. As demonstrated by higher levels of HNE in comparison to MDA, according to Sommerburg
3. Study on creatinine and uric acid
The studied population consisted of 10 patients with endogenous both hypercreatininemic hyperuricemic and ten normal subjects. The measurement of total proteins in RBC ghosts and HNE and MDA are conducted on blood samples of patients. The increase of MDA and HNE levels represent the elevated activities of oxidative stress in human body. It can be seen from Figures 6 and 7 that the increase of protein concentration in the membrane of the blood samples is associated with the rise of MDA and HNE levels. From the ratio between either MDA levels (μM) or HNE levels and RBC membrane proteins (g/l), we deduced the following results on the basis of Figures 8 and 9 in which we have divided samples into 3 groups according to their blood reactive oxygen species (ROS) levels. According to this kind of grouping, we can observe that both uric acid and creatinine levels have influence on the ratio either of MDA/protein or HNE/protein contents in RBC membranes, demonstrating their protective role against oxidative stress at low concentrations (lower than 5 mg/dL, for uric acid) and slight oxidizing power at high concentrations (higher than 1.1 mg/dL, for creatinine), as previously evaluated by Qasim and Mahmood [2] and even more powerful oxidant (HNE) could act similarly. In each plot, “a valley” was observed. In this valley, it is seen that there is a dramatically fall in μmole concentration of MDA per mg of RBC proteins which signifies low oxidative stress activity. Thus, it suggests that if the concentration of creatinine and uric acid is within medium range of uric acid concentration, each of these substances would strongly express their protective role toward oxidative stress. In Figures 6–9 one can observe that the recovery of the curve is smooth and starts from the proximal area of Sample knot, suggesting that at this point both Creatinine and uric acid gradually loss their protective abilities as their concentration in the RBC membranes increase. The described results confirm the observation on MDA levels are similar for the action of uric acid and creatinine on ROS levels with almost parallel patterns [16, 17, 18]. An hypothetic explanation for this paradox could be that a rise in uric acid concentration represents an attempted protective response by the host [6]. Probably HNE data in the same experimental conditions should parallel the similar effects of ROS on the oxidation of longer chain lipids in human RBC membranes. The relationship between oxidation of long chain fatty acids and the concentration of both uric acid and creatinine blood levels according to their paradoxical action on oxidation of this kind of RBC constituents probably are attributable to multiple mechanisms of interaction of several constituents of ROSs mixture generated in human organisms with the molecular structures of RBC membranes as carbonylated proteins. This hypothesis shall be investigated in further research also on other structural components of membrane. At present, we have only data on total RBC membrane proteins.
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F6.png)
Figure 6.
Effect of blood uric acid levels on RBC membrane MDA. Each result is the main plus/minus standard error on the mean of ten independent experiments. Results are evaluated by one-way ANOVA and Bonferroni post-hoc test according to Graphpad prism 5.0.*
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F7.png)
Figure 7.
Effect of blood uric acid levels on RBC membrane HNE. Each result is the mean plus/minus standard error on the main of ten independent experiments. Results are evaluated by one-way ANOVA and Bonferroni post-hoc test according to GraphPad prism 5.0. *
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F8.png)
Figure 8.
Effect of blood creatinine levels on RBC membrane MDA. Each result is the mean plus/minus standard error on the main of ten independent experiments. Results are evaluated by one-way ANOVA and Bonferroni post-hoc test according to Graphpad prism 5.0. *
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F9.png)
Figure 9.
Effect of blood creatinine levels on RBC membrane HNE. Each result is the mean plus/minus standard error on the main of ten independent experiments. Results are evaluated by one way ANOVA and Bonferroni post-hoc test according to GraphPad prism 5.0. *
The data from this research suggest that nitrogen metabolism, mainly creatinine, acts upon cellular lipid metabolism, as this chemical in itself is a reductant compound but at high intracellular concentrations it works as an oxidizing product, as described by Qasim and Mahmood [2]. Creatinine metabolism can interact with uric acid excretion by kidneys. Hyperuricemia damages kidneys where creatinine is excreted by humans. The interaction between concentrations of creatinine and uric acid, that is powerful scavenger of singlet oxygen, slows down the activity of oxidative stress in human erythrocyte membranes. The contemporaneous modulation of both creatinine and uric acid metabolism and their anatomical and functional consequences could modulate MDA and HNE levels. Only if the concentration of these 2 substances overcomes thresholds, they will begin to express their harmful both oxidative and reductive activities.
4. Study on PUFAs
Currently, in the literature, there are only partial discussions on the role of lipids and their oxidation products as intermediates of their membrane structural damage and/or on the protective role in the same structures [19, 20]. Elevated levels of triglycerides are associated with atherosclerosis and predispose to cardiovascular disease [21]. Oxidative stress, i.e., an altered balance between the production of free radicals and antioxidant defenses [22]. The peroxidation of n-3 and n-6 polyunsaturated fatty acids (PUFAs) and of their metabolites is a complex process. It is initiated by free oxygen radical-induced abstraction of a hydrogen atom from the lipid molecule followed by a series of nonenzymatic reactions that ultimately generate the reactive aldehyde species 4-hydroxyalkenals (HNE). Some authors show that high doses of dietary n-3 PUFAs, as well as long-time treatments, affect human RBC susceptibility to lipid peroxidation by changes in fatty acid composition content. According to experimental data, the accumulation of the alkenals in RBC membrane could be produced either by partial PUFA oxidation contained in glycerides and plasma glycerides and by glycerides into recycled plasma membrane in RBC neogenesis (Figure 10). According to these hypotheses, the increased charge of triglycerides in plasma forces its metabolism toward either incorporation in cell membrane of degradative oxidation. This last pathway induces increase of oxidative product such as alkenals and MDA. Furthermore, free alkenals can be dissolved in lipid membrane bilayer degrading their structures. This last process favors increased level of macromolecular assemblies in circulation that enhances microcirculation damage. Such last data could be studied in following works.
![](http://cdnintech.com/media/chapter/78519/1512345123/media/F10.png)
Figure 10.
Graphical resume of main metabolic steps of fatty acids in cell membrane.
5. Conclusions
In the first study, it is evident that after
Acknowledgments
The authors would like to acknowledge Prof. Domenico Sturino for his mother language support and technical revision of manuscript.
ORAC | Oxygen Radical Absorbance Capacity, or the oxygen radical absorption capacity |
AAPH | 2, 2′-azobis (2-amidinopropane) dihydrochloride |
RBC | red blood cell |
MDA | malondialdehyde |
HNE | 4-hydroxynonenal |
PUFAs | polyunsaturated fatty acids |
EPA | eicosapentaenoic acid |
DHA | docosahexaenoic acid |
ROS | reactive oxygen species |
CM | the visible absorbance spectrum method evaluating the absorption of methemoglobin |
References
- 1.
H VisioliH F, Galli C, Plasmati E, H ViappianiH S, H HernandezH A, Colombo C, Sala A. Olive phenol hydroxytyrosol prevents passive smoking-induced oxidative stress Circulation. 2000; 102(18):2169-2171 - 2.
Qasim N, Mahmood R. Diminution of Oxidative Damage to Human Erythrocytes and Lymphocytes by Creatinine: Possible Role of Creatinine in Blood. PLoS One. 2015;10(11):e0141975 - 3.
Mufidah M, Ermina P, Gemini A, Marianti AM, Lukman M, Rusdi M, et al . Lipid Peroxidation Inhibitory Activityin vitro ofMezzetia parvi-flora Becc. Wood Bark Polar Extract. Pharmacogn J. 2017;9(2):171-175 - 4.
Gallo G, Martino G. In vitro action of 2.2’azobis2 amidinopropan dihydrochloride, red wine polyphenols, resveratrol and catechin on anion permeability for chloride in human red blood cell. Free Radicals and Antioxidants. 2014;4(2):13-17 - 5.
Gallo G, Bruno R, Taranto A, Martino G. Are Polyunsaturated Fatty Acid Metabolites, the Protective Effect of 4-hydroxytyrosol on Human Red Blood Cell Membranes and Oxidative Damage (4-hydroxyalkenals) Compatible in Hypertriglyceridemic Patients?. Pharmacogn Mag. 2017;13(Suppl 3):S561-S566 - 6.
Sautin YY, Johnson RJ. Uric acid the oxidant–antioxidant paradox. Nucleosides Nucleotides Nucleic Acids. 2008;27(6-7):608-619 - 7.
P Palozza , E Sgarlata, C Luberto, E Piccioni, M Anti, G Marra, F Armelao, P Franceschelli, G M Bartoli n-3 fatty acids induce oxidative modifications in human erythrocytes depending on dose and duration of dietary supplementation. Am J Clin Nutr 1996;64(3):297-304 - 8.
Gallo G, Martino G, Carino AR. Spinning, oxidative damage and hemolysis in athletes. Free Radic Antioxid 2013;3:61-66 - 9.
Coccia R, Spadaccio C, Foppoli C, Perluigi M, Covino E, Lusini M, et al. The effect of simvastatin on erythrocyte membrane fluidity during oxidative stress induced by cardiopulmonary bypass: A randomized controlled study. Clin Ther 2007;29:1706-1717 - 10.
Frémont L, Belguendouz L, Delpal S. Antioxidant activity of resveratrol and alcohol-free wine polyphenols related to LDL oxidation and polyunsaturated fatty acids. Life Sci 1999;64:2511-2521 - 11.
Gallo G, Mazzulla S, Martino G. Scavenger enzymes and natural reducing compounds roles in oxidative stress relieving of mammalians. Rec Res Dev Physiol 2012;5:159-173 - 12.
Paiva-Martins F, Fernandes J, Rocha S, Nascimento H, Vitorino R, Amado F, et al. Effects of olive oil polyphenols on erythrocyte oxidative damage. Mol Nutr Food Res 2009;53:609-616 - 13.
Riahi Y, Cohen G, Shamni O, Sasson S. Signaling and cytotoxic functions of 4-hydroxyalkenals. Am J Physiol Endocrinol Metab 2010;299:E879-E886 - 14.
Sommerburg O, Grune T, Hampl H, Riedel E, van Kuijk FJ, Ehrich JH, et al. Does long-term treatment of renal anaemia with recombinant erythropoietin influence oxidative stress in haemodialysed patients? Nephrol Dial Transplant 1998;13:2583-2587 - 15.
McGowan MW, Artiss JD, Strandbergh DR, Zak B. A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 1983;29:538-542 - 16.
Rutkowski P, Słominska EM, Szołkiewicz M, Aleksandrowicz E, Smolenski RT, Wołyniec W, et al . Relationship between uremic toxins and oxidative stress in patients with chronic renal failure.Scand J Urol Nephrol. 2007;41(3):243-248 - 17.
Buranakarl C, Trisiriroj M, Pondeenana S, Tungjitpeanpong T, Jarutakanon P, Penchome R, et al . Relationships between oxidative stress markers and red blood cell characteristics in renal azotemic dogs. Res Vet Sci. 2009;86(2):309-313 - 18.
Onyesom I. Synergistic effect of alcohol and fructose administration on blood urate and biochemical indices of insulin resistance in albino rabbits. Indian J Med Res. 2006;124(6):715-717 - 19.
Richard D, Kefi K, Barbe U, Bausero P, Visioli F. Polyunsaturated fatty acids as antioxidants. Pharmacol Res 2008;57:451-455 - 20.
Ambrozova G, Pekarova M, Lojek A. Effect of polyunsaturated fatty acids on the reactive oxygen and nitrogen species production by raw 264.7 macrophages. Eur J Nutr 2010;49:133-139 - 21.
Pejic RN, Lee DT. Hypertriglyceridemia. J Am Board Fam Med 2006;19:310-316 - 22.
Chang T, Wu L. Methylglyoxal, oxidative stress, and hypertension. Can J Physiol Pharmacol 2006;84:1229-1238