**2.2 Study on 4-hydroxytyrosol**

The assay (ALDetectTM *Lipid Peroxidation* assay kit**)** is designed to measure either MDA alone (in hydrochloric acid) or MDA in combination with 4-hydroxyalkenals (in methanesulfonic acid).

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 2 molecules of reagent R1 to yield a stable chromophore with maximal absorbance at 586 nm. For simultaneous determination of MDA and 4-hydroxyalkenals, one must use the procedure utilizing methanesulfonic acid (MSA) as the acid solvent. The procedure in which HCl is used will only detect MDA, since the 4-hydroxyalkenals do not form a chromophore with reagent R1 under those conditions. Interestingly, in addition to the 586 nm chromophore, 4-hydroxyalkenals gave a second chromophore with maximal absorbance at 505 nm and alkanals produced a single chromophore with maximal absorbance at 505 nm. The concentration of reducing agent is chosen according to literature data [12]. Reducing agent is provided from Sigma Chemical Co. (St. Louis, MO, USA) and used at the final concentration of 80 μM for 3 h at 37°C.In vitro 4 hydroxytyrosol treatment to evaluate the membrane lipids reducible by this phenol.

#### *Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

As described in **Figure 4a** and **b** the level of oxidation products of membrane lipids are referred as μM of MDA or HNE. MDA levels are always ten times those of HNE as referred in right-hand scale. In **Figure 4a** and **b** the scales are quite different (for instance: 0.00 /0.85 in A and 0.000/0.033 in B for MDA). We can also observe that MDA levels of pathological samples are slightly higher than control ones but HNE levels of pathological samples are four times those of control ones. After the 4 hydroxytyrosol action both lipid metabolites substantially decrease. The mean values of total membrane protein levels in our casuistry are described in **Figure 4**. It is evident that for the two dosed substances each group of data is significantly different (*P* < 0.001) from the other ones. In **Figure 5** are reported the total levels of MDA + HNE per milligram of total membrane proteins of RBC ghosts. As described the total oxidized lipid levels in RBC ghost membranes almost doubled in pathological samples in comparison to control ones. This shows that even if total membrane proteins dramatically increase in hypertriglyceridemic patients, also total oxidized lipids sufficiently increase in such samples so that ratio between patients RBC oxidized lipids to membrane proteins is always doubled. Such partial compensation of lipids in comparison to proteins from RBC membrane could be explained as an attempt of RBC metabolism to partially compensate ghost structure damages by oxidative stress. The ratio of total oxidized lipids to membrane proteins before and after 4-hydroxytyrosol treatment are referred in **Figure 5**. After 4-hydroxytyrosol treatment, the ratio of oxidized lipids to total membrane proteins decreases even more, but the values in our controls are always lower than in hypertriglyceridemic patients. The mean values of ratio of oxidized lipids (MDA + HNE) and membrane proteins in our casuistry are described in **Figure 5**. It is evident that the two groups of data are significantly different (*P* < 0.001). Neither Vitamin E nor Vitamin C are used because their ultraviolet–visible (UV–Vis) spectra superimpose to HNE determination method interfering with their evaluation. The structures most exposed to the damaging action of radicals are the lipid structures constituting the cellular and nuclear membranes that are subjected to destructuring. One of the most sensitive sites to damage caused by ROS is the plasma membrane, in particular the target is at the level of polyunsaturated fatty acids. The oxidation by ROS of lipids is called peroxidation. Alkenals are products of lipid peroxidation while hydroxytyrosol is a powerful reducing agent. This explains the decrease in the parameters of alkenals.

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 *et al*. [14] The RBC membrane preparations are obtained increasing the efficiency of Fe2+ removal with an higher concentration of EDTA (100 mM). Spectrophotometric method [15] to measures free HNE in human RBCs is optimized for the determination of alkenals in membranes. Other kinds of reducing agents, such as Vitamin E, are not tested because their UV–Vis spectra superimpose to N-methyl-2 phenylindole product spectra. In membranes of hypertriglyceridemic patients, the levels (triglycerides, total membrane proteins, total aldehydes and their ratio) are higher than in control samples. All the data decrease relevantly (*P* < 0.001) after the 4-hydroxytyrosol treatment, especially those of alkenals. In summary, lipid peroxidation is divided into two stages. Lipid Hydroperoxide is produced in the Early stage while Aldehydes (4-HNE, MDA) is produced in the Late stage. Lipid peroxidation consists of the radical peroxidation of a polyunsaturated fatty acid, with the formation of the lipoperoxide radical and its subsequent fragmentation into short-chain aliphatic and malondialdehyde compounds. The oxidation of arachidonic acid occurs by means of two enzymes: lipooxygenase and cyclooxygenase. The reaction sequence can take

**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* P *< 0.001 by two ways ANOVA and Bonferroni* post hoc *test. \**P *< 0.05; \**\*P *< 0.01, and \*\*\**P *< 0.001.*

place under physiological conditions involving the arachidonic acid, contained in the membrane phospholipids, and is triggered by the superoxide anion, which is transformed into water producing the C-13 radical of arachidonic acid, which is followed by the radical in C-11. This, in the presence of oxygen, generates the radical peroxide or, by transferring the radical nature to another molecule, is transformed

*Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

#### **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* P *< 0.001 by two-ways ANOVA and Bonferroni* post hoc *test.* \*P *< 0.05; \*\**P *< 0.01 and \*\*\**P *< 0.001 from the others.*

into stable hydroperoxide. The radical cycloperoxide, in the presence of oxygen and due to the effect of heat, can fragment, producing malondialdehyde (MDA) and aliphatic compounds of a radical nature. Peroxidation, like all chain reactions, consists of an "initiation", a "propagation" and a "termination" stage. The consequences of an intense peroxidation of biological membrane lipids are: loss of biological membrane fluidity, reduction of membrane potential, increased permeability to H+ ions and other ions and rupture and release of cellular content to the outside.

### **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

#### **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.\** p *< 0.05, \*\** p *< 0.01 and \*\*\**p *< 0.001.*

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 [16–18]. 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

*Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

**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. \** p *< 0.05, \*\** p *< 0.01 and \*\*\**p *< 0.001.*

**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. \**p *< 0.05 \*\** p *< 0.01 and \*\*\**p *< 0.001.*

**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. \** p *< 0.05, \*\** p *< 0.01 and \*\*\**p *< 0.001.*

research also on other structural components of membrane. At present, we have only data on total RBC membrane proteins.

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

*Reducing Compounds Roles in Oxidative Stress Relieving of Human Red Blood Cells DOI: http://dx.doi.org/10.5772/intechopen.99977*

#### **Figure 10.**

*Graphical resume of main metabolic steps of fatty acids in cell membrane.*

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.

### **5. Conclusions**

In the first study, it is evident that after *in vitro* oxidative damage of the membrane, red wine polyphenol extracts are as effective reducing agents also on CM indicator of membrane protein damages. Consequently, both red wine and polyphenol extracts both alone and mixed among them efficiently relieve the effects of oxidative stress. Regarding the study on 4-hydroxytyrosol. All the data decrease relevantly (*P* < 0.001) after the 4-hydroxytyrosol treatment, especially those of alkenals. As for the creatinine and uric acid studies they this study suggest that at low concentrations of creatinine and uric acid there is oxidative stress in human erythrocyte membrane, but if creatinine and uric acid have slowed down to sufficient limits there is also oxidative stress, as demonstrated by MDA and HNE levels; only if the concentration of these two substances overwhelms a threshold. 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 plasma membrane recycled RBC.
