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?

Abstract

Background: Increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are demonstrated in plasma of uremic patients. A study showed that the comparison of erythrocytes of healthy and diseased patients (obese, hypertensive, and Type 2 diabetics) with age is associated to a disturbed oxidant/antioxidant balance when obesity is associated with hypertension. 4-hydroxytyrosol is shown to significantly protect red blood cells (RBCs) from oxidative damage (4-HNE). In literature, there are partial discussions on the role of lipids and their oxidation products. The products of degradation of membrane proteins are observed as self-consisting products without interrelations with membrane lipids.

Objective: The aim of this study is to evaluate the role of polyunsaturated fatty acid (PUFA) metabolites on oxidative damage (4-hydroxy-alkenals) in RBCs of hypertriglyceridemic patients after membrane treatment with 4-hydroxytyrosol.

Materials and methods: The authors optimize the isolation of RBC ghosts and spectrophotometric method to measure free 4-hydroxyalkenals in human RBC membranes and investigated the effect on oxidative damage in human erythrocyte membranes and in vitro 4-hydroxytyrosol treatment to evaluate the membrane lipids reducible by this phenol.

Results: Plasma triglyceride levels in patients are clearly higher than in controls. Moreover, total membrane proteins data are similar to previous described. The normalized alkenals levels are significantly enhanced in hyperlipemic patients in comparison to normoglyceridemic controls. After the 4-hydroxytyrosol action, lipid metabolites substantially decrease. The ratio of oxidized lipids (MDA + HNE) and membrane proteins data are similar to previously described ones.

Conclusion: 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.

Summary: Hypertriglyceridemia induces oxidative stress in human red blood cell (RBC) membranesOxidative stress causes increased plasma membrane total protein concentration and hydroxynonenal and malondialdehyde levelsThe authors optimize the isolation of RBC ghosts and spectrophotometric method to measure free 4-hydroxyalkenals in human RBC membranesAfter the reduction with 4-hydroxytyrosol, oxidized lipid concentration significantly decrease. Abbreviations used: RBC: Red blood cell; MDA: Malondialdehyde; HNE\HAE: 4-hydroxyalkenals; LPO: Lipid peroxidation; ROS: Reactive oxygen species; ORAC: Oxygen Radical Absorbance Capacity.

Keywords: 4-hydroxyalkenals; 4-hydroxytyrosol; hypertriglyceridemia; lipid peroxidation; malondialdehyde; oxidative stress.

Figure 1

Proposed molecular mechanism of formation of hydroxynonenal-histidine adducts, according to Uchida and Stadtman, 1992

Figure 2

Scheme of the procedure used to evaluate ethanolamine phospholipid-alkenal Michael adducts by gas chromatography-mass spectrometry as their ethanolamine-alkenal residue, according to Bacot et al., 2007

Figure 3

Plasma biochemical data of controls and hypertriglyceridemic patients (mean ± standard deviation). The mean values of triglyceride plasma levels. It is evident that data of the two groups are significantly different for P < 0.01 by one-way ANOVA and Bonferroni post hoc test. *P < 0.05; **P < 0.01 and ***P < 0.001

Figure 4

The quantitative assay of total proteins per blood ml from red blood cell ghosts. Mean values of proteins in red blood cell purified membranes. It is evident that data of the two groups are significantly different for P < 0.01 by one-way ANOVA and Bonferroni post hoc test. *P < 0.05; **P < 0.01 and ***P < 0.001

Figure 5

Human red blood cell membrane concentration of malondialdehyde and hydroxynonenal (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

Figure 6

Ratio of oxidized lipids (malondialdehyde + hydroxynonenal) 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

Figure 7

Mechanisms of 4-hydroxyalkenal production. Reactive oxygen species, reactive oxygen species. (a) Nonenzymatic generation of 4-hydroxy-2E-hexenal and 4-hydroxy-2E-nonenal (4-hydroxynonenal) from n-3 and n-6 polyunsaturated fatty acids, respectively. (b) lipoxygenase-initiated peroxidation of n-6 polyunsaturated fatty acids and generation of 4-hydroxynonenal and 4-hydroxy-2E,6Z-dodecadienal

Figure 8

Stages of lipid peroxidation (modified by authors scheme of Abd El-Aal HAHM. Lipid Peroxidation End-products as a Key of Oxidative Stress: Effect of Antioxidant on Their Production and Transfer of Free Radicals. In: Catala A, editor. Biochemistry, Genetics and Molecular Biology “Lipid Peroxidation”. Winchester (GB): InTech; 2012. p. 63-88)

Figure 9

4-hydroxytyrosol ultraviolet–visible absorbance spectrum peak at 280 nm (Klen TJ. Olive fruit phenols in olive oil processing: the fate and antioxidant potential. Thesis, University of Nova Gorica, Nova Gorica, Slovenia; 2014)

Figure 10

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