Pharmacognosy Magazine

: 2017  |  Volume : 13  |  Issue : 51  |  Page : 561--566

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?

Giuseppe Gallo1, Rosalinda Bruno2, Adele Taranto3, Guglielmo Martino1,  
1 Department of Biology, Ecology and Earth Sciences, University of Calabria, Rende (CS), Italy
2 Department of Pharmacy and Health Sciences and Nutrition, Ecology and Earth Sciences, University of Calabria, Rende (CS), Italy
3 Regione Calabria ASP Cosenza, Hospital Giovanni Iannelli, Cetraro (CS), Italy

Correspondence Address:
Giuseppe Gallo
Via P. Bucci, Building 4C, Rende (CS) 87030


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. Abbreviations used: RBC: Red blood cell; MDA: Malondialdehyde; HNE\HAE: 4-hydroxyalkenals; LPO: Lipid peroxidation; ROS: Reactive oxygen species; ORAC: Oxygen Radical Absorbance Capacity.

How to cite this article:
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?.Phcog Mag 2017;13:561-566

How to cite this URL:
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?. Phcog Mag [serial online] 2017 [cited 2022 Dec 9 ];13:561-566
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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.


As described by Sommerburg et al., patients with end-stage renal failure undergoing hemodialysis are exposed to oxidative stress. Increased levels of malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are demonstrated in plasma of uremic patients, indicating accelerated lipid peroxidation (LPO).[1] According to Breusing et al.,[2] human plasma samples exposed to UVA irradiation at different doses (0, 15 J, 20 J) where analyzed for MDA, 4-HNE, and isoprostanes contents, result in a variation of free HNE by HPLC analysis in an interlaboratory validation of methods of LPO measurement. A study of Constantin et al. investigated the effects of aging on the carbonyl stress and glutathione antioxidant defenses in red blood cells (RBCs) of obese Type 2 mellitus diabetic patients with/without hypertensive complications. The results showed that compared to RBCs of healthy controls, in obese Type 2 diabetics, aging is associated with: (i) an increase in the concentration and expression of carbonylated proteins, a marker of oxidative stress; (ii) a decrease of both nonenzymatic and enzymatic endogenous glutathione defences; (iii) a severely disturbed oxidant/antioxidant balance when obesity is associated with hypertension.[3] 4-hydroxytyrosol is shown to significantly protect RBCs from oxidative damage in a dose-dependent manner (20–3 μM).[4] 4-hydroxytyrosol inhibits 4-HNE-induced apoptosis, characterized by increased production of reactive oxygen species (ROS).[5]

4-hydroxytyrosol shows a substantial degree of inhibition against ascorbate-Fe 2+ induced LPO in rat liver microsomes.[6] In a previous study of Gallo and Martino [7] on RBC membranes damages by oxidant agents, authors demonstrated the conformational alterations and polyphenols repairing in the same structure. Among the known risk factors of atherosclerosis-associated oxidative stress are hyperlipidemia and/or hyperglycemia.[8],[9],[10] 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.[11],[12] Furthermore, the products of degradation of membrane proteins are observed as self-consisting products without interrelations with membrane lipids. In this manuscript, we examine the role of triglycerides overload on degradation of membrane lipids in the presence of other reducing agents (protective effect of 4-hydroxytyrosol).[13],[14],[15] The studied membrane model is the RBC that is damaged from ROS.[16] The RBC is an experimental model of essential cellular structure. Hypertriglyceridemia denotes high blood levels of triglycerides, the most abundant fatty molecules in organisms. Elevated levels of triglycerides are associated with atherosclerosis and predispose to cardiovascular disease.[17] Oxidative stress, i.e., an altered balance between the production of free radicals and antioxidant defenses.[18] 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 (HAE). 4-HNE has been implicated in various pathophysiological interactions due to their chemical reactivity and the formation of covalent adducts with macromolecules. The progressive accumulation of adducts alters normal cell functions that lead to cell death. At low and noncytotoxic concentrations, these molecules may function as signaling molecules in cells. This paper addresses the origin and cellular functions of 4-HAE.[19] The scavenging activity is studied after the “in vitro” treatment with 4-hydroxytyrosol that is classified as a phytochemical compound expressing strong antioxidant properties. The Oxygen Radical Absorbance Capacity index [20] for 4-hydroxytyrosol totaled (Trolox equivalent [TE] antioxidant capacity) 40,000 μmol TE/g that is about ten times greater than green tea at least twice compared to CoQ10. One study showed that a low dose of 4-hydroxytyrosol in rats reduces the consequences of the side effects of oxidative stress induced by smoking.[21] 4-hydroxytyrosol is shown to significantly protect RBCs from oxidative damage (LPO [4-HNE]). The authors [7] studied the protective effect of 4-hydroxytyrosol on human RBCs and oxidative damage so, the intent is to examine the role of triglycerides overload on degradation of membrane lipids in the presence of other reducing agents (protective effect of 4-hydroxytyrosol). For instance, in complete eukaryotic cells, the following damages can happen: 4-HAE are endogenous ligands for peroxisome proliferator-activated receptor-δ. 4-HNE affects p38 mitogen-activated protein kinases, c-Jun N-terminal kinase, protein kinase Cβ and -δ, cell cycle regulators, epithelial growth factor receptor, microRNAs, and the nuclear factor-κB [Figure 1].[22] Cytotoxic effects furthermore result from covalent interactions of 4-HNE with aminophospholipids and distort the bilayer structure of membranes and consequently alter their barrier and transport functions [Figure 2].[23],[24]{Figure 1}{Figure 2}

 Materials and Methods


The studied population consisted of 10 patients with endogenous hypertriglyceridemia and ten normal controls who are recruited from the Health Center of the University of Calabria, Italy. All patients followed traditional local diet. The diagnosis of endogenous hypertriglyceridemia is based on the peroxidase-coupled method for the colorimetric determination of serum triglycerides.[25]

Blood samples

After fasting, blood is sampled on ethylenediaminetetraacetic acid (EDTA)-coated tubes, spun down at 1550 g for 10 min, plasma and leukocyte layers removed, and the packed RBCs layer used for the subsequent assays.

RBCs layer is lysed in 5 mM hypotonic phosphate buffer pH 8, containing in origin 1 mM EDTA (to complex all interfering cations, authors simplified the method increasing EDTA concentration at 100 mM) and 0.03 mM phenylmethylsulphonyl fluoride, followed by 20 min centrifugation at 16,000 g, the RBCs ghost sediment are resuspended by swirling while adding back sufficient of the fresh hypotonic buffer to reconstitute the original volume. The ratio of erythrocytes to washing solution is 1/40. The ghosts are washed 3 times subsequent to the hemolysis by sedimenting in a swinging-bucket rotor (16,000 g for 30 min), till free of hemoglobin. Aliquots of this should be taken for sample replicates, as well as for protein determination. 200 μL portions are required for use in this assay. Sample homogenates should be made as concentrated as possible. The concentration of protein in the homogenate should be determined. It is recommended that 0.2 μL of a homogenate containing 15–60 mg/mL protein be assayed for initial studies in a previously untested biological sample. The supernatant should be kept on ice before analyze or, if not analyzed immediately after preparation, frozen at − 70°C for longer storage.[3]

 Assay of Total Proteins in Red Blood Cells Ghosts

The determination of protein concentration is performed by the Biuret method.[26] The principle on which is based on the reaction of the peptide bond of proteins with Cu 2+ ions. In alkaline solution, the formation of an indigo colored complex: the intensity of the color is directly proportional to the protein concentration. The standard used is bovine serum albumin from Sigma. Procedure: Samples prepared by plugging the tubes are shaken, and after 45 min it reads the absorbance of each sample at 540 nm on Shimadzu UV 2100 spectrophotometer.

Spectrophotometric analysis of free hydroxynonenal

Assay procedure for malondialdehyde ([concentrated hydrochloric acid] and malondialdehyde + hydroxyalkenals [concentrated methane sulfonic acid procedure])

All reagents are from Enzo Life Science. The standards (HNE standard: (E)–Hydroxynonenal IkB inhibitor) are prepared in clean glass test tubes or polypropylene microcentrifuge tubes. Preferably, standards should be run in triplicate. Add 200 μL of sample to a clean glass test tube or polypropylene microcentrifuge tube. Unknowns should be run in triplicate. Add 650 μL of diluted N-methyl-2-phenylindole reagent. Mix gently by vortexing the sample. Add 150 μL concentrated (12 N) HCl or methanesulfonic acid reagent. Mix well and stopper the tube. Incubate at 45°C for 60 min. Centrifuge turbid samples (e.g., 15,000 × g for 10 min) to obtain a clear supernatant. Transfer the clear supernatant to a cuvette. Measure the absorbance at 586 nm.[27]

In vitro 4-hydroxytyrosol treatment

The concentration of reducing agent is chosen according to literature data.[4] 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.

Statistical analysis

For statistical analysis, one-way ANOVA and Bonferroni post hoc test are used. Results are given as mean ± standard error of the mean on at least ten undependent determinations.


Authors studied the effect of oxidative stress of RBC ghosts by the method of Gèrard-Monnier et al.[27] The diagnostic criteria for endogenous hypertriglyceridemia are as follows: total plasma triglyceride level > 200 mg/dL [Figure 3]. So that triglyceride plasma levels in patients are neatly higher than in controls. The mean values of triglyceride plasma levels in our casuistry are described in [Figure 3]. It is evident that data of the two groups are significantly different (P P P P [19] As demonstrated by higher levels of HNE in comparison to MDA, according to Sommerburg et al.[1] The RBC membrane preparations are obtained increasing the efficiency of Fe 2+ removal with an higher concentration of EDTA (100 mM). Spectrophotometric method (25) to measures free 4-HAE in human RBCs is optimized for the determination of alkenals [Figure 8] 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.{Figure 7}{Figure 8}

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 Acknowledgements

The authors would like to acknowledge Prof. Domenico Sturino for his mother language support and technical revision of manuscript.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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