Detection of oxidative stress biomarkers in myricetin treated red blood cells

Abstract

Homeostasis is a key characteristic of cellular lifespan. Its maintenance influences the rate of aging and age related disorders. Only certain flavonoids have been shown to alter homeostasis of red blood cells in the course of aging. It has been demonstrated that myricetin possesses both antioxidant and pro-oxidant properties. The objective of this study was the determination of the membrane bound oxidative stress biomarkers (Na⁺, K⁺-ATPase, Ca²⁺-ATPase, and Na⁺, H⁺ exchanger) activity in myricetin treated red blood cells during human aging. The study was carried out on clinically relevant blood samples obtained from 92 healthy subjects between the ages of 20–79 years. The subjects were divided into three age groups, young (18–35 years), middle (36–60 years) and old (>60 years). The effects of myricetin were evaluated by detecting Na⁺, K⁺-ATPase, Ca²⁺-ATPase, and Na⁺, H⁺ exchanger activities by co-incubating the red blood cells in the presence of myricetin (10⁻⁸ M to 10⁻³ M final concentration). The results showed significant (p < 0.001) age dependent decline in the activities of Na⁺, K⁺-ATPase, and Ca²⁺-ATPase and elevation in the activity of Na⁺, H⁺ exchanger as compared to the respective young controls. In vitro administration of myricetin significantly attenuated the deleterious effect of oxidative stress in red blood cells from all three age groups. We believe that these findings are novel and they will help in further research against oxidative stress in red blood cells, thereby this study has remarkable scope in medical science.

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Introduction

Flavonols belong to the large natural antioxidant group of flavonoids.¹ Flavonoids show both anti-oxidative as well as pro-oxidative properties depending on their intracellular concentration. Myricetin (3,5,7,3′, 4′,5′-hexahydroxylflavone) is a natural flavonol, found in many fruits, vegetables, berries, medicinal herbs and other plants.² It is well known for its nutraceutical value. The chemical uniqueness of myricetin mediates a direct antioxidative effect by the catechol groups in ring B forming a semiquinone radical after oxidation; the 4′-OH group of ring B and 3-OH group forming a quinine methide after oxidation and the keto group in combination with 3-OH or 5-OH group chelating redox active metal ions.³ The motivation behind choosing this flavonoid is its structural characteristic of donating proton that may alter the activity of ion transporters in cell membrane (Fig. 1). Myricetin is highly effective with respect to scavenging of reactive oxygen species (ROS) and provides protection against oxidative stress.⁴ It has also shown anti-inflammatory⁵ and anti-mutagenic⁶ activities. Several polyphenols including myricetin have been reported to increase the life span.^7,8 Recently, it has been reported that a methylated derivative of myricetin enhances life span in Caenorhabditis elegans.⁹

Fig. 1 Chemical structure of myricetin. The antioxidant property is due to three OH-groups in ring B.

Aging is a part of natural life cycle of an organism which is linked with morphological, biochemical and functional impairments of the body. Aerobic cells are frequently exposed to free radicals in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS).¹⁰ These free radicals damage biomolecules such as carbohydrates, proteins, lipids, and nucleic acid.¹¹ To provide protection against ROS/RNS, organisms develop endogenous antioxidant defense arrangements that comprise of primary and secondary antioxidants.

Human red blood cells (RBCs) survive in the circulatory system for approximately 115 days. However in practice there is considerable difference in the life span of human erythrocytes, this value may vary between 70 to 140 days.¹² Red blood cells membrane consist of several membrane bound enzymes which help in ionic transport across cell membrane.^13,14 Ionic transport is essential for the maintenance of viable RBCs in the blood. Several metabolic pathways are involved in the water and solute balance in RBCs and cellular volume is regulated by the monovalent cation concentration.¹⁵ The transport of ion across cell membrane is regulated by several enzymes, including Na⁺, K⁺-ATPase, Ca²⁺-ATPase and Na⁺, H⁺ exchanger.^16,17 Na⁺, K⁺-ATPase is a key protein that regulates the cell volume of red blood cells, which is fundamental for avoiding hemolysis, and has huge impact on the deformability of RBCs which is necessary to withstand blood pressure and to pass through narrow vessels.¹⁸ The red blood cell Ca²⁺-ATPase is a highly regulated transporter involved in maintaining Ca²⁺ homeostasis vital for cellular metabolic function.¹⁹ Impairment in ion transporter activity and deregulation of homeostasis has been linked with RBCs membrane fluidity and susceptibility of membrane towards oxidative damage.^20,21 Activities of ATPases are modulated by minor changes in the surrounding micro-environment.²² Emerging findings suggest the link between oxidative stress and the ion exchangers.^23,24 In recent years, Na⁺, K⁺-ATPase has demonstrated significance in oxidative stress related disease states, including obesity, atherosclerosis, heart failure, uremic cardiomyopathy, and hypertension.²⁵ Oxidative stress by ROS at membrane level also disturbs the inherit integrity of ion exchangers which subsequently alters their functions.^25–27 Many other metabolic elements such as calcium may alter ion exchangers independently of oxidative stress.^28,29 Recently, our group has also established the correlation of NHE activity with Na⁺, K⁺-ATPase and Ca²⁺-ATPase activity with respect to human age.¹⁷

Since, there are several questions remaining concern with the antioxidative effect of myricetin in red blood cells, we investigated the effect of myricetin using in vitro red blood cell membrane as a model followed by spectroscopic analysis for detection of oxidative stress biomarkers, Na⁺, K⁺-ATPase and Ca²⁺-ATPase, as shown in Scheme 1.

Scheme 1 Schematic representation of methodology adapted for analysis of Na⁺, K⁺-ATPase, Ca²⁺-ATPase in blood samples.

Variations in intracellular pH have long been suggested to be important in various metabolic processes. The inactivation of various nutrient transports has been shown previously to influence aging.³⁰ Tightly coupled exchange of Na⁺ for H⁺ occurs across the surface membrane of virtually all living cells. Na⁺, H⁺ exchanger is ubiquitously expressed in RBCs plasma membrane that plays a crucial role in intracellular pH and cell volume homeostasis by catalyzing an electro-neutral exchange of extracellular sodium and intracellular hydrogen.³¹ For years, the underlying molecular entity was unknown and the full physiological significance of the exchange process was not appreciated, but much knowledge has been gained in the last two decades. To investigate the effect of myricetin on Na⁺, H⁺ exchanger, we used in vitro packed red blood cells (PRBCs) as a model to study proton efflux, as shown in Scheme 2.

Scheme 2 Schematic representation of methodology adapted for analysis of Na⁺, H⁺ exchanger in blood sample. PRBCs, packed red blood cells.

Results and discussion

The results of our study showed a significant (p < 0.001) age dependent decline in Na⁺, K⁺-ATPase activity in mixed red blood cells population within the groups. In vitro administration of myricetin caused significant (p < 0.05) up-regulation of Na⁺, K⁺-ATPase activity at 10⁻⁵ M to 10⁻³ M as compared to their respective controls in all three age group RBCs. Myricetin at 10⁻⁶ M showed significant (p < 0.001) up-regulation in enzyme activity only in middle and old age groups as compared to their respective controls. The effect was insignificant at 10⁻⁷ M and 10⁻⁸ M myricetin in all three age groups as compared to their respective controls (Fig. 2).

Fig. 2 Effect of myricetin administration (10⁻⁸ M to 10⁻³ M final concentration) on red blood cells Na⁺, K⁺ ATPase activity in young (<35 years; n = 37), middle (36–60 years; n = 30) and old (>60 years; n = 25) age group. Na⁺, K⁺-ATPase activity was expressed in terms of micro mole of Pi released per h per mg membrane protein at 37 °C. Data are expressed as mean ± SD. $, p < 0.01; $$, p < 0.001 compared with young control and *, p < 0.01; **, p < 0.001 compared with the respective control group. M, molar.

Fig. 3 showed a significant (p < 0.001) age dependent decrease in Ca²⁺-ATPase activity in mixed red blood cell population among groups. Co-incubating red blood cells with different concentration (10⁻⁸ M to 10⁻³ M final concentration) of myricetin causes variation in Ca²⁺-ATPase activity in all three age groups. In vitro treatment of myricetin at 10⁻³ M and 10⁻⁴ M showed significant (p < 0.01) up-regulation of Ca²⁺-ATPase activity in all three age groups compared to their respective controls but significance (p < 0.001) was more pronounced in old age group. Myricetin at 10⁻⁵ M and 10⁻⁶ M showed up-regulated activity only in old age group as compared to its control. The effect was insignificant at 10⁻⁵ M and 10⁻⁸ M myricetin treated red blood cells in young and middle age groups compared to respective control.

Fig. 3 Effect of myricetin administration (10⁻⁸ M to 10⁻³ M final concentration) on red blood cells Ca²⁺-ATPase activity in young (<35 years; n = 37), middle (36–60 years; n = 30) and old (>60 years; n = 25) age groups. Ca²⁺-ATPase activity was expressed in terms of micro mole of Pi released per h per mg membrane protein at 37 °C. Data are expressed as mean ± SD. +, p < 0.01; ++, p < 0.001 compared with young control and *, p < 0.01; **, p < 0.001 compared with the respective control group. M, molar.

Na⁺, H⁺ exchanger activity significantly (p < 0.01) elevated in middle age group as compared to young age group. Significance was even higher (p < 0.001) in the old group as compared to the young control. In vitro treatment of myricetin provides significant fortification against oxidative stress in all age groups. Myricetin caused significant (p < 0.001) inhibition of Na⁺, H⁺ exchanger activity compared to respective control at 10⁻³ M and 10⁻⁵ M in all three age group red blood cells. The effect was also significant (p < 0.01) at 10⁻⁶ M in young and old age groups but insignificant at 10⁻⁷ M and 10⁻⁸ M in all three age groups compared to their respective controls (Fig. 4).

Fig. 4 Effect of myricetin administration (10⁻⁸ M to 10⁻³ M final concentration) on red blood cells Na⁺, H⁺ exchanger activity in young (<35 years; n = 37), middle (36–60 years; n = 30) and old (>60 years; n = 25) age group. Na⁺, H⁺ exchanger activity was expressed as proton efflux μmol per L RBC per h at 37 °C. Data are expressed as mean ± SD. #, p < 0.01; ##, p < 0.001 compared with young control and *, p < 0.01; **, p < 0.001 compared with the respective control group. M, molar.

A major development over past two decades has been of the realization that free radical mediated oxidative damage is associated with a variety of health problems, such as cancer, neurodegenerative diseases and aging.³² In this study, we propose that myricetin has remarkable antioxidant properties which are evident by up-regulation of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities, while inhibiting Na⁺, H⁺ exchanger activity. Age dependent decline in the activity of ATPases and increase in Na⁺, H⁺ exchanger during human aging may be a compensatory response of the individual to an increased oxidative stress.¹⁶ Our result shows that myricetin modulates ion transporters in red blood cells. Although the exact mechanisms are not fully understood, however we hypothesize that myricetin acts on ion transporters via indirect mechanisms. The antioxidant scavenging competence of myricetin is connected with the presence of hydroxyl groups (OH) in the B-ring and it may have metal binding sites for ion exchange between the 5-hydroxy and 4-carbonyl group, or between 3′- and 4′-hydroxyl group, thereby chelating metal ions.³³ One possible mechanism is the incorporation of myricetin in red blood cell membrane, which may change properties of ion transporters and its activities through non-covalent interactions. It may lead to the change in conformation of ion exchangers. Another possible mechanism is that myricetin may act on phospholipid bilayer, which are easy target of ROS because of unsaturated fatty acids. It has been shown that change in bilayer thickness, fluidity and head group content of membrane affect the activity of ion transporter.^34,35 Ion transporter activities depend on properties of cell membrane, and thus inhibition or stimulation by myricetin may cause some changes in physico-chemical properties of the membrane.³⁶ We suggest that the antioxidant action of myricetin alone is not adequate to facilitate cellular aging, but various interactions with distinct intracellular pathways are required. To the best of our knowledge, with this study, we are the first to present concentration dependent effect of myricetin in human red blood cells by detecting Na⁺, K⁺-ATPase, Ca²⁺-ATPase and Na⁺, H⁺ exchanger biomarkers during aging. Protective effect of myricetin has been reported against oxidative stress biomarkers which supports our study.^37,38 A microarray-based pathway showed that activation of the antioxidant response element is involved in myricetin-induced modulation of gene expression in human hepatoma cells.³⁹ Epidemiological studies suggest that dietary flavonoids ameliorate membrane structure and function against oxidative stress during human aging,^40–42 which are in accordance with our results.

Bioavailability of dietary constituents is a critical mediator of health benefits,⁹ dose dependent myricetin mediated protection against aging has been studied. We report here that myricetin can ameliorate oxidative stress in red blood cells during human aging. Our findings clearly show that myricetin has potent antioxidant properties that consequently influence cellular aging (Fig. 5).

Fig. 5 Role of myricetin in red blood cell metabolism. Eukaryotic cells display enzymes (Na⁺, K⁺-ATPase and Ca²⁺-ATPase) and Na⁺, H⁺ exchanger on red blood cell membrane. The maintenance of Na⁺ and Ca²⁺ is mediated through Na⁺, K⁺-ATPase and Ca²⁺-ATPase. The Na⁺, H⁺ exchanger reduces cell acidosis by extruding H⁺ from the cells by exchanging H⁺ for Na⁺ leading in turn, to Na⁺ overload, and subsequent Na²⁺ exchange for Ca²⁺. Aerobic cells produce ROS as a by-product of their metabolic processes. ROS cause oxidative damage to plasma membrane under conditions when the antioxidant defense of the body gets overwhelmed. During normal aging, Na⁺, K⁺-ATPase and Ca²⁺-ATPase declines while Na⁺, H⁺ exchanger goes up as a function of human age. In vitro co-incubation with myricetin causes significant increase in ATPase activity and inhibition of Na⁺, H⁺ exchanger.

ROS are one of the products of oxidative reactions which react with polyunsaturated fatty acids (PUFA) of red blood cell membranes, result in lipid peroxidation and alter membrane fluidity.⁴³ ROS can influence the activities of ATPases and Na⁺, H⁺ exchanger. We have reported in our previous studies that Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities decrease as a function of human age,¹⁶ while Na⁺, H⁺ exchanger activity elevates during human aging.¹⁷ Na⁺, K⁺-ATPase is a dimeric integral membrane enzyme that belongs to P-type ATPases. This ATPase catalyzes the transport of 3Na⁺ ion outside the cell and 2K⁺ ion in the cell, there by generating an asymmetric gradient across the plasma membrane.⁴⁴ A study by Lei et al. demonstrated that myricetin enhances Na⁺, K⁺-ATPase activity to protect against D-galactose induced cognitive impairment.⁴⁵ Ca²⁺-ATPase also belongs to P-type ATPases which participate as an integral part of the Ca²⁺ signaling mechanism for eukaryotic cells and crucial component of cell function.⁴⁶ Na⁺, H⁺ exchanger generates a permissive pH level that is critical for the development of mitogenic responses. Also, it has been reported that deletion of Na⁺, H⁺ exchanger in mice causes neurological defects, and the growth and viability of those mice was greatly reduced; therefore Na⁺, H⁺ exchangers play an important role in cell growth, differentiation and aging.⁴⁷ Health benefits of myricetin in various diseases have been reported^4,48 but little is known about its role in human aging particularly for red blood cells membrane transporters. Authors believe that this study is very novel and it will help in further research on human aging.

Experimental

Materials and methods

Reagents. Myricetin, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonicacid) and ATP were purchased from Sigma (St. Louis, MO, USA). Bovine serum albumin (BSA), ouabain, imidazole were purchased from Himedia Laboratories (India). All the other chemicals used were of analytical grade.

Selection of subjects. The study was carried out on 92 normal healthy subjects of both the sexes which were divided into three age groups, young (18–35 years; 37 subjects), middle (36–60 years; 30 subjects) and old (>60 years; 25 subjects). The criteria of selection were based on our previously published data.⁴⁹ Briefly, the subjects were screened for diabetes mellitus, asthma, tuberculosis or any other major illness. None of the subjects were smokers or under any medication. They were neither taking any dietary supplement. All the subjects gave their informed consent for the use of their blood samples for this study. The protocol of study was in conformity with the guidelines of the Ethical Committee of Amity University Uttar Pradesh, Noida, India. The University guidelines were concerned with the following observations (i) pro forma should be developed and filled by volunteers, which should mention the health status of the volunteers (ii) biosafety for use of myricetin (iii) committee suggested inclusion of three age groups (iv) medical practitioner should be involved during the collection of blood from volunteers. The committee has unanimously cleared the experiments both from scientific and ethical view points.

Isolation of packed red blood cells and preparation of erythrocyte ghost. Human venous blood samples (10 mL) from different healthy volunteers were obtained by venipuncture in sterile polystyrene tubes containing heparin. The blood was centrifuged at 1800 × g for 10 min at 4 °C. The packed red blood cells (PRBCs) were obtained after removal of plasma, buffy coat. The red blood cells were washed twice with cold phosphate buffer saline (PBS: 0.9% NaCl, 10 mM Na₂HPO₄, pH 7.4). Erythrocyte ghosts from leukocyte free red blood cells were prepared by osmotic shock procedure as described in an earlier report⁵⁰ and protein content was determined using the method of Lowry et al.⁵¹

Determination of Na⁺, K⁺-ATPase activity. The Na⁺/K⁺-ATPase activity was measured as described earlier.¹⁶ The final assay mixture contained 0.4 to 0.9 mg membrane protein per mL, 140 mM NaCl, 20 mM KCl, 3 mM MgCl₂, 30 mM imidazole (pH 7.25), ±5 × 10⁻⁴ M ouabain and 6 mM ATP. Incubation was carried out for 30 min at 37 °C; and the reaction was stopped by adding 3.5 mL of a solution containing 0.5 M H₂SO₄, 0.5% ammonium molybdate and 2% SDS. The amount of liberated inorganic phosphate was estimated following an earlier reported method.⁵² The Na⁺, K⁺-ATPase activity is expressed in terms of micro mole of Pi released per h per mg membrane protein at 37 °C.

Determination of Ca²⁺-ATPase activity. The Ca²⁺-ATPase activity was assayed as described earlier,¹⁶ where 2.25 mL of the assay mixture contained 80 mM NaCl, 15 mM KCl, 3 mM MgCl₂, 18 mM Tris–HCl (pH 7.4), 0.1 mM ouabain, 0.1 mM EGTA, 0.2 mL of the membrane containing 0.4 to 1.5 mg protein per mL and ±0.2 mM CaCl₂. The reaction was initiated by the addition of 0.1 mL of 30 mM ATP. After 30 min at 37 °C, the reaction was stopped by adding 3.5 mL of a solution containing 0.5 M H₂SO₄, 0.5% ammonium molybdate and 2% SDS. The amount of liberated inorganic phosphate was estimated following an earlier reported method.⁵² Ca²⁺-ATPase activity is expressed in terms of micro mole of Pi released per h per mg membrane protein at 37 °C.

Determination of Na⁺, H⁺ exchanger activity. Na⁺, H⁺ exchanger activity in the isolated erythrocytes was estimated in terms of amiloride-sensitive H⁺-efflux from acid loaded cells as reported previously.¹⁷ The activity of the anti-port is extrapolated by the difference in hydrogen efflux rates from acid loaded erythrocytes in the absence and presence of the inhibitor amiloride. Briefly, 0.2 mL of packed erythrocytes were suspended into a 3.8 mL solution containing 150 mmol L⁻¹ NaCl, 1 mmol L⁻¹ KCl, 1 mmol L⁻¹ MgCl₂, 10 mmol L⁻¹ glucose and were incubated at 37 °C for 5 min under magnetic stirring. The cell suspension was brought to pH 6.35–6.45 within 10 min using 0.2 mol L⁻¹ HCl solution in 150 mmol L⁻¹ NaCl. DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonicacid) was added (0.2 mmol L⁻¹ final concentration) and the pH of the medium was brought to 7.95–8.00 using 0.05 mol L⁻¹ NaOH solution in 150 mmol L⁻¹ NaCl. In a parallel experiment, amiloride (0.5 mmol L⁻¹ final concentration) was added with DIDS. Thereafter, the first minute proton efflux was registered. The rate of NHE, expressed in μmol L⁻¹ of cells per h, derives from the difference in rates of medium acidification in the absence (ΔpH₁) and presence (ΔpH₂) of amiloride, corrected by the buffer capacity of the incubation medium (b), the cell volume in the suspension and the incubation time. Erythrocyte hemolysis was assessed by measurement of the hemoglobin released from cells, relative to the total cellular content. Following incubation, samples were centrifuged at 2000 × g and the hemoglobin concentration of supernatants was determined spectrophotometrically.

In vitro treatment with myricetin. Effect of myricetin on red blood cells membrane Na⁺, K⁺-ATPase, Ca²⁺-ATPase was studied by co-incubating red blood cell membrane (0.8–1.5 mg of protein) with myricetin (10⁻⁸ M to 10⁻³ M) in Krebs ringer phosphate buffer (KRP) containing 5 mmol L⁻¹ glucose (KRP-G), pH 7.4 for 1 hour at 37 °C, prior to assay. After incubation, RBCs membrane were washed twice with PBS at room temperature and subjected to assay. In parallel control experiments, red blood cell membrane was incubated without myricetin. RBCs membrane protein concentration was assayed according to Lowry.⁵¹ The in vitro effect of myricetin on Na⁺, H⁺ exchanger was investigated as follows: blood was washed 2–3 times with Krebs ringer phosphate buffer (KRP) containing 5 mmol L⁻¹ glucose (KRP-G), pH 7.4. Erythrocytes were then suspended in 4 volume of KRP-G containing 5 mmol L⁻¹ glucose. The effect of myricetin was evaluated by co-incubating RBCs in the presence of myricetin (10⁻⁸ M to 10⁻³ M) (final concentration) for 60 min at 37 °C with mild shaking. We have selected flavonoid concentrations in the present study following a previous report in human RBCs.⁵³ Erythrocytes were again washed two to three times with KRP, pH 7.4 and finally; packed red blood cells (PRBCs) were used for the assay. In parallel, for control experiments, blood was incubated without myricetin.

Statistical analyses. All statistical analyses were performed using the Statistical Package for Social Science (SPSS, version 20.0 for Windows). The effect of myricetin was analysed using multivariate ANOVA (MANOVA). Post hoc testing for within group comparison (control and +flavonoid) for young, middle, and old. A probability (p) value of less than 0.05 was considered statistically significant.

Conclusions

Myricetin modulates both ATPases and Na⁺, H⁺ exchanger activity during aging in humans. Damage of the plasma membrane occurs directly through interaction with membrane components such as Na⁺, H⁺ exchanger, ion dependent ATPase, ion channels and indirectly as a consequence of excess cytosolic damage. Inhibiting function of ion dependent ATPase leads to disturbance in ion homeostasis resulting in impaired signal transduction, altered cellular metabolism, changes in cell membrane permeability and integrity, an elevation in membrane fluidity and disturbances in vital function which finally leads to aging and other age related diseases. Despite this it can be suggested that myricetin may be used as an alternative for available therapeutic strategies for aging and age related disorders. Further research work should be directed towards finding mechanism underlying this effect. These findings emphasize the need to establish age-dependent reference values of dietary flavonoids for oxidative stress biomarkers in different populations and in studies involving their role in different disease conditions.

Acknowledgements

PKM and PK acknowledge Amity University Uttar Pradesh, Noida, India for providing necessary research facility and PKM also acknowledge support by fellowship (Science without Borders-Level A) from coordination of Improvement of Higher Education Personnel (CAPES), National Counsel of Technological and Scientific Development (CNPq), Brazil.

Notes and references

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