Posts Tagged ‘Red blood cell’

Alteration in Reduced Glutathione level in Red Blood Cells: Role of Melatonin

Author: Shilpa Chakrabarti, PhD

List of abbreviation:
DTNB- 5,5- dithiobis,2-nitrobenzoic acid
t-BHP- Tertiary butyl hydroperoxide
GSH-Reduced glutathione
GSSG- Oxidised glutathione

Objective: The study was taken up to see the effect of melatonin on the alteration of reduced glutathione level in red blood cells.

Pineal melatonin is involved in many physiological functions, the most important among them being sleep promotion and circadian regulation. This pineal product exhibits characteristic diurnal rhythm of synthesis and secretion, which attains its peak at night followed by a gradual decrease during the daytime. Melatonin detoxifies highly toxic hydroxyl and peroxyl radicals in vitro, scavenges hydrochlorous acid, as well as peroxynitrite. It has also been reported to increase the synthesis of glutathione and of several antioxidant enzymes [1].

Method: The present study was undertaken to understand the modulation of intracellular reduced glutathione (GSH) by melatonin in human red blood cells according to the oscillatory circadian changes in levels of this hormone.We have also studied the dose-dependent effect of melatonin on GSH in erythrocytes obtained from blood at two different times, subjected to oxidative stress by incubating with tert-butyl hydroperoxide (t-BHP) [2]. We used t-BHP as pro-oxidant [3]. Erythrocyte GSH was measured following the method of Beutler [4]. The method was based on the ability of the –SH group to reduce 5,5- dithiobis,2-nitrobenzoic acid (DTNB) and form a yellow coloured anionic product whose OD is measured at 412 nm.

A suspension of packed red blood cells in phosphate-buffered saline (PBS) containing glucose was treated with melatonin taken at different concentrations. A stock solution (10mM) of melatonin was prepared in absolute ethanol; further dilutions (100 uM–10 nM) were done with PBS. The concentration of ethanol was alwaysThe in vitro effect of melatonin was evaluated by incubating erythrocytes with melatonin at different doses (10 uM –1 nM final concentration) of melatonin for 30 minutes at 37°C. After washing the erythrocytes with the buffer, to remove any amount of the compound, and finally, packed erythrocytes were used for the assay of GSH. In parallel control experiments, blood was incubated with ethanol (final concentration not more than 0.01% (v/v)) but without melatonin.Oxidative stress was induced in vitro by using tert-butyl hydroperoxide both in presence and absence of melatonin. Use of TBHP is in accordance with the published reports [5].

Results and Discussion: The experiment demonstrated that erythrocyte GSH level increased in nocturnal samples which highlights the role of endogenous melatonin in the circadian changes in cellular glutathione level. Exogenous melatonin demonstrated a protective effect against t-BHP-induced peroxidative damage in both diurnal and nocturnal samples, the effect being more pronounced in aliquots containing very low concentration of melatonin (10 nM – 1 nM) [6]. Melatonin was found to inhibit GSH oxidation in a dose-dependent manner.

Melatonin has been found to upregulate cellular glutathione level to check lipid peroxidation in brain cells [7]. We may say that the incubation of the red cells with melatonin for an extended period (more than 30 minutes) may not have the same effects on the level of glutathione in these cells [12]. Melatonin may act as pro-oxidant in the cells exposed to the indoleamine for longer time. Also, the half-life period of pineal melatonin is for 30 to 60 minutes, as reviewed by Karasek and Winczyk [11].The recycling of glutathione in the cells depends on an NADPH-dependent glutathione enzyme system which includes glutathione peroxidise, glutathione reductase, and γ-glutamyl-cysteine synthase forming a meshwork of an antioxidative system. The stimulatory effect of melatonin on the regulation of the antioxidant enzymes has been reported [8].Since melatonin has an amphiphilic nature, its antioxidative efficiency crosses the cellular membrane barriers in a non-receptor-mediated mechanism. Another explanation of melatonin’s antioxidative activity may be based on its role in the upregulation of some antioxidant enzymes directly. Blanco et al had reported that glutathione reductase and glutathione peroxidase, the major constituents of the glutathione-redox system being stimulated by melatonin [9]. The plasma GSH/GSSG redox state is controlled by multiple processes, which includes synthesis of GSH from its constitutive amino acids, cyclic oxidation and reduction involving GSH peroxidase and GSSG reductase, transport of GSH into the plasma, and the degradation of GSH and GSSG by γ-glutamyltranspeptidase. The increase in erythrocyte GSH concentration after melatonin administration can be related Blanco et al’s report on the known stimulation of γ-glutamylcysteine synthase,a rate-limiting enzyme in reduced glutathione synthesis, by melatonin [10].

Conclusion: On the basis of our study, we may conclude that melatonin affects the glutathione level in red blood cells in a circadian manner. The rhythmic pattern of glutathione level confirms the relationship between physiological melatonin and erythrocyte GSH level and pharmacological dosage of the drug. The role of melatonin as an antioxidant and its activity in relation to these biomarkers has been studied in the above experiments.

Key words: Glutathione, circadian rhythm,, melatonin, biomarkers, oxidative stress


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2. A. V.Domanski, E. A. Lapshina, and I. B. Zavodnik, “Oxidative processes induced by tert-butyl hydroperoxide in human red blood cells: chemiluminescence studies,” Biochemistry (Moscow), vol. 70, no. 7, pp. 761–769, 2005. http://link.springer.com/article/10.1007%2Fs10541-005-0181-5
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5. P. Di Simplicio, M. G. Cacace, L. Lusini, F. Giannerini, D. Giustarini, and R. Rossi, “Role of protein -SH groups in redox homeostasis—the erythrocyte as a model system,” Archives of Biochemistry and Biophysics, vol. 355, no. 2, pp. 145–152, 1998.
6. S. Chakravarty and S. I. Rizvi., “Day and Night GSH andMDA Levels in Healthy Adults and Effects of Different Doses ofMelatonin on These Parameters” International Journal of Cell Biology, vol. 2011, pp. Article ID 404591.http://www.hindawi.com/journals/ijcb/2011/404591/9CDay+and+Night+GSH+andMDA+Levels+in+Healthy+Adults+and+Effects+of+Different+Doses+ofMelatonin+on+These+Parameters”>
7. S. R. Pandi-Perumal, V. Srinivasan, G. J. M. Maestroni, D. P. Cardinali, B. Poeggeler, and R. Hardeland, “Melatonin: nature’s most versatile biological signal?” FEBS Journal, vol. 273, no. 13, pp. 2813–2838, 2006.http://onlinelibrary.wiley.com/doi/10.1111/j.1742-4658.2006.05322.x/full
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10. R. A. Blanco, T. R. Ziegler, B. A. Carlson et al., “Diurnal variation in glutathione and cysteine redox states in human plasma,” American Journal of Clinical Nutrition, vol. 86, no. 4, pp. 1016–1023, 2007. http://www.ncbi.nlm.nih.gov/pubmed/17921379
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12. A. Krokosz ,J. Grebowski, Z. Szweda-Lewandowska et al., ” Can melatonin delay oxidative damage of human
erythrocytes during prolonged incubation?” Advances in Medical Sciences, vol. 58, no. 1, 2013.http://www.researchgate.net/publication/236614971_Can_melatonin_delay_oxidative_damage_of_human_erythrocytes_during_prolonged_incubation


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Melatonin and its effect on acetylcholinesterase activity in erythrocytes

Author: S. Chakravarty, PhD


Objective: The study was conducted to see the effect of melatonin on the activity of acetylcholinesterase in red blood cells.

Mammalian red blood cells contain membrane-bound acetylcholinesterase which acts as biomarkers of oxidative imbalance. Melatonin is a powerful free radical scavenger and upregulates several antioxidant enzymes to reduce oxidative stress. Being an effective antioxidant, it may initiate variation in erythrocyte acetylcholinesterase activity.

The study was carried out on twenty-nine subjects of both sexes who gave their informed consent for the use of their blood samples for the study (Chakravarty and Rizvi, 2011a). The red cells isolated from blood collected at two different timings of the day, viz., 10:00 a.m. and 10:00 p.m.,were subjected to in vitro treatment with melatonin in a dose-dependant manner followed by the assay of enzyme activity (Ellman et al., 1961).

Acetylcholinesterase (AChE) is also found on the red blood cell membranes, where it constitutes the Yt blood group of antigen, which is a blood-group determining protein. AChE has the features of a secreted rather than a transmembrane protein because it lacks long hydrophobic stretches, other than that which forms the signal peptide (Li et al., 1991). Besides, acetylcholinesterase activity in erythrocytes may be considered as a marker of central cholinergic status (Kaizer et al., 2008). AChE shows highest activity in the immature rat brain is at 6.00 a.m. and lowest after midnight, which undergoes a reversal after attaining maturity (Moudgil and Kanungo, 1973). The enzyme also exhibits annual changes in its activity (Lewandowski, 2008). Acetylcholinesterase activity has been used to for studying the activity pattern of human erythrocytes (Prall et al., 1998). Free radicals and increased oxidative stress have been found to reduce AChE activity (Molochkina et al., 2005). This indicates that melatonin may have some relation with the circadian rhythmicity of acetylcholinesterase activity.

The concentration-dependant assay of AChE activity in red cells bear close relation with the circadian rhythm in humans thus sharing a similar conclusion with that mentioned by Moudgil and Kanungo (Moudgil and Kanungo, 1973). The effect of melatonin on enzyme functions in erythrocytes follows rhythmic modulation with day/night cycle. The samples obtained in the morning exhibit significantly higher activity of acetylcholinesterase than those obtained during the night-time. The samples collected at two different timings of the day show different response to in vitro melatonin treatment. The rise in AChE activity is more pronounced at low doses of melatonin. Our results indicate significant increase in acetylcholinesterase activity in diurnal as well as nocturnal blood samples at different concentrations of exogenous melatonin (Rizvi and Chakravarty, 2011). At supraphysiological doses, the enzyme activity exhibits no significant change, owing to the prooxidative influence exerted by melatonin (Marchiafava and Longoni, 1999).

Acetylcholinesterase activity is affected by the hydrophobic environment of the cell membrane and depends on the plasma membrane fluidity and surface charge of the cell (Klajnert et al., 2004).  The activity of AChE depends largely on the biophysical features of membrane. Oxidative stress decreases the fluidity of membrane lipid bilayer, thus affecting its normal functions (Goi et al., 2005).  Such are the ill-effects of oxidative radicals that tend to increase with aging. The decrease in AChE correlates significantly with age-induced oxidative stress (Jha and Rizvi, 2009).  On the basis of our study we conclude that melatonin modulates acetylcholinesterase activity in erythrocytes. The rhythmicity observed in the activity of acetylcholinesterase in response to the melatonin confirms our opinion on the relationship between the enzyme function, pineal secretion and pharmacological dosage of the indole antioxidant.


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Day and Night Variation in Melatonin Level affects Plasma Membrane Redox System in Red Blood Cells

Author: Shilpa Chakravarty, PhD

Melatonin is a well-established antioxidant and sleep-regulating hormone. Over the past fifty years, its efficiency as a regulator of circadian rhythm and several other physiological functions has been studied extensively in different species. As a free-radical scavenger, melatonin has shown its activity in coordination with its circadian nature. One of the most important biomarkers of oxidative stress studied in red blood cells is the plasma membrane redox system (PMRS).

As a part of the research activity, PMRS activity has been summarised in this article. The experiments with PMRS and ascorbate free-radical reductase (AFR reductase) have been conducted in vitro.

The study was carried out on 61 healthy individuals of both sexes (aged 20-30) having no acute or chronic diseases (such as diabetes mellitus, asthma, or tuberculosis) or any organ dysfunction and had not taken any medication. Blood samples were collected at two different timings at 10:00AM and 10:00PM.  Red blood cell-membrane, was in retrospect a good experimental system to try to extract and isolate membrane proteins for biochemical assays. Two factors that have favoured it for experimental use are availability and simplicity. Results from its study have been replicated in every other mammalian cell type, and in some crucial points, the patterns shown by RBC
proteins have led the way to such interpretations of extensive physiological studies.

PMRS transfers electrons from extracellular substates to intracellular electron acceptors incorporating AFR reductase. An increase in PMRS activity indicates the ability of the cell to combat oxidative damage.The aging of human red cells may well be attributed to free radical induced oxidative damage. Maintenance of redox state of sulphydryl residues and reduction of lipid hydroperoxides at the expense of electron donors, such as ascorbate and NADH, is essential for normal energy metabolism in the cell. The neutralisation of oxidants also involves some membrane proteins that comprise the PMRS. The rise in PMRS activity is required to maintain a balanced NAD+/NADH ratio that is essential for normal energy metabolism. It leads to cell survival and membrane homeostasis under stress conditions and during calorie restriction in eukaryotes. The day and night variation in PMRS activity shows that the antioxidative behaviour of melatonin is also influenced by its circadian mode of action. While melatonin is an effective antioxidant against cellular toxicity, it also increases the PMRS activity in red blood cells at night. During the day, when the pineal secretion is low, the PMRS activity is also suppressed.

However, if subjected to in vitro treatment with melatonin, at such a concentration that lies close to the maximal melatonin level in the plasma (maximal secretion of melatonin occurs during the scotopic phase of the day), PMRS increases in red blood cells. This shows that the circadian nature of the hormone not only pertains to its pineal production but also to exogenous administration of the drug.


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