Prabhanshu Kumar

l-Cysteine Efflux in Erythrocytes As A Function of Human Age: Correlation with Reduced Glutathione and Total Anti-Oxidant Potential

Thiol compounds such as cysteine (Cys) and reduced glutathione (GSH) play an important role in human aging and age-related diseases. In erythrocytes, GSH is synthesized by glutamic acid, cysteine, and glycine, but the rate of GSH synthesis is determined only by the availability of L-cysteine. Cysteine supplementation has been shown to ameliorate several parameters that are known to degenerate during human aging. We have studied L-cysteine efflux in vitro in human erythrocytes as a function of age by suspending cells in solution containing 10 mM L-cysteine for uptake; later cells were re-suspended in phosphate-buffered saline (PBS)-glucose to allow efflux. Change in the free sulfhydryl (-SH) concentration was then measured to calculate the rate of efflux. The GSH/oxidized glutathione (GSSG) ratio was taken as a control to study the oxidation/reduction state of the erythrocyte. The total anti-oxidant potential of plasma was measured in terms of ferric reducing ability of plasma (FRAP) values. We have shown a significant (p<0.0001) decline in the efflux of L-cysteine in erythrocytes during human aging, and the GSH/GSSG ratio decreases as a function of human age. The decline in L-cysteine efflux during aging correlates with the decrease in GSH and the FRAP value. This finding may help to explain the shift in the redox status and low GSH concentration that might determine the rate of L-cysteine efflux observed in erythrocytes and an important factor in the development of oxidative stress in erythrocytes during aging.

Quercetin-modulated erythrocyte membrane sodium-hydrogen exchanger during human aging: correlation with ATPase's.

Abstract Content: Quercetin uptake by erythrocytes is rapid. The sodium-hydrogen exchanger (NHE) is a secondary active transporter, regulating intracellular pH, Na+ concentration and cell volume. Objective: The aim of present study was to investigate NHE as a function of human age and effect of quercetin on its activity. The NHE activity was correlated with erythrocytes ATPases. Materials and methods: We analyzed normal, healthy subjects of both sexes (20–82 years). NHE activity was estimated in terms of amiloride-sensitive H+-efflux from acid-loaded cells. Results: A significant age-dependent increase in NHE activity was observed during aging in humans. Concentration (10−3 M to 10−8 M)-dependent in vitro treatment with quercetin causes inhibition of NHE activity. The Na+/K+ -ATPase (r = 0.8882) and Ca2+-ATPase (r = 0.9540) activities positively correlated with it. Discussion and conclusion: The present data show an additional mechanism where dietary flavonoids may exerts beneficial effect during aging.

Epigallocatechin-3-Gallate Protects Erythrocyte Ca 2+ -ATPase and Na + /K + -ATPase Against Oxidative Induced Damage During Aging in Humans

PURPOSE The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. METHODS Human erythrocyte membrane bound Ca(2+)-ATPase and Na(+)/K(+)-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10(-7)M to 10(-4)M) to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10(-5) M final concentration). RESULTS We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca(2+)-ATPase and Na(+)/K(+)-ATPase during aging in humans. We have detected a significant (p < 0.001) decreased activity of Ca(2+)-ATPase and Na(+)/K(+) -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. CONCLUSION Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca(2+)-ATPase and Na(+)/K(+) -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

Age-dependent detection of erythrocytes glucose-6-phosphate dehydrogenase and its correlation with oxidative stress.

Abstract Context: Glucose-6-phosphate dehydrogenase (G6PD) is an important enzyme of hexose monophosphate shunt, involved in the biosynthesis of reduced nicotinamide adenine dinucleotide phosphate hydrogen (NADPH). Objective: This study was designed to investigate age-dependent changes in human erythrocyte G6PD activity. The G6PD activity pattern was correlated with reduced glutathione (GSH) and total antioxidant potential in terms of FRAP (ferric reducing ability of plasma) value. Materials and methods: We analyzed normal, healthy subjects of both sexes between the ages of 20 and 80 years. G6PD activity was determined by Burties method. Results: We observe a significant age-dependent decrease in G6PD activity (p < 0.0001). It was positively correlated with GSH (r = 0.5706) and total antioxidant potential (r = 0.7723) as a function of human age. Discussion and conclusion: Our findings on erythrocyte G6PD and their correlation with GSH and FRAP provide evidence of a higher oxidative stress in old age population.

Prospects and advancements in C-reactive protein detection.

C-reactive protein (CRP) is one of the earliest proteins that appear in the blood circulation in most systemic inflammatory conditions and this is the reason for its significance, even after identification of many organ specific inflammatory markers which appear relatively late during the course of disease. Earlier methods of CRP detection were based on the classical methods of antigen-antibody interaction through precipitation and agglutination reactions. Later on, CRP based enzymatic assays came into the picture which were further modified by integration of an antigen-antibody detection system with surface plasma spectroscopy. Then came the time for the development of electrochemical biosensors where nanomaterials were used to make a highly sensitive and portable detection system based on silicon nanowire, metal-oxide-semiconductor field-effect transistor/bipolar junction transistor, ZnS nanoparticle, aptamer, field emission transmitter, vertical flow immunoassay etc. This editorial attempts to summarize developments in the field of CRP detection, with a special emphasis on biosensor technology. This would help in translating the latest development in CRP detection in the clinical diagnosis of inflammatory conditions at an early onset of the diseases.

Influence of Dietary Capsaicin on Redox Status in Red Blood Cells During Human Aging.

PURPOSE Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is a major pungent compound found in hot peppers of the plant genus Capsicum. In vitro effects of dietary capsaicin on redox status in red blood cells during human aging have been explored. METHODS Total antioxidant potential of capsaicin was evaluated using Ferric reducing ability of plasma (FRAP) assay. GSH was measured as per standard protocol. The in vitro effect of capsaicin was evaluated by incubation of the cells in the assay medium with 10(-5)M capsaicin (final concentration) for 60 min at 37°C. RESULTS Treatment with capsaicin (10(-5)M) caused a significant (p < 0.01) increase in GSH level in all age groups. Reduced glutathione (GSH) / Oxidized glutathione (GSSG) ratio measures the redox status of the red blood cell. Significant increase in GSH level due to capsaicin, shift the GSH/GSSG ratio, thus alters the redox status of the cell. CONCLUSION The results conclusively prove the efficacy of the antioxidant property of capsaicin and its role in modulating the redox status of red blood cells. This evidence suggests that dietary factors that act as antioxidants to increase GSH level may contribute to a protective effect against age related diseases. This antioxidant effect may, in part, explain the high consumption of capsicum in certain regions of the world.

Multi-target detection of oxidative stress biomarkers in quercetin and myricetin treated human red blood cells

Quercetin and myricetin are important dietary flavonoids with potential health benefits and interfere with reactive oxygen species metabolism. The objective of this study was multi-target spectroscopic analysis of oxidative stress biomarkers (malondialdehyde (MDA), glutathione (GSH) and sulfhydryl (–SH) groups) in quercetin and myricetin treated red blood cells (RBCs) during human aging. The study was carried out on clinically relevant blood samples obtained from 105 healthy subjects between the ages of 18–82 years. The subjects were divided into three age groups, young (18–35 years), middle (36–60 years) and old (>60 years). Oxidative stress was induced in vitro by incubating RBCs with 10−5 M tert-butyl hydroperoxide (t-BHP). The effects of flavonoids were evaluated by detecting MDA, GSH and –SH groups by co-incubating the RBCs in the presence of flavonoid (10−8 M to 10−5 M final concentration) and t-BHP. The GSH/GSSG ratios were estimated to demonstrate the antioxidant power of the RBCs. The results showed elevated MDA levels (p < 0.001) after incubation with t-BHP as compared to a control. The GSH and –SH groups significantly (p < 0.001) decreased when incubated with t-BHP. In vitro administration of both flavonoids significantly attenuated the deleterious effect of oxidative stress in erythrocytes from all age groups. We showed a significant (p < 0.0001) negative correlation (r = −0.8334) between GSH/GSSG during human aging. We believe that these findings are novel and will help in the fast screening of new chemical molecules which may help against oxidative stress in RBCs, and thereby have tremendous scope in medical diagnostics and therapeutics.

Prospects of electrochemical immunosensors for early diagnosis of preeclampsia

Preeclampsia is a vascular multisystem disorder that accounts for varying degree of morbidity and mortality of mother and the fetus. This can be significantly averted if diagnosed at an early (18‐20 weeks) stage of gestation, as there is no known way to prevent preeclampsia. In spite of extensive work on biomarker discovery, the existing method for its detection is mostly based on colorimetric immunoassays whose sensitivity is ranging in nanomolar range. Further, it has also been observed that change in the expression of a single biomarker is not sufficient to diagnose this condition. So, for early diagnosis (by 18‐20 weeks), an immuno‐diagnostic platform with detection limits in picomolar range and beyond along with the ability to do simultaneous detection of multiple analyte would be of great importance. A nano‐immunosensors with an electrochemical readout system can be a potential alternative that promises for the ultrasensitive detection of analyte with high specificity as well as suitability for on‐site analysis. Coupling the lateral flow technology with immunosensors would make it feasible to detect more than one biomarker simultaneously on a microchip. This review intends to summarize the potential preeclampsia biomarkers, limitations of existing diagnostic methods along with the recent advancements, and prospects to develop electrochemical immunosensors for early clinical diagnosis.

Age-related changes in erythrocyte membrane sulfhydryl groups and β-D-glucuronidase activity

Background: Aging is associated with oxidative stress but its underlying mechanisms remain poorly understood. The aim of this study was to investigate alterations in human erythrocyte membrane sulfhydryl groups and levels of &bgr;‐D‐glucuronidase during aging. Methods: The study was carried with a cohort of 52 normal, healthy subjects of both sexes. They were divided into three groups: young, middle‐aged and old. Blood samples were taken, and membrane sulfhydryl groups and &bgr;‐D‐glucuronidase activity were measured within 24 hours. Results: Results showed significantly (p<0.001) decreased sulfhydryl group levels as a function of human age. A significant (p< 0.001) decrease in erythrocyte membrane &bgr;‐D‐glucuronidase activity was also observed in subjects in the ‘old’ group. Conclusions: We conclude that aging is associated with systemic oxidative stress that is able to influence the integrity of cell membranes and their antioxidant capacity. Evaluation of sulfhydryl groups and &bgr;‐D‐glucuronidase activity provides a useful and early indication of structural and functional alterations of the red blood cell membrane during human aging.

Detection of oxidative stress biomarkers in myricetin treated red blood cells

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.