A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens.

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca++ extrusion pumps. Although much of our knowledge about these Ca++ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca++ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca++ efflux using extracellular fluorescent Ca++ indicators. We optimized Ca++ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca++ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca++ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca++ pump and should facilitate screens for specific inhibitors of this essential transport activity.

Protective effect of rutin against thiram-induced cytotoxicity and oxidative damage in human erythrocytes.

Thiram is a fungicide that is used to prevent fungal diseases in seeds and crops and also as an animal repellent. The pro-oxidant activity of thiram is well established. Rutin is a flavonoid glycoside present in many fruits and plants and has several beneficial properties, including antioxidant effects. We have previously shown that thiram causes oxidative damage in human erythrocytes. The present study was designed to evaluate the protective effect of rutin against thiram-induced damage in human erythrocytes. Treatment of erythrocytes with 0.5 mM thiram for 4 h increased the level of oxidative stress markers, decreased antioxidant power and lowered the activity of antioxidant and membrane bound enzymes. It also enhanced the generation of reactive oxygen and nitrogen species (ROS and RNS) and altered the morphology of erythrocytes. However, prior treatment of erythrocytes with rutin (0.5, 1 and 2 mM) for 2 h, followed by 4 h incubation with 0.5 mM thiram, led to a decrease in the level of oxidative stress markers in a rutin concentration-dependent manner. A significant restoration in the antioxidant power and activity of antioxidant enzymes was observed upon the treatment of erythrocytes with 1 and 2 mM rutin. Pre-incubation with rutin lowered the generation of ROS and RNS which will reduce oxidative damage in erythrocytes. The thiram-induced changes in cell morphology and activity of membrane-bound enzymes were also attenuated by rutin. These results suggest that rutin can be used to mitigate thiram-induced oxidative damage in human erythrocytes.

Synthesis, Structure and Biological Activity of Indole-Imidazole Complexes with ZnCl2: Can Coordination Enhance the Functionality of Bioactive Ligands?

The ability of the indole-imidazole hybrid ligands to coordinate with the Zn(II) ion and the resulting structures of this new class of coordination compounds were analyzed in order to determine their structural properties and biological functionalities. For this purpose, six novel Zn(II) complexes, [Zn(InIm)2Cl2] (1), [Zn(InMeIm)2Cl2] (2), [Zn(IniPrIm)2Cl2] (3), [Zn(InEtMeIm)2Cl2] (4), [Zn(InPhIm)2Cl2] (5) and [Zn2(InBzIm)2Cl2] (6) (where InIm is 3-((1H-imidazol-1-yl)methyl)-1H-indole), were synthesized by the reactions of ZnCl2 and the corresponding ligand in a 1:2 molar ratio in methanol solvent at an ambient temperature. The structural and spectral characterization of these complexes was performed using NMR, FT-IR and ESI-MS spectrometry and elemental analysis, and the crystal structures of 1-5 were determined using single-crystal X-ray diffraction. Complexes 1-5 form polar supramolecular aggregates by utilizing, for this purpose, the N-H(indole)∙∙∙Cl(chloride) intermolecular hydrogen bonds. The assemblies thus formed differ depending on the distinctive molecular shape, which can be either compact or extended. All complexes were screened for their hemolytic, cytoprotective, antifungal, and antibacterial activities. The results show that the cytoprotective activity of the indole/imidazole ligand significantly increases upon its complexation with ZnCl2 up to a value comparable with the standard antioxidant Trolox, while the response of its substituted analogues is diverse and less pronounced.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Wild garlic extract reduces lipid peroxidation in terbuthylazine-treated human erythrocytes.

Background: Among other negative effects, herbicides induce oxidative stress, leading to lipid peroxidation and protein oxidation. Therefore, there is a growing need to identify natural compounds with sufficient antioxidant capacity and mitigate the negative effects of herbicides without side effects.Objective: Our study aimed to examine the protective effect of the phenolic extract of wild garlic (WG) leaves on terbuthylazine-treated erythrocytes.Material and methods: In human erythrocytes treated with the herbicide terbuthylazine (4.5 mg/L) alone and a combination of terbuthylazine and WG extract, we measured malondialdehyde (MDA) and haemoglobin (Hb) concentrations and the antioxidant activities of CuZn superoxide dismutase (SOD1; EC 1.15.1.1) and catalase (CAT; EC 1.11.1.6) in vitro.Results: In comparison with terbuthylazine, WG extract reduced the concentrations of MDA and Hb from 59.69 to 43.45 nmol/gHb (27%, p < 0.001) and 165.08 to 128.64 g/L (22%, p < 0.05), respectively. Catalase activity was induced for samples treated with both WG extract and terbuthylazine compared with terbuthylazine alone (p < 0.05).Conclusions: The results demonstrated that WG may reduce the toxicity of terbuthylazine, and the erythrocyte membrane may be the primary site of phenolic action. Therefore, the lipid peroxidation intensity could be a biomarker of oxidative damage caused by terbuthylazine and the protective effect of WG.

Synthesis and Characterization of Mesoporous Mg- and Sr-Doped Nanoparticles for Moxifloxacin Drug Delivery in Promising Tissue Engineering Applications.

Mesoporous silica-based nanoparticles (MSNs) are considered promising drug carriers because of their ordered pore structure, which permits high drug loading and release capacity. The dissolution of Si and Ca from MSNs can trigger osteogenic differentiation of stem cells towards extracellular matrix calcification, while Mg and Sr constitute key elements of bone biology and metabolism. The aim of this study was the synthesis and characterization of sol-gel-derived MSNs co-doped with Ca, Mg and Sr. Their physico-chemical properties were investigated by X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis (SEM/EDX), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), X-ray fluorescence spectroscopy (XRF), Brunauer Emmett Teller and Brunauer Joyner Halenda (BET/BJH), dynamic light scattering (DLS) and ζ-potential measurements. Moxifloxacin loading and release profiles were assessed with high performance liquid chromatography (HPLC) cell viability on human periodontal ligament fibroblasts and their hemolytic activity in contact with human red blood cells (RBCs) at various concentrations were also investigated. Doped MSNs generally retained their textural characteristics, while different compositions affected particle size, hemolytic activity and moxifloxacin loading/release profiles. All co-doped MSNs revealed the formation of hydroxycarbonate apatite on their surface after immersion in simulated body fluid (SBF) and promoted mitochondrial activity and cell proliferation.

Polysaccharide from Lycium arabicum: Structural Features, in Vitro Antioxidant Activities and Protective Effect against Oxidative Damage in Human Erythrocytes.

In this research work, a water-soluble polysaccharide (LAP) isolated from the fruits of Lycium arabicum was investigated. LAP contains carbohydrates (82.45±1.23 %), protein (1.56±0.21 %), and uronic acids (3.56±0.34 %). The analysis of the monosaccharide composition revealed the presence of rhamnose, arabinose, galactose, glucose and mannose in a molar ratio of 4.7 : 1.5 : 1 : 8.7 : 16.4 : 5.6. The extracted polysaccharide (PS) was considered as heterogeneous and highly branched by interpreting its GC/MS, FT-IR and NMR data. Crystallinity of LAP was inferred from its X-ray diffractometry (XRD) and Scanning Electron Microscopy (SEM) analysis. LAP exhibited an interesting stability at high temperatures (∼254 °C) and in a wide range of pH (3-9) deduced, respectively, from its DSC and zeta potential analysis. LAP displayed a strong antioxidant activity at low concentrations evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging, ferric reducing activity power (FRAP), free radical scavenging ability, superoxide radical-scavenging and hydroxyl radical-scavenging abilities. Inhibition of erythrocyte hemolysis and lipid peroxidation was also assessed. In 5 h, LAP treatment allowed the protection of the damaged erythrocytes caused by AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), to reduce the level of malondialdehyde (MDA) as well as to increase the reduced glutathione (GSH) level.

A Human Erythrocyte-based Haemolysis Assay for the Evaluation of Human Complement Activity.

The complement system consists of at least 50 proteins that serve as one of the first lines of defence against foreign, or damaged, cells and invading microorganisms. Its dysregulation underlies the pathophysiology of many different diseases, which makes functional assays of complement activity crucial; they are, however, underutilised. Standard haemolysis assays for the analysis of complement function employ sensitised non-human erythrocytes (e.g. from the sheep, guinea-pig or rabbit), the use of which raises animal welfare concerns. To provide an alternative to the use of such animal-derived products for complement function assays, we developed a method that employs modified human erythrocytes to evaluate the activity of complement pathways. Human erythrocytes were subjected to various chemical and/or proteolytic treatments involving 2,4,6-trinitrobenzene sulphonate (TNBS) and pancreatin. Haemolysis assays demonstrated that sequential treatment with TNBS and pancreatin resulted in significantly greater complement-mediated haemolysis, as compared to TNBS or pancreatin treatment alone. Evidence that lysis of the modified erythrocytes was complement-mediated was provided by the chelation and subsequent restoration of calcium in the plasma. Thus, such modified human erythrocytes could be used as an alternative to animal-derived erythrocytes in haemolysis assays, in order to evaluate complement activity in human plasma during, for example, the screening of patients for complement deficiencies and other abnormalities in a clinical setting.

Effect of the Solvent on Propolis Phenolic Profile and its Antifungal, Antioxidant, and In Vitro Cytoprotective Activity in Human Erythrocytes Under Oxidative Stress.

Propolis is a natural bee product with various beneficial biological effects. The health-promoting properties of propolis depend on its chemical composition, particularly the presence of phenolic compounds. The aim of this study was to evaluate the relationship between extraction solvent (acetone 100%, ethanol 70% and 96%) and the antifungal, antioxidant, and cytoprotective activity of the extracts obtained from propolis. Concentrations of flavonoids and phenolic acids in the propolis extracts were determined using ultrahigh-performance liquid chromatography. The antioxidant potential of different extracts was assessed on the basis of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free-radical-scavenging activity, Fe3+-reducing power, and ferrous ion (Fe2+)-chelating activity assays. The ability of the extracts to protect human red blood cell membranes against free-radical-induced damage and their antifungal activity was also determined. The results showed that the concentration of flavonoids in the propolis extracts was dependent on the solvent used in the extraction process and pinocembrin, chrysin, galangin, and coumaric acid were the most abundant phenols. All extracts exhibited high antioxidant potential and significantly protected human erythrocytes against oxidative damage. On the other hand, the antifungal activity of the propolis extracts depended on the solvent used in extraction and the fungal strains tested. It needs to be stressed that, to the best of our knowledge, there is no study relating the effect of solvent used for extraction of Polish propolis to its phenolic profile, and its antifungal, antioxidant, and cytoprotective activity.

Microfluidic assemblies designed for assessment of drug effects on deformability of human erythrocytes.

Extreme deformability of human erythrocytes is a prerequisite for their ability to squeeze through narrow capillaries of the blood microcirculation system. Various drugs can modify this deformability and consequently provoke circulation problems. We demonstrate that microfluidic assemblies are very convenient platforms for in vitro study of the associated processes. Two types of microfluidic channels were designed to quantitatively investigate modifications of erythrocyte deformability induced by hydrogen peroxide, ethanol and pentoxifylline based on transit velocity measurements. With a high sensitivity our microfluidic assemblies show that hydrogen peroxide decreases erythrocyte deformability in a dose-dependent manner. Then, results on ethanol resolve a biphasic nature of this reactant on the deformability of single erythrocyte cells. Results on pentoxifylline provide evidence that, similar to ethanol, also this medical drug has a double-sided effect on the erythrocyte deformability, i.e. increasing the deformability at low concentrations, while decreasing it at higher ones. Taken together, our microfluidic designs propose a potent measurement method for the erythrocyte deformability, as well as providing a perspective to evaluate effects of drugs on it.

Proton-controlled synthesis of red-emitting carbon dots and application for hematin detection in human erythrocytes.

The Red-emitting nitrogen-doped carbon dots (N-CDs) are synthesized using o-phenylenediamine by a one-step method, and can serve as a fluorescent probe for "turn off" detection of hematin in human red cells. The red-emitting N-CDs can be obtained only in acidic conditions and the emission of the red-emitting N-CDs is pH-dependent, indicating proton-controlled synthesis and emission. The red-emitting N-CDs are 2.7 nm in mean size and have a uniform dispersion and exhibit a high quantum yield (12.8%) and great optical properties. The developed sensing system for hematin displays a linear response from 0.4 to 32 µM with a detection limit of 0.18 µM. Importantly, this fluorescent probe demonstrates a good potential practicability for the quantitative detection of hematin in complex matrixes. Graphical abstract ᅟ.

The role of OAT2 (SLC22A7) in the cyclic nucleotide biokinetics of human erythrocytes.

The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The Km -values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 µM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 µM. The uptake was characterised with apparently low affinity and similar Km -values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real Km -values for uptake were 100-200 µM for cGMP and 50-150 µM for cAMP. The established OAT2-substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a Km of 854 ± 98 µM. Unexpectedly, co-incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed Km -values were 399 ± 44 and 259 ± 30 µM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1-substrate para-aminohippurate (PAH) showed some uptake (Km -value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport.

1H-indazole molecules reduced the activity of human erythrocytes carbonic anhydrase I and II isoenzymes.

Carbonic anhydrase (CA) is an important metabolic enzyme family closely related to many physiological and pathological processes. Currently, carbonic anhydrase inhibitors are the target molecules in the treatment and diagnosis of many diseases. In present study, we investigated the inhibitory effects of some indazole molecules on the CA-I and CA-II isoenzymes isolated from human erythrocytes. We showed that human CA-I and CA-II activities were reduced by of some indazoles at low concentrations. IC50 values, Ki constants, and inhibition types for each indazole molecule were determined. The indazoles showed Ki constants in a range of 0.383 ± 0.021 to 2.317 ± 0.644 mM, 0.409 ± 0.083 to 3.030 ± 0.711 mM against CA-I and CA-II, respectively. Each indazole molecule exhibited a noncompetitive inhibition effect. Bromine- and chlorine-bonded indazoles were found to be more potent inhibitory effects on carbonic anhydrase isoenzymes. In conclusion, we conclude that these results may be useful in the synthesis of carbonic anhydrase inhibitors.

Ankyrin exposure induced by activated protein kinase C plays a potential role in erythrophagocytosis.

BACKGROUND: In physiological and pathological conditions activated protein kinace C (PKC) has been observed in the erythrocytes. Externalization of ankyrin followed by Arg-Gly-Asp (RGD)/integrin recognition also triggers erythrophagocytosis. In the present study, to test whether activated PKC is associated with ankyrin exposure in erythrophagocytosis. METHODS: Phorbol 12-myristate-13-acetate (PMA)-induced PKC activation and ankyrin phosphorylation were tested, and under different treatment conditions the subpopulation of erythrocytes with ankyrin exposure and the levels of intracellular calcium were analyzed by flow cytometry. RESULTS: Results showed that treatment of erythrocytes with PMA in a calcium-containing buffer led to ankyrin exposure. In the absence of extracellular calcium, no ankyrin exposure was observed. PKC inhibition with calphostin C, a blocker of the PMA binding site, completely prevented the calcium entry, protein phosphorylation and ankyrin exposure. PKC inhibition with chelerythrine chloride, an inhibitor of the active site, diminished the level of ankyrin-exposing cells and ankyrin phosphorylation; however it even led to a higher percentage of cells with increased levels of calcium than with PMA treatment alone. Although PKC was activated and ankyrin phosphorylation occurred, no ankyrin exposure was observed in the absence of extracellular calcium. CONCLUSION: Analyses of results suggested that PMA induces calcium influx into the erythrocytes, leading to the activation of calcium-dependent enzymes and the phosphorylation of membrane proteins, ultimately inducing ankyrin exposure and erythrophagocytosis. This study may provide insights into the molecular mechanisms of removing aged or diseased erythrocytes.

Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform.

For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Chlorpyrifos and lambda cyhalothrin-induced oxidative stress in human erythrocytes.

Pesticides are one of the most potentially harmful chemicals introduced into the environment, and their adverse impacts on non-target organisms can be significant. The present study was conducted to shed light on effects of locally used insecticides chlorpyrifos (CPF) and lambda cyhalothrin (LCT) on oxidative stress biomarkers in human erythrocytes. The activity of catalase (CAT), superoxide dismutase (SOD), and protein contents as well as the levels of malondialdehyde (MDA) and osmotic fragility (OF) were measured in human erythrocytes exposed to CPF at concentrations of 0, 100, 500, 1000, and 2000 ppm and LCT at concentrations of 0, 100, 300, 600, and 800 ppm for 1 h and 3 h at 37°C. MDA levels and OF of erythrocytes were significantly higher in erythrocytes incubated with CPF and LCT at increasing concentrations of both insecticides and increased incubation time. However, erythrocyte CAT and SOD activities were decreased at all concentrations of CPF and LCT tested. Protein oxidation products were decreased at lower doses of CPF (100 and 500 ppm); at higher doses (1000 and 2000 ppm), total protein content was increased compared with control. In contrast LCT was associated with decreased in protein contents at all the concentrations. These results clearly demonstrated that CPF and LCT can induce oxidative stress in human erythrocytes ( in vitro).

Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes.

Antioxidant properties of thio-caffeine derivatives: Identification of the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine as antioxidant and highly potent cytoprotective agent.

A series of nine thio-caffeine analogues were synthesized and characterised by NMR, FT-IR and MS spectroscopic methods. Molecular structures of four of them were determined using single crystal X-ray diffraction methods. The antioxidant properties of all compounds, at concentration ranges from 0.025 to 0.1mg/mL, were evaluated by various chemical- and cell-based antioxidant assays. Human erythrocytes were used to examine in vitro haemolytic activity of all compounds and their protective effect against oxidative haemolysis induced by AAPH, one of the commonly used free radical generator. All compounds studied showed no effect on the human erythrocytes membrane structure and permeability with the exception of 8-(phenylsulfanyl)caffeine. Among the nine caffeine thio-analogues tested, the newly synthesized 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine possessed exceptionally high antioxidant properties. Moreover, it protects human erythrocytes against AAPH-induced oxidative damage as efficiently as the standard antioxidant Trolox. Therefore, 8-[(pyrrolidin-1-ylcarbonothioyl)sulfanyl]caffeine may have a significant cytoprotective potential caused by its antioxidant activity.

The free heme concentration in healthy human erythrocytes.

Heme, the prosthetic group of hemoglobin, may be released from its host due to an intrinsic instability of hemoglobin and accumulate in the erythrocytes. Free heme is in the form of hematin (Fe(3+) protoporphyrin IX OH) and follows several pathways of biochemical toxicity to tissues, cells, and organelles since it catalyzes the production of reactive oxygen species. To determine concentration of soluble free heme in human erythrocytes, we develop a new method. We lyse the red blood cells and isolate free heme from hemoglobin by dialysis. We use the heme to reconstitute horseradish peroxidase (HRP) from an excess of the apoenzyme and determine the HRP reaction rate from the evolution of the emitted luminescence. We find that in a population of five healthy adults the average free heme concentration in the erythrocytes is 21±2µM, ca. 100× higher than previously determined. Tests suggest that the lower previous value was due to the use of elevated concentrations of NaCl, which drive hematin precipitation and re-association with apoglobin. We show that the found hematin concentration is significantly higher than estimates based on equilibrium release and the known hematin dimerization. The factors that lead to enhanced heme release remain an open question.

Dietary indicaxanthin from cactus pear (Opuntia ficus-indica L. Mill) fruit prevents eryptosis induced by oxysterols in a hypercholesterolaemia-relevant proportion and adhesion of human erythrocytes to endothelial cell layers.

Toxic oxysterols in a hypercholesterolaemia-relevant proportion cause suicidal death of human erythrocytes or eryptosis. This process proceeds through early production of reactive oxygen species (ROS), release of prostaglandin (PGE2) and opening of PGE2-dependent Ca channels, membrane phosphatidylserine (PS) externalisation, and cell shrinkage. The present study was the first to reveal that a bioavailable phytochemical, indicaxanthin (Ind) from cactus pear fruit, in a concentration range (1.0-5.0 µM) consistent with its plasma level after a fruit meal, prevents PS externalisation and cell shrinkage in a dose-dependent manner when incubated with isolated healthy human erythrocytes exposed to an oxysterol mixture for 48 h. Dietary Ind inhibited ROS production, glutathione (GSH) depletion, PGE2 release and Ca2+ entry. Ind alone did not modify the erythrocyte redox environment or affect other parameters. Ex vivo spiking of normal human blood with the oxysterol mixture for 48 h induced eryptosis, resulting in the production of ROS and decreased levels of GSH, which was prevented by concurrent exposure to 5 µm-Ind. The adherence of eryptotic erythrocytes to the endothelium causes vascular tissue injury. Erythrocytes isolated from blood incubated with the oxysterol mixture plus 5 µm-Ind did not adhere to endothelial cell monolayers. Eryptotic erythrocytes may contribute to thrombotic complications in hypercholesterolaemia. Our findings suggest the positive effects of diets containing Ind on erythrocytes in hypercholesterolaemic subjects.