Erythrocyte Calcium Research Articles

Genetic variations in human ATP2B4 gene alter Plasmodium falciparum in vitro growth in RBCs from Gambian adults

BackgroundPolymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown.MethodsUsing a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygotes (carriers of the minor alleles, T/T (variant), heterozygote T/C and wildtype C/C (ancestral)) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank.ResultsRed blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ± 159 and 4261 ± 283], for homozygotes, heterozygotes and wildtypes respectively, p < 0.0001) and slower rates of calcium expulsion (calcium t½ ± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p < 0.0001). Growth of a Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p < 0.01). Genotype group did not affect parasite adhesion in vitro or var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50 = 122uM CI: 110–134) confirming its sensitivity to calcium channel blockade.ConclusionThe data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.

Cellular Mechanism by Which Genetic Variations in Human ATP2B4 Gene Protect Against Severe Malaria

Background: Recent genome-wide association and cross-sectional studies have identified novel human genetic polymorphisms associated with alterations in severe malaria risk. Some of the strongest associations have been identified in the ATP2B4 gene. ATP2B4 codes for PMCA4b, the primary regulator of erythrocyte calcium concentration. Methods: To investigate the cellular mechanisms by which polymorphisms in ATP2B4 alter malaria pathogenesis, we performed in vitro assays that model malaria pathogenesis on red blood cells (RBCs) collected from individuals (n=96) with and without copies of ATP2B4 variant haplotype. Findings: We found reduced rates of PMCA4b expression (p<0.0001), calcium expulsion (p<0.0001) and Plasmodium falciparum growth in RBCs from homozygote carriers of the mutant haplotype (p<0.01). We were unable to detect any difference in adhesion or var-gene expression in malaria infected RBCs with and without the variant ATP2B4 haplotype. Interpretation: This data is consistent with the conclusion that polymorphisms in ATP2B4, protect against severe falciparum malaria by controlling parasite density rather than adhesion and invasion. Funding: This work was supported by the Medical Research Council Unit The Gambia at London School of Hygiene and Tropical Medicine (MRCG at LSHTM) PhD fellowship. We acknowledge core funding to the MRC International Nutrition Group through MCA760-5QX00 from the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement. Declaration of Interest: None to declare. Ethical Approval: The study proposal was reviewed and approved by the Scientific Coordinating Committee of the Medical Research Council Unit The Gambia at London School of Hygiene and Tropical Medicine (MRCG at LSHTM) and ethical approval was granted by the Joint Gambia Government/Medical Research Council Ethics committee (SCC number 1421).

The content of chemical elements in the blood and lung tissue in pulmonary tuberculosis patients

Objective — to learn the content of certain micro­and macro elements in serum and erythrocytes of blood, as well as in the affected and unchanged parts of the removed lung preparation in the first diagnosed TB patients.Materials and methods. 61 patients with the first diagnosed infiltrative (45 persons) and disseminated (16 persons) pulmonary tuberculosis were examined. The control group consisted of 50 healthy, similar in age and gender.The content of chemical elements in erythrocytes (iron, copper, zinc, magnesium, manganese, calcium, potassium and sodium), blood serum (iron, copper, zinc, magnesium and calcium) and in the preparation of the removed lung tissue (iron, copper, zinc, magnesium, manganese, calcium, potassium and sodium) were determined on a C 115 atomic absorption spectrophotometer.Results and discussion. The content of zinc and calcium in serum, as well as iron, magnesium, calcium and potassium in erythrocytes of tuberculosis patients is significantly lower than in the control group, while the content of copper, manganese and sodium in erythrocytes of patients was higher compared with healthy persons. Changes in the content of chemical elements in preparations of the affected areas of the lungs, mainly, correlate with the content of chemical elements in the blood of TB patients. All this indicates the presence of metabolic disturbances in patients, dysmicroelementosis syndrome and associated hypoxia as a result of tuberculosis intoxication.Conclusions. In patients with first diagnosed infiltrative and disseminated pulmonary TB in serum and erythrocytes with a specific affected process of the lung tissue, pronounced dysmicroelementosis was observed, both in the direction of decreasing and increasing the content of chemical elements in serum and erythrocytes in the preparations of the affected lung region, which affects the course of a specific process. A decrease in the iron content in erythrocytes is, to some extent, the cause of hypoxia, which is characteris­tic of these forms of TB, and an increase in the content of copper and manganese in erythrocytes (hemo­globin synthesis) is a compensatory reaction. The results of the study provide a basis for a more adequate approach to pathogenetic therapy, both due to a selected diet and microelement preparations. However, despite all this, the issue of chemical elements in the blood of patients with tuberculosis requires further careful study.

Sex hormone intake in female blood donors: impact on haemolysis during cold storage and regulation of erythrocyte calcium influx by progesterone.

Sex hormone intake in blood donors may affect the quality of red blood cell (RBC) products via modulation of RBC function and predisposition to haemolysis during cold storage. The aims of this study were to evaluate the association between female sex hormone intake and RBC storage outcomes, and to examine possible mechanisms by which sex hormones interact with RBCs. Sex hormone intake by race/ethnicity and menopausal status, and association analyses between hormone intake and donor scores of storage, osmotic or oxidative haemolysis, were evaluated in 6,636 female donors who participated in the National Heart, Lung and Blood Institute's RBC-Omics study. A calcium fluorophore, Fluo-3AM, was used to define RBC calcium influx in response to exogenous sex hormones or transient receptor potential cation (TRPC) channel drugs. Sex hormone intake was more prevalent in premenopausal women from all racial groups (18-31%) than in postmenopausal women (4-8%). Hormone intake was significantly (p<0.0001) associated with reduced storage haemolysis in all females, reduced osmotic haemolysis in postmenopausal donors (23.1±10.2% vs 26.8±12.0% in controls, p<0.001), and enhanced susceptibility to oxidative haemolysis in premenopausal women. In vitro, supraphysiological levels of progesterone (10 μmol/L), but not 17β-oestradiol or testosterone, inhibited calcium influx into RBC and was associated with lower spontaneous haemolysis after 30 days of cold storage (0.95±0.18% vs 1.85±0.35% in controls, p<0.0001) or in response to a TRPC6 activator. Sex hormone intake in female donors is associated with changes in RBC predisposition to haemolysis. Menstrual status and the type of hormone preparation may contribute to differences in haemolytic responses of female RBCs to osmotic and oxidative stress. Progesterone modulates calcium influx into RBC via a mechanism that may involve interactions with membrane TRPC6 channels.

Evaluation of H-800/FUS-100 automatic urine analyzer performance

Abstract Objective Automated urine analysis is usually preferred for laboratories with intensive workload. The aim of this study was to evaluate the performance of the automated urine analyser H-800/FUS-100 and detect the error sources. Materials and methods One thousand four hundred fifty nine fresh urine samples were analyzed with H-800/FUS-100 automated systems. The urine sediment of the samples with discrepant strip and microscopy results were confirmed by manual microscopy. Precision and carry over studies were performed. Results The discrepancy is detected in a ratio of 5.89% between chemical analysis (H-800) and microscopic analysis (FUS-100) of the device. A total of 86 discrepant samples were detected. Fifty six of 86 were erythrocyte discrepancies (65.1%) and 30 of 86 were leukocyte discrepancies (34.9%). The results of carry over analysis for erythrocyte and leukocyte were 21.85% and 13.64%, respectively. Conclusions Sixteen (1.09%) of 1459 patients’ results in FUS-100 were discrepant with manual microscopy. Commonly, yeasts and crystals affected erythrocyte counts and calcium oxalate and amorphous crystals affected the leukocyte counts. Images should be reviewed for every sample when automated systems are used for urine analysis. Especially if discrepancy is detected between chemical and microscobic analysis, the results should also be confirmed with manual microscopy.

Erythrocyte Membrane Bound ATPase and Antioxidant Enzyme Changes Associated with Vascular Calcification is Reduced by Sodium Thiosulfate

Sodium thiosulfate (STS), a cyanide antidote has been reported to possess antioxidant and calcium chelation effects, useful for the treatment of renal failure due to vascular calcification and urolithiasis. The present study investigated the in vivo modulatory effects of STS on erythrocyte calcium, phosphorous levels, lipid peroxidation, antioxidant enzyme and membrane ATPase activities (Ca2+, Na+K+, Mg2+ and 5'' nucleotidase) in an adenine induced model of vascular calcification in rats. Adenine (0.75%) was supplemented through the diet for 28days, which resulted in significantly (P<0.05) increased circulating calcium and phosphorous product and oxidative stress within the RBCs, as measured from lipid peroxidation and reduced antioxidant enzymes. The membrane ATPase activities were altered (increased Ca2+, Na+K+ ATPase and decreased Mg+ ATPase, 5' nucleotidase) compared to the rats fed on normal diet. STS (400mg/kg) given orally was effective in establishing a normalcy in the RBC alterations. This effect was more pronounced, when STS was given from day 28 to day 49 after induction of calcification, instead of day 0 to day 28. These findings may benefit to evaluate the effectiveness of STS therapy in patients with chronic renal failure associated with increased circulating calcium and phosphorous product that leads to stiffening of vascular smooth muscles of aorta, due to calcium deposition.

Relationship between intracellular calcium and morphologic changes in rabbit erythrocytes: Effects of the acylated and unacylated forms of E. coli alpha-hemolysin

Alpha-hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic Escherichia coli strains, whose expression correlates with the severity of the infections produced. HlyA is synthesized as a protoxin, ProHlyA, that becomes matured to the active form in the cytosol by hemolysin-C–directed fatty acylation at the ε-amino residues of Lys 564 and Lys 690, before export from the toxin-producing bacteria. This posttranslational modification is remarkable because the nature of the protein is changed by the lipidic moiety from a benign protein to a frank toxin.In the present work, we demonstrated for the first time that, despite being hemolitically inactive, ProHlyA induced cellular morphologic changes in rabbit erythrocytes. A discocyte-to-echinocyte transformation was triggered by the protoxin in the absence of any accompanying increase in intracellular-Ca2+ levels. In addition, the Ca2+-influx kinetics in HlyA-treated erythrocytes indicated that the active toxin induced an elevation in intraerythrocyte-Ca2+ content in a biphasic manner, with an initial rise in Ca2+ that depended on the association of the protein with the target membrane and a second increment corresponding to an activation of purinergic channels. The first increase was sufficient to trigger a discocyte-echinocyte-spherocyte transition in erythrocyte shape, though the subsequent rise mediated by purinergic signalling was essential for the occurrence of hemolysis. The results presented here provide new insights into the mechanism of action of this toxin.

Active Plasma Membrane P-type H+-ATPase Reconstituted into Nanodiscs Is a Monomer

Plasma membrane H(+)-ATPases form a subfamily of P-type ATPases responsible for pumping protons out of cells and are essential for establishing and maintaining the crucial transmembrane proton gradient in plants and fungi. Here, we report the reconstitution of the Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 into soluble nanoscale lipid bilayers, also termed nanodiscs. Based on native gel analysis and cross-linking studies, the pump inserts into nanodiscs as a functional monomer. Insertion of the H(+)-ATPase into nanodiscs has the potential to enable structural and functional characterization using techniques normally applicable only for soluble proteins.

Calcium-phosphate metabolism parameters and erythrocyte Ca 2+ concentration in autosomal dominant polycystic kidney disease patients with normal renal function

IntroductionThe aim of this study was to assess calcium-phosphate metabolism of autosomal dominant polycystic kidney disease (ADPKD) patients with a special consideration to the following serum parameters: calcium (Ca2+), inorganic phosphate (Pi), parathyroid hormone (PTH) and intracellular erythrocyte calcium ([Ca2+]i) concentrations.Material and methodsThe study included 49 adult ADPKD patients (19 males, 30 females) aged 36 ±11 years with normal renal function and no diagnosis of diabetes as well as 50 healthy controls (22 males, 28 females) matched for age and gender. Serum concentrations of sodium (Na+), potassium (K+) and magnesium (Mg2+) ions and Pi were determined with an indirect ion-selective method, while Ca2+ concentration was measured with a direct ion-selective method. The PTH was detected using a radioimmunometric method. [Ca2+]i concentration was determined with the Ca2+ sensitive fluorescent dye Fura-2 method.ResultsIn the ADPKD group, when compared to controls, the following concentrations were significantly higher: serum Ca2+ (1.18 ±0.06 mmol/l vs. 1.15 ±0.06 mmol/l, p = 0.0085), [Ca2+]i (146.9 ±110.0 nmol/l vs. 96.5 ±52.7 nmol/l, p = 0.0075), serum Na+ (139.4 ±2.7 mmol/l vs. 138.5 ±2.1 mmol/l, p = 0.060, borderline significance), and PTH (15.5 ±6.8 pg/ml vs. 13.6 ±5.3 pg/ml, p = 0.066, borderline significance), while serum Mg2+ was significantly lower (0.81 ±0.09 mmol/l vs. 0.85 ±0.05 mmol/l, p = 0.021). In the ADPKD group we observed significant negative correlations of PTH with Ca2+ serum concentrations (Rs = –0.32, p = 0.025) and with estimated glomerular filtration rate (Rs = –0.31, p = 0.033).ConclusionsThe erythrocyte Ca2+ concentration is elevated in ADPKD patients with normal renal function. It may result from a dysfunction of mutated polycystins which can affect various aspects of electrolyte metabolism.

Distinguishing patients with glossopyrosis from those with oropyrosis based upon clinical differences and differences in saliva and erythrocyte magnesium

ObjectiveBurning mouth syndrome (BMS) is a complex of clinical symptoms defined by burning sensations in the oral cavity without observed oral pathology. Clinically two patient groups within BMS were distinguished, one with burning limited to the anterior tongue (glossopyrosis) and the other with burning in multiple mouth regions, including tongue, lips, palate, gums and cheeks (oropyrosis). Biochemical differences between these two groups could assist in distinguishing them. DesignEighty-three patients with BMS, 47 with oropyrosis and 31 with glossopyrosis were studied. Measurements of zinc, copper, magnesium and calcium in blood plasma, erythrocytes and parotid saliva were obtained in patients and in normal subjects and mean levels were compared. ResultsClinical history differentiated patients into categories of oropyrosis and glossopyrosis. Erythrocyte and saliva levels of magnesium were significantly lower in patients with glossopyrosis than in patients with oropyrosis or in normal volunteers whereas levels of zinc and calcium were similar. ConclusionsThese data suggest that patients with glossopyrosis not only differ clinically from those with oropyrosis but also exhibit magnesium deficiency as manifested by lower than normal magnesium levels in saliva and erythrocytes. Lingual burning in patients with glossopyrosis is consistent with hyperalgesia and neurogenic inflammation observed in patients and animals with magnesium deficiency and in magnesium deficient tissues. These results suggest a possible biochemical mechanism for pyrosis in patients with glossopyrosis.

Ca2+-Mg2+-dependent ATP-ase activity in hemodialyzed children. Effect of a hemodialysis session

In the course of chronic kidney disease (CKD) the intracellular erythrocyte calcium (Cai2+) level increases along with the progression of the disease. The decreased activity of Ca2+-Mg2+-dependent ATP-ase (PMCA) and its endogenous modulators calmodulin (CALM), calpain (CANP), and calpastatin (CAST) are all responsible for disturbed calcium metabolism. The aim of the study was to analyze the activity of PMCA, CALM, and the CANP-CAST system in the red blood cells (RBCs) of hemodialyzed (HD) children and to estimate the impact of a single HD session on the aforementioned disturbances. Eighteen patients on maintenance HD and 30 healthy subjects were included in the study. CALM, Cai2+ levels and basal PMCA (bPMCA), PMCA, CANP, and CAST activities were determined in RBCs before HD, after HD, and before the next HD session. Prior to the HD session, the level of Cai2+ and the CAST activity were significantly higher, whereas bPMCA, PMCA, and CANP activities and the CALM level were significantly lower than in controls. After the HD session, the Cai2+ concentration and the CAST activity significantly decreased compared with the basal values, whereas the other parameters significantly increased, although they did not reach the levels of healthy children. The values observed prior to both HD sessions were similar. Cai2+ homeostasis is severely disturbed in HD children, which may be caused by the reduction in the PMCA activity, CALM deficiency, and CANP-CAST system disturbances. A single HD session improved these disturbances but the effect is transient.

Indices of hydromineral metabolism in tyulka (Clupeonella cultriventris; Clupeiformes, Clupeidae) introduced in the Rybinsk Reservoir in comparison to aboriginal and marine fish species

The parameters of hydromineral exchange and the upper limit of salinity within the historical range of tyulka are typical of stenohaline freshwater fishes. These features assume freshwater origin of this species. Compared to the aboriginal species, tyulka introduced and established in the Rybinsk Reservoir has a higher level of potassium in the plasma, lower contents of water and potassium in muscles, lower content of calcium in erythrocytes, and lower contents of magnesium in plasma, erythrocytes, and muscles. High levels of sodium in the tyulka erythrocytes reflect elevated oxygen-carrying capacity of blood, indicating increased oxygen demand of this species. The content of magnesium in the tyulka erythrocytes is 2–3.7 times lower than in the indigenous species. This shows lower affinity of hemoglobin to oxygen in tyulka. The study reveals that strong external impacts are accompanied by very fast desalination of tyulka blood. In turn, this determines increased vulnerability to unfavorable environmental factors.

The endogenous modulators of Ca2+-Mg2+-dependent ATPase in children with chronic kidney disease (CKD)

Calcium homeostasis is disturbed in many ways in the course of chronic kidney disease (CKD). The concentration of free cytoplasmic calcium in erythrocytes is increased. Maintenance of a high concentration gradient (between the cystoplasmic and extracellular space) is possible only due to a finely tuned cooperation between many regulating systems in the cytoplasmic membranes and cell organelles. The aim of our study was to evaluate the activity of Ca(2+)-Mg(2+)-dependent ATPase (PMCA), calmodulin and calpain-calpastatin (CANP-CAST) system in erythrocytes of CKD children treated conservatively in the stages II-IV. A total of 36 patients with CKD were enrolled in the study. Group A contained patients with CKD stage II; group B with CKD stage III; and group C with CKD stage IV. The control group D consisted of 30 healthy subjects. In the serum, we determined the following: intact parathormon, total calcium, creatinine; in the red blood cells: free cytosolic calcium concentration (Ca(i)(2+)), activity of Ca(2+)-Mg(2+)-transporting ATPase (PMCA), basal PMCA (bPMCA), calmodulin (CALM), CANP, CAST. In all groups, Ca(i)(2+) concentrations were significantly higher, whereas PMCA and bPMCA activity were lower than in the controls. CANP concentrations in group A were elevated compared to the controls, whereas in groups B and C they were significantly lower. In group C, the mean CAST activity reached the highest values. CALM concentrations were decreased versus controls in all groups of patients. The intracellular Ca(i)(2+) homeostasis is disturbed in children with CKD and aggravates the deterioration of renal function as well. The reasons for the progressing increase of erythrocyte calcium concentration are multifactorial. Undoubtedly, the decreased PMCA activity, the calmodulin deficiency and the dysregulated CANP-CAST system are responsible for that phenomenon. The impact of many other biological modulators, creating a network defending the cell against the calcium accumulation, cannot be excluded.

Magnesium in Canine Epilepsy

Epilepsy is the expression of a functional paroxysmal neural impulses disturbance where neurons discharge excessive nerve impulses with a high frequency and abnormal synchrony. Investigations were conducted on a total of 45 dogs diagnosed with epilepsy aged between 3 months and 10 years. A number of 40 clinically healthy dogs were controll group (group M). Were determined plasma and erythrocyte magnesium, calcium, inorganic phosphate, potassium and sodium by molecular absorption spectrophotometry (colorimetric methods). In dogs with primary and secondary epilepsy has recorded a negative balance of magnesium with a statistically significant decrease of plasma and erythrocyte magnesium and also a negative correlation between attacks frequency-plasma and erythrocyte magnesium; the dogs with status epilepticus recorde the lowest levels of plasma and erythrocyte magnesium. The hypomagnesaemia, hypocalcemia and hyperphosphatemia recorded in dogs with secondary epilepsy are the important biochemical changes of magnesium deficiency syndrome with neuromusculare symptoms.

Reversal of ionoregulatory disruptions in occupational lead exposure by vitamin C

In order to investigate the toxic effects of lead during occupational exposure to this metal and the antidotal efficacy of ascorbic acid directed against these toxic effects, various artisans in Abeokuta, Nigeria, who have been shown to be occupationally exposed to lead were supplemented daily with 500 mg ascorbic acid for 2 weeks. Ca 2+-Mg 2+-ATPase activity in erythrocyte membrane, as well as calcium and magnesium concentrations in plasma, erythrocytes, erythrocyte membrane and urine of the artisans were determined before and after ascorbic acid supplementation. The 2-week ascorbic acid administration resulted in the reversal of lead-induced accumulation of calcium and magnesium in the erythrocyte membranes of the artisans. Ascorbic acid also reversed lead-induced inhibition of erythrocyte membrane Ca 2+-Mg 2+-ATPase. Urinary excretion of calcium and magnesium was not affected by ascorbic acid. There may be some scope in introducing ascorbic acid as an intervention strategy for the prevention and therapy of lead intoxication, especially in cases where the subjects cannot be removed from the source of lead exposure.

Magnesium, calcium and trace elements distribution in serum, erythrocytes, and hair of patients with chronic renal failure.

Disturbance of mineral status in patients with chronic renal failure (CRF) is one of many complications of this disease. Trace elements analysis in hair is sometime used by clinicians for a diagnosis of mineral status. In the present study concentration of magnesium and other trace elements was determined in serum, erythrocytes, and hair of patients with CRF undergoing hemodialysis (n = 31) and with impaired renal function but non-dialyzed (n = 15). Measurements of mineral content were performed by the atomic absorption spectrometry method (AAS). In serum of hemodialyzed patients as well as in erythrocytes and hair we found significantly increased levels of almost all tested elements, especially for Mg, Al, and Cr, compared to the control group. No significant differences were observed between these groups only in the Cd content in the examined samples. However, a significant correlation between its concentration in serum and erythrocytes was only found in the case of this element. Hair analysis reflected well the changes of mineral distribution in patients with CRF and may be used to diagnose these anomalies, in particular, with regard to Ca, Mg, Fe, and Cr. However, a strong variability of the concentration for these elements was found. In conclusion, our results confirm that renal failure as well as dialysis provoke imbalances of elemental status in physiological fluids and tissues, which should be monitored.

Disturbed erythrocyte calcium homeostasis and adenine nucleotide dysregulation in canine phosphofructokinase deficiency

Muscle-type phosphofructokinase deficiency (PFKD) causes a hemolytic disorder and exertional myopathy in humans and dogs. In humans, PFKD is accompanied by a disturbed calcium homeostasis and associated adenine nucleotide dysregulation, which may potentiate the erythroenzymopathy associated with this inherited disorder. This study shows that canine PFKD also manifests these erythrocyte abnormalities. Compared to normal, healthy red cells, PFK-deficient erythrocytes contain lower concentrations of ATP and higher concentrations of IMP and calcium, the latter as per a calcium indicator dye. Adenosine monophosphate deaminase (AMPD) enriched 5000-fold from canine erythrocytes adsorbs to immobilized calcium–calmodulin and the interaction between these two proteins activates AMPD through a Kmapp effect. This behavior is similar to that of the human erythrocyte enzyme and provides a potential contributing mechanism for accelerated adenine nucleotide turnover in canine PFKD. We propose that adenine nucleotide replacement strategies could benefit the erythroenzymopathy in human and canine PFKD and that the dog model of this disorder is an appropriate vehicle for further elucidating this hypothesis.

Divalent Cations Increase Lipid Order in Erythrocytes and Susceptibility to Secretory Phospholipase A 2

Elevated concentrations of intracellular calcium in erythrocytes increase membrane order and susceptibility to secretory phospholipase A 2. We hypothesize that calcium aids the formation of domains of ordered lipids within erythrocyte membranes by interacting directly with the inner leaflet of the cell membrane. The interface of these domains with regions of more fluid lipids may create an environment with weakened neighbor-neighbor interactions that would facilitate phospholipid migration into the active site of bound secretory phospholipase A 2. This hypothesis was investigated by determining the effects of seven other divalent ions on erythrocyte membrane properties. Changes in membrane order were assessed with steady-state fluorescence spectroscopy and two-photon microscopy with an environment-sensitive probe, laurdan. Each ion increased apparent membrane order in model membranes and in erythrocytes when introduced with an ionophore, suggesting that direct binding to the inner face of the membrane accounts for the effects of calcium on membrane fluidity. Furthermore, the degree to which ions affected membrane properties correlated with the ionic radius and electronegativity of the ions. Lastly, erythrocytes became more susceptible to enzyme hydrolysis in the presence of elevated intracellular levels of nickel and manganese, but not magnesium. These differences appeared related to the ability of the ions to induce a transition in erythrocyte shape.

Elevated Cardiac Troponin T in Hemodialysis Patients Receiving More Intravenous Iron Sucrose

Elevated cardiac troponin T (cTnT) has been associated with shorter survival in hemodialysis patients. Moreover, intravenous (IV) iron treatment has been held responsible for oxidative stress and accelerated atherosclerosis in these patients. In the present study, we investigated the relationship between cTnT concentration, IV iron treatment, and parameters of iron status. In addition, parameters of oxidative stress, inflammation, and atherosclerosis were evaluated. Predialysis blood samples of 78 chronic hemodialysis patients were analyzed for cTnT, malondialdehyde, creatine kinase (CK), and CK‐isoenzyme MB (CK‐MB). In addition, the mean value of predialysis serum samples collected during the last year, were considered for homocysteine, ferritin, iron, iron binding capacity, blood cell counts, blood urea nitrogen, creatinine, albumin, erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP), calcium, phosphate, iPTH, cholesterol, and triglyceride. The quantity of IV iron sucrose administered during the last two years was counted from the patients' files. Echocardiography, all events related to ischemic heart disease, and urine volume were also recorded. Elevated cTnT levels (≥ 0.10 ng/mL) were found in 18 patients (23.1%). The amount of iron administered was 2264 ± 1871 mg with a range 0–7000 mg. Patients with elevated cTnT levels received more IV iron than those with normal cTnT (3692 ± 1771 vs. 1761 ± 1595 mg, p < 0.001). The serum ferritin level was higher in patients with elevated cTnT (median levels; 477 vs. 288 ng/mL; P < 0.05). Patients with elevated cTnT were longer on dialysis compared to those with normal levels (median times; 35.5 vs. 15 months, P < 0.01) and regression analysis identified the amount of administered iron as an independent factor for elevated cTnT (P < 0.01). Intravenous iron treatment and high ferritin concentration are related to high cTnT level, which has previously been incriminated as a survival marker in hemodialysis patients.

Arachidonic acid increases intracellular calcium in erythrocytes

Recently, we have measured in erythrocytes a voltage-modulated and dihydropyridine-inhibited calcium influx. Since arachidonic acid and other polyunsaturated fatty acids influence the activities of most ion channels, we studied their effects on the erythrocyte Ca 2+ influx. It was measured on fresh erythrocytes, isolated from healthy donors, using the fluorescent dye Fura 2 as indicator of [Ca 2+] i. AA (5–50 μM) and EPA (20–30 μM) stimulated a concentration-dependent increase in [Ca 2+] i, deriving from extracellular calcium (1 mM), without affecting the intra- and extracellular pH and membrane voltage. The Ca 2+ influx rate varied from 0.5 to 3 nM Ca 2+/s in the presence of AA and from 0.9 to 1.7 nM Ca 2+/s with EPA. The Ca 2+ influx elicited by AA and EPA was not inhibited by dihydropyridines, while cyclooxygenase inhibitors were effective and PGE1 or PGE2 did not produce any effect. We conclude that AA could activate an erythrocyte voltage-independent Ca 2+ transport via an intermediate product of cyclooxygenase pathway; however, a direct interaction with the membrane lipid–protein cannot be excluded.