Intraerythrocytic Concentration Research Articles

Tenofovir-Diphosphate and Emtricitabine-Triphosphate Adherence Benchmarks in Dried Blood Spots for Persons with HIV Receiving Tenofovir Alafenamide and Emtricitabine-based Antiretroviral Therapy (QUANTI-TAF).

QUANTI-TAF aimed to establish tenofovir-diphosphate/emtricitabine-triphosphate (TFV-DP/FTC-TP) adherence benchmarks in dried blood spots (DBS) for persons with HIV (PWH) receiving tenofovir alafenamide/emtricitabine (TAF/FTC)-based antiretroviral therapy (ART). During a 16-week pharmacokinetic study, PWH received TAF/FTC-based ART co-encapsulated with an ingestible sensor to directly measure cumulative (enrollment to final visit) and 10-day adherence. At monthly visits, intraerythrocytic concentrations of TAF/FTC anabolites (TFV-DP/FTC-TP) in DBS were quantified by LC-MS/MS and summarized at steady-state (week 12 or 16) as median (IQR). Linear mixed-effects models evaluated factors associated with TFV-DP/FTC-TP. 84 participants (86% male, 11% female, and 4% transgender), predominantly receiving bictegravir/TAF/FTC (73%) enrolled. 92% completed week 12 or 16 (94% receiving unboosted ART). TFV-DP for <85% (7/72), ≥85%-<95% (9/72), and ≥95% (56/72) cumulative adherence was 2696 (2039-4108), 3117 (2332-3339), and 3344 (2605-4293) fmol/punches. All participants with ≥85% cumulative adherence had TFV-DP ≥1800 fmol/punches. Adjusting for cumulative adherence, TFV-DP was higher with boosted ART, lower BMI, and in non-Blacks. FTC-TP for <85% (14/77), ≥85%-<95% (6/77), and ≥95% (57/77) 10-day adherence was 3.52 (2.64-4.48), 4.58 (4.39-5.06), and 4.96 (4.21-6.26) pmol/punches. All participants with ≥85% 10-day adherence had FTC-TP ≥2.5 pmol/punches. Low-level viremia (HIV-1 RNA ≥20-<200 copies/mL) occurred at 60/335 (18%) visits in 33/84 (39%) participants (range: 20-149 copies/mL), with similar TFV-DP (3177 [2494-4149] fmol/punches) compared with HIV-1 RNA <20 copies/mL visits (3279 [2580-4407] fmol/punches). We propose PK-based TFV-DP (≥1800 fmol/punches)/FTC-TP (≥2.5 pmol/punches) benchmarks in DBS for PWH receiving unboosted TAF/FTC-based ART with ≥85% adherence. In the setting of high adherence, low-level viremia was common.

TRANSMEMBRANE ION AND WATER TRANSPORTS IN ERYTHROCYTE VOLUME HOMEOSTASIS: AN OVERVIEW OF THE PHYSIOLOGIC PROCESSES

Background: The intraerythrocytic concentrations of ions (Na+, K+, Cl- and HCO3-) play key roles in maintaining erythrocyte volume homeostasis. Anisosmotic and isosomotic changes of these ion concentrations challenge erythrocyte volume to either shrink or swell, thereby sending signals to activate regulatory volume mechanisms that are mediated by ion transporters. Ion fluxes directed inwardly or outwardly with obliged water movements do restore the erythrocyte volume to its steady state. These physiological phenomena prevent the erythrocyte from becoming overhydrated or dehydrated with the consequence of intravascular haemolysis or senescent changes associated with eryptosis. Objectives: To review the literature on the physiological processes associated with transmembrane ion and water transports during erythrocyte volume homeostasis. Method: Offline and online libraries were searched with indexing tools using keywords derived from the subject area of review. Conclusions: The review has highlighted the physiological concepts involved in erythrocyte volume homeostasis in relation to the engaged transmembrane ion and water transport systems, which can influence experimental designs to study ion and water channel blockers and channelopathies of erythrocytes.

Concentrations of Intra-erythrocyte Folate, Serum Vitamin B12, and Hemoglobin in Women of Childbearing Age and Associated Factors

Objective: The aim was to verify the relations among intra-erythrocyte folate, serum vitamin B12, and hemoglobin concentrations in women of childbearing age and their association with several socioeconomic and demographic variables, nutritional status, and food consumption in the city of Recife, Northeastern Brazil.Methods: This was a cross-sectional study with a random sample of 1,210 women of childbearing age from the city of Recife. Concentrations of serum vitamin B12 and intra-erythrocyte folate were analyzed by immunoassay and hemoglobin concentrations by automated determination. A questionnaire was used to collect socioeconomic and demographic data of nutritional status and a food frequency questionnaire was used to evaluate food consumption.Results: Reductions in hemoglobin concentrations were found in 141 women (12.0%; 95% confidence interval (CI), 10.3–13.9), serum vitamin B12 in 81 women (7.0%; 95% CI, 5.7–8.6), and only 1 woman had a reduction in intra-erythrocyte folate concentrations. No women had a concomitant reduction of intra-erythrocyte folate, serum vitamin B12, and hemoglobin concentrations. However, 13 women (1.1%; 95% CI, 0.6–1.9) had a concomitant deficiency of hemoglobin and vitamin B12. Intra-erythrocyte folate concentrations were higher in women of a higher age and income. Hemoglobin concentrations were lower in smokers, and serum vitamin B12 concentrations did not show a significant variation in relation to socioeconomic, demographic, and anthropometric parameters. There was no strong correlation between food consumption and biochemical concentrations evaluated.Conclusion: Despite the strategies to eradicate anemia, this nutritional deficiency was dominant. Serum vitamin B12 deficiency outpacing intra-erythrocyte folate is worrying due to the lack of programs to fight against vitamin B12 insufficiency. Age and income were directly related to intra-erythrocyte folate concentrations. Food consumption suggests that there is a homeostatic control to maintain equilibrium at biochemical concentrations.

Iron Deficiency Induces Faster Sickling Kinetics in Transgenic Sickle Mice

Sickle cell disease (SCD) is caused by polymerization of deoxygenated hemoglobin‐S (HbS) within the red blood cell (RBC). The kinetics of hemoglobin polymerization are dependent on the intra‐erythrocytic concentration of HbS; specifically, the time from deoxygenation to polymerization is inversely related to HbS concentration30. We hypothesized that iron‐deficient erythropoiesis would produce RBCs with decreased intracellular Hb concentration and prolonged sickling times which would reduce the severity of SCD.To interrogate this hypothesis, we studied Townes mice that express humanized alpha globin, gamma globin, and either beta‐S (Townes‐SCD) or normal beta globin (Townes‐Control). RBCs from these Townes‐SCD mice have similar sickling kinetics to RBCs from human volunteers with SCD. To induce iron deficiency, we fed mice either an iron‐restricted diet (3 ppm iron) or iron‐normal diet (48 ppm iron) introduced at the time of weaning. After two months on the experimental diets, we measured RBC parameters using an Advia 2120 hematology analyzer and measured in vitro RBC sickling kinetics at 3% oxygen. Analysis of RBC parameters revealed that SCD mice on the iron‐restricted diet had lower hematocrit (32% vs 42.2%, P &lt;0.0001), and there were trends toward lower hemoglobin, mean corpuscular volume, mean cell hemoglobin, and mean corpuscular hemoglobin concentration. Surprisingly, the in vitro sickling kinetic assay showed a significantly shortened time to 50% sickled in the Iron Restricted group (t50: 96.6 vs 136.5 min., p&lt;0.0001).At this two‐month time point, this study provides insights into the hematologic changes that occur during the initial onset of iron deficiency anemia in Townes‐SCD mice. We were surprised to find that the rate of sickling increased in the iron‐restricted mice; however, at two months of iron deficient diet, we have yet to see a substantial fall in the MCHC, a major determinant of the sickling delay time. Furthermore, these mice may have experienced compensatory changes in the red cell that adversely affected sickling such as a decrease in hemoglobin F levels, or a shift in the Hb‐ O2 dissociation curve that favors releasing oxygen at lower partial pressures. To address these questions, we will investigate the hemoglobin composition and the oxygen dissociation curve at the remaining four‐ and six‐month timepoints. We aim to determine the intermediate and long‐term potential of iron deficiency to delay sickling and reduce the severity of organ damage in the context of SCD. This may inform a strategy of optimizing iron intake as a potential treatment for SCD.Support or Funding InformationSupport from NIAID Intramural funding and NIH Division of Veterinary ResourcesScholarship Award from the Saudi Arabian Cultural Mission (SACM)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of erythrocyte biomarkers in amyotrophic lateral sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. It has been hypothesised that red blood cells (RBCs) may be involved in the disease process by the release of damaging molecules. The aim of this ex vivo study is to compare RBCs biochemical and hemorheological parameters between ALS patients and healthy donors to identify novel biomarkers of the ALS disease. We included 82 ALS patients and 40 gender age-matched healthy donors. We performed quantification of erythrocyte aggregation and deformability, nitric oxide (NO) efflux from RBCs, acetylcholinesterase (AChE) enzyme activity and intraerythrocytic concentration of nitrite, nitrate and S-nitrosogluthatione (GSNO). Erythrocyte deformability and AChE activity were increased in patients with ALS in comparison to healthy donors. NO efflux from RBCs and concentration of intraerythrocytic nitrite were lower in ALS patients. In patients, we found that for higher NO range of values the respiratory function is worse and that for higher AChE range of values the RBCs nitrite content increase. The results of the present study indicate that NO efflux from RBCs and RBCs AChE should be further explored as potential biomarkers for ALS.

Determination of Concentrations of Azathioprine Metabolites 6-Thioguanine and 6-Methylmercaptopurine in Whole Blood With the Use of Liquid Chromatography Combined With Mass Spectrometry

Background6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are used in many autoimmune diseases and after solid-organ transplantation. Their properties are mediated by active metabolites, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine (6-MMP). The most common adverse effects are myelo- and hepato-toxicity. The aim of the study was quantification of 6-TG and 6-MMP, with the use of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) in solid-organ transplant recipients. MethodsIn 33 patients, kidney transplant recipient (n = 25) and liver transplant recipient (n = 8) intra-erythrocyte concentrations of 6-TG and 6-MMP were measured with the use of LC/MS/MS. ResultsThe mean concentration of 6-TG was 205.35 ± 157.62 pmol/8 × 108 red blood cells (RBC); median concentration of 6-MMP was 1064.1 (35.78–11,552.9) pmol/8 × 108 RBC. There were no correlations between 6-TG levels and peripheral blood parameters (white blood cell count, WBC; hemoglobin, Hb concentration; PLT, blood platelet count) or alanine aminotransferase activity (AlAT) activity. Relationships between 6-MMP concentrations and peripheral blood parameters (WBC, Hb, PLT) or AlAT activity have not been found. Subgroups with leukopenia, anemia, thrombocytopenia, and liver dysfunction did not differ in concentrations of 6-TG or 6-MMP. We have observed a negative correlation between daily azathioprine dose and WBC count (r = −0.37, P = .04). ConclusionsRelationships between concentrations of azathioprine metabolites and myelotoxicity or hepatotoxicity have not been confirmed. Further studies on larger groups of patients would be helpful in a more accurate understanding of the impact of azathioprine metabolites on parameters of bone marrow and liver function.

Hemolysis interferences on frequently required stat analysis: a French multicentric study

Hemolysis should lead to changes in test results. Our study evaluated the impact of hemolysis on 26 blood measurements of stat biochemistry markers (sodium, potassium, chloride, urea, creatinine, glucose, total protein, calcium, magnesium, inorganic phosphorus, uric acid, C-reactive protein, total bilirubin, ASAT, ALAT, LDH, creatine kinase, alkaline phosphatase, γ glutamyl-transferase, lipase, alcohol, iron, β hCG, troponins, natriuretic peptides) determined with 13 different types of instruments in 17 hospital laboratories. Four pools of samples (collected from lithium heparin or EDTA or sodium fluoride tubes, according to the measured parameters) were overloaded with five increasing concentrations of whole blood lysate (final concentration from 0 to 2.000 mg/dL). Replication was performed for each assay, average values were calculated and differences between results with and without lysate were analyzed. A difference exceeding the square root of the sum of both squared analytic and biologic imprecisions for each analyte, was judged to be significant. Except homogeneous and expected impact of hemolysis on certain parameters like potassium, LDH... (due to their intra-erythrocyte concentration) a heterogeneous effect was found for other parameters, according to the analyzer and/or to the methodology. In summary, this study confirms the importance of mastering the measurement of the hemolysis and leads to several recommendations: (i) biologists should have a good knowledge of the impact of hemolysis on the measurements they perform, depending on their chosen analyzers; (ii) if an interference is noticed, it is recommended to add to the result a relevant comment and to check that the comment is properly edited in the laboratory computer software and appears on printed and transmitted results.

Evaluation of oxidative stress in the anemia of dogs with chronic kidney disease.

Anemia and systemic oxidative stress may occur in dogs with chronic kidney disease (CKD). Only scarce information regarding the intraerythrocytic redox status under these conditions is available at this time. The aim of this study was to evaluate the indicators of oxidative stress and intraerythrocytic antioxidant defense in dogs with anemia of CKD. Thirty dogs with CKD in stages 3 or 4 with nonregenerative anemia (HCT ≤37%) were compared to 20 healthy dogs. Complete blood count, reticulocyte %, blood smear evaluation, intraerythrocytic concentrations of total (GSHt), reduced (GSH), and oxidized glutathione (GSSH), and activities of glutathione peroxidase, glutathione reductase and superoxide dismutase (SOD), as well as plasma concentrations of thiobarbituric acid-reactive substances (TBAR) were determined. Anemia of CKD dogs was nonregenerative (reticulocytes ≤0.2% with scarce anisocytosis and poikilocytosis). Intraerythrocytic GSSH and SOD, and plasma TBAR were higher in dogs with CKD. There was a positive correlation between the creatinine concentration and TBAR, and negative correlations between creatinine concentration and HCT, as well as between HCT and TBAR. In CKD dogs with a higher degree of anemia, SOD levels were higher and GSSH concentrations were lower. Despite the evidence of increased systemic oxidative stress, the compensatory response of SOD and the sustained intraerythrocytic concentrations of GSSH in CKD dogs with anemia indicated that the erythrocytes maintained the antioxidant defense. There was no strong evidence that oxidative stress was associated with higher degrees of anemia in dogs with CKD.

Paper-Based Assay for Quantification of HbS in Blood of Sickle Cell Disease Patients

Introduction: Sickle cell disease (SCD) is a common inherited blood disorder caused by sickle hemoglobin (HbS) which, unlike normal adult hemoglobin (HbA), becomes insoluble and polymerizes under hypoxic conditions. Patients with SCD experience chronic hemolytic anemia, episodic pain crises and abnormal blood flow to critical organs that cumulatively result in significant illness and shortened lifespans for many. The severity of SCD varies significantly between patients, but for individuals the rate of adverse events is strongly correlated with intraerythrocytic concentration of HbS (%HbS). High per test costs and long turnaround times make conventional laboratory methods (e.g. Hb electrophoresis, HPLC, IEF) impractical for quantifying %HbS in real-time (e.g. during transfusion therapy). The objective of this study was to demonstrate that %HbS in blood could be quantified using our recently developed rapid, low-cost paper-based SCD assay [1].Methods: Blood samples were obtained from SCD and sickle cell trait (SCT) patients at the Texas Children’s Hematology Center (Houston, TX). To perform the SCD assay a 20μL droplet of blood mixed with Hb solubility buffer (1:10 by volume) was dropped on chromatography paper. The resulting blood stain was digitized with a flatbed scanner (Canon USA Inc, Melville, NY) and analyzed using a custom image analysis code (The MathWorks Inc, Natick, MA). Conventional Hb electrophoresis was performed with the semi-automated Sebia Hydrasys 2 Scan system (Sebia Inc, Norcross, GA).Results: The difference in transport of Hb through the paper produced a blood stain with two parts: the area of the initial drop where polymerized HbS is retained (center spot) and the area where soluble Hb is wicked laterally (peripheral ring). The relative color intensity of the center spot and peripheral ring is related to the blood sample %HbS (Fig. 1). The image analysis code automatically isolates and calculates the ratio of the average color intensities of each area (S-index). A series of reconstituted blood samples with artificially adjusted %HbS from 0 to 60% was used to calibrate the assay so that %HbS could be estimated based on blood stain color intensities (Fig. 2a). The values of %HbS estimated for patient samples using our paper-based SCD assay and actual values measured using conventional Hb electrophoresis were highly correlated with R2 = 0.898 (Fig. 2b). The estimated and actual %HbS values also showed strong agreement with the standard deviation of the difference between the two measurements = 5.5 %HbS (Fig. 2c). The majority of the differences between actual and estimated %HbS (96.67%) are within 2 standard deviations of the mean of the differences. The assay could be performed in under 35 minutes and multiple assays could be performed and analyzed in parallel. The cost of consumable materials and reagents for the paper-based SCD assay is less than $0.03.Conclusions: This study demonstrates the feasibility of using our recently developed paper-based assay to quantify %HbS in blood samples in real-time. The ability to rapidly, inexpensively measure %HbS will be particularly useful for monitoring the effectiveness of chronic transfusion or hydroxyurea therapy for long-term control of HbS content in blood of SCD patients. The ability to measure %HbS in real-time could also potentially facilitate more aggressive prophylactic therapy to intervene rapidly and significantly reduce the rate of life-threatening complications in SCD patients, including stroke. [Display omitted] [Display omitted] Acknowledgments: This work was supported in part by a 2012 NIH Director’s Transformative Research Award (NHLBI R01HL117329, PI: SSS).

Telaprevir-S isomer enhances ribavirin exposure and the ribavirin-related haemolytic anaemia in a concentration-dependent manner

The standard-of-care for the treatment of genotype-1 chronic hepatitis C is based on the combination of direct acting antivirals, such as boceprevir and telaprevir, with ribavirin and pegylated-interferon alfa. These triple regimens give a higher response rate than dual therapy, but on the other hand show a more than 10% higher rate of anaemia. Not enough focus has been given to the interaction between telaprevir and RBV. In this work, we aimed to study and deepen this relationship by comparing ribavirin plasma and intra-erythrocytic concentrations at one month of triple and dual therapy (17 vs. 119 patients). Moreover, we determined telaprevir isomers concentrations and tested them for correlation with ribavirin concentrations and haemoglobin loss at one month of treatment. Finally, all drugs concentration data were tested for their correlation with the renal function during treatment.The comparisons of ribavirin concentration and toxicity data were repeated on a sub-group of 9 patients who had been treated 1year before with dual therapy and then re-treated with triple therapy.The observed ribavirin plasma and intra-erythrocytic concentrations in triple therapy were significantly higher compared to dual therapy, both in whole group and sub-group comparison. Ribavirin concentrations were significantly correlated to the haemoglobin loss and telaprevir-S isomer concentrations (r2=0.317 Pvalue=0.023 and r2=0.388 Pvalue=0.008, respectively). Renal function had a significant decrease from the baseline value, but was not significantly correlated with drugs concentrations.These results highlight for the first time that, in the context of triple therapy with telaprevir, ribavirin exposure is related to the telaprevir-S isomer plasma concentration.We conclude that the addition of telaprevir to the dual therapy increases ribavirin exposure and haemoglobin loss: this effect could probably be managed through the therapeutic drug monitoring of ribavirin and telaprevir-S concentrations.

The co-administration of telaprevir increases ribavirin plasma and intra-erythrocytic concentrations, causing higher onset of anemia

The co-administration of telaprevir increases ribavirin plasma and intra-erythrocytic concentrations, causing higher onset of anemia

Poor Response to Thiopurine in Inflammatory Bowel Disease: How to Overcome Therapeutic Resistance?

A 24-year-old woman (53 kg) with a 5-year history of steroid-dependent ulcerative colitis with mild and extensive ulcerations presented to the gastroenterology clinic for symptom recurrence. She was given 100 mg/day (1.9 mg · kg−1 · day−1) azathioprine (AZA)5 for 1 month, after which the dose was increased to 125 mg/day (2.3 mg · kg−1 · day−1). Four months later, the patient was tapered off steroid therapy. Her symptoms persisted after 7 months of AZA therapy, however, and she experienced gastrointestinal side effects. The patient was switched to another thiopurine drug, 6-mercaptopurine (6-MP), at 75 mg/day (1.4 mg · kg−1 · day−1), which was well tolerated but similarly ineffective (8 stools daily). A brief course of steroid therapy rapidly produced a substantial but short-lived clinical improvement. To understand this patient's unresponsiveness to 2 thiopurine agents, we quantified thiopurine metabolites (1) 1 year after initiating AZA therapy. Intraerythrocyte concentrations of 6-thioguanine nucleotides (6-TGNs) were low (132 pmol/8 · 108 erythrocytes; therapeutic interval, 230–400 pmol/8 · 108 erythrocytes), and 6-methylmercaptopurine ribonucleotides (6-MMPRs) were very high (11 666 pmol/8 · 108 erythrocytes; therapeutic interval, <5800 pmol/8 · 108 erythrocytes). A second quantification of thiopurine metabolites 3 months later confirmed these results (6-TGNs, 127 pmol/8 · 108 erythrocytes; 6-MMPR, 26 304 pmol/8 · 108 erythrocytes). The patient had an unusual and extremely high thiopurine S -methyltransferase (TPMT) activity in erythrocytes [61.5 nmol · h−1 · (mL erythrocytes)−1; reference interval, 8.5–15 nmol · h−1 · (mL erythrocytes)−1]. The lack of clinical efficacy for 6-MP, together with the evidence of pharmacologic resistance, prompted discontinuation of 6-MP therapy. Thereafter, we administered the tumor necrosis factor-α (TNF-α) antagonist adalimumab, but we quickly replaced it with infliximab, which has a …

Immunophilin-loaded erythrocytes as a new delivery strategy for immunosuppressive drugs

Cyclosporine A (CsA) and tacrolimus (also known as FK506) are natural compounds with immunosuppressive activity that have improved the outcome of organ transplantation. Unfortunately, both drugs are characterised by high pharmacokinetic variability, poor bioavailability and high toxicity. Until now, no optimal method to deliver immunosuppressant drugs into circulation has been developed. Here we propose the use of engineered erythrocytes as a drug delivery system for the release of immunosuppressants in circulation in order to modify their pharmacokinetic and restrain toxic effects. After administration, FK506 and CsA mainly distribute within erythrocytes owing to the presence into these cells of immunophilins that bind the drugs with very high affinity (FKBP12 for FK506 and cyclophilin A for CsA); therefore, a new strategy aimed to increase the amount of FK506/CsA carried by erythrocytes by increasing the intra-erythrocytic concentration of the respective binding proteins has been developed. We manufactured recombinant forms of human FKBP12 and cyclophilin A to be loaded into RBC through a hypotonic dialysis and isotonic resealing procedure. Erythrocytes loaded with 3.5 ± 1.3, 7.5 ± 3.1 and 15.5 ± 0.4 nmol FKBP12 were able to bind 3.5 ± 1.5, 6.0 ± 1.9 and 11.4 ± 2.9 μg FK506 per millilitre RBC, respectively, while RBC loaded with 4.0 ± 0.6, 5.0 ± 0.8 and 15.9 ± 2.4 nmol of cyclophilin A could bind 8.9 ± 3.4, 12.2 ± 3.5 and 17.0 ± 3.2 μg CsA. Thus, both engineered RBC were demonstrated able to bind up to an order of magnitude more drug than corresponding native erythrocytes (1.0 ± 0.3 μg FK506 and 3.2 ± 0.3 μg CsA). Moreover, FK506 released from FKBP12–RBC is able to be up-taken by T lymphocytes and inhibit IL-2 expression in vitro as free administered drug. In summary, our results indicate that diffusible immunosuppressants could be entrapped into red cells (thanks to the loading of the respective target protein) and suggest that immunophilin-loaded RBC could be employed as potential delivery system for immunosuppressive agents.

Changes of intra-erythrocyte calcium concentration during cardiopulmonary bypass in pediatric patients

Objective To study the changes of intra-erythrocyte calcium concentration during cardiopulrnonary bypass( CPB ).Methods 26 pediatric patients undergoing surgical repair of ventricular or atrial sepal defect under CPB were enrolled in this prospective study. Heparinized venous blood samples were taken from the central vein before CPB, at the beginning of CPB and after CPB, respectively. The concentrations of intra-erythrocyte [ Ca2+] were measured by fluorescent probes (Fluo-3-AM), while plasma [Ca2+] were determined by blood gas analyzer. Results Before CPB and after CPB, the concentrations of plasma[ Ca2+]were ( 1.24±0.06) mmol/L and (1.28±0.08) mmol/L, respectively. It remarkably decreased at the beginning of CPB (0.99±0.05) mmol/L. There were no significant differences in the levels of intra-erythrocyte[ Ca2+ ] concentration among the three time points[ (138±72) nmol/L vs (145±70) nmol/L vs (115±83) nmol/L]. Conclusion CPB doesn't change the intra-erythrocyte calcium concentrations. Key words: Cardiopulmonary bypass; Erythrocyte; Intracell calcium content

Simultaneous analysis of 13C‐glutathione as its dimeric form GSSG and its precursor [1‐13C]glycine using liquid chromatography/isotope ratio mass spectrometry

Determination of glutathione kinetics using stable isotopes requires accurate measurement of the tracers and tracees. Previously, the precursor and synthesized product were measured with two separate techniques, liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). In order to reduce sample volume and minimize analytical effort we developed a method to simultaneously determine (13)C-glutathione as its dimeric form (GSSG) and its precursor [1-(13)C]glycine in a small volume of erythrocytes in one single analysis. After having transformed (13)C-glutathione into its dimeric form GSSG, we determined both the intra-erythrocytic concentrations and the (13)C-isotopic enrichment of GSSG and glycine in 150 microL of whole blood using liquid chromatography coupled to LC/IRMS. The results show that the concentration (range of micromol/mL) was reliably measured using cycloleucine as internal standard, i.e. with a precision better than 0.1 micromol/mL. The (13)C-isotopic enrichment of GSSG and glycine measured in the same run gave reliable values with excellent precision (standard deviation (sd) <0.3 per thousand) and accuracy (measured between 0 and 5 APE). This novel method opens up a variety of kinetic studies with relatively low dose administration of tracers, reducing the total cost of the study design. In addition, only a minimal sample volume is required, enabling studies even in very small subjects, such as preterm infants.

Thiopurine S-Methyltransferase (TPMT) Gene Polymorphism in Brazilian Children With Acute Lymphoblastic Leukemia: Association With Clinical and Laboratory Data

The frequency of allele variants of gene TPMT*2, *3A, *3B, and *3C was estimated in a population of 116 Brazilian children with acute lymphoblastic leukemia. The association between genotype and clinical and laboratory data obtained during chemotherapy maintenance phase and the correlation of intraerythrocyte concentration of 6-mercaptopurine metabolites (6-tioguanine nucleotide nucleotides and methylmercaptopurine) were analyzed. A multiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was used in DNA amplification. Twelve patients presented TPMT gene mutation, all in heterozygous form. The most frequent allele variation was TPMT*3A (3.9%), followed by *3C (0.9%), *2 (0.4%), and *3B (0%). There was no significant association between clinical and laboratory data and the presence of mutation in TPMT gene. Of the 36 patients who were monitored for 6-mercaptopurine metabolite levels, only 1 had the mutation. In this patient, high 6-tioguanine nucleotide and low methylmercaptopurine concentrations were found. Event-free survival (EFS) for the whole group was 73.4%. There was no significant difference in event-free survival in the comparison between the groups with and without mutation (P = 0.06).

Oxidative stress and intraerythrocytic concentrations of sodium and potassium in diabetic dogs

Oxidative stress parameters; thiobarbituric acid reaction substances (RBC-TBARS), catalase (RBC-CAT) and reduced glutathione (RBC-GSH) and the intraerythrocytic concentrations of electrolytes; sodium and potassium (RBC-Na and RBC-K) were determined in 18 well- controlled (WC) and 22 poorly-controlled diabetic mellitus (DM). Dogs with DM had significant higher blood glucose concentration (P < 0.001), haemoglobin A1c (P < 0.01) and fructosamine (P < 0.001) compared to normal healthy dogs (n = 19). Diabetic dogs in both groups had higher RBC-CAT (P < 0.05) while RBC-TBARS were higher significantly only in poorly-controlled DM group (P < 0.05). The RBC-K was significantly higher in both DM groups (P < 0.001). No changes in RBC-GSH and RBC-Na were found between DM and control healthy dogs. By linear regression analysis, the relationship were found between degree of diabetic mellitus and RBC-CAT, RBC-TBARS, RBC-Na and RBC-K. The relationship was also found between oxidative stress parameters and intraerythrocytic K+. The results suggest that in diabetic dogs, oxidative stress occurs which related to the severity of disease and may affect potassium homeostasis.

Novel method for measurement of glutathione kinetics in neonates using liquid chromatography coupled to isotope ratio mass spectrometry

A novel analytical method using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) was developed for measuring the fractional synthesis rate (FSR) of glutathione (GSH) in neonates after infusion of [1-(13)C]-glycine as a tracer. After transformation of GSH into GSSG, its dimeric form, the intra-erythrocytic concentration and (13)C-isotopic enrichment of GSH were determined using 200 microL of blood. The results showed that, using LC/IRMS, the concentration (range of micromol/mL) was reliably measured using norvaline as internal standard with precision better than 0.1 micromol/mL. In addition, the (13)C-isotopic enrichment measured in the same run gave reliable values with excellent precision (with standard deviation (sd) lower than 0.3 per thousand) and accuracy (measured between 0 and 2 Atom % Excess (APE)). The inter-assay repeatability of delta(13)C of norvaline used as internal standard with in vivo samples was assessed at -26.07 +/- 0.28 per thousand with coefficient of variance (CV) at 1.1%. The FSR calculated either with GSH or GSSG showed similar results with slightly higher values for GSSG (41.6 +/- 4.7 and 46.5 +/- 4.4, respectively). The slightly lower FSR of GSH is probably due to interfering compounds in the biological matrix. Successfully used in a clinical study, this rapid and reliable method opens up a variety of kinetic studies with relatively low administration of tracer infusates, reducing the total cost of the study design. The small volume of blood needed enables studies even in extremely small subjects, such as premature infants, as reported in this study.

Changes in arterial, mixed venous and intraerythrocytic concentrations of ions in supramaximally exercising horses

Horses experience major perturbations in acid-base balance during supramaximal exercise. Ion movement in and out of erythrocytes (RBCs) is believed to be important in maintaining acid-base balance but it is unclear as to the extent to which this happens, nor how it affects single measurements of ion concentrations in arterial and venous blood. To clarify the role RBCs play in mitigating perturbations in acid-base balance during high speed exercise in horses, and to describe associated differences in arterial (a) and mixed venous (v) concentrations of key ions. Six exercise-trained Thoroughbreds galloped to fatigue at speeds calculated to have an oxygen demand that was 115% of the VO2max. Blood samples (a and v) were collected pre-exercise, during warm-up, at fatigue, and immediately post exercise. Packed cell volume (PCV), pH, PCO2, and plasma concentrations of bicarbonate (HCOP3-), chloride (Cl-), sodium (Na+), potassium (K+), and lactate (Lac-) and strong ion difference (SID) were determined, and RBC concentrations of Lac- and electrolytes calculated for each sample. Data were analysed using a 2-way ANOVA for repeated measures testing for effects of sampling time and site (P<0.05). Plasma and RBC [Cl-] were increased with hypercapnoea and acidaemia. [HCO3-]v was greater than pre-exercise values at fatigue, although [HCO3l]a was lower. Hyperkalaemia and decreased RBC [K+] were evident at fatigue, as was an increased RBC [Na+]. Plasma [K+] started to decrease as soon as exercise ceased and Na+ began to move back onto RBCs in exchange for K+. Concentrations of all measures of Lac- rose from fatigue to post exercise. The SID decreased with exercise and was higher in v at fatigue and post exercise, reflecting the decrease in pH. RBCs act as a repository for lactate, and therefore the increase in PCV facilitates the maintenance of the muscle to plasma Lac- diffusion gradient during exercise. This serves to keep intramuscular [Lac-] lower than it would otherwise be and, because of the link between Lac- accumulation, pH decrease and the onset of fatigue, may help delay the onset of fatigue.

Hemoglobin autoxidation and regulation of endogenous H 2O 2 levels in erythrocytes

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H 2O 2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H 2O 2 and superoxide in the erythrocyte were used to estimate H 2O 2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 × 10 −7 s −1 and an endogenous H 2O 2 and superoxide concentrations of 5 × 10 −11 and 5 × 10 −13 M, respectively, lower than previous estimates.