ZOOMICS: comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days

The use of omics technologies in human transfusion medicine has improved our understanding of the red blood cell (RBC) storage lesion(s). Despite significant progress towards understanding the storage lesion(s) of human RBCs, a comparison of basal and post-storage RBC metabolism across multiple species using omics technologies has not yet been reported, and is the focus of this study

AltitudeOmics: Red Blood Cell metabolic adaptation to high altitude hypoxia

Red blood cells (RBCs) are key players in systemic oxygen transport. RBCs respond to in vitro hypoxia  through  the so-called  oxygen-dependent  metabolic  regulation,  which  involves  the competitive  binding  of  deoxyhemoglobin  and  glycolytic  enzymes  to  the  N-terminal  cytosolic domain  of  band  3.  This  mechanism  promotes  the  accumulation  of  2,3-DPG,  stabilizing  the deoxygenated state of hemoglobin, and cytosol acidification, triggering oxygen off-loading through the  Bohr  effect.  Despite  in  vitro  studies,  in  vivo adaptations  to  hypoxia  have  not  yet  been completely elucidated. Within  the  framework  of  the AltitudeOmics  study,  erythrocytes  were  collected  from  21 healthy volunteers at sea level, after exposure to high altitude (5260m) for 1, 7 and 16days, and following  reascent  after  7days  at 1525m.  UHPLC-MS  metabolomics  results  were  correlated  to physiological and athletic performance parameters. Immediate  metabolic  adaptations  were  noted as early as a few hours from ascending  to >5000m, and maintained for 16 days at high altitude.  Consistent with the mechanisms elucidated in vitro, hypoxia promoted glycolysis and deregulated the pentose phosphate pathway, as well purine catabolism, glutathione homeostasis, arginine/nitric oxide and sulphur/H2S metabolism. Metabolic adaptations were preserved one week after descent, consistently with improved physical performances in comparison to the first ascendance, suggesting a mechanism of metabolic memory

Development and validation of an ultra?performance liquid chromatography quadrupole time of flight mass spectrometry method for rapid quantification of free amino acids in human urine

An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-qTOFMS)method using hydrophilic interaction liquid chromatography was developed and validated for simultaneous quantification of 18 free amino acids in urine with a total acquisition time including the column re-equilibration of less than 18 min per sample. This method involves simple sample preparation steps which consisted of 15 times dilution with acetonitrile to give a final composition of 25 % aqueous and 75 % acetonitrile without the need of any derivatization. The dynamic range for our calibration curve is approximately two orders of magnitude (120-fold from the lowest calibration curve point) with good linearity (r2 ? 0.995 for all amino acids). Good separation of all amino acids as well as good intra- and inter-day accuracy (<15 %) and precision (<15 %) were observed using three quality control samples at a concentration of low, medium and high range of the calibration curve. The limits of detection (LOD) and lower limit of quantification of our method were ranging from approximately 1–300 nM and 0.01–0.5 µM, respectively. The stability of amino acids in the prepared urine samples was found to be stable for 72 h at 4 °C, after one freeze thaw cycle and for up to 4 weeks at ?80 °C. We have applied this method to quantify the content of 18 free amino acids in 646 urine samples from a dietary intervention study. We were able to quantify all 18 free amino acids in these urine samples, if they were present at a level above the LOD. We found our method to be reproducible (accuracy and precision were typically <10 % for QCL, QCM and QCH) and the relatively high sample throughput nature of this method potentially makes it a suitable alternative for the analysis of urine samples in clinical setting

Erythrocytes retain hypoxic adenosine response for faster acclimatization upon re-ascent

Faster acclimatization to high altitude upon re-ascent is seen in humans; however, the molecular basis for this enhanced adaptive response is unknown. We report that in healthy lowlanders, plasma adenosine levels are rapidly induced by initial ascent to high altitude and achieved even higher levels upon re-ascent, a feature that is positively associated with quicker acclimatization. Erythrocyte equilibrative nucleoside transporter 1 (eENT1) levels are reduced in humans at high altitude and in mice under hypoxia. eENT1 deletion allows rapid accumulation of plasma adenosine to counteract hypoxic tissue damage in mice. Adenosine signalling via erythrocyte ADORA2B induces PKA phosphorylation, ubiquitination and proteasomal degradation of eENT1. Reduced eENT1 resulting from initial hypoxia is maintained upon re-ascent in humans or re-exposure to hypoxia in mice and accounts for erythrocyte hypoxic memory and faster acclimatization. Our findings suggest that targeting identified purinergic-signalling network would enhance the hypoxia adenosine response to counteract hypoxia-induced maladaptation

Оценка эффективности комбинированного лечения ишемической болезни сердца – аортокоронарное шунтирование, трансплантация аутологичных мононуклеаров костного мозга: результаты рандомизированного, слепого, плацебо контролируемого исследования

Introduction. Despite resounding success in treatment of patients with coronary heart disease (CHD), researchers are yet unable to significantly reduce mortality in this disease. With this in mind, there are ongoing studies everywhere, which are aimed at investigating new techniques in order to boost the efficiency of existing standards. One of such promising techniques is cell/regenerative therapy with autologous bone marrow mononuclear cells (ABMMCs). However, even though ABMMCs have been studied for more than 10 years, there are no unambiguous data yet on several issues. Objective: to evaluate the outcome of ABMMC transplantation during coronary artery bypass grafting (CABG) surgery in combined treatment of CHD. Materials and methods. The data of 408 patients admitted to the clinic from 2013 to 2016 for planned surgical treatment of CHD were analyzed. The work included 117 people based on the design of the study. Patients were randomized in 3 groups: Group 0 (control group) – CABG surgery and intramyocardial injection of 0.9% NaCl solution, Group 1 – CABG surgery and intramyocardial injection of ABMMCs, Group 2 – CABG surgery, intramyocardial and intra-graft injection of ABMMCs. The dynamics was assessed 12 months later – functional class of angina pectoris and heart failure, echocardiography, speckle tracking (assessment of the degree of myocardial deformation), treadmill test, 6-minute walk test, daily ECG monitoring, quality of life questionnaires, coronary angiography. Qualitative indicators were calculated using the Pearson’s chi-squared test and Fisher criteria. Quantitative indicators were calculated using the Kruskal–Wallis and Wilcoxon tests. Factor analysis was used to identify certain severity factors and to study data homogeneity. Discriminant analysis was performed to investigate the leading characteristics that determine differentiation between the groups. For analysis of variance, taking into account various factors, the model of variance analysis for dependent samples – Repeated Measures ANOVA – was used. Results. In the observation groups, an improvement in both systolic and diastolic myocardial function was universally noted. A six-minute walk test showed statistically significant increase in Groups 1 and 2 compared with the control Group 0 – 315.06 ± 17.6 (433.54 ± 20.6), Group 1 – 319.8 ± 24.5 (524.4 ± 28.7), Group 2 – 329.9 ± 25.3 (452.7 ± 29.7) meters. A significant decrease in the functional class of exertional angina pectoris in Groups 1 and 2 was noted unlike in the control group. The percentage of functioning coronary shunts after a 12-month follow-up period was 87.6% in Group 0. In Groups 1 and 2, this ratio was 96.2% and 97.3%, respectively. Predictors of overall effectiveness were identified: smoking, initial diastolic myocardial dysfunction, left ventricular ejection fraction. Conclusion. In addition to surgical treatment of coronary heart disease, ABMMC transplantation can improve myocardial contractility, boost exercise tolerance, and increase the duration of the functioning of coronary shunts at the follow-up period of 12 months. The study showed the need for stage-by-stage analytical calculations with the aim of possible correction of further work.Введение. Несмотря на несомненные успехи в лечении пациентов с ишемической болезнью сердца (ИБС), пока не удается добиться существенного снижения смертности при данном заболевании. Учитывая это, в настоящий момент повсеместно ведутся работы в отношении исследования новых методик с целью увеличить эффективность уже существующих стандартов. Одной из таких перспективных методик является клеточная/регенеративная терапия аутологичными мононуклеарами костного мозга (АМНКМ). Однако несмотря на то что АМНКМ исследуются на протяжении более 10 лет, к настоящему моменту не получено однозначных данных по ряду вопросов. Цель. Провести оценку результатов трансплантации АМНКМ при выполнении операции аортокоронарного шунтирования (АКШ) в комбинированном лечении ИБС. Материалы и методы. Проанализированны данные 408 пациентов, поступивших в клинику с 2013-го по 2016 г. для планового хирургического лечения ИБС. В работу включено 117 человек согласно дизайну исследования. Проведена рандомизация в 3 группы: группа 0 – операция АКШ и интрамиокардиальное введение 0,9% раствора NaCl – контрольная группа, группа 1 – операция АКШ и интрамиокардиальное введение АМНКМ, группа 2 – операция АКШ, интрамиокардиальное и внутришунтовое введение АМНКМ. Через 12 месяцев выполнена оценка динамики – функционального класса стенокардии напряжения и сердечной недостаточности, ЭхоКГ, speckle tracking (оценка степени деформации миокарда), тредмил-теста, теста с 6-минутной ходьбой, суточного мониторирования ЭКГ, опросников качества жизни, коронарографии. Качественные показатели рассчитаны при помощи критериев Пирсона (χ2 ) и Фишера. Количественные показатели – критерии Краскела–Уоллиса и Вилкоксона. Для выявления определенных факторов тяжести и исследования однородности данных – факторный анализ. Для исследования ведущих характеристик, определяющих дифференцировку между группами, проведен дискриминантный анализ. Для анализа дисперсии с учетом различных факторов использовали модель дисперсионного анализа для зависимых выборок – Repeated Measures ANOVA. Результаты. В группах наблюдения повсеместно отмечено улучшение как систолической, так и диастолической функции миокарда. Тест с 6-минутной ходьбой показал статистически значимый прирост в группах 1 и 2 по сравнению с контролем: группа 0 – 315,06 ± 17,6 (433,54 ± 20,6), группа 1 – 319,8 ± 24,5 (524,4 ± 28,7), группа 2 – 329,9 ± 25,3 (452,7 ± 29,7) метра. Отмечено значимое уменьшение функционального класса стенокардии напряжения в группах 1 и 2 по сравнению с контрольной группой. Процент функционирующих коронарных шунтов через 12 месяцев наблюдения составил 87,6% в группе 0, в группах 1 и 2 это соотношение составило 96,2% и 97,3% соответственно. Выявлены предикторы общей эффективности: курение, исходная диастолическая дисфункция миокарда, фракция выброса левого желудочка. Заключение. Трансплантация АМНКМ в дополнение к хирургическому лечению ИБС позволяет улучшить сократительную способность миокарда, увеличить толерантность к физическим нагрузкам, увеличить продолжительность функционирования коронарных шунтов в срок наблюдения 12 месяцев. Проведенное исследование показало необходимость проведения этапных аналитических расчетов с целью возможной коррекции дальнейшего выполнения работы

Metabolic linkage and correlations to storage capacity in erythrocytes from glucose 6-phosphate dehydrogenase-deficient donors

Objective: In glucose 6-phosphate dehydrogenase (G6PD) deficiency, decreased NADPH regeneration in the pentose phosphate pathway and subnormal levels of reduced glutathione result in insufficient antioxidant defense, increased susceptibility of red blood cells (RBCs) to oxidative stress, and acute hemolysis following exposure to pro-oxidant drugs and infections. Despite the fact that redox disequilibrium is a prominent feature of RBC storage lesion, it has been reported that the G6PD-deficient RBCs store well, at least in respect to energy metabolism, but their overall metabolic phenotypes and molecular linkages to the storability profile are scarcely investigated. Methods: We performed UHPLC-MS metabolomics analyses of weekly sampled RBC concentrates from G6PD sufficient and deficient donors, stored in citrate phosphate dextrose/saline adenine glucose mannitol from day 0 to storage day 42, followed by statistical and bioinformatics integration of the data. Results: Other than previously reported alterations in glycolysis, metabolomics analyses revealed bioactive lipids, free fatty acids, bile acids, amino acids, and purines as top variables discriminating RBC concentrates for G6PD-deficient donors. Two-way ANOVA showed significant changes in the storage-dependent variation in fumarate, one-carbon, and sulfur metabolism, glutathione homeostasis, and antioxidant defense (including urate) components in G6PD-deficient vs. sufficient donors. The levels of free fatty acids and their oxidized derivatives, as well as those of membrane-associated plasticizers were significantly lower in G6PD-deficient units in comparison to controls. By using the strongest correlations between in vivo and ex vivo metabolic and physiological parameters, consecutively present throughout the storage period, several interactomes were produced that revealed an interesting interplay between redox, energy, and hemolysis variables, which may be further associated with donor-specific differences in the post-transfusion performance of G6PD-deficient RBCs. Conclusion: The metabolic phenotypes of G6PD-deficient donors recapitulate the basic storage lesion profile that leads to loss of metabolic linkage and rewiring. Donor-related issues affect the storability of RBCs even in the narrow context of this donor subgroup in a way likely relevant to transfusion medicine. © 2018 Reisz, Tzounakas, Nemkov, Voulgaridou, Papassideri, Kriebardis, D&apos;Alessandro and Antonelou

Transfusion of Anaerobically or Conventionally Stored Blood After Hemorrhagic Shock.

BackgroundResuscitation from hemorrhagic shock (HS) by blood transfusion restores oxygen (O2) delivery and provides hemodynamic stability. Current regulations allow red blood cells (RBCs) to be stored and used for up to 42 days. During storage, RBCs undergo many structural and functional changes. These storage lesions have been associated with adverse events and increased mortality after transfusion, increasing the need for improved RBC storage protocols. This study evaluates the efficacy of anaerobically stored RBCs to resuscitate rats from severe HS compared with conventionally stored RBCs.Methods and resultsRat RBCs were stored under anaerobic, anaerobic/hypercapnic, or conventional conditions for a period of 3 weeks. Hemorrhage was induced by controlled bleeding, shock was maintained for 30 min, and RBCs were transfused to restore and maintain blood pressure near the prhemorrhage level. All storage conditions met current regulatory 24-h posttransfusion recovery requirements. Transfusion of anaerobically stored RBCs required significantly less RBC volume to restore and maintain hemodynamics. Anaerobic or anaerobic/hypercapnic RBCs restored hemodynamics better than conventionally stored RBCs. Resuscitation with conventionally stored RBCs impaired indices of left ventricular cardiac function, increased hypoxic tissue staining and inflammatory markers, and affected organ function compared with anaerobically stored RBCs.ConclusionsResuscitation from HS via transfusion of anaerobically stored RBCs recovered cardiac function, restored hemodynamic stability, and improved outcomes