Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disorder, characterized clinically by a progressive loss of memory, cognitive function, ability to care for oneself and psychiatric symptoms. First-line agents for the treatment of AD are ChE inhibitors (DON, GAL), whose modest clinical efficacy and the high incidence of dose-limiting toxicities limit their clinical utility. In addition to AD, ChE inhibitors (PYR) are used for other medical conditions, such as myasthenia gravis (MG). Furthermore, ChE inhibitors (PYR) are used by military personnel prophylactically if impending exposure to chemical warfare agents, e.g., soman, is suspected. The purpose of this research project was to understand the effect of age on the in-vitro sensitivity of ChE inhibitors in human RBCs and plasma. Understanding possible covariates, such as age and gender, may assist in optimizing dosing regimens of ChE inhibitors and/or developing newer ChE inhibitors with better adverse effect profiles. Plasma PHYS concentrations were measured by a validated HPLC-FD method. RBC AChE activity and plasma BuChE activity were measured by a modified Ellman’s colorimetric method using the model substrates, acetylthiocholine and butyrylthiocholine, respectively. The kinetics of RBC and plasma ChE activity followed Michaelis-Menten kinetics. Acetylthiocholine was found to be a nonselective substrate (RBC AChE Km = 73 μM; plasma BuChE Km = 117 μM); while butyrylthiocholine was a selective substrate for plasma BuChE (RBC AChE Km = 130,000 μM; plasma BuChE Km = 72 μM). For the following studies, RBC AChE activity was measured using acetylthiocholine as the substrate and plasma BuChE activity was measured using butyrylthiocholine as the substrate. This research project was performed in two parts: First, mechanistic studies of PHYS, PYR, DON and GAL, explored and determined the mechanism of in-vitro inhibition of RBC AChE and plasma BuChE inhibition, as well as the in-vitro degradation of PHYS in human whole blood, plasma and RBC. PHYS was rapidly degraded in human whole blood, RBC and plasma and followed Michaelis-Menten kinetics but its degradation clearance - scaled to whole blood clearance - was only predicted to account for 4-6% (i.e., 195-261 ml/min) of the reported total body clearance for PHYS (4500 ml/min). RBCs were responsible for 60% of the whole blood clearance while plasma accounted for 40% of the whole blood clearance. Inhibition results indicated that both PHYS and PYR were nonselective and rapid suicide ChE inactivators. PYR inactivated RBC AChE more rapidly at low concentrations and inactivated plasma BuChE more rapidly at high concentrations, but inactivated both more rapidly than PHYS. PHYS was a more potent inactivator than PYR with a Ki for RBC AChE of 0.011 μM and 0.063 μM, respectively, and 0.023 μM and 0.036 μM, respectively for plasma BuChE. DON was found to be a noncompetitive inhibitor for RBC AChE (Ki,noncomp = 114 μM), but a competitive inhibitor for plasma BuChE (Ki,comp = 213 μM). GAL was found to be a competitive inhibitor for both RBC AChE (Ki,comp = 66 μM) and plasma BuChE (Ki,comp = 358 μM). The second part involved a clinical study with ten young and nine elderly healthy subjects, balanced for gender, who donated blood for an in-vitro study in order to assess any age- and gender-related differences in in-vitro sensitivity to RBC AChE and plasma BuChE inhibition to all four ChE inhibitors. Elderly adults were found to be 2-3-fold less sensitive compared to the young adults for PHYS (BuChE Ki,pss; 0.010 and 0.015 μM, young and elderly, respectively) and PYR (AChE Ki,pss; 0.12 and 0.25 μM, young and elderly, respectively) only, while neither DON nor GAL showed any age-related differences in sensitivity. The observed differences for PHYS and PYR may be due to kinetic differences in ChE inactivation between young and aged adults, rather then a difference in binding affinities/potencies. These carbamate ChE inhibitors, presumably, have a slower decarbamoylation rate in younger adults than elderly adults, which leads to the observed difference in in-vitro sensitivity. The above in-vitro results were consistent with results of a meta-analysis: In a study by Knapp et al. (1991), young males (n=6), receiving 18 mg, 24 mg and 30 mg PHYS tablets, showed similar ex-vivo plasma BuChE sensitivity to (28 %/(ng/ml)) as the in-vitro sensitivity for young males in the current study (33 %/(ng/ml)). On the other hand, in the study by Men (2004), elderly males (n=8) and females (n=8), receiving 6.7 μg/kg PHYS as 30-minute infusion, showed similar ex-vivo RBC AChE sensitivity (12 %/(ng/ml)) as the in-vitro sensitivity for elderly subjects in the current study (9.7 %/(ng/ml)). This suggests that in-vitro measurement of ChE sensitivity is predictive of ex-vivo sensitivity in clinical studies. The study results suggest that elderly adults may require a 2-3-fold higher blood concentration than young adults to achieve the same ChE inhibition. This may explain why for epistigmine, an investigational carbamate ChE inhibitor for the treatment of AD, the maximum tolerated dose observed in young adults (40 mg single dose) was lower than for older adults (90 mg/day). Higher sensitivity in young adults prevented further dose escalation, while all elderly subjects tolerated higher doses. This research may have implications for other diseases and conditions, most notably MG and as a prophylaxis of nerve gases poisoning. As patients with MG age, they may become less sensitive to PYR, the most common symptomatic treatment for MG, and an increase in dose may be required. Further, older military personnel assigned to receive PYR, may require increased doses to achieve the targeted 10% RBC AChE inhibition, necessary to protect against nerve gas poisoning.

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas. / Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

<p>УВОД: Diabetes mellitus (DM) је водећа глобална епидемија 21. века, сложена болест коју карактерише поремећај метаболизма и хронична хипергликемија, која доводи до развоја микроваскуларних и макроваскуларних компликација. Повишене вредности гликемије у ДМT2 доводе до поремећаја ћелија крви и њихових параметара. Истраживања су показала да хема-толошки параметри имају допринос у настанку оштећења васкуларног ендотела и учествују у развоју дегенеративних промена и путем других механизама код пацијената са дијабетесом. Циљ истраживања је да се утврде могуће промене параметара комплетне крвне слике (ККС) у зависности од гликорегулације, дужине трајања болести и њихова повезаност са микро и макро ангиопатским комликацијама код пацијената са шећерном болести тип 2 (ДМT2). МЕТОДЕ: Студија је била проспективна у трајању од једне године, од 2016. до 2017. године. Истраживањем је обухваћено укупно 137 испитаника, од којих 90 болује од ДМТ2, а 47 је здравих, оба пола, старијих од 40 година, спроведена у Дому здравља &ldquo;Др Милорад Мика Павловић&rdquo;, Инђија, Србија. Да би се утврдила могућа корелација између параметара ККС, гликорегулације у ДМT2 и дегенеративних компликација, испитаници су подељени на више начина: на групу оболелих од ДМT2 и групу здравих; групе оболелих са вредностима HbA1c&le;7% и оне са вредностима HbA1c &gt;7%, као и на групе са и без дегенеративних компликација. Коришћењем стандардних биохемијских поступака анализирани су параметри ККС, параметри гликорегулације, липидни статус, а вршена су и антропометријска мерења. Подаци од пацијената прикупљени су путем упитника и електронског картона пацијента. РЕЗУЛТАТИ: Утврђена је статистички значајна разлика између група са и без шећерне болести за WBC, еозинофилне гранулоците, хемоглобин, MCH, MCHC, SE у 2016. год, неутрофилне гранулоците, моноците, RDW, PDW, SE у 2017. год. У групи чија је дужина трајања ДМT2 већа од 6 година утврђене су повишене вредности MCHC и PDW. У односу на гликорегулацију уочене су значајне разлике у PMDW, великим тромбоцитима и RDW у групи са HbA1c&gt;7%. У односу на дегенеративне микроваскуларне компликације постоје значајне разлике у броју лимфоцита и неутрофилних гранулоцита, а за макроваскуларне компликације у вредностима PDW-а. ЗАКЉУЧАК: На основу добијених резултата нашег истраживања може се закључити да постоји повезаност између појединих хематолошких параметара и гликорегулације, обољевања од шећерне болести као и повезаност са компликацијама код пацијената са ДМТ2.</p> / <p>UVOD: Diabetes mellitus (DM) je vodeća globalna epidemija 21. veka, složena bolest koju karakteriše poremećaj metabolizma i hronična hiperglikemija, koja dovodi do razvoja mikrovaskularnih i makrovaskularnih komplikacija. Povišene vrednosti glikemije u DMT2 dovode do poremećaja ćelija krvi i njihovih parametara. Istraživanja su pokazala da hema-tološki parametri imaju doprinos u nastanku oštećenja vaskularnog endotela i učestvuju u razvoju degenerativnih promena i putem drugih mehanizama kod pacijenata sa dijabetesom. Cilj istraživanja je da se utvrde moguće promene parametara kompletne krvne slike (KKS) u zavisnosti od glikoregulacije, dužine trajanja bolesti i njihova povezanost sa mikro i makro angiopatskim komlikacijama kod pacijenata sa šećernom bolesti tip 2 (DMT2). METODE: Studija je bila prospektivna u trajanju od jedne godine, od 2016. do 2017. godine. Istraživanjem je obuhvaćeno ukupno 137 ispitanika, od kojih 90 boluje od DMT2, a 47 je zdravih, oba pola, starijih od 40 godina, sprovedena u Domu zdravlja &ldquo;Dr Milorad Mika Pavlović&rdquo;, Inđija, Srbija. Da bi se utvrdila moguća korelacija između parametara KKS, glikoregulacije u DMT2 i degenerativnih komplikacija, ispitanici su podeljeni na više načina: na grupu obolelih od DMT2 i grupu zdravih; grupe obolelih sa vrednostima HbA1c&le;7% i one sa vrednostima HbA1c &gt;7%, kao i na grupe sa i bez degenerativnih komplikacija. Korišćenjem standardnih biohemijskih postupaka analizirani su parametri KKS, parametri glikoregulacije, lipidni status, a vršena su i antropometrijska merenja. Podaci od pacijenata prikupljeni su putem upitnika i elektronskog kartona pacijenta. REZULTATI: Utvrđena je statistički značajna razlika između grupa sa i bez šećerne bolesti za WBC, eozinofilne granulocite, hemoglobin, MCH, MCHC, SE u 2016. god, neutrofilne granulocite, monocite, RDW, PDW, SE u 2017. god. U grupi čija je dužina trajanja DMT2 veća od 6 godina utvrđene su povišene vrednosti MCHC i PDW. U odnosu na glikoregulaciju uočene su značajne razlike u PMDW, velikim trombocitima i RDW u grupi sa HbA1c&gt;7%. U odnosu na degenerativne mikrovaskularne komplikacije postoje značajne razlike u broju limfocita i neutrofilnih granulocita, a za makrovaskularne komplikacije u vrednostima PDW-a. ZAKLJUČAK: Na osnovu dobijenih rezultata našeg istraživanja može se zaključiti da postoji povezanost između pojedinih hematoloških parametara i glikoregulacije, oboljevanja od šećerne bolesti kao i povezanost sa komplikacijama kod pacijenata sa DMT2.</p> / <p>BACKGROUND: Diabetes mellitus (DM) is the leading global epidemic of the 21st century,a complex disease characterized by metabolism disorders and chronic hyperglycaemia, that leads to the development of microvascular and macrovascular complications. Elevated blood glucose level in T2DM lead to disturbance of blood cells and its parameters. Previous studies have reported that haematological parameters have contributed to the development of vascular endothelial damage and are involved in the development of degenerative changes through other mechanisms in patients with diabetes. The aim of the research is to determine possible changes in the complete blood count (CBC) parameters depending on glycemic control, the duration of the disease and their association with micro and macroangiopathic complications in patients with Type 2 diabetes mellitus (T2DM). METHODS: The study was prospective from 2016. to 2017. year. The study included a total of 137 subjects, 90 with T2DM and 47 healthy, of both gender over the age of 40 years, from the Health Care Center &quot;Dr Milorad Mika Pavlović&quot; Indjija, Serbia. The subjects were divided into several ways, in order to notice the possible correlation between the CBC parameters and glucose control in T2DM, with and without T2DM, two groups with HbA1c&le;7% and with HbA1c&gt;7%, and with and without complications. We analysed CBC parameters, parameters of glycoregulation, lipid status using standard biochemical methods, performed anthropometric measurements and collected patients data by questionnaire and electronic patient card. RESULTS: There were statistical difference between group with T2DM and healthy subjects for WBC, eos, Hgb, MCH, MCHC, ESR in 2016. and neutro, mono, RDW, PDW, ESR in 2017. In the group of T2DM patients with duration of disease longer than 6 years we found elevated value of MCHC, PDW. In relationship to glycoregulation, significant differences in PMDW, large platelets and RDW were found in the group HbA1c&gt;7%. According to degenerative complications significant differences were revealed in lympho, neutro in the group with microvascular complication, and PDW in the group with macrovascular complications. CONCLUSION: Based on the resuluts of our research, it can be concluded that there is an association between particular haematological parameters and glycoregulation, diabetes mellitus, as well as relationship with degenerative complications in patients with T2DM.</p>

<p>Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. </p><p>Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state <i>in vitro</i> and <i>in vivo</i>. </p><p>Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p>

<p>The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.</p><p>Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.</p>

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods. Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo. Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids. An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

L’anémie est fréquente chez les patients pédiatriques en postopératoire de chirurgie cardiaque. Malgré le nombre important de patients transfusés, le taux d’hémoglobine (Hb) pour lequel les bénéfices surpassent les risques est inconnu chez ces patients. Récemment, Lacroix et al. ont démontré qu’une stratégie transfusionnelle restrictive n’était pas inférieure à une stratégie libérale en ce qui concerne le développement ou la progression du syndrome de défaillance multiviscérale (SDMV) et la mortalité chez les patients de soins intensifs pédiatriques (SIP).Devant le manque d’évidence, une analyse de sous-groupes des patients en postopératoire de chirurgie cardiaque de l’étude Transfusion Requirements in Pediatric Intensive Care (TRIPICU) a été réalisée. L’objectif de cette étude était de déterminer l’impact d’une stratégie transfusionnelle restrictive comparée à une stratégie libérale sur l’acquisition ou l’aggravation du syndrome de défaillance multiviscérale (SDMV) chez les enfants en postopératoire de chirurgie cardiaque. Cette étude n’a pas démontré de différences statistiquement, ni cliniquement significatives du nombre de patients ayant acquis ou aggravés un SDMV, ni des issues secondaires entre les stratégies transfusionnelles restrictive et libérale. L’analyse de sous-groupes permet de générer une hypothèse de recherche et les résultats devraient être confirmés par un essai randomisé contrôlé. / Anemia is frequent in pediatric patients following cardiac surgery. Despite frequent transfusions, the optimal hemoglobin threshold where benefits surpass risks is still unknown for these patients. Recently, Lacroix et al. showed that a restrictive transfusion strategy was not inferior to a liberal strategy concerning the development or progression of multiple organ dysfunction syndrome (MODS) and mortality in pediatric intensive care patients. In the absence of evidence, the aim of this study was to determine the impact of a restrictive versus a liberal transfusion strategy on new or progressive multiple organ dysfunction syndrome (MODS) in children following cardiac surgery. We conducted a subgroup analysis of the postoperative cardiac surgery patients of the Transfusion Requirements in Pediatric Intensive Care Unit (TRIPICU) study. Our study showed no statistically and clinically significant differences in the number of patients who acquired or worsened MODS, nor secondary outcomes between a restrictive and a liberal transfusion strategy. This subgroup analysis generates a research hypothesis that should be confirmed by a randomized controlled trial.

L’anémie est fréquente chez les patients pédiatriques en postopératoire de chirurgie cardiaque. Malgré le nombre important de patients transfusés, le taux d’hémoglobine (Hb) pour lequel les bénéfices surpassent les risques est inconnu chez ces patients. Récemment, Lacroix et al. ont démontré qu’une stratégie transfusionnelle restrictive n’était pas inférieure à une stratégie libérale en ce qui concerne le développement ou la progression du syndrome de défaillance multiviscérale (SDMV) et la mortalité chez les patients de soins intensifs pédiatriques (SIP).Devant le manque d’évidence, une analyse de sous-groupes des patients en postopératoire de chirurgie cardiaque de l’étude Transfusion Requirements in Pediatric Intensive Care (TRIPICU) a été réalisée. L’objectif de cette étude était de déterminer l’impact d’une stratégie transfusionnelle restrictive comparée à une stratégie libérale sur l’acquisition ou l’aggravation du syndrome de défaillance multiviscérale (SDMV) chez les enfants en postopératoire de chirurgie cardiaque. Cette étude n’a pas démontré de différences statistiquement, ni cliniquement significatives du nombre de patients ayant acquis ou aggravés un SDMV, ni des issues secondaires entre les stratégies transfusionnelles restrictive et libérale. L’analyse de sous-groupes permet de générer une hypothèse de recherche et les résultats devraient être confirmés par un essai randomisé contrôlé. / Anemia is frequent in pediatric patients following cardiac surgery. Despite frequent transfusions, the optimal hemoglobin threshold where benefits surpass risks is still unknown for these patients. Recently, Lacroix et al. showed that a restrictive transfusion strategy was not inferior to a liberal strategy concerning the development or progression of multiple organ dysfunction syndrome (MODS) and mortality in pediatric intensive care patients. In the absence of evidence, the aim of this study was to determine the impact of a restrictive versus a liberal transfusion strategy on new or progressive multiple organ dysfunction syndrome (MODS) in children following cardiac surgery. We conducted a subgroup analysis of the postoperative cardiac surgery patients of the Transfusion Requirements in Pediatric Intensive Care Unit (TRIPICU) study. Our study showed no statistically and clinically significant differences in the number of patients who acquired or worsened MODS, nor secondary outcomes between a restrictive and a liberal transfusion strategy. This subgroup analysis generates a research hypothesis that should be confirmed by a randomized controlled trial.

Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas. / Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.