RESUMO
Transfusion effectiveness of red blood cells (RBCs) has been associated with duration of the storage period. Storage-dependent RBC alterations lead to hemolysis and release of toxic free heme, but the increase of free heme levels over time is largely unknown. In the current study, an apo-horseradish peroxidase (apoHRP)-based assay was applied to measure levels of free heme at regular intervals or periodically in supernatants of RBCs until a maximum storage period of 42 days. Free heme levels increased with linear time-dependent kinetics up to day 21 and accelerated disproportionally after day 28 until day 42, as determined with the apoHRP assay. Individual time courses of free heme in different RBC units exhibited high variability. Notably, levels of free hemoglobin, an established indicator of RBC damage, and those of total heme increased with continuous time-dependent linear kinetics over the entire 42 day storage period, respectively. Supernatants from RBC units with high levels of free heme led to inflammatory activation of human neutrophils. In conclusion, determining free heme in stored RBCs with the applied apoHRP assay may become feasible for testing of RBC storage quality in clinical transfusion medicine.
Assuntos
Preservação de Sangue , Heme , Humanos , Peroxidase do Rábano Silvestre , Eritrócitos , HemóliseRESUMO
Cell-free hemoglobin (CFH), a pro-oxidant and cytotoxic compound that is released in hemolysis, has been associated with nephrotoxicity. Lung transplantation (LuTx) is a clinical condition with a high incidence of acute kidney injury (AKI). In this study, we investigated the plasma levels of CFH and haptoglobin, a CFH-binding serum protein, in prospectively enrolled LuTx patients (n = 20) with and without AKI. LuTx patients with postoperative AKI had higher CFH plasma levels at the end of surgery compared with no-AKI patients, and CFH correlated with serum creatinine at 48 h. Moreover, CFH levels inversely correlated with haptoglobin levels, which were significantly reduced at the end of surgery in LuTx patients with AKI. Because multiple other factors can contribute to AKI development in the complex clinical setting of LuTx, we next investigated the role of exogenous CFH administration in a mouse model of mild bilateral renal ischemia reperfusion injury (IRI). Exogenous administration of CFH after reperfusion caused overt AKI with creatinine increase, tubular injury, and enhanced markers of renal inflammation compared with vehicle-treated animals. In conclusion, CFH is a possible factor contributing to postoperative AKI after LuTx and promotes AKI in an experimental model of mild transient renal ischemia. Targeting CFH might be a therapeutic option to prevent AKI after LuTx.
Assuntos
Injúria Renal Aguda , Hemoglobinas , Transplante de Pulmão , Traumatismo por Reperfusão , Animais , Camundongos , Injúria Renal Aguda/diagnóstico , Creatinina/química , Haptoglobinas/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Transplante de Pulmão/efeitos adversos , Reperfusão/efeitos adversos , Traumatismo por Reperfusão/metabolismoRESUMO
Quantitative analysis of oxylipins in blood samples is of increasing interest in clinical studies. However, storage after sampling and transport of blood might induce artificial changes in the apparent oxylipin profile due to ex vivo formation/degradation by autoxidation or enzymatic activity. In the present study we investigated the stability of free (i.e. non-esterified) and total oxylipins in EDTA-plasma and serum generated under clinical conditions assessing delays in sample processing and automated transportation: Free cytochrome P450 monooxygenase and 5-lipoxygenase (LOX) formed oxylipins as well as autoxidation products were marginally affected by storage of whole blood up to 4 h at 4 °C, while total (i.e. the sum of free and esterified) levels of these oxylipins were stable up to 24 h and following transport. Cyclooxygenase (COX) products (TxB2, 12-HHT) and 12-LOX derived hydroxy-fatty acids were prone to storage and transport induced changes due to platelet activation. Total oxylipin patterns were generally more stable than the concentration of free oxylipins. In serum, coagulation induced higher levels of COX and 12-LOX products showing a high inter-individual variability. Overall, our results indicate that total EDTA-plasma oxylipins are the most stable blood oxylipin marker for clinical samples. Here, storage of blood before further processing is acceptable for a period up to 24 hours at 4 °C. However, levels of platelet derived oxylipins should be interpreted with caution regarding potential ex vivo formation.
Assuntos
Oxilipinas/sangue , Adulto , Preservação de Sangue , Coleta de Amostras Sanguíneas/métodos , Ácido Edético/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxilipinas/análise , Plasma/química , Soro/química , Adulto JovemRESUMO
We present an optical setup capable of mirroring an arbitrary, potentially time-varying, polarization state of an ultrashort laser pulse. The incident beam is split up in two and the polarization of one beam is mirrored by reflection off a mirror in normal incidence. Afterwards, both beams are recombined in time and space such that two collinear ultrashort laser pulses with mutually mirrored polarization, i.e., laser-pulse enantiomers, leave the setup. We employ the Jones formalism to describe the function of the setup and analyze the influence of alignment errors before describing the experimental implementation and alignment protocol. Since no wave plates are utilized, broadband pulses in a large wavelength range can be processed. In particular, we show that the setup outperforms broadband achromatic wave plates. Furthermore, since the two beams travel separately through the optical system they can be blocked independently. This opens the possibility for circular dichroism, ellipsometry, and anisotropy spectroscopy with shot-to-shot chopping and detection schemes as well as chiral coherent control applications.
RESUMO
Trihalide anions are linear molecules that can be photodissociated with ultraviolet (UV) light. Whereas deep-UV excitation leads to three-body dissociation, for near-UV excitation just one molecular bond is cleaved, which notionally opens up the possibility for different fragmentation patterns. Here, we explore whether the dihalide fragment is formed as an anionic or neutral species and whether heteronuclear trihalides can lead to two different dihalides. The analysis is based on pronounced wavepacket dynamics induced by femtosecond UV pulses and associated both with the initial trihalide and the nascent dihalide species. For the trihalide anions I3-, Br3-, IBr2-, and ICl2- (point group D∞h), as well as for I2Br- and I2Cl- (point group C∞v) in dichloromethane solution, we identify dihalide fragments by their characteristic vibrational wavenumbers, which we achieve from globally fitting the vibrational wavepacket oscillations, considering a wavelength-dependent phase. No signature from neutral species is found right after excitation, hence there is only one diatomic product in D∞h trihalides. For the investigated C∞v trihalides, which could allow a homonuclear and a heteronuclear product, only the homonuclear one is observed. Since dihalide anions are unstable intermediates, their absorption and the ground-state bleach of the trihalide anion show a biexponential recovery for all samples due to recombining fragment pairs. The rate of the electron transfer yielding a neutral dihalogen and an atomic anion, a prerequisite for the recombination, gives rise to the biexponential behavior; fast recombination is mediated by vibrational excess energy, while slow recombination occurs for cooled-down dihalogens. These data reveal the fragmentation and recombination dynamics from a time-domain approach rather than frequency-domain vibrational spectroscopy and contribute to the in-depth comprehension of these versatile model molecules.
RESUMO
Free heme is released from hemoproteins during hemolysis or ischemia reperfusion injury and can be pro-inflammatory. Most studies on nephrotoxicity of hemolysis-derived proteins focus on free hemoglobin (fHb) with heme as a prosthetic group. Measurement of heme in its free, non-protein bound, form is challenging and not commonly used in clinical routine diagnostics. In contrast to fHb, the role of free heme in acute kidney injury (AKI) after cardiopulmonary bypass (CPB) surgery is unknown. Using an apo-horseradish peroxidase-based assay, we identified free heme during CPB surgery as predictor of AKI in patients undergoing cardiac valve replacement (n = 37). Free heme levels during CPB surgery correlated with depletion of hemopexin (Hx), a heme scavenger-protein. In mice, the impact of high levels of circulating free heme on the development of AKI following transient renal ischemia and the therapeutic potential of Hx were investigated. C57BL/6 mice were subjected to bilateral renal ischemia/reperfusion injury for 15 min which did not cause AKI. However, additional administration of free heme in this model promoted overt AKI with reduced renal function, increased renal inflammation, and reduced renal perfusion on functional magnetic resonance imaging. Hx treatment attenuated AKI. Free heme administration to sham operated control mice did not cause AKI. In conclusion, free heme is a predictor of AKI in CPB surgery patients and promotes AKI in transient renal ischemia. Depletion of Hx in CPB surgery patients and attenuation of AKI by Hx in the in vivo model encourage further research on Hx therapy in patients with increased free heme levels during CPB surgery.
Assuntos
Injúria Renal Aguda , Hemopexina , Traumatismo por Reperfusão , Animais , Humanos , Camundongos , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Ponte Cardiopulmonar/efeitos adversos , Heme , Hemoglobinas/metabolismo , Hemólise , Hemopexina/química , Hemopexina/metabolismo , Isquemia/complicações , Rim/metabolismo , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/etiologiaRESUMO
Photochemical reactions in solution often proceed via competing reaction pathways comprising intermediates that capture a solvent molecule. A disclosure of the underlying reaction mechanisms is challenging due to the rapid nature of these processes and the intricate identification of how many solvent molecules are involved. Here combining broadband femtosecond transient absorption and quantum mechanics/molecular mechanics simulations, we show for one of the most reactive species, diphenylcarbene, that the decision-maker is not the nearest solvent molecule but its neighbour. The hydrogen bonding dynamics determine which reaction channels are accessible in binary solvent mixtures at room temperature. In-depth analysis of the amount of nascent intermediates corroborates the importance of a hydrogen-bonded complex with a protic solvent molecule, in striking analogy to complexes found at cryogenic temperatures. Our results show that adjacent solvent molecules take the role of key abettors rather than bystanders for the fate of the reactive intermediate.