Donor variation in stored platelets: Higher metabolic rates of platelets are associated with mean platelet volume, activation and donor health.

BACKGROUND: Platelets (PLTs) differ in glycolytic activity, resulting in rapid acidification of 'poor' storing PLT concentrates (PCs) in plasma, or depletion of glucose when stored in PLT additive solution (PAS). We aimed to understand why PLT glycolysis rates vary between donors and how this affects storage performance. STUDY DESIGN AND METHODS: Buffy coats from donors <45, 45-70 and >70 years were selected and single-donor PCs in plasma or PAS-E were prepared. PCs were stored for 8 days at 22 ± 2°C and sampled regularly for analysis. Mitochondrial activity was analyzed with an Oroboros oxygraph. Age groups, or subgroups divided into quartiles based on glucose consumption, were analyzed with ANOVA. RESULTS: In each comparison, PCs of the different groups were not different in volume and cellular composition. PLTs with the highest glucose consumption had a higher initial mean platelet volume (MPV) and developed higher CD62P expression and Annexin A5 binding during storage. Higher glycolytic activity in these PLTs was not a compensation for lower mitochondrial ATP production, because mitochondrial ATP-linked respiration of fresh PLTs correlated positively with MPV (R2  = 0.71). Donors of high glucose-consuming PLTs had more health-related issues. Storage properties of PCs from donors over 70 were not significantly different compared to PCs from donors younger than 45 years. CONCLUSIONS: High glucose-consuming PCs developing higher activation levels, not only displayed enhanced mitochondrial activity but were also found to contain larger PLTs, as determined by MPV. Storage performance of PLTs was found to be associated with donor health, but not with donor age.

Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Hepatitis E virus (HEV) infection has emerged as a global health problem. However, no approved medication is available, and the infection biology remains largely elusive. Electron transport chain (ETC), a key component of the mitochondria, is the main site that produces ATP and reactive oxygen species (ROS). By profiling the role of the different complexes of the mitochondrial ETC, we found that pharmacological inhibition of complex III, a well-defined drug target for the treatment of malaria and Pneumocystis pneumonia, potently restricts HEV replication. This effect demonstrated in our HEV models is equivalent to the anti-HEV potency of ribavirin, a widely used off-label treatment for patients with chronic HEV. Mechanistically, we found that this effect is independent of ATP production, ROS level, and pyridine depletion. By using pharmacological inhibitors and genetic approaches, we found that mitochondrial permeability transition pore (MPTP), a newly identified component of ETC, provides basal defense against HEV infection. HEV interferes with the opening of the MPTP. Furthermore, inhibition of the MPTP attenuated the anti-HEV effect of complex III inhibitors, suggesting that the MPTP mediates the antiviral effects of these inhibitors. These findings reveal new insights on HEV-host interactions and provide viable anti-HEV targets for therapeutic development.-Qu, C., Zhang, S., Wang, W., Li, M., Wang, Y., van der Heijde-Mulder, M., Shokrollahi, E., Hakim, M. S., Raat, N. J. H., Peppelenbosch, M. P., Pan, Q. Mitochondrial electron transport chain complex III sustains hepatitis E virus replication and represents an antiviral target.

Cross Talk between Nucleotide Synthesis Pathways with Cellular Immunity in Constraining Hepatitis E Virus Replication.

Viruses are solely dependent on host cells to propagate; therefore, understanding virus-host interaction is important for antiviral drug development. Since de novo nucleotide biosynthesis is essentially required for both host cell metabolism and viral replication, specific catalytic enzymes of these pathways have been explored as potential antiviral targets. In this study, we investigated the role of different enzymatic cascades of nucleotide biosynthesis in hepatitis E virus (HEV) replication. By profiling various pharmacological inhibitors of nucleotide biosynthesis, we found that targeting the early steps of the purine biosynthesis pathway led to the enhancement of HEV replication, whereas targeting the later step resulted in potent antiviral activity via the depletion of purine nucleotide. Furthermore, the inhibition of the pyrimidine pathway resulted in potent anti-HEV activity. Interestingly, all of these inhibitors with anti-HEV activity concurrently triggered the induction of antiviral interferon-stimulated genes (ISGs). Although ISGs are commonly induced by interferons via the JAK-STAT pathway, their induction by nucleotide synthesis inhibitors is completely independent of this classical mechanism. In conclusion, this study revealed an unconventional novel mechanism of cross talk between nucleotide biosynthesis pathways and cellular antiviral immunity in constraining HEV infection. Targeting particular enzymes in nucleotide biosynthesis represents a viable option for antiviral drug development against HEV. HEV is the most common cause of acute viral hepatitis worldwide and is also associated with chronic hepatitis, especially in immunocompromised patients. Although often an acute and self-limiting infection in the general population, HEV can cause severe morbidity and mortality in certain patients, a problem compounded by the lack of FDA-approved anti-HEV medication available. In this study, we have investigated the role of the nucleotide synthesis pathway in HEV infection and its potential for antiviral drug development. We show that targeting the later but not the early steps of the purine synthesis pathway exerts strong anti-HEV activity. In particular, IMP dehydrogenase (IMPDH) is the most important anti-HEV target of this cascade. Importantly, the clinically used IMPDH inhibitors, including mycophenolic acid and ribavirin, have potent anti-HEV activity. Furthermore, targeting the pyrimidine synthesis pathway also exerts potent antiviral activity against HEV. Interestingly, antiviral effects of nucleotide synthesis pathway inhibitors appear to depend on the medication-induced transcription of antiviral interferon-stimulated genes. Thus, this study reveals an unconventional novel mechanism as to how nucleotide synthesis pathway inhibitors can counteract HEV replication.

Cutaneous Mitochondrial PO2, but Not Tissue Oxygen Saturation, Is an Early Indicator of the Physiologic Limit of Hemodilution in the Pig.

BACKGROUND: Hemodilution is a consequence of fluid replacement during blood loss and is limited by the individual ability to compensate for decreasing hemoglobin level. We tested the ability of a novel noninvasive method for measuring cutaneous mitochondrial PO2 (mitoPO2) to detect this threshold early. METHODS: Anesthetized and ventilated pigs were hemodynamically monitored and randomized into a hemodilution (n = 12) or a time control (TC) group (n = 14). MitoPO2 measurements were done by oxygen-dependent delayed fluorescence of protoporphyrin IX after preparation of the skin with 20% 5-aminolevulinic acid cream. Tissue oxygen saturation (StO2) was measured with near infrared spectroscopy on the thoracic wall. After baseline measurements, progressive normovolemic hemodilution was performed in the hemodilution group in equal steps (500 ml blood replaced by 500 ml Voluven; Fresenius Kabi AG, Germany). Consecutive measurements were performed after 20-min stabilization periods and repeated 8 times or until the animal died. RESULTS: The TC animals remained stable with regard to hemodynamics and mitoPO2. In the hemodilution group, mitoPO2 became hemoglobin-dependent after reaching a threshold of 2.6 ± 0.2 g/dl. During hemodilution, hemoglobin and mitoPO2 decreased (7.9 ± 0.2 to 2.1 ± 0.2 g/dl; 23.6 ± 2 to 9.9 ± 0.8 mmHg), but StO2 did not. Notably, mitoPO2 dropped quite abruptly (about 39%) at the individual threshold. We observed that this decrease in mitoPO2 occurred at least one hemodilution step before changes in other conventional parameters. CONCLUSIONS: Cutaneous mitoPO2 decreased typically one hemodilution step before occurrence of significant alterations in systemic oxygen consumption and lactate levels. This makes mitoPO2 a potential early indicator of the physiologic limit of hemodilution and possibly a physiologic trigger for blood transfusion.

Non-invasive monitoring of mitochondrial oxygenation and respiration in critical illness using a novel technique.

INTRODUCTION: Although mitochondrial dysfunction is proposed to be involved in the pathophysiology of sepsis, conflicting results are reported. Variation in methods used to assess mitochondrial function might contribute to this controversy. A non-invasive method for monitoring mitochondrial function might help overcome this limitation. Therefore, this study explores the possibility of in vivo monitoring of mitochondrial oxygen tension (mitoPO2) and local mitochondrial oxygen consumptionin in an endotoxin-induced septic animal model. METHODS: Animals (rats n = 28) were assigned to a control group (no treatment), or to receive lipopolysaccharide without fluid resuscitation (LPS-NR) or lipopolysaccharide plus fluid resuscitation (LPS-FR). Sepsis was induced by intravenous LPS injection (1.6 mg/kg during 10 min), fluid resuscitation was performed by continuous infusion of a colloid solution, 7 ml kg(-1) h(-1) and a 2-ml bolus of the same colloid solution. MitoPO2 and ODR were measured by means of the protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT). Kinetic aspects of the drop in mitoPO2 were recorded during 60s of skin compression. ODR was derived from the slope of the mitoPO2 oxygen disappearance curve. Measurements were made before and 3 h after induction of sepsis. RESULTS: At baseline (t0) all rats were hemodynamically stable. After LPS induction (t1), significant (p < 0.05) hemodynamic changes were observed in both LPS groups. At t0, mitoPO2 and ODR were 59 ± 1 mmHg, 64 ± 3 mmHg, 68 ± 4 mmHg and 5.0 ± 0.3 mmHg s(-1), 5.3 ± 0.5 mmHg s(-1), 5.7 ± 0.5 mmHg s(-1) in the control, LPS-FR and LPS-NR groups, respectively; at t1 these values were 58 ± 5 mmHg, 50 ± 2.3 mmHg, 30 ± 3.3 mmHg and 4.5 ± 0.5 mmHg s(-1), 3.3 ± 0.3 mmHg s(-1), 1.8 ± 0.3 mmHg s(-1), respectively. At t1, only mitoPO2 showed a significant difference between the controls and LPS-NR. In contrast, at t1 both LPS groups showed a significantly lower ODR compared to controls. CONCLUSION: These data show the feasibility to monitor alterations in mitochondrial oxygen consumption in vivo by PpIX-TSLT in a septic rat model. These results may contribute to the development of a clinical device to monitor mitochondrial function in the critically ill.

Cutaneous mitochondrial respirometry: non-invasive monitoring of mitochondrial function.

The recently developed technique for measuring cutaneous mitochondrial oxygen tension (mitoPO2) by means of the Protoporphyrin IX-Triplet State Lifetime Technique (PpIX-TSLT) provides new opportunities for assessing mitochondrial function in vivo. The aims of this work were to study whether cutaneous mitochondrial measurements reflect mitochondrial status in other parts of the body and to demonstrate the feasibility of the technique for potential clinical use. The first part of this paper demonstrates a correlation between alterations in mitochondrial parameters in skin and other tissues during endotoxemia. Experiments were performed in rats in which mitochondrial dysfunction was induced by a lipopolysaccharide-induced sepsis (n = 5) and a time control group (n = 5). MitoPO2 and mitochondrial oxygen consumption (mitoVO2) were measured using PpIX-TSLT in skin, liver and buccal mucosa of the mouth. Both skin and buccal mucosa show a significant mitoPO2-independent decrease (P < 0.05) in mitoVO2 after LPS infusion (a decrease of 37 and 39% respectively). In liver both mitoPO2 and mitoVO2 decreased significantly (33 and 27% respectively). The second part of this paper describes the clinical concept of monitoring cutaneous mitochondrial respiration in man. A first prototype of a clinical PpIX-TSLT monitor is described and its usability is demonstrated on human skin. We expect that clinical implementation of this device will greatly contribute to our understanding of mitochondrial oxygenation and oxygen metabolism in perioperative medicine and in critical illness. Our ultimate goal is to develop a clinical monitor for mitochondrial function and the current results are an important step forward.

Measuring Mitochondrial Oxygen Tension during Red Blood Cell Transfusion in Chronic Anemia Patients: A Pilot Study.

In light of the associated risks, the question has been raised whether the decision to give a blood transfusion should solely be based on the hemoglobin level. As mitochondria are the final destination of oxygen transport, mitochondrial parameters are suggested to be of added value. The aims of this pilot study were to investigate the effect of a red blood cell transfusion on mitochondrial oxygenation as measured by the COMET device in chronic anemia patients and to explore the clinical usability of the COMET monitor in blood transfusion treatments, especially the feasibility of performing measurements in an outpatient setting. To correct the effect of volume load on mitochondrial oxygenation, a red blood cell transfusion and a saline infusion were given in random order. In total, 21 patients were included, and this resulted in 31 observations. If patients participated twice, the order of infusion was reversed. In both the measurements wherein a blood transfusion was given first and wherein 500 mL of 0.9% saline was given first, the median mitochondrial oxygen tension decreased after red blood cell transfusion. The results of this study have strengthened the need for further research into the effect of blood transfusion tissue oxygenation and the potential role of mitochondrial parameters herein.

Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion.

BACKGROUND: Intravascular red cell hemolysis impairs nitric oxide (NO)-redox homeostasis, producing endothelial dysfunction, platelet activation, and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or microparticles and hemolysis, which we hypothesized could impair vascular function and contribute to the putative storage lesion of banked blood. METHODS AND RESULTS: We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell-free and microparticle-encapsulated hemoglobin, which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator NO. Using stopped-flow spectroscopy and laser-triggered NO release from a caged NO compound, we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies, we show that hemoglobin, even at concentrations below 10 µmol/L (in heme), produces potent vasoconstriction when infused into the rat circulation, whereas controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red blood cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage-related hemolysis. CONCLUSIONS: The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis.

Validation of the protoporphyrin IX-triplet state lifetime technique for mitochondrial oxygen measurements in the skin.

Mitochondrial oxygen tension can be measured in vivo by means of oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Here we demonstrate that mitochondrial PO(2) (mitoPO(2)) can be measured in the skin of a rat after topical application of the PpIX precursor 5-aminolevulinic acid (ALA). Calibration of mitoPO(2) measurements was done by comparison with simultaneous measurements of the cutaneous microvascular PO(2) This was done under three different conditions: in normal skin tissue, in nonrespiration skin tissue due to the application of cyanide, and in anoxic skin tissue after the ventilation with 100% nitrogen. The results of this study show that it is feasible to measure the mitoPO(2) after the topical application of ALA cream by means of the PpIX-triplet state lifetime technique.

In Vivo and Ex Vivo Mitochondrial Function in COVID-19 Patients on the Intensive Care Unit.

Mitochondrial dysfunction has been linked to disease progression in COVID-19 patients. This observational pilot study aimed to assess mitochondrial function in COVID-19 patients at intensive care unit (ICU) admission (T1), seven days thereafter (T2), and in healthy controls and a general anesthesia group. Measurements consisted of in vivo mitochondrial oxygenation and oxygen consumption, in vitro assessment of mitochondrial respiration in platelet-rich plasma (PRP) and peripheral blood mononuclear cells (PBMCs), and the ex vivo quantity of circulating cell-free mitochondrial DNA (mtDNA). The median mitoVO2 of COVID-19 patients on T1 and T2 was similar and tended to be lower than the mitoVO2 in the healthy controls, whilst the mitoVO2 in the general anesthesia group was significantly lower than that of all other groups. Basal platelet (PLT) respiration did not differ substantially between the measurements. PBMC basal respiration was increased by approximately 80% in the T1 group when contrasted to T2 and the healthy controls. Cell-free mtDNA was eight times higher in the COVID-T1 samples when compared to the healthy controls samples. In the COVID-T2 samples, mtDNA was twofold lower when compared to the COVID-T1 samples. mtDNA levels were increased in COVID-19 patients but were not associated with decreased mitochondrial O2 consumption in vivo in the skin, and ex vivo in PLT or PBMC. This suggests the presence of increased metabolism and mitochondrial damage.

Non-invasive versus ex vivo measurement of mitochondrial function in an endotoxemia model in rat: Toward monitoring of mitochondrial therapy.

Mitochondrial function has been predominantly measured ex vivo. Due to isolation and preservation procedures ex vivo measurements might misrepresent in vivo mitochondrial conditions. Direct measurement of in vivo mitochondrial oxygen tension (mitoPO2) and oxygen disappearance rate (ODR) with the protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) might increase our understanding of mitochondrial dysfunction in the pathophysiology of acute disease. LPS administration decreased mitochondrial respiration (ODR) in vivo but did not alter mitochondrial function as assessed with ex vivo techniques (high resolution respirometry and specific complex determinations). PpIX-TSLT measures in vivo mitoPO2 and ODR and can be applied non-invasively at the skin.

Rejuvenation of stored human red blood cells reverses the renal microvascular oxygenation deficit in an isovolemic transfusion model in rats.

BACKGROUND: Storage of red blood cells (RBCs) results in various biochemical changes, including a decrease in cellular adenosine triphosphate and 2,3-diphosphoglycerate acid. Previously it was shown that stored human RBCs show a deficit in the oxygenation of the microcirculation in the gut of anesthetized rats. In this study, the effect of RBCs on rat kidney oxygenation and the effect of rejuvenation of stored RBCs on their ability to deliver oxygen were investigated. STUDY DESIGN AND METHODS: Washed RBCs, derived from leukoreduced RBCs stored in saline-adenine-glucose-mannitol, were tested in an isovolemic transfusion model in rats after hemodilution until 30 percent hematocrit (Hct). The cells were derived from RBCs stored for up 3 days or from RBCs stored for 5 to 6 weeks with or without incubation in Rejuvesol to rejuvenate the cells. Renal microvascular oxygen concentrations (microPO(2)) were determined by Pd-porphyrin phosphorescence lifetime measurements. RESULTS: Isovolemic transfusion exchange of 5- to 6-week-stored RBCs resulted in a significantly larger decrease in renal microPO(2) than RBCs stored for up to 3 days: 16.1 +/- 2.3 mmHg versus 7.1 +/- 1.5 mmHg, respectively (n = 5). Rejuvenation of stored RBCs completely prevented this deficit in kidney oxygenation. The differences in oxygen delivery were not due to different recoveries of the human RBCs in the rat circulation. CONCLUSION: This study shows that the storage-induced deficit of human RBCs to oxygenate the rat kidney microcirculation at reduced Hct is completely reversible. Prevention of metabolic changes during storage is therefore a valid approach to prevent this deficit.

Influence of fluid resuscitation on renal microvascular PO2 in a normotensive rat model of endotoxemia.

INTRODUCTION: Septic renal failure is often seen in the intensive care unit but its pathogenesis is only partly understood. This study, performed in a normotensive rat model of endotoxemia, tests the hypotheses that endotoxemia impairs renal microvascular PO2 (microPO2) and oxygen consumption (VO2,ren), that endotoxemia is associated with a diminished kidney function, that fluid resuscitation can restore microPO2, VO2,ren and kidney function, and that colloids are more effective than crystalloids. METHODS: Male Wistar rats received a one-hour intravenous infusion of lipopolysaccharide, followed by resuscitation with HES130/0.4 (Voluven), HES200/0.5 (HES-STERIL 6%) or Ringer's lactate. The renal microPO2 in the cortex and medulla and the renal venous PO2 were measured by a recently published phosphorescence lifetime technique. RESULTS: Endotoxemia induced a reduction in renal blood flow and anuria, while the renal microPO2 and VO2,ren remained relatively unchanged. Resuscitation restored renal blood flow, renal oxygen delivery and kidney function to baseline values, and was associated with oxygen redistribution showing different patterns for the different compounds used. HES200/0.5 and Ringer's lactate increased the VO2,ren, in contrast to HES130/0.4. CONCLUSION: The loss of kidney function during endotoxemia could not be explained by an oxygen deficiency. Renal oxygen redistribution could for the first time be demonstrated during fluid resuscitation. HES130/0.4 had no influence on the VO2,ren and restored renal function with the least increase in the amount of renal work.

Targeted disruption of the protein kinase C epsilon gene abolishes the infarct size reduction that follows ischaemic preconditioning of isolated buffer-perfused mouse hearts.

OBJECTIVE: Activation of protein kinase C (PKC) isoforms is associated with the cardioprotective effect of early ischaemic preconditioning (IP). PKC consists of at least 10 different isoforms, encoded by separate genes, which mediate distinct physiological functions. Although the PKC-epsilon isoform has been implicated in preconditioning, uncertainty remains. We investigated whether preconditioning still occurs in a mouse line lacking cardiac PKC-epsilon protein due to a targeted disruption within the pkc-epsilon allele. METHODS: The isolated buffer-perfused hearts from knockout mice lacking PKC-epsilon (-/-) and sibling heterozygous mice (+/-), with a normal PKC-epsilon complement, were preconditioned by 4 x 4 min ischaemia/6 min reperfusion. Hearts then underwent 45 min of global ischaemia followed by 1.5 h of reperfusion. RESULTS: In PKC-epsilon (+/-) hearts ischaemic preconditioning reduced infarction volume as a percentage of myocardial volume (24.3+/-4.5 vs. 41.3+/-4.7%, P<0.001). In contrast, in PKC-epsilon (-/-) hearts preconditioning failed to diminish infarction (36.4+/-2.9 vs. 38.8+/-4.5%). Surprisingly however, although preconditioning did not reduce infarct size, it did enhance contractile recovery in PKC-epsilon (-/-) mice (43.1+/-3.9 vs. 24.9+/-5.1%, P<0.05), similar to the level seen in PKC-epsilon (+/-) hearts (35.2+/-3.9 vs. 20.9+/-5.0%, P<0.05). CONCLUSIONS: These data suggest that PKC-epsilon is essential for the reduction in infarction that follows early ischaemic preconditioning, but is not associated with the improvement in functional recovery.

Excitation pulse deconvolution in luminescence lifetime analysis for oxygen measurements in vivo.

Oxygen-dependent quenching of phosphorescence has been proven to be a valuable tool for the measurement of oxygen concentrations both in vitro and in vivo. For biological measurements the relatively long lifetimes of phosphorescence have promoted time-domain-based devices using xenon arc flashlamps as the most common excitation light source. The resulting complex form of the excitation pulse leads to complications in the analysis of phosphorescence lifetimes and ultimately to errors in the recovered pO2 values. Although the problem has been recognized, the consequences on in vivo phosphorescence lifetime measurements have been neglected so far. In this study, the consequences of finite excitation flash duration are analyzed using computer simulations, and a method for the recovery of phosphorescence decay times from complex photometric signals is presented. The analysis provides an explanation as to why different calibration constants are reported in the literature and presents a unified explanation whereby calibration constants are not solely a property of the dye but also of the measuring device. It is concluded that complex excitation pulse patterns without appropriate analysis methods lead to device-specific calibration constants and nonlinearity and can be a potent source of errors when applied in vivo. The method of analysis presented in this article allows reliable phosphorescence lifetime measurements to be made for oxygen pressure measurements and can easily be applied to existing phosphorimeters.

Cutaneous respirometry by dynamic measurement of mitochondrial oxygen tension for monitoring mitochondrial function in vivo.

Progress in diagnosis and treatment of mitochondrial dysfunction in chronic and acute disease could greatly benefit from techniques for monitoring of mitochondrial function in vivo. In this study we demonstrate the feasibility of in vivo respirometry in skin. Mitochondrial oxygen measurements by means of oxygen-dependent delayed fluorescence of protoporphyrin IX are shown to provide a robust basis for measurement of local oxygen disappearance rate (ODR). The fundamental principles behind the technology are described, together with an analysis method for retrievel of respirometry data. The feasibility and reproducibility of this clinically useful approach are demonstrated in a series of rats.

Direct sGC activation bypasses NO scavenging reactions of intravascular free oxy-hemoglobin and limits vasoconstriction.

AIMS: Hemoglobin-based oxygen carriers (HBOC) provide a potential alternative to red blood cell (RBC) transfusion. Their clinical application has been limited by adverse effects, in large part thought to be mediated by the intravascular scavenging of the vasodilator nitric oxide (NO) by cell-free plasma oxy-hemoglobin. Free hemoglobin may also cause endothelial dysfunction and platelet activation in hemolytic diseases and after transfusion of aged stored RBCs. The new soluble guanylate cyclase (sGC) stimulator Bay 41-8543 and sGC activator Bay 60-2770 directly modulate sGC, independent of NO bioavailability, providing a potential therapeutic mechanism to bypass hemoglobin-mediated NO inactivation. RESULTS: Infusions of human hemoglobin solutions and the HBOC Oxyglobin into rats produced a severe hypertensive response, even at low plasma heme concentrations approaching 10 µM. These reactions were only observed for ferrous oxy-hemoglobin and not analogs that do not rapidly scavenge NO. Infusions of L-NG-Nitroarginine methyl ester (L-NAME), a competitive NO synthase inhibitor, after hemoglobin infusion did not produce additive vasoconstriction, suggesting that vasoconstriction is related to scavenging of vascular NO. Open-chest hemodynamic studies confirmed that hypertension occurred secondary to direct effects on increasing vascular resistance, with limited negative cardiac inotropic effects. Intravascular hemoglobin reduced the vasodilatory potency of sodium nitroprusside (SNP) and sildenafil, but had no effect on vasodilatation by direct NO-independent activation of sGC by BAY 41-8543 and BAY 60-2770. INNOVATION AND CONCLUSION: These data suggest that both sGC stimulators and sGC activators could be used to restore cyclic guanosine monophosphate-dependent vasodilation in conditions where cell-free plasma hemoglobin is sufficient to inhibit endogenous NO signaling.

Microvascular and mitochondrial PO(2) simultaneously measured by oxygen-dependent delayed luminescence.

Measurement of tissue oxygenation is a complex task and various techniques have led to a wide range of tissue PO(2) values and contradictory results. Tissue is compartmentalized in microcirculation, interstitium and intracellular space and current techniques are biased towards a certain compartment. Simultaneous oxygen measurements in various compartments might be of great benefit for our understanding of determinants of tissue oxygenation. Here we report simultaneous measurement of microvascular PO(2) (µPO(2) ) and mitochondrial PO(2) (mitoPO(2) ) in rats. The µPO(2) measurements are based on oxygen-dependent quenching of phosphorescence of the near-infrared phosphor Oxyphor G2. The mitoPO(2) measurements are based on oxygen-dependent quenching of delayed fluorescence of protoporphyrin IX (PpIX). Favorable spectral properties of these porphyrins allow simultaneous measurement of the delayed luminescence lifetimes. A dedicated fiber-based time-domain setup consisting of a tunable pulsed laser, 2 red-sensitive gated photomultiplier tubes and a simultaneous sampling data-acquisition system is described in detail. The absence of cross talk between the channels is shown and the feasibility of simultaneous µPO(2) and mitoPO(2) measurements is demonstrated in rat liver in vivo. It is anticipated that this novel approach will greatly contribute to our understanding of tissue oxygenation in physiological and pathological circumstances.

Angeli's salt counteracts the vasoactive effects of elevated plasma hemoglobin.

Plasma hemoglobin (Hb) released during intravascular hemolysis has been associated with numerous deleterious effects that may stem from increased nitric oxide (NO) scavenging, but has also been associated with reactive oxygen species generation and platelet activation. Therapies that convert plasma oxyHb to metHb, or metHb to iron-nitrosyl Hb, could be beneficial because these species do not scavenge NO. In this study, we investigated the effects of Angeli's salt (AS; sodium α-oxyhyponitrite, Na2N2O3), a nitroxyl (HNO) and nitrite (NO2(-)) donor, on plasma Hb oxidation and formation of iron-nitrosyl Hb from metHb and on the vasoactivity of plasma Hb. We hypothesized that AS could ameliorate hemolysis-associated pathology via its preferential reactivity with plasma Hb, as opposed to red-cell-encapsulated Hb, and through its intrinsic vasodilatory activity. To test this hypothesis, we infused (n=3 per group) (1) cell-free Hb and AS, (2) cell-free Hb+0.9% NaCl, (3) AS+3% albumin, and (4) 3% albumin+0.9% NaCl (colloid controls for Hb and AS, respectively) in a canine model. Co-infusion of AS and cell-free Hb led to preferential conversion of plasma Hb to metHb, but the extent of conversion was lower than anticipated based on the in vivo concentration of AS relative to plasma Hb. This lower metHb yield was probably due to reactions of nitroxyl-derived AS with plasma components such as thiol-containing compounds. From a physiological and therapeutic standpoint, the infusion of Hb alone led to significant increases in mean arterial pressure (p=0.03) and systemic vascular resistance index (p=0.01) compared to controls. Infusion of AS alone led to significant decreases in these parameters and co-infusion of AS along with Hb had an additive effect in reversing the effects of Hb alone on the systemic circulation. Interestingly, in the pulmonary system, the decrease in pressure when AS was added to Hb was significantly less than would have been expected compared to the effects of Hb and AS alone, suggesting that inactivation of scavenging with AS reduced the direct vasodilatory effects of AS on the vasculature. We also found that AS reduced platelet activation when administered to whole blood in vitro. These data suggest that AS-like compounds could serve as therapeutic agents to counteract the negative vasoconstrictive consequences of hemolysis that occur in hemolytic anemias, transfusion of stored blood, and other diseases. Increases in metHb in the red blood cell, the potential of AS for neurotoxicity, and hypotension would need to be carefully monitored in a clinical trial.