Characterization of pepcan-23 as pro-peptide of RVD-hemopressin (pepcan-12) and stability of hemopressins in mice.

Hemopressins ((x)-PVNFKLLSH) or peptide endocannabinoids (pepcans) can bind to cannabinoid receptors. RVD-hemopressin (pepcan-12) was shown to act as endogenous allosteric modulator of cannabinoid receptors, with opposite effects on CB1 and CB2, respectively. Moreover, the N-terminally elongated pepcan-23 was detected in different tissues and was postulated to be the pro-peptide of RVD-hemopressin. Currently, data about the pharmacokinetics, tissue distribution and stability of hemopressin-type peptides are lacking. Here we investigated the secondary structure and physiological role of pepcan-23 as precursor of RVD-hemopressin. We assessed the metabolic stability of these peptides, including hemopressin. Using LC-ESI-MS/MS, pepcan-23 was measured in mouse tissues and human whole blood (~50 pmol/mL) and in plasma was the most stable endogenous peptide containing the hemopressin sequence. Using peptide spiked human whole blood, mouse adrenal gland and liver homogenates demonstrate that pepcan-23 acts as endogenous pro-peptide of RVD-hemopressin. Furthermore, administered pepcan-23 converted to RVD-hemopressin in mice. In circular dichroism spectroscopy, pepcan-23 showed a helix-unordered-helix structure and efficiently formed complexes with divalent metal ions, in particular Cu(II) and Ni(II). Hemopressin and RVD-hemopressin were not bioavailable to the brain and showed poor stability in plasma, in agreement with their overall poor biodistribution. Acute hemopressin administration (100 mg/kg) did not modulate endogenous RVD-hemopressin/pepcan-23 levels or influence the endocannabinoid lipidome but increased 1-stearoyl-2-arachidonoyl-sn-glycerol. Overall, we show that pepcan-23 is a biological pro-peptide of RVD-hemopressin and divalent metal ions may regulate this process. Given the lack of metabolic stability of hemopressins, administration of pepcan-23 as pro-peptide may be suitable in pharmacological experiments as it is converted to RVD-hemopressin in vivo.

Biodistribution PET/CT Study of Hemoglobin-DFO-89Zr Complex in Healthy and Lung Tumor-Bearing Mice.

Proteins, as a major component of organisms, are considered the preferred biomaterials for drug delivery vehicles. Hemoglobin (Hb) has been recently rediscovered as a potential drug carrier, but its use for biomedical applications still lacks extensive investigation. To further explore the possibility of utilizing Hb as a potential tumor targeting drug carrier, we examined and compared the biodistribution of Hb in healthy and lung tumor-bearing mice, using for the first time 89Zr labelled Hb in a positron emission tomography (PET) measurement. Hb displays a very high conjugation yield in its fast and selective reaction with the maleimide-deferoxamine (DFO) bifunctional chelator. The high-resolution X-ray structure of the Hb-DFO complex demonstrated that cysteine ß93 is the sole attachment moiety to the αß-protomer of Hb. The Hb-DFO complex shows quantitative uptake of 89Zr in solution as determined by radiochromatography. Injection of 0.03 mg of Hb-DFO-89Zr complex in healthy mice indicates very high radioactivity in liver, followed by spleen and lungs, whereas a threefold increased dosage results in intensification of PET signal in kidneys and decreased signal in liver and spleen. No difference in biodistribution pattern is observed between naïve and tumor-bearing mice. Interestingly, the liver Hb uptake did not decrease upon clodronate-mediated macrophage depletion, indicating that other immune cells contribute to Hb clearance. This finding is of particular interest for rapidly developing clinical immunology and projects aiming to target, label or specifically deliver agents to immune cells.

[Safety Evaluation of Cellular-type Artificial Blood Based on Pharmacokinetic Analysis and Its Use in Medical Gas Delivery].

Hemoglobin vesicles (HbVs) in which human hemoglobin is encapsulated in a phospholipid bilayer membrane (liposome) have been developed as artificial red blood cells. Although the effectiveness of HbVs, including their physicochemical characteristics and pharmacological effects, has been reported, data on the pharmacokinetic properties of HbVs are limited. Previously, we developed two kinds of radiolabeled HbV, 125I-HbV and 3H-HbV, in which the internal hemoglobin and lipid membranes were labeled with 125I and 3H, respectively. Using these isotope-labeled HbVs, we clarified the detailed pharmacokinetic properties of HbVs in healthy animals and experimental animal disease models of hemorrhagic shock, chronic cirrhosis, and hyperlipidemia. This review describes our previous results regarding the pharmacokinetic properties of HbVs, and we discuss the safety and usefulness of HbVs from the viewpoint of their pharmacokinetic characteristics. Furthermore, we have modified HbVs by employing them as a carbon monoxide (CO) carrier because the hemoglobin inside HbVs reversibly binds to CO, resulting in CO-bound HbVs (CO-HbVs). Here we report the potential of CO-HbVs for the treatment of intractable inflammatory disorders based on their therapeutic efficiency in experimental animal models.

Renal Toxicodynamic Effects of Extracellular Hemoglobin After Acute Exposure.

Plasma hemoglobin (Hb) is elevated in some hematologic disease states, during exposures to certain toxicants, and with the use of some medical devices. Exposure to free Hb can precipitate oxidative reactions within tissues and alter the normal physiological function of critical organ systems. As kidney structures can be highly sensitive to Hb exposures, we evaluated the acute dose dependent renal toxicologic response to purified Hb isolated from RBCs. Male Hartley guinea pigs (n = 5 per group) were dosed with 0.9% saline (2 ml), 15, 75, 150, or 300 mg of purified Hb, infused over a 2-h period. The primary endpoints of this study were to define toxicokinetic parameters after increasing doses of purified Hb, identify clinically recognized and experimental markers of acute kidney injury (AKI), and determine relevant toxicological parameters and potential causes of renal toxicity in this model. Experimental findings demonstrated a dose dependent increase in Cmax after a 2-h infusion, which correlated with an elevation in serum creatinine, renal Kim-1 mRNA expression and increased urinary Kim-1. Renal NGAL mRNA expression and urinary NGAL excretion were also increased in several groups, but these parameters did not correlate with exposure. Iron increased in the renal cortex as Hb exposure increased and its deposition colocalized with 4-hydroxy-nonenal and 8-Oxo-2-deoxyguanosine immune reactivity, suggesting oxidative stressors may contribute to AKI in animals exposed to Hb. The results presented here suggest that Cmax may effectively predict the risk of AKI in normal healthy animals exposed to Hb.

Hypoxic tumor therapy by hemoglobin-mediated drug delivery and reversal of hypoxia-induced chemoresistance.

Chemotherapy has become a critical treatment for many cancer types. However, its efficacy is hindered by chemoresistance and limited drug accumulation induced by the hypoxic tumor environment. Therefore, there is an urgent need for useful strategies to alleviate tumor hypoxia and enhance chemotherapy response in solid tumors. Herein, we report the development of a multifunctional liposome simultaneously loading an oxygen carrier (hemoglobin, Hb) and an anti-tumor drug (doxorubicin, DOX) to enhance chemotherapeutic effects against hypoxic tumors. The liposomes, DOX-Hb-lipo (DHL), showed efficient loading of oxygen and site-specific oxygen delivery into tumors, inducing the reversal of tumor hypoxia. Furthermore, the O2 interference capacity increased the uptake of the drug into hypoxic cancer cells, inducing a remarkably increased toxicity of the drug against cancer cells. Interestingly, the obtained DHL showed a significantly enhanced internalization into cancer cells and accumulation in tumors compared to DL (DOX loaded liposomes without Hb), while the enhanced effect did not occur in normal cells. The specific delivery of DHL into cancer cells should be attributed to the mediation of Hb on the surface of the liposomes. In addition, DHL considerably increased reactive oxygen species (ROS) production in a hypoxic environment and promoted the ROS-mediated cytotoxicity of DOX. Based on the elevated drug accumulation in the tumor sites, increased internalization into cancer cells and enhanced oxygen levels in tumor regions, DHL reversed hypoxia-induced chemoresistance and exhibited stronger antitumor effects. Thus, DHL might be a promising alternative strategy for cancer treatment.

Hemoglobin as a Smart pH-Sensitive Nanocarrier To Achieve Aggregation Enhanced Tumor Retention.

Natural proteins have been greatly explored to address unmet medical needs. However, few work has treated proteins as natural pH-sensitive nanoplatforms that make use of the inherent pH gradient of pathogenic sites. Here, hemoglobin is employed as a smart pH-sensitive nanocarrier for near-infrared dye IR780, which disperses well at normal tissue pH and exhibits aggregation at tumor acidic milieu. The pH-sensitive hemoglobin loaded with IR780 shows higher uptake by cancer cells at tumor acidic pH 6.5 than normal tissue pH 7.4. In vivo and ex vivo studies reveal that the hemoglobin nanocarrier exhibits distinct retention kinetics with remarkably prolonged residence time in tumor. Hemoglobin is then proved to be a potent pH-sensitive nanocarrier for cancer diagnosis and treatment.

Preparation of artificial red cell and its application on alleviation of tumor hypoxia.

Hemoglobin-based oxygen carriers were developed as an alternative for blood transfusion. However, the research progress for their further clinic applications was slow in recent several years. Hypoxia is found in most solid tumors, which is responsible for the tumor formation, increased metastasis, drug resistance during therapeutic process as well as poor patient survival. In this work, novel hemoglobin (Hb) loaded nanoliposomes, as artificial red cells for oxygen delivery, were optimized by screening various types of phospholipids and analyzing different mole ratio of phospholipid to cholesterol. The nanoliposomes presented a high encapsulating efficiency to hemoglobin and also significantly enhanced its stability. The obtained hemoglobin loaded nanoliposome (HLL) could be lyophilized for long term storage. HLL did not cause significant cell death in the concentration range of 0-100µg equivalent Hb/mL under normoxia and hypoxia incubation conditions, suggesting the low cytotoxicity and high biocompatibility of HLL. Importantly, HLL could efficiently accumulate into subcutaneous and deep orthotopic tumors, inducing a significant decrease of hypoxia-inducible factors 1α subunits (HIF-1α) in the tumors and remarkably reduced expression of vascular endothelial growth factor (VEGF). The study of acute and chronic toxicity indicated that HLL did not induce obvious damage to main organs of mice after intravenous injections with total Hb dose of 120mg/kg. We presented a promising method for relieving the hypoxia degree in solid tumors and down-regulating HIF-1α protein by directly delivering oxygen into tumors, which will be very helpful for subsequent cancer therapy.

Acute 40% exchange-transfusion with hemoglobin-vesicles in a mouse pneumonectomy model.

OBJECTIVES: Hemoglobin vesicles (HbVs) function as a red blood cell (RBC) substitute and are composed of purified hemoglobin encapsulated in a phospholipid bilayer membrane. The performance of HbVs as a substitute for RBC transfusions was examined in a mouse model of pneumonectomy following acute 40% exchange-transfusion with HbVs. METHODS: Before performing left pneumonectomies, 40% of the blood volume of mice was replaced with a) lactated Ringer's solution (control), b) 5% recombinant human serum albumin (rHSA), c) mouse RBCs shed in rHSA (mRBCs/rHSA), or d) HbV suspended in rHSA (HbV/rHSA). We compared postoperative a) survival, b) functional recovery, and c) histopathological, immunohistochemical, and inflammatory responses among the study groups. RESULTS: In the HbV/rHSA and mRBC/rHSA groups, all mice survived ≥7 days after pneumonectomy, whereas 100% of the control mice died within a few h and 50% of mice in the rHSA group died within 24 h after pneumonectomy. Immunohistochemical staining for hypoxia-inducible factor-1α showed that hepatic and renal hypoxic injuries were prominently mitigated by HbV and mRBCs. CONCLUSIONS: The oxygen-carrying performance of HbV was similar to that of mRBCs, even with impaired lung functions following pneumonectomy. HbV infusion did not interfere with the recovery from surgical injury. In the near future, HbVs could be used clinically as a substitute for the perioperative transfusion of RBCs, when or where donated RBCs are not immediately available.

US-localized diffuse optical tomography in breast cancer: comparison with pharmacokinetic parameters of DCE-MRI and with pathologic biomarkers.

BACKGROUND: To correlate parameters of Ultrasonography-guided Diffuse optical tomography (US-DOT) with pharmacokinetic features of Dynamic contrast-enhanced (DCE)-MRI and pathologic markers of breast cancer. METHODS: Our institutional review board approved this retrospective study and waived the requirement for informed consent. Thirty seven breast cancer patients received US-DOT and DCE-MRI with less than two weeks in between imaging sessions. The maximal total hemoglobin concentration (THC) measured by US-DOT was correlated with DCE-MRI pharmacokinetic parameters, which included K(trans), k ep and signal enhancement ratio (SER). These imaging parameters were also correlated with the pathologic biomarkers of breast cancer. RESULTS: The parameters THC and SER showed marginal positive correlation (r = 0.303, p = 0.058). Tumors with high histological grade, negative ER, and higher Ki-67 expression ≥ 20% showed statistically higher THC values compared to their counterparts (p = 0.019, 0.041, and 0.023 respectively). Triple-negative (TN) breast cancers showed statistically higher K(trans) values than non-TN cancers (p = 0.048). CONCLUSION: THC obtained from US-DOT and K(trans) obtained from DCE-MRI were associated with biomarkers indicative of a higher aggressiveness in breast cancer. Although US-DOT and DCE-MRI both measured the vascular properties of breast cancer, parameters from the two imaging modalities showed a weak association presumably due to their different contrast mechanisms and depth sensitivities.

In vivo biodistribution and oxygenation potential of a new generation of oxygen carrier.

Natural giant extracellular hemoglobins (Hbs) from polychaete annelids are currently actively investigated as promising oxygen carriers. Their powerful oxygenating ability and their safety have been demonstrated in preclinical studies, motivating their development for therapeutic and industrial applications. HEMARINA-M101 (M101) is derived from the marine invertebrate Arenicola marina. It is formulated as a manufactured product designated HEMOXYCarrier(®) (HEMARINA SA, France). The aim of the present study was to unveil the fate of M101 after a single intravenous (i.v.) injection in mice. For this purpose, M101 was tagged with a far-red fluorescent dye. Repeated non-invasive fluorescent imaging revealed a rapid diffusion of M101 in the whole body of animals, reaching all the examined organs such as brain, liver, lungs and ovaries. Functional M101 was circulating in bloodstream for several hours, without inducing any obvious side-effects. Last, a single i.v. injection of M101 in mice bearing human-derived subcutaneous tumors demonstrated the ability of this Hb to reduce hypoxia in poorly vascularized tissues, thus supporting the biological relevance of M101 oxygen release to vertebrate tissues. Altogether, these results further encourage the development of M101 as an oxygen carrying therapeutic.

Scintigraphic imaging with a peptide glucuronide in rabbits: 99mTc- exorphin C glucuronide.

A peptide glucuronide (Exorphin C glucuronide) was labeled with 99mTc using glucoheptonate (GH) as a bifunctional chelating agent. Scintigraphic imaging was performed in male Albino rabbits. Exorphin C glucuronide showed rapid and efficient labeling with 99mTc using glucoheptonate as a bifunctional chelate. Results demonstrated that 99mTc-GEG may be a useful new type of glucuronide derivative of peptides for diagnosis of some cancer diseases.

Evaluation and modification of N-trimethyl chitosan chloride nanoparticles as protein carriers.

N-Trimethyl chitosan chloride (TMC) nanoparticles were prepared by ionic crosslinking of TMC with tripolyphosphate (TPP). Two model proteins with different pI values, bovine serum albumin (BSA, pI=4.8) and bovine hemoglobin (BHb, pI=6.8), were used to investigate the loading and release features of the TMC nanoparticles. TMC samples with different degrees of quaternization were synthesized to evaluate its influence on the physicochemical properties and release profiles of the nanoparticles. Sodium alginate was used to modify the TMC nanoparticles to reduce burst release. The results indicated that the TMC nanoparticles had a high loading efficiency (95%) for BSA but a low one (30%) for BHb. The particle size and zeta potential were significantly affected by the BSA concentration but not by the BHb concentration. Nanoparticles of TMC with a lower degree of quaternization showed an increase in particle size, a decrease in zeta potential and a slower drug-release profile. As for the alginate-modified nanoparticles, a smaller size and lower zeta potential were observed and the burst release of BSA was reduced. These studies demonstrated that TMC nanoparticles are potential protein carriers, and that their physicochemical properties and release profile could be optimized by means of various modifications.

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates.

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.

Regulation of surface CD163 expression and cellular effects of receptor mediated hemoglobin-haptoglobin uptake on human monocytes and macrophages.

Local dysregulation of iron metabolism is suggested to contribute to atherosclerotic lesion development through hemoglobin scavenging pathways. We evaluated the effects of CD163-mediated uptake of hemoglobin-haptoglobin (HbHp) complexes on surface CD163 and intracellular heme oxygenase-1 expression and the secretion of pro- and antiinflammatory cytokines by macrophages. We found that increased availability of HbHp complexes triggers the upregulation of surface CD163, and also results in a dose-dependent secretion of IL-6 and IL-10.

Interaction of heme proteins with poly(propyleneimine) dendrimers in layer-by-layer assembly films under different pH conditions.

With the isoelectric point at pH 7.4, hemoglobin (Hb) has net positive surface charges at pH 5.0 and overall negative charges at pH 9.0, and is essentially neutral at pH 7.0. The fifth-generation poly(propyleneimine) (PPI) dendrimer is usually positively charged in aqueous solution. The {PPI/Hb}n films under different pH conditions have been successfully fabricated on various solid surfaces by the layer-by-layer assembly technique, and the growth of films was monitored by ultraviolet-visible (UV-vis) spectroscopy, quartz crystal microbalance (QCM), and cyclic voltammetry (CV). Not only was the negatively charged Hb at pH 9.0 alternately adsorbed with positively charged PPI onto solid substrates by electrostatic attraction between them, but the positively charged Hb at pH 5.0 was also successfully assembled with like charged PPI into layer-by-layer {PPI/Hb(pH 5.0)}n films. For the latter, the localized electrostatic interaction or the charge reversal of proteins on PPI surface may be the main driving force. For {PPI/Hb(pH 7.0)}n films, however, the hydrophobic/hydrophilic interaction may play a more important role in the assembly, making the amount of adsorbed Hb even less than that of {PPI/Hb(pH 5.0)}n films. For comparison, negatively charged catalase (Cat) at pH 8.0 was used to assemble layer-by-layer films with positive PPI, but {PPI/Cat}n films showed quite different properties from {PPI/Hb}n films. UV-vis and infrared (IR) spectroscopy, QCM, ellipsometry, and voltammetry were utilized to characterize the {PPI/protein}n films. The results suggest that the proteins in the multilayer films retain their near-native structure and display good voltammetric response for heme Fe(III)/Fe(II) redox couples at underlying pyrolytic graphite (PG) electrodes. Electrocatalysis of oxygen and hydrogen peroxide based on direct electrochemistry of heme proteins at {PPI/protein}n film electrodes was also demonstrated.

Neutral and anionic liposome-encapsulated hemoglobin: effect of postinserted poly(ethylene glycol)-distearoylphosphatidylethanolamine on distribution and circulation kinetics.

To prepare long-circulating liposomes, poly(ethylene glycol) (PEG)-lipid is usually mixed with other lipid components before vesicle formation. PEG-lipids can also be postinserted in the outer layer of liposomes after the preparation. In this study, PEG-distearoylphosphatidylethanolamine was incorporated by postinsertion technique into liposome-encapsulated hemoglobin (LEH) carrying neutral or negative charge. Postinsertion technique improved the encapsulation efficiency of hemoglobin from about 0.0017 to 0.017 (hemoglobin/phospholipid, molar ratio) for a similar lipid composition. Thus, neutral, anionic, PEG-neutral, and PEG-anionic LEHs were made and labeled with technetium-99m to follow their biodistribution. A small dose of LEH (approximately 15 mg of phospholipid) was injected intravenously in rabbits, and its distribution was monitored by blood sampling, gamma camera imaging, and tissue radioactivity counting on necropsy. The 24-h blood levels of neutral, PEG-neutral, anionic, and PEG-anionic LEHs were 14, 40.3, 13.1, and 35.7% of injected dose, respectively; calculated T(1/2) values of circulation were 8.9, 19.3, 9.6, and 16.5 h, respectively. PEGylation also influenced accumulation of LEH in the reticuloendothelial system. Liver uptake of neutral LEH dropped from 52.1 to 19.1%, whereas that of anionic LEH came down from 35.3 to 11.5% on PEGylation. In contrast, PEGylation increased the spleen uptake by 8.5- and 2.5-fold for neutral and anionic LEH, respectively. The results demonstrate that PEGylation by postinsertion not only improves the circulation t1/2 of LEH but also enhances hemoglobin content inside the vesicles for better oxygen-carrying capacity.

Heme and lipid peroxides in hemoglobin-modified low-density lipoprotein mediate cell survival and adaptation to oxidative stress.

Low-density lipoprotein (LDL) oxidation mediated by a variety of catalysts in atherosclerotic lesions plays a crucial role in the genesis and evolution of atherosclerotic plaques. In this study we focused on oxidative properties of hemoglobin (Hb)-modified LDL because Hb is present in atherosclerotic lesions. Under low oxygen tensions Hb was previously found to modify apolipoprotein B100 with covalent binding of Hb fragments and formation of electronegative LDL particles (LDL-). Here we show that HbLDL is highly susceptible to oxidation, but is not cytotoxic to vascular cells, as was found for LDL- isolated from human plasma. HbLDL and LDL- have similar levels of oxidized lipid products and low uptake rates; however, the virtual absence of HbLDL-induced toxicity depends on a marked adaptive oxidative stress response. This was evidenced by a time- and dose-dependent induction of heme oxygenase (HO-1). Cell survival was significantly decreased in the presence of HO-1 inhibitor, tin protoporphyrin (SnPPIX). HO-1 induction by HbLDL increased resistance of cells to toxic doses of hemin or t-BuOOH. The high sensitivity to oxidation and HO-1 induction was largely dependent on lipid hydroperoxides and heme associated with HbLDL. Reduction of pre-existing lipid peroxides using ebselen delayed HbLDL kinetics and inhibited HO-1 induction. Moreover, heme inactivation or its degradation inhibited HO-1 induction and provided an additive inhibitory effect to ebselen. We conclude that Hb-catalyzed reactions may modulate vascular cell survival and oxidative stress adaptation due to the presence of peroxides and heme, thus providing a possible mechanism for the evolution of atherosclerotic and hemorrhagic lesions.

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes.

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.

Randomized trial of diaspirin cross-linked hemoglobin solution as an alternative to blood transfusion after cardiac surgery. The DCLHb Cardiac Surgery Trial Collaborative Group.

BACKGROUND: Risks associated with transfusion of allogeneic blood have prompted development of methods to avoid or reduce blood transfusions. New oxygen-carrying compounds such as diaspirin cross-linked hemoglobin (DCLHb) could enable more patients to avoid allogeneic blood transfusion. METHODS: The efficacy, safety, hemodynamic effects, and plasma persistence of DCLHb were investigated in a randomized, active-control, single-blind, multicenter study in post-cardiac bypass surgery patients. Of 1,956 screened patients, 209 were determined to require a blood transfusion and met the inclusion criteria during the 24-h post-cardiac bypass period. These patients were randomized to receive up to three 250-ml infusions of DCLHb (n = 104) or three units of packed erythrocytes (pRBCs; n = 105). Further transfusions of pRBCs or whole blood were permitted, if indicated. Primary efficacy end points were the avoidance of blood transfusion through hospital discharge or 7 days postsurgery, whichever came first, and a reduction in the number of units of pRBCs transfused during this same time period. Various laboratory, physiologic, and hemodynamic parameters were monitored to define the safety and pharmacologic effect of DCLHb in this patient population. RESULTS: During the period from the end of cardiopulmonary bypass surgery through postoperative day 7 or hospital discharge, 20 of 104 (19%) DCLHb recipients did not receive a transfusion of pRBCs compared with 100% of control patients (P < 0.05). The overall number of pRBCs administered during the 7-day postoperative period was not significantly different. Mortality was similar between the DCLHb (6 of 104 patients) and the control (8 of 105 patients) groups. Hypertension, jaundice/hyperbilirubinemia, increased serum glutamic oxalo-acetic transaminase, abnormal urine, and hematuria were reported more frequently in the DCLHb group, and there was one case of renal failure in each group. The hemodynamic effects of DCLHb included a consistent and slightly greater increase in systemic and pulmonary vascular resistance with associated increases in systemic and pulmonary arterial pressures compared with pRBC. Cardiac output values decreased more in the DCLHb group patients after the first administration than the control group patients. At 24 h postinfusion, the plasma hemoglobin level was less than one half the maximal level for any amount of DCLHb infused. CONCLUSIONS: Administration of DCLHb allowed a significant number (19%) of cardiac surgery patients to avoid exposure to erythrocytes postoperatively.

The absorption of iron, calcium, phosphorus, magnesium, copper and zinc in the jejunum-ileum of control and iron-deficient rats.

The effects of iron deficiency on the absorption of different dietary sources of iron were studied, together with the interactions between iron, calcium, phosphorus, magnesium, copper and zinc in the jejunum-ileum of control and iron-deficient rats. In this study, three perfusion solutions containing different iron sources: ferric citrate, haemoglobin, and equal parts of ferric citrate and haemoglobin were used. In addition, the same perfusion solutions were used with and without 2,4-dinitrophenol, an inhibitor of oxidative phosphorylation. Iron absorption in anaemic rats was greater than in the controls, except after perfusion with solutions containing haemoglobin. The absorption of calcium, copper and zinc in iron-deficient animals was not significantly affected, while the absorption of phosphorus and magnesium increased, with respect to animals in the control group. After perfusion with solutions containing haemoglobin, the absorption values of calcium, copper and zinc were lower than after ferric citrate in both groups (control and iron-deficient rats).