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Stroma-free hemoglobin (SFH) contains a trace amount of phospholipids, which has been implicated in the toxic reactions associated with SFH. We analyzed stromal phospholipids by high-performance liquid chromatography and found that SFH contained small quantities of lysophosphatidylcholine (LPC), which is known to be capable of producing a defect in endothe-lium-dependent arterial relaxation, in addition to major classes of constituent phospholipids in red cell membrane. LPC content was determined to be 1.65 nmol/ml (hemoglobin 8.1 g/dl). To evaluate the role of these stromal phospholipids in SFH-induced vasoconstriction, we next examined the effect of lipids on vascular tone in rabbit aortic strips. Preincubation with the crude lipid extract or the LPC purified from SFH significantly inhibited acetylcholine-induced relaxation in phenylephrine-precontracted tissues. The LPC-induced inhibition was reversed by incubation of the tissues in the absence of lipids, indicating the functional integrity of endothelium. From these results, we propose a possibility that LPC, a component of stromal phospholipids, is a candidate for vasoconstrictive factors present in SFH.

The effects of pyridoxalated hemoglobin polyoxyethylene conjugate (PHP) and other hemoglobin-related substances on arterial blood pressure (ABP) and heart rate (HR) were examined. Infusion of PHP and other hemoglobin-related substances elevated ABP and increased HR. The degree of the increase in ABP and HR did not differ among the groups. There were species' differences in responses to PHP. Pretreatment with various blockers did not abolish the elevation of ABP. The pretreatment of NG-monomethyl-l-arginine dose-dependently reduced the elevation of ABP but did not completely abolish it. PHP also elevated ABP in conscious rats. The magnitude of the ABP elevation was significantly smaller than in the anesthetized rats. Results indicate that inhibition of nitric oxide (NO) might be responsible in part for the elevation of ABP, and the degree of the elevation would be dependent on the degree of contribution of NO to the regulation of ABP.

This study reports some recent work dealing with the stabilization of the tetramers of hemoglobin. It is shown that by using a variety of diacids, it is possible to increase the P50 above that of stroma free hemoglobin. In order to lengthen the retention times in the circulatory system, the stabilized hemoglobins were complexed with both hydroxyethyl starch polymers and polyol tetronic polymers. The resulting hemoglobin-polymer compounds were then freeze-dried. It was possible to reconstitute the powder by the addition of physiological saline when needed. The methods presented here appear to be appear to be as effective as using pyridoxal phosphate but at a fraction of the cost.

Analytical methods for phospholipids and blood group antigen were developed using HPLC-UV and EIA, respectively, for the quality control of SFH. These methods had sensitivities superior to those of conventional assays. In addition, the HPLC method could isolate PS, which might play an essential role in blood coagulation. The above analytical methods were applied to evaluate three representatives SFH preparation methods, 36,000xg centrifugation followed by the filtration with a 0.22 um filter, ultrafiltration with a 100 kd membrane, and filtration with the BMM-40 nm membrane. Both the BMM method and the ultrafiltration method were shown to remove more than 99.7% of phospholipids, and more than 99.99% of blood group antigen. On the other hand, the centrifugation preparation still contained much of both components. The BMM filter was originally developed as a virus removal filter. The virus removal efficiency of the BMM-40 nm was examined with a genetic technique, the PCR method. The BMM-40 nm was demonstrated to remove HB viruses with an efficiency of eight log or more. These results indicate that the BMM is a useful filter to prepare a stroma-free, virus-reduced hemoglobin solution.

Effects of a newly introduced modified hemoglobin, pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), and stroma-free hemoglobin (SFH) on cardiac rhythm and contractility were examined in isolated rat hearts. Hearts were perfused with HEPES-buffered Krebs solution containing 6% PHP, 6% SFH, or 6% hydroxy ethylstarch (HES). Arrhythmia was not observed in PHP-perfused hearts, but marked sinus bradycardia occurred in SFH-perfused hearts. Ventricular contractility assessed by left ventricular pressure and pressure-volume relationship in both PHP and SFH-perfused hearts were well maintained during perfusion. There was no significant difference to the coronary flow resistance between hearts perfused with PHP and HES solutions, indicating that PHP has little vasoactive effect. The flow resistance in SFH-perfused hearts decreased significantly when compared with other groups. Results demonstrate that PHP solution can preserve cardiac function and have no toxic effects in isolated rat hearts.

We developed a method to determine whole complement hemolytic activity (CH50) in rat plasma using a modification of the method of Mayer(1). Plasma CH50 level in 30 male Sprague Dawley rats was 1000 +/- 200 units. Following infusions with human and rat stroma-free hemoglobin or polyhemoglobin solutions, CH50 did not change significantly in rats when compared to albumin infused rats. On the other hand, CH50 significantly decreased in the group receiving membrane stroma and bacterial endotoxins.

A negative chronotropic effect of bovine stroma-free hemoglobin was identified in spontaneously contracting myocardiocytes derived from neonatal rats. This model allowed for the evaluation of direct effects of hemoglobin solutions without the influence of hemodynamic reflexes. Significant slowing of myocardiocyte beating rates were observed from 0.8 to 11.6 g.dl-1 stroma-free hemoglobin solution. Polymerization of hemoglobin into soluble crosslinked polyhemoglobin markedly reduced its negative chronotrophic effects by more than 50% at most concentrations assayed. Chronotropic potency (% change in beating rate from baseline per g.dl-1 was significantly higher for stroma-free hemoglobin when compared to polyhemoglobin and albumin solutions. Colloid osmotic potency (% change in beating rate from baseline per mmHg) however, was similar for stroma-free hemoglobin and polyhemoglobin but significantly lower for albumin solutions. This negative chronotropic effect may in part contribute to the transient bradycardia observed following hemoglobin infusion.

Pulmonary vascular responsiveness to norepinephrine(NE), angiotensin-II (ANG-II), acetylcholine(ACh) and nitroglycerin (NG) was examined in isolated perfused rat lungs. The lungs were perfused with pyridoxalated hemoglobin polyoxyethylene conjugate (PHP), a newly introduced modified hemoglobin, stroma free hemoglobin (SFH) or hydroxyethyl starch (HES) at a constant flow rate. Perfusion pressure (PP) and intra-tracheal pressure (ITP) were measured. Effects of intrapulmonary arterial injection of NE, ANG-II, ACh and NG on PP were examined. NE and ANG-II produced a dose dependent increase in PP in all groups. The response to NE in PHP or SFH perfused group was significantly greater than that in HES-perfused group. ACh induced a dose dependent decrease in PP in HES and PHP groups, while in the SFH group, ACh produced a dose dependent increase in PP. NG produced a dose dependent decrease in all groups. Results showed that stroma free hemoglobin (SFH) inhibits relaxation induced by endothelium-derived relaxation factor (EDRF), but PHP dose not exert inhibitory action on vascular relaxation induced by EDRF.

One hundred years of study of stroma-free hemoglobin have generated much information regarding its scope and limitations as an oxygen-carrying blood substitute. The most important development in recent years is the development of a truly stroma-free hemoglobin solution. At this time, we know that SFH can readily bind, transport, and unload oxygen. It does not appear to be nephrotoxic or significantly antigenic. It may not be converted to methemoglobin in the body in significant amounts. Its short in vivo half-life may have important advantages.

A simple kinetic test with visual observation of hemoglobin solutions under 4 - 10x magnification was used to detect and roughly characterize a rapid formation of fine fibrous inhomogeneities in agitated "stroma-free" hemolyzates (SFH). In parallel SFH samples stored motionless for months, no such precipitate was observed. Hydrodynamic conditions are necessary to provoke a stepwise aggregation of small amounts of unstable filamentous nonhemoglobin molecules originating mostly from the stromata of erythrocytes and from constituents of other lysed blood cells. Numerous screening experiments mentioned here failed to remove significantly the "fiber-forming" substances from SFH or to prevent their precipitation. Development of a hydrodynamically stable and better purified SFH seems to be a prerequisite for further progress in the field of infusable SFH and its chemically modified variants (MSFH).

The major problems requiring solution before full-scale evaluation of stroma-free hemoglobin solution (SFHS) in a clinical environment are its high oxygen affinity and relatively short intravascular retention time. Through postmanufacture chemical modification of SFHS we have been able to improve both properties as determined in in vitro and in vivo studies. Permatization of SFHS with pyridoxal 5'-phosphate (PLP) does not affect oxygen-carrying capacity and improves the P50 and intravascular retention. This chemically modified hemoglobin is worthy of further evaluation.

(1988). An Overview of the Chemical Modification of Stroma Free Hemoglobin. Biomaterials, Artificial Cells and Artificial Organs: Vol. 16, No. 1-3, pp. 71-75.

In recent years there has been considerable interest in the use of hemoglobin solution as a volume expander. This solution would be able to both pick-up and off-load oxygen as well as expand the circulating value. It is extremely important to know whether or not hemoglobin itself as well as stroma is antigenic, since an affirmative answer would preclude its use as a plasma expander. To obtain this answer, the following study was carried out. A group of white rabbits were challenged over a 21-day period with either saline solution, hemolysates containing various concentrations of stroma, or stroma-free hemoglobin solution. We found that stroma produced marked increase in gamma to globulin concentrations, phospholipid concentrations, and clotting times. In addition, marked thrombocytopenia was also noted. In contrast we found no such changes in animals given either saline solution or stroma-free hemoglobin solution. The data indicate that the stroma is antigenic but that hemoglobin solution without stroma is not.

In an anaesthetized dog an exchange transfusion was carried out with stroma-free haemoglobin solution. The total circulating blood volume was kept constant. The heart-syncronous changes in the thoracic electrical impedance (Zo) were measured before and after the exchange transfusion. Zo consists of a parallel connection of a tissue impedance (Zt) and a blood impedance (Zb). With the aid of this model for Zo the relative variations in Zb (delta Zb/Zb) were calculated from the relative variations in Zo (delta Zo/Zo). The marked decrease of delta Zb/Zb during the experiment can only be explained by the fact that apart from the heart-synchronous changes in vascular volume the impedance changes are also caused by flow dependent changes in the electrical conductivity of blood caused by variations in orientation of the erythrocytes.

A coronary vasoconstrictor effect of human stroma-free hemoglobin (SFH) was identified in isolated rabbit hearts perfused with Krebs-Henseleit buffer or whole rabbit blood at a constant coronary flow rate. In buffer-perfused hearts, SFH in concentrations of 5 to 200 mg/dl produced dose-related increases of coronary perfusion pressure. At a concentration of 150 mg/dl, SFH, equilibrated with CO to form carboxyhemoglobin, caused an increase in perfusion pressure (55 +/- 7 mmHg), similar to that observed with oxyhemoglobin (57 +/- 6 mmHg); addition of potassium ferricyanide to form methemoglobin reduced the increase of perfusion pressure to 34 +/- 5 mmHg (P less than 0.05). The vasoconstrictor activity could not be eliminated by dialyzing against the perfusion buffer. Human SFH prepared by different methods had similar vasoconstrictor activity. Rabbit SFH and human SFH were equi-effective in the rabbit heart. Less constrictor activity of SFH was evident in rat and guinea pig heart. Polymerized, pyridoxalated SFH had greatly reduced constrictor effect compared with unmodified or pyridoxalated tetramer SFH. In blood-perfused hearts, increasing plasma hemoglobin to 1.6 +/- 0.1 g/dl, without changing total hemoglobin or arterial O2 content, increased coronary perfusion pressure by 36 +/- 13 mmHg (P less than 0.05). We conclude that stroma-free hemoglobin solutions exert a coronary vasoconstrictor effect that is unrelated to O2 delivery.

A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.

To determine if tolerance to severe anemia (Hct less than or equal to 10%) might be improved with stroma-free hemoglobin solution (SFH), 15 swine were placed on normothermic right heart bypass (RHBP) for evaluation of stroke volume (SV), coronary blood flow (CBF), arterial-coronary sinus oxygen content difference S(a-cs)O2, and myocardial oxygen consumption (MVO2) during a control period at a hematocrit (Hct) level of 30%. These 15 animals, divided into three equal groups, subsequently underwent exchange transfusion either to a Hct of 5% using 7% SFH (group 1), or to a Hct of 5% (group 2) or 10% (group 3) using 7% bovine albumin solution. All tests were repeated during these experimental conditions. Myocardial performance after albumin solution exchange was sustained on RHBP in only 1 of 10 animals. SFH animals (group 1) had a significant drop in SV at 14 torr after exchange (20 +/- 3 ml vs 10 +/- 4 ml, p less than 0.025), but this 50% performance level could be sustained. CBF rose and MVO2 fell in all groups, although the statistically nonsignificant mean differences were less with SFH. S(a-c)O2 fell significantly (p less than 0.05) with albumin solution (group 2 7.3 +/- 1.4 vs 2.2 +/- 0.2, group 3 8.9 +/- 2.0 vs 3.8 +/- 1.0), and nonsignificantly with SFH (5.6 +/- 0.7 vs 4.1 +/- 1.4). Although myocardial performance decreased with SFH, the authors believe these comparative results support the use of SFH at an Hct of 5%.

The contribution of the reticuloendothelial system (RES) in pyridoxalated stroma-free hemoglobin (SFH-P) clearance may be insignificant. The magnitude of this is not at present clear. Any compromise of RES function would militate against its potential benefit as an oxygen-carrying resuscitation fluid. The relationship between lethal hemorrhagic shock resuscitation with SFH-P and subsequent host depression was examined in a rat model. Host tolerance to a standard intra-abdominal polymicrobial septic challenge was assessed 5 days after hemorrhagic shock. Shock resuscitation with pyridoxalated stroma-free hemoglobin was equal to or better than all other resuscitation groups evaluated. Tolerance to a standard septic challenge 5 days after resuscitation was no different between resuscitation groups. There appears to be no compromise to host defense in general in tolerating intra-abdominal sepsis 5 days following shock resuscitation with pyridoxalated stroma-free hemoglobin.

Stroma Free Hemoglobin (SFH) and Perfluorotributylamine (FC-43) have been extensively studied as oxygen carriers. Therefore, capitalizing on the role of oxygen reversing sickling, and thus decreasing resistance, this study was undertaken to investigate possible differences in the rheological effects of mixtures of FC-43 and SFH at different ratios. We used the positive pressure cell filtration system (PPCFS) of Lessin and Kurantsin- Mills. FC-43 was purchased from Alpha Therapeutics Corporation, and SFH was prepared by the method of Rabiner et al. The FC-43/SFH mixtures were made on a volume basis in ratios of 1:1 and 1:2. Fresh sickle cell anemia (HbSS) blood was used to prepare 0.2% cell suspensions which were deoxygenated, by bubbling with moist N2 for one hour. Studies of the rheological effects of FC-43/SFH mixtures on sickled erythrocytes were determined by obtaining filtration measurements on the following:- 1. sickle cells in ambient air; 2. deoxygenated sickle cells; 3. deoxygenated sickle cells + oxygenated FC-43/SFH (1:1 v/v); 4. deoxygenated sickle cells + oxygenated FC - 43/SFH (1:2 v/v). The results showed that treatment of deoxygenated sickle erythrocytes with oxygenated SFH/FC-43 mixtures decreased the flow resistance of the deoxygenated cells. Secondly, the SFH/FC-43 (1:2 v/v) mixture caused greater decrease in flow resistance than the 1:1 mixture.Therefore it is reasonable to conclude that the mixtures of SFH/FC-43 (1:2 v/v) ratio appear to have a additive effect in reducing the flow resistance i.e. increasing the filterability of sickled erythrocytes.

Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity. (C) 2008 Elsevier B.V. All rights reserved.

Poskitt et al reported that stroma of red cell membrane induced complement activation in vitro (1). On the other hand, Wilson and Thomas (2) thought that it was hemoglobin and not the stroma which causes complement activation via the alternative pathway. Infusion of stroma-free Hb resulted in complement activation in dogs but not in other species (3). The objectives of this research are to evaluate the effects of Hb and polyHb on complement activation.

A new infusion solution prepared from stroma-free, crosslinked and pyridoxalated hemoglobin is described. The characteristics of which have been adapted to the functions of native, intra-erythrocyte hemoglobin. The solution contains 8.5 per cent weight per volume of glutardialdehyde modified hemoglobin with a mean molecular weight (Mn) of 200,000 Dalton. The non-crosslinked fraction constitutes 15 per cent of the total hemoglobin. The p50 of 27 torr at pH 7.4 (plasma pH) corresponds to the value for the intra-erythrocyte hemoglobin at pH 7.25. The oncotic pressure is adjusted to 28 torr by the addition of 2.5 per cent weight per volume of human albumin. The methemoglobin content is less than 5 per cent of the total hemoglobin and remains constant for one year. The relative viscosity of the 8.5 per cent hemoglobin solution is 3.0 and, thus, is lower than the viscosity of blood. The intravascular half-life was determined in chimpanzees as about 16 hours. On the basis of available data, this hemoglobin solution may be suitable for clinical use as oxygen carrying plasma substitute.