Towards a novel haemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, vasoactivity and renal filtration

Blood transfusion is still a critical therapy in many diseases, traumatic events and war battlefields. However, blood cross-matching and storage may limit its applicability, especially in Third World countries. Moreover, haemoglobin, which in red blood cells is the key player in the oxygen transport from lung to tissues, when free in the plasma causes hypertension and renal failure. This investigation was aimed at the development of a novel haemoglobin-based oxygen carrier with low vasoactivity and renal filtration properties. Human haemoglobin was chemically conjugated with polyethylene glycol (PEG) under either aerobic or anaerobic conditions, following different chemical procedures. The resulting PEGylated haemoglobin products were characterized in terms of oxygen affinity, cooperativity, effects of protons and carbon dioxide concentration, and oxidation stability, and were transfused into rats to evaluate vasoactivity and renal filtration. A deoxyhaemoglobin, conjugated with seven PEG and seven propionyl groups, which we called Euro-PEG-Hb, did not produce profound hypertension, was 99% retained within 6 h, and exhibited oxygen binding properties and allosteric effects more similar to human haemoglobin A than the other tested PEGylated haemoglobin derivatives, thus appearing a very promising candidate as blood substitute.     

Characterization of the near infrared absorption spectra of cytochrome aa3 and haemoglobin for the non-invasive monitoring of cerebral oxygenation

Near infrared (IR) spectroscopy can give continuous, direct information about cerebral oxygenation in vivo by providing signals from oxygenated and deoxygenated haemoglobin and cytochrome aa3. Due to a lack of precise spectral information and uncertainties about optical path length it has previously been impossible to quantify the data. We have therefore obtained the cytochrome aa3 spectrum in vivo from the brains of rats after replacing the blood with a fluorocarbon substitute. Near infrared haemoglobin spectra were also obtained, at various oxygenation levels, from cuvette studies of lysed human red blood cells. Estimates of optical path length have been obtained. The data were used to construct an algorithm for calculating the changes in oxygenated and deoxygenated haemoglobin and oxygenated cytochrome aa3 in tissue from changes in near IR absorption.     

Blood physiological resources and their role in physiological adaptation of the body to blood loss (based on the experimental data and calculations)

In white rats, loss of about 66 +/- 3% of the blood volume results in an abrupt drop of the BP and arrest of the lung respiration. Introduction of a plasma substitute in the volume equal to the lost blood resulted in an increase of the BP and restoration of rhythmical respiration in 9 out of 14 experimental rats. Haematocrit after the blood loss and subsequent administration of the substitute was about 15%. Previously obtained data show that the Ht of about 15% decreases the oxygen transport to tissues. However, an increase in the lung ventilation, a shift of the haemoglobin dissociation curve to the right, an increase of oxygen extraction from the blood, and an increase in the minute blood volume by 50-100%, allow a sufficient oxygen transport to the organism tissues to be ensured at the Ht of about 15%. Thus, the reserve of the blood respiration function of the blood remaining after a life-threatening blood loss plays an important physiological role in replenishing the volume of the lost blood with the plasma substitute and in survival of the organism.     

Purification, crystallisation and preliminary X-ray study of haemoglobin from Crocodilis palustris and Crocodilis porosus

The unsolved three-dimensional structure of crocodile haemoglobin and its prospects as a blood substitute have led us to initiate the purification and crystallisation of haemoglobin molecules from crocodile species (Crocodilis palustris or mugger and Crocodilis porosus or salt water crocodile). The work has resulted in the prevention of polymerisation of naked haemoglobin molecules using N-ethylmaleimide or iodoacetamide. The purified monomeric haemoglobin molecule of C. porosus was crystallised in two different forms and X-ray diffraction data were collected up to 2 A resolution for both forms. Form I: a=53.62, b=53.55, c=103.77 A; beta=93.35 degrees, space group P2(1), Z=2. Form II: a=71.30, b=54.70, c=80.00 A; beta=106.4 degrees, space group P2(1), Z=2. Structure solution and rigid body refinement of form I data resulted in a model with R(free)=0.42 and R=0.35.     

Uptake of homologous haemolymph protein by salivary glands of Chironomus thummi

Radioisotopically labelled homologous protein fractions were injected into the haemolymph of Chironomus larvae. Among four different protein fractions, fraction II appears to enter the salivary gland and accumulates in the glandular lumen. Lack of blood pigmentation in the gland and secretion lumen indicates that the haemoglobin is probably not secreted, but a portion of the haemoglobin molecule or a fraction co-precipitating with haemoglobin in fraction II is being transported by the gland. Since only a portion of fraction II is taken up and secreted, the specificity of the process suggests to us a molecular basis for the activity which must ultimately be reflected in the genes of the transporting tissue.     

Brain oxygen transport related to levels of fetal haemoglobin in stable preterm infants.

The relative amount of regional cerebral oxygen transport was compared between different preterm infants by performing measurements of cerebral blood flow velocity, mean arterial blood pressure, whole blood viscosity and haemoglobin content for each individual. In addition the percentage of fetal haemoglobin was determined. On 25 occasions measurements of fetal haemoglobin and cerebral oxygen transport have been performed prior to and following a blood transfusion with adult red blood cells. Comparison of the data for cerebral oxygen transport suggests that the actual amount of cerebral oxygen transport is lowest at fetal haemoglobin levels below 30% and will increase progressively as soon as the percentage of fetal haemoglobin rises about 30%. Thus, at increasing fetal haemoglobin levels, cerebral haemodynamic mechanisms in the human neonate cause elevations of regional cerebral blood flow and oxygen transport. The found increase of cerebral blood flow and oxygen transport at high fetal haemoglobin levels will minimize the impeded dissociation and delivery of oxygen to brain tissues.     

The interaction of triethyltin with components of animal tissues

1. The distribution of triethyl[(113)Sn]tin chloride in the rat, guinea pig and hamster is not uniform, the highest concentrations being in rat blood and the liver of all three species. 2. Subcellular fractionation of rat liver, brain and kidney shows that triethyltin binds to all fractions to different extents. In the liver of the rat and guinea pig the supernatant fraction contains the largest amount and the highest specific concentration; this triethyltin is bound to a non-diffusible component. 3. Rat haemoglobin is responsible for the binding of triethyltin in rat blood (2 moles of triethyltin/mole of haemoglobin). Haemoglobins from other species have much less affinity for triethyltin. 4. A variety of other proteins do not bind triethyltin.     

Biases incurred from nonrandom repeat testing of haemoglobin levels in blood donors: Selective testing and its implications

To help prevent anaemia, it is a requisite for blood donors to undergo a haemoglobin test to ensure levels are not too low before donation. It is therefore important to have an accurate testing device and strategy to ensure donors are not being inappropriately bled. A recent study in blood donors used a selective testing strategy where if a donor's haemoglobin level is below the level required for donation, then another reading is taken and if this occurs again, a third and final reading is used. This strategy can reduce the average number of readings required per donor compared to taking three measurements for all donors. However, the final decision‐making measurement will on average be higher than a single measurement. In this paper, a selective testing strategy is compared against other strategies. Individual‐level biases are derived for the selective strategy and are shown to depend on how close a donor's true haemoglobin level is to the donation threshold and the magnitude of error in the testing device. A simulation study was conducted using the distribution of haemoglobin levels from a large donor population to investigate the effects different strategies have on population performance. We consider scenarios based on varying the measurement device bias and error, including differential biases that depend on the underlying haemoglobin level. Discriminatory performance is shown to be affected when using the selective testing strategies, especially when measurement error is large and when differential bias is present in the device. We recommend that the average of a number of readings should be used in preference to selective testing strategies if multiple measurements are available.     

Effect of venesection on calf blood flow in polycythaemia.

Calf blood flow at rest and during postocclusive reactive hyperaemia was measured using an electrocardiogram-triggered plethysmograph in 14 patients with polycythaemia (nine with primary disease and five with polycythaemia secondary to cyanotic heart disease) before and after a course of venesection. The mean packed cell volume was reduced from 0.57 to 0.47, and whole-blood viscosity fell by 50% at low shear rates. Venesection did not affect rest flow, but peak flow was increased by 18%. The increase in peak flow failed to compensate for the reduced haemoglobin content of the blood, calculated haemoglobin delivery being reduced by 23% at rest and 10% during reactive hyperaemia. These results indicate that while venesection improves blood viscosity, this does not necessarily lead to improved delivery of oxygen to the tissues.     

Seasonal changes in the blood parameters of two major carps, Labeo rohita (Ham.) and Cirrhina mrigala (Ham.).

Seasonal changes in blood properties of two Indian major carps, L. rohita and C. mrigala, were investigated. Haematocrit values, haemoglobin concentrations and erythrocyte numbers showed seasonal fluctuations, being high from September to November and again in April-May, and low from January to March. Males had generally higher blood values than the females.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

FV blood group and haemoglobin type versus haematological and blood chemical parameters in young Swiss bulls

A relationship between the FV blood group phenotype and 4 out of 45 haematological and blood chemical parameters--red cell number, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and serum iron--has been demonstrated in young bulls of three Swiss cattle breeds. There was also a relationship between haemoglobin type and 7 out of 45 haematological and blood chemical parameters (haemoglobin concentration, red cell number, MCV, MCHC and red cell concentrations of K+ and Na+ and their sum). In addition to expanding the species in which there is an effect of haemoglobin phenotype on MCV to include cattle, these data also demonstrate a significant correlation between their FV phenotype and MCV.     

Observations on the blood of the Chinese silver carp Hypophthalmichthys molitrix (Valenciennes)

The ranges and mean values for several blood parameters of adult specimens of Hypophthalmichthys molitrix were determined in order to establish a standard haematological profile for future investigations. Several significant correlations were established, the most important being between haematocrit, haemoglobin and erythrocyte counts. The data are discussed in relation to the general biology of this species.     

Development of a combined setup for simultaneous detection of total and glycated haemoglobin content in blood samples

An original setup for analysis of glycated haemoglobin (HbA1c) in blood is reported. The construction employed a combination of the piezoelectric biosensor for glycated haemoglobin and the flow-through photometric sensor for total haemoglobin (Hb). The modification of gold electrodes with 3-aminophenylboronic acid (APBA) as a specific ligand was studied; the chemisorbed conjugate of APBA with a long-chain thiocompound provided the best affinity for HbA1c. The effect of various operating parameters, such as flow rate and instrumental setup, was optimised. The total haemoglobin content was analysed as absorbance of the haemoglobin-cyanide derivative at 540 nm. Only one standard (calibrator) diluted in various ratio was necessary for calibration and 1 microl of blood was sufficient for analysis. The full range of HbA1c content (4-15%) in blood can be analysed; the working ranges of total and glycated haemoglobin were 50-2000 and 10-90 microg/ml, respectively. The developed method was successfully evaluated on blood samples collected from diabetics.     

Haemoglobin types in peripheral blood and bone marrow cells in rats after a single blood loss.

The authors studied quantitative changes in the synthesis of haemoglobin types in rat peripheral erythrocytes and in the cells of the bone marrow erythrocytic series after a single blood loss. They demonstrated that raised synthesis of given haemoglobin types could be found in the peripheral cells 5-7 days after blood loss. These changes could already be detected in the bone marrow cells 24 hours after blood loss. The question of the probable site and level of the origin of altered haemoglobin synthesis is discussed.     

Some haematological responses of normal and splenectomized rainbow trout Salmo gairdneri to a 12% blood loss

Anaemia can be induced in Rainbow trout by removing 12 % of the blood volume by cardiac puncture. Recovery from such an anaemia has been monitored in both normal and splenectomized fish. After the bleeding there is a decrease in red cell numbers, haematocrit and blood haemoglobin concentration. Although recovery in normal trout was not complete within 27 days, which was the period of the observations, it was seen as a replenishing of red cell numbers, haematocrit and blood haemoglobin concentration. Mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), both depressed by the bleeding, did not appear to recover during the experimental period.
Splenectomy reduced erythropoietic intensity. After bleeding all the parameters studied were more depressed than those found in normal trout, and recovery was not noted. Blood haemoglobin and red cell numbers did not have the same post-bleeding pattern as normal individuals; MCH and MCHC values remained markedly depressed.
Splenic involvement was further demonstrated by bleeding trout for a second time from both normal and splenectomized groups. In contrast to the response of normal animals, there was a further deterioration of red cell numbers and blood haemoglobin concentration in splenectomized individuals.     

FV blood group and haemoglobin type versus haematological and blood chemical parameters in young Swiss bulls

A relationship between the FV blood group phenotype and 4 out of 45 haematological and blood chemical parameters — red cell number, mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), and serum iron — has been demonstrated in young bulls of three Swiss cattle breeds. There was also a relationship between haemoglobin type and 7 out of 45 haematological and blood chemical parameters (haemoglobin concentration, red cell number, MCV, MCHC and red cell concentrations of K⁺ and Na⁺ and their sum). In addition to expanding the species in which there is an effect of haemoglobin phenotype on MCV to include cattle, these data also demonstrate a significant correlation between their FV phenotype and MCV.     

A mathematical model for the computation of carboxyhaemoglobin in human blood as a function of exposure time.

A mathematical model is developed for the carbon monoxide (CO) uptake by the blood by taking into account the molecular diffusion, convection, facilitated diffusion and the non-equilibrium kinetics of CO with haemoglobin. The overall rate for the combination of CO with haemoglobin is derived by including the dissociation of CO from carboxyhaemoglobin (COHb). The resulting coupled system of nonlinear partial differential equation with physiologically relevant initial, entrance and boundary conditions is solved numerically. A fixed point iterative technique is used to deal with nonlinearities. The concentration of COHb in the blood is computed as a function of exposure time and ambient CO concentration. The COHb levels computed from our model are in good agreement with those measured experimentally. Also, results computed from our model give better approximation to the experimental values compared with the results from other models. The time taken by the blood COHb to attain 95% of its equilibrium value is computed. The COHb concentration in the blood increases with the increase in ventilation rate, association rate coefficient of CO with haemoglobin and total haemoglobin content in the blood, and with the decrease in dissociation rate coefficient of CO with haemoglobin and mean capillary blood PO2. It is found that the COHb level in the blood is not affected significantly because of endogenous production of CO in the body under normal condition. However, the effect may be significant in the patients with haemolytic anaemia.     

Chemical kinetics measurements on the reaction between blood and ozone

The pseudofirst-order ozonization rate constant of whole bovine blood has been measured in comparison to that of free haemin. The free prosthetic group haemin (which has also the central iron atom in the oxidized form) shows k values in the range of 0.20-0.03 s(-1) while the haeme groups inside haemoglobin protein and contained in the whole blood sample show slightly lower k values, just in the range of 0.10-0.02 s(-1). It has been found that ozone even with whole blood reacts specifically with haemoglobin of the red cells because it is adsorbed selectively on the iron atoms of the haeme prosthetic groups of haemoglobin. The absorption implies the oxidation of the central iron atom of the haeme groups with formation of methaemoglobin followed by an oxidative fission of the haeme rings. The other blood components do not exert any significant protection to the reaction between ozone and haemoglobin, which appear extremely specific and selective like the reaction between CO or HCN and haemoglobin. By analogy with the behaviour of these other gases ozone may be classified as a blood poison. The results of this work are discussed in the frame of the risks connected to the ozonotherapy and autohaemotherapy involving the blood ozonization of human or animal subjects and the re-injection of ozonized blood into the bodies.     

Basic haematological values in antelopes--IV. A comparison and concluding remarks

Comparison of mean erythrocyte counts, mean haematocrit values, mean haemoglobin content and mean corpuscular volume of the red cell is presented for 21 species of antelopes (four species being excluded because of the small number of animals investigated). A direct relationship of haematocrit values and haemoglobin content and an inverse relationship of red cell counts and mean corpuscular volume of the erythrocytes were noted. The significance of an increased total surface area for oxygen exchange is discussed and values obtained in the red blood picture are compared with those of domesticated animals (cattle, goat and sheep) taken from the literature. Mean leukocyte counts were found to be in the human range with the exception of two species, but significantly lower than in domesticated artiodactylids. Problems in assessing the general health, the age, the effect of diet and of environment in captivity are discussed. Problems of methodology, especially of blood sampling, are given comparing results in manually restrained and in sedated animals.