[Haemoglobin hyperpolymers, a new type of artificial oxygen carrier -- the concept and current state of development]. / Hämoglobin-Hyperpolymere, künstliche Sauerstoffträger eines neuen Typs -- Konzept und aktueller Stand der Entwicklung.

In the clinical setting, artificial oxygen carriers are needed when a patient has a tissue oxygen deficiency which he/she can not automatically compensate. There are two quite different situations where this might occur: (1) Heavy blood loss (e. g., following an accident) and (2) insufficient perfusion (e. g., as a result of arteriosclerosis or myocardial infarction) or anaemia, both without blood loss. In the first instance, an iso-oncotic oxygen-transporting plasma expander is required, whereas in the second instance a (hypo-oncotic) so-called blood additive is needed. This second type of situation also presents the greater range of very important indications. Experimental work has shown that, in comparison to erythrocytes, dissolved haemoglobin is able to release oxygen more rapidly (effective plasmatic transport), while at the same time also facilitating oxygen release from erythrocytes (mediator function). Blood additives occur naturally in lower forms of life (e. g., earthworm) where they can be found in the form of giant oxygen-carrying molecules. Using these natural forms as a basis, new oxygen-transporting blood additives were designed and developed (so-called haemoglobin hyperpolymers: HP (3)Hb) which exhibit a strong oxygen affinity (half saturation partial pressure p (50) = 16 Torr) and high cooperativity (n (50) = 2.1). One product has, up until now, been produced aseptically on a small technical scale and consists of highly purified, polymerised and pegylated porcine haemoglobin which is free of monomers and oligomers, with a mean molecular weight of approximately 800 kDa. It is sufficiently low in endotoxin (< 0,029 EU/mL), blood plasma compatible, and - at an effective concentration of 3 g/dL in blood plasma - causes only minor increases in oncotic pressure or viscosity. The product has a shelf-life of up to 2 years and is administered as a carbonyl derivative. Its half-life in the conscious rat is 30 h. This product was found to prevent death in rats where acute lung injury was induced using oleic-acid. In human self-experiments this product was repeatedly administered: No effects on blood pressure and heart rate, no increase in blood transaminase concentration and no immunological reaction were seen; the latter was also not found in selected sensitive mice. Furthermore, the blood additive is universally applicable as an oxygen transporter, since, when mixed with a conventional plasma expander, it can also be used to treat an oxygen deficiency occurring together with blood loss.

[An extended evaluation of a neuroleptanesthesia for the guinea pig with analysis of mixed expiratory gases during spontaneous breathing. Effects of fasting on the cardiorespiratory system and metabolism]. / Eine erweiterte Evaluation der Neurolept-Anästhesie für Meerschweinchen mit einer Analyse gemischt-exspiratorischer Gase während Spontanatmung. Wirkung des Fastens auf das kardiorespiratorische System und den Metabolismus.

The artificially ventilated guinea pig was frequently used for neurophysiological and respiratory studies. This species is also preferable for an evaluation of hemoglobin based artificial oxygen carriers, because its oxygen hemoglobin binding is very similar to that of man. But the narcosis of this animal-species is very difficult, because of cardiorespiratory depression induced by conventional procedures. The following intraperitoneal administered neuroleptanesthesia was proved in guinea pigs: 0.2 mg Fentanyl (Janssen/D), 10 mg Droperidol (Janssen/D) and 400 mg Urethan in 10 ml isotonic sodium chloride solution per kg body weight. Our new animal model with a special valve system enables to assess the gas exchange under spontaneous breathing, cardiovascular and the acid-base parameters. The vital parameters of animals were stable over 6 hours and very close to those of awake animals, especially the arterial average blood pressure. For that reason, this established neuroleptanesthesia of guinea pigs is preferable for research purpose. The fasted animals show significantly decreased values of arterial blood pH (7,345 vs. 7401), of heart frequency (244 vs. 277 min(-1)), and of ventilation value (167 vs. 205 ml/min) compared to non-fasted animals.

[Development of a flexible cardiorespiratory monitor based on induction plethysmography]. / Entwicklung eines flexiblen kardio-respiratorischen Monitors auf Basis der Induktionsplethysmographie.

Induction plethysmography (IP) utilizes changes in the inductance of sinusoidal wires embedded in elastic bands placed around the chest and abdomen to detect volume changes in the two compartments. These changes can be attributed to respiration or heart beat. To date, most applications have been tailored to an investigation of respiration. More sensitive systems have been employed for the detection of cardiac activity. The wires within the bands, which function as the coil in a resonant circuit, are excited by an oscillator. Among other factors, the inductance of the coil depends on the cross-sectional area of the coie, and changes with respiration in coils placed around the chest and abdomen. Using LabView software, the biosignals obtained undergo an analog-to-digital conversion prior to processing. The system was calibrated using the isovolume method. In 10 adults, IP was tested against a pneumotachograph (PNT) in different body positions (standing, sitting, supine, prone). Correlation between tidal volumes measured with IP and PNT was of r > or = 0.96 on average, recalibration being done after each change in position. The absolute mean error ranged between 3.7 and 8.5%, depending on body position. The smallest error (3.7%) and greatest agreement between the two methods was found in the supine position (93.3% of the IP measurements within +/- 10% of the PNT measurements). An IP application that could be used to collect data over the long term and which is in good agreement with PNT was developed by employing a "virtual instrument" (VI, LabView) for flexible data acquisition and data processing. Agreement was best when the volunteer adopted a supine position. A smaller correlation was found in standing or seated subjects. This might be due to the fact that in the latter two positions, the respiratory system may have more than 2 degrees of freedom, and thus cannot be adequately monitored by only two bands around the thorax and abdomen. Signals produced by cardiac activity were detectable on the surface of the body.

[A miniaturized, highly sensitive polarimeter as detector in an implantable glucose probe. II. Opto-electronic amplification and processing of measuring signals]. / Ein miniaturisierbares, sehr empfindliches Polarimeter als Detektor einer implantierbaren Glukosesonde. II. Opto-elektronische Verstärkung und Verarbeitung der Messsignale.

There is a clinical requirement for an implantable telemetric probe for monitoring glucose levels in humans. This probe can measure the glucose content of the intercellular tissue fluid, which reflects glucose levels in the blood. The lifespan of such an implantable probe should be maximal, so that presumably only physical measuring detectors, but not aging-sensitive bio-sensors can be considered. We are in the process of developing a very sensitive miniaturised detector based on polarimetry, capable of determining the measuring parameter--the spatial orientation of the in-plane vibration of a polarised light beam--with high accuracy. This is necessary for our purpose, since the physiological and pathological glucose levels modify in in-plane vibration by only a tiny angle of rotation. The high level of accuracy is achieved by various specific mechanisms both of the measuring parameters and the electric signal. Two suitable optoelectronic amplification methods are described. The first makes use of the ratio of the signal provided by the intensity of two consecutive beams, derived from the original light beam with the aid of a beam splitter. In this way, the sensitivity of determining the spatial position of the in-plane vibration of the polarised light beam can be increased by up to 50-fold in comparison with a "simple" polarimetry. The second method requires two very closely approximated (quasi united) or actually united beams from two sources, which are both "fixed-phase" time-coupled and quantitatively periodically intensity-modulated in opposite sense. Together with the already-mentioned ratio of the intensity signals of two consecutive beams, a periodically modulated signal generated from the individual signals is derived from this quasi-unified beam that enables the use of a phase sensitive rectifier-amplifier (lock-in amplifier) with its enormous amplification factor and noise elimination in the following circuitry.

[Miniaturization capable, very sensitive polarimeter as detector of an implantable glucose probe. I. Optical amplification of the measuring value]. / Ein miniaturisierbares, sehr empfindliches Polarimeter als Detektor einer implantierbaren Glukosesonde. I. Optische Verstärkung der Messgrösse.

From a clinical point of view, an implantable telemetric probe for monitoring the blood glucose profile is highly desirable. It should be capable of monitoring the blood glucose level continuously or at regular brief intervals, if necessary requirement-controlled. Apart from blood, measurement can also be made in intercellular tissue fluid, for example, in subcutaneous connective and fatty tissue, because this fluid accurately reflects blood glucose levels after only a brief, but negligible, time lag. Since the functional lifespan of an implantable probe is of decisive importance, only physical sensors, but not bio-sensors can be considered. We are in the process of developing a very sensitive miniaturised detector based on polarimetry, capable of determining the measuring parameter--the spatial orientation of the in-plane vibration of a polarised light beam--with extreme accuracy. This is a very important point, since the physiological and pathological glucose levels modify the in-plane vibration by only a very tiny angle of rotation. The high level of accuracy is achieved by various specific optical amplification mechanisms, and amplification of the electric signal. Two purely optical amplification methods are described here. Simple linear elongation of the optical path of a laser beam within the sample, resulting in a proportional amplification of the measuring signal, is obviously strictly limited in an implantable probe. We therefore developed a technique that preserves the polarisation state of the light beam during reflection. This technique makes possible multiple passage of the light beam through the fluid being sensed, thus elongating the optical path by "folding" the light beam without the need to enlarge the measuring cuvette. In a second possibility, enlargement of the rotation angle can be achieved by reflecting the light beam from a suitable surface, when the orthogonal components of the polarised light beam are reflected to different extents.

A model of stepwise isovolaemic blood exchange in anaesthetised, spontaneously breathing rats to evaluate the oxygen transport efficiency of artificial oxygen carriers.

Our research pursues the production of hypo-oncotic artificial oxygen carriers, based on artificial covalently cross-linked hyperpolymeric mammalian haemoglobins. To evaluate their in vivo efficiency in oxygen delivery to the tissue we developed a small animal model of stepwise isovolaemic blood exchange in anaesthetised, spontaneously breathing rats. With the aid of a two-way respiratory micro valve for small animals the overall oxygen uptake by the tissue of the animal can be determined. Measurements of oxygen contents in arterial and mixed venous blood and of some further blood parameters together with known oxygen-binding characteristics of artificial and native oxygen carriers, permits the determination of the way the two oxygen carriers contribute to the overall oxygen uptake. These so-called partial oxygen net to transport rates (i.e. partial oxygen uptakes), related to the corresponding intravascular mass flow of the transporters, are characteristic measures of the efficiency of the oxygen transporter, the so-called oxygen transport quality. Other biological indicators for an adequate oxygen supply are oxygen-dependent changes of ventilation, cardiac output, heart rate, and systemic vascular resistance. The performance of artificial oxygen carriers is elucidated by a comparison with experimental results from the analogous treatment of rats with non oxygen-transporting plasma expanders.

A model for evaluation of artificial oxygen carriers regarding circulation, respiration and metabolism in anesthetized spontaneously breathing guinea pigs.

The evaluation of artificial oxygen carriers requires experiments with suitable animals. Many investigators do this with the classical laboratory animal, the rat, but it has a quite different oxygen pressure of half saturation (p50 = 36 mmHg) from that of humans (26 mmHg). It was demonstrated that induced changes of the p50 value in animals provokes substantial changes in important cardiovascular parameters. Therefore, we decided to develop a guinea pig model for evaluation of artificial oxygen carriers, because it has a p50 of about 24 mmHg that is very similar to that of man. We found an anesthesia combination using fentanyl/droperidol/urethane to be very suitable for the narcosis, because important cardiovascular and respiratory parameters remain normal. Our model allows assessment of arterial pressure, oxygen uptake, carbon dioxide release, cardiac output, total peripheral resistance, shock index, blood lactate level, and, in particular, it allows to differentiate the oxygen transport in blood. We evaluated two hemoglobin-based oxygen carriers: bovine and porcine hemoglobin polymerized with glutaraldehyde in isoncotic solution (n = 3). Control experiments (n = 4) were done with an isotonic albumin solution. The protocol comprised a so-called exchange phase (I) with different degrees of hemodilution and a so-called time phase (II), an observation period with a hematocrit of 10% over about 3 hours. In the control group substantial changes in mean arterial pressure, total peripheral resistance, shock index, oxygen uptake and of blood lactate level were seen. All these effects were prevented by the artificial oxygen carriers tested. The carriers proved to be very effective, as small quantities in blood effectively restored these parameters, presumably via synergetic effects. Moreover, the experiments clearly revealed the limitation of hemodilution, at least in guinea pigs: below a hematocrit of 20% all parameters mentioned above changed significantly. The animal model presented proved to be appropriate for the evaluation of artificial oxygen carriers.

A new measuring device for non-invasive determination of oxygen partial pressure and oxygen conductance of the skin and other tissues.

About fifty percent of the oxygen consumption of the skin is supplied by diffusion through the surface. This portion of the skin oxygen supply becomes of high importance in case of arterial occlusion. The oxygen permeation coefficient (P) of the upper layers and the oxygen pressure field within the skin determine the diffusive oxygen uptake from the outside. To our knowledge, the permeation coefficient (P) until now was only estimated by indirect methods of little practicability (Baumberger et al., 1951; Eberhard et al., 1978). An oxygen partial pressure of the skin is conventionally measured by modified CLARK type electrodes. A disadvantage of this so-called transcutaneous electrode is its oxygen consumption and the fixed coupling of the consumption with the oxygen pressure to be determined. Therefore the measurement always induces a systematic error (the so-called stirring effect) which depends, among other factors, on the oxygen availability of the skin under the electrode. The new device combines a consumption-free oxygen partial pressure detector on the basis of luminescence quenching by oxygen with an independently working specific oxygen consumer realized by an active galvanic chain (silver-lead element). The chain permits setting any oxygen mass flow (mO2) in a certain range by varying the electrode current choosing different resistors within the electrical circuit. According to the diffusion law, the surface oxygen pressure (ePO2) being measured is a linear function of the oxygen flow (mO2) directed to the cathode: ePO2 identical to -(1/P).(mO2/A) + icPO2; A: area under the cathode. The intracutaneous oxygen partial pressure (icPO2) is a virtual quantity defined by the equation given. Only by using an active electrode different oxygen mass flows can be set and so the oxygen conductance of the upper skin layers can be assessed. First experiments on human skin in the gluteal region of an adult delivered an estimated value of the permeation coefficient (P): 2.2.10(-5) ml O2 (STPD)/(atm.s.cm2) at 42 degrees C skin surface temperature; the intracutaneous partial pressure obtained was 5.5 kPa (41 mmHg) (STPD: "standard temperature pressure dry" conditions of the gas). At 42 degrees C skin temperature no burning occurs. The determined O2-conductance is in the same range as estimated formerly (see above). The intracutaneous oxygen partial pressure determined seems to be a realistic value of the tissue at 42 degrees C. By in vitro measurements with technical membranes the new device and procedure was validated giving precise values of the oxygen conductance. Hence the new method may be used for calibration of the oxygen flux optode (Holst et al., 1993). The O2-permeation coefficient (P) could be an important parameter for evaluating dermatological applications (which attempt to increase P) in the treatment of local dermal oxygen deficiency. The intracutaneous oxygen partial pressure found is a reasonable value for a surface temperature of 42 degrees. Because of the independence of the O2-partial pressure determined and the O2-consumption the new device exhibits no stirring effect and it provides more insight into the limitations of conventional transcutaneous oxygen measurement.

[A new microprocedure for continuous and non-consuming determination of cellular oxygen uptake based on fluorescence quenching]. / Ein neues Mikroverfahren zur kontinuierlichen und verbrauchsfreien Bestimmung der zellulären Sauerstoffaufnahme auf Basis der Fluoreszenzlöschung.

The oxygen uptake in a cell suspension can be measured by various methods using manometric, paramagnetic or photometric techniques, oximetry, mass spectrometry or radiospectrometry. Easy-to-apply Clark-type electrochemical (polarographic) sensors are by far the most commonly used devices in medical applications. One of their drawbacks is the fact that they consume oxygen and may cause systematic errors when measuring oxygen uptake. Since the beginning of this century, concentration dependent quenching luminescence by oxygen has been used in a number of experimental settings. Using this analytical approach it is possible to detect oxygen without consuming it. We report about a new method of assessing cellular oxygen uptake using the luminescence quenching by oxygen. In an 850 microliters oxygen-tight microchamber, a fluorescent dye (tetraphenylporphyrin) adsorbed on a monolayer of gas chromatographic beads is separated from a cell suspension by a silicone membrane. An active electrochemical electrode integrated within the chamber is used to calibrate the fluorescence signal. Fluorescence is generated by green light (wavelength lambda = 546 nm), the intensity of the emitted red fluorescent light (lambda > 630 nm) is measured with a photomultiplier tube. As the first application of this new method, the oxygen uptake of human lymphocytes was determined. The cells were prepared using a routine separating technique-gradient centrifugation in Ficoll. For methodological reasons, all experiments were carried out at a temperature of 22 degrees C. In 7 consecutive measurements, an oxygen uptake of 2.81 +/- 0.85 mmol O2/10(11) lymphocytes/h was found. In less concentrated suspensions this figure is higher--an effect known as the "crowding phenomenon"--which means that with increasing cell concentration the specific oxygen uptake rate decreases. Our values for cellular oxygen uptake are higher than those in the literature. Since most reported studies on lymphocytes were done at 37 degrees C, a larger difference between our values and those in the literature would be expected. This may in part be attributed to different cell concentrations, separation techniques, etc. Another explanation might be the fact that electrochemical oxygen sensors used by other authors consume oxygen. Cell suspensions investigated with polarographic sensors therefore need to be stirred. Slow stirring reduces the values of oxygen uptake, so that a high stirring frequency is needed to obtain correct results (so-called stirring effect of polarographic electrodes). High stirring frequencies, however, destroy cells, which might be another factor explaining the results reported above. Our new method based on fluorescence quenching consumes no oxygen, and is therefore independent of stirring.

Molar masses and structure in solution of haemoglobin hyperpolymers--a common calibration of size exclusion chromatography of these artificial oxygen carriers.

We are developing artificial oxygen carriers for medical use, based on synthetic polymers--so-called hyperpolymers--obtained by cross-linking mammalian haemoglobins. One requirement with respect to the polymers is that they should not increase the oncotic pressure of blood remarkably--this can be realized by high molecular weights of the polymers with a narrow distribution. They may act as a oxygen transporting blood additive, and--in combination with a plasma expander--as a blood substitute. Another important and desired property of the artificial oxygen carrier is a low viscosity, which--first--is due to a high degree of uniformity of the polymer size (or molar mass) distribution and--second--is influenced by the so-called structure in solution of the haemoglobin hyperpolymers. In this paper former determinations of molar masses--with size exclusion chromatography (SEC)--and of the structure in solution--using viscometric measurements--of hyperpolymers of human haemoglobin, synthetized with glutaraldehyde and with bis(thioisocyanato) benzenesulfonic acid as cross-linkers, were extended to hyperpolymers of bovine and pyridoxylated porcine haemoglobin, cross-linked with glycolaldehyde. These determinations were done by applying a new iterative procedure. Within a range of error all SEC calibration curves found were the same for all hyperpolymers investigated. So-called MARK-HOUWINK or structure in solution diagrams (logarithm of intrinsic viscosity versus logarithm of molar mass) yield equal straight lines for all the haemoglobin polymers. The first derivatives of these lines are the MARK-HOUWINK exponents which has a mean value of 0.38. These results indicate that there exists a common SEC calibration line for all different polymer haemoglobins produced with comparable preparative procedures. This calibration line differ significantly from that of native globular proteins--haemoglobin hyperpolymers are less compact--so a calibration of SEC with globular proteins for the determination of molar masses of haemoglobin polymers is erroneous. Furthermore, the structure in solution of the hyperpolymers is clearly different from that of flexible, randomly coiled, linear artificial polymers: hyperpolymers are more compact. A possible explanation is that the hyperpolymers--according to a great number of functional amino groups of haemoglobin-contain many intra-polymeric cross-links, and thus are at least import branched polymers or even macromolecular networks of the constituting haemoglobin "monomers".

[Perspectives of a respiratory function analysis]. / Perspektiven einer respiratorischen Funktionsanalyse.

The necessity to develop procedures to assess respiratory functions which are independent of collaboration, non-invasive, specific, differentiating and sensitive, is stressed. New measuring methods, like main stream detection for oxygen and carbon dioxide as well as continuous assessment of pulmonary impedance are permissive for this purpose. In addition new characteristic quantities are defined. By using these new tools it is shown that the Tiffeneau-maneuver effects the properties of the bronchial system substantially. Examples of application show clearly that single measurements are not sufficient for characterization of the bronchial system. Instead continuous measurement during a certain closed period are proposed to get a so-called bronchogram. The increased sensitivity possibly makes provocation tests with its risks needless. Experimental results reveal longterm instabilities of the bronchial system, which may be discovered by a longterm-bronchogram in analogy to a longterm electrocardiogram. A new detector for the skin is presented; it allows the determination of an intracutaneous oxygen partial pressure as well as the oxygen conductivity of the skin. All results offer possibilities of functional analysis as mentioned above.

[A simple method for improvement of the measurement of respiratory impedance in humans by increasing oropharyngeal impedance using cheek and mouth floor clamps]. / Eine einfache Möglichkeit zur Verbesserung der Bestimmung der respiratorischen Impedanz des Menschen durch Vergrösserung der oropharyngealen Impedanz mittels Wangen- und Mundbodenklammer.

It is known, that the upper airway (oropharyngeal) impedance (Z(op)) influences the measured input impedance of the respiratory system (Zrs). This investigation deals with simple methods reducing the artefact caused by Z(op). The following methods have been examined: (1) Supporting the cheeks with fingers, (2) supporting the cheeks and the floor of the mouth with fingers and (3) using a new mechanical clamp for the cheeks and the floor of the mouth. The effect of these procedures on Zrs, were investigated on two subjects. Also Z(op) has been estimated during a voluntary closure of the glottis (VALSALVA manoeuvre). The impedance has been assessed as well with forced sine shaped oscillations as with the pulsatile technique. All three procedures show, compared to measurements without any support, an increase of the Zrs, especially at high values. The highest increase has been obtained while using the clamp (up to 50%). The varied impedance must be placed in parallel to Zrs, because Z(op) increases even more than Zrs. So, by applying the clamp the subjects is better coupled to the measuring system. The clamp is easy to handle, leads to more exact values, and increases the inter- and intra-individual reproducibility.

[An innovative procedure of oxygen detection in medicine, biology, environmental research and biotechnology based on luminescence quenching]. / Ein neuartiges Verfahren der Sauerstoffdetektion für Medizin, Biologie, Umweltforschung und Biotechnik auf Basis der Lumineszenzlöschung.

For most (aerobic) animal organisms, oxygen is a mandatory and global substrate. The accurate measurement of oxygen is therefore of importance in the fields of medicine, biology, environmental research and biotechnology. The fact that oxygen is not readily soluble in aqueous media makes its detection more difficult. In contrast to the technique of polarography, the use of luminescence quenching by paramagnetic oxygen, does not consume the oxygen. Another problem of oxygen detection in connection with respiration is the need for very short response times. A third problem, which is associated with luminescence itself, is the fading of the dyes, which results in long-term signal instability. The last two problems can be optimally resolved by adsorbing the luminescence dye onto chromatographic materials--in particular hydrophobic material--having a very large internal surface area, and using the decay time in accordance with the Stern-Volmer equation as oxygen signal. For this, continuous evaluation of the signal is necessary. The carrier material doped with dye is incorporated in a single-grain layer. For measurements in liquids, the detector layer is protected by a black silicone membrane. Two designs are possible for the oxygen detector: (I) a special form using glass fibres, and (II) a miniature detector utilizing optoelectronic solid state technology. Both fluorescence and phosphorescence can be employed, the dye used being excited by light, obviating the need for quartz. The detector layers may be either of high sensitivity for small oxygen concentrations, or have equal sensitivity over the entire oxygen concentration range. There is an optimal figure for the specific amount of adsorbed dye. Application examples are given for respiration and for the determination of oxygen uptake by suspended cells.

[Experiments aimed at developing an implantable and continuously functioning glucose sensors based on polarimetry]. / Experimente zur Entwicklung eines implantierbaren und dauernd funktionsfähigen Glukose-Sensors auf Basis der Polarimetrie.

In vitro and in vivo experiments devised with the aim of developing a permanently implantable glucose sensor based on polarimetry are described. It was found that in ultrafiltrated human blood plasma the overall optical rotation was 94% specific for glucose, and that polarisation photometry yielded a sufficiently sensitive signal for in vivo glucose detection. The three types of capillary membrane intended for implantation that we tested, revealed an in vitro response time to glucose concentration of 10 minutes; when implanted, they maintain this over a period of weeks, during which time the same glucose concentrations can be measured daily in ultrafiltrated capillary fluid as in the blood of the animals (guinea pigs). The drop in glucose concentration induced 20 minutes after a single administration of insulin can also be detected in both fluids. The experiments described indicate that the development of an implantable polarimetric glucose sensor is possible.

[A respiratory microvalve for spontaneously breathing anesthetized small animals]. / Ein Mikro-Atemstromventil für spontan atmende narkotisierte Kleintiere.

For the accurate functional analysis of the gas exchange in the lungs or evaluation of artificial oxygen carriers in spontaneously breathing anaesthetized small animals, we developed a new respiratory micro-valve. The body of the valve is made of aluminium, and the flaps are made of silicone rubber. The maximum flow rate in a rat measured with a pneumotachograph and the micro-valve was an average of 19.9 ml/s during inspiration, and 17.8 ml/s during expiration. The pressure measured in the tracheal tube was -0.85 during inspiration, and +0.39 cm H2O during expiration; the end-expiratory pressure in the tube was zero. In two experiments with anaesthetised rats lasting 4-5 hours, ventilation, oxygen uptake, carbon dioxide release and the respiratory exchange ratio were 638 ml/min/kg, 21.7 ml O2(STPD)/min/kg, 16.6 ml CO2(STPD)/min/kg, and 0.77, respectively. There was no significant change in any parameter during the experiment. The micro-valve increases the dead space by approximately 35%, but this is well tolerated by the rats, which compensate by increasing their tidal volume by about 10 to 15%. The major advantage of using the micro-valve in comparison with other methods is the fact that the true difference between inspiratory and mean mixed expiratory gas can be measured with great accuracy. The micro-valve can readily be adjusted for optimal use with a range of animals.

Divinyl sulfone cross-linked hyperpolymeric human haemoglobin as an artificial oxygen carrier in anaesthetized spontaneously breathing rats.

The production of hyperpolymer haemoglobins, exhibiting sufficiently low colloid osmotic pressure and sufficiently low viscosity is possible, even in concentrations, and therewith oxygen transport capacity, high enough to supply an organism adequately with oxygen. Such hyperpolymers, when infused, are tolerated by anaesthetized rats in acute blood exchange experiments. Ex vivo determinations of plasma colloid osmotic pressure and both, plasma and whole blood kinematic viscosity during blood exchange showed, that corresponding properties found in vivo were refound within the animal. Furthermore we could show that hyperpolymers produced from desoxygenated human haemoglobin with divinyl sulfone as a crosslinker take part in tissue supply of oxygen to a substantial degree (about 50%) without and with increased inspiratory oxygen fraction, demonstrating the principal ability of hyperpolymers to transport oxygen in blood and to deliver it to tissues.

Influence of the polymerization step alone on oxygen affinity and cooperativity during production of hyperpolymers from native hemoglobins with crosslinkers.

The aim of this study was to find out how the polymerization per se changes oxygen affinity (P50) and cooperativity (n50) of various soluble huge hyperpolymers prepared from native hemoglobins by crosslinking. Increase of cooperativity would be expected considering natural hemoglobin networks. Those hyperpolymers with molecular weights of some 10(6) g/mol are candidates for artificial oxygen-carrying blood additives rather than volume substitutes. Human and bovine hemoglobin reacted with several crosslinkers (2,5-diisothiocyanatobenzenesulfonate (DIBS); 4,4'-diisothiocyanatostilbene-2, 2'-disulfonate (DIDS); 1,3-butadiene diepoxide (BUDE); glutaraldehyde (GDA)) in concentrated (case 1) and diluted (case 2) hemoglobin solutions. With high concentration hyperpolymer and with low concentration only monomer products were obtained. P50 and n50 of the products were determined at pH = 7.4, PCO2 = 40 mmHg, temp. = 37 degrees C. The difference of properties in both cases are regarded as the influence of polymerization per se. Considering this difference we found with almost all combinations of hemoglobin and crosslinker an increase of O2 affinity, with DIBS and DIDS cooperativity was not changed and with BUDE and GDA it was decreased. As compared with native hemoglobin loss of cooperativity is considerable in any combination and condition, but comparing human and bovine hemoglobin the first seems to maintain better cooperativity. In contrast bovine hemoglobin as compared with human hemoglobin maintains better or even decreases its O2 affinity upon reaction with the crosslinkers forming both, monomer and hyperpolymer products, especially in the deoxy state. DIBS and DIDS react very similarly.(ABSTRACT TRUNCATED AT 250 WORDS)

The proton Bohr factor of native and crosslinker treated hemoglobins--its possible significance for the efficacy of hemoglobin based artificial oxygen carriers.

Especially the (alkaline) proton Bohr effect seems to provide an important self regulating mechanism of the organism to deliver specifically oxygen into tissues suffering from O2 deficit. In this way these tissues switch from aerobic to anaerobic metabolism, get lactacid, thereby shifting oxygen hemoglobin binding curve to the right and thus facilitating the oxygen release. The higher the absolute value of the proton Bohr factor (: delta logP50/ delta pH) is the better this mechanism works. To get one characteristic number the proton Bohr factor at pH 7.1 is taken. This pH in blood is about a lower limit for organism and human blood has at this pH its maximum proton Bohr factor which is about -0.5. When designing a hemoglobin based artificial oxygen carrier such a high or even a higher proton Bohr factor should be aimed at. But bringing human hemoglobin into extracellular milieu decreases the said proton Bohr effect down to -0.31; about the same values have bovine and porcine hemoglobin under these conditions. Before native hemoglobin can be used as an artificial oxygen carrier outside the red blood cells, they must be crosslinked; otherwise they are cleared quickly by the kidneys. Reaction of human and bovine hemoglobin with the crosslinkers DIBS (2,5- diisothiocyanatobenzenesulfonate) and DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonate) decreases the proton Bohr effect once again substantially down to about -0.1 irrespective of the degree of polymerization (monomer and hyperpolymer). Proton Bohr factors of reaction products from various hemoglobins and different crosslinkers evaluated from measurements of other investigators largely confirm the findings of this study.(ABSTRACT TRUNCATED AT 250 WORDS)