Wolfgang Barnikol

A novel, non-invasive diagnostic clinical procedure for the determination of an oxygenation status of chronic lower leg ulcers using peri-ulceral transcutaneous oxygen partial pressure measurements: Results of its application in chronic venous insufficiency (CVI)

The basis for the new procedure is the simultaneous transcutaneous measurement of the peri-ulceral oxygen partial pressure (tcPO2), using a minimum of 4 electrodes which are placed as close to the wound margin as possible, additionally, as a challenge the patient inhales pure oxygen for approximately 15 minutes. In order to evaluate the measurement data and to characterise the wounds, two new oxygen parameters were defined: (1) the oxygen characteristic (K-PO2), and (2) the oxygen inhomogeneity (I-PO2) of a chronic wound. The first of these is the arithmetic mean of the two lowest tcPO2 measurement values, and the second is the variation coefficient of the four measurement values. Using the K-PO2 parameter, a grading of wound hypoxia can be obtained. To begin with, the physiologically regulated (and still compensated) hypoxia with K-PO2 values of between 35 and 40 mmHg is distinguished from the pathological decompensated hypoxia with K-PO2 values of between 0 and 35 mmHg; the first of these still stimulates self-healing (within the limits of the oxygen balance). The decompensated hypoxia can be (arbitrarily) divided into “simple” hypoxia (Grade I), intense hypoxia (Grade II) and extreme hypoxia (Grade III), with the possibility of intermediate grades (I/II and II/III). Measurements were carried out using the new procedure on the skin of the right inner ankle of 21 healthy volunteers of various ages, and in 17 CVI (chronic venous insufficiency) wounds. Sixteen of the 17 CVI wounds (i.e., 94%) were found to be pathologically hypoxic, a state which was not found in any of the healthy volunteers. The oxygen inhomogeneity (I-PO2) of the individual chronic wounds increased exponentially as a function of the hypoxia grading (K-PO2), with a 10-fold increase with extreme hypoxia in contrast to a constant value of approximately 14% in the healthy volunteers. This pronounced oxygen inhomogeneity explains inhomogeneous wound healings, resulting in the so-called mosaic wounds. The hypoxia grades found in all of the chronic wounds was seen to be evenly distributed with values ranging from 0 to 40 mmHg, and therefore extremely inhomogeneous. In terms of oxygenation, chronic wounds are therefore inhomogeneous in two respects: (1) within the wound itself (intra-individual wound inhomogeneity) and (2) between different wounds (inter-individual wound inhomogeneity). Due to the extreme oxygen inhomogeneity, single measurements are not diagnostically useful. In healthy individuals the oxygen inhalation challenge (see above) results in synchronised tcPO2 oscillations occurring at minute rhythms, which are not seen in CVI wounds. These oscillations can be interpreted as a sign of a functioning arterial vasomotor system. The new procedure is suitable for the routine characterisation of chronic wounds in terms of their oxygen status, and correspondingly, their metabolically determining (and limiting) potential for healing and regeneration. The oxygen characteristic K-PO2 can furthermore be used as a warning of impending ulceration, since the oxygen provision worsens over time prior to the demise of the ulcerated tissue, thus making a controlled prophylaxis possible.

Complete healing of chronic wounds of a lower leg with haemoglobin spray and regeneration of an accompanying severe dermatoliposclerosis with intermittent normobaric oxygen inhalation (INBOI): a case report.

A new healing procedure has been developed on the basis of the successful treatment of therapy-resistant hypoxic (and practically anoxic) leg ulcerations located within a heavy dermatoliposclerosis. The procedure involves an initial intra-ulceral application of haemoglobin followed by the intermittent administration of normobaric oxygen via inhalation. Haemoglobin is capable of externally supplying the granulating wound bed with oxygen at low partial pressure in a physiological manner, like a micro lung, so that oxidative stress can be avoided. A long-term daily administration of oxygen from within – including the peri-ulceral skin – is achieved by intermittent normobaric oxygen inhalation (INBOI) regularly throughout the day in the form of 1-hour sessions. Using this combined healing treatment during haemoglobin applications the ulcerations healed within about 1 month, and subsequently with INBOI therapy within further approx. 4 months the peri-ulceral skin regenerated as far as the oxygenation status was concerned: The peri-ulceral transcutaneous oxygen partial pressure (tcPO2) of zero (measured during breathing of normal air) rose to a satisfactory value of approx. 35 mmHg. After 28 months of treatment, the completely hypoxic and degenerated skin on the leg had practically returned to normal with a PO2 of 45 mmHg. Furthermore, the skin dermatoliposclerosis regressed. The skin regeneration was long-lasting, which was probably related to cellular tissue regeneration with an increase in the capillary density, whereby it had to be maintained by regular oxygen inhalation (INBOI maintaining treatment). By unintended intra-individual therapy variations it is evidenced that local hypoxia was the reason for skin degeneration: 3 x 1 h oxygen inhalation were sufficient for the healing treatment; 2 x 1 h sufficed for maintenance, whereas 2 x 0.5 h did not. The new procedure carries practically no risks, is simple, cheap and effective. Whereas the application of haemoglobin requires professional supervision, the oxygen inhalation can be carried out at home following initial guidance and monitoring by a physician. Using this novel method, the therapy-resistant ulceration could be closed within 5 months, during which daily outpatient care was only necessary for 1 month. The successful outcome of the treatment in terms of improvement of oxygen supply can monitored at any time using peri-ulceral tcPO2 measurements, whereby, due to the inhomogeneity of the values, measurements at a minimum of two locations at the wound edge are strongly recommended and more measurements at more skin locations would be preferable. Besides its use in the healing of ulcers, the new procedure is also suitable for the prevention of ulceration development (prophylactic INBOI treatment) in skin rendered susceptible due to the presence of hypoxia. Here, peri-ulceral transcutaneous oxygen partial pressures of below 10 mmHg should be considered as being critical and are an indication for a prophylactic oxygen inhalation treatment. The new procedure may also be suitable even before the peri-ulceral oxygen partial pressure falls below 10 mmHg. Four measures for rehabilitation, conservation, and prevention with regard to a healed chronic wound are proposed.

Ein Mikro-Atemstromventil für spontan atmende narkotisierte Kleintiere - A Respiratory Micro-Valve for Spontaneously Breathing Anaesthetized Small Animals

Zur genauen Funktionsanalyse des Gasaustausches der Lunge oder zur Validierung der Sauerstofftransporteigenschaften künstlicher Sauerstoffträger an spontan atmenden narkotisierten Kleintieren ist ein Mikro-Atemstromventil erforderlich. Das hier vorgestellte Mikro-Ventil besteht aus Aluminium, und die benötigten Klappen werden aus Silikonkautschuk hergestellt. Die unter Anwendung des Mikroventils pneumotachographisch gemessenen maximalen Atemströme einer Ratte lagen im Mittel während der Inspiration bei 19,9 ml/s und während der Exspiration bei 17,8 ml/s. Dabei entstand im Tubus des Tieres inspiratorisch ein negativer Druck von 0,85 und exspiratorisch ein positiver Druck von 0,39 cm Wassersäule; endexspiratorisch ist der Tubusdruck gleich null. Während zweier Versuche mit Ratten von 4 bis 5 Stunden Dauer werden für die Ventilation 638 ml/min/kg, für die Sauerstoffaufnahme 21,7 ml O2(STPD)/min/kg, für die Kohlendioxidabgabe 16,6 ml CO2(STPD)/min/kg und für den Respiratorischen Quotienten 0,77 gemessen, ohne signifikante Änderung einer der Meßgrößen während der Versuche. Der Totraum vergrößert sich durch das Ventil um ungefähr 35 %, was die Tiere gut tolerieren und durch Steigerung des Atemzugvolumens um 10 bis 15 % kompensieren können. Im Vergleich zu anderen Methoden besteht der wesentliche Vorteil darin, daß die tatsächliche Fraktionsdifferenz der Atemgase zwischen Inspirationsund mittlerem Exspirationsgasgemisch mit großer Genauigkeit meßbar wird. Das vorgestellte Mikro-Atemstromventil läßt sich leicht an das betreffende Tier anpassen.

On the dissociation of human hemoglobin

SummaryA model, which takes into account the molecular explanation of the oxygen dissociation curves, is constructed for the dissociation into subunits of human haemoglobin. The model is characterized by the symmetrical dissociation of the Hb molecule into dimer subunits, each with an α- and a β-chain, as well as the further dissociation into unimers. Moreover, a stabilising intermediary substance,Z, must be assumed, in order to explain quantitatively the unusual molecular weight concentration dependence. TakingSchachman andEdelsteins (1966) expermental results as a basis, the parameters of the model are then determined with the aid of an electronic computer.ZusammenfassungIm Hinblick auf die molekulare Deutung der Sauerstoffbindungskurven wird ein Modell für die Dissoziation des Human-Hämoglobins aufgestellt. Das Modell ist charakterisiert durch den symmetrischen Zerfall des Hb-Moleküls in dimere Untereinheiten mit je einer α- und einer β-Kette und die weitere Dissoziation zu Unimeren. Ferner muß eine stabilisierende Zwischen-substanzZ angenommen werden, um den außergewöhnlichen Verlauf der Konzentrationsabhängigkeit des Molekulargewichtes quantitative zu deuten. Auf der Grundlage der experimentellen Ergebnisse vonSchachmann u.Edelstein (1966) werden die Parameter des Modells mit Hilfe einer elektronischen Rechenanlage ermittelt.

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.

Zur Interpretation der O2-Bindungskurve des Human-Hämoglobins

SummaryA hypothesis for the hemoglobin oxygen reaction after the procedure of “minimal model” is developed, which is able to explain quantitatively the dependence of molecular weight and of the O2 dissociation curve on the hemoglobin concentration. After this hypothesis the tetramer hemoglobin dissociates symmetrically into its dimer and monomer subunits. Each of these subunits has a specific O2 affinity, which is the same for every stage of their oxygenation. An essential aspect of this hyothesis, moreover, is the effect of a low molecule intermediary substance, Z, which stabilizes the tetramer. The parameter values obtained from the numerical calculations allow the Z substance to be identified with Ca++ and/or Mg++. By taking into consideration the complex-forming tendency of these bivalent ions, the other known properties of the O2 dissociation curve can be easily qualitatively explained. Because of the great importance of this complex-forming property, we call the model a coordination hypothesis.ZusammenfassungEs wird nach der Methode des einfachsten Modells eine Hypothese für die Hämoglobin-Sauerstoff-Reaktion entwickelt, die es erlaubt, u. a. die Abhängigkeit der O2-Bindungskurve und des Molekulargewichts von der Hämoglobinkonzentration zu beschreiben. Nach dieser Hypothese zerfällt das tetramere Hämoglobin symmetrisch in seine dimeren und monomeren Untereinheiten. Jeder dieser Untereinheiten kommt eine spezifische, für alle Oxygenierungsstufen gleiche O2-Affinität zu. Ein wesentlicher Bestandteil der Hypothese ist ferner die Wirkung einer niedermolekularen, das tetramere Hämoglobin stabilisierenden Zwischensubstanz Z. Die aus den numerischen Rechnungen erhaltenen Werte der Parameter geben Anlaß, die Z-Substanz mit Ca++ und/oder Mg++ zu identifizieren. Unter Berücksichtigung der Tatsache, daß diese zweiwertigen Ionen Komplexe bilden, lassen sich die weiteren bekannten Eigenschaften der O2-Bindungskurve qualitativ zwanglos deuten. Wegen der großen Bedeutung der Komplexbildung bezeichnen wir diese Modellvorstellung als Koordinationshypothese.

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.

Ein unabhngiges Verfahren zur Messung der Zusammensetzung von Gasgemischen mit Hilfe der Gaschromatographie

SummaryThe volume fraction of gas mixtures may be measured gas chromatographically by application of the 100% method, provided they can be separated. In case of gases it is not necessary to calibrate with another method. The specific character of the detector signals can be eliminated by measurements with pure components. The procedure is compared with the Scholander method. In case of O2/N2 mixtures the results agree within reproducibility, but in case of O2/CO2/N2 mixtures a systematic deviation of the CO2 fractions to smaller values is found as concerns the Scholander method. A gas mixture with 5.6 Vol% CO2 shows a difference of approximately 0.3 Vol%. The gas chromatographic method is almost 100 times as sensitive as the Scholander method. High fractions may be measured without problems and the method is not restricted to O2 and CO2.

On the Accuracy of an Improved Method for the Measurement of O2-Dissociation-Curves According to Niesel & Thews,1961

During the first part of its journey to tissue oxygen is carried by blood. The greatest part thereby is bound chemically to hemoglobin. The molecular mechanism of this binding gives the so-called O2-dissociation-curve its typical, sigmoidal form. The O2-dissociation-curve is the function between O2-saturation and O2 -parfial-pressure. This curve is of great importance for the O2-supply of ‘he tissues. It is important for practice as well as for formulating a theory in this field. Within the last few years it has been discovered, that the O2-dissociation-curve is influenced by organic phospha-tes, e.g. 2,3-DPG (CHANUTIN, CURNISH, 1967; BENESCH, BENESCH, 1967). Because of this new discovery a correlation to metabolism and also new therapeutic possibilities have been found. The influence of a low molecular substance on the O2-dissociation-curve could be deduced from a theo-retical analysis of the O2-hemoglobin-equilibrium, too (BARNIKOL, THEWS, 1969).

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 netto 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.