Summary
We succeeded in determining the O2 saturation of Hb in capillaries of the rabbit myocardium with a cryo-microphotometric method. Myocardial samples (2–3 mm Ø) were removed in situ with a pair of nitrogen-cooled copper tongs, and rapidly frozen in Freon 13. The samples were sectioned into 10–12 μm s;ices in a cryotome at about −60°C. Hemoglobin absorption spectra were measured on the capillary sections of the frozen slices at −100°C in a vacumm-isolated microscope cooling chamber which we developed for the UMSP 1 (C. Zeiss, Oberkochen). The spectra were subdivided into three basic components (oxygenated Hb, desoxygenated Hb, and desoxygenated dehydrated Hb) with a weighted multicomponent analysis (Lübbers and Wodick, 1969). The basic components were measured in Hb solutions. The accuracy of the method was tested both with Hb solutions inserted into glass capillaries, and Hb droplets. O2 saturations of the Hb solutions were measured at room temperature. Subsequently, both test solutions were frozen. HbO2 saturation measured in the glass capillaries at low temperatures agreed well with those recorded at room temperature. HbO2 saturation of the sectioned hemoglobin droplets was found to be systematically increased with low HbO2 saturations as compared with the records made at room temperature. This systematic error was determined and the intracapillary HbO2 saturations were corrected accordingly. Measurements on samples of the rabbit myocardium showed that the histogram of capillary HbO2 saturations has a maximum at saturations of 20–30%. The lowest saturation values were found to be between 0 and 10%, but these values were recorded less frequently. The arterial HbO2 saturation in the femoral artery was about 98%.
Similar content being viewed by others
Literatur
Blackman, M., Lisgarten, N. D.: The cubic and other structural forms of ice at low temperature and pressure. Proc. roy. Soc. A239, 93 (1956)
Branton, D., Moor, H.: Fine structure in freeze-etched allium cepa L. root tips. J. Ultrastruct. Res.11, 400–411 (1964)
Cain, D., Davies, R. E.: Rapid arrest of metabolism with melting freon. In: Rapid mixing and sampling techniques in biochemistry. B. Chance, R. H. Eisenhardt, Q. H. Gibson and K. K. Lonberg-Holm (Eds.). New York: Academic Press 1964
Contan, J. B., Kamerling, S. E.: Studies in the chlorophyll series. Evidence as to structure from measurements of absorption spectra. J. Amer. chem. Soc.53, 3522 (1931)
Estabrook, R. W.: Low temperature spectrum of cytochrome c. Biochim. biophys. Acta (Amst.)19, 184 (1956)
Estabrook, R. W.: Spectrophotometric studies of cytochromes cooled in liquid nitrogen. In: Haematin enzymes. J. E. Falk, R. Lemberg and R. K. Morton (Eds.). Oxford-London-New York: Pergamon Press 1961
Fernandez-Moran, H.: Low temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II. Ann. N. Y. Acad. Sci.85, 689–713 (1960)
Fleckenstein, A., Janke, J., Davies, R. E., Krebs, H. A.: Chemistry of muscle contraction. Nature (Lond.)174, 1081 (1954)
Grunewald, W.: Der Einfluß des deaquatisierten Hämoglobins auf die kryophotometrisch bestimmte HbO2-Sättigung. Pflügers Arch.335, R4 (1972)
Grunewald, W., Lübbers, D. W.: Fixation of the O2 saturation of the hemoglobin in the capillary blood by rapid freezing. Pflügers Arch.307, R12 (1969)
Grunewald, W., Lübbers, D. W.: Application of rapid freezing methods for measurements of intracapillary hemoglobin saturation in tissue slices. Pflügers Arch.316, R2 (1970a)
Grunewald, W., Lübbers, D. W.: Methode zur Bestimmung der lokalen intrakapillären Hämoglobin-Sättigung. Naturwissenschaften57, 134 (1970b)
Grunewald, W., Lübbers, D. W.: First systematic measurements of the intracapillary HbO2-saturation in the rabbit myocardium with the cryophotometric method. Pflügers Arch.347, R43 (1974)
Keilin, D., Hartree, E. F.: Effect of low temperature on the absorption spectra of haemoprotein with observation on the absorption spectrum of oxygen. Nature (Lond.)164, 254–259 (1949)
Kreuzer, F., Hoofd, L. J. C.: Facilitated diffusion of oxygen in the presence of hemoglobin. Respir. Physiol.10, 542–558 (1970)
Laué, D.: Ein neues Tonometer zur raschen Äquilibrierung von Blut mit verschiedenen Gasdrucken. Pflügers Arch. ges. Physiol.254, 142–143 (1951)
Lübbers, D. W., Niesel, W.: Über die Auswertung von spektralen Veränderungen in biologischen Objekten. Naturwissenschaften44, 59–60 (1957)
Lübbers, D. W., Wodick, R.: The examination of multicomponent systems in biological materials by means of a rapid scanning photometer. Appl. Optics8, 1055–1062 (1969)
Lübbers, D. W., Wodick, R.: Schnelle Photometrie komplizierter biochemischer Mehrkomponentensysteme. Z. Analyt. Chem.261, 271–280 (1972)
Luyet, B. J.: On the growth of the ice phase in aqueous colloids. Proc. roy. Soc. B147, 434 (1957)
Merryman, H. T.: Mechanics of freezing in living cells and tissues. Science124, 515–521 (1956)
Miyamoto, Y., Moll, W.: Measurements of dimensions and pathway of red cells in rapidly frozen lungs in situ. Respir. Physiol.12, 141–156 (1971)
Mommaerts, W. F. H. M.: Is adenosin triphosphate broken down during a sinlge muscle twitch? Nature (Lond.)174, 1088 (1954)
Mommaerts, W. F. H. M., Schilling, M. O.: Interruption of muscular contraction by rapid cooling. Amer. J. Physiol.182, 579–593 (1955)
Moor, H.: Die Gefrier-Fixation lebender Zellen und ihre Anwendung in der Elektronenmikroskopie. Z. Zellforsch.62, 546–580 (1964)
Moor, H., Mühlethaler, K.: Fine structure in frozen-etched yeast cells. J. Cell Biol.17, 609–628 (1963)
Moor, H., Riehle, U.: Snap-Freezing under high pressure: a new fixation technique for freeze-etching. 4th Europ. Reg. Conf. Electron. Microsc., Rome 1968, pp. 33–34
Scholander, P. F.: Oxygen transport through hemoglobin solutions. Science131, 585–590 (1960)
Staub, N. C.: Gas exchange vessels in the cat lung. Fed. Proc.20, 107 (1961)
Staub, N. C., Storey, W. F.: On the possibility of freezing living lung rapidly. Physiologist3, 151 (1960)
Stephenson, M. D.: Ice crystal growth during the rapid freezing of tissues. J. biophys. biochem. Cytol.2, 45–52 (1956)
Thorell, B., Chance, B., Legallais, V.: Microspectrophotometry of cytochromes in the single cell at room and liquid nitrogen temperatures. J. Cell Biol.26, 741–746 (1956)
Weigelt, Ch., Schneider, S., Grunewald, W., Lübbers, D. W.: Determination of HbO2 gradients in thin layers—method to quantify facilitated diffusion. Pflügers Arch.339, R4 (1973)
Wilson, D. F.: Effect of temperature on the spectral properties of some ferrocytochromes. Arch. Biochem. Biophys.121, 757–768 (1967)
Wittenberg, J. B.: The molecular mechanism of hemoglobin-facilitated oxygen diffusion. J. biol. Chem.241, 104–114 (1966)
Wodick, R.: Die Auswertung von Mehrkomponentensystemen der Kurzzeitspektrophotometrie. Dissertation, Marburg (Lahn) 1968
Wodick, R., Lübbers, D. W.: Quantitative Analyse von Reflexionsspektren und anderen Spektren mit inhomogenen Lichtwegen an Mehrkomponentensystemen mit Hilfe der Queranalyse, I. Hoppe-Seylers Z. physiol. Chem.354, 903–915 (1973)
Wodick, R., Lübbers, D. W.: Quantitative Analyse von Reflexionsspektren und anderen Spektren mit inhomogenen Lichtwegen an Mehrkomponentensystemen mit Hilfe der Queranalyse, II. Hoppe-Seylers Z. physiol. Chem.354, 916–922 (1973)
Wollenberger, A., Ristan, O., Schoffa, G.: Eine einfache Technik der extrem schnellen Abkühlung größerer Gewebestücke. Pflügers Arch. ges. Physiol.270, 399–342 (1960)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Grunewald, W.A., Lübbers, D.W. Die Bestimmung der intracapillären HbO2-Sättigung mit einer kryo-mikrofotometrischen Methode angewandt am Myokard des Kaninchens. Pflügers Arch. 353, 255–273 (1975). https://doi.org/10.1007/BF00584288
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00584288