Comp. Biochem. Physiol., 1975, Vol. 5OA,pp. 273 to 276. Pergamon Press. Printed in Great Britain
SPECTROPHOTOMETRIC MEASUREMENT OF HEMOGLOBIN OXYGEN SATURATION IN THE OPOSSUM DIDELPHIS
CLAYTON PETTY,* NICHOLAS C. BETHLENFALvAY,t AND THOMAS BAGEANT~ Departments
and Hematology, Brooke Army Medical Center, Ft. Sam Houston, Texas 78234, U.S.A. (Received 5 November
A spectrophotometric method can be used to measure oxyhemoglobin dissociation in opossums at optical densities of 500 and 592 nm. 2. Opossum hemoglobin spectrogram reveals a double-humped curve for oxygenated hemoglobin and a single-humped curve for reduced hemoglobin. 3. The Ps,, calculated by the above method at pH 7.4, 35°C and 40 mm Hg P, CO2 is 35.0 mm Hg of oxygen. Abstract-l.
INTRODUCTION THE SPECTROPHOTOMETRICmeasurement of the oxygen dissociation curve in man has proven to be as accurate (Nahas, 1951; Rand et al., 1966; Deibler et al., 1958) as the manometric technique
of Van Slyke & Neil1 (1924). Developmental research of the opossum (Didelphis virginiana) is expanding and a spectrophotometric method to study hemoglobin saturation would be advantageous in the examination of the respiratory functions of marsupial blood. MATERIALS AND METHODS Four female opossums were trapped and confined in the laboratory for 3-4 days prior to study. A 15-ml sample of cardiac blood was collected from each opossum into a sterile 20-ml glass syringe whose dead space had been filled with heparin. The syringe was immediately placed in ice and the blood studied within 1 hr of collection. Hemoglobin, red blood cell count and 2,3diphosphoglycerate (2,3-DPG) was measured on each blood sample. Aliquots of blood were equilibrated in a sterile glass tonometer flask (Instrumentation Laboratories 237 tonometer) at 37°C for 20 min. Totally saturated hemoglobin was obtained by exposure to 100% oxygen and unsaturated hemoglobin was obtained by adding sodium hydrosulfite to 1 ml of blood (Refsum, 1957). Hemoglobin saturation points that fell on the straight portion of the sigmoid dissociation curve were obtained by * Present address: Division of Anesthesiology, College of Medicine, University of Arizona, Tucson, Arizona 85724. t Present address: Department of Clinics, Fitzsimmons General Hospital, Denver, Colorado 80240. $ Department of Anesthesia and Hematology, Brooke Army Medical Center, Ft. Sam Houston, Texas 78234.
equilibrating blood samples with 4.0% oxygen-6.0% carbon dioxide-90.0% nitrogen and 5.9% oxygen-6.0% carbon dioxide-88.1% nitrogen gas mixtures. Following equilibration, a 3-ml portion was withdrawn anaerobically and used for simultaneous determination of blood gases and optical density spectrum. Hemoglobin and 2,3-DPG was not measured after tonometry as previous studies have shown no alteration (Carden & Petty, 1973). Blood gases and pH were measured at 37°C using microelectrodes (Radiometer) calibrated immediately prior to use. Oxygen and carbon dioxide values were read after 2 min and immediate recalibration allowed correction for any minor electrode drift during measurement. Gases used to calibrate the oxygen and carbon dioxide electrodes were recalculated daily using a mercurial barometer pressure reading corrected for temperature, latitude and water vapor pressure. Oxygen tension was corrected for the gas/blood difference (1.04 times original reading) and pH to 7.40 using the following formula (Severinghaus, 1966): A log P, OS = 0.48 A pH+0.00138
Temperatures in the tonometer and blood gas electrode water baths were checked daily with a National Bureau of Standards thermometer and maintained at 37.O”C. Hemoglobin was determined by the cyanmethemoglobin method. Concentration of 2,3-DPG was measured using an enzymatic, ultraviolet method and reagents supplied by the Sigma Chemical Company (St. Louis, MO.). The dead space of a l-ml glass syringe was filled with Triton X-100 (Diebler et al., 1958) and then filled anaerobically with the appropriately treated blood sample. The syringe was gently rotated to ensure complete hemolysis and was then attached tip down into a 0.075-mm cuvette (Beckman Co.). The cuvette was filled carefully to avoid any bubbles and checked for § Base excess calculations were taken from the blood gas calculator of Severinghaus (1966) and represented a minute change in A log P, O2 determinations. 273
CLAYTON PETTY,NICHOLASC. BETHLENFALVAY ANDTHOMASBAGEANT
red blood cell debris. A similar cuvette was filled with plasma obtained from the initial blood sample and treated in the same manner with Triton X-100. The plasma blank and hemolyzed sample were placed in a Beckman Acta C III recording spectrophotometer. A optical density spectrum from 450 to 700 nm was recorded for each blood sample using a chart speed of 10 nm/in., a scan speed of 2 nm/sec and a slit width adjusted to give a 500 dynode reading at 550 nm. An optical density spectrum was obtained for each opossum on a totally saturated hemoglobin, an unsaturated hemoglobin and two different partially saturated hemoglobin samples. METHODS The isobesic point [same molar extinction coefficient for reduced and saturated hemoglobin (Siggard-Andersen et al., 1962)] and wavelength for optimum separation of oxygenated and reduced hemoglobin were determined for each spectrogram. Using these two points and the formula described by Gordy & Drabkin (1957) for the Lambert-Beers laws of absorption the per cent hemoglobin saturation could be determined for the two The saturated hemoglobin samples. intermediate calculated per cent saturation was plotted against the corrected oxygen tension at pH 7.4, 37°C and 40 mm Hg P, CO, for the two samples of each opossum. In order to allow comparison of opossums the P, 0, was read at two points common to all animals, 33 per cent saturation and 43 per cent saturation. The saturation lines constructed for each opossum fell below the hemoglobin 50 per cent saturation point (P& It was assumed that the oxygen saturation curve for opossums
was linear between 33, 43 and 50 per cent saturation and a straight line was extended to the 50 per cent intercept to obtain an oxygen tension commonly used for comparison with other species. RESULTS
Table 1 lists the red blood cell data for each opossum. The hemoglobin, red blood cell count and calculated red blood cell volumes compare well with other laboratory data (Mays & Loew, 1968; Timmons & Marques, 1969). Values for hemoglobin concentration for the four animals appears satisfactory for determining accurate hemoglobin saturation by spectrophotometry (Rand
et al., 1967) since the normal value for opossum hemoglobin concentration is less than humans. The mean blood oxygen tension at 33 per cent saturation was 29.4 mm Hg and at 43 per cent saturation was 35.1 mm Hg. Extension of the line connecting the high and low saturation points to intercept at the 50 per cent saturation level gave a mean Pj,, of 39.1 mm Hg (see Table 2). The hemoglobin spectrograms were almost identical for each opossum. The isobesic point was found to be 500 nm and the optimal separation of oxygenated and reduced hemoglobin to be at a wavelength of 592 run (see Figs. 1 and 2). The familiar double-humped curve for saturated hemoglobin and the single-humped curve for reduced hemoglobin seen in humans and other mammalians was found in opossums. Tonometry required longer in opossum blood (20 min) for equilibration than in human blood (8 min; Bageant & Petty, 1973) under duplicate experimental conditions. DISCUSSION
Opossum blood has been shown to have a sigmoidshaped oxyhemoglobin dissociation curve with a P,, of approximately 35 mm Hg at 35°C (pH and P, CO, unknown; Scott, 1938) using the Van Slyke method. Correction of the Pso value of 39.1 mm Hg at 37°C found in our paper to 35°C was done by using the following formula: A log P 0, = 0.0240AT (Severinghaus, 1966) where T is temperature and 0.0240 is a constant derived from six experimental studies. The value of P,, at 35°C was found to be 35.0 mm Hg which is exactly the same value as determined by Scott (1938). Thus the P5,, value for the opossum is greater than that of man, but similar to that of the cat and rabbit. The use of the spectrophotometer for determining hemoglobin saturation has gained popularity over the years, and especially the Van Slyke manometric method because of the small sample size required and the rapid determination time. Opossum blood can readily be subjected to present spectrophotometric methods with 500 and 592 nm being used as wavelengths for determining hemoglobin saturation.
Table 1. Red blood cell data
Hemoglobin Total erythrocytes/mm3 ( x 106) (g/100 ml)
Mean cell Mean corpuscular volume hemoglobin W) (W g)
Mean corpuscular hemoglobin 2,3-DPG concentration (pmoles/ml packed red blood cells) (%)
1 2 3 4
13.0 9.0 13.0 10.0
4.3 4.0 4.5 4.1
80 74 78 75
28 23 25 24
36 29 32 30
3.63 2.83 2.69 3.05
;P + SE.
Measurement Table 2. Relationship
of oxygen tension to hemoglobin P* CO, Per cent saturation
oxygen saturation in the opossum saturation
at pH 7.4, 37°C and 40 mm Hg
Per cent saturation
Per cent saturation
1 2 3 4
33 33 33 33
28.8 29.2 31.0 28.8
43 43 43 43
36.0 34.0 34.5 36.0
50 50 50 50
41.1* 37.6* 37.0* 40.8*
R + S.E.
* Values at intercept from extension of original data points.
0% Hemoglobin Saturation
100% Hemoglobin Saturation
Fig. 1. Optical density spectra for fully oxygenated and totally reduced hemoglobin. Point A (500 run) represents the isobesic point and 592 nm the wavelength for optimum separation of oxygenated and reduced hemoglobin.
0% Hemogbbin Sahrratbn
100% Hemoglobin Saturation
27% Hemoglobin Saturation
-....“‘44% Hemoglobin Sahntti
Fig. 2. Representative
for fully oxygenated, hemoglobin.
CLAYTONPETTY, NICHOLAS C.
BETHLENFALVAYAND THOMAS BAGEANT
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REFSUMH. E. (1957) Spectrophotometric
determination of hemoglobin oxygen saturation in hemolyzed whole blood by means of various wavelength combinations. Scar& J. clin. Lab. Invest. 9, 19&193. SCOTT W. J. (1938) Gas transport by the blood of the opossum, Didelphys virginiana. J. cell camp. Physiol. 12, 391401. SEVERINGHAUS J. W. (1966) Blood gas calculator. J. appl. Physiol. 21, 1108-1116. SIGCARD-ANDERSENO., JORGENSENK. & NAERAA N. (1962) Spectrophotometric determination of oxygen saturation in capillary blood. &and. J. clin. Lab. Invest. 14, 298-302.
TIMMONSE. H. & MARQUESP. A. (1969) Blood chemical and hematological studies in the laboratory-confined, unanesthetized opossum, Didelphis virginiana. Lab. Anim. Care 19, 342-344.
VAN SLYKED. D. & NEILLJ. M. (1924) The determination of gases in blood and other solutions by vacuum extraction and manometric measurement. J. biol. Chem. 61, 523-573. Key Word Index-Oxyhemoglobin 2,3-diphosphoglycerate (2,3-DPG); virginiana).
dissociation; P,,; opossum; (Dideiphis