determination of the saturation of hemoglobin inhboz and hb in a study of the ,penetration of...

SPECTROPHOTOMETRIC STUDIES

XII. OBSERVATION OF CIRCULATING BLOOD IN VIVO, AND THE DIRECT DETERMINATION OF THE SATURATION OF HEMOGLOBIN IN

ARTERIAL BLOOD*

BY DAVID L. DRABKIN AND CARL F. SCHMIDT

(From the Departments of Physiological Chemistry and Pharmacology, School of Medicine, University of Pennsylvania, Philadelphia)

(Received fbr publication, September 19, 1944)

Uncertainty still exists concerning a fundamental point in hemoglobin physiology, the degree of saturation of arterial blood at sea level. Values for this quantity (per cent of oxyhemoglobin, HbOz) in man, reported in the more recent literature (5-7), are as low as 93.0 per cent and yield an average of 95.0. Such values for percentage saturation have been used in deducing the oxygen tension (~02) of arterial blood, usually read off from previously determined oxygen dissociation curves which relate saturation to tension. On typical dissociation curves (8) 93.0 and 95.0 per cent HbOz correspond respectively to approximately 65 and 80 mm. of Hg of arterial ~02. If these values are correct, a difference (ApOz) of appreciable magnitude (average = 20 mm. of Hg) exists between alveolar and arterial oxygen tensions. This ApOz has been accepted as a physiological phenomenon, and has been explained by the hypothesis that “oxygen equilib- rium is not attained unt.il after passage through the lung capillaries” (9). It may be pointed out at once that this indirect procedure for obtaining the arterial ~02 is not precise. Th e portion of the dissociation curve which applies to arterial blood at sea level is asymptotic; so that an increase in arterial saturation by 2 per cent (from 95 to 97 per cent) would markedly reduce, and an increase of 3 per cent (from 95 to 98) would practically erase the A ~02 (8). Thus, the question whether the values of arterial saturation, as obtained by current methods, are too low becomes a vital one.

The usual gasometric technique (10) for determining the percentage of saturation of hemoglobin with oxygen is indirect. Two separate analyses,

* The work described in this paper was done under a contract, recommended by the Committee on Medical Research, between the Office of Scientific Research and Development and the University of Pennsylvania. Permission for publication has been granted. Preliminary reports were presented at the May 16, 1944, meeting of the Physiological Society of Philadelphia, and abstracts have appeared (1, 2). At the same meeting reports were made upon related investigations of sources of error in the gasometric determination of oxygen saturation by Roughton, Darling, and Root (3), and the determination of arterial oxygen tension by Comroe (4). The results of the independent investigations mere mutually concordant.

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70 SATURATION OF ARTERIAL HEMOGLOBIN

oxygen content and oxygen capacity, are required. The oxygen content usually is determined fairly promptly after the blood, unexposed to air, is drawn, whereas an indefinite interval of time (often 1 hour or longer) may elapse before the analysis of oxygen capacity is performed upon an aliquot which has been equilibrated with air in a tonometer. Since blood is not an inert system, it may well be questioned whether the original sample and the subsequent aliquot are strictly comparable. This issue has ceased to be academic in view of reports (11-13) that normal blood may contain substances (other than HbOz) which, after treatment with dithionite (NazSzOd), combine with CO, thereby increasing the CO-combining capacity. One such substance may be methemoglobin, MHb. It is known that MHb, when present in freshly drawn blood samples, may rather quickly revert to HbOe (through Hb) when the blood sample is allowed to stand (14). Thus in the usual analysis of percentage saturation of blood samples containing a small amount of MHb, the latter may change to HbOz. Opportunity for reversion is small in the promptly performed estimation of oxygen content, but it is much greater in the longer interval involved in the oxygen capacity determination. Such a phenomenon could account for an oxygen capacity too high relative to the oxygen content, and as a result the value of percentage saturation would be too low. Factors of this type have been investi- gated and discussed fully by Roughton and his associates (3) .lv2

In seeking additional information upon t,he saturation of hemoglobin we have turned to the spectrophotometric analysis of arterial blood. The optical technique is particularly appropriate for the direct quantitative determination of two or more species (in the present work, HbO2 and Hb) in a solution (16,17). The Drabkin and Austin special cuvette of 0.007 cm. depth and chamber volume of 0.021 ml. (18) was utilized. This cuvette was designed for and has been used successfully in the measurement of absorption spectra of blood hemolyzed by saponin and of concentrated solutions of hemoglobin unexposed to environmental gases (16, 18). The technique has been extended to a study of turbid systems such as whole, unhemolyzed blood (19) and the measurement of mixtures of intracellular HbOz and Hb in a study of the ,penetration of Na2S204 and ascorbic acid into erythrocytes (20). A cuvette of similar design (but of about twice the depth) has been adapted by Lowry, Smith, and Cohen (21) to a photoelectric, filter photometer, for the estimation of HbOz-Hb in samples of

1 The mechanism of MHb reversion has been studied by one of us (D. L. D.), and will be published separately. The study revealed that the disappearance of MHb from blood is inhibited by fluoride and iodoacetate, and that the inhibition by fluoride can be removed by pyruvate. Reference may be made to Kiese’s recent report (15) upon the mechanism of MHb reduction.

2 Roughton, F. J. W., Darling, R. C., and Root, W. S., to be published.


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D. L. DRABKIN AND C. F. SCHMIDT 71

cutaneous blood, hemolyzed and slightly diluted by the addition of a solution of saponin and ammonia. In this communication we shall report (1) the successful performance of continuous spectrophotometric observation of circulating arterial blood in viva in the dog, and (2) the determination of hemoglobin saturation in dog and man by direct spectrophotometry on undiluted arterial samples, hemolyzed without exposure to air, and measured within 2 to 3 minutes after withdrawal from the subject. The arterial saturation was found to be consistently of the order of 98 per cent in both sets of determinations. As far as we know, direct spectrophotometric observation of circulating blood has not been reported previously. The closest approach to the present technique is the ingenious work of Kramer and his colleagues (22, 23), who made photoelectric measurements upon transilluminated blood vessels, utilizing the difference in light transmission of HbOz and Hb over a broad spectral range of red and near infra-red. Outgrowths of Kramer’s technique are the method of Matthes and Gross (24) and Millikan’s oximeter (25).

Methods

The subjects of the experiments were six healthy, mongrel dogs weighing 8.6 to 12.5 kilos, and five young men, 17 to 23 years of age. In the latter group four were normal and one a controlled diabetic, maintained with insulin (subject R. C. L., Table II). Four of the subjects were non- smokers ; one (L. J. D., Table II) had abstained from smoking for a period of 3 days prior to the experiment, a precaution taken to avoid the presence of HbCO (3).2 The dogs were in the postabsorptive state, and the human subjects were instructed to partake of only a very light breakfast, the arterial punctures being performed 4$ to 5 hours thereafter.

Xpectrophotometric Observation of Circulating Blood-The 0.007 cm. cuvette (18) was filled with isotonic saline3 and then introduced into the stream of one femoral artery of the dogs, which were anesthetized with nembutal(40 mg. per kilo intraperitoneally) and aligned on a board beneath the optical bench of the spectrophotometer. The arrangement is described in Fig. 1 and the accompanying legend. Clotting was prevented by previ- ous intravenous injection of a 5 per cent solution (100 mg. per kilo) of du Pont’s pontamine fast pink4 (same as chlorazol fast pink 28 (26)) and

3 A volume of approximately 0.2 ml. is required to fill the cuvette and its entry and exit capillaries. The saline is not essential, but is helpful in establishing immediate homogeneous blood flow after the circulation is diverted through the cuvette. It should be emphasized that in all the measurements upon hemolyzed (saponized) blood, the sample was admitted only into scrupulously clean and thoroughly dry cuvettes.

4 The synthetic anticoagulant pontamine fast pink was found to have an absorption curve with a slight inflection at 562 rnr and a maximum at 521 rnp. The respec-


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periodic injection (at approximately half hour intervals) of 0.5 ml. quantities of heparin. With heparin alone circulation through the cuvette could be maintained for only approximately 5 minutes before the flow was interrupted by deposition of fibrin in the chamber. The arrangement of connections (Insert 2, Fig. 1) from the artery to cuvette provided for arrest-- ing the flow through the chamber, when desired, and by-passing it through. a second channel to the artery. This permitted periodic trapping of samples at different phases of the respiratory cycle, and the evaluation of the effect of this factor on hemoglobin saturation, disclosed in some of Kramer’s studies (22, 23). To allow for rapid changes from room air to 100 per cent or 10 per cent oxygen, in these experiments a cannula was inserted in the trachea. The respiratory rate of the nembutalized animals was 10 to 12 per minute. Samples of blood were removed periodically for erythrocyte count and for independent spectrophotometric analysis of hemoglobin by- the usual technique (27) with the 1 cm. cuvette upon diluted, hemolyzed blood as HbG, Hb(HbOz + Xa&04), and total pigment as cyanmethemo-. globin, MHbCN.

Collection and Preparation of Hemolyzed Arterial Blood, Unexposed ta Environmental Gases-To avoid the possibility of equivocal results due to use of pontamine fast pink and turbid systems, and to assure full precision of spectrophotometric measurement, the following procedure was adopted. Small tonometers of 6 to 15 ml. capacity, provided at each end with the usual double bore stop-cocks, permitting discard of the blood which first, flows through the entry tube, were employed as collection vessels. A solution containing 30 mg. of oxalate and 50 mg. of saponin (Merck, purified)’

tive millimolar E values at these wave-lengths are 10.9 and 20.4, with a molecular weight of 928 (based on the value for chlorazol fast pink (26)). It may be calculated, with a value of 8 per cent of body weight in gm. for blood volume in ml., that the original concentration of the dye in the blood in our experiments should be approximately 1.3 m&f per liter. The concentration of total hemoglobin (Table I) is of the order of 10 mM per liter. When these respective concentrations were duplicated in vitro (i.e. dye added to drawn blood), the absorption spectra upon clear solutions, (diluted 1:lOO) yielded results predictable from the contributions to absorption of the two components, dyestuff and HbOz. On the other hand, blood samples withdrawn from the animals following injection of the dye, upon dilution to 1: 100 for spectrophotometry by the usual technique in 1 cm. cuvettes, showed only a smal1 influehce on the absorption spectrum of HbOz. In samples w,ithdrawn early after

. . . . . indectlon of the dye the characteristic ratios of e 578 mP: E 562 mP and E 542 m,,: E 562 me were changed from 1.75 and 1.69 respectively to 1.70 and 1.62. As the experiment pro- gressed (2, 3, and 4 hours after injection), restoration towards the normal ratio was observed. The water-soluble dye promptly spills over into urine, but the impression was gained that this accounts for only a fraction of that removed from the circulation. A precise study of the rate of disappearance of pontamine fast pink from the blood was not undertaken.


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(quantities sufficient for approximately 4 ml. of blood, the size of the usual sample) was evenly air-dried on the walls of the tonometers. In this opera-

FIG. 1. Arrangement for continuous direct spectrophotometric observation of circulating blood in Go. The alignment of the Bausch and Lomb spectrophotometer assembly, permanently set up in a dark room, is shown. The observer, reading through the eyepiece, E. P., of the spectrometer, is completely shielded from the light, L, by enclosing the lamp housing, L. H., and accessory biprisms (which split t,he light into two parallel, optical paths) in an additional large box shielding. Insert

1, a rectangular monochromatic, L’, photometric field (of two halves), P. F., of 20 A in width is defined by a diaphragm in E. P. Different wave-lengths are brought into

position by rotation of a drum, W. D., translated into rotation of the prism (housed within P. H.) from red, 7, to blue, b, or vice versa, about the pivotal point, P. The

photometer scale, P. S., after matching the half fields, is read at R. Insert 2 illus-

trates the introduction of the Drabkin and Austin special cuvette into the femoral artery, F. A. The arrangement of clamps is shown. The passage of blood through the cuvette may be arrested and the circulation by-passed by closing the clamps at the entry and exit capillaries of the cuvette and opening the clamp, B. P., in the by- passing channel. The connections at the clamp sites between the glass capillaries are of small bore, pure gum tubing.

tion care was exercised to obtain a fine, thoroughly dry deposit with no evidence of trapping of air bubbles. The tonometers were then connected by means of clean pure gum tubing to small leveling bulbs and filled with


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thoroughly clean mercury5 and were repeatedly evacuated to insure freedom from traces of trapped air. Before connection was made by means of a 2 cm. length of pure gum tubing to the cannula or needle in the artery, the tonometers were completely filled with mercury, including the entry tube. In the anesthetized dogs the blood was delivered through a glass cannula inserted in the femoral artery. In man (at rest, and supine) the arterial blood was obtained under local procaine anesthesia by the method of femoral puncture, by use of cut down lumbar puncture needles (with stilus), gage 19. With the stilus withdrawn, a slow, free flow of blood was provided. The needle was allowed to remain in the artery for periods of about 1 hour, during which time samples were drawn at intervals into the tonometer collection vessels, with the level of the mercury reservoir adjusted to exert only very slight negative pressure. In all cases the first flow of blood was dis- carded through the side tube of the tonometer. Samples in which suspicion existed of the possible trapping of minute bubbles of air were not used.

The blood samples were usually collected in three to four spurts, timed t’o synchronize with “end” inspiration or “end” expiration, kymographically recorded in both dog and man. By gentle shaking the blood sample was quickly hemolyzed. The blood in the entry tube of the tonometer was removed with a pipe-stem cleaner and replaced by the hemolyzed solution. After quick establishment of connection between tonometer and entry capillary of the dry, thoroughly clean special cuvette, the sample was transferred to the latter by adjusting the reservoir to very slight positive pressure. Enough of the hemolyzed sample was transferred to allow for appreciable overflow into the exit capillary of the cuvette. Since the volume of the chamber of the latter is 0.021 ml. and the volume of the capillary tubes approximately 10 times greater, the optical chamber of the cuvette is completely washed out, thereby assuring that the specimen within has been transferred without contamination with environmental gases. The overflow of blood in the capillary entry and exit tubes effectively seals the chamber: so that oxygenation of the contained sample will not occur for periods of time up to 1 hour (18). Since the element of time has been considered as possibly essential in the present problem, it should be stated that spectrophotometric measurement was begun within 2 to 3 minutes and completed within 6 minutes after withdrawal of the blood from the subject.

The remainder (about 3 to 3.5 ml.) of the hemolyzed blood was transferred from the tonometer to a flat weighing dish. After exposure to air in this

6 In one instance failure to observe the elementary precaution of using scrupulously clean tubing and mercury resulted in the formation of appreciable amounts of changed pigment, recognized spectroscopically as MHb from the character of the absorption spectrum and the effects of addition of Na&Oa (conversion to Hb), addition of KCN (conversion to MHbCN), and change in pH (17).


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container (in which drainage difficulties are not encountered (3)2 portions were reread spectrophotometrically in the 0.007 cm. cuvette with and without addition of NazSz04. An aliquot was diluted and determined in the 1 cm. cuvette as HbOz and, after conversion, as Hb and MHbCN.

A’pectrophotometric Technique, and Error of Method-For details of the technique readers are referred to a recent review (16) and to earlier papers in this series, particularly upon the spectrophotometry of turbid systems (19) and the method of Austin and Drabkin (17) for the determination of two or more species in a single solution. In the latter technique, here applied to mixtures of HbOz and Hb, accuracy is increased by measurements at several characteristic wave-lengths, rather than being limited to two spectral regions. In the present work calculations were based upon E values6 at 600, 578, 562, and 542 rnp, with the wave band or spectral interval limited to 2 rnp, and total pigment concentration determined by conversion to MHbCN’ (27). Measurements were also made at wave-lengths of 630,555 (maximum of Hb), and 505 rnp (an isobestic point in the absorption curves of HbOz and Hb). Readings at 505 rnp are not highly accurate, but can serve as a check upon total pigment concentration (Table I, last column). An example of the procedure and calculation employed is furnished in Fig. 2. At the characteristic wave-lengths, E values must be known or must be determined for the individual species, HbOz and Hb. The summation of the total change in absorption, ZAE, at these wave-lengths between HbOt and Hb is designated &. The summation of the partial change, Z,, is the change in absorption from HbOz to the particular mixture, JJ, of the two pigments measured. The ratio, r = 21,:& = fraction of Hb, and 1 - r = fraction of HbOz. (1 - r) X 100 = percentage saturation.

Mean e values have been established with sufficient precision for hemolyzed dog blood to be used as absorption constants. The mean E values, obtained in the present experiments (recorded in Fig. 2), agree very closely with values previously obtained upon hemolyzed dog blood in the 0.007 cm. cuvette (18), and differ only very slightly from the absorption constants upon diluted hemolyzed blood, measured in the 1 cm. cuvette (27). Therefore, with saponized dog blood valid interpretations could have been

B As heretofore, our E values are for a concentration of 1 mM per liter (in the case of hemoglobin, referred to an equivalent weight of 16,700) and a depth of 1 cm. Thus,

E = (l/(c X d)) X log I,/Z, where the concentration c is expressed in rnM per liter, the depth d in cm., the original intensity I0 is 1.0, and the intensity of transmitted light I is expressed as a fraction of unity.

’ In the spectrophotometric determination of total pigment concentration upon aliquots converted to MHbCN, e = 11.5 at 540 rnp is used. This constant has been established for preparations from hemolyzed, washed dog erythrocytes (27). The same constant was used in the present measurements for hemolyzed, human blood. Evidence for the validity of this procedure will be furnished in a subsequent report.


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obtained from measurements limited to the experimental sample, containing HbOz and Hb, by utilizing the previously established (18) E values for individual pigments. However, a sufficiently large number of analyses was not available for the precise establishment of constants for HbOz and Hb in

16-

14-

12-

IO- -l -

SE .2- a : _

TIT - -Qu - w 6-

nin mp

FIG. 2. Absorption spectrum curves obtained from measurements on hemolyzed (saponized) dog whole blood in the 0.007 cm. cuvette, with an example of the method used in the direct spectrophotometric determination of percentage saturation. Curve HbOz (heavy, solid line), based on mean E values for fully oxygenated blood from six normal animals in the present study. The absorption constants (mean e values) for HbO?, at the indicated characteristic wave-lengths used in the estimation of saturation, are given in the column headed by 0.96. Curve Hb (broken line), based on mean B values upon aliquots of the samples used for Curve HbOt, after deoxygenation by means of solid dithionite, Na2SZ04 (0.1 mg. per ml.). The mean B values, used in the estimation of saturation, appear in the column headed by 4.03. Curve M (light, solid line), sample from Dog 4, partially deoxygenated in a tonometer by COZ and I’&, total pigment concentration (as MHbCN) = 9.81 mM per liter. Appended data show that the curve rebresents a mixture of 0.756 HbOz and 0.244 Hb. The percentage saturation of the sample is therefore 75.6. For symbols, see the text under “Methods.”

the blood of man. Hence the procedure was adopted of determining for each individual hemolyzed blood sample (1) the original absorption spectrum (of the portion transferred directly into the 0.007 cm. cuvette), (2) the spectrum after the remainder had been fully oxygenated (read undiluted in the 0.007 cm. cuvette, and also read diluted 1: 100 in the 1 cm. cuvette as a check upon complete oxygenation), and (3) the spectrum of an aliquot


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treated with solid NazSz04 (read both undiluted and diluted). The rede- termination of the sample in the 0.007 cm. cuvette after procedures (2) and (3) yields the c values for HbOz and Hb respectively, from which the values Of ZAcHbOl-Hb, Or % are obtained for each sample. The individual 21, values (Table II, third column) were employed in the calculation of percentage saturation in the arterial blood of man. Results (or con- clusions), however, would not be materially altered if the mean value Z:T = 16.38 and the mean e values for HbC& and Hb had been used. With the dog blood, mean E values, obtained in the present work, which yielded mean ZT = 15.89, were used, since practically ident,ical results were obtained by the individual treatment of each sample.

Based upon the deviation of E values from the mean in the establishment of absorption constants, or the determination of hemoglobin concentration (as MHbCN), Drabkin and Austin (27) concluded that the accuracy of the spectrophotometric method was rtO.5 per cent, and greater in t.heir hands than the accuracy attained in parallel analyses by the gasometric technique. In the determination of mixtures, under ideal conditions, practically as high accuracy was obt,ained as for individual pigments (17). In the present work the accuracy may bc expected to be of a somewhat lower order, since analyses were carried out upon mixtures in which the proportion of one component is very small in comparison with the other. On the other hand the need for volumetric manipulations (measurement of volume of blood and its dilution) with their possible errors is eliminated in the use of the 0.007 cm. cuvette. A conservative estimate would be that the analytical error is within &l .O per cent, a value well beyond the agreement of analytical results such as J?& (in human blood) = 16.38, standard deviation = 0.04 & 0.01, and the concordance of results presented in Table I.

Results

Observations on Circulating Blood. General Findings-( 1) A homogeneous circulation of the diverted blood flow through the cuvette chamber was maintained, permitting continuous visual measurements up to periods of 4 hours. (2) With thoroughly clean cuvettes the film of blood rapidly filled the entire cuvette chamber and uniform photometric fields were rapidly established and maintained. Streaming eJects were totally absent. The gratifying absence of streaming or channeling, which would have viti- ated the application of photometry, appears remarkable in view of the thin- ness of the chamber, the dept.h of which is approximately only 9 times the diameter of the red blood cell. (3) The passage of the blood through the cuvette was pulsatory and synchronous with the pulse. This phenomenon, probably related to the introduction of a rigid chamber into the otherwise


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78 SA’kURATION OF ARTERIAL HEMOGLOBIN

distensible artery, did not interfere with spectrophotometry. In the ex- tension of this technique automatic recording would be profitable. (4) Owing to the presence of pontamine fast pink and lack of exact information concerning its level of maintenance in the blood stream, some uncertainty exists in the extrapolation of the true spectra from the data obtained in the

TABLE I

Agreement in Total Pigment Concentration (“Total Hemoglobin”), Measured Spectrophotometrically by Different Procedures

In all cases the values are in mM per liter (referred to the equivalent weight of 16,700) in the original, undiluted blood. Concentration is calculated from e (upon the sample measured) divided by appropriate, corresponding E (the standard, or mean value, used as an absorption constant). The standard e values employed were 11.50 for MHbCN at 540 rnp (same for diluted as undiluted), 15.13,8.73, and 14.62 for HbOz (diluted 1:lOO) at 578, 562, and 542 rnp respectively, 15.08, 8.62, and 14.75 for HbOn (undiluted, whole blood) at 578, 562, and 542 rnp respectively, 13.45 for Hb at 555 rnp (same for diluted as undiluted), and 5.22 at 505 rnp, an isobestic point for HbOz, Hb, and mixtures of the two.

Blood Source

Dog 3

“ 3

“ 4

-

Treatment

Diluted, 1 cm. cuvette Undiluted,1 0.007 cm.

cuvette

Diluted, 1 cm. cuvette Undiluted,% 0.007 cm.

cuvette

Diluted, 1 cm. cuvette Undiluted,1 0.007 cm.

cuvette

MHbCN

10.33

11.38

9.78

Total pigment measured as

HbOr’ Hbt

10.27 10.33 10.38 10.35

11.33 11.47

9.74 9.70

11.40 11.41

9.78 9.68

-

- Isobestically

* Blood sample completely oxygenated. t Blood sample deoxygenated by means of Na&Oa. $ Hemolyzed (saponized), oxalated whole blood. $ The hemolyzed, arterial sample, unexposed to air, taken at end inspiration. (1 The hemolyzed, arterial sample, unexposed to air, taken at end expiration.

10.44, HbO** 10.33, Hbt 10.33, MS

11.35, HbOz* 11.47, Hbt 11.35, MI[

9.72, HbOz* 9.72, Hbt 9.72, MI1

turbid state (19, 20). However, reliance may be placed upon a direct comparison of the extinction data obtained under varying conditions, and the agreement of results with those found on hemolyzed blood. The base- line of comparison was afforded by assuming that the spectrum yielded when the animal was under 100 per cent oxygen represented complete saturation (100 per cent HbOz). A simplified extrapolation procedure (20) was used.


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The following pertinent data were secured. (1) In the circulating blood the concentration of pigment remained relatively constant (order of 5 per cent) over a period of several hours. (2) Intravenous injection of

TABLE II

Direct Spectrophotometric Determination of Saturation of Hemoglobin in Arterial Blood

Species

Dog 31

“ 4t.

“ 5 “ 6

_

-

Total pigment*

m-d per 1.

10.33 11.47 11.35

9.72 9.78

10.54 9.87

zTt Fraction Fraction Hb, r = +/zT) t

HbOz, l--r

15.89 0.018 0.982 15.89 0.013 0.987 15.89 0.045 0.955 15.89 0.017 0.983 15.89 0.034 0.966 15.89 0.015 0.985 15.89 0.013 0.987

* Average............................................ 98.5§ .

Man, W. K.

‘I H. R.

“ H. G. S.9

“ R. C. L.** “ L. J. D.tt

8.03 16.4111 0.019 0.981 98.1 8.19 16.3711 0.020 0.980 98.0 8.63 16.3911 0.015 0.985 98.5 8.55 16.3411 0.017 0.983 98.3 8.57 16.4111 0.007 0.993 99.3 8.14 16.4611 0.009 0.991 99.1 7.76 16.3611 0.010 0.990 99.0 9.12 16.3211 0.018 0.982 98.2

Average............................................ 98.6

per cent

98.2 98.7 95.5 98.3 96.6 98.5 98.7

Respiratory phase at sampling

End inspiration “ “

“ expiration “ inspiration “ expiration

Undetermined “

End expiration “ “ “ “ “ “

“ inspiration “ expiration “ “ “ “

* Total pigment determined as MHbCN. To obtain gm. per 100 ml., multiply the values by factor 1.67.

i For symbols see the text under “Methods.” $ Nembutal anesthesia. $ Values at end expiration omitted from the average. II Mean Z, in man = 16.38; standard deviation = 0.04 f 0.01. 7 Oxygen tension (~OZ), determined by Dr. J. H. Comroe, Jr., ((4); unpublished

work of Comroe and Dripps) upon an arterial sample withdrawn within several minutes of the sample used for spectrophotometry, yielded the value 98.5 mm. of Hg.

** A controlled diabetic, taking insulin. tt A mild smoker, who abstained from smoking for 3 days prior to femoral

puncture.

adrenalin (0.05 mg.) produced hemoconcentration of the order of 10 to 15 per cent, definitely observable within 4 minutes and maximal at 7 minutes after injection. (3) The oxygen saturation was maintained at a level of 97 to 98 per cent during the inhalation of room air. When this was changed


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to 10 per cent oxygen, the percentage saturation promptly fell to 75 to 80. (4) Samples trapped (see “Methods”) at end expiration consistently showed a lower saturation (95 to 96 per cent) than corresponding samples (trapped several minutes later) at end inspiration (98 per cent saturation) in these nembutalized animals breathing ten to twelve times per minute.

Observations on Hemolyzed (Oxalated, Saponized) Arterial Blood-The data recorded in Table I upon the agreement of total pigment concentration, measured as HbOz (after oxygenation), Hb (after deoxygenation with NazSz04), MHbCN (after conversion with ferricyanide and cyanide), and isobestically at 505 rnp indicate that the spectrophotometric technique does not reveal the presence in measurable amount of MHb (besides HbOz and Hb) in freshly drawn dog arterial blood, determined promptly. 14s an added check in several instances (both in dog and human) KCN alone was added to the freshly drawn arterial blood, exposed to air. No change in spectrum could be demonstrated.

The data on the direct spectrophotometric determination of arterial saturation are collected in Table II. The values are uniformly high (98.0’ to 99.3 per cent), and average 98.5 per cent in the dog and 98.6 per cent in man. The influence of respiratory phase, observed in the circulating blood, is confirmed by the measurements upon the hemolyzed samples from anesthetized dogs. This effect, however, was not demonstrable in unanesthetized man.

DISCUSSION

If the present results are correct, the conclusion must be drawn that the values for hemoglobin saturation in arterial blood at sea level have been underestimated by the indirect gasometric procedure. The causes for error probably reside, not in the manometric technique per se, but in the nature of the blood and its manipulation. This is in accord with the recent study of sources of error in the estimation of saturation by Roughton, Darling, and Root (3),2 who conclude that hemoglobin saturation is underestimated by a,bout 2 per cent in the gasometric method. According to them, the average value for hemoglobin saturation in arterial blood should be raised from 95 to 97 per cent. Dr. Boothby kindly has permitted us to quote corroborative evidence obtained by him and Dr. Robinson in man with the oximeter (25). It was customary to set the oximeter to read 95 per cent saturation when the subjects breathed room air. Boothby and Robinson, however, found that when the oximeters are set to read 100 per cent with the subjects breathing oxygen the saturation upon change to breathing room air is consistently 96 to 98 per cent.

Since percentage saturation has been employed in estimating oxygen tension from the dissociation curve, it is of interest that our values of the


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order of 98.0 and 98.5 per cent saturation correspond on a typical curve for man (8) to ~02 values of 97 mm. and 100 mm. of Hg respectively. In one of the men (H. G. S., Table II), whose arterial saturation was 99.1 per cent, an independent determination of ~02 by Dr. Comroe (4) yielded a value of 98.5 mm. With an improved technique Comroe and Dripps (unpublished work) have recently obtained an average value of 97.1 mm. for arterial PO% The independent analytical procedures therefore are in close accord. The ApOz between alveolar air and arterial blood would appear to be vir- tually erased whether the ~02 is deduced from our values for saturation or obtained by direct determination.

The effect of phase of respiration on arterial oxygen saturation, demonstrated in the anesthetized dogs, confirms Kramer and Sarre’s finding (23), also in the anesthetized animal. The same factor may be operative in unanesthetized man, but of a magnitude too small to be measured. Further observations are contemplated.

It may be proper to raise the question whether pure HbOz has ever been available for study. Perhaps the pigment in preparations from completely oxygenated blood is always contaminated with a minimal amount of MHb. When solutions prepared from fresh, fully oxygenated normal blood are treated with cyanide, the spectrophotometric technique fails to disclose changes in the direction suggesting the formation of MHbCN. On this basis the conclusion may be drawn that if preformed MHb is present its concentration must be of the order of 0.5 per cent of the total pigment, or lower, an amount beyond t,he possibilities of differentiation by present techniques. Roughton and his colleagues (3)2 have confirmed Ammundsen (13) that after treatment with dithionite (EazSz04) there is an average increase in CO capacity of the order of 3 per cent. This is a perplexing problem. The 3 per cent cannot represent MHb alone, since 1.0 to 1.5 per cent should be readily detectable by the spectrophotometric methods used in the present study (17). Other substances besides MHb have CO capacity after reduction, e.g., ferrohemin and nitrogenous ferroporphy- rins (28). It is unfortunate in the evaluation of this phenomenon that parallel determinations cannot be made of 02 capacity after NazSz04, owing to the formation of oxidants from the latter. The possibility remains that part of the effect on CO capacity may be an artifact. It would also be desirable to accumulate sufficient data for a comparison of the behavior of arterial and venous blood in this connection. Most of the analyses have been performed upon venous blood, although Roughton et al. (3)” state that this makes little difference.

The factors which influence the equilibria HbOz + Hb G MHb in blood are indeed complicated. MHb may disappear from blood after it is drawn (14). On the ot,her hand, MHb may be formed in blood and blood solutions


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on standing (17, 27). Roughton and associates (3)2 found an increase in 02 capacity (without Na&,04) in blood standing at room temperature. In the best experiments this effect amounted to 1.1 per cent in terms of total pigment, and was interpreted to represent the quantity of the total MHb which had reverted to HbOz (through Hb). Whereas many hemoglobin derivatives combine with CO, very few (only Hb, under normal pH conditions in blood) can unite with 02. We believe that the 1.1 per cent may approach the value for total MHb in Roughton’s samples, mainly of venous blood.

We are indebted to Miss H. Lorraine Leidy and Dr. H. H. Pennes (De- partment of Pharmacology) for technical assistance, and particularly to Dr. H. D. Bruner (Harrison Department of Surgical Research) for perform- ing the femoral arterial punctures in man.

SUMMARY

The Drabkin and Austin special cuvette of 0.007 cm. depth (18) has been ulitized to extend the spectrophotometric technique to the study of blood in viva and to the determination of arterial saturation.

1. The continuous spectrophotometric observation of circulating blood has been successfully performed in nembutalized dogs.

2. The hemoglobin saturation of hemolyzed arterial blood has been determined in dog and man by direct spectrophotometry. The analyses were performed within 2 to 3 minutes after the blood was withdrawn from the subject. The saturation was uniformly high in both species. It varied from 98.0 to 99.3 per cent, and had average values of 98.5 and 98.6 per cent in the dog and man respectively.

3. The phase of the respiratory cycle was found to be a factor in the value of arterial saturation in the anesthetized dogs, but not in unanesthetized man.

4. The significance and interpretation of the findings have been discussed.

BIBLIOGRAPHY

1. Drabkin, D. L., and Schmidt, C. F., Am. .I. Med. SC., 208, 133 (1944). 2. Drabkin, D. L., Schmidt, C. F., Bruner, H. D., and Pennes, H. H., Am. J. Med.

xc., 208, 135 (1944). 3. Roughton, F. J. W., Darling, R. C., and Root, W. S., Am. J. Med. SC., 208, 132

(1944). 4. Comroe, J. H., Jr., Am. J. Med. SC., 208, 135 (1944). 5. Lennox, W. G., and Gibbs, E. L., J. Cl&. Invest., 11, 1155 (1932). 6. Keys, A., and Snell, A. M., J. C&n. Invest., 17, 59 (1938). 7. Cullen, S. C., and Cook, E. V., Am. J. Physiol., 137, 238 (1942). 8. Bock, A. V., Field, H., Jr., and Adair, G. S., J. Biol. Chem., 69, 353 (1924).


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9. Bock, A. V., Dill, D. B., Edwards, H. T., Henderson, L. J., and Talbott, J. H., J. Physiol., 68, 277 (1929-30).

10. Van Slyke, D. D., and Neill, J. M., J. BioZ. Chem., 61, 523 (1924). Van Slyke, D. D., J. BioZ. Chem., ‘73, 121 (1927).

11. Ammundsen, E, and Trier, M., Acta med. Xcand., 101, 451 (1939). 12. Taylor, D. S., and Coryell, C. D., J. Am. Chem. Xoc., 60, 1177 (1938). 13. Ammundsen, E., J. BioZ. Chem., 138, 563 (1941). 14. Wendel, W. B., J. CZin. Invest., 18, 179 (1939). Cox, W. W., and Wendel, W. B.,

J. BioZ. Chem., 143, 331 (1942). Heubner, W., and Stuhlmann, M., Arch. exp. Path. u. Pharmakol., 199, 1 (1942).

15. Kiese, M., Biochem. Z., 316,264 (1944). 16. Drabkin, D. L., in Glasser, O., Medical physics, Chicago, 967 (1944). 17. Austin, J. H., and Drabkin, D. L., J. BioZ. Chem., 112, 67 (1935-36). 18. Drabkin, D. L., and Austin, J. H., J. BioZ. Chem., 112, 105 (1935-36). 19. Drabkin, D. L., and Singer, R. B., J. BioZ. Chem., 129, 739 (1939). 20. Drabkin, D. L., Proc. Am. Sot. Biol. Chem., J. Biol. Chem., 140, p. xxxiv (1941). 21. Lowry, 0. H., Smith, C. A., and Cohen, D. L., J. BioZ. Chem., 146, 519 (1942). 22. Kramer, K., 2. Biol., 96, 61 (1935). 23. Kramer, K., and Sarre, H., 2. Biol., 96, 76, 89, 101 (1935). 24. Matthes, K., and Gross, F., Arch. exp. Path. u. Pharmakol., 191, 369, 381, 391,

523, 706 (1939). 25. Millikan, G. A., Rev. Scient. Instruments, 13, 434 (1942). 26. Quick, A. J., The hemorrhagic diseases and the physiology of hemostasis, Spring-

field and Baltimore, 98, 106 (1942). 27. Drabkin, D. L., and Austin, J. H., J. BioZ. Chem., 112, 51 (193536). 28. Drabkin, D. L., J. BioZ. Chem., 146, 605 (1942). by guest on M

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David L. Drabkin and Carl F. SchmidtARTERIAL BLOOD

SATURATION OF HEMOGLOBIN INDETERMINATION OF THE

BLOOD IN VIVO, AND THE DIRECTXII. OBSERVATION OF CIRCULATING

SPECTROPHOTOMETRIC STUDIES:

1945, 157:69-84.J. Biol. Chem.

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