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Inert gas measurements of coronary blood flow
Inert
Gas Measurements
of Coronary
Blood Flow* FRANCIS J. KLOCKE,
M.D., DOUGLAS R.
Buffalo,
ROSING, M.D. and DAVID E. PITTMAN, M.D. New York
T
HE USE OF inert gas technics to measure coronary blood flow has been an important part of quantitative studies of ventricular function and myocardial metabolism since the nitrous oxide technic was adapted to the coronary circulation in the late 1940’5~-~ The present communication is intended to summarize several principles which are agreed to be important in performing such measurements. Of particular interest are problems that arise when coronary flow is heterogeneously distributed within the heart, as is certainly the case in coronary artery disease6s6 and as is probably the case in most other situations.7
100 gm./min. For a variety of reasons investigators sometimes prefer to perform measurements during periods of desaturation or “washout,” that is, during periods in which inert gas previously dissolved in the myocardium is leaving the heart by way of venous blood.8 These approaches may be divided into two types, depending on the manner in which test gas is delivered to the myocardium before the washout. In the first case, delivery of gas is accomplished by having the subject breathe a mixture containing the test gas. He is returned to room air at time zero, and flow per unit volume of tissue is calculated from the ensuing change in venous gas concentration, the tissue-blood partition coefficient and the area between the arterial and venous desaturation curves. In the second case, the subject breathes room air at all times, and saturation is accomplished by injecting saline in which the test gas has been dissolved into the left ventricle or a coronary artery. The saline is discontinued at time zero and, when the test gas has a low solubility in blood, recirculation is minimal and the arterial gas concentration drops quickly to negligible levels. The pattern of myocardial gas elimination is then obtained from the coronary venous washout curve or precordial monitoring of myocardial radioactivity. Frequently used examples of this approach include the 85Kr and 133Xe methods developed by Herd,g Cohen,lO and Ross” and their co-workers. The saline is usually administered as a sudden single injection, although longer saturation periods are theoretically feasible. If the data are plotted semilogarithmically against time and myocardial
GENERAL APPROACHES Although inert gas methods vary in their details, all are ultimately derived from the Fick principle and involve measurements of the difference between concentrations of arterial and venous (or tissue) gas during a period of myocardial uptake or release. In the original nitrous oxide (NzO) technic, arterial and coronary sinus concentrations are followed during a period of breathing Nz0.1e4 The rate of coronary flow is obtained by dividing the change in tissue concentration of NzO by the mean arterial-venous NzO difference. The change in tissue concentration is not measured directly but is calculated from the change in venous N20 concentration and the tissue-blood partition coefficient. The arterial-venous difference is obtained by integrating the area between the arterial and venous curves. The quotient represents flow per unit volume of tissue and is conventionally expressed as ml./
* From the Department of Medicine, State University of New York at Buffalo School of Medicine, Buffalo, N. Y. This study was supported by U. S. Public Health Service Research Grant HE-09587 from the National Heart Institute and by grants from the Heart Association of Western New York and the United Health Foundation of Western New York. Address for reprints: Francis J. Klocke, M.D., Buffalo General Hospital, 100 High Street, Buffalo, N. Y. 14203. 548
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Gas
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of Coronary
perfusion is truly homogeneous, the venol~s (or precordial) washout curve should decay as a single exponential. Many workers assume that this is the case and calculate flow per unit volume from the slope of the straight line which best fits the semilogarithmically plotted data for the first few minutes of desaturation. OthersI find that the slope of the semilogarithmically plotted curve is not constant and, by “curvepeeling” procedures, treat the data as rcpresenting a number of homogenous compartments arranged in parallel. Still others13 avoid exponential treatments and calculate flow per unit volume of distribution of tracer from the mean transit time of the tracer through the system. This latter approach also avoids the assumption that end-capillary blood and tissue are in equilibriurn for partial pressure of the test gas (see the following). In any event? the major difficulty in the clinical application of these technics is that by nonuniformity of myocardial presented flow. Of particular interest are areas with flows per unit volume which are lower than the average flow per unit volume for the entire tissue. The expected effects of these areas on measured gas differences are illustrated in Figure 1. In measurements during test gas saturation, areas of low flow prolong the time required for the
DURING GAS
540
Flow
arterial-venous gas difference to become negduring desaturation, ligible. l1 In measurements areas of low flow are cleared of test gas more slowly than the areas of “normal” flow and produce increased concentrations of tissue and venous gas in the latter portion of the washout. In washouts after gas breathing, this results in a small but persistent venous-arterial gas difference. In washouts after dissolved gas it causes the semilogarithmically infusion, plotted venous (or tissue) washout curve to deviate clearly from a single exponential. Additional complications that arise when concentration of tissue gas is not uniform at the completion of the period of administration of gas will be discussed later. ANALYTICAL
TECHNICS AND
METHODS OF SATURATION
These observations help to define two crucial requirements for measuring coronary blood flow when flow is distributed unevenly within the heart. 1. The method of gas analysis employed must be capable of quantitating small venous-arterial (or tissue-arterial) gas di$erences. In our own laboratory, we have found it advisable to perform analyses of blood gas with a specially designed thermal conductivity gas chromatograph.15
AFTER
AFTER
BREATHING
GAS
BREATHING
GAS
INFUSION
yp7 :kVEN.(UNIF.FLOW) ; ,I bEN.(NON-UNIF $1 ,r
FLOWi
:. :. \ it i\
VEN.(OR (UNIF.
& .:\ VEN.
(UNIF.
FLOW)
‘:\,~::,“,I”,“,::“‘“” / \
‘\ :
’ \ :.. \
Figure 1. VOLUME
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APRIL
TISSUE) FLOW 1
FLOW 1 ‘\._
Effete of nonuniform myocardiat bloodflow on measurrdinert ,gas mchange. See text for details. 1969
550
Klocke
The chromatograph is more sensitive and specific than Van Slyke analyses and can be used for virtually any gas. It differs from conventional chromatographs primarily by being housed in a water bath at constant temperature instead of in an insulated air oven. The improved temperature control minimizes “baseline noise” and thereby provides an improved signal-to-noise ratio. The detector is a commercially available sealed thermistor unit, and is suitable for operation at only 1 to 2O C. above ambient temperature. The flow control system is also commercially available and, like the rest of the unit, is immersed in the water bath, which is kept at 24.0’ C. with a heater and proportional temperature controller. Blood samples are collected in 2.0ml. glass syringes, and their dissolved gases are extracted in a modified Van Slyke apparatus. ls The extracted gases are then compressed into a glass sampling loop and introduced into the chromatograph carrier gas stream. If desirable, substances that remove water vapor and CO2 are included in the tubing
“Control”
et al. conrlecting the sampling loop to the chromatograph. Concentrations of gas are usually quantitated from the heights of the gas peaks on the chromatograms. Representative chromatograms are shown in Figure 2. Limits of detection for most gases have been 10e5 to 10W6 ml. Since each analysis requires only 2 ml. of blood, a large number of points can be obtained for each saturation or desaturation curve, thus improving curve resolution. In addition, since several gases can be measured in a single blood sample, a variety of gases can be studied simultaneously. 2. The technic by which test gas is delivered to the myocardium must be adequate for areas of lowjlow as well as for areas of normal flow. In considering the saturation of an organ with an inert gas, it is helpful to think of the process in terms of attaining the same partial pressure (that is, the same gas tension) in the tissue as in incoming arterial blood. The rate at which tissue gas tension approaches the arterial gas tension will vary importantly with flow per unit volume of
tk-1.3~
SAYPUS: 2.0
IO-•mt
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BATH
CARRIER FLOW
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:
WHOLE
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GAS : 02
RATES
( ML/MIN.
1:
3.1 REF. COL. - 3.0 STR.SPL. EXH.- 6.6 SAYP.
SAMPLE
A,N2
t
ML
TEMP.
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COLUMN
-
:
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CHARCOAL,
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DETECTOR:
He-0.5x IO-‘ml
7.OMA,Arl.
A
t
Samp. W
A,Ne
XI
A,Nz
t S$P. .
Records illustrating the analysis of trace amounts of helium (He) and hydropn (HZ) in blood. Analytical conditions are shown on the upper right. The record on the upper left is from a blood sample containing neither He nor H2 and serves to verify that the blood contained no extractable substance that could have produced a peak interfering with those of He or Hz. The large amount of Or extracted from the blood does not appear on the record because 02 was employed as the carrier gas. COs is not seen because a CO2 absorbent was included in the tubing connecting the gas sampling loop to the chromatograph sample inlet. (A = argon, Ns = nitrogen.) (Reproduced by permission of the publisher from Klocke, F. J.16) Figure
2.
THE
AMERICAN
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OF CARDIOLOGY
Gas Measurements tissue (F/V) and with the tissue-blood partition coefficient (x). The rate would also be affected by a diffusion limitation to gas exchange. However, in this section of our discussion, we shall assume that tissue-blood gas exchange is limited by perfusion only, that is, that partial pressure equilibrium between tissue and blood is always reached at the capillary level. For purposes of illustration, consider a situation in which myocardial saturation is initiated by a sudden, ‘
of Coronary
551
Flow
Prn(OR V) AS X OF
Pa
ing equation: P m(orv)t = PA1 -
Ib
epkt)
TIME
where : P m(orv) = partial
pressure of inert gas in myocardial tissue (or venous blood) at time t; P, = partial pressure of inert gas in arterial blood; and k= rate constant of saturation, which = (F/W/X
Figure 3 expresses these relations graphically for rate constants ranging from 1.20 to 0.02. In considering this material, it is important to keep in mind the distinctions among gas tension, concentration and content. Concentration is the product of partial pressure and solubility and, at any given partial pressure, the relative concentrations of a gas in tissue and blood will depend on the relative solubilities of the gas in tissue and blood, that is, on the tissue-blood partition coefficient. The gas content of an area of tissue is in turn the product of tissue gas concentration and tissue volume. As will be discussed, the effects of areas of low flow on desaturation curves obtained from measurements of venous gas concentration can differ significantly from the effects of the same areas on curves obtained from precordial measlu-ements of myocardial gas content. The importance of appropriate analytical methods and technics 01 saturation can be illustrated by another example. Assume tissue specific gravity to be 1.0 and consider a “two-compartment heart” in which 80 per cent has a perfusion of 80 ml./100 gm./min. and 20 per cent (perhaps representing an area of scar tissue) has a perfusion of only 5 ml./100 gm./min. If the tissue-blood partition coefficient is 1.0 in both VOLUME23, APRIL 1969
2'0
(min.)
Figure 3. Expected approach of tissue (or venous) gas tension to arterial gas tension in homogeneously perfused areas of thr heart following a “square-wave” innement in arterial ga5 tension. Each curve corresponds to the rate constant of saturation shown adjacent to it. When the tissue-blood partition coefficient is 1 .O, each rate constant also reprtsents flow in m.L/gm./min. Pm(orv) = myocardial (or venous) gas tension; P, = arterial gas tension.
the corresponding rate constants arc areas, 0.80 and 0.05. Over-all flow is 65 ml./100 gm./ min., and the blood draining the area of lower flow constitutes only 1.5 per cent of total flow. Thus, even if saturation is complete, the area of lower flow will be overlooked in a venolls washout curve if analytical methods cannot detect venous-arterial differences of less than 1.5 per cent of the initial venous and arterial concentrations. If the wash-in period does not allow for full saturation of the area of lower flow, the situation is even more difficult. If the approach shown in Figure 3 is employed, the gas tension in the area of lower Aow at the end of a 10 minute saturation will be only 39 per cent of that in the area of higher flow, and the area of lower flow will not be evident in a venous washout curve unless analytical methods can detect venous-arterial differences of less than 0.6 per cent of the initial venous and arterial concentrations. In addition, since venous blood in favor of the area of higher is “weighted” flow, the measured venous concentration at the beginning of desaturation will be an inaccurate index of both the tissue concentration in the area of lower flow and the volume-averaged tisslle concentration for the cantir? heart.
552
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When myorardial gas content, rather than venous gas concentration, Is measured, the situation is somewhat different and reflects the fact that the area of lower flow constitutes a greater fraction of total heart volume than its venous drainage does of total coronary flow. If the saturation period were adequate to attain the same partial pressure in the area of lower flow as in arterial blood, the area of lower flow would comprise 20 per cent of the total gas content at the beginning of desaturation, and its determination in a tissue washout curve would be relatively easy. If the wash-in period were only 10 minutes, the gas concentration achieved in the area of lower flow would be only 39 per cent of that in the area of higher flow, and the gas content of the area of lower flow would constitute 8.9 per cent of the total gas content at the beginning of desaturation. As the wash-in period is shortened more and more, tissue gas tension does not reach the arterial tension in either compartment, and gas content in the area of lower flow forms a progressively smaller percentage of total gas content at the beginning of desaturation. Additional and perhaps very significant complications that are introduced by cardiac adipose tissue will be discussed below. It seems clear, however, that the relations among analytical sensitivity, method of saturation, partition coefficients and method of monitoring tissue-blood desaturation serve to define a “threshold” or “minimal detectable” flow per unit volume when Aow is heterogeneously distributed. The higher this threshold, the greater is the chance of ignoring an area of low flow and of overestimating total flow. In view of the importance of adequate saturation, one Could consider performing measurements during a prolonged period of saturation rather than during a conventional period of desaturation. Analytical limitations might be especially troublesome, however, since the evaluation of areas of low flow would involve the measurement of small differences between gas concentrations which are higher than at comparable times during desaturation. One additional approach to this problem is to measure flow from nitrogen (Nz) desaturation curves.l7 Since Nz is breathed normally, complete myocardial saturation at the partial pressure of Nz in room air is ensured. No preliminary breathing of gas is required, and the patient is merely switched from room air to a mixture containing 21 per cent OZ and 79 per
et al. cent of some other inert gas such as argon. Washouts of this type before and after embolic occlusion of a portion of the left coronary artery in a closed chest dog are shown in Figure 4. Figure 5 summarizes similar data in 7 dogs. As advantageous as NZ appears theoretically, however, it suffers practically from the extreme precautions that must be taken to exclude “contamination” of sampled blood with small amounts of dissolved Nz originating from ambient air. If this obstacle can be overcome, it should be of considerable value. ADDITIONAL FACTORS IN MEASURING SMALL VENOUS-ARTERIAL (OR TISSUE-ARTERIAL) GAS DIFFERENCES From this discussion, it is evident that areas of myocardium having a low flow are represented in inert gas desaturation curves as small but persistent venous-arterial (or tissue-arterial) gas differences. In focusing attention on these differences, one must attempt to exclude the possibility that they originate from cardiac adipose tissue, from “contamination” of sampled coronary venous blood by right atria1 blood, or from “contamination” of precordially monitored myocardial gas content by tracer contained in the chest wall or lungs. In the case of adipose tissue, the rate constant of saturation for commonly employed inert gases is probably small because of low flows per unit volume and high tissue-blood partition coefficients. This observation implies that the partial pressure of test gas in adipose tissue at the completion of a conventional saturation period is considerably less than the partial pressure of the test gas elsewhere in the heart. Although a paucity of information about the perfusion of cardiac adipose tissue makes it difficult to speak quantitatively, venous washout curves would be expected to have two significant advantages over precordial counting technics: (1) Because of the large adipose tissue-blood partition coefficients, the concentration of test gas in blood draining adipose tissue will be only a small fraction of that in the adipose tissue itself. (2) Because adipose tissue has a low flow per unit volume, its venous drainage should constitute a smaller fraction of total coronary flow than its volume does of total heart volume. In the case of 133Xe, preliminary reports of autoradiographic studies suggest that tracer does reach adipose tissue after a single intracoronary injection of the dissolved gas.‘8 THE AMERICAN JOURNALOF CARDIOLOGY
Gas
Measurements
of Coronary
POST-
C
PRE
- INFARCTION
--Q--= -+-
? z 3
553
Flow INFARCTION
100
ARTERIAL = VENOUS
--O--=
ARTERIAL
+:
VENOUS
"1
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I
4
I
I
I
8
,
I
12
20
12
TIME(min.1
TIME (min.)
,
0
1
16
0
TlME(min.1
TIME (min.)
Figure 4. Arterial and oenous nitrogen (Nz) desaturation curves in a closed chest dog before and after productton of an acute myocardial infarction by embolic occlusion of the left anterior descending coronary artery. The venous-arterial Ns difference is prolonged after the infarction.
The possibility of right atria1 contamination of sampled coronary venous blood has troubled many workers. Recent studies in our laboratory in closed chest dogslg indicate that right atria1 admixture is unlikely when the tip of the sampling catheter is inserted at least 1.5 cm. into the coronary sinus. The situation in man seems more variable, and on some occasions we have documented right atria1 contamination proximal to the point where the posterior interventricular vein enters the coronary sinus. Our current practice in human studies is to exclude right atria1 admixture in each case by studying coronary venous Hz concentrations during an intravenous infusion of dissolved Hz. Since Hz is nearly quantitatively eliminated in the lungs, over 95 per cent of the infused Hz never reaches the myocardium, and the sampled coronary venous Hz concentration is extremely small unless right atria1 admixture is present. The frequency with which signzjkant amounts of radioactive gas are delivered into the systemic circulation during ’ ‘selective’ ’ coronary arterial injections secllls difficult to estimate. The same applies to the possibility of counting tracer contained in the lungs. Of particular interest in the latter regard is delayed tracer elimination from alveoli with VOLUME
23,
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1969
low ventilation-perfusion ratios.“I Such alveoli may accumulate a surprising amount of an insoluble tracer from incoming blood and discharge it slowly into expired air. 24PRE ,NT.?R‘TION i p~ST-,Nr.%Rcrlm
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.05
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Summary of jindings in 7 dogs studied in the Figure 5. manner shown in Figure 4. Mean differences in N* concentration between venous and arterial blood are plotted against time before and after embolic occlusion of a portion of the left coronary artery. Vertical lines represent k-1 S.E.M.
Klocke et al.
554
F’inally, in measuring small venous-arterial differences, a test gas with a low solubility in blood has the advantage of having a more constant arterial concentration after the first few minutes of desaturation than a test gas with a high solubility in blood. Figure 6 illustrates this point by contrasting the simultaneous desaturations of helium (a = 0.008) and NzO (= = 0.42) in a model experiment. OTHER CONSIDERATIONS Several considerations which can be acknowledged only briefly have been covered in previous I-
-
:
---a._-
: NITROUS OXIDE
HELIUM
O TIM
E(min.1
Figure 6. Model experiment illustrating dijerencc~ in intrt gas dcsaturation cwues related to dzserent patterns of arterial desnturntion. Using an anesthetized, spontaneously ventilating dog, blood was pumped from a femoral artery through a magnetically stirred chamber at a rate of 176 ml./min, and returned to the dog through a femoral vein. Since the volume of the chamber was 210 ml., the flow through it was equivalent to 84 ml./100 ml./min. After a 20 minute period in which the dog breathed a gas mixture containing 10 per cent helium (He) and 3 per cent nitrous oxide (NrO), he was returned to room air and “arterial” and “venous” blood were sampled immediately upstream and downstream to the chamber. Because of the greater solubility of NrO in blood, the arteria1 NrO concentration decreased more slowly than the arterial He concentration.26 Elimination of NrO from the chamber was therefore slower than elimination of He, even though total areas between the respective venous and arterial desaturation curves agreed within 5 per cent. Calculated flows were 81 ml./100 ml./min. for He and 85 ml./100 ml./min. for NrO.
One involves the degree to which the measured gas exchange is representative of gas exchange throughout the heart. In studies employing venous blood, samples are usually obtained through a coronary sinus catheter, and measurements reflect only those areas whose drainage is included in the samples. In studies before and after experimental interventions, it is at least theoretically possible that the intervention alters the pattern of venous drainage and the proportional representation in sampled blood of areas with different perfusions. In similar fashion, measurements made by precordial counting represent only that portion of the heart “seen” by the collimated detector. Another difficulty is the dependence of numerical flow calculations on tissueblood partition coefficients. Measurements of partition coefficients in disease states have been reported only occasionally26 and more are needed. When employing simultaneous arterial and venous gas analyses, corrections for the time required for the blood to traverse the coronary bed are usually not attempted. When calculating flow from exponential analyses, an additional question is whether the measured exchange has been limited to a significant degree by diffusion. This is a particularly fascinating problem since the presence of a diffusion limitation for inert gas exchange would imply the coexistence of unusually large diffusion limitations for other substances of physiologic interest. Attempts to assess the importance of all these considerations in man are complicated by the lack of a primary standard of measurement against which inert gas data can be compared. Finally, in considering the various limitations of inert gas technics mentioned throughout this discussion, it should be emphasized that virtually all of them were recognized and described by those who pioneered in the development of the technics. reviews.‘3r21-“4
SUMMARY Several factors which are of interest when measuring coronary blood flow by inert gas technics are reviewed. The major problems in these measurements arise when flow is not uniform throughout the heart. Methods of delivery and analysis of gas must be adequate to quantitate persistent venous-arterial (or tissue-arterial) differences produced by areas of low flow. The effects of these areas on measured gas exchange can vary with different THE
AMERICAN
JOURNAL
OF CARDIOLOGY
Gas
Measurements
experimental approaches. Methods of calculating flow from desaturation curves also vary and sometimes involve assumptions concerning the distribution of flow within the heart. Potential probIerns are also presented by right atria1 “contamination” of sampled coronary venous blood, by extracardiac “contamination” of precordially monitored myocardial gas content, and by the contribution to measured gas exchange of cardiac adipose tissue. All these considerations are particularly important in measurements in disease states. ACKNOWLEDGMENT The material contained in this article has been developed during the course of collaborative work with Drs. Robert C. Koberstein, David G. Greene and Ivan L. Bunnell. It is a pleasure to acknowledge their assistance.
REFERENCES 1. ECKENHOFF,J. E., HAFKENSCHIEL,J. H., LANDMESSER,C. M. and HARMEL, M. Cardiac oxygen metabolism and control of the coronary circulation. Am. J. Physiol., 149: 634, 1947. 2. GOODALE, W. T., LUBIN, M., ECKENHOFF,J. E., HAFKENSCHIEL,J. H. and BANFIELD,W. G., JR. Coronary sinus catheterization for studying coronary blood flow and myocardial metabolism. Am. J. Physiol., 152: 340, 1948. 3. ECKENHOFF,J. E., HAFKENSCHIEL,J. H., HARMEL, M. H., GOODALE, W. T., LUBIN, M., BING, R. J. and KETY, S. S. Measurement of coronary blood flow by the nitrous oxide method. Am. J. Physiol., 152: 356, 1948. 4. BINc, R. J., HAMMOND,M. M., HANDELSMAN,J. C., POWERS, S. R., SPENCER,F. C., ECKENHOFF,J. E., GOODALE, W. T., HAFKENSCHIEL, J. H. and &TY, S. S. The measurement of coronary blood flow, oxygen consumption and efficiency of the left ventricle in man. Am. Heart J., 38: 1, 1949. 5. SULLIVAN, J. M., TAYLOR, W. J., ELLIOTT, W. C. and GORLIN, R. Regional myocardial blood flow. J. Clin. Invest., 46: 1402, 1967. 6. KLOCKE, F. J., KOBERSTEIN,R. C., PITTMAN,D. E., BUNNELL,I. L., GREENE, D. G. and Rosmc, D. R. Effects of heterogeneous myocardial perfusion on coronary venous HZ desaturation curves and calculations of coronary flow. J. Clin. Invest., 47: 2711, 1968. 7. KIRK, E. S. and HONE, C. R. Nonuniform distribution of blood flow and gradients of oxygen tension within the heart. Am. J. Physiol., 207: 661, 1964. 8. GOODALE, W. T. and HACKEL, D. B. Measurement of coronary blood flow in dogs and man from rate of myocardial nitrous oxide desaturation. Circulation Res., 1: 502, 1953. 9. HERD, J. A., HOLLENBERG,M., THORBURN, G. D., KOPALD, H. H. and BARGER, A. C. Myocardial blood flow determined with krypton 85 in unanesthetized dogs. Am. J. Physiol., 203: 122, 1962. VOLUME 23, APRIL 1969
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Flow
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10. COHEN, L. S., ELLIorr, CV. C. and GORLIN, R. Measurement of myocardial blood flow using krypton 85. Am. J. Physiol., 206: 997, 1964. 11. Ross, R. S., UEDA, K., LICHTLEN, P. R. and REES, J. R. Measurement of myocardial blood flow in animals and man by selective injection of radioactive inert gas into the coronary arteries. Circul&ion Res., 15: 28, 1964. 12. BASSINGTHWAIGHTE,J. B., STRANDELL, T. and DONALD, D. E. Coronary clearance of intraarterial xenon and antipyrine. Clin. Res.. 14: 424, 1966. 13. ZIERLER, K. L. Equations for measuring blood flow by external monitoring of radioisotopes. Circulation Res., 16: 309, 1965. 14. SAPIRSTEIN,L. A. and OGDEN, E. Theoretic limitations of the nitrous oxide method for the determination of regional blood flow. Circulation Res., 4: 245, 1956. 15. KLOCKE, F. J. Measurement of trace amounts of inert gases in blood by gas chromatography. In: Lectures on Gas Chromatography, 1966, p. 75. Edited by MATTICK, L. R. and SZYMANSKI,H. A. New York, 1967. Plenum Press. 16. FARHI, L. E., EDWARDS, A. W. T. and HOMMA, T. Determination of dissolved Nz in blood by gas chromatography and (a - A)Ns difference. J. Appl. F’hysiol., 18: 97, 1963. 17. ROSING,D. R. and KLOCKE, F. J. Myocardial nitrogen washout curves before and after coronary occlusion. Texas Rep. Biol. & Med., 25: 495, 1967. 18. SHAW, D., FRIESINGER,G. C., PITT, A. and Ross, R. S. Macro-autoradiography of XeI33 in the myocardium. Fed. Proc., 25: 401, 1966. 19. KOBERSTEIN,R. C., PITTMAN, D. E. and KLOCKE, F. J. Right atria1 admixture in coronary venous blood. Am. J. Physiol., in press. 20. BRANDI, G., FAM, W. M. and MCGREGOR, M. Measurement of coronary flow in local areas of myocardium using xenon 133. J. A#. PhysioI., 24: 446, 1968. 21. ROWE, G. G. The nitrous-oxide method for determining coronary blood flow in man. Am. Heart .I., 58: 268, 1959. 22. KETY, S. S. Theory of blood-tissue exchange and its application to measurement of blood flow. In: Methods in Medical Research, Vol. 8, p. 223. Edited by BRUNER, H. D. Chicago, 1960. Year Book Publishers. 23. GREGG, D. E. and FISHER,L. C. Blood supply to the heart. In: Handbook of Physiology, Sect. 2, Circulation, Vol. 2, chap. 44, p. 1517. Edited by HAMILTON,W. F. and Dow, P. Washington, D. C., 1963. American Physiological Society. 24. ROWE, G. G., CASTILLO, C. A., APONSO, S. and CRUMPTON, C. W. Coronary flow measured by the nitrous-oxide method. Am. Heart .I., 67: 457, 1964. 25. KOZAM, R. L., CUBINA, J. M., LANDAU, S. and LUKAS, D. S. Variables affecting solubility of nitrous-oxide in blood and myocardium. Clin. Res., 12: 187, 1964. 26. KETY, S. S. The theory and applications of the exchange of inert gas at the lungs and tissues. Pharmacol. Rev., 3: 1,1951.
Gas Measurements
of Coronary
Blood Flow* FRANCIS J. KLOCKE,
M.D., DOUGLAS R.
Buffalo,
ROSING, M.D. and DAVID E. PITTMAN, M.D. New York
T
HE USE OF inert gas technics to measure coronary blood flow has been an important part of quantitative studies of ventricular function and myocardial metabolism since the nitrous oxide technic was adapted to the coronary circulation in the late 1940’5~-~ The present communication is intended to summarize several principles which are agreed to be important in performing such measurements. Of particular interest are problems that arise when coronary flow is heterogeneously distributed within the heart, as is certainly the case in coronary artery disease6s6 and as is probably the case in most other situations.7
100 gm./min. For a variety of reasons investigators sometimes prefer to perform measurements during periods of desaturation or “washout,” that is, during periods in which inert gas previously dissolved in the myocardium is leaving the heart by way of venous blood.8 These approaches may be divided into two types, depending on the manner in which test gas is delivered to the myocardium before the washout. In the first case, delivery of gas is accomplished by having the subject breathe a mixture containing the test gas. He is returned to room air at time zero, and flow per unit volume of tissue is calculated from the ensuing change in venous gas concentration, the tissue-blood partition coefficient and the area between the arterial and venous desaturation curves. In the second case, the subject breathes room air at all times, and saturation is accomplished by injecting saline in which the test gas has been dissolved into the left ventricle or a coronary artery. The saline is discontinued at time zero and, when the test gas has a low solubility in blood, recirculation is minimal and the arterial gas concentration drops quickly to negligible levels. The pattern of myocardial gas elimination is then obtained from the coronary venous washout curve or precordial monitoring of myocardial radioactivity. Frequently used examples of this approach include the 85Kr and 133Xe methods developed by Herd,g Cohen,lO and Ross” and their co-workers. The saline is usually administered as a sudden single injection, although longer saturation periods are theoretically feasible. If the data are plotted semilogarithmically against time and myocardial
GENERAL APPROACHES Although inert gas methods vary in their details, all are ultimately derived from the Fick principle and involve measurements of the difference between concentrations of arterial and venous (or tissue) gas during a period of myocardial uptake or release. In the original nitrous oxide (NzO) technic, arterial and coronary sinus concentrations are followed during a period of breathing Nz0.1e4 The rate of coronary flow is obtained by dividing the change in tissue concentration of NzO by the mean arterial-venous NzO difference. The change in tissue concentration is not measured directly but is calculated from the change in venous N20 concentration and the tissue-blood partition coefficient. The arterial-venous difference is obtained by integrating the area between the arterial and venous curves. The quotient represents flow per unit volume of tissue and is conventionally expressed as ml./
* From the Department of Medicine, State University of New York at Buffalo School of Medicine, Buffalo, N. Y. This study was supported by U. S. Public Health Service Research Grant HE-09587 from the National Heart Institute and by grants from the Heart Association of Western New York and the United Health Foundation of Western New York. Address for reprints: Francis J. Klocke, M.D., Buffalo General Hospital, 100 High Street, Buffalo, N. Y. 14203. 548
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perfusion is truly homogeneous, the venol~s (or precordial) washout curve should decay as a single exponential. Many workers assume that this is the case and calculate flow per unit volume from the slope of the straight line which best fits the semilogarithmically plotted data for the first few minutes of desaturation. OthersI find that the slope of the semilogarithmically plotted curve is not constant and, by “curvepeeling” procedures, treat the data as rcpresenting a number of homogenous compartments arranged in parallel. Still others13 avoid exponential treatments and calculate flow per unit volume of distribution of tracer from the mean transit time of the tracer through the system. This latter approach also avoids the assumption that end-capillary blood and tissue are in equilibriurn for partial pressure of the test gas (see the following). In any event? the major difficulty in the clinical application of these technics is that by nonuniformity of myocardial presented flow. Of particular interest are areas with flows per unit volume which are lower than the average flow per unit volume for the entire tissue. The expected effects of these areas on measured gas differences are illustrated in Figure 1. In measurements during test gas saturation, areas of low flow prolong the time required for the
DURING GAS
540
Flow
arterial-venous gas difference to become negduring desaturation, ligible. l1 In measurements areas of low flow are cleared of test gas more slowly than the areas of “normal” flow and produce increased concentrations of tissue and venous gas in the latter portion of the washout. In washouts after gas breathing, this results in a small but persistent venous-arterial gas difference. In washouts after dissolved gas it causes the semilogarithmically infusion, plotted venous (or tissue) washout curve to deviate clearly from a single exponential. Additional complications that arise when concentration of tissue gas is not uniform at the completion of the period of administration of gas will be discussed later. ANALYTICAL
TECHNICS AND
METHODS OF SATURATION
These observations help to define two crucial requirements for measuring coronary blood flow when flow is distributed unevenly within the heart. 1. The method of gas analysis employed must be capable of quantitating small venous-arterial (or tissue-arterial) gas di$erences. In our own laboratory, we have found it advisable to perform analyses of blood gas with a specially designed thermal conductivity gas chromatograph.15
AFTER
AFTER
BREATHING
GAS
BREATHING
GAS
INFUSION
yp7 :kVEN.(UNIF.FLOW) ; ,I bEN.(NON-UNIF $1 ,r
FLOWi
:. :. \ it i\
VEN.(OR (UNIF.
& .:\ VEN.
(UNIF.
FLOW)
‘:\,~::,“,I”,“,::“‘“” / \
‘\ :
’ \ :.. \
Figure 1. VOLUME
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TISSUE) FLOW 1
FLOW 1 ‘\._
Effete of nonuniform myocardiat bloodflow on measurrdinert ,gas mchange. See text for details. 1969
550
Klocke
The chromatograph is more sensitive and specific than Van Slyke analyses and can be used for virtually any gas. It differs from conventional chromatographs primarily by being housed in a water bath at constant temperature instead of in an insulated air oven. The improved temperature control minimizes “baseline noise” and thereby provides an improved signal-to-noise ratio. The detector is a commercially available sealed thermistor unit, and is suitable for operation at only 1 to 2O C. above ambient temperature. The flow control system is also commercially available and, like the rest of the unit, is immersed in the water bath, which is kept at 24.0’ C. with a heater and proportional temperature controller. Blood samples are collected in 2.0ml. glass syringes, and their dissolved gases are extracted in a modified Van Slyke apparatus. ls The extracted gases are then compressed into a glass sampling loop and introduced into the chromatograph carrier gas stream. If desirable, substances that remove water vapor and CO2 are included in the tubing
“Control”
et al. conrlecting the sampling loop to the chromatograph. Concentrations of gas are usually quantitated from the heights of the gas peaks on the chromatograms. Representative chromatograms are shown in Figure 2. Limits of detection for most gases have been 10e5 to 10W6 ml. Since each analysis requires only 2 ml. of blood, a large number of points can be obtained for each saturation or desaturation curve, thus improving curve resolution. In addition, since several gases can be measured in a single blood sample, a variety of gases can be studied simultaneously. 2. The technic by which test gas is delivered to the myocardium must be adequate for areas of lowjlow as well as for areas of normal flow. In considering the saturation of an organ with an inert gas, it is helpful to think of the process in terms of attaining the same partial pressure (that is, the same gas tension) in the tissue as in incoming arterial blood. The rate at which tissue gas tension approaches the arterial gas tension will vary importantly with flow per unit volume of
tk-1.3~
SAYPUS: 2.0
IO-•mt
l+3min_rl
BATH
CARRIER FLOW
\r”/’
:
WHOLE
6LOOb
25.3’C.
GAS : 02
RATES
( ML/MIN.
1:
3.1 REF. COL. - 3.0 STR.SPL. EXH.- 6.6 SAYP.
SAMPLE
A,N2
t
ML
TEMP.
COL.
COLUMN
-
:
4 FT. ACTIVATED
Samp. Inj
CHARCOAL,
‘/S”O.D.
DETECTOR:
He-0.5x IO-‘ml
7.OMA,Arl.
A
t
Samp. W
A,Ne
XI
A,Nz
t S$P. .
Records illustrating the analysis of trace amounts of helium (He) and hydropn (HZ) in blood. Analytical conditions are shown on the upper right. The record on the upper left is from a blood sample containing neither He nor H2 and serves to verify that the blood contained no extractable substance that could have produced a peak interfering with those of He or Hz. The large amount of Or extracted from the blood does not appear on the record because 02 was employed as the carrier gas. COs is not seen because a CO2 absorbent was included in the tubing connecting the gas sampling loop to the chromatograph sample inlet. (A = argon, Ns = nitrogen.) (Reproduced by permission of the publisher from Klocke, F. J.16) Figure
2.
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Gas Measurements tissue (F/V) and with the tissue-blood partition coefficient (x). The rate would also be affected by a diffusion limitation to gas exchange. However, in this section of our discussion, we shall assume that tissue-blood gas exchange is limited by perfusion only, that is, that partial pressure equilibrium between tissue and blood is always reached at the capillary level. For purposes of illustration, consider a situation in which myocardial saturation is initiated by a sudden, ‘
of Coronary
551
Flow
Prn(OR V) AS X OF
Pa
ing equation: P m(orv)t = PA1 -
Ib
epkt)
TIME
where : P m(orv) = partial
pressure of inert gas in myocardial tissue (or venous blood) at time t; P, = partial pressure of inert gas in arterial blood; and k= rate constant of saturation, which = (F/W/X
Figure 3 expresses these relations graphically for rate constants ranging from 1.20 to 0.02. In considering this material, it is important to keep in mind the distinctions among gas tension, concentration and content. Concentration is the product of partial pressure and solubility and, at any given partial pressure, the relative concentrations of a gas in tissue and blood will depend on the relative solubilities of the gas in tissue and blood, that is, on the tissue-blood partition coefficient. The gas content of an area of tissue is in turn the product of tissue gas concentration and tissue volume. As will be discussed, the effects of areas of low flow on desaturation curves obtained from measurements of venous gas concentration can differ significantly from the effects of the same areas on curves obtained from precordial measlu-ements of myocardial gas content. The importance of appropriate analytical methods and technics 01 saturation can be illustrated by another example. Assume tissue specific gravity to be 1.0 and consider a “two-compartment heart” in which 80 per cent has a perfusion of 80 ml./100 gm./min. and 20 per cent (perhaps representing an area of scar tissue) has a perfusion of only 5 ml./100 gm./min. If the tissue-blood partition coefficient is 1.0 in both VOLUME23, APRIL 1969
2'0
(min.)
Figure 3. Expected approach of tissue (or venous) gas tension to arterial gas tension in homogeneously perfused areas of thr heart following a “square-wave” innement in arterial ga5 tension. Each curve corresponds to the rate constant of saturation shown adjacent to it. When the tissue-blood partition coefficient is 1 .O, each rate constant also reprtsents flow in m.L/gm./min. Pm(orv) = myocardial (or venous) gas tension; P, = arterial gas tension.
the corresponding rate constants arc areas, 0.80 and 0.05. Over-all flow is 65 ml./100 gm./ min., and the blood draining the area of lower flow constitutes only 1.5 per cent of total flow. Thus, even if saturation is complete, the area of lower flow will be overlooked in a venolls washout curve if analytical methods cannot detect venous-arterial differences of less than 1.5 per cent of the initial venous and arterial concentrations. If the wash-in period does not allow for full saturation of the area of lower flow, the situation is even more difficult. If the approach shown in Figure 3 is employed, the gas tension in the area of lower Aow at the end of a 10 minute saturation will be only 39 per cent of that in the area of higher flow, and the area of lower flow will not be evident in a venous washout curve unless analytical methods can detect venous-arterial differences of less than 0.6 per cent of the initial venous and arterial concentrations. In addition, since venous blood in favor of the area of higher is “weighted” flow, the measured venous concentration at the beginning of desaturation will be an inaccurate index of both the tissue concentration in the area of lower flow and the volume-averaged tisslle concentration for the cantir? heart.
552
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When myorardial gas content, rather than venous gas concentration, Is measured, the situation is somewhat different and reflects the fact that the area of lower flow constitutes a greater fraction of total heart volume than its venous drainage does of total coronary flow. If the saturation period were adequate to attain the same partial pressure in the area of lower flow as in arterial blood, the area of lower flow would comprise 20 per cent of the total gas content at the beginning of desaturation, and its determination in a tissue washout curve would be relatively easy. If the wash-in period were only 10 minutes, the gas concentration achieved in the area of lower flow would be only 39 per cent of that in the area of higher flow, and the gas content of the area of lower flow would constitute 8.9 per cent of the total gas content at the beginning of desaturation. As the wash-in period is shortened more and more, tissue gas tension does not reach the arterial tension in either compartment, and gas content in the area of lower flow forms a progressively smaller percentage of total gas content at the beginning of desaturation. Additional and perhaps very significant complications that are introduced by cardiac adipose tissue will be discussed below. It seems clear, however, that the relations among analytical sensitivity, method of saturation, partition coefficients and method of monitoring tissue-blood desaturation serve to define a “threshold” or “minimal detectable” flow per unit volume when Aow is heterogeneously distributed. The higher this threshold, the greater is the chance of ignoring an area of low flow and of overestimating total flow. In view of the importance of adequate saturation, one Could consider performing measurements during a prolonged period of saturation rather than during a conventional period of desaturation. Analytical limitations might be especially troublesome, however, since the evaluation of areas of low flow would involve the measurement of small differences between gas concentrations which are higher than at comparable times during desaturation. One additional approach to this problem is to measure flow from nitrogen (Nz) desaturation curves.l7 Since Nz is breathed normally, complete myocardial saturation at the partial pressure of Nz in room air is ensured. No preliminary breathing of gas is required, and the patient is merely switched from room air to a mixture containing 21 per cent OZ and 79 per
et al. cent of some other inert gas such as argon. Washouts of this type before and after embolic occlusion of a portion of the left coronary artery in a closed chest dog are shown in Figure 4. Figure 5 summarizes similar data in 7 dogs. As advantageous as NZ appears theoretically, however, it suffers practically from the extreme precautions that must be taken to exclude “contamination” of sampled blood with small amounts of dissolved Nz originating from ambient air. If this obstacle can be overcome, it should be of considerable value. ADDITIONAL FACTORS IN MEASURING SMALL VENOUS-ARTERIAL (OR TISSUE-ARTERIAL) GAS DIFFERENCES From this discussion, it is evident that areas of myocardium having a low flow are represented in inert gas desaturation curves as small but persistent venous-arterial (or tissue-arterial) gas differences. In focusing attention on these differences, one must attempt to exclude the possibility that they originate from cardiac adipose tissue, from “contamination” of sampled coronary venous blood by right atria1 blood, or from “contamination” of precordially monitored myocardial gas content by tracer contained in the chest wall or lungs. In the case of adipose tissue, the rate constant of saturation for commonly employed inert gases is probably small because of low flows per unit volume and high tissue-blood partition coefficients. This observation implies that the partial pressure of test gas in adipose tissue at the completion of a conventional saturation period is considerably less than the partial pressure of the test gas elsewhere in the heart. Although a paucity of information about the perfusion of cardiac adipose tissue makes it difficult to speak quantitatively, venous washout curves would be expected to have two significant advantages over precordial counting technics: (1) Because of the large adipose tissue-blood partition coefficients, the concentration of test gas in blood draining adipose tissue will be only a small fraction of that in the adipose tissue itself. (2) Because adipose tissue has a low flow per unit volume, its venous drainage should constitute a smaller fraction of total coronary flow than its volume does of total heart volume. In the case of 133Xe, preliminary reports of autoradiographic studies suggest that tracer does reach adipose tissue after a single intracoronary injection of the dissolved gas.‘8 THE AMERICAN JOURNALOF CARDIOLOGY
Gas
Measurements
of Coronary
POST-
C
PRE
- INFARCTION
--Q--= -+-
? z 3
553
Flow INFARCTION
100
ARTERIAL = VENOUS
--O--=
ARTERIAL
+:
VENOUS
"1
Oh---x--
F
I
4
I
I
I
8
,
I
12
20
12
TIME(min.1
TIME (min.)
,
0
1
16
0
TlME(min.1
TIME (min.)
Figure 4. Arterial and oenous nitrogen (Nz) desaturation curves in a closed chest dog before and after productton of an acute myocardial infarction by embolic occlusion of the left anterior descending coronary artery. The venous-arterial Ns difference is prolonged after the infarction.
The possibility of right atria1 contamination of sampled coronary venous blood has troubled many workers. Recent studies in our laboratory in closed chest dogslg indicate that right atria1 admixture is unlikely when the tip of the sampling catheter is inserted at least 1.5 cm. into the coronary sinus. The situation in man seems more variable, and on some occasions we have documented right atria1 contamination proximal to the point where the posterior interventricular vein enters the coronary sinus. Our current practice in human studies is to exclude right atria1 admixture in each case by studying coronary venous Hz concentrations during an intravenous infusion of dissolved Hz. Since Hz is nearly quantitatively eliminated in the lungs, over 95 per cent of the infused Hz never reaches the myocardium, and the sampled coronary venous Hz concentration is extremely small unless right atria1 admixture is present. The frequency with which signzjkant amounts of radioactive gas are delivered into the systemic circulation during ’ ‘selective’ ’ coronary arterial injections secllls difficult to estimate. The same applies to the possibility of counting tracer contained in the lungs. Of particular interest in the latter regard is delayed tracer elimination from alveoli with VOLUME
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low ventilation-perfusion ratios.“I Such alveoli may accumulate a surprising amount of an insoluble tracer from incoming blood and discharge it slowly into expired air. 24PRE ,NT.?R‘TION i p~ST-,Nr.%Rcrlm
-: _.....
< ti e a;k uvz ‘23 %i* Ow
s;
16
: : 1
i
:
: ‘8
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12-
$kS
ofm 4;s _ 5
_ 6-
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I1 4
6
12
16
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TIME (min.) P(
.Ol
.05
.05
.Ol
Dl
.Ol
Summary of jindings in 7 dogs studied in the Figure 5. manner shown in Figure 4. Mean differences in N* concentration between venous and arterial blood are plotted against time before and after embolic occlusion of a portion of the left coronary artery. Vertical lines represent k-1 S.E.M.
Klocke et al.
554
F’inally, in measuring small venous-arterial differences, a test gas with a low solubility in blood has the advantage of having a more constant arterial concentration after the first few minutes of desaturation than a test gas with a high solubility in blood. Figure 6 illustrates this point by contrasting the simultaneous desaturations of helium (a = 0.008) and NzO (= = 0.42) in a model experiment. OTHER CONSIDERATIONS Several considerations which can be acknowledged only briefly have been covered in previous I-
-
:
---a._-
: NITROUS OXIDE
HELIUM
O TIM
E(min.1
Figure 6. Model experiment illustrating dijerencc~ in intrt gas dcsaturation cwues related to dzserent patterns of arterial desnturntion. Using an anesthetized, spontaneously ventilating dog, blood was pumped from a femoral artery through a magnetically stirred chamber at a rate of 176 ml./min, and returned to the dog through a femoral vein. Since the volume of the chamber was 210 ml., the flow through it was equivalent to 84 ml./100 ml./min. After a 20 minute period in which the dog breathed a gas mixture containing 10 per cent helium (He) and 3 per cent nitrous oxide (NrO), he was returned to room air and “arterial” and “venous” blood were sampled immediately upstream and downstream to the chamber. Because of the greater solubility of NrO in blood, the arteria1 NrO concentration decreased more slowly than the arterial He concentration.26 Elimination of NrO from the chamber was therefore slower than elimination of He, even though total areas between the respective venous and arterial desaturation curves agreed within 5 per cent. Calculated flows were 81 ml./100 ml./min. for He and 85 ml./100 ml./min. for NrO.
One involves the degree to which the measured gas exchange is representative of gas exchange throughout the heart. In studies employing venous blood, samples are usually obtained through a coronary sinus catheter, and measurements reflect only those areas whose drainage is included in the samples. In studies before and after experimental interventions, it is at least theoretically possible that the intervention alters the pattern of venous drainage and the proportional representation in sampled blood of areas with different perfusions. In similar fashion, measurements made by precordial counting represent only that portion of the heart “seen” by the collimated detector. Another difficulty is the dependence of numerical flow calculations on tissueblood partition coefficients. Measurements of partition coefficients in disease states have been reported only occasionally26 and more are needed. When employing simultaneous arterial and venous gas analyses, corrections for the time required for the blood to traverse the coronary bed are usually not attempted. When calculating flow from exponential analyses, an additional question is whether the measured exchange has been limited to a significant degree by diffusion. This is a particularly fascinating problem since the presence of a diffusion limitation for inert gas exchange would imply the coexistence of unusually large diffusion limitations for other substances of physiologic interest. Attempts to assess the importance of all these considerations in man are complicated by the lack of a primary standard of measurement against which inert gas data can be compared. Finally, in considering the various limitations of inert gas technics mentioned throughout this discussion, it should be emphasized that virtually all of them were recognized and described by those who pioneered in the development of the technics. reviews.‘3r21-“4
SUMMARY Several factors which are of interest when measuring coronary blood flow by inert gas technics are reviewed. The major problems in these measurements arise when flow is not uniform throughout the heart. Methods of delivery and analysis of gas must be adequate to quantitate persistent venous-arterial (or tissue-arterial) differences produced by areas of low flow. The effects of these areas on measured gas exchange can vary with different THE
AMERICAN
JOURNAL
OF CARDIOLOGY
Gas
Measurements
experimental approaches. Methods of calculating flow from desaturation curves also vary and sometimes involve assumptions concerning the distribution of flow within the heart. Potential probIerns are also presented by right atria1 “contamination” of sampled coronary venous blood, by extracardiac “contamination” of precordially monitored myocardial gas content, and by the contribution to measured gas exchange of cardiac adipose tissue. All these considerations are particularly important in measurements in disease states. ACKNOWLEDGMENT The material contained in this article has been developed during the course of collaborative work with Drs. Robert C. Koberstein, David G. Greene and Ivan L. Bunnell. It is a pleasure to acknowledge their assistance.
REFERENCES 1. ECKENHOFF,J. E., HAFKENSCHIEL,J. H., LANDMESSER,C. M. and HARMEL, M. Cardiac oxygen metabolism and control of the coronary circulation. Am. J. Physiol., 149: 634, 1947. 2. GOODALE, W. T., LUBIN, M., ECKENHOFF,J. E., HAFKENSCHIEL,J. H. and BANFIELD,W. G., JR. Coronary sinus catheterization for studying coronary blood flow and myocardial metabolism. Am. J. Physiol., 152: 340, 1948. 3. ECKENHOFF,J. E., HAFKENSCHIEL,J. H., HARMEL, M. H., GOODALE, W. T., LUBIN, M., BING, R. J. and KETY, S. S. Measurement of coronary blood flow by the nitrous oxide method. Am. J. Physiol., 152: 356, 1948. 4. BINc, R. J., HAMMOND,M. M., HANDELSMAN,J. C., POWERS, S. R., SPENCER,F. C., ECKENHOFF,J. E., GOODALE, W. T., HAFKENSCHIEL, J. H. and &TY, S. S. The measurement of coronary blood flow, oxygen consumption and efficiency of the left ventricle in man. Am. Heart J., 38: 1, 1949. 5. SULLIVAN, J. M., TAYLOR, W. J., ELLIOTT, W. C. and GORLIN, R. Regional myocardial blood flow. J. Clin. Invest., 46: 1402, 1967. 6. KLOCKE, F. J., KOBERSTEIN,R. C., PITTMAN,D. E., BUNNELL,I. L., GREENE, D. G. and Rosmc, D. R. Effects of heterogeneous myocardial perfusion on coronary venous HZ desaturation curves and calculations of coronary flow. J. Clin. Invest., 47: 2711, 1968. 7. KIRK, E. S. and HONE, C. R. Nonuniform distribution of blood flow and gradients of oxygen tension within the heart. Am. J. Physiol., 207: 661, 1964. 8. GOODALE, W. T. and HACKEL, D. B. Measurement of coronary blood flow in dogs and man from rate of myocardial nitrous oxide desaturation. Circulation Res., 1: 502, 1953. 9. HERD, J. A., HOLLENBERG,M., THORBURN, G. D., KOPALD, H. H. and BARGER, A. C. Myocardial blood flow determined with krypton 85 in unanesthetized dogs. Am. J. Physiol., 203: 122, 1962. VOLUME 23, APRIL 1969
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Flow
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10. COHEN, L. S., ELLIorr, CV. C. and GORLIN, R. Measurement of myocardial blood flow using krypton 85. Am. J. Physiol., 206: 997, 1964. 11. Ross, R. S., UEDA, K., LICHTLEN, P. R. and REES, J. R. Measurement of myocardial blood flow in animals and man by selective injection of radioactive inert gas into the coronary arteries. Circul&ion Res., 15: 28, 1964. 12. BASSINGTHWAIGHTE,J. B., STRANDELL, T. and DONALD, D. E. Coronary clearance of intraarterial xenon and antipyrine. Clin. Res.. 14: 424, 1966. 13. ZIERLER, K. L. Equations for measuring blood flow by external monitoring of radioisotopes. Circulation Res., 16: 309, 1965. 14. SAPIRSTEIN,L. A. and OGDEN, E. Theoretic limitations of the nitrous oxide method for the determination of regional blood flow. Circulation Res., 4: 245, 1956. 15. KLOCKE, F. J. Measurement of trace amounts of inert gases in blood by gas chromatography. In: Lectures on Gas Chromatography, 1966, p. 75. Edited by MATTICK, L. R. and SZYMANSKI,H. A. New York, 1967. Plenum Press. 16. FARHI, L. E., EDWARDS, A. W. T. and HOMMA, T. Determination of dissolved Nz in blood by gas chromatography and (a - A)Ns difference. J. Appl. F’hysiol., 18: 97, 1963. 17. ROSING,D. R. and KLOCKE, F. J. Myocardial nitrogen washout curves before and after coronary occlusion. Texas Rep. Biol. & Med., 25: 495, 1967. 18. SHAW, D., FRIESINGER,G. C., PITT, A. and Ross, R. S. Macro-autoradiography of XeI33 in the myocardium. Fed. Proc., 25: 401, 1966. 19. KOBERSTEIN,R. C., PITTMAN, D. E. and KLOCKE, F. J. Right atria1 admixture in coronary venous blood. Am. J. Physiol., in press. 20. BRANDI, G., FAM, W. M. and MCGREGOR, M. Measurement of coronary flow in local areas of myocardium using xenon 133. J. A#. PhysioI., 24: 446, 1968. 21. ROWE, G. G. The nitrous-oxide method for determining coronary blood flow in man. Am. Heart .I., 58: 268, 1959. 22. KETY, S. S. Theory of blood-tissue exchange and its application to measurement of blood flow. In: Methods in Medical Research, Vol. 8, p. 223. Edited by BRUNER, H. D. Chicago, 1960. Year Book Publishers. 23. GREGG, D. E. and FISHER,L. C. Blood supply to the heart. In: Handbook of Physiology, Sect. 2, Circulation, Vol. 2, chap. 44, p. 1517. Edited by HAMILTON,W. F. and Dow, P. Washington, D. C., 1963. American Physiological Society. 24. ROWE, G. G., CASTILLO, C. A., APONSO, S. and CRUMPTON, C. W. Coronary flow measured by the nitrous-oxide method. Am. Heart .I., 67: 457, 1964. 25. KOZAM, R. L., CUBINA, J. M., LANDAU, S. and LUKAS, D. S. Variables affecting solubility of nitrous-oxide in blood and myocardium. Clin. Res., 12: 187, 1964. 26. KETY, S. S. The theory and applications of the exchange of inert gas at the lungs and tissues. Pharmacol. Rev., 3: 1,1951.