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A STUDYOF THE RELATIONSHIP BETWEENRADIOACTIVESODIUMCLEARANCEANDDIkECTLY MEASURED

BLOODFLOWIN THE BICEPS MUSCLEOFTHE DOG

BY THEODOREC. PRENTICE, ROBERTR. STAHL, NORMANA. DIAL, ANDFRANKV. PONTERIO

(From the Division of Surgery, Army Medical Service Graduate School, Walter Reed ArmyMedical Center, Washington, D. C.)

(Submitted for publication October 18, 1954; accepted December 8, 1954)

Since the introduction by Kety in 1949 (1) ofthe radioactive sodium clearance method for meas-uring regional blood flow in muscle, much workusing this method has been published. The re-sults have been varied, and conflicting in manyrespects. Some have supported the contentionthat the rate of sodium clearance is a good indexof local muscle blood flow (2, 3), others have not(4, 5). Perhaps the dominant conclusions onedraws from existing data are that: 1) There isa wide range of values for sodium clearance ratefrom muscle in the resting human; 2) there is alarge overlap between clearance rates from anormal group and from a group with knownperipheral vascular disease; and 3) neverthelessfor any given individual those influences such asexercise, drugs, and temperature, which have beennoted to increase or decrease muscle blood flow,usually cause parallel changes in the rate of so-dium clearance from muscle.

The problem arises then as to whether the widerange of clearance values noted in normals, andthe considerable overlap found between normaland pathologic material, reflect actual variation inregional flow rates or are due to inherent errorsin the clearance method as an index of flow.

To further elucidate this problem and to testas rigorously as possible the accuracy and de-pendability of the sodium clearance method as ameasurement of total blood flow, a series of directand simultaneous comparisons between total bloodflow through muscle and sodium clearance there-from was carried out.

Any conclusions concerning blood flow as re-lated to radioactive sodium clearance from thisstudy must of necessity refer to total blood flow

since that was the quantity measured. Effectiveblood flow, which refers to the fraction of totalblood flow going through the capillaries, cannotbe directly measured at the present time. There-fore any conclusions relating the clearance rate ofradioactive sodium to effective blood flow (capil-lary circulation) are in the realm of speculation.

METHODS

Ten experiments were performed to determine the re-lationship between the cearance rate of radioactive so-dium from an injected intramuscular site and the bloodflow through the muscle. Dogs used in this experiment,ranging in weight from 12.7 to 23 Kg., were anesthetizedwith Sodium Nembutal.@

A large incision was made in the skin of the foreleg ex-tending from the anterior superior surface of the shoulderjoint, distally and somewhat medially, to the elbow joint.The skin and subcutaneous tissue were reflected and theunderlying muscle was exposed. Several large muscles,serratus ventralis, brachiocephalicus, pectoralis super-ficialis, and pectoralis profundus, were divided and re-flected so as to expose the biceps muscle. This also al-lowed for exposure of the brachial artery and vein whichwere about one centimeter medially and along the longi-tudinal axis of the muscle. The brachial artery and veinand their branches and tributaries were carefully dissectedand freed of their connective tissue attachments. In mostof the animals three pairs of nutrient vessels supplied thebiceps muscle (Figure 1). The artery and vein whichentered the middle portion of the muscle on its medialaspect were the largest in caliber and these vessels werethe ones used for perfusion. The smaller pairs of vesselsat the proximal and distal portions of the muscle wereligated. All other branches of the brachial artery andtributaries of the brachial vein. were ligated and divided.A cannula (Cv) was placed in the brachial vein and thisvein was ligated just distal to the point where the veinfrom the biceps muscle joined it This made possible thecollection of the venous outflow from the biceps muscle.The experimental dogs received heparin approximately

545

T. C. PRENTICE, R. R. STAHL, N. A. DIAL, AND F. V. PONTERIO

FIG. 1. SCHEMATICDIAGRAM OF LOCAL ANATOMYAND DISSECTION

4.5 mg. per Kg. of body weight just prior to the inser-tion of the venous cannula. A cannula (CA) was placedin the brachial artery which was ligated just distal tothe arterial branch to the biceps. The arterial cannulawas then connected to the perfusion pump with Tygontubing.

In each experiment the venous outflow from the bicepsmuscle was measured immediately after cannulation ofthe brachial vein and just prior to the cannulation of thebrachial artery. In all instances a relatively large rateof flow was found. These values, however, were notreported since they did not appear directly relevant tothe experiment and ligation of numerous branches of thebrachial artery would tend to divert more blood to thebiceps muscle.

The nerve supply to the biceps was left intact exceptin Experiment No. 2 when it was inadvertently severedduring dissection.

Both the tendon of origin and tendon of insertion of thebiceps muscle were severed. This was necessary so asto obliterate any blood supply reaching the muscle byway of the tendons. At this point in the dissection theentire muscle could be lifted and completely separatedfrom the extremity except for the arterial branch, ve-nous tributary, and the nerve supplying the biceps. Theproximal tendon, the tendon of origin, was sutured to-gether again. The more distal tendon, the tendon of in-sertion, was short; the tendinous portion of the musclewas clamped with a small hemostat The hemostat wasanchored to the extremity with thread so as to maintainanatomic relations and relative muscle stretch.

Donor dogs were anesthetized, heparinized (approxi-mately 4.5 mg. per Kg. of heparin), and bled into anopen reservoir. In all but Experiment Nos. 1, 3, and 4this blood was passed through an oxygenating device.The oxygenating device for the blood consisted of aVigreaux distillation column to which a glass joint andside tube was attached near the bottom. Oxygen was al-lowed to enter through this side tube. Blood was pouredinto the column at the top and bubbles of oxygen passedup through the tube as the column of blood descended.This method of oxygenation resulted in very little hemoly-sis as demonstrated by the plasma hemoglobin values be-fore and after passage through the oxygenating column.Prior to this passage of the blood through the column,5 to 10 ml. of heparin was added. This blood was thenplaced in two open reservoirs A and B (Figure 2).

A Phipps and Bird Infusion Pump was utilized to es-tablish a relatively constant blood flow through the mus-cle (Figure 2). This pump pushes a standard 50 ml. hy-podermic syringe at an adjustable constant speed. Thusa steady flow of blood to the biceps muscle could be main-tained for a desired period of time. The syringe (S)was filled with blood from reservoir A by changing theposition of the stopcock at D. While the syringe wasbeing filled, flow to the isolated muscle could be main-tained by gravity from reservoir B by changing positionof Stopcock E.

Pressure within the system was recorded with aStatham Pressure Transducer, Model P23A (c), whichwas placed into the perfusion circuit through a T-tube atF. The pressure was recorded through a Grass Bridge

546

RELATIONSHIP BETWEENNA22 CLEARANCEAND BLOODFLOW

Amplifier on a direct-writing ink oscillograph. Meanpressures were obtained by planimetric integration.

Temperature of the room was recorded continuouslyon a Brown Recording Thermograph.

Blood samples for analysis of hemoglobin and per cent0, saturation were obtained just proximal to the arterialcannula through the three-way stopcock at G.

Radioactive sodium, Na2, 5 microcuries in 0.1 ml. iso-tonic NaCl, was injected approximately one centimeterinto the biceps muscle through a No. 26 gauge needle.

Occasionally a small reflux of the injected material wouldappear at the injection site after the withdrawal of theneedle. This was removed with gauze moistened withsaline and the surface then blotted with dry gauze.

A lead-shielded Nuclear Scintillation Counter, ModelNo. DS1 and a Nuclear Scaler, Model No. 165, were

used to follow the disappearance rate of the radioactivesodium from the muscle. The scintillation assenbly was

placed over the injection site one inch from the surfaceof the muscle. No additional shielding of the apertureof the scintillation tube was used to eliminate beta radia-tion. (Previously at this distance there was no differencein counting rate between samples counted with an alumi-num absorber adequate to eliminate beta radiation andthose counted without the aluminum absorber.) Sufficientsample and background counts were measured to assure

a standard deviation of 2 per cent or less for all butthe weakest samples. Rarely, late in an experiment, theone minute counting rate had fallen to a level producinga standard deviation of 5 per cent.

After injection of the Nan and placement of the scintil-lation counter repeated one-minute counts were recorded

with fifteen-second intervals between each count. Usuallyeight successive counts were recorded and the intervalof time over which these counts were taken constituteda clearance period. Occasionally as few as five or as

many as nine counts were used, depending upon the re-

producibility of the volumes of the two-minute collectionsof venous blood during the clearance period.

Blood flow through the biceps muscle was measuredby collecting the venous outflow from the biceps musclethrough a cannula in the brachial vein. During all clear-ance periods the venous blood was collected for two-minute intervals in 10 milliliter volumetric mixing cyl-inders. Thus the sum of these two-minute volumes was

determined during the total time required for a clear-ance period. The mean venous outflow was then calcu-lated in milliliters per minute. Later when the weightof the biceps muscle was determined, the mean venous

outflow was expressed in milliliters per minute per 100grams of muscle. The sum of the volumes of blood col-lected for two-minute intervals during a clearance pe-

riod was determined and the mean flow per two-minuteinterval was calculated for the entire clearance period.All clearance periods which contained two-minute vol-umes of blood which deviated more than ten per centfrom the mean for that period were discarded. This was

taken as evidence of an inconstant flow during the clear-ance period.

The biceps muscle preparation in this experiment hada completely isolated circulation. Fresh donor dog'sblood was infused into the muscular arterial branch sup-

plying the muscle and the total venous outflow from themuscle was collected and discarded. Consequently, in

1=0=t -1._-

A- Blood ReservoirB- Blood Rervoir(grovity flow)C- Statham Pressure Transducer

Model P23AC^Arteroll CannulaCV-Venous CannulaD- Three-way StopcockE- Three-way StopcockF- T-tubeG- Three-way StopcockS- Standard 50ml. Hypoderric

Syrige

FIG. 2. SCHEMATIC DIAGRAM SHOWINGPERFuSION APPARATUSAND ITS RELATIONSHIP TOPERFUSEDMUSCLE

547

548 T. C. PRENTICE, R. R. STAHL, N. A. DIAL, AND F. V. PONTERIO

TABLE I

Summary of the data from the experiments where Naruwas injected intramuscularly-Each horisontal row of figuresin a given experiment refers to the data obtained during one clearance period in that experimen

deviatianIMber Mmbi- offIowt

of one- nop von- from momClearance nminte Mean bi- One outp- Nomn per- flov for F2am bin.- Toqnrtor

wmsber rate of Kef- Comete cope von- flow In fuaeio intire caontint globin corn- ring ofof r &22 in tim detormin- one out- ./mi.AO0 pressure oleara n of ptrfueat in tint of pe- IVe room

experi- %per in Ing clear- flow in a. Of In M. of period % fuete In In degreemt mte minutes ance rete al./minute _mel morou4 In - maturatln s. ehrebeit .I_ Arkg

1 94.5 42.2 31.2 7 .7 4.3 100 5.7 Oqgeating device

3.8 17.9 8 1.1 6.7 135 7.0

3.7 18.4 8 2.1 12.8 206 3.0 8498993.5 4

4.7 U.4 8 2.0 12.2 215 4.0

3.1 22.0 8 1.1 6.7 168 2.7

2 Dfore0 9142 102.9 23.6 5 .7 4.0 118 9.0 0ft 2 94.0 2 Denerwatod.3.7 18.4 6 1.0 5.7 176 5.2 78-4

4.6 .14.7 7 1.3 7. 4 234 0 92.2 27

3 95.8 U12.6 26.3 8 .7 2.7 79 1.5 Oxygenating devio,

not need.3.3 20.6 9 1.2 4.7 124 5.5

3.2 21.3 8 1.5 5.9 161 8.8 768294.8 29

3.9 17.4 8 1.8 7.0 186 7.1

2.7 25.3 8 .8 3.1 100 5.2

3.7 18.4 7 1.8 7.0 177 3.4.93.0 96

87.0 31.9 36.1 9 .8 4.0 86 5.0 0 ting devic

4.1 16.6 8 1.3 6.5 133 2.3 71-81

3.6 18.9 8 1.8 9.0 190 5.7

.4.6 14.7 8 2.2 11.0 246 3.687.3 690

S Afe 02 90-6 122.8 24.4 8 .8 3.2 82 3.8 2

4.4 15.4 8 1.3 5.2 117 4.0

3.9 17.4 8 1.7 6.8 139 2.3 74804.0 17.0 8 2.1 8.4 144 1.4 90.1 4G01.9 36.1 6 1.1 4.4 139 0

Before 02 96.4 56 After Oj2 96.9 U4

2.7 25.3 8 .8 . 4.41 94 3.8

4.2 16.2 8 1.3 7.2 120 0

5.0 13.5 8 1.8 10.0 155 1.6 75-8094.8 60

5.3 12.7 9 2.1 11.7 14 4.2

5.3 12.7 9 2.2 12.2 208 3.139,.3 53

Dofore 02 89.9 27 ~~~~~~~~~~~~~~~~AfterO93.0 6

2.7 25.3 8 .7 2.6 93 5.14 2

4.3 15.8 8 1.2 4.4 U6 5.6 80-834.6 U.7 8 1.7 6.3 165 3.0

90.9 465.3 12.7 9 1.9 7.0 201 7.6

This veiue in the m_zini deviatio of individ1a two-minu vinon oolloctlaon volanee for tho mom of entwo-minute collectLon voluoc for the entire clrmne period, exreed in per oent.

549RELATIONSHIP BETWEENNA22 CLEARANCEAND BLOODFLOW

TABLE I-Continued

Meziumdeviation

Numbr Neow bi- Of flovof one- cepe van- f|rmman

Clearanoe Minute Mean bl- ona ot- em per flow for Plasm h_o- TempratureNuer .ra of alf.. oonts cope vSa- flov in fuai& entire Oqgea content globin oo- range of

of 2t In tims deteruin- cue out- ml./min.AOO premwe cleareano of perfuate In tent of per- the romoperi- % per in ing clear- flow in a. Of In M. period % fuate in in degrees

im int ute uinut no rate .l.MiUt wale scl in S saturatim M. % Fabrmebeit Iawrk8 3.3 20.6 8 .8 2.6 54 3.8 Before 02 94 5 2

4.9 13.8 8 1.3 4.2 85 9.3 72-77

6.5 10.3 8 1.7 5.5 116 466.4 10.5 8 2.0 6.5 137 8.3 1 96 9 5

9 2.1 32.7 8 .8 3.5 93 6.6 Before 02 89.1 9After 02 94.8 22

3.1 22.0 8 1.2 5.2 130 2.4 275794.6 U.7 8 1.6 7.0 186 1.8

95.3 1925.4 12.5 7 2.1 9.1 157 6.7

10 3.7 18.4 9 1.5 5.6 105 9.6 Before 02 81.0 4

3.7 18.4 7 1.4 5.2 117 4.8 2 88.889.9 660

4.6 U.7 9 1.8 6.7 93 5.5 74-793.5 19.5 9 1.6 5.9 146 4.2

4.1 16.6 7 1.8 6.7 89 7.9

3.6 18.9 7 1.6 5.9 71 1.885.2 222

IS4RDC AID AonIC 0sCLEz

IA 2.6 26.3 8 .8 2.6 56 5.0 Before 02 84.4 6After 02 94.8 45

3.7 18.4 8 1.4 4.5 59 2.1 leohanic for 7573-79. minutes priortQ.4.5 15.1 8 1.8 5.8 98 4.3 90.4 415 injection of Wa.

6.2 10.8 8 2.2 7.1 93 3.1

2A 1.5 45.9 10 .8 2.9 59 6.9 efors02 694 11

3.8 17.9 8 1.3 4.6 75 3.1 9 73-8 lchampi for 75minute. prior to

5.2 13.0 9 1.6 5.7 11646.2 i9741njection of X22.

3A .1.4 49.2 8 .8 3.3 54 4.9 Before 02 64.2 4

2.5 27.4 7 1.2 5.0 84 2.0 2 76-80 Ischamio for 80minutes prior to3.5 19.5 8 1.8 7.5 165 4492.1 158jetion of 1a22.

* h vaue is the mimem deviation of individual tw-minte vanon collection volumes for the me of all

two-minute collection voilmem for the entire clearance period, expressed in per cont.

this preparation the "recirculation" of radioactive sodiumthrough the muscle was completely eliminated.

It was frequently noted during the later portions of anexperiment that the clearance rate for any given bloodflow would progressively decrease. When the same rateof blood flow was maintained for two or three clearanceperiods in the late phase of the experiments, progressive"flattening" 1 of the slope of disappearance of Na' often

1 The term "flattening" refers to a progressive decreasein the rate of clearance of radioactive sodium as a func-tion of a given rate of flow.

was observed. When this "flattening" phenomenon wasobserved, the results from these latter clearance periodswere not included in the data in Table I.

No reinjections of radioactive sodium were performedin any of these experiments. It had previously beenlearned that a high rate of flow for a long time intervalwas usually required to completely remove all of theradioactive sodium from the muscle and thus obtain acount which was even near the original backgroundcount.

Collateral blood flow, which could have inadvertently


resulted from failure to ligate small arterial branches andvenous tributaries, was checked by placing a hemostat on

the perfusion tube which was connected to the arterialcannula. Absence of venous outflow during this proce-

dure was accepted as evidence of an isolated circulationto the biceps muscle. Either small arterial branchesjoining the brachial artery distal to the cannula or smallvenous tributaries joining the brachial vein proximal tothe venous cannula would have resulted in a flow of bloodfrom the venous cannula. If evidence for collateral ves-

sels was found, and the vessels could not be obliterated,the experimental data were considered invalid and notreported.

At the termination of each experiment India Ink was

injected into the arterial cannula. The ink flowed rela-tively easily through the muscle vasculature and drainedfrom the venous cannula. No evident swelling of the

10

9

z

z

O (L

U.ezZ.

0~~~~.

iL~~~~~~~~

*-

muscle was produced by perfusion of either the bloodor the colloidal suspension of India Ink. The bicepsmuscle was then removed and sectioned with a scalpel.In each experiment the muscle was found to be diffuselyand homogeneously darkened. The muscle was thenweighed. Section of neighboring muscles in the forelegwith a scalpel revealed no evidence of darkening with thecolloidal suspension.

Experiment Nos. IA, 2A, and 3A were performed inthe usual way except that the muscle was purposelymade anoxic and ischemic. After cannulation of the ves-

sels, and prior to the onset of perfusion and injection ofthe radioactive sodium intramuscularly, the muscle was

allowed to remain without any blood supply for a pe-

riod of 75 to 80 minutes. Then the perfusion and in-jection of the Na' were carried out in the usual way.

Experiment Nos. 1B, 2B, and 3B were performed to

y 2.5+0.22xSy= 0.9r = 0.575

Innervated Biceps MuscleoDenervated Biceps MuscleAhnevated Biceps Muscle After

75-80min.Without Blood Flow.These Points Are Not Included InThe Statistical Analysis.

Line of RegressionLimits of Standard Error of Estimate

A

0

A

A

MEANVENOUSOUTFLOWFROMTHE BICEPS MUSCLEIN ml. PER MINUTE PER lOOgm. OF MUSCLE

FIG. 3. LINEAR RELATIONSHIP BETWEENCLEARANCEOF RADIOACTIVE SODIUMFROM

INJECTED SITE IN MUSCLEAND BLooD FLOWTHROUGHTHE MUSCLEThe wide variation in clearance rate for any given flow is evident.

550


test the closeness of correlation between flow rate andclearance of Na from muscle following its injection intothe arterial supply to that muscle. They differed fromthe other experiments in the following respects. Insteadof injecting the radioactive sodium intramuscularly, Na'(25 microcuries in 0.5 ml. in Experiment Nos. 1B and2B and 20 microcuries in 0.4 ml. in Experiment No. 3B)was injected into the perfusion tube just proximal to theintra-arterial cannula. In Experiment Nos. 1B and 2Bthe clearance periods were much longer than in previousexperiments. In Experiment No. 3B the clearance pe-riods were of the usual length. At the termination ofeach of these experiments the biceps muscle was injectedwith India ink and then removed without changing theoriginal position of the scintillation tube. In each in-stance the counts after removal of the muscle were veryclose to background and it was concluded that there was

very little contamination of the tubing and cannulae distalto the injection site.

The oxygen saturation of the blood used for perfusionwas determined by the method of Gordy and Drabkin (6).

The plasma hemoglobin concentration of the blood usedfor perfusion was determined by the method of Creditor(7).

RESULTS

Relationship between blood flow and clearancerate of radioactive sodium from an intramus-cular injection site in muscle wuith an adequatesupply of oxygenated blood

The over-all results of the correlation betweenthe clearance rate of radioactive sodium from an

injected site in the biceps muscle and the bloodflow through the muscle can be seen in Table Iand Figure 3. A linear relationship as determinedstatistically was noted between clearance and flow.However, the large standard error of estimateabout the line of regression prevented accurateestimation of flow from a given clearance value.

The linearity of the line of regression was

tested by the analysis of variance about the line us-

ing the method of Fisher (8). The deviationsfrom linear regression were not significant-theywere fully accounted for by the variations withinthe arrays; so linearity was indicated. The equa-

tion found for the line of regression was y = 2.5 +.22 x. The standard error of estimate about thisline of regression was 0.9. Thus 99 per cent ofthe clearance values for a given flow would fallbetween plus or minus 2.7 per cent (2.7 per centreferring to units of the ordinate rather than percent variation about the line). The coefficient ofcorrelation was 0.57. This can be considered a

Ioopx9000c8000(7000C6000(

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100009000

80007000

6000

5000

4000

30001

2000

L F -2mIi minJoogm T=25.2K -.027

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4 F-70ml./minJ1009m T=z132K-.053

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0 z 4 6 8

TIME IN MINUTES10 12 14

FIG. 4. PRoGREssivE INCREASE IN EXPONENTIALSLOPES OF DISAPPEARANCEOF NA! FROM INJEcTED SrIEIN MUSCLEAS A FUNCTION OF INCRaASING PERFUSIONRATES

Slope 5 illustrates the "flattening" effect seen in laterphases of some experiments.

significant correlation since the value of "z", de-rived from the coefficient of correlation, exceededthree times the standard error of "z".

Figure 4 demonstrates typical slopes of disap-pearance of radioactive sodium from an injectedsite in the biceps muscle with increasing rates ofperfusion during successive clearance periods.(Taken from Experiment No. 7.) It can beseen that clearance rate and blood flow rise con-

cordantly during the first four clearance periods.However, progressive decrease in clearance rate,as a function of flow, was observed during thelast two clearance periods even though the flowwas maintained relatively constant. Data fromthe fifth and sixth clearance periods of this ex-

periment were not included in the analysis of theresults. Similar terminal clearance periods fromthose other experiments which demonstrated"flattening" were likewise not included.

To further clarify, in a quantitative way, the re-

lationship between clearance rate of radioactive

551


sodium from the muscle and perfusion rate the fol-lowing calculations were made. The degree of in-crease or decrease in the values of clearance rateand blood flow between successive clearance pe-riods in each experiment was calculated. Thesevalues were expressed as ratios of the clearanceand flow, respectively, of each successive clearanceperiod to its predecessor. The change in flow wasthen divided by the change in clearance for eachinterval between clearance periods, thus giving aquantitative estimate of the relationship betweenchange in flow and change in clearance. The re-sults of these computations are shown in Table II.In 50 per cent of the instances the degree of changein clearance and flow found between successiveclearance periods were within plus or minus tenper cent of each other.

Progressive increase in the ratio between thechange in flow and the change in clearance be-tween successive clearance periods was acceptedas evidence of "flattening." This trend was notevident in the data selected for analysis. In in-stances where it was noted, invariably in the later

clearance periods of an experiment, these datawere deleted.

The flow produced by the infusion pump waspulsatile in character having about 40 beats perminute. The pulse pressure varied between 8 and18 millimeters of mercury, independent of the totalpressure within the system.

There was no obvious relationship noted be-tween the ambient temperature and the results ofany of the experiments.

In view of the previous reports that elevatedplasma hemoglobin produces vasodilatation, thehemoglobin concentration in the perfusating plasmawas measured. No relationship was found be-tween levels of plasma hemoglobin and the resultsof the experiments.

Relationship between blood flow and clearance rateof radioactive sodium from an intramuscularinjection site in muscle follozing a 75 to 80-minute period of ischemia and anoxia

The data from three experiments in which ra-dioactive sodium was injected into an ischemic

TABLE II

Table of ratios indiating the quantitative changes in dcearance rates (C) and fiw rates (F) during successive dearanceperiods, and the rettioxnship of change in flow rate to change in clearance rate

Clearanceperiods

determiningratio

RatioCr/Cr

1 2/1 1.733/2 .974/3 1.275/4 .66

2 2/1 1.273/2 1.24

3 2/1 1.273/2 .974/3 1.225/4 .696/5 1.37

4 2/1 2.163/2 .884/3 1.28

5 2/1 1.573/2 .894/3 1.025/4 .47

6 2/1 1.553/2 1.194/3 1.065/4 1.00

RatioFt/Fi

1.561.91

.95

.55

1.421.30

1.741.251.19

.442.26

1.621.381.22

1.621.311.23

.52

1.641.381.171.04

Ft CSFr * Ci

.901.96

.75

.83

1.121.05

1.371.28

.97

.641.65

.751.56

.95

1.031.471.201.11

1.061.161.101.04

Numberof

experi-ment

Clearanceperiods

determiningratio

7 2/13/24/3

8 2/13/24/3

9 2/13/24/3

10 2/13/24/35/46/5

RatioCG/Ci1.601.071.15

1.481.33

.98

1.481.481.17

1.001.24

.761.17

.88

Ischemic and AnoxicIA 2/1 1.42

3/2 1.224/3 1.38

2A 2/1 2.533/2 1.37

3A 2/1 1.783/2 1.40

Ratio Ft Cs

F2/Fl Fi Ci

1.691.431.11

1.611.311.18

1.481.351.30

.931.29

.881.13

.88

muscle1.731.281.22

1.591.24

1.511.50

1.061.34

.96

1.09.98

1.2

1.0.91

1.11

.931.041.16

.961.00

1.221.05

.88

.63

.91

.851.07

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and anoxic muscle are recorded in the latter partof Table I. In each of these experiments the mus-cle was completely without blood flow for a pe-riod of 75 to 80 minutes. The relationship be-tween clearance rate of radioactive sodium andrate of blood flow in this preparation, followingthis period of ischemia, was very similar to thatfound in the first ten experiments in which themuscle had an adequate supply of oxygenatedblood (Figure 3). During the first clearance pe-riod of Experiment Nos. 2A and 3A, during whichthe rate of blood flow was low, the clearance rateof radioactive sodium was less than that seen inany oxygenated muscle for a similar range ofblood flow. However, the subsequent clearancevalues for any given blood flow for these experi-ments, as well as all the clearance values in Ex-periment No. 1A, fell within the scatter of datafor the first ten experiments. The data from thesethree experiments are not included in the statisticalanalysis of the data for the first ten experiments.

Relationship between blood flow and clearancerate of radioactive sodium from muscle follow-ing intra-arterial injection of Na22 into the nu-trient artery supplying the muscle

Three experiments were performed in whichthe clearance rate of radioactive sodium from thebiceps muscle was determined following the in-jection of Na22 into the perfusion tube immediatelyproximal to the arterial cannula (Table III).In two of these experiments (1B and 2B) an at-tempt was made to maintain the perfusion rate ata constant level until the clearance rate of Na22from the muscle had reached a constant value.In Experiment No. 2B it was necessary after thefirst thirty minutes, during which time the flowwas maintained constant at a level of 4.0 ml. perminute per 100 Gm. of muscle (maximum devia-tion 4.4 per cent) to refill the syringe on the pump.After this was accomplished the flow was main-tained at a slightly lower level, 3.4 ml. per minuteper 100 Gm. of muscle (maximum deviation 9.8per cent). The values recorded for the firstclearance period in the data in Table III are theaverage of these two values. The maximum de-viation from mean two-minute flow was calcu-lated using the mean of all two-minute collectionvolumes throughout the entire 53.5 minutes. In

both experiments the disappearance rate of radio-active sodium from the muscle could be sepa-rated into two exponential components with widelyvariant rate constants. The results are illustratedin Figures 5 and 6. In both of these experiments,when a constant rate of clearance appeared tohave been reached, the perfusion rate was raised.In Experiment No. 1B the perfusion rate wasraised from 5.9 to 9.5 ml. per min. per 100 Gm.and in Experiment No. 2B from 3.7 to 5.7 ml. permin. per 100 Gm. In neither instance was thereany resultant increase in the clearance rate subse-quent to the increase in perfusion rate. In 2B theclearance rate during this period of increased flowactually decreased slightly. The values for thelast clearance periods in lB and 2B were recordedunder "Component No. 2" in Table III since theydiffered little from the slower component.

In Experiment No. 3B perfusion was started ata low rate, 3.7 ml. per min. per 100 Gm., and wasincreased stepwise during four subsequent clear-ance periods to a maximum of 10 ml. per min.per 100 Gm. Table III shows the results. Therewas a rise in the clearance rate during the secondclearance period as compared to the first, con-comitant with the rise in perfusion rate. How-ever, during successive clearance periods there-after the clearance rates decreased progressivelyin spite of increasing perfusion rates. During thelast two clearance periods a relatively constantperfusion rate of 10 ml. per min. per 100 Gm. wasmaintained and the clearance rates continued todecrease, going from 3.9 per cent in the fourthclearance period to 2.7 per cent in the last.

DISCUSSION

The results of the intramuscular injection ex-periments, namely, the wide range of blood flowrepresented by any given clearance rate of radio-active sodium, tend to minimize the value of thismethod as an index of total muscle blood flow.

There are several factors which may be re-sponsible in greater or lesser degree for this lackof correlation between rate of clearance of radio-active sodium from an intramuscular injection siteand total blood flow through the muscle. Thesecan be divided conveniently into physiological fac-tors and anatomical factors.

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Physiological factors

The presence of effective shunts at the arteriolarlevel which by-pass a fraction of the arterial bloodaround the capillary circulation could be impor-tant in producing the observed lack of correlationbetween total blood flow and radioactive sodiumclearance. Since, presumably, radioactive sodiumis cleared from the injected site only by bloodflow at the capillary level, the radioactive sodiumclearance would correlate only with that fractionof total blood flow not shunted. What quantita-tive significance shunts actually possess is notknown at the present time and therefore theirinfluence on the present data cannot be evaluated.

Pappenheimer, Renkin, and Borrero (9) haveshown from their data on osmotic transients thatat low blood flows the diffusion rate of sodium.chloride is flow limited. The question arises as

to whether, at higher levels of blood flow, theexchange of sodium between the vascular systemand extracellular space may be limited by perme-

ability factors and the diffusion rate of sodiumrather than by blood flow per se. Jones (10) pre-

sented evidence that the exchange rates in varioustissues of several inert gases, having an eightfoldrange of relative diffusion rates, were approxi-mately the same. These results led to the viewthat diffusion and transcapillaric permeability were

not factors limiting the inert-gas-exchange rateand that in fact blood flow determined the ex-

change rate. The linearity of our clearance/flowrelationship and the absence of any consistent re-

duction in increment of clearance rates at higherblood flows suggest that radioactive sodium ex-

change in these experiments was not limited athigher levels of blood flow by diffusion or perme-

ability factors.Although sodium is freely diffusible it is cer-

tainly not inert and consequently is subject to suchinfluences as electric potential gradient and chem-ical combination. What effect these influences hadon our results cannot be assessed.

It is now known that muscle fibers are notimpermeable to sodium and hence that an energy-

using transport system must be operative to driveoutward the sodium that enters by diffusion andthus to keep it in its primarily extracellular loca-tion (11, 12). That this "sodium pump" may

have been injured via anoxia and other factors

during the- dissection was a possibility. How suchinjury would effect our clearance values was notknown. However, it was suspected that by allow-ing a portion of radioactive sodium to diffuse intothe muscle fibers the amount available for clearancemight have been decreased. The three anoxicexperiments were carried out to measure theeffects of complete absence of blood supply over a75 to 80-minute interval on the clearance of radio-active sodium. The data from these experimentsdid not indicate any obvious effect of anoxia onthe clearance rate of radioactive sodium as a func-tion of flow. In light of these findings, as well asthe fact that we were perfusing with relativelywell-oxygenated blood, it would appear that an-oxia was not a significant contributing factor tothe wide scatter of data.

Anatomical factors

The anatomical factors which could possibly af-fect the correlation between clearance rate andflow have been discussed by others. They in-volve principally the vascularity of the site selectedfor injection, the degree of tissue injury producedby the injection, and the amount of distortion andcompression of local tissues, produced by the in-jected- fluid. It has been shown recently (13)that the clearance rate is dependent upon the vol-ume of fluid injected into the muscle; larger vol-umes result in lower clearances. The presentstudies shed no further light on this general as-pect of the problem but it seems likely that theselocal anatomical factors may be of importance inproducing some of the discrepancies in correlation.

The clearance rate following intra-arterial in-jection of radioactive sodium was measured inthree experiments to observe its correlation withblood flow. By injecting radioactive sodium ioninto an artery, a large portion of the injected ma-terial can be localized in the region supplied by thevessel due to the rapid exchange which occurs withthe local sodium pool. The subsequent rate ofremoval (clearance) of this residual material canthen be measured. Dobson, Warner, Pace, Fin-ney, and Johnston (14) reported the originalstudies using this method and found that "therate of removal is not constant and is much morerapid than it is in the case of local injections.The variation of the removal rate with time can

556


be explained on the basis of differing degrees ofvascularity in the tissue."

If the diffusion rate of sodium is very rapid oversuch tissue and cell barriers as may exist, com-pared to the rate of movement of blood throughthe capillaries, then the clearance rates followingintra-arterial injection should reflect vascularity oftissue. The quantitative aspects of blood flow asdetermined by clearance rates of intra-arterially in-jected Na22 could be handled analogously to themethods of Jones (10) using inert gases except fortwo correction factors to allow for 1) the differingconcentration of sodium between blood and tissueand 2) the fact that sodium is limited to the so-dium space. The above interpretation would leadone to believe that in Experiments 1B and 2Bthere were two differently vascularized areas oftissue within the muscle; the one with a hightime constant having a relatively high rate of flowand the one with a low time constant having arelatively low rate of flow for a given total bloodflow to the muscle. One would have expected,however, that in Experiments 1B and 2B an in-crease in the remaining slow component shouldhave occurred subsequent to an increase in totalblood flow. That this did not occur would tendto invalidate the conclusion that the clearancerates of sodium from muscle following intra-ar-terial injection were dependent only upon bloodflow to the part.

In Experiment 3B, except for the second clear-ance period, the clearance rates decreased progres-sively in spite of increasing blood flow. Interpre-tation here is more difficult. These results couldoccur 1) because clearance rate was dependentupon other factors as well as blood flow, and 2)that during clearance periods 1 and 2 most of theradioactive sodium was removed from the morehighly vascularized area and subsequent decreasesin clearance were accounted for by increasing pre-dominance of clearance from the less vascularizedarea.

SUMMARYAND CONCLUSIONS

Ten experiments were performed to establishthe correlation between the clearance rate of radio-active sodium from an intramuscular injection siteand total blood flow through the biceps muscle ofthe dog. A linear relationship was found toexist between these two variables. However, the

scatter of data around the line of regression wasso great that the clearance rate of radioactive so-dium could not be utilized as an accurate measureof total blood flow.

Three similar experiments were performed af-ter a 75 to 80-minute interval during which timethe biceps muscle was completely deprived ofblood supply. Comparison of results from thesethree experiments with those of the previous tenrevealed no obvious difference in the relationshipbetween clearance and flow. These results,coupled with the fact that the muscle was perfusedwith relatively well oxygenated blood, were con-sidered as evidence that anoxia was not a signifi-cant contributing factor to the wide scatter ofthe data in the first ten experiments.

Three experiments were performed in an at-tempt to establish the correlation between theclearance rate of radioactive sodium from muscle,subsequent to its intra-arterial injection, and bloodflow through the muscle. The data from theseexperiments did not support the concept that theclearance rate of radioactive sodium under thesecircumstances was solely dependent upon bloodflow.

ACKNOWLEDGMENTS

We are grateful to many for their help and advice.Wewish to express our sincere appreciation and thanksto Dr. Robert W. Clarke, Chief of Department of Surgi-cal Physiology, Army Medical Service Graduate School,Walter Reed Army Medical Center, for his guidanceand interest in our project; to Dr. Lewis Sokoloff, Na-tional Institutes of Health, for his suggestions regard-ing the experimental preparation; to Dr. Audie Lubin,Department of Psychology, Walter Reed Army MedicalCenter, for his help with the statistical analysis of thedata; and to Mrs. Ann Marsh and Sgt. Willard RGauthier for their technical assistance.

REFERENCES

1. Kety, S. S., Measurement of regional circulation bythe local clearance of radioactive sodium. Am.Heart J., 1949, 38, 321.

2. Wisham, L H., Yalow, R. S., and Freund, A. J., Con-sistency of clearance of radioactive sodium fromhuman muscle. Am. Heart J., 1951, 41, 810.

3. McGirr, E. M., The rate of removal of radioactivesodium following its injection into muscle and skin.Clin. Sc., 1952, 11, 91.

4. Miller, H., and Wilson, G. M., The measurement ofblood flow by the local clearance of radioactivesodium. Brit Heart J., 1951, 13, 227.

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5. Semple, R., McDonald, L., and Ekins, R. P., Radio-active sodium (Nak) in the measurement of localblood flow. Am. Heart J., 1951, 41, 803.

6. Gordy, E., and Drabkin, D. L., A simplified spectro-photometric technique applicable to standard equip-ment for the determination of 02 saturation ofblood. Am. J. M. Sc., 1951, 221, 231.

7. Creditor, M. C., The quantitative determination ofplasma hemoglobin by the benzidine reaction. J.

Lab. & Clin. Med., 1953, 41, 307.8. Fisher, R. A., Statistical Methods for Research Work-

ers, 11th ed., Edinburgh, Oliver and Boyd, 1950.9. Pappenheimer, J. R., Renkin, E. M., and Borrero,

L. M., Filtration, diffusion and molecular sievingthrough peripheral capillary membranes: A con-

tribution to the pore theory of capillary perme-ability. Am. J. Physiol., 1951, 167, 13.

10. Jones, H. B., Respiratory system: Nitrogen elimina-tion in Medical Physics, Vol. 2, Otto Glasser,Ed., Chicago, Yearbook Publishers, Inc., 1950, p.

855.11. Ussing, H. H., Transport of ions across cellular

membranes. Physiol. Rev., 1949, 29, 127.12. Steinbach, H. B., Sodium extrusion from isolated

frog muscle. Am. J. Physiol., 1951, 167, 284.13. Warner, G. F., Dobson, E. L., Pace, N., Johnston,

M. E., and Finney, C. R., Studies of human periph-eral blood flow: The effect of injection volume on

the intramuscular radiosodium clearance rate. Cir-culation, 1953, 8, 732.

14. Dobson, E. L., Warner, G. F., Pace, N., Finney, C.R., and Johnston, M. E., Clearance rates follow-ing intra-arterial injection of radioactive sodiumas a measure of regional blood flow. FederationProc., 1953, 12, 33.

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