vasodilator effects of substance present in with the...

Vasodilator Effects of a Substance Present in

NormalHuman Urine in Comparison with the

Effect ofMethacholine and Sodium Nitrite

A Statistical Analysis of Reliability of theMethod of Assay

By HAROLD D. GREEN, M.D., J. MAXWELL LITTLE, Ph.D., CLIFFORD G. GADDY, M.D.,LEWIS T. FRANKLIN, M.D., AND HOWARD H. WAYNE, B.A.

This paper presents a method for bioassay of vasotropic substances. Data on the accuracy of themethod are presented in terms of the response to repeated injections of methacholine. Some of thefactors affecting the response to methacholine are analyzed. The peripheral vasodilator effect of a

nondialyzable substance present in human urine is demonstrated and the effectiveness of thissubstance is related to that of methacholine.

A PREVIOUS paper' from this laboratorydescribed the temporary decline in ar-terial pressure produced by the intra-

venous injection of a substance present in nor-mal urine. It was suggested that this substanceprobably acts by causing peripheral vasodila-tion.4 * In this paper we describe a method forassaying vasotropic substances by injectingthem intra-arterially while recording the ve-nous outflow from the isolated hind limb of thedog. This paper demonstrates by means of thisassay technic that the vasodepressor substancein urine has a direct vasodilator effect. Thesuitability of this method of assay of vaso-tropic substances is discussed and certain of thefactors which lead to variability in the respon-ses to intra-arterial injections are analyzed.This method of assay has the advantage that

From the Department of Physiology and Pharma-cology, Bowman Gray School of Medicine of WakeForest College, Winston-Salem, N. C.

Supported by grants from the Life InsuranceMedical Research Fund, the Roche Anniversary fundand Grant H-487 from the National Heart Institute,U. S. Public Health Service.

Preliminary reports of this work were presentedat the 1948 and 1949 meetings of the American Physi-ological Society.' 2

During the study, L. T. F. was a fellow of the LifeInsurance Medical Research Fund.

* For additional literature on the urinary de-pressor substance the reader may refer to references3 and 14 to 16.

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repeated injections may be made in the sameanimal and only 0.2 to 0.8 ml. of urine is re-quired whereas 2 to 10 ml. are required whenthe assay is made by intravenous injections.3

METHODSa. Operative Preparation and Flowmeter Equip-

ment. The general arrangement of the animal andequipment are shown in figure 1. Twenty-five dogsweighing 7 to 22 Kg. (average 13.6) were anes-thetized with 30 mg. per kilogram of sodium pento-barbital.* The femoral artery and vein of one hindleg, usually the left, were dissected free. The veinwas prepared for peripheral cannulation while asmall superficial arterial branch was prepared forcentral cannulation, the former to be used for venousoutflow and the latter for the intra-arterial injec-tions. Dissection was carried through from the fem-oral triangle to the sciatic sheath. Tourniquets werepassed through and around the medial and lateralmuscle masses and tightened so as to occlude allcollateral venous and arterial channels,5' 6 whileexcluding the femoral artery and vein and the fem-moral and sciatic nerves. Cessation of venous out-flow following occlusion of the femoral artery wasused as evidence of effective occlusion of collateralcirculation. In the opposite leg, the femoral arterywas cannulated for registration of mean arterialpressure and the femoral vein for return of the bloodobtained from the perfused leg. In many experiments,a pressure regulator7 was connected to a cannulainserted into a carotid artery to assist in stabilizingthe arterial pressure. Coagulation of the blood wasprevented by heparin; the initial dose of 5 mg. per

* Kindly furnished by Premo Pharmaceutical Lab-oratories, New York, N. Y.

Circulation, Volume V, April 1959

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VASODILATOR EFFECTS OF A SUBSTANCE IN URINE

kilogram was followed by 1.6 mg. per kilogramevery half hour thereafter.

The rate of outflow was measured by the piston-type of volume recorder shown in figure 2. Thismeter operates on the Gaddum5 principle of a con-stant cycle length in which the height.of each strokeindicates the rate of flow. The cycle length of 10seconds used approximately two and a half secondsfor filling and seven and a half seconds for draining.This timing allowed completion of both the fillingand emptying phases during each 10 second cycle.The meter contained an L stopcock rotated by a

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FIG. 1. Connections for measurement of flow inthe isolated hind limb of the dog. A, aorta; VC,vena cava; FA, femoral artery; FV, femoral vein;S, tuberculin syringe for injection of drugs; T,tourniquet; VM, venous pressure manometer; FM,venous outflow meter; IG, blood reinfusion system;and HgM, mercury manometer for recording perfu-sion pressure. Shaded portion represents path ofblood during flow measurement. When flow measure-ments are not being made, venous return can bedirected from left into right femoral vein by rotatingstopcocks.

solenoid which in turn was actuated from a relayconnected with the laboratory timing circuit. Whenrotated to the A position the L stopcock connectedthe reservoir which collected blood from the fem-oral vein with the measuring tube in which was apiston recorder. When rotated to the B position thestopcock connected the measuring tube with a con-stant level outflow device which caused the pistonrecorder to return to the same point at the end ofeach stroke. With this device, there was never anyoutflow blood which was not measured. The recorderused had a range of 0 to 20 ml. per 10 seconds. Thiswas quite adequate since, under control conditions,

most of the hind legs studied had control flows of2 to 6 ml. per cycle. Thus the meter had amplecapacity to handle the increased flow seen duringthe experimental period. The over-all apparatuswas calibrated by running fluid through it at variousconstant rates and measuring the amplitude ofdeflection. The calibration was linear. The apparatusis described more fully elsewhere.9' PP. 71-73; 10The blood leaving the flowmeter was collected

and reinfused into the dog every four minutes; thevolume reinfused each time was approximately 100ml. Blood for the initial filling of the pressure regula-tor and flowmeter and for providing a reserve ofblood was obtained from a separate dog.

b. Control Experiments. In six control experimentsmethacholine, dissolved in pyrogen-free physiologicsaline (0.2 pg. per ml.), was given in amounts of0.2 to 0.8 ml. over a period of 20 seconds; controlinjections of the saline were given in like volume.In these control experiments flows and responses to

INK

FIG. 2. Diagram of the flowmeter used in recordingthe venous outflow. See text for description.

the drug injections were recorded for one to two andthree-quarters hours in the intact preparation; fol-lowing this the vagi were severed and the carotidarteries ligated and flow responses recorded for twoto six additional hours; the femoral and sciatic nerveswere then cut as high as possible, and the flow re-sponses were followed for an additional three andone-half hours. In experiment 2 the dog died at thetime of nerve section; in experiment 3 the dog sur-vived one-half hour and in experiment 4, one andone-quarter hours. In experiments 3 and 4 only oneinjection of methacholine was made after the nervesection. In experiments 5 and 6, flow responses wererecorded for two and three and one-half hours inthe intact preparation; after this the femoral andsciatic nerves were sectioned as high as possible andflow responses followed for three and three and one-half hours respectively. The total hours of perfusionwere: experiment 1, nine and one-half hours; ex-periment 2, four and one-half hours; experiment 3,six and three-quarters hours; experiment 4, fiveand three-quarters hours; experiment 5, seven hours;.and experiment 6, five hours.

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GREEN, LITTLE, GADDY, FRANKLIN AND WAYNE

'c. Method of Preparation of Urines. All urinespecimens were collected in autoclaved flasks frommale subjects after careful cleansing of the penisand after discarding the initial voided urine. Partof the urine, indicated as P, was stored unaltered;another portion, F, was filtered through a Seitzfilter; a third portion, D, was dialyzed in sterileviscose cellophane bags through frequent changesof distilled water at 4 to 9 C. for 24 hours; a fourthportion, FD, was both filtered and dialyzed. Allsamples were either used immediately, or werestored at -20 C. until used. Representative sampleswere found pyrogen-free using the rabbit as testanimal. All equipment used in the preparation andstorage of the urine was thoroughly washed andthen rinsed with pyrogen-free saline before auto-

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X 602 40

200

arterial injections of 0.4 ml. of urine and to twoinjections of 0.4 ml. of methacholine chloride(0.2 pg. per ml.) The flowmeter had a cyclelength of 10 seconds (6 cycles per minute).The calibration on the left gives the flow per

cycle. The calibration on the right gives theflow per minute represented by the amplitudeper stroke of the flowmeter (that is, the flowper cycle X 60/cycle length in seconds). Dur-ing the control period the flow was 2.4 ml.per cycle, that is, 14.4 ml. per minute. Within2 to 3 cycles after the intra-arterial injectionwas started, the venous outflow had begun to

50

40 d

30 220?10 a0

FIG. 3. Typical record of increases in flow produced by intra-arterial injection of a urine prepara-tion and of 0.08 ug. of methacholine. See text for description.

claving. All specimens of urine were examined forclarity and frequent checks were made of the urinein various stages of preparation by inoculating theurine on tryptose phosphate broth and thioglycolatemedia. Cultures were read after 48 hours. Sterile,pyrogen-free saline, handled in the same manneras the urines, served as controls. Urines prepared inthe above manner were studied for their vasomotoractivity by intra-arterial injection into dogs as

described above for methacholine. The potency ofthe urine was expressed in terms of the amount ofmethacholine required to give a similar vasodilatorresponse.

RESULTSA. Method for Measuring Flow Curve

Figure 3 shows a typical flow curve in whichis recorded the response to two alternate intra-

increase. It reached maximums of 5.5 to 6.4ml. per cycle (33 to 38 ml. per minute) in fivecycles after the start of the injections and re-

turned approximately to control values in seven

cycles with the urine and in 10 cycles with themethacholine.The responses to injections were analyzed

as follows:(1) AF (increment in flow) = MF (maxi-

mum flow) - CF (control flow).(2) Per cent A F (percentile increment in

A F X 100flow) =.

CF

Selection of the highest flow cycle during theresponse to the drug may lead to some vari-ability, since the cycle length of the flowmeter

TIME- 10 Sec./cycle

_~~~~~~~~~~I I

U M U M

_~~~~~~~~~~~~~-Dialysed Urine (60 D)_ ~~~~~~~~~~M-Methacholine 0.2,ugm>/m1.

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usually used was 10 seconds. However, thiseffect was minimized by making each of theinjections at approximately the same momentin the flowmeter cycle. The method of calcula-tion was checked also by integrating the flowthroughout the vasodilator effect. This methoddid not give any more constant response thanthe use of the single greatest cycle. Since use ofthe highest cycle is much quicker and appearedto be equally as accurate we have expressedall the results in this paper in this manner.It was found by experience that a responseequal to a 100 to 200 per cent increase in flow(per cent AF) was most convenient for use asthe change was of sufficient magnitude to bemeasured easily, yet not so great that the ef-fect lasted an excessive length of time.

B. Responses to Intra-arterial Injection of SalineControl injections of the saline used in pre-

paring the methacholine solution were givenin volumes equivalent to those of the urine andreference solutions in most of the experiments.In most cases 0.4 ml. of the solution causedeither no increase in flow, or a minimal, briefincrement amounting to a percentile incrementin flow (per cent AF) of 15 to -60. In one ex-periment saline caused percentile incrementsof flow of 90 to 147, comparable with those pro-duced by the urine; this experiment was dis-carded.

C. Reference Vasotropic SubstancesIt was assumed that the response to a given

amount of vasotropic substance would varyfrom animal to animal, but that the ratio ofthe response to substance A and the responseto substance B might be somewhat moire con-stant from animal to animal. If this were shownto be true then the potency of substance Bcould be expressed in terms of the amount ofsubstance B required to give the same vaso-dilator effect as a standard amount of refer-ence substance A. Two reference substanceswere examined, sodium nitrite and methacho-line. It was found that 0.2 to 0.4 ml. of a 10per cent solution of sodium nitrite gave per-centile increases in flow ranging from 34 to584 (average 138) in the nine experiments inwhich the substance was used; and, that 0.4

ml. of a solution containing 0.2 pg. per ml. ofmethacholine gave percentile increases in flowof 26 to 688 (average 162) in the 10 experimentsin which it was used. The flow curves for so-dium nitrite and urine were similar but sodiumnitrite, upon repeated injection, tended to causemethemoglobinemia. Methacholine induced re-sponses the duration and contour of which weresimilar to those caused by the vasodilator sub-stances in urine. (See fig. 3.) Furthermore, thetime interval between the drug administrationand the beginning of response, the magnitudeof response and the duration of response did notchange markedly as the experiment progressedand no tachyphylaxis was noted to repeatedinjections of the methacholine.

D. Control Experiments using Methacholine(1) Magnitude of Control Flows. As shown in

table 1, the control flows ranged from 13 to 39ml. per minute in the six intact preparations.The animal weights varied from 10 to 13 Kg.Since the perfused mass of the legs was notdetermined, no attempt was made to relate theflows to the mass of the extremity.

(2) Variability of Control Flows in the ControlExperiments. In the six intact preparations(table 1) control flows decreased steadily inthree dogs, falling 25 to 67 per cent; in one theflow rose 100 per cent; and in the remainingtwo control flows did not change significantly.The coefficient of variation for the controlflows in these experiments ranged from 9 to24 per cent (average 9 per cent). Mean arterialpressure in the various experiments ranged be-tween 80 and 116 (average 101 mm. Hg); thecoefficient of variation for mean arterial pres-sure in these six control experiments rangedbetween 1 and 11 per cent (average 6 per cent).Since, in five of the six experiments, mean ar-terial pressure fluctuated only i 5 mm. Hg,the changes in control flow could not be cor-related with the fluctuations in mean arterialpressure. Additional injections of anesthetic hadto be given at irregular intervals. These in-jections (2 ml. of 3 per cent sodium pento-barbital) increased the control flow 30 per centto 400 per cent and the increase in flow per-sisted for 5 to 10 minutes.

(3) Effects of Vagal Section, Carotid Artery

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Ligation and Femoral and Sciatic Nerve Sectionon Control Flow in the Control Experiments.After a series of injections either the vagusnerves were sectioned and the carotid arteriesligated to minimize pressoreceptor influences,or the femoral and sciatic nerves were cut toeliminate nerve impulses going to the perfusedleg. In one instance section of the vagus nervesand ligation of the carotid arteries caused thecontrol flow to decrease almost immediately.In the three other experiments leg flow de-creased after section of the vagus nerves andligation of the carotid arteries, but the resultswere less clear cut because the control flow wasdeclining at the time of the section. However,these procedures usually increased the vari-ability of the perfusion pressures, the controlflows, and maximum flows and the changes inflow. Following section of the femoral andsciatic nerves the control flows increased by150 to 430 per cent in three of the four experi-ments in which prior section of the vagus nervesand ligation of the carotid arteries had beenperformed. The remaining dog of this groupdied at the time of nerve section. Section of thefemoral nerves in the two intact animals caused32 per cent and 100 per cent increases respec-tively in control flow.

(4) Variation of Response to Repeated Injec-tions of Alethacholine in the Control Experi-ments. In order to determine the reliability ofthe assay method, we made repeated injec-tions of the same quantity of methacholine ineach of the six control experiments. The resultsare shown in table 1, part I. Column A identi-fies the experiment; B gives the animal weight,C the number of similar injections, D the per-fusion pressure (mean arterial pressure), E thecontrol flow (CF), F the maximum flow (MF),G the maximum increment in flow (AF), and

H the percentile increment inflow (CF X 100 =

per cent A F) in the intact preparations.In the six intact animals (table 1), the maxi-

mum flows tended to vary slightly less than thecontrol flows (coefficients of variation 4 to 16per cent), but the increment in flow (AF) variedmore than either the control flow or the maxi-mal flow, that is, the coefficients of variability

for AF were 4 to 41 per cent. When the resultswere expressed as per cent AF rather than asAF, the coefficients of variability were increasedin three of the six experiments on the intactdogs, and were essentially unchanged in three(range 4 to 48 per cent).

(5) Effect of Severing the Vagus Nerves, Liga-tion of the Carotid Arteries, and Section of theFemoral and Sciatic Nerves on the Variabilityof the Response to Methacholine. The possibilitywas considered that changes in control flowdue to variations in the activity of the reflexhomeostatic mechanisms might be playing apart in causing the variability in response tosuccessive injections. Therefore, as noted above,in several of the experiments (a) the vagi weresevered and the carotid arteries ligated (col-umn I, table 1); (b) the femoral and sciaticnerves were severed (experiments 5 and 6,column J, table 1), or (c) both procedures werecarried out successively (experiment 1, columnJ, table 1). As shown in table 1, these proce-dures decreased the variability in two experi-ments and increased the variability of thechanges in flow in two experiments.

(6) Relationship of Response to VasomotorTone in the Extremity in the Control Experi-ments. In attempting to determine some of thecauses of variability of response to the metha-choline, we analyzed the effects of changes ofvasomotor tone upon the responses. Compari-sons of pairs of injections, in the intact dogs,in which the perfusion pressure was constantbut the control flow had varied, fairly consist-ently indicated that the response was greaterthe lower the control flow, that is, the higherthe control vasomotor tone. During the in-crease of vasomotor tone that followed vagaland carotid section the responses tended to bedecreased, and during the decrease in vaso-motor tone that followed sciatic or femoralnerve section the responses tended to beincreased. However, the change was not con-sistent enough to be taken as indicating adefinite relationship. During the periods of in-creased flow which followed injections of addi-tional amounts of anesthetic, the incrementsin flow in response to methacholine usuallywere less than those recorded just prior to theinjection of anesthetic.

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572 VASODILATOR EFFECTS OF A SUBSTANCE IN URINE

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(7) Variability after Omitting Data in WhichControl Flows Varied Considerably from theMean for the Group. In view of the above notedeffects of variations in control vasomotor tone,we selected from the data in table 1 onlythose observations in which mean arterial pres-

sure and control flows were relatively constant,that is, in which vasomotor tone was relativelyunchanged, and again computed the variabil-ity. This selection resulted in approximatelya 30 per cent decrease in variability.

E. Vasodilator Effects of Urine and VariousUrinary Extracts(1) Typical Flow Response to Urine. Figure

3 reproduces two flow responses to the intra-arterial injection of 0.4 ml. of urine. As may beseen by comparison with the other two re-

sponses, the amplitude of the flow incrementand the duration of the effect in this experi-ment is about the same as that noted with0.08 gg. of methacholine.

(2) Variability of Response to Repeated Injec-tions of Urine into the Same Animal. The effectsof repeated injections of the same quantity(0.4 ml.) of the same dialyzed urine prepara-

tion (60D) were studied in three intact animals,(table 1, part II, columns A-H), and again inthe same animals after cutting the femoral andsciatic nerves (columns K and L). Two of theseanimals (5 and 6) were the same as those usedfor testing methacholine; in these the injec-tions of the urine preparation and methacholinewere given alternately. There was no system-atic variation in response which would suggesttachyphylaxis. The magnitude and the vari-ability of the responses to the urine prepara-tion were of the same order as those noted withmethacholine. Section of the sciatic and femoralnerves increased slightly the variability of theresponse to the urine preparation in all threeexperiments.

(3) Variation in Vasodilator Effect of theUrine in Different Experiments. Plain sterileurine was injected intra-arterially in doses of0.1 to 0.4 ml. in 16 intact animals. The per-centile increment in flow (per cent AF) in thevarious experiments in response to 0.4 ml. ofurine varied from 6 to 410 (average 180). Whendifferent urines were tested in the same animal

they varied considerably in their potency.Urines from some human subjects possessedno vasodilator effects. Such urines were elim-inated from the study. Injection of the sameurine preparation into each of several legs gaveresponses which varied quite widely; the varia-tion between leg preparations was, however,of the same order as that noted for metha-choline.

(4) Relationship of Vasodilator Effect of Urineto That of the Reference Substances. Because ofthe wide variability of the response of both thereference substances and urine from test ani-mal to test animal, we made repeated alternate

TABLE 2.-Comparison of responses to 0.4 ml. of adialyzed urine preparation (60D) and to 0.4 ml. of asolution of 0.2,Og. of methacholine per ml. All responsesare expressed as percentile increment in flow (per centzXF).

Intact Animals

Urine Preparation Methacholine

Exp. A B C D E F RatioNo. C/F

Number Numberof In- Range Av. of In- Range Av.

jections jections

5 16 103-410 167 8 110-287 182 0.926 3 152-246 190 3 184-198 192 0.997 15 96-357 211 15 147-366 233 0.918 5 26-63 48 6 26-65 48 1.09 3 146-182 160 3 109-122 115 1.39

Av. 1.06

In all experiments the injections of urine prepara-tion and methacholine were given alternately.

injections of the same preparation of urine, andof methacholine in each of five dogs' legs. Table2 shows the result of this comparison, using ineach case 0.4 ml. of a dialyzed urine prepara-tion (60D) and 0.4 ml. of the standard metha-choline solution (0.2 ,ug. per ml.). The responsesto both methacholine and urine varied con-siderably from time to time in each of the ex-periments. However the ratio of the response tomethacholine to the response to urine remainedrelatively constant so long as pairs of injec-tions, made near the same time in the experi-ment, were compared. Furthermore, as shownin table 2, despite the considerable fluctuationin the various responses from time to time in thesame experiment, and from experiment to ex-

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periment, the ratios of the average response tothe urine preparation, to the average response tothe methacholine solution were relatively con-stant from experiment to experiment.

(5) Relationship of Dose to Response. In sixexperiments the responses were sufficientlystable so that an approximation of the dose-response relationship was attempted. Plots ofthe data from these six experiments were madeon linear and on single and double logarithmpaper. In general over the range of 0.2, 0.4,0.8 and 1.6 ml. the closest approximations tostraight lines were obtained on double log-arithm paper. Figure 4 shows a plot for the datafor a typical experiment. The slopes were such

METHACHOLINE 0.281g/mi.

0

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DOSE IN ML

FIG. 4. Dose-response relationship for methacho-line, plotted on log-log paper, with response expressedas percentile increase in flow.

that the percentile increments in flow were

related to the dose according to the formulaRx = K.D, where x varied between 1 and 3.The average value for x was 1.8.

(6) Effect of Filtration and Dialysis on Vaso-dilator Activity of Urine. Urines, prepared as

described under Methods, were injected intotest preparations. The vasodilator responses ofthe treated preparations were related to thaton the original untreated urine. In seven experi-ments six different urines were tested. Theaverage of the results of 15 sets of injections ofeach of the four kinds of urine preparationswere as follows: filtered urine possessed 80 per

cent, dialyzed urine 80 per cent, and filteredand dialyzed urine 70 per cent, of the vaso-

dilator activity of the untreated urine. In allbut one of the 15 trials, the vasodilator effect

of the plain urine was as great as or greater thanthat of the treated urines. When pairs of re-sponses to filtered and dialyzed urine were com-pared the latter was on the average 23 per centless effective. However this average differencewas not statistically significant and in four ofthe 15 pairs of injections the dialyzed urinegave a greater response than the filtered urine.

DISCUSSIONUsing the Dumke and Schmidt bubble flow-

meter as a measure of control femoral arteryblood flow, Kemp, Paul and Hines"1 foundaverage flows of 40 ml. per minute and 74 ml.per minute for two dogs. Higher average flowsof 120 ml. per minute and 106 ml. per minutefor two different dogs were later described.The lower flows previously recorded by theseauthors were thought by them to be the resultof experimental error in that they had notwaited a sufficient time for stabilization. Siems,Kosman and Osborne,'2 using a modified Ledenbubble flowmeter, noted average control flowsof 42 ml. per minute. Richardson and co-work-ers,'3 using the electromagnetic flowmeter,found control flow values of 59 to 65 ml. perminute. In our series the average control flowsranged from 9 ml. per minute to 35 ml. perminute or approximately one-half of that foundby other investigators. Our lower flows mayhave been due: (1) to the use of smaller ani-mals (the dogs in our control experimentsweighed 10 to 13 Kg.); (2) to perfusion of asmaller fraction of the leg mass, or (3) to de-velopment of a shocklike state with vaso-constriction as a result of trauma incident tothe preparation of the leg for flow measurement.We have been unable to find any literature

primarily devoted to study of blood flow re-sponses to methacholine with which to compareour results. An injection of 0.4 ml. of metha-choline solution (0.08 pug.) produced a widerange of change of flow in the intact controlexperiments (+44 per cent to +287 per cent).The variation in response was considerablyless within a single animal, but still such as togive coefficients of variation of 4 to 48 per cent.The possible causes of the variability in flow

following drug injections are numerous. Theyinclude changing vasomotor tone in the per-

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fused leg due to spontaneous alterations in theactivity of the vasomotor center and to theoccasional injections of additional anesthetic,fluctuations in viscosity, alterations in meanarterial pressure, variations in the rate of druginjection, and development of refractoriness tothe drug. No obvious refractoriness to eitherthe methacholine or the urine preparations wasnoted. The rate and time of the drug injectionswas kept as constant as possible. Eliminationof the effects of variations in activity of thepressor receptor organs and of the direct in-fluence of the vasomotor center upon the leg bysection of the femoral and sciatic nerves did notsignificantly reduce the variability of responseto methacholine. The variability was consid-erably reduced by selection of experiments inwhich vasomotor tone was fairly constant. Thissuggests that part of the variability was due toirregular fluctuations in the concentration ofcirculating vasotropic substances.The range of variation of the responses to

methacholine was fairly large with the assaymethod described in this paper, but it was lessthan that obtained by recording the effectsof vasodilator drugs on mean arterial pressure.Furthermore, when the vasodilator effect ofurine was related to that of methacholine inpairs of injections given closely together in thesame experiment, the measure of relative po-tency of the urine was much more constant.Since the method also demonstrates the pre-cise peripheral vasodilator effects of the drug,we believe this procedure possesses advantagesover registration of mean arterial pressure inthe study of vasotropic substances.A previous report' has shown the presence of

a depressor substance in human urine whichmay be the same as that described by Woll-heim,'4' 15 but probably different from that de-scribed by Westerfeld and co-workers.'6 Thevasotropic principle causes an increase in car-diac output and a decrease in mean arterialpressure.4 This paper demonstrates that urineand urine extracts possess a direct peripheralvasodilator action. This vasodilator effect isprobably responsible, at least in part, for thedepressor effect of urine.The vasodilator effect is not due to the pres-

ence of pyrogenic substances since it is obtained

with preparations of urine which are demon-strated to have been sterile throughout theirprocess of handling and, furthermore, such prep-arations do not induce pyrogenic reactionswhen injected into rabbits. Pyrogen-free saline,handled in the same way as the urine, also doesnot develop significant vasodilator properties.A vasodilator effect equivalent to that in-

duced by 0.08 ,ug. of methacholine injectedintra-arterially is produced by 0.4 ml. of plainurine obtained from many but not all subjects.The dose of urine or methacholine usually hasto be increased roughly as the square (average1.8 power) of the response, when the responseis expressed as the percentile increase in maxi-mum flow per cycle of the flowmeter. The vaso-dilator substance is reduced in potency by only20 to 30 per cent by filtration and dialysis.This loss may be due to adsorption.

SUMMARYWe have studied the blood flow response to

methacholine injected intra-arterially in an iso-lated, blood-perfused leg. In a series of six con-trol experiments, graphic and statistical stud-ies were made of control flows, and of thechanges in flow produced in the intact and inthe denervated isolated leg by intra-arterialinjections of methacholine. The responses torepeated injections of the drug varied consid-erably (coefficients of variation were 18 to 48per cent). No tachyphylaxis to methacholinewas demonstrated.

Section of the vagus nerves and ligation ofthe carotid arteries consistently reduced thecontrol flows; section of the sciatic and femoralnerves and injections of the anesthetic regularlyincreased control flow, particularly if performedwhen vasomotor tone was high as a result of thepreceding procedures. Neither removal of theactive reflex homeostatic mechanisms (sectionof the vagi and ligation of the carotid arteries)nor section of the sciatic and femoral nervesupplying the perfused leg decreased the vari-ability of the flow responses to methacholine.Blimination of data in which control flow var-ied considerably from the mean of the groupresulted in a significant decrease in the vari-ability of the responses to methacholine.A vasodilator substance is present in normal

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human urine which cannot be attributed tobacterial contamination. The substance is onlypartially removed by Seitz filtration and dialy-sis through cellophane bags in distilled water.Its action appears to be peripheral, since itproduces its effect when given directly intra-arterially in the intact or denervated extremity.No tachyphylaxis is noted to repeated injec-tions. The concentration of this substancevaries considerably in different urine specimens.On the average the amount present in 0.4 ml.of urine is approximately equivalent in vaso-

dilator effect to that of 0.08 gg. of methacholine.Despite the variability of the responses to

methacholine and to urine, the ratio of theresponse to urine and the response to metha-choline in closely adjacent pairs of injectionsremained quite constant. This method appears

to offer certain advantages over registration ofthe effects of vasotropic substances o01 mean

arterial pressure in the bioassay of vasotropicsubstances, particularly when their potency isrelated to that of a standard vasotropic sub-stance.

ACKNOWLEDGMENTSThe authors are indebted to Dr. William McCall,

Mr. M. F. Parsons, Mr. Delmar Bland, AIr. WilliamKeck and 1\Ir. George Barrett for valuable assistancein these experiments.

REFERENCES1 GADDY, C. G., GREEN, H. D., AND LITTLE, J. MI.:

Peripheral vasodilator effect of a substancepresent in normal human urine. FederationProc. 7: 39, 1948.

2 FRANKLIN, L., WAYNE, H. H., LITTLE, J. MW., ANDGREEN, H. D.: Factors affecting constancy ofvasomotor response to repeated intra-arterialinjections of mecholyl and of epinephrine. Fed-eration Proc. 8: 50, 1949.

LITTLE, J. M., GREEN, H. D., AND BUMGARNER,J. I.: Heat lability of a depressor substancepresent in human urine; effects of section ofvagus nerves, ligation of carotid artery and ofautonomic blockade upon depressor response.Am. J. Physiol. 155: 345, 1948.

'GREEN, H. D., LITTLE, J. M., HESTER, J., ANDHILDERMAN, H.: Vasodilator substance presentin urine. Federation Proc. 6: 114, 1947.7 COSBY, R. S., AND RADZOW, K. H.: Dynamicsof collateral circulations. Am. J. Physiol. 140:726, 1944.

6 : Circulatory system: methods. In 0. Glasser,Ed.: Medical Physics, vol. II. Chicago, YearBook Publishers, 1950. P. 208.

7 LAMSON, P. D., AND DE TURK, W. E.: Studies onshock induced by hemorrhage. Method forthe accurate control of blood pressure. J. Phar-macol. & Exper. Therap. 83: 250, 1945.

8GADDUM, J. H.: An outflow recorder. J. Physiol.67: 16 P, 1929.

GREEN, H. D.: Circulation blood flow measure-ment. In V. R. Potter, Ed-in-Chief: Methodsin Medical Research, vol. I. Chicago, YearBook Publishers, 1948. P. 66.

10 , DENISON, A. B., Jr., FRANKLIN, L., ANDWAYNE, H.: A system composed of a pump,outflow meter, and oxygenator calpable of per-fusing an organ at either a constant head ofpressure or a constant rate of blood flow, re-gardless of change in flow resistance in theorgan. J. Lab. & Clin. AMed. 36: 485, 1950.

1 KEMP, C. R., PAUL, W. D., AND HINES, H. M\1.:Studies concerning effect of deep tissue heaton blood flow. Arch. Phys. Med. 29: 12, 1948.

12 SIEMS, L. L., KOSMAN, A. J., AND OSBORNE, S. L.:A comparative study of short wave and micro-wave diathermy on blood flow; the role of thesomatic and sympathetic nerves in the vascularresponse to deep tissue heating. Arch. Phys.Med. 29: 759, 1948.

13 RICHARDSON, A. W., IMIG, C. J., FEUCHT, B. L.,AND HINES, H. M.: The relationship betweendeep tissue temperature and blood flow (luringelectromagnetic irradiation. Arch. Phys. Med.31: 19, 1950.

14 WOLLHEIM, E.: Eine neue koipereigene blut-drucksenkende Substanz und ihre Bedeutungfuir die essentielle Hypertonie. Acta med. Scan-dinav. 91: 1, 1937.

15 -: Gefasserweiternde Stoffe des Harns und Renin.Acta physiol. Scandinav. 10: 126, 1945.

16 WESTERFELD, W. W., WEISIGER, J. R., FERRIS,B. G., JR., AND HASTINGS, A. B.: The productionof shock by callicrein. Am. J. Physiol. 142:519, 1944.

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FRANKLIN and HOWARD H. WAYNEHAROLD D. GREEN, J. MAXWELL LITTLE, CLIFFORD G. GADDY, LEWIS T.

the Method of Assaythe Effect of Methacholine and Sodium Nitrite: A Statistical Analysis of Reliability of

Vasodilator Effects of a Substance Present in Normal Human Urine in Comparison with

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