767

TABLE III—RELATIVE SUBCUTANEOUS POTENCIES OBTAINEDBY VARIOUS WORKERS

SUBCUTANEOUS ASSAYS

Three series of assays were made with aqueous solutions.In the first series stilbcestrol, hexcestrol, diencestrol, andpotassium hexoestrol sulphate were compared ; in thesecond and third series cestrone, hexcestrol, and dienoestrolwere compared with stilboestrol. The preparation of the animals and the preparation and

scoring of the smears were the same as described above.The aqueous solutions of stilboestrol, hexoestrol,

dienoestrol, and cestrone were prepared by dilutingwith distilled water stock solutions in absolute alcohol ;aqueous dilutions were prepared every day, and stocksolutions freshly made for each test. The stock solutionswere stored in a refrigerator during the period of thetest, and the aqueous dilutions were similarly storedbetween the times of dosing. Aqueous solutions of potas-sium hexoestrol sulphate were prepared freshly each day.The total dose for each animal was divided into six

equal parts, and each part was given in a volume of0-5 ml. on the morning and afternoon of three successivedays. Smears were made on the fourth and fifth days.The results were treated as described above.

Results.—Three series of assays were made, eachseries consisting of a large number of observations. Inthe first series hexoestrol, dienoestrol, and potassiumhexcestrol sulphate were compared with stilbcestrol, thepotency of which was assigned the value 100. In thesecond and third series cestrone was substituted for

potassium hexoestrol sulphate.The results obtained in the three series of assays are

given separately in table II to show the order of theagreement which may be expected in separate assaysof this type. The mean potencies compared with thevalues obtained by Kemp and Pedersen-Bjergaard(1943) and by Campbell et al. (1939) are given in table III.In all cases the results are shown relative to stilbcestrol,which has been assigned the value 100.

DISCUSSION

Our findings that the relative potencies of stilboestrol,diencestrol, and hexcestrol given by mouth are 100,68, and 10 do not agree with those of Campbell et al.(1939), who found hexcestrol almost as active as stil-bcestrol. They do, however, agree with those of Kempand Pedersen-Bjergaard (1943).This is the more interesting since the mode of admini-

stration used by us duplicated that used by Campbellet al., but differed from that used by Kemp andPedersen-Bjergaard. It seems from this that the modeof administration-i.e., solvent, arrangement of doses,&c.-may have less influence on relative potency thanhas been assumed. It is also interesting to note that ourresults are in close agreement with the clinical resultsreported by Bishop et al. (1948).

Similarly, our results for the relative subcutaneouspotencies show close agreement with those obtained byKemp and Pedersen-Bjergaard, though they differ fromthose reported by Campbell et al.

SUMMARY

Large-scale experiments designed to assess the cestro-genic activity of some synthetic oestrogens have beenmade with ovariectomised adult female rats.

Hexcestrol given by mouth has only a tenth ofthe activity of stilboestrol, dienoestrol two-thirds, andpotassium hexcestrol sulphate a fourteenth. Potassiumhexcestrol sulphate was given in aqueous solution ;the others were dissolved in arachis oil.

Injected subcutaneously in aqueous solution hexoestrolhas about two-thirds, dienoeatrol less than a third, andpotassium hexcestrol sulphate a hundredth of the activityof stilboestrol. Œstrone has about the same activityas diencestrol.

REFERENCES

Bishop, P. M. F., Kennedy, G. C., Wynn-Williams, G. (1948)Lancet, Nov. 13, p. 764.

Campbell, N. R., Dodds, E. C., Lawson, W., Noble, R. L. (1939)Ibid, ii, 312.

Emmens, C. W. (1938) J. Physiol. 94, p. 22.Gaddum, J. H. (1933) Spec. Rep. Ser. med. Res. Coun., Lond.

no. 183.Kemp, T., Pedersen-Bjergaard, K. (1943) Acta path. microbiol.

scand. 20, 552.

RATE OF FLOW OF VENOUS BLOOD INTHE LEGS

MEASURED WITH RADIOACTIVE SODIUM

H. PAYLING WRIGHTB.Se., Ph.D. Lond., L.M.S.S.A.

RESEARCH ASSISTANT, OBSTETRIC UNIT

S. B. OSBORNB.Sc. Lond., A.Inst.P.

PHYSICIST

DENISE G. EDMONDSTECHNICAL

ASSISTANT

UNIVERSITY COLLEGE HOSPITAL AND MEDICAL SCHOOL,LONDON

VENOUS thrombosis has long been known to beinfluenced by three main factors, commonly known asVirchow’s triad-changes in the constitution of theblood, damage to the blood-vessels, and disturbances ofthe circulation. The conception of phlebitis as a factorin thrombosis was introduced by John Hunter (1793),and Hayem (1889) first recognised the role played bythe platelets. Baillie (1793), Laennec (1819), and othersemphasised stasis of the blood as a factor in thrombosis,and Lancereaux (1875) postulated this law : " Throm-boses are always found at the level of the points wherethe blood has the greatest tendency to stasis, that is,at the limit of the action of the forces of cardiac propulsionand thoracic aspiration." Though the first two ofVirchow’s three factors are now well established experi-mentally and recognised in clinical practice, the partici-pation of the third has hitherto remained largelyinferential.The introduction of radioactive tracers, however, has

made possible the direct measurement of the rate ofblood-flow in a limb by, introducing such a tracer atone point and determining its arrival at some other

point with a suitably placed Geiger-Muller (G-M) countersensitive to the type of radiation emitted. In the

present study radioactive sodium, Na24, was used todetermine the rate of flow of venous blood up the legin normal people. The findings are interesting andfurnish valuable controls for comparative studies invarious pathological states later.

Tracer.-A radioactive isotope of sodium, Na24, wasprepared by the neutron irradiation of ’ Analar ’ NaCl,which becomes, after neutron capture, partially convertedinto N.a24Cl. The dry crystals were first assayed todetermine their activity ; usually, on arrival, 1 g.contained 1-2 millicuries of Na24, corresponding toabout 5 atoms of radioactive isotope per 1010 atoms ofstable Na. The crystals were made into sterile solutionso that 1 ml. contained 10-20 microcuries. Immediatelyafter delivery this concentration corresponded to about1% NaCl ; but, since the half-life of Na24 is only 14-8hours, it was necessary, on successive days, to use

higher concentrations of saline solution to achieve thesame specific activity of Na24. This solution, which

768

Fig. 1—Block diagram of apparaicus.

never exceeded a concentration of 5%, was used forintravenous injection. ,

Apparatus.-The apparatus * consists essentially ofa gamma-ray G-M counter, carried on an adjustablegantry. The counter is inserted into a tubular cylindricallead block with a slot in the lower surface. All ra,ysexcept those from vertically below are therefore subjectto absorption by 4 cm. of lead, the thickness of the

cylindrical wall. The counter is arranged so that theslot in the screen lies transversely over the femoralvein in the groin, and is pressed down as close to thebody as possible without impeding the venous flow.Each pulse from the G-M counter is recorded on a motor-driven kymograph drum in such a way that anysignificant change in counting-rate becomes immediatelyevident. Fig. 1 is a block diagram of the apparatus, andfig. 2 shows a typical tracing obtained in a normal personand the method by which it has been interpreted.

- INVESTIGATION

In the present study we examined 121 normal peopleaged 17-48 years, most of them falling within the20-30 years age-group. The majority were studentsand probationer nurses leading active lives. No personwith clinically observable varicose veins was included.The flow time was measured with the subject in the

supine position, care being taken to ensure that thefoot was at the same level as the heart, and thatthe head was not raised. The subjects were rested for atleast 20 min. under a warm electric blanket so thattheir foot temperature approximated as nearly as possibleto that of patients in bed-i.e., 33°C. To see whetherskin temperature had any pronounced effect on the

foot-groin time, 10 people were .examined on two occa-sions, first in the way just described and again withthe foot and leg immersed to the knee for 20 min. ina water-bath at 45-48°C.A prominent vein on the dorsum of the foot was

selected for injection, and a cuff, raised to 40 mm. Hg,was placed on the ankle above. A fine-gauge needle(no. 20) was inserted into’the vein, without an injectionbeing made. At this moment the drum was started and,after the " background " had been recorded for 15-20 sec.,1 ml. of Naz4Cl solution was injected into the vein asrapidly as possible. The duration of the injection,about 3 sec., was recorded on the tracing. Flow timeshave been measured from the start of the injectionperiod, since it is obvious that the solution first enteringthe vein will normally be the first to pass under theG-M counter. At the moment the radioactive materialreached the groin the counter indicated an increase incounting-rate, and this was recorded immediately as achange of slope in the tracing. The moment of changewas usually clear-cut, and, from a knowledge of the timeof injection, the time of arrival at the groin, and the* For a detailed account of the circuit and of the recording device

used in this study, together with a discussion of the accuracyof recording and analysing the data obtained, see Osbornand Wright 1948.

length of the leg (measured from the internal malleolusto the inguinal ligament over the femoral triangle),the average rate of flow could be calculated. It wasfound most convenient to express the result as foot-

groin time in seconds, after correction for length of legto a standard of 80 cm.

RESULTS .

The foot-groin times for the 121 normal people areshown in fig. 3. The average flow time for the groupwas 18 ± 0-9 sec. The mode of the group was at 13 sec.It can be seen that the observations form a skew curvewith a range of 4-50 sec. When abscissae are logarithmsof the flow times, however, the data fit a normal distri-bution curve (x2 = 11-65 ; P = 0-074). This type oflog-normal curve is often found in biological data

(Gaddum 1945).The data obtained have been further analysed for

statistical differences between (1) males and femalesand (2) right and left legs, as follows :

There is thus no significant difference between these

groups, nor does any group differ significantly from theseries as a whole.

Finally it was shown that the results obtained in the10 people examined in the standard manner and againafter vasodilatation had been induced by immersion in’hot water showed no significant change when analysedby Fisher’s (1944) method. The times observed were asfollows :

Subject A B C D E F G H I KFlow time (see.) :At standard temp... 11 13 13 8 8 11 13 15 11 15

After immersion.. 10 11 10 4 10 13 11 14 15 20

DISCUSSION

The present study offers a direct and objective methodof measuring the rate of blood-flow in a particularsection of the vascular system. The method avoidssubjective errors by either the patient or the observer;but there remain certain other possible errors whichneed consideration. Since these are discussed more

fully elsewhere (Osborn and Wright 1948), it need onlybe stated here that the combination of the errors intro-duced both by the random fluctuations of the "back-ground " and by the fitting of a line to the slope ofthe kymograph tracing may be about 1 sec. in flow

Fig. 2-Typical tracing obtained in a normal person, and method ofinterpreting it.

769

times of 10 sec. or less and may increase up to 3 or 4 sec.in cases with long flow times of about 50 sec.The end-point, when the slope of the tracing changesfrom that of the " background " to the new rate

produced by the Na24Cl arriving under the G-M counter,is very clear when 10 microcuries or more is used, butwith less amounts the concentration may be insufficient

to produce so pronounced an alteration of slope. The

injected solution is considerably diluted as it passes upthe vein, especially in cases with a slow flow. This isshown by the fact that with a slow flow the change ofslope is similar to that obtained when a more dilutesolution of Na24CI is injected in a person with a rapidflow, since the angle of the slope depends solely on thequantity of Na24Cl under the counter window. The timeat which the slope changes, however, is independent ofthe concentrationof N a2 4Cl used,provided it raisesthe count above the

"background"sufficiently to be

detected; themagnitude of thechange of slope isthe only variable v

depending on the <:concentration of Itthe radioactive (;:

material at the q

counter. q-

Afurtherfeature :::: of interest is thepersistence of theraised counting-rate at the groinafter the arrival ofthe Na24Cl. Fig. 2illustrates thisclearly. It can beseen that, com-

pared with theduration of the

injection (about 3sec.), a long timeis required for the

&mdash;-.-.-.<Bi<-ttr<)-’t-<!t-

SECONDS

Fig. 3-Foot-groin times of blood-flow in121 normal people.

blood containing Na24Cl to pass the counter. We believethat the reasons for this are (1) that the radioactivematerial is diluted by blood flowing into it from tributaryveins as it passes up the leg ; and (2) that the viscousflow- causes the blood in the centre of the vein to travelalong much more rapidly than that at the periphery.By the time all the Na24Cl has passed through the veinunder the G-M counter the first part of the injectedNa24CI may well have become mixed with the circulationand be returning under the counter in the femoralartery. In this way the slope often trails off only slowlytowards that of a new " background " level.The observed range. of flow times in the present

series of normal people under standard conditions ofrest and temperature has shown a wider variation thanwas expected by comparison with the recorded figuresof other workers. Blumgart and Weiss (1927) used aradioactive substance (radon) in their observation ofarm-arm times in normal and in diseased persons. Theydid not record any foot-arm measurements. Thompsonet al. (1928), using vital brilliant red, found a range offoot-arm times from 30 to >75 sec., whereas Smithet al. (1940), using sodium cyanide, reported a range of20 to >70 sec. in normal people for foot-carotid time.It is difficult to compare our average time of 18 sec.

with those reported by these workers, since our measure-ment is of a direct foot-groin time, whereas other methodsgive foot-heart or lung-heart-artery times, and the time

proportions of such a circuit can at present be estimatedonly approximately. Nevertheless it seems clear fromthe figures obtained by subtracting the arm-carotidfrom the foot-carotid times in the same person thatour figure of 18 sec. is of the same order as those foundby the indirect methods.The much larger range of observed times in our series

may be due to the sensitivity of the method. In suchtests as that with sodium cyanide, where the end-pointis determined by the observer noting a dilatation of thealse nasi followed by a deep inspiration, it is likelythat a proportion of cases will be noted as " no end-point," owing to a poor reaction to the drug ; suchcases would therefore be recorded as having exceededthe upper limit. In our entirely mechanical method ofrecording, however, even end-points with a minimalchange of slope can be detected, and consequently a caseshowing no end-point is rare.An increase of circulation-rate when the temperature

of a limb had been raised was recorded by Kvale andAllen (1939) and by Smith et al. (1940). Significantchanges were noted by these workers when the skintemperature rose from 20 to 33&deg;C. We obtained no

significant alteration of flow time, however, in the

group examined at 33&deg;C and again immediately afterimmersion in water at 45&deg;C. The range of temperaturechange was about 10&deg;C in both cases, but it is possiblethat our lower temperature, which was definitely" warm," had already evoked considerable vasodilatationand little further change took place when the temperaturewas raised to 45&deg;C. Smith’s lower figure of 20&deg;C’,however, might be considered as " cold," and a muchgreater vasodilatation would follow a rise over their

temperature range. This suggestion is borne out by theobservations of Roth et al. (1940) on the degree ofvasodilatation produced in the extremities of limbs atdifferent temperatures. We chose 33&deg;C as a standardtemperature because it approximates closely to the skintemperature of persons comfortably warm in bed, andour observations on normal people have been collectedand analysed primarily to be a basis for the assessmentof possible changes observed in pathological conditionsin patients confined to bed. The extension of this workis in progress.

SUMMARY

A direct method of measuring the rate of venousblood-flow in the leg with the radioactive isotope Na24is described.In 121 normal people the mean foot-groin flow time

under standard conditions was 18 &plusmn;0-9 sec. The extreme

range of the observations was 4-50 sec.The accuracy of the method is discussed and a com-

parison made with the findings by other methods ofmeasuring rates of blood-flow.We wish to thank Prof. W. C. W. Nixon, director of the

Obstetric Unit, University College Hospital Medical School,for his interest and support; Mrs. M. Merrington, of theGalton research department, University College, London, foradvice and help in the statistical analysis ; and the students,nurses, and colleagues who volunteered as subjects for thisinvestigation.

REFERENCES

Baillie, M. (1793) Trans. Soc. Improv. med. chir. Knowledge, 1, 119.Blumgart, H. L., Weiss, S. (1927) J. clin. Invests 4, 15.Fisher, R. A. (1944) Statistical Methods for Research Workers.

Edinburgh; p. 121.Gaddum, J. H. (1945) Nature, Lond. 156, 463.Hayem, G. (1889) Du sang et des alt&eacute;rations anatomiques. Paris.Hunter, J. (1793) Trans. Soc. Improv. med. chir. Knowledge, 1, 18.Kvale, W. F., Allen, E. V. (1939) Amer. Heart J. 18, 519.Laennec, R. T. H. (1819) De l’auscultation m&eacute;diate. Paris.Lancereaux, E. (1875) Trait&eacute; d’anatomie pathologique. Paris ;

vol. I, p. 604.Osborn, S. B., Wright, H. P. (1948) Brit. J. Radiol. (in the press).Roth, G. M., Horton, B. T., Sheard, C. (1940) Amer. J. Physiol.

128, 782. Smith, L. A., Allen, E. V., Craig, W. M. (1940) Arch. Surg. 41, 1366.Thompson, W. O., Alper, J. M., Thompson, P. K. (1928) J. clin.

Invest. 5, 605.