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THE JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA VOLUME 25, NUMBER 2 MARCH, 1953

Determinationf theAcoustic ropertiesf Blood nd ts Components*EDWIN L. CASTENSEN,KAX L, AXD HEKrAN P. SCHWANMoore School f ElectricalEngineering nd Departmentof PhysicalMedicine, Universityof Pennsylvania,Philadelphia4, Pennsylvania

Measurements f absorption nd velocity of sound n blood,plasma,and solutions f albumin and hemo-globin have been carried out in the frequency range 800-3000 kc and temperature range 5--45C.Theabsorption epartsonly slightly rom a linear dependencepon requency.Absorption or the varioussolu-tions is in direct proportion o protein content. t is concluded hat the acousticpropertiesof blood arelargely determinedby the proteinswhich t contains.

LINICALnterestnhighrequencyoundsherapeutic agent is continually increasing.Awide range of applicationshave been suggestedor itsuse . From physical considerations,t appears thatultrasound ortuitously combinesadequate depth ofpenetrationn tissues ith wavelengthshortenoughopermit sharp beaming, thus making it possible oprovide a localizedheating n the deep body tissues'.An investigationof the acousticpropertiesof thebiologicalmediummay be expected, irst, to provideaquantitativebasis or the phenomenologicalescriptionof the heating processes nd, second, o lead to anunderstanding f the mechanismof absorption.Measurementsof the absorption and velocity ofsound n someof the solid issues ave been eported nthe literature -.Blood was chosen o begin this in-vestigationbecauset containscellsand in this sensessimilar o body tissues. et, it is sufficiently qmogene-ous that it can be measuredwith a relatively highdegreeof accuracy.

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0 5 I0 15 20 25 30 gm/100 ccPROTEIN CONTENT OF SOLUTIONFro. 1. Absorption of sound n human blood versusvolume concentration of red cells.

* Aided by a grant from the National Foundation or InfantileParalysis, Inc. Der Ultraschall n der Medizin (Hirzel, Zurich, 1949).2 H. P. Schwan,and E. L. Carstensen, . Am. Med. Assoc.149, 121 (1952).3 R. Pohlman, Physik. Z. 40, 159 (1939). T. Hater, Naturwissenschaften5, 285 (1948). G. D. Ludwig, J. Acoust.Soc.Am. 22, 862 (1951).GR. Esche,Akust. Beih. 1, 71 (1952).'

EXPERIMENTAL PROCEDUREAbsorption measurementswere carried out by atwo-transducerpulse technique which has been de-scribed n detail previously. The unusual eatureof themeasurement s that transducerseparation s main-tained constant,and continuouslyvarying amounts ofwater are substitutedor test iquid in the path between

the transducers. his is accomplished y using a two-chamber test vesselwith water on one end separatedfrom the test liquid on the other end by a thin plasticwindow. The transducersare located on an assemblysuch that the source is in the water and receiver is inthe test liquid. Substitutionof water for test liquid isachieved by moving the entire transducerassemblyalong the axis of the test vessel. By maintaining'constant transducerseparation, t is possible o avoiddifficultieswhich arise from complexvariations n thefield near a transducer. This technique s particularlysuited to measurements f water svlutionsor liquidswith characteristic mpedance approaching that ofwater. Errors arising rom reflections t the interfacebetween he two liquids, the transducer aces,and thesidesof the test vesselare eliminated argely by use ofpulsing techniquesand directional transducers.Re-fraction of the sound beam at the interface between theliquids presents the possibility of error. However,experimental heckshave shown hat theseerrorscanbe made negligible,even for poorly matched liquids,by careful alignment of the transducers o providenormal incidence of the sound wave at the interface.The over-all error of the absorptiondeterminations sestimated to be approximately+10 percent or 0.02db/cm, whichever s the larger.

To determinephasevelocity, the wavelengthn testliquid is measured y comparinghe phaseof a directsignal rom the oscillatorwith that of the rf output ofthe receiver as its position s varied relative to thesource.The accura_cyf velocity measurementsorbiological ubstances asof the orderof +0.5 percent.Both water and the test liquid were placed undervacuum beforemeasuremento remove a part of thenormally dissolved ases. he temperaturewas main-tained constant to within a few tenths of a degree?H. P. Schwan,and E. L. Carstensen, lectronics, uly, 1952,p. 216-220.s H. Born, Z. Physik 120, 383-(1943).286

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8/3/2019 1953 Determination of the Acoustic Properties of Blood and Its Components

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ACOUSTIC PROPERTIES OF BLOOD 287

centigrade. t was necessaryo circulateblood duringmeasurements,o prevent sedimentationof red cells.EXPERIMENTAL RESULTS

Exploratory absorption measurementswere madeusing he red cell esiduesof centrifuged uman blood.The result of this work is illustrated in Fig. 1. Theresidues, ontainingabout 85 percent red cells, werediluted with plasma and curves of absorption as afunction of concentration f red cells n plasma wereobtained. The curves are straight lines, indicatingnegligible nteractionamong he absorbers p to maxi-mum concentration. These data are shown as a functionof frequency in Fig. 2. It is apparent that the ab-sorption n plasma is not negligiblebut actually ac-counts for a significantportion of the absorption nwhole blood. The high absorption n plasma is anindication that the cells, as such, are at least notsolely responsible or the absorption.Other measure-ments, which were carried out, have shown that thecell membrane alone can account for a maximum ofone-tenth of the absorptionobserved or red cells.On the other hand, it is interesting o examine thedata of Fig. ! in terms of protein concentration f thesolutions.Actual protein determinationswere not madeon the samplesused n thosemeasurements. owever,the lower abscissa f Fig. 2 indicates oughly he pro-tein concentration of the solution as estimated on thebasis of generally acceptedvalues of protein contentof plasmaand red cells.Extrapolationof the absorptioncurvesgives to a first approximation ero absorptionfor zero protein concentration.Actually, if proteinswereprincipally esponsibleor the absorption,t wouldbe anticipated that the extrapolated curves wouldintercept the zero protein concentrationaxis at thevalue of the absorption or water. This is the case orthe 800 kc and 1200 kc curves.The high intercept forthe 2400kc curvemay be explained y the independentbehaviorof plasmaat high frequencies s indicated nsubsequent iscussion.n reality, severaldifferentpro-teins are involved in this comparison. he red cellprotein is almost entirely hemoglobin,while roughly60 percent of the total plasma protein is albumin.These two proteins are similar. Both have molecularweightsof the order of 70 000 and axis ratios between1'5 and 1'9. All of the remainingplasma proteins,principally the globulins, are orders of magnitudeheavier han albumin.Yet, the data of Fig. 1 provideastrong indication that the proteins of blood are re-sponsibleor its absorption.A more extensive investigation was subsequentlyconducted o confirm he importanceof the proteinin the absorption.Plasma and red cell concentrate

9The pointsabove3 mc, as shown n Fig. 3 were obtainedatthe Franklin nstitute through he cooperationf Dr. George.Cathers. Each circle represents single measurementon onesampleof red cellsor plasma.This will providean indicationofbehaviorat frequencies igher than otherwiseobtained n thisinvestigation.

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Fro. 2. Absorptionof sound n human blood.were obtained from horse blood by sedimentation.Sodiumcitrate solutionwas added to prevent coagula-tion. The protein concentration f each solutionwasdetermined y the Kjeldahl method.Proteinconcentra-tion or plasma amplesangedrom5.4 to 6.0 g/100cc,and for red cell concentratesrom 32 to 33 g/100 cc. naddition, a 7 percent solutionof human hemoglobinin dextroseand water and a 12.5 percent solution ofhuman serum albumin in distilled water were ob-tained. Absorption for these solutionswas measuredover the frequency ange rom 800 to 3000 kc/secandfor temperaturesrom 5 to 45C.The data are sum-marizedbriefly n Figs. 3-5. The measured bsorption,in db/cm, has been converted o absorptionper unitquantity of proteinpresent n the solutionby dividingby the measured rotein content.The contributionofwater alone to the absorptions considered egligible.The data for all the solutionsare presentedas afunctionof frequency t 10C,20C,40C n Figs. 3,4, and 5, respectively. t 40C Fig. 5) it is readilyseenthat there is no significantdeparture of any of thesolutions rom their averageas indicatedby the solidline. The samesituationholds n generalat 10Cand20C (Figs. 3 and 4). The agreement mong the datafor the various solutionsshows hat absorption s adirect function of protein concentration.The pro-portionality s the same or both albuminand hemo-globinwithin the limits of error.This relationappearsto holdwhether he protein s in solution r containedin the cells.An interesting eparture rom the relationoccursnthe caseof plasmaat low temperaturesnd high fre-quencies s shown n Figs. 3, 4, and 7. The excessabsorptionn plasma elative to the other solutionsmay be caused y the presence f the larger plasmaproteins.

0R. B. Pennell,ndW. C. Smith, . Hematology, 380 1949).This methoduses 6 percentdextrose olution s a medium orthe hemoglobin. ix percent dextrosealone has negligibleab-sorption.

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8/3/2019 1953 Determination of the Acoustic Properties of Blood and Its Components

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288 CARSTENSEN, LI, AND SCHWAN

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Fro. 3. Absorptionof sound n protein solutions -10C.The absorptiondeparts only slightly from a lineardependence pon frequency n the range measured.For clarity, the data of Figs. 3, 4, and 5 have beenpresentedn Fig. 6 without the experimental oints.The slopeof the absorption ersusrequency urves nlog og presentations approximately .2.The temperaturecoefficient f absorption s smallbut definitelynegative.Averages f absorption or allthe solutions re plotted as a function of temperaturefor 1, 2, and3 mc n Fig. 7. The independentehavior fplasmaat low temperaturesnd high frequenciess

also indicated.The velocityof soundwasdeterminedor the varioussolutions escribed reviously.Typical data are shownin Fig. 8, which gives the velocity of sound n 6.2percent and 12.5 percent solutions f albumin indistilled water, as well as a curve for water alone. Ingeneral, dditionof protein o a solvent as he effect

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Fro. 4. Absorption f soundn proteinsolutions-20C.

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Fro. 5. Absorptionof sound n protein solutions -40C.of increasing elocity of sound.For all solutionsmeas-ured, this ncreasewas found to go n direct proportionto the proteinconcentration,veraging oughly m/secper gm/100 cc protein. Velocity measurementsor allsampleswere performedat both 800 and 2400 kc. Nodispersionwas observedn this range.

SUMMARYOn the basisof this nvestigation,t carlbe concluded

that the acousticpropertiesof blood are determinedlargely by the proteinswhich t contains.Absorptionof soundhas beenshown o be directly proportional othe protein concentration hether n solutionor con-tained within cells. Although the similar molecules

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Fro. 6. Averageabsorption ersusrequencyor proteinsolutionsred cells,hemoglobin, lbumin).

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8/3/2019 1953 Determination of the Acoustic Properties of Blood and Its Components

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ACOUSTIC PROPERTIES OF BLOOD 289

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TEMPERATURE CFro. 7. Absorptionversus emperature or protein solutions.Solid curve s average or red cells,hemoglobin nd albumin. Theindependent ehaviorof plasma s indicatedby dotted line.

albuminand hemoglobin avesimilarabsorption,hereis some ndication hat the largerplasmaproteins avesomewhat different characteristics.The authorswish to acknowledgehe generous elpand guidance f Dr. Robert B. Pennell,of Sharpeand

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TEMPERATUREFro. 8. Velocity of sound n albumin solutions.

Dohme, Inc., Glenolden,Pennsylvania,who suppliedthe materials or this investigation nd assistedhroughmany helpful discussions;Dr. George I. Cathers,formerly of the Franklin Institute, who performed hepreliminary measurementsmentioned n the text andsupplied elpfuladviceat the initiationof this work;Dr. John G. Reinhold, Hospital of the University ofPennsylvania,who performed the protein determina-tions on blood samples;and Dr. Eugene Ackermanand Mr. John Parnell for helpful discussions.THE JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA VOLUME 25, NUMBER 2 MARCH, 1953

Effects of Sonic Vibration on the Proteolytic Activity of Pepsin*G.oo. M. NAIMARK AND WILLIAM A. M OSH.Biochemical esearch oundation,Newark, Delaware

(ReceivedDecember3, 1952)Pepsinpreparationswere sonically reated for variousperiodsof time in a 9-kc magneto-striction scil-lator with the temperaturemaintainedat 13C-16Cduring treatment. After sonic reatment the residualproteolyticactivity of the enzymewasdetermined y measuringhe turbidity decrease uring he digestionof an albuminsubstrate.t was ound hat dilutesolutions f Merck U.S.P. pepsinwere apidly nactivatedby sonic reatment whereashighly concentrated olutionswere refractory to ultrasonicdestruction.Sonicirradiationof a pure Armour crystallinepepsinsolutionyieldedslight enzyme nactivationonly. In no in-stancewas enzyme activation observed n this study.

INTRODUCTION

A LIMITEDumberfpapersaveeportedheffects of sonic and t/ltrasonic vibration on thebiologicalactivity of enzyme systems. It has beenshown that oxidases re usually inactivatedby sonictreatment,'- althoughHaas* successfullyreparedan

* Presentedat the Symposiumon Acoustics nd Chemistry atWestern Reserve University, May 21-23, 1952. Now at StrongCobb and Compariy, nc., 2654 LisbonRoad,'Cleveland4, Ohio. Naimark, Klair, and Mosher, J. Franklin Inst. 250, 279-299,402-408 (1951).2 R. J. Christensen nd R. Samisch,Plant Physiol.9, 385-386{1934).

active cytochromeoxidase using sonic techniquesReductase nd amylase,on the other hand, were foundto be highly resistant to inactivation by vibrationalwaves while catalaseswere unaffectedby such treat-ment unlesssufficientlydilute2 Chambers ound8 thata M. Matsudaira and A. Sato, Tohoku J. Exptl. Med. 22, 412-416 (1934).4 M. Kasahara and T. Yoshinare,Z. Kinderheilk. $9, 462464(1938).5 R. Wurmser and S. Filitti-Wurmser, Compt. rend. soc.biol.128, 475-476 (1938).6 Grabar, Voinovitch,and Prudhomme,Biochim. et Biophys.Acta 3, 412-416 (1949).* E. Haas, J. Biol. Chem. 148, 481-493 (1943).a L. A. Chambers,J. Biol. Chem. 117, 639-649 (1937).

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