THE

JOURNAL

OF THE

ACOUSTICAL

SOCIETY

OF AMERICA

VOLUME

25, NUMBER

MARCH,

1953

Determination theAcoustic of Properties Blood its Components* of and

EDWIN L. CASTENSEN, KAX L, AXD HEKrAN P. SCHWAN Moore School ElectricalEngineering of and Department PhysicalMedicine, Universityof Pennsylvania, of Philadelphia4, Pennsylvania

Measurements absorption of and velocityof soundin blood,plasma,and solutions albuminand hemoof globin have been carried out in the frequency range 800-3000 kc and temperature range 5--45C.The absorption departsonly slightlyfrom a linear dependence uponfrequency. Absorption the varioussolufor tions is in direct proportionto protein content. It is concluded that the acousticpropertiesof blood are largelydeterminedby the proteinswhich it contains.

Absorption measurements were carried out by a two-transducerpulse technique which has been described detail previously. The unusual in 7 featureof the measurement that transducerseparationis mainis penetration tissues in with wavelengths shortenough to tained constant,and continuouslyvarying amounts of permit sharp beaming, thus making it possibleto water are substituted testliquid in the path between for This is accomplished using a twoby providea localizedheatingin the deep body tissues the transducers. '. An investigationof the acousticpropertiesof the chamber test vesselwith water on one end separated biological mediummay be expected, first, to providea from the test liquid on the other end by a thin plastic quantitative basis the phenomenological for description window. The transducersare located on an assembly of the heating processes and, second,to lead to an such that the source is in the water and receiver is in the test liquid. Substitutionof water for test liquid is understanding the mechanism absorption. of of achievedby moving the entire transducerassembly Measurementsof the absorption and velocity of along the axis of the test vessel. By maintaining soundin someof the solidtissues have beenreportedin 'constant transducerseparation, is possible avoid it to the literature Blood was chosen begin this in- difficultieswhich arise from complexvariations in the 3-. to vestigationbecause containscellsand in this sense it is field near a transducer. This techniqueis particularly a similarto body tissues. Yet, it is sufficiently hqmogene- suited to measurements water svlutionsor liquids of ous that it can be measuredwith a relatively high with characteristicimpedance approachingthat of degreeof accuracy. water. Errors arisingfrom reflections the interface at betweenthe two liquids,the transducer faces,and the sidesof the test vesselare eliminatedlargely by use of pulsing techniquesand directional transducers. Rewide range of applications have been suggested its for use From physical considerations, appears that . it ultrasoundfortuitously combinesadequate depth of
0.8 I

LINICAL inhigh increasing.A interest frequency a as therapeutic agent is continually sound

EXPERIMENTAL

PROCEDURE

fraction of the sound beam at the interface between the

z 0.4-o
o 0.2--

liquids presents the possibility of error. However, experimental checks have shownthat theseerrorscan be made negligible,even for poorly matched liquids, by careful alignment of the transducers provide to
normal incidence of the sound wave at the interface.

o
VOLUME

20

,o

60

8o

,oo
IN PLASMA

liquid is measured comparing phaseof a direct by the signalfrom the oscillator with that of the rf output of PROTEIN CONTENT OF SOLUTION the receiveras its positionis varied relative to the Fro. 1. Absorption of soundin human blood versus source.The accura_cy velocity measurements of for volume concentration of red cells. biological substances of the orderof +0.5 percent. was Both water and the test liquid were placed under * Aided by a grant from the National Foundationfor Infantile Paralysis, Inc. vacuum beforemeasurement remove a part of the to Der Ultraschall der Medizin (Hirzel, Zurich, 1949). in normallydissolved gases. The temperature was main2 H. P. Schwan,and E. L. Carstensen, Am. Med. Assoc. J.
0 5 I0 15 20 25 30 gm/100 cc

CONCENTRATION

OF RED CELLS

The over-all error of the absorptiondeterminations is estimated to be approximately+10 percent or 0.02 db/cm, whicheveris the larger. To determine phasevelocity,the wavelength test in

149, 121 (1952). 3R. Pohlman, Physik. Z. 40, 159 (1939). T. Hater, Naturwissenschaften 285 (1948). 35, G. D. Ludwig, J. Acoust.Soc.Am. 22, 862 (1951). GR. Esche,Akust. Beih. 1, 71 (1952).'
286

tained constant to within a few tenths of a degree

?H. P. Schwan,and E. L. Carstensen, Electronics, July, 1952,

p. 216-220.

sH. Born, Z. Physik 120, 383-(1943).

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ACOUSTIC

PROPERTIES

OF

BLOOD
/CENTRIFUGEff

287

centigrade.It was necessary circulateblood during to measurements, prevent sedimentationof red cells. to
EXPERIMENTAL RESULTS

7.0 5.0

/I
3.0 2.0

BLOOD /'NORMAL HUMAN I

/// / IOOMPuTED*

Exploratory absorption measurements were made usingthe red cellresiduesof centrifuged human blood. The result of this work is illustrated in Fig. 1. The residues, containingabout 85 percent red cells, were diluted with plasma and curves of absorptionas a function of concentration red cellsin plasma were of obtained. The curves are straight lines, indicating negligible interactionamongthe absorbers to maxiup
mum concentration. These data are shown as a function

1.0
0.7

0.5
0.3 0.2

o.i 007

/
0.3 0.5 0.7 I 2 3

DR

- EldiTS MEASUREM BY
GEORGE CATHERS

of frequency in Fig. 2. It is apparent that the ab sorption in plasma is not negligiblebut actually accounts for a significantportion of the absorptionin whole blood. The high absorptionin plasma is an Fro. 2. Absorptionof soundin human blood. indication that the cells, as such, are at least not solely responsible the absorption.Other measurefor were obtained from horse blood by sedimentation. ments, which were carried out, have shown that the Sodiumcitrate solutionwas added to prevent coagulacell membrane alone can account for a maximum of tion. The protein concentration each solutionwas of one-tenthof the absorptionobserved red cells. for determined the Kjeldahl method.Proteinconcentraby On the other hand, it is interestingto examine the tionfor plasma samples ranged from5.4 to 6.0 g/100cc, data of Fig. ! in termsof protein concentration the of and for red cell concentrates from 32 to 33 g/100 cc. In solutions. Actual protein determinations werenot made addition, a 7 percentsolutionof human hemoglobin on the samples used in thosemeasurements. However, in dextroseand water and a 12.5 percent solution of the lower abscissa Fig. 2 indicates of roughlythe pro- human serum albumin in distilled water were ob5 7 I0 20

0.05

FREOUENCY

(MEGACYCLE$

SEC )

wereprincipally responsible the absorption, would for it be anticipated that the extrapolated curves would intercept the zero protein concentrationaxis at the value of the absorptionfor water. This is the casefor the 800 kc and 1200 kc curves.The high intercept for the 2400kc curvemay be explained the independent by behaviorof plasmaat high frequencies indicatedin as subsequent discussion. reality, severaldifferentproIn teins are involved in this comparison. The red cell protein is almost entirely hemoglobin, while roughly 60 percent of the total plasma protein is albumin. These two proteins are similar. Both have molecular weightsof the order of 70 000 and axis ratios between 1'5 and 1'9. All of the remainingplasma proteins, principally the globulins, are orders of magnitude heavierthan albumin.Yet, the data of Fig. 1 providea strong indication that the proteins of blood are responsible its absorption. for A more extensive investigation was subsequently conducted confirmthe importance the protein to of in the absorption.Plasma and red cell concentrate
9The pointsabove3 mc, as shownin Fig. 3 were obtainedat the FranklinInstitute throughthe cooperation Dr. George of I. Cathers. Each circle represents single measurement one a on sampleof red cellsor plasma.This will providean indicationof behaviorat frequencies higher than otherwise obtainedin this
investigation.

tained. Absorptionfor these solutionswas measured basis of generally acceptedvalues of protein content over the frequency rangefrom 800 to 3000 kc/secand of plasmaand red cells.Extrapolationof the absorption for temperatures from 5 to 45C.The data are sumcurvesgives to a first approximationzero absorption marizedbrieflyin Figs. 3-5. The measured absorption, for zero protein concentration.Actually, if proteins in db/cm, has been convertedto absorption per unit

tein concentration

of the solution as estimated

on the

quantity of proteinpresent the solution dividing in by by the measured protein content.The contribution of water aloneto the absorption considered is negligible. The data for all the solutionsare presentedas a functionof frequency 10C,20C,40Cin Figs. 3, at 4, and 5, respectively. 40C(Fig. 5) it is readilyseen At that there is no significantdeparture of any of the solutions from their averageas indicatedby the solid line. The samesituationholdsin generalat 10Cand 20C(Figs. 3 and 4). The agreement amongthe data for the various solutionsshowsthat absorptionis a direct function of protein concentration.The proportionalityis the samefor both albuminand hemoglobinwithin the limits of error.This relationappears to holdwhetherthe proteinis in solution contained or
in the cells.

An interesting departure from the relationoccurs in the caseof plasmaat low temperatures high freand quencies shownin Figs. 3, 4, and 7. The excess as absorption plasmarelative to the other solutions in may be caused the presence the largerplasma by of
proteins. 0 B. Pennell, W. C. Smith, Hematology (1949). R. and J. 4,380
This methoduses 6 percentdextrose a solution a mediumfor as the hemoglobin. percent dextrosealone has negligibleabSix
sorption.

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288

CARSTENSEN,

LI,

AND

SCHWAN

P- PLASMA

P- PLASMA

R- CELLS RED
H- HEMOGLOBIN
z 0.03
o

R- CELLS RED

- 0.03
o

A-ALBUMIN
/_

:]HEMOGLOBIN

ALBUMIN
/-

E 0.02

0.02
E

I
I

TEMP.-IOC

z O.01
z

,o O.Ol

a :/
I
2

TEM P.-40'G
i
3

I
i

I
2

I
3

FREOUENGY

IN

MC

FREQUENCY

MG

Fro. 3. Absorption soundin protein solutions of --10C.

Fro. 5. Absorptionof soundin protein solutions--40C.

The absorptiondepartsonly slightly from a linear dependence upon frequencyin the range measured. For clarity, the data of Figs. 3, 4, and 5 have been presented Fig. 6 without the experimental in points. The slopeof the absorption versus frequency curves in log log presentation approximately is 1.2. The temperaturecoefficient absorptionis small of but definitelynegative.Averages absorption all of for the solutions plotted as a function of temperature are for 1, 2, and3 mc in Fig. 7. The independent behavior of

of increasing velocity of sound.For all solutions measured, this increase was found to goin direct proportion to the proteinconcentration, averaging roughly m/sec 4 per gm/100 cc protein. Velocity measurements all for samples were performedat both 800 and 2400 kc. No dispersion was observed this range. in
SUMMARY

plasmaat low temperatures high frequencies and is

also indicated.

On the basis this investigation, carlbe concluded of it that the acousticpropertiesof blood are determined The velocityof sound wasdetermined the various largelyby the proteinswhich it contains. for Absorption solutions described previously. Typical data are shown of soundhas beenshownto be directly proportionalto in Fig. 8, which gives the velocity of soundin 6.2 the protein concentration whetherin solutionor conpercent and 12.5 percent solutions albumin in tained within cells. Although the similar molecules of distilled water, as well as a curve for water alone. In

general, additionof proteinto a solvent the effect has

0.03
IOOC 20C 30 C 40 C

P-PLASMA
0.03

H-HEMOGLOBIN A-ALBU I N M

R-RED CELLS
0.02

0.02

0.01

O.OI

,//
I
I
FREQUENCY

TEMR -20C
I
i

I
2

I
3

I
2
MC FREQUENCY IN MC

Fro. 4. Absorption sound proteinsolutions of in --20C.

Fro. 6. Averageabsorption versus frequency protein for solutions (red cells,hemoglobin, albumin).

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ACOUSTIC

PROPERTIES
1600

OF

BLOOD

289

0.0.5 P--PLASMA

AVERAGES

FOR RED
1550

z
o

0.04

R,\
3 MC ..,,,,,.. .

CELL, HEMOGLOBI N,

AN IN D ALBUM
SOLUTIONS

12.5% ALBUMEN

_
j

j6.2%
J

ALBUMEN

....DISTILLED WATER

1500

0.03

0.02

_2 MC
i
i0

1450

//

o. O.Ol
o

1401

I
IOaC

I
20C
TEMPERATURE

I
$0C

I
40C

I
20
TEMPERATURE

i
30
C

i
40

Fro. 8. Velocity of sound in albumin solutions.

Dohme, Inc., Glenolden,Pennsylvania,who supplied formerly of the Franklin Institute, who performedthe preliminary measurements mentionedin the text and

Fro. 7. Absorptionversus temperature protein solutions. the materialsfor this investigation for and assisted through Solid curveis averagefor red cells,hemoglobin and albumin. The many helpful discussions; Dr. George I. Cathers, independent behaviorof plasmais indicatedby dotted line.

albuminandhemoglobin havesimilarabsorption, there helpfuladvice the initiationof this work; at is some indication that the largerplasma proteins have supplied Dr. John G. Reinhold, Hospital of the University of somewhat different characteristics. Pennsylvania,who performed the protein determinaThe authorswish to acknowledge generous the help tions on blood samples;and Dr. Eugene Ackerman and guidance Dr. Robert B. Pennell,of Sharpeand and Mr. John Parnell for helpful discussions. of

THE

JOURNAL

OF THE

ACOUSTICAL

SOCIETY

OF AMERICA

VOLUME

25, NUMBER

MARCH,

1953

Effects of Sonic Vibration on the Proteolytic Activity of Pepsin*

G.oo. M. NAIMARK AND WILLIAM A. M OSH. Biochemical Research Foundation,Newark, Delaware (ReceivedDecember3, 1952)

Pepsinpreparations were sonicallytreated for variousperiodsof time in a 9-kc magneto-striction oscillator with the temperature maintainedat 13C-16C during treatment.After sonictreatment the residual proteolytic activity of the enzymewasdetermined measuring turbidity decrease by the duringthe digestion of an albuminsubstrate. wasfoundthat dilutesolutions Merck U.S.P. pepsin It of wererapidlyinactivated by sonictreatment whereashighly concentrated solutions were refractory to ultrasonicdestruction.Sonic irradiationof a pure Armour crystalline pepsinsolutionyieldedslightenzymeinactivationonly. In no instancewas enzyme activation observedin this study.

active cytochromeoxidase using sonic techniques Reductase and amylase,on the other hand, werefound 4 effects of sonic and t/ltrasonic vibration on the to be highly resistant to inactivation by vibrational were unaffectedby such treatbiologicalactivity of enzyme systems. It has been waveswhile catalases ment unlesssufficientlydilute2 Chambersfound8 that shown that oxidases usually inactivatedby sonic are a M. Matsudaira and A. Sato, Tohoku J. Exptl. Med. 22, 412treatment, althoughHaas* successfully '- preparedan

INTRODUCTION

A LIMITED of number have the papersreported

416 (1934).

* Presentedat the Symposium Acoustics on and Chemistryat Western ReserveUniversity, May 21-23, 1952. Now at StrongCobband Compariy, Inc., 2654LisbonRoad, 'Cleveland4, Ohio. Naimark, Klair, and Mosher,J. Franklin Inst. 250, 279-299, 402-408 (1951). 2R. J. Christensen and R. Samisch, Plant Physiol.9, 385-386 {1934).

4 M. Kasahara and T. Yoshinare,Z. Kinderheilk. $9, 462464 (1938).

5 R. Wurmserand 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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