Ultrasound in Med. & Biol.. Vol. 4, pp. 125-130. Pergamon Press, 1978. Printed in Great Britain

ULTRASONIC ABSORPTION IN THE H U M A N

BREAST CYST LIQUIDS J. LANG and

R. ZANA

C e n t r e d e R e c h e r c h e s s u r les M a c r o m o l 6 c u l e s , 6, r u e B o u s s i n g a u l t , 6 7 0 8 3 S t r a s b o u r g - C e d e x , F r a n c e

and B. GAIRARD, G. DALE and CH. M. GROS S6nologie-RadiologieCentrale, Hospices Civils, 67005 Strasbourg-Cedex, France (First received 22 February 1978; and in final form 12 June 1978)

Abstract--The ultrasonic absorption of samples of cyst liquids (CL) of the human breast has been measured as a function of frequency (1.67-115 MHz), pH (1-13), dilution and temperature (25 and 37"C). The absorption behavior of these liquids is very similar to that of protein solutions. It is shown that the absorption of CL at physiological pH arises essentially from the protein contained in these liquids and, in fact, can be evaluated to within a factor 1.5, from that of serum albumin in similar condition. The origin of this absorption is discussed. Key words: Human breast cysts, Ultrasonic absorption, Protein, Proton transfer. 1. INTRODUCTION

As the ultrasonic methods used in medical diagnosis b e c o m e more and more sophisticated (see, for instance, Wells, 1977), the knowledge, if not the full understanding, of the ultrasonic absorption properties of biological tissues gains importance. The absorption of ultrasound in biological tissues has been the subject of a number of investigations (Wells, 1977 (and references therein); Hussey, 1975; Lele et al., 1976; Sobel et al., 1977). For our part we have started a few years ago a systematic investigation of the ultrasonic absorption of solutions of biomolecules such as proteins (Zana and Lang, 1970; Lang et al., 1971; Zana et al., 1972) and nucleic acids (Lang and Cerf, 1969; Sturm et al., 1971). Indeed a large part of the attenuation of homogeneous biological tissues has been shown to be an absorption occurring at the molecular level and involving proteins and nucleic acids (Pauly and Schwan, 1971; Smith and Schwan, 1971; Carstensen and Schwan, 1959a, b). We have later extended our measurements to the study of a biological fluid: the human amniotic liquid (Zana and Lang, 1974). The ultrasonic absorption of amniotic liquid was determined in the 1-15 MHz range and was shown to arise essentially from the proteins contained in this fluid at least at physiological pH. UMB Vol. 4, No, 2--C

This paper is to report the absorption of ultrasound in a somewhat more complex biological fluid namely the breast cyst liquid (abbreviated herein as CL). In addition to characterizing the ultrasonic absorption of CL, our work was also intended to check whether significant differences exist between the absorption of cancerous and noncancerous C t ' s . Before going into the results a few words must be said about the C L composition. In a recent report Koehl et al. (1976a) have shown that these liquids may be considered as aqueous solutions of proteins containing various ions: Na ÷, K ÷, CI-, Ca 2+ and Mg 2+. The proteins are essentially mucoproteins (about 70%) and proteins from the serum (albumins). Their average protein content is 26g/1, with large variations, however, from 12 to 43g/1. In two instances, one of them associated with an intracystic cancer, the protein content was even above 80g/1. The electrolyte content and composition were also found to undergo large variations. (Koehl et al., 1976b).

2. EXPERIMENTAL PROCEDURE

The ultrasonic absorption measurements were performed by means of the standard pulse method (accuracy: ---2%) and the ultrasonic interferometer (accuracy: --+5%) 125

126

J. LANG et al.

used in our previous studies (Lang and Cerf, 1969; Lang et al., 1971). The investigated samples of cyst liquids have been numbered in the order of decreasing protein content (see Table 1). The patients from whom these liquids were obtained are referred to by the same numbers. Patients 4 and 6 were regularly examined for the control of their cyst disease. Patients 2, 3, 5 and 7 were examined for the first time. Patient I was 77 years old, but the six other patients were all between 45 and 52 years old. Patients 2, 3, 6 and 7 were in their premenopausal period and patients 1 and 4 in their postmenopausal period. Patient 5 had been hysterectomized. The diagnosis and location of the cysts were performed by means of the echographic technique. The sample of CL was obtained by puncturing the cyst under echographic control. It must be pointed out that the puncture is the usual treatment for this disease in our hospital. The ultrasonic measurements were performed, usually within 24 or 48 hr, only when a volume of at least 30cm 3 was available. Note, however, that no significant changes of the ultrasonic properties were observed after several days of storage at 5°C. The protein, glucose, Na +, K +, C1- and P contents of the CL samples were determined by means of classical procedures (Koehi et al., 1976b) by using an automatic analyser (Technicon SMA). The proteins were titrated with biuret, the glucose with neocuproine, Cl with mercury thiocyanate, P with molybdate. The sodium and potassium contents were determined by flame photometry and the Ca 2÷ and Mg2÷ contents by atomic absorption spectrometry.

3. RESULTS

Figure 1 shows the ultrasonic relaxation spectra of six CL samples, and a result at a single frequency for an additional sample, at 37°C and pH's between 7.2 and 8.2. The quantity a [ f 2 (a = ultrasonic absorption coefficient in cm-1; f = frequency in sec -I) is plotted against the frequency. The results clearly indicate the existence of relaxation processes in the range 4-115MHz. Three remarks may be made concerning these results: (1) The value of a l l 2 is strongly dependent on the sample investigated. We have given in Table 1 the composition of the seven CL samples and it appears that ~t/f 2 roughly increases as the protein content of the sample

300

--~~

N~

g ,oo_

-7 ?o I 4

7

5

I

1

I0

J

15

20

f,

t

[

I

[

3 0 40 5 0 MHz

70

Fig. 1. Ultrasonic relaxation spectra of samples of cyst

Cyst liquids

Proteins Glucose Na ÷ K* CI Ca 2÷ P Mg2+

(g/l) (g/l) (rag/I) (mg/I) (rag/l) (mg/l) (mg/l) (rag/l)

80 1 130 4 100 100 37 30

1 94 0.71 138 3.5 101 91 21 21.3

2 84 i.08 100 62 52 81 33 39.2

150

liquids at 370C. Sample number: (O) I; (A) 2; (Fq) 3; (V) 4; (*) 5; (©) 6; ( I ) 7. The pH of the samples ranged from 7.2 to 8.2. The dotted line shows the a / f 2 of water in the frequency range investigated.

Table 1. Composition of the investigated samples of human blood plasma and CL's (communicated by Dr Koehl)*

Plasma

I

I00

3

4

5

6

7

46

37

26

19

130 6.5 79 115

122 36 72 77 21

27 0.8 45 96 6 110 14 12.2

30 84 26 174 62 28.3

18 124 12 130

30.2

21.5

*The blank spaces correspond to values which have not been measured. The minimum detectable concentrations of glucose, P and Mg3+ are, respectively, 50 rag/I, 5 mg/I and 0.02 mg/l.

Ultrasonic absorption in human breast cyst liquids

127

increases, although there are large fluctua- of about -0.01 deg -I in the range 25--40°C. On tions, in particular with CL 3. the other hand it can be seen that down to (2) The decrease of Ot[f 2 at increasing f 1.7MHz, in both instances, there is no does not resemble that found for single inflexion of the a l l 2 vs f curve towards the relaxation frequency systems. A spectrum of frequency axis, thereby indicating that the relaxation frequencies appears to charac- spectrum of relaxation times extends well terize the CL's. Such a behavior has always below 1 MHz, as for hemoglobin (Edmonds, been found for protein solutions at physio- 1962; Edmonds et al., 1970; Schneider et al., logical pH's (Edmonds, 1962; Edmonds et al., 1969). Also, the plots log a against log f have 1970; Kessler and Dunn, 1969; Zana et al., been found to be linear in the whole 1972; O'Brien and Dunn, 1972; Schneider et frequency range 1.7-115MHz, yielding at al., 1969) and biological tissues (Wells, 1977, 25°C: and references therein; Lele et al., 1975). a = 8.8 x 10-3 fl.42 for CLI and (3) There is no striking difference between a = 6.6 x l0 -3 fL4~ for plasma, the spectra for the CL sample 1 which was obtained from a cancerous cyst and the CL where a and f are expressed in cm -~ and Hz. sample 2 which was obtained from a cyst It must be recalled that very similar expresbelieved to be benign but which had similar sions have been obtained for minced and protein and electrolyte contents, in spite of homogenized beef and lamb liver (Pauly and the fact that concerous liquids are rather Schwan, 1971; Wells, 1977, p. 122; Goss and turbid with a brown color as in the case of Dunn, 1974). cyst liquid 1 in contrast to the non-cancerous The change of the ultrasonic absorption of liquids which are much less colored and most CL1 upon dilution by water is shown on Fig. of the time transparent. 3. The experiments have been performed at The relaxation spectrum of the CL sample 25°C and at a fairly low frequency (2.81 MHz) 1 (referred to as CL1) has also been deter- in order to start from large a l f 2 values. The mined at 25°C in a frequency range extending absorption ot/f2 is plotted as a function of the down to 1.7 MHz (at 37°C the measurements volume fraction ¢b of CL1 in the mixture were performed only above 3.9MHz). The H20-CL1. Within the experimental unresults at 25 and 37°C are plotted on Fig. 2, certainly the plot ~tlf 2 vs ~ may be considered where we have also given the relaxation as linear. Again, this result is very similar to spectra for human blood plasma determined that found for protein solutions, where Ol]f 2 as part of this work at these two tempera- increases linearly with the protein concentures. Both for the plasma and CLI, an in- tration up to at least 50g/1 (Carstensen and crease of T brings about a decrease of a l f 2, Schwan, 1959b; Kessler and Dunn, 1969; with a temperature coefficient d l n a / f 2 / d T Zana and Lang, 1970).

T E o

500

0

o

I

1

I

1

1

1

I

1

I

2

3

5

7

I0

20

30

50

",;'0

f,

I .:9 I00

MHz

Fig. 2. Ultrasonic relaxation spectra of CLI (O and Q) and human blood plasma ([] and II) at 25°C (solid

lines) and 37°C (dotted lines).

J. LANG et al.

128

jto3

500

% T i:: u

400 --

~

.500

e

/"

200

o tO0

were not taken at the same pH but at pH between 7.2 and 8.2. 4. DISCUSSION

Q

T E --0,2

All the results presented in the preceding section show that the ultrasonic absorption behavior of CL samples is similar to that of protein solutions as far as the effects of frequency, concentration and pH are concerned. This is particularly clear when comparing the relaxation spectra of CL1 and human blood plasma (see Fig. 2) or the variation a / f with pH for CL1 and, for instance, a solution of protein such as bovine serum albumin (Zana and Lang, 1970). In fact the results obtained in this study appear to indicate that the ultrasonic absorption of CL samples can be evaluated semi-quantitatively from our previous results on protein solutions as follows. The proteins found in CL are mostly mucoproteins, the absorption of which has never been investigated thus far. However the results of Figs I and 2 show that the absorptions of human plasma (which contains essentially albumins) and of CLI and CL2 do not differ much in the whole frequency range, both at 25 and 37°C. It may therefore be concluded that the specific absorption (a/f2)~p (absorption per unit concentration) of mucoproteins must be fairly close to that of albumins. In a previous study (Zana et al., 1972) the specific absorption of bovine serum albumin was reported to be equal to 4.4xlO-JTcm-~sec21g-: at pH 5-8, and 2.81 MHz. From this value the absorption of

~"

--0.1



-

/o 0

I

I

I

0.2

0.4

0.6

0.8

t.O

Fig. 3. Effect of dilution with water on the absorption of CL1 at 25°C, 2.81 M H z and pH 7.1.

A further check that the absorption behavior of CL's is qualitatively similar to that of proteins solutions is provided by the results of Fig. 4 where we have shown the effect of pH on the absorption of a mixture H20--CLI at • = 0.25 (four-fold dilution). The a/[ 2 vs pH curve for CL1 is strikingly similar to the analogous curves determined for globular proteins (compare the curve in Fig. 4 with Fig. 1 in Zana et al., 1972). Both curves for CL1 and globular proteins a l l 2 go through two maxima: one in the acid range at around pH 3, the other in the alkaline range at about pH 11.3, but Ot/f 2 is almost independent of pH in the range 4.5-8.5. From this latter result it is clear that the comparison of the absorptions of the various CL samples made on Fig. 1 retains its validity, even though the samples

%

//i

300

T

E

200

0.2

T E m

....-."'"'\....,..,..._.jj.

O

O.P rJ

I00

I I

--H20 I I 2

3

I

I

4

5

1 6

I

I

I

I

I

I

J

7

8

9

IO

H

p2

~3

pH Fig. 4. Effect of pH on the ultrasonic absorption of C L I diluted four times, at 25°C and 2.81 MHz.

Ultrasonic absorption in human breast cyst liquids

the CL1 sample in the same condition can be calculated to be: 1017¢X/f2 = 1017(Ot/f)l-t20+ 4.4 X 94 = 436 cm -~ sec 2, in excellent agreement with the experimental result: 470cm -t sec 2 of Fig. 2. Of course, such a good agreement will not be found for the other samples since, as noted above, otlf2 does not increase smoothly with the CL protein content. Nevertheless it may be stated that the absorption of CL samples at physiological pH can be calculated to within a factor 1.5 from the specific absorption of bovine serum albumin at the same frequency, pH and temperature, and from the CL protein content. The possible contribution of the electrolytes present in CL to the measured absorption has not been considered in the above calculations because uni-univalent salts such as NaCI or KC1 as well as divalent-univalent salts (MgCI2 or CaCI2) are known to give rise to a negligible absorption (Stuehr and Yeager, 1965 (and references therein)). On the other hand magnesium and calcium phosphates, although they are more absorptive than the above salts at equivalent concentration, would give rise to a negligible absorption at the maximum concentration at which they are found in CL (about 0.002 M), in the frequency range investigated. As for proteins the absorption maxima at around pH 3 and ll, observed with CL are most likely due to proton transfer reactions involving the protonatable side chains of the protein residues (Zana and Lang, 1970; Zana et al., 1972). The origin of the absorption below the absorption maxima and at physiological pH is still unclear however. Several mechanisms have been put forward (Zana et al., 1972), the most recent one involving proton exchanges between protonable protein residues with pKo's not too far apart (White and Slutsky, 1972). These reactions are similar to those involved in the absorption of solutions of certain nucleotides (Lang et ai., 1974). One cannot, however, exclude the possible contribution of two other processes: (l) Hydration equilibria involving the protein peptide bonds and, (2) Motion of residue side chains and of coiled parts of the protein molecule (this type of process is responsible for the excess absorption of polymer solutions in organic

129

solvents and may be also operative in water (see Zana, 1976, and references therein)). These two processes are characterized by absorption increasing linearly with concentration. Moreover, the first one would be almost pH independent while the second one should show some dependence on pH. These characteristics are similar to those found for protein solutions as well as CL and amniotic liquid. The results presently available do not permit us to find out which, if any, of the three above processes is the predominant contributor to the ultrasonic absorption of protein solutions. Finally it must be added that some of the investigated CL samples contained a fairly large amount of suspended particles which did not settle down even after periods of several days, although some of them were rather large (0.5 mm), probably because of a density very close to that of the suspending liquid. These particles may have contributed to the apparent absorption measured in the present work by back-scattering the sound waves. Although this effect may explain in part the large fluctuation of the ~tlf2 vs protein content it does not affect the essential conclusion reached in this work. Finally it must be recalled that in our study of amniotic liquid we have clearly shown that filtration, which eliminated suspended particles, resulted in a decrease of ultrasonic absorption. A similar effect may be expected upon filtration of cyst liquids. 5. CONCLUSIONS

The measurements reported in this study of cyst liquids of the human breast lead to the same conclusion as that of the study of amniotic liquid. For these two biological fluids the ultrasonic absorption at physiological pH is essentially determined by their protein content. Acknowledgements---The authors are pleased to thank Dr C. Koehl (Laboratoire de Biochimie, Pr. Mandel, Facult6 de M6decine, Strasbourg), for communicating the compositions of the cyst liquids prior to publication.

REI~IgIRENCES Bamber, J. G., Fry, M., Hill, C. and Dunn, F. (1977) Ultrasonic attenuation and backscattering by mammalian organs as a function of time after excision. Ultrasound Med. Biol. 3, 15-20. Carstensen, E. and Schwan, H. (1959a) Absorption of sound arising from the presence of intact cells in blood. J. Acoust. Soc. Am. 31, 185-189. Carstensen, E. and Schwan, H. (1959b) Acoustical pro-

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perties of hemoglobin solutions. J. Acoust. Soc. Am. 31,305-31 I. Edmonds, P. D. (1962) Ultrasonic absorption of hemoglobin solutions. Biochim. Biophys. Acta 63, 216--219. Edmonds, P. D., Bauld, T. J., Dyro, J. and Hussey, M. (1970) Ultrasonic absorption of aqueous hemoglobin solutions. Biochim. Biophys. Acta 200, 174-177. Goss, S. and Dunn, F. (1974) Ultrasonic Symposium Proceedings, p. 65. Hussey, M. (1975) In: Diagnostic Ultrasound, An Introduction to the Interactions between Ultrasound and Biological Tissues. Blackie, Glasgow. Kessler, L. and Dunn, F. (1969) Ultrasonic investigation of the conformal change of bovine serum albumin in aqueous solutions. J. Phys. Chem. 73, 4256-4263. Koehl, C., Gairard, B. and Abecassis, J. (1976a) Etude de la composition prot6ique et 61ectrolytique des liquides de kystes mammaires. Senologia 2, 46. Koehl, C., Gairard, B. and Abecassis, J. (1976b) Unpublished results. Lang, J. and Cerf, R. (1969) Absorption ultrasonore dans des solutions d'acide d6soxyribonucl6ique. Etude de la d6naturation alcaline. 3". Chim. Phys. Physicochim. Biol. 66, 81-87. Lang, J., Tondre, C. and Zana, R. (1971) Effect of urea and other organic substances on the ultrasonic absorption of protein solutions. J. Phys. Chem. 74, 374-379. Lang, J., Sturm, J. and Zana, R. (1974) Ultrasonic absorption in aqueous solutions of nucleotides and nucleosides. II. Kinetics of proton exchange in adenosine 5'-monophosphate. J. Phys. Chem. 78, 8086. Lele, P., Mansfield, A., Murphy, A., Namery, J. and Senapati, N. (1976) Tissue characterization by ultrasonic frequency-dependent attenuation and scattering. N B S Special Publication 453, 167-196. O'Brien, W. D. and Dunn, F. (1972) Ultrasonic absorp-

tion mechanisms in aqueous solutions of bovine hemoglobin. J. Phys. Chem. 76, 528-533. Pauly, H. and Schwan, H. P. (1971) Mechanism of absorption of ultrasound in liver tissue. J. Acoust. Soc. Am. 50, 692-699. Schneider, F., Muller-Landau, F. and Mayer, A. (1969) Acoustical properties of aqueous solutions of oxygenated and deoxygenated hemoglobin. Biopolymers 8, 537-544. Smith, A. and Schwan, H. P. (1971) Acoustic properties of cell nuclei. J. Acoust. Soc. Am. 49, 1329--1330. Stuehr, J. and Yeager, E. (1965) In: Physical Acoustics (Edited by Mason, W. P.), Vol. II, Part A, pp. 398--401, Academic Press, New York. Sturm, J., Lang, J. and Zana, R. (1971) Ultrasonic absorption of DNA solutions: Influence of pH. Biopolymers 10, 2639--2643. Wells, P. T. (1977) In: Biomedical Ultrasonics, Academic Press, New York. White, R. and Siutsky, L. (1972) Ultrasonic absorption and relaxation spectra in aqueous bovine hemoglobin. Biopolymers 11, 1973-1984. Zana, R. (1976) In: Encyclopedia of Polymer Science and Technology, Supplement Vol. l, pp. 607-611, Wiley, New York. Zana, R. and Lang, J. (1970) Effect of pH on the ultrasonic absorption of protein solutions. J. Phys. Chem. 74, 2734-2735. Zana, R. and Lang, J. (1974) Interaction of ultrasound and amniotic liquid. Ultrasound Med. Biol. 1,253-258. Zana, R., Lang, J., Tondre, C. and Strum, J. (1972) Interaction of ultrasound with proteins and nucleic acids in solutions. In: Interactions of Ultrasound and Biological Tissues (Edited by Reid, J. M. and Sikov, M. R.), pp. 21-29. U.S. Dept HEW Publication 738008, BRH/DBE 73-I.

Ultrasonic absorption in the human breast cyst liquids.

Ultrasound in Med. & Biol.. Vol. 4, pp. 125-130. Pergamon Press, 1978. Printed in Great Britain ULTRASONIC ABSORPTION IN THE H U M A N BREAST CYST L...
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