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Dependence of ultrasonic attenuation of liver on pathologic fat and fibrosis: Examination with experimental fatty liver and liver fibrosis models
Ultrasound in Meal. & Biol. Vol. 18, No. 8. pp. 657-666, 1992 Printed ill the U.S.A,
0301-5629/92 $5.00 + ,00 (c) 1992 Pergamon Press Lid.
OOriginal Contribution DEPENDENCE OF ULTRASONIC ATTENUATION OF LIVER ON PATHOLOGIC FAT AND FIBROSIS: EXAMINATION WITH EXPERIMENTAL FATTY LIVER AND LIVER FIBROSIS MODELS K. S u z u ~ , *
N . HAYASHI, t Y. SASAKI, t M . KONO, t A. KASAHARA, t H . FUSAMOTO, t Y. IMAI * a n d T. KAMADA t
tFirst Department of Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan, and *Research and Development Engineering, Process Instrumentation Division, Shimadzu Corporation, Kyoto 604, Japan (Received 23 October 1991; in final form 26 May 1992) Abstract--To clarify the effect of the pathological state of the liver on ultrasonic attenuation, we produced two experimental rabbit models. The influence of fat on ultrasonic attenuation was examined using a fatty liver model without liver fibrosis, and that of fibrosis on attenuation using a liver fibrosis model without fatty infiltration. Ultrasonic data were obtained in vivo directly from the liver, and an acoustic attenuation coefficient slope was obtained by the spectral difference method. Tissue components of the liver, namely the total lipid, hydroxyproline and water contents, were measured precisely by quantitative methods. We revealed that ultrasonic attenuation depends mainly on fatty infiltration of the liver and to a lesser extent on fibrosis, but not on the water content.
Key Words: Attenuation, Ultrasound, Rabbit liver, Experimental fatty liver model, Experimental liver fibrosis model, Fat content, Collagen content, Water content.
INTRODUCTION
ture. A positive correlation between attenuation and fibrosis has been reported (Afschrift et al. 1987; Duerinckx et al. 1988; Lin et al. 1988). However, some authors found little or no elevation of attenuation with fibrosis alone (Bamber et al. 1981; Taylor et al. 1986; Wilson et al. 1987), while others reported that the attenuation value varies with the etiology of the liver cirrhosis (Maklad et al. 1984). There are the following possible reasons for such contradictory findings: i. In a human study, fibrotic livers often exhibit varying degrees of fatty infiltration, and the fatty infiltration itself elevates attenuation, making interpretation of the attenuation results complicated. 2. In most human studies, the contents of fat and fibrosis were not quantitatively measured. Liver specimens were only classified into 3 or 4 groups based upon the microscopic evaluation of the fat and fibrosis by pathologists. In addition, precise grading is not always possible for a small biopsy specimen. 3. The influence of the abdominal wall on the ultrasonic beam and acoustic attenuation has not been thoroughly investigated, and they may affect the attenuation measurement.
Conventional gray scale B-mode ultrasound of the liver is a well-established modality in the diagnosis of focal lesions within the liver. However, diffuse parenchymal diseases of the liver, such as liver cirrhosis and fatty infiltration, are difficult to differentiate on the basis of B-mode imaging alone (Gosink et al. 1979; Sandford et al. 1985). Furthermore, sonographic grading of these diseases is not precise enough to predict the severity of the pathological changes (Needleman et al. 1986). The attenuation of ultrasound in the liver has received considerable attention as a quantitative ultrasonic tissue characterization technique. A positive correlation between fatty infiltration of the liver and attenuation has been reported by many researchers, both in vitro (Bamber et al. 1981; Lin et al. 1988) and in vivo (Afschrift et al. 1987; Duerinckx et al. 1988; Taylor et al. 1986; Wilson et al. 1987). However, as to the influence of fibrosis of the liver on ultrasonic attenuation, there has been some disagreement in the literaAddress correspondence to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan. 657
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4. A negative correlation between the water content of the liver and in vitro attenuation has been suggested (Bamber et al. 1981), but the water content was not measured in other studies. 5. The results of in vitro studies may be different from those of in vivo ones and thus difficult to interpret; the vasculature of resected specimens has collapsed and thus contains less blood, of which attenuation is low. On the other hand, postmortem specimens contain microbubbles, which may elevate attenuation. In the present study, to clarify the effect of the pathological state of the liver, namely fibrosis and fat, on ultrasonic attenuation, we used two experimental animal models. One is a liver fibrosis model without fatty infiltration and the other a fatty liver model without liver fibrosis. Ultrasonic data were obtained in vivo directly from the liver through an incision in the abdominal wall, and the determined attenuation coefficient was compared with the contents of fat, fibrosis and water, measured by quantitative methods. MATERIALS AND M E T H O D S
Materials Twenty-four male albino rabbits weighing 2.02.8 kg were used. Experimental fatty liver model (n = 6). Six rabbits were intravenously administered 80 mL/kg of 10% Intrafat (Daigo Nutritive Chemicals, Osaka, Japan) daily for 6 to 9 weeks. Intrafat is a fat emulsion for intravenous infusion, containing soybean oil 10.0 g, phospholipid 1.2 g and glycerol 2.5 g per 100 mL. Experimental liverfibrosis model (n -- 12). Equal volumes of carbon tetrachloride and olive oil were emulsified, and the rabbits received weekly subcutaneous injections of 0.2 mL/kg. Subcutaneous injections of 0.2 mL of 10% phenobarbital were given simultaneously to induce fibrosis in a shorter period (McLean et al. 1969). The rabbits were treated for 3 to 8 months to induce varying degrees of fibrosis. It is well-known that the administration of carbon tetrachloride causes acute liver damage, especially necrosis and steatosis (Cameron and Karunaratne 1936). However, these changes disappear in a short period (Cameron and Karunaratne 1936; Jorgensen et al. 1974; Parker and TuthiU 1986). Therefore, in order to avoid the influence of acute liver damage on ultrasonic attenuation, liver fibrosis model rabbits were subjected to ultrasonic data acquisition 10 days after the last administration of carbon tetrachloride. Normal controls (n = 6). Six rabbits which received no injections were used as controls.
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Instrumentation The equipment consisted of an A-mode transducer (nominal center frequency 10 MHz, 7 mm in diameter) (Shimadzu Corp., Kyoto, Japan), a pulserreceiver (Shimadzu Corp.), an A/D converter (R/ 390AD; SONY-TEKTRONIX, Tokyo, Japan), a personal computer (PC-9801 VM2; NEC, Tokyo, Japan) and an oscilloscope (T912; TEKTRONIX, Beaverton, OR, USA). A block diagram of the system is presented in Fig. 1. The focal length of the transducer was 16 mm, and the - 2 0 dB bandwidth extended from 6.3 MHz to 13.6 MHz, as was found from the spectrum of the signal reflected from a glass reflector immersed in water. A pulser-receiver was used to excite the transducer and linearly amplify the returning radio frequency (RF) waveforms. The output signals were digitized at a sampling rate of 60 MHz with a resolution of 10 bits with the A/D converter, transferred to the memory of the personal computer, and stored on a floppy disk. Nonlinear processing, such as log-amplification or sensitivity-time control gain was performed on the waveforms during data acquisition. Off-line analysis was applied to the waveforms using the personal computer. Data acquisition A rabbit was anesthetized with pentobarbital, and then its liver was exposed through a midline incision in the abdomen. The transducer was placed on the liver using a holder. Scanning gel was used to keep the transducer in contact with the liver. Ultrasonic waveforms from the liver were monitored with the oscilloscope. Care was taken to avoid specular echoes, such as from large vessels and biliary ducts, during data acquisition. One hundred A-lines were obtained moving the transducer, and each A-line was separated by 1-2 mm. Acoustic attenuation measurement It has already been shown that acoustic attenuation in soft tissues increases nearly linearly with frequency (Pauly and Schwan 1971). Although it is reported that attenuation does not exhibit absolutely linear dependence on frequency (Parker et al. 1988), the attenuation coefficient slope still provides an excellent parameter for practical bandwidths (Ophir et al. 1984). Many studies use the attenuation coefficient slope as a parameter of attenuation, and it is convenient to use the attenuation slope for a comparison with other studies. In this study, we used an acoustic attenuation coefficient slope obtained by the spectral difference method (Kuc 1980). The attenuation computation is shown schematically in Fig. 2. Two non-overlapping data segments were obtained
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for the waveforms. Both the near data segment and far data segment were Hamming windowed and fast Fourier transformed to yield power spectra. Then, the spectral difference between two segments was computed. It was assumed that the spectral difference was linear in the frequency range of 6.3-11.7 MHz, and the slope was determined by the least squares regression. The Frequency Dependent Attenuation (FDA) coet~cient slope was obtained by dividing the slope value by twice the distance between two segments.
It is known that the transducer focusing and diffraction affect the attenuation measurement. Beam correction using measured power spectra obtained from a perfect reflector, or tissue mimicking phantom at the same distance with the data segment of the target tissue has been proposed; however, remaining range-dependent bias errors were reported even after the beam correction (Insana et al. 1983; Wilson et al. 1987). In this study, beam correction was not performed. Instead, the distance of data segments from
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Fig. 2. Schematic presentation of the ultrasonic attenuation coefficient calculation. Two data segments are obtained for digitized ultrasonic signals (upper part). The signals inside each region of interest (ROI) are Hamming windowed, fast-Fourier transformed and then averaged to yield the corresponding averaged power spectra (middle part). By subtracting two averaged power spectra, a spectral difference was obtained (lower part), and a slope value was estimated by means of least squares regression. The Frequency Dependent Attenuation (FDA) coefficient slope, denoted by 8, was obtained by dividing the slope value by twice the distance between two segments.
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the transducer was fixed to avoid range-dependent attenuation estimation error. In addition, the two data segments were positioned so that diffraction effects were insignificant. The location of the two data segments was determined by a phantom experiment, in which a tissue mimicking graphite-gelatin phantom was used. First, attenuation of the phantom was measured by broadband insertion-loss technique (Ophir et al. 1984), which is a more accurate way of estimating attenuation. The transducer was positioned so that its beam was perpendicular to a glass reflector surface situated at the focus. FDA of the phantom was determined from the difference of the power spectrum of received echo signal with and without the phantom interposed between the transducer and the glass reflector. Second, FDA was also estimated from the backscattered echo signal from the phantom using the spectral difference method. Fifty A-lines, each of them separated by a sufficient distance to insure independent measurements, were acquired from the phantom by laterally moving the transducer. The length of the data segments (4.3 us, corresponding to 3.2 ram) was chosen to be short enough so that attenuation of acoustic energy within the region could be ignored. As to the separation of the two data segments, larger separation is preferable to reduce attenuation estimation error, but separation is limited by the size of the rabbit liver. In this study, separation of the two data segments was determined to be 15 #s, corresponding to 11.3 ram. FDA was computed by the spectral difference between averaged power spectrum of segment pairs with varying distance from the transducer. We found that segment pairs beginning 7.5-11 mm from the transducer were optimal, since computed FDA from these data coincided with that determined by insertion-loss techniques. In the rabbit experiment, FDA was calculated with the near data segment beginning 10 mm from the transducer and the far data segment beginning 21.3 mm from the transducer.
Assessment of the pathological state of the liver After ultrasonic data acquisition, a rabbit was sacrificed by means of an overdose injection ofpentobarbital, and then its liver was excised immediately. The liver was divided into several pieces, which were then subjected to histological examination and measurement of tissue components, namely, fat, fibrosis and water content. The histological examination was done at the location where data acquisition was performed. The histological slices were stained in hematoxylin and eosin, Sudan III and Azan-MaUory, and then examined by an experienced pathologist.
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Evaluation offatty infiltration of the liver Liver tissue was homogenized, and total lipid content of the liver was extracted using chloroform and methanol (Folch et al. 1957). This was expressed as the total lipid content per g wet weight of the liver.
Evaluation offibrosis of the liver For estimation of the collagen content of the liver specimens, the hydroxyproline content of the liver was measured by the method of Blumenkrantz (Blumenkrantz and Asboe-Hansen 1973). Liver tissue was homogenized and hydrolyzed overnight in 6 N HC1 in a sealed tube at 118°C. The hydrolysate was evaporated and then dissolved in a buffer. A sample containing hydroxyproline was oxidized with periodic acid. Color development was performed with p-dimethylaminobenzaldehyde and the absorbance was read at 565 nm.
Measurement of the water content of the liver After the wet weight of a liver specimen had been determined, the specimen was lyophilized at - 4 0 ° C for 48 h and then weighed. The water content of the liver was determined as follows: (wet weight - dry weight) × 100 (%). wet weight
Statistical methods Values are expressed as mean +_ standard deviation (SD). The unpaired t-test was used to compare the total lipid content, hydroxyproline content, water content and FDA between the experimental groups. Simple linear regression was performed to assess the relationship between FDA and the total lipid content, between FDA and the hydroxyproline content, and between FDA and the water content. RESULTS The livers of rabbits intravenously administered fat emulsion showed mild-to-moderate fatty infiltration, with some predisposition to the pericentral area (Fig. 3). Severe fatty infiltration, i.e., such that more than 60% of the hepatocytes contained fat droplets, was not produced in this experimental model. Apparent fibrosis was not observed. The livers of rabbits given chronic carbon tetrachloride injections showed pericentral and periportal fibrosis. Rabbits subjected to chronic carbon tetrachloride administration for more than six months showed the formation of nodules surrounded with thin fibrotic septa, indicating that liver cirrhosis was induced (Fig. 4). However, apparent fatty infiltration was not observed in Sudan III
Ultrasonic attenuation of liver • K. SuzuKI et
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Fig. 3. Photomicrograph of a hematoxylin and eosin stained liver sample obtained from a rabbit intravenously administered a fat emulsion. Large fat droplets can be observed mainly in the pericentral area.
stained sections (picture not shown). Apparent necrosis was not observed in the liver fibrosis model, either. The total lipid content of the liver in the fatty liver and liver fibrosis models is given in Fig. 5. The
fatty liver model shows a significantly elevated total lipid content (95.7 + 19.2 mg/g liver) compared with the normal controls (45.0 _+ 7.3 mg/g liver, p < 0.01) and the liver fibrosis model (39.1 _+ 5.5 mg/g liver, p <
Fig. 4. Photomicrograph of an Azan-Mallory stained liver sample obtained from a rabbit treated with carbon tetrachloride for more than six months. Regenerating nodules surrounded with thin fibrotic septa can be observed.
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Fig. 5. Total lipid content in the livers of the experimental fatty liver model and the fibrosis liver model of rabbits. The fatty liver model rabbits show a significantly elevated total lipid content. The total lipid content of the liver fibrosis model is not increased. Values are means _+ SD; • = p < 0.01, unpaired t-test.
0.01). As was suggested on histological examination o f Sudan III stained liver sections, the total lipid content was not elevated in the liver fibrosis model. The hydroxyproline content of the liver is given in Fig. 6. The liver fibrosis model showed a significantly elevated hydroxyproline content ( 1.49 _+ 1.11 mg/g liver) compared with the normal controls (0.22 ___0.1 mg/g liver, p < 0.01) and the fatty liver model (0.37 +- 0.18 mg/g liver, p < 0.01). In agreement with the histological findings for Azan-Mallory stained sections, the hydroxyproline content in the fatty liver model was not elevated. The water content o f the liver is given in Fig. 7. The water content in the liver fibrosis group (75.5 + 2.6%) was significantly elevated compared with normal controls (72.4 + 2.0%, p < 0.05) and the fatty liver group (66.0 +_ 3. 1%, p < 0.01 ). The water content in the fatty liver model was significantly lower than in the normal controls (p < 0.01). Here, we have obtained a fatty liver model without liver fibrosis and a liver fibrosis model without fatty infiltration. This indicates that we can evaluate the influence of fatty infiltration on FDA using the fatty liver model and normal controls, ignoring the influence of fibrosis. In addition, we can also evaluate the influence of liver fibrosis on FDA using the liver fibrosis model and normal controls, ignoring the influence of fatty infiltration. Figure 8 shows the FDA level in three experimental groups. The liver fibrosis group shows a tendency
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Fig. 6. Hydroxyproline content in the livers of the experimental fatty liver model and the fibrosis liver model of rabbits. The hydroxyproline content of the liver fibrosis model is increased, whereas that of the fatty liver model is not increased. Values are means _+ SD; * = p < 0.01, unpaired t-test. of elevated FDA (0.446 _+ 0.092 d B / c m / M H z ) compared with normal controls (0.374 + 0.060 d B / c m / MHz), but it does not reach a significant level (p <
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0.10). The fatty liver group exhibits larger FDA (0.688 + 0.110 d B / c m / M H z ) than the liver fibrosis group (p < 0.001) and normal controls (p < 0.001). Figures 9 and 10 show the results for the experimental fatty liver model and normal control groups. Figure 9 shows the relationship between the total lipid content and FDA in the fatty liver group including 0.8
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Fig. 10. Relationship between the attenuation coefficient value and the water content in the rabbit fatty liver model and normal controls. There is a negative correlation between the two. Y = -0.0355X + 2.988, r = -0.795, p < 0.01.
normal controls. FDA increases with the total lipid content of the liver. There is a good correlation between the total lipid content and FDA (r = 0.881, p < 0.001). In Figure 10, the water content of the liver shows an inverse relationship with FDA (r = -0.795, p < 0.01). However, in this experimental group, fatty infiltration of the liver is accompanied by decreased water content (correlation coefficient between total lipid content and water content is r = -0.856, p < 0.001, data not shown). It is suggested that FDA mainly depends on fat, since the correlation between FDA and fat is of greater significance. However, dependence of FDA on water content is unclear. There is the possibility that, to some extent, a negative correlation between FDA and water may reflect a negative correlation between fat and water. Figures I 1 and 12 show the results for the liver fibrosis model and normal controls. Figure 11 shows a good correlation between the hydroxyproline content and FDA (r = 0.868, p < 0.001). In Fig. 11, the livers with more than 1.0 mg/g liver ofhydroxyproline were cirrhotic. It is notable that both cirrhotic livers and non-cirrhotic livers are on the same regression line. This suggests that FDA elevation in fibrotic liver depends on the a m o u n t of collagen, not on the structure of the liver. Figure 12 exhibits a positive relationship between FDA and water content (r = 0.593, p < 0.01). The results in Fig. 12 are confusing, since water is essentially non-attenuating. In this experimental group, the water content is correlated with the hydroxyproline content (r = 0.595, p < 0.01, data not
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shown). Therefore, the inverse relationship between FDA and water content seems to reflect the fact that fibrotic liver contains increased water. DISCUSSION We have produced an experimental fatty liver model without fibrosis and an experimental liver fibrosis model without fatty infiltration. We evaluated fatty infiltration, fibrosis and the water content quantitatively, and clarified the dependence of acoustic attenuation on each tissue component of the liver. It is known that infusion of fat emulsion causes nutritional fatty liver, and large fat droplets are observed mainly around the pericentral area (Sherlock 1989). Our fatty liver model exhibits fat droplet distribution that closely resembles human nutritional fatty liver and exhibits no apparent fibrosis. Therefore, our fatty liver model is a suitable model for human nutritional fatty liver. In this study, the fatty liver model showed a 2- to 3-fold increase in the total lipid content compared to normal controls, while Bamber et al. (1981) reported that severe human fatty liver showed a 3- to 4-fold increase in fat. Therefore, our fatty liver model may correspond to mild-to-moderate human fatty infiltration of the liver. The histological examination also revealed mild-to-moderate fatty infiltration. Chronic administration of carbon tetrachloride is most frequently used to induce experimental liver cirrhosis model. It causes progressive fibrosis, from thin strands of fine fibers to apparent liver cirrhosis, according to the dose and duration of the administration (Cameron and Karunaratne 1936; McLean et al.
Volume 18, Number 8, 1992 1969; Tamayo 1983). In this liver cirrhosis model, features that closely resemble human liver cirrhosis, such as regenerating nodules surrounded by connective tissue septa, portal hypertension, ascites, and splenomegaly are observed (McLean et al. 1969; Tamayo 1983). In the case of the liver fibrosis model, the 6 liver cirrhosis rabbits showed an hydroxyproline content of 1.3-3.3 mg/g liver. On the other hand, normal rabbits showed an hydroxyproline content of less than 0.4 mg/g liver. These results agree with the report that human cirrhotic livers showed a 4- to 7-fold increase in the collagen content above normal (Tamayo 1983). Furthermore, the lipid content of the liver fibrosis model was not increased. Our experimental fatty liver model and liver fibrosis model seem to be adequate models of human fatty liver and liver fibrosis, respectively. When we compare Fig. 9 and Fig. 11, we can notice that FDA in fatty liver increases up to 0.8 dB/ cm/MHz, whereas that in the liver fibrosis model increases at most about 0.6 dB/cm/MHz. As mentioned above, our experimental fatty liver model shows mild-to-moderate fatty liver, but the experimental liver fibrosis model shows liver cirrhosis, which is a severe form of liver fibrosis. Therefore, it is suggested that fat has a larger effect on FDA than fibrosis. This result is in agreement with many other human studies, both in vivo and in vitro. Virtual unanimity exists as to the positive relationship between ultrasonic attenuation and the fat content of the liver. Attenuation of ultrasound is the sum of the energy losses due to reflection and scattering as well as absorption. In addition to the fact that fat itself absorbs ultrasound, fat
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Ultrasonic attenuation of liver • K. Suzulo et aL
droplets in the liver cause scattering. Therefore, the elevated attenuation of fatty infiltration may be due to both increased absorption and increased scattering of ultrasound. Furthermore, increased fatty infiltration leads to decreased water content, which is essentially non-attenuating for our range of parameters. This fact may also contribute to high FDA in fatty liver. Our study also clarified that FDA depends on fibrosis of the liver, but the dependence was rather low compared with that on fatty infiltration. This result agreed with the in vitro study of Linet al. (1988) and with the in vivo study of Wilson et al. (1987), both involving human liver. However, some studies revealed the absence of correlation or only weak correlation (Maklad et al. 1984; Taylor et al. 1986). Since the dependence of FDA on fibrosis is rather weak, it may be masked by imprecise estimation of both acoustic attenuation and the histological state. It seems reasonable that FDA increases with fibrosis, since collagen tissue has a different acoustic impedance from liver parenchyma and thus may act as an acoustic scattering structure. Furthermore, in our experimental model, liver fibrosis is accompanied by increased water content. That may be another reason why dependence of FDA on fibrosis is rather low. In human liver, Bamber et al. (1981) also reported that fibrotic liver is accompanied by increased water content. As to the influence of the water content on the attenuation, water content measurement should not be disregarded in an attenuation study, since water is a non-attenuating medium for ultrasound and is the largest component of the liver. Many authors have mentioned the importance of water (Afschrift et al. 1987; Bamber et al. 1981 ; Maklad et al. 1984; Wilson et al. 1987); however, only Bamber et al. (198 I) measured the water content of human liver in vitro. This is probably due to the difficulty in obtaining a sufficient volume of biopsy liver tissue to measure the water content. Bamber et al. (1981) quantitatively measured the water, collagen and fat contents of excised human fibrotic livers and fatty livers. Bamber et al. (1981) reported a positive correlation between fat content and attenuation, a positive correlation between collagen content and attenuation, and a negative correlation between water and attenuation. However, their conclusion as to the dependence of the attenuation coefficient on the tissue components was different from ours. They suggested that the positive correlation between attenuation and fat content was the result of the inverse relationship between the fat and water content, since the correlation between attenuation and water was of greater sig-
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nificance than that between attenuation and fat. Furthermore, they concluded that there is a barely significant relationship between attenuation and fibrosis after the correction of water content. Although the relationship between attenuation and each tissue component showed the same tendency with the results of Bamber et al. (1981). we concluded that attenuation depended mainly on fat and, to a lesser extent, on fibrosis. This discrepancy may be due to several differences between the two studies. The fibrotic liver tissue in their study was post-mortem metastatic liver tumors, and that in our study was an experimentally induced liver fibrosis model. We think that our liver fibrosis model is closer to in vivo human fibrotic livers, most of which are caused by chronic hepatitis or alcohol. Attenuation was measured in vitro in their study, but in vivo in our study. As to the dependence of attenuation on water content, it is difficult to assess, since water content changes with the change of fat content and collagen content. In a human study, Afschrift et al. (1987) measured FDA of normal liver of patients with chronic renal failure before and after hemodialysis. Although the patients had lost an average of 2.9% of their weight by withdrawal of fluid, FDA after hemodialysis was not significantly different from that before hemodialysis. It is suggested that a change in water content without a change in fat or fibrosis has a rather small effect on FDA. Regarding the clinical value of acoustic attenuation, it cannot discriminate liver fibrosis from fatty liver; however, it may be of value in cases such as the monitoring of fatty infiltration during the treatment of fatty liver or evaluation of the progress of fibrosis during follow-up of chronic hepatitis. On the other hand, acoustic attenuation increases with both fatty infiltration and fibrosis, which are the two most important findings observed in diffuse liver disease. Therefore, it can also be used as a means of screening for diffuse liver disease, although the pathology is unspecified. REFERENCES Afschrifi, M.; Cuvelier, C.; Ringoir, S.; Barbier, F. Influence of pathological state on the acoustic attenuation coefficient slope of liver. Ultrasound Med. Biol. 13:135-139; 1987. Bamber, J. C.; Hill, C. R.; King, J. A. Acoustic properties of normal and cancerous human liver--II. Dependence on tissue structure. Ultrasound Med. Biol. 7:135-144; 1981. Blumenkrantz, N.; Asboe-Hansen~ G. A quick and specific assay for hydroxyproline. Anal. Biochem. 55:288-291 ; 1973. Cameron, G. R.; Karunaratne, W. A. E. Carbon tetrachloride cirrhosis in relation to liver regeneration. J. Pathol. Bacteriol. 42:1-21; 1936. Duerinckx, A.; Rosenberg, K.; Hoers, J.; Aufrichtig, D.; Cole-Beuglet, C.; Kanel, G.; Lottenbcrg, S.; Ferrari, L. A. In vivo acoustic
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attenuation in liver: Correlations with blood tests and histology. Ultrasound Med. Biol. 14:405-413; 1988. Folch, J.; Lee, M.; Stanley, G. H. S. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509; 1957. Gosink, B. B.; Lemon, S. K.; Scheible, W.; Leopold, G. R. Accuracy ofultrasonography in diagnosis ofhepatocellular disease. Am. J. Roentgenol. 133:19-23; 1979. Insana, M.; Zagzebski,J.; Madsen, E. Improvements in the spectral difference method for measuring ultrasonic attenuation. Ultrason. Imaging 5:331-345; 1983. Jorgensen, M.; Norgaad, T.; Faarup, P. Functional structure of the cirrhotic rat liver. Acta Pathol. Microbiol. Scand. 82:13-20; 1974. Kuc, R. Clinical application of an ultrasound attenuation coefficient estimation technique for liver pathology characterization. IEEE Trans. Biomed. Eng. BME-27:312-319; 1980. Lin, T.; Ophir, J.; Potter, G. Correlation of ultrasonic attenuation with pathologic fat and fibrosis in liver disease. Ultrasound Med. Biol. 14:729-734; 1988. Maklad, N. F.; Ophir, J.; Balsara, V. Attenuation of ultrasound in normal liver and diffuse liver disease in vivo. Ultrason. Imaging 6:117-125; 1984. McLean, E. K.; McLean, A. E. M.; Sutton, P. M. Instant cirrhosis. Br. J. Exp. Pathol. 50:502-506; 1969. Needleman, L.; Kurtz, A. B.; Rifkin, M. D.; Cooper, H. S.; Pasto, M. E.; Goldberg, B. B. Sonography of diffuse benign liver disease: Accuracy of pattem recognition and grading. Am. J. Roentgenol. 146:1011-1015; 1986.
Volume 18, Number 8, 1992 Ophir, J.; Shawker, T. H.; Maklad, N. F.; Miller, J. G.; Flax, S. W.; Narayana, P. A.; Jones, J. P. Attenuation estimation in reflection: Progress and prospects. Ultrason. Imaging 6:349-395; 1984. Parker, K. J.; Tuthill, T. A. Carbon tetrachloride induced changes in ultrasonic properties of liver. IEEE Trans. Biomed. Eng. BME-33:453-460; 1986. Parker, K. J.; Asztely, M. S.; Lerner, R. M.; Schenk, E. A.; Waag, R. C. In vivo measurements of ultrasound attenuation in normal or diseased liver. Ultrasound Med. Biol. 14:127-136; 1988. Pauly, H.; Schwan, H. P. Mechanism of absorption of ultrasound in liver tissue. J. Acoust. Soc. Am. 50:692-699; 1971. Sandford, N. L.; Walsh, P.; Matis, C.; Baddeley, H.; Powell, L. W. Is ultrasonography useful in the assessment of diffuse parenchymal liver disease? Gastroenterology 89:186-191; 1985. Sherlock, S. Diseases of the liver and biliary system. Oxford: Blackwell Scientific Publications; 1989. Tamayo, R. P. Is cirrhosis of the liver experimentally produced by CC14 an adequate model of human cirrhosis? Hepatology 3:112-120; 1983. Taylor, K. J. W.; Riely, C. A.; Hammers, L.; Flax, S.; Weltin, G. Quantitative US attenuation in normal liver and in patients with diffuse liver disease: Importance of fat. Radiology 160:6571; 1986. Wilson, L. S.; Robinson, D. E.; Griffiths, K. A.; Manoharan, A.; Doust, B. D. Evaluation of ultrasonic attenuation in diffusediseases of spleen and liver. Ultrason. Imaging 9:236-247; 1987.
0301-5629/92 $5.00 + ,00 (c) 1992 Pergamon Press Lid.
OOriginal Contribution DEPENDENCE OF ULTRASONIC ATTENUATION OF LIVER ON PATHOLOGIC FAT AND FIBROSIS: EXAMINATION WITH EXPERIMENTAL FATTY LIVER AND LIVER FIBROSIS MODELS K. S u z u ~ , *
N . HAYASHI, t Y. SASAKI, t M . KONO, t A. KASAHARA, t H . FUSAMOTO, t Y. IMAI * a n d T. KAMADA t
tFirst Department of Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan, and *Research and Development Engineering, Process Instrumentation Division, Shimadzu Corporation, Kyoto 604, Japan (Received 23 October 1991; in final form 26 May 1992) Abstract--To clarify the effect of the pathological state of the liver on ultrasonic attenuation, we produced two experimental rabbit models. The influence of fat on ultrasonic attenuation was examined using a fatty liver model without liver fibrosis, and that of fibrosis on attenuation using a liver fibrosis model without fatty infiltration. Ultrasonic data were obtained in vivo directly from the liver, and an acoustic attenuation coefficient slope was obtained by the spectral difference method. Tissue components of the liver, namely the total lipid, hydroxyproline and water contents, were measured precisely by quantitative methods. We revealed that ultrasonic attenuation depends mainly on fatty infiltration of the liver and to a lesser extent on fibrosis, but not on the water content.
Key Words: Attenuation, Ultrasound, Rabbit liver, Experimental fatty liver model, Experimental liver fibrosis model, Fat content, Collagen content, Water content.
INTRODUCTION
ture. A positive correlation between attenuation and fibrosis has been reported (Afschrift et al. 1987; Duerinckx et al. 1988; Lin et al. 1988). However, some authors found little or no elevation of attenuation with fibrosis alone (Bamber et al. 1981; Taylor et al. 1986; Wilson et al. 1987), while others reported that the attenuation value varies with the etiology of the liver cirrhosis (Maklad et al. 1984). There are the following possible reasons for such contradictory findings: i. In a human study, fibrotic livers often exhibit varying degrees of fatty infiltration, and the fatty infiltration itself elevates attenuation, making interpretation of the attenuation results complicated. 2. In most human studies, the contents of fat and fibrosis were not quantitatively measured. Liver specimens were only classified into 3 or 4 groups based upon the microscopic evaluation of the fat and fibrosis by pathologists. In addition, precise grading is not always possible for a small biopsy specimen. 3. The influence of the abdominal wall on the ultrasonic beam and acoustic attenuation has not been thoroughly investigated, and they may affect the attenuation measurement.
Conventional gray scale B-mode ultrasound of the liver is a well-established modality in the diagnosis of focal lesions within the liver. However, diffuse parenchymal diseases of the liver, such as liver cirrhosis and fatty infiltration, are difficult to differentiate on the basis of B-mode imaging alone (Gosink et al. 1979; Sandford et al. 1985). Furthermore, sonographic grading of these diseases is not precise enough to predict the severity of the pathological changes (Needleman et al. 1986). The attenuation of ultrasound in the liver has received considerable attention as a quantitative ultrasonic tissue characterization technique. A positive correlation between fatty infiltration of the liver and attenuation has been reported by many researchers, both in vitro (Bamber et al. 1981; Lin et al. 1988) and in vivo (Afschrift et al. 1987; Duerinckx et al. 1988; Taylor et al. 1986; Wilson et al. 1987). However, as to the influence of fibrosis of the liver on ultrasonic attenuation, there has been some disagreement in the literaAddress correspondence to: Norio Hayashi, M.D., First Department of Medicine, Osaka University Medical School, 1-1-50 Fukushima, Fukushima-ku, Osaka 553, Japan. 657
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4. A negative correlation between the water content of the liver and in vitro attenuation has been suggested (Bamber et al. 1981), but the water content was not measured in other studies. 5. The results of in vitro studies may be different from those of in vivo ones and thus difficult to interpret; the vasculature of resected specimens has collapsed and thus contains less blood, of which attenuation is low. On the other hand, postmortem specimens contain microbubbles, which may elevate attenuation. In the present study, to clarify the effect of the pathological state of the liver, namely fibrosis and fat, on ultrasonic attenuation, we used two experimental animal models. One is a liver fibrosis model without fatty infiltration and the other a fatty liver model without liver fibrosis. Ultrasonic data were obtained in vivo directly from the liver through an incision in the abdominal wall, and the determined attenuation coefficient was compared with the contents of fat, fibrosis and water, measured by quantitative methods. MATERIALS AND M E T H O D S
Materials Twenty-four male albino rabbits weighing 2.02.8 kg were used. Experimental fatty liver model (n = 6). Six rabbits were intravenously administered 80 mL/kg of 10% Intrafat (Daigo Nutritive Chemicals, Osaka, Japan) daily for 6 to 9 weeks. Intrafat is a fat emulsion for intravenous infusion, containing soybean oil 10.0 g, phospholipid 1.2 g and glycerol 2.5 g per 100 mL. Experimental liverfibrosis model (n -- 12). Equal volumes of carbon tetrachloride and olive oil were emulsified, and the rabbits received weekly subcutaneous injections of 0.2 mL/kg. Subcutaneous injections of 0.2 mL of 10% phenobarbital were given simultaneously to induce fibrosis in a shorter period (McLean et al. 1969). The rabbits were treated for 3 to 8 months to induce varying degrees of fibrosis. It is well-known that the administration of carbon tetrachloride causes acute liver damage, especially necrosis and steatosis (Cameron and Karunaratne 1936). However, these changes disappear in a short period (Cameron and Karunaratne 1936; Jorgensen et al. 1974; Parker and TuthiU 1986). Therefore, in order to avoid the influence of acute liver damage on ultrasonic attenuation, liver fibrosis model rabbits were subjected to ultrasonic data acquisition 10 days after the last administration of carbon tetrachloride. Normal controls (n = 6). Six rabbits which received no injections were used as controls.
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Instrumentation The equipment consisted of an A-mode transducer (nominal center frequency 10 MHz, 7 mm in diameter) (Shimadzu Corp., Kyoto, Japan), a pulserreceiver (Shimadzu Corp.), an A/D converter (R/ 390AD; SONY-TEKTRONIX, Tokyo, Japan), a personal computer (PC-9801 VM2; NEC, Tokyo, Japan) and an oscilloscope (T912; TEKTRONIX, Beaverton, OR, USA). A block diagram of the system is presented in Fig. 1. The focal length of the transducer was 16 mm, and the - 2 0 dB bandwidth extended from 6.3 MHz to 13.6 MHz, as was found from the spectrum of the signal reflected from a glass reflector immersed in water. A pulser-receiver was used to excite the transducer and linearly amplify the returning radio frequency (RF) waveforms. The output signals were digitized at a sampling rate of 60 MHz with a resolution of 10 bits with the A/D converter, transferred to the memory of the personal computer, and stored on a floppy disk. Nonlinear processing, such as log-amplification or sensitivity-time control gain was performed on the waveforms during data acquisition. Off-line analysis was applied to the waveforms using the personal computer. Data acquisition A rabbit was anesthetized with pentobarbital, and then its liver was exposed through a midline incision in the abdomen. The transducer was placed on the liver using a holder. Scanning gel was used to keep the transducer in contact with the liver. Ultrasonic waveforms from the liver were monitored with the oscilloscope. Care was taken to avoid specular echoes, such as from large vessels and biliary ducts, during data acquisition. One hundred A-lines were obtained moving the transducer, and each A-line was separated by 1-2 mm. Acoustic attenuation measurement It has already been shown that acoustic attenuation in soft tissues increases nearly linearly with frequency (Pauly and Schwan 1971). Although it is reported that attenuation does not exhibit absolutely linear dependence on frequency (Parker et al. 1988), the attenuation coefficient slope still provides an excellent parameter for practical bandwidths (Ophir et al. 1984). Many studies use the attenuation coefficient slope as a parameter of attenuation, and it is convenient to use the attenuation slope for a comparison with other studies. In this study, we used an acoustic attenuation coefficient slope obtained by the spectral difference method (Kuc 1980). The attenuation computation is shown schematically in Fig. 2. Two non-overlapping data segments were obtained
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for the waveforms. Both the near data segment and far data segment were Hamming windowed and fast Fourier transformed to yield power spectra. Then, the spectral difference between two segments was computed. It was assumed that the spectral difference was linear in the frequency range of 6.3-11.7 MHz, and the slope was determined by the least squares regression. The Frequency Dependent Attenuation (FDA) coet~cient slope was obtained by dividing the slope value by twice the distance between two segments.
It is known that the transducer focusing and diffraction affect the attenuation measurement. Beam correction using measured power spectra obtained from a perfect reflector, or tissue mimicking phantom at the same distance with the data segment of the target tissue has been proposed; however, remaining range-dependent bias errors were reported even after the beam correction (Insana et al. 1983; Wilson et al. 1987). In this study, beam correction was not performed. Instead, the distance of data segments from
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Fig. 2. Schematic presentation of the ultrasonic attenuation coefficient calculation. Two data segments are obtained for digitized ultrasonic signals (upper part). The signals inside each region of interest (ROI) are Hamming windowed, fast-Fourier transformed and then averaged to yield the corresponding averaged power spectra (middle part). By subtracting two averaged power spectra, a spectral difference was obtained (lower part), and a slope value was estimated by means of least squares regression. The Frequency Dependent Attenuation (FDA) coefficient slope, denoted by 8, was obtained by dividing the slope value by twice the distance between two segments.
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the transducer was fixed to avoid range-dependent attenuation estimation error. In addition, the two data segments were positioned so that diffraction effects were insignificant. The location of the two data segments was determined by a phantom experiment, in which a tissue mimicking graphite-gelatin phantom was used. First, attenuation of the phantom was measured by broadband insertion-loss technique (Ophir et al. 1984), which is a more accurate way of estimating attenuation. The transducer was positioned so that its beam was perpendicular to a glass reflector surface situated at the focus. FDA of the phantom was determined from the difference of the power spectrum of received echo signal with and without the phantom interposed between the transducer and the glass reflector. Second, FDA was also estimated from the backscattered echo signal from the phantom using the spectral difference method. Fifty A-lines, each of them separated by a sufficient distance to insure independent measurements, were acquired from the phantom by laterally moving the transducer. The length of the data segments (4.3 us, corresponding to 3.2 ram) was chosen to be short enough so that attenuation of acoustic energy within the region could be ignored. As to the separation of the two data segments, larger separation is preferable to reduce attenuation estimation error, but separation is limited by the size of the rabbit liver. In this study, separation of the two data segments was determined to be 15 #s, corresponding to 11.3 ram. FDA was computed by the spectral difference between averaged power spectrum of segment pairs with varying distance from the transducer. We found that segment pairs beginning 7.5-11 mm from the transducer were optimal, since computed FDA from these data coincided with that determined by insertion-loss techniques. In the rabbit experiment, FDA was calculated with the near data segment beginning 10 mm from the transducer and the far data segment beginning 21.3 mm from the transducer.
Assessment of the pathological state of the liver After ultrasonic data acquisition, a rabbit was sacrificed by means of an overdose injection ofpentobarbital, and then its liver was excised immediately. The liver was divided into several pieces, which were then subjected to histological examination and measurement of tissue components, namely, fat, fibrosis and water content. The histological examination was done at the location where data acquisition was performed. The histological slices were stained in hematoxylin and eosin, Sudan III and Azan-MaUory, and then examined by an experienced pathologist.
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Evaluation offatty infiltration of the liver Liver tissue was homogenized, and total lipid content of the liver was extracted using chloroform and methanol (Folch et al. 1957). This was expressed as the total lipid content per g wet weight of the liver.
Evaluation offibrosis of the liver For estimation of the collagen content of the liver specimens, the hydroxyproline content of the liver was measured by the method of Blumenkrantz (Blumenkrantz and Asboe-Hansen 1973). Liver tissue was homogenized and hydrolyzed overnight in 6 N HC1 in a sealed tube at 118°C. The hydrolysate was evaporated and then dissolved in a buffer. A sample containing hydroxyproline was oxidized with periodic acid. Color development was performed with p-dimethylaminobenzaldehyde and the absorbance was read at 565 nm.
Measurement of the water content of the liver After the wet weight of a liver specimen had been determined, the specimen was lyophilized at - 4 0 ° C for 48 h and then weighed. The water content of the liver was determined as follows: (wet weight - dry weight) × 100 (%). wet weight
Statistical methods Values are expressed as mean +_ standard deviation (SD). The unpaired t-test was used to compare the total lipid content, hydroxyproline content, water content and FDA between the experimental groups. Simple linear regression was performed to assess the relationship between FDA and the total lipid content, between FDA and the hydroxyproline content, and between FDA and the water content. RESULTS The livers of rabbits intravenously administered fat emulsion showed mild-to-moderate fatty infiltration, with some predisposition to the pericentral area (Fig. 3). Severe fatty infiltration, i.e., such that more than 60% of the hepatocytes contained fat droplets, was not produced in this experimental model. Apparent fibrosis was not observed. The livers of rabbits given chronic carbon tetrachloride injections showed pericentral and periportal fibrosis. Rabbits subjected to chronic carbon tetrachloride administration for more than six months showed the formation of nodules surrounded with thin fibrotic septa, indicating that liver cirrhosis was induced (Fig. 4). However, apparent fatty infiltration was not observed in Sudan III
Ultrasonic attenuation of liver • K. SuzuKI et
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Fig. 3. Photomicrograph of a hematoxylin and eosin stained liver sample obtained from a rabbit intravenously administered a fat emulsion. Large fat droplets can be observed mainly in the pericentral area.
stained sections (picture not shown). Apparent necrosis was not observed in the liver fibrosis model, either. The total lipid content of the liver in the fatty liver and liver fibrosis models is given in Fig. 5. The
fatty liver model shows a significantly elevated total lipid content (95.7 + 19.2 mg/g liver) compared with the normal controls (45.0 _+ 7.3 mg/g liver, p < 0.01) and the liver fibrosis model (39.1 _+ 5.5 mg/g liver, p <
Fig. 4. Photomicrograph of an Azan-Mallory stained liver sample obtained from a rabbit treated with carbon tetrachloride for more than six months. Regenerating nodules surrounded with thin fibrotic septa can be observed.
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Volume 18, Number 8, 1992
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0.01). As was suggested on histological examination o f Sudan III stained liver sections, the total lipid content was not elevated in the liver fibrosis model. The hydroxyproline content of the liver is given in Fig. 6. The liver fibrosis model showed a significantly elevated hydroxyproline content ( 1.49 _+ 1.11 mg/g liver) compared with the normal controls (0.22 ___0.1 mg/g liver, p < 0.01) and the fatty liver model (0.37 +- 0.18 mg/g liver, p < 0.01). In agreement with the histological findings for Azan-Mallory stained sections, the hydroxyproline content in the fatty liver model was not elevated. The water content o f the liver is given in Fig. 7. The water content in the liver fibrosis group (75.5 + 2.6%) was significantly elevated compared with normal controls (72.4 + 2.0%, p < 0.05) and the fatty liver group (66.0 +_ 3. 1%, p < 0.01 ). The water content in the fatty liver model was significantly lower than in the normal controls (p < 0.01). Here, we have obtained a fatty liver model without liver fibrosis and a liver fibrosis model without fatty infiltration. This indicates that we can evaluate the influence of fatty infiltration on FDA using the fatty liver model and normal controls, ignoring the influence of fibrosis. In addition, we can also evaluate the influence of liver fibrosis on FDA using the liver fibrosis model and normal controls, ignoring the influence of fatty infiltration. Figure 8 shows the FDA level in three experimental groups. The liver fibrosis group shows a tendency
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Fig. 6. Hydroxyproline content in the livers of the experimental fatty liver model and the fibrosis liver model of rabbits. The hydroxyproline content of the liver fibrosis model is increased, whereas that of the fatty liver model is not increased. Values are means _+ SD; * = p < 0.01, unpaired t-test. of elevated FDA (0.446 _+ 0.092 d B / c m / M H z ) compared with normal controls (0.374 + 0.060 d B / c m / MHz), but it does not reach a significant level (p <
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Ultrasonic attenuation of liver • K. SuzuKI et al.
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0.10). The fatty liver group exhibits larger FDA (0.688 + 0.110 d B / c m / M H z ) than the liver fibrosis group (p < 0.001) and normal controls (p < 0.001). Figures 9 and 10 show the results for the experimental fatty liver model and normal control groups. Figure 9 shows the relationship between the total lipid content and FDA in the fatty liver group including 0.8
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normal controls. FDA increases with the total lipid content of the liver. There is a good correlation between the total lipid content and FDA (r = 0.881, p < 0.001). In Figure 10, the water content of the liver shows an inverse relationship with FDA (r = -0.795, p < 0.01). However, in this experimental group, fatty infiltration of the liver is accompanied by decreased water content (correlation coefficient between total lipid content and water content is r = -0.856, p < 0.001, data not shown). It is suggested that FDA mainly depends on fat, since the correlation between FDA and fat is of greater significance. However, dependence of FDA on water content is unclear. There is the possibility that, to some extent, a negative correlation between FDA and water may reflect a negative correlation between fat and water. Figures I 1 and 12 show the results for the liver fibrosis model and normal controls. Figure 11 shows a good correlation between the hydroxyproline content and FDA (r = 0.868, p < 0.001). In Fig. 11, the livers with more than 1.0 mg/g liver ofhydroxyproline were cirrhotic. It is notable that both cirrhotic livers and non-cirrhotic livers are on the same regression line. This suggests that FDA elevation in fibrotic liver depends on the a m o u n t of collagen, not on the structure of the liver. Figure 12 exhibits a positive relationship between FDA and water content (r = 0.593, p < 0.01). The results in Fig. 12 are confusing, since water is essentially non-attenuating. In this experimental group, the water content is correlated with the hydroxyproline content (r = 0.595, p < 0.01, data not
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shown). Therefore, the inverse relationship between FDA and water content seems to reflect the fact that fibrotic liver contains increased water. DISCUSSION We have produced an experimental fatty liver model without fibrosis and an experimental liver fibrosis model without fatty infiltration. We evaluated fatty infiltration, fibrosis and the water content quantitatively, and clarified the dependence of acoustic attenuation on each tissue component of the liver. It is known that infusion of fat emulsion causes nutritional fatty liver, and large fat droplets are observed mainly around the pericentral area (Sherlock 1989). Our fatty liver model exhibits fat droplet distribution that closely resembles human nutritional fatty liver and exhibits no apparent fibrosis. Therefore, our fatty liver model is a suitable model for human nutritional fatty liver. In this study, the fatty liver model showed a 2- to 3-fold increase in the total lipid content compared to normal controls, while Bamber et al. (1981) reported that severe human fatty liver showed a 3- to 4-fold increase in fat. Therefore, our fatty liver model may correspond to mild-to-moderate human fatty infiltration of the liver. The histological examination also revealed mild-to-moderate fatty infiltration. Chronic administration of carbon tetrachloride is most frequently used to induce experimental liver cirrhosis model. It causes progressive fibrosis, from thin strands of fine fibers to apparent liver cirrhosis, according to the dose and duration of the administration (Cameron and Karunaratne 1936; McLean et al.
Volume 18, Number 8, 1992 1969; Tamayo 1983). In this liver cirrhosis model, features that closely resemble human liver cirrhosis, such as regenerating nodules surrounded by connective tissue septa, portal hypertension, ascites, and splenomegaly are observed (McLean et al. 1969; Tamayo 1983). In the case of the liver fibrosis model, the 6 liver cirrhosis rabbits showed an hydroxyproline content of 1.3-3.3 mg/g liver. On the other hand, normal rabbits showed an hydroxyproline content of less than 0.4 mg/g liver. These results agree with the report that human cirrhotic livers showed a 4- to 7-fold increase in the collagen content above normal (Tamayo 1983). Furthermore, the lipid content of the liver fibrosis model was not increased. Our experimental fatty liver model and liver fibrosis model seem to be adequate models of human fatty liver and liver fibrosis, respectively. When we compare Fig. 9 and Fig. 11, we can notice that FDA in fatty liver increases up to 0.8 dB/ cm/MHz, whereas that in the liver fibrosis model increases at most about 0.6 dB/cm/MHz. As mentioned above, our experimental fatty liver model shows mild-to-moderate fatty liver, but the experimental liver fibrosis model shows liver cirrhosis, which is a severe form of liver fibrosis. Therefore, it is suggested that fat has a larger effect on FDA than fibrosis. This result is in agreement with many other human studies, both in vivo and in vitro. Virtual unanimity exists as to the positive relationship between ultrasonic attenuation and the fat content of the liver. Attenuation of ultrasound is the sum of the energy losses due to reflection and scattering as well as absorption. In addition to the fact that fat itself absorbs ultrasound, fat
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Ultrasonic attenuation of liver • K. Suzulo et aL
droplets in the liver cause scattering. Therefore, the elevated attenuation of fatty infiltration may be due to both increased absorption and increased scattering of ultrasound. Furthermore, increased fatty infiltration leads to decreased water content, which is essentially non-attenuating for our range of parameters. This fact may also contribute to high FDA in fatty liver. Our study also clarified that FDA depends on fibrosis of the liver, but the dependence was rather low compared with that on fatty infiltration. This result agreed with the in vitro study of Linet al. (1988) and with the in vivo study of Wilson et al. (1987), both involving human liver. However, some studies revealed the absence of correlation or only weak correlation (Maklad et al. 1984; Taylor et al. 1986). Since the dependence of FDA on fibrosis is rather weak, it may be masked by imprecise estimation of both acoustic attenuation and the histological state. It seems reasonable that FDA increases with fibrosis, since collagen tissue has a different acoustic impedance from liver parenchyma and thus may act as an acoustic scattering structure. Furthermore, in our experimental model, liver fibrosis is accompanied by increased water content. That may be another reason why dependence of FDA on fibrosis is rather low. In human liver, Bamber et al. (1981) also reported that fibrotic liver is accompanied by increased water content. As to the influence of the water content on the attenuation, water content measurement should not be disregarded in an attenuation study, since water is a non-attenuating medium for ultrasound and is the largest component of the liver. Many authors have mentioned the importance of water (Afschrift et al. 1987; Bamber et al. 1981 ; Maklad et al. 1984; Wilson et al. 1987); however, only Bamber et al. (198 I) measured the water content of human liver in vitro. This is probably due to the difficulty in obtaining a sufficient volume of biopsy liver tissue to measure the water content. Bamber et al. (1981) quantitatively measured the water, collagen and fat contents of excised human fibrotic livers and fatty livers. Bamber et al. (1981) reported a positive correlation between fat content and attenuation, a positive correlation between collagen content and attenuation, and a negative correlation between water and attenuation. However, their conclusion as to the dependence of the attenuation coefficient on the tissue components was different from ours. They suggested that the positive correlation between attenuation and fat content was the result of the inverse relationship between the fat and water content, since the correlation between attenuation and water was of greater sig-
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nificance than that between attenuation and fat. Furthermore, they concluded that there is a barely significant relationship between attenuation and fibrosis after the correction of water content. Although the relationship between attenuation and each tissue component showed the same tendency with the results of Bamber et al. (1981). we concluded that attenuation depended mainly on fat and, to a lesser extent, on fibrosis. This discrepancy may be due to several differences between the two studies. The fibrotic liver tissue in their study was post-mortem metastatic liver tumors, and that in our study was an experimentally induced liver fibrosis model. We think that our liver fibrosis model is closer to in vivo human fibrotic livers, most of which are caused by chronic hepatitis or alcohol. Attenuation was measured in vitro in their study, but in vivo in our study. As to the dependence of attenuation on water content, it is difficult to assess, since water content changes with the change of fat content and collagen content. In a human study, Afschrift et al. (1987) measured FDA of normal liver of patients with chronic renal failure before and after hemodialysis. Although the patients had lost an average of 2.9% of their weight by withdrawal of fluid, FDA after hemodialysis was not significantly different from that before hemodialysis. It is suggested that a change in water content without a change in fat or fibrosis has a rather small effect on FDA. Regarding the clinical value of acoustic attenuation, it cannot discriminate liver fibrosis from fatty liver; however, it may be of value in cases such as the monitoring of fatty infiltration during the treatment of fatty liver or evaluation of the progress of fibrosis during follow-up of chronic hepatitis. On the other hand, acoustic attenuation increases with both fatty infiltration and fibrosis, which are the two most important findings observed in diffuse liver disease. Therefore, it can also be used as a means of screening for diffuse liver disease, although the pathology is unspecified. REFERENCES Afschrifi, M.; Cuvelier, C.; Ringoir, S.; Barbier, F. Influence of pathological state on the acoustic attenuation coefficient slope of liver. Ultrasound Med. Biol. 13:135-139; 1987. Bamber, J. C.; Hill, C. R.; King, J. A. Acoustic properties of normal and cancerous human liver--II. Dependence on tissue structure. Ultrasound Med. Biol. 7:135-144; 1981. Blumenkrantz, N.; Asboe-Hansen~ G. A quick and specific assay for hydroxyproline. Anal. Biochem. 55:288-291 ; 1973. Cameron, G. R.; Karunaratne, W. A. E. Carbon tetrachloride cirrhosis in relation to liver regeneration. J. Pathol. Bacteriol. 42:1-21; 1936. Duerinckx, A.; Rosenberg, K.; Hoers, J.; Aufrichtig, D.; Cole-Beuglet, C.; Kanel, G.; Lottenbcrg, S.; Ferrari, L. A. In vivo acoustic
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attenuation in liver: Correlations with blood tests and histology. Ultrasound Med. Biol. 14:405-413; 1988. Folch, J.; Lee, M.; Stanley, G. H. S. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509; 1957. Gosink, B. B.; Lemon, S. K.; Scheible, W.; Leopold, G. R. Accuracy ofultrasonography in diagnosis ofhepatocellular disease. Am. J. Roentgenol. 133:19-23; 1979. Insana, M.; Zagzebski,J.; Madsen, E. Improvements in the spectral difference method for measuring ultrasonic attenuation. Ultrason. Imaging 5:331-345; 1983. Jorgensen, M.; Norgaad, T.; Faarup, P. Functional structure of the cirrhotic rat liver. Acta Pathol. Microbiol. Scand. 82:13-20; 1974. Kuc, R. Clinical application of an ultrasound attenuation coefficient estimation technique for liver pathology characterization. IEEE Trans. Biomed. Eng. BME-27:312-319; 1980. Lin, T.; Ophir, J.; Potter, G. Correlation of ultrasonic attenuation with pathologic fat and fibrosis in liver disease. Ultrasound Med. Biol. 14:729-734; 1988. Maklad, N. F.; Ophir, J.; Balsara, V. Attenuation of ultrasound in normal liver and diffuse liver disease in vivo. Ultrason. Imaging 6:117-125; 1984. McLean, E. K.; McLean, A. E. M.; Sutton, P. M. Instant cirrhosis. Br. J. Exp. Pathol. 50:502-506; 1969. Needleman, L.; Kurtz, A. B.; Rifkin, M. D.; Cooper, H. S.; Pasto, M. E.; Goldberg, B. B. Sonography of diffuse benign liver disease: Accuracy of pattem recognition and grading. Am. J. Roentgenol. 146:1011-1015; 1986.
Volume 18, Number 8, 1992 Ophir, J.; Shawker, T. H.; Maklad, N. F.; Miller, J. G.; Flax, S. W.; Narayana, P. A.; Jones, J. P. Attenuation estimation in reflection: Progress and prospects. Ultrason. Imaging 6:349-395; 1984. Parker, K. J.; Tuthill, T. A. Carbon tetrachloride induced changes in ultrasonic properties of liver. IEEE Trans. Biomed. Eng. BME-33:453-460; 1986. Parker, K. J.; Asztely, M. S.; Lerner, R. M.; Schenk, E. A.; Waag, R. C. In vivo measurements of ultrasound attenuation in normal or diseased liver. Ultrasound Med. Biol. 14:127-136; 1988. Pauly, H.; Schwan, H. P. Mechanism of absorption of ultrasound in liver tissue. J. Acoust. Soc. Am. 50:692-699; 1971. Sandford, N. L.; Walsh, P.; Matis, C.; Baddeley, H.; Powell, L. W. Is ultrasonography useful in the assessment of diffuse parenchymal liver disease? Gastroenterology 89:186-191; 1985. Sherlock, S. Diseases of the liver and biliary system. Oxford: Blackwell Scientific Publications; 1989. Tamayo, R. P. Is cirrhosis of the liver experimentally produced by CC14 an adequate model of human cirrhosis? Hepatology 3:112-120; 1983. Taylor, K. J. W.; Riely, C. A.; Hammers, L.; Flax, S.; Weltin, G. Quantitative US attenuation in normal liver and in patients with diffuse liver disease: Importance of fat. Radiology 160:6571; 1986. Wilson, L. S.; Robinson, D. E.; Griffiths, K. A.; Manoharan, A.; Doust, B. D. Evaluation of ultrasonic attenuation in diffusediseases of spleen and liver. Ultrason. Imaging 9:236-247; 1987.