MAGNETIC RESONANCE IN MEDICINE 19,
124-135 ( 1991)
In Vivo NMR, Biochemical, and Histologic Evaluation of AlcoholInduced Fatty Liver in Rat and a Comparison with CC14 Hepatotoxicity JOHND. HAZLE, * PONNADAA. NARAYANA, * 7tAND HAROLDA. DUNSFORDS
* Department of Radiology, The University of Texas Medical School, 6431 Fannin Street, Suite 2.132, Houston, Texas 77030, and $University of Texas Medical Branch, Department of Pathology, G-43, Galveston. Texas 77550 Received November 2, 1989; revised May 14, 1990 Magnetic resonance imaging (MRI) and spectroscopy (MRS) were used to follow the time course of ethanol-induced fatty liver in a group of 10 rats fed a diet containing I290 alcohol (ethanol) over a 5-week period. The MR data consisted of TI-weighted images, in vivo 'H spectra, and in vivo T , relaxation measurements. Changes in short TR images as a result of fatty accumulation were noted only as a slight increase in liver intensity relative to surrounding muscle. A poorly correlated ( r = 0.54) increase in water T I with time was observed. No statistically significant changes in lipid T Iwere found. MRS derived lipid content was compared with biochemically derived total lipids and histology. MRS determined liver lipids were found to increase linearly with time ( r = 0.9 1). Biochemically derived lipid content also increased with prolonged exposure to ethanol ( r = 0.96). The averagesof MRS derived lipid content agreed well with the average changesin biochemically determined total lipid concentration. Histologic examination revealed slight to moderate changes in fatty accumulation with significant variation in the group at the end of the study. On an individual basis the MRS and histologic evaluation were highly correlated ( r = 0.94). 0 1991 Academic Press, Inc. INTRODUCTION
The acute effects of ethanol on liver metabolism are well known ( 1,2). The oxidative metabolism of ethanol into acetaldehyde generates an abundance of reducing equivalents which tends to slow oxidative metabolism, thereby hampering electron transport. A secondary result of the reduced state of the liver is to inhibit the oxidation of fatty acid residues because of the decreased injection of acetyl coenzyme-A into the tricarboxylic acid (TCA) cycle. This leads to an accumulation of lipids, normally in the form of triglycerides, in the liver. A tertiary contribution to fatty accumulation is the result of increased lipolysis in adipose tissue due to hormonal changes (2, 3 ) . It would be clinically usefully to noninvasively distinguish fatty liver associated with early stage alcoholism from the more serious advanced stages which include hepatitis and cirrhosis. The differentiation of the various stages is to date determined either by indirect blood enzyme analysis or by invasive needle biopsy ( 4 ) . Although blood enzyme analysis is noninvasive, it is also nonconclusive in many cases. Needle biopsy
t To whom correspondence should be addressed. 0740-3194191 $3.00 Copynght 0 1991 by Academic Press, Inc. All nghts of reprcductiunIn any form reserved
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is conclusive; however, it must be performed under anesthesia. A small, but significant, mortality is associated with this procedure. X-ray computed tomography has been used in diagnosis as a supplemental procedure, but is not considered a conclusive diagnostic procedure ( 5 ) . As a result of the noninvasive nature of magnetic resonance (MR) it should be possible to follow progression or regression of this disease in individuals over long periods of time. Also, because in vivo studies do not require sacrifice of the animal for biochemical examination fewer animals are necessary to obtain a given statistical confidence. We recently demonstrated that the progression of liver disease associated with carbon tetrachloride ( CC14) hepatotoxicity in the rat may be successfully monitored using MRI and in vivo 'H magnetic resonance spectroscopy (6). The CCl, model was chosen to simulate ethanol-induced hepatitis and cirrhosis, stages difficult to achieve in rats by administering alcohol alone. The early, dramatic increase in liver triglycerides associated with C C 4 hepatotoxicity assessed using biochemical and histologic techniques were well correlated with the results of MR examination. Hypointense nodules in TI-weighted images and a decrease in MRS derived liver lipid content seemed to distinguish cirrhosis from fatty infiltration. The current study was initiated primarily to determine if the more subtle changes associated with ethanol-induced fatty liver could also be monitored using MR techniques. To this end, MR imaging, in vivo high resolution, MRS and in vivo T I measurementsof water and lipids were performed and correlated with biochemically derived total lipid content and histology. MATERIALS AND METHODS
Twenty-five male Sprague-Dawley rats weighing approximately 300 g were fed ad libitum a liquid diet containing 12% ethanol by volume for a period of 5 weeks (7). The animals were divided into two groups: Group 1 consisted of 10 animals that served as the MR study group and Group 2 consisted of 15 animals serving as weekly total lipid and histology specimens. MR studies were camed out weekly on all Group 1 animals. Anesthesia was induced using pentobarbital at a dose of 60 mg/kg. Images, in vivo 'H spectra, and in vivo TI measurements were made on a 2-T imager/spectrometer (8). The receiver coil was a saddle-shaped surface coil mounted on a heating block with a 95% D 2 0 sample imbedded under the coil to serve as a reference for lipid quantification ( 9 ) . A 15-cmdiameter slotted tube resonator was used as the transmitter. Short TR images (TE = 40 ms and TR = 700 ms) were obtained using standard spin-warp imaging techniques. Localized TI measurements were made using a stimulated echo (STE) localization (10-12) sequence preceded by a nonselective inversion pulse. The in vivo T , measurements were analyzed using an iterative technique ( 1 3 ) . Inversion delay intervals, T I , were varied from 10 ms to 10 s and all data were acquired using the following parameters: TE = 50 ms, TM = 10 ms, TR = 7000 ms, spectral width = 800 Hz, 1024 points, and eight averages. In vivo spectra for lipid quantification were also acquired using STE localization. Water suppression was achieved using a chemically selective inversion pulse centered on water with the inversion delay T I set to the T I null value determined for that
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particular animal ( 14). The inverted water magnetization was allowed to relax back into the transverse plane prior to executing the STE localization pulses, similar to the WEFT techniques ( 1 5 ) , thereby achieving maximum water suppression while minimally affecting other resonances. All acquisition parameters were the same as those for the T I relaxation measurements, with the exception of TR being reduced to 2500 ms. In order to quantitate the MRS determined lipid content, a 95% D,O sample was imbedded in the surface coil block. The reference also allowed for correction of dayto-day variations in scanner performance. A reference spectrum for each animal and time point was obtained by electronically moving the voxel into the 95% D20sample and obtaining a nonsuppressed spectrum. The integrated area under the lipid peak from the liver was then normalized relative to the integrated water peak from the reference. The machine sensitivity was found to to be very stable, with a standard error over the 5-week experiment of approximately 8%. Liver samples were removed immediately after euthanasia, histology specimens were obtained, and the remaining liver was quick frozen in liquid nitrogen. The liver samples were stored at -70°C prior to total lipid extraction. Lipid extraction was carried out following the technique of Folch et al. (16) using a ch1oroform:methanol solvent. Briefly, liver samples were extracted using a 20-fold volume of 2: 1 chloroform: methanol solvent during homogenization. The solution was paper filtered to remove rough debris. A 0.2-vol of water was added to remove hydrophilic components. The solution was then centrifuged at 2500 rpm for 20 min to separate fractions. The upper hydrophilic fraction was removed by suction and the interface washed three times. The lower fraction was dried to constancy and weighed. Total lipid concentration was expressed as milligrams of residual lipids per gram of liver. Histology slides were prepared by fixing slices of liver tissue in 10%buffered formalin, dehydrating in graded alcohol, and imbedding in paraffin blocks. The blocks were sectioned at 6 Fm. Sections were stained with hematoxylin and eosin ( H & E). The slides were examined and photomicrographs taken on a Nikon photomicroscope. Semiquantification of fatty change was based on examination of four slides per liver per animal aided by an ocular micrometer with a grid divided into 100 squares. Grading was performed using an ascending scale of 1 to 4 based on the following: I+, fatty change involving a few cells around the central vein; 2+, fatty change involving hepatocytes around the central vein extending one-third of the way toward the portal area; 3+, fatty change extending from the central vein to two-thirds of the way to the portal area; and 4, fatty change extending from the central vein to the portal area. A statistical analysis was performed using the Student’s t-test to determine if differences in the experimental time points were significant from those of the controls. All means were tested at the 95% confidence level ( p < 0.05). The significance of regression slopes was also tested using t-statistics at the 95% confidence level ( 17). RESULTS
The quality of localization is demonstrated in Fig. 1 by simultaneously showing (A) a standard short TR image and (B) a localized image from the same animal. The landmarks on the standard image include the spinal cord (SC), sternum (ST), and a partial volume of the stomach (SM). Due to the relatively homogeneous nature of
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FIG. 1. Axial (A) standard 2D spin-warp and (B) STE localized images of normal rat liver. The following anatomical landmarks are identified: spinal cord (SC), sternum (ST), and stomach (SM). A partial volume of stomach was included in the localized image for anatomical landmarking. The stomach was avoided in the spectroscopic studies. Acquisition parameters were TE = 40 ms, 700 ms, 10 cm FOV, and four averages. The mixing time T Mfor the STE image was 10 ms.
the liver, a partial volume of stomach was included in the images for anatomical landmarking. It should also be noted that the slice thickness for the localized image was 15 mm, while the slice thickness for the standard image was 5 mm. The increased effect of partial volume averaging is clearly demonstrated in the localized image. For spectroscopic studies the stomach was avoided. Both images were acquired with TE = 40 ms, TR = 750 ms, FOV = 10 cm, and four averages. The localized image was acquired with TM = 10 ms. Weekly MR images for a typical rat are shown in Fig. 2. The images were acquired from approximately the same location in each examination. Apparent differences in positioning as judged from different partial volumes of stomach visible are due in most part to the feeding status of the animal at the time of examination, i.e., the volume of the stomach content. The short TR images show a slight to moderate increase in liver intensity relative to surrounding skeletal muscle over the course of these studies. Only diffuse fatty infiltration is noted with no nodule formation as observed with more severe hepatotoxins (18, 19). The TI relaxation data for both water and lipids were found to be monoexponential. In a few cases the lipid recovery appeared to overshoot the equilibrium value. This phenomenon was seen less than 25% of the time and is typical of cross-relaxation behavior in solutions (20). The average water and lipid T I values as a function of ethanol administration are given in Table 1. The water T , shows a slight increase with time; however, the data were poorly correlated ( Y = 0.54) and only three of the five time points were different from those of the control. Control lipid T I values were not obtained due to unreliably small lipid signals in nonsuppressed spectra in this group of rats. The control lipid TI values from a separate experiment (6) using the same strain of rats and NMR acquisition parameters were used as a best estimate for the control lipid T I for this group. The lipid T I values increased from a control value of 283 ms to 385 ms on Day 8. This was followed by a decrease to 285 ms on Day 15,
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FIG.2. Short TR axial images of the liver of a typical rat obtained weekly for 5 weeks. The progression of fatty liver is demonstrated by a slight overall increase in liver intensity with increased exposure to ethanol. ( A ) Control, ( B ) 1 week, (C) 2 weeks, (D) 3 weeks, (E) 4 weeks, and ( F ) 5 weeks.
different fron the Day 8 value, but not significantly different from that of the control, after which time no significant changes were seen. A typical set of water-suppressed spectra used in the calculation of lipid /reference ratios is shown in Fig. 3. The spectra were normalized using the reference so that changes in lipid peak ( 1.25 ppm) area correspond to changes in MRS observable lipids. The lipid methylene resonance is seen at 1.25 ppm, with the residual water peak at 4.75 ppm. The variation in the amplitude of the residual water peak in the different plots reflects the degree of water suppression achieved in a given session and does not affect the lipid quantification. The use of a reference permits consistent estimation of the lipid signal irrespective of changes in water suppression efficiency. A plot of average MRS derived lipid/reference values as a function of ethanol administration is given in Fig. 4a. The lipid/reference ratios ranged from 0.8 for control animals to 7.5 by Day 35. A generally smooth upward trend with good correlation was observed ( r = 0.9 1 ).
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TABLE 1 Water and Lipid T , Relaxation Data Relaxation time (ms) Water
Lipid
Days of ethanol
Average
St. error
Average
St. error
Control 7 days 15 days 22 days 29 days 36 days
907 1061" 950 11 18" 1014 1053a
36 52 54 86 84 71
283 385 a 285 320 298 313
38 12 25 14 17
14'
Averages that are significantly different from control ( p < 0.05). From a previous study; see text for details.
Average total lipid content determined biochemically using the Folch technique is shown in Fig. 4b. Again, a well-correlated upward trend ( r = 0.94) was found as the lipid concentration increased from the control value 43 mg/g to 74 mg/g on Day 35. Histology slides of typical normal, 8, 15,2 1, and 35 day alcohol animals are shown in Fig. 5. Sections of liver from Day 8 showed a mild degree of fatty change in hepatocytes around the central vein. Fatty change was in the form of accumulations of lipid within the vesicles in the cytoplasm (Fig. 5B). The number of hepatocytes with fatty change increased at the later time points extending out from the central vein to the portal area by the end of the fifth week. A progressive increase oflipid accumulation in nonparenchymal cells was also noted (Fig. 5C). At least one animal from each
L 8
6
4
2
0
-
2
-
4
PPm FIG. 3. Typical set of water-suppressed spectra acquired weekly from one of the Group 1 animals. These spectra were used to determine MRS observable lipid content. The control spectrum is at the bottom of the figure with the weekly spectrum progressingupward. The lipid methylene resonance appears at approximately 1.25 ppm, while the residual water signal is seen at 4.75 ppm. The spectra were acquired using TE = 50 ms, TM = 10 ms, TR = 2500 ms, 1024 points, and 128 averages.
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)
Days of Ethanol
FIG.4. Changes in liver lipid content are demonstrated by various methodologies. (a) MRS determined lipid changes relative to the 95% D20 reference, (b) total lipid content as determined using the Folch technique expressed in milligrams extracted lipids per gram liver, and (c) histologically determined lipid content. Error bars indicate one standard error of the mean. The number of animals is given in parentheses, and time points different ( p < 0.05) from control are shown with an asterisk.
time period for days 15 through 35 demonstrated hepatocytes with macro aggregates of fat giving the appearance of lipocytes (Fig. 5D). The average histology grade as a function of days on the protocol is shown in Fig. 4c. The correlation of these data was found to be slightly worse ( r = 0.8 I ) on average than the MRS and biochemically derived lipids, but still showed an upward trend. In order to determine the correlation between the MR and biochemistry or histology results for individual animals, MRS derived lipid content was plotted against lipid concentration (Fig. 6 ) and histology grade (Fig. 7). On an individual basis the MRS and biochemically determined lipids were not well correlated ( r = 0.52). However, the histology grade did appear to be very consistent with the MRS determined lipid content with a high degree of correlation ( r = 0.94). The fatty changes observed by all three experimental methods with this regimen of ethanol are much less severe than those observed as a result of CC14 administration (6). The relative changes in MRS observable lipids for this study are compared to the changes observed during CC4 administration by plotting the lipid / reference ratios from the two studies on the same scale (Fig. 8). Presenting the data in this manner elucidates the more dramatic changes in MRS visible lipids as a result of CC14 (Days 2 through 29) or CC14 phospholipase-D (CC4 PLD) administration relative to alcohol. The lipid/reference ratios increased by as much as a factor of 6 in the alcohol study. Similar changes in the biochemically derived lipid concentrations for the two
+
+
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FIG. 5. Typical histology slides of liver sections from normal and ethanol-treated rats. All slides were stained with hematoxylin and eosin and magnified to 320X. Central veins are indicated by C and portal areas by P. (A) Normal liver section. ( B ) Liver section obtained after 8 days on the alcohol diet showing mild microvesicular fatty changes (arrows). (C) A slight increase is noted in hepatocytes (small arrow) and nonparenchymal cells (large arrow on Day 1 5 ) . ( D ) Microvesicular fatty change (small arrow) and macro accumulations of fat in hepatocytes producing “lipocyte-like” cells (large arrow) were observed after 2 1 days. (E-F) Marked fatty change around the central vein extending to the portal areas (P) was visible after 35 days on the alcohol diet (arrows).
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A
A
A
A
A 60
A
A
A A
I
A
100
80
Lipid Concentration (rng/g)
FIG. 6 . MRS determined lipid content for the individual Day 35 rats against biochemically derived lipid concentration. Only a moderate degree of correlation was found ( I = 0.52). FIG. 7. Average MRS lipid content for the 35 day rats against histologic grade along with the +95% confidence limits. The values in parentheses indicate the number of animals for each histology grade. A correlation coefficient of 0.94 was obtained.
studies is seen from Fig. 9. In this case the maximum increase of lipid concentration for the CC14 PLD study is a factor of approximately 4, while the maximum increase from alcohol administration is only about factor of 1.5. Although the histologic changes for the two studies were not graded (semiquantitatively) on the same scale, the changes associated with CC14 ( 6 ) are much more severe when histology slides from the two studies are compared. When the quantified MRS observable lipids as a function of biochemically determined total lipid concentration from both studies were compared, no correlation was found. This indicates that for a given lipid concentration both studies gave approxi-
+
A-A
CCI,
+ -+
FtOH
-
0--0
CCI
7-
V
EIOH
cn 190,
@
Days on Protocol
@
Days o n Protocol
FIG. 8. Relative changes in MRS determined lipid/reference ratios acquired during this study and a previous study evaluating the effects of C C 4 ( 6 ) . FIG. 9. Biochemically obtained lipid concentrations for this study and a previous study evaluating the effects of CCll (6) demonstrate the significantlylesser effects of alcohol on liver lipid content. The error bars represent one standard error.
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mately the same change in MRS determined lipid content. Regardless of differences in the mechanisms which induce fatty liver for alcohol or C C 4 ,the MRS observable lipid fraction appears to be the same in each case. DISCUSSION
Although the effects of moderate durations of ethanol administration are well known ( 1 - 4 ) , the induction of fatty liver disease has not been thoroughly evaluated using in
vivo MR techniques. We chose the alcohol model for fatty liver because the relatively slow accumulations of lipids in the liver (compared to other hepatotoxins, such as carbon tetrachloride) would allow us to determine if subtle changes in liver lipid content could be followed over a period of time with MR. The short TR images demonstrated only mild changes in liver intensity relative to surrounding tissue. This slight increase in overall liver intensity was noted in the final weeks of the 5-week experiment with no observable structural changes. This is not unexpected since the duration of alcohol administration was not long enough to induce the inflammatory and fibrotic changes normally associated with later stage disease, potentially detectable with MRI. No inflammatory or fibrotic changes were noted in the histologic examination of these animals. It does appear, however, that MRS relaxation measurements could potentially be of interest in the study of more chronic ethanol hepatotoxicity. Although the only significant changes in water or lipid TI appeared as a higher value for lipid TI on Day 8, there did appear to be a trend of increasing water T Iwhich could manifest itself as useful indicator in experiments of longer duration. An acute increase in water or composite liver TI has been reported for CC14hepatotoxicity and is usually associated with changes in intracellular “free” water content ( 6 , 18, 19, 21, 22). Prolonged administration of ethanol would be expected to produce inflammatory changes which are believed to result in increased “free” water content and possibly increased water T I . Average quantitative changes in liver lipids determined using MR spectroscopic techniques were well correlated with changes in biochemically derived lipid concentrations. Average MRS findings also agreed well with histologic lipid content grading. On an individual basis, MRS results did not correlate well with biochemical results (Fig. 6 ) . The sensitivity of the biochemically derived concentration could no doubt be improved using chromatographic techniques to separate the various components. However, the individual MRS results were well correlated (Y = 0.94)with the histologic data (Fig. 7). This is not unexpected as the biochemical lipid extraction has a substantial baseline, the average control lipid concentration was 48 mg/g, and the lipid concentration increased only up to 80 mg/g by the end of the study. Individual changes could easily be masked by experimental uncertainty. The mechanism of lipid accumulation in the liver during alcohol administration using this diet is felt to be primarily due to the replacement of alcohol for carbohydrates as the main energy source in the liver cell. This results in the generation of excess reducing equivalents from the oxidation of alcohol in the form of NADH, which tends to slow down fatty acid oxidation ( I ). The decreased fatty acid oxidation leads to enhanced synthesis of lipoproteins and triglycerides which also contribute to the fatty change (23). Hormonally induced lipolysis in adipose tissue giving rise to increased
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supplies of fatty acids to the liver also contributes to the generation of fatty liver ( 3 ) . Although the extent of fatty change during alcohol administration is known to be influenced by diet (24, 2 5 ) , the liquid diet chosen for our studies has been shown not to affect lipid content (7). On the other hand, the mechanism of lipid accumulation in the liver as a result of CC14 insult is felt to be due to the production of the trichloromethyl radical ( 2 3 ) . Lipid peroxidation induced by this mechanism leads to the destruction of microsomal proteins, including cytochrome P-450 (26, 2 9 ) . These changes lead to the inability of the liver cells to synthesize lipoproteins which are needed to remove tnglycerides from the cell. Histopathologically this is seen as an accumulation of triglycerides in the cytoplasm of hepatocytes, especially around the central vein ( 3 0 ) . Further evidence to support the position that the mechanisms of fatty liver induction are different for the two compounds is provided by the observation that C C 4enhances the injury initiated by alcohol in rats (30-32). In fact, the enhanced fibrogenesis has suggested to some investigatorsthat EtOH alone may not be sufficient to cause cirrhosis in humans, but may require a propagator such as CC4 (31) . ACKNOWLEDGMENTS The authors thank Dr. John H. Hams, Jr., for his continued support, Drs. Robert Guynn and Paul Rosevear for use of laboratory facilities to perform the total lipid extractions, Dr. John Hunt for assistance in preparing the histology specimens, and Mr. David Menill for assistance in performing the lipid extractions. This work was supported in part by the John S. Dunn Foundation and NIH Grant 34635. REFERENCES 1. C. S. LEIBER,Med. Clin. N. Amer. 668, 3 (1982). 2. C. S. LIEBER,in “Biochemical Mechanisms of Liver Injury” (T. F. Slater, Ed.), Academic Press, New York, 1978. 3. M. C. GEOKAS, C. S. LIEBER,S. FRENCH,AND C. H. HALSTED,Ann. Internal Med. 95, 198 ( 1981 ). 4. E. ALBANO, G. POLI,A. TOMASI,L. GORIA-GATTI, AND M. U. DIANZANI, in “Pathophysiology of the Liver,” Excerpta Medica, Amsterdam, 1988. 5. THECLINICAL NMR GROUP(ABERDEEN), Clin. Rudiol. 38,495 ( 1987). 6. J. D. HAZLE,P. A. NARAYANA, AND H. A. DUNSFORD, Mugn. Reson. Med. 15, 21 1 ( 1990). 7. C. S. LIEBER AND L. M. DECARLI, Alcoholism 10, 550 (1986). 8. P. A. NARAYANA, J. L. DELAYRE, AND L. K. MISRA,Magn. Reson. Med. 3,549 (1986). 9. J. D. HAZLE,P. A. NARAYANA, AND W. A. KUDRLE,J. Magn. Reson. 83, 595 ( 1989). 10. J. GRANOT,J . Mugn. Reson. 70,488 (1986). 11. R. KIMMICH AND D. HOEPFEL,J. Mugn. Reson. 72, 379 (1987). 12. J. FRAHM,K. D. MERBOLDT, AND W. HANICKE, J. Magn. Reson. 72, 502 (1987). 13. R. GERHARDS AND W. DIETRICH, J. Mugn. Reson. 23, 21 (1976). 14. P. A. NARAYANA, E. F. JACKSON, J. D. HAZLE,L. K. FOTEDAR, M. V. KULKARNI, AND D. P. FLAMIG, Magn. Reson. Med. 8, 151 (1988). 15. S. L. PATTANDB. D. SYKES,J. Chem. Phys. 56,3182 (1972). 16. J. FOLCH,M. LEES,AND G. H. SLOANE-STANLEY, J. Bid. Chem. 226,497 ( 1957). 17. J. H. ZAR,“Biostatistical Analysis,” 2nd ed., Prentice-Hall, Englewood Cliffs, NJ, 1984. IS. D. D. STARK,H. M. BASS,A. A. Moss, B. R. BACON,J. H. MCKERROW,C. E. CANN,A. BRITO,AND H. 1. GOLDBERG, Radiology 148, 743 (1983). 1Y. A. V. RATNER,E. A. CARTER, G. M. POHOST,AND J. R. WANDS,Ale. Clin. Exp. Re.?.10,24 1 ( 1986). 20. J. C. GORE,M. S. BROWN,AND I. M. ARMITAGE, Mugn. Reson. Med. 9,333 ( 1989). 21. B. R. ROSEN,E. A. CARTER,AND I. L. PYKETT,Radiology 154,469 (1985).
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22. M. BRAUER,R. A. TOWNER, 1. RENAUD, E. G. JANZEN, AND D. L. FOXALL, Magn. Reson. Med. 9, 229 (1989). 23. R. BLOMSTRANDAND L. L. DRAGER, L$e Sci'. 13, 1131 (1973). 24. C. S. LIEBERAND L. M. DECARLI,Alcoholism (NY) 6, 523 ( 1982). 25. H. TSUKAMOTO, S. J. TOWNER,L. M. CIOFALO, AND S. W. FRENCH, Hepatofogy 6,814 ( 1986). 26. R. 0. RECKNAGEL AND E. A. GLENDE, JR., Crit. Rev. Toxicol. 2,263 ( 1981). 27. G. D. NADKARNI AND N. B. DSOUZA,Biochem. Med. Metab. Bid. 40,42 ( 1988). 28. E. A. GLENDE,JR., A. M. HRUSZKEWYCZ, AND R. 0. RECKNAGEL, Biochem. Pharmacol. 25, 2163 (1976). 29. U. FLANDER, W. HAAS,AND H. KRONER,Exp. Mol. Pathol. 36,34 (1982). 30. J. V. BRUCKNER, W. F. MACKENZIE, S. MURALIDHARA, R. LUTHRA,G. M. KYLE,AND D. ACOSTA, Fundam. Appl. Toxicol. 6, 16 (1986). 31. A. BOSMA,A. BROUWER, W. F. SEIFERT, AND D. L. KNOOK,J. Pathol. 156, 15 (1988). 32. M. YOUNES, W. REICHL,A N D c . P.SIEGERS, Xenobiotica 13, 47 (1983).
124-135 ( 1991)
In Vivo NMR, Biochemical, and Histologic Evaluation of AlcoholInduced Fatty Liver in Rat and a Comparison with CC14 Hepatotoxicity JOHND. HAZLE, * PONNADAA. NARAYANA, * 7tAND HAROLDA. DUNSFORDS
* Department of Radiology, The University of Texas Medical School, 6431 Fannin Street, Suite 2.132, Houston, Texas 77030, and $University of Texas Medical Branch, Department of Pathology, G-43, Galveston. Texas 77550 Received November 2, 1989; revised May 14, 1990 Magnetic resonance imaging (MRI) and spectroscopy (MRS) were used to follow the time course of ethanol-induced fatty liver in a group of 10 rats fed a diet containing I290 alcohol (ethanol) over a 5-week period. The MR data consisted of TI-weighted images, in vivo 'H spectra, and in vivo T , relaxation measurements. Changes in short TR images as a result of fatty accumulation were noted only as a slight increase in liver intensity relative to surrounding muscle. A poorly correlated ( r = 0.54) increase in water T I with time was observed. No statistically significant changes in lipid T Iwere found. MRS derived lipid content was compared with biochemically derived total lipids and histology. MRS determined liver lipids were found to increase linearly with time ( r = 0.9 1). Biochemically derived lipid content also increased with prolonged exposure to ethanol ( r = 0.96). The averagesof MRS derived lipid content agreed well with the average changesin biochemically determined total lipid concentration. Histologic examination revealed slight to moderate changes in fatty accumulation with significant variation in the group at the end of the study. On an individual basis the MRS and histologic evaluation were highly correlated ( r = 0.94). 0 1991 Academic Press, Inc. INTRODUCTION
The acute effects of ethanol on liver metabolism are well known ( 1,2). The oxidative metabolism of ethanol into acetaldehyde generates an abundance of reducing equivalents which tends to slow oxidative metabolism, thereby hampering electron transport. A secondary result of the reduced state of the liver is to inhibit the oxidation of fatty acid residues because of the decreased injection of acetyl coenzyme-A into the tricarboxylic acid (TCA) cycle. This leads to an accumulation of lipids, normally in the form of triglycerides, in the liver. A tertiary contribution to fatty accumulation is the result of increased lipolysis in adipose tissue due to hormonal changes (2, 3 ) . It would be clinically usefully to noninvasively distinguish fatty liver associated with early stage alcoholism from the more serious advanced stages which include hepatitis and cirrhosis. The differentiation of the various stages is to date determined either by indirect blood enzyme analysis or by invasive needle biopsy ( 4 ) . Although blood enzyme analysis is noninvasive, it is also nonconclusive in many cases. Needle biopsy
t To whom correspondence should be addressed. 0740-3194191 $3.00 Copynght 0 1991 by Academic Press, Inc. All nghts of reprcductiunIn any form reserved
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is conclusive; however, it must be performed under anesthesia. A small, but significant, mortality is associated with this procedure. X-ray computed tomography has been used in diagnosis as a supplemental procedure, but is not considered a conclusive diagnostic procedure ( 5 ) . As a result of the noninvasive nature of magnetic resonance (MR) it should be possible to follow progression or regression of this disease in individuals over long periods of time. Also, because in vivo studies do not require sacrifice of the animal for biochemical examination fewer animals are necessary to obtain a given statistical confidence. We recently demonstrated that the progression of liver disease associated with carbon tetrachloride ( CC14) hepatotoxicity in the rat may be successfully monitored using MRI and in vivo 'H magnetic resonance spectroscopy (6). The CCl, model was chosen to simulate ethanol-induced hepatitis and cirrhosis, stages difficult to achieve in rats by administering alcohol alone. The early, dramatic increase in liver triglycerides associated with C C 4 hepatotoxicity assessed using biochemical and histologic techniques were well correlated with the results of MR examination. Hypointense nodules in TI-weighted images and a decrease in MRS derived liver lipid content seemed to distinguish cirrhosis from fatty infiltration. The current study was initiated primarily to determine if the more subtle changes associated with ethanol-induced fatty liver could also be monitored using MR techniques. To this end, MR imaging, in vivo high resolution, MRS and in vivo T I measurementsof water and lipids were performed and correlated with biochemically derived total lipid content and histology. MATERIALS AND METHODS
Twenty-five male Sprague-Dawley rats weighing approximately 300 g were fed ad libitum a liquid diet containing 12% ethanol by volume for a period of 5 weeks (7). The animals were divided into two groups: Group 1 consisted of 10 animals that served as the MR study group and Group 2 consisted of 15 animals serving as weekly total lipid and histology specimens. MR studies were camed out weekly on all Group 1 animals. Anesthesia was induced using pentobarbital at a dose of 60 mg/kg. Images, in vivo 'H spectra, and in vivo TI measurements were made on a 2-T imager/spectrometer (8). The receiver coil was a saddle-shaped surface coil mounted on a heating block with a 95% D 2 0 sample imbedded under the coil to serve as a reference for lipid quantification ( 9 ) . A 15-cmdiameter slotted tube resonator was used as the transmitter. Short TR images (TE = 40 ms and TR = 700 ms) were obtained using standard spin-warp imaging techniques. Localized TI measurements were made using a stimulated echo (STE) localization (10-12) sequence preceded by a nonselective inversion pulse. The in vivo T , measurements were analyzed using an iterative technique ( 1 3 ) . Inversion delay intervals, T I , were varied from 10 ms to 10 s and all data were acquired using the following parameters: TE = 50 ms, TM = 10 ms, TR = 7000 ms, spectral width = 800 Hz, 1024 points, and eight averages. In vivo spectra for lipid quantification were also acquired using STE localization. Water suppression was achieved using a chemically selective inversion pulse centered on water with the inversion delay T I set to the T I null value determined for that
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particular animal ( 14). The inverted water magnetization was allowed to relax back into the transverse plane prior to executing the STE localization pulses, similar to the WEFT techniques ( 1 5 ) , thereby achieving maximum water suppression while minimally affecting other resonances. All acquisition parameters were the same as those for the T I relaxation measurements, with the exception of TR being reduced to 2500 ms. In order to quantitate the MRS determined lipid content, a 95% D,O sample was imbedded in the surface coil block. The reference also allowed for correction of dayto-day variations in scanner performance. A reference spectrum for each animal and time point was obtained by electronically moving the voxel into the 95% D20sample and obtaining a nonsuppressed spectrum. The integrated area under the lipid peak from the liver was then normalized relative to the integrated water peak from the reference. The machine sensitivity was found to to be very stable, with a standard error over the 5-week experiment of approximately 8%. Liver samples were removed immediately after euthanasia, histology specimens were obtained, and the remaining liver was quick frozen in liquid nitrogen. The liver samples were stored at -70°C prior to total lipid extraction. Lipid extraction was carried out following the technique of Folch et al. (16) using a ch1oroform:methanol solvent. Briefly, liver samples were extracted using a 20-fold volume of 2: 1 chloroform: methanol solvent during homogenization. The solution was paper filtered to remove rough debris. A 0.2-vol of water was added to remove hydrophilic components. The solution was then centrifuged at 2500 rpm for 20 min to separate fractions. The upper hydrophilic fraction was removed by suction and the interface washed three times. The lower fraction was dried to constancy and weighed. Total lipid concentration was expressed as milligrams of residual lipids per gram of liver. Histology slides were prepared by fixing slices of liver tissue in 10%buffered formalin, dehydrating in graded alcohol, and imbedding in paraffin blocks. The blocks were sectioned at 6 Fm. Sections were stained with hematoxylin and eosin ( H & E). The slides were examined and photomicrographs taken on a Nikon photomicroscope. Semiquantification of fatty change was based on examination of four slides per liver per animal aided by an ocular micrometer with a grid divided into 100 squares. Grading was performed using an ascending scale of 1 to 4 based on the following: I+, fatty change involving a few cells around the central vein; 2+, fatty change involving hepatocytes around the central vein extending one-third of the way toward the portal area; 3+, fatty change extending from the central vein to two-thirds of the way to the portal area; and 4, fatty change extending from the central vein to the portal area. A statistical analysis was performed using the Student’s t-test to determine if differences in the experimental time points were significant from those of the controls. All means were tested at the 95% confidence level ( p < 0.05). The significance of regression slopes was also tested using t-statistics at the 95% confidence level ( 17). RESULTS
The quality of localization is demonstrated in Fig. 1 by simultaneously showing (A) a standard short TR image and (B) a localized image from the same animal. The landmarks on the standard image include the spinal cord (SC), sternum (ST), and a partial volume of the stomach (SM). Due to the relatively homogeneous nature of
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FIG. 1. Axial (A) standard 2D spin-warp and (B) STE localized images of normal rat liver. The following anatomical landmarks are identified: spinal cord (SC), sternum (ST), and stomach (SM). A partial volume of stomach was included in the localized image for anatomical landmarking. The stomach was avoided in the spectroscopic studies. Acquisition parameters were TE = 40 ms, 700 ms, 10 cm FOV, and four averages. The mixing time T Mfor the STE image was 10 ms.
the liver, a partial volume of stomach was included in the images for anatomical landmarking. It should also be noted that the slice thickness for the localized image was 15 mm, while the slice thickness for the standard image was 5 mm. The increased effect of partial volume averaging is clearly demonstrated in the localized image. For spectroscopic studies the stomach was avoided. Both images were acquired with TE = 40 ms, TR = 750 ms, FOV = 10 cm, and four averages. The localized image was acquired with TM = 10 ms. Weekly MR images for a typical rat are shown in Fig. 2. The images were acquired from approximately the same location in each examination. Apparent differences in positioning as judged from different partial volumes of stomach visible are due in most part to the feeding status of the animal at the time of examination, i.e., the volume of the stomach content. The short TR images show a slight to moderate increase in liver intensity relative to surrounding skeletal muscle over the course of these studies. Only diffuse fatty infiltration is noted with no nodule formation as observed with more severe hepatotoxins (18, 19). The TI relaxation data for both water and lipids were found to be monoexponential. In a few cases the lipid recovery appeared to overshoot the equilibrium value. This phenomenon was seen less than 25% of the time and is typical of cross-relaxation behavior in solutions (20). The average water and lipid T I values as a function of ethanol administration are given in Table 1. The water T , shows a slight increase with time; however, the data were poorly correlated ( Y = 0.54) and only three of the five time points were different from those of the control. Control lipid T I values were not obtained due to unreliably small lipid signals in nonsuppressed spectra in this group of rats. The control lipid TI values from a separate experiment (6) using the same strain of rats and NMR acquisition parameters were used as a best estimate for the control lipid T I for this group. The lipid T I values increased from a control value of 283 ms to 385 ms on Day 8. This was followed by a decrease to 285 ms on Day 15,
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FIG.2. Short TR axial images of the liver of a typical rat obtained weekly for 5 weeks. The progression of fatty liver is demonstrated by a slight overall increase in liver intensity with increased exposure to ethanol. ( A ) Control, ( B ) 1 week, (C) 2 weeks, (D) 3 weeks, (E) 4 weeks, and ( F ) 5 weeks.
different fron the Day 8 value, but not significantly different from that of the control, after which time no significant changes were seen. A typical set of water-suppressed spectra used in the calculation of lipid /reference ratios is shown in Fig. 3. The spectra were normalized using the reference so that changes in lipid peak ( 1.25 ppm) area correspond to changes in MRS observable lipids. The lipid methylene resonance is seen at 1.25 ppm, with the residual water peak at 4.75 ppm. The variation in the amplitude of the residual water peak in the different plots reflects the degree of water suppression achieved in a given session and does not affect the lipid quantification. The use of a reference permits consistent estimation of the lipid signal irrespective of changes in water suppression efficiency. A plot of average MRS derived lipid/reference values as a function of ethanol administration is given in Fig. 4a. The lipid/reference ratios ranged from 0.8 for control animals to 7.5 by Day 35. A generally smooth upward trend with good correlation was observed ( r = 0.9 1 ).
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I29
TABLE 1 Water and Lipid T , Relaxation Data Relaxation time (ms) Water
Lipid
Days of ethanol
Average
St. error
Average
St. error
Control 7 days 15 days 22 days 29 days 36 days
907 1061" 950 11 18" 1014 1053a
36 52 54 86 84 71
283 385 a 285 320 298 313
38 12 25 14 17
14'
Averages that are significantly different from control ( p < 0.05). From a previous study; see text for details.
Average total lipid content determined biochemically using the Folch technique is shown in Fig. 4b. Again, a well-correlated upward trend ( r = 0.94) was found as the lipid concentration increased from the control value 43 mg/g to 74 mg/g on Day 35. Histology slides of typical normal, 8, 15,2 1, and 35 day alcohol animals are shown in Fig. 5. Sections of liver from Day 8 showed a mild degree of fatty change in hepatocytes around the central vein. Fatty change was in the form of accumulations of lipid within the vesicles in the cytoplasm (Fig. 5B). The number of hepatocytes with fatty change increased at the later time points extending out from the central vein to the portal area by the end of the fifth week. A progressive increase oflipid accumulation in nonparenchymal cells was also noted (Fig. 5C). At least one animal from each
L 8
6
4
2
0
-
2
-
4
PPm FIG. 3. Typical set of water-suppressed spectra acquired weekly from one of the Group 1 animals. These spectra were used to determine MRS observable lipid content. The control spectrum is at the bottom of the figure with the weekly spectrum progressingupward. The lipid methylene resonance appears at approximately 1.25 ppm, while the residual water signal is seen at 4.75 ppm. The spectra were acquired using TE = 50 ms, TM = 10 ms, TR = 2500 ms, 1024 points, and 128 averages.
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HAZLE, NARAYANA, AND DUNSFORD
)
Days of Ethanol
FIG.4. Changes in liver lipid content are demonstrated by various methodologies. (a) MRS determined lipid changes relative to the 95% D20 reference, (b) total lipid content as determined using the Folch technique expressed in milligrams extracted lipids per gram liver, and (c) histologically determined lipid content. Error bars indicate one standard error of the mean. The number of animals is given in parentheses, and time points different ( p < 0.05) from control are shown with an asterisk.
time period for days 15 through 35 demonstrated hepatocytes with macro aggregates of fat giving the appearance of lipocytes (Fig. 5D). The average histology grade as a function of days on the protocol is shown in Fig. 4c. The correlation of these data was found to be slightly worse ( r = 0.8 I ) on average than the MRS and biochemically derived lipids, but still showed an upward trend. In order to determine the correlation between the MR and biochemistry or histology results for individual animals, MRS derived lipid content was plotted against lipid concentration (Fig. 6 ) and histology grade (Fig. 7). On an individual basis the MRS and biochemically determined lipids were not well correlated ( r = 0.52). However, the histology grade did appear to be very consistent with the MRS determined lipid content with a high degree of correlation ( r = 0.94). The fatty changes observed by all three experimental methods with this regimen of ethanol are much less severe than those observed as a result of CC14 administration (6). The relative changes in MRS observable lipids for this study are compared to the changes observed during CC4 administration by plotting the lipid / reference ratios from the two studies on the same scale (Fig. 8). Presenting the data in this manner elucidates the more dramatic changes in MRS visible lipids as a result of CC14 (Days 2 through 29) or CC14 phospholipase-D (CC4 PLD) administration relative to alcohol. The lipid/reference ratios increased by as much as a factor of 6 in the alcohol study. Similar changes in the biochemically derived lipid concentrations for the two
+
+
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131
FIG. 5. Typical histology slides of liver sections from normal and ethanol-treated rats. All slides were stained with hematoxylin and eosin and magnified to 320X. Central veins are indicated by C and portal areas by P. (A) Normal liver section. ( B ) Liver section obtained after 8 days on the alcohol diet showing mild microvesicular fatty changes (arrows). (C) A slight increase is noted in hepatocytes (small arrow) and nonparenchymal cells (large arrow on Day 1 5 ) . ( D ) Microvesicular fatty change (small arrow) and macro accumulations of fat in hepatocytes producing “lipocyte-like” cells (large arrow) were observed after 2 1 days. (E-F) Marked fatty change around the central vein extending to the portal areas (P) was visible after 35 days on the alcohol diet (arrows).
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HAZLE, NARAYANA, AND DUNSFORD
A
A
A
A
A 60
A
A
A A
I
A
100
80
Lipid Concentration (rng/g)
FIG. 6 . MRS determined lipid content for the individual Day 35 rats against biochemically derived lipid concentration. Only a moderate degree of correlation was found ( I = 0.52). FIG. 7. Average MRS lipid content for the 35 day rats against histologic grade along with the +95% confidence limits. The values in parentheses indicate the number of animals for each histology grade. A correlation coefficient of 0.94 was obtained.
studies is seen from Fig. 9. In this case the maximum increase of lipid concentration for the CC14 PLD study is a factor of approximately 4, while the maximum increase from alcohol administration is only about factor of 1.5. Although the histologic changes for the two studies were not graded (semiquantitatively) on the same scale, the changes associated with CC14 ( 6 ) are much more severe when histology slides from the two studies are compared. When the quantified MRS observable lipids as a function of biochemically determined total lipid concentration from both studies were compared, no correlation was found. This indicates that for a given lipid concentration both studies gave approxi-
+
A-A
CCI,
+ -+
FtOH
-
0--0
CCI
7-
V
EIOH
cn 190,
@
Days on Protocol
@
Days o n Protocol
FIG. 8. Relative changes in MRS determined lipid/reference ratios acquired during this study and a previous study evaluating the effects of C C 4 ( 6 ) . FIG. 9. Biochemically obtained lipid concentrations for this study and a previous study evaluating the effects of CCll (6) demonstrate the significantlylesser effects of alcohol on liver lipid content. The error bars represent one standard error.
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mately the same change in MRS determined lipid content. Regardless of differences in the mechanisms which induce fatty liver for alcohol or C C 4 ,the MRS observable lipid fraction appears to be the same in each case. DISCUSSION
Although the effects of moderate durations of ethanol administration are well known ( 1 - 4 ) , the induction of fatty liver disease has not been thoroughly evaluated using in
vivo MR techniques. We chose the alcohol model for fatty liver because the relatively slow accumulations of lipids in the liver (compared to other hepatotoxins, such as carbon tetrachloride) would allow us to determine if subtle changes in liver lipid content could be followed over a period of time with MR. The short TR images demonstrated only mild changes in liver intensity relative to surrounding tissue. This slight increase in overall liver intensity was noted in the final weeks of the 5-week experiment with no observable structural changes. This is not unexpected since the duration of alcohol administration was not long enough to induce the inflammatory and fibrotic changes normally associated with later stage disease, potentially detectable with MRI. No inflammatory or fibrotic changes were noted in the histologic examination of these animals. It does appear, however, that MRS relaxation measurements could potentially be of interest in the study of more chronic ethanol hepatotoxicity. Although the only significant changes in water or lipid TI appeared as a higher value for lipid TI on Day 8, there did appear to be a trend of increasing water T Iwhich could manifest itself as useful indicator in experiments of longer duration. An acute increase in water or composite liver TI has been reported for CC14hepatotoxicity and is usually associated with changes in intracellular “free” water content ( 6 , 18, 19, 21, 22). Prolonged administration of ethanol would be expected to produce inflammatory changes which are believed to result in increased “free” water content and possibly increased water T I . Average quantitative changes in liver lipids determined using MR spectroscopic techniques were well correlated with changes in biochemically derived lipid concentrations. Average MRS findings also agreed well with histologic lipid content grading. On an individual basis, MRS results did not correlate well with biochemical results (Fig. 6 ) . The sensitivity of the biochemically derived concentration could no doubt be improved using chromatographic techniques to separate the various components. However, the individual MRS results were well correlated (Y = 0.94)with the histologic data (Fig. 7). This is not unexpected as the biochemical lipid extraction has a substantial baseline, the average control lipid concentration was 48 mg/g, and the lipid concentration increased only up to 80 mg/g by the end of the study. Individual changes could easily be masked by experimental uncertainty. The mechanism of lipid accumulation in the liver during alcohol administration using this diet is felt to be primarily due to the replacement of alcohol for carbohydrates as the main energy source in the liver cell. This results in the generation of excess reducing equivalents from the oxidation of alcohol in the form of NADH, which tends to slow down fatty acid oxidation ( I ). The decreased fatty acid oxidation leads to enhanced synthesis of lipoproteins and triglycerides which also contribute to the fatty change (23). Hormonally induced lipolysis in adipose tissue giving rise to increased
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supplies of fatty acids to the liver also contributes to the generation of fatty liver ( 3 ) . Although the extent of fatty change during alcohol administration is known to be influenced by diet (24, 2 5 ) , the liquid diet chosen for our studies has been shown not to affect lipid content (7). On the other hand, the mechanism of lipid accumulation in the liver as a result of CC14 insult is felt to be due to the production of the trichloromethyl radical ( 2 3 ) . Lipid peroxidation induced by this mechanism leads to the destruction of microsomal proteins, including cytochrome P-450 (26, 2 9 ) . These changes lead to the inability of the liver cells to synthesize lipoproteins which are needed to remove tnglycerides from the cell. Histopathologically this is seen as an accumulation of triglycerides in the cytoplasm of hepatocytes, especially around the central vein ( 3 0 ) . Further evidence to support the position that the mechanisms of fatty liver induction are different for the two compounds is provided by the observation that C C 4enhances the injury initiated by alcohol in rats (30-32). In fact, the enhanced fibrogenesis has suggested to some investigatorsthat EtOH alone may not be sufficient to cause cirrhosis in humans, but may require a propagator such as CC4 (31) . ACKNOWLEDGMENTS The authors thank Dr. John H. Hams, Jr., for his continued support, Drs. Robert Guynn and Paul Rosevear for use of laboratory facilities to perform the total lipid extractions, Dr. John Hunt for assistance in preparing the histology specimens, and Mr. David Menill for assistance in performing the lipid extractions. This work was supported in part by the John S. Dunn Foundation and NIH Grant 34635. REFERENCES 1. C. S. LEIBER,Med. Clin. N. Amer. 668, 3 (1982). 2. C. S. LIEBER,in “Biochemical Mechanisms of Liver Injury” (T. F. Slater, Ed.), Academic Press, New York, 1978. 3. M. C. GEOKAS, C. S. LIEBER,S. FRENCH,AND C. H. HALSTED,Ann. Internal Med. 95, 198 ( 1981 ). 4. E. ALBANO, G. POLI,A. TOMASI,L. GORIA-GATTI, AND M. U. DIANZANI, in “Pathophysiology of the Liver,” Excerpta Medica, Amsterdam, 1988. 5. THECLINICAL NMR GROUP(ABERDEEN), Clin. Rudiol. 38,495 ( 1987). 6. J. D. HAZLE,P. A. NARAYANA, AND H. A. DUNSFORD, Mugn. Reson. Med. 15, 21 1 ( 1990). 7. C. S. LIEBER AND L. M. DECARLI, Alcoholism 10, 550 (1986). 8. P. A. NARAYANA, J. L. DELAYRE, AND L. K. MISRA,Magn. Reson. Med. 3,549 (1986). 9. J. D. HAZLE,P. A. NARAYANA, AND W. A. KUDRLE,J. Magn. Reson. 83, 595 ( 1989). 10. J. GRANOT,J . Mugn. Reson. 70,488 (1986). 11. R. KIMMICH AND D. HOEPFEL,J. Mugn. Reson. 72, 379 (1987). 12. J. FRAHM,K. D. MERBOLDT, AND W. HANICKE, J. Magn. Reson. 72, 502 (1987). 13. R. GERHARDS AND W. DIETRICH, J. Mugn. Reson. 23, 21 (1976). 14. P. A. NARAYANA, E. F. JACKSON, J. D. HAZLE,L. K. FOTEDAR, M. V. KULKARNI, AND D. P. FLAMIG, Magn. Reson. Med. 8, 151 (1988). 15. S. L. PATTANDB. D. SYKES,J. Chem. Phys. 56,3182 (1972). 16. J. FOLCH,M. LEES,AND G. H. SLOANE-STANLEY, J. Bid. Chem. 226,497 ( 1957). 17. J. H. ZAR,“Biostatistical Analysis,” 2nd ed., Prentice-Hall, Englewood Cliffs, NJ, 1984. IS. D. D. STARK,H. M. BASS,A. A. Moss, B. R. BACON,J. H. MCKERROW,C. E. CANN,A. BRITO,AND H. 1. GOLDBERG, Radiology 148, 743 (1983). 1Y. A. V. RATNER,E. A. CARTER, G. M. POHOST,AND J. R. WANDS,Ale. Clin. Exp. Re.?.10,24 1 ( 1986). 20. J. C. GORE,M. S. BROWN,AND I. M. ARMITAGE, Mugn. Reson. Med. 9,333 ( 1989). 21. B. R. ROSEN,E. A. CARTER,AND I. L. PYKETT,Radiology 154,469 (1985).
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22. M. BRAUER,R. A. TOWNER, 1. RENAUD, E. G. JANZEN, AND D. L. FOXALL, Magn. Reson. Med. 9, 229 (1989). 23. R. BLOMSTRANDAND L. L. DRAGER, L$e Sci'. 13, 1131 (1973). 24. C. S. LIEBERAND L. M. DECARLI,Alcoholism (NY) 6, 523 ( 1982). 25. H. TSUKAMOTO, S. J. TOWNER,L. M. CIOFALO, AND S. W. FRENCH, Hepatofogy 6,814 ( 1986). 26. R. 0. RECKNAGEL AND E. A. GLENDE, JR., Crit. Rev. Toxicol. 2,263 ( 1981). 27. G. D. NADKARNI AND N. B. DSOUZA,Biochem. Med. Metab. Bid. 40,42 ( 1988). 28. E. A. GLENDE,JR., A. M. HRUSZKEWYCZ, AND R. 0. RECKNAGEL, Biochem. Pharmacol. 25, 2163 (1976). 29. U. FLANDER, W. HAAS,AND H. KRONER,Exp. Mol. Pathol. 36,34 (1982). 30. J. V. BRUCKNER, W. F. MACKENZIE, S. MURALIDHARA, R. LUTHRA,G. M. KYLE,AND D. ACOSTA, Fundam. Appl. Toxicol. 6, 16 (1986). 31. A. BOSMA,A. BROUWER, W. F. SEIFERT, AND D. L. KNOOK,J. Pathol. 156, 15 (1988). 32. M. YOUNES, W. REICHL,A N D c . P.SIEGERS, Xenobiotica 13, 47 (1983).
