FUNDAMENTAL AND APPLIED TOXICOLOGY 4, 819-826 (1984)

Hepatic Steatosis in Rats Fed Diets with Varying

Concentrations of Sucrose
BRUCE R. BACON, C. H. PARK,* ELVIN M. FOWELL,
AND CHRISTINE E. MCLAREN
Departments of Medicine and *Pathology, Case Western Reserve University School of Medicine
at Cleveland Metropolitan General Hospital, Cleveland, Ohio 44109
Hepatic Steatosis in Rats Fed Diets with Varying Concentrations of Sucrose. BACON, B. R.,
PARK, C. H., FOWELL, E. M., AND MCLAREN, C. E. (1984). Fundam. Appl. Toxicol. 4, 819-
826. The role of dietary sucrose concentrations in the development of hepatic steatosis in rats
was investigated. Twelve groups of weanling male Sprague-Dawley rats received semipurified
diets with different sucrose concentrations ranging from 20 to SW (w/w); one group received a
cereal-based chow diet. Rats were sacrificed after 3 weeks and body weight, liver/body weight
ratio, plasma alanine aminotransferase concentration, hepatic triglyceride concentration, and
liver morphology (light and electron microscopy) were determined. Body weight and liver/body
weight ratio were decreased in rats receiving 40-50 or 25-3596 dietary sucrose compared to tats
receiving 20% sucrose or chow. Plasma alanine aminotransfemse concentrations were within
normal limits. Hepatic triglyceride concentration was significantly increased in rats receiving 40-
50 and 25-351 dietary sucrose compared to rats receiving 20% dietary sucrose or chow. Light
microscopy showed hepatic steatosis in a periportal distribution at all concentrations of dietary
sucrose. Both the frequency and the severity of the steatosis were increased with increasing dietary
sucrose concentrations. Electron microscopy from selected livers with increased hepatic triglyceride
concentrations revealed increased lipid spheres and increased smooth endoplasmic reticulum
without prominent Golgi apparatus or GERL complex. It is concluded that high dietary sucrose
concentrations are responsible for the development of hepatic steatosis. Semipurified diets with
high dietary sucrose concentrations such as the AIN-76A diet (50% sucrose) should not be used
in animal studies in which increased triglyceride deposition could influence experimental outcome.
8 1984 Society of Toxicology.
Carefully defined purified diets with known
amounts of nutrients, vitamins, and minerals
have been recommended for use in animal
studies of toxicity and carcinogenicity. The
National Research Council Committee on
Laboratory Animal Diet has emphasized the
need to use such defined diets in order to make
valid comparisons of studies in different lab-
oratories (Newberne et al., 1978). For this rea-
son, the American Institute of Nutrition (AIN)
Committee on Standards for Nutritional
Studies has recommended a purified diet
I To whom correspondence and requests for reprints
should be sent: Division of Gastroenterology, Cleveland
Metropolitan General Hospital, 3395 Scranton Rd.,
Cleveland, Ohio 44 109.
composed of commercially refined proteins
(casein), carbohydrates (sucrose, cornstarch),
fat (corn oil), and fiber (cellulose) with added
mineral and vitamin mixtures and supple-
mental choline and methionine (AIN-76A
Purified diet) (Bieri et al., 1977; Bieri, 1980).
Since the introduction of this diet there have
been reports of excessive bleeding (Roebuck
et al., 1979; Medinsky et al., 1982), nephro-
calcinosis (Nguyen and Woodard, 1980; Med-
insky et al., 1982), and fatty liver (Medinsky
et al., 1982; Hamm et al., 1982) in rats fed
the AIN-76A diets. Excessive bleeding has
been prevented by increasing the level of vi-
tamin K added to the diet (Bieri, 1980). Prob-
lems with the development of fatty liver appear
to be related to the high sucrose concentration
819
0272X)590/84 $3.00
Copyci&t Q 1984 by the society of Toxicology.
All rights of reproduction in any fom esemecl.
820 BACON ET AL.
used in the diet (Hamm et al., 1982), and
make the use of such a diet questionable in
animal studies that require normal hepatic
morphology and/or function. The current
study was designed to determine more pre-
cisely the role of dietary sucrose concentration
in the development of hepatic triglyceride ac-
cumulation.
METHODS
Diets. Twelve experimental diets were prepared and
kindly donated by ICN Nutritional Biochemicals (Cleve-
land, Ohio). The formulation of these 12 experimental
diets is shown in Table 1. Purina Lab Chow (Ralston
Purina Company, Chicago, Ill.) was the closed formula
cereal-based diet used as the control diet.
Animals. One hundred forty-eight weanling (50-75 g)
male rats of the Sprague-Dawley strain were purchased
from &&Miller Laboratories, Inc. (Allison Park, Pa.).
Rats were housed in polyethylene cages with stainless steel
wire tops and were allowed diet and tap water ad lib. Rats
were weighed at the beginning of the study and at weekly
intervals thereafter. Sixteen rats were sacrificed at the be-
ginning of the study period to provide baseline data. Them
were 10 rats in each of the 12 experimental groups and
12 in the group which received chow (control group). Rats
TABLE 1
FORMULATION OF EXPERIMENTAL DIETS (W)
Group Sucrose Cornstarch Casein Alphacel
1 50 15 20
2 45 15 20
3 40 15 20
4 40 20 20
5 40 20 25
6 40 25 20
7 35 15 20
8 35 20 25
9 30 15 20
10 25 15 25
11 20 20 30
12 20 20 25
13 Purina Lab Chow
5
10
15
10
5
5
20
10
25
25
20
25
The remaining 10% of each diet consisted of corn
oil-51, AIN mineral mix-3.5% AIN vitamin mix-
1.0% DL-methionine-O.396, and choline bitartrate-
0.2%. Diet No. 1 is the recommended formulation of the
American Institute of Nutrition (AIN-76A) (Bieri et al..
1977; Bieri, 1980).
were fed the diets for 21 days and then fasted overnight
prior to sacrifice, which was by exsanguination via cardiac
puncture while under light ether anesthesia. The liver was
excised, weighed, and portions from the right lobe were
saved for light microscopy, electron microscopy, and
analysis of triglyceride concentration.
Lighf microscopy. Blocks of liver were fixed in 10%
(w/v) neutral buffered fotmalin, dehydrated with a graded
series of ethyl alcohol, and embedded in paraffin. Sections
(5 pm) were prepared and stained with hematoxylin and
eosin. Frozen sections of selected livers were stained with
oil red 0 to assess hepatic neutral lipid (Luna, 1968).
The histologic sections from all rat livers were evaluated
blindly by one of the investigators (C.H.P.). Rat liver his-
tology was evaluated for the presence or absence of hepatic
steatosis and for the presence of any other hepatic ab
normality. For the purpose of this study, we have graded
the presence of steatosis as follows: none-no macroves-
icular fat seen anywhere within the entire section of liver
tissue, mild-less than 10% replacement with fat, mod-
erate- 10 to 25% replacement with fat, and severe-greater
than 25% replacement with fat.
Electron microscopy. Blocks of selected livers were fixed
(4-6 hr) in 2.5% (w/v) glutaraldehyde, postfrxed in osmium
tetroxide, dehydrated in a graded series of acetone, and
embedded in Araldite (R. P. Cargille Laboratories, Inc.,
Cedar Grove, N.J.). Sections (1 pm) were stained with
methylene blue and viewed by light microscopy. Repre-
sentative areas were further sectioned (ultrathin) for elec-
tron microscopy. Ultrathin sections were stained with
uranyl acetate and lead citrate.
Hepatic triglyceride concentration. Liver was stored fro-
zen (-20C) until the assay was performed. Hepatic lipids
were extracted with chloroform/methanol (2: 1) according
to the method described by Folch et al. (1954) with the
modification of Bligh and Dyer (1959). Phospholipids were
separated and removed by batch chromatography with
silicic acid (Schotz and Recknagel, 1960). Triglycerides
were determined by glycerol analysis alter saponification
of the nonphospholipid fraction (Pinter et al.. 1967) and
are expressed as milligrams triglyceride per gmm liver
(wet wt).
Plasma alanine aminotransferase. Blood was drawn by
fuged. The plasma was assayed for alanine aminotrans-
ferase (ALT) concentration according to the method of
I&men et al. (1955), with the modification of Henry et
al. (1960) using a Beckman Trace III spectrophotometer
(Beckman Instruments, Inc., Fullerton, Calif.).
Statistical methods. Data were analyzed using BMDP
computer programs (Dixon, 1977). For each variable, one
way analysis of variance was used to test for statistically
significant differences between the means of the 13 diet
groups ( 12 experimental, 1 control) and between the means
of 4 groups determined by dietary sucrose concentration
(40-50% sucrose, 25-3596 sucrose, 20% sucrose, and chow).
When the hypothesis of equality of variances was rejected
HEPATIC STEATOSIS DUE TO DIETARY SUCROSE 821
TABLE 2
EFFECT OF DIETARY SUCROSE CONCENTRATION ON RAT GROWTH, PLASMA ALT,
AND HEPATIC TRIGLYCERIDE CONCENTRATIONS
Group
I
2
3
4
5
6
7
8
9
10
11
12
13
a Means f SE.
n
IO
10
10
10
10
10
9
10
10
10
10
8
12
MY
weight at
sacrifice (g)
199.8 * 8.6
196.8 f 4.3
206.2 f 4.0
194.5 f 7.3
201.5 + 2.4
196.3 zk 5.6
207.0 + 8.5
202.9 + 7.3
226.0 + 6.4
198.5 2 6.8
216.8 f 3.6
238.1 k 4.4
227.6 + 8.7
Liver/body
weight (%)
3.50 k 0.16
3.20 f 0.05
3.40 1 0.09
3.59 f 0.09
3.40 f 0. IO
3.35 2 0.06
3.60 f 0.09
3.76 f 0.14
3.39 + 0.04
3.58 + 0.14
3.79 zk 0.1 I
3.73 f 0.10
4.08 f 0.10
Plasma Hepatic
ALT triglyceride
(IU/liter) (mg/g liver)
45.4 * 2.2 21.9 f 5.7
37.9 + 2.2 25.8 + 4.1
35.3 + 2.1 30.1 f 5.3
38.6 f 1.1 20.3 k 3.6
43.5 + 2.5 20.9 + 4.1
40.8 + 2.1 24.2 + 4.3
36.8 + 2.8 12.9 f 2.9
31.2 + 2.1 27.9 + 6.9
28.6 f 2.9 20.7 + 5.8
23.3 + 1.6 20.8 + 5.7
33.8 f 3.4 7.0 f 1.5
33.2 2 2.6 10.1 + 2.4
41.9 f 3.1 8.0 f 1.5
at the 0.05 significance level by Levenes test, the Brown-
Forsythe analysis of variance statistic was used. To assure
an overall significance level of 0.05, the Bonferoni multiple
comparisons procedure was applied to test for pairwise
differences between the means (Ahfi and Azen, 1979).
RESULTS
During the course of the 3-week study pe-
riod two rats died (one each from Groups 7
and 12) and one rat (from Group 12) had
lost considerable weight by the time of sacrifice
and was found to have pneumonia.
For the sixteen weanling rats that were sac-
rificed at the beginning of the study period
the following results were obtained (mean
f SE): body weight, 59.4 f 1.3 g; liver/body
weight, 3.20 f 0.05%; plasma ALT, 28.4 + 3.3
III/liter; and hepatic triglyceride concentra-
tion, 5.2 + 0.5 mg/g. No hepatic steatosis was
seen by light microscopy.
The mean (*SE) for each variable (body
weight, liver/body weight ratio, plasma ALT,
and hepatic triglyceride concentration) for rats
from all 13 diet groups is shown in Table 2.
One-way analysis of variance showed that the
means of the 13 diet groups differed signifi-
I- P<O.OOl .-I
B
+ P<O.OOl __(
+ K0.001 +
40-50 25-35 20 Chow
(n-80) (n-39) (n-19) (n-12)
SUCROSE CONCENTRATION IN DIETS (%)
FIG. I. Effect of dietary sucrose concentrations on rat
growth (A) and liver/body weight ratio (B). Weanling rats
received diets with variable sucrose concentrations for 3
weeks before sacrifice. Each value represents the mean
f SE from the particular dietary group. Differences be-
tween dietary groups that were not significant are not
shown.
a22 BACON ET AL.
- p<o.o01-,
A 5Or
mm F---- D<O.OOl ----I
SUCROSE CONCENTRATION IN DIETS (%)
FIG. 2. Effect of dietary sucrose concentrations on he-
patic triglyceride concentration. Weanhng rats received
diets with variable sucrose concentrations for three weeks
before sacrifice. Each value represents the mean + SE
from the particular dietary group. Differences between
dietary groups that were not significant are not shown.
cantly for each variable. In order to determine
differences as related to changes in sucrose
concentration, the 13 diet groups were further
divided into 4 groups by sucrose concentration
as follows: 40-50% sucrose (n = 60), 25-35%
sucrose (n = 39), 20% sucrose (n = 18), and
chow (n = 12). One-way analysis of variance
showed that the means of the four diet groups
differed significantly for each variable. Results
of multiple comparisons between the four di-
etary groups for body weight and liver/body
weight ratio are shown in Fig. 1; results for
hepatic triglyceride concentration are shown
in Fig. 2. Only significant differences are noted.
The mean hepatic triglyceride concentration
was not significantly different between rats
which had been fed chow or the diet with 20%
sucrose. In contrast, the mean hepatic tri-
glyceride concentration for rats receiving 40-
50% dietary sucrose and 25-35% dietary su-
crose was three times greater and 2/2 times
greater, respectively, than the mean for rats
receiving chow (Fig. 2). There were significant
differences between the four dietary groups
for plasma ALT concentration, but all mean
values were within normal limits.
By light microscopy, hepatic steatosis was
observed in 50 of the 60 rats (83%) which
received diets with 40-50% sucrose and 26 of
the 39 rats (67%) which received 25-35% di-
etary sucrose. In contrast, hepatic steatosis was
observed in only 6 of the 18 (33%) and 1 of
the 12 (8%) rats from the 20% sucrose and
chow diet groups respectively. The severity of
the steatosis was greater in the rats which re-
ceived the diets with the higher sucrose con-
centration (Table 3). The accumulation of the
hepatic steatosis was invariably predominantly
periportal in distribution (Fig. 3), with most
of the periportal hepatocytes containing ma-
crovesicular fat. Occasionally, the lipid drop
lets were so large that the nuclei were com-
pressed to the periphery of the hepatocyte.
Centrilobular hepatocytes contained numer-
ous microvesicular lipid droplets in the cy-
toplasm (Fig. 4). The oil red 0 stain performed
on frozen sections confirmed the presence of
neutral lipid within the cytoplasmic vacuoles.
No other significant hepatic pathology was
identified.
Electron microscopic examination of peri-
portal hepatocytes of selected livers with in-
TABLE 3
EFFXY OFDIETARY SUCROSE CONCENTRATION ON HISTOLOGICGRADINGOFHEPATICSTEATOGIS
Dietary sucrose
concentration
(%) n
Histologic grading of hepatic steatosis (No. of rats)
None Mild Moderate Severe
40-50 60 10 30 13 7
25-35 39 13 18 5 3
20 18 12 6 0 0
Chow 12 11 1 0 0
None-no macrovesicular fat seen anywhere within the entire. section of liver tissue, Mild-less than 109b replacement
with fat, Moderate- 10 to 25% replacement with fat, and Severe-greater than 25% replacement with fat.
HEPATIC STEATOSIS DUE TO DIETARY SUCROSE
823
FIG. 3. Light microscopy of rat liver. This photomicrograph (hematoxylin and eosin stain) is of liver
tissue from a rat that had been fed a 40% sucrose diet for 3 weeks. Macrovesicular fat can be seen in a
periportal distribution (P = portal triad, C = central vein). X105.
creased triglyceride concentrations showed electron-lucent spaces (Fig. 5). Mitochondria
numerous large electron-lucent lipid spheres showed marked variability in size and shape
distributed throughout the cytoplasm. Some (Fig. 5) and smooth endoplasmic reticulum
lipid spheres were heterogenous containing was increased with both vesicular and non-
areas of finely granular osmiophilic material vesicular forms present (Fig. 6). Primary and
thought to be triglyceride (Peterson, 1974) and secondary lysosomes were scarce. Golgi zones
PIG. 4. Light microscopy of rat liver. This photomicrograph (hematoxylin an eosin stain) is of liver tissue
from a rat that had been fed a 40% sucrose diet for 3 weeks. Both microvesicular (arrows) and macrovesicular
fat can be seen in this mid- and centrilobular zone (C = central vein). X340.
824
BACON
FIG. 5. Electron microscopy of rat hepatocyte. This
electron micrograph is from a rat that had been fed a 25%
sucrose diet for 3 weeks. Multiple heterogenous lipid
spheres can be seen containing finely granular osmiophilic
material (triglyceride, arrows) and electron-lucent spaces.
Mitochondria showed marked variability in sixe and shape
x3700.
were not prominent and vacuoles containing
very low density lipoprotein (VLDL) within
Golgi zones were not found. No saccules con-
ET AL.
taming VLDL were found in the area of the
sinusoidal plasma membrane.
DISCUSSION
In the present study, we have demonstrated
both biochemical and morphological evidence
of increased hepatic steatosis in male Sprague-
Dawley rats fed the AIN-76A diet (containing
50% sucrose) and similar semipurified diets
containing lesser amounts of sucrose. Only
when the dietary sucrose concentration was
reduced to 20% did the hepatic triglyceride
concentration approximate that found in rats
which were fed the cereal-based control diet
(Fig. 2). This suggests that a dietary sucrose
concentration of 20% may be the upper limit
allowable in a semipurified diet to avoid sig-
nificant hepatic steatosis. Even at this dietary
concentration of sucrose (20%), 6 of 18 rats
still had a histologically identifiable slight in-
crease in hepatic steatosis. This level (20% su-
crose) is far lower than the amount recom-
mended by the Ad Hoc Committee on Stan-
dards for Nutritional Studies (Bieri et al.,
FG 6. Electron microscopy of rat hepatocyte. This electron micrograph is from a rat that had been fed
a 25% sucrose diet for 3 weeks. Electron-lucent lipid spheres can be seen in close proximity to mitochondria.
Also, there is an increased amount of vesicular smooth endoplasmic reticulum (arrows). X 13,500.
HEPATIC STEATOSIS DUE TO DIETARY SUCROSE 825
1977). Our study confirms and extends the
findings reported by Medinsky et al. ( 1982)
who demonstrated increased hepatic steatosis
in a periportal distribution in male Fischer-
344 rats fed three different lots of the AIN-
76A diet prepared by two different suppliers.
Rats received the diets for up to 8 weeks and
progressively increasing amounts of periportal
lipidosis were observed with increasing lengths
of time that the rats received the diets. Ad-
ditionally, it has been shown that if sucrose
is replaced entirely by cornstarch the hepatic
steatosis does not appear (Hamm et al., 1982).
We observed slight but significant differ-
ences in body weight and liver/body weight
ratio between groups, such that with decreas-
ing dietary sucrose concentration there was
increasing body weight and liver/body weight
ratio after the 3-week feeding period (Fig. 1A
and B). These findings are consistent with what
was found by the Ad Hoc Committee on
Standards for Nutritional Studies. In their re-
port, the body weight and the liver/body
weight ratio were greater in male Sprague-
Dawley rats fed the cereal-based chow diet
than in rats fed the AIN-76A diet for 2 1 days
(Bieri et al., 1977). This is in contrast to what
was observed by Medinsky et al. (1982), who
noted no change in body weight between male
Fischer-344 rats that were fed either the AIN-
76A diet or a control chow diet. The liver/
body weight ratio however was greater for rats
fed the AIN-76A diet compared to controls
(Medinsky et al., 1982). These differences be-
tween studies may be related to changes in
rat strains or perhaps to different lengths of
time the rats were receiving the various diets.
In our own study, the differences in body
weight and liver/body weight ratio were sta-
tisticallly significant between the dietary
groups (Fig. 1 A and B) but the relative changes
were actually quite small.
Hepatic steatosis can be caused by many
alterations in hepatic triglyceride synthesis and
secretion. An increase in synthesis without a
concomitant increase in secretion can result
in lipid accumulation in hepatocytes. Peri-
portal hepatic steatosis can be seen in Kwash-
iorkor, a human condition which results from
severe protein deficiency. A major cause of
fatty liver in Kwashiorkor is thought to be
defective hepatic removal of very low density
lipoprotein (VLDL), presumed to be due to
decreased synthesis of VLDL apoproteins
(Hoyumpa et al., 1975). Also, dietary defi-
ciencies of methionine and other lipotropic
factors can cause hepatic steatosis. Adequate
protein concentration (20-30% casein) and
methionine and choline supplementation were
provided in all of our experimental diets, thus
these deficiencies could not be considered to
be contributory to the development of the he-
patic steatosis.
The mechanism whereby a high dietary su-
crose concentration causes periportal hepatic
steatosis in unknown. High sucrose diets in
conjunction with hepatotoxins and in genet-
ically obese Zucker rats have resulted in the
formation of hepatic steatosis. Novikoff et al.
(1974) have demonstrated that hepatic stea-
tosis (two times control triglyceride concen-
tration) could be produced by feeding 0.25%
chlorophenoxyisobutyrate (clofibrate) to rats
which were receiving a diet containing 60%
sucrose. This increase in hepatic triglyceride
concentration however, may have resulted
from the high dietary sucrose concentration
alone. In contrast, severe hepatic steatosis (9
to 20 times control) was produced when 1 .O%
erotic acid was added to the high sucrose diet.
The addition of erotic acid clearly led to sig-
nificant hepatic steatosis. Ultrastructural
studies showed the presence of prominent
Golgi apparatus with numerous saccules and
vacuoles containing VLDL, increased smooth
endoplasmic reticulum, and increased lyso-
somes forming the GERL complex. These ul-
trastructural findings suggested that there was
accumulation of both triglyceride and VLDL
without an abnormality in the secretory ap-
paratus. In our study with increased dietary
sucrose we did not find evidence of prominent
Golgi apparatus or the GERL complex.
Smooth endoplasmic reticulum however, was
increased, suggesting an increase in synthesis
of triglyceride without a concomitant increase
826
BACON ET AL.
in secretion. Finally, genetically obese Zucker DIXON, W. J. (1977). BMDP: Biomedical Computer Pro-
rats (homozygous recessive, ff) developed
grams. Univ. of California Press, Los Angeles.
marked hepatic steatosis after receiving a diet
FOLCH, J., LEES, M., AND SLOANE, S. G. H. (1954). A
containing 60% sucrose for 1 week, whereas
simple method for the isolation and purification of total
lean litter-mates (FF and/or Ff) did not develop
lipids from animal tissues. J. Biof. Chem. 226, 497-
509.
the heDatic steatosis (Novikoff, 1977). HAMM, T. E., RAYNOR, T., AND CAVISTON, T. (1982).
In conclusion, the major significance of our
findings is that the AIN-76A semipurified diet
is not suitable for toxicologic studies which
may depend on normal hepatocellular integ-
rity. It appears from our work and from that
of Hamm et al. (1982) that the high sucrose
concentration in the diet is responsible for the
development of the hepatic steatosis. Satis-
factory growth and liver/body weight ratios
apparently are insufficient parameters for use
in judging the adequacy of new purified or
semipurified diets. At the very least, histologic
evaluation of body tissues should be included
in order to identify obvious histopathologic
alterations that may be related to dietary com-
position.
ACKNOWLEDGMENTS
The authors thank Dr. Anthony S. Tavill for critically
reviewing the manuscript, Joan Skiba for expert secretarial
assistance, and Pat Griffin and Linda Hrabak for skilled
technical assistance. This research was supported in part
by the Cuyahoga County Hospital Foundation and by
U.S. Public Health Service Grants ROl AM 31505 and
T32 HL 07147. Dr. Bacon was a Postdoctoral Research
Fellow of the American Liver Foundation and is a Teach-
ing and Research Scholar of the American College of
Physicians.
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