Drug and Alcohol Dependence, 19 (1987) 333-344

Elsevier Scientific Publishers Ireland Ltd.

333
ANIMAL MODELS OF CHRONIC ALCOHOL INGESTION: THE LIQUID
DIET
LEIGH C. WARD
Alcohol Research Unit, Department of Biochemistry, University of Queensland, St Lucia
QLD 4067 (Australia)
(Received November 13th, 1986)
SUMMARY
A suitable animal model that mimics the effects of chronic alcohol intake
in man has long been sought. The ethanol-containing liquid diet, although
often finding favour in a variety of experimental studies, has recently been
criticised for being nutritionally inadequate. Results of this study indicate
liquid diets are capable of maintaining the growth of female, but not male,
rats at rates comparable to those of similar animals fed on commercially
available rodent pellets or on laboratory-prepared pelleted diets. In addition
preliminary data are reported indicating the suitability of this model for
inducing tolerance to alcohol.
Key words: Alcohol ingestion - Animal models - Liquid diet
INTRODUCTION
The pathophysiological consequences of excessive alcohol consumption
have for many years provided the stimulus for much basic biomedical
research. Owing to technical, logistic or ethical issues, most of this research
cannot be conducted in human beings; consequently animal models for
alcohol-related human disease have been sought [ 11. Whilst many individual
technical variations exist, four primary procedures for administration of
ethanol may be identified; (i) ingestion of solutions of alcohol as drinking
fluid [e.g. 21, (ii) inhalation of ethanol vapour [e.g. 31, (iii) administration
via intragastric intubation [e.g. 41 and (iv) provision of a liquid diet contain-
ing all nutrients including alcohol [e.g. 51.
0376-8716/87/$03.50
0 1987 Elsevier Scientific Publishers Ireland Ltd.
Printed and Published in Ireland
334
Various species of animals including dogs [ 61, guinea-pigs [ 71, rats [ 81,
mice [3] s baboons [9], pigs [lo] and turkeys [ll] have been used. Of the
possible combinations of procedure and species, one which has found
particular favour, following the pioneering work of Lieber and De Carli
[ 5,8] , is the rat fed on an ethanol-containing liquid diet. This model produces
a high level of alcohol consumption, tolerance and withdrawal behaviour and
is amenable to nutritional manipulation [ 121. Recently, however, the liquid
diet procedure has been criticised for being nutritionally inadequate for the
growth of rats [ 13-161, thus providing a confounding influence in the study
of the effects of alcohol per se when this model is used. Indeed, in 1982,
Lieber and De Carli [ 171, had recommended modifications to their original
dietary formulation.
The purpose of this report is to present a comparison of a number of liquid
diet formulations used in the authorss laboratory with isoenergetic control
diets and with commercially available rodent diets. The utility of the diets
was primarily assessed by their effects upon growth rate from post-weanling
to early maturity, and food intake, since it is these parameters of dietary
efficacy which have received most criticism [14-161. The usefulness of
these diets as models of alcohol-related disease, with particular regard to
protein metabolism, has been reported elsewhere [ 18-251.
MATERIALS AND METHODS
Experimental animals and housing
In all experiments rats of the Wistar strain from an outbred colony es-
tablished at the Central Animal Breeding House, University of Queensland
were used. Animals were non-litter mates and had from weaning been
maintained on commercial rodent diet (Table I, Barastok Pty. Ltd., Brisbane,
Australia) prior to each feeding trial. All animals were housed in plastic
rodent cages with free access to food and water. Environmental conditions
in the animal house were 12 h light-12 h dark (the light period 07.00-19.00
h) and 24 2 2C.
Feeding trial protocol
At the commencement of a feeding trial rats were weighed and the required
number was selected from those whose body-weights most closely approxi-
mated the mean. The selected rats were allocated randomly to the dietary
regimes as indicated in Table I. The number of animals per treatment varied
from 3 to 6 depending upon the experiment, and animals were caged in-
dividually or in pairs as indicated (Table I). The experimental diets were
prepared with the compositions listed in Tables I and II as previously de-
scribed [ 18,20,26] . Ethanol-containing diets were prepared daily by blending
ingredients in a Waring blendor and were stored at 4C until used. The caloric
density of all liquid diets was 1.5 kcal ml-.
In all experiments the rats were introduced to the experimental diets
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331
gradually over 4 days so that the ethanol concentration increased progres-
sively; 10,15, 20-27% energy. Each animal was allowed access to 60 ml of
diet each day and water was provided ad libitum. In paired-feeding experi-
ments, control rats were allowed free access to water and to the amount of
diet eaten by the ethanol-fed partner. The liquid diets were supplied from
bottles fitted with nipple feeders to minimise spillage and loss of ethanol
by evaporation (< 5% d-l ). The food intakes and weight gains of the rats
were measured at the time of feeding (16.00 h) at least every second day.
For comparison, three experiments, in which the growth rate of rats fed
on pellet-based diets, either commercially available (expts. VII and IX, Tables
I and II) or laboratory prepared (expts. VII, Tables I and II) are included in
this study. The study designs were similar to those described above and
details may be found in references [ 22,261,
In two experiments, from the series VI, blood alcohol concentrations were
determined in heparinized blood samples obtained via cardiac puncture at
the end of the feeding trial. Blood samples were obtained at 13.00 h, the
mid-point of the light period. Ethanol concentrations were determined by
the enzymatic procedure described elsewhere [27].
In another experiment from this series alcohol tolerance was assessed by
resistance to hypothermia following an acute intraperitoneal dose of ethanol
(0.20 g 100 g-l body-weight) [28]. Rectal temperature was measured using
a rectal probe [29].
Data were analysed by analysis of variance and the significance of difference
by Scheffe test or, for pair-feeding experiments by paired t-test.
RESULTS
The mean daily caloric intakes varied, between 39.1 and 63.0 kcal d-
rat- for animals receiving the liquid control diets and between 39.7 and
50.4 kcal d- rat- for those fed on the ethanol-containing diets (Table II).
Since the fat content of the diet was held constant (expts. I, II and III) it
was not possible to distinguish whether the increased food intake (expts.
III-l) was a response to the decreasing concentration of protein or to the
increasing carbohydrate content of the diet. For example, a 16% increase in
carbohydrate and concomitant 60% fat in protein concentration elicited a
28% increase in caloric intake (comparison of experiments I and III).
Consumption of the Sustagen-based diets (expts. IV, V and VI) was
generally less than that of the casein-based diets (diets I, II and III). It is
unclear whether this reflects the slightly higher protein concentration of
these diets, the markedly (3-fold) lower fat content or an undetermined
palatability factor [30]. Irrespective of the absolute magnitude of total
caloric consumption, the intakes of protein and carbohydrate by control
rats fed on the high protein diets (expts. II-VI) were remarkably similar;
6.8-7.3 and 27.0-31.4 kcal d-l rat-, although fat intake varied 4-fold
(3.9-16.5 kcal d-l rat-). Rats fed on the low-protein diet (I), although
338
exhibiting the highest total caloric intake, and concomitant fat intake, were
only able to achieve approx. 50% of the protein intake of the high-protein
fed animals.
The presence of ethanol in the diet elicited a slight (Z--20%), but significant
(P < 0.05 in expt. I) decrease in total caloric consumption by rats fed ad
libitum when compared with their controls (expts. I--IV), a difference
which was eliminated by pair-feeding (expts. V and VI).
Notwithstanding the feeding regime adopted, a similar quantity of ethanol,
ranging from 10.7 to 13.6 kcal d-l, was ingested by all ethanol-fed rats. In
all cases these intakes represented 27% of total calorie intake.
The total caloric intake of rats fed on the liquid-based normal protein
diets was, in all cases, less than that of similar animals fed on a commercially
available rodent diet (expts. VII and IX) whilst intake of the low-protein
liquid diet was greater than a comparable pellet diet (expts. I and VII).
The mean daily calorie intakes for Cday periods during the feeding trials
are presented in Table III. Food consumption remained essentially constant
or increased only slightly (expts. I and III) during the trials. In most experi-
ments food intake was lower during the first 4-day period than subsequent
periods, presumably reflecting an adaptation period to the diets.
The mean daily growth rates of the rats each of these 4-day periods are
presented in Table IV. The growth rates of the female control rats were
similar in all experiments, ranging from 1.9 to 3.4 g 6 and in all cases were
greater than those of similar animals fed a commercial laboratory diet
(expt. VIII). Furthermore, female rats fed on the low-protein but liquid
diet were also able to maintain a higher growth rate than rats fed on the
same diet in pellet form (2.2 and 0.6 g d- respectively; comparison of
expts. I and VII). These observations suggest improved assimilation of the
diet when in liquid form. This view is supported by the more efficient food
conversion exhibited by rats fed on liquid diets when compared to pellet-fed
animals (Table IV). The growth rates of male rats (expt. V) were slightly higher
(3.7 g d-l ) than those of female rats (1.9-3.4 g d- ) receiving liquid diets,
but lower (P < 0.05) than those of male rats fed on the laboratory pellet
diet (5.0 g d- ).
The growth rate of animals fed on the Sustagen-based diets were lower,
but not significantly so, than those of animals fed on similar diets in which
the protein source was casein (comparison of expts. IV and VI with II and
III).
All animals were able to gain weight when fed on the diets which contained
alcohol, although the rates of growth varied depending upon the formulation
of the diet. The rates of growth of ethanol-fed rats increased as the protein
concentration of the diet increased (expts. I to III) but were always lower
than those of the matched control animals. In contrast, rats receiving the
Sustagen-based diets containing ethanol all gained weight at rates comparable
with or greater than, those of their appropriate controls, irrespective of
whether ad libitum or pair-feeding regime was used (expts. IV to VI). In
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341
general the growth rates of all animals were lower during the first 4-day
period than during the subsequent days of the trials, presumably a reflection
of the period of adaptation to the experimental conditions.
Blood alcohol concentrations were low and highly variable, ranging from 0
to 0.26 mg m-l (5.4 mM). It should be noted, however, that these represent
concentrations present 6 h after the main feeding period of rodents. Similar
observations have been made by Ritzmann and Tabakoff [28] who observed
a precipitous decline in blood alcohol during the light period, from 2 mg
ml- (43 mM) during feeding to zero during daylight hours.
The results of the hypothermia resistance test are presented in Table V.
The rats which had received the ethanol-containing diet exhibited smaller
decreases in rectal temperature following an acute dose of ethanol than
control animals. The mean difference was significant within 15 min of
injection and was maintained for at least 120 min.
DISCUSSION
Circero [ 121 had suggested that the following criteria should be applied
when assessing the adequacy of any particular model in alcohol studies: (i)
alcohol should be self-administered, via the oral route and achieve a blood
concentration which elicits an effect; (ii) tolerance should develop after
prolonged administration; (iii) withdrawal behaviour should be observable;
(iv) physiological or biomedical complications should arise. It is not man-
TABLE V
THE EFFECT OF CHRONIC ETHANOL CONSUMPTION UPON RECTAL
TEMPERATURE FOLLOWING AN ACUTE DOSE (0.2 g 100 g- BODYWEIGHT)
OF ETHANOL
Significance of difference; a u b, P < 0.05; c u d, P < 0:Ol; e u f, P < 0.001.
Time after
injection
(min)
Rectal temperature (C)
Ethanol-fed
N=6
Controls
N=6
0 37.7 + 0.9
15 37.6 ? 0.3a
30 36.9 + 0.4a
45 36.6 + 0.5
60 36.5 ? 0.5
75 36.3 2 0.6e
90 36.3 ? 0.6e
105 36.1 ? 0.5e
120 36.1 + 0.5e
37.5 ? 0.3
36.1 ? 0.5b
35.2 ? 0.4b
34.5 + 0.3d
33.8 + 0.6d
33.2 ? 0.4f
32.7 ? 0:5f
32.8 ? 0.4f
32.9 + o.5f
Atmospheric temperature 22C.
342
datory that the model should meet all of these criteria, rather it should
meet those most relevant to the objectives of the particular study [ 121.
Clearly, the provision of alcohol to the subject animal as a liquid diet
fulfills the requirement for self-administration via the oral route (criterion i).
Blood alcohol concentrations fluctuate markedly in response to the eating
pattern of the animals and to the circadian rhythm, but concentrations of
up to 2 mg ml- (43 mM) have been reported [ 21. The present diets, whilst
not achieving such high blood concentrations, do elicit effects [18-251
which, with due regard to confounding variables such as nutritional adequacy
(discussed below), may be attributed to alcohol.
The present model has not been extensively evaluated to determine
whether a high degree of tolerance to ethanol develops following chronic
administration of the liquid diet. The limited data presented herein suggests
that, by the criterion of resistance to ethanol-induced hypothermia, tolerance
may develop. Liquid diet regimes have certainly been shown by others to
elicit tolerance in rodents [ 28.31,32] ,
No attempt has been made to validate the models reported here by the
criteria of alcohol dependence and withdrawal phenomena. Freund in 1969
[33] demonstrated that mice developed dependence and withdrawal
symptoms within 3 days of receiving a liquid diet containing ethanol.
A large body of literature attests to the suitability of the liquid diet
model as a means for induction of alcohol-related physiological complications.
Many such complications may be multi-factorial in origin and, although an
animals model generally provides no difficulty in controlling most con-
founding variables, the nutritional adequacy of liquid diets has remained
contentious. Indeed, the adverse effects of ethanol until quite recently have
been often related to nutritional deficit rather than to alcohol per se [34,35] .
One advantage of the liquid diet, however, is that the composition of the diet
can be readily manipulated, not only to mimic a particular experimental
nutritional state, but also to overcome perceived nutritional inadequacy.
The latter assumes the existence of an appropriate measure of nutritional
adequacy, for example, the ability of the diet to meet the well-established
nutritional requirements of the experimental species or the ability of the diet
to support the growth of experimental animals at a rate equal to or better
than, that of the animals usual diet.
In the present experiments food consumption by the rats of the experi-
mental diets met between 70 and 100% of the protein and between 50 and
250% of the fat requirements of a growing female rat according to the
recommendations of the U.S. National Research Council [ 361. The carbohy-
drate content of the diet varied reciprocally with the fat content but was
always sufficient to meet needs. It should be noted that the more recent
recommendations of the NRC [ 371 have suggested a protein content of 13%
of total energy is nutritionally adequate, a concentration easily exceeded by
the present diets. Rao and Larkin [ 153 have observed that the metabolic
changes that ethanol elicits in the liver may lead to enhanced protein
343
utilization for gluconeogenesis so that, despite ingestion of the required
amount of protein, insufficient is available for growth. Furthermore, the
presence of ethanol in the diet may lead to malabsorption [38], further
reducing the biological availability of nutrients. Nitrogen balance studies
with the liquid diets have indicated, however, that malabsorption does not
occur to a significant degree [ 181 although altered protein metabolism is
characteristic of ethanols physiological actions [ 19-251.
The micronutrient (vitamins and mineral) content of the diets is more
than adequate to meet requirements [36,37] and Porta et al. [ 391 concluded
that if the ethanol concentration was less than 30% of calories (27% in the
present study) micronutrient deficiency was unlikely. Although the data
presented herein are for feeding trials of only 21 days duration, no indica-
tions of micronutrient deficiency have been observed in experiments lasting
up to 60 days (unpublished observations). If micronutrients are in excess
of requirement higher ethanol concentrations may be tolerated [39] , an
observation supported by Lieber and DeCarli [ 171.
Ultimately the adequacy of the diet is determined by its ability to sustain
growth in a way comparable to commercial diets. The diets presented here
are clearly able to attain this objective for growing female rats. They do not,
however, promote a growth rate in male rats comparable to that of animals
maintained on the commercial diet although the rate of weight gain is
similar to or greater than that of male rats fed other commonly used liquid
diet formulations [ 14,401. Whilst modifications to liquid diets may be made
to achieve exceptionally high growth rates in male rats [ 161 these may be
considered abnormal and thus do not provide a firm basis for comparison.
It is unlikely that an animal model will be developed that mimics totally
all the characteristic effects of alcohol ingestion in man. The liquid diet
model provides for the examination of the development of tolerance, with-
drawal and biomedical effects of excessive alcohol intake. Caution should,
however, always be exercised in applying conclusions drawn from such animal
studies to the effects of alcohol in man.
ACKNOWLEDGEMENTS
The experimental work reported here was, in part, financially supported
by the National Heart Foundation of Australia. The author acknowledges
the assistance of co-workers cited in Refs. 18-27.
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