The effects of a liquid ethanol diet on nutritional status and fluid balance in the rat

Mariann R. Piano,*, James Artwohl1,, Shann Dixon Kim,2 and Gerry Gass,3

Department of Medical–Surgical Nursing, University of Illinois at Chicago, 845 S. Damen (M/C 802), Chicago, IL 60612,
1 University of Illinois at Chicago, 845 S. Damen, Chicago, IL 60612,
2 School of Kinesiology, University of Illinois at Chicago, 909 W. Roosevelt Road, Chicago, IL 60608 and
3 Baxter Laboratories, Round Lake, Illinois, USA

Received 9 August 2000; in revised form 16 January 2001; accepted 29 January 2001


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The liquid ethanol diet is a widely used method of ethanol administration. The purpose of this study was to evaluate fluid balance using a multitude of physiological parameters (electrolytes, osmolality, total serum proteins, fluid intake/output and body weight), during and after the introduction of liquid ethanol diet. Animals were randomized into four different dietary protocols (two control and two ethanol groups) and were placed in metabolic cages for 16 days. Serum electrolytes, as well as the above parameters, were measured before, during and 1 week after the introduction of 9% (v/v) ethanol-containing diet (Lieber–DeCarli: LD). After the first night on 9% (v/v) ethanol LD, animals had significantly decreased diet consumption, urine output and body weight. However, a major finding of this study was that, during the habituation phase, the electrolyte values remained within the normal range for rats and, in particular, serum sodium was not altered at any time point measured in this study. Based upon the findings from this study, it is recommended that body weight be carefully monitored as a measure of the animal's equilibration and physiological adaptation during the initiation of a liquid ethanol diet, since neither the serum sodium nor calculated osmolality values were changed. Our results also highlight the need to offer water to animals during the habituation phase of ethanol consumption. This is because ethanol rats that were offered water ad libitum lost less weight than groups that did not receive water ad libitum, despite consuming the same amount of LD diet.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animal models of ethanol consumption have been used extensively over the last several decades to study the medical consequences of ethanol consumption (Erikson, 1969Go; Bode et al., 1986Go; Lieber and DeCarli, 1989bGo; Jaatinen et al., 1994Go; Pandey et al., 1996Go). These models have been the most efficient means by which to study the effects of alcohol, under controlled or modified nutritional conditions (e.g. high vs low fat or protein). Models of ethanol consumption or exposure are varied and include: ethanol-containing liquid diets, ethanol gastric gavage, ethanol inhalation, and intraperitoneal injection of ethanol (Keane and Leonard, 1989Go; Sampson et al., 1997Go; Juarez and Barrios de Tomasi, 1999Go; Iimuro et al., 2000Go). Most of these models allow the investigator to administer different concentrations of alcohol and, therefore, simulate specific clinical conditions, such as alcohol-induced liver injury or alcoholic heart muscle disease (Iimuro et al., 2000Go; Kim et al., 2001Go).

Of the aforementioned models, the ethanol-containing liquid diet is one of the most commonly used. These liquid diets are nutritionally complete, and have been shown to produce a high level of ethanol consumption (Lieber and DeCarli, 1982Go, 1989aGo,Lieber and DeCarli, bGo; Ward, 1987Go; Pandey et al., 1996Go; Sampson et al., 1997Go; Piano et al., 1999Go). However, these diets have been criticized because, despite equal energy values between the control and alcohol diets, there are differences between the groups in the pattern of diet consumption, the initial weight gain and the total weight gain by the animals. With regard to the latter, an especially vulnerable time is during the habituation phase of the liquid diet. The taste of ethanol is aversive to rats, and the introduction of alcohol is associated with decreased food intake and weight loss, potentially important non-experimental variables. Therefore, during this phase, the animals are gradually introduced to progressively increasing concentrations of ethanol.

Moderately restricted food intake has been reported to affect the secretion patterns of a number of hormones, which include renin, cortisol, thyroid hormone, and insulin (Naim et al., 1980Go; Oliveira de Souza and Masur, 1981Go; Palou et al., 1981Go; Hill et al., 1985Go). In addition, moderate food restriction can impair physiological function in organs such as the heart, liver, and central nervous system (Landsberg and Young, 1982Go; Bodnar et al., 1990Go; Brady et al., 1990Go; Hilderman et al., 1996Go; McKnight et al., 1996Go). Moreover, this type of diet could affect the animal's hydration status because the combination of decreased liquid diet consumption and the diuretic effects of ethanol could lead to dehydration. To our knowledge, no studies have examined whether decreases in food consumption and weight loss are accompanied by changes in hydration or electrolyte status. Furthermore, no study has investigated daily fluid intake, urine output and body weight, which are important measures of hydration status during or after the habituation phase to a liquid diet, nor have any changes with respect to other measures of fluid status, such as serum electrolytes and osmolality, been investigated. Therefore the purpose of this study was to characterize the fluid and nutritional status of rats using multiple physiological measures, during and after the introduction of an ethanol liquid diet.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Experimental design and animals
Virus-free, male Sprague–Dawley rats (Charles River, Portage, MI, USA) were used in all experiments. Animals were randomly assigned to one of four diet groups (n = 8), which received the 1982 nutritionally complete Lieber and DeCarli (LD) liquid ethanol or control diet (Bioserve, Frenchtown, NJ, USA). In the 1982 formulation, 18% of the calories are derived from protein, 35% from fat and 47% from carbohydrate (Lieber and DeCarli, 1982Go). In terms of the caloric profile, the diet provides a total of 1000 kcal/l (the latter is the theoretical value, the actual kcal/l can vary between 1000 and 1013 kcal/l per batch preparation). There were two ethanol groups and two control groups. One ethanol group received an LD ethanol (9% v/v) diet as their sole source of food and water and was designated as the EtOH group. A second group also received the LD ethanol diet, however, this group was given water ad libitum and designated the EtOH + water group. Two control groups received the control LD diet in which maltose-dextrin was isocalorically substituted for ethanol. One control group received the LD control diet and water ad libitum, and was designated the CON + water group. The second control group received the LD control diet, but was pair-fed to the EtOH group (i.e. this control group received the same number of calories consumed by the EtOH group the night before). This control group also was given water ad libitum and therefore was designated the CON pair-fed + water group. Except for the CON pair-fed + water group, all animals were allowed to consume their respective LD diet ad libitum. LD diet was monitored daily and animals were allowed 24 h access to their LD diet and water. Fresh diet was provided daily between 16.00 and 17.00.

The animals in the ethanol groups were gradually introduced to a final concentration of 9% (v/v) (13 g ethanol/kg/day) over a 9-day period designated the habituation phase. The habituation phase was as follows: the first 3 nights the animals received 100 ml of LD control diet, provided in 150 ml graduated plastic bottles. This was followed by 3 nights of 3% (v/v) of ethanol in the diet, after which the ethanol concentration was increased to 6% (v/v). After 3 nights of consuming 6% (v/v), the ethanol was increased to the final concentration of 9% (v/v) for 7 days.

Blood collection and processing
Blood was collected for electrolyte determination, in the morning of days 1 (baseline), 10 (1 day after the introduction of 9% v/v) and 16 (1 week after the introduction of 9% v/v) in all animal groups. On days 1 and 10, rats were anaesthetized with ether and 2 ml of blood were obtained from the right retro-orbital plexus with a 1 mm non-heparinized microhaematocrit tube (Scientific Products, McGaw Park, IL, USA). This blood volume represents <9% of the animal's total blood volume. Others have shown this volume of blood removal is not associated with marked haemodynamic changes (Sorg and Bruckner, 1964Go). The final blood sample was obtained at the end of the protocol when animals were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.), and exsanguinated via the descending abdominal aorta.

All blood samples were centrifuged (4°C, 1000 g, 30 min) and serum was removed. Blood ethanol levels (BELs) were measured with the Sigma Diagnostics ethanol kit (333-UV; Sigma Chemical, St Louis, MO, USA) on days 10 and 16 in both EtOH groups. Serum sodium (Na+) and potassium (K+) were measured using the Ciba Corning Na+/K+ Analyzer (Model M614/2 Ciba Corning Na+/K+ Analyzer, Medfield, MA, USA). Serum total protein, glucose, and blood urea nitrogen (BUN) were measured in all blood samples with the biuret technique, hexokinase method and modified urease technique, respectively (Ciba Express 550 Chemistry Analyzer, Medfield, MA, USA). Plasma osmolality was directly measured with the freezing-point depression technique (3 MO, Advanced Micro Osmometry, Norwood, MA, USA). Calculated serum osmolalities were determined according to the following equations:

Statistical analysis
All data are expressed as means ± SEM. Comparisons among groups were made using two-way ANOVA (Sigma Stat, Jandel Scientific, CA, USA). If significant differences were found, a Tukey's post-hoc test was used to determine which groups were different from each other (P < 0.05).


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Body weight and fluid intake
Over the 16-day protocol, the pattern of, and absolute, weight gain were different among the four groups (Table 1Go). In all groups there was a significant increase in body weight at day 10 compared to baseline. The CON + water group gained weight over the entire protocol, for a mean weight gain (MWG) of 67 g (or 24% increase over the baseline body weight). The body weights on day 16 of the CON + water group were significantly greater than the other three groups (P < 0.05). By day 16, the EtOH and EtOH + water groups lost 27 and 17 g, respectively, from day 10. This resulted in a MWG over the 16-day protocol of only 2 g in the EtOH group, and 7 g in the EtOH + water group. Since the CON pair-fed + water group's intake was adjusted to that of the EtOH group, this group had similar changes in body weight. However, their weight loss was less severe, and by the end of the 16-day protocol, the CON pair-fed + water had a MWG of 20 g.


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Table 1. Changes in body weight (g) and the mean net weight gain (MWG; g) from baseline to day 16 during the habituation phase of ethanol consumption
 
All groups consumed a large amount of LD diet at baseline (Table 2Go). In both ethanol groups, a significant decrease in the diet consumption was found at day 10, compared to baseline (P < 0.05). By day 16, the diet consumption of the ethanol groups had increased, but was significantly less than the CON + water group. The CON pair-fed + water and EtOH + water groups began to consume water at day 10. These two groups continued to drink water until the end of the study. By day 16, the EtOH + water group drank more than twice as much water as the CON pair-fed + water group. The CON + water group did not drink any water at any time during the protocol.


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Table 2. Liquid diet (ml) and water intakes (ml), urine output, and mean net water intake in all groups during the habituation phase of ethanol consumption
 
At baseline, urine output (U/O) was similar among all groups (Table 2Go). At day 10, urine output was significantly decreased in the EtOH and the CON pair-fed + water groups compared to their respective baseline values (P < 0.05). In both these latter groups, urine output remained significantly decreased at the end of the study, compared to baseline. Urine output was similar between the CON + water and EtOH + water groups and did not significantly change during the 16-day protocol. By day 16, the mean net water intake (mNWI) (oral intake minus urine output) was significantly higher in the CON + water compared to the other diet groups.

Serum electrolytes and osmolality
No significant differences were found among groups in serum sodium at any point in the 16-day protocol (Table 3Go). In all groups, the BUN had significantly decreased by day 10, without further significant changes at day 16. The serum osmolality measured on day 10 was significantly greater in the EtOH group compared to that of the CON pair-fed + water group, and by day 16, this parameter had increased further in the EtOH group (P < 0.05). The calculated osmolality was not different among the groups at any time point. The osmolal gap was significantly increased in the EtOH group at days 10 and 16. On day 10, the BELs (samples drawn at 07.00) were similar between the EtOH and EtOH + water groups. However, in both groups the BELs were significantly increased at day 16 (Table 3Go).


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Table 3. Serum electrolyte and osmolality levels during the habituation phase of ethanol consumption
 
Total serum proteins (TSP) were measured in all groups. In all groups over time the TSP values remained unchanged and ranged from 6.1 ± 0.2 to 6.4 ± 0.2. The only exception was that in the EtOH group: a significant decrease was found in the TSP value (5.0 ± 0.6) at 1 week after 9% (v/v) compared to the earlier times and other groups.

Urine sodium levels were significantly increased in the EtOH group at day 16 compared to day 10 and baseline levels, and compared to the other diet groups (Table 4Go). The urine potassium levels were significantly increased in the EtOH group by day 10, and remained high at day 16 compared to the other diet groups (Table 4Go).


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Table 4. Urine electrolytes in diet groups during the habituation phase of ethanol consumption
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
To our knowledge, this is the first evaluation of the hydration status, using a multitude of physiological parameters, in the rat during and after the introduction of an ethanol liquid diet. This study reaffirms the need for an habituation period, since there is a clear decrease in liquid diet intake and a weight loss, which is associated with the progressive increase in the diet's alcohol concentration. In the light of the weight loss, a major finding of this study was that during the habituation phase the electrolyte values remained within the normal range for rats and, in particular, serum sodium was unaltered at any time point measured in this study (Charles River Technical Bulletin, 1984Go; Riley and Cornelius, 1989Go).

Along with serum sodium, we measured total serum proteins (TSP), which are usually elevated in dehydration. Overall, we found no changes in TSPs in any of the groups, except for a significant decrease in TSPs in the EtOH group by day 16. We believe that the lower value in the EtOH group is related to a catabolic physiological state that probably developed as a result of decreased diet consumption, as well as metabolic adaptation to increased ethanol consumption (Pirola and Lieber, 1972Go). Interestingly, the EtOH + water group had TSP values similar to the control groups. This group drank large quantities of water, which may have offset the effects of decreased diet consumption.

Another important indicator of the hydration status is plasma osmolality. In the present study, the measured plasma osmolality was significantly increased in the EtOH group on day 10, with a further increase on day 16. These increases in measured osmolality paralleled the progressive increases in the EtOH group's BELs. We attribute the increase in the measured osmolality to ethanol, because ethanol is an ineffective osmole. This means that ethanol can contribute to the total osmolality, but not to the tonicity of the blood. (Other solutes/osmoles contributing to the ineffective osmolality include glucose and BUN.) Serum tonicity determines the overall hydration status, and most importantly, affects the shift of water between the extra- and intracellular fluid compartments (Oster and Singer, 1999Go). The measured plasma osmolality values were determined by the direct measurement of osmolality with an osmometer. Direct osmometry is influenced by the presence of ineffective osmoles, such as ethanol (Walker et al., 1986Go). Interestingly, the calculated osmolalities (Osmolcalc) of these groups were not significantly different from the values in the other groups at any time. We believe that the calculated value reflects more accurately the hydration status, because the relative contributions of significant ineffective osmoles, such as ethanol and glucose, are calculated as part of the equation.

When evaluating the hydration status, serum electrolytes, total serum proteins and osmolality must be interpreted along with fluid intake and output, as well as body weight. Our findings demonstrate that the EtOH + water and CON pair-fed + water groups did consume water that could have been related to thirst or a behavioural response to the presence of water.

As expected, urine output was related to fluid consumption. For example, the animals in the EtOH group had significantly decreased urine output on day 10, which corresponded to their decrease in fluid intake. Among a variety of species, rats have been found to be very efficient at renal water conservation (Toth and Gardiner, 2000Go).

Changes in body weight are also one of the parameters used to evaluate the hydration status and are helpful in estimating the magnitude of the fluid loss. However, decreases in body weight in the absence of changes in other hydration parameters, such as serum sodium, tonicity and serum proteins, indicate that the weight loss may be due to other physiological mechanisms. Both the ethanol groups and the CON pair-fed + water group lost weight. The weight loss may have been in part related to their water intake, but it could also have been related to a decrease in total caloric intake and metabolic adaptation to ethanol. The minimum daily intake of calories required to maintain a young, adult rat's body weight is 150–160 kcal/kg/day (or 40–45 kcal/day for a 300 g rat) (Johnson et al., 1990Go). The LD diet at the 9% (v/v) ethanol concentration level provides ~1.0–1.4 kcal/ml liquid diet. The CON + water group consumed considerably more calories than the minimum requirement (~120 kcal/day), which was reflected by a weight gain of 63 g by the end of the protocol. The ethanol-fed rats in the study fell below this level on days 10–13 (~33–46 kcal/day). Perhaps because ethanol is not utilized as effectively as an energy source, the rats may have been in negative energy balance for a slightly longer period of time (Lieber, 1991Go). We have found that, if animals consuming the 9% (v/v) ethanol LD diet are allowed to drink for longer periods of time (e.g. more than 1 month), ethanol LD diet consumption progressively increases, such that animals consume an average of 60–70 ml of diet per day, or 85–100 kcal per day and weight gain is well within normal limits (data not shown). Therefore, at these later times the animals will have become accustomed to the diet.

We also measured BUN levels. Over the course of the protocol, all diet groups had decreased BUN levels, which we believe was probably related to the quality of the protein found in the liquid diet, rather than ethanol per se. Because of the high quality of protein used in the commercial diet, BUN levels would decline because fewer metabolic by-products from protein metabolism (e.g. urea) would be produced.

Our laboratory has used the 1982 LD formulation (Bioserve, NJ, USA) for over 10 years. When prepared according to the manufacturer's directions, the final ethanol concentration is 6.7% (v/v). We have modified this, such that the animals receive 9% (v/v). Over the last several decades, investigators have used higher concentrations of ethanol, with many laboratories using 10%, rather than lower concentrations of ethanol (2–5% v/v) (McMillen, 1997Go). Initially, we performed a series of ethanol-tasting experiments to establish which concentration would allow for optimal drinking, as well as body growth. Given the different types of rodent strains, some of which are ethanol-preferring, it is recommended that investigators perform their own series of ethanol-tasting experiments to determine which concentration of ethanol will produce a high level of ethanol consumption.

For this study, a related question concerned the adaptation of rats to the ethanol liquid diet. A physiological definition of adaptation has been provided by Weisbroth et al. (1997), who stated that equilibration or adaptation is the time it takes the body to return to within one standard deviation of the control group(s). Based upon the data from the present study, the variable that should be monitored as an indicator of adaptation should be body weight, since clinical chemistry parameters were not changed. Other animal studies using rats have utilized food or water deprivation to maintain animals at ~80% of their ad libitum feeding weight, which is considered neither unethical nor excessive deprivation (Riley and Cornelius, 1989Go).

In summary, regardless of the concentration, the animals will require an habituation phase. Based upon the findings from this study, it is recommended that body weight is carefully monitored as a measure of the animal's equilibration and physiological adaptation, since neither serum sodium nor calculated osmolality values were changed. Using our protocol, rats require 4–6 weeks to adapt to the full-strength diet (data not shown). At this time, body weights are usually within one standard deviation of the LD control rats. Our results also highlight the need to offer water to animals during the habituation phase of ethanol consumption. The EtOH + water group lost less weight, compared to the EtOH-only group, and yet they consumed the same amount of LD diet. We recommend providing animals with water during the habituation phase; however, supplemental water is probably not required after this time. In previous studies, we offered supplemental water to the EtOH and CON groups that had been on the LD protocol for several months and none of the animals from either group drank water. Although no diet regime is perfect, we believe that the liquid diet protocol can be safely and effectively used in rats to examine the effects of long-term ethanol consumption on a number of different physiological systems. How soon the animals adapt and begin to gain weight may depend on the strain of rodent, sex and final concentration of ethanol in the diet.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study was supported by the National Institutes of Alcohol Abuse and Alcoholism AA 11112 (M.R.P.) and by the Office of Research Development at the University of Illinois (M.R.P).


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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