Growth, liver lipid and blood amino acids in rats fed ethanol with an adequate diet

Drug and Akohol Dependence, 22 (1988) 49- 54 Elsevier Scientific Publishers Ireland Ltd.

49

Growth, liver lipid and blood amino acids in rats fed ethanol with an adequate diet* James

D. Shoemaker

and Willard

J. Visek**

University of Illinois College of Medicine, 506 S. Mathews Urbana-Champaign, Illinois 61801 K%S.A.) (Received February 20th, 1988)

The weight, histology and RNA, DNA, protein and lipid content of the liver and arterial and portal plasma amino acid concentrations were determined in male Sprague-Dawley rats fed a liquid diet which met AIN-76A standards with 36oh of the calories supplied by ethanol. The dietary components of the dry mixture in percentages by weight included 20% casein, 22% sucrose, 43ob dextrin, 5% corn oil, vitamins, minerals and other dietary factors. For feeding these were suspended in distilled water containing 2.5ob xanthan gum with or without ethanol to supply 1 kcallml. The feeding method employed perforated neoprene discs floated on top of the suspended diet to control evaporative losses. Animals were pair fed or ad libitum fed for 8- 10 weeks. Gain/feed ratios were virtually identical for ethanol-fed rats and their pair-fed controls. Ethanol intake of ad libitum fed rats averaged 14.8, 10.3 and 7.4 g/kg BW/day after 1. 5 and 10 weeks, respectively. No chemical or histological evidence of liver fat accumulation or significant differences in arterial or portal amino acid concentrations were detected in animals fed ethanol. The lack of apparent ethanol toxicity is discussed in relation to the results of others and to our earlier report of increased erotic acid excretion by ethanol-fed rats. Key words: ethanol; steatosis; cirrhosis: rats; xanthan 8 Im; plasma amino acid

Introduction

Experimental studies have shown that as the concentrations of protein, vitamins and minerals of diets fed with ethanol approach dietary requirements, the biochemical and histological changes indicating liver injury become less severe. Lieber [l] demonstrated four-fold accumulation of lipid in the livers of rats fed a diet containing 36% of calories as ethanol for 24 days. He later showed [2] that this lipid accumulation was dietary fat dependent and minimal with 10 -25% of calories supplied by fat and 36?4~ from ethanol. In earlier work Porta [3] stressed the importance of choline in preventing steatosis in rats found only a transient *Partially reported in Fed. Proc., 43 (3) (1984) 495. **To whom all correspondence should be sent. 0376-8716/88/$03.50 OElsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

increase in liver lipid after 8 weeks of feeding diets supplying 36Ok, of calories from ethanol and containing 2.0 g/l of choline chloride, 8 times the amount fed by Lieber [l]. Both of these laboratories used mineral mixtures [4] which contained no zinc, now recognized as a dietary essential and needed for alcohol metabolism 151.More recently Shorey [6,7] formulated alcohol-containing liquid diets using AIN-76A [8] vitamin and mineral mixtures and carrageenan for suspension [8]. We are reporting studies with rats fed a slight modification of this formulation with xanthan gum as the suspending agent and a novel method of feeding. This report contains data on liver weight, histology, RNA, DNA, protein and lipid content with arterial and portal plasma amino acid concentrations from these studies.

50

Methods

Diet formulation Previous investigators [1,6] have used the polysaccharide carrageenan to thicken alcoholcontaining liquid diets. Carrageenan increases viscosity hut is not a true suspending agent like xanthan gum, a polysaccharide (mol. wt., 2 x 106-50 x lo6 Dal produced by Xanthomonas campestris and commonly used for emulsifying foods and cosmetics [17]. For use in diets 260 g of xanthan gum were added slowly to 18 1 of autoclaved glass-distilled water at 100°C during constant mixing (Blakeslee Model F-30 mixer, G.S. Blakeslee Co. ChicagoJLl. These suspensions were stored at 4OC and portions were used to prepare fresh liquid diet daily. The diets contained concentrations of protein, vitamins and minerals recommended by the American Institute of Nutrition (AIN-76A) [8]

Table I.

with one-half of the recommended concentration of fiber replaced by the xanthan gum (Table Il. The sucrose content was twice that used by others [7]. Batches of the dry ingredients mixed in 6 kg quantities were stored at - 10°C. For each feeding day, 260 g or 170 g of dry ingredients for the control and ethanol containing diets, respectively, were mixed with 450 ml of the xanthan gum suspension. Sixty-five ml of ethanol were then added to the appropriate mixture, and the volumes were adjusted to 1 1 with double distilled water. The calculated caloric densities were: control diet, 0.956 Cal/g and ethanol diet, 0.983 Cal/g. Nutrient-to-calories ratios were virtually identical. The diets were fed in wide-mouth (7 cm diameter) 4 oz glass ointment jars equipped with metal screw caps with a hole 3.2 cm in diameter. Neoprene discs 3 mm in thickness with a single 2 mm hole in the center and allowing

Composition of liquid diets. (Mean f S.E.M.)

Component

Control gn

Ethanol Calories (%)

Casein (85% protein)” Corn 0iP Ethanol’ Cellulose” Xanthan gumd AIN-76A vitamin mix’ Choline bitartrate’ Methionine’ AIN-76A mineral mix” Sueroses Dextrinh

52 13 0 6.5 6.5 2.6 0.52 0.78 9.1 56.3 112.5

20.7 11.6 0 0.3 tr 22.5 44.9

Total Water

259.8 toll

100 -

-

% Nonwater wt. 20 5 0 2.5 2.5 1 0.2 0.3 3.5 21.7 43.3 100

a Teklad, Harlan Sprague - Dawley Co., Madison, WI. b Mrs. Tucker’s Pure Corn Oil, Anderson Clayton Foods, Dallas, TX. cU.S. Industrial Chemical Company, Tuscola, IL. dKelco K8B12-lK, Kelco Division of Merck and Co., Rahway, NJ. cDyets, Inc., Bethlehem, PA. ’ Ajinomoto U.S.A., Raleigh, NC. g Great Western Sugar Co., Denver, CO. hStaley Food Service, Elk Grove Village, IL.

gn (I)

52 13 51.4 6.5 6.5 2.6 0.52 0.78 9.1 56.3 22.5 221.2 to 11

Calories (0~)

20.7 11.7 36 0 0.3 tr 22.5 8.9 100 -

% Nonwater wt. 23.5 5.9 23.2 2.5 2.5 1.17 0.23 0.35 4.1 25.4 10.2 100

51

about 1 mm of clearance between their perimeter and the inside diameter of the jars were placed on top of the diet offered. The animals obtained food by depressing the discs. Uneaten portions averaged 95.2 f 5.4% of their original ethanol content 24 h after feeding as determined by gas chromatography described below. Animals and housing Two experiments were performed, each using 20 randomly assigned male SpragueDawley rats (Harlan - Sprague Dawley, Madison, WI). Experiment I had 9 control and 11 ethanol-fed rats with an initial weight of 274 + 4.1 g (mean + S.E.M.1 which were fed for 10 weeks. The feed allowance for each control animal was based upon the mean consumption by the ethanol-fed group during the previous day. During Experiment II, ten control and ten ethanol-fed rats with an initial weight of 170 f 2.0 g (mean f S.E.M.1 consumed the diets ad libitum for 8 weeks. Each experiment allowed 3 days for the animals to become accustomed to the purified diet in dry form. Then increments of 12% in ethanol calories per day were added until the total calories supplied by ethanol equaled 36%. The rats were confined to individual stainless-steel wire bottom cages in rooms with a 12-h light/dark cycle and a temperature maintained at 24 OC. Plasma amino acid and ethanol analysis At the end of pair-feeding in Experiment I, the animals were anesthetized without fasting and 1 - 2 ml of blood were drawn from the portal vein into a heparinized syringe. Another blood sample was also drawn by cardiac puncture. Immediately after collection, the blood samples were individually mixed with 200 pl of 40/o NaF and centrifuged at 500 x g for 5 min. For deproteinization 500 p.l of plasma were added to 1 ml of ice cold 7.5Ok1sulphosalicylic acid in lithium citrate buffer (pH 1.8) [9] and centrifuged at 4800 x g for 15 min. The supernatant was stored at - 70 OC (Beckman Instruments application DS561) until analyzed for amino acids by ion exchange chromatography (Model 119CL, Amino Acid Analyzer, Beckman

Instruments, Palo Alto, CA) according to the manufacturer’s instructions for physiological fluids. Blood and dietary ethanol analyses were based upon standards prepared by adding 20 ~1 of n-butanol to 180 4 of deproteinized plasma or filtered liquid diet. Two microliters of preparations for analysis were injected directly onto a 2 mm ID gas chromatography column packed with 3% OV-17 on Chromosorb W(HP) (Pierce Chemical Co., Rockford, IL) maintained at room temperature with nitrogen as the carrier gas flowing at 30 ml/min. Ethanol content was calculated from the peak area in relation to the nbutanol standard curve. Sacrafice protocol Forty-five minutes before sacrifice, five animals per group from experiment I were injected i.p. with 5 &i of 6[14C]orotic acid (spec. act. 40-60 mCi/m mol New England Nuclear Corp., Boston, MA) in 500 ~1of bicarbonate buffer (pH 7.4). The livers were weighed and separate samples were taken for histology and homogenization. Samples for histology were preserved in 2% glutaraldehyde in 0.1 M sodium phosphate, pH 7.4. Homogenates prepared immediately with a Kinematica Polytron (Brinkman Instruments, Westbury, NY) in 1.5 ml of homogenization buffer per gram of tissue [lo] were stored at - 70°C until analyzed for dry weight, total fat, RNA, DNA and total protein. All operations were performed at 4OC except as noted. Total dry weights of liver were determined upon 200 mg aliquots of the homogenates (0.5 ml) in tared aluminum weighing dishes held for 24 h at 7OOC). Two hundred mg aliquots were also treated with 6 ml of 1.0 N HClO, and centrifuged for 15 min at 4800 x g. The pellets were resuspended twice in 6 ml of 0.2 N HClO, and recentrifuged at 4800 x g each time. Lipids were then extracted with 2.0 ml of 1.0 N potassium acetate in absolute ethanol, twice with 4.0 ml chloroform :methanol (1: 1) and once with 2.0 ml of absolute ethanol. Each of these extractions included rehomogenization followed by centrifugation at 4800 x g for 10 min. The solvent extracts, pooled and evaporated overnight

52 Table II. The effects of a liquid casein based diet supplying 36% of calories from ethanol (calculated at 7 kcal/g), upon weight gain, feed intake and gain/feed of rats. (Mean f S.E.M.). Control

Ethanol

I. Pa+feeding e3cpetiment (60 &ysl Weight gain (g) 126.0 f 25.0 Feed intake (cal160d) 4354.0 f 180 Gain/feed (mg/cal) 28.9 f 4.9 No. of animals 9

122.0 f 14 4255.0 f 143 28.7 + 2.4 11

II. Ad libitum feeding experiment 151duysl Weight gain (g) 203.6 f 8.0’ Feed intake hxl/51 d) 3444.0 f 8gb (Gain/feed (mglcal) 59.0 f 1.2 No. of animals 10

168.7 f 6.7’ 3095.0 f 47” 54.6 f 1.4’ 10

t = 3.34, P < 0.01. b t = 3.18, P< 0.01. et= 2.35, P < 0.05. l

in tared vials at 70°C were weighed

(Table II). Experiment II rats fed 36% of total calories as ethanol for 8 weeks gained 1’7% less weight than their counterparts fed the same basal diet without ethanol. Both of these groups were fed ad libitum (Table II). This difference in weight gain was associated with 10% lower food intake and 7% less efficient utilization of food for growth. Liver weight, RNA, DNA, protein and lipid contents are shown in Table III. There was

Table III. The weight and RNA, DNA, protein and lipid of livers and the [l’C]orotate uptakes for rats fed ethanol. Liver component

for total

lipid.

The fat-free precipitates suspended overnight in 5.0 ml of 1.5 N HClO, and centrifuged were resuspended for two extractions in 2.5 ml 0.5 N HClO, at 70°C for 20 min. The protein remaining was solubilized in 4 ml of 1 N KOH for 15 min at 70°C. Incorporation of 6[14C]oroticacid was based upon radioactivity in the RNA and DNA fractions as described by Glazer and Weber [ll]. Radioactivity in 2.5 ml aliquots of the RNA and DNA extracts mixed with Aquasol II (New England Nuclear Corp., Boston, MA) was assayed with a Beckman LK 9000 Scintillation Counter. RNA, DNA and protein content of the liver homogenates were determined calorimetrically according to Cerotti [12], Hubbard [13] and Lowry [14], respectively. Results

Experiment I animals showed identical gainfeed ratios. Being 100 g heavier in initial weight than Experiment II animals and pair fed they had showed somewhat lower gain-feed ratios, as expected. Feed intake, weight gain, and ethanol intake for both replicates are shown

Body wt.(g) Liver wt, g/kg body wt. Pair-fed, Expt. I Ad libitum fed, Expt. II

Control w) 388

Ethanol WI

f 32 (1-I)

32.1

i

(7) 38.23 f

373

1.25

30.65 -c

0.56

0.74’

00) 34.41 f

0.39’

(10)

(10) Liver lipid, mg/g wet wt. Pair-fed, Expt. I Ad libitum fed, Expt. II

86

f

86

(7) f

3.2 1.7

91

f

1.9

90

(9) * (10)

1.6

*

1.10

(10) Liver lipid, % dry wt. Pair-fed. Expt. I Ad libitum fed, Expt. II

[*‘C)Orotic acid, dpmhng

30.4

f

Protein/DNA, mglkg

1.64

(7) 27.3 + (10) 1830

1.1

f 400

RNA

31.8

(9) 28.7 f (10) 2820

(5) 78.9

*

5.90 f

0.87

f 700 (5)

17

67.4

(7) RNA, mg/g wet wt.

2 17 120)

0.50

(7)

+ (9)

5.82 + (9)

11

0.55

RNA. mg/liver

77.8

f (7)

6.6

68.6

f (9)

6.5

DNA, mg/liver

28.7

k (7)

4.0

23.5

f (9)

3.18

2.26 f (7)

0.49

1.58 f (9)

0.26

Protein, g/liver

‘t = 4.55, P < 0.001.

53

histological evidence of abnormal accumulation of lipid in livers of ethanol-fed animals either at 8 or 10 weeks. The difference in G[“C]orotic acid uptake by RNA was not significant. No changes in liver lipid or histology were seen due to ethanol feeding. There were no significant changes in plasma amino acids due to ethanol (Table IV). Two deproteinized plasma samples from each of two ethanol-fed rats sacrificed at the end of Experiment I averaged 68 mgldl ethanol. Their ethanol intake in the previous 24 h averaged 4.4 g/kg body wt. These results are consistent with previous reports [7]. Discussion

Some commercially available ethanol-containing diets have been found to lack essential

Table IV. S.E.M.).

nutrients [5]. The adequacy of nutrient supply and comparable nutrient-to-calorie ratios are essential if reliable assessments of diet ethanol interactions are to be made. Traditional gravity or ball-bearing activated feed delivery systems may also lead to inaccuracies because of viscosity associated problems with some suspending agents used to suspend diets. These difficulties and evaporative losses were circumvented by the methodology we have described here. The toxicity of ethanol in diets meeting nutritional requirements has been a topic of controversy. Our data show that ethanol alone, in the absence of a known dietary deficiency, did not cause steatosis in rats in 8 or 10 weeks, confirming the report by Porta et al. [3]. In some of our other studies with ethanol, excre-

Plasma amino acids (qrol/B of pair-fed rats of Experiment I fed casein diets with or without ethanol (Mean k

Amino acids (pmol/i)

Taurine Threonine Serine Glutamic Acid Glutamine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Br Ch/Arom” Methionine Cystine Histidine Lysine Arginine Ornithine Citrulline

Portal

Arterial Control In = 5)

Ethanol (n = 4)

Control (n = 4)

Ethanol (72= 5)

183 f 12 246 + 14 164 2 10 51k 9 431 + 47 349 f 42 128 f 24 393 f 40 193 f 19 90-c 9 157 + 19 99-t 9 672 6 72* 6 2.65 78k 7 26k 2 522 4 296 + 23 782 9 372 3 42k 6 75 f 22

252 + 74 251 + 33 153 f 13 42 zt 15 522 + 36 293 f 79 120 f 13 336 f 31 224 f 32 95 k 11 147 f 26 142 + 28 69* 3 70* 7 2.21 842 9 352 2 52k 3 332 f 19 952 3 452 3 35k 1 552 7

262 f 30 361 f 30 350 r 70 99 f 16 401 + 69 623 + 67 273 + 21 880 -1-102 312 k 30 172 + 20 277 -c 42 158 f 18 126 f 14 812 8 2.68 123 f 13 222 4 100 f 9 486 f 41 144 f 15 52k 3 652 2 158 f 19

240 k 34 328 f 34 :226 k 31 90 -e 20 ‘437 f 41 819 f 72 :219 k 20 693 f 89 :300 f 40 152 + 22 ~260f 43 173 f 37 110 f 19 139 f 50 2.52 111 + 13 24k 2 99* 3 434 f 28 1232 8 58k 4 53 + 11 165 2 10

‘Br Ch/Arom, ratio of branch chain amino acids valine, isoleucine and leucine divided by the aromatic amino acids tyrosine and phenylalanine.

54

tion of urinary erotic acid and erotic acid uptake into nucleic acid was measured because damage by hepatotoxins such as carbon tetrachloride and galactosamine increases orotate production [16]. We also found that exposure to ethanol significantly increased orotate excretion and that this could be prevented by adding lactulose to the diet. Lactulose lowers pH of the intestinal content and suppresses ammonia absorption [15]. The histology, lipid content, feed intake and weight gain data argue against direct toxic effects of ethanol in our present study although increased orotate excretion by ethanol indicates that pyrimidine metabolism is affected as is also seen with ammonia intoxication or treatment with carbon tetrachloride and galactosamine [15]. In a previous study by Miller et al. [‘7],rats fed ethanol containing diets ad libitum consumed 25% less feed and gained 75% less than their controls. The increased willingness of rats to consume the diet described here may change with age, sucrose content or the method of feeding. Previous reports have cited changes in hepatic tissue amino acids as an explanation of urea cycle defects due to ethanol [18]. Neither portal nor arterial blood amino acids were consistently changed by ethanol intake in our studies. The plasma urea cycle amino acids arginine, ornithine and citrulline likewise did not change. Therefore, an effect of ethanol on portal or arterial nitrogen compounds is an unlikely explanation of erotic aciduria due to ethanol [15]. Zinc, intrinsic to many enzymes notably alcohas received attention hol dehydrogenase, because its dietary supply may influence ethanol toxicity. The diet fed by DeCarli and Lieber [2] was deficient in zinc by NAS-NRC standards [5]. The more recent formulation of Shorey’s group [7] and the diets used in our studies contained the AIN-76A [8] mineral mix-

ture which exceeds the dietary requirement of rats for zinc. With the diet fed here, ethanol feeding did not alter liver size, histology, or the content of lipid RNA, DNA or protein in the pair-fed animals. References

1

C.S. Lieber, D.P. Jones and L.M. DeCarli, J. Clin. Invest., 44 (1965) 1009- 1021. 2 C.S. Lieber and L.M. DeCarli, Am. J. Clin. Nutr., 23 (1970) 474 - 478. E.A. Porta, OR. Koch, C.L.A. Gomez-Dumm and W.S. Hartcroft, J. Nutr., 94 (1968) 437 - 446. D.M. Hegsted, R.C. Miiis, C.A. Elvehjem and E.B. Hart, J. Biol. Chem., 138 (1941) 459- 466. R.E. Stuii and J.D. Russell, Drug Alcohol Depend., 7 (1981) 393-394. C.K. Erickson, R.L. Shorey and T.K. Li, Drug Alcohol Depend., 6 (1980) 13- 14. 7 S.S. Miller, M.E. Goldman, C.K. Erickson and R.L. Shorey, Psychopharmacology, 68 (1980) 55- 59. 8 Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutrition Studies. J. Nutr., 107 (1977) 1340-8. Second Report of the American Institute of Nutrition Ad Hoc Committee on Nutritional Studies. J. Nutr., 110 (1980) 1726. 9 A. Mondino, G. Bogiovanni. S. Fumero and L. Rossi, J. Chromatogr., 74 (1972) 255- 263. 10 D.L. Pierson and J.M. Brien, J. Biol. Chem., 255 (1980) 7891- 7895. 11 P.I. Glazer and G. Weber, J. Neurochem., 18 (1971) 1569- 1567. 12 G. Cerotti, J. Biol. Chem., 214 (1955) 59-70. 13 R.W. Hubbard, W.T. Matthew and D.A. DuBorwik, Anal. Biochem., 38 (1970) 190-201. 14 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265- 275. 15 W.J. Visek and J.D. Shoemaker, J. Am. Coll. Nutr., 5 (1986) 153- 166. 16 J.D. Shoemaker, The erotic aciduria of chemical hepatotoxicity. Doctoral Thesis, University of Illinois, Urbana, IL, 1984. 17 Kelco Xanthan Gum Book, 2nd edn., Kelco, Division of Merck, and Co., Chicago, IL, 1980. 18 M.A. Petit and I. Barral-Alix, Biochem. Pharmacoi., 28 (1979) 2591- 2595.