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1、Fatty Liver in the Rat after ProlongedIntake of Ethanol with a NutritionallyAdequateNew Liquid Diet1a LEONOREM. DECARLI ANDCHARLES S. LIEBER3 Liver Disease and NutritionUnit, Second (Cornell) MedicalDivision, BellevueHospitaland Departmentof Medicine,Cornell University Medical College, New York, New

2、 York ABSTRACTTo determinewhetherprolongedethanolintakecan producea fatty liver, even whenassociatedwith a diet containingadequateamountsof protein,min erals and vitamins,rats were given liquiddiets containing18% of caloriesas casein, supplementedwithmethionine(0.3mg/kcal)andcystine(0.5mg/kcal),chol

3、ine (0.25 mg/kcal),fat (35%of total calories),adequatemineralsand vitaminsand, in the control diet, 47% of the caloriesas carbohydrates.A littermateof each control rat was pair-fedwiththe samediet in whichcarbohydrateshadbeen isocaloricallyre placedwithalcoholto the extentof 36%of the totalcalories.

4、Thesedietsassured continuedgrowth in all animalsand normalliver in the controls,whereasin the rats fed withalcohol,fattyliver developed,whichwas evidentboth morphologicallyand on chemicalanalysis;after 24 days of alcohol, hepatictriglycrideshad increasedon the averagesixfoldand cholesterolestersfive

5、fold,comparedwiththoseof the con trols. Thesestudiesdemonstratethatprolongedalcoholintakecanproducea fatty liver even when given with a diet with nutritionallyadequatecontentof protein,vita mins and minerals,and an amountof fat less thanthat of the averageAmericandiet. There is a widespreadbelief th

6、at when alcohol is ingestedwith an adequatediet, it producesno liver damage.This concept waschallengedwhenwe showedprevi ously that both in manand in rats, fatty liver could be produced by prolonged alco hol intake despite diets with adequatecon tent in nutrients(1,2).To overcome the naturalaversion

7、 of rats for alcohol, totally liquid diets, containingin one formula the necessarynutrients,as wellas alcohol, were used. Our formerpurifieddiet con taineda completeaminoacid mixtureas a substitutefor protein,sucrose as carbo hydrateand an amount of fat comparable to that of an average Americandiet

8、(43% of total calories).Withsuch a diet, iso- calorie substitutionof carbohydrateswith ethanolto the extent of 36% of total cal ories resulted, after 24 days, in an average 7- to 8-fold increaseof hepatictriglycr ides (1,2).The presentstudy was under takentodeterminewhetherasimilar ethanoleffect cou

9、ld be demonstratedwith a diet containing,insteadof amino acids, a protein(casein),and instead of sucrose, adextrin-maltosemixturewhichmore closely resembles carbohydratescommonly found in food. The fat contentof the diet was also reducedto 35%of the calories. MATERIALSAND METHODS Male ratsof a Sprag

10、ue-Dawleystrain (CD)were used in 11 groups of 2 litter- mateseach.They were maintainedwith a commerciallaboratoryration * and tap waterad libitumuntilthey had reached a weight of 100 to 150 g, at which time they were housed in individual wire-bottom cagesand given liquiddiets in drinking tubes as th

11、e only source of food and water. The overall compositionof the new diet is shown in figure 1, together with our pre vious formula,to facilitatecomparisonof the changes made. The compositionof the ethanoland control diets was as follows: casein5(supplementedwithmethionine 0.3 mg/kcal,andcystine0.5 mg

12、/kcal), 18% of the total calories; fat, 35% of total calories;adequatevitaminsand minerals; Receivedfor publicationJuly28, 1966. i Presentedatthe50thAnnualMeetingofthe FederationofAmericanSocietiesforExperimental Biology,AtlanticCity, New Jersey,April,1966.(Fed erationProc.,25: 304, 1966). *Thisinve

13、stigationwassupportedin partby Pub lic HealthServiceResearchGrantsno. AM-06284,no. AM10-893 andno. AM-09536fromthe NationalInsti tuteof ArthritisandMetabolicDiseases. 3Dr.LieberisrecipientofPublicHealthService ResearchCareerDevelopmentAwardK3-AM22.590 fromthe NationalInstituteof Arthritisand Metabol

14、ic Diseases. 4 PurinaLaboratoryChow, RalstonPurinaCompany, St. Louis. Vitamin-freeMicropulverizedCasein,Nutritional BiochemicalsCorporation,Cleveland. J. NUTRITION, 91: 67 331 by guest on February 26, 2011Downloaded from 332LEONOREM. DECARLI AND CHARLESS. LIEBER 100 so 60 Calories (o

15、ffertali 40 20 Corn oil Amino ac co Fig. IComposition of liquid diets fed to the rats; amino acid, vitamin and mineral con tentwasas describedpreviously(22).Gray sectionbelow cornoil:ethyl-linoleate, 2 mg/kcal.Casein supplemented with methionine,0.3 mg/kcaland cystine, 0.5 mg/kcal. andinthecontroldi

16、et,carbohydrate,8 47%of the total calories.In the ethanol formula,carbohydratewasisocalorically replaced to the extent of 36% of the total calories. The choline contentand the calculated methioninecompositionwere0.25and 1.5 mg/kcal,respectively, which is equiva lent to concentrationsof 0.1 and 0.6%

17、in solid diets, amountsfoundto be optimal fortherat(3).Thedietscontained 1 kcal/ml,anadequatewater-to-calorie ratio for the rat (4). Sodium carrageenate7 (2 g/liter)was used to stabilize the liquid diet. Animalswere fed in groups of 2 litter- mates each : one rat was given the control diet, whilethe

18、otherreceivedthesame regimenin whichdextrin-maltose,to the extentof 36%of calories,was isocalori cally replacedwith ethanol.Loss of etha nol by evaporationwas negligiblein the diets kept in the graduateddrinkingtubes (5) for periods of up to 48 hours. The diet was changed every 24 hours and, in some

19、 experiments,every 12 hours. Ethanolwas introducedintothedietgradually.The final concentrationof 50 g/literof ethanol was achieved on the fifth day of feeding. During the first 2 days, the animalswere given the liquid diet with 30 g/literetha nol which was increasedto 40 g/literfor the third and fou

20、rth days. Observation dur ing the initial days indicatedin each pair of littermateswhichof the animalshad the lowest spontaneousfood intake.This rate-limitingrat(whichwas usuallythe one given ethanol)received the liquid diet ad libitumandthecorrespondinglitter- matewas fed isocaloricamountsof the ot

21、her diet on the following day. During the 24 hours precedingkilling of the rats, the diets were given in three divided doses at approximately8-hour intervals. The body weight of the rats was deter mined at least twice a week. After 24 days, theanimalsweredecapitatedand blood wascollectedfromtheneckv

22、esselsin heparinizedtubesandplasmawasob tained by immediatecentrifugationin the cold. The liver was quicklyexcisedand approximatelyone gram was weighedin to tubescontainingchloroform :methanol (2:1).The plasmaandremainingliver were stored at ”18. Total hepaticlipids were extracted(6) and quantitated

23、by the method of Amenta (7).An aliquot of the total lipid extract, containingapproximately20 mgof fat, was evaporatedundernitrogento a vol ume of about 0.5 ml, and applied to a 0.5- mm thick silica gel chromatoplate(8) and developedin hexane:diethylether:acetic acid (83:16:1).The triglycrides,choles

24、 terol esters and free cholesterol were eluted by the method of Goldrick and Hirsch (9); triglycrideswere quantitatedby determi- Dexin,generouslysuppliedby Dr. Singleton,Bur roughs Wellcomeand Company,Tuckahoe,New York. 7Viscarin,Marine Colloids, Inc., P.O. Box 70, Spring field, New Jersey. by guest

25、 on February 26, 2011Downloaded from FATTYLIVERAFTERETHANOLANDADEQUATEDIET333 nationof ester linkagesby the procedure of Snyder and Stephens(10),and choles terolandcholesterolestersweredeter minedby theprocedureof Searcyand Bergquist(11).Plasmaalcoholwasde terminedby themethodof Bonn

26、ichsen (12).Samplesofhepatictissuewere fixed in 10% neutralformalinuntil they were processedfor histologicalexamina tion. The resultsobtainedfrom each animal werecomparedwiththecorresponding valuesin the pair-fedcontrollittermate. The means(SE) and individualdiffer ences were calculatedand the degre

27、e of significancewas determinedby Students i test (13). RESULTS Twenty-fourdays of isocaloricreplace ment of carbohydrateby ethanolresulted in a significantincreaseof total hepatic lipids to 96.8 6.6 mg/g,comparedwith only 46.1 1.3 mg/g(P 0.001)in the controls.Thisincreaseinhepatictotal lipids resul

28、tedprimarilyfrom a change in hepatic triglycridesand cholesterol esters as indicatedin figures2 and3, respec tively.Theaveragetriglycrideincrease was almost sixfold, from10.8 0.64 to 56.8 4.6 mg/g,whereas the average in creasein cholesterolesterswas fivefold, from 0.59 0.05 to 2.92 0.14 mg/g. Free c

29、holesterolchangedonly slightlyas indicatedin figure 3. Alcohol levels, determinedon the plas matakenat the timeof killing,varied DEXTRIN-MALTOSE (controll ETHANOL (36* call 60 50 ” 40 mg/gm (wet wt) SE 20 10 - o1” Fig. 2Hepatictriglycrideconcentrationin rats pair-fed for 24 days with liquiddiets con

30、 tainingeitherdextrin-maltose(control)or iso caloricamountsof ethanol(36%of calories). Cholesterol mg/gm liver (wet weight) SE DEXTRIN-MALTOSE CONTROL DIET ISOCALORIC ETHANOL DIET o1” FREECHOLESTEROL ESTERIFIED CHOLESTEROL Fig. 3Hepatic cholesterolconcentrationin rats pair-fed for 24 days with liqui

31、d diets containingeitherdextrin-maltose(control)or isocaloricamountsofethanol(36%of calories). by guest on February 26, 2011Downloaded from 334LEONOREM. DECARLI AND CHARLESS. LIEBER widely with a mean of 145.1 23.7 mg/ 100 ml. With both diets, rats continuedto grow, withanaveragedail

32、yweightgainof 3.05 g. Withhematoxylinandeosinstains, hepaticmorphologywas foundto be nor mal in controls, whereas fat accumulation was evident in the rats given alcohol, sub stantiatingthe chemicaldeterminations. DISCUSSION The purposeof the presentpaper was to determinewhetherprolongedethanol intak

33、ecan result in fatty liver, despitea dietadequateinprotein,vitaminsand minerals.With conventionalfeeding tech niques, that is, incorporationof alcohol in drinking water and administrationof solid food, rats limitedtheir alcohol intake,in most studies,to 20%of total caloriesor even less (14, 15); onl

34、y in one study was the intake as high as 30% of total calories (16).With these techniques,however,as long as the dietaryintakewas adequate, no pronouncedaccumulationof fat in the liver was detected(14-16).This lack of steatosis is not unexpected,since 20% of totalcaloricintakeas alcoholis a very low

35、 dose which, in our laboratory, not only failed to produce an appreciablefatty liver, but also did not result in any significant blood level of alcohol (2).No blood alco hol levels were reportedin the one study in which 30% of total calories as alcohol failedto producea fattyliver(16).To determinewh

36、etherhepaticsteatosiscan be producedin ratswith ethanolin the absenceof dietarydeficiency,an experi mental method was needed to increase the amountof alcohol consumedby the rats. Alcohol, when given acutely withoutfood in a large single dose by gastric tube, was found to produce fat accumulationin t

37、he liver(17-22)butthemechanismsIn volved in these acute experimentsdo not necessarilyapply to more prolongedalco hol intakesuchas observedin chronic alcoholic patients(2,22,23).In our pre vious studies(1,2,24),prolongedintake ofsubstantialamountsofalcoholwas achieved by overcomingthe naturalaver sio

38、n of rats for alcohol by incorporationof theethanolin a completelyliquiddiet. This previous diet, however, contained,as a substitutefor protein, an expensive ami- no acid mixture.The presentstudy dem onstratesthatasimilareffectcanbe obtainedwithalcoholwhentheamino acids are replaced by casein, enric

39、hed with methionineand cystine. To eliminatepos sible directeffects of sucroseon hepatic lipidmetabolismwhichhavebeende scribed by some (25, 26) but not observed by others (27),sucrose was replacedby a mixtureof dextrin-maltose.The diet was furtherchangedby decreasingits fat con tent from 43 to 35%

40、of total calories, an amountlessthanthatoftheaverage Americandiet (28).This decreasein fat contentallowed an increaseof the carbo hydratefrom41 to 47%in the control diet; in the ethanoldiet this resultedin a doublingof theremainingcarbohydrate after incorporationof the ethanol. In addi tionto a comp

41、ositioninall knownre quired nutrientsin amountsconsideredto be adequateor optimalfor the rat(29), the qualityof the diet was evidencedby the continuedgrowth of the rats as well as normalhepaticfat contentand morphol ogy in the controls. Lipotrope contentof our diet (0.25 mg of choline and 1.5 mg of

42、methionine/kcal, includingthemethioninepresentin ca sein) was equivalentto the amount of lipo- tropicsubstancesreportedby Klatskinet al. (30)and Best et al. (31)to fully pro tect against fatty liver developmentin rats given a choline-deficientdiet, with or with out ethanol in drinking water. The amo

43、unt of choline was also reportedby others to be optimal for the rat (3). Since the possi bility hasbeenraisedthatethanolmay increasecholinerequirements(30),and since the ethanol intake in the present ex perimentswasgreaterthanthatinthe studies of Klatskin et al. (30),the possi bility hasto be consid

44、eredthatcholine requirementsmayhavebeenincreased even further.It is unlikely, however,that simpleenhancementof cholinerequire ments could fully explain our results, since in one of our previousstudies,massive choline supplementationwith 20 times the amountpresentin our diet failed to fully protectag

45、ainststeatosis;hepatictriglyc ride accumulation,although reduced, still representeda threefoldincreasecompared with the controls (24). It is therefore likely by guest on February 26, 2011Downloaded from FATTYLIVERAFTERETHANOLANDADEQUATEDIET335 thatethanolproducessteatosisthrough effe

46、cts other thanor in additionto those relatedto lipotrope metabolism.The pres ent study demonstratesthatthis hepatic steatosiscan be produced by ethanoleven whenouroriginalformula(1,2,24)is modified to more closely resemble conven tionaldiets by replacingsucrose by dex trin-maltose,aminoacids by prot

47、ein,and by decreasing the fat content to an amount less thanthatof theaverageAmerican diet. This improvedprocedurefor the ex perimentalproductionof a fatty liver on prolonged alcohol ingestionis proposed as a convenientand inexpensivetool for fur ther studies of the pathogenesisand pos siblythetreat

48、mentandpreventionof alcoholic liver disease. ACKNOWLEDGMENTS The authorsare gratefulto Dr. T. P. Almy for his continuousinterestand sup port; to Mrs. N. Lohse and BarbaraSmol fortheirexcellenttechnicalassistance, and to Mrs. M. Wilson and PamelaMay- nard for the preparationof the diets. LITERATURECI

49、TED 1. Lieber,C. S., D. P. Jones,J. Mendelsonand L.M. DeCarli1963Fattyliver,hyper- lipemiaand hyperuricemiaproducedby pro longedalcoholconsumption,despiteade quatedietaryintake.Trans.Assoc.Amer. Physicians,76: 289. 2.Lieber, C. S., D. P. Jonesand L. M. DeCarli 1965Effectsof prolongedethanolintake: P

50、roductionoffattyliverdespiteadequate diets. J. Clin. Invest.,44: 1009. 3.Treadwell,C. R.1945Growthandlipo- tropism.I. The dietaryrequirementsof me- thionine,cystine,and choline. J. Biol. Chem., 160: 601. 4.Morgan,A. F., L. Brinner,C. B. Plaaand M. M. Stone1957Utilizationof calories from alcoholand w

51、inesand theireffects on cholesterolmetabolism.Amer.J.Physiol., 389: 290. 5.Richter,C. P.1926A studyof the effect of moderatedoses of alcoholon the growth andbehaviorof the rat.J. Exp.Zool., 44: 397. 6.Folch, J., M. Lees and G. H. S. Stanley1957 A simple methodfor the isolationand purifi cationof tot

52、allipidsfromanimaltissues. J. Biol. Chem., 226: 497. 7.Amenta,J.S.1964A rapidchemical methodfor quantificationof lipids separated by thin-layerchromatography.J. Lipid Res., 5: 270. 8.Randerath,K.1963Thin-layerChroma tography.Academic Press, Inc., New York. 9.Goldrick,B., andJ. Hirsch1963A tech nique

53、 for quantitativerecovery of lipids from chromatoplates.J. Lipid Res., 4: 482. 10.Snyder,F., andN. Stephens1959A sim plifiedspectrophotometricdeterminationof estergroupsinlipids.Biochim.Biophys. Acta, 34: 244. 11.Searcy, R. L., and L. M. Bergquist1960A newcolorreactionforthequantitationof serumchole

54、sterol.Clin. Chim. Acta, 5: 192. 12.Bonnichsen,R.1963Ethanoldetermina tionwithalcoholdehydrogenaseandDPN. In:MethodsofEnzymaticAnalysis,ed., H-U. Bergmeyer.AcademicPress, New York, p. 285. 13.Snedecor,G. W.1956StatisticalMethods Appliedto ExperimentsinAgricultureand Biology, ed. 5. The Iowa State Co

55、llege Press, Ames. 14.Klatskin,G., H. M. Gewinand W. A. Krehl 1951Effects of prolongedalcohol ingestion on the liverof the ratunderconditionsof controlledadequatedietaryintake.YaleJ. Biol. Med., 23: 317. 15.Mallov,S.1955Effectof chronicethanol intoxicationon liverlipidcontentof rats. Proc. Soc. Exp.

56、 Biol. (U.S.),88: 246. 16.Scheig, R., N. M. Alexanderand G. Klatskin 1966Effectsof prolongedingestionof glu cose or ethanolon tissuelipidcomposition and lipidbiosynthesisin rat.J. LipidRes., 7: 188. 17.Mallov,S., and J. L. Bloch1956Role of hypophysisand adrenalsin fattyinfiltration of liver resultin

57、gfromacuteethanolintoxi cation.Amer. J. Physiol.,184: 29. 18.DiLuzio,N. R.1958Effect of acuteetha nolintoxicationon liverandplasmalipid fractionsof the rat.Amer. J. Physiol.,194: 453. 19.Brodie, B. B., W. M. Butler,Jr., M. G. Horn ing, R. P. Maickeland H. M. Maling1961 Alcohol-inducedtriglycride depositionin liverthroughderangementof fat transport. Amer. J. Clin. Nutr.,9: 432. 20.Horning,M. G., M. Wakabayashiand H. M. Maling1963Biochemicalprocessesin volvedinthesynthesis,accumulationand release of triglyc