Gut 1992; 33: 1099-1104

Effects of ethanol, acetaldehyde and cholesterylesters on pancreatic lysosomes

J S Wilson, M V Apte, M C Thomas, P S Haber, R C Pirola

AbstractRecent studies indicate that altered lysosomalfunction may be involved in the early stages ofpancreatic injury. Chronic consumption ofethanol increases rat pancreatic lysosomalfragility. The aim of this study is to deter-mine whether the lysosomal fragility observedafter chronic ethanol consumption is mediatedby ethanol per se, its oxidative metaboliteacetaldehyde or cholesteryl esters (substanceswhich accumulate in the pancreas after ethanolconsumption). Pancreatic lysosomes fromchow fed rats were incubated for 30 minutes at37°C with ethanol, acetaldehyde or phos-phatidylcholine vesicles containing cholesteryloleate. Lysosomal stability was then assessedby determination of: (a) Latency - that is, theper cent increase in lysosomal enzyme activityafter addition of Triton X-100 and (b) Super-natant activity - that is, the proportion oflysosomal enzyme remaining in the super-natant after resedimentation of lysosomes.Acid phosphatase, N-acetyl glucosaminidase,3-glucuronidase and cathepsin B were assayedas lysosomal marker enzymes. Lysosomesincubated with homogenising medium alone orequivalent volumes of phosphatidylcholinevesicles without cholesteryl oleate were usedas controls. Cholesteryl oleate at concentra-tions of 15 and 20 mM increased pancreaticlysosomal fragility as shown by decreasedlatency and increased supernatant enzyme. Incontrast, ethanol (150 mM) and acetaldehyde(5 mM) had no effect on lysosomal stability invitro. These results suggest that increasedpancreatic lysosomal fragility observed withethanol may be mediated by cholesteryl esteraccumulation rather than by ethanol oracetaldehyde.(Gut 1992; 33: 1099-1104)

Pancreatic ResearchGroup, Department ofGastroenterology, PrinceHenry and Prince ofWales Hospitals,University ofNew SouthWales, Sydney, NewSouth Wales, AustraliaJ S WilsonM V ApteM C ThomasP S HaberR C PirolaCorrespondence to:Dr J S Wilson, Department ofGastroenterology, BlacketBuilding, Prince of WalesHospital, Randwick, NSW2031, Australia.

Accepted for publication28 November 1991

Abuse of ethanol is a known association ofpancreatitis. ' The mechanisms whereby ethanolexerts its pancreatotoxic effects, however, are

unknown. Over the past decade, digestiveenzyme activation by lysosomal hydrolases(particularly cathepsin B)2 has been implicated as

the initial event in three forms of experimentalpancreatitis.3 Furthermore, recent studies fromthis laboratory have shown that experimentalethanol administration increases the fragility ofrat pancreatic lysosomes.6The increase in pancreatic lysosomal fragility

could result from a direct effect of ethanol on

lysosomal membranes. In vivo and in vitroanimal studies have shown that ethanol alters themorphology and/or function of membranes ofhepatocytes,7'9 erythrocytes,"' intestinal micro-villi," and brain microsomes. `

Alternatively, the increased lysosomalfragility may be mediated by acetaldehyde. Thishighly reactive metabolite of ethanol has beenshown to form adducts with liver plasma mem-branes which result in activation of the comple-ment sequence. 11 14 In addition, Tillotson et al"have shown that acetaldehyde increases mem-brane permeability and fluidity as well as inhibit-ing Na+-stimulated glucose transport in ratintestinal microvillus membranes.An additional possibility is that cholesteryl

esters alter lysosomal stability. Fat dropletaccumulation in acinar cells has been observed asthe earliest morphologic alteration in the pan-creas of alcoholics.` 116 This phenomenon hasbeen reproduced in a rat model of ethanolfeeding'7 and subsequent lipid analyses indicatedthat accumulated cholesteryl esters wereresponsible for the morphologic changesobserved.'7 8 These compounds (Fig 1) areknown to disrupt biologic membranes in vitro'9 °2and therefore have the potential to mediate theincreased pancreatic lysosomal fragility observedin ethanol fed rats.The aim of this study was to determine

whether ethanol induced fragility of pancreaticlysosomes is mediated by ethanol, its oxidativemetabolite acetaldehyde or cholesteryl esters. Tostudy this question, the stability of pancreaticlysosomes was assessed after exposure in vitro toethanol, acetaldehyde, and the cholesteryl ester,cholesteryl oleate.

Methods

EXPERIMENTAL ANIMALSChow fed male Sprague-Dawley rats weighing300-350 g were used in all experiments. Eachanimal was killed in the fed state by decapitation.A portion of the tail of the pancreas was thenquickly excised, trimmed of adipose and connec-tive tissue and used immediately.

ISOLATION OF PANCREATIC LYSOSOMESPancreatic lysosomes were isolated by a modifi-cation of the method of Ignarro et a12' as pre-viously described.6 Briefly, approximately 100mg pancreatic tissue from each animal washomogenized in a medium containing 0.25 Msucrose, 1 mM ethylenediaminetetraacetic acid(EDTA) and 25 mg% soybean trypsin inhibitorin a Dounce homogeniser (using five up anddown strokes of the 'B' pestle). The resultingcrude homogenate was then centrifuged at 600 gfor 10 minutes to remove unbroken cells andnuclear debris. The resulting pellet was resus-pended, rehomogenised and centrifuged asabove. Supernatants were combined and centri-

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Wilson, Apte, Thomas, Haber, Pirola

Fatty acid

Figure 1: Cholesteryl esters consist ofa cholesterol moleculeesterified at the 3 position with afatty acid moiety. They can beincorporated into biologic membranes where they may exert adisordering effect (see text).

fuged at 16500 g for 25 minutes to obtain alysosome enriched pellet. Electron microscopicanalysis of the pellet revealed the presence ofzymogen granules as well as lysosomes. Thepellet was then gently resuspended in 10 mlhomogenising medium and used for incubationwith ethanol, acetaldehyde, or cholesteryl oleate.A lysosome enriched fraction rather than apure lysosomal preparation was used becausepreliminary experiments indicated a loss oflysosomal stability with prolonged preparativeprocedures.

PREPARATION OF PHOSOPHOLIPID VESICLESExperiments examining the effect of cholesterylesters on the stability of isolated pancreaticlysosomes were carried out using cholesteryloleate incorporated into phosphatidylcholinevesicles. This was done in order to avoid phaseseparations during the incubation process.Vesicles were prepared by the method ofChobanian, Tall, and Brecher.22 Egg yolklecithin (phosphatidylcholine) and cholesteryloleate were dissolved in chloroform:methanol(2:1) and mixed together in a molar ratio of 25:1as Forrest and Cushley'9 have reported that amaximum of 5 mol% of cholesteryl ester can beincorporated into phosphatidylcholine vesicles.The lipid mixture was evaporated under N2,lyophilised overnight and resuspended in 250FiM NaCI - 25 FtM Tris buffer (pH 7.4).Unilamellar vesicles were prepared by sonicationas described by Barrow and Lentz23 in a nitrogenatmosphere using a Heat Systems Cup HornSonicator (Heat Systems Ultrasonics Inc, NY,USA) at an output setting of 4, for 55 minutes at25-35°C. Care was taken to place the vial in thecentre of the 'cavitation umbrella' of the CupHorn. After sonication, the sample was centri-fuged at 150 000 g for one hour to yield a faintlyopalescent supernatant consisting of a homo-geneous population of cholesteryl oleate vesicles.

CHARACTERISATION OF VESICLES

(a) SizingLipid vesicles were sized by photon correlationspectroscopy as described by Barenholz et al124using a Malvern Autosizer II C. The meandiameter of the vesicles was 605 (15) A. Thepolydispersity index of the preparation was 0 33.

(b) Electron microscopyThe vesicle preparation was diluted 1:4 with

NaCl-Tris buffer and examined by electronmicroscopy using: (i) Negative staining withsodium phosphotungstate as described by Tallet al,25 and (ii) Platinum coating of vesicles on acarbon free hydrophilic grid as described byHuang.2'

(c) Cholesteryl oleate concentrationRecovery of added labelled cholesteryl oleate(cholesteryl [1-_4C] oleate, specific activity 52mCi/mmol), was used to estimate the concentra-tion of cholesteryl oleate in the liposome prepara-tion.

(d) Freefatty acid concentrationThe concentration of free fatty acids in thevesicle preparation was determined by thin layerchromatography followed by gas chromato-graphy mass spectroscopy. The vesicle prepara-tion was freeze dried overnight and resuspendedin chloroform:methanol (2:1) at a concentrationof 20 mg lipid/ml solvent. This preparation wasthen applied to standard silica gel thin layerchromatography plates together with phosphati-dylcholine, cholesteryl oleate, and oleic acidstandards. Plates were developed in chloroform:methanol:water (65:25:4 v/v/v) and the bandsvisualised by iodine staining. The free fatty acidband was identified and scraped off the plate.Free fatty acids were then eluted three times withmethanol acidified to pH 3 by the addition of afew drops of acetic acid. Free fatty acids weremethylated by the method of Howard-Black27using excess etherial diazomethane and thenidentified and quantified using gas chromato-graphy mass spectroscopy. The vesicle prepara-tion was thus found to contain 13.9 [ig palmiticacid and 11.5 [ig oleic acid/ml. The maximumconcentration of free fatty acid in the incubationmixture was calculated to be 50 FtM.

INCUBATION OF LYSOSOMES WITH ETHANOL ANDACETALDEHYDEIsolated pancreatic lysosomes were incubatedwith ethanol or acetaldehyde in tightly sealedtubes for 30 minutes at 37°C. The concentrationsof ethanol and acetaldehyde in the incubationmedium were 150 mM and 5 mM respectively.Lysosomes incubated with the homogenisingmedium alone were used as controls.

INCUBATION OF LYSOSOMES WITH CHOLESTERYLOLEATEIsolated pancreatic lysosomes were incubatedwith phosphatidylcholine vesicles containingcholesteryl oleate for 30 minutes at 37°C. Theconcentration of cholesteryl oleate in the incuba-tion medium ranged from 10 to 20 mM. Controlincubations used equivalent amounts of phos-phatidylcholine vesicles without cholesteryloleate.

INCUBATION OF LYSOSOMES WITH FREE FATTYACIDIn some experiments, lysosomes were incubated

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Effects ofethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes

with oleic acid which had been emulsified in 250FtM NaCI - 25 FtM Tris buffer (pH 7.4) bysonication under nitrogen as described by Nagaiet a128 using a Heat Systems Cup Horn Sonicatorat an output setting of four for 10 minutes at25°C. Lysosomes were then incubated at 37°Cfor 30 minutes with 50 ,uM oleic acid; thisconcentration of oleic acid corresponded to themaximum amount of free fatty acid present inthe liposome preparation. Lysosomes incubatedin the presence of buffer alone were used ascontrols.

ASSESSMENT OF LYSOSOMAL STABILITYLysosomal stability was assessed in two ways:(i) Determination of latency - that is, the per-centage increase in lysosomal enzyme activity ofthe incubated sample after the addition ofTriton X-100 (0.1% final concentration).Latency was calculated as follows:(enzyme activity after Triton)-(enzyme activity before Triton)

enzyme activity before Triton

Increased lysosomal fragility is indicated by adecrease in latency. (ii) Determination of theproportion of total enzyme released into thesupernatant after resedimentation of lysosomesfrom the incubated samples by centrifugation at16500 g for 25 minutes. Increased lysosomalfragility is indicated by an increase in super-natant enzyme. There was no increase in lyso-somal enzyme activity after the addition ofTriton X-100 to the supernatant suggesting thatsupernatant enzymes were in the free form - thatis, not membrane bound.The presence of zymogen granules in the

lysosome enriched fraction did not interfere withthe assessment of lysosomal stability as this wasmeasured using enzyme markers specific forlysosomes (vide infra).

LYSOSOMAL MARKER ENZYMESThe lysosomal enzymes N-acetyl glucosamini-dase, acid phosphatase and ,B-glucuronidasewere assayed fluorimetrically using 4-methyl-umbelliferone substrates.29 Activity wasdetermined by measuring the amount of 4-methylumbelliferone released after incubationwith the enzyme source. Cathepsin B wasassayed fluorimetrically by the method ofMcDonald and Ellis,30 as modified by Salujaet al,3' using CBZ-Arg-Arg-,3-naphthylamide asthe substrate. Activity was determined from the

Effect ofethanol (150 mM) and acetaldehyde (5 mM) on pancreatic lysosomal latency and onthe proportion oflysosomal enzyme released into the supernatant as indices oflysosomal stability(Methods). Results are expressed as per cent ofcontrol values and are presented as means(SEM) (four experiments)

Ethanol (150 mM) Acetaldehyde (5 mM)

Supernatant SupernatantLysosomal enzyme Latency enzyme Latency enzyme

CathepsinB 115 1(20.4) 101-5 (0.8) 104.9(8.9) 101 9(0 9)N-acetyl glucosaminidase 117 4 (8.0) 175.5 (44.9) 123-8 (7.2) 75.6 (19.4)Acidphosphatase 106-5(10-6) 113.5(5.2) 112-7(37.8) 99-6(7.6)I-glucuronidase 117 5 (14-6) 94-7(15-6) 109.2(15.6) 89.5 (10.5)

No significant differences were found between the groups for each of the four marker enzymesstudied.

rate of change of fluorescence as a result of therelease of (-naphthylamide after incubation withthe enzyme source. All lysosomal enzyme assayswere linear with respect to time and proteinconcentration. Triton X-100 (0.1% final concen-tration) was added to the reaction mixture inorder to determine the total amount of lysosomalenzyme present in each lysosomal fractionstudied. Tissue protein concentrations weredetermined by the method of Lowry et al.32

STATISTICAL ANALYSISData were expressed as means (SEM). Differ-ences between samples incubated with ethanol,acetaldehye or cholesteryl oleate and controlsamples were analysed by Student's t test forpaired comparisons.33

MATERIALSAll chemicals were of analytical reagent grade.Cholesteryl oleate and phosphatidylcholine(99.9% pure) were purchased from the SigmaChemical Company, St Louis, MO, USA, andeluted as single bands when chromatographed onthin layer chromatography plates. Methyl-umbelliferone substrates were also purchasedfrom Sigma Chemical Company, St Louis, MO,USA. CBZ-Arg-Arg-,3-naphthylamide waspurchased from BACHEM, California, USA.Ethanol (96%) was obtained from AjaxChemicals, Australia and acetaldehyde (99%pure) was purchased from BDH Ltd, Poole,England. Cholesteryl [1-'4C] oleate was obtainedfrom Amersham, Bucks, England.

Results

EFFECT OF ETHANOL AND ACETALDEHYDE ONLYSOSOMAL STABILITY

Lysosomal latencyThe Table indicates the latency results obtainedwith control, ethanol and acetaldehyde incu-bated lysosomes. For all four lysosomal markerenzymes studied, there was no significant differ-ence between the latency of lysosomes incubatedwith ethanol or acetaldehyde when comparedwith control values.

Proportion oflysosomal enzymes in supernatantThe Table also indicates the proportion of lyso-somal enzymes released into the supernatantafter sedimentation of incubated pancreaticlysosomes. Again for all four marker enzymesstudied, lysosomes incubated with ethanol oracetaldehyde showed no difference in the pro-portion of lysosomal enzyme released into thesupernatant when compared with control values.

EFFECT OF CHOLESTERYL ESTER ON LYSOSOMALSTABILITY

Lysosomal latencyFigure 2 (a-d) depicts the effect of increasingconcentrations of cholesteryl oleate on lysosomal

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Wilson, Apte, Thomas, Haber, Pirola

10 15 20

N-Acetyl glucosaminidase

H p*** p < 001,**** p < 0005

10 15 20

Acid phosphatase 80

60

40

3-GlucuronidaseD

**** p < 0.005

20

010 15 20 10 15 20

Cholesteryl ester (mmol/1) Cholesteryl ester (mmol/1)

Figure 2 (a-d): Effect ofcholesteryl ester on pancreatic Iysosomal latency as an index oflysosomal stability (Methods). Resultsare expressed as per cent ofcontrol values and are presented as means (SEM) (four experiments). Control preparations containedphosphatidylcholine vesicles without cholesteryl ester.

10 15 20

N-Acetyl glucosaminidase

3000 B

**** p < 0.005

10 15 20

Acid phosphatase ,B-Glucuronidase300-C

p < 0.05, * Do** p<002 T ** -*<00,

O p < 0.005400

, 200-

200100

ccCD

~~~~~~~~~~~~~~~~100

0 0

10 15 20 10 15 20

Cholesteryl ester (mmol/1) Cholesteryl ester (mmol/l)

Figure 3 (a-d): Effect ofcholesteryl ester on proportion ofpancreatic lysosomal enzyme released into the supernatant as an indexoflysosomal stability (Methods). Results are expressed as per cent ofcontrol values and are presented as means (SEM) (fourexperiments). Control preparations contained phosphatidylcholine vesicles without cholesteryl ester.

ACathepsin B

B

*** p < 001,**** p < 0.005

o 150

0

0

C.)U

a)50

-J

50

0

Co-c)J

Cathepsin B- 150-a

004-

0o 100

EN

50-c

c)m

0n0.

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Effects ofethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes

latency. With all lysosomal enzymes studied,there was a progressive decrease in latency withincreasing concentrations of cholesteryl oleatewhen compared with phosphatidylcholine con-trols. This decrease in latency was statisticallysignificant with cholesteryl oleate concentra-tions of 15 and 20 mM.

Per cent oflysosomal enzymes in supernatantFigure 3 (a-d) indicates the percentages oflysosomal enzymes released into the supernatantafter incubation of isolated pancreatic lysosomeswiih phosphatidylcholine vesicles (with andwithout cholesteryl oleate) and subsequentresedimentation of lysosomes. Paralleling thefindings observed with lysosomal latency (Fig 2),cholesteryl oleate (15 and 20 mM) resulted insignificant increase in per cent of supernatantenzyme.

EFFECT OF FREE FATTY ACID ON LYSOSOMALSTABILITYFree oleic acid (50 FtM) did not alter rat pan-creatic lysosomal stability in vitro. Using acidphosphatase as a lysosomal marker enzyme,values for latency and supernatant enzyme(calculated as a per cent of control values andexpressed as means (SEM)) were as follows:latency - 143.6 (38'7)%; supernatant enzyme -96.4 (2.8)% (four experiments).

DiscussionExperimental ethanol administration results inincreased fragility of pancreatic lysosomes6 andthe simultaneous accumulation of cholesterylesters in the pancreas.7 18 Ethanol inducedfragility of pancreatic lysosomes has beenobserved in the absence of any morphologicevidence of pancreatic damage making itunlikely that it is a secondary phenomenon.'7 8This study shows that cholesteryl oleate candisrupt pancreatic lysosomes in vitro suggestingthat cholesteryl esters may be responsible for thelysosomal fragility observed in vivo. In contrast,ethanol and acetaldehyde did not have anyapparent deleterious effect on lysosomalstability.The concentrations of cholesteryl ester seen to

increase lysosomal fragility in vitro (15 and20 mM) were greater than those observed in therat pancreas in vivo (5 mM).'8 The requirementfor higher concentrations to disrupt pancreaticlysosomes may reflect the fact that, in an in vitrosystem, the period of incubation of isolatedlysosomes with cholesteryl ester is necessarilylimited (30 minutes in our study) whereas in theprevious in vivo study,6 lysosomes were exposedto increased levels of cholesteryl esters for amuch longer period (up to four weeks).The effect of cholesteryl ester on pancreatic

lysosomal latency appeared more pronouncedthan the effect on supernatant enzyme (Figs 2, 3)although significant changes in the proportion ofsupernatant enzyme were also seen. A somewhatsimilar result was observed in vivo where chronicethanol administration was associated withdecreased lysosomal latency but unaltered values

for proportion of supernatant enzyme.6 Thisdifferential effect on the two parameters oflysosomal stability may indicate that the pre-dominant effect of chronic ethanol administra-tion and of cholesteryl ester is to increase thepermeability of pancreatic lysosomal membranesrather than to completely disrupt the organelles.Thus, enzyme substrates may have greateraccess to enzymes within the lysosomes resultingin increased pre Triton enzyme activities anddecreased latency (Methods). Complete disrup-tion of lysosomes also occurs, but to a lesserextent resulting in less marked release ofenzymeinto the supernatant.

It is unlikely that the effect of cholesterylesters on pancreatic lysosomes is an artefactrelated to release of free fatty acids duringpreparation or incubation of phosphatidyl-choline vesicles. The maximum concentration offree fatty acids to which the lysosomes wereexposed (50 FM) did not disrupt pancreaticlysosomes after in vitro incubation.The mechanism whereby cholesteryl esters

decrease the stability of pancreatic lysosomes isnot known. There have been only limited studieswhich directly address the effect of cholesterylesters on the stability of biologic membranes.Forrest and Cushley'9 have reported thatincorporation of cholesteryl esters increases thepermeability of model membranes to ions 10fold. Chronic ethanol administration to rodentshas been shown to increase the cholesteryl estercontent9 and to decrease the microviscosity ofhepatic plasma membranes.7 In addition, there isevidence from the work of Hamilton and Small34that cholesteryl oleate molecules incorporatedinto model membranes are aligned with thecarbonyl group close to the aqueous interfaceand the sterol ring and fatty acyl chain parallel tothe acyl chains of phospholipid, thereby exertinga disordering effect.

It is conceivable that the observed destabilis-ing effect of cholesteryl esters on lysosomescould be an indirect one, possibly mediated bydisruption of zymogen granules in the lysosomeenriched fraction and release of their contents.This is an unlikely explanation, however, for tworeasons. First, pancreatic zymogen granules con-tain inactive precursors of the digestive enzymes(proteases, phospholipases) likely to be injuriousto lysosomal membranes. Second, a proteaseinhibitor was used to counter any autoactivationwhich may have occurred during isolation.The concentration of ethanol used (150 mM)

was much higher than that usually found inthe blood of ethanol fed animals or alcoholicindividuals. Despite such a relatively high con-centration there was no demonstrable alterationin pancreatic lysosomal stability. Similarly, theacetaldehyde concentration used (5 mM),although much higher than that detected in vivo(0-25 itM),35 failed to produce any evidence oflysosomal damage. The lack of an acute effect ofethanol or acetaldehyde on pancreatic lysosomesin vitro, however, does not exclude the potentialpancreatotoxicity of these compounds in vivo.For example, acetaldehyde has been shown toform circulating cytotoxic adducts with serumalbumin.37 Furthermore, chronic ethanol con-sumption has recently been shown to increase

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1104 Wilson, Apte, Thomas, Haber, Pirola

the synthesis of pancreatic digestive enzymes" 3and ethanol4 4' and acetaldehyde42 have beenshown to inhibit pancreatic exocrine secretion.These latter changes could contribute to theincreased glandular content of digestive enzymesobserved after ethanol feeding'843 and may pre-dispose the gland to autodigestion.The pathogenesis of alcoholic pancreatitis can

be approached in two ways: (i) by studyingfactors influencing the individual susceptibilityto the disease or (ii) by studying constant effectsof ethanol on the pancreas. It is well known thatonly a minority of alcoholics are afflicted withclinical pancreatitis and the issue of individualsusceptibility to alcoholic pancreatitis hasrecently been reviewed.44 In essence, however,the predisposing factors remain unknown.44 Thestudy reported in this paper has adopted thelatter approach and has explored one of theconstant metabolic effects of ethanol on thepancreas - namely, increased lysosomal fragility,which may render the organ susceptible toautodigestion by facilitating contact betweenpancreatic digestive enzymes and lysosomalhydrolases.

This study has shown that cholesteryl esterscan disrupt pancreatic lysosomes in vitro andthus supports the notion that cholesteryl estersmay mediate the increased pancreatic lysosomalfragility observed after experimental ethanoladministration. Ethanol induced lysosomalfragility may permit release of lysosomalproteases and their subsequent contact withdigestive enzymes, thereby predisposing thepancreas to autodigestion. The implication isthat cholesteryl esters may play an important rolein the pathogenesis of alcohol induced pancreaticinjury.This study was supported by the National Health and MedicalResearch Council of Australia. The authors are indebted to theBiomedical Mass Spectrometry Unit of the University of NewSouth Wales.

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22 Chobanian JV, Tall AR, Brecher PI. Interaction betweenunilamellar egg yolk lecithin vesicles and human highdensity lipoprotein. Biochemistry 1979; 18: 180-7.

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cholesteryl esters on pancreatic lysosomes.Effects of ethanol, acetaldehyde and

J S Wilson, M V Apte, M C Thomas, P S Haber and R C Pirola

doi: 10.1136/gut.33.8.10991992 33: 1099-1104 Gut

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(1949)Pancreas and biliary tract

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