Effect of ethanol on lipoprotein synthesis and fecal sterol excretion

NUTRITION RESEARCH, Vol. 5, pp. 45-56, 1985 0271-5317/85 $3.00 + .00 Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

EFFECT OF ETHANOL ON LIPOPROTEIN SYNTHESIS AND FECAL STEROL EXCRETION

Joanne E. Cluette, M.S., John J. Mulligan, Ph.D., Richard Noring, B.S., Frank D. Igoe, B.S. and

Jerome L. Hojnacki, Ph.D.* Graduate Biochemistry Program,

Department of Biological Sciences, University of Lowell, Lowell, Massachusetts 01854

ABSTRACT

The effect of variable doses of ethanol on plasma lipoprotein composition, lipoprotein synthesis and fecal sterol excretion was examined in male, atherosclerosis susceptible squirrel monkeys. Primates were divided into three groups: 1) Controls fed isocaloric l iquid diet; 2) Low Ethanol monkeys given l iquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. Circulating high density lipoprotein (HDL) free cholesterol and phospholipid, very low density-low density lipoprotein (VLDL-LDL) total cholesterol, and total plasma cholesterol and triglyceride were signif icantly elevated in High Ethanol primates compared to the other treatments. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups. There were no significant differences in serum glutamate oxaloacetate transaminase. High Ethanol primates also had signif icantly greater HDL f~e cholesterol specific act iv i ty following intravenous injection of H mevalonolactone compared to the other groups while radioactive VLDL-LDL free cholesterol was elevated in both High and Low Ethanol animals. Although, total fecal bile acid mass was signif icantly greater in both alcohol treatment groups compared to Controls, fecal neutral sterol specific act iv i ty was only higher in monkeys fed the high ethanol diet. This study provides evidence that ethanol at 24% of calories: 1) raises HDL cholesterol levels by enhancing lipoprotein synthesis; 2) increases the fecal output of newly synthesized cholesterol without causing l iver dysfunction; and 3) maintains a constant relative distribution of cholesterol among lipoprotein classes.

KEY WORDS: Alcohol, lipoproteins, fecal sterols, bile acids

*To whom correspondence should be addressed.

45

46 J.E. CLUETTE et al .

INTRODUCTION

A number of epidemiological studies have demonstrated that alcohol consumption is associated with an elevation in high density l ipoprotein (HDL) cholesterol and reduced coronary artery occlusion ( I -5 ) . HDL's ant i -athero- genic properties may in turn be related to i t s role in removing cholesterol from peripheral t issue and transport of the sterol to the l i ve r for excretion in b i le (6). Schwartz et a l . (7) and more recently Portman et a l . (8) substantiated th is hypothesis by showing that HDL free cholesterol was the preferred substrate for b i l i a r y cholesterol and b i le acids in humans and nonhuman primates. Despite th is information, three aspects of the ethanol-HDL cholesterol-excret ion hypothesis need fur ther c l a r i f i ca t i on . These include: i ) the dose-response relat ionship between alcohol consumption and HDL elevations; 2) the metabolic mechanisms(s) responsible for th is increase; and 3) the quant i tat ive effect of ethanol on neutral sterol and b i le acid excretion.

Ethanol dose in most c l i n i ca l t r i a l s evaluating HDL levels in alcoholic and non-alcoholic subjects has often been reported as ounces/week with no reference to the percentage of total dietary calories (1,2,4,5). By contrast, experimental studies with animals show that ethanol at 26% of calories in pigs (9) and 36% of calories in rats (10) and monkeys (11) causes a s ign i f i cant increase in the concentration of c i rcu la t ing HDL. These levels, however, are considerably higher than the ethanol calor ic content of the average American diet estimated at 5-10% by Burton (12), 7% from dietary recall data in the Mult ip le Risk Factor Intervention Tr ia l (13) and 4-5% for American men aged 40-59 in the Lipid Research Cl inics Program Prevalence Study (14)

Secondly, the underlying mechanism(s) responsible for the ethanol related increase in HDL have not been f u l l y elucidated. Two current hypotheses suggest that alcohol may raise HDL levels by: I) d i rect st imulat ion of hepatic l ipoprotein synthesis and secretion secondary to ethanol's induction of microsomal enzyme ac t i v i t y (3,13); and/or 2) by enhancing extrahepatic l ipoprotein l ipase ac t i v i t y which in turn promotes transfer of surface components from very low density l ipoproteins (VLDL) and chylomicrons during l i po lys i s to nascent HDLR part ic les (3,15). HDL~, which may be secreted d i rec t l y by the l i v e r , is the~ converted to mature spherical HDLoby the lec i th in cholesterol acyl transferase reaction (15-17). Taskinen et ~ I . (18) demonstrated increased l ipoprotein l ipase ac t i v i t y and elevated HDL~ cholesterol concentrations in male alcoholics and have thus provided evidence~n support of the l a t t e r conjecture. However, only two rat studies using ethanol at 36-37% of calories have provided ind i rect experimental data substantiat ing the enhanced synthesis hypothesis (19,20).

F ina l ly , experiments involving ethanol's effect on b i le acid and neutral sterol excretion have resulted in equivocal f indings. For example, Lefevre et a l . (21) and Lakshmanan and Veech (22) demonstrated hepatic cholesterol accumulation, decreased 7 ~-hydroxylase ac t i v i t y and decreased b i le acid synthesis and excretion in rats fed dietary ethanol at 36-37% of calories while Boyer (23) reported increased b i le sa l t secretion in rats at the same ethanol leve l . Maddrey and Boyer (24) showed ei ther inh ib i t ion or st imulat ion of b i le acid secretion at 28% of calories depending on the blood ethanol level . More recently, Topping et a l . (9) reported increased HDL levels, increased fecal b i le acids and a higher fecal b i le acid/neutral sterol ra t io in pigs fed ethanol at 26% of calor ies. This is in agreement with c l i n i ca l t r i a l s which show that: I ) low HDL levels may represent a fundamental l i p i d abnormality in women with cholesterol gallstone disease who have also suffered a myocardial in farc t ion (25); and 2) moderate alcohol intake in healthy indiv iduals raises HDL levels, reduces the b i le cholesterol saturation index and may thus protect against both coronary heart disease and gallstone formation (26).

Atherosclerosis susceptible squirrel monkeys represent a nonhuman primate species idea l ly suited to c l a r i f y the ethanol-HDL cholesterol-excret ion re lat ionship because the i r plasma l ipoproteins are par t i cu la r ly responsive to

ETHANOL LIPOPROTEINS FECAL STEROL 47

dietary perturbations (27), their lipoprotein metabolic pathways have been well characterized (8), and because they can be taught to drink solutions of ethanol (28). The squirrel monkey also has pathological relevance in that these primates develop extensive atherosclerosis similar to what has been reported in humans (27,28). Furthermore, l ike humans, HDL cholesterol is the preferred substrate for b i l iary cholesterol and bile acids in this species (8).

The present study was thus designed to determine the effect of variable doses of ethanol fed to squirrel monkeys over a chronic period on synthesis of HDL cholesterol and on fecal cholesterol and bile acid excretion.

MATERIALS AND METHODS

Fifteen yearling male Bolivian squirrel monkeys with an average weight of 592 • 32g were purchased from South American Primates, Inc. (Miami, FL) and randomly assigned to three treatment groups consisting of 5 monkeys/group: 1) Controls fed isocaloric, chemically defined Juvenile Primate Liquid Diet #19 purchased from BioServ, Inc. (Frenchtown, NJ); 2) Low Ethanol animals fed l iquid diet with 100 proof vodka substituted isocalorically for carbohydrate at 12% of total calories; and 3) High Ethanol monkeys given l iquid diet plus vodka substituted isocalorically and representing 24% of total calories. Diet #19 had a caloric density of 0.87 Kcal/ml, a caloric distribution of 18.4% protein, 29.4% fat, and 52.2% carbohydrate, and a polyunsaturated/saturated fatty acid ratio of 1.00. Monkeys were housed in individual cages at our Primate Research Center and given 80 ml of diet in graduated glass Richter drinking tubes twice daily in the morning and late afternoon. Animals were weighed bi-weekly.

At monthly intervals blood was collected from the femoral vein of fasted monkeys into tubes containing EDTA. Na~ and plasma was isolated following low speed centrifugation at 4~ Serum g?~tamate oxaloacetate transaminase (SGOT) was monitored using the Reitman-Frankel method (Dade Diagnostics, Inc., Miami, FL) and plasma triglyceride was measured with Sclavo reagent kits (Sclavo Diagnostics, Wayne, NJ). After precipitation of lower density lipoproteins by addition of heparin-manganese (30), HDL and plasma total cholesterol were measured colorimetrically (31). Very low density-low density lipoprotein (VLDL-LDL) cholesterol was estimated by difference. A separate aliquot of the HDL supernatant was then extracted (32) and free cholesterol was measured by gas l iquid chromatography using a column packed with 3% SP-2250 on 100/120 Supelcoport (Supelco Inc., Bellefonte, PA), under isothermal conditions (267~ and with cholestane as an internal standard. Free plus cholesteryl ester cholesterol were measured in a similar manner following saponification with 33% KOH (32). Esterified cholesterol mass was calculated by difference and corrected to cholesteryl linoleate. Free and esterified cholesterol mass in the VLDL-LDL pellet was measured in an identical manner. All lipoprotein, plasma cholesterol and triglyceride values were corrected for analytical variation using Monitrol (Dade Diagnostics, Inc., Miami, FL) as an external reference standard.

After one year of treatment, fasted, unanesthetized monkeys ~ere injected intravenously via the saphenous vein with 183-250 uCi of RS-(5- H) mevalonolactone (specific act ivi ty 13.8 Ci/mmol) (New England Nuclear, Boston, MA) dissolved in physiological saline. Blood samples were collected at 5, 20, 40 min., 1,2,3,4,5,6 hr. and plasma was isolated by low speed centrifugation. Monkeys were allowed free access to l iquid diet beginning after the 40 min. time point. Barter and Connor (33) used a similar procedure to evaluate HDL free and esterified cholesterol synthesis in humans.

HDL and VLDL-LDL were isolated as described above and immediately extracted with chloroform:methanol (2:1 v/v) (34). Free and esterified cholesterol in each lipoprotein fraction were separated by thin-layer chromatography (TLC) (35), scraped into sc int i l la t ion vials containing Aquasol cocktail (New England Nuclear, Boston, MA) and radioactivity was measured with

48 J.E. CLUETTE et al .

a liquid scint i l lat ion spectrometer. HDL and VLDL-LDL cholesterol and cholesteryl ester mass and radioactivity were corrected to a plasma volume calculated at 4% of body weight and specific activi ty measurements were then expressed as a percentage of the injected dose. HDL~ and HDL subfractions were separated in the two hour plasma sample by #olyacryla~ide disc gel electrophoresis (36). After v isual iz ing bands with Amido Black B, HDL fract ions were subjected to scanning densitometry, gels were cut, digested with NCS so lub i l i ze r (Amersham Corp., Arl ington Heights, IL) and rad ioac t iv i ty was monitored as described above.

After the 6 hr. time point, metal trays were placed beneath individual cages and fecal samples were collected for an 18 hr. period (24 hr. from the i n i t i a l in ject ion time). Fecal matter was pooled and one gram aliquots were extracted by the Folch procedure (34). The fecal l i p i d extract was then dried under nitrogen, resuspended in chloroform methanol (1:1 v /v ) , and al iquots were taken for TLC (37), neutral sterol (31) and total b i le acid mass which was measured co lor imet r ica l ly using 3 ~-hydroxysteroid dehydrogenase (Worthington Biochemicals, Freehold, NJ) (37,38). S i l ica gel G TLC plates were developed in n-butanol:water:glacial acetic acid ( I 0 : I . I : I . I ) for 3.5-4 hr. Samples were co-chromatographed with sodium taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC) (Sigma Chemical Co., St. Louis, MO), cholesterol (Applied Science, State College, PA) and several neutral sterols (coprostanol, dihydrocholesterol, coprostan-3-one) (Sigma Chemical Co., St. Louis, MO). Radiolabeled TCDC and TDC co-migrated as one band which was scraped and counted as described above. TC constituted a second band and cholesterol plus the other neutral sterols were scraped and counted as a th i rd f ract ion.

Data was expressed as the mean plus or minus the standard error of the mean. Mean values for Ethanol (Low, High) and Control groups were analyzed for s ign i f i can t differences (P<O.05) by analysis of variance and Duncan's mult iple range test .

RESULTS

Averaged over 12 months of treatment, there were no significant differences in SGOT between Control (88 • 6 International units/ml), Low (82 • 4) and High (90 • 4) Ethanol primates. There was also no significant difference in the mean body weights between the Control (825 • 69g), Low (890 • 74) and High (675 • 62) groups recorded just prior to the metabolic study.

As shown in Table 1, High Ethanol monkeys circulated significantly more VLDL-LDL, HDL and total plasma cholesterol and had higher plasma triglyceride levels than Control and Low Ethanol primates. However, the percent distribution of cholesterol among the lipoprotein fractions was identical for the three groups.


TABLE 1

Effect of Alcohol Consumption on Lipoprotein Total Cholesterol and Plasma Triglyceride

Measurement Very Low Density - Low Density Lipoprotein

Cholesterol

High Density Lipoprotein Cholesterol

Total Plasma Cholesterol

Plasma Tr iglycer ide

Control Treatment Groups Low Ethanol High Ethanol

(12%) (24%)

104• ,c 97• c 131• c (45) D (44) (46)

129• c 124• c 155• c (55) (56) (54)

233• c 221• c 286• c (I00) (I00) (IOO)

86• c 94• c I06• c

avalues represent means • SEM from 12 monthly measurements for 5 monkeys/group bexpressed as mg/dl. Values in parentheses represent re lat ive d is t r ibu t ion of cholesterol among

cl ipoprotein classes. High ethanol mean s ign i f i can t ly d i f ferent (P<O.05) from means of other groups with superscript c.

HDL free cholesterol was s ign i f i can t ly higher in the High Ethanol group as was HDL phospholipid whereas there was no difference between the groups in HDL cholesteryl ester mass (Table 2).

TABLE 2

Effect of Alcohol Consumption on High Density Lipoprotein (HDL) Composition

Treatment Groups Control Low Ethanol High Ethanol

(12%) (24%) Constituent Free Cholesterol 39• a'b 36• b 49• b

Cholesteryl Ester 148• 134• 152•

Phospholipid 203• b 185• b 240• b

Free Cholesterol/ 0.19 0.20 0.20 Phospholipid Ratio

avalues represent means • SEM from 12 monthly measurements for 5 monkeys/group bexpressed as mg/dl. High ethanol mean s igni f icant ly di f ferent (P<O.05) from means of other groups with superscript b.

50 J.E. CLUETTE et al.

The pattern of incorporation of 3H mevalonolactone into HDL free and esterified cholesterol is shown in Figs. I and 2, respectively. High Ethanol monkeys incorporated signif icantly more labeled precursor into HDL free cholesterol than Control and/or Low Ethanol monkeys beginning at two hours after injection and continuing for the next 4 hours (Fig. 1). Significantly more radioactivity remained in this fraction in High Ethanol animals compared to Controls even after 24 hours. By contrast, the curvilinear pattern and levels of HDL cholesteryl ester specific act iv i ty were similar for the three treatment groups (Fig. 2).

o4 el l~ [ l ~ - . ~ ~ 2a

ve

, m �9 - �9

2 I 4 s 6 ~4

C~ Cu4 ro ;

~k Lew [ t hau~

H N I I k | l kaN I

:0 4 S 4 H

ICmo (Ne r l )

FIG. 1

In vivo synthesis of HDL free cholesterol following injection of 3H mevalonolactone. Time points represent mean • SEM for 5 monkeys/group. S indicates significant difference (P<O.05) between High Ethanol and Control monkeys and SS difference between High Ethanol vs. both Control, Low Ethanol groups.

FIG. 2

In vivo synthesis of HDL chol~steryl ester following injection of H mevalonolactone. Time points represent mean • SEM for 5 monkeys/group.

There were no significant differences between the groups in the densitometric scans of HDLp and HDLR subfractions or in the relative distribution of radioactivit~ between th~ HDL subclasses. The HDL~/HDL~ ratio range for the three treatments was 5.8-8.7. For all groups, approximately 39% and 61% of the radioactivity appeared in the HDL 2 and HDL 3 subclasses, respectively.

Synthesis of VLDL-LDL free plus esterified cholesterol is shown in Fig. 3. Although the pattern ef incorporation and ranking of treatments (High Ethanol highest, Control lowest) was similar to HDL free cholesterol synthesis (Fig. 1), there were no significant differences between the groups when the data was expressed on a specific act iv i ty basis. However, both Low and High Ethanol primates synthesized signif icantly more VLDL-LDL free cholesterol compared to Controls at 3, 4, and 5 hours after injection when data was calculated on a radioactivity basis (DPM/plasma volume) (Fig. 4). Significant differences between Control and High Ethanol treatments were also noted for this parameter at 6 hours.


O 0 r

_;z

2 | 4 S I 24

I~me (h,mrs)

c~O e,mreo L ~ |tlmeeO

D O n l Ih Etb***~

' ! .

i.i

I i 2 4 s I 14

1,m, cb~rJ )

FIG. 3 FIG. 4

In vivo synthesis of VLDL-LDL total 3 cholesterol following injection of H mevalonolactone. Time points represent mean • SEM for 5 monkeys/group expressed as specific act ivi ty (DPM/mg) calculated2as percent of injected dose x 10- ,

In vivo synthesis of VLDL-LDL free 3H cholesterol following injection of mevalonolactone. Time points represent mean • SEM for 5 monkeys/group expressed as plasma radioactivity (DPM) calculated aslpercent of injected dose x 10 -~. S indicates significant difference (P<O.05) between High Ethanol and Control monkeys. SS indicates significant difference between both High and Low Ethanol groups vs. Controls.

The appearance of radiolabeled neutral sterols and bi~e acids in the feces of primates during the f i r s t 24 hours after injection of H mevalonolactone is shown in Table 3. Neutral sterol specific act ivi ty was significantly higher in the High Ethanol group compared to the other two treatments, while total bile acid mass was elevated in fecal samples from both alcohol treated groups. Total bile acid specific act iv i ty (TC + TCDC + TDC) was similar for the three treatments.


TABLE 3

Effect of Alcohol Consumption on Neutral Sterol and Bile3Acid Excretion

Following Injection with H Mevalonolactone

Control

Fecal Constituent Neutral Sterol Specific 64,242• a'b Activity (DPM/mg)

Treatment Groups Low Ethanol High Ethanol

~12%) (24%)

85,860• b 140,106• b

Bile Acid Specific 18,286• 46,892• 28,165• Activity (DPM/mg)

Bile Acid Mass 1.9• b'c 3.3• c 3.1• b (mg/g feces)

~Values • SEM for 5 represent means monkeys/group. High ethanol mean signif icantly different (P<O.05) from means of other groups

cwith superscript b. Low ethanol mean signif icantly different (P<O.05) from means of other groups with superscript c.

DISCUSSION

Results from this study provide the f i r s t experimental evidence that increased HDL cholesterol levels resulting from ethanol consumption (Table 1) are at least in part due to enhanced synthesis (Fig. 1). Data in Fig. 1 substantiate previous rat metabolic in vivo (19) and l iver perfusion (20) studies which showed that dietary ethanol at 36-37% of calories may increase the rate of incorporation of newly synthesized l ip id into HDL which thus suggests enhanced de novo lipoprotein synthesis. The present study is unique, however, in elucidating a lower dose (24%) at which this ethanol effect occurs in nonhuman primates, and in providing the f i r s t in vivo experimental verification that alcohol consumption specifically stimulates synthesis of HDL's free cholesterol component (Fig. 1).

The hypothesis that alcohol may induce hepatic microsomal enzyme act iv i ty which in turn may promote synthesis and secretion of HDL (3,13,39) is further substantiated by our results. As noted earl ier, over 60% of the newly synthesized sterol plus sterol ester were isolated in the HDL 3 subclass. HDL 3 represents a nascent HDL particle which may originate via direct secretion from the l iver and intestine (15,17), or from surface components of chylomicrons and VLDL during their intravascular l ipolysis (3,15,16). Recent evidence suggests that this subfraction is selectively elevated in men consuming moderate amounts of alcohol (1). Whereas normally only 40% of the total body cholesterol synthesis in squirrel monkeys occurs in the l iver (40), i t is conceivable that dietary ethanol might increase this capacity by stimulating the proliferation of smooth endoplasmic reticulum which is responsible for the manufacture of lipoproteins prior to secretion (41,42). Thus, besides increased synthesis and secretion of HDL 3, one might predict that as a result of the induction of hepatic microsomal enzymes, other lipoproteins would be produced in greater amounts (41). Figure 4 indicates that at both levels of ethanol intake, more labeled free cholesterol was found in the VLDL-LDL fraction compared with that of Control monkeys. Significant differences between the groups were not detected when data was reported on


specific activity basis because of the greater experimental variabil ity in free and esterified cholesterol mass monitored in this fraction (Fig. 3).

I t appears that alcohol may concurrently and non-selectively enhance the hepatic synthesis and secretion of both the lower and high density lipoproteins particularly when administered at 24% of calories (Figs. 1 and 4). This would account for the proportional increase in the VLDL-LDL and HDL total cholesterol mass in the High Ethanol group which resulted in a relative lipoprotein cholesterol distribution identical to the other treatments and thus a stable lipoprotein profile (Table 1). Further evidence of a stable profile is suggested from the HDL free cholesterol/ phospholipid ratio which was unchanged in the three primate groups (Table 2). Alternations in this ratio in circulating lipoproteins have been used as an index of functional abnormalities observed in a number of disease states (43).

Although our data suggests that increased lipoprotein synthesis may contribute to elevations in HDL and VLDL-LDL cholesterol (Table 1), ethanol- induced modifications in clearance of lipoproteins from the plasma compartment may also influence the concentration of these macromolecules (44,45). Studies in progress in our laboratory which will be reported separately are d~signed to ~aluate whether the 12% and 24% ethanol doses alter the removal of H LDL and

C HDL cholesteryl ester from circulation. The second major finding from this study was that consumption of ethanol

at 24% of calories may promote fecal excretion of newly synthesized cholesterol. As noted in Table 3, High Ethanol monkey s had greater fecal output of radiolabeled neutral sterols than the other groups. Although there were no significant differences between the groups in total fecal bile acid specific activity, total fecal bile acid mass was higher in both alcohol groups (Table 3) which is similar to what has been observed in pigs fed ethanol at 26% of calories (9). Bile acid specific activity for the Low Ethanol monkeys was not statist ically different from the other groups despite their apparently higher mean because of the wide range of values for this parameter (Table 3). This in turn may be due to the 18-hour fecal collection period during which time individual animal to animal variations in bowel evacuation and/or fecal bile acid output may have become manifest.

Enhanced fecal excretion of radiolabeled cholesterol is consistent with a recent finding that consumption of 39g of alcohol daily by humans causes a significant increase in HDL cholesterol, a decrease in the bile cholesterol saturation index and putative protection against cholesterol gallstone formation (26). Our data suggest that the mechanism responsible for this decrease in bil iary saturation may be an ethanol induced increase in output of newly synthesized cholesterol. We were unable to distinguish whether fecal radiolabeled sterols (Table 3) were derived primarily from direct hepatic secretion of newly formed cholesterol or uptake of recycled lipoprotein labeled cholesterol. Previous studies have indicated that approximately 30% of the bil iary bile acids and cholesterol may be derived from the former and 70% from the latter primarily in the free cholesterol form (7,10). More specifically, HDL free cholesterol appears to be the preferred lipoprotein source for bil iary cholesterol and bile acids in both squirrel monkeys and humans (7,8). Elevations in HDL total (Table 1) and free (Table 2) cholesterol and the greater output of radiolabeled neutral sterols (Table 3) in the High Ethanol primates are at least in part consistent with the hypothesis that this lipoprotein may transport cholesterol from peripheral tissues to the liver for removal (6) and that alcohol may enhance this process (26).

Taken together, results from this study are s igni f icant in: I) document- ing a lower dose (24% of total calories) at which dietary ethanol increases HDL free cholesterol levels: 2) elucidating a mechanism by which this increase occurs (synthesis); and 3) describing the potent ial ly beneficial effect of alcohol in promoting fecal output of newly synthesized cholesterol. Furthermore, evidence is presented which suggests that the squirrel monkey may


represent a nonhuman primate model ideally suited for examining the mechanisms of alcohol-induced alterations in lipoprotein metabolism-biliary cholesterol excretion without the confounding problem of hepatic dysfunction as evidenced by normal SGOT levels in the ethanol treated groups. In addition, these primates may have great cl inical relevance to man in that both species preferentially u t i l ize HDL free cholesterol as the major source for bilia~y cholesterol and bile acids and that the in vivo patterns of incorporation of ~H mevalonolactone into HDL free (Fig. i) and esterified (Fig. 2) cholesterol are similar to what has been reported for humans (33).

ACKNOWLEDGMENTS

This work was supported by grants from the Alcoholic Beverage Medical Research Foundation at The Johns Hopkins University School of Medicine. The authors wish to express their sincere thanks to Mary Hojnacki and Carol Martin for preparation of the manuscript.

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Accepted for publication September 24, 1984

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Effect of ethanol on lipoprotein synthesis and fecal sterol excretion