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Applied nutritional investigation

Effect of the type of dietary fat on biliary lipid composition and bilelithogenicity in humans with cholesterol gallstone disease

María Dolores Yago, Ph.D.a,*, Victoria González, M.D., Ph.D.b, Pilar Serrano, M.D., Ph.D.b,Rafael Calpena, M.D., Ph.D.b, María Alba Martínez, Ph.D.a,

Emilio Martínez-Victoria, Ph.D.a, Mariano Mañas, Ph.D.aa Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, Granada, Spain

b Department of Surgery, Faculty of Medicine, University of Alicante, Alicante, Spain

Manuscript received January 9, 2004; accepted June 16, 2004.

bstract Objective: The effect of the type of dietary fat on bile lipids and lithogenicity is unclear. This studycompared the effects of two dietary oils that differed in fatty acid profile on biliary lipid composition inhumans.Methods: Female patients who had cholesterol gallstones and were scheduled for elective chole-cystectomy were studied. For 30 d before surgery, subjects were kept on diets that contained oliveoil (olive oil group, n � 9) or sunflower oil (sunflower oil group, n � 9) as the main source of fat.Gallbladder bile and stones were sampled at surgery. After cholecystectomy, duodenal samples werecollected by nasoduodenal intubation during fasting and after administration of mixed liquid mealsthat included the corresponding dietary oil. Duodenal and gallbladder bile samples were analyzedfor cholesterol, phospholipids, and total bile acids by established methods. Individual bile acidconjugates in gallbladder bile were measured by high-performance liquid chromatography. Gall-stones were analyzed by semiquantitative polarizing light microscopy.Results: Despite marked differences in the absolute concentration of biliary lipids and total lipid content,manipulation of dietary fat ingestion did not influence the cholesterol saturation or the profile ofindividual bile acids in gallbladder bile obtained from patients who had gallstones. All but one subjecthad mixed cholesterol stones. A cholesterol saturation index of hepatic bile in fasted cholecystectomizedpatients was similar in both dietary groups and indicative of supersaturation. In response to the test meal,the cholesterol saturation index decreased significantly in patients given the olive oil diet, reaching valueslower than one at 120 min postprandially. In contrast, hepatic bile secreted by patients who consumedsunflower oil appeared supersaturated (cholesterol saturation index �1.5) throughout the experiment.Conclusions: Our results suggest that the type of dietary fat habitually consumed can influence bilecomposition in humans. In gallbladder, this influence was noted in the presence of more concen-trated bile in the olive oil group. However, this was not translated into a modification of cholesterolsaturation, which is likely due to the fact that cholesterol gallstones were present by the time thedietary intervention started. The finding that a typical postprandial variation in hepatic bile litho-genicity occurred only in olive oil patients was revealing. While keeping in mind the methodologiclimitations of this part of the study, some gastrointestinal and metabolic mechanisms for this effectare discussed. © 2005 Elsevier Inc. All rights reserved.

Nutrition 21 (2005) 339–347www.elsevier.com/locate/nut

eywords: Dietary fat; Biliary lipids; Cholesterol saturation; Gallstone disease; Cholecystectomy

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ntroduction

The overall effect of the type of dietary fat on bile lipidsnd lithogenesis in humans is unclear, although epidemio-

This study was supported by the Spanish CICYT.* Corresponding author. Tel.: �34-958-248321; fax: �34-958-248326.

pE-mail address: mdyago@ugr.es (M.D. Yago).

899-9007/05/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.nut.2004.06.028

ogic, clinical, and animal studies have indicated an impor-ant role for this dietary component. A key limitation ofnimal studies is the existence of marked differences iniliary lipid homeostasis from those in humans, i.e., mostnimal species readily convert any excess cholesterol to bilecids, so biliary cholesterol concentration seldom ap-

roaches the saturation point [1].

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340 M.D. Yago et al. / Nutrition 21 (2005) 339–347

As for the human studies, the investigation of the effectf dietary fats on biliary composition has yielded equivocalesults. A first reason is the small number of availabletudies, possibly because of the difficulty and risks of sam-le collection. Second, gender and inherent differences inetabolism at the time of dietary intervention may affect

he outcome. The third reason concerns the variety of di-tary protocols, including differences in the amount of di-tary fat and in the duration of treatment. Thus, consumingor 2 to 4 wk “prudent” diets with less cholesterol andaturated fat and more polyunsaturated fat than the previousnormal” diets tended to increase biliary cholesterol satu-ation in healthy women [2] but induced no change inallbladder bile of healthy men despite modifications in theelative proportion of biliary lipids [3]. Dietary supplemen-ation with �-3 fatty acids over 6 wk decreased the choles-erol saturation index (CSI) in healthy subjects [4]. A sim-lar effect was found in lithiasic patients in association withn increased proportion of long �-3 fatty acids and a de-reased proportion of �-6 fatty acids in biliary phospholip-ds [5]. In another study in patients who had gallstone, nohange in lipid composition or decrease in cholesterol sat-ration was observed after supplementing the diet withinoleic acid or soybean lecithin for 3 wk [6]. Although theffects of polyunsaturated fat rather than the typical West-rn diet have been investigated in several studies, the influ-nce of monounsaturated fats has been even less explored,lthough Schlierf et al. [7] found no significant difference iniliary lithogenicity in healthy males who received for 2 wkwo lipid-lowering diets, one rich in polyunsaturated fattycids and the other rich in oleic acid.

The present study compared the effects of two dietaryils that differ markedly in fatty acid profile (virgin olive oilnd sunflower oil) on biliary lipid composition in humanubjects. These oils are used preferentially in our geo-raphic area. Further, olive oil is a major component of theediterranean diet, and its role in human health currently is

eing actively debated. The study group, which comprised8 female patients who had gallstones and have been re-erred to elective cholecystectomy, was kept on the exper-mental diets for 30 d before surgery. This design madeossible the collection of fasting gallbladder bile and stonesat surgery). In addition, fasting and postprandial hepaticile (postcholecystectomy) levels were examined to rule outhat a possible influence of the dietary intervention wasasked by the occurrence of pre-existing gallstones.

aterials and methods

ubjects

Eighteen non-smoking, non-alcoholic women who hadholelithiasis, showed current clinical signs or symptoms,nd were scheduled for elective surgery (cholecystectomy)

ere selected within the population served by the Hospital s

linico, University of Alicante (Alicante, Spain). None ofhem had undergone previous pancreatic, gastric, or biliaryract surgery. Cases of choledocholithiasis and asymptom-tic cholelithiasis were excluded from the study. The sub-ects had no history of systemic or gastrointestinal disease.one was receiving medication known to influence gastro-

ntestinal secretions, motility, or postprandial hormonal re-ponses. The study was conducted in conformance with theelsinki Declaration and the experimental protocol was

pproved by the ethics committee of the Hospital Clinico.ll subjects gave written consent after being fully in-

ormed of the nature and procedures of the study. Patientsere randomized into two experimental groups, an oliveil group and a sunflower oil group, according to theirietary habits and specifically to the type of dietary fatabitually consumed before the study. This informationas gained from a dietary history interview performed at

he beginning of the study. Each group comprised nineatients and had the following characteristics (mean �tandard error): 54.4 � 4.04 y of age and 27.3 � 1.2g/m2 for body mass index for the olive oil group and1.7 � 3.85 y of age and 28.4 � 8.0 kg/m2 for theunflower oil group. These parameters showed no signif-cant differences between groups.

xperimental protocol and diets consumed before surgery

Subjects from both groups were told at the start of thetudy to consume their habitual diets for the 30-d periodmmediately before surgery, with these requirements: 1) thenly source of dietary fat used to prepare their meals had toe olive oil (olive oil group) or sunflower oil (sunflower oilroup); 2) subjects had to avoid eating food items high inaturated fat (butter, sausages, etc.). Although patients withallstone are usually very inclined to adhere to dietaryecommendations, compliance with the diets was checkedy completion of four 7-d dietary records. These wereollected during weekly visits to the hospital, the data wereuantified, and the energy and nutrient intakes were as-essed with our computer program Nutrition and HealthInstitute of Nutrition and Food Technology, University ofranada, Spain; General Asde, Valencia, Spain). The data-ase of this program is the Spanish Food Compositionables [8]. As shown in Table 1, the two dietary groupsiffered primarily in relation to intakes of polyunsaturatednd monounsaturated fats, whereas the composition of theest of the diet was similar.

ollection of gallbladder bile and stone samples

All patients fasted from 12 AM and underwent surgeryetween 9 and 10 AM. Gallbladders were removed with aaparoscopic cholecystectomy procedure. Bile samples were

tored as aliquots at �80°C until analyzed for biliary lipid

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341M.D. Yago et al. / Nutrition 21 (2005) 339–347

omposition. Gallstones were preserved under sterile con-itions for further microscopic examination.

ostcholecystectomy study

The experiments were performed 48 h after surgery, oncet had been confirmed that subjects had recovered gastroin-estinal motility. The test meals (pH 6.33, 294 mosm/L)ontained 1 kcal/mL and were composed of 17% energy asrotein, 30% as fat, and 53% as carbohydrate, in addition toitamins and minerals. The meals were isoenergetic andsonitrogenous, contained no measurable amounts of cho-esterol and phospholipids, and were prepared by mixing theeparate modular components lactalbumin, maltodextrins,nd a vitamin-mineral mixture (Edda Modular, Ibys Nu-rición, Madrid, Spain). Olive oil was added to the mealiven to the olive oil group and sunflower oil to the sun-ower oil group. The fatty acid composition of the liquideals (Table 2) was determined according to the method ofepage and Roy [9] using a Hewlett Packard HP 5890eries II chromatograph (Palo Alto, CA, USA) and a Su-elco capillary column (Supelco Inc., Bellefonte, CA, USA)mpregnated with an Sp 2330 FS that was 60 m long, 32 mmn inner diameter, and 20 mm thick.

Each subject was studied on 2 consecutive days and aftert least 8 h of fasting. The participants were intubated withradiopaque two-lumen nasoduodenal tube that enabled

eparate aspiration of gastric and duodenal contents. Duo-enal content was collected at the distal aspiration site,hich was placed in the third or fourth duodenal segment.dequate positioning of the tube was confirmed by radio-

ogic control at different times throughout the investigation.uodenal samples (2 mL) were taken immediately before

nd at 30, 60, 120, and 180 min after beginning the inges-ion of the liquid meal (200 mL ingested over 30 min). Theomplete feeding and sampling procedure was repeated onhe second experimental day, with each individual receiving

able 1aily energy and nutrient intake by gallstone patients during the 30-dietary adaptation period before cholecystectomy*

Olive oilgroup

Sunflower oilgroup

nergy (kcal/d) 1623.5 � 153.3 1485.5 � 297.9nergy ratioProtein (%) 18.6 � 1.7 18.2 � 2.1Carbohydrate (%) 39.4 � 2.5 38.8 � 3.9Fat (%) 41.6 � 2.5 42.6 � 3.3

atty acids (g/d)Monounsaturated fatty acids 40.1 � 2.6 26.2 � 2.9Polyunsaturated fatty acids 8.3 � 0.8 19.8 � 2.5Saturated fatty acids 20.6 � 3.8 18.8 � 3.2

* Subjects consumed diets containing virgin olive oil or sunflower oil ashe main source of dietary fat. Data are presented as mean � standardeviation of four 7-d dietary records per subject (nine subjects per group).

he same meal as the day before.

ample analysis

Gallbladder bile and duodenal samples were analyzed forholesterol, phospholipid, and total bile acid concentrationsfter removing protein and pigments [10]. Cholesterol lev-ls were measured by the CHOD-PAP method (Boehringerannheim, Mannheim, Germany). An enzymatic colori-etric test (Boehringer Mannheim) was used to determine

hospholipid concentration. Total bile acid concentrationas measured by the 3�-hydroxysteroid dehydrogenaserocedure [11].

A modification of the technique described by Tietz et al.12] was used to measure individual glycine and taurine bilecid conjugates in gallbladder bile by reverse phase high-erformance liquid chromatography with an isocratic sol-ent system. The chromatograph (Beckman Instruments,ullerton, CA, USA) was equipped with a WatersBondapak octadecylsilane column (30 cm long, 3.9 mm in

nner diameter, 10-�m particles; Waters Associates, Mil-ord, MA, USA). Detection was accomplished at 200 nm.he mobile phase (flow rate 2 mL/min) was 10% acetoni-

rile and 90% of a mixture of methanol and 0.1 M mono-asic potassium phosphate (60:40, v/v, pH 4.50). Beforese, the solvent was filtered through a 0.45-�m filter (typeV, Millipore, Bedford, MA, USA). An elution profile of

onjugated bile acid standards (Sigma, St. Louis, MO,SA) was obtained by injecting 20 �L of a standard meth-

nolic mixture that contained 20 �g of each bile acid. Welso established a standard curve for each standard bile acidy plotting concentration injected versus eluted peak area.efore analysis, gallbladder bile samples were diluted 1:3

v/v) in isopropanol, heated for 10 min at 75°C, centrifugedt 450g for 15 min and the supernatant filtered through aelman Acrodisc filter (0.45-�m pore, Pall Gelman Sci-

nces, Ann Arbor, MI, USA). Of this solution, 100 �L wasvaporated under nitrogen and redissolved in methanol. Thenjection volume was 20 �L.

Gallstone sections (20- to 30-�m thick) were analyzedy semiquantitative polarizing light microscopy. Sections

able 2atty acid composition of the liquid test meals administered toholecystectomized patients*

Olive oilgroup

Sunflower oilgroup

leic acid (18:1) 61.89 � 2.00† 29.03 � 0.66inoleic acid (18:2�-6) 5.06 � 0.12† 42.16 � 1.70onounsaturated fatty acids 63.08 � 1.95† 29.67 � 0.66

olyunsaturated fatty acids 8.19 � 0.14† 44.62 � 1.55aturated fatty acids 29.03 � 1.86 25.93 � 1.93/S 2.51 � 0.23 2.99 � 0.30

U/S, ratio of unsaturated to saturated fatty acids* Patients were kept on diets with olive oil or sunflower oil as the main

ource of dietary fat for 30 d before surgery. Results are expressed as gramser 100 g of total fatty acids. Values are mean � standard error (n � 6 foroth groups).

† P � 0.05 versus sunflower oil group.

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342 M.D. Yago et al. / Nutrition 21 (2005) 339–347

ere prepared by the Microscopy Service of the Universityf Granada (Granada, Spain). Briefly, gallstones werembedded in polyester resin (Estratil A-116, Glaspoloposites, Valencia, Spain) for 30 min under vacuum and

hen stored in the freezer for 24 h. Next, samples were cutnto 2- to 5-mm sections, polished, and mounted on glasslides impregnated with polyester resin. After drying, sec-ions were reduced to a thickness of approximately 300 �mn a diamond cut-off saw. Sections were then ground andolished to the final thickness.

ata analysis and statistical evaluation

The CSI was defined as the ratio of the actual molarercentage of cholesterol in a given sample to that sample’saximum capacity for cholesterol solubility. The latter was

alculated according to the method of Metzger et al. [13]. Inallbladder bile samples, maximum cholesterol solubilityas also obtained from Carey’s critical tables [14]. Unlesstherwise stated, results are expressed as mean � standardrror. Statistical analysis was done with SPSS 11.0.1 for

indows (SPSS, Inc., Chicago, IL, USA). Differences be-ween dietary groups in fatty acid composition of meals andn gallbladder and hepatic bile parameters were tested forignificance by the independent samples Student’s t test. Fortatistical comparisons within groups (above or below fast-ng values) in the postcholecystectomy study, a one-waynalysis of variance was done. P � 0.05 was consideredtatistically significant.

esults

allbladder bile and gallstones

Table 3 presents the composition of gallbladder bilepecimens obtained at surgery. There was a significantly (P

0.05) greater concentration (millimoles per liter) of cho-esterol and bile acids in the olive oil group than in theunflower oil group. The concentration of phospholipidsas slightly higher in the olive oil group. As a consequence,allbladder bile from patients who consumed olive oil had aigher total lipid content (11.48 � 1.22 g/dL versus 7.77 �.59 g/dL in the sunflower oil group, borderline significanceith P � 0.063 with nine subjects in each group). When the

omposition of bile was expressed relative to total biliaryipids (molar percentages), we found that the proportions ofholesterol and bile acids were similar in both groups,hich contrasts with a significantly (P � 0.05) lower molarercentage of phospholipids in the olive oil group. Amonghe linkage coefficients, only the molar ratio of cholesterolo phospholipids was found to differ between groups (P �.05), with higher values in the olive oil group. Theseifferences, however, did not translate into bile lithogenic-ty, as shown by comparable values of cholesterol saturation

CSI; Table 3). c

Bile acid composition of gallbladder bile is shown in Fig. 1.o significant differences were noted in the proportion of sixajor individual bile acid conjugates. The molar ratios of

lycine-to-taurine conjugates and dihydroxylated-to-trihy-roxylated bile acids were similar in the olive oil group (3.19

0.44 and 1.69 � 0.07, respectively) and the sunflower oilroup (3.81 � 0.74; 2.04 � 0.18, respectively; n � 9 for bothroups in all parameters). Values for total (glycine plus taurineonjugates) cholic acid were 37.33 � 1.02 mol% for the oliveil group, and 33.40 � 1.74 mol% for the sunflower oil groupnd those for chenodeoxycholic acid were 43.52 � 1.09 mol%or the olive oil group and 41.17 � 3.10 mol% for the sun-ower oil group (n � 9 for both groups in all parameters).

Gallstones from all patients were analyzed. Using a 70%inimum cholesterol content to define mixed cholesterol, the

ollowing results were obtained. In the sunflower oil group,ne of nine patients had a pure cholesterol stone and the restad mixed cholesterol stones composed of: cholesterol andodium palmitate (one of eight), cholesterol and bilirubin (fivef eight), and cholesterol, bilirubin, and sodium palmitate (twof eight). All patients in the olive oil group had mixed choles-erol stones composed of: cholesterol and sodium palmitateone of nine), cholesterol and bilirubin (five of nine), andholesterol, bilirubin, and sodium palmitate (three of nine).

ostcholecystectomy study

No differences were noted between groups in the percent

able 3iliary lipid composition of gallbladder bile sampled at the time of

urgery*

Olive oilgroup

Sunflower oilgroup

otal bile acidsmM/L 142.23 � 15.05† 87.95 � 20.42mol% 66.47 � 0.56 60.37 � 3.11

holesterolmM/L 17.60 � 1.78† 9.92 � 1.94mol% 8.33 � 0.52 7.54 � 1.20

hospholipidsmM/L 54.06 � 6.00 42.55 � 7.94mol% 25.20 � 0.25† 32.09 � 2.43

inkage coefficientsCholesterol/bile acids 0.126 � 0.009 0.132 � 0.027Phospholipids/bile acids 0.379 � 0.006 0.553 � 0.072Cholesterol/phospholipids 0.331 � 0.021† 0.236 � 0.034

SI‡ 0.835 � 0.052 0.857 � 0.167SI§ 1.056 � 0.069 1.013 � 0.176otal lipids (g/dL) 11.48 � 1.22 7.77 � 1.59

CSI, cholesterol saturation index* Patients were kept on diets with either olive oil or sunflower oil as theain source of dietary fat for 30 d prior to surgery. Data are mean �

tandard error (n � 9, for both groups).† P � 0.05 versus sunflower oil group.‡ Calculated according to Metzger et al. [13].§ Calculated according to Carey [14].

omposition of biliary lipids in fasting duodenal samples

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343M.D. Yago et al. / Nutrition 21 (2005) 339–347

Fig. 2A–C). Values for cholesterol saturation (CSI) werelso comparable (Fig. 3C). Feeding did not modify theolar percentage of phospholipids in any group, and thereere no significant differences between them (Fig. 2C). No

ppreciable changes from fasting values were recorded forhe molar percentage of bile acids in the sunflower oil groupfter the test meal in contrast to a significant (P � 0.05)ncrease at 60 min postprandially in the olive oil group (Fig.A). However, this different response did not result inignificant differences between groups during the course ofhe experiment (Fig. 2A). Molar percentage of cholesterolFig. 2B) remained relatively constant in the sunflower oilroup after food intake, whereas a clear decrease was notedn the olive oil group (P � 0.05 versus values before theeal). At 60 and 120 min, cholesterol values were signifi-

antly (P � 0.05) lower compared with those in the sun-ower oil group.

The molar ratios of cholesterol to phospholipids and ofholesterol to bile acids (Fig. 3A,B) followed a similar trendfter the ingestion of the test meals, i.e., a lack of changesn the sunflower oil group and a decrease in the olive oilroup. Compared with the fasting level, values in this groupecreased significantly (P � 0.05) at 60 min for the ratio ofholesterol to bile acids (Fig. 3B) and at 120 min for the

ig. 1. Bile acid composition of gallbladder bile in gallstone patients after30-d intake of diets that contained virgin olive oil or sunflower oil as theain source of dietary fat. Bile was sampled at surgery (cholecystectomy).ata are presented as mean � standard error (n � 9/group). gc, glycocholic

cid; gcdc, glycochenodeoxycholic acid; gdc, glycodeoxycholic acid; tc,aurocholic acid; tcdc, taurochenodeoxycholic acid; tdc, taurodeoxycholiccid.

atio of cholesterol to phospholipids (Fig. 3A). At this time, g

his parameter differed significantly (P � 0.05) betweenroups (Fig. 3A). As measured by the CSI (Fig. 3C), hepaticile secreted by patients in the sunflower oil group appearedupersaturated throughout the experiment (mean CSI �.5), whereas the degree of cholesterol saturation in bilebtained from patients in the olive oil group decreased fromfasting level of 2.051 � 0.290 (n � 18, supersaturated) tosignificantly (P � 0.05) lower value of 1.048 � 0.152 (n18) at 60 min postprandially and to a minimum of 0.8210.201 (n � 18) at 120 min (P � 0.05) that was indicative

f undersaturated bile. CSI showed a significant (P � 0.05)ifference between dietary treatments at 120 min after theeal.

iscussion

Manipulation of dietary fat ingestion did not influencehe cholesterol saturation of gallbladder bile sampled at theime of surgery. To a certain extent, this was an expectednding because our subjects were patients with pre-existingallstones. Data on the effects of dietary fats and fatty acidsn gallbladder bile in patients with established cholelithiasisre very limited. In one study, fish oil decreased the CSI by5% after a 5-wk treatment period [5]. In contrast, dietaryupplementation with linoleic acid for 3 wk did not alter theipid composition or cholesterol saturation [6], in goodgreement with the results of the present study. The occur-ence of a similar CSI in our two groups is also in line withhe homogeneity in the composition of the stones. Concern-ng the profile of individual bile acids in gallbladder bile, webserved no clear influence of the type of fat ingested. Bothroups of patients displayed a modest “cheno” profile, i.e.,ile had slightly more chenodeoxycholic acid than choliccid. This is a common feature in patients who have cho-esterol gallstone. In comparison, cholic species predomi-ate in healthy human bile [1,15].

When gallbladder bile composition was analyzed inerms of absolute concentrations of lipids, marked differ-nces were found between our study groups. Concentrationsf cholesterol and total bile acids were enhanced in patientsho received the olive oil diet. Gallbladder bile from thelive oil group also had considerably higher total lipidontent. A decreased total lipid concentration has been re-orted in samples of gallbladder bile collected from patientsho had cholesterol gallstones [16]. It is unclear why this

eature was noted only in our patients who received theunflower oil diet. One possibility is that gallbladders in theunflower oil group absorbed fluid less efficiently than didhose in the olive oil group. Gallbladder fluid absorption issually determined indirectly by calculating the ratio ofallbladder to hepatic bile for bile acid concentration. Un-ortunately, we were unable to collect paired gallbladdernd hepatic samples at surgery, but the concentrations ofile acids (as non-absorbable bile solutes) and total lipids in

allbladder bile provide a rough estimate [17]. A proposed

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344 M.D. Yago et al. / Nutrition 21 (2005) 339–347

echanism for differential gallbladder fluid absorption ishat the activity of the membrane-bound enzyme Na�-K�

denosine triphosphatase, the key factor in this process, isffected by dietary-induced alterations in phospholipid acylhain composition, as shown in other tissues [18]. Whetherhis is also true for epithelial cells of the gallbladder mem-rane awaits investigation. It cannot be excluded that theseesults are, other than an expression of gallbladder fluidbsorption, at least partly influenced by differences in he-atic bile composition. However, our observation that thebsolute concentration of bile acids in fasting duodenalamples obtained after cholecystectomy from patients whoeceived olive oil (data not shown) was significantly lowerhan in those who received sunflower oil (although thepposite was found in gallbladder bile) seems to support the

ig. 2. Time course of changes in molar percentages of bile acids (A), choatients after the administration of a liquid meal. Subjects were kept on d0 d before surgery and were given meals that included the correspondingar). Values represent mean � standard error of two experiments on each sualue. #Significantly different (P � 0.05) from the olive oil group.

rst hypothesis. i

Previous work in our laboratory [19] investigated theffects of feeding dogs diets high in olive oil or sunfloweril on bile secretion. The animals, with intact gallblad-ers, were prepared with chronic biliary cannulas andecretion was studied at rest and after food intake. Pat-erns of biliary response to food suggested a greaternvolvement of the gallbladder in animals fed the oliveil diet, as most clearly shown by a marked increase inile acid output soon after eating compared with a con-inuous decrease in dogs fed sunflower oil. These differ-nces [19] could not be explained by changes in bile flowate, which was similar during the initial postprandialours, but by dramatic differences in the concentration ofile acids in the bile secreted. Although this might be inart a consequence of differences in gallbladder empty-

(B), and phospholipids (C) in duodenal samples from cholecystectomizedcontained olive oil or sunflower oil as the main source of dietary fat for

ro time denotes fasting values. Meals were ingested within 30 min (black� 9/group). *Significantly different (P � 0.05) from the respective fasting

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345M.D. Yago et al. / Nutrition 21 (2005) 339–347

ile after olive oil feeding (as shown in the present study)s also compatible with the results in dogs.

The investigation of biliary homeostasis is very difficulto perform in humans. In the second part of this study,epatic bile was obtained from cholecystectomized patients.he rationale for these experiments may be questionedecause there is the concept that gallbladder bile aspirated aturgery after an overnight fast is much more representativef the situation of the bile salt pool and other biliary lipids.e agree in part with this idea but also believe that exam-

nation of hepatic bile composition after cholecystectomyas crucial in this study to confirm that a possible effect ofietary fat type on biliary lipids was detected. Analysis ofnly gallbladder bile could have masked the influence of theietary intervention because of the presence of established

ig. 3. Time course of changes in molar ratios of cholesterol to phospholiprom cholecystectomized patients after administration of a liquid meal. Subf dietary fat for 30 d before surgery and were given meals that included th0 min (black bar). CSI was calculated in these samples according to thewo experiments on each subject (n � 9/group). *Significantly different (P �he olive oil group. CSI, cholesterol saturation index.

allstones. We collected hepatic bile samples by duodenal r

ntubation. This technique does not allow measurement ofiliary lipid output unless a non-absorbable marker is used,ut this approach would have complicated even more anlready demanding study. However, duodenal samplingrovided a mechanism by which the proportion of biliaryipids (relative to total lipids) in hepatic bile could be quan-ified, offering interesting data that would otherwise beifficult to obtain in humans in vivo.

No differences were noted in molar percentages, molaratios, or cholesterol saturation (CSI) between our groups ofasted cholecystectomized patients. CSI in all patients wasndicative of cholesterol supersaturation. This is a well-ecognized physiologic phenomenon [21–24] that resultsrom the curvilinear relation between cholesterol and bilecid output [23,24]. During overnight fasting, which inter-

and cholesterol to bile acids (B) and in the CSI (C) in duodenal samplesre kept on diets that contained olive oil or sunflower oil as the main sourceponding oil. Zero time denotes fasting values. Meals were ingested withinof Metzger et al. [13]. All values represent the mean � standard error offrom the respective fasting value. #Significantly different (P � 0.05) from

ids (A)jects wee corresmethod

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upts the enterohepatic circulation, bile acid flux rate is very

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346 M.D. Yago et al. / Nutrition 21 (2005) 339–347

ow and bile becomes supersaturated with cholesterol. Con-ersely, accelerated recirculation of intestinal bile acids inesponse to a meal increases bile acid output and hepaticile reverts to a less saturated state. This trend, which haseen observed in healthy, lithiasic, and cholecystectomizedubjects [21–24], was, surprisingly, non-existent in our pa-ients in the sunflower oil group (Figs. 2 and 3). Withoutiliary output data, this finding is amenable to more thanne interpretation. The postprandial response observed inhis study is consistent with an attenuated increase in bileow and bile acid output in the sunflower oil group com-ared with a normal response in the olive oil group. Sus-ained output of bile acids in response to a meal depends onhe return of bile acids to the liver, which, in cholecystec-omized patients, is strongly influenced by intestinal motil-ty and transit time [23,24]. The ingestion (chronic, acute, oroth) of the two types of fat used in this study might haveroduced different feeding motility patterns through differ-ntial stimulation of humoral agents, in turn affectingholeresis. A second possibility is that patients in the sun-ower oil group have normal bile acid secretion in response

o food but that this is coupled with a supranormal choles-erol secretion rate and persistence of cholesterol supersat-ration of bile. In the rat, a species lacking the gallbladder,iets rich in �-6 polyunsaturated fatty acids versus saturatedats have been shown to increase the secretion of cholesterolnto bile [25,26]. Biliary cholesterol is derived principallyrom circulating lipoproteins [1,27], mainly high-densityipoprotein [1,28,29]. This particle is captured by the he-atic scavenger receptor class B type I [30,31]. Interest-ngly, diets rich in �-6 polyunsaturated fatty acids upregu-ate hepatic expression of scavenger receptor class B type I32] and enhance binding of high-density lipoprotein toiver membranes [33] compared with saturated and mono-nsaturated fats, respectively. This explains the loweringffect of plasma high-density lipoprotein cholesterol byietary �-6 polyunsaturated fatty acids, an effect not con-istently shared by monounsaturated fatty acids [34,35].ccording to this metabolic hypothesis, our results in the

unflower oil group may have been the result of increasedransfer of cholesterol back to the liver and subsequentlyore secretion into bile. It remains unknown why choles-

erol hypersecretion occurred only postprandially. It seemslear that the nature of the delivery and route of transit ofholesterol from the blood through the liver into bile need toe clarified.

Duodenal sampling in the present work was conducted8 h after surgery, so we should caution readers that theesults of the postcholecystectomy study might reflect ab-ormal postoperative changes. However, it is more likelyhat the differences found in biliary lipid composition afterhe test meals can be explained by changes in dietary fatntake. There are two main reasons for this. First, cholecys-ectomy was performed by laparoscopic means. This pro-edure is usually accompanied by a very short recovery of

astrointestinal motility [36,37]. Before the experiments,

e confirmed in all patients by clinical observations thatostoperative atony was non-existent. Second, our patientsolerated to a high degree not only the test meals but also theest of the food supplied (once each experiment was com-leted) to suit their daily nutritional requirements.

In conclusion, the present results indicate that type ofietary fat (olive oil versus sunflower oil) affects bile com-osition in humans with established cholelithiasis. Althoughanipulation of dietary fat ingestion did not influence the

holesterol saturation or the profile of individual bile acidsn gallbladder bile, there were marked differences in termsf absolute concentrations of lipids and total lipid content.fter cholecystectomy, cholesterol saturation of hepatic bileecreased typically in response to a test meal only in pa-ients given the olive oil diet and remained unchanged, i.e.,upersaturated, in those given sunflower oil. Further re-earch is required to define the precise mechanisms of theeported effects and to confirm whether a similar behaviorccurs in healthy human subjects.

cknowledgments

The authors are grateful to the Spanish Ministry of Sci-nce and Technology for the research grant and to theniversity of Granada and Junta de Andalucía for personal

upport of M. A. Martinez and M. D. Yago.

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