Effects of dietary cholesterol on cholesterol and
bile acid homeostasis in patients with
cholesterol gallstones.
F Kern Jr
J Clin Invest.
1994;
93(3)
:1186-1194.
https://doi.org/10.1172/JCI117072
.
We examined changes in cholesterol and bile acid metabolism produced by dietary
cholesterol in gallstone subjects and matched controls. Healthy women were recruited and,
after confirming the presence or absence of radiolucent gallstones, they were studied on
regular diets and again on the same diet supplemented with five eggs daily for 15-18 d.
Studies included plasma lipids, lipoproteins and apolipoproteins, dietary records,
cholesterol absorption, cholesterol synthesis, plasma clearance of chylomicron remnants,
biliary lipid composition, and secretion and bile acid kinetics. On low cholesterol, gallstone
subjects absorbed a slightly lower fraction of dietary cholesterol, synthesized more
cholesterol, and had smaller bile acid pools and faster fractional turnover rate (FTR) of bile
acids. On high cholesterol, the fraction of cholesterol absorbed decreased in both groups
and cholesterol synthesis decreased, especially in the gallstone group. Biliary cholesterol
secretion increased in the gallstone group only. FTR of bile acids did not change in either
group. Bile acid synthesis and pool tended to increase (P = NS) in the controls, but in
gallstone subjects, synthesis and pool size decreased. We concluded that in gallstone
subjects cholesterol and bile acid homeostasis is significantly altered, and that increasing
dietary cholesterol increases biliary cholesterol secretion and decreases bile acid synthesis
and pool, changes associated with cholesterol gallstone formation.
Research Article
Find the latest version:
Effects
of Dietary
Cholesterol
onCholesterol and Bile Acid
Homeostasis
in
Patients with Cholesterol Gallstones
FredKern,Jr.
Divisionof Gastroenterology and Hepatobiliary Research Center, University of Colorado School ofMedicine, Denver, Colorado 80262
Abstract
We examined changes in cholesterol and bile acid metabolism produced by dietary cholesterol in gallstone subjects and matched controls. Healthy women were recruited and, after
confirming thepresenceorabsence of radiolucentgallstones,
they werestudied onregulardiets andagainonthesamediet
supplementedwith fiveeggsdailyfor 15-18 d. Studies included
plasma lipids, lipoproteins and apolipoproteins, dietary
rec-ords, cholesterol absorption, cholesterol synthesis, plasma clearance ofchylomicronremnants, biliary lipid composition,
and secretion and bile acid kinetics. On lowcholesterol,
gall-stonesubjectsabsorbedaslightlylower fraction ofdietary
cho-lesterol, synthesized more cholesterol, and had smaller bile acid pools and faster fractional turnover rate (FTR) of bile
acids. Onhigh cholesterol,the fraction of cholesterol absorbed decreased in bothgroupsandcholesterolsynthesis decreased,
especiallyinthegallstone group.Biliarycholesterol secretion
increasedinthegallstonegrouponly. FrRof bile acidsdid not
change ineithergroup.Bile acidsynthesisandpooltendedto
increase (P= NS)inthe controls, but in gallstone subjects,
synthesisandpool size decreased. We concluded that in
gall-stonesubjectscholesterol and bile acid homeostasisis signifi-cantly altered,and thatincreasingdietarycholesterol increases
biliarycholesterol secretion and decreases bile acidsynthesis
andpool, changesassociated with cholesterolgallstone
forma-tion.(J. Clin.Invest.1994.93:1186-1194.) Keywords: choles-terol metabolism - cholesterol, dietary * cholelithiasis * bile acids and salts-lipids, biliarysecretion
Introduction
Cholesterol gallstones occur predominantly in Western cul-turesandaregenerallybelievedtobe relatedtodiet, especially
excesscholesterol,but theeffect ofdietarycholesterolon
choles-terol in the bile has not been clearly established. Published
studies, each involving only asmall number ofsubjects, are
contradictory (1-5), showingthatdietarycholesterol either
in-creasesordoesnotaffect cholesterol saturation ofgallbladder
bile. The rateofbiliarysecretion of cholesterol has notbeen described inmostof these reports. Inarecentstudywefound
AddresscorrespondencetoDr. F.Kern,Jr., Division of Gastroenterol-ogyand Hepatobiliary Research, University ofColorado School of
Medicine,Denver, CO 80262.
Receivedfor publication4May1993and inrevisedform 27 Sep-tember 1993.
that increasing dietary intake of cholesterol - 1,600 pmol
(610 mg)perday increasedthesecretion of allbiliary lipids-bileacid, phospholipid, and cholesterol-but didnotalter the cholesterol saturation index (6). The homeostatic and regula-tory responses todietary cholesterol, including secretion of cho-lesterolinto bile,have beenextensively studied inrecent years, but suchstudies have not beenpublishedinpatientswith
gall-stones. The homeostatic andregulatory responses, including cholesterolabsorption,cholesterolsynthesis,cholesterol esteri-fication, biliaryexcretion ofcholesterol,LDL receptoractivity, andbiosynthesis of bile acids,arecomplexandare knownto
vary considerably amongindividuals(7-15). In recent years noninvasivetechniques, notrequiringlengthyhospitalization for the accurate measurement of these processes, have been developed.
The purposeof thisstudy was to test thehypothesisthat the metabolicresponses todietarycholesterol aredifferentin sub-jects withandwithout gallstones,andthat,whendietary choles-terolis increased, biliary secretion ofcholesterol isgreater in thosewith gallstones.Thehypothesiswasconfirmed,and other important metabolic differencesbetweengallstoneand control subjectswere identified. These includea 44% greater rate of sterol synthesis by freshly isolated peripheral mononuclear cellsduringintake of theregulardiet andadecrease in bile acid synthesisandpoolin responsetoenhanceddietarycholesterol intake ingallstonesubjects. Bile acid kinetics did notchange significantlyincontrol
subjects
fedthesamediet.Methods
Subjects.This studywasapproved by the HumanSubject Committee of the University of Colorado School of Medicine. Allsubjectswere
paidvolunteers and gave written informed consent.Eightwomenwith asymptomatic radiolucent gallstones and eightwomencontrolsubjects without gallstones werestudied. In so far as possible, control subjects were matched to gallstone subjects for age, weight, and body mass index(BMI)'-weight/height squared (Table I). Themean(±SD) age ofthecontrol andgallstonesubjects was 34.8±3.1 and 46.9±3.4 yr, respectively (P<0.05) (TableI).The meanweightof the controls was 72.7±4.1 kgandgallstonesubjects, 71.0±4.5. Four subjects in each groupwereobese;i.e., their weightwas >120%of ideal body weight ( 16).The mean BMI was28.0±7.1kg/mi2in controls and 27.7±1.6 in gallstonesubjects.
Thesubjectswerehealthy and were not taking medications. None had had symptoms ofbiliarytractdisease for at least 3 mo. Appropriate laboratory studieswereperformedtoexcludehematologic,liver, bili-ary tract, andmetabolic disease.Most hadnormal plasmalipids and lipoproteins, including plasma total, LDL and HDL cholesterol, and triglyceride. Two women ineach group had total plasma cholesterol levels above5.18
,M
(200 mg/dl). Plasma apolipoproteins A-I and B1.Abbreviations usedinthispaper:BMI, bodymassindex; CSI, choles-terolsaturationindex; FTR,fractionalturnover rate.
J.Clin. Invest.
C The AmericanSocietyforClinicalInvestigation,Inc.
0021-9738/94/03/1186/09 $2.00
TableL Characteristics ofStudy Subjects
CONTROL (n=8) GALLSTONE (n=8) Characteristic Mean±SEM Range Mean±SEM Range P
Age 34.8±3.1 27-48 46.9±3.4 32-60 <0.05 Weight(kg) 72.7±4.1 59-93 71.0±4.5 49-95 NS
Idealweight (%) 123.7±7.6 94-157 121.5±6.4 95-156 NS BMI(kg/ht2) 28.0±2.1 20.7-36.5 27.7±1.6 22.8-34.9 NS
were also normal. Experimental subjects had gallstones identifiedon
ultrasound examination and shown by oral cholecystographyto be radiolucent and noncalcified. Oral cholecystograms and/orultrasound
failed to show stones in the controlsubjects.
Study protocol. Each subjectwasstudiedonherregular diet and again after 15-18 donthesamedietsupplemented with five eggsaday. Plasmalipids and apolipoproteins A-IandB, measured by standard clinical laboratorytechniques,weredetermined three times before be-ginning the control period studies and againatthecompletion of each studyperiod. The followingweremeasuredattheendof eachdietary period: cholesterolabsorption,cholesterolsynthesisandesterification,
plasma clearance ofretinyl palmitate, bile acid kinetics, composition of duodenalbileafter gallbladder stimulation, and biliary lipid secretion.
Diet.TheClinical Research Center dietician took a careful dietary history and instructed the subjects in the maintenance of a diary of daily food intake ( 17). The diet and diarywerereviewed periodically by thedietician, who calculated with the aid of a computer program (Food ProcessorII,ESHA Research,Salem, OR) the mean daily intake ofcalories, cholesterol, saturated,mono-andpolyunsaturated fat, and other nutrients. The additional five eggswerepreparedand eatenin anymannerpreferred by the subjects. Since the reported cholesterol
contentof eggs varies, wemeasured cholesterol inlocally purchased large eggs. Thecholesterol was extracted from the yolks, hydrolyzed, and derivatized, and theconcentration was measuredby gel liquid chromatography. Themean was604.9±34.2 Amol(233.5±13.2 mg) peregg.
Procedures. All procedures have been in use in our laboratory for a numberof yearsandhave beendescribed (6, 18). Cholesterol absorp-tionwasmeasuredusingtheisotope ratio method ( 19) which required thesimultaneous oraladministration of2 gCiof[4-'4C]cholesterol
and theintravenous administration of2gCi[1 ,2-3H]cholesterol. After 48 and 72 h, samples of plasma weredrawn, the isotope ratio was
determined,and the percent cholesterol absorbed was calculated. Cho-lesterol synthesis wasestimated from the incorporation of [2-
'4C]-acetate(New England Nuclear, Boston, MA) into sterols by freshly isolatedperipheral mononuclear cells (20). Plasma clearance ofretinyl palmitatewasmeasuredaspreviously described (6, 18). Retinyl
palmi-tate wasgiven by mouth and plasma concentration of retinyl palmitate wasmeasured at intervals for 24 h. The concentration was plotted againsttime, andtheareaunder thecurve wastakenas thehepatic clearance ofchylomicronremnants(6, 18, 21, 22).
Biliarylipid composition and secretionratesweremeasuredby
es-tablished procedures and methods, described in detail byusand others (23, 24). After insertion ofanasoduodenaltube,asample ofduodenal content,"hepatic bile,"wasobtained andgallbladdercontractionwas
stimulated byanintravenousinfusion oftheoctapeptide ofcholecysto-kinin (CCK;Squibb CCK-8, 0.02,g/kgperh)and asample of
con-centrated bile"gallbladder bile"wascollected. Bile aciddistribution was measured by gel liquid chromatography andthe concentration of bileacid, phospholipids,andcholesterolwasdetermined bypreviously
described methods.Thecholesterol saturation of eachsamplewas cal-culated. A liquid formula testmeal, containing [14C]PEG 4000 as
nonabsorbablemarker,was theninfusedintraduodenally for 8h.
Sam-pleswerecollectedfromtheduodenumcontinuallyandpooledat 30-min intervals. Theconcentration of bileacid, phospholipid,
choles-terol, and`4Cradioactivity was determined in all samples bydescribed methods ( 18, 21 ) and the secretion rates of each lipid was calculated. Bileacid kinetics were measured using stable isotopes and gas chro-matography/massspectrometry analysis of serum bile acids (25). 13-cholic and13-chenodeoxycholic acids (Merck, Sharp&Dohme, Mon-treal) were given orally, and blood samples were obtained daily at the sametime for 4-5 d. Bile acids were extracted and derivatized, and molar ratios of labeled to unlabeled bile acids in each sample were determined by selected ion monitoring. The fractional turnover rate (FTR),pool sizes, and synthesis rates of the primary bile acid were calculated (25). Thedeoxycholate pool size was determined by com-paring the intensity of its ion to the intensity of the chenodeoxycholate ion (6).
Statistical methods.Results are reported as the means±SEM. Dif-ferencesin means between control and gallstone subjects and in the percent change from low to high cholesterol diet were analyzed by Student'sttest.Differences withingroupsonandoff the
high-choles-terol diet were analyzed by paired t tests. The relationships between variables were analyzed by linear regression.
Results
Differences
between the controls and gallstone subjects
Dietaryintake. Thedietary intake of cholesterol, calories, and fat, and the ratio of polyunsaturated to saturated fatty acids and other nutrients (not shown) were similarinthe twogroups in both study periods (Table II). Cholesterol intake in the
con-trol subjectswas 585±85 ,umol (226±33 mg) on the regular diet, compared with 548±91
Amol
(211±35 mg) in the gall-stone group (P = NS). On the high-cholesterol diet, it was2,978±40
,mol
(1,150±16 mg) in the controls and 3,010±50 ,umol (1,162±19 mg) in the gallstone group (P= NS).Plasma lipids. Lipids and apolipoproteins were similarin the two groups of subjects (TableIII).
Cholesterol absorption.On the low-cholesteroldietthe per-centcholesterol absorption tended to be less in the gallstone subjects (52.7±1.8% vs. 60.4±3.9% in the controls; P=0.08); on thehigh-cholesterol intake, mean absorption was similar in the two groupsof subjects (43.1±1.4 vs. 49.0±4.3). The abso-lute amountof cholesterol absorbed (dietary cholesterol multi-plied by percent absorbed) was 20% less in the gallstone sub-jects on the control diet (282.5±44 vs.
353.5+55,
P< 0.05) and 11%Iless(P=NS) on the high-cholesterol diet. Fig. 1shows the changes infraction absorbed for individual subjects produced by the high-cholesterol diet.TableII. MeanDailyDietary Intake
Calories Cholesterol Fat P/S*
'smot g
Controlsubjects
Low-CH diet 1716±145 585±85 68±7 0.61±0.05
High-CH diet 1568±96 2978±41 76±5 0.59±0.04
P NS < 0.0001 NS NS
Gallstonesubjects
Low-CH diet 1425±168 548±92 57±11 0.68±0.09
High-CH diet 1343±71 3010±50 66±5 0.65±0.08
P NS <0.0001 NS NS
Values aregiven asmean±SEM. Noneofthedifferencesbetween
Table III. Plasma Lipids
Cholesterol Apolipoproteins
Total LDL HDL TGL A-I B
mM mg/dl mg/dl
Controlsubjects
Low-CH diet 4.38±0.24 2.78±0.21 1.08±0.09 103.8±15.3 133.7±9.0 83.1±8.5 High-CHdiet 4.52±0.25 2.89±0.20 1.13±0.08 96.1±13.7 140.0±11.4 80.0±7.5
P NS NS NS 0.07 NS NS
Gallstonesubjects
Low-CH diet 5.03±0.30 3.22±0.22 1.33±0.16 97.0±13.7 155.4±12.8 80.5±4.9 High-CH diet 4.81±0.37 3.05±0.22 1.28±0.11 93.6±13.0 154.7±14.4 80.1±5.5
P NS NS NS 0.05 NS NS
Values aregiven as mean±SEM. Neither thedifferences between mean valuesfor control and gallstonesubjectsnorthe differences between percentchange isstatistically significant.Abbreviations: CH, cholesterol;TGL,triglycerides.
Cholesterolsynthesis. The rateofcholesterolsynthesiswas consistently higher in the gallstone subjects on both diets (Fig. 2). On the low-cholesteroldiet itwas73.4±2.7 pmol/ I07 cells per h in thegallstonegroup and 47.6±2.6in thecontrols(P
<0.001),and onhighcholesterol it wasrespectively58.3±3.5 vs. 44.3±2.4 (P < 0.03).Chylomicronremnant clearance was the same in bothgroups on both diets.
Biliary lipids. On the low-cholesterol diet, the cholesterol saturation index (CSI)and the secretionrateofbiliary lipids were the same in the two groups (Table IV). The molar percent cholesterol in gallbladderbile was slightly higherinthe gall-stone subjectsand the rate ofbiliary secretion of cholesterol was slightlyhigherin the control group, butneitherdifference wassignificant. On thehighcholesterolintake,the molar per-cent cholesterol was 42% higher in the gallstone group (7.92±1.38 vs. 5.58±0.41; P=0.08), but the secretion rate of cholesteroldidnotdiffersignificantlybetween the groups. Bile acid andphospholipidsecretion rates as well as the CSI of he-paticandgallbladder bileweresimilar. The mean distribution of bile acids ingallbladderbile did not differ between groups.
Bile acid kinetics. Bile acid kinetics differed in the two groups(TableV). FTRof bothbile acidsweregreaterinthe
gallstone subjects on both diets. The pools of both bile acids were smallerinthosewithgallstones onbothdiets, especially thehigh cholesterol diet; on the high cholesteroldiet(P=0.08
forchenodeoxycholic acid,<0.025for cholicacid,<0.05for deoxycholic acid, and<0.02 for total bileacid). The synthesis rates ofeach primary bile acid was slightly, but not signifi-cantly,smaller in the gallstone group on both diets. The total bile acidsynthesiswas 42%smaller inthiegallstone subjectson
thehigh cholesterol diet (P<0.07).
Responses to
the
high-cholesterol diet
Plasma lipids. No plasma lipid or apolipoproteinwas altered by 15-18 dofthehigh-cholesteroldiet except theplasma tri-glyceride, which decreasedslightlyonthehigh-cholesteroldiet in both groups(Table II). Neither total norLDLcholesterol increased.
Cholesterol absorption
andsynthesis.
The percent choles-terol absorption decreasedin almost every subjectwhen the cholesterol intake was increased(Fig. 1).The mean decreased from 60.1±3.9% to 49.0±4.3%(P<0.01) in the controls andCONTROL GALLSTONE
CONTROL
100
75
0 a.
a-0 CL
50
25
0
P<o.005
GALLSTONE
100
75
50
25
0
Low High
Chol Chol
100
p<0.001
0
0
0 E a.
75
50
25
0
Low High
Chol Chol
Figure1.Fraction of cholesterol absorbed in control and gallstone subjectsonboth diets. The high-cholesterol diet caused a highly sig-nificant decrease in both groups. Each point represents a single subject.
NS
100
75
0
50
25
0 Low High
Chol Chol
p<0.001
Low High
Chol Chol
Figure2.Cholesterol synthesis measured by incorporation of labeled
TableIV.Biliary Lipids
CSI Secretion
Hep GB BA PL CH BA PL CH
M% gmol/kg perh
Control subjects
Low-CHdiet 1.563 1.158 77.60 16.67 5.54 26.86 5.60 1.88
(0.248) (0.060) (1.08) (0.82) (0.35) (5.64) (0.90) (0.26)
High-CH diet 1.601 1.178 75.08 19.09 5.58 25.70 5.67 1.85
(0.175) (0.067) (1.78) (1.16) (0.41) (5.28) (0.66) (0.22)
P NS NS NS NS NS NS NS NS
Gallstonesubjects
Low-CH diet 2.000 1.180 75.47 17.61 6.94 19.57 4.24 1.60
(0.445) (0.093) (1.71) (1.07) (0.82) (3.24) (0.42) (.18) High-CH diet 1.427 1.237 73.46 18.60 8.12* 20.73 5.27 2.01
(0.335) (0.478) (0.55) (0.68) (1.25) (2.08) (0.95) (0.39)
P NS NS NS NS 0.08 NS NS < 0.05
Values are given as mean±SEM. Abbreviations: BA, bile acid; CH, cholesterol; GB, gallbladder; Hep, hepatic; PL, phospholipid. * Differs from molar percent cholesterol in control group on high-cholesterol diet 0.05 < P < 0.08. The difference in percent change in molar percent cholesterol between the controls (< 1.0%) and the gallstones subjects (25%) was not quite significant. There are no other statistically significant differences between control and gallstone subjects.
from 52.7±1.8% to 43.1±1.3% (P < 0.001) in the gallstone patients. The rate of sterol synthesis (Fig. 2) did not change significantlyin the controls (47.3 pmol/ I07 cells per h on the low diet, 43.2 on the high diet), but in the gallstone group synthesis decreased from 73.4±3.0 to 58.3±3.9 (P< 0.001). Thedifferencebetween the groupsinpercent changein absorp-tion and synthesis was not significant. The rate ofsynthesis decreasedastheamountofcholesterol absorbedincreased in thegallstonegrouponly (r=0.72,P<0.05) (datanotshown). Retinyl palmitateclearance rate was unaffectedbydiet in ei-ther group.
Biliary lipids. The high cholesterol diet didnotsignificantly influence biliary lipidcompositionorsecretion in control sub-jects (Fig.3 and TableIV).Inthegallstonesubjects, however,
thedietcausedanincreaseintherateofcholesterol secretion in mostpatients(1.60±0.18
,umol/h
on the lowdiet, 2.01±0.39on the high,P< 0.05). Therewas also anincrease in mean
molar percent cholesterol (from 6.84±0.82 to 7.92±1.33, P <0.08),inthesamegroup, but theCSIof gallbladderbiledid notchangesignificantly(1.1 vs. 1.27). Therewere no
signifi-cantdifferences inrateof secretion of bile acidorphospholipid ineithergroup(Fig.4).
Bile acid kinetics
Dietarycholesterolaffectedbile acidkinetics quite differently inthe two groups(Fig.4and Table V). In the controls, there was atrend toward an increase in bile acid synthesis and pool size. Cholic acidand totalbile acidsynthesisincreased in sixof
TableV. Bile AcidKinetics
Chenodeoxycholicacid Cholic acid Total bile acid
Deoxycholic
FTR Pool Synthesis FTR Pool Synthesis acid pool Pool Synthesis
days-' jm/kg gm/kgper d days-' um/kg um/kgper d jim/kg jm/kg jim/kgper d
Controlsubjects
Low-CH diet 0.157±0.014 20.58±2.36 3.24±0.23 0.165±0.028 33.22±8.76 5.71±0.90 13.50±2.21 72.38±6.44 8.95±0.84
High-CHdiet 0.168±0.013 24.52±3.57 3.58±0.30 0.189±0.022 35.16±5.85 6.50±0.52 18.03±1.86 74.25±9.78 10.07±0.50
Percentchange 11.6±9.3 8.1±10.3 20.5±10.3 30.8+20.7 8.1±6.5 15.8±11.5 52.5±36.1 7.9±8.9 16.7±10.7
P NS NS NS NS NS NS NS NS NS
Gallstone
Low-CH diet 0.275±0.050 14.33±1.94 3.48±0.31 0.309±0.063 21.50±2.88 5.91±0.61 9.37±1.49 44.87±5.06 9.25±0.82 High-CH diet 0.284±0.072 13.23±2.44 2.91±0.33 0.321±0.068 18.21±3.53 4.71+0.85 8.11+1.36 39.55±6.16 7.62±0.94
Percentchange 0.2±5.0 -9.3±8.9 -11.9±6.8 2.6±6.5 -20.3±9.0 -21.0±7.5 -10.8±13.1 -14.7±8.1 -17.3±6.9
P NS NS NS NS 0.05 <0.05 NS 0.05 <0.05
Controlvs.gallstone
(Pvalue)
Low-CH diet <.05 0.08 NS <.05 NS NS NS NS NS
High-CH diet 0.08 0.08 NS 0.08 <0.025 NS <0.05 <0.02 0.07
Percentchange* NS NS <0.05 NS <0.05 <0.05 NS NS <0.05
GALLSTONE
NS
12
10
0
E 8
4
0
Low High Chol Chol
CA SYN
p<O.05
15
10*
5'
0o
Low High
Choi Chol
TOTAL
BA SYNp<O.05
Low High
Chol Chol
12'
0
E
8
4
0
4'
2
0
E
0
NS
12
8
4.
0 Low High
Choi Chol
NS
4
2
0
NS
40
0
0 E
Low High Chol Chol
p<O.05
Low High Low High Chol Chol Chol Chol
Figure 3. Biliary secretionratesof bileacid (top),phospholipid (middle),andcholesterol (bottom)inbothgroupsonboth diets.
Therewas aslight, but significant, increase intherateofcholesterol secretioninthegallstone subjectsonthehigh-cholesteroldiet. No othersignificant changeswerefound.Thereisawidespread ofall datapoints.
30
20
10
0o
CA
POOL
p<0.05
TOTAL BA
POOL
80
60s
40
20
0
Low High
Choi Choi
p<0.05
Low High Chol Chol
Figure 4. Top: Cholic acid(CA) and total bile acid (BA)synthesisin
thegallstone subjectsonthelow-andhigh-cholesteroldiets.Synthesis decreasedinallsubjects butone.Eachpoint isanindividualsubject. Bottom:Cholic andtotal bile acidpool also decreasedonthe high-cholesterol diet.
the controls differed significantly from the slightdecrease in
bile acidsynthesisinthegallstonegroup(P<0.05).
Themajor differencesincholesterol and bile acid
metabo-lismcaused by theadded dietarycholesterolinthetwogroups are summarized in Fig. 5. Cholesterol absorption efficiency
decreased 18% in eachgroup.Cholesterolsynthesis decreased
21 % ingallstonesubjects andonly7% incontrols. Molar
per-cent cholesterol in bile and biliary cholesterol secretion in-creased 17% and26%,respectively,inthegallstonegroupand decreasedslightlyincontrols.Totalbileacidsynthesis and pool
decreased 17%and 15%,respectively,ingallstone subjects and increased 17% and 8%, respectively, incontrols.
theseeight subjects,but thechangeswerenotstatistically
signifi-cant. Inthegallstone subjects (Fig. 4),however,thecholic acid
pool decreased from 21.5+2.88
gmol/kg
to 18.21±3.53 (P< 0.05), and its synthesis rate decreased from 5.91±0.61
,gmol/kg
per dto4.71±0.85, P< 0.05). Therewere similarsignificantdecreasesintotal bileacidsynthesisandpool.There
were noconsistentorsignificant changesinchenodeoxycholic acidpoolorsynthesis,indeoxycholicacidpoolorinthe FTR
ofeither bile acid. Theslightincrease inbileacid synthesisin
Discussion
Although the pathogenesisofcholesterol gallstones is complex
and involvesanumber ofrecognizedsteps,the first, the hepatic
secretionof bile thatcontainsexcesscholesterol, isanecessary conditionforgallstone formation. Known risk factors for the
secretion ofexcess cholesterol into bile include age, obesity, femalegender,andfemalesexhormoneadministration(26). Inaddition, factorsthatinclude total calories and cholesterol
CONTROL
60 NS 60I
0 0
0
E
=-40
20
40
20
0o
Low High Chol Chol
0
percent
30
-20
10
-
0--10
20
--30
-p<0.05 p=0.06
p=0.08 p=0.07
Chol Chol Mol% Chol Tot Tot
Abs Syn Chol Sec BA BA
Eff Syn Pool
Figure 5.Thisbargraphsummarizesthemajorchangesinducedby
thehigh-cholesterol diet in bothgroupsof subjects. Controlsare
shown inopenbars and gallstone subjects in black bars.In the gall-stonesubjects, dietary cholesterol produceda greaterdecreasein
cho-lesterolsynthesis,anincrease inmolar percent cholesterolin bile,an
increase in cholesterolsecretedinbile,anddecreases incholicacid andtotalbile acid synthesis.
mayberiskfactors contributingtothehigh prevalence of cho-lesterol gallstones in the Western world (27). Epidemiologic studies support this belief, but direct studies oftheeffect of dietary cholesterol onbiliary lipid compositionand secretion andonbile acid metabolismarelimited.Table VI,which sum-marizesthe reportedstudies of dietary cholesterol, shows that biliary cholesterol saturation increased in three and did not changeintwoof five studiesinwhichsubjectswerefed high-cholesteroldiets.
The goalofthisstudy was togaininsight intothe pathogene-sis of cholesterol gallstones bystudying thehomeostaticand regulatory responsestriggeredbydietarycholesterol in groups ofsubjects with and without radiolucent gallstones. Using methods developed in the past 15 yr, westudiedmostofthese responses, except LDL receptor activity, in nonhospitalized, healthy individuals. The homeostatic responses to increased dietary cholesterol includedecreaseinintestinal absorption of cholesterol, inhibition ofcholesterol synthesis, down-regula-tion ofLDLreceptoruptake, increaseincholesterol
esterifica-tion, increase in biliary secretion ofcholesterol, and an increase in hepatic conversion of cholesterol to bile acids (8, 12, 27-29).These responses varygreatlyamong animalspecies and in
manthere is marked individual heterogeneity in each response. Itseemsreasonabletohypothesizethatindividuals with choles-terolgallstones who eat a high cholesterol diet mightsecrete
into the bilemorecholesterol than those withoutstones.
All ofthe subjects were women simply because we were
unabletoattractmalevolunteers. Nonewastakingfemalesex
hormonesorother medications known to affecthepatobiliary function or lipid metabolism. They were well matched for weight and body mass index, variables thought to influence cholesterol and bile acid metabolism. The controls were
younger than thegallstonegroup(Table I). There were, how-ever, nosignificant correlations in either group of subjects (or the groups combined) between age and any measured variable including cholesterol absorption and synthesis, biliary lipid composition and secretion, CSI, bile acid synthesis, pool size, andturnover.These results differ from those reported by Val-diviesoetal. (30) and by Einarsson et al. (31 ), perhaps because thenumber of our subjects is small and the range in age was limited, possibly notgreat enough to allow the detection of differences.
We found major metabolic differences between subjects with radiolucent gallstones and controls. First, those with gall-stones synthesized more cholesterol than controls and even
when synthesis was inhibited by feeding cholesterol, it re-mained higher in gallstone subjects. Second, in response to
increased dietary cholesterol, gallstonesubjects, but not con-trols, secretedmorecholesterol into bile.Third,onthe regular or low-cholesterol diet, therateof bile acidsynthesis did not differ between groups, but in response to dietary cholesterol bile acid synthesis and pool decreased inwomenwith gallstone. Bileacidpool size was smaller and FTR of bothprimarybile acids was faster in gallstone subjectsonboth diets.
Biliary lipids did not differ clearly between controls and gallstone subjects on either diet. The molar percent cholesterol in stimulated (gallbladder) bile was slightly higher in the
gall-stone group on the high cholesterol intake (7.9 vs. 5.6%, P
= 0.08, Table IV), but there were no other differences that
approached statistical significance.
A number ofinvestigators have called attentiontothe fre-quent similarity of biliary lipids in normal and gallstone pa-tients, emphasizingthe importanceoffactors other than bile lithogenicityin gallstoneformation (32). Althoughtherateof biliarycholesterol secretion isusuallygreater inpatientswith
Table VI.
Effects
ofDietaryCholesterolonBiliary CholesterolSubjects Cholesterol indiet Biliarycholesterol Authors n Status Low High Saturation Secretion
mg
Dam et al.(1) 9 normal 500 1500-2500 nochange
DenBestenetal.(2) 10 normal 0 750 increase
GrundyandMok(3) 2 obese 0 3000 increase increase
Anderson andHellstrom(4) 12 6normal6HLP 300 1500 nochange
Leeetal.(5) 19 7normal 12gallstone 500 1000 increase
gallstones, it wasnot in this small group of subjects. Biliary cholesterol secretion did increase, however, in gallstone sub-jects on thehigh cholesterol diet,but not in the controls. In fact,there were noeffects ofthe high cholesteroldietonbiliary lipids in the control subjects as noted in Table IV. Increases in dietary cholesterol of 500-750 mg/d causedanincreaseinCSI in twostudiesof normal subjects (3, 5), but in two other stud-ies increasesof1,000-2,000 mg/d had no effect ( 1, 4). In our earlierstudy(6),anincrease of 800 mg/d had no effect on CSI. Thediet-inducedincreaseinbiliarycholesterolsecretionin the gallstone subjects, butnotin thecontrols, suggests that dietary cholesterol might be important in the pathogenesis of choles-terolgallstonesand supports thehypothesis that hepatic metab-olism of cholesterol in gallstone patients differs from those without stones.
Although thefactors that regulate biliarycholesterol
secre-tionare notknown forcertain, anumber of studies have sug-gestedthatmostbiliarycholesterol is derived frompreformed rather than newlysynthesized cholesterol (33-39). Our finding of virtuallyidentical specific activities ofbiliaryand plasma cholesterol is consistent with that conclusion. Robins et al. (38)suggestedthattheprecursor cholesterol forbiliary
secre-tion is transported directly fromplasma through the plasma membraneofthe livercell to thebile canaliculus without
enter-ingtheinteriorofthe cell. On the otherhand,Nervietal.(39) foundareciprocal relationshipbetweensecretion ofcholesterol in VLDL particles and in the bile, suggesting regulation by intracellular mechanisms. These, and otherinvestigators, be-lieve that thesize of metabolic "pools"orcompartments deter-minesthefate ofhepatic cholesterol.
Meancholesterol
synthesis
was44% greater in thegallstone subjects, with little overlapbetween groups(P<0.001)(Fig. 2). As notedearlier (6, 18), themethod usedto measure choles-terolsynthesis,
theincorporation
of labeledacetateinto sterols byperipheralbloodmononuclearcells, isavalid reflection of therelative rateofhepatic
cholesterolsynthesis
under allcir-cumstancesstudied, which include
starvation,
cholesterol feed-ing, cholestyramineandlovastatinadministration,
type II fa-milialhypercholesterolemia,etc.Therateofcholesterol synthe-sis isknowntoincrease withbodymassindex,
asitdid inthe gallstonesubjects
inthis study, buttherewas nodifferenceinBMIorbodyweightbetween the groups. Itcanbeconcluded, thereforethat the cholesterolsynthesisrateisgreater in individ-ualswith radiolucent gallstone. Although cholesterol synthesis
wasdiminished by the high cholesterol diet in both groups, it remained greater in the gallstone group, suggesting a basic dif-ference in the regulation of cholesterol metabolism.
Theactivity ofhepatic 3-hydroxy-3-methylglutaryl CoA re-ductase, therate-limitingenzyme in cholesterolsynthesis, has beenstudiedingallstoneandcontrolpatients by six laborato-riessince 1975 (40-47) (Table VII). Although all usedliver tissue obtainedatsurgery andessentially thesameassay tech-niques, the reported results varied: four groups of investigators reportedsignificantly greateractivityin subjectswith choles-terolgallstones thanin thosewithoutstones(40,41, 43, 44); onegroup found no change (42); the sixthgroup, in three studies in threegroups of patients, found no change in two
studies (45, 47) and a significant decrease in activity in the third study (46).The reasonfor the differentresultsfrom this laboratoryisnotclear. Nervietal. (48) measured cholestero-genesis fromlabeled acetate byliver slices obtainedatsurgery in normalweightgallstone and controlsubjects.They found highercholesterolsynthesis inboth maleandfemale gallstone subjects,buttheirgroupsweresmall, sixin each group, and the intragroup differenceswerewide (sixfold)and notstatistically significant. Thus, most ofthe published studies supportthe conclusionthatcholesterolsynthesis is higherin gallstone pa-tientsthan incontrols.Although increased cholesterogenesis is consistent withtheobservationthathepatic cholesterol, espe-cially cholesterol ester, is increased ingallstone patients (40, 41),itsrole,ifany,inthepathogenesisofcholesterol gallstones isnot known.
3-Hydroxy-3-methylglutaryl CoAreductaseactivity inthe liverandothertissues, including peripheralblood monocytes, is regulated by cellular uptakeofcholesterol in chylomicron remnantsand otherlipoprotein fractions, especiallyLDL, and bytheconcentration of certain hydroxylated sterols, by-pro-ductsofcholesterol
synthesis,
which inhibitcholesterol synthe-sis (49). NeitherLDL receptoractivitynorhydroxysterol pro-ductionwasmeasured inthesesubjects,butplasmaLDL lev-els, a major determinant of LDL receptor activity, and remnant uptake,measured byretinylpalmitateclearance, were the same in gallstone and control subjects. Although theamountof cholesterol absorbedinthe gallstone subjectswas slightly less than in the controls on both diets (Fig. 1), the differenceappearstoosmallto accountfor suchalarge
differ-encein cholesterol synthesisonthebasisof diminished feed-back inhibition. This mechanism cannot be excluded,
how-TableVII.Hepatic3-Hydroxy-3-methylglutarylCoenzyme A Reductase Activity in Patients with Cholesterol Gallstones
Controls Gallstones Reductase*
Author Year n %IBW n %IBW Control GS P
Salenetal.(40) 1975 9 108 12 107 61 77 0.01
Coyneetal.(41) 1976 4 ? 6 ? 113 153 0.01
Carullietal.(42) 1980 17 89 25 102 58 63 NS
Maton et al.(43) 1980 10 92 6 124 14 32 0.001
Key et al. (44) 1980 6 105 12 121 113 220 0.01
Ahlbergetal.(45) 1981 10 1040 23 105t 25 21 NS
Angelinetal. (46) 1982 10 155t 18 100 61 38 0.05
Reihneretal.(47) 1991 13 95t 22 97t 109 104 NS
Abbreviations:GS, gallstones;IBW, ideal body weight. *Activity=pmol/mg microsomalprotein/min. *Relative body weight((kg/ht[cm]
ever,and should be explored in future studies. It is possible that thesynthesisofinhibitoryhydroxylatedsterols, such as 25- or 26-OH-cholesterol, a normal by-productof cholesterol synthe-sis, isless in thegallstone subjects.Little is known ofthe regula-tion of thesynthesis of these sterols, but recent studies suggest thatthey serveassubstratesforcholesterol 7-a hydroxylase, the rate limitingenzyme in bileacid synthesis (49, 50). In sum-marythemechanism underlying a more rapid rate of choles-terolsynthesisin gallstonepatients isnotknown.
Bile acid kinetics. On a regularor low-cholesterol diet the gallstonesubjectshad morerapidFTR (P<0.05) and smaller (P = NS) bile acid pools (Table V), as has been described repeatedly(2, 6)sinceVlahcevic and co-workersfirst reported it in 1971 (5 1 ). The causeofthesekinetic differences has been debated,butnotsatisfactorily explained.Thefinding ofsimilar rates of bile acid synthesis is consistent with earlier studies (5 1 ).Cholesterol 7-a hydroxylaseactivity in microsomes iso-latedfrom liverbiopsy tissuewasreported to be lower in
gall-stonepatientsthanin controls in somestudies (40,41, 43) but notin all (47). The effect of dietary cholesterol on bile acid kineticsin such patients has notpreviously been studied. In normalsubjects,andinrats, amodestincreaseindietary choles-terol has little or no effect on bile acid synthesis, as in our
controls, whereas a very large increase in cholesterol intake may increase synthesis of bileacids in both species(26, 52-54). Thedecreaseinsynthesis and pool in the gallstones sub-jectsingesting high cholesterolwasnotanticipated,but thereis
arationalbiologic explanation, proposedbelow.
Itis generally believedthat thefate of hepatic free choles-terolis largely determined by the size of various intracellular metabolic pools or functional compartments of free choles-terol. As noted above, cholesterol for biliary secretion origi-natespredominantly from preformed cholesterol. Onthe other hand,anumberof linesof evidencesuggestthat30-40% ofthe substratefor bile acid synthesis, regulated bytherate-limiting enzyme, cholesterol 7a-hydroxylase, is derived fromthe intra-hepaticsynthesis ofcholesterol.
The FTRofthebile acids didnotchangeonthehigh choles-terol dietand the synthesis rate decreased;thus thebile acid pool must
necessarily diminish,
according
toclassicalisotope
dilution kinetics which states that
synthesis
rate equals pool multiplied by FTR. (Therefore, pool =synthesis
. FTR.)When synthesis decreaseswithout achangein turnoverrate, pooldecreases,accounting forthedecreasedbile acidpool in gallstonepatients, especiallythosefedanincreasedamountof dietarycholesterol.
Insummary, themetabolicresponsesto
dietary
cholesterol inpatients withcholesterolgallstonesobserved in this study, theincreaseinbiliarycholesterolsecretionanddecreaseinbile acidsynthesis,
areinternally consistentandprovide
arational,
physiologically
soundbasis forthesecretion ofsupersaturated
bile that isa necessary
requisite
for gallstone formation. Fur-thermore, theysupport theepidemiologic
studieslinking
di-etary cholesteroltogallstonesandstrongly
support theconten-tionthat thereare major metabolic differencesin cholesterol metabolisminpatientswithgallstones.
Acknowledgments
The author thanksCarolMcKinley, R.N., for able assistancewith the
clinicalstudies,RadeneShowalter forexperttechnicalassistance,and Mary LuJones, R.D., for assistance withdietarycontrol.
This work wassupported by National Institutes of Healthgrant
ROI DK31765.Additionalsupportfor thesestudieswasprovidedby
the Hepatobiliary Research Center of the University of Colorado School of Medicine (National Institutes of Health grant 5P30 DK349 14) and the GeneralClinical Research CenterProgramof the DivisionofResearch Resources,National Institutesof Health (grant 5MRO1 RR00051).
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