The effects of pregnancy and female sex steroids on ...

THE EFFECTS OF PREGNÀNCY AND FEMALE SEX STEROIDS

ON GALLBLADDER EMPTYING BILTARY LIPID OUTPUT ÀND

SMALL BOÚ{EL TRANSIT TIME

by

MICHAEL J. LA!{SON. . MBBS, BSC (MED) FRÀCP

DEPÀRTMENT OF MEDICINE3, THE UNIVERSITY OF ÀDELAIDE

THESIS SUBMITTED TO THE UNIVERSITY OF

ÀDELAIDE FOR THE DEGREE OF DOCTOR OF MEDICINE

I

\Ó.'¡s'¡áeJ ñ cr*Ã rtl 1-l \arf 6 .

CONTENTS

PAGE

I

I

SUMMARY

STATEMENT

ACKNOVüLEDGEMENTS

CHAPTER I:

I.

I

I

A

B

C

D

E

. LITERATURE REVIE!'Ù

GaIl-stone Incidence

GaIIstone Cl-assif icat ion

Biliary LiPid Biochemistry in

Chol-esterol Gallstone Disease

The EnterohePatic Circulation

The Rol-e I qf the Gatlbladder in 24

Gal-lstone Format i'on

Female Sek Steroids and Smooth 25

Muscl-e MotilitY

Female Sex Steroids and BiIiarY 28

Lipid Secretion

Gastrointestinal- Peptides and 37

Gall-bl-adder Mot ilitY

Neuronal Determinants of 4L

ExtrahePatic BiJ-iarY MotiJ-itY

The RelationshiP of Gastric 44

Emptying to GaLl-bladder Emptying

II: CURRENT KNOWLEDGE AND UNANS!{ERED

QUESTIONS REGARDING GÀI,LBLADDER

MOTOR FUNCTION

Gall-bladder' Funct ion - Current 49

Concepts in PregnancY

I

5

6

8

9

9

L2

LB

I. F.

I G

I. H

I I

I. J

CHAPTER

II. À

II. B Unanswered Questions lìegarding

Gallbl-adder lrtrnct i-on

Thes i-s Aims

52

53II. C.

PAGE

CHAPTER III: METHODS ÀND RESULTS 55

Galtbtadder Function in the Human 57

Female: Effects of the OvulatorY :

Cycle, Pregnancy and Contraceptive

Steroids

Gallbladder and Small Intestinal 74

Regulation of Biliary Lipid Secretion

During Intraduodenal Infusion of

Standard Amino Acid and Liquid Formula

St imuli

The effeét's of Chronic Oestrogen 87

Administration on BiJ-iarY LiPid

Secretion, BiIe Acids and Gallbladder

Function in Post-Menopausal Women

Orocaecal Tlansit Tjne i¡ Pregrnancy 98

III. À.

III. B.

III. C.

III. D.

III. E

CHAPTER

. Co-ordination of Gastric and Gall-

bladder Emptying After Ingestion of

a Regular Meal

IV: DISCUSSION

PregnancY, Female Sex Steroids and

Gallbladder Function

The Effect of GaLIbl-adder Emptying

on Biliary LiPid Secretion

Onocaecal fransit Tjrre jrt kegnancy

108

lzs

L29

118

119IV.

IV.

IV.

À.

B.

c.

IV. D. Gastric and GalLbLadder Errtptying 136

PAGE

V

CHAPTER V: CLINICAL IMPLICATIONS OF ALTERED

GALLBLADDER EMPTYING AND BILIARY

LIPID SECRETION IN PREGNANCY

A. THE ROLE OF MORNING SICKNESS

(i) Clinical features of morning sickness

(ii) Diet and cholestenol gall-sbone formation

L42

THE POTENTIAL RELATIONSHIP OF DISEASES OF THE

ALIMENTARY SYSTEM TO ALTERATION IN BILIARY

LIPIDS IN PREGNANCY

L46

Mucosal protective effects of biliary cholesterol

Cholecystitis. in Pregnancy

Reflux oesophagitis and pepbic ul-cer disease

151

V-B

(i)(ii )

(iii )

CHAPTER VI: FINAL DISCUSSION

APPENDICES: A

F

BIBLIOC.RAPHY

ReaI Time Ultrasound Method for 15g

Determining GaIIbl-adder VoIume.

CharacLeristics of Infusates 161

Comparison of MeLhods for Measuring L62Gallbladder EmpLying.

Reported Studies of Biliary Bile 163

Acid Secretion in Subjects wibh

InLacL Gal-l-bladders and Subjects

SLaLus Post CholecystectomY.

I@asr:renent of Sma1t BoI/{tel TYansit L64Tiræ by the Hydrogen Breath llest

trÞasr:renent of Sen¡n tnmn Pancreatic t.nPollpeptide

'1.7r

B

L

D

E

1

SUMMARY

THE EFFECTS OF PREGNANCY AND FEMALE SEX STEROIDS ON GALLBLADDER

EMPTYING, BILIARY LIPID OUTPUT AND SMALL BOWEL TRANSIT TIME

In tlestern pqpulations gallstones occur in approximately 10 percent of men

and 20 percent of women by the age of 65. The majority of gallstones

are predominantì-y composed of chol-esterol. The mechanisms leading to

chol-esteroì- gallstone formation are poorJ-y understood, but prequisites

incl-ude supersaturation of biliary J-ipids with excess chol-esterof' the

presence of nucfeating factors and the retention of precipated choÌesterol-

crystals. The greater incidence of galÌstones in üIomen is probabì-y

reÌated to hormonal- factors.

Risk factors in blomen for gallstone formation incfude pregnancy and

the ingestlon of oral contrace.pþ1ve steroids. The increased probability

of gaÌlstones correlates with the number of pregnancies and women who take

oral contraceptive steroids or conjugated oestrogens double their risk of

deveJ-oping gal-lslones. The mechanisms by which pregnancy and oral

contraceptive steroids increase the risk of cholesterol- gallstones are

poorly understood but severaÌ mechanisms which include increased biJ-iary

chol-esteroL secretion and retention of precipitated cholesterol crystals

have been implicated. A factor contributing to bil-iary cholesterol

saturation in pregnancy may be the observed decrease in the numben of

ent,erohepatic cycles during pregnancy. Thj-s observation coul-d be caused

by slow transit of bife acids through the smal-l intestine, perhaps

secondary to pnogesterone or. other neurohormonaf effecls on small intestinal

muscle. Femal-e steroid hormones and pregnancy may also infl-uence gallstone

formation by aLtering the motility of the gallbJ-adder.

The aims of thi-s thesis wene to (a) quantitate gallbladder volumes throughout

the day in non-pregnant and pregnant subiects as well- as in subjects

taking oral contraceptive steroids or oestrogens aì-one, (b) assess the

2

infLuence of gaJ.Ibladder volume and small intesLine transiL time on

biliary 1ipid composition (c ) sfuay Iipid composition of gallbJ-adder bile

in women taking oral con$ugated oestrogens (d) assess orocaecal- transit time

in pregnancy and (e) examine the relationship between gastric emptying

and gallbladder emptying and time to nefilJ-ing'

Abdominal ultrasound was used to measure gallbladden volume throughout I

the day and night and during ingesbion of standard meals in pregnanl and

postpartum t¡tomen and oral contraCeptive users. Results b.Iere compared with

a control gnoup who were studied in bolh the follicular and lubeal phases

of the menstrual_ cycle. Increases in gal-lbladder volume in pregnancy

were correlated with serum progesterone. Evidence of al-tered gallbladder

motility in pregnancy was found.r The gallbladder of pregnancy was sluggish'

Fasting volume, the residual- volume after meals and the volume remaining

in the gallbladder throughout Lhe- day doubl-ed during pregnancy and these

changes cornelated with increases j.n serum progesterone. In contrasl'

emptying of the gallbJ-adder was not altered by the phase of the ovulaLory

cycle or by the ingestion of oral conLraceptive steroids. Gallbladder

refj-lling in the day did not occur in normal subjecLs ingesLing three

sLandard meals per daY.

The role of lhe enterohepatic circul-ation in biliary l-ipid secreLion was

studied as this may be an important mechanism by which altered motility of

the gal-Ibl-adder during pregnancy and the ingestion ofl conLraceptive steroids

predispose to cholesterol saturated bil-e and gaJ-J-stone formaLion' The raLe

ofl biliary secreLion was measured in human female volunLeers during naso-

gastnic infusion of both weak and poLent stimuli of gallbl-adder contracbility

(amino acids and fat respectively) and upper smal-l inLesLinal bile was

simul-taneousJ-y collected. Changes in Lhe enterohepatic circulaLion wene

monitored using abdominal ultrasound Lo quanitabe gal-Ibladder voLume and

breaLh hydrogen levels after adminisLration of bhe non-absorbable carbohydrate

lactulose to estimabe smal-l- intestine transiL ti.me. Serum levels of

J

pancreatic polypeptj-de were measured during each test. Continuous

i-ntraduodenal infusion of a sofution of amino acids that is known to

maximally stimulate pancreatic secretion uias less potent in stimulating

contraction of the galtbladder than intraduodenal- infusion of fat" Bil-e

was relatively more saturated with choÌesterol- when the gal-lbJ-adder

contracted at a sLower rate and the smal-I intestine transit time was slow.

The effects of Premarin (Ayerst), a mixture of conjugated oestnogens '/

prepared from the urine of pregnant maresr oh galJ-bl-adder emptying and

bil-iary ì-ipid secretion in postmenopausal vlomen were studied using

the techniques described above. No difference was found in biliary lipid

secretion or gal-J-bladder emptying on on off Premarin.

The lactulose breath test was used to measure orocaecal- transit time

throughout pregnancy and in the,pôstpartum period. The results were compared

with serum progestenone. Orocaecal transit time was deJ-ayed in late pregnancy

and returned to normaÌ postpanlum.- The pattern was similar to the pattern

of the sluggish gallbladder of pregnancy suggesting a common neurohormonaf

mechanism.

To investigate if the rate of emptylng of solids from the stomach controll-ed

the rate ofl gallbladder emptying and time to refilJ-ing, the relationship

bebween gallbladder and gastrlc emptying rates was studied in healthy

volunteers ¡ Fotlowing the ingestion of a standard radioactively l-abell-ed

meal gastric emptying was measured scintigraphical-ly while gallbladder vol-umes

i^rere morìitored sonÍgraphical-ly until gatlbladder refill-ing occurred. The rate

of gallbladder emptying in normal volunteers after a regulan meal was

dependent upon the rate of gastric emptying of the meal. Therefore' some of the

delay in gallbladder emptyin{ seen in late pregnancy could be due to deJ-ayed

gastric emptying.

The findings of this study provide valuable information on normal biliary

physiology and a plausibl-e rationabfe for pregnancy as a gallstone risk

factor by demonslrating the pnesence during gestation of prolonged periods

4

of gallbJ-adder stasis. The aetiology of this stasis is unlikeJ-y to be

related to high circulating oestrogen levels and is more likeJ-y due

to progesLerone effects.

Supporting evidence for the latter hypothesi-s comes from the observation

of the 1ack of influence of exogenous oestrogens on gallbladder and biliary

Iipid kinetics. The daLa also suggests.that conbraceptive sberoids are1

more likely to predispose to cholel-ithiasis by inducing changes in biliary

l-ipid metabolism rather than changes in galJ-bladder function.

5

STÀTE}4ENT

This thesis contains no material which

has been accepted for any other degree or

diploma in any Univers.ity and does not contain

any material previouqly published or written

by another person, except where due reference

is made to such material in the text.

DR. MICHAEL J. LAWSON,GE UNIT,

QUEEN EI ,.{A8EfH HOSPITALWOürivltLE,5011

45 0222

/

i, r-¡!N, :! +aràf

6

ACKNOVüLEDGEMENTS

The work contained in this thesis was carried out

during the tenure of a temporary research fellowship

in the Department of Medicine, the University of

Colorado, Health Sciences Centre, Denver, Colorado, USA.

I am deepJ-y indebted to Professor Fred Kern Jr. for

allowing me to work in his laboratory and for his

j-nvaluable guidance. I am extremely grateful to

Dr. Gregory Everson for direction and val-uab.Ie assistance

in study design, ultrasound techniques and anal-ytical'

methods. I am especially gratefuJ- for his consent to

use some of the results in, Chapter III which were

generated prior to my commencement as a Research fe.]-l-ow'

I al-so thank Dr. Craig Fausel for technical assistance

with some of the oestrogen experiments described in

Chapter III. I am grateful to Dr. Bob Hall- of the

Department of Maths and Computer Studies, South Austrafian

Institute of Technology for vafuable statistical- advice

and to Drs. Mal-col-m V'Ihiting and Michael Horowitz for

perusing the manuscript.

I wish to thank Mrs. Radene Showal-ter and Mrs. Carof

McKinley for expert technical assistance and preparation

of il-l-ustrations. The photographic work was carried out

by Mr. G. Hadaway to whom I am grateful,

I wish to thank l"lrs. Barbara Raymond who helped in the

enormous task of typing the manuscript and Dr. Stephen Rofe

who contributed his time in heJ-ping with the word

processing aspect for the preparation of this thesj-s.

7

Finally, I wish.to thank my Supervisors Dr. J. Toouli

and Dr. A. Kerr Çrant for heJ-pfuJ- criticisms and care-

fu1 perusal of the manuscript, which made the preparation

of this thesis both an educational and pleasurable

experience.

This thesis would not have been possible without the

staunch and unending support of my wife He1en and my

children Peter and Emily.

/

L

B

CHAPTER I: LITERATURE REVIEIV

Gallstone Incidence

Gallstone Classif ication

A

B

( i) Radiology

(ii) X-ray Diffraction of Stone Powders

( iii)Chemical AnalYsis

C. giliary Li pid Biochemistrv in Cholesterol

Gal.l-stone Disease

D. The EnterohePatic Circul-at ion

(i)

(ii)The role of

Interupt ionon Biliary

RoIe of the

the Gallbladder and Sma]-].

of the EnterohePatic CYcJ-eLipids

Intest ine

Effect

The cal-l-Úladder in Gal-l-stone Format ion

1

E

F

G

H

Fema.l-e Sex Steroids and Smooth Muscl-e MotilitY

( i ) Non Biliary Sr.nooth Muscle

(ii) Gal-lbladder and Small Intestinal Motility

Female Sex Steroids and Biliary LiPid Secretion

Gastrointest inal- Pe ptides and Gal]-bladder Motil-itY

( i ) Cholecystokinin

( ii) Pancreatic PoIYPePtide

( iii)Moti].in

I Neuronal- Determinants o f Extrahepatic Biliarv Motility

(i) Infl-uence of the parasympathetic autonomicnervous sYstem

(ii) Influence of the sympathetic nervous system

J . The Rel-at ionshi of Gastric EmPtYing to Gal-]-b1adder

Emptyinq

9

ii

LITERATT]RE RHÆE\^I

A. C,AI;I,STO{E INCIDENCE

Iltre incidence of gallstones is 1Ot irr lTren and 20? in \¡toûEn by the age of 50

to 65 (CIeIand 1953). In the USA gallstones are responsible for about

5001000 operations each year and approxirnately 81000 deaths. The direct

costs have been estimated to be rnore than two billion dollars (Kern 19751.

B. C.AI;I,STINE g.ASSIFICATTGT

C,allstones can be classified by tho following three nrethods:

(i) Radioloqy

Stones can be classified as:

(a) Radiopaque - This feature accounLs for 20% of stones and inclicates a

relative calciun content of greater than 4% by v'zeight.

(b) p.adiolucent - Accounts for tlre renaining 80t of stones indicating a

reJ-atively lower calciurn content.

(ii) X-ray Diffraction of Stone Povders

Using x-ray por,,rler diffractionr' the c4rstalline crcnstituerrts of gallstones

rernoved frcrn the gallbladder can be broadly classified as choLesterol and

calcir¡n salts (Si:tor ard lrlooley 19731. On this basis there are three nrai¡r

types of stones:

(a) clrolesterol (<5* calciunt) 608

(b) nixed stones 27?

(c) pure calcium 13ã

By this method it was found that men are just as likely as wonìen to form

pure calcir:rn stones but both calculi of cholesterol and those of

cholesterol with calcium salts.are nrcre ccrÌtncn i¡¡ r'ucnen-

(iii) Cholesterol vs Pigment

Gallstones can be classified fron direct chemical- analysi-s as chol-esterol

or pignrent (Trotman, Petrella,-Soloway et aI 1975) -

(a) Pigrnent

Understanding of the pattrogenesis of pigment stones has not progressed very

far. Tkre lack of clear understanding of their chernical corpositì-on has

retarded research progress. Although certain clinical associations are

noted below nrost pigrment stones in the Western worl-d and in the Orj-ent

occur in their absence.

(b) Cholesterol

Factors thought to be irrportant in the aetiology of cholesterol- gal-lstones

include the following:

l-. Infection

2. Bile Stasis

3. Changes in the conposition of hepatic bile

Originally it was thought'that infection of the gallbladder wall coul-d

/

!ü'ù

J

T

tII

I

allcx¿ bile salts to Þpass¡¡¡ely eþsçrþed -Leavi¡g ¡ç Lalrvgl

IncidenceMorphology

Compos it ion

clinicar features ::::i:":'::;:i:":::,steroids, conditions

11_

CHOLESTEROL VS PIGMENT STONES

CHOLESTEROL

70-908Multiple, 2-30mm indiameter, smooth orfaceted, laminated orcrystalline on crosssect ion

60-edåCholesterolscholesterol, mono-

hydrate, anhydrouscholesterof and

cholesterol-.IIaccount for 7IZ of thetota.l- crystallinematerial in gaÌlstones(Sutor and Wooley,1971 ).Other - pigment,precipitated biLe acids,mucoproteins, otherproteins or ca-Lcium

salt s

PIGMENT

10- 308

MultipJ-e, 2-5mrn , 1

irregular or smooth,black or brown, amorphousor crystalline.

Black - confined togallbJ-adder. Bilirubinpolymer.Brown - found inconìmon bile duct. CaLcium

bi]-irubin -

Bilirubin 35å (range 10-50)Bil-e acids 2

Chol-esterol 2

Cal-cium 9

Carbonate 7

AIso heavy metals,proteins,sul-phates, magnesium

saÌts, cal-cium soaps,pol-ypymoJ-e poJ-ymers and

mucin.

Associated with cirrhosis,chronic hemolyticanaemia - black type,

!lr¡l

I

t

reducing bile sal-t pool- stasis and infections' ( ascaris, cl onorchis,

typhoid) - brown type

T2

cholesterol in the gallbladder to nucleate and form chol-esterol- gallstones

but this no\^/ seems unlike1y. In add.ition to the rol-e of infection in

providing potential nucleating agents it is likely ttrat infection and

inflanrnation of the galì-bl-adder allow precipitation of calcium sal-ts to

initiate or accelerate gal-J-stone formation.It is noh/ generalÌy accepted

that gallstones may form without infection and biliary lipid research has

concerned itsel-f with the latter two aspects.

C BILIARY LIPID BIOCIIEIV1ISTRY IN CHOLESTM.OL GALLSTONE DISEASE

Si-nce 1968 progress has been made in r:nderstanding the pathogenesis of

cholesteroJ- gallstones but many uncertaintj-es remaj-n. The cl-arification of

the physicaf state of the lipids in bile in the nlid 60's and the

demonstration of the ìrrportance of the rel-ative nrol-ar percentage of each

lipid were key findings (A&nirandland Smafl 1968).

Gall-stones a-re fornred from organic conponents of bile.The major organic

components of bile are bj-le acids, chofesterol and phosphoJ-ipid.Bì-1e salts

are formed in the liver from chol-esterol. The rate firniting reaction in

this sequence is the 7-a1pha hydroxylation of cholesterol- (Mendefsohn,

Mendel-sohn, Staple 1965). Bile salt slmthesis may be regulated by a

negative feedback system such that bile sal-ts returning to the liver

inhibit 7-alpha hydroxylase, and the incorporation of acetate and

meval-onate into cholesterof (Shefer, Hauser, Bekersky et al- 1970). Removal

of these inhibitions may increase bile sal-t synthesis several fold. Both

cholesterol and phosphoJ-ipid are insolubl-e in water, but they are sol-uble

in bile because it is a micellar solution. Phosphofipid (l-ecithin) has a

hydrophobic and a hydrophilic portion sirnilar to bil-e acid and is present

in bil-e in high concentrations (350-600nìg/100ÍLI) (Figure l-). It greatly

oçands the size of bile acid rnicelles and increases their capacity to

I

13

Biìe saltLeci thi n

Choì esterol

}.IIXED BILE ACID-LICITHIN MICELLE

FIGURE I

Diagram showing the accepted structure of bile sal-t-lecithin (phospholipid) micelles-

I

L4

sol-ubilise cholesterol-. These lipids are present normalJ-y in the rnolar

rati-os of bile sal-ts 75: phospholipid 20: cholesterol 5. I¡ühen the

cholesterol percentage is increased to greater than l-0?, either because of

increased cholesterof secretion or decreased bil-e acid secretion,

chol-esterol may precipitate in the gallbladder and initiate stone

formation. It is no\^/ recognised that cholesterol gal-lstones will form only

vrhen bile is supersaturated with cholesterol-, as it contains rnore

chol-esterol- in a single phase solution than can be solubilized in the bile

salt - phospholipid miceì-les present. ltre water insoluble chol-esterol is

maintained in solution wj-t]. bile salts, providing a sufficient

concentration of the swel-ling anphipath. Phosphoì-ipid is present to

increase the size of the rnixed rnicelles. Excess phospholipid disrupts the

micellar structure. The inter-rel-ationship of the concentrations of these

three J-ipids is nrost clearly demonstrated using the trj-angufar co-ordinates

described by A&nirand and Small- (1968). This representation assumes that

the water content is fixed at 90? by weight. A zone of concentratj-ons for

these three lipids in which a Iiquid rnicellar phase is maintained can be

demonstrated (Figure 2). Ho'o/ever, there has been considerabJ-e di-sagreement

as to the exact definition of the l-ine of supersaturation and its

significance (Metzger, Heymsfield, Gnrndy 1972),

/

15

À

_O/OBILE ACID

&zoH Admirond 6 SmollH Holzboch, el ol

fllffiÏll Meloslobil e. SupersoluroledZones"

"ud

to

90 80 70 60 50 40PERCENT BILE ACIDS

FIGURE 2:

The relative molar proportion of biliary lipids plottedon triangular co-ordinates. The axes represent thepercentage of the total moles of bile acids, lecithin(phospholipids) and cholesterol constituted by eachof these components which total 100 percent. The limitsof cholesterol solubility are shown as defined byAdmirand and smarl (1968) and Horzbach,ii.:c.'.:,31sze:.s-;i :.t al (1973)

with the metastable supersaturated zone between them.

/

/

Aùnirand and Smal-I (1968) focussed attention on the liver as the site of

active bile secretion and biliary tipid production. They hlzpothesized thrat

a difference in the ability to solubilize cholesterol must exist between

human control bil-es and those from chol-esterol gallstone pati-ents and that

cholesterol insol-ubility probably represented the first step in gallstone

formation. Ttrey and subsequently others (MeLzger, Adler, Heymsfì-e1d et al,

1973) found that in patients with cholesterol gallstones, bile secreted by

the liver is supersaturated witJ^ cholesterof. In the same patients, bile

stored in the gal-Ibladder is l-00% saturated with cholesterol in micefl-ar

sol-ution and also contains cholesteroJ- crystals. They postulated tl-at

duri-ng storage in the gallbladder ,the su¡rersaturated bile was "seeded" or

nucleated by precipitated, bile pigment or other material, and that

chol-esterol- crystals precipitated frorn sofution and ini-tiated gallstone

formation.

Many investigators have found supersatr:rated or lithogenic bil-e rn

gallstone pati-ents and have identified the basis for the abnormal biliary

lipid conposition i-n several- groups of patients who a-re at high risk of

developing stones. For exanple, obese subjects secrete excessive biliary

cholesterol (Bennion and Grr.rndy 1975, Grundy, Duane, Adler et al- 1974) and

patients with il-eal resection, disease or blpass secrete reduced anrounts of

bile sal-ts (Cohen L97I). In both disorders the rel-ative molar percent

cholesterol in bil-e is increased and the incidence of gaÌIstones is high.

Vlahcevic and co-workers (Vl-ahcevic, Bell, Buhac et aI 1970¡ SweÌl, Bel-I,

Vl-ahcevic 1971) were the first to report a smalf bile salt pool size in

patients wj.th chol-esterol gallstones and to inplicate this small pool size

T7

in the pathogenesis of stones. They and others (Mok, Von Bergmann, Grundy

1977) hlpothesized that the bile salt pool size was small because of an

overly active negative feedback regulation of bile salt slmthesis. This

presumed defect in regulation, thought by sorne to be genetic, was proposed

primarily because other rnechanisms causing a small- bile salt pool size

could not be demonstrated. Specifically, the patients did not lose excess

bil-e sal-ts in the stool and they had normal hepatic slmthetic function.

A nurnber of other observations of biliary lipid conposition and bile salt

pool size cast sorne doubt u¡ron the inportance of the hypothesis just

described in the pathogenesj-s of stones.

The observations are;-

l-. Both hepatic bil-e secreted in the fasting state and bile stored

overni-ght in the gallbladder, can be supersaturated with cholesterol in

normal- subjects without gallstones (Metzger, Adler, Helzmsfield et a1

L973).The large overlap in biliaqz li-pid composition between gallstone

patients and controls suggests that supersaturated bife alone is not.

sufficlent for gallstone formation.

2. Smal-l bile salt pool sizes are also found in many subjects without

gallstones. Indeed, some investigators find no difference in mean pool size

between control and gallstone patients (Northfiel-d and Hofmann 7975).

These findings raise an inportaht question.VrThrat factors in addition to

supersaturated bile are necessarlz for gallstone formation?.

D. TTM ENTFR.OHEPATIC CIRCULATION

(i) Thre RoIe of the Gall-bl-adder and Smal-l- Intestj-ne:

The enterohepatic circulati.on (EIC) of bil-e occurs via the liver, biliary

tree, gallbladder, jejunum, il-eum and portal vein. The secretion of

cholesterol- and l-ecithin into bil-e is dependent upon bil-e acid secretion

which is in turn dependent on an intact EHC-

In heatthy individuals, il-eal bile acid absorption frsn the intestinal-

Iumen (I1¡ag and Phill-ips 1974) and hepatic bil-e acid extracton fron portal-

blood are highJ-y efficient (Reichep and Paumgartner 1980). Because bile

acid pool size is constant in the steady state and. no bile is stored in the

liver, the rate of hepatic bile acld secretion is directly proportionaL to

the frequency of enterohepatic cycling, which is largely deterrnined by the

rate of derivery of bile acid frorn the extrahepati-c bil-iar1z tree to the

terminal ileum. The delivery of bile acid to the terrninal- ileum in its ttlrn

is detennined by two mechanical pumps, the gallbladder and smalf intestine-

The gallbladder, in its reservoir capacity, determines the delivery of bile

acid to the intestinal- lumen. BiIe acid, the major conponent of biÌiar1z

secretion, is required for fat digestion and absorption thrrough the

formation of rnicelles. During fasting about 50? or nore of the bil-e acid

pooJ- is stored in the gallbladder and bite acid secretion into the

intestine is relatively Ìow (Metzger, Adler, Heysmfield et al 1973¡

Northfiel-d and Hofmann 1975¡ Mok, Von Bergrmann, Grundy 1980). During

feeding the gallbl-adder contracts and bile acid secretion into the

intestine j-ncreases in relation to the degree of gallbladder enptying,

whichrin turn, is primariJ-y a function of the content of the nreal (Rock'

Malmud, Fisher 198I; Ladas, Isaacs, Mur?hy et al- 1984). High fat rneals

ti

19

induce maxjmum galJ-bladder contraction. To integrate and optirnise the

process of digestion, gastric enptying of solids should regulate

gallbladder contraction through the release of hunroral nediators from upper

small bowel- and pancreas. Increased storage of bile acid in the

gallbladder or slowed smatl intestinal transit could slow the delivery of

bile acid to the terrninal- ileum and fower the bil-e acid secretion rate.

Biliary bite acid secretion rate is l-ower when stimufated by interrnittent

feeding (Mok, Von Bergmann, Grundy I9l9; Northfj-eld and Hofmann 1975¡

LaRusso, Szcepanik, Hofmann Ig77ì Mok, Von Bergmann, Grundy 1980; La Russo'

Hoffman, Hofmann et aI Ig75) than by continuous intraduodenal infusion

(Grundy and Metzger 19']2; Mok, Von Bergmann, Grundy I979,Yon Berg'mann'

Mott, Howard 1980, Grundy, Von Berg:mann, Grundy L979, Grundy, Duane, Adler

et aI 1914; Bennion and Gn:ndy 1975; Mabee, Meyer, Den Besten et af 19'76'

Valdivieso, Pal-ma, Nervi et aI |g':g, Mok, Von Bergmanrt, Gmndy 1978) of a

standard J-iquid formula. Because úite "ontains refatively more cholesterof

when bile acid secretion rate is ]ow, the l-ower rates of bile acid

secretion during gallbladder storage and slowed intestinal- transit coul-d be

expected to increase cholesterol saturation of bile'

The importance of the gallbladder and smal-l intestinal transit in

regulating the enterohepatic cycJ-e and pool size is suggested by several

observations: -

I. Sonre (Shaffer and small L977, Redinger L976) but not all (Adler,

MeLzger, Grundy L974¡ Shaffer,Braasch, smal-l I9l2), investigators have

noted a decrease in pool size after cholecystectomy, attributed to nearly

continuous cycling of bile acid's and riearly continuous inhibition of

hepatic bil-e acid slmthesis. These studies are difficult to interpret

because the storage firnction of the galJ-btadder before surgery was usually

not knornrn.

¿U

2. fn the dog (Parkin, Srnith, Johnston l-973) and in man (Inberg and Vuorio

1969¡ Faberberg, Grevsten, Johansson et a] 1970) truncal- vagotonqz was

fol-l-owed by increase in gallbladder vol-ume and increase in bile acid pool

size. Gallbl-adder nrctor response to standard stimuli seemed uninpaired by

vagotony but Inberg and Vuorio (1969) noted a larger residual volure in the

gallbJ-adder after a fatty meal. Stenpel and Duane (1978) more recently

found that the poor size of both primarlz bile acids' (cholic acid and

chenodeoxychol-ic acid) increased after vagoton¡z in eight male patients-

Ttrey also noted a decrease in cholesterol saturation of bile after vagotorny

and a significant inverse correlation between pool size and change in nrolar

¡rercent cholesterol. Their findings suggested that vagotonlz does not

predis¡lose to cholesterol- gallstoneq, but others have pro¡rosed that

retention and stratification of bile in the gallbladder results in certain

Iayers having a disproportj-onal percentage of cholesterol- (Thrurebon 1966¡

Nakayama and Van Der Linden 1975). Leáding to nidus formation, precipitation

of cholesterol and an increased incidence of galJ.stones.

3. In patients with coel-iac disease Low-Beer, Heaton, Heaton et aI (1971)

described enlarged gallbtadders and decreased gallbladder contractility

after a fatty meal-, with decreased bile acid turnover and increased bile

acid pool size. The changes \iùere attributed to dirninished CCI( release by

damaged smal-I intestinal mucosa and reduced stjmulation of the

gallbladder.

4. Hepner (Lg75) reduced gallbl-adder enrptying in healthy subjects by

feeding a 95å carbohydrate diet.for several days and then studied bile acid

kinetics. He found a similar increase in pool si-ze of both chol-ic acid and

chenodeoxychoric acid and a decrease in the secondarlz bite acid deo>cychoric

acid pool size. The traditional turnover rate of both prinrary bj-Ie acids

decreased slightty but significantly during the o<peri¡nental diet. The

2I

slmthesis rates v,/ere unchanged. A sjmilar diet in cholecystectornised

patients had no effect on bile acid kinetics.

5. Duane and Hanson (1978) used:indocyanine green as a marker of

gaJ-Ibladder content and rel-ated its rate of enptying after a standard meal-

to bile acid kinetics. They for:nd significant negative correlations between

rates of gallbladder enptying and pooJ- sizes of the primary bil-e acids plus

significant positive correlations between the traditional tr:rnover rate of

each bile acid and gallbladder enptying rates. Gal-l-bl-adder enptying was not

significantJ-y reJ-ated to synthesis rates of elther bile acid but slmthesis

rates did correlate positively with intestinal transit tirre measr:red by the

breath hydrogen method (Bond and Levitt 1975i Mok, Von Bergrnann, Grundy

L977). They also found an inverse Çorrelation between pool size and cycling

frequency.

6.Recent stuôies in man (Ir4ok, Von Bergmann, Gn-rndy 1980) indi-cate that

considerable amounts of bile secreted at night by pass the gallbladder

contrarlz to earlier belief. Sirnilar conclusions had been reached by

Stanley (I9lO) on the basis of his studies on the faecal excretion of bile

acids i-n obese subjects undergoing a prolonged fast and by Small et al-

(SmaII, Dowling, Redinger 1972) in their primate studies. Von Berg;mann et

al- (Von Bergmann, Mok, Grundy 1916) described the use of bili-rubin output

after stimul-ation of the gaÌlbladder to estimate ga1J-bladder storage

capacity and reported 37-86? of the bile acid pool in the gal-Ìbl-adder after

an overnight fast.

7.Small intestinal transit is an inportant deterrninant of bil-e acid cycling

frequency (Einarsson, Grundy, Hardison i-979) and thus can alter the rate of

biliary l-ipid secretion (Valdivieso, Palma, Nervi et al- L979¡ Mok, Von

Bergmann, Grundy Ig77). Faster sinalÌ intestinal transit increases bile acid

22

cycling frequency and the rate of bitiary lipid secretion. Slow small

intestinal transit woul-d slow delivery of bile acid to the terrninal ileum

and l-ower bile acid secretion rate. Because bil-e contains nx¡re cholesterol

when bile acid secretion rate is low (Northfiel-d and Hofmann 1975;

Lindblad, Lundholm, Schersten 1977¡ Wagner, Trotman, Soloway 1976, Nilsson

and Schersten 1969 i Ì(etzger, Adler, Helzmsfield et al 1973), the lower

rate of bile acid secretion during slow intestinaf transit would be

oçected to j-ncrease cholesterol saturation of bile. Small- intestinal

transit tjme is related to the type of stimulus infused j-nto the bowel

l-umen. Ruckebush and Fioranronti (1915) observed that different tlzpes of

equicaloric test neals produced d-ifferent durations of disruptions of

myoeì-ectrical- activity in the rat smal-1 intestine. Longer lasting digestive

activity was noted with oleic acidt conpared with arnino acids or glucose- De

Weaver, Eeckhout, Vantrappen et al- (l-978) showed in the dog that the

dr.ration of disruption of tJ-e sm.il-I intestine nigrati-ng myoeJ-ectric conplex

after a meal depends much more on the physicochernical conqrosition of the

food that on its volume or amount of calories. Therefore the physiochenical

composition of food may regulate smal-l- bowel motor activity and in turn

bitiary lipid secretion.

Thus an abnormality of motor function of the gal-lbladder coul-d result in a

decrease in the nunrber of enterohepatic circulations causing a decrease in

bile salt secretion rates leaving relatively greater amounts of cholesterol

in bite increasing the chance of crystat and stone formation- During

pregnancy the total bil-e acid pool is increased (Kern, Everson, De Mark et

al- 1981). pooling of bile acidd within the smalt intestine and gallbladder

coul_d contribute to an expansion of the total bile acid pooJ-.

During pregnancy, total bil-e acid pool size and gallbladder volurne are

increased (Kern, Everson, De Mark et al- 198f; Braverman, Johnson, Kern

rit

23

1980; Ylostalo, Kirkjnen, Heikkinen 1982). In addition, the size of the

bile acid pool directly correlates with the increases in gallbladder volunre

(I{ern , Everson, De },fark et al 1981 ). It is likely t}nt a hormone,

probabì-y proc;esterone, inhibits srnooth nn¡scle contraction, and irduces the

j¡rcrease in gallbtadder volume. Subsequently bile acids are sec1uestered in

the gallbladder, slowing their enterohepatic circulation, secondarily

stilrn:Iating bile acid slntlresis and æçanding the biLe acid pool to a new

steady state level. Ilov¡ever total bile acid pool size is significantly

i¡creased in the first trimester at a ti¡re '.vhen gall-bladder volume is not

significantJ-y increased. Ttl¡s in early pregnancy, the j¡crease<l bil-e acid

pool is either an effect of one or more hormonal or nretairolic changes in

bile acid synthesis or bile acid poo] sequestration is occurring in another

site. i

The rnain non-gallbladder site rvithiir tlre enterohepatic circulation where

bile acicl sequestration can occur is the s¡nall intestine. Aùrinistration of

propa.ntlreline brcrnide to healthy subjects resufts in prolonged

giastrointestinal transit tjme and expa.nsion of bile acid pool size (Duane

ancl Bond 1930). In aclclition, orocaecal transit is reversibly prolonged in

both the thiro trj-nester of pregnancy (i^Ji.ald, Van Thiel, Floechstetter et aI

19t)2) and in the luteal phase of the ovulatory cycle (i'Iald, Van

Thielriloechstetter et aI 1981 ). 'Ihus it is possj-bIe thr,at gastrointestinal

t¡ansit is proJ-onged in earty pregrìancy and therefore contributes to the

increased bile acid pool by sequestering bile acids rvithrin the intestinal

Iunen.

24

(ii)fnterruption of the Enterohepatic Cycle - Effect on Biliarlz Lipids

Thr:rebon (1962) by careful experiments on patients with occludable connìon

duct T-tubes denronstrated that interruption of the EIC for one to three

hours produced a decrease in lecithin and bil-e salt concentration. However

chol-esterol- secretion by tJ:e l-iver was unchanged causing the formatj-on of

bil-e saturated with cholesterol. Refeeding of bile collected during the

interruption returned cholesterol saturation of bile to normal. Thurebon

concl-uded that later in the storage cycle, the gallbl-adder fills with

hepatic bil-e that has changed its conposition due to natural interruption

of the EIC creati-ng favourable conditions for layer formation,

stratification and cholesterol- gallstone formation. Possibl-e causes for

this interruption courd lncl-ude dietarlz factors (e'gt predorninance of

carbohydrates) or proJ-onged periods-of fasting. Other observers have shov¡n

that interruption of the EX{C by fasting (causing sequestering of the bile

acid pool in the gallbladder) increases the chotesterol- saturation of bile

(Metzger, Ad.ì-er, Heysmfield et al- l-973¡ Mok, Von Bergmann, Grundy l97B)

E. TT{E ROLE OF TTIE GALLBI"ADDM. IN CHOLESTEROL GALLSTONE FORMATION

A stasis theory for gallstone formation was first suggested by Mecke1 von

Helmsbach (1865). The idea that inconpJ-ete gallbladder enptying contributed

to cholesterol galJ-stone formation hras supported by Montgonery (1866) and

Ord (1879). They showed that cholesterol- stones could form fron spherolJ-ths

which resulted frorn layering of liquids of different density in the

gallbladder. Steinman (faae) and Rous,McMaster and Drurlz (1924) felt

that nucl-eation or nidus formatj-on by bile pigrment spheroliths \^Ias

encouraged by gallbl-adder stasis, producing larninated gallstones. They also

suggested that inpaired gallbladder enptying allowed stratification within

I

25

the gallbladder of sol-ids of differing specific gravi-ty, thus favouring the

aggregation and subsequent additional layering of stones in a rnilieu

containing proteinaceous material or layers of bile. Poor galJ-bladder

enptyj-ng or biliary lipid rnixing could prevent the natural passage of early

rninute stones into the duodenum (Rains 1962).

In recent years however, nxcst enphasis has been on biliary lipid

conposition and there have been few studies of the gallbl-adder in rel-ation

to the pathogenesi-s of stones. Nevertheless, it is apparent that the

galtbladder must be invofved in the formation of gallstones for several

reasons:

(a) Chol-esterol- stones are rare after cholecystectorny

(b) Precipitation of chol-esterol cqystals from a suþrsaturated solution

requires time (Ho1an, HoÌzbach, Hermann et al- IgTg). BiIe retention in the

gallbJ-adder must be sufficientJ-y plolonged not only for crystal formatj-on

but for crystal agglorneration and stone formation.

(c) If gallbladder contraction is conpl-ete, lithogenic bile and cholesterol

crystals, if present, wil-I be expelJ-ed into the duodenum where they are

harml-ess. Stasis of bil-e j-n the gallbJ-adder, because of i-neffective or

inconpÌete contraction, might be the additional necessary condition for

galJ-stone formation.

F. FEMALE SÐ( STROIDS ANID SIVX]CITTI MUSCLE MOTILITY

(i) Non-Biliary Snrooth Muscle

There is good evidence that the ovarian hormones, oestrogen and

progesterone, can affect the motor activity of extra-uterine smirottr muscle.

Van v'Iagenan and Jenkins (1939) de¡n<onstrated of the

26

ureters of nronkeys in the irnnediate post partum period. Hund1ey, Diehl and

Diggs (cited Kumar 1962) showed decreased ureteric peristaltic activity in

non-pregnant wonen given progesterones. Activity increased with oestrogens.

Sjmil-ar results were found with in vivo intestinal motility studies by

Tsutsulopulos (cited by Yoshida and Mori 1969). Îøenty years l-ater Kumar

(1962) denxrnstrated that progesterone in vitro had a marked inhibitory

effect on the spontaneous contractility of ureter, large bowel and stornach.

Schatzman (1961) studied the guinea pig ileum and concluded that a decrease

in muscl-e tone occurred with the a&ninistration of progesterone or

oestradiol-. The previously conflicting resul-ts with oestrogens r,¡/ere

probably rel-ated to the high concentrations used. Yoshida and Mori (1969)

found two different level-s of oestrogen effects on in vitro intesti-nal

motility. Vrlith lower concentrations.'there \^ras an excitatory effect and with

higher concentrations an inhibitory effect. Nagler and Spiro (1961) studied

oesophageal rotility by manornetry jn_ control-s and pregrnant vvonìen.

Non-propulsive motor activity r^/as seen Íìore consistently in pregnant

subjects. This finding led the way for fi:rther investigations of the

effects of sex steroi-ds on gastrointestinal snrooth muscle ín human

subjects '

(ii) Caftbladder and Small Intestinal Motility

There is evidence that contraceptive steroids increase biliarlz cholesterol

secretj-on in wornen (Bennion, Mott, Howard l-980; Bennion, Grinzberg, Garrick

et al- 1976; Pertsernilidis,Paneveliwalla, Ahrens L974),but information on

the affect of female sex hormones and.pregnancy on gallbladder function is

scant. In separate chol-ecystographic studies Gerdes and

Boyden (1938) and Potter (l-936) suggested that inpaired gallbladder

emptying and concentrating ability occur late in pregnancy. Nil-sson and

Stat-t-in (1967 ) also used oral cholecystography to neasure gallbladder

/

zt

erptying after bol-us injection of chol-ecystokinin (CCK) in both phases of

the menstrual cycle in each of 10 subjects. They found slower enptying in

the luteal phase in eight of them. However the phase of the cycle was not

accurately documented by serum progesterone levels.

Pregrnancy and female sex steroid intake are major risk factors for

chol-esterol cholel-ithiasis (Bennion and Grr-rndy I97B¡ Kern, Erfling, Si-nx¡n

et al I97B¡ Frieùnan, KanneÌ, Da¡¡¡l¡er L966). In the pathogenic seguence of

cholesterol cholelithiasis proposed by SmaJ-J- (Small- 1980; Small 1974;

Shaffer and Smal-l- 1976) a prerequisite for cholesterol gallstone formation

is the hepatic secretion of lithoÇenic bil-e. rn addition major

physiochernical changes, incJ-udj-ng nucl-eation (Hol-an, Holzbach, Hermann et

aI 1979), crystal formation (Sefaghat and Grundy l-980; Walton 1967), and

adherance of crystal- must occur in the gallbtadder to generate cholesterol

gal-lstones. Because these changes.require tjrne, retention of lithogenic

bile in the gallbladder is necessarlz for these events to occur. If

gaÌlbladder enptying u/ere prompt and conplete, lithogenic bil-e and any

cholesterol crystals that may have formed would pass uneventfully into the

duodenum, preventing the sequence leading to gallstones. Previous studies

in pregnant women have suggested that the volume of the gallbladder is

increased and its contraction is sluggish (Braverman, Johnson, Kern l9B0;

Potter 1938; Gerdes and Boyden 1938). Gallbladder emptying in the l-uteal

phase may be impaired (Nilsson and Stattin 1967 ) but \^/olnen taking oral

contraceptive steroids seem to have normal gaìlbladder volumes and emptying

after a liquid meal (Braveïman, Johnson, Kern l-980). However enptying

during continuous intraduodenal arnino acid infusion is slower (I(ern,

Everson, De Mark et aI I9B2). Ttre latter finding inplies that gallbl-adder

contractiJ-ity rnight be irrpaired in vvorrìen tal<ing contraceptive steroids.

/

Progesterone and oestrogen l-evels increase progressively in pregnancy- rt-

¿ó

appears that progesterone rather than oestrogen causes the inhihition of

gallbladder enptying and the delay in orocaecal transit tine is noted

in the third trinester of pregnancy (Wald, Van Thiel-, Hoechstetter et al-

l9B2). Studies in experlrnental anjmals suggest that oestrogens actually

enhance srnooth nmscle activity. Ryan and Pellecchia (l-982) showed that

oestrogen pretreatnent in the in vitro mal-e gminea pig gallbladder

significantly decreased tl-e threshold acetycholine requirernents for

contraction. Progesterone had the opposite effect. Datta, Hey and Pleuvrlz

(1974) found that oestradiol- pretreatment causes a dose-dependent increase

in intestinal propul-sion when aùn-inistered to oopherectornised mice. A

rnixture of progesterone and oestradiol had no significant effect. There are

no studies in man to confirm or refute these experirnental findings of

gallbladder and intestinal smooth muscl-e excitation by oestrogen.

G. FEMALE SEX STM.OTDS AI\D BILIARY LIPID SECRETION

BiIe of \ivollten taking birttr control'p-iIls has often been shown to be

considerabJ-y nore saturated with cholesterol than that of other tn/olnen

(Benni-on, Mott, Howard l-980; Kern, Everson, De Mark et aI I9B2) - It has

been established that the higher the chol-esterol saturation anÐng patients

without gallstones, the greater the prevalence in the popuJ-ation from which

the patients were chosen (Redinqer and Small 1972). For instance the Masai

of East Africa and the Japanese have a very low cholesterol saturation and

as a consequence a low prevalence of cholesterol- gallstones, ulLrereas young

North American fndian Vùomen have a high prevalence of su¡rersaturated bil-e

(Thistl-e and shoenfie]-d, 1971-) and an exceptionally high prevalence of

gallstones(Sanpliner, Bennett, corness l-970). Tttus, the higher the

cholesterol saturation, the greater the risk of gallstones.

According to some epiderniological studies, \^/omen on birth control pitls

(Boston Collaborative Drug Surveil-lance Progranrne 1973) and postmeno-

pausal h/ontqn (Boston ColÌaborative Drug Surveillance Progranrne 1974) on

oestrogens have a risk of surgically documented gallstone disease that is

2.0 to 2.5 times tJ.at of control- groups. Hcxuever more recently the Royal

College of General Practitioners' oral contraceptive study (1982) suggested

I

29

that the use of oral contraceptì-ves confers no increased risk but simply

accelerates gal-J-bJ-adder disease onJ-y in htomen susceptible to ib. In

addition, this study suggested that the acceleration may be associated

with the dose of oestrogen in the combined oral contraceptives. These

Ìatter new observations may be due to currenl|tlow doserroestrogen

contnaceptives. I'High doserr contraceptives are prescribed onJ-y when the

l-ower dose oestnogen contraceptives prove inadequate. Many of the

physioJ-ogicaf changes in biJ-iary lipids observed with high dose oestrogen

preparations may not occur with lower doses. In addition most studies

performed in different phases of the menstrual- cycle have failed to show

significant differences in bil-iary l-ipid output and in particul-ar' the

cholesterof saturation index (Kern, Everson, De Mark et al 1981 , illiJ-l-j-ams,

Scallion, McCarthy 1980; !r/hiting,, Down, Iitlatts 1981).

A few studies have examined the effects of oestrogens on biliary i-ipids

in humans.

Oestrogens have several effects on hepatic biliary lipid metabolism whlch

resuft in the formation of lithogenic bile. These effects incl-ude the

foì-lowing: -

1 . I¡,lomen taking oraf contraceptive steroids have an increase in

cholesterol saturatlon index and the proportion of women with

supersaturated bil-e is increased (Bennion, Ginsberg, Garrick et

at 1976; Vrlhite, Howat, Schoefield 1976; Pertsemilidis, Panveli-wall-a,

Ahrens 19'14). 'l'he mechanism is probably by hepatic enhancement c¡f

choÌesterol- secretion in bife (Bennion, Mott, Howard 1980).

2. An increase in molar percénLage chol-esterol in T-bube bile from

three bromen taking Premari-n and Provera (conjugated equine oestrogens

30

Íedrox)4grogesterone acetate) al-ong with a decrease in cholicacid slmthesis

and pool size (Pertsemilidis, Panveliwalla, Ahrens 1974) -

3. Reduced bil-e acid synthesis and secretion in rats (Davis and Kern L976i

Davis and Kern 1977¡ Stranentinoli, DiPadova, Jualano et al 1981) and

hamsters (gorurorris,,Colme, Chung et aI 1977) so that bile becores

supersaturated

4. In hamsters the rate lirniting enzynìe 7-a1pha-hydro>q¿l-ase which converts

cholesterol to bile acids is reduced by ethinyl oestradiol (Bonnorris,

Co1me, Chung et al- 1977) -

5. The physical characteristics o{ the lipid conposition of the hepatic

rnicrosomal nre¡rbrane, tJle site of cholesterol conversion to bile acids is

al-tered in the rat by ethinyl oestradiol (Davis and Kern 1977) -

Suggestive evidence of an oestrogen effect coItES from the folJ-owing

obsen¡ations:-

I. Bennion, Drobny, Knowl-er et al-. (f978) found a smal-Ier total bil-e acj-d

pool and a smaller cholic acid pool in Indian and white !úofiìen cornpared with

nen. Bil-iar1z cholesterol saturation \^/as inversely correlated witl

chenodeoxycholic acid pool size.

2. Bennion, KnowJ-er, l4ott et al-. (1979) studied biliary J-ipids and bile

I

ü

tT

I

31

acid kinetics in young Pima Indian boys and girls before, during and after

puberty. Prior to puberty the bile acid pool size was the same in both

sexes. It becanre l-arger in males but not females at the tirne of puberty'

Biliary lipid saturation increased in both sexes especially females at

puberty and correl-ated positively with urinary oestrogen excretion.

3. Bennion (1977) stud'ied one young r¡/oman before and after bilateral

oophorectorny. After operation the bile acj-d pooJ- size doubl-ed without

change in distribution of bile acids and the rnol-ar percent cholesterol- in

the bile decreased.

4. fn male vol-unteers a seven day course of a 30ug oral dose of ethinyl

oestradj-ol caused a significant rise in the cholesterol- saturation index of

gallbladder bil-e (Anderson, Jamesr. McDonald et aI r9B0). However more

recently, Do,*rn, V{triting, Watts et al (1983) showed that the daily ingestion

of 30ug ethinyloestradiol alone in women had no significant effect on

Iithogenic index. The reason for this sex difference in uncl-ear' but it may

be related to the 503 fal-l in the l-evels of circulating testosterone which

al-so accontpanied the use of ethinyl- oestradiol- in men'

criticism has been directed at the anjmal- studies because of the large

doses of oestrogens required to produce biliarlz J-ipid changes. Also

physiological- changes in natural sex hormones during a normal menstrual

cycle have been shown to have l-ittle (Low-Beer, !Ùicks, Heaton et al- L977)

or no effect (Bennion, Grinzberg, Garrick et al 1981; Whiting, Down, watts

:{r\j

''r)

I

l'

$

I

l

)aJZ

I

l-981, Kern, Everson, Delvlark et aI l9B1; lrlilliams, Scaflion, l4cCarthy

re80).

hlith a variety of slmthetic oestrogen/progestin mixtures Bennion' lbtt arid

noward(I98g) shor¡red that contraceptive steroid use còuld increase both the

molar percent cholesterol in human female gatlbladder bile and hepatic

secretion of cholesterol. Kern, Everson, De l4ark et aI (l-982) showed the

same changes in healthy young $/onÊn taking a contracepti-ve steroid

cqnbination of 50ug of nestranol and lmg of norethnidrone.

Recently Do\,rm, l'ühriting, watts et aI (1983) found evidence in hea]-thy young

h¡ornen that the progestagen, norçfestrel and not the oestroçfen' ethinyÌ

oestradiol is responsible for the iricrease in bile chol-esterol saturation

which acconpanies the use of oral contraceptives. Ttrey suggested that the

overall- oestrogen to progestagen potency ratio of an oral contraceptive may

be rnore irrportant in detennining its effect on the bilia4z cholesterol

saturation index than the dose of each sex hormone'

The origin of the increased chol-esterol secreted into bile is unl<nor,n, but

there are five main possibilities:-

(i) increased dietarlz intake

(ii) enhanced absorPtion

(iii) increased slmthesi's

(iv) increased hepatic uptake of J-ipoprotein cholesterol

I

I{r

T

33

(v) decreased breakdown of cholesterol to bile acids

The first two possibilities seem unlikely. In rats the rate of cholesterol

slmthesis is greater in females than in males. (ltukherjee and Gupta 1967;

carlson, Mitchell, Goldfarb l97B). Preginancy markedJ-y stimuJ-ates hepatic

cholesterol slmthesis in the rat and the saguines fuscj-ollis nronkey

(Feingold, Wi-ley, Moser et al I9B3). Oestrogten aùninistration stirrmlates

the activity of hepatic 3-hydroxy 3-methylg1utar1zl coenzlnle A reductase'

the rate lirniting enz)nìe in cholesterol synthesi-s. However, Turley and

Dietschy QgTg) have clearly shov¡n in rats that the rate of bifiary

cholesterol secretion is independent of the rate of hepatic cholesterol

synthesj-s. Other studies in rats show that a large dose of ethinyl

oestradiot (5mg,/kg/day) increases the specific bj-nding of low density

lipoprotein (LPL) by rat liver menrbráne (Kovanen, Brown, c'oldstein 1979)

and greatly augments the uptake and catabolism of plasma LDL (chao'

Vfindler, Chen et aI 1919). Ttrose findings may be relevant to the

observation that ethinyr oestradiol (150ug'/day) increases the turnover rate

of serum cholesterol in human subjects (Nestel-, Hirsch, Couzens 1965) and

to studies suggesting that plasma ì-ipoproteins serve as the major source of

biliary cholesterol (Schwartz, Halloran, VJ-ahcevic et al I97B; Schwartz'

Berman, Vlahcevic et al- l97B). It seems possible therefore that the excess

biliary cholesterol secreted by users of contraceptive steroids could be

derived from accelerated hepatic uptake and catabolism of plaslna LDL

cholesterol, but direct study of this problem is needed.

tI

l

I

34

The relation between serum lipids and lipoproteins and biliarlz lipid and

bile acid corposition and secretion has been studied.Patients with type fV

hyperlipidaernia appear to have an increased incidence of cholesterol

gallstones (XadziolkarNil-sson, Schersten 1977), Einarsson and co-workers

have described an increase in bil-e acid slmthesis and pooJ- size, especia]-ly

cholic acid (Einarsson, Hellstrsn, KaIIner 1974) increased hepatic HI{G CoA

reductase activi-ty (Angel-in, Einarsson, Hellstron 1976) and increased

chofesterol satr:ration of bil-e. Pregnancy (Williams, Sj-mons, Turtl-e 1976)

and oral- contraceptive steroids (Odell and Mol-itch L974) are associated

with sirnilar changes in serum tipids - an increase in triglyceride

concentration, a slight variabl-e i4crease in serum cholesterol and an

increase in very low density lipoproteins. Elevated high density

lipoprotein (HDL) fevels have been associated wj-th a fower incidence of

gallstone disease (Scragg, Mclrtichäel-, Seamark 1984). The effect of

contraceptive steroids on HDL Ìevels varies with their conposition,

oestrogens causing an increase and progestagen a decrease (Bradley'

Wingerd, Petitti et al 1978). Schwartz, Berman, Vlahcevj-c et at (1978)

reported that HDL chofesterol- is the major precursor for biliarlz

cholesterol and bile acid synthesis,Ì:ut this issue cannot be regarded as

conpletely settled. Further¡¡s¡s the role of lipoprotein remnants and the

rate of metabolism of serum J-ipi-ds in biliary lipid synthesis and secretion

have not been adeguatelY studied.

The possibility of al-tered gallbtadder ftmction in response to oestrogens

has been incomptetel-y studie¿. tn addition effecls of oestrogens upon

gal1bladder storage capacity, concentrating abil-ity and contractil-ity have

not been weÌI chanacterised.

The gatlbÌadder has enormous concentrating capacity (Makhl-ouf 1979) which

has not been studied in intact man except, by choì-ecystography. Concentration

of bile is dependant upon sodium and water absorption' probabLy mediated

35

by the putative sodium pump Na+, K+ ATPase (Van Os and SJ-egers 1971).

Na+. K+ ATPase is an enzyme which suppJ-ies lhe energy for the movement

of sodium out of the hepatic cel-l- into the canalicular bife and is the

putative mediator of bile acid independant flow (Schwartz' Lindenmayer'

All_en 1975, Erlinger, Dhumeaux, Berthel-ot et al- 1970; charney, silva,

Besarab et al- 1974; Simon, Sutherland, Accatino 1977)'

Sodiurn enters gal-l-bl-adder epithel-ial- cells passivel-y across the luminaf

membranes down an electrochemical- gnadient maintained by Na+ exLrusion

across the basol-ateral- membrane by a Na+ K+ - ATP+se pump. Movement of

water is believed to be passive and- secondary to active sol-ute movement.

Cyclic Amp (c Amp) has been propoq"å t" a second messenger for the effects

ofl several mediators (fiood and Svanvik 1983). Secretin and extracts

of isl-et cell tumours bul not CCK'.or gastrin have been reported to

inhibit fl-uid absorption by the isol-ated rabbit gal-l-bladder (Leyssac'

Bukhave and Frederiksen 1974). VIP is a potent stimulant of c Amp

production by human gall-bladder cel-l-s (Dupont, Broyart, Bnoery et aI 1981)

VIp infused intravenousl-y in the cat inhibits gal-l-bladder i/later and

eJ-ectroJ-yte transport and reverses its direction to a net secretion'

Femal-e sex hormones in pharmacological doses have been reported to inhibit

gallbladder fluid absorption j-n vitro (France, Men on, Reay et at 1977)'

36

It is not known v¡trether gallbladder function is influenced by different

phases of the ovulatory cycle. Riegel, Ravdin, Morrison et a1(1935)

studied the corposition of gallbladder bile in \ÀTcrllen at term and found

changes consistent with irrpaired gallbladder concentrating fr:nction' This

effect could be due to oestrogen or progesterone. Historical reports

suggest that bil-e becones dilute in the later phases of preqnancy (Riegel'

Ravôin, !trorrison et aI 1935; Gerdes and Boyden l-938; Potter 1936) ' In the

pregnant hamster there is decreased bile racid independant bil-e ft'ow with an

associated fall in Na*K+-ATPase activity:in liver honngenates (Reyes and

Kern 1970). oestrogen a&ninistratlon qls-o decreases the bile acid

independent fraction of bile and hepatic Na+K*-ATPase activj-ty (Reichen and

Paumgartner L977) -

It is possible that the enlarged gallbladder noted in pregnancy by Kern'

Everson, De Mark et aI (r98l-) coufd be primaril-y due to reduced water

absorption without increased retention of bile acids, al-though this seems

unlikely. Ttre significance of the increased volume of the gallbl-adder in

pregnancy can be interpreted correctJ-y only with additional information

about whether bire acid mass retained in the garlbradder is arso

increased

iì

37

H. GASTROI}MESTINAL PEPTIDES A}JD GALLBLADDER. ¡4CIILITY

Gastrointestinaf peptide effects on extrahepatic bili-ary notil-ity are

multipJ-e and their integration is conplex. It has been generally accepted

that CCK is the candidate hor¡none for gallbladder contraction- (Broden

1958, Sturdevant, Stern, Resin et aI 1973).

( i ) Chol-ecystokinrn

In I92B Ivy and Oldberg used the name cholecystokinin (CCK) to describe the

active pri-nciple responsibfe for the-contraction of the gallbladder' Sone

15 years later Harper and Ra¡rer (1943) showed that intestinal extracts

stimulated secretion of pancreatic enzlnìes and named their active factor

pancreozlzrnin. subseguentJ-y, Jorpes and Mutt (1968) at the Karol-inska

Institute, Stockholm showed that these active principles (CCK and

pancreozlzrnin) were one and the same regulatory peptide capable of eliciting

both contraction of the galtbladder and secretion of pancreatic enzlzmes'Ttte

original peptide of CCK contained 33 anino acids (CCK-33) ' Subsequent'

studies demonstrated that only the octapeptide (CCK-B) located at the

carboxy-terrninus of CCK-33 was required for contraction of the gaj-lb1adder

and relaxation of the sphincter of Oddi. In addition, CCK-B was shown to be

3 to 10 times more potent than CCK-33 in stimulating contraction of the

gallbladder. ccK rel-eased frorn the small intestinal I cells may regulate

gallbtadder contraction (lrliener, Inoue, Fagan et aI tg8r) in response to

Iurninal- stimuti. Guinea pig galibladder contraction in response to CCK is a

result of a direct action on sn¡coth muscl-e cells (Yau, Makhlouf ' Edr¡¡ards et

aI l_973 ) . CCK-induced small bowel- contraction is nrediated via ner:ral

pathways (Hedner 1970). Continued release of CCK frorn upper small bowel may

be stjmulated by slow gastric enptying of solids wLrich may in turn control

3B

the rate of gallbladder contractions. During feeding the gallbladder

contracts all-owing bile acids to enter the intestine to enmlsify fat and

transport cholesterol in rnicelles. Gal-Ibladder contraction is dependent on

the content of the ¡neal- which rel-eases CCK from the jejunum and it shoul-d

therefore relate d.irectly to the rate of delivery of ingested rneals into

the upper smal-l intestine. Itrus factors controlling the rate of gastrlc

enptying could indirectly control the rate of gallbladder enptying.

Thre sphincter of oddi has the potential for altering bile partitioning

between the galtbladder and smal-l intestine. Meltzer (1917) advanced the

concept of reciprocal innervation to oçIain the simultaneous contraction

of the gallbladder and relaxation of the sphincter in res¡ronse to

intraduodenal- magnesium sulphate. 4¿civlaster and Elman (1926) also

demonstrated that sphincter resistance decreased as the gallbl-adder

contracted and that differential- pressures betwen the extra and intra

hepatic biliarlz tree and the gatlbladder could account for gallbladder

refilJ-ing (elman and McMasLer 1926). It is relevant to note from previous

studies that in addition to its direct gatlbladder effect, CCK reÌaxes the

sphincter of Oddi (Sandbl-om, Voegtlin, Ivy 1935, Hong, Magee, Crewdon

19s6).

The role of CCK in co-ordinating galJ-bladder contraction and sphincter of

Oddi rel-axation is well established (V'la1sh I97B). Guinea pig gallbladder

contraction in response to CCK is a result of a direct acl-ion on smooth

muscle cel-ls. The sphincter of Oddi in the dog (Sandbl-om, Voegtlin, fvy

1935) cat (Behar and Biancani 1978) baboon (La Morte, Gaca, VÙise et a1

l9B0) and man (V{hrite and Bourde 1970) relaxes in response to CCK, probabJ-y

by the action of nonchol-inergic, nonadrenergic inhibitory neurons (Behar

and Biancani 1980). However other studies have demcnstated that CCK

increases muscular activity in the sphincter of Oddi in o¡rosstlln a¡td rabbit-

39

species ,(Becker and Ploody I97B; Sarles, Bidart, Devaux et aI 1976; Honda,

Toouli, Dodds et aI f983).

Autonornic control- of the gallbladder plays an inportant role in

potentiation of the pressure effect of CCK and the maintenance of

gallbladder tone (Pall-in and Skoglund 1964).

(ii) Pancreatic PoJ-lpeptide

pancreatic PoÌlzpeptide (PP), a 36 arnino acid polypepti-deris found almost

exclusively in the pancreas (Lonovics, Devitt, Watson et al 19Bl). In vivo

studies j-n humans, dogs and ttre pig have demonstrated that PP

ad¡ninistration results in gaì-lbladder relaxation with increased storage of

bil-e (Lin and Chance L974; Adrian,'.l,Litchenere, Sagor et aI I9B2; Greenberg,

McCIoy, Adrian et al I97B; Greenberg, McCloy, Chadr¿ick et al 1979) -

Although the effects of PP on the biJ-iarlz tree are opposite to ccK' both

peptides rise in parallel after a meal and purified CCK is an effective

hunroral rel-easer of PP in humans (Lonovj-cs, Guzman, Devitt et al- l-980).

( iii )Motilin

Motilin, a pollzpeptide hormone of 22 anrino acid residues has been isolated

frorn the duodenum and jejunum of man and several animals (dogs, Pi9s,

baboons). It is not sjnúl-ar in structure to any mernber of either gastrin or

secretin farnifies of hornrones (Domschke l-977)-

Motilin is released during Phase III and IV activity of the interdigestive

(fasting) myoelectrical conrplex and in man is associated with bile flow

into the duodenum (Keane, Dillagno, Dozois et al- 1980). Periodic

contractions of the gall-blaclder occur in close association with the

interdigestive rnigrating contractions in the stornach and both can

,1

40

simultaneously be reproduced by exogenous aùninistration of nrotil-j-n. (Itoh

and Takahashi 19BI).Thus motilin may regulate biliarlz motility in the

period between rneals.

Pancreatic Pollzpeptide and nrotil-in have litt1e effect on in vitro

preparations of human, rabbit and dog gallbladder muscle strips (Poneranz'

Davison, Shaffer 1983, Lonovics, Devitt, Rayford et al- 1979). Ttterefore

both pp and nrotilin appear to exert their respective actions at sites

remote fron the gallbladder without directly affecting galJ-bladder muscle-

(iv) opioids

Receptors that bind opioids are found on mucosal celÌs, snooth muscle fibres

and on both the body and ternrinals.of nerve cells of the gnrt. At present

there is little data on the distribution of either cells that produce

endogenous opioids or cells that containoplcids receptors within the biliary

tree. Thus, the physiotogical rofe of opioids in reg-ulating biliary

motility is undefined.

Hrptying of the gallbladder during an infusion of CCK-B and adnrinistration

of either saline, morphine, enkephal-in or naloxone (an opioid-antagonist)

has been evaluated in man by real time ultrasonography (t{orobetz, Baker,

McCallum et al IgB2). Saline and naloxone did not alter the normal pattern

of enptying of the gal-Ibladder during intravenous infusion of CCK-8. On the

other hand, enptying during the. infusion of CCK-B was conpletely blocked by

nrorphine or enkephalin. After administration of nrorphine or enkephalin, the

gal1bJ-adder assumed a round shape and its volunre significantly increased-

Vlhen naloxone \^Ias adrninistered after morphine or enkephalin, the

gallbladder enptied rapidly. These findings suggest that the opioids

stinn:Iate the sphincter of Oddi and the pressure of the sphincter

4T

increases. As a result the resistance to the flow of bile out of the .

gallbladder and conrnon bile duct into the duodenum increases. The

gallbladder cannot enpty even though it contracts. Blockade of the opioid

effect by naloxone relaxes the sphincter and the contracting gallbJ-adder

enpties quickly.

I NET]RONAL DSTERMINATVTS OF ÐflRAHEPATIC BILIARY I\OIILITY

(i) Influence of the parasynpathetic autonornic nervous system-

Autonornic contributions to gallbJ-adder tone have been exarnined by nerve

stirrml-ation and pharmacologic methods. Tkre paras]fipathetic nervous system

is clearly involved in the maintenance of gatJ-bladder tone (Pallin and

SkogJ-und 1964) i conversely vagotomy decreases resting galJ-bladder pressure

(Liedberg 1969) Galtbladder pressirie can be elevated by intravenous

acetylcholine and pilocarpine and this effect is blocked by atropine in all-

animal species studied (Halpert and Lewis 1930; Winkelstein and Achsner

1924; Winkelstein and Aschsner 1926; Menguyr Harlenbeck, Bollman et al

r95B).

Paraslnrpathetic receptors appear to be present in the gallbJ-adder and the

paraslznrpathetic system probably interacts with hornronal stimul-i in

regul-ating gall-bladder tone.

Foesel and Sewing (1978) showed that contraction of the gallbladder caused

by a stimulation of its intrinsic paras)¡rTpathetic fibers coul-d be enhanced

42

by a sublhreshol-d concentration of CCK. This observation strongly favoured

a contibuting role of cholinergic innervation in the control of gallbladder

motor function. yau and Youther (1984) nx¡re recently performed direct

transmural fiel-d stimul-ation on guinea pig in vitro gallbladder

preparations and showed acetlychoÌj-ne-mediated contraction. The

contractions were sensitive to atropine and tetrodotoxin, indicating that

the activity of choJ-inergic neurons \^/ere responsible for the muscle

contraction.

In humans,Rock, l4almud and Fisher (1981) showed that sham feeding resulted

in enptying of 50å of galJ-bladder vo'lume. Ttre effect was abolished by

atropine. The anxcunt of bite in the, fasting galJ-bladder is doubl-ed in

humans after truncal vagotomy conpared with selective vagotomy and the

anrount of residuat bile after enptyinÇ is greater (Inberg and vuorio 1969).

Whril-e human gallstones placed in a canine galtbJ-adder usually d'issolve

pronptly one study has shov¡n that truncal vagotomy significantly retards

dissolution (Barnett and Hilburn 1966). Sequestrati-on of bil-e into a large

stagnant gal-Ibladder could result in diminished bil-e acj-d secretion and

increased cholesterol saturation of bile through interruption of the

enterohepatic circul-ation. However, studies have sho'¡¡n a decrease in

biliary chofesterol saturation when the gallbladder leaves the

enterohepatic circul-ation either by disease or surgery (Shaffer and Small

1977; Redinger 1976).

Westphal, Gleichmann and Joik (1931) .reported that electrical- stjmulation

of the vagus could increase water absorption by the dog gallbladder' TLreir

resul-ts were not consistent and nìore recently vagal stimulation in the cat

fail-ed to influence the rate of fluid transport in the gallbladder using a

peifusion technique (Bjorck, Jansson and Svanik 1983) '

43

The influence of vagotomy on the sphincter of Oddi is contnoversial. Some papers

have reported sphincter refaxation. (Dowling I97I¡ Beneventano, Rosen'

Schein 1969). In dogs results are variabl-e (Amdrup and Griffith f970)

while in the rabbit, vagotonqz does not change electrornyographic patterns

(Gerolarni and Sarles L977 ) . In the conscious dog, nretacholine has been /reported to elicit spasm of the colTrnon duct (Lin 1975)

More importantly CCK appears to inhibit phasic contractions and decrease

spincter tone by stjmulation of non-adrenergic non-cholinergic i-nhibitory

neurones (Behar and Biancani 1980) -

(ii) Infl-uence of the slznpathetic àutonomic nervous system

Slzmpathetic stj-mulation by way of the right sptancLuric nerve has been

demonstrated to cause a relaxation of the gal-lbladder and this effect is

most readi-ly seen on a gallbl-adder that is in a contracted state either

induced hormonaly or neuraly. Such adrenergic pathways are nrediated via

beta receptors (Persson Ig12). However the exact functj-onaJ- implications of

this are still unclear.

It has al-so been found that electrical stimulation of the splanchnic nerves

stimulates the rate of water absorption by the gallbladder of the cat' an

effect which can be abol-ished by alpha adrenergic receptor bl-ockade. The

precise site of action of the slznpathetic nerves on water transport by the

gallbladder is urknown. \:art from a possibte direct effect on mucosal

cells, sympathetic nerves may act on l-ocal ganglia to inhibit rel-ease of a

neurotransrnitter which inhibits water absorption. A possible transrnitter

released by locat reflexes is vas6fi&iY&.ina1 peptide (VIP).Vasoactive intestinal-

peptide is a hornrone whrich is a rnember of the secretin farnily of

44

gastrointestinal hormones. Vasoactive intestj¡rai peptide may fi:nction

physiologically to inhibit contraction of the gallbladder. In several

animals (gnrinea pig, o¡lossum, cat and rabbit), physiotogic concentrations

of VIp inhibit the contraction of the srnootlt muscl-e of the gall-bladder

induced by acetylcholine and CCK (Lonovics, Devitt, Rayford et al- 1979) - fn

add.ition, VIP has been isolated fron nerve fibres within the muscfe layers

of the gatJ-bladder of both man and cat (Sund1er, A-l-umots, Hakanson et al

re71) -

The effect of pregnancy, the nrensti'ua1 cycle or female sex steroids on the

autonorn-ic nervous system has not been studied with regard to

gastrointestinal smooth muscl-e activity. In addition no studies have

exarnined the interaction of female horrnones with cholinergic or adrenergic

receptors. Therefore it remains a.possibitity that these hormones produce

some of their effects through autonornic pathways and receptors.

J. TTIE RM,ATIONSHIPS OF GASTR.IC EMPTYING TO GALLBI"ADDM. H\4PTYING

For a thorough understanding of post prandial gallbladder enrptying it is

inrportant to exarnine the conplex regnrtatory mechanism of gastric enptying which

are poorJ-y rnderstood.

The enptying of solids and liguids frorn the human stomach is tightly

regnrlated by chenroreceptive nrechanisms that are capable of sel-ective

response to meal conponents. For exanple, these nrechanisms respond

strongly to long chain but not short chain,fatty acids (Hunt and Knox

1968), to trlptophan but not glycine (Stephens, WooJ-sen, Cooke 1975),and

more strongl-y to HCI than citric acid (Hunt and Knox 1969). The mechanisms

are not only selective but are also quantitative, regutating the quantity

of acj-d entering the duodenum, independent of its concentration (Hunt and

45

Knox 1962) and reg'ulating the number of calories enptying from the stornach

per minute despite wide variations in the cal-orie density of liquid mass

(Hunt and Stubbs 1975). Although relevant chemoreceptors have been

Iocal-i-sed in the proximal small intestine (Cooke I97l) l-itt1e is knov¿-r of

receptor nechanisms through wLrj-ch they control the flow of nutrients frorn

the stomach.

Solids and liguids entering the duodenum rnight trigger mechanisms that

inhibit propul-sive forces. For exanple, both neuronal and hornronal

nrechanisms are known to be capable of regulating the tone of the stomach

(Stephens, Woolsen, Cooke 1976). Thus, the release of intestinal hormones

or the triggering of nervous reflexes by chenroreceptors in the intestine

might slow gastric enptying by djminishing gastri-c tone and thereby

reducing gastroduodenal- pressure gçadients. The final mechanism may

operate through the vagus nerve. Phasic contraction of the antrum is known

to be inhibíted via vagal- mehanlsms by fat, trlzptophan, glucose, or acid in

the duodenum (Kel-l-y and Code l-969; Meyer and Jones 1974; Roze, Couturier,

Chariot et al- I9l7; Thomas, Crider, Morgan 1934). Such inhibition might

reduce propulsive punping by the antrum.

As a motor unit the stomach is classicaly considered as having two

fi.rnctional areas,consisting of a proximal receptacle the fundus and body,

and a distal- punp the antrum. The antrum rnixes and churns gastric contents

and propels them into the duodenum..

Antra1 contractions deterrnine the delivery rate of gastric contents

into the dudenum. Frequency, direction and velocity of antral- contractj-ons

are determined by the gastric slow waves and their superinposed spike

activity. Spike activity is required for contractions to occut (Cooke

ì1

46

19751.

i,ti>te<1 solid liqricì. nea-Is rùay sepclral-e inLo different ptrysical phases i'ùricir

r¡glz each erçty frorn the sto¡iracìr at a¡r indepenclent ra'te' |'icluii-ìs ]eave tþe

stcrnach nore rapicily tl-lall solicis (ileaciing, Toihill, ì''Iclougtrlin et al 19761'

Às tþe1z are oi¡ltiecl into Lile fluocic-¡r.u1r 'ife:' are contiiruously r-eplacecl b1t

nei.;Iy seccetecl gastric juices, liru:i c':>q:osing'tìre reiirainiix; scJ-ic'Ìs to fresir

gastric secre'tions. Liquic',s of Ìri-gh solute conterrt have sl-ot'ret: gar';tric

o-nptying rates tlnn isotonic J-ic;uicl,s (cool'-e 19751. I-io"'¡e-ver tlæ,v qterrerallT

exn¡:ty frcrit tìle sto¡ach at ra-te.s rrci:e raJ-'i-c-t tlìa:'ì fai's or solicls (l'core'

Cristiart, Ocl':,ulrì 19:--11 ).

DigestiÌrlesohc'sclr¡:t;'sllc'\'rer¡-'ro:ai:lyi;ccaus;r-:i:Ì-teatr'ri:t'o:ia-ctsasa-

sie.¿ingi a¡¿la::atUs. SuS¡x:nc-,c-ci -¡rar--tiCleS Ca-rr pass i:rrrouc,Ìr tnr: ¡1'lc-rrrus ollJ-y

i"rjrer-r ìlrey hane irea:r grounii Cior,¡¡i'ct> ¡nrLicles }ess'ijram l-¡il¡t j-n clia;;'retr:r

(i:Þyer, ohashi, .j-e.iÏi et al 1931 ). i']ariicles greater tha;-r -J'iis size are

returned io the antn¡n for furiJrer grilc]inE (ï'l'einer, Graharn' R'eeCy et al

1931 ) . i,lon-d-igestairle sol iCs ,-tå Oto,rght to remaiu in tire stor¡acli r¡-rl-il

tlre resL o¡ li-re rrreal j-s erirptie,r. ,Iiolvever, Ca:nill-eri, l-ìror'm ancl r'aiagelacla

( 1 986 ) usìr-rg non-ciígestil¡l-e laltelled bran rece¡tl)¡ shot¡ed iJrat eiçt}zì-ng o'i

tirese particles is also a function of a¡rtral p=rist:'Isis' \''iÏrile tì-ie

Ial¡eltecj Ì:ra' r..¡as selectiveJ-y retained Ì:y the s'tolr,ach rela'cive to liqriri'

it cl.icì aptlear 'co øriptir at s'iirúlar rates to oiùer solid ccrrr¡;onent:; of Lhe

nrc¡l r¿hich LncÌ l¡cc¡r grorurd j-nto sllall i'rrrticles by the antn:¡':l' In suÌ¡Ì-v¡rt

(¡f ijrese frnd.il.lgs liclt, iìeic1, 1Þylor et aI (ga2) ol'-'served si¡rilar ea¡rtyi-ug

patterns for both larger non-digesti-bte solids and' titurable liver culæs'

If gastric onptying of solicls is res¡ronsi]¡Ie for contj¡rued stj"nulati on of

galJ-biarÌ1er conbraction furen gaIIJ:I¿rdCer re-fill-ing sirould occur rvhen

gastric errptying of sol-ids is; ccxlçIete'

/

47I{ore recently King, Adam, PryCe et aI (1984) have drav¡n attention to the

function oi antral contractions and tLre role tlnt peristaltic activity of

'bhe ctistal stcxnach plays in regulating gastric an¡:tying of solids. Using

real-ti¡ne ultrasound tlrey shorved tlrat ernptyir-rg of gastric crontents tlrcugh

the pyloms terdeci to occr-rr j-n short episocles rvhen the terminal arttrum,

pylorus ancl cluodern:m ryere rela.xed. Ttre authors c.oncluded that this pa.ttern

of nrovernent v,as unlil<ely ¡o be related Lo antral ¡reristalsis or to lce

attrilcuLecl to constant in-uragastric pressure alone. Itrey also obsen¡ed

tlrat r:icst gastric ¡reristalti c cycles vüere accctilpanied by retrognade flot'¿ at

tire Pirlorus.

It is lrcssible iluri a¡i' cÌraäges in ga}IÌ:ladcler einptlrj¡c; se'en r¡iti-r increasei

enc-ìor;eucus a:rcÌ, a¡>lcvj:enolts fcnle ser: ste::oicis relt be seconCary L<-; clerrges in

ças-i::ic anptiri¡q. Ijo';.'eve:: recmt s'tudies sr-rggest tirat gastric e.i'ptying is

not al-terec- i-n eitl:r::r iire seccnC b:1res'Ler of preçrralcl' (Scirade, Pelel'anos,

1.a'¡¡1¡e et aLl 198¿¡| or tire l-utea.l pirase of the ovulatory crTcle (Ilorol.'itz,

t\Þdi.crrr, (l:atterton et al- 19Lr4).

.fi-rus in the last centurythe search for ijre aetiology of gallstones has

goire fuiJ- circl-e. Initial-J-y the'eause of gall-stone foraation vras thought to

relate to irflarLi-iLlation of Ure qallblad-der i+all and inccrnplete gallbLadc]-er

eq:t1ri¡g. I',tren i'c l¿as re¡.lisei that biliary cholesterol- satr:ration v.as

vital for stone fonr,aticn, attentiorr r.¡as directed to the liver. l'he role of

altereC gaJ-lbtaCder motiLity in the genesis of litJrog'eni-c l¡ile and.

cÍrolesterol choleliihiasis is stil-l relatively une>q,olored artcl atternpts to

descri]:e lùis as¡rect of biliary physiology rvill improve or:r unclerstanding

of the nultifac-uorial pathogenesis of cholesterol cholelitiriasis.

i',luch re¡rains to i¡e ]earned a,bout the effects of fernale se>< hornones on

biliarl¿ rnotility and the metabolism of l:iIe acids and cholesterol.

48

CHAPTER II: CURRENT KNOhILEDGE ÀND

UNANSWERED QUESTIONS REGARDING

GALLBLADDER MOTOR FUNCTION

A. Gallbladder Function Current Concepts in

Preqnancy

B. Unanswered Questions Reqardinq Gallbladder

Function

C. Thesis Aims

L

l

ì

I

49

CL]RRENIT KNOI¡ILEDGE AI{D QUESTIONS T]NA}TSV{HìED

A. GALLBI"ADDER FUNCTION - CURREITI CONCEPTS IN PREGNA}¡CY

Studies by the Gastroenterology Unit of the University of Col-orado have

already shovun that after the l3th week of pregnancy (i) the size of the

galJ-bl-adder in the fasting state is twice that of non-pregnant control-s (2)

the tjme of contraction stjmulated by a standard liquj-d meal, is longer

than in controls (3) the maxj¡num percent emptied after the meal is less and

(4) the resj-dual volume after maximum emptying is nearly three times as

great as in controls. rn early pregnancy (first 12 weeks) the only

significant alteration 1s slower emptying after the liqui-d meal. These

alterations in gallbladder function could contribute to precipitation of

cholesterol crystals and their retention in the gallbladder. F\rrther, they

could account in part for the increased bile acid pool sj-ze and for sorne of

the changes in bife acid slmttresis observed in pregnancy.

The interrel-ation of the various conqronents of the enterohepatic

circulatj-on (EIC) of bife acids is exceedingly conplex. However, to

understand chofesterol gallstone formatj-on it is apparent that knowledge of

mechanism altering bj_Ìe acid EFIC is inportant.

The storage of a J-arge proportion of bile acid pool in the gallbladder

ù:ring the night would be oçected to produce fasting hepatic bil-e that isnore lithogenic, raùtereas the storage of only a small propgrtion of the pool

would probably be associated with less lithogenic fasting hepatic bil-e. Inthe latter situation most of the bile acid pool would be nrore or less

continuously circulating thror.rgh the intestine and 1iver, causing

inhibition of bile acid synthesis and a smaller pool size, as fouhd in many

50

patients after cholecystectorny.

If a large portion of the pool is stored in the gallbladder overnight,

synthesis woufd be stjmulated and the pool size increased. On the other

hand, synthesis rate and pool size rnight be deterrnined largely by tJ:te

cornpleteness of gallbladder enptying. If the gal-J-bladder enptied completely,

a large load of bile acid eventually returning to the liver woufd be

expected to suppress synthesis, but if galtbladder errptying were inconpJ-ete

and a substantial portion of the bile acid pool was retained, fess would

return to the l-iver and synthesis would be stjmulated- Results from

Colorado have strongly suggested a central role for the gallbladder in the

,enterohepatic circulation of bile acids and bile acid kinetics and sup¡rort

the hlpothesis that altered gallbladder function in pregnancy nright play an

im¡rortant role in cholesteroJ- gallstone formation (Kern, Everson, De Mark

et a1 19Bl).

The hypothesis described assumes that bile acid absorption efficiency'from

the small intestine is the sarne du¡ing pregnancy, that intestinal transit

of bile acids is not significantly altered and that there is no effect of

pregnancy on the capacity of the liver to respond appropriately to changes

in intracellular bile acid. concentration. Alterations in the intestinal

bile acid absorption or hepatic response are not suspected,but if

experinrental resul-ts i-ndicated otherwise then direct studies would be

necessa-ry. fntestinal absorption of bile acids can be studied by ileal

perfusion, but techniques for direct studies of hepatic response to changes

in intracelluLar bil-e acid concentration in human subjects do not now

exist. Intestinal transit tìrne rnight be slowed as pregnancy progresses by a

progesterone effect or other neurohormonal alteration. It j-s als<-l

conceivable that gallbladder enptying could be inrpaired by reduced gastric

enptylng by food stjmulus to CCK release attd/or reduced production of CCK

/

5l_

by ttre intestinal mucosa. Since the gallbladder response to

intraduodenally-infused arnino acid is diminished in pregrnancy, gastric

enptying may not be an irrportant factor (Kern, Everson, De Mark et al I9B2)

At the tjrne of the present studi-es, rel-iable means for measuring CCK

were not generally available. CCK (or its octapeptide) cannot be

gj-ven to pregnant wonen for ethicaf reasons butrif necessarlz to study the

question of insufficient endogenous CCK vs irrpaired gallbladder response,

it can be given to the other groups being studied, consj-sting of normal

vùornen and those on oral contraceptive steroids

A hlzpothesis of this thesis is that the increased size of the gallbladder

after an overnight fast and after a meal in the latter two-thirds of

pregnancy has two separate causes: (a) decreased concentration of bile,

probably due to inhibition of gallbladder epithelial Na+K+ ATPase activity

resulting from high serum oestrogen concentrations and (b) decreased tone

and contractility of the gallbladder'.due to its exposure to high

concentrations of progesterone.

It is essential to know whether the-increased gallbladder volurne contains

an increased amor:nt of bile acid or whether the volu¡ne increase is due

entirely to water. Oral chol-ecystography has shown decreased concentration

of contrast material by the galJ-bladders of pregnant \^/olnen (Gerdes and

Boyden 1938). One study has suggested that oestrogens inhibit galJ-bladder

mucosal Na+K+ ATPase activity (France, Menon, Reay et aL 1-977 ), the enzlnìe

responsible for its absorption of Na* and water. The concentration of

gallbladder bile ca¡not be neasured directl-y but can be estimated from

simultaneous nìeasure¡nents of volume changes by ultrasound and bile acid

output into the duodenum. The latter can be calculated from data obtained

\,rith the noñ:ábsoibable mâiker téchnique dur-ing Çallbladder contraction.

+

I11j¡

rlql

'u¡

,i

52

Progesterone is a snrooth muscle relaxant. Both pregnancy and progesterone

aùninistration have been shov¡n to inprove gallbladder emptying in several

animal species. This feature of gallbladder physiology wiII be studied in

human subjects by nreasuring the effect of aùn-inistration of progesterone

containing componnds on gal-lbladder emptying in response to various stimul-i

and by correlating gallbladder motor activity with serum proqesterone

Ievels during pregnancy.

B. TJNANSI/ÌERED QT]ESTIONS REGARDING GALLBI,ADDM FUNCTION

fn this thesis the planned studies r¡/ere designed to answer several

questions about gallbladder function, the relation of gallbladder function

to the bile acid kinetics of the enterohepatic circulation, the nrechanism

of observed changes, and the detailed character of alterations associated

with pregnancy and female sex hornxrnes. The following questions were

exarrined: .

(f) fn pregnancy, is there more bile in the gallbladder throughout the day

than in non-pregnant controJ- subjects?

(2) Does ad¡ninistration of contraceptive steroids and oestrogen alone

affect gallbladder function in human subjects?

(3) Do increased volumes of the gallbladder influence biliarlz lipid

con-position?.

(4) Is gallbladder bile in the fastj-ng state less concentrated in

flenopausa]- women before and after taking oestrogens?

/

I

!The increased pool size of the primarlz bile acids and the decreased number

I

53

of enterohepatic cycJ-es dr:ring pregnancy couÌd be caused in part by slow

transit of bile acids through the small intestine, perhaps seconda4z to a

progesterone or other neurohormonal effect on small intestinal snx¡oth

muscle. Thus an additional question asked in thls thesis htas:-

(5) Is small intestinal transit tjme proJ-onged in pregnancy?

As the work of this thesis developed it was observed that the gallbladder

fail-ed to refill in normal subjects ingesting three meals per day. A

pro¡rosed nechanism for this observation \^/as that the sJ-ow enptying of

solids from the stomach into the upper smal-I bowel- controlled gallbJ-adder

enptying and refilllng. Therefore a further question asked \^/as:-

(6) What is tJ.e rel-ationship between the rate of gastric erçtying and

gallbladder emptying?

C TTIESIS AIMS

The aims of this thesis were designed to answer the preceding guestions and

a-re as follows:-

(I) To measure gallbladder storage and enptylngthrroughout the day and night

during ingestion of meals in four similar groups of heaì-ttty, non-obese

v/omen: (a) controls in the follicular and luteal phases of the ovulatory

cycle (b) pregnant wonen (c) post-partum h/onen (d) contraceptive steroid

users.

(2) To investigate the rol-e of the gallbÌadder and small intestine in

:*!il

I,I

tI

;

I

t

regulating the rate of biliarlz lipid secretion and biliary lipid

54

coq)osition.

(3) To deterrnine if chronic aùninistration of'the conjugated

equino-oestrogen premarin alters gallbladder fuction, biliarlz lipid

conposition and secretion in post n€nopausal women.

(4) To deterrnine if gastrointestinal t¡ansit time is prolonged in early

pregnanc)z and to neasure serj-al changes in gastrointestinal transit by

studying the same i-ndividual ttrroughout pregnancy and post-partum.

(5) To measure the ti¡e course of gall-bladder enrptying and refilling after

ingestion of a standard breakfast, to rel-ate the rate of gal-lb1adder

enptying to the rate of gastric emptying of solids, and to rel-ate

gallbladder refilling to both corpletion of gastric emptying of solids and

reduction in hunoral stimulation.

/

55

CHAPTER III: METHODS ÀND RESULTS

A. Gallbtadder Function in the Human Female:

Effect of the Ovulatorv Cvcle, Preqnancy

and Contraceptive Steroids

B

(i) Methods (a) Subjects

(b) Ànalytical Techniques

(c) Indices of Ga].]-bladder Function

(d) Analysis of Data

(ii) Results (a) Effects of the Ovulatory Cycle

(b) Effects of PregnancY

(c) Effects of Contraceptive Steroids

Gal-Ibladder and Small- Intestina]. Requlation of

Methods ( a)

(b)

Subj ect s

Gallbladder Concentration and

Emptying Af,ter IV CCK

Analytical Techniques

Biliary Lipid Secretion

Biliary Lipid Secretion During Intraduode¡gl

Infusion of Standard AminoAcid and Liquid Formula

St imul-i

(i) Methods (a) Subjects and Procedures

(b) Analytical- Techniques

(c) Analysis of Data

(ii) Results (a) Biliary Lipid Secretion

(b) Gallbladder Emptying

(c) Small Bowel Transit Time

(d) Human Serum Pancreatic Polypeptide

C. The Effects of Chronic Oestrogen Administration on

Biliarv Lipid Secretion, Bile Acids and Gallblad<ler

Function in Post-Menopausal blomen

(c)

(d)

(i)

( ii ) Results

56

(e) Bile Acids

(f) Gallbladder Volume and Emptying

with Standard Meals

(g) Analysis of Data

( a) GalJ-bladd'er EmPtYing

(b) Gallbladder Concentration

(c) Biliary Lipid Secretion

(d) Bile Acids

- Transit Time in PregnancYD. Orocaecal

(i) Methods (a)

(b)

(c)

( ii ) Results

Subj ect s

Orocaecal Transit Time

Analysis of Data

E. Co-ordination of Gastric and Gallbladder

Emptyin q After Inses tion of a Reqular Meal

( i) Methods

(ii) Results

(a)

(b)

(c)

(d)

(e)

(f)

(a)

(b)

(c)

Subj ect s

Gastric Emptying

Gatlbladder EmptYing

Gastrointestinal Transit Time

and Serum HPP

Ànalytical Techniques

Analysis of Data

Gallbladder EmPtYing

Gastric EmptYing

Serum HPP

/

57

MSTTTODS AT{D RESTILTS

A. GALLBLADDER. FUNCTION IN ITIE HT]MAN FEMALE: EFFECT OF TT]E OWI,ATORY CYCLE,

PREGNA}TCY AI{D CO}üIRACPTIVE STROIDS

( i) ¡,ETTIODS

(a) Subjects

The characteristics of subjects are shor¡¡n in Tab1e 1. There v/as no

difference in height, weight, percent ideal weight (Metropolitan Life

Insurance Conpany.statisticafsull-etin, f960) age, history of contraceptive

use, or history of pregnancy between foll-icular and l-uteal phase controls.

Although the pregnant group was heavier than the control group' the

difference \^/as consistent with hoimal-fetoplacental developnent. Conpared

with controls,.half as many pregnant \^/olnen had prior history of taking

contraceptive steroj-ds,whil. ari"å as many had had a previous pregnancy.

Wonen taking contracepti-ve steroids were slightly but not significantly

heavier than control-s. Alt of them had been taking contraceptive steroids

continuously for 0.5-6 yr (median 3 yr) and none had been pregnant-

No subjects had any known illness and none was taking any medication

(except contraceptive steroids). Al-I subjects had normal fasting serum

Ievels of aspartate amino transferase, alkaline phosphatase, and

bil-irubin.

Eleven studies were done in 9 wonen taking contraceptive steroids. Ttre

phase of the ovulatory cycle in controls was determined from nenstrual

\

TABLE I

CHARACTERISTICS OF SUBJECT S

Percentideal body

, weightaHeight ( cm ) I^leighr (kg ) %

Prior HistorvPouGr n Age(yr) csb% pB"% prog(nglml)

CONTROLS

AllFPLP

Pregnant [ntorend

AllTM1Tt42TM3

Contrace pt ivee

ST eroid UsersAll

palues givenphase, LP=lutadetermined f.

lable existssteroid use.in all trimeshad been Èaki

164 .4!5 . L764.4!5 .6ß4 .3!4 . s

165 . 016rc7 .6!6Læ ,7!5ß5 . r!7

52.5!4 .752 . O!4 .353.015.2

5s.s!7 .

56.556 .565.0

94.3!7.4% .4!7 .495.2!7 .6

26.91¡. o27 .O!4.026 .8!3 .2

27 .9!Z . Z2r.7t3 .O22 .5!3 .72L.3t2 .6

22TT11

227

108

675088

33332050

(Pg).

273020

557T6038

4.st6.oo.7!0.4s.o1o.z

(-rlco

IU

22

!:9.15.r:5.

269

65..029'.858 .8

!70.7

a

-J',l*. /r110. Ia:L7.4I:25 .6

9 168.81 57.6!5.4 97.B!4.7 25.4!3.g 0

as mean t SD. Abbreviations: n=number of subjects, Prog=progesterone, FP=folliculareal phase, TMI=first trimester, TMz second trimester, TM3=third trimester,rom height-wei ght tabl f the Metropolitan Life Insurance C ompany. No comparableeo

bPtfor pregnant subjects. oportion with a previous histo y of contraceptive (cs)cPropor tion wiEh a previous history of Pregnancy One sub ject was studiedters and I subject was studied in the s econd and third tri

rd

ng contraceptive steroids continuously for 0.5-6 yr.mester. eAll subjects

59

history (ovulation \^/as considered as occr:rring 15 days before nenstruation)

and a single serum progesterone level. It was 0.15 - I.7 ng/nù in the

fol-licular phase, and 2-I7ng/ml in the luteal phase. Al-1 subjects had

regular cycles, l-l- were in the follicular and l-1 in the luteal phase of the

ovulatory cycle. The duration of pregnancy vüas deterrnined from the date of

the last normal ovulatory cyc1e. Sevän subjects were in the first

trirrester, 10 in the second, and B in the third. The contraceptive sterolds

used in this study contained Ìmg of the progestational conpound

norethindrone, and eitJrer 35, 50 or B0mg of the oestrogenic corn¡ronent,

mestranol. Studies were done on days 12 Lo 2I of pill adrninistration.

At 8.00 p.m. the evening before study, subjects ingested half a turkey

sandr¡'rich and 250 ml of whole ndl-k, and then fasted overnight. The day of

study their diet contained 2238 caI, l.5Z protein and 46? fat. They ate at

8.30 a.m., 12.00 noon, 5.00p.m. and 8.00 p.m. Breakfast consisted of one

egg, t\^¡o slices of bacon, two pieces of buttered toast, 100 ml orange

juice, 40 ml low fat nlilk, and 20O ml- of coffee or tea (610 cal, I2e",

protein, 44s" faL) A.l-J- subjects ingested the entire diet as nronitored by a

dietitian

(b) Analytical Techniques

Gallbladder size and enptying were deterrnined by real tjrne ultrasonography

(see þpendix). Ttrey were obtained during fasting at 15 min after the start

of breakfast, and eveq/ 5-10 nin for 90 min. Ttrereafter, sonographs \tere

taken hourly until rnidniqht.

Subjects ate breakfast upright, reclined at a 15-30 degree angle for the

next 90 min, but they engaged in normal physical activity the rest of the

day

60

(c) Indices of Gallbladder .t'unction

Gallbladder volumes brere calcul-ated from sonographs. Fasting volume (FV)

was lhe vol-ume befone breakflast. Residual galì-bladder volume (RV) was the

lowest volume achieved in the first 90min after breakfast' The pencent of

emptying (%E) was equal to (I-RV/FV) x 100%. The average hourly vol-ume (HV)

v¡as the average of gal-lbladder volumes mêasured every hour from 11.00 am to

midnight.

Although changes in gal-Ibladden vofume can result from bil-e entering the

gallbladder, blle leaving the gallbladder, absorption of water, and

secretion of water, onJ-y gallbladder vol-ume was measured. Emptying is defined

as bhe net decrease in volume that occurs over time. Gal-lbladder emptylng

was assumed to obey the finst-order exponential function, VL/Vo - e-(bb)'

where Vt is gallbl-adder volume at time t, Vo is initial gallbladden volume

and b is the rate constant of emptying. Since commencing this study another

group using the more easily reproducible radionuclide scanning technique

with j-ntravenous 99t.-eUlOR, has shown that gal-J-bÌadder emptying after a mixed

sotid/tiquid meal- is best describeà úy a double exponential function sinlilar

to the observations reported here (,Baxter, Grime, Critchl-ey et aI 1985)'

Rate constants of galJ-bladder emptying after breakflast were calculated from

lnllinear regression of gaIÌUf.Oa"" vofume vs time. Serum pnogesterone levels

were measured by radioimmunoassay at Endocrine Sciences (Tarzana, California).

(d) Analysis of Data

Group differences hrene evaluated by Studentrs t-test for unpaired data. The

relationship of indices of gallbladder function to other variables were

evaÌuated by J-ì-near negression analysis. Group differences in slopes of

regression lines were evaluated by F statistics using the extra sum of

squares principle (Draper and Smith 1966) -

It was intended to eval-uate each control subject in both phases of the

óvulatory cycle, each pregnant subject in each trimester o f pregnancy and

/

6

postpartum and each subject taking contraceptive steroids before and after

contraceptive use. Most subjects however decl-ined repeated studies. OnIy 2

pregnant \^¡omen were studied in more than one trinester of pregnancy, and

only 5 were studied post partum. No control was studied in both phases of

the ovulatory cycle and no contracentivl steroid user \^¡as studied before

starting this agent. Accordingly, differences in galtbladder function

induced by the ovulatory cycJ-e, pregnancy, or contraceptive steroids were

anal-ysed by conparing group mean vaÌues, a less sensitive technique to

detect differences than paired analysis.

(ii) RESTILTS

(a) Effects of the Ovul-atory Cycle

Al1 indices of gallbladder frmction \^/ere similar in the fol-licu1ar and

luteal phases of the ovulatory cycle (Table 2). Neither the day of the

ovulatory cycle nor serum progesterone level- correlated with any index of

gallbladder function in the control ?roup. Accordingly, data frorn control-

subjects were pooled for conparison in other groups.

(b) Effects of Pregnancy

Fasting, residual and average hourly volunes were increased in all

trirnesters of pregnancy (Table 2). In addition, gallbladder volume was

significantly greater in pregnant \,vornen at each hour of the day (Figure 3).

TLrus, bile retention in tfie gallbladder $/as greater in pregnancy at all

tirnes. Fasting and residual gallbladder volume (Figure 4A and 48) increased

linearly during pregnancy from the first to third trinrester. Thre l-inear

and residual gallbladder volunes corre lated directly

and significantly with week of pregnancy and serum progesterone levels

INDICES OF GALLB

FV(ml )

t7 .2!5.216.81s .818.014. 5

NS

TABLE IT

FUNCTION DURI

Earl

0.003

0.006

0 .00s

0. o2z1o. oo:NS

NG INGESÎION OF

Indices'of

-1b (min )v LaÈe

A REGULAR DIET

gal lbladder funcEion

"LE,"L)Group n

CorltrolsAlrFBLT

IPrdgnant r,romen..

LADDER

RV(ml)

4.2!4.3!4.7!

NS

21

9 .z!3.9Qs.oot6.7!2.5<0.049:7!4.5

(0. oor0 7t3.5

.0014o

5 .O!4.7NS

8 0, 02zto. oo¡0 0.02010.0067 0.02410. oo5

NS

0.00910.0020.ool!o.ooz0.01110.002

NS

HV( ml )

3.511.¿. o1r.¡ . r1r.

NS

221111

74.L!7L.2!77.0!

NS

1

9.3

7.2

6.6

L2.3

1

IIS+

NS

69 .9N

7 L.8N

70.8

1

01

S1

S+

0.005

0.022!NS

0.0251NS

0.0261NS

'0.01810.003NS

1

L2.214.98.6

683

Alla 25

7

10

8

@. oorraceptive-steroid users

9 23.8!5 .2< 0.01

I0I010+

30. 5 Ol

PTM1

PTM2

PTM3

p

onE

AltP

<0.23.7<0.

33.7

32.4

016.349.0019.0

0.005<0 .0

0.007N

N0.00/+

0.00150.002

0 .002

0.002

7.0(0.7.4

<0.8.3

<0.

7.4o

5

orN

1: .0Q01:3.gg6:3.0901-3 .4001c

<0.005

o.01.010.002NS

N67 .0

80.2115.8 3NS

st¡. g

NS,

Values given as mean Ivolume, b=raEê consEanEwomen. The p value undphases. All oÈher p val

SD. AbbreviaEions: See legend forof . emptyirg r -

"/.[=percenE ãmptied,er the conErol data refers Eo theues refer Eo the comparison of the

Table l. FV=fasÈing volume, RV=ÍêsidualHV=hourly volume. ãTotal sÉudies j,n-iZ-comparison of follicular and luEealrespective group to all conErols.

63

(Figures 5A and 58).

Average hor:rly volunes did not correlate significantJ-y witJ- either week of

pregnancy or serum progesterone levels (Fign:res 4C and 5C).

Gallbladder enrptying after breakfast is shown in Fig'ure 6. T\^/o rates of

gaÌlbladder emptying were identified. The initial rate of emptying to 50%

of fasting volune, was identical in pregnant and control subjects

(-0.022/nin and -0.022/nrin respectively; p: NS)- The second rate of

entptying in the pregnant group was one-half that of the control group

(-0.004/min vs. -0.009/min, respectiveJ-y; F(n1-2, n2-2) 8.5f4; p (0.005).

Four women \^/ere studied in the third trimester of pregnancy and again after

delivery. Fasting, residual,and hourly volunre decreased in the postpartum

period in each case (Tabl-e 3). Gallbladder vol-umes returned toward normal

as soon as 2 weeks postpartum. In al1 subjects only one rate of enptying

was observed after breakfast (Tabte 3) One subject, KLic, was studied at 18

weeks of pregnancy, 2 days postpartrìrô, and 4 weeks postpartum. At 2 days

postpartum, aII gallbladder volumes-were Iarge and enrptying was extremely

slow (Tabl-e 3). Throughout the day her gallbladder achieved only 48?

enptying. This marked degree of bile retention and slow enptying rate

approached the normal- range 4 weeks fater.

(c) effects of Contraceptive Steroids

Fasting gaì-ì-bladder volume was increased in this group, but residual and

hourly volurnes were not (Table 2). Early and late rates of gallbladder

enrptying were alnost identical to control (early =-0.022/ntn and late:

-0.0l0/nLin)., The anx¡unt of c-restrcx¡en in Lhe preparation did not correl-ate

64

mlt2

4

Pregnont (ZSI r¡Gontrol þZl æ

[ ,,,rem t SEM

t.t

"rl_. i\

t

lt

f,'+

Jt¡*

t¡-J-_¡,

08AM IO I2PM

FIGURE 3:

2 ...4

Time

681012

The hourLy gallbladder volumes ofpregnant and control subjects areplotted againsE Iime of day. The solidbars on the time axis represent periodsof meal ingestion. The difference ingallbladder volumes between conCroland pregnant subjecEs hras significantaE each hour of the day and nighc (* =

p (0.0f). Note the very small change

in volume after meals. SEM = standarderror of the mean.

/

,Ì!.

65

A.

ml50-

40

30-

20-

ro-

aa

a

a

a

a a a

aaa a

aa

a

a

a

a

a

a

o

a

!=.48P..o2

FIGURE 4A:

to 20 30 40

Week of. Preg noncy

Fasting gallbladder volumes are plottedagainst week of pregnancy. Fasting volumes(panet À) significantly correlated withweek of pregnancy.

E = mean t standard deviation of controls-

66

RESIDUAL . VOLUMEB.

m20

o

a

aaa

aa

a

ao a

a aa aaaB

I

30

aa

a

aa

a4 r- .54

p<.01

ìo ?oWeek of Pregnoncy

40

Fiqure 48: Residual gallbladder volumes areplotted against week of pregnancy.Residual volumes (pane1 B)

significantì-y correlated with week

of pregnancy.

-rt = mean + standard deviation of

controls.

'lll.Flriþi:1

67

cml

ró

r =.o9p=ns

arta

Ia a a

I a

aa

aa

aOaaa a

o

a

a

oa

aa

a

4

I

40I

30¡

20¡

r0

Figure 4Cz

Week of Pregnoncy

Àverage hourly gallbladder volumes are

plotted against week of pregnancy.

Average hourly volumes (panel C) were

not significantly correlated with week

of pregnancy.

r = mean t standard deviation of controls

A.

ml

68

FASTING VOLUME

50-

40-

20-

r0-

oa

a

a aa a

a

aaa

aa

30 a

oo . a a

tra

aa

otj

o

@

a

oo

r=.46p<.o2

20 40 óo 80 ro0 120 t40

Serum Progésterone (ng/ml)lóo t80 200

Figure 5A: Fasting galJ-bladder volumes of pregnant

(a) and control (0) subjects E:" plotted

against serum progesterone leveJ-s.

Fasting volumes directly correlated with

serum progesterone levels-

69

B

ml

tó

4

a

a a

a

ao

aa

t2

a

a8a

a

a

aa

o@oo

@

Q.^ú'o%

aa a

a

a

a

a

1

ø

o

r -_ .57P < .ooz

20 40 óo

erum

BO ì00 r 0 140 ìóo lB0 20c

Progeiterone (ng /ml.)

Residual volumes of pregnant ( O )

and control (O) subjects arê

plotted against serum progesterone

levels. Residual volumes directly

correlated with serum progesterone

IeveIs.

S

Figure 58:

a

70

c.mlró-

a

r=.28P= ns

aaa

a

at2-

aa

a

aa

a

a

aat

a

a

aooo

8-

4-

aa

a

o.a

o

o9O

#20 40

Figure 5C:

óo

Serum

80 100 120 r40 tóo r80 200Progesterone (nglml)

Àverage hourly gáÌJ_bladder voLumes ofpregnant (o) and control (O) subjectsare plotted agáinst serum progesteroneLevels. In pregnant women averagehourly volumes did not directJ-y correlatewith serurn progesterone J.eveÌs.

m

30b= -.O22 min-l

7I

50

Time (min

Pregnont (ZS) o<Control (22) æ{ ^^ro*

r sEM

oEtr

9-I

b= -.0O4 min-l

b= -.009 min-l

70 90 ilo

b= -.O22min-l

3to 30

FIGURE 6:

î

The gallbladder volumes of pregnanC(a) and control (O) subjects before andafCer breakEast are plotted againsC Cime.The late rate of gallbladder emptying,calculated by In/linear regression ofgallbladder volume vs. time, is slower inpregnanc sub jecEs ( F ( r,t _Z ,n2_21

B ' 513;

p ( o.oo5).

.-

38.0

34.7

28.3

48.7

22.0

15.8

9.9

26.L

20.6

5L.2

22.0

15.1

6.8

8.2

16. I5.5

13.5

6.3

7.8

3.0

0.021

0.020

0.012

0.014

0.025

0.013

0.020

0.016

0.023

0.006

0.025

2.1 '

5.1

5.0

26..3

5.5

ÎABLE' III

INDICES OF GALLBLADDER FUNCTION IN POSTPARTUM I^IOMEN

l.leek of SEudy FV (ml)

SubjecE "Pre Post Pre

Indices of. gallbladder funcEion

RV (ml) HV (ml) B(min-1

PosE Pre Post Pre PosE Pre PosE

)

AMil

DFiC

MSco

SMac

KLic

34

28

37

39

18

8

0

0

0

3

L

9

8

4

0

4

4.0{N

0

13.9 5.1

3.3 26.3

3.2

Abbreviations:' See legend for Table 2; Pre = week of pregnancy, PosE = ldêêk afEer delivery

\

73

oo a

af

o

a

a o ox

ot

^ o

o Control. Pregnonl

'cs^ Postportum

30

AKLic

/oEf

I->loTq,o)o(,

oa

Io

ao

.Ooo

o

FIGURE I z

x

oo

T

oa

. ro 20.

Residuol Volume (ml)

¡d

"u

Average hourly vt¡Iumes ( HV ) are plottedagainsr resiclual volumes (RV) from all63 sLudics (22 controls, 25 Pregnantwomen, 5 postpartum women' and ll contra-ceptive sceroicl users) - The linear relation-ship with sul-rjccI Kl,ic (-) was HV = 0' 78 RV +

0.3 (r=0.88, p < 0-001) and without KLic(--) it eras HV 0.69 RV + 0.6 (r=O'72,

p ( 0.001). CS = rdomên taking contraceptives'Eéro id s'.

I

I

74

with any index of gal-lbladder function-

In alt 63 studies, hourly and residual volumes h/ere sirnilar (Figure 7)

Ttrus, t11e residual volure after the norning nreal- approximated the hourly

vol-ume the remainder of the daY.

B. GALLBI"ADDER. AI\D SMALL INTESTINAL REGUI,ATION OF BILIARY LIPID SECRETION

DI.]RING I}üIRADUODMüAL INFUSION OF STAT\DÀRD A}4INO ACID A}üD LIQUID FORMUI'A

ST]IqULT

(i) Methods

(a) Subjects and Procedures

Five healthy non-obese \^/ornen', aged 2I Lo 28 were each studied twice- After

they fasted overnight, a triple lumen polyvì-nyJ- tube was passed through the

nose and positioned in the duodenum by fluoroscopy with genital shielding

so that the proximal infusion orifice was adjacent to the anpulla of Vater,

12 cm from the distal- coll-ecting orj-fice. Thre third lumen was filled with

mercury frorn proximal to distal orifice and seal-ed at both ends to aid in

fluoroscopic A-ui-dance. After the tube was in position, either a rnixed am-ino

acid solution with 5mg BSPr/IOO ml as marker or li-quid fornml-a with

beta-sj-tosterol as marker was continuously infused through the proximal-

lumen (See Appendix, Tabte Ia). The amino acid sol-ution cont-ained 5?

(wt,/vo1) gJ-ucose and 4 .3% wL/vol of rnixed amino acids (Shaffer and SmaII

Lg77-). Each litre of liquid formula.contaj-ned l.32 g of powdered skinrned

milk, Bl-0 ml- of distil-]ed water, I4O ml- of polycose (Ross Laboratories,

Cofumbus, OH) and 550 ml of corn oil. The rnixture was sonicated with a

sonifier cel-] disrupter (Branson Sonic Power, Plainview NY) for 30 min just

beforc infusion. The resultant uniform emulsion was agitated every 30 min

..L

ili'tli¡

T

li

:1

*

II

I

75

throughout the study. The concentration of beta-sitosterol- in the infusate

\^/as fiìeasured in aliquots taken every hour during the period of infusion and

was constant. Liquid formul-a contains 40% of calories as fat. It has three

tj¡res nrore carbohydrate, tkrree times more calories and is slightly nore

hypertonic than the amino acid solution-

In earlier studies , ]10-20e. of subjects beca¡ne nauseated and vornited during /

infusion of the arnino acid solution, (Kern, Everson, De lt¡lark et aI l9B1).

To ensure paired data, each subject was first studied with arnino acid so

that only those completing the amino acid infusion without incident were

studied with liquid formula. TWo of seven subjects did not conplete the

anr-ino acid study because of nausea and vonr-iting. Thre interval between the

two infusions was 5-l-0h. Duodenaf'bile was continuously aspirated from the

distal orifice at a rate of O.5ml-/rnin and each 30-min sanple was treated

separately. At the bedside, du.nlicate aliquots of each sanple were

extracted with 2:l- chloroform,/nrethanol for phospholipj-d measurenent whil-e

other aliquots were refrigerated at 4 degreesC for analysis of cholesterol,

bile acid and markers. ReaI time sonographs of the gallbJ-adder were

obtained every 30 rnin by using an ADR model 2l3l- real-time scanner with a

33.5m Hz multiplexed linear array transducer, l-3.5cm length(Advanced

Diagnostic Research corp, Terçe, AZ) (See epp,endix). Bl-ood samples for

measurement of human pancreatic pollpeptide were drawn when sonographs were

obtained.

SmaII intestinal transit was nìeasured by the factulose breath test (Bond

and Levitt 1975) (See eppendix). At the 5th h of study J-Og of l-actulose in

t00m1 of water was given as a bol-us through the distal- orifice- Breath

sanples for measurement of hydrogen \^/ere collected before and every 15 rnin

for 4 h after the lactufose was given-

76

(b) Ana1ytì-cal Techniqes

Infusate and each 30 min sanple were analysed for bile acid, phospholipid,

cholesterol- and marker. Bile acid concentration was neasured

spectrofluoronretrically by the enzymatic method of Talalay (Hurlock andI

Talalay 1957), phospholipid colorirnetrically by ttre method of Bartl-ett

(Bartlett 1959) and BSP colori¡retrically (Seligson, Marino, Dodson 1957).

Cholesterol- and beta-sitosterol \¡/ere measured by gas-liquid chromatography

by using coprostanot (5-beta-chol-esterol-3-ol) as internal- standard (Kern'

Eriksson, Curstedt et al 1977). The arnino acid sol-ution contained no

cholesterol- or phospholipid. Li-quid formula contaj-ned chol-esterol (

0.lunrol,/nù) and phospholipid ( !.]unrol/mf ) The cholesterof and phospholipid

concentration of each duodenal sanpJ-e was corrected for infusate

cholesterol and phospholipid (Sée þpendix) .

Based on previous studies (Grundy and trletzger L972, Grundy, Ahrens, Salen

1968) it was assumed that negligible cholesterol- and beta-sitosterol

absorption occurred over the l-2crn perfused segment of bowel.

Gallbladder volume was measured from each gallbladder sonograph (see

append.ix). Serum human pancreatj-c polypeptide (HPP) level was deternrined

by radioinrmrnoassay (see appendix) .

Srnall- bowel- transit tirne was defíned as the time of rise in breath hydrogen

concentration fron base line' (see appendix). In each ex¡rerJrnent, this was

subjectively evaluated by eight observers blinded to the resul-ts of

galJ-bladder enptying and biliary lipid secretion. Transit tirne was

deterrnined fron the appearance of the plot of hydrogen concentration (parts

per rniJ-lion) Vs, tilrie, and re¡rorted as the nrea¡r (+/- SD) of these eight

77

estimations

(c) Analysj-s of data

Differences in gallbladder enptying, small bowel transit tjme serum levels

of human pancreatic poJ-lpeptide, mean bil-iary lj-pid secretion rates and

mean molar percent cholesterol during the two infusions were analysed by

paired t tests. Data frorn afl five subjects were included in all

statistical analyses. Biliarlz lipid secretory relationships during each

infusion were evaluated by linear and non-linear regression analysis;

linear; y:ax + b; non-l-inear; y = x/(b + ax), talhere y and x are hourì-y

lipid secretory rates and a and b are constants-

Investigators have usualÌy ignored biliary lipid oqtput during the first 4

h of study, because of its fluctuations and have calculated secretion rates

only after this period. Accordingly the results of this study were divided

into the first 4 and last 6 h.

RESULTS

(a) Biliary Iipid Secretion

With both infusions, secretion rates were fower in the l-ast 6 h than in the

first 4 h due to the high secretion rates in the first 2 h after initiating

gallbladder contraction. During both the first 4 and last 6 h of study, the

mean hourly secretion rates of bile acid, phospholipid and cholesterol

showed considerable variability but were greater during the liquid formula

than during the arnino acid infusion (Table 4) -

TABLE IV

BILIARY LIPID SECRETION RATES

BA

LF AA

ch

O First 4h

AA

t,81S*¡O¡1,895+8492,353+82tL,479:470

902=343

pmol /h

1o.ozs

1o.oz

7 6L!27 ZA868119799511 7gO1601139190811035

4gL! 9534s!2L8546.!245 .._

246!rL9r+alros

LF

lltlgn1. oo¡1¡sor, oz¡l¡go

6æ=357ogtlu t

576!nt6ß!L¿+4aor-1r azoatlu o654!206

PL

¡¡mol /h

( 0.00s

Z- o. oor

AA

ß7!23ßz!7 8rgtlt trrslss

sslzt

stlzstglzgw!z+r95:7 2+sltl

¡:rnof /h

< 0.0s

LF

L57282303t22198

L2345

P

j++

it

24233

zt7131222

9359

{@

t Last 6h

I2345

t,zsaI 382L,258+ 50St,zø5i 399L,382: 861

gg0rt ,020

2,0843,2052,6903,8884,527

95L1,151

71+0t,042L,745

z¡B! e3rg:1 s2zogjros207!ßsrsllts+

1tJ11

oqlzt1 36j38zos!¿rtrzaJ¡r-razlo g

P <0 .0s

BA, total bile acid; PL, phospholipid; CH, cholesterol; AA, amino acid infusion;LF, liquid formula infusion.osecretion rates measured during the firsf 4h of infusion of stimulus

Secretion rates measured during the last 6h of the infusion of stimulus. The higher.secretio' raiås during the firãt 4h of. stimulus infusion are due to the high secretionrates that occur in tñe first Zlt af.ter initiation of gallbladder contrac{ion..

t9h

The rates of bile acid and phospholipid secretion were increased more than

that of chol-esterol during liquid formul-a infusion, resulting in a lower

nrolar percent cholesterot than during the arnino acid infusion in four of

five subjects during the first 4h (p ç 0.05) and in all subjects during the

l_ast 6h (p < 0.05) (Figure B). The increments in chol-esterol secretion

coi-ncident with increments in either bile acid or phospholipid secretion

were far smaller during liquid formula infusion (Figure 9 A{). For these

reasons, biliarlz lipid secretory rel-ationships were quite different with

the two infusj-ons. More chol-esterol was secreted ¡rer nr-icrorole bile acid or

phospholipid during the arn-ino acid infusion. Ttre best fit of the conrbined

data (both arnino acid and liquid formula for chol-esterol vs. bile acid,

cholesterol vs. phospholipid or phospholipid vs bil-e acid) was the equation

for a rectangular hlperbola, y:x/, (b + ax) (TabÌe 5). However as bile acid

secretion rate increased there was considerable divergence of cholesterol

and phospholipid secretion, CH Sec-max : 298 and PL Sec : 5555 (Tab1e 5) '

As bile acj-d secretion increased t-here was little change in the ratio of

phospholipì-d to bile acid but the ratio of cholesterol to bile acid

decreased considerablY.

(b) Gallbladder HnPtYing

In each subject galJ-bladder enptying was more compl-ete with liquid formula

infusion during both the first 4 (p.<0.05) and Iast 6h (p<0.02) (Figure

l0). Gallbl-adder volumes cluring amìno acid infusion fluctuated considerably

and there were periods of apparent refilling. On the other hand, during

liquid fornml-a infusion gallb.Iadder emptying \'\ras pronrpt and more complete'

and showed li-ttl-e fluctuation (Figure tl)

In atl- studies including both amino acid and l-iquid formula infusions,

there were 23 periocls, exclurlìng tle initial hour in wh'ich gallbladder

/

80

A. 0 to 4 HOURS B. 4 ro l0 I-IOURS

CHMd 9b

1-

2-

rrcung B:

6-

CH,$d %ó-

2-

AA LF AA I-f

The mean molar percenE cholesterol(CH mol %) hras lower with li-quidformula during Ehe firsE 4 (panel A)and last 6h (panel B) of infusion. AA,amino acid infusion; LF, liquid formulainfusion; Different slrmbols representdj-fferent subjects

1-

..,,.i$iï$!,i , . ,&?&ff[fi..

B1

pM/hA

I ,t tt

,,I ,-

,

ld: Çfl =.Oó9 BA- 9.95, r - .8ó

LF: CH =.018 BA + 84.23 , t -.15

8000

AAr---.-t2"1'

IFCH J

I

2000 4000 óooo

BA {¡¡Mlh I

Figure 9A: Hourly secretion rates of

biLe acid (BA) are plotted

The sol-id regression l-ines

of the data to the equation

y and x are secretion and a

chofesterol (CH)

against each other.

represent the fit

y=ax + b, where

and b are

constants- The 952 confidence interval for

the slope of each regression is given by the

dotted lines. Increments in BA secretion were

associated with greater increments of CH

secretion during AA infusion (p=52) than

during LF infusion (n=57).

/

82

ttw/hB

CH

t

Figure 98:

ftI

AAt,t , t

It I, _----e

ê

tF

aa

aa

taaÀÀ:

LF:

CH -.318 PL+ 13.81, ¡-.87

CH - .09ó PL+76.78, r-.16

500 r000 r500

PL lpwth I

Hourly secretion rates of cholesterol- (CH)

and phosphoJ-ipid ( PL ) are plotted against

each other. Increments in PL secretion

were associated with greater increments of

CH secretion during AA infusion (n=47)

than during LF infusion ( n=58 ).

/

r'1 .. ti..*i j-:

trM.lhrsoo c

rooo

B3

a

aa

aa aa aaa a tF

AA: P[- .21 gA- 95.25,,r-.fö[i-: P[-.15 BA+ 22ó.58, Í =.71

2000

BA (øMlh )

Hour1y secretion rates of bile acid (BÀ)

and phospholipid ( PL ) are Plotted

against each other. Approximately the

same relationship was observed between

PL and BÀ secretion as for PL and CH

during infusion of either AA (n=47) or

LF (n=58 ).

a

a

a

a

,aa

aaaa

aa 1

Pt aa

a

u

aaa,

soo

a

AA,a

t

Fiqure 9C:

B4

TABLE V

REGRESSION ANALYSIS OF SECRETORY RELATIONSHIPS

OF COMBINED DATA FROM AMINO ACID AND LIQUIDFORMULA INFUSIONS

oLqua C rons

y=xl(b+ax) y=.**b

CH-BA T RSS$ 2.19

298

2.ro

269

4.77

5,555

2.55

2.38

4.26

CH_PL

PL-BA

ch Sec llmax

RSS

Ch Sec max

RSS

PI- Secnì¿t x

CH, cholesEerol; BA, bile acid; PL, phospholipid.oy="* + b describes a linear relationship beEween x andv.

v describes a hyperbolic relaEionshipb+ax

between x and y.f fh" secretion raEe (micromoles /hour) of Ehe Iipid toEhe left of the hyphen is always plotted on che y=axiswhile thaL of the lipid t.o Ehe righE of Ehe hyphen isploEted on Ehe x-axis.$nSS, residual sum of sqaures x 10-5. The equation EhaEbeEEer fics the daEa is the one wiEh Ehe lower RSS.

Jl Sec-^., is Ehe maximum secreEion raEe (micromolars/hour) ofy-axis'^lipid as the secreEion ra[e of Ehe x-axis lipidapproaches infinity.Sec---- equals I : a in the equation, y = x/(b + ax).max'.

x

ti

85

Aml B

O-4 Hours

AA LF

4 -lO Hours 1

LF

Gallb1adder volume (millitres)during both the first 4 (Pane]- A)

and last 6h (panel B).

AA

¡IGURE 10:

O()

ml Subject I

ml

¡0 I

FIGURE ]-I:

Subject 2

26Time (t' )

Subject 3_-__--AA

-tFt îç,r.i: j: ?¡l ¡ ! r

li ,q,lif.,ii{.,i | ¿

?¡i¡lri?

,¡,a,.

î,oI

òaa.o I

öoJ

þ\^^ ?

ai

2 2ór0

Subject 4 Subject 5

,t¡'¡

6t0 6loTime (¡ )

Gallbladder volume is plottedagainst time. Volumes during bothamino acid (dotted line) and liquidformula (solid line) infusion are shown

for each subject.

rft.'ì f I

¡I l'r't

/

-.¡:j;¡ii.r.:,

87

enptying \,\¡as 5ml or greater. During 22 of these periods there hlas a

concornitant increase in bil-e acid output.

(c) Small Bowel Transit Tinre

In four of five subjects, sma1l bowel- transit tjme was slower ù:ri-ng arnino

acid infusion (Tabl-e 6).

(d) Human Senrm Pancreatic PoJ-lzpeptide

fn four of five subjets levels of pancreatic polypeptide were two to

threefold hi-gher during both the first 4 and l-ast 6 h with liquid formula

infusion (Tabfe 7). Subject 5 had no change in pancreatic polypeptide

Ievels from basel-ine with either infusion. Nonethefess, this subject had

findings which were sjrnilar to. tþe rest of the group: increased gallbladder

enptying, faster intestinal- transit and increased bile acid secretion in

response to liquid formula. In the other subjects, the pattern of the

pancreatic polypeptide response was that of a sustained elevation from base

line with minimal fluctuations.

C. TTIE EFFECTS OF CHRONIC OESTROGH\ ADMINISTR¡IIION ON B]LIARY LIPIDS,BILE

ACIDS AI\TD GALLBIÄDDM. FUNCIION ]N POST-ME}JOPAUSAL h]OMEN

(i) Methods:

/

(a) Subjects

BB

TABLE VI

SMALL INTESTINAL TRANSIT JIMES DURING

INFUSION OF STIMULI

Transit time, min

Subjects AA LF

Mean!SD

1

2

3

Y!Y+9ol o991 8

240! gI45! 0

47!L545! 0

2t! 8

68J B

3ot oI5

P (o.r:

Mean l standarcl cleviation of che transittime as cle[ermined by eight observers'oNo hydtogen rise from base line was

derected ãfc". 240 min. Since this subject

1

89

TABLE VII

HTJMAN PANCREATIC POLYPEPTIDE RESPONSE TO

INFUSION OF STIMULI /

HPP, pBlmlFirst 4h LasÈ 6h

SubjecE AA LF AA LF

1 g]^lzr

2 1501603191161264!LO422t!2621871 80

26! 3

g¡1ro-LO8!22

iozl:s2r13

424!94198159

2ß!86zæ!97:ajrr

3

4

5

91136

$!ßzB! 7

t 56!361

rlir

P ( o. ozs ( o. oos

HPP, human pancreatic polypepticle; AA,infusion; LF, liquid formula inft¡sion.

amino acid(mean+SD)

90

Nine post nenopausal wonten, aged 28-47 were studied off and on Premarl¡(Ayerst)

They had been taking conjugated equine oestrogens for at least 4 weeks

(nrean dose l-.39 mg dail-y) but no other drugs. To ensr:re paired data each

subject was first studied at the end of four weeks oestrogen abstínence and

again after six weeks of oestrogen intake.

(b) Gallbladder concentration and enptying after fV CCK

On day I of the study a triple l-u¡nen tube was positioned by fluoroscopy

after an overnight fast. The proximal- infusion orifice was adjacent to the

anpulla of vater, 12 cm from the distal- orifice. After the tube was

positioned, distill-ed water with the nondiffusable marker BSP (5 mgrlnù) was

infused at a rate of 3-4 mlrlnin. Duodenal bite drained continously by

gravity frorn the distat orifice: The gallbladder was stimulated to contract

by continuous fV CCK-B infusion .(.02 ug/kg/h) over 90 rnin and gallbladder

bile was aspirated for cholesterol saturation index, bile acid distribution

by GLC and cumul-atlve bil-irubin output. Sj-multaneously, the arnount of bil-e

oçelJ-ed and the rate of gallbladder errptying vüas estimated by reat time

ultrasound. Five min sanples of bile were coll-ected for 90 rnin. Aliquots

were taken frorn vortexed samples and the residual bile was retr:rned to the

duodenum.

(c) Analytical- Techniques

Each 5 rnin sanple was analysed for bilirubin by the method of Rand and Di

Pasqua (L962).

The concentration of bilirubin in gallbladder bite was determined as

follows.

by

91

The 5 rnin collections of duodenal bile are anal-ysed for bilirubin output

bili (nqlsnù ¡ = (B)Dx (M)I x R

(M)D

where (M) D= duodenal marker concentration (B) D = duodenal bil-irubin

concentration, (tq)I : infused marker concentration R= mLs markers solutions

infused over 5 min. Since gallbtadder enptying follows a first order decay

fimction,the cumulatj-ve exponential function Bt : ç(l-e-b(t-t.I,) ) describes

the output of galJ-bladder bil-irubin into the duodenum, where Þcumulative

rnilligrams of bilirubin put out at tjrne t, TL time tag in billirubin

output, C:total mitlì-grams of bilirubin enpti-ed, and b=the rate constant at

which galJ-bladder contents enter the duodenum (when galJ-bJ-adder errptying is

conplete b: the rate constant of gallbtadder enrptying). Ttre linear

parameter, C and the two non-l-inear parameters, b and TL are estjmated frorn

conputer analysis of the plot of cumul-ative bil-irubin output versus time.

Since the amount of bilirubin ejected from the gall-bladder, C and the

volume (VE) of bile ejected from the gallbl-adder can be calcul-ated one can

estimate the concentration of gallbladder bilirubin (B) GB by calculating

(B) GB = CNE

(d) Biliary Lipid Secretion

Liquid formul-a (see appendix) was infused following 90 rnin of the IV CCK-B

stimulus. The formul-a was infused over B h through the proximal port with

beta-sitosterol as a marker. Duodenal bile was continuously aspi-rated from

the distal- orifice at a rate of 0 - 5 ml-rzrnin and each 30 rnin sanple was

analysed for biliarlz lipids. (see alTerrlix) Real tinre sonograhs of the

gal-lbladder were obtained every 30 min and vol-unres nìeasured (see

/

i)

92

appendix).

On day 3 another sanple of gallbladder bile was col-lected for lithogenic

index deterrnined by the nethod of Hegardt and Dam(1971) and Ho1zbach,

Marsh, Oleszewski et al(1973).

(e) BiIe Acids

A sanple of bil-e obtained after gal-lbladder stimulation by CCK-B was added

to nethanol and 4N NaOH with 5-beta cholanic acid as an internal- standard

and hydroì-yzed. The bile acids were then extracted, methylated with

diazonrethane (Back, Sjovalì-, Sjovall 1974) or with dinrethoxlpropane (Ali

and Javitt 1970) and trimethylsilyl or acetate derivatives (Makita and

Wel-l-s 1963, Roovers, Evrard, Vanderhactlrelg68) were prepared. GIÆ was

performed on 6ft glass columns aL 220 degrees C with 18 HiEff BBP (Applied

Science Laboratories, Inc., State College, Pa. ) on IO0/L20 mesh gas chrom Q

with helium as the carrier gas at 30 ml/min (Kern, Eriksson, Curstedt et al

L971).

(f) CattUladder volume and enptying with standard rneals

On day 5 of the study gallbladder storage and ernptying throughout the day

and night \^/as measured sonographically during ingestion cf 3 standard meals

(as described previously) .

(S) anafysis of data;

Data are expressed as rean +/- SD. Differences betr,r¡een control and

treatment periods were evaluated by the Wilcoxon signed rank test.

/

93

(ii) RESULTS:

(a) Gallbladder enptying

Tabte 8 shows the neasurenents of galJ-bladder vol-ume and enptying. Ttrere

was rapid gallbladder enptying over the first 30 min of the CCK-B infusion

but no significant differences occurred between the on and off oestrogen

periods. Slower changes in galJ-bladder volume v/ere seen after a ¡nixed

solid-Iiguid meal, but there \^/ere no significant changes in enptying rates,

fasting, residual or hourly vofumes witJ: chronì-c oestrogen ingestion.

(b) callbladder concentration

There h/as no apparent effect of oestrogens on bilirubin in gallbladder biLe

(Figure. l.'2).

(c) Biliarlz J-ipid secretion

The mean lithogenic indices of gallbladder bile in the on and off oestrogen

periods were sjrnilar (FiWre 13). There \^/ere no significant effects of

oestrogen on the mean hourì-y secretj-on rate of any biliary lipid (Fig'ure

14).

(d) Bile acids \^¡ere assessed by GLC for distribution of cholic acid,

chenodeoxycholic acid and deoxycholic acid. Tkre small anrounts of secondarlz

bite acids (5 to 12å) were disregarded in calculating percentages of the

major bile acids. There \^/ere no changes in the bile acid distribution

associated with the use of oestrogens.

ü1t

J

I

r

.- + _4€Ê- j*Æ'ËL-

I7!7 NS 2!2 .059 t .049 NS

GAL

MEAL

off

IV-CCK 8

off

TABLE VIII

LBLADDER EMPTYING KINETICS IN POST-MENOPAUSAL V'IOMEN

ON AND OFF PREMARIN

FV(ml- ) RV(mI )-'lb(min')

19 19 6!6 .034 t .030

16 1 5 NS' 5 t 5 NS .O25 t .016 NS

18t7 3i2 .055 ! .029

tgÈ

On

On

FV = Fasting Volume

RV = Residual Volume

b = Rate Constant of Emptying

NS = Not Significant (mean+SD)

95

Gollbladder BilirubinConcentrqtion (mg/ml)

ì

i

dï.i offon

FIGURE L2

Gal-Ibladder biLirubin concentrations on and off premarin.

I

r

96

Lithogenic lndex

HepaticBile

Gqllblqdder :

Bile

offon

7

2

l. ì.0

off on

FIGURE 13

Lithogenic index of fasting hepatic and gallbl-adder bileon and off Premarin.

\\--â

7-

ç

97

40

off on offon off

FIGURE 14

Biliary lipid secretion on and off Premarin. BA: Bile acid;PL: Phospholipid; CH: Chol-esterol.

Biliary Lipid Secretion ( lmoles/ Lg / h)

PL CH

I

BA

4l5

2

I

on

/\+1

a-.1ç

7<4---

ç/

9B

D. OROCAECÀL TRANSIT T]]"18 IN HUMAN PREGMI{CY

(i) r"grrioDs:

(a) Subjects

Fifty nine stuòies v¡ere perfornred (Table 9) in 27 healthy non-obese wLrite

r^roflren whose ages ranged frorn L6-34. Of B subjects initially studied in the

first trimester, 4 were subsequently stud.ied in the second trimester, 5 in

the third trimester and 5 postpartum. Of B subjects initially studied in

the second trimester 6 were studied in the third trimester and 5

postpartum. Seven of 1l- were initialJ-y studied in the third trirnester and

¡rostpartum. t .

(b) Gastrointestinal transit tinte

Thj-s was measured by the l-actulose hydrogen breath test (Bond and Levitt

1975; La Brooy, Mal-e, Beavis et al 1983; SoJ-omons, Viteri, Hanil-ton 1977)

(see Appendix).

Fasting serum progesterone level-s vùere deternined by radioirrrnunoassay at

Endocrine Sciences' Tarzana, Cal-ifornia-

(c) Anal-ysis of data

Group data were eval-uated by Student's t-test for unpaired data,and serial

studies by paired t-test.

(ii) RESULTS:

/

l4ean gastrointestinal- transit tine was 99 min in the first tri¡rester, I25

99

TABLE IX

DISTRIBUTION OF OROCAECAL TRANSIT TIME

STUDIES

Initi-alStudy Serial- Studies

TM1 TM2 TM3 PP

TMl

TM2

TM3

TOTALS

(8) 4 5

5

7

5

6(B)

B 12

11

22 17

ABBREVIATIONS: TM1 = first trimester, 7-13 weels; TMZ = second,trimester , 15-26 weeks; TM3 = third trimester, 27-39 weeks;PP = postpartum, 2-12 weeks. ( ) = period of initial study.

100

nin in the second trirnester, I37 min in tJ.e third tri¡nester and 75 rnin

postpartum (Table 10). There was considerable variation within each study

group but variation was sjrnilar between study groups, the range of the

percent coefficient of varj-ation being 39 to 44%. The sigrnificance of

differences in transit time between study groups was evaluated by both

Student's t-test for r:npaired data (S-test) and paired t-test for serial

studies (p-test), (Table l-1). Ttre results of these 2 statistical analyses

were identical. There was a sigrnificant increase in transit tinre frorn first

to second trjmester, a slight but insignificant increase frorn second to

third trinrester, and a marked decline postpartum. There \^/as no significant

difference between first tri¡nester and postpartum transit tinre. An exanple

of serial- studies of breath hydrogen curves for one subject is shcjwn in

Figmre l-5. There was a sharp sustäi-ned rise in breath hydrogen at 45

rninutes in the first trinrester, 90 rninutes in the third trimester and 30

rninutes postpartum

Serial- transit ti¡re studies of r.n/omen who were initialty studied in the

first trirrester is shov¡n in Figure 16. Transit time is expressed as the

percent of maximum transit time observed for each individual. This vafue i-s

plotted against the week of pregnancy or week postpartum- In each case, the

maximum transit time was achieved in ei-ther the second or third trimester

frorn l-5 to 40 weeks. In 4 of the 5 subjects transit time decreased

postpartum.

The relationship of transit tjJne to serum progesterone is demonstrated in

Table 12. Thre increment in transit tfune is greatest as the prq¡esterone

increases frorn I to B0 ng/fiI. There is no further incrernent in transit

time as progesterone Levels increase frorn 80 Lo 230ng/nl. However, the wide

variation of transit tjlrre within each range of serum progesterone precludes

accurate detennination of a "threshold" lcvel of progesterone, above which

l

L01

no firrtfÞr i¡¡crease i¡r transit tiÍe occurs.

hlt¡en nrouth to caecum transit tine was plotted against serun progesterone

for al-I tri¡resters and post partun, there vras no significant correlation

(figrr:re 17). Tterefore there ís only a trend for transj-t tine toj¡rcrease with serun progesterone with any significant relationship

¡nssiblï nnsl<ed by biological variation.

I

L02

TABLE X

MEAN AND VARIATION OF OROCAECAL TRANSIT

TI}48 FOR EACH TRTMESTER OF PREGNANCY AND

THE POSTPARTUM PERIOD

StudyPeriods OROCAECAL TRANSIT TI}4E

cv (%){ SD

39

4B

5B

33

TM1

TM2

TM3

8

12

22

(min )

99

125

137

75

39

3B

42

44PP 17

ABBREVIATIONS: N = Total number of subjects studied,

1 = meanr SD = Standard deviation, CV - coefficient ofvariation ( SO + X x 100%)' TM1 - first trimester'TM2 = second trimesterr TM3 = third trimester, and

PP = postpartum.

/

r_03

TABLE XI

STATIST]CAL ANALYSIS OF DIFFERENCES ]N OROCAECAL

TRANSIT TIME BETI,üEEN STUDY PERIODS

TM2

Study Period B

TM3

df

(.oo5( .05

32 NS

NS9

PP

23 NS

S Period A

TM1

S-Test

P-Test

TM2

S-Test

P-Test

TM3

S-Test

P-Test

df

1B .025

.05

BdfBt

1

2B

4 4NS

27

B (.001

< .005

<.001

?7

15

ABBREVIATIONS: df = degrees of flreedom, P = probabiJ-ity value,

S - test = Student's t-test of Lhe difflerence between study periods

A and B, and P-test=paired t-test of the diflference in serialstudies between study periods A and B.

104

lpp'n)

0

cÌ- - - -r) TM3

PP /t

,

I, TM

.t

IIIIII

a

tc

^

^

^ ta

- - . r-t- - -: -i..'- .. - _cI

_

aI

'r-:

0 ó0Time lmin!

r20

FIGURE 15

Example of serial lactulose hydrogen breathtests for one subject. ppm is the concentrationof H, in exhaled breath.. Lactulose (I0gm) wasadmiñistered at time = 0.

";ç\{:1,.-r i1: .a -.1.,*ffii¡r,, ,ì;,:..:'¡i..lffi'

80

%ro0

óo

40

105

20 30

Wk of Pregnoncv

20

lo 40 4 8

wk

FIGURE 16

Serial measurements ol orocaecaL transit in women

who were iniLialJ-y studied in Lhe fj-rst trimester.7" of maximum transit time Ís plolted on the ordinate-In each case maximum transit time occurred in latepregnancy. First Lrimester and posLpartum transÍttimes were similar.

PP

I

106

TABLE XII

CHANGES IN MEAN OROCAECAL ÌRANSIT TIME

I¡¡ITH INCREASES IN SERUM PROGESTERONE

LEVELS

Subjects Progesterone(nglrnl )

Transit Time(min )

N

Postpartum

Pregnant

15

3 20-40

15 40-80

13 81 - 230

72+33'113 + 35

132 + 57

122 + 50

1

Serum for measurement of progesterone level was

obtained in only 46 or the 59 sbudies. Serum was

not obtained in two TMl , three TM2, six TM3 andtwo PP studies.

/

r07

GIT

lminf24

t20

o a

Pregnont .Post Porlum \ì

r=.22P=NS

oo

O

o

o

aa

OOa

O

aI

0 0.8 20 50

FIGURE L7z

Oo

oo()o

oooo

o o

a() ooaa

o o

r50 200

Progesterone (ng /ml)

Mouth to caecum transit time(GIT) is plotted against serum

progesterone for all subjects-

108

E.CO-ORDINATION OF GASTR.IC AND GALLBLADDER M4E{TYING ATIIM. INGESTTON OF A

REGT]I"AR MEAL

(i) MHIHoDS:

(a) Subjects

οel-ve healthy non-obese voJ-unteers, 6 men aged 23.8 +/- 2.9 yr (x +/- SD)

and 6 ü/onìen, aged,27-O +/- 2.5 yr were studied. Ultrasound exarnination of

the ga1-J-bladder excluded gaÌlstones in aII subjects. Since a small- amount

of radioactive material was used formeasuring gastric enptying, \^/omen rú/ere

studied only dr:ring menstruation- i

(b) Gastric enptyi-ng

After an overnight fast subjects ingested a standard breakfast of one egg

l-abelled with 200uCi of 99m Tc-sulphr.rr colloid, 2 sl-ices of bacon, 2 pieces

of toast with butter, 24omr of 22 fat (wt,/vol-) milk, and l0g of lactulose

in IOOml of water. The meaf contaj-ned 6l-0 cal- . 44e. faL, and 12? protein.

Most of the fat is associated with solid conponents of the meal and even if

partially emulsifj-ed, would enpty from the stomach with the sol-id phase

(Ji-an, Vigneron, Najea et al l-:gï2). The labelled egg $/as prepared by rnixing

rar^r egg with 2OOuCi of 99m Tc-suJ-phur colloid in an al-urninium foil tray;

the labelled egg was scrarnbl-ed in and subsequently eaten from the foil

tray. To determine binding of 99m Tc-sulphr:r coll-oid to the egg, it was

rnixed vigorously with fresh gastric juice obtained at endoscopy frorn a

separate subject. The nixture was incubated at 37 degrees C for 6 h, rnixed,

109

centrifuged., and radioactivity in the supernatant fiquid and in the solid

phase \^/as assayed. More than 95% was in the sôIid phase v¡hich is sirnilar

to the findings of I(roop, Long, Al-avi et a1(I979) -

( c )Gal1bl-adder enptYing

Subjects fasted overnight and real-time galtbladder sonographs \^/ere

obtai-ned, fasting every 5-l-0 min for the first 90 min after ingestion of

the meal (15 rnin allowed for eating), and then every 30 rnin until the

gallbladder refilled to at least 702 of its fasting volume. Gastric

scintiscans were taken at the end of ingestion of the meal and every

S-1gmin for the next 2 h. Gallbladder sonographs and gastric scintiscans

during this 2h period were obtained with the patient sitting upright'

Subjects were then moved to a bed.where they lay supine, with the head of

the bed elevated 30 degrees for the remainder of the study.

(d) Gastrointestinal Transit tiirre and serum HPP

Breath sanpJ-es for hydrogen analysis were obtained fasting and every 15 min

for 4 h after conpJ-eting the meal-. Venous blood for measurement of

pancreatic pollzpeptide level- was drawn through an indwelling venous

cathetêr fasting and every 30 rnin throughout the study'

(e) Analytical techniques

Gall-bladder volume was fiìeasured from gallbladder sonographs (see appendix);

To conpare results betwen individuals, volume rnlas expressed either as

percent of fasting volune or. as percent of volume irnnediately after meal-

ingestion. Ttre time of initiation of gal-Ibladder refiJ-ling was deterrnied

frorn inspection of each plot of volume vs tjrne-

I

110

Gastric enptying obeyed a zeto-otder functi6¡-(Headilg, Tothill, Mclouglr-lin

et al, L976\.

DPtr4txI00=kt+b

DPMo

r¡ihere DpM is disintegrations per nin, DPMo is gastric DPM at corpletion of

the nreal, DMPI is DPM at time t, k is the constant of enptying, b is the

intercept at t=o and l-00 is the conversion factor for ¡rercent. k was

calculated frorn linear regression of (DPMtÆPlaO) x 100 vs time.

The plot of breath hydrogen vs time was evaluated by two

gastroenterologists blinded to the protocol and results of the study'

Estjmates varied in only 2 cases and by only 15 rnin each.

(f) Analysis of data

Data are expressed as nìean +/- SD. bitf"rences between. ren and wornen were

evaluated by Student's t-test. Correlations of various pararneters vvere

determined by bivariate regression analysis

(ii) RESULTS:

There was no difference in gallbladder enptying, gallbJ-adder refil-Iing,

gastric enptying, or HPP response between men and \^/omen (Table 13)

AccordingJ-y, data from all- 12 studies \^/ere pooled for subsequent analysis'

The mean orocaecaf transit time of the women was twice that of the

men, 13.6 +/- 20.2 anð,37.5 +/- 22.'7 min, respectively. The transit tirnes

in these rúr'omen, who were studied early in the follicular phase of their

ovulatory cycles, are similar to those previously reported by WaJ-d et al-'

using a similar technique in healthy young l^rollten in the foll-icular phase of

the ovulatory cycle, 97 +/- 3I min (Vrlald, Van Thiel, Hoechstetter et al

19Br).

TABLE XIII

COMPARISONìJ OF MEASUREMENTS MADE IN MEN AND I^JOMEN

xlsDMen (6)

26.5!s.L

-0 . 01 slo . oo¡--0.00710.002

¿252:7 8

333169

-0.3s10.r0

LzO!Tß32s!5?

37 .5!22 ..7

l^lomen ( 6 )

-0.01¡10. oo¡

-o .0o5to. oor

246!62337!37

-o.3oto. rr

1 15188

33817 1

7 3 .6!20 .2

P

Gallbladder responseFV (mI)

_1b (min -)

fasE..

s lowRefilling time (min)

initial70% FV

Gastric emptying rate (7" per min)HPP response

integraEed ( pg/mf/min)time Eo basal (min)

GastrointesEinal transit (min)

n.o!5.7 NS

NS

NS

NS

NS

NS

NS

.NS0 .025

PPP

x I SD = *""n 1 standard deviaEion; p= probability value; FV = fasting volume; b=rate

consEant of emptyi.g; HPP=human pancreatic polypeptide; NS=noE significanE

\

LL¿

(a) Gallbladder enPtYing

Íhe ti¡ne course of gallbladder enptying and refiIling for all subjects is

shovm in Fignrre l-8. A In/J-inear pJ-ot of gallbladder volune vs tjrne from 0

to I20 min revealed two phases of enptying, an initial fast phase frorn 0 to

30 nin and a second slow phase frorn 30 to 120 rnin. TLre initial fast rate of

enptying was 0.0L5 +/- 0.003 rnin -1, wtrj-Ie the second slow rate was 0-006

+/- 0.001 rnin -I, the gal-Ibladder remained tonically contracted r:ntil

refilling began aL 249 +/- 67 rnin (l'igure l9). The time interval from

initiation of galtbÌadder refilting to achieving 70% fastì-ng vofume ranged

frorn 30 to 180 min (86-l- +/- 52.I; f +/- SD) -

(b) Gastric enptying

The zero order rate constant of gastric enptying of sol-ids after rneaf

ingestion was 0.35 +/- 0.L18 per mil and the first order rate constant of

the second slow phase of gallblader enptying was 0-006 +/- 0.001 per rnin

(Tabl-e 13, Fig,r:re 20).Substituting the mean tjme of initiation of

gatlbladder refilling (249 min) into thre eguation defining the rate of

gastric enptying, it can be seen that when gallbladder refifling begins'

only 13% of so|-ds remain in the stomach. fn addition, using the zero-order

rate constant, gastric enptying woul-d be conrplete at 286 min, I h before

the gallbl-adder refilled to 70% of original fasting volume 335 rnin'

ùrocaecaf transit tinre di-d not correlate with fast and sl-ow rates of

gaJ-lbladder enptying, rate of gastric enptying, time to initiate

gallbladder refilling or tirne of gallbladder refil-Iing to 70ø of fasting

volume

Ttre correlation between the percent of bile remaining in the galÌbladder

and percent of solids remaining in the stornach for each tirne point was

highly significant (Figure 21)-

:iií

ri!'l'¡t;¡

{

II

I

r

%

HPP

1_13

r80

Time (

80

ó0

40

20

,T

Breokf osl ó0

FIGURE 13 :

/

/ JI

II

ür¡t

J

I

{ iÞGB

ì20

{ I0r

min

240 300 3ó0 420

)

Time-course oI gallbladcJer volume (GB) and

human parìcreaCic polypeptide resPonse (HPP)

in all subjects. Galtbladder volume isexpressecl as percent of fasEing volume and

HPP as Percent of peak level. VerEical bar

Ehrough symbol indicaces mean ! SEM'

r

IT4

Time tocommence

GBref illing

rh xt sD

1

..I

ü't,r

mtn400

300

200

o

I

FIGURE T 9:

Time tocom pleteTO ro GBref illing

Time for refilling of gallbladderafEer breakfast

I

t

al

I

115

...T T'ö----0

%ì00

80

ó0

40

o ix t SEM'-ð_.5J..J..tr

-6....l

t.. -¿." J-...¿ -''--ð

I

q

'l 015 30 45 ó0

Time (min7s 90 lo5 t20

FIGURE 23: Time-course of gallbladder emptying and

gastric emptying of solids for alI subjects.Galll¡ladder emptying ( closed circles ) isexpressed as percenE of fasring gallbladdervolume and gastric emptying (open circles)as percent of DPMs present in the stomach atcomple'Eion of the meal. Gastric emptyingobeyed zero-order kinetics and gallbladderemptying first-order kinetics.

I

I

116

FIGURE 2 1 z

"" REMAIN¡NG lN

?" REMAINING

GALLBLADDER VS

IN STOMACH

roo o

o

r=.93Slope =.9O

P << .o0l

LC)

T!o

€oo

o).E.EoE0)ú.

f

1Oo

B a

o o

oo

050ó070o-/o Kemolnlng

80 90

in Stomoch

oo

o

50

or00

Correlation between the percent ofbil-e remaining in the gallbladder and

percent of original- solids remainingin the stomach for the seventeen timepoints observed.

t17

(c) Serum HPP

Plots of HPP level- and gallbladder vol-ume vs time are shown in Figiure 17.

The initial fast phase of gallbladder enptying is acconpanied by a rapid

rise and early peak in HPP response. The slow phase of gallbladder enptying

and tonic Aallbladder contraction is associated with a sustained el-evation

in HPP leve1s. Ttre return of HPP level-s to basel-ine roughly corresponds to

the time of initiation of gallbladder refilJ-ing.

The integrated HPP response did not correlate with rates of gallbJ-adder

enptying, gastic enptying, or GTT. Thre initial peak HPP l-evel- did not

correfate with the initiaf fast rate of galJ-bladder enptying. The

integrated HPP level dr.rring the slow phase of gallbladder enrptying and

tonic aallbladder contraction did not correl-ate with the average

gal-Ib1adder volume during the same- interval of tinre.

1L8,

CHAPTER IV: DISCUSSION

A. Presnancv, Female Sex Steroids and

Gallbladder Function

B

(i) Effects of the Ovulatory Cycle and

Contraceptive Steroids

(ii) Effects of Pregnancy

(iii) Effects of Chronic Oestrogen Intake

The Effect of Gallbladder Emptying on

Bitiary Lipid Secretion

Or.ocaecal - Transit Time in Pregnancy

Gastric and Gall-bladder t Ln

c

D

I

119

DISCUSSION

A. PREGNANCY, FEMAT,E SEX STEROIDS AI']D GÀLLBI,ADDER FUNCTION.

Pregnancy causes greater than normal retention of bil-e in the gallbl-adder

throughout the day and night. The retention is maximal at a ti¡ne when bil-e

is rel-atively supersaturated wj-th cholesterol-. Such changes could

contribute to the increased incidence of cholesterol cholelithiasis

associated with muJ-tiparity.

(i) nffects of the ovulatory cycle a4d contraceptive steroids

Fasting, residual- and hourl-y volume were sirnilar in t-l.e fol-l-icular and

luteal phases of the ovulatory cyc.lés. Rates of enptying after breakfast

were the same in both phases of the ovulatory cycle. Contracepti-ve steroids

increased fasting gallbladder volumes only.

Nilsson and Stattin (1967) used oral cholecystography to measure

gallbladder emptying after bolus injection of cholecystokinin in both

phases of the cycle in each of I0 subjects, and they found slower enrptying

in the l-uteal phase in B of them. However, the phase of the cycle was not

documented by serum progesterone level, as in the present study, and 5 of

these B had a l-0% decrease in enptying in the luteal phase. Nonetheless

this data indicates that the efevated progesterone levels associated with

the l-utea1 phase do not significantly alter ei-ther gallbladder vofumes or

enptying during i-ngestion of regular meals.

(ii) Effects of pregnancy

12a

Assessment of the data in the way that has been performed in this section

of the thesj-s requires further explanation. In general, the regression of

data j-s perfornred to study the rel-ationship between a predictor variabfe

and a response variabl-e. Ttre regression anal-ysis sunrnarises the refatíon

between the response and preðictor variabl-es and rneasures the correl-ation

between variables. Before regressing data it is irrportant to plot and

exarn-ine data to check for outl-iers and to define nonl-inear patterns in the

data. It is atso irrportant to look for patterns suggesting that the data

shoul-d be transformed to a different scale, such as loqarithrlic, to produce

a better fit, to tl:re data. Itre sinple linear-regression nrodel assumes that

the relationship between x and y can be sunrnarised as a straight-line

graph. However, if the data do not seem to lie on a line or if the

residual-s show a cl-ear pattern, tfre, l-i.r"ar ¡nodel- may not be consistent with

.the data. One way to deal with this problem is to attenpt to fj-t the data

to a particular crrrve, such as a troþatithnic, sinusoidal, or exponential

curve, nonlinear regression, as discussed by Bliss (1970) and Snedecor and

Cochran (1980). According to C,odfrey (1985) if part of the data seems to

lie along one line and part along another l-ine, the data can be stratified.

Using this technique, a different line is fitted to each segment of the

data, In this case it is assumed that a linear relationship hoJ-ds within

each stratum, al-though t.l.e exact relationship may not be the sanre frorn

stratum to stratum. Such a nrodel- is call-ed a piecewise linear nx¡del-. If a

sinple l-inear regression is used to study the rel-ationship between initial-

gallbladder enptying and pregncnclr there is l-ittl-e difference between

controls and pregnant subjects. However, it j-s appropriate and valid to

use a logarithrnic scale for gallbladder vol-ume and to apply a piecewise

regression. The result is that a significant difference is demonstrated

between pregnant subjects and controls in the second slower phase of

gallbladder enptying (Figure 6). Again this rel-ationship would be rnissed

if an alternative to a sirrple linear regression \^/as not used.

¡i

I2I

Fasting and residual vol-umes \^¡ere larger than control values in every

trjmester of pregnancy, and they increased J-inearJ-y dr:ring pregnancy.

For fasting volume a correlation of r = .48 (e = .016) is obtained and the

line y = 20.17 + 0.450 x is the l-ine of best fit for sinple linear

regression. Given the relative large variation between ind-ividuals, thi-s

indicates an increase in volune over the pregnancy. The guadratic

relationship does not significantly inprove the fit over the linear (R2

changes from 0-226 Lo 0.257) and the equation y:10.89 + 1.36x - 0.019x2

gives the best quadratic fit with a maxj¡num volune at 36 r,^/eeks' Ttris does

not support the hypothesis of no increase in volume in the third trimester.

An exponential growth n¡¡de1 woufd bêhave similarly-

For the residual- volume a correlatiòn of r : .54 (P : .006) is obtained and

the line y : 3 -'78 + .232 x is the line of best fit for sirrple linear

regression. Given the relatì-ve J-arge variation between individuals this

indicates an increase in volume over the pregnancy-

The quadratic relationship again does not significantly inprove the fit

over the linear (n2 changes from .290 Lo .308) and the equation y = -48 +

.55x - .0065x2 gives the best quadratic fit with a maxjmum vol-u¡ne aL 42

weeks. This does not support the hypothesis of no increase in volume in

the third trimester. An exponential growth model- woul-d behave similarly-

Vlhren the volumes in the tlree tri-mesters are conpared the means are ( 23.7 ,

33.3 and 32.1) for fasting volumes and (6.4, 9.5, 10-5) for residual

volumes. Statistically significant differences are shov¡n by one way

analysis of variance to occur at the l-evels of P : .064 (F= 3 -l.2) for

fasting vol-umes and P : 0.116 (F= 2.38) for residual volumes neither of

122

which quite reaches the connonly used value of .05 for statistical-

significance.

Hourly volumes frorn tl a.m. r:ntil rnidnight were larger than those of

control-s throughout pregnancy, without progressive change (Figure 4C). The

failure to find a progressive increase vùas due to large hourly volumes in 2

first-trinester subjects (Fignrre 4C). Because the progesterone level of

these two subjects was appropriate for the week of pregnancy, the vari-ation

in hourly volumes is probably due to individual- dj,fferences in galJ-bladder

srnooth-muscle res¡rcnse to increases in progesterone-

As fastinq, residual and hourly vol-umes return toward normal in the

postpartum period, it i-s probabJ-e thit most of the changes in gatJ-bladder

vol-ume during pregnancy are transient. !{hether vol-umes return to prepartum

val-ues in a given individual cannet be deterrnined from this study as no

subject was studied before pregnancy.

Only the last phase of galJ-bladder enptying after breakfast was sfower Ín

pregnant women. The slower late phase of enptying may be due to inpaired

gallbladder contractility or slower gastric enptying. The late rate of

gallbladder enptying correlates with gastric emptying of solids- Thus it is

possible that the sfower rate of gallbladder enptying after breakfast in

pregnant subjects i-s partì-al-ty due to slow gastric enptying of solj-ds-

An overall sunrnary of the gall-bladder enptying curves would be l-ess li-keJ-y

to contrast the differences in gallbladder enptying between controf and

pregnant subjects. The earJ-y rapid phase of gallbJ-adder enptying is a

reproducible finding as can be seen j-n Chapter III, Section E when a

different control group of volunteers \^/ere studied including males and

females. This early response is most likely vagatly innervatecl. Fisher,

1

]-23

Rock and l'la1nnrd (19861 have recentJ.y shovm in hurnan volunteers that intact

vagus nela/es and cholinergic pathvrays are required in order for the

ga1-Ibladder to respond to sh¡am feeding. T'hey found that the maxirnal

curn:Iative gallblaclcler anrptyjlg response to sham feedilg vras 44%, vrhich r,ras

not significantly different frqn the respoonse to beth,anechol and to

ingestion of a test n'¿eal of steak and potato. (holinergic blockage vrittrI

atropine eli¡¡irnteo the enptying reslænse to sham feecling and sham feeding

did not sti:rmlate gallbladder e-nptying 1n patients i.rith vagotorry.

A biphasic response has been reported for gastric erptying of digestiJcle

soticl meals v¡hen neasurernents are tal<en cluring rneal ingestion r'¡ith a 'Iag'

phase precetìing a l-j-near oiptying phr,ase (Col-Iins, Flororvitz, Cook et aI

1983 ) . Tlrerefore our earl-y phase of gallbladder enptying is unl-i]<ely to be

rel-ated to rapiC initial solid gastric ottptying.

Because tlre honnonal changes of pregr-rancy are ccxitplex (Iilo¡:¡:ert and I\rchs

1977) it is not possil:Ie to identify v¡ith certainty ttre nediator or

rediators of the alterations in galLbl¿r.dier function. It is also not

possibl-e to :--.:clucle a c-ìirect or inclirect neuronal influetlce produced iry

ÌïeEl^ranc:,,/. In aclCition neuronal chamges coul-a rnodify ani¡ honnor¡al effects

124

on motility. Nevertheless, progesterone, a knov¿r inhibitor of snxcoth

muscle contraction (Cohen l-980; Sonrlzlo and Sonqzlo l-970; Schultze and

Chri-stensen ISBO; Fj-sher, Roberts, Grabowski et al- I9lB) is a likely

candidate. The data shows a ôirect correl-ation of fasting and residual

volurne with serum progesterone concentrations. Thj-s is

consistent with, but not proof of progesterone mediation. As there was a

highly siginificant direct correlation between serum progesterone and week

of pregnancy, and the correlation of each of these variabl-es with fasting

and residual volume were identical, it cannot be concluded that

progesterone caused the effects- The changes in indices of gallbladder

function could be due to other, unnìeasured factors in pregnant women-

In sunrnarlz, the phase of the ovulätory cycle did not affect gallbladder

function, but pregnancy caused retention of bil-e in the gallbladder

throughout day and night. Prolonged contraceptive steroid use induced an

increase in fasting (overnight) but not daytime bile retention-

The results of this study indicate that in pregnant women, gallbladder bile

retention is lncreased throughout the day and night. Bil-e retention is

greatest in the second and third trimeters, a time when gal-Ìbl-adder bj-Ie is

most lithogenic (Kern, Everson, De Mark et al- 19BI). Thus, gallbladder bil-e

retention could contribute to the pathogenesj-s of cholesterol gallstones by

allowing more tinre for nucl-eation and pecipitation of cholesterol

crystal-s.

(iii) Effects of chroni-c oestrogen intake

This study has sho¡¡¡n that oestrogen is unlikely to be responsibl-e for the

increase in bile cholesterof saturation which acconpanies the use of oral

contraceptives.

t25

The results of the effects of clrronic oestrogen intak-e on cholesterol

secretion, cholesterol saturation index and gal-Ibladder concentrating

ability reporbed shov¡ no sigrnificant change v¡ith the doses used. One

possible r€¿se¡ for tkre lack of response is failure of absorption of the

conjugated oestrogens. Pre¡rnrin contains equilin sulphate (252)and as v¡ell

as other conjugated ccnrpourds, includes sulptr,ate triester of estrone túSzl

17 alpha tihydroecluihn (1 5u ) , 17 alpha d.ihydroequilenin and 1 7 beta

dihydroequilenin (15S). After oral aùninistration of Prqnarin to

postinenopausaf v¡ornen equitin sulptrate and oestrone sulphate appear in the

serum as unconjugated equilin and oestrone (Bhawrani, SarCa, ir'oolever

1981 ). T'hese findhgs indicate a-l:sorption of oral- Pre¡narin.

In the study re¡iortecl here rthe gallbladder contractil-e res¡:onse to

intravenous CCK, a núxed meal and intraduodernl ì-iquid fcxr,ula v¡zrs

unaffecbed by chrronic oestrogen aùninistration. Oestrogen pretreatmeni j-n

anj¡ral-s has previously been re¡rcrted to erùrance the in vivo contractility

of the oesophiag-us, antn-r'n ¿ncl colon (Bruce and Eehsudi 1979 ). oestrc4len

pretreai:;rent has not been shor^,'n to increase contractile responses in the il

vitro ç¡:inea pig gallbladder, but it significantly decreases the thresholcl

acetylc¡oline dose requiraments (Lffan and Pellecchia 1982l.. It is possibJ-e

tirat the gallbladder is rel-atively less sensitive than other tissues to thr

excitatory effects of oestrogen.

B. 'I]]E EIIÞ'}]CI OF GAIJ,E|T,ADDM{ Eù{HTYING CN] BT,IAIìY LIPID SECRETICN

The observatiori in pregnancy of supersaturated bile and delayed gallbtadde

onptying raised the qrestion of the role of the gallbladder j¡r controlJ-ing

biJ-iary cholesterol secretion. Secretion studies using different strength

sti¡mli we¡e desigrred to study thre effect of inconplete and alnucst ccnplet

, L26gallbladder enptying on biliary cholesterol secretion.

Il'lhen previous secretion studies are ccnrparecl it can be shov¡n tLnt bile acid

secretion rates durJlg continuous intraduodenal amino acid infusion

(Shaffer arrd Smal-I 1977¡ Kern, Everson, De l4ark et al 1981 ) are l-ess tl¡an

those neasured during c-ontinuous intraduodenal liquid formul-a infusion

(Grurüiy and lbtzger 1972; Ivlok, Von Bergmarrn, h:ndy 1979; Bennion and /Grundy 1975¡ Mabee, I"Ieyer, Den Besten et al- 1977; Valdivieso, Palma, I'Iervi

et I 1979; l{cJ<, Von Bergnrann, Gn:rrly 1978) and sjmil-ar to that measr:red

during intermittent lic-iuid formula feedings (t'iok, r/on Bergnrann, Grr-rndy

1979, Northfield and Flofnnnn 1975i La Russo, Szczeperrik, Iìofnnrrr 1977; Itok,

Von Bergnann, Grurrcìy 1980; La Russo, Hoffnnnrr, Hofinann et al 1975') and inpost cholec¡¿stectonry sr-rbjeets (Shaffer and Small 1972; I/rey, Bonorris, i,larks

et al- 1980) (see Thble 3a+ Appendi:<).

I'lhen similar groups of subjects are ccn'rpared bile acid secretion rates

ù:rhg tiquid formula infusion are Íìore th,an trvice those during a¡lino acid

j-nfusj-on. In the present stucly durilg licruid forin¡rla infusiorr the rate of

biì-iary lipid secretion r.ns higher, gallbladder onptyilg \./as pronpt and

nìore corq)Iete, and sinall in'bestinal transit \,ãs faster. It can læ

concluCed that tl're higher rate of biliary lipid secretion achievecl during

liguid formul-a in this ancl other studies is due to j-ncreased enterohepartic

q¡cling of bile acj-d causecì by more ccnplete galJ-bl-adder er,ptying and

faster snrall intestinaÌ transit. Bil-iary lipid secretion rate during amino

acid infusion is sulmaxirnal, due to inoonplete gallbladder emptyilg and

sl-ower intestinat transit.

BiJ-iary lipid ccrnposition is dependent on the ra'te of biliary bile acid

seqretion. In patients rvith bil-e fistulae the nrrl-ar percent chol-esterol- of

T¿I

bile increases as the bile acid secretion rate drops (Lindblad, Lundholm,

Schersten L977; Wagner, Trotjnan, Soloway 1916; Nilsson and Schersten 1969).

In subjects with gallbtadders, fasting sequesters part of the bil-e acid

pool- in the gallbladder, induces a lower bile acid secretion rate

(Northfiel-d and Hofmann L975¡ Mok, Von Bergmann, Grundy 1980; WLzger,

AdÌer, Heysmfield et al 1973) and increases cholesterol- saturation of bil-e

(Northfield and Hofmann L9l5; Me|zger, Adler, Helzmsfield et al L973).

When sirnilar groups of subjects from different studies are conpared' the

nolar percent cholesteroJ- during continuous liquid formula infusion (2.9

Hcward l9B0) is less than that ùring continuous arnino acid infusion

(4.3 +/- .5å Kern, Everson, De Mark et al 19Bl). In the present study a

sirnilar difference has neen sfrovå between the two infusates in the sanre

individual-. It can be concluded that the decreased molar percent

chol-esterol during liquid fornmla infusion is the result of a higher rate

of bil-e acid secretion due to nìore conplete gallbladder enptying and more

rapid smaLl intestinal- transit.

Since the rnrlar percent chol-esterol of bile varies with bile acid secretion

rate the rel-ationship of cholesterol secretion to bil-e acid secretion must

necessarily be curvilinear over the futl range of bile acid secretion

rates. However, if only a portion of the range of bile acid secretion rates

is exarnined, the relationship between bile acids or phospholipid and

chol-esterol may appear linear. !{hren the data in t}ris study fron amino acid

and liquid formula are combined the resulting relationship for bile acids

and cholesterol is curvilinear (Figu-re 9). If the data frqn each infusion

is exarnined separately then tÌ.e relationships appear linear as only a small-

portion of the range of bile salt secretion rates is being represented

(Figr:re 9). Ttris may explain d.iscrepancies shor,vn in other studies where

/

I2g

sone authors found linear relationships (Kern, Everson, De Mark et aI t9B1;

La Russo, Szczepanlk, Hofmann 1977; La Russo, Hoffinann, Hofmann et aI 1975;

Einarson, Grundy, Hardison 1979)while others showed them to be curvil-inear

(Lindblad, Lundholm, Schersten 1977; Wagner, Trotman, SoÌoway 1976) -

Gal-Lbl-adder volune fl-uctuated during infusion of both arnino acids and

Iiquid formula but large fluctuations (10-20m1) \^/ere seen only with arnino

acid infusion- Dr:ring the studlz bil-e from ttre gallbl-adder was often

e>çelled Ínto the duodenum to join the stream of hepatic bil-e. When this

occurs the measr:red secretional rate may not accurately determine the

hepatic secretion rate.

Faster small intestinal- transit increases the frequency of bile acid

recycJ-ing and increases bil-e r.iå ="".etion rates. In this study faster

intestinal transit occr:rred yith. Iiquid formula which resulted in greater

biliary lì-pid secretion rates.

Since subject 4 produced hydrogen in response to lactul-ose during liquid

formul-a infusion, the l-ack of increase in breath hydrogen during the arnino

acid infusion is most consistent with a markedly prolonged transit time.

However, there may be other explanations for the l-ack of hydrogen response.

The nasoduodenal tube may have slipped back into the stomach, the subject

may have been colonised with colonic bacteria incapable of making hydrogen

or this may represent day to day variation. Ttre first seems unlikely as

bj-Ie was continuously aspirated during the study and there was no drop in

secretion rate in the 4 hours after lactul-ose was given. The second is afso

unlikety as a dramatic change in colonic flora would have had to occr:r

within 1 week without the a&ninistration of drugs or antibiotics. The third

is extrenely unlikely as the day to day variations in transit tine neasured

r29

by this technique have been defjled and are rn:kìjmal (La Brooy, Male, Beavis

et at 1983), es¡:ecially with i¡rtraduodenal i¡rfusj-on of l-actu-lose (Bond and

Levitt, L977). Subject l, who had the biggest difference in gal-lbladder

emptying with the j¡lfusi-ons, had no change i¡r transit tilrÞ. Interestingly,

shre had a sna-l-ler increase i¡ bil-e acid secretion corgnred with thre rernaìning

subjects.

The more corplete gaJ-lbladder emptyilg and more rapid intestj¡ral transit

during liquid formul-a infusion are probably related to greater stjrrn:-l-us

for rel-ease of hr¡noral- mediators of gal-tbladder contraction and j¡rtestj¡a1

peristalsis. However it has been shov¡n that CCK can enfrìnce cholinergic

innvervation of the gal-J-bladder ( Foesel- and Sewj¡rg 7978 ) arìd therefore sqne

of the observed changes could be nediated by neurogenic ¡nthways. Pancreatic

potlpeptide reLease frcrn the pancreas paral.lels that of cholecystolcinin

and nrotilin (Floyd 1980; Keang,: DìMagno, Dozois et al- 1980), two hormones

thought to be responsibl-e for gallbJ-adder emptying and snall intesti¡al-

peristalsis. Reliable assays for the fatter two hormones were not available

but the two to threefol-d greater rise i¡l serum pancreatic pollpeptide i¡r four

of five subjects during tiquid formu-Ia i¡rfusion j¡tdicates greater hunoral.

stimul-ation.

Ttrese secretion studies h,ave shown that incornplete gallbladder emptyirg and

slower intestj¡ral- transit decrease the enterohe¡ntic cycling of bile acids

and j¡rcreases the nrolar percent cholestcrol of bile.

C. OROCAECAL TRANSIT T]]'IE IN PREGMNICY

FTqn thre secretion study reported here and from work by ottrers (Einanson,

Grundy, Hardison L979¡ Mok, Von Bergrnann, C.rundy 1977¡ Valdivieso, Pa-lrna,

¡üerr/i et aI 1919) it is apparent that snnl-I i¡ltestj¡ral tr¿rnsit tirre is an

irrçnrtant determj¡rant of the rate of cycling of biliarlr lipids and thus

,1

130

biliary lipid secretion rates.

In t.l-e first trjmester of pregnancy, an increase in bile acid pool size

without an increase in galtbladder vol-une has been previously reported

(Kern, Everson, De Mark et al IgSl). This pronpted the sequentia] study of

gastrointestinal transit in pregnancy to deterrnine if prolonged transit

ti¡ne coul-d e>çIain the expanded bil-e salt pool of the first tri¡nester of

pregnancy.

The data in this thesis has shov¡n that gastrointestinal transit is not

significantly prolonged in the first trimester of pregnancy. It is

prolonged in the second and third trimester and decreased postpartum,

approximating first trirnester valueé. Ttre postpartum fevels of progesterone

meas¡red in this study are conparabl-e to those occurring in the folÌicufar

phase of t-l-e ovulatory cycle. Likei,vise, the transit tinres of .wonen stud-ied

in the first trimester or postpartum period in this study are similar to

those observed in the folU-cutar phase of the ovulatory cycle or postpartum

by other groups usì-ng sjrnifar techniques (Wal-d, Van Thiel, Hoechstetter et

aI 1982; Wald, Van Thiel, Hoechstetter et al 19BI)

There is a threshold Ìevel of serum progesterone above which no further

prolongation of gastrointestinal transit occurs. The concordance between

studies of the relationship of smal-l intestinal transit and gallbladder

volume to progesterone levels suggests, but does not prove, that

progesterone may be the medj-ator of tJ.e alterations in motility observed in

pregnancy. Obviously, other hornrones, such as oestrogens, prolactin or

other hormones that increase during pregnancy, may correl-ate with the

observed changes j-n transit tinre and gall-bl-adder vof.ume. In addition

alterations in neuronal input rnay play a role in sore of the observed

changes.

i

131

r''bre recently Carride, Prokop, T?oncale et. aI l19B¡') studied s¡nall

intestinal transit scintigraphicalJ-y in healthy volunteers r+ith

99:r-technetir.rn-diethylenetrj-ar'rine i;en-ta acetic acid. Sublects ingeste-J the

scintigraphic solution v¡ith isosmotic Iactulose. The arrival of tfre

radionuclide label at the caecum correlated v¡eII rvitkr the rise in l¡reattt/

hydroc;en (r=.77 ) arld the transit ti.'res v¡ere sj-nil-ar for the scintigraphic

¡mtlrod (73 + 6.5 min; x +SEÞt) ancl ttre lactulose breath test (75.I + 8.3

rnin). Ino.ividr:al variations in snnl-l intestinal transit tj¡ne in Caride et

al's stucly r,rere si-gnificantly correlatecl vrith individual varia'tions in

Eastric en'çtyirrc;. Boncl and Levitt (i975) shov¡ed that indivicl-r:al varj-aticn

i:r sr¡atl intestinal t¡ansit. tùne, as fæasllrec1 by ilre l-aclulose hyclrogien

i¡reath test, coul-cÌ be siginificantly rectr-rced l:y infusing lactulose clirectÌy

into the ciuo.iem¡n via a transpylori-c tube. Ttrese findings sr-rggest that

smal-I intestinat transit tin" .r= measurecl by the factulose hrea-th test is

sic;rri-ficantly affecbed by the rate of gastric ernptying. I-Ior.rever, caution

is incicatecl before assurninq th,a'i transpl'le¡ic intul:al-ion is necessary for

acc;rate ìrêâsrrr(trrêrìt of srnall intesti:ral transit ti'ne. Di-fferences in

gastric mpLyi,rg are ual-:-kely to sicr,nificarrtlr.¡ affect the orocaecal transit

tj-rle rcsults j:-l tho first and second tri'ncs+-er of pregrnncl' aS liçti<i

elältying is not affected ity tÌre luteal phase of tÌre nrerrstruaf rycle or

preErna-ncy up to the second tri"nester (FtorowiLz, l.laddern, Clntterton <lt aI

1984i Schacie, Pe1el,:anos, I'ar-lxe et a.l 1984). Ilov¡ever as input of a rneal-

into the smal-l i:ov¡el is variable and ö,epends on the gastric anptying rate,

it is an advantage to quantitate both gastric ernptying and orooaecal

transit tjrne sinrultaneously. tÞlayed gastric ernptying is an unlik-ely car-r-se

of the prolonged gastrointestir¡al 'bra¡sit tj¡re occurring in late pregnancy

The delayecl transit ti-rne is prolxrJrly a result of prolongecl snall boi,vel

transit ti¡e due to inhibition of sma1l intestinal srx¡oth rrtuscle

LJ¿

contraction by fsriale steroid ho::aones, rost }il<-ely progesterone. Ilov¡ever ¿

neuronal effect of pregrnncy on orocaecal- transit car¡not be e-xcluC.ed.

I;r Brooy, i',la1e, Beavis et' al- (1983) have shov¡n t-hat the lactulose breath

test ca¡r be rade rTìore repraluci-ble by including a 1ic1uid. meal. They shovrer

t-nat variation in transit ti¡res betv¡een i¡dividuals v;as corrsiderable r¿ith

aII doses of lactulose ranging frorn 10 to 20gm (nean coefficient of 1

variation of 18.5 to 28.32 respectively). The addition of lactulose to aIiquid meal containi-ng carl:ohydrate, fat and, protej-n clesreased the

ooefficient of variation to 10t i-rr for-rr subjects studiecl. It is

therefore possiJole that I could have ìrnprovecl the r¡aria.bil-ity in pregnant

subjects by includjng a liquicl meal :rith the l-actulose close.

The ideal tecirnj-qrre to investiEa-ue intestinal transit shoula monitor tÌre

rrrcvenent of the liquicì or chlanous contenLs throuEh tlre lul-¡en ';¡j-thout

r-:.isturbing the phys-i-oJ-ogiôal- con-urol of gastr:ointestinal mo't-iIity. l,1ost

technic¡res used ¡e .-iuantitate transit tire to the caecuin have inadecluacies.

Tire use of barir¡'n as an ar-tificial ir,eal- to stucÌy sri'alI bolvef Lr¿r¡sit Ìras

-lÍn-i'tations ( j-n partic'uJ-ar in pr:e.;ri-nnc1' ) "encl tirc resufts cannot be

e;<'t-ra1rcJ-ateC to other rnea'ìs. Ìietncxls using non-absorJ:abl-e nüerkers l-ille

irolyethylene glyco1 (PiG) rec¡:ire Cre use of an intr¿rlr-r.ninal- Lr-rh: 'to

retrieve sarçJ-es frorn a given lccation in the intestine. this prcrìuces

stj¡:uli th,at are not '.rsually present a;rd cÂuses scme pa.tient disccxnfort.

i-Ìoi.rc:ver, the deter':rination of lrycrrogen in the i:reath after the ingestion of

Iactul-ose is non-j¡vasive. Tlre test does depencl on the presence of

bacteria in the large íntestine to riretabol-ise l-actulose. .P't l-east 5-t of

the ¡:opulation, however cannot met¿rl:rolise the sugar Ì:ecause they lacl< the

proper bacterial strains in the colon. llydrogen pro<luction rlay also be

re<luced because of lov¡ cofonic pll (Perman, l.lodlers, Olson 1981 ). It is

theoretically possibl-e tlrat a re.rluctio¡r in bicarbonate rich bile flory couli

J.JJ

Iead to a fall- in caec;tl ¡-rii i:esul'ting irl a Cecreasecl or even absent

i-ri'cit'crr* peal.., folloiving lactrilose ir:rqestion. .*ile flo.,v i¡r preçnancy' is

proì:aì:ly recuced and tiN: l:icar-l¡onate ccncen'ti'ation of bile Cecreasecl þr

cjilution. 'rlrese pertuÌ:atiol-rs .,¡ould l:eqxie nore pronounced as i)reqi),anc)'

pro:jiresses anCi r¡1ây e:ì?lain rihir scr,te ,iregrranL t¡cmen beccrne non-hyd:rogert

proclucers IaLe:: il p::egiranry.

/

I;ùrooir, ihle, B,-rvis e': al t19C3) ìravr¡ suggestecì that variations of srnall

bo..¡el- :no'i-ilitir in the fastec]. sta'te pi:oÞ.ì:J-y account for the relatively poor

rc¡-.i:oc1ucìì:ility of dre l-actul-ose breatll test j-n, or l:etrveen indiviCuals.

irio!,€vern their results ca¡¡ot be e>:t::a'.colated- to ¡:regnant r'nnen r'¡lrose

gastroi:rtest-inal- sr¡,coth n:-iscle is ',rncjer the influence of ccrlplex

neurohoi-rrcnal ciranges. Such varia.+;-i c.rr of orocaecal Lransit. tir.e in

i-,recj¡ancy has not i:een stuäreC. D:s¡rite 'lil.,ely individual varia,Ì¡ility' tlre

studies descz'iJred in tiris thes;is rr:r:e stil-'l- ai-¡le to dsnonstrate significant

seconcl ¿rnd ilirci tri-nester ilif ferences, Crre problern in interpreting breaiÌl

h)..irocjen cl,eril'ecl orocaecal Lransj-t tirr.e rneas,-rr€rireots is -th,at tire results

r-ir,, ìLo-u necessaril-r' relate tc ii-:e trinsit of focc 'Crro:Eh the sirall

ir:Lesti.ne. r-i-.,erefo::e it- i-s :i-n':or;¿lt 'to llo','r -if tl¡.e b::ea-dr Ìryiroç;eir '¡na,l'-

ccr;:clalcs,,¡iürilie tir.ic'Crat "thc ìrc¡r-ri oi: tl:e i:'.l]-i-' of the i;eal reaches the

caecll:r. if tlie ir-ltcial h;,cz'o_rel lral-. c,c-'cs ncìt correspoticl r'riLh iire heaci of

tirc r,leal r:cachj-:tg l-re cecc.r,i: i-!rc-u ¡r.:r:ireti;s i-eaìi þclrogeri excreLion r'ny ì:: a

nore precise niæ.sure of l'norri:h'to caecr:ii transit til.e. P-ead, i'iiles, Eisher

et aI (1930 ) cor{nrec'l tire ti:a;rsit t:i-i,r.e of an isotopicalty IaJ¡eIIeil ni><ecl

r,r¡al- rvj-th lrreatn hylrogen o:cretion in 14 trealtfrlz volunteers by estirratinE

raciicractiviLy over ijte caecun. 'IÌrcrc \'ras a Ìrighly siErr-ificernL correlaLion

treL',¿een tlre tj-r,c of the increase j-n r--¡rdj-oacti-vj-ty (t=.82i p < .001 ) over

tlre ca.cct-un ancl tlre tiine of ile seconc-ia-r1z incrr:ase in h1'd¡6gen olcretion

¡orocìr-rcecl Ìty ì-actulose feninn't¿r1-ion. n 9 ouL of 14 sujrjects, tìre seconcìar1z

i¡rcrease in Ìry,:ir.oçieir er:cr:etion occuri:eji al- the sanÌ.: ti¡le as the incre¿rse ill

.I-t'f

lrj

l-34

caecal radio¿rctivitlz sugt stjfì{j tj"at ¡neasüreri€nt of itre¿rth }ry-dro<;en is a

useí:ul- r,e'cl:oc-l to indicate r¡hen a rirell en"ters tlre colon. -[n 10 subjects tìte

rrEìxirr.¡r¡¡¡ coturt over tÌte caeç:un, coñ'ected for dec=y of i:ire is;oto¡-e, ¿¿j'tairreri

the level- of the initial g.astric counis suggesting tÌrat atl oi 'ijre i:'c¿rl

residue ,,./¿ìs present in the caecui¡L afLer f-assaie tlrrouEh i:he srnall

i¡rtestine, Ítere 'ivâ3 â Ìlighly sic;nifiærtt correlation (r = .66; ir < .01 )

i:et'¿een tlre tiri.e tirat the rileu{iJrrl]m concentra-tion of Ìly-clrocJen gas vIas /

excreteC in tlre lrrea-tÌr j¡r'rhese sui¡ects ¿Lno tlle tine that'clie rna::irutrt

a¡io-.ur-¡ of radicactivi-ty r.¡as recorCec-'ì- over the cêêcuÌ.ir. In 7 out of 10

stij-rjec'us, i¡ea,1i ìrydror;er-r excreLion occurreri a't ìj-re sarnei:j¡le as iÌre to'cal-

racic-.,aci:ivity l+as recortled over 'che caecl-:r,l an1 i:resur-r,tably inclj-ca'Leci tÌre

ti-irc ü.nt. aI-ì_ of LÌ:Le rneal resicÌue ira-s enierec- tfìe colotr.

:i,he close corresyrcnr,ì."n"" ùLo,=en caeca-l racioaciivity ancl ll.easurrerilent o-í

l¡reatir hyclror;err suggests Lini- tlre incr:ease in hyCiro,¡en e>''cretiot-t imy

incìicate the tine l,¡hen 'rhe heacì of the nreal- enters the caecum rvirile in at

leas'¿ 50% of the s;u:jects ¡:e;ù: hyc-ì-ræ;en excretion indicates ÌJle 1-ine ','iieir

iìie l'>t'lli of the rle¿.l entered- tl:'.e colon. If tlis is Lhe cese' Lhen-the-

ol¡ser-v¿itir:rr i-ira-t tirese t.^¡o i:i-l,es a,re closellr correl-ateti s'''r;4:orts t-'re i'ci:a

'L'raL l'iu.asu¿-rlr:nt- c¡f l-ìle increæ,:;e in Ì¡r'e¿L'tlr ili'i¡otatt exci:eti-on o;:ovide:; a-il

-ì-i'ic.Le< o-i iJie irensit ti¡,Le of -ehe ireail ¿l.s rvell as'Lire Lrull.: of t-ìre i¡cal .

iiov,cver, in viei..;, of tire reCeltt ciat¿r of i'trlagelac.;r, iìocnrtson, Srov;n et ¿rl

(198¿1) ì-his ol¡ser-.¡¿ltion is r¡-restior-rtbl.e. ri\r:cival of chyr,e j-nio tlre cofon

is infruencec:r' as :imch by tìre gastic anptizing ra'te as it is by i niestirr'ar

treursit, ¿ur¿[ can-¡ro't- be inCLivicìualise,i ]:y lieasr:ring the cu¡:nulative en'try oÍ

narker into the caecun. Isotopic neilrods for intestirnl transit ii¡ie lurve

several aclvantages over ]:re¿rtÌr hycìrcgen cstj¡rai-ions. The lactul-ose Cose

use.j for hyc-ircçj'en ìtrea'ch testing can cause dose de¡:errlent chang,es in

Earstrointestinal transit (LaBrcoy, liale, lleavis et aI 1983). 'Itre

,orocltiction of h1rlroç1err in the intestine is dependent on Lìre type of

III

¡

þ

_trtl

i

135

loacierial flora present. Tìre a:rpunt of non-absori:a:1e carbohl'olrette

iirgesteo 'úre cìay J:ej=ore tlte test i:J-us oral l:ncteri¿r can alter lrasal l-e'¿els

tÌ're follo,,+ilg iiay (P,eacr, I'iiles, Fis;her c:i al 1940, lttcritpson, O'jirien,

l..c(Þr:thiz eL aI 1979).

iligestibte fo:rj ld.¡eltecl rvi"ch a g¿urtro¿ì ernil-ting rilariier (eg 99"n Tc-egg)

ca¡u1ot -r(1easlìre transi'c tj:Lie of solicìs Lrecause the food carrier is i

hi.c'L::olyseiJ.. 'rhe <jevelopnent of 131 I la.oeltecl fibre by ifülagela<ìa'

ir..ol:ertsor-r, Bror.,m et aI (19S4) llas provic-le<i a useful- ¡rarker for stlxlil'- of

¡.ansl;ort of sr:Iic'l-s throug{rout ijle sriell ancl large l';ov;el . ':-'he use of o-r-ral

ciâi:riâ lal:elleii iLr¿'.rl<ers iras cie;äosLratecl. tint, unlil-'e -':lie sicraacil, tlle small-

L<¡1.¡etl crrr-x)s rìo't ciiscrirlina'b.e betr,¡æn iite solicl aurcì. Iir¡r'l.icl- ccrnponents of

ch¡r¡¡s. Ij: Ì:tiJr li'l¿r.rjlers are, J::gesteci- sj.u',ultaneously the lic¡rici ccrirDonent

cripties -i'¿rs;i-c:r j-rc¡i l-rre =tart¡,cri a¡rcr: arLives at- tire câe-clTrl firs't but liroür

soU-ci a:rr-j -lic-¡rrid. r.rarl.,ers ,¡awel a-Iong ìjre sr¡n-l I l¡o'"'lef at si:nilar s¡:necis -

li-hese resulis ',.¡c¡-rl-d suggest LÌrat ccntrary 1-o 'the conclusions of lìeacl-r

i,ilesrFislieretal(1980)'"Íirsi-a-r::iva-l"ofci'ryt':e'LotirecolonisnoLa

::ehaì-rle frrecr,ictojt oF tlte subse;1'.rent ti:ansit sTect::lrrr of the Ì¡ull-- oj= cÌr1zne

¿,¡ii' U,,:.:rr:fore tÌtc ìrldr<4en brea.t'lt test c¡sxlct bc u:;c'j- to i¡'nntif)'ijle

i;ans-r-'ì: 'i::ì:rx: oi -C:e 'cr-ri:ire i,c¿l} .

tle fiiniitr;s ir-r tÌris s;'c'-rd.y a-re con:;j-st.:nt i¡irir c¡'tlre¿'s'cuclies r';'Ìlicir:;Ìror"

c.:e3re:;sio¡loisitootÌrn-lsclccontractihLizÌ-ly'fcrivrÌei-torr'oncs'Tt-lgerreral'

prcçester:onc rcduceci cr¡ntractiJ-ity ',,.nile oestrcr;cn ir¿rs art o<citatori/ ei'Fect

on tÌ-le sðJrìc tissue. T'Lcse Íinitinçs suçEest uÌnt- tl-re pr:olongation of

gastroi:ttestj-nal. 't¡::ursi t cccurring j.n -l-atr: prcÐîan'ìc-v is a result cf iJre

i-r-rc:<ra¡;clì ¡;'ìrys io1,:1 j.c¿r I concentr:¿r'b-ion of prog'es-terone'

I1re a¡,¡nrent l¿tcl'- of pro:luction of lilrdt**"t in 0 of E first' I of 12

seconii, a¡ii 4 o:f 22 t\ird trinester sul-rjects l:E)z lf,f i:el-atcv-l to r¡nrlied

ÍII

i

þ

¡J1ü

136

prolongation of -transit. I:ad sarr.rples Tnen ta-l'.en for 6 or B hours, it is

pno¡ab1e that the hyclroEerr peal< lror-rld have ]reen c-,étectec. Ttre fincìirtEs that

al-l- stuclies r.¡ith laclt of trlrdro<¡en proil-uction occurreci in la-te pregnanq/

su¡4nrts tÌris j¡rterpretation. Horvever, LaB'rooy, i:iAle, Ëeavis et al (1983)

have cle¡ronstrated that lcn¡ hydrogen producers on occasions excreted no

hydrog^¡ at all in expirecl air after lactulose. This fincìi-ng indicates

that scxne subjects are occasiornl non-proclucers of hydrcr;en. rhis could /æ

iæcause tlrey krarbour onry snarl mrnÏ:ers of hydroge*n - prociucing bacteria in

the col-on, which, lvith the norrnal shifts of ba.cterial flora, beccme

depleted at tj:-¡es.

;ìlürough it is unlikel-y, Iacl< of hycrrcael proc'ì.uction nay also be cltto to

pr:egineriqy-irclucecl ch.anges in colonic ]:acteria. Breaui hyCiroEen irrccluction

is tlre net resulb of hydrog¡err proclucLion ancì. iri'Ctogsn cc,nsui:ilrtion }ry

colonic l:acteria. Since hirc-,rogen coirsulincì ¡act¿:ria. e;<ist (I-€Vitt'

Uastings, Berggrol 1974) it is possible l-jrat preqnancy indttces a ch,arrgie i-n

colonic ba-cteria favor:-rinq hl4irogen consurip'tion over hydì-roc;etl ¡:'i'cr-uction.

Ilor..rever, tlrere aïe nc oç;'eriraenLlt cleta. to ei tircr rc-fu"e ol: sl-lilÐor-t iìis

Ìriz¡rcth.esis. rf cita'r;es in colonic fl-ora cc.-rlr::ry:r iir.r:::e¡rla¡:c1i, 1ieen'tite

absence of i-re¿r{h irlzdror*. at 2/:0 ilin ryoulcl l;: CiiÍficurlt- i:c rntclri;ret.

fnis stuiV and. ¡revious stuclies of Eal-Iblaäõ.e:: ft':rction in pregnanL r'¡cxircrt

suggest bhat cira¡rges in orocaecal ü:ansit or ç;aÌ1i:IailCr:r .¡oltune c¿¡ru:ot

account for the obsen¡erf increase in bile aci.j i:col size tha.t occurs in

early pregrnncy. ir-Ìrus, t-lre increase in ]:ile acic pooJ- r.rzrlz bo relatecl to a

direct effect of fe¡nre horn¡cnes u¡nn the Ìrepai:ic syn'thesì-s of bil-e acic-ls'

D. GAS'IT{TC lll,Ð GA]:.J,EI,Ai-ìLìIIP, EI'IFTIYIÌ{G

I

Ì

The finding of cieJ-ayecì and ìlcrcrnplete gallblaclder enptying in p::egrnncy

r37

after a rnixed solid liquid nreal could have resulted frcrn impairecl gastric

ernptying of solicls resulting in cliministred'neurohn¡r¡oral stj¡auti fron the

upper jejgnum. ttrenefore it was inportant to study stornach anrl gallbladder

simultar¡eor.rsly after a si¡nilar neal. In tÌÞ pnesent studlz it tns bee¡r

shov¡n Ulat, after ingestion of a st¿ndard neaÌ containjng 40es of. calories

as fat, the sc,cond slovr phrase of galllaclder ønptying ruas related to gastric

erçtying of solids. callbladder refilling @an v¡hen 138 of solids '1

rernai¡red in tlre stcrnach. Ttris suggests that the concentration of fat and

protein that enters the intestiral h¡nen after 50 min is probably

i¡sufiicient to fully stùrn:late i¡rtesti¡al or pancreatic honnones, or botl]

and naintain gallbladder contraction. Support for this is the drop to

baseli¡e serum I-IpP levels at the tinre r^¡hen 138 of sol-ids rsnained in the

stonach. The initial rapid rate of gaì-Ibladder erptying lray represent rragal

ùne::¡ation. Èoct., tia1rTrud; Fisher (1981 ) have shor^¡n that stra'n feeCing can

resul-t in erptyilg of 50e¿.of gallbladder voh¡ne. Tn addition atropile

allov¡s the gallbladcler to acccnnrodate larger i¡fused volures (Schoetz, La

t,trrte, Vlise et aI 1981 ). Therefore contj:ruei vagal simu-l-ation could play a

r"ole i¡ ¡rnj¡tai¡ing tìre gallbladcler crcntracteC post pnandially.

Si¡rce colrnencing tl-ris study it has been shov¡n tllat gastric elçtying is

biphr,asic v¡ith an early "Iag" pLr,ase being dsnonstrated if crcunting conti¡ues

tLrroughout the nreal. This lag phase is variable rvith a me¿ux of around 20

nún (61lins, IiorortlLz, Cool< et aI 1983). As 15 min v¡as alloi'¡ed for

i¡gestion of the test nieal this lag phase was not ds¡ronstrated. The gastric

enptying studies in this thesis were ¡ærfonned r¿itlt a ¡nsterior camera to

allor,¡ abdcrnirnl- ultrasor:¡d to be performed anteriorly. Alc]<ernans, Jacrcbs,

Srout et aI ( 19S4 ) usi¡rg a similar sized nreal slror¡¡ed that in enptying

curves obtained frsn a caflera posterior to the subject, a lag phase could

not be distingrished. In tlreir studies tlre geanetric nean of bottt

anteriorly and posteriorly obtainerl cr:rves shor'¡ed tl-re laqr prhase cloarly.

138

Hcrvlever, usirig a bran fabelled meal Ba;<terrGrirer'fritchley et al (1985)

'failed to stpr,r, a lag pLrase using an anteridr canera vrhich according to the

studies of Aklcernans, Jaoobs, Srr{rt et aI (1984) shoul-d strov¡ an exaggeratecl

lag,$rase. Malagelada, P,obertson, hcovrn et aI (1984) initially shou¡ed a lag

phase in only 2 an&- of 6 tealtlry patients using labelled bnan and an

anterior camera. Later sh¡dies nþre ænsistently denr¡nstraÈed duat prases

(Calnill-eri, Borol.n, l4alagelada 1986). It dæs seern like1y that by using/

cr-rJ-y one canera an "early erçtying" phase can be arbificially introduced.

IIohever, Akl<ernans, Jaæbs, Srrr¡t et aI (1984) have shoçvn by a dual-headed

cainera teckyrigue that after gastric enqltyjxg actr.rally begins, gastric

ernptyi:lg rates neasr:red by tlre two cameras are parallel. Therefore, tlre

assessn¡ent of the slope of the post lag solid anptying pbase is not int¡al-id

usjng one caflìera brt where ¡rossible tlre gecnetric nean of gastric ernptying

should have been used. '' :

Tlrere are cbvic¡¡sJ-y several potential j¡raccuracies that rnay reduce the

sensitj-vity ancl specificity of gastric ernptying tests using radionuclides.

These pnoblems nray relate to the instn¡nents, the types of Ia-i:ellec1 solids

and tlre constitrrents of ineal-s, the arntcrny of the patients studiecl,

analysis of gastric enptying data and tlre plrysical properties of the

raclionuclides thenrselves. For exanple, an fuqrortant guestion of the stìrdy

reported lrere is vùether tlre radiqruclicle r.rixed witkr the scrarnbì-ed egg

re¡nains bound with extracellular or intracellular fat. tr'byer, l4ayer, Jehn

et aI (1986) have rec-ently shcn^n tlnt nrost of tlre jntrac-elluÌar fat ernpties

with the solicl food phase rvhereas nost of the o<tracellul-ar fat enpties as

an oil phase. Iìor,everr. both intrac.'ellular and extracellualr fat appear to

leave the stqnach at the same rate. It is also uncertain as to hov¡ rmrch of

ttre tectunetiun label is bourd to fat and hov¡ rm¡ch is bourd to pnotein and

vrùrether ttris is inportant, ín interpreting the gastric ønptying results.

139

Ttris stucþ also shows th¡at tonic aal-lbladcler crcntraction is maintained for

4 hor¡rs after the starda:rd breal<fast. This explains the " flat "

gallbladder voltrne cr.ua/e obsenzecl i¡r earlier studies vùrere regular meals

rvere eaten at 3.5 to 5.0 hourly intenzals

Unfortunately a reliable raclioj¡rnrnrnoasay for HPP but not. for CtK or /

no'tilinrvras ørly available at the tjme of performance of these studies. The

ËlPP response \,,,as biphasic, as reported. ÍIPP levels dropi:ed to Ï¡asal valt',es

at approxi-rnately the time of initiation of gallblacicler refilling a¡d dicl

not increase suÌ:secluently¡ suggesting tirat tonic gaJ-Ibladder contraction is

rrraintained by huriroral stjmulation in response to gastric erçtying of

solicls. In acldition, IIPP, scnetimes referred to as "ãnticholecystokinin!:

horrnone" (Sarles, Flage, Iau$ier et aI 19791 is not res¡nnsible for

gal-IbJ-adcier refilling after ingestion of a meal. C,alIblac1c1er refilling is

rx¡st likely a ¡:a-ssive process which occurs when ernptyìng is no longer

sti¡iulated-.

,'rrr interesting ol¡servation i,yhicìr cìeserves iurrtjrer investiga'tion, is ijre

ti.,'ofoliì slov.rer GlIr' irr inenstruating w,rr,en cont5n-reci to nten. As v¡crnen t'¡ere

rreirstn-rai-ilg at .Lire ti¡re of the stucþ it is 1:oss-ì-bì-e Lllat nenstr-ua.I

sym¡rtcrns strppressed rnotility thrrough a central ntech¿msnt.

r¡lb.Idr Van Thiel, Fioechstetter et al 1981 reported. tllat GIT in r'¡cnren is

prolongecl in the luteal phase of the ovulatory rycle presunably because of

the inqreased ]evel- of .progesterone. Iracl subjects in the luteal phase of

the ovulatory rycle been sbrdied it could be anticipated tl¡at the

difference i¡r GIT betv¡een nen and hrcren could have been even greater.

It is conch¡ded ttrat gastric ernptying of the sofid porLion of a meaL

.[

140

containing fat induces and majntains tonic gallbladder contracti-on,

probably throtrgh conti¡rued releasê of hr¡¡oral mediators, frcrn small bovrel

and pancreas. Vlhen gastric anptying is co'nplete, hr.unoral- sti¡rmlation ceases

arrd the garlbradder refilrs. Ttris gestro-biliary integration herps naintain

rnixing of bile acids with ingested fat for optirnal fat ingestion,and

absorption. AlÈered gallbladder enptying rnay be secrcndary to altered

gastric enptying. - /

.,¡

u-'rÈI

i

i

I

{t'

NII

I

t

T4T

CHAPTER V

CLINICAL IMPLICATIONS OF ALTERED GALLBLADDER EMPTYING

AND BILIARY LIPID SECRETION IN PREGNANCY

A. THE ROLE OF MONNING SICKNESS

Clinical feabures of morning sickness

Diet and cholesteroì- gallstone formatj-on

B. THE POTENTIAL RELATIONSHIP OF DISEASES OF THE

ALIMENTARY SYSTEM TO ALTERATION IN BILIARY

LIPIDS IN PREGNANCY

I{rcosal protpcti,re effeöts of bil-iary cholesterol

(i)(ii )

(i)

(ii)

(iii)

ûhoJ-ecystitis in pregnancy

Reflux -oesoþhâgitis and peptic ulcer di-sease

T

I

t

t!

,ir¡l

I i .: i-.r *$: .:.,,;, '..lli;,,..¡¡,.-.;ii ;ns#S+*N,1iffi'+ffiAng' ]

r42

g,INICãL ]]..IPLICATICb]S OF ÀLTRÐ GAI;LBT,ADDER BIVIHTYING Æ{D BILIAPJ LTPID

SECRSMCTT TN PREG'IA}']CY

A. TÍIB ROI,tr OF IqSRNING SIC]TVESS

(i) Clinical features of norni¡g sickness

Pregrnncy v¡itkr its attendant physical horrnor¡al and physiologì-c aÌterations,

significantly affects the functj-oning of the gnrt at many leve1s. It

displaces stsnach and srnall- j¡ltestine and mobilises portions of the colon

frcnl thei-r custcrnary anatcxnic l-ocations; it alters irxrtility of oesopiragus'

stcxnach, srrrall i¡rtestine, colon and biliary tree; it irnpairs appropriate

furrctionÍng of the l-ov¡er oesopf,agdt sphincter; it decreases gas'cric

acidity; it is associated rvitLr ch,anges j¡r the selecti"¡e absor¡rtion

mechanisrn of snal-l- intestine and. col-on and probably in pancreatic secretion

as l'¿el-l- and it at'tenuates tire bod-y's response to such crises as

inflanrnati on, perforation and obstruction.

Ì.Lausea, with or ivitlrout vorniting is a ccnrnon, suggestively cliagii-rostic, sel-f

Iimiting slnnpton of ezrrly pregrìa-ncy. In its nxcst j¡nocous forrn j-t is

"mornilrg sickness" oçeriencect as nausea by as Íìany as 50eu and as vcr.riting

by appr:oxiinately 30% of \¡¡Gnen during their first trimester (FainveaLlrer

1968; ScLrer 1965). Both tlrese conditions generally arise during the first

12-'14 v¡eeks of pregnanry. It i; felt furpically u¡nn arising often before

any food has béen ingestecl. It nay persist at 14 weeks in as many as 40e;,

Ìry 16 v¡eelcs in less than 20% ancl by 20 r^¡eel<s in less than 10S (Diggory arrl

Tbrnl<i¡rson 19621.

:lN!r¡

tII

¡

!

l-¡* J

In morning sicÌ<ness, no specific,gastrointestj¡ral l-esion hâs been

denxrnstrated. I..lzrry theories have been advanced. Ttre literature h,as

centred on four general areas; allergy to ¡rrtative placental or serninal

toxins (none isolated); alteration in ga.strointestinal motility (no good

correlatj-on dsnonstratecl); lrormonal ey.cesses or deficiences acting dírectly

on the gut or on the central nervous system (poor correlation beb¡¡een

hormonal titres and clinical symptcnrs: Soules 1980); and psyctroscrnatic

factors (no correlation between vcrniting and personality profil-es::Palmer

19731. Ttre changes in orocaecal transit tjrne and gallbladder ernptying occurr:ed

in late pregnancy arrd no chanEes in Eastric enpting have been denons'urated

in the first trimester. frrerefore, it is r¡niil<ely tliat rnotility

clisturbances j-nduceC. by pregnan.y *l explain tlre slzrnptcrns of nrcrning

sici,,ness.?r central cause for r,orrring:sicktress seems rrore like1y ancl the

clecreasecl inciclence of severe vcxniti:rg in recent years ha-s been linl<ed to a

npre relaxed aititucle to pregnanal;'availabil-ity of aborLion and birth

control, as v¡eII as less restrictive obstetrical attitudes and regimes

(Jacdcs and Janor'tlLz 1965).

ii ) Diet. arrd clrolester':I qallstone fomation

Several studi es lrave sÌror,¡-r a¡r i¡lcrease in chol-esterol saturation of hurnart

hepa.tic and gallbì-adder bile after an eight to 16 hour fast (t,ietzcJer I

êdler, Heymsfield et aI 1973i I'lilliarns, Þlcrse, lncnornld et aI 1977; l3Ioch,

Thorntcn, Fleaton 1980). FastinE j-s associatecl rvith the storage of a part of

the bile-acid pool j¡r the gallblaclder, a clecrease i:r the rate of biliarl¿

bile-acicl secretion, vrithout paialleJ- diminution in the biliary secretion

of chotesterol and the lrepa.tic production and secretion of su¡:ersa'turatecl

biLe. Ttris effect rnight l:e accentuated by previous bile-acicl deficiency or

Ð(cess cLrolesterol secretion or both. I'/hile high cholesterol intak'e (Lten

Besten, Connor, BeIl 1973) and ovemeight appear to play an Jmportant role

I

in galtstone fo::nation, short term, Iov¡ calorie cliets Con't appear to

effert bilian¿ cholesterol saturation (Schlierf, Schellenlcerg, Stehl et aI

1981 ).In addition, prolonged fasting, gireater tÌtan 16 hours significantl-y

decrease bile litJ:ogenicity. Ttrerefore if nornirtg sicJ<ness plays a rol-e in

gallstone formation it is unlikely to be through an effect on total

caloric intal<e alone, reducing gallbladder erptying. A lrþre likely

gallstone risk factor frcrn rrprning sickness is the effecbs of nreal tlpe and

meal frequency on gallbladder erçtying. Ttre importance of gallbladcler

ernptyilg in the fornation of gallstones v¡as dsnonstratecì by llessing,

Bories, ltunstlinger et al- (1983). They showed ttrat patients fed þr total

larenteral nutrition for 6 v¡eel<s developed biliary stasis ancl gallstones.

Vlnile it is unlikely that v.rcrnen r¡ith. n"orning sicJ<ness woul-d undergo

pu:olongecl fastìlg, meal frec¡:ency i-s 1i}<ely to l¡e recluced, Capron,

Delarnere, Eev-ve et a1 (1981 ) studiecl 115 patients with radiolucent

gal-Istones. ltre cluration of fastìng r'/a-s neasured frcrn the end of the l-ast

meal of the cr,ay until the beginning of the next neal (bre¿l<fast or lunch).

In the youngest patie-nts (20-35 years) the dr:ration of overnight fast-ing

r¡as sig'rlifj-cantly longer in bhe ¡:atients vrith gallstones tiran in aEe ani

se>< matcheci controls.

Therefore, in pregrnncy prol-ongecl o-zatn-ight fasting in the first trinester

dne to rnorning sickness coul.f contriJ¡ute to other risk factors for

gallstone formation by jrcreasing cholesterol saturation of Ì¡ile and

reducing gallbladder enptying.

/ln alterrntive e>çJ-anation for a ç;ossible role of norning sickness i-n

chol-esterol gallstone fornutj-on'may be'via a change in dietarlr prefeg nce

r,ririch might increase biliary cJ:olesterol saturation. A beneficial- effect

of v¡treat fibre, in the form of bran, on the chrolesterol satr:ration of bile

lras Leen obselvetl by rnost (Porrare, IJeaton, IÐv/-B€€r- et aI 1976¡ i".bDougall,

Yalqzmyshyn, VIaIJ<er et aI 1978¡ l^Iatts, Jablonski, Toouli 19781 but not all

/

145

\,'¡orkers (Tarpila, Ì'liettinen, Lfe-tsaranta 1978). This decrease i¡r biliary

cholesterol- saturation v¡ith dietary fibre is obsen¡ed only l^dren the bile is

initially supersaturated. Therefore lack of dietary fibre crculcl be

res¡>onsJ-ble for sqne of the bil-iary lipid changes in pregnanqy.

Orr epideniological and e4perjnrental grcurds, Heaton (1973) postulated th,at

the consurnption of refined carbohydrates (]¡etter dessibed as fj-bre

depleterl foods) favours tLre secretion of bile supersaturateC with

cholesterol and hence i¡rcreases the risk of gaLlstones. This h1n:othesis

iras rore recently been supported Ì:y the e,-,tensive epidoiriol-ogical study of

Scragg, i¡Ici,lichael anct Searnarl< (1984). ft Lns al-so been argiued tb,at the

rnture of refinecl foods, especialÌy refined sugars, is such that they

inevi'Eably inflate the intal<e of energy. Ttrornton, lfunett and Hea.ton

(19S3) sho..vecl that. a Ciet rich irr r:"iirr.o carbohydrate resulted i¡r

Eal-Ibladder bil-e i¡eing more saturatecl l+ith cholesteroi "crni-rutn

i v¡ith a diet

erxcluding refined foocls. i{ovreverr'tåe refined cliet contained nnrch l-ess

fibre. i'ierner, Enn'rett, arrd Heaton (1984) showed no effect of a si;< ',.¡eeli ac-l

libitun ãiet' supplernentecl with refined sucrose, on the clrolesterol

saLuration irrler,r of fasting gallblaCder bile or bili-arty tipid secretioir

ra-tes. 'Ihese rece¡rt results suggestei 'EhaL a refined. diet is ¡xrthø9-enic

onllr in as ir-rch as it. is ck:ficient in cii-e-uar-1' fibre. I-lo\'/ever, as tÌre Ìrigìr

sr-lcr-ose ciiet adverselir affecteC ¡:Iasnra lipicls, it is ¡lossible that in thc

long tenn, it night j¡rcrease the risl., of gallstones. Irs prergne.ncy

progiresses past tJ:e first trimester, !./crren are inore likely to se'ek high

fibre foals to counteract j.nrpendilg constipation. Flo\orever, in the first

trj¡Llester nalrsea nray mal<e pregnant r,/crTen ai¡oid tlre bloating and abdoninal

disccrnfort of unrefinecl carrbohyclrates fuhich could then leacl to an increased

risk of gallstone forrnation as a result of j:rcreased bitiary lipid

secret,ion

,1

The above results suggiest tlnt certain cliets n'ny predispose to biliarl

r46

clrolesterol saturation. The nech,a¡rism rnay be via biliary cholesterol

saturation of tbrrough jrnpaired gallbladder eaptying. Data in útapter TïI

h,as skrorvn tlnt amlno acids are less potent than a rnjxed liquricl forrm.rla j¡r

aausing gallbladder ønptlzing. Gallbladder enptyi¡g in hrrrnn strk¡ects Lr,as

also been studied after meals of differerrt ccrnposition (Rocl<, l'{alrrud,

Fisher 1981 ). Gallbladder anptying after a rnixeC. solid nreal i s decreased

significantty conpared to a lic¡rid nreal for 75 mins after neal ingestion.

By 90 nrin, tLre maxi¡ral gallbladder ernptying responses are sjmilar. The

rnxi¡nal gal-Ibladcler erptying responses to 5% ancl 50sø glucose ancl to amino

acids are similar l¡:t all are less tha-n the rnaxi¡ra1 response to a rilixed

IiqLlici rneal. Therefore, galllcladder ernptying is affecbed by the physical

ancì che¡aical ccxnposition of ingested- nreals anci arry chanEe il dietary

preference could resul-t in altered- 1and scxnetinres inccriplete gallbladd.er

enptying. Ttre subsequent biJ-iary stasis coulcl preCispose to galtstone

form¿rtion

B. THE POIIT.'rI'IAL PJ{,AIIOdS;I]P OF DISAASES OF 'IIf,I ALIÌ'[XÍ'I!]FJ SYSlE.i TO

ÀLIELÌ\TIG'] ]f{ B[.IAl']J LIPIÐS l,i PF-t-rGl?il(a-

(i) liucosal protective ei-i'ects of bi.liarl' cÌroleste.r:c¡l-

In e,-lcerirnental anin'als, bile salts have caused dari',age to tÌre sr¡all and

Iarge intestine, stcmach a'rd gal-lÌ:l-acider and in rnan they have beeu

irnplicatect in the pathogenesis o'F several gaslrointestir¡,al inflan'rnatory

states such as gasbric ulcer ancì oesophagitis.

The rn:cosa of the gallbladder, ocso¡:hagus, stcrnach and cìuodenun are

presentecl ¡,.¡irrr* the problon of a potentially injurious solution of harmful

bile salts whiclr are povJerful cletergents able l-o peneLraLe lipicl bilayers

and at high concentrations solubilise rTemlcrane phosplrolipids ultùnately

L+t

causing nrsrbrane cìamage.

As ¡.¡el,l- as represeniilg an exeetory substance, drolesterol rnay also have

an i:nportant fi:nstion in bile (together rvith plrospholipids) of preverrtìng

the darnaging effects of bile salts on the biliary and j¡ltestinal

epittrelir-un. Bile salts placecl in the canine galJ.blaCder cause haerorrhagic

drolecystitis but tkre onset of inflalmration can be delayed by addhg

cholesterol (Riegel, Pørvdil, Johnston 1931 ), F\rrther evidence for the

pnotective effect of biliary cholesierol ccrips fron a studlz on the effects

of dihyCroxy bile acids on the hr¡nan jejr:m:rn (Broor, Slota, Àn¡ipn 19e0).

It v¡as sho',.in that bile salts induced secretion of v¡ater and electrolytes

into thre intestinal h¡nen (as opposed io the norn'a1 absorption of v¡ater a-rrl

electrolytes) but tlrat this effect coul-d be bl-ocl-,ed. by adCing cholesterol

to the bile salt solutions. 'ihese observations suggest thrat biliary

cholesterol has a role in preventinb Life salt j¡rducecl nmcosal damage.

Bi1iary su¡:ersaturation by cholesterol in fasting and preçnanry may protect

the biliarl¿ ancl- j¡testinal- epihreliul frcin the toxic effects of bile

sal-ts.

(ii ) drolecl-stitis j-n pregnar-rcy

Despite the prior evidence tLr,at pre¡rnnclz preclisposes to gallstones, the

incidence of cholerystitis is Iow, around .02- .078 (Sparlcnan 1957; lIam1i:r,

Eartlett, Smith 1951 ). This does noL exceed the rate in non-pregnant r"/cmen

of the same age Troup and in faét, parity ancl age clo not influence ttre

occurence of sltmptons.

It is frosslble agaln tiut clrolesterol Ìras a protectlve actlon and desplte

significant gallbladder stasis clrolesterol crystal-s and epithelial- damage

'1

148

are not rore likely to occur. Ttrere are several pnssible oçIanations for

this apparent protective action. One possibility is th,at bile sal-ts are

bound to cLrolesterol- and plrosphotipids by liçophilic association within

miv,ed rn-icelles and are therefore less able to r<-rct r'¡ith ihe epitheliurn

along v¡hich they are bei¡g transpori:ecl. Another is th,at absorption of

cholesterol by the gallbladder an¡1 incorporation j¡¡to mernbranes, alters tlre

cellular phl¡siolog)a and norphology of the biliarlr epitheliurn.

(iii) Refh:x oesophagitis and peptj-c ul-cer disease

iYoaclly clefined, reflux oesophagitis is the constellation of slroptons

and/or cÐnsequences to the oesophaçrs i'¡hicir result frcre contact of gastri c

or intestinal contacts r,./i1jr the oespptngeal rimcosa. Reflux oesophagj-tis

leads to Lhe substernal ì:urnìreg disccxnfort of heartburn. rhis slzmptcrn has

been reported in 30-70? of pregnancies, occuring daily i¡r as nnrry as 25e;

(A'tlay, Gillison, Iiorton 1973; ltrebel , Irornes, Castell 1976'). Onset is

usually in ijre seconcl trinester aurcl frec¡uency ancl ìntensity often escalates

a-s tire pregrìarìcy proceeds, only to vanisli a-fter delivery. Ir-lI pregnanl,

v¡crûen, irres¡:ective of hea,r-ubun-r syrnç:tcxirs, tend to exhibit nrore

non-propul-sive rrotor activitl' of Lhe oesoirir.egus l¡iLh clecreasal i"rave

anplitud.e ¿urd. sl-o.,ver ireristal-Lic qrcccì (i'i.agler and Spi-ro 1951 ).

Àlthough a source of discqnfort, heartl¡urn is usually not a serious

s¡anptor. Pe¡rtic oesophageal stricture in pregnancy is rare (Swirùroe,

ûoclrrane, tíisharb 1981 ), as is L¡ae¡norrhagic oesophagitis.

Patients rvith reflux ancl regurgitation often cqrr'lent tlnt the fluid tastes

l:itter ratler tlran sour and is ye1low or green in colour due to the

Ifresënce of cluoclerlal- contents. It h¿rs been knov¡n for scle tj.lre tlraL Ì:ilt:

salts in concentrations for:nd in gastric contents will cause perreabiJ-ity

"1

r49

cha:eges in 'the oesoph,agearl nn-rcosa (Safaie-ShLrazi, DenEesten, Z:tJ<e 1975,1.

l'he lack of severe ccrnplications of reflu.x oescphagitis in pregnancy could-

be due to increased binding of bile salts as a resulb of a relative e]<cess

of biliary chol-esterol. In addition, bile salts are irore likely to be

ciilutecl out in gallJrladder bile and 1m¡:aired gallblaclder erptying rray lead

to less bile salts enterilg the duodenur¿ at any one ti¡re point.

Sìrnilarly to oesophageal disease, the ccxnpJ-icatj-ons of peptic ulcer ciiseasr:

are rare in pregnancy. In fact, peptic ulcer disease itself is distincLly

rare in pregr-rancy (Sandr,¡eissn Poclo1slry, Saì-tz:rtein l9¿¡3) vriih tìrc condifion

terx-iinç¡ to :ir'tprove slzn:ptcxi-atically in at leas't 80eà of cases as pre/jnanc)¡

Ì)rf,Ð.resses (Clark 1953). Ttp rarity of tire concli'ticn ar-rcl- its ì-r-.,r¡:rovernent

during pregnancy have been linl-,ed to crjminished. gastric aciclity a-nd 'che

irealingr effects of oestra;en. iloryever, it is ¡rossible 'ijrat the <ia,rnaging

efects of reflu,-,ed bile acicls rr-Ely l:e reduce+ì by ililution and Lhe presence

of greater ar.punts of biliary cholesterol.

I

CHAPTER VI:

150

FINAL DISCUSSION

151

FINAL D]SCUSSTON

Chapter III of this study has ' shown that biliary lipid secretion

rates are dependent on stimulation of gallbladder enptying and small

intestinal transit. Re¡rcrted differences in flrean secretion rates between

groups of subjects fron different studies or between different groups of

subjects within the same study may be due to differences in stinmlation of

gallbladder enrptying and intestinal transit. Monitoring gal-lbladder

enptying and intestinal transit during biÌiary U-pid secretion studies is

inportant for the i-nterpretation of results. fn addition in healthy

individuals gal-Ibladder enptying and small intestinal transit affect

biliarlz lipid conposition. I ,

Tn the control- group of women studied by ultrasound aftêr a rnixed nreal the

initial rate of gallbladder errptying after breakfast was approximately 40?

of that previously reported using a smal-1 volume liquid meal (Braverman,

Johnson, Kern t9B0). The tiquid meal contained 520 cal- (35% faL, I2Z

protein). Increasing the volume, fat and amount of sol-id in a meal sfows

the enptying of both liquids and solids from the stomach (Moore, ChrÍ-stian'

Coleman L9B1; Malagelada 1917, Moberg and Carlberger 1974, Kroopr Long,

A-lavi et al- 1979, Cortot, PhiJ-lips, Malagelada l-981). Thê results of

studying gastric and gallbladder emptying sjmul-taneously suggest that the

J-arge volume high fat, rnixed solid liquid meal described probably enptied

slowIy frorn the stomach, reducing the rate of release of neurohunroral

mediators of gallbladder contraction from the upper smal-l- bowel and

pancreas, and resul-ted in the slower rate of galJ-bladder enptying.

Different rates of gastric enptying of liquids and sol-ids may have caused

the two phases of gallbladder enptying observed in this study, although

. r52

vagal- i¡rren¡ation could also e>.¡plain tlre initial rapid phase and contribute

to prolongec-l galJ-bl-acid-er contraction.

ttris study has sho,l.n tiiat gatlbladder refilling C.oes not occur until 6-7

hours after breal<fast alone and that this long pe::iocl of tonic ga-Ilbladder

contraction (or IaC< of gallbtadder refilling) is related to slorv enptyirtg;

of sol-ids from the stornach.Eecause nteals i,/ere ingested at 3-5 hour /j¡tervals i:y ..^¡6r*tr in the control group the galli:laclder dic- not refill..

this al so accornts for the cl-ose agireerent of resiclual and hourly voluiircs

i.e, tire s:¡allest galJ-Ì:1ac-lder volu¡*e a-chievec'l after l-rreal:fast approxir.rated

{-he gal-lblac-ìder volume for tlie resb of tJre daiz.

'Ilrc lacli of si.gnifica:-rt gallbladder refilling before ancl after -ure noon anC

evening meal-s in atl grou.os væ.s due to tire ccrnpositi-on anc-l frequencl' of

irlgestion of tl:e rneais. t

In arf.lition to the chnnges observed in gallbladcier enptyirtg,

ga-strointestinal transit tirne is p::olonge<j- ín late pregnanc)¡- It d-ecreases

;:os-i-partu:n to the level previously re¡:orte,f for Lhe follicul-ar phase of tÌre

o..-ulai:ory ryc1e. ,9ottr oroc¿recaf transit tiirie a:,rd serurL ¡trogos-terone levels

rise ;,.roçiressively Jretrreen prog'esterorre lc.¡s1s or 0 to iìOnç;/r:rl , -1'his Cior:s

not a*<cl-uc1e, holever, the possiiril-ity Lii;ri- c'tìrer hon:ones or neuronal

clturges ¡-rod-uced ciuri:r.-J pr:egnanc)¡ rnigïtt r:cr.iiate 'anescl effccts. Tt ¿ri¡. ¿'rs

tl:¿lì: r-l-re increased bile acid pool s-ize occu'rring in earrly prcrJnallcl is not

seconclary to sequestration of bile aci Cfi ei'ïlrer .,.¡j-tirin ga1-ll:J-ai,öer or smal I

intestine. Insteacì the increaseC irile acicl ¡:coJ- is proì:erbly a clirect

r.e-t¿bolic or lrorrrtonal effect on bile acici. sintSresis.

I5J

-t-"he effects on bilia-z-y' lipid secretion anC gall-bladdler function seen cltiring

pre?i-lancy v.'ere not reprsluceil in post menoinusal r,¡cr¿en tal.-ing cirronic :

oestrogten therapy. Ilovrever, the results are appU-c.l'-:1e only to tle doses of

oestrogen usecl in this stucþ. The other studies re¡:orted here i:',tplicate

progesterone as tlre rost l-il<eJ-y honrone to be causing C,isttubance of

biliary tipici secretion and snrooth rmscle fi¡:ction jn the Eal-Ibladder ancl

gasi=ointestinal tract.

'Ihe stucr,ies descrj-T¡erf in this thesis strongllr support a Sratlrog-enetic role

for gal-Iìcl-aclder sLasis in ga.J-Ìstone fornntion.I?-ecent observations frc¡r

aninal studies supçorb this hypothesis. The prairie cioE is a relativeli'

EoorJ anirnal rnociel for studying certain as¡;ects of cholesterol gallstone

fonnation. The prairie Ccg'increases cholesterol ab,sorption r','i'ch increasecl.

ilietary clrol-esterol- intal<è and secretes this excess cholesterol- into

he-patic l¡ile lvhich is stored in tl-re gallbJ-ailder (ilc1.er and lleriÐes+-en 1977).

Hithin a fevr days cholesterol- gallstones begin to fc::rn. Freceecling stone

fo::¡retion tÌre gatlbl-aclder voh¡ne enlarges dite to stasis (I'ieyer, Ðentresten,

G\rrnell- 1978, Flutton, Sieverlr, Vennes eì: al 1982).

liphincterotcny (surgice.l rencval of the specialisei :ansculatr-ire 'Jrat

contro-ls bile flov¡ frorn the ccrnnon cuct) al'ìo',.:s j'ree drainagc of tire

gall-blarcicel: anc'I. prevents galJ-stone fonraiion, cvê-n lviren bile 'i s

su1>e.rsaturateii- rrj-th chol-esterol.

I'lcllo'øing sphincterotcnry gallblailcler voltu,res rer.lainecl significantlLy srnaller

tiran in sham operated anir:nì-s. Atropine a-h:olisìres 'Lhe p::otective effects of

sphi¡cterotcxr¡z and qal-istones ¿rre al-Ior,¡ec1 to forr,. (llubton, Sievert, Vennes

et aI 19821

Does stasis play a role i¡r cholesterol choleliLlriasis in rnan? ilessing atrd

his colleaçues (I-'iessing, Eories, I{unstlinç¡er et a1 1983) reportcd tlut

154

alrnost ìralf of 23 patients on long tenn 1:arenteral hirperalirentation in

r"'¡lrc¡¡ no gallh-.rlarclder abno:¡nalities v¡ere initially ilenonstrated devel-o,oecì

bj-J-iary "sludge" l,¡h-ich is a miz,tu::e of biliary pignr:nt a¡rcl clrolesterol

c4zstals. In al-I instances these observatiors 'rJere rLraile a rninimirnrm of

LÌrree r.¡eeks after inib.iation of total- parenteral nutrib.ion (TÐ:l). In six

of their sJ-trdge positive pa.tients tÌro subsequently developed

chpl-elithiasis, four had norrnal ultrasonography studies cìuring the initial

one to tlrree rveeks of TFN. In a-ddition, F,osl1m, Pitt, I.kr¡n et aI (1983)

reported a Ìriglrer than eçected j¡cidence of EallblaclCer C'isease incJ-r-rding

acalculou,s c?nleq¡stitj-s during the course of prolonged 1?Î'i. These stu-cli es

st-::onEly suggest a patlrogenetic rol-e for gallbladder st:rsis.

Stucies of gaì-1bl¿¡.cicì.er oiq;tying in pa.tients rriih gial-lstones give variable

::esul-ts. ilortirfir:,-d, K.u¡:f¿¡, ì'ziuCgal ei al {1980) using scinti-graphy found

a¡r incre¿isec sensitivity to intravenous CG{ in rumy çra-Ilstone patients.

(k¡nversefv rrishrer, StelzJ, Roci', et aI (1982) shcriecl a. nonnaf res¡ronse to

CfIi but a dirninislred response to an ingestedr rni:rei rieal-. They shovred titat

tlris cìj-r-ni¡iisiied respcnse v¡as lrot clue to slci.¡er gastric anptyûrg. In other

sti;Cics çalì-r;laäicr erpLyinE Ìns i:cen shorn: tc i:e cicla-'icrl (Shaffer,

ì';cC:::rcni-i, D:99;rn 1t30) or non:al (V'an ,;erEe iíenegcui;..:li, llcfl:rrnn 1973,

rlori:.irfielCi anrcì lloñirane, 1975, iloll ancl Grr:rrc4z, 1978) - Ttrese ciiscrei::urcies

c¡-i¡roi l¡c reconciled at -j-ris sta'Ee. 'lhey rlo noL a,o¡-,eau: 'to ¡:efate to tìte

ege or se>: of tÌre patien'ts stu<Lieci. I.iiret-her -these changes represent Lhe

ca,.rse or -ulre resu-l-t of ga-Ilstones has not lrcen establishecl.

Scrne clinical situations suggest that galJ-icladcler stasis alone is not a

sufficient prerequisiLe to gallbl-aclcler clisease. Coeliac sprue is

characteriseC frecluently by gallÌ:Iadder enlargorent associatexl l¡ilh

sluggish contractility (Lov¡-Ecer, Iieaton, lieaton et aI 1971 , Low-Beer,

Flarvey, Davies et aI i975). Yet no unusual suscepLibility to clrolesterol

155

cholelithiasis Ìras been reported in these coeliac sprue patients. Possibly

the reason is tlrat i-he intestiral- l-esion produces an r:ndersatr:rated bil-e.

A sj-lnilar situatio:r o<ists i'or truncal vagoto-.ny pa'-tients (Gl-anviÌIe and

D:thie 1964, In-berg anC. Vuorio 1969, I{ielsen 19641.

l-uture studies of garstrointestj¡aÌ riotility in pregrnnry lvill need to

concen'trate on relatively non-invasive techniques. l-.l:rtlrer refinernent pf

the innovative ultrasourd techn-ique of líing, Adam, Pr1rcle et aI (1g1o4l ålrf,

yield valuabl-e new da-ta on ciranges in transpyloric fluid movenent as

precj¡ancy prqjresses. T[-re scatter of cìata be'tv¡een trines-ters sho',,¡n in iJ:ri¡

'bhesis dexionstrates the need for sequentJ-al sttrdies on the sane irdiviiìual:

to detect siqrrificant chr,anges betr.¡een trj-rnesters. À reliable ultrasound

techn-i-que for measuring gastric enptying v¡ould be an iCeal rnelhod for

ron-j¡.v¿rsive sec¡rential- studi es j:r pregranci/.

Poteniial proiclenrs lvithr hydrogren breath testing irave læen outl-inecl. I\rLhe:

stuclies of transit tjnle in pregr-nnry should includle a tncuth r.,'ash to

elir,rin¿,te oral bacterial hy-drogen procl-uction. fn aCdition, a- J-iquid neal

e,s reccÈiìrirenceci 3-j1' I,e lìr:oo;r, iãl-e Bcavis et al- {1983) cor-r.l-ci iiriprove

rei;rorjllc:Lbiì-il-1'. Lecause of the tncertainty as,i io'tire reeson:ior Ìùe lacl:.

of ]-.rea.-ti:L ìri,.ci:rcr_¡oi pr:odrrctj-olr i;-l sc';,ntirirrf tri¡:s'i-e;'sr::>jects rt rr.ay Ì:e

ilÌore i):recis;e tc ccnsicìer an afienntj-ve fo:r-rl of i:::ansit 'tji'le nieastuernc:r't.

iost r¡iehio:isr of irte¿:.sursnent use ra.c],'ionucl-icles i--,,ut i-ncre: recenLly IieIIor',',

Ecrocly, tririlli¡-rs et a-l (1985) liave äescrj-l=rcl an accrrfrete neasurement oi

orccaeq¡l transit- rrsinE the appeararrce in ver-rol-ls l:Ioccl o-i tÌre sulphur

ccxir¡:onent of oral-Iy i:rqested sul¡:hasalazine. Ttris urethod may prove useiul-

in the str-rdy of 1-ransit tir-,e j:r pregrr,ant \.ictnen, buL the need for frec¡rent

btood sarnpliirg l,ioulcl be a sic¡nificant c'ìrarvback¡ Il- should also be notd

th,at tirere is a cl-ose correlation hjetweên the appearance of Ì:reath hydroger

frcrn orarl lactufose ancl the appearance of suI1:hap1-ridine in the venous

blooci after ingiestion of sulpìrasalazi¡re.

In future studies of tire effects of prr-¡rr,ancy on orocaecal r-crotility' gut

lror¡rones r,iricit change c]ur-ing pregrlanclz shoulci be crcrrelateci vlith oilserverÌ

rnotilit-y chranc¡es. T'Ìie digesl-ive ca¡rzrcily of the grut j-s increaseci cìu-ring

precrÌncy ancl lactationr. Àntral ancì plasrna levels of gastrin lrave been

slror.¡n to be el-evatct in lactat-ing rats (Lichterìberger,!Trier, 1979\ arfl,

tlrere is a significant suckÌing-rel-ated effect on scrnatostatin tevel-s ín

pigs (tlvrns- i,loberg-, Ðriksson, Blcxnquist et at 1984). Scx¡ntostatin levels

r.¡or-r.kl b: of pa.rticular i¡rte::est in pregnancy as ì:rfusion of sc¡natostati-n

lras beerr reportecì to inhibit lùe gallbladder ernl:tying resi:onse to

hl.perLonic Alucose achinistereci, orally (Johansson, I'oIIIærg, Ilfenciic et aI

1931)

In ì:he fu'i:ure it v,'ou1c1 k¡e l¡orthl'¡hil-e systor'atically studying al teration j-n

gallbtaäcer fi:nction in'other special pa.tient groups. l's in other stuäies

of the trhl'=1oto"ic and re-udrolic al-terations occurring crirrir:,g nonral

pregnancy, alteratìons in gal-lbiaclci.er ärotility iuncLion, if clernonstra.i¡l-e ir

otjrer Ìrigii risì, Eroups, i;o¿Ici l:c'_rn1i}.-eJ_1r -to be an isolated abnonu,.Ii'ty lru

one ¡nssiì:Iy aurernl.lc: t-o cire-rr,peu-uic rinnipulatj-on ar-xl ther:e.r-'ore ,.,/or'uìi

:.no'.,'i¡g al-nut.

I

I

t

L57

APPENDICES

A. Real- Time Ultrasound Method for Determining

Galtbladder Volume

B. Characteristics of Infusates

C. Compari-son of Mebhods for Measuring Gallbladder

Emptying

D. Report ed Studies of Bil-iarv Bile Acid Secretion

In Subjecls with Intact Gallb1adders and Subjects

Status Post Cholecvstectomy

E Measurement of Smatl Bowel Transit Time

by the Hydrogen Breath Test

F. l4e asurement of Serum Human Pancreatic

Polypeptide

L

I

158

REÀL TIME ULTRASOUND METHOD FOR DETERMINING GALLBLADDER

VOLUME

Sum of cyJ-inders method: This method was original-ly

used but not val-idated, in cholecystographic studies

of gallbladder vol-ume. Volume is cal-culated from

sonograms by dividing the gallbladder image into

a series of cyJ-inders of egual height (h). A transparent

grid of paral-J-e1 lines, with constant distance (h)

separating lines, is placed over the galJ-bJ-adder image.

The longitudinal axis of the gallbladder image is

positioned perpendicul-ar-,to the grid of paralJ-eJ- l-ines.

Util-ising the formu.la för volume of a cylinder, the

formul-a for gal-J-bJ-adder volume becomes:-

V n ITa.l_ l_

i= l- (r):h

2

4

where h. and d. are the respective heightl- l-

of the ith cyJ-inder, and n represents the

cylinders in the series. But since hl is

between paral-Iel lines on the grid and is

each cylinder in the series, the formul-a

vol-ume is: -

and diameter

total number of

the distance

the same for

for gallbladder

(2)V 0.78s h ,Lur'

i=rI

I¡Ihen calculating volume with this method onJ-y sonographs

of the longitudinal projection are used. Vrlhen the

gallbladder is curved, ês it often is, the longitudinal-

projection wiJ.l not pass through the central axis of

the gallbladder (Figure la). Displacement of the

159

,longitudinal- projection from the central axis causes the

diameter measured from the longitudinal projection to

be small-er than the true galJ-bl-adder diameter (Figure 1b ) .

To correct for this, a factor E is introduced:-

tr- AP dlameter + width2 (3)

x 1

Since this correction factor

measurement of diameter, the

vofume becomes: -

Greatest diameber on longi-

tudinal projection

applies only to the

formula for gal-ì-bl-adder

V 0.7B5hE 2 d (4)

Irlhen one applies cornection factor Ei_ one assumes that

the displacement of the longitudlnal proiection from

the cenlral axis is uniform throughout the length of

the gallbladder. This is not always true, especiall-y

in Ìongr trârrohi tapering segments of the gall-bladder,

where volume is smal-l-. The volume in these areas would

n 2l_

i=l

be overestimated slightJ-y, but the effect on

volume is minimal. AJ-so, in situations ùIhere

total

the J- ongi -

thetudinal axis is paral-J-eI to, but

central axis, the caÌcul-ated GB

underestimaLe GB volume .in areas

the effect on total volume woutrd

displaced from,

volume woul-d tend

of tapering.

be small-.

to

Again,

rov

No correction factor for magnification ( as is used in

cholecystographic volume determination) is necessary

for ultrasound measurements. The measurement and

cafculation ( using a hand calculator ), of volume by the

corrected sum of cylinders method requires approximately

5 minutes for each PhotograPh-

,l

161

APPENDIX

TABLE IA

CHARACTERISTICS OF INFUSATES

Liquid formula Amino Acid Solution

ItII

Composition, glðtcHo

Protein

Calories. cat/ml 1.3

Osmolality, mosmol/litelr: 543

Marker Ê .titosterolInfusion rate, ml/min 3 .2!0.2

6.414.L4.7

Fat FaE 0Glucose 5.0Amino Acid

o.4

420

BSP

3.7!o.s

4.3L

cHo, carbohydrate. osmolality was measured by fteëzí-ngpoint lowering.

Amino Acid Gm/Litre

LysineTryptophanPhenylaJ-anineMethionineThreonineLeuCineIsol-euCineVal-ine

5.52.O

5.58.53.87.93.76.1

.a - ---.=<-r

COI"IPARISON OF METHODS FOR ING GALLBLADDER EYPTYING

TABLE ?A

MEASUR

AUTHOR

DUANE I^IE. & HANSON KC. L978

ENGLERT E.& CHIU Si^I. L966

SexM:F

ll:013:2

[.le i100-1 60

114-180

2L-62

26-68

hË Age S E imulustest meal

" fatty"meal

90 min CCK-IV(1r.7 Ivy u/rg/min)Total dose asbolus over 2 min

ccK-.004

Liquid formula

"fatEyl'meal

ICG marker dilutionr131-ropanic acidscintigraphy

Techni

ll

il

Real Time US

Real Time US

Mean b( min-1 )

-.02/+t

-.0385

-.0347

- . 0115

-.010M,- . 010F

-.03014ì-.027F-.040M,-.032F

-.0523

-.02

8 :0 L3L-2.0I 23-65

4:0 t3t-L7 5 26-49

10:9 20-68 CCK - ..001 Crick Tc-HIDA scinEigraphyHarPerRaPer

ccK-.002 rr

SPELLMAN SJ, SHAFFER EA.,ROSENTHALL L. 1979

BRAVERMAN D, JOHNSON M &KERN F. 1980

PALFRAMNA & MErRE HB, 1979

POrN,,1

0: II

14: 0

9 5-1 30

,|

L8-27

23-4L

'': -:l

163

-TABLE 3a

REPORTED STUDIES OF BILIARY BILE ACID SECRETION

IN SUBJECTS I^IITH INTACT GALLBLADDERS AND SUBJECTS

STATUS POSTCHOLECYSTECTOMY

BiIe'acid secreElonr¡mol/h

MEAN RANGE

'tl,i,)

SubjecEs with gallbladders o

Liquid formula, continuous

Liquid formula, inl-erniEfenE

Amino acid, continuous

2,20O

r,650

l, r50

1 ,550-2 ,92O

| ,47O-2,015

1 ,07 3-L ,236

Subjects posccholecysLectomy I I ,390 7 ,149-L,588

All da.ta were converEed coTmot/h by using the publishedweighEs of subjects and where applicable, by assuming anav"i.g. molecuÍar r^reighC of conjugaCed bile- actds of 500. Meanis the average of repõrced conLiol means; numbers ln parenEhesesare references.oThe only crlEerion for inclusion in Ehis headlng was Èhal Èhegroup served as a control populatton i.e., Ehey had no llver orgallbladder disease. Most rrçontrolrr populaËLons brere qulEehererogeneous; age ranges 18-87, sex rarios (male:female)from 0:L Eo l:O;-perceñc.of ideal body weighc from 86 co 2817".

SEudies erere done by duodenal perfusion Eechnique afcerpaEienEs had complecely recovered from surgery.t

I

;

I

iÌi

I

]-64

I4easr:re¡rent of Snaf]- Bor,,¡el TTansit Tirne by the Hydroqen Breath Test

Orocaecaf transit tjrre was neasured by the lactufose hldrogen breath

test (Bond ard Levitt, 1975; Bond and Levitt 1977¡ La Brooy, Ma]-g

Beavis et al- 1983; Solcnons, Viteri, Hamil-ton 1977). Sr:bjects raere

advised to avoid excess intake of starch polysacctr,aride the day before 1

the test. After an overnight fast, subjects irgested 10 grams of factufose

i¡ 100nù of water. Breath sam¡rles for neasure¡nent of hydrogen concentration

r,,¡ere obtai¡red fasting and eveÐr 15 minutes for 4 hor:rs. Breath hydrogen

concentration was neasured with a Quj¡rtron mode.I S gas chronatograph (Quintron

Instrr:rnents Inc. lutifwaukee, Wisconsi¡r) usi¡rg argon as carrier gas at lBmI ¡:er

rni¡rutes, a molecufar sieve coJ-tmn,' and thermal- conductivity detector.

Orocaecaf transit tirre was defined as the tirre .of the first sustai¡red

rise in breath hldrogen concentrati-on frqn base.U¡re. If after 4 hours no

rise in breath hydrogen occurred, the transit tj-rne was recorded as 24O m-i¡rutes.

In each study of pregnant vvonen, transit tjrre was estirnated by 3 observers

bl-inded to the str:dy protocoJ- and design. Each subject's transit tjlrre

was reported as the nean of the 3 estjrnates. TTansit tine varied aÍìong

observers ín 242 of str:dies but j¡r each case by onJ-y 15 rnilutes. In the

secretion study fess observations were avail-abl-e and eight observers were

used. Bond and Levitt (1975) validated the H, breath test technique as

a Íþasure of srnal-I j¡ltestj¡ra-l transit by simultaneously neasuring the

appearance of non-aborbed PEG at the il-et¡n wj,th a rise i¡r breath H, Produced

frqn the intesti¡ral- bacterial- netabolism of ingested lactu-l-ose.

I4easr:renent of Serun Hunan Pancreatic Polypeptide

Sert¡n hr¡nan pancreatic pollpeptide(HPP) levef was determined by radio-

j¡rmunoassay in the taboratory of Dr. Ian TayJ-or, at tLre Centre for Ufcer

Research and Education, Veterans Adminístration Centre Vùadsworth

Hospital, Los Àngeles, California. l4ean i¡tra ard iìterassay precision

1t

I1

I

¡

i

I

I

i

I

165

h¡ere. 4å ar¡d 88 respectively. Recgvery of HPP added to serun deviated

by no npne than 178 frqn tle ocpected values otner the range of 40-340

pg/m1 senm. HÙman pancreatic pol-y¡nptide was plotted agailst tÍrÞ

and tt¡e integrated response was rneasured frqn the area urder tte HPP

curye.

CaLcu-l-ation of BiJ-ian¡ Cholesterol and Phospholipid i¡ Secretion Studies

Ite cholesterol ard phospholipid concentration of each duodenaL sanple

was corrected for jnfi¡sate cholesterol and phospholipid using the

calculation: -

166

tCH I net tCH ] ( tCHlr IBeta 5lo )D

lCHl net

IBeta 5lr

: Concentration of cholesterof in duodenal

bile sample due to biliary secretion

( micromoles /míl-l-i]-itre )

: Measured choJ-esteroJ- in duodenal- samples

: Infusate chol-esterol concentration

: Infusate beta-sitosterol concentration

: Duodenal- sampJ-e beta-sitosterol- concentration

(or [PL]O net) is used in subsequent

calculations of secretion rates (as IX]o,

see below)' by the standard marker di.l-ution

equat ion : -

X sec = [X]¡ [M]I, R. 60 min

lMl r>

Secretion rate of a particufar biliary J-ipid,

X, (micromoles per hour).

Duodena.I concentration of X (micromoles

per millilitre )

Duodenal- concentration of marker (micromoles

per mì-IIititre ) .

Infusate marker concentration

Rate of. infusion (mil-l-il-itres per min)

lcHlo

lcHlr

I Beta

IBeta

lcHlo

sìrs1o

net

Xsec

lxlt

tMl D

tMl IR

j-61

B.

Short Axis Projection

FIGURE IA

Diagram showing relationships of rtarr centralaxis of the gallbladder, 'rl'r, longiEudinalaxis from sonogram and ttct', short axis fromsonogram. B. Sketch of ultrasound longitudinalprojection (-----) superimposecl uPon actualsíze (-) of gallbladder. The short axis projectionis also shown with diameter rrdrr (d=At' diameEer +

width) /2. rrd, is the diameEer which is measured

from Ehe longitudinal projection.

\\

{z,ttd\

tltIt

f

l'aII

idII

l.t

I¡

IIIIIII

I

II

tIIrOtI

l!IItI

I

-ti -

F,

,t

ldt.It

_t-

T7I

B]BLIOGRAPHY

172

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]-73

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t78

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