Contraceptives on Bile Lipid Composition

Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from

Guit, 1983, 24, 253-259

Effect of synthetic oestrogens and progestagens in oral contraceptives on bile lipid composition

R H L DOWN, M J WHITING, J McK WATTS, AND W JONES From the Departments ofSurgery and Obstetrics and Gynaecology, Flinders Medical Centre, and Flinders University ofSouith Australia, NSW, Australia

SUMMARY The prevalence of cholesterol gall stones in young women has increased since the introduction of oral contraceptives. The synthetic female sex hormones used in these preparations, increase the degree of cholesterol saturation in bile. To determine whether oestrogens, progestagens, or both, are responsible for the change in biliary cholesterol saturation index, a prospective randomised, controlled study was performed. A significant increase in the cholesterol saturation index of bile was observed when either 30 ug ethinyloestradiol plus 150 ,g norgestrel (p=001) or 50 ,ug ethinyloestradiol plus 250 gg norgestrel (p<0.01) were ingested daily for two months. No change in the cholesterol saturation index was observed when 30 ,g ethinyloestradiol alone, or 30 ,ug ethinyloestradiol plus 2.5 mg norethisterone were used. The mechanism for the increase in cholesterol saturation index did not appear to involve bile acid metabolism. These results indicate that the progestagen, norgestrel, and not as previously thought the oestrogen, ethinyloestradiol, is responsible for the increase in cholesterol saturation of bile which accompanies the use of oral contraceptives. http://gut.bmj.com/

Epidemiological studies'-"' have suggested that relative time periods of cholesterol unsaturation to there must be a causal relationship between female supersaturation during the day in favour of sex hormones and gall-stone disease. Gall stones are cholesterol supersaturation within the gall bladder twice as common in females once puberty4 has been and lead to cholesterol crystal formation and gall- reached. After the menopause2-3 this sex difference stone growth.'6 gradually diminishes. Similarly, pregnancy3 and the Studies'7-25 have been performed in reproductive on September 24, 2021 by guest. Protected copyright. use of synthetic female sex hormones for contra- women to investigate the effect of female sex ception>-9 or the prevention of postmenopausal hormones on the cholesterol saturation index of symptoms"' have been shown to increase the bile. Physiological changes in natural sex hormones incidence of gall-stone disease. during a normal menstrual cycle have been shown to Approximately 85% of gall stones are composed have no effect in four separate studies,'7-20' and a predominantly of cholesterol. It is now recognised small effect in a study by Low-Beer et al.2' The that cholesterol gall stones will form only when bile withdrawal of natural female sex hormones by is supersaturated with cholesterol." Conversely, oophorectomy in one woman reduced the dissolution of cholesterol gall stones may occur cholesterol saturation index of bile.22 The ingestion when bile becomes unsaturated with chol- of synthetic female sex hormones in oral contra- esterol. 12-14 In 1973 Thomas and Hofmann'5 intro- ceptive doses has been shown to increase both the duced the cholesterol saturation index (CSI) to cholesterol saturation index of bile and the propor- quantify the degree of cholesterol saturation in bile. tion of women with supersaturated (CSI >1.0) Any substance or factor that increases the bile.2>25 It has been claimed that oestrogen is cholesterol saturation index of bile may alter the responsible for these changes.2$28 The evidence, however, is circumstantial. Studies in rats,29 Address for correspondence: Discipline of Surgical Science. Facultv of monkeys, 30 and baboons3' which used oestrogen Medicine. The Universitv of Newcastle. David Maddison Clinical Sciences unit than Building. The Royal Newcastle Hospital. Newcastle. New South Wales. doses up to 1000 times per weight greater Australia 2300. that used in oral contraceptives have shown that Received for publication 1 June 1982 oestrogens can alter the lipid composition and the '53 Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from

254 Down, Whiting, Watts, and Jones bile acid kinetics of bile, but not always in favour of biliary cholesterol saturation index, the bile acid cholesterol supersaturation. Clearly, species differ- profile, the total bile acid pool size, and the level of ences exist. We therefore decided to investigate in circulating serum progesterone. humans, firstly, whether contraceptive doses of synthetic oestrogens or progestagens were BIL1E SAMPLING responsible for the reported rise in the biliary Duodenal collections of gall-bladder bile were made cholesterol saturation index and, secondly, whether exactly 12 hours after the ingestion of a standard this could be attributed to an effect on bile acid meal - two slices of bread and butter and a glass of metabolism. milk. The subjects swallowed an AN-20 weighted duodenal tube (Anderson Products Inc, Oyster Bay, Methods New York, USA). Gall-bladder bile samples were aspirated from the duodenum after a slow intra- The original objective was to evaluate the effect on venous injection of 1 U/kg Pancreozymin (Boots Co the biliary cholesterol saturation index of equivalent Ltd, Nottingham, England). A 2 ml sample of the pharmacological doses of the synthetic female sex most concentrated bile was immediately sent to the hormones commonly used in oral contraceptives: laboratory for lipid analysis. Excess bile was 30 jig ethinyloestradiol alone, 150 ,ug norgestrel returned to the duodenum to avoid depletion of the alone, 30 ,g ethinyloestradiol plus 150 ug bile acid pool. norgestrel, and 30 ,ug ethinyloestradiol plus 2.5 mg norethisterone. Fears were raised about possible BILE LIPID ANALYSIS side effects from the use of 150 ,ug norgestrel alone The biliary lipids (cholesterol, total bile acids, and because this dose was five times that currently used phospholipids) were assayed using fresh bile for in progestagen alone oral contraceptives. A brief solvent extraction as described by Whiting et al.'8 pilot trial showed that the use of this dose of Assays were performed in several batches as norgestrel alone for longer than one month caused samples became available during the study. The persistent nausea and breakthrough uterine cholesterol saturation index was calculated by the bleeding. Therefore, the part of the study dealing method of Thomas and Hofmann'5 using the with norgestrel alone had to be replaced. A 'high' cholesterol equilibrium saturation limit of Holzbach http://gut.bmj.com/ oestrogen dose oral contraceptive, 50 ,ug ethinyl- et al.32 oestradiol plus 250 ,ug norgestrel, was chosen Individual bile acids in the gall-bladder bile because most of the oral contraceptives used in the samples were deconjugated and quantified by gas original study of Bennion et a123 contained 50 ,ug liquid chromatography as described by Whiting and ethinyloestradiol. Watts.33 Individual bile acid profiles were deter- We undertook a prospective controlled trial in 10 mined by expressing the relative concentrations of

healthy female volunteers, aged 19 to 35 years, the individual bile acids as moles per cent. on September 24, 2021 by guest. Protected copyright. known to have normal oral cholecystograms. Intra- uterine contraceptive devices were used as necessary BILE ACID POOL SIZE to prevent pregnancy. The study was approved by The total bile acid pool size was measured by the the Hospital's ethics committee. simplified isotope dilution technique of Duane et The design of the study was as follows: there were al.3 Two minor modifications were used. Firstly, five phases - a physiological control menstrual cycle the 10 ml intravenous injection of radioactive and four oestrogen/progestagen phases. The order cholate was always given half an hour before eating of participation in each phase was randomised. In the standard evening meal. Secondly, the addition each oestrogen/progestagen phase, the prescribed of non-radioactive sodium cholate to the vial of dose (see above) of the synthetic female sex radioactive cholic acid proved essential to prevent hormone(s) was ingested daily from the fifth to the major losses from the solution of radioactive cholate 26th day of the 28-day cycle for two cycles. When a by absorption to glass surfaces. 14C-carboxyl cholic control menstrual cycle followed an oestrogen/ acid (specific activity 50-60 mCi/mol; the Radio progestagen phase, two physiological menses were Chemical Centre, Amersham, England) and (2,4- allowed to elapse before bile sampling to ensure 3H) cholic acid (specific activity 14 Ci/mmol; New complete elimination of the synthetic hormones and England Nuclear, Boston, USA) were purchased as allow ovulation to start again. Gall-bladder bile and sterile solutions in ethanol. Each isotope was blood samples were taken on the 23rd and 26th day repackaged in a 100 ml sterile injection vial of 0.9% of the second month in each of the oestrogen/ sodium chloride with 175 meq/1 sodium bicarbonate, progestagen phases, and on the third, 13th, and 21st 0.2 mg/ml cold carrier sodium cholate and either 2.5 day of the control menstrual cycle to measure the ,Ci 'C cholate or 12.5 ,uCi 3H cholate per 10 ml. Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from

Effect ofsynthetic oestrogens and progestagens in oral contraceptives otn bile lipid composition 255

SERUM PROGESTERONE Control 30 ,ug EE Control 30 yug EE Ten millilitres of blood were obtained by 2.5 mg Nor venepuncture with each bile sample. The serum was separated and snap frozen at -70(C for subsequent 1*0- I batch hormone analysis. The serum progesterone was determined by radioimmunoassay after hexane extraction. 08-

STATISTICS x 06- A precise measure of the cholesterol saturation of -a W duodenal bile requires multiple samples. 18 35 There- .c c fore, the average of the two or three cholesterol 0 saturation index measurements in each phase of the study was used for statistical analysis. The 2L- Control Control 50 EE U, Mu Wilcoxon's signed rank test for paired samples was a 250 jug Ng used to analyse all data. 0 .c Results 1-0- Seven of the subjects completed all phases of the study, while the other three were able to participate 08- only in a control phase and two oestrogen/ progestagen phases because they left the area. The 0.6 control menstrual cycles proved to be ovular in all 0.80 Median 0-96 0-80 Median 0.87 subjects by measurement of serum progesterone P=0.01 P< 0.01 levels. The oestrogen/progestagen combinations induced an anovular cycle in all subjects except one. This subject ovulated while taking 30 ,ug ethinyl- Fig. 1 Effect in normal women ofdifferent synthetic female sex hormone combinations on cholesterol saturation alone. The daily dose of synthetic female http://gut.bmj.com/ oestradiol index ofgall-bladder bile. Mean CSI during normal sex hormone(s) in each phase was tolerated well by menstrual cycle (control) in each subject has been compared all women except one who developed early morning with mean CSI during last week ofsecond 'month' of nausea and vomiting while taking 30 ,ug ethinyl- synthetic female sex hormone ingestion. oestradiol plus 150 ,g norgestrel. None of the EE: ethinyloestradiol, Nor: norethisterone, Ng: norgestrel. women had major changes in their weight during the year of study. on September 24, 2021 by guest. Protected copyright. CHOLESTEROL SATURATION OF GALL-BLADDER while only one of the 10 subjects had bile super- BILE saturated in cholesterol during her normal The average cholesterol saturation index of gall- menstrual cycle, this increased to five of the 10 bladder bile in each subject's control menstrual subjects while taking 30 jig ethinyloestradiol plus cycle was compared with that in each oestrogen/ 150 ,g norgestrel. progestagen phase of the study (Fig. 1). Variation Comparisons of the subject's gall-bladder bile between the multiple cholesterol saturation index cholesterol saturation index in each of the values obtained for each subject in each phase of the oestrogen/progestagen phases of the study are study was less than we found in a previous study,18 shown in Fig. 2. A significant increase in the median the mean range of values being 0*17±0*04 (SEM). cholesterol saturation index was observed when 150 No significant change in the cholesterol saturation jig norgestrel was added to 30 ,ug ethinyloestradiol index occurred when the subjects ingested a daily (p<0.02). An increase in the cholesterol saturation dose of 30 ,ug ethinyloestradiol alone or 30 ,ug index was also observed between the 30 ,ug ethinyl- ethinyloestradiol plus 2.5 mg norethisterone. A oestradiol alone and the 50 ,g ethinyloestradiol plus significant increase in the median cholesterol 250 ,g norgestrel but it did not quite reach saturation index was observed, however, when the significance, possibly because of the smaller sample subjects ingested a daily dose of both 30 ,ug size. No significant difference in the cholesterol ethinyloestradiol plus 150 ,ug norgestrel (p=0.01), saturation index was found between 30 jig ethinyl- and 50 ,ug ethinyloestradiol plus 250 jig norgestrel oestradiol plus 150 ,ug norgestrel and 50 ,ug ethinyl- (p<001). Another important observation was that oestradiol plus 250 gg norgestrel, nor 30 ,ug ethinyl- Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from

256 Down, Whiting, Watts, and Jones

30 pig EE 30 Mg EE 30 pu EE 30 ,ug EE Control 30 pig EE Control 30 pig EE 2-5 mg Nor 50 pig Ng 2 5 mg Nor 10-

1 0- 8-

0 8- 6-

06- -4 W U) 0-76 Median 0 75 0 76 Medicn 098 0 5.9 Median 7.0 5.8 Medion 5.0 P=NS P 0-02 E E P=NS P=NS

a, ci 2? .--a 30Opg EE 50 Oug EE 30pg EE 50 ug EE ° Control 30 pg EE Control 50 pug EE 0 250 pg Ng I1150 pg Ng 250 pig Ng n 150 pg Ng 250 pig Ng 8 _A 10- a- ffi1~~~~~a 1*0 8-

08 6-

0.6 0.76 Median 0.85 1 0o95 Median 0.87 4- P=NS P=NS 5 6 Median 4 8 6.0 Medion 5-4 P=NS P =NS Fig. 2 Comparison ofeffect ofingestion ofdifferent syntheticfemale sex hormone combinations on cholesterol Fig. 3 Effect in normal women ofdifferent synthetic saturation index ofgall-bladder bile in normal women. female sex hormone combinations on total bile acid pool http://gut.bmj.com/ Mean oftwo bile CSI measurements taken during the last size. Total bile acid pool size (in moles) during a normal week ofsecond month ofingestion ofeach syntheticfemale menstrual cycle (control) has been compared with that sex hormone combination have been compared in each during the last week ofthe second month ofsyntheticfemale subject. EE: ethinyloestradiol, Nor: norethisterone, Ng: sex hormone ingestion. EE: ethinyloestradiol, Nor: norgestrel. norethisterone, Ng: norgestrel. on September 24, 2021 by guest. Protected copyright. oestradiol alone and 30 ,ug ethinyloestradiol plus 2.5 Epidemiological studies5-9 have shown that the use mg noresthisterone. of oral contraceptives has changed the prevalence and incidence of gall-stone disease. More recent BILE ACID METABOLISM biochemical studies2~25 have shown that some oral The effect of the synthetic female sex hormones on contraceptives increase the cholesterol saturation the bile acid pool size is shown in Fig. 3. None of the index of bile and thus its lithogenic potential. These synthetic female sex hormones had a significant studies had limitations. Initially, up to five effect on the size of the total bile acid pool. There commercial brands of oral contraceptives were used, was no significant change in the profile of individual and these contained several different synthetic bile acids except for a small decrease (median 32.5% female sex hormones (50 ,ug ethinyloestradiol plus to 28-0%) in the percentage of chenodeoxycholic 500 ,ug norgestrel, 50 ,ug mestranol plus 1.0 mg acid in the bile of women taking 30 ,ug ethinyl- norethindrone, 50 jig ethinyloestradiol plus 1.0 mg oestradiol alone (p<0.01). ethyndiol diacetate, and 0-1 mg mestranol plus 1-0 mg ethyndiol diacetate). It was, therefore, Discussion impossible to determine whether the observed change in the cholesterol saturation index was an The proportion of women of child-bearing age who oestrogenic or progestagenic effect. Secondly, these use oral contraceptives in the USA, Europe, and 'high dose' oestrogen contraceptives are now Australia had increased from a few per cent in 1962 prescribed only where lower dose oestrogen contra- to 20-35% by 197336 and may still be increasing. ceptives prove inadequate. It is possible that the Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from Effect of s nthleti oestrogens atnld progestagenis ini oral contraceptives otn bile lipid cottnposition 257 effects which have been observed with high dose cholesterol saturation index of bile and oestrogen oral contraceptives do not occur with the norethisterone did not, could be related to the lower dose preparations. especially as most differing properties of synthetic progestagens. It can studies'7-2' have shown that the physiological be calculated from the figures of Dickey and Stone38 changes in natural female sex hormones during a that, while 150 Mg norgestrel and 2.5 mg normal menstrual cycle do not alter the cholesterol norethisterone are approximately equivalent doses saturation index of bile. of progestagen, norgestrel is four to five times more The daily ingestion of 30 ,ug ethinyloestradiol androgenic and seven times more anti-oestrogenic alone in this study had no significant effect on the than norethisterone. Furthermore, norethisterone degree of cholesterol saturation in bile even though has a mild oestrogenic effect which norgestrel lacks. the cycles were anovular. This suggests that the As norgestrel is a potent anti-oestrogen and yet increase in the biliary cholesterol saturation index androgenic, an oral contraceptive which contained which was observed with ethinyloestradiol plus ethinyloestradiol 30 ,g plus 150 Mg norgestrel would norgestrel cannot be attributed to ethinyloestradiol, have a lower oestrogen to progestagen potency ratio nor the suppression of physiological oestrogens, than ethinyloestradiol 30 Mg plus 2.5 mg progesterone, luteinising hormone, or follicular noresthisterone.39 The overall oestrogen to stimulating hormone. Only one other study on the progestagen potency ratio of an oral contraceptive effect of 30 Mug ethinyloestradiol has been carried may, therefore, be more important in determining out, and it was performed in men, not women.37 It its effects on the biliary cholesterol saturation index showed that this dose of ethinyloestradiol resulted in than the dose of each sex hormone. This theory a significant rise in the bile cholesterol saturation already has some support as an inverse correlation index after seven days' treatment. The reason for has been shown between the oestrogen to this sex difference is unclear, but it may be related to progestagen ratio of oral contraceptives and the risk the 50% fall in the levels of circulating testosterone of developing gall stones.6 Future studies into the which also accompanied the use of ethinyloestradiol effect of sex hormones on bile should consider not in men.: only the doses of oestrogen and progestagen, but The importance of the increase in the biliary also the overall oestrogen to progestagen potency cholesterol saturation index which was observed ratio.

with the combined oral contraceptives containing None of the oestrogen/progestagen combinations http://gut.bmj.com/ 30 Mg ethinyloestradiol plus 150 Mg norgestrel, and used in our study significantly changed the total bile 50 Mug ethinyloestradiol plus 250 Mig norgestrel, is acid pool size or the bile acid profile of bile, except highlighted by the fact that the number of women for ethinyloestradiol alone, which decreased the with bile supersaturated in cholesterol increased proportion of chenodeoxycholic acid. Other from 10% during a normal menstrual cycle to 50% studies'9 22 23 have also found that both endogenous while on therapy. Furthermore, the addition of and exogenous female sex hormones are associated also with a decreased proportion of chenodeoxycholic 150 ,ug norgestrel to 30 gg ethinyloestradiol on September 24, 2021 by guest. Protected copyright. resulted in a significant increase in the biliary acid in bile. It is not clear, however, whether this cholesterol saturation index. It is surprising, change is related to an increase in the cholesterol therefore, that the addition of a pharmacologically saturation index of bile. Our results also show that equivalent dose of another progestagen, increases in the biliary cholesterol saturation index norethisterone, to 30 ,ug ethinyloestradiol did not can occur with combination oral contraceptives have a similar effect. These findings indicate that without concomitant changes in bile acid norgestrel, and not ethinyloestradiol, was composition, and support the more recent study of responsible for increasing the degree of cholesterol Bennion et al.4 ) They were unable to show a change saturation in bile. The theory2628 that the lithogenic in total bile acid pool size or bile acid composition effect of oral contraceptives on bile is soley because with oral contraceptives, even though the biliary of oestrogens is, therefore, no longer tenable. It is cholesterol saturation index increased. Instead, they still possible, however, that higher doses of ethinyl- showed that the increase in the biliary cholesterol oestradiol or other synthetic oestrogens - for saturation index was because of hepatic enhance- example, mestranol - might affect the biliary ment of cholesterol secretion into bile. cholesterol saturation index, or that oestrogens are In conclusion, our study has shown that the necessary for norgestrel to exert its effect. The latter progestagen, norgestrel, and not the oestrogen, possibility might have been answered if side effects ethinyloestradiol, is responsible for the increase in had not prevented subjects from participating in a bile cholesterol saturation which accompanies the 150 Mug norgestrel alone phase of this study. use of oral contraceptives.225 It also suggests that The reason why norgestrel affected the this side effect is not a property of all progestagens. Gut: first published as 10.1136/gut.24.3.253 on 1 March 1983. Downloaded from

258 Down, Whiting, Watts, and Jones If future studies confirm these findings, it may be 13 Dowling RI-. Chenodeoxycholic acid therapy of gall- possible to manufacture oral contraceptives which stones. C'lin Gastroenterol 1977; 6: 141-63. do not subject women to an increased risk of 14 Hofmann AF, Thistle JL, Klein PD, Szczepanik PA, gall-stone formation. Yu PY. Chemotherapy for gallstone dissolution. II. Induced changes in bile composition and gallstone response. JAMA 1978; 239: 1138-44. We would like to thank Schering AG, Berlin, West 15 Thomas PJ, Hofmann AF. A simple calculation of the Germany, and the National Health and Medical lithogenic index of bile: expressing biliary lipid Research Council of Australia for generous grants composition on rectangular coordinates. Gastro- that have made this study possible. We would also enterology 1973; 65: 698-700. like to thank Sr G Watherston for help in collecting 16 Small DM. Cholesterol nucleation and growth in the bile samples, Mr V Jarvinen and Dr G White for gallstone formation. N Engl J Med 1980; 302: 1305-7. their help with the bile and hormone analyses, and 17 Bennion LJ. Ginsberg RL. Garnick MB, Bennett PH. Ms E Couper and Mrs D McVicar for typing the Effects of the normal menstrual cycle on human gallbladder bile. N Engl J Med 1976; 294: 1187. manuscript. 18 Whiting MJ. Down RHL. Watts JMcK. Precision and accuracy in the measurement of the cholesterol saturation index of bile. Lack of variation due to the menstrual cycle. Gastroenterology 1981; 80: 533-8. 19 Kern F, Everson GT. DeMark B. McKinley C. Showalter R. Ertling W. Braverman DZ. Szczepanik- van Leeuwen P, Klein PD. Biliary lipids. bile acids and gallbladder function in the human female. J Clin Invest References 1981; 68: 1229-42. 20 Williams CN, Scallion SM. McCarthy SC. Bile acid 1 Robertson HE. 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