Plasma deoxycholic acid concentration is elevated in postmenopausal women with newly diagnosed breast cancer

Abstract

Objective: High concentrations of plasma deoxycholic acid (DCA) are found in human breast cyst fluid and it has been hypothesised that this may be related to risk of breast cancer. The aim of this pilot study was to ascertain whether plasma bile acid concentrations were greater in women with breast cancer.

Design: A case–control study comparing postmenopausal women with breast cancer with healthy controls was conducted.

Subjects: Twenty Caucasian postmenopausal breast cancer patients were recruited at the time of diagnosis together with 20 healthy controls matched for age and body mass index. Exclusion criteria included any treatment for breast cancer, use of hormone replacement therapy in the last 12 months, diabetes mellitus, a history of liver or gall bladder disease or abnormal liver function.

Measurements: Fasting plasma bile acid concentrations were determined by gas–liquid chromatography/mass spectrometry.

Results: The mean plasma DCA concentration was 52% higher (P=0.012) in patients with breast cancer compared with controls.

Conclusion: These results support the hypothesis that DCA may be involved in the aetiology of breast cancer.

Sponsorship: V Costarelli was the recipient of a scholarship from Harokopion Institution, Athens.

Introduction

It has been proposed that deoxycholic acid (DCA), which is derived from the bacterial degradation of the primary bile acid cholic acid (CA) in the colon, may be involved in the aetiology of breast cancer (Hill et al, 1971; Lewis & Heaton, 1999). DCA is found in human breast cyst fluid at concentrations about 50 times greater than those in plasma (Raju et al, 1990; Javitt et al, 1994). DCA is mutagenic (Watabe & Bernstein, 1985), has a co-carcinogenic activity (Kawasumi et al, 1988) and promotes the growth, oestrogen receptor and oestrogen-regulated proteins of MCF-7 human breast cancer cells (Baker et al, 1992). The formation of DCA from CA in the colon is inhibited by low pH (van Munster et al, 1994). Colonic pH is influenced by fermentation of carbohydrate in the colon, to short chain fatty acids but also by ammonia release from bacterial degradation of protein. Reddy et al, (1998) found the intake of starch and dietary fibre to be negatively associated with the faecal concentration of DCA and faecal pH. Several studies have reported differences in faecal bile acids excretion or in the composition of the major faecal bile acids in breast cancer patients (Murray et al, 1980; Papatestas et al, 1982; Owen et al, 1986), but the results have been equivocal. However, these studies can be criticised as the disease process or treatment may have affected liver function.

The plasma bile acid profile has shown to reflect the intestinal bile acid profile (van Faassen et al, 1997). The aim of the present study was to ascertain whether plasma concentration of DCA was greater in newly diagnosed patients with breast cancer compared with healthy controls matched for age and body mass index.

Subjects, materials and methods

Subjects

Caucasian postmenopausal women with newly diagnosed breast cancer were recruited from the Oncology Clinics of Guy's and Charing Cross hospitals. Healthy postmenopausal women were recruited from the staff and relatives of staff and students of King's College London and from the general public, using an advertisement in a national newspaper The Guardian. Exclusion criteria included any treatment for breast cancer, use of hormone replacement therapy in the last 12 months, diabetes mellitus, a history of liver or gall bladder disease or abnormal liver function. A blood sample (10 ml) was collected after an overnight fasting in the morning from both cases and controls for the measurement of plasma bile acid concentrations and for assessment of liver function using routine clinical chemistry (serum total protein, albumin, total bilirubin, alkaline phosphatase, aspartate transaminase, gamma-glutamyl transferase). Subjects completed a questionnaire concerning their medical history. Body weight was recorded on a beam balance in minimum indoor clothing and height was measured without shoes with a stadiometer. Information about the patient's stage of cancer was obtained from their medical records.

The subjects gave informed written consent and the protocol was approved by the Research Ethics committees of King's College, Guy's and St Thomas's Hospitals and the Riverside Health Authorities. Ten of the 40 patients who were initially recruited to the study had abnormal liver function tests, five were suffering from diabetes, and five had had breast cancer in the past. As a result, only 20 of the cases met the inclusion criteria and only plasma from these subjects and their respective controls was analysed for bile acids. Cases were matched by age (mean 60, range 50–75 y) and body mass index (mean 26.1, s.d. 3.6 kg/m2) with postmenopausal women. Out of the 20 breast cancer patients recruited in the study, four were diagnosed with breast cancer grade I, 12 with breast cancer grade II and four with grade III. Details of the subjects are given in Table 1.

Table 1 Age, body mass index (BMI) and indices of liver function in 20 newly diagnosed postmenopausal women compared with age and BMI matched controls

Laboratory methods

Blood was collected from an anticubital vein using the vacutainer technique into tubes containing EDTA as anticoagulant for analysis of bile acids. Plasma samples from cases and their respective controls, were analysed in the same batch. Plasma bile acids were analysed by gas chromatography/mass spectroscopy (Clayton & Muller, 1980). Briefly an internal standard of nordeoxycholic acid (Catalogue no. N2000, Steraloids, Newport, Rhode Island, USA) was added to the plasma sample and bile acids were deconjugated with choline glycine hydrolase (Sigma-Aldrich catalogue C4018, Poole, Dorset, UK) and extracted with alkaline XAD-2 resin. The bile acids were recovered by eluting sequentially with 2 ml of a mixture of hexane:chloroform:methanol (1:1:1 by volume) and twice with 2 ml methanol. The bile acids were methylated with diazomethane and trimethyl silylated with Tri-Sil (catalogue no. 48999, Pierce, Rockford, IL, USA) and analysed on a Hewlett Packard 6890 mass spectrometer using a 25 m BPX35, 0.22 mm internal diameter, 0.25 µ film thickness (SGE Europe, Milton Keynes UK). The initial column temperature was 80°C, which was held at this temperature for 2 min and then increased at 100°C/min to 200°C for 4 min then increased at 2°C/min to 300°C and held at that temperature for 4 min. The injector temperature and transfer line temperatures were set at 250°C. Bile acids were identified by their mass spectra and quantified in relation to internal standard.

Statistical analysis

A sample size of 20 subjects had statistical 85% power to detect a 1 s.d. difference in plasma DCA concentration between groups at P<0.05. Comparisons between cases and controls were made using a paired sample t-test.

Results

Table 2 shows the plasma bile acid concentrations in the cases and the controls. Plasma deoxycholic acid concentrations were 52% higher (P=0.012) in patients with breast cancer than in controls. The ratio of plasma DCA/CA tended to be higher in the cases compared to the controls but the difference was not statistically significant (P=0.105). Ursodeoxycholic acid, which is a bacterial metabolite of chenodeoxycholic acid, was detected in 14 cases and 15 controls did not differ significantly. No other significant differences were noted.

Table 2 Plasma bile acids concentrations (µmol/l) in 20 postmenopausal women with breast cancer compared with age and BMI matched controls

Discussion

The aim of this pilot study was to ascertain whether postmenopausal breast cancer patients have higher plasma concentrations of DCA than healthy controls. Lithocholic acid (LCA) is the other major secondary bile acid synthesised in the colon. However, the absorption of LCA from the colon is low and only trace amounts are present in plasma in the free form and most are present in the sulphated form (Arias et al, 1994). There are several possible explanations for the higher plasma concentrations of deoxycholic acid. Plasma bile acids are rapidly cleared from circulation by the liver and one cause of elevated plasma bile acid concentrations is liver disease (Salen & Batta, 1999). However, this is an unlikely explanation in the present study as liver function was normal. It is known that obesity, which is a risk factor for breast cancer in postmenopausal women (Schindler, 1998), results in increased bile acid output. However, in the present study, subjects were matched for body mass index. Fasting plasma bile acid concentrations were measured in the present study as there is a large increase following meals containing at least 15 g of fat. In contrast, fasting plasma bile acid concentrations did not differ significantly on a day-to-day basis over a 6 week period; the mean within-subject deviation for DCA was 0.43 µmmol/l (Costarelli & Sanders, 2001).

The results of this study support the hypothesis that DCA is associated with an increased risk of breast cancer. The study had adequate statistical power to detect the difference detected: with a sample size of 20, the study had a 90% power to detect a 38% difference in plasma DCA concentration compared with an observed difference of 52%. It is not possible to conclude from the present study whether the higher concentration of deoxycholic acid preceded the breast cancer or whether it was a consequence of the disease process, since no estimates of relative risk using conditional logistic regression were calculated due to the small sample size. These findings support the concept of a relationship between intestinally derived bile acids and risk of breast cancer, but require confirmation from prospective case–control studies. Future studies should also consider both determining fasting and postprandial plasma concentrations of bile acids in cases and controls as well as determining the concentrations of deoxycholic in breast cyst aspirates of cases and controls.

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Acknowledgements

We thank the women who agreed to participate in this study. We also thank Professor Ian Fentiman and Mr Dudley Sinnett from Guy's and Charing Cross Hospitals respectively, for their valuable assistance in the recruitment of the breast cancer patients.

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Correspondence to V Costarelli.

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Keywords

  • plasma deoxycholic acid
  • breast cancer
  • secondary bile acids

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