domain: chim sdv

text:
Lydie Humbert

PhD Dominique Rainteau

Noshine Tuvignon

Claude Wolf

Philippe Seksik

PhD Frédéric Carrière
email: carriere@imm.cnrs.fr

René Laugier

Postprandial bile acid levels in intestine and plasma reveal altered biliary circulation in chronic pancreatitis patients

Keywords: Bile acids, Cholangiohepatic shunt, Chronic pancreatitis, Exocrine pancreatic insufficiency, Lipidomics

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Chronic pancreatitis (CP) is an ongoing inflammatory disorder associated with the loss of exocrine and endocrine pancreatic parenchyma and its replacement by fibrotic tissue. It results in various nutritional deficit linked to maldigestion subsequent to exocrine pancreatic insufficiency (EPI) and diabetes mellitus subsequent to pancreatic endocrine insufficiency.

CP is mainly induced by alcohol abuse in western countries (1). Other toxic and/or metabolic agents, gene mutations regulating the pancreatic enzyme-activating cascades and their inhibitors are also factors associated with a hereditary form of the disease [START_REF] Büchler | Chronic pancreatitis: novel concepts in biology and therapy[END_REF][START_REF] Etemad | Chronic Pancreatitis: Diagnosis Classification, and New Genetic Developments[END_REF]. The first symptom is commonly the abdominal pain. Over the first five years of course, complications as pseudocysts or bile duct stenosis can occur. Occurrence of pancreatic calcifications is a lately observed diagnostic key. Because the clinical symptoms remain vague and non-specific in many CP patients, biological tests are required [START_REF] Duggan | Chronic pancreatitis: A diagnostic dilemma[END_REF] for the diagnostic as well as the follow up.

In CP patients, fat digestion is more severely impaired than carbohydrate and protein digestion, and steatorrhea is usually a major symptom. The decrease in fat absorption is explained by a decrease in human pancreatic lipase (HPL) secretion and duodenal pH due to the reduced pancreatic bicarbonate secretion [START_REF] Carrière | Quantitative study of digestive enzyme secretion and gastrointestinal lipolysis in chronic pancreatitis[END_REF][START_REF] Youngberg | Comparison of gastrointestinal pH in cystic fibrosis and healthy subjects[END_REF]. The decrease in duodenal pH impairs the activity and stability of residual HPL. It also promotes the precipitation of bile acids and thus a further deterioration of intestinal lipid absorption [START_REF] Zentler-Munro | Effect of intrajejunal acidity on lipid digestion and aqueous solubilisation of bile acids and lipids in health, using a new simple method of lipase inactivation[END_REF][START_REF] Zentler-Munro | Effect of intrajejunal acidity on aqueous phase bile acid and lipid concentrations in pancreatic steatorrhoea due to cystic fibrosis[END_REF]. It was shown that human gastric lipase (HGL) secretion and contribution to lipolysis are increased in CP patients, but not sufficiently to compensate for the loss of the prominent pancreatic lipase [START_REF] Carrière | Quantitative study of digestive enzyme secretion and gastrointestinal lipolysis in chronic pancreatitis[END_REF].

The current treatment of EPI is enzyme replacement with porcine pancreatic extracts.

Fat absorption can be improved with enteric-coated formulations of these exogeneous pancreatic enzymes, associated also with proton pump inhibitors to increase the duodenal pH and enhance enzyme stability and activity [START_REF] Sander-Struckmeier | Retrospective analysis to investigate the effect of concomitant use of gastric acid-suppressing drugs on the efficacy and safety of pancrelipase/pancreatin (CREON(R)) in patients with pancreatic exocrine insufficiency[END_REF][START_REF] Vecht | Efficacy of lower than standard doses of pancreatic enzyme supplementation therapy during acid inhibition in patients with pancreatic exocrine insufficiency[END_REF][START_REF] Dimagno | Gastric acid suppression and treatment of severe exocrine pancreatic insufficiency[END_REF]. In some patients with severe EPI, pancreatic enzyme replacement therapy (PERT) may not lead to the reduction of steatorrhea suggesting other mechanisms to interplay with the reduced lipase secretion. Although exogenous lipases act correctly in the small intestine, it can be suspected that other factors affecting the absorption of lipolysis products are limitant. Bile acid (BA) levels for instance were rarely adressed in CP patients [START_REF] Regan | Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment[END_REF]. The BAs are critical for the digestion and absorption of fats [START_REF] Hofmann | The Function of Bile Salts in Fat Absorption. The Solvent Properties of Dilute Micellar Solutions of Conjugated Bile Salts[END_REF][START_REF] Hofmann | The Intraluminal Phase of Fat Digestion in Man: The Lipid Content of the Micellar and Oil Phases of Intestinal Content Obtained During Fat Digestion and Absorption[END_REF]. Due to their amphiphilic properties, they are found adsorbed at the surface of oil-in-water emulsions where they regulate the lipolytic activity of the pancreatic lipase-colipase complex.

BAs are also found in the form of mixed micelles with other bile lipids (cholesterol, phosphatidylcholine) and lipolysis products (free fatty acids, 2-monoglycerides). The micellar co-solubilization of lipolysis products by BA is required for removing these products from the lipid-water interface and for ensuring their diffusion in the aqueous milieu of the gut towards the enterocytes. In the absence of bile secretion, fat absorption is impaired [START_REF] Hofmann | Bile Acids: The Good, the Bad, and the Ugly[END_REF].

Apart from their function in fat absorption and regulation of cholesterol homeostasis, BAs are increasingly being appreciated as complex metabolic integrators and signaling factors. They have become attractive therapeutic targets for metabolic disorders and useful markers of hepatobiliary and intestinal diseases [START_REF] Thomas | Targeting bile-acid signalling for metabolic diseases[END_REF]. Individual BA molecular species from various biological samples (blood plasma, feces, urine) can now be separated and quantified in the clinical laboratory using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The BA profiles are used for a sensitive diagnosis of cholestasis [START_REF] Humbert | Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients[END_REF]. BA levels and profiles in patients with EPI have not been fully investigated with the LC-MS/MS profiling method whereas BA malabsorption in patients with CP was fully evidenced by the radiolabeled synthetic bile acid standard test [START_REF] Reid | A Multicenter Prospective Study to Investigate the Diagnostic Accuracy of the SeHCAT Test in Measuring Bile Acid Malabsorption: Research Protocol[END_REF]. In the present study we have detailed the post-prandial individual BA level variations in both the duodenum and plasma of CP patients and healthy volunteers (HV) after a standard test meal.

MATERIALS AND METHODS

Ethical aproval

The duodenal content and blood samples used in this study were collected in the framework of two clinical studies (mrtm02-01 for HV and mrtm03-01 for CP patients; Principal Investigator: Prof René Laugier, MD, PhD) that were not initially designed for the analysis reported here. The clinical study protocols were accepted on January 17, 2003

CP patients and heathy volunteers

Two groups of 6 HV (mean age: 30 ± 3 years) with no history of pancreatic disease and 6 patients with severe CP (mean age: 53 ± 8 years) were selected for this study. The severity of each patient's pancreatic insufficiency was estimated from the medical record and steatorrhea level in the absence of treatment by pancreatic extracts (see Table S1 in Supplemental Data). All selected CP patients had high levels of steatorrhea (8 to 45 g/day).

All were documented with pancreatic calcification. Patients with common biliary duct stenosis had been treated by endoscopy within the 5 years preceeding the study. Some patients were also treated for stenosis after the present study. Only one patient (G.R.) had highly elevated liver enzymes, presumably due to severe hepatic disease.

Test meal

The mixed solid/liquid meal used for the in vivo experiments contained 80 g string beans, 90 g beef meat, 70 g fried potatoes, 10 g butter, 15 mL olive oil and water for a total volume of 700 mL. The string beans, beef meat and fried potatoes were passed individually into a mincer with 2-mm holes before they were mixed . The total amount of neutral fat given as triglycerides (TG) in the meal was measured to 31.8 ± 2.9 g after extraction of total lipids with chloroform-methanol and TG quantification using thin-layer chromatography coupled to flame ionization detection [START_REF] Cavalier | Validation of lipolysis product extraction from aqueous/biological samples, separation and quantification by thin-layer chromatography with flame ionization detection analysis using O-cholesteryl ethylene glycol as a new internal standard[END_REF]. A nonabsorbable marker was added to the meal (5 g PEG 4000) to measure the gastric emptying rate and correct the duodenal volumes based on the PEG recovery rates [START_REF] Carrière | Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans[END_REF].

Experimental device for collecting samples

After fasting overnight, the patients and volunteers were intubated with a doublelumen duodenal tube (outside diameter 5 mm) and a separate single-lumen gastric tube (outside diameter, 3 mm). The tubes were placed under fluoroscopy and their positions were checked by analyzing the pH of their contents. The distal end of the duodenal tube with the occluding balloon was located at the ligament of Treitz (see Figure S1 in Supplemental Data).

During the sampling period, the subjects stayed in bed with continuous duodenal aspiration by connection of the duodenal tube to vacuum (around -10 mbar). Aspiration was started a few minutes before the meal and the occluding balloon was inflated (10 mL air). After collecting basal gastric and duodenal samples, the test meal was introduced into the stomach via the gastric tube using a 50 mL syringe, during a period of 5 minutes.

The duodenal fluid was collected continuously for 15-minute periods by aspiration upstream of the occluding balloon, The parameters measured in each sample were: i) volume and pH (to the nearest 0.5 mL and 0.1 pH unit respectively), ii) PEG 4000 concentration, iii) pancreatic lipase activity and iv) individual BA concentrations. In order to prevent the proteolytic inactivation of lipase before the assay, 1 mL glycerol and 40 µL protease inhibitors were added immediately to 1 mL duodenal sample before storage at -20°C. The solution of protease inhibitors was prepared by dissolving a pellet of Complete ™ protease inhibitor mix (Roche). in 2 mL distillated water. All samples were kept at -80°C before they were analyzed.

Blood sample collection.

Blood samples were collected before and after the meal for a total duration of 240 minutes. All samples were kept at -80°C before they were analyzed.

Measurement of PEG 4000 in gastric and duodenal samples.

The PEG 4000 concentration was measured using the turbidimetric method developped by Hyden [START_REF] Hydén | A turbidimetric method for determination of higher polyethyleneglycols in biological materials[END_REF] as modified by Malawer and Powell (22).

Assay of pancreatic lipase activity

The enzyme activity of human pancreatic lipase (HPL) in duodenal contents was measured by the pHstat technique as previously reported [START_REF] Carrière | Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans[END_REF][START_REF] Erlanson | Tributyrin as a substrate for determination of lipase activity of pancreatic juice and small intestinal content[END_REF]. Lipase levels were expressed in international units per ml (1 international unit = 1 µmole of fatty acid released per minute from the standard substrate, tributyrin). Lipase outputs were expressed either in total units or mg of active lipase based on the known specific activity (8000 U/mg) of a pure HPL acting on tributyrin under similar condition.

Use of PEG for correcting the volume of dudenal aspirates

A correction factor accounting for the partial recovery in duodenal aspirates of the nonabsorbable marker PEG 4000 was calculated for the whole period of measurement by dividing the total amount of marker initially added in the meal by the recovered amount at the end of the experiment.

Estimation of HPL duodenal outputs

The duodenal output of HPL was estimated from the assay of enzyme activity in duodenal samples, the volume of duodenal aspirates and the correction factor based on the recovery of PEG.

Preparation of bile acid standards for LC-MS/MS analysis

Concentrated BA calibration solutions were prepared in methanol (1mg/ml) and stored in a sealed container at -20°C. These stock solutions were diluted to obtain calibration solutions ranging from 31.3 ng/ml to 31.3 µg/ml. Cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA), hyocholic acid (HCA) and the corresponding glyco-and tauro-conjuguates were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). 3-sulfate derivatives of the BAs were a generous gift from Dr J. Goto (Niigita University of Pharmacy and Applied Life Science, Japan). 23-nor-5β-cholanoic acid-3α,12β diol, muricholic acid derivatives and glyco-and tauro-derivatives were purchased from Steraloids Inc. (Newport, USA).

Bile acid analysis by LC-MS/MS

An internal standard solution (2 µL of 23-nor-5β-cholanoic acid-3α,12β diol at 1mg/ml) was added to plasma samples (500 -1000 µL) and duodenal aspirates (100-200 µL).

Proteins were precipitated by addition of ammonium carbonate 0.4 M for 30 min at 60°C [START_REF] Humbert | Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients[END_REF]. The clean-up procedure was achieved by centrifugation (4000 g for 10 min) followed by a solid-phase extraction (SPE). Reverse phase Chromabond C 18 cartridges (100 mg; Macherey-Nagel, Düren, Germany) were pre-washed by 5 ml methanol and 5 ml water, successivel,y before the sample was loaded onto the cartridge. The subsequent steps were processed on a vacuum manifold designed for SPE. The cartridge was rinsed successively with 20 ml water, 10 ml hexane to discard neutral lipids and again with 20 ml water. BAs were finally eluted by methanol. The eluates were dried under a nitrogen stream at 50 °C and the residue dissolved in 150 µl methanol. Five microliters were injected into the HPLC-MS/MS equipment.

The chromatographic separation of BAs (see Figure S2 in Supplemental Data) was carried out on a reverse phase column (Restek C18 Pinnacle II (250mm x 3.2mm, 5µm), Restek, Lisses, France) thermostated at 35°C (Agilent 1100 HPLC, Agilent Massy, France).

The column was initially equilibrated with a 65/35 (v/v) mixture of solvent A (15 mM aqueous ammonium acetate, pH 5.3) and B (methanol). BA elution was achieved by increasing proportion of solvent B from 65/35 to 95/5 (v/v). Simultaneously, the flow rate was increased from 0.3 to 0.5 mL/min for 30 min.

The HPLC column eluates were infused into the ESI source of a triple quadrupole mass spectrometer (QTRAP 2000, Applied Biosystems-SCIEX, Concord, Ontario, Canada).

Electro-spray ionisation was set in the negative mode. Nebulizer, curtain and heater nitrogen gas were set at 40, 20 and 40 (arbitrary units), respectively. The temperature for the evaporation gas (nitrogen) was set at 400°C. The ion spray, declustering and entrance potentials were set at -4500V, -60 V and -10 V, respectively. The MS/MS detection was operated with a unit resolution in the multiple reaction monitoring (MRM) mode. The dwell time for each transition was set at 70 ms. MRM was performed by examination of the transition reactions from precursor conjuguated BA to product fragment-ions after collision induced dissociation (CID): the sulfite (m/z=80, SO 3 fragment-anion cleaved from taurine) and glycine moieties (m/z= 74) respectively, for tauroconjugated and glycoconjugated BAs. Sulfo-conjuguates were identified by the sulfuric anion (m/z = 97, HSO 4 ). No specific fragment-ions are observed for unconjugated BAs, except those corresponding to loss of water and these fragments are not reliable for quantification. Therefore, we used selected ion monitoring (SIM) mode for quantifying unconjugated BAs, with mono-, di-and trihydroxylated BAs scanned at m/z of 375, 391 and 407, respectively. 23-nor-5β-cholanoic acid-3α,12β diol (m/z = 377) was used as the internal standard (IS) for normalization. The method for BA analysis was validated according to Humbert et al. [START_REF] Humbert | Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients[END_REF], who previously checked that similar values were obtained with 3 different IS (23-nor-5β-cholanoic acid-3α,12 α diol, ursodeoxycholic-2,2,4,4-d4 acid and lithocholic-2,2,4,4-d4 acid) with distinct hydrophobicity and retention times.

Statistics

All the data were expressed as means±SD unless stated otherwise. The data obtained in the various series of experiments were compared using paired and unpaired Student's ttests. The data obtained at time 30 min after meal administration were also analyzed using principal component analysis (PCA) and discriminant analysis (DA) using an Orthogonal-Partial Least Square (O-PLS) statistical model (SIMCA14 software package (Umetrics, Umeå, Sweden).

RESULTS

Variations in the duodenal pH

In HV, the duodenal pH values remained in the 4.1-7.4 pH range during the meal digestion, with a mean value of 6.10 ± 0.8 (Figure 1A). In CP patients, the duodenal pH decreased rapidly after 30 min, and then remained very low with mean pH values ranging between pH 2 and pH 3.5 (Figure 1A). The pH differences between CP patients and HV were significant (P<0.05) from 30 to 180 min.

HPL duodenal concentration and outputs

Pancreatic lipase outputs during the whole digestion period were estimated from the recovery of duodenal contents. The HPL activities recorded in HV were in the 320-5250 U/mL range (i.e. 40-656 µg/mL of active HPL), whereas HPL activity in CP patients was extremely low and never greater than 12 U/mL (1.5 µg/mL; Figure 2B). From the cumulated outputs of HPL, it was observed that pancreatic lipase secretion was almost abolished in CP patients (0.7 ± 0.2 mg vs. 116.7 ± 68.1 mg in HV; P<0.005).

Total bile acid concentrations in duodenal contents

In HV, the BA concentration in duodenal contents was increased immediately after the meal intake and reached a maximum value (14.8 ± 12.7 mM) after 30 min (Figure 2A). It then decreased and leveled off around 3-4 mM after 120 minutes. Except during the 75-90 min period, mean BA concentration found in human duodenal contents was above the average critical micellar concentration (CMC) of the main BAs found in human bile (24) (around 4 mM; see Table S2 in Supplemental Data). In CP patients, the BA concentration in duodenum was much lower than in HV. A maximum value of 3.9 ± 5.6 mM was observed at 45 min (Figure 2A). Except at 30 and 45 minutes, the total BA concentration remained below the CMC.

The ratio of primary BA (7α hydroxylated in the liver) over secondary BA (produced from primary BA by the intestinal microbiote after 7α-dehydroxylation) was considerably higher in CP patients (38.09 ± 48.1) than in HV (4.15±2.37; P<0.15) (Figure 2B). The ratio ranged from 19.3 to 72.8 in CP patients as compared with 4.1 to 7.1 in HV .

Bile acid duodenal outputs

Total BA outputs in duodenal contents over the post-prandial period (Table 1) were much lower in CP patients (1.00 ± 0.89 mmoles; 0.47 ± 0.42 g) vs. HV (5.52 ± 4.53 mmoles; 2.62 ± 2.14 g). The ratio of primary to secondary BA was also much higher in CP patients (38.1 ± 39.4) than in HV (4.2 ± 1.9). The examination of the detailed BA composition (expressed as total BA mole %) revealed a significant decrease in TCDCA (P<0.015), TDCA (P<0.025), GLCA-3-sulfate (P<0.02), TLCA-3-sulfate (P<0.01), UDCA-3-sulfate (P<0.035), LCA-3-sulfate (P<0.05), GUDCA-3-sulfate (P<0.03), GUDCA (P<0.035), GHDCA (P<0.005), GCA (P<0.015) and TLCA (P<0.05) in CP vs. HV (Table 1). Only traces of glucuronated conjugates (<0.01% of total BAs) were detected and were not taken onto account for comparison of BA profiles between CP and HV. For comparison, total sulfated BAs accounted for around 0.04% (CP) to 0.3% (HV) of total BAs in duodenal contents.

Bile acid concentrations in blood plasma

The BA concentration in blood plasma was in the µM range, that is three orders of magnitude lower than duodenal BA concentration (mM range; Figure 2). After meal intake, the total BA concentration in plasma of CP patients showed a peak (3.88 ± 2.32 µM; Figure 3C) at 30 min. The peak contrasted with the decreased BA concentration in HV (1.92 ± 1.56 µM; Figure 2C). In HV plasma BA concentration increased only lately to 2.32 ± 1.55 µM at 60 min (Figure 2C) before decreasing. BA concentrations in HV and CP patients reached similar values at 120-180 min.

The analysis of individual circulating BAs revealed a higher primary to secondary BA ratio in CP patients. The largest difference in the ratio was observed at 90 min (Figure 2D).

The comparison of the area under the plasma concentration curve (AUC, µmoles.L -1 .min) for individual BAs was not statistically different between the HV and CP groups (Table 2), except for the conjugated ursodeoxycholic acid (UDCA and GUDCA) significantly lower in CP patients (P<0.05). The decreased ursodeoxycholic conjuguates were unexpected regarding a commonly found increase in prolonged cholestasis [START_REF] Smith | Endogenous ursodeoxycholic acid and cholic acid in liver disease due to cystic fibrosis[END_REF]. Analysis of AUCs showed a significantly higher (P<0.05) proportion of primary BA in CP patients (87.1 ± 6.5 mol % of total BAs) as compared with HV (67.1 ± 17.0 mol % of total BAs). Only traces of glucuronated conjugates (<0.01% of total BAs) were detected in plasma of CP and HV, while total sulfated BAs accounted for around 0.7% (HV) to 1.5% (CP) of total BAs in plasma.

Simultaneous changes in individual bile acid concentrations in duodenal contents and blood plasma

The variations with time in individual BA duodenal concentrations were similar to those observed with total BAs (Figure 2A), as illustrated by the kinetics of GCA (Figure 3A), a major BAs comprised in the human bile. Variations in other BAs are reported in Supplemental Data (Figure S3). The main deviation between HV and CP patients was observed 30 min after meal with mean GCA duodenal concentrations of 2,012 µM and 616 µM, respectively (Figure 3A). Then, mean GCA duodenal concentration in HV decreased to values similar to CP patients after 60 min. In CP patients, only weak changes in the GCA duodenal concentration were observed (Figure 3A).

Interestingly, opposite variations in GCA concentration were observed in blood plasma (Figure 3B). The plasma concentration of GCA increased clearly in CP patients and peaked at 30 min (mean concentration of 0.72 µM) but in HV the mean GCA concentration remained low (mean 0.15 µM). Plasma GCA concentration of CP patients returned to low HV values after 60 min.

Since the main differences between CP patients and HV were observed at 30 min, we focused on duodenal (Figure 3C) and plasma (Figure 3D) concentrations of individual BAs in duodenal and blood samples at 30 min (TCDCA, TDCA, TCA, GUDCA, GCDCA, GDCA, GCA). For each individual BA, the duodenal concentration measured in CP patients was significantly lower (P<0.05) than in HV (Figure 3C). Conversely, plasma concentrations in CP patients were higher with significant differences (P<0.05) for the abundant BAs, GCDCA and GCA (Figure 3D).

Conjugated BA levels

Both glyco-and tauro-conjugated BA levels were much lower in CP patients (0.6 ± 0.7 and 0.4 ± 0.5 mmoles, respectively) than in HV patients (3.3 ± 3.7 and 2.2 ± 2.7 mmoles, respectively). The glyco-to tauro-conjugated BA ratio in CP (2.5 ± 1.7) and HV (1.5 ± 0.7)

were however not significantly different.

Unconjugated BA levels

Unconjugated BA levels (UDCA, CDCA, DCA, CA) were measured at a low level (<25 µM) in the duodenal contents of both HV and CP patients (Table 1; 0.01 to 1.5 % of total BA) except for a CP patient (C.K.) with particularly high concentrations of CDCA, DCA and CA (646, 293 and 124 µM, respectively at 30 min). This outlier patient has an impact on the significativity of the difference with the control HV group (Table 1). Unconjugated BA showed higher contributions in the plasma than in the bile for both HV (28,5 ± 24,2 % of total BAs) and CP patients (18,2 ± 15,4 % of total BAs). However no significant difference between the two groups was found including for the CP patient showing the particularly high duodenal concentrations of unconjuguated BAs.

BA levels in patient with alcoholic liver disease

Only one CP patient at out six had highly elevated liver enzymes, presumably due to alcoholic liver disease (patient 4 (G.R.); see Table S1 in Supplemental data). Total bile acid outputs (0.66 mmoles) in this patient were not significantly different from those found in the five other patients with severe chronic pancreatitis and were lower than those observed in HV.

Primary to secondary BA molar ratio in this patient was the highest found among all CP patients, in both duodenal contents (55 to 190) and blood plasma (10 to 110), but this finding did not change the fact that all CP patients showed a higher primary to secondary BA ratio than the healthy volunteers group.

DISCUSSION

The availability of LC-MS/MS analytical methods for BA in the clinical laboratory allows the investigation of BA secretion and circulation. The comparison of healthy volunteers (HV) and chronic pancreatic (CP) patients, using samples of duodenal contents and blood plasma collected during test meal experiments contributes to a detailed understanding of the condition. We selected CP patients with a severe pancreatic exocrine insufficiency authentified by a low duodenal pH value (Figure 1A) resulting from weak bicarbonate secretion, and extremely low pancreatic lipase (HPL) levels (Figure 1B). On average, HPL secretion in CP patients (0.7 ± 0.2 mg) represented only 0.6 % of control outputs (116.7 ± 68.1 mg in HV). The HPL outputs were found in the range previously reported for severe CP patients (5) and healthy subjects [START_REF] Carrière | Quantitative study of digestive enzyme secretion and gastrointestinal lipolysis in chronic pancreatitis[END_REF][START_REF] Lengsfeld | Physiology of gastrointestinal lipolysis and therapeutical use of lipases and digestive lipase inhibitors[END_REF], respectively.

Bile acids in healthy volunteers

The data on BA analysis with HV confirmed concentrations and outputs previously reported in healthy humans under fed conditions. Total output (2.62 g; 5.52 mmoles) during the duration of the meal (3 h) was close to the 3 g BA amount estimated for the pool of BAs circulating 4 to 12 times per day in the human GI tract [START_REF] Carey | The enterohepatic circulation[END_REF]. Furthermore, the peak in duodenal concentration of BAs observed 30 min after meal ingestion was typical of a normal gallblader emptying [START_REF] Carey | The enterohepatic circulation[END_REF][START_REF] Clarysse | Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states[END_REF]. While highly variable intestinal concentrations of BAs under fed conditions have been reported in literature, ranging from 0.5 mM (29) to 37 mM (30), there is an agreement for duodenal concentrations recorded 30-60 min after meal and following the gallblader emptying: 14. (high-fat meal) reported by Clarysse et al. [START_REF] Clarysse | Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states[END_REF]. The mean duodenal BA concentration measured presently after 30 min in HV (14.8 ± 12.7 mM, n=6; Figure 2A) was therefore consistent with previous data. BA profiles were also in the normal range with conjugated BAs >90% but low levels of unconjugated and sulfated BAs (Tables 1 and2). The primary to secondary BA ratio in duodenal contents of HV (c.a 5.5) was close to previously reported values for healthy subjects under fasting (5.5) and fed (8.2) conditions [START_REF] Clarysse | Postprandial evolution in composition and characteristics of human duodenal fluids in different nutritional states[END_REF].

BAs were found at a low concentration (µM range) in the peripheral blood circulation, as resulting from the normal highly efficient uptake by the liver [START_REF] Monte | Bile acids: chemistry, physiology, and pathophysiology[END_REF]. Plasma BA composition was also similar to previous report with healthy subjects [START_REF] Humbert | Bile acid profiling in human biological samples: comparison of extraction procedures and application to normal and cholestatic patients[END_REF][START_REF] Schmid | Bile Acid Metabolome after an Oral Lipid Tolerance Test by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)[END_REF]. It is noticeable that the contribution of unconjugated BA to total BAs found in plasma is much higher (28,5 ± 24,2%;

Table 2) than in duodenal contents (around 0.06 %; Table 1). These higher plasma concentrations of unconjugated BAs can be explained by their slower first-pass hepatic clearance, which disproportionately increases their concentration in plasma versus conjugated BAs. Therefore, the measurement of the relative amounts of conjugated versus unconjugated BAs in plasma is not a good indicator of their relative proportions in the whole-body BA pool or in other compartments.

Bile acids in chronic pancreatitis patients

In addition to the deficit in pancreatic bicarbonate and enzyme secretions, total BA concentration in duodenal contents was decreased c.a 5-fold in CP patients as compared to HV (Figure 2A). No peak of BA concentration in duodenal contents, that could reflect gallblader emptying, was observed at 30 min. The maximum concentration was observed with a delay of 15 min compared to HV. Overall, the total BA output in the duodenum was severely reduced in CP patients (0.47 g; 1.00 mmoles). It amounted only 18% of BA output in HV (2.62 g; 5.52 mmoles; Table 1). As a results, BA duodenal concentration was often found below the average CMC of the main bile salts found in human bile and duodenal contents (Table 3). It is worth noticing that we analyzed complete duodenal samples and therefore, low BA levels in CP could not be explained by some BA precipitation at low pH as reported in previous studies [START_REF] Regan | Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment[END_REF][START_REF] Go | Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man[END_REF]. This point will be further discussed.

As in healthy subjects, conjugated BAs represented >90% of total bile BAs and very low levels of unconjugated BAs and sulfated BAs were measured (Table 1). The glyco-to tauro-conjugated BA ratio was not changed compared to HV, indicating that no selective precipitation of glyco-conjugated BA at low pH occurred in CP patients compared to previous reports [START_REF] Regan | Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment[END_REF][START_REF] Go | Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man[END_REF]. We therefore re-analyzed these previous studies by comparing BA concentrations and pH levels with those we measured here. In the paper by Go et al. [START_REF] Go | Disturbances in fat digestion induced by acidic jejunal pH due to gastric hypersecretion in man[END_REF], which reports BA precipitation at low jejunal pH in one patient with gastrin-producing islet cell tumor, gastric hypersecretion and steatorrhea, BA precipitation occurred at very low pH values (≤2. [START_REF] Carrière | Quantitative study of digestive enzyme secretion and gastrointestinal lipolysis in chronic pancreatitis[END_REF]), what could be reproduced in vitro. Only 48% of BA where present in the micellar phase. This finding is probably the first one that led to the explanation of low BA levels in CP patients by acid precipitation. However, the sum of BA levels in all phases was only 23% of that recorded in normal subjects, which shows a reduced secretion of BAs independently from a loss by acid precipitation and is in agreement with our own observations. It was shown in this study that the precipitated BAs were largely the glycine dihydroxy conjugates (around 70%). In the paper by Regan et al. on patients with advanced acquired EPI [START_REF] Regan | Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment[END_REF], the lowest pH values (between 3 and 4) and BA concentrations were not as low as in Go's study and in our work. Indeed, at the BA secretion peak, the concentration in the micellar phase was 8.1 ± 0.6 mM in EPI patients versus 9.3 ±0.4 mM in normal subjects, which indicates that BA secretion was not drastically reduced in these patients compared to the CP patients with severe EPI enrolled in our study. Although a pH-dependent lowering of BA concentration in the micellar phase was observed, its impact on BA levels was limited and the patients involved in this study had clearly much higher BA levels than those we included in our study. Nevertheless, this publication and its conclusions have certainly led to an overestimation of the impact of acid precipitation on BA levels in EPI patients, and have delayed the recognition that low BA in these patients could be also explained by an altered BA circulation.

On a relative basis, primary BAs (CA and CDCA conjugates) were increased but secondary BAs (DCA derivatives) decreased in CP patients, which is consistent with a reduced bacterial conversion of primary to secondary BA in the intestine or fecal loss of BA due to intestinal bile acid malabsorption. In any case, this finding suggests changes in the enterohepatic circulation of bile acids [START_REF] Carey | The enterohepatic circulation[END_REF] and this is supported by the fact that the increased primary to secondary BA ratio observed in duodenal contents (Figure 2B) was also observed in the peripheral blood circulation (Figure 2D). Intestinal BA malabsorption is one hypothesis to explain these findings: it would lead to the fecal loss of total BA pool, depletion of secondary BA formed in the intestine and would derepress hepatic production of newly synthesized primary BAs. We have no information however on the fecal loss of BA in these CP patients and cannot confirm whether it could account for the 5-fold depletion in BA pool observed CP patients versus HV.

Another hypothesis could be a bypassed BA circulation via the cholehepatic shunt, a pathway which is minored under the normal physiological conditions (37) by the extensive enterohepatic recycling of BAs from intestine. Indeed, a remarkable particularity in CP patients was a sharp increase in plasma BA concentration 30 min after meal ingestion (Figure 2C) and the absence of BA concentration peak in duodenal contents corresponding to gallbladder emptying, which is consistent with a shunt of BA circulation. The cholehepatic shunt represents the fraction of BAs absorbed by cholangiocytes lining the biliairy ducts and gallbladder, which is recycled back to hepatocytes before reaching the intestinal lumen [START_REF] Xia | Bile acid interactions with cholangiocytes[END_REF].

BA absorption by cholangiocytes involves specific BA transporters like the Apical Sodium

Bile Acid Transporter (ASBT) and heteromeric organic solute transporter (OST) [START_REF] Xia | Bile acid interactions with cholangiocytes[END_REF][START_REF] Lazaridis | Rat cholangiocytes absorb bile acids at their apical domain via the ileal sodium-dependent bile acid transporter[END_REF][START_REF] Ballatori | OSTalpha-OSTbeta: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia[END_REF]. The cholehepatic shunt pathway might be favored in severe CP patients due to the drastic acidification of intestinal contents. Indeed, using a mouse model of cystic fibrosis (Cftr -/-), Debray et al. have shown that increased duodenal acidity results in attenuation of gallbladder contraction through the overexpression of vasoactive intestinal peptide (VIP), which leads to an increase in cholecystohepatic shunting of BAs and reduced levels of secondary BAs [START_REF] Debray | Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies[END_REF].

Cholecystectomy reversed those changes which provided evidence that the gallblader rather than the bile duct epithelia may be the major site of BA re-absorption in the cholehepatic shunt. In our study, the rise in plasma BAs observed at the 30-minute time point in the CP patient group suggests that the gallbladder contraction after meal intake occurs properly but BAs may be re-absorbed before reaching the duodenum. The cholehepatic shunt has also been proposed as a protection mechanism for the liver [START_REF] Debray | Defects in gallbladder emptying and bile Acid homeostasis in mice with cystic fibrosis transmembrane conductance regulator deficiencies[END_REF][START_REF] Boyer | A cholecystohepatic shunt pathway: does the gallbladder protect the liver?[END_REF], particularly in response to bile duct obstruction. Indeed, it has been shown that this pathway provides an alternative route for continuation of hepato-cholangiocyte flux of BAs in bile duct-ligated rats [START_REF] Lamri | Immunoperoxidase localization of ursodeoxycholic acid in rat biliary epithelial cells. Evidence for a cholehepatic circulation[END_REF][START_REF] Alpini | Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats[END_REF].

Based on these findings, we hypothesized that the low BA levels observed in the duodenal contents of CP patients (Figure 2A) might be explained by bile duct obstruction, which could eventually lead to a cholehepatic shunt of BA secretion, rather than by BA precipitation at low pH or intestinal BA malabsorption. We checked the medical history of the CP patients selected for this study (Table S1). They all had alcohol abuse-related chronic pancreatitis. Four of them were diagnosed for stenosis of the main bile duct before they were involved in this study and underwent an endoscopic treatment in the years preceeding the study (1998)(1999)(2000)(2001)(2002). The two other CP patients were diagnosed for main bile duct stenosis after the study (2005)(2006). None of them had cholecystectomy. A cholehepatic shunt of bile secretion in these patients could therefore be explained by the main bile duct stenosis encountered in chronic pancreatitis [START_REF] Cahen | Long-term results of endoscopic drainage of common bile duct strictures in chronic pancreatitis[END_REF].

The cholehepatic shunt being usually considered a minor pathway, it might not be sufficient to explain the extensive duodenal depletion in BAs in CP patients. As indicated before, there is no evidence that the precipation of BAs at low pH [START_REF] Hofmann | Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions[END_REF] could explain this depletion. Alternatively, BA secretion by the liver might be reduced in CP patients by a feedback control of the liver BA synthesis and transport [START_REF] Telbisz | Recent advances in the exploration of the bile salt export pump (BSEP/ABCB11) function[END_REF]. Measurements of the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19) would have helped distinguishing between cholehepatic shunting of BAs and intestinal BA malabsorption (elevated C4, reduced FGF19 ( 48)) as an explanation for the BA phenotype in CP patients. Unfortunately, we did not have enough plasma samples to perform these assays.

Test meal and plasma bile acid analysis as a potential diagnosis tool for the follow up of EPI and biliairy duct stenosis

Using a test meal and BA analysis in both duodenal contents and blood plasma, we found a clear anti-correlation between duodenal and plasma BA concentrations in the time frame (30 min after meal ingestion) corresponding to gallbalder emptying (well seen in HV).

In HV, high BA concentrations in duodenal contents corresponded to low BA concentrations in plasma, while in CP patients, low BA concentrations in duodenal contents corresponded to high BA concentrations in plasma (Figure 4A). In CP patients, the primary to secondary BA ratio also increased in duodenal contents and blood plasma. A discriminant analysis performed with the Orthogonal-PLS statistical model has establish a S-plot for the circulating blood plasma BAs assayed at 30 minutes after meal intake. The S-plot revealed the most discriminative biomarkers for CP patients (DCA, UDCA, CDCA) out of 17 plasma BA (Figure 4B; Additional analysis can be found in Figures S4 to S8 in Supplemental Data).

Therefore these three plasma BA assay may be used as a quantitative appreciation of EPI complications related to bile acids. BA profiling could also be used in the follow up after endoscopical treatment of main bile duct stenosis in parallel with the assay of hepatic enzyme levels in cholestatic plasma. It should be noted that the plasma bile acid biomarkers in this study (Figure 4, Supplemental Figures S4 to S8) were identified following a test meal in patients that were intubated with a duodenal occluding balloon near the ligament of Treitz (Figure S1). Additional studies are required to determine if these plasma bile acid biomarkers would be predictive in patients without the duodenal occluding balloon since this latter is restricting the movement of luminal contents towards the distal intestine and may distort the pattern (post-prandial rise) in BAs present in plasma.

In conclusion, the data presented here show that duodenal BA levels can be drastically reduced in patients with severe CP. Low levels of BAs in intestinal contents have already been described in cases of pancreatic insufficiency. This was usually attributed to BA precipitation at low pH, particularly that of glyco-conjugated BAs [START_REF] Zentler-Munro | Effect of intrajejunal acidity on lipid digestion and aqueous solubilisation of bile acids and lipids in health, using a new simple method of lipase inactivation[END_REF][START_REF] Zentler-Munro | Effect of intrajejunal acidity on aqueous phase bile acid and lipid concentrations in pancreatic steatorrhoea due to cystic fibrosis[END_REF][START_REF] Regan | Reduced intraluminal bile acid concentrations and fat maldigestion in pancreatic insufficiency: correction by treatment[END_REF][START_REF] Hofmann | Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions[END_REF], what we did not observed here. Our results strongly suggest that low duodenal BA levels in the CP patients involved in our clinical study results from the bile duct stenosis, that may lead to a cholehepatic shunt of BA. Enzyme replacement therapy based on the oral administration of pancreatic extracts might not be sufficient to restore fat absorption in these patients, since BAs are essential for the micellar solubilization of lipolysis products and absorption by enterocytes [START_REF] Hofmann | Bile acids: chemistry, pathochemistry, biology, pathobiology, and therapeutics[END_REF]. BA duodenal concentrations were often below CMC (Figure 2A) and formation of mixed BA-lipolysis product micelles could be seriously impaired. Also, since

BAs have antimicrobial effects, their defficiency in the small intestine of CP patients can promote bacterial growth and changes of intestinal microbiota [START_REF] Hofmann | How bile acids confer gut mucosal protection against bacteria[END_REF].

The analytical method describe here for measuring BAs in plasma samples can be used easily to obtain additional information on EPI patients and identify whether their exocrine pancreatic insufficiency is coupled with changes in BA secretion and enterohepatic circulation. The "cholehepatic shunt" signature obtained from plasma BA analysis might also be useful for orientating the diagnosis towards intra-hepatic or extra-hepatic cholestasis, with more specific markers than those currently used in clinics such as levels in hepatic enzymes (γ-glutamyl transpeptidase, alkaline phosphatase, transaminases). The increase in primary to secondary BA ratio could also be used as a secondary marker. In connection with this potential diagnosis tool, it is now essential to develop new treatments for improving fat absorption in EPI patients and one can think about a co-administration of pancreatic enzymes and BAs. BA replacement therapy has already been used for improving fat absorption in patients using dessicated ox bile or the synthetic conjugated BA cholylsarcosine [START_REF] Lillienau | Physicochemical and physiological properties of cholylsarcosine. A potential replacement detergent for bile acid deficiency states in the small intestine[END_REF][START_REF] Schmassmann | Cholylsarcosine, a new bile acid analogue: metabolism and effect on biliary secretion in humans[END_REF][START_REF] Gruy-Kapral | Conjugated bile acid replacement therapy for short-bowel syndrome[END_REF].

Other approaches are using the formulation of pancreatin with a semi-solid self-microemulsifying excipient (Gelucire ® 44/14) to improve pancreatic lipase activity and intestinal absorption [START_REF] Shlieout | Oral pharmaceutical compositions of lipase-containing products, in particular of pancreatin, containing surfactants[END_REF][START_REF] Lukovac | Gelucire 44/14 improves fat absorption in rats with impaired lipolysis[END_REF]. These studies could serve as a basis for further improvement of EPI treatment.

TABLES

  by the Institutional board of the ethics committee CCPPRB (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Hôpital d'Adultes de la Timone, Marseille). Test meal experiments and sample collection were performed from March to July 2003 at "Centre de Pharmacologie Clinique et d'Etudes Thérapeutiques (CPCET, Hôpital d'adultes de la Timone, Marseille, France)" after written informed consents were obtained from all CP patients and HV. The study conformed to the standards set by the Declaration of Helsinki, except for registration in a database.

  5 ± 8.8 mM (mean ± SD, n =5) reported by Hernell et al.(30), 14.7 ± 8.0 mM (mean ± SD, n = 16) by Tangerman et al. (31), 15.8 ± 5.6 mM (mean ± SD, n = 5) by Ladas et al. (29), 16.2 ± 1.5 mM (mean ± SE, n = 13) by Rautureau et al. (32), 14.5 ± 9.4 (mean ± SD, n = 12) by Fausa et al. (33), and 8.3 mM (normal meal) to 11.9 mM

Figure 1 :

 1 Figure 1: Variations with time in duodenal pH (A) and HPL concentration (B). Values are

Figure 2 :

 2 Figure 2: Bile acid (BA) levels in duodenal contents and blood plasma. (A) Variations with

Figure 3 :

 3 Figure 3: Comparison of main bile acid (BA) concentrations in duodenal contents and blood

Figure 4 :

 4 Figure 4: Discrimination of severe chronic pancreatitis (CP) patients and heathy volunteers (HV) based on bile acid (BA) analysis. (A) Correlation between BA duodenal and blood plasma concentrations measured 30 min after meal ingestion. Only main conjugated BA found in both duodenal contents and blood plasma are displayed. Values are means (n=6 for CP patients and n=6 for HV). (B) S-plot of the circulating blood plasma BAs 30 minutes after ingestion of the test meal. The plot illustrates the selection of the most significant biomarkers among of the various plasma BA (n=17) in an O-PLS model calculated to discriminate CP patients and HV. The BA levels measured in plasma by LC-MS/MS are "Pareto" scaled. The vertical axis in the S-plot (p(corr)) represents the correlation between BAs and the score of CP patients and HV. The horizontal axis (p(1)) is the contribution of the distinct BAs estimated by the O-PLS-DA model. The S-plots identifies BAs with the best prediction capability and the largest variation between the CP group (high unconjuguated DCA, UDCA, CDCA) and the control group (high GDCA, GCA, TCDCA). R2X[1] = 0.617, i.e. 61.7 % of

Figure S2 :

 S2 Figure S2: Separation and identification of bile acid standards by LC-MS. The chromatogram is obtained by merging the detection by parent ion/fragment ion pairs of the various bile acids (see Materials and Methods in the article).

Figure S3 (

 S3 Figure S3 (continued): Variations with time of total and individual BA concentrations in duodenal contents and plasma. Open symbols correspond to CP patients while close symbols correspond to healthy volunteers (HV).

Figure S3 (

 S3 Figure S3 (continued): Variations with time of total and individual BA concentrations in duodenal contents and plasma. Open symbols correspond to CP patients while close symbols correspond to healthy volunteers (HV).

Figure S3 (

 S3 Figure S3 (continued): Variations with time of total and individual BA concentrations in duodenal contents and plasma. Open symbols correspond to CP patients while close symbols correspond to healthy volunteers (HV).

Figure S4 :

 S4 Figure S4: Principal Components Analysis (PCA) showing the separation of chronic pancreatitis patients based on the plasma BAs assayed 30 minutes after the test meal.CP patients are characterized by their low duodenum lipase activity (HPL range 2-12 U) and healthy control (HV) by the high duodenum lipase activity (HPL range 320-3066 U). Scores of CP patients (white hexagonal symbols are numbered from 14 to 20D) and controls (light grey to black hexagonal symbols are numbered 1D0 to 13D) are projected on the factorial plan formed by the 2 first principal components t(1) and t(2). The principal components t(1) and t(2) comprises 39,4% and 22,2%, respectively, of the blood plasma BAs levels variance. Loadings in the PCA are also projected on the same factorial plan for 17 non-zero out of 25 blood plasma BAs detected and assayed individually in the plasma by LC-MS/MS (black filled circle symbols). The scores of CP patients (white hexagonal symbols) are projected in areas (right) suggesting the enrichment in tauro-and glyco-conjuguated BAs (TCA, TCDCA, TDCA, GCA, GDCA, GCDCA) relative to the control scores. Conversely, the scores of control plasmas (grey hexagonal symbols) are projected in the (left) areas indicating a relative enrichment as regard to CP patients in unconjuguated BAs (CA, CDCA, DCA, MCA, UDCA). The minor species glycoconjuguated UDCA are also enriched in control at 30min after meal.

Table 1 .

 1 Bile acid outputs (µmoles) and distribution (expressed as mole % of total BA) in

	duodenal contents of healthy volunteers (n=6) and patients with severe chronic pancreatitis
	(n=6) after intake of a liquid-solid test meal. Abbreviations: CA, Cholic acid ; CA-3S, Cholic
	acid 3-sulfate; CDCA, Chenodeoxycholic acid; CDCA-3S, Chenodeoxycholic acid 3-sulfate;
	DCA, Deoxycholic acid; DCA-3S, Deoxycholic acid 3-sulfate; GCA, Glycocholic acid;
	GCDCA, Glycochenodeoxycholic acid; GDCA, Glycodeoxycholic acid; GHCA,
	Glycohyocholic acid; GHDCA, Glycohyodeoxycholic acid; GLCA, Glycolithocholic acid;
	GLCA-3S, Glycolithocholic acid 3-sulfate; GUDCA, Glycoursodeoxycholic acid; GUDCA-
	3S, Glycoursodeoxycholic acid 3-sulfate; HCA, Hyocholic acid; HDCA, Hyodeoxycholic
	acid; LCA, Lithocholic acid; LCA-3S, Lithocholic acid 3-sulfate; MCA, Muricholic acid;
	TCA, Taurocholic acid; TCDCA, Taurochenodeoxycholic acid; TDCA, Taurodeoxycholic
	acid; THCA, Taurohyocholic acid; THDCA, Taurohyodeoxycholic acid; TLCA,
	Taurolithocholic	acid;	TLCA-3S,	Taurolithocholic	acid	3-sulfate;	TUDCA,
	Tauroursodeoxycholic acid; TUDCA-3S, Tauroursodeoxycholic acid 3-sulfate; UDCA,
	Ursodeoxycholic acid; UDCA-3S, Ursodeoxycholic acid 3-sulfate;
		Healthy volunteers (controls)	Chronic Pancreatitis patients
	Bile acids
			Output		Total BA %	Output	Total BA %	% of
			(µmoles)		(mole %)	(µmoles)	(mole %)	controls
	TUDCA	53.02 ± 58.93	0.78 ± 0.79	0.67 ± 0.53	0.07 ± 0.03	1.27
	THDCA	10.04 ± 14.32	0.08 ± 0.12	0.54 ± 0.75	0.02 ± 0.04	5.42
	TCDCA	777.22 ± 618.97 14.56 ± 2.99 101.68 ± 94.82	8.78 ± 3.15	13.08

Table 2 .

 2 Kinetics (AUC, µmoles.L -1 .min) and composition (mole %) of bile acids in serum of

	healthy volunteers (n=6) and patients with severe chronic pancreatitis (n=6) after intake of a
	liquid-solid test meal.
		Healthy volunteers (controls)	Chronic Pancreatitis patients
	Bile acids	AUC	Total BA %	AUC	Total BA %	% of
		(µmoles.L -1 .min)	(mole %)	(µmoles.L -1 .min)	(mole %)	controls
	TUDCA	0.00	-	0.00	-	-
	THDCA	0.00	-	0.00	-	-
	TCDCA	17.29 ± 12.50	5.93 ± 1.56	18.99 ± 12.38	6.70 ± 4.47	109.81
	TDCA	4.19 ± 3.66	1.54 ± 0.67	3.57 ± 3.30	1.11 ± 1.05	85.19
	THCA	0.00	-	0.00		-
	TCA	6.51 ± 5.84	1.98 ± 0.78	12.92 ± 12.44	5.27 ± 5.63	198.41
	GLCA-3S	0.73 ± 1.34	0.24 ± 0.34	1.42 ± 0.96	0.44 ± 0.26	193.32
	TLCA-3S	0.09 ± 0.13	0.03 ± 0.04	0.80 ± 1.05	0.27 ± 0.32	891.11
	UDCA-3S	0.00	-	0.00	-	-
	CDCA-3S	0.00	-	0.00	-	-
	DCA-3S	0.10 ± 0.19	0.03 ± 0.04	0.18 ± 0.27	0.05 ± 0.07	182.25
	LCA-3S	0.00	-	0.00	-	-
	CA-3S	0.02 ± 0.04	0.01 ± 0.02	0.00	-	0.00
	TUDCA-3S	0.00	-	0.05 ± 0.08	0.03 ± 0.04	-
	GUDCA-3S	0.72 ± 1.22	0.36 ± 0.47	1.58 ± 1.89	0.73 ± 1.02	218.94
	GLCA	0.36 ± 0.72	0.10 ± 0 .19	0.02 ± 0.06	0.01 ± 0.01	6.66
	GUDCA	8.81 ± 8.30	3.77 ± 2.21	0.82 ± 1.37	0.21 ± 0.34	9.27
	GHDCA	0.00		0.00		-

Table 3 .

 3 Critical micellar concentrations of the main bile acids found in duodenal contents.CMC values are from Roda et al.[START_REF] Roda | The influence of bile salt structure on self-association in aqueous solutions[END_REF] and were determined by surface tension measurements, using 0.15 M Na + solution for bile salt dispersion. Occurrences in duodenal contents (mol % of total BAs) are based on data obtaind with healthy subjects (HV).

		Occurrence in duodenal contents
	Bile acids		CMC (mM)
		(mol % of total BAs)
	GCA	15.02	10
	GDCA	12.55	2
	GCDCA	26.19	1.8
	GUDCA	2.33	4
	TCA	19.13	6
	TDCA	7.8	2.4
	TCDCA	14.56	3
	TUDCA	0.78	2.2
	Weighted average		4.1

Table S2 :

 S2 Critical micellar concentrations of various bile acids. CMC values are from Roda et al. (1) and were determined by surface tension measurements, using pure water or 0.15 M Na + solution for bile salt dispersion. The main BAs found in duodenal contents of healthy subjects (HV) are shown in blue. It is inferred from the table that in HV the arrangement of BA is probably micelles but dispersed monomers in CP patients (see Results).

		Bile acids	CMC (mM) H 2 O 0.15 M Na +	Occurrence in duodenal contents (mol % of total BAs)
		CA	13	11		0,04
		DCA	10	3			<0,01
		CDCA	9	4			0,02
		81.6 HCA HDCA	58.0 17 14	8 6	70.2	74.0	62.0	91.6
	Clinical data	Alcoholic chronic UCA UDCA	Alcoholic chronic 60 19	Alcoholic chronic 39 7	Alcoholic chronic	Alcoholic chronic <0,01	Alcoholic chronic
		pancreatitis, pancreas divisum, main bile duct stenosis GCA GDCA GCDCA GUCA GUDCA	pancreatitis, main bile duct stenosis treated by endoscopia 12 6 6 35 12	pancreatitis, main bile duct stenosis treated by endoscopia 10 2 1,8 30 4	pancreatitis, main bile duct stenosis treated by endoscopia, 15,02 pancreatitis, pancreas divisum, main bile duct stenosis 12,55 26,19 2,33	pancreatitis, treated for mesenteric vein ischemia without digestive
		treated by endoscopia, type I TCA TDCA	since 1998, diabetes 10 6	since 2000, diabetes 6 2,4	diabetes, hepatic disease	treated by endoscopia since 2002, 19,13 7,8	resection, polyneuropathy due to alcohol
		diabetes, TCDCA	7	3			diabetes 14,56
		dyslipidemi TUCA	52	40
		a, obesity, TUDCA	8	2,2		0,78
		Chronic
		obstructive
		pulmonary
		disease
	Pancreatic	Yes	Yes		Yes	Yes	Yes	Yes
	calcification
	Steatorrhea	>7	45		8	>7	23	>7
	(g/day fecal
	fat)
	AST	21 (N)	23 (N)	19 (N)	97 (high,	19 (N)	17 (N)
	(IU/mL)					>2N)
	ALT	12 (N)	48 (N)	10 (N)	304 (high,	18 (N)	15 (N)
	(IU/mL)					4N)
	ALP	66 (N)	104 (N)	59 (N)	528 (high,	52 (N)	66 (N)
	(IU/mL)					>4N)
	GGT	100 (high,	160 (high,	11 (N)	577 (high,	22 (N)	21 (N)
	(IU/mL)	2N)	3N)			12N)

). R2X[1] = 0.617, i.e. 61.7 % of the BA total variability.

ACKNOWLEDGEMENTS

Our thanks are due to Dr Christine Audebert and all the staff of Centre de Pharmacologie Clinique et d'Evaluation Thérapeutique (CIC-CPCET), Assistance Publique -Hôpitaux de Marseille for their assistance during clinical investigations.

SUPPLEMENTAL DATA

Postprandial bile acid levels in intestine and plasma reveal altered biliary circulation in chronic pancreatitis patients

Lydie Humbert 1 , Dominique Rainteau 1,2* , Noshine Tuvignon 3,4 , Claude Wolf 5 , Philippe Seksik 1,6 , René Laugier 3,4* and Frédéric Carrière

Figure S8: Analysis of 30 min post-prandial plasma BAs in Chronic Pancreatitis patients (white hexagonal symbols) and Healthy Volunteers (light grey to black hexagonal symbols, corresponding to increasing lipase activity). The BAs profile is modeled according to an

Orthogonal-Partial Least Square regression as a function the duodenal lipase activity and duodenal pH (framed labels in the upper right quadrant). The statistical regression identifies a predictive component representing the correlations with duodenum lipase activity and pH (horizontal axis). The orthogonal component for variations not correlated with duodenal lipase and pH (vertical axis) represents a small fraction of the plasma BAs variance (1,9% vs of 31,3% for the predictive component). The separation of CP patients (solid line) and healthy volunteers (dashed line) is shown horizontally but for an outlier control (see arrow). The detected outlier control has a duodenal pH [START_REF] Youngberg | Comparison of gastrointestinal pH in cystic fibrosis and healthy subjects[END_REF][START_REF] Cavalier | Validation of lipolysis product extraction from aqueous/biological samples, separation and quantification by thin-layer chromatography with flame ionization detection analysis using O-cholesteryl ethylene glycol as a new internal standard[END_REF] and lipase activity (720 U/mL) in agreement with other healthy volunteers. Method: The biplot shows scores of patients and control projected in the same factorial plan. The biplot reveals that unconjuguated BAs (UDCA, HDCA, DCA, CA, DCA-3S) are associated with high duodenal pH and lipase in the healthy volunteers group. Conversely, Chronic Pancreatitis (CP) patients identified by empty hexagonal symbols for their low duodenal lipase activity show the higher loadings (i.e., postprandial levels) of major tauroconjuguated (TCDCA, TCA, TDCA) and glycoconjuguated BAs (GDCA, GCA, GCDCA), which results in a higher total plasma post-prandial BA concentration.