Obesity is an important risk factor in the development

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1 Changes in Gallbladder Bile Composition and Crystal Detection Time in Morbidly Obese Subjects After Bariatric Surgery Ulf Gustafsson, 1 Lisbet Benthin, 2 Lars Granström, 1 Albert K. Groen, 3 Staffan Sahlin, 1 and Curt Einarsson 2 The aim of the present study was to elucidate the mechanisms of development of cholesterol crystals and gallstones during weight reduction in obese subjects. Twenty-five morbidly obese, gallstone-free subjects underwent vertical-banded gastroplasty. Gallbladder bile was collected at the time of the operation via needle aspiration and months after the operation via ultrasound-guided transhepatic puncture of the gallbladder. The mean weight loss was 17 kg. Two patients developed gallstones and 10 patients displayed cholesterol crystals in their bile. In patients with a follow-up time of less than 2 months (n 13), cholesterol saturation increased from 90% to 114% but tended to decrease in the patients with a follow-up time of more than 2 months. The extraction of the concanavalin-a binding fraction from gallbladder bile obtained after weight reduction in 7 patients prolonged crystallization detection time from 6 to 10 days. The hexosamine concentration, a marker for mucin, was increased by about 100% in bile obtained in 6 of 7 patients after weight reduction. In conclusion, the results indicate that crystallization-promoting compounds (mucin) are of great importance in the development of cholesterol crystals and gallstones in obese subjects during weight reduction, probably because of defective gallbladder emptying. (HEPATOLOGY 2005;41: ) Obesity is an important risk factor in the development of cholesterol gallstones. Cholesterol-supersaturated gallbladder bile is considered to be a prerequisite but not the only factor of importance in cholesterol crystal and gallstone formation. 1,2 According to several studies, the hypersecretion of cholesterol from the liver is the most common cause of saturated bile in humans. In addition, in the obese subject, the hypersecretion of cholesterol is considered to be the main cause of an increased risk of gallstone disease. 3-5 Abbreviations: BMI, body mass index; VBG, vertical-banded gastroplasty; CDT, crystal detection time; CABF, concanavalin-a binding fraction. From the 1 Department of Surgery, Danderyd Hospital, Karolinska Institutet, Stockholm, Sweden; the 2 Division of Gastroenterology and Hepatology, Department of Medicine, Karolinska Institutet at Huddinge University Hospital, Stockholm, Sweden; and the 3 Department of Gastroenterology, Academic Medical Center, Amsterdam, The Netherlands. Received April 28, 2004; accepted February 28, Supported by grants from the Swedish Research Council (K X-4793), the Swedish Society of Medicine (Bengt Ihres fond), and Karolinska Institutet, Stockholm, Sweden. Address reprint requests to: Dr. Ulf Gustafsson, M.D., Ph.D., Department of Surgery, Danderyd Hospital, SE Danderyd, Sweden. ulf. gustafsson@ds.se; fax: (46) Copyright 2005 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep Potential conflict of interest: Nothing to report. During rapid weight loss, the risk for cholesterol gallstone formation increases further in obese subjects Patients with the highest body mass index (BMI) before weight loss and those who lose weight most rapidly seem to be at the highest risk for gallstone formation. This finding was true in subjects on a weight-reducing diet as well as those subjects undergoing surgical treatment. The mechanism for gallstone formation during rapid weight loss is not quite clear, although different factors have been suggested. According to several studies, cholesterol saturation of bile increases during weight reduction. 3,6,12-14 Increased amounts of pronucleating proteins and mucin in the gallbladder, which may predispose to gallstone formation, have also been observed. 6,13,15,16 Poor gallbladder emptying and gallbladder stasis, which allows crystals to grow, have been reported during rapid weight loss by means of certain very low caloric diets. 14,17 However, most of these studies have been performed on bile samples obtained by duodenal drainage, leading to dilution of the bile and contamination with duodenal content. In the present study, we have had the unique possibility of receiving pure gallbladder bile from the same patient before and after weight reducing surgery for analysis. We wanted to get answers to the following questions: 1322

2 HEPATOLOGY, Vol. 41, No. 6, 2005 GUSTAFSSON ET AL Table 1. Clinical Data of 25 Obese Patients Before and After Weight Reduction Sex (F/M) Age, yrs (Range) Body Mass Index, kg/m 2 BMI/Reduction kg/m 2 /month S/Cholesterol (mmol/l) S/Triglycerides (mmol/l) Before After Before/After Before After Before After Group I (n 13) ( 2 months follow-up) Group II (n 12) ( 2 months follow-up) 9/4 41 (26-56) * / (27-48) * NOTE. All values are expressed as the mean SD. Abbreviations: F, female; M, male. *P.0001 (significantly different before and after weight reduction). P.05 (significantly different before and after weight reduction). P.005 (significantly different between group I and group II). 1. Does gallbladder bile become more saturated with cholesterol during weight reduction? 2. Is the crystal detection time shortened? 3. Does the extraction of the concanavalin-a binding fraction affect crystal detection time? 4. Is the biliary concentration of mucin increased? Patients and Methods Materials. Concanavalin-A Sepharose and Sepharose-4B were obtained from Pharmacia (Uppsala, Sweden). D- (1-14 C) glucosamine hydrochloride (7.6 mci/ mmol) was purchased from New England Nuclear Corp. (Boston, MA). Patients. Altogether, 25 morbidly obese patients (20 females and 5 males) were included in the study. They all had a BMI greater than 34, and they all had been admitted for vertical-banded gastroplasty (VBG) for obesity. Basic data are given in Table 1. No clinical or laboratory evidence of intestinal, kidney, or thyroid disease or addiction to alcohol or narcotics was present. A few of the obese patients displayed slight hyperlipoproteinemia; some had slightly elevated aminotransferase levels, whereas other liver function tests were within normal limits. None of the patients had gallstone disease on preoperative ultrasound examination. Informed consent was obtained from each patient before operation. The ethical aspects of the study were approved by the Ethical Committee at Karolinska Institutet, Stockholm. Experimental Procedure. All patients were hospitalized in the surgical ward, where laboratory tests and a clinical investigation were performed. All operations were performed between 8:00 and 10:00 A.M. after an overnight fast. After establishment of pneumoperitoneum or opening of the abdomen, the gallbladder was completely emptied of bile with a sterile needle and syringe to avoid possible stratification of bile. 18 During open operative procedures, the gallbladder was carefully examined to exclude gallstones. A VBG was then performed. After 1.1 to 7.3 months, the patients were again admitted to the hospital. Laboratory tests and a clinical investigation were performed, and bile was collected via ultrasound-guided transhepatic puncture of the gallbladder. We did not observe any complications after the puncture, compared with a study by Hussaini et al. in which 16% of the patients experienced transient pain and/or ultrasonographic abnormalities. 19 Two patients had developed small floating gallstones, as detected by the ultrasound examination. Gallbladder bile was transported to the laboratory for analysis. Analysis of Biliary Lipids and Calculation of Cholesterol Saturation. A portion of the bile was extracted with chloroform methanol (2:1, v/v); the chloroform phase was analyzed for cholesterol using an enzymatic method, 20 and phospholipids were analyzed using the method of Rouser et al. 21 Total bile acid concentration was determined using an enzymatic method 22 in another portion of bile. The relative concentrations of cholesterol, bile acids, and phospholipids were expressed as the molar percentage of total biliary lipids. The cholesterol saturation of bile was calculated according to Carey 23 and was expressed as a percentage. Analysis of Cholesterol Crystals and Crystal Detection Time. Bile samples were examined for typical rhomboid monohydrate cholesterol crystals by polarizing light microscopy on prewarmed slides. Crystal detection time (CDT) was determined using the method of Holan et al. 24 with minor modifications. 25 Extraction of Concanavalin-A Binding Fraction. The extraction of the concanavalin-a binding fraction (CABF) was performed essentially as described by Keulemans et al. 26 Fresh gallbladder bile was centrifuged for 10 minutes at 1,000g. Two portions of 3 ml were used. From one of these portions, CABF was removed using concanavalin-a Sepharose-4B. The concanavalin-a Sepharose beads had been prewashed as described by Keulemans et al. The beads were pelleted for 5 minutes at 1,000g and the supernatant was removed completely. The bile was incubated with 1.5

3 1324 GUSTAFSSON ET AL. HEPATOLOGY, June 2005 ml packed concanavalin-a Sepharose beads for 2 hours at 20 C under gentle rotation. After centrifugation for 15 minutes at 500g, the supernatant was collected. As a control, the other portion of bile was added to Sepharose-4B and subjected to the same procedure. Supernatants from concanavalin-a and control Sepharose were passed through a 0.22 m Millipore filter into sterile tubes, which were incubated at 37 C. To determine CDT, samples of 10 L were examined daily for cholesterol monohydrate crystals under a light microscope with polarized light. CDT was defined as the day on which the first cholesterol crystal was observed. Analysis of Gallbladder Mucin and Protein. Gallbladder mucin was determined by assaying the hexosamine content according to a modification of the method of Boas 27 as previously described. 28 According to this method, correction for losses is made by using the recovery of added C-14 labeled glucosamine. Total proteins were measured according to the method of Bradford 29 as previously described. 30 Measurement of Bile Acid Composition. Aliquots of bile were hydrolysed in 1 mol/l potassium hydroxide at 110 C for 12 hours. The deconjugated bile acids were extracted with ethyl ether after acidification to ph 1 with hydrochloric acid. After preparation of the methyl trimethylsilyl ethers, the bile acids were further analyzed via gas liquid chromatography. A Hewlett Packard gas chromatograph (Hewlett Packard, Böblingen, Germany) equipped with a HP1 column was used. Statistical Analysis. Data were presented as the mean SD. The statistical significance of the difference was evaluated via repeated Student t tests. A correlation between parameters was tested by estimating Spearman s rank correlation coefficient. Results Loss of Weight. The time interval between the VBG and the follow-up varied between 1.1 and 7.3 months. The patients were divided into two groups of roughly equal size: group I had a follow-up time of less than 2 months, and group II had a follow-up time of more than 2 months. The average weight loss of group I was 13 kg (range: 4-27) with a mean weight loss of 1.9 kg per week; the average weight loss of group II was 22 kg (range: 4-44) with a mean weight loss of 1.0 kg per week. Biliary Lipid Composition and Cholesterol Saturation. The results of biliary lipid analysis are summarized in Table 2. The mean molar percentage of cholesterol increased during the weight reduction from to (P.005) in group I and decreased from to (P.05) in group II. There was no significant correlation between the extent of weight reduction and the changes in mean molar percentage of cholesterol in bile in the two groups. The mean molar percentage of bile acids increased significantly in group II, whereas the molar percentage of phospholipids decreased. The biliary lipid concentration increased during weight reduction from g/dl to g/dl in the whole series of patients (P.0005) as well as in group II (P.005). Before weight reduction, cholesterol saturation exceeded 100% in 9 of 25 obese patients. During weight reduction, cholesterol saturation increased significantly from 90% 24% to 114% 29% (P.005) in group I. In group II, on the other hand, weight loss was associated with a reduced cholesterol saturation in 9 of 12 patients, but mean cholesterol saturation of the whole group was not significantly reduced (Fig. 1). Table 2. Biliary Lipid, Cholesterol Saturation, CDT, and Occurrence of Cholesterol Crystals Before and After Weight Reduction Group I (<2 Months Follow-up) (n 13) Group II (>2 Months Follow-up) (n 12) Before After Before After Cholesterol (mmol/ml) * Cholesterol (mol%) * Bile acids (mol%) * Phospholipids (mol%) * Lipid concentration (g/dl) * Cholesterol saturation (%) * Nucleation time (in days), median (range) 28 (4-41) 6 (3-32) 17 (9-41) 7 (3-31) Crystals/no crystals 0/13 6/7 0/11 4/7 NOTE. Values are expressed as the mean SD. Crystals number of patients positive for crystals; no crystals number of patients negative for crystals. *P.005 (significantly different before and after weight reduction). P.05 (significantly different before and after weight reduction).

4 HEPATOLOGY, Vol. 41, No. 6, 2005 GUSTAFSSON ET AL Fig 1. Change in cholesterol saturation in patients before and after weight reduction operation. Solid lines represent the patients in group I ( 2 months weight reduction); individual symbols represent the patients in group II ( 2 months weight reduction). CDT and Occurrence of Cholesterol Crystals. Cholesterol crystals were present in 6 gallbladder bile samples of the 13 patients in group I and in 4 of 11 patients in group II after weight loss. There was no significant relationship between the percentage of weight loss and the presence of crystals in bile. Two patients had developed small floating stones. The median CDT decreased from 28 to 6 days in group I. In group II, the CDT was determined in only 5 of the 12 patients; the CDT decreased in 4 of 5 of these patients. In the 10 patients displaying cholesterol crystals, the mean CDT was reduced from 24 to 7 days (P.05) after weight loss. The effect of extraction of the CABF on CDT was studied in the gallbladder bile samples of 7 patients of group I after weight reduction. As can be seen in Fig. 2, Fig 3. Hexosamine concentration before and after weight reduction in gallbladder bile of 7 patients from group I. crystal formation was delayed in all 7 samples. The mean CDT increased from 6 to 11 days (P 0.05). Cholesterol saturation of bile was not increased in these patients during weight loss. The CDT was measured relative to sepharose control, not the original sample. Lipid content, cholesterol saturation index, and hexosamine were not analyzed in relation to extraction of the CABF. Mucin Concentration. The concentration of hexosamine, a marker for mucin, was determined in the gallbladder bile of 7 patients in group I before and after weight reduction. The hexosamine concentration was increased in 6 of the patients and was unchanged in 1; the mean concentrations were g/g and g/g lipids or and when expressed as micrograms per milliliter (P.05) (Fig. 3). Total Protein Concentration. The concentration of total protein was determined in the gallbladder bile of 7 patients in group I before and after weight reduction. The total protein concentration was increased in 6 of the patients and was decreased in 1; the mean concentrations Fig 2. CDT with ( ) and without ( ) the CABF in gallbladder bile of 7 patients from group I after weight reduction. Fig 4. Total protein concentration before and after weight reduction in gallbladder bile of 7 patients from group I.

5 1326 GUSTAFSSON ET AL. HEPATOLOGY, June 2005 Table 3. Bile Acid Composition in 25 Obese Patients Before and After Weight Reduction Group I (<2 Months Follow-up) (n 13) Group II (>2 Months Follow-up) (n 12) Before After Before After Cholic acid (%) * Chenodeoxycholic acid (%) Deoxycholic acid (%) Ursodeoxycholic acid (%) * Lithocholic acid (%) NOTE. Values are expressed as the mean SD. *P.05 (significantly different before and after weight reduction). were mg/ml before and mg/ml after weight reduction (P.05) (Fig. 4). The total protein concentration also increased when the total protein concentration was related to bile acid concentration to correct for varying degrees of bile acid concentration in the gallbladder; the mean values were mg protein/mg bile acid before and mg protein/mg bile acid after (P.05). Bile Acid Composition. The bile acid composition is summarized in Table 3. Cholic acid, chenodeoxycholic acid, and deoxycholic acid were the dominant bile acids. Only small amounts of ursodeoxycholic acid and lithocholic acid were found. Weight loss did not induce any changes in bile acid composition in patients belonging to group II. The proportions of cholic acid decreased in 8 of 11 patients investigated in group I. No correlation was obtained between the percentage of deoxycholic acid and cholesterol saturation. Plasma Lipids. The plasma lipids did not differ significantly between the two groups. Plasma cholesterol before surgery was mmol/l, and plasma triglycerides were mmol/l. When comparing plasma lipids before and after surgery (n 15), plasma cholesterol decreased significantly (P.05) from 5.4 to 4.9 mmol/l, and plasma triglycerides decreased significantly (P.01) from 2.1 to1.4 mmol/l. Discussion The present study comprised 25 morbidly obese subjects undergoing VBG. None of the subjects had gallstones in the gallbladder at the time of the operation. During the postoperative follow-up period lasting from 1.1 to 7.3 months (mean: 14.5 weeks), 2 patients developed gallstones, constituting 8% of the patients or 0.6% of incidence of gallstones per week. Because the weight loss averaged 1.9 kg and 1.0 kg per week in the two groups, respectively, the incidence of gallstones is in reasonable agreement with previously published studies. 10,31 Ten patients (40%) had cholesterol crystals in the gallbladder bile at follow-up. Bile was obtained directly from the gallbladder both at the time of the operation and at follow-up. This model offers a unique possibility of studying changes of the bile composition during the formation of cholesterol crystals and gallstones. In only one previous study was gallbladder bile obtained from obese patients before and after weight reduction. 15,16 However, these patients developed symptomatic gallstones, and the second bile sample was obtained at cholecystectomy. In patients with a follow-up period of less than 2 months, gallbladder bile became more saturated with cholesterol, whereas in those with a longer follow-up period there was a tendency toward less-saturated bile. These findings agree with some previous studies showing that the cholesterol content of bile is relatively high during the most rapid weight loss period, probably because cholesterol is mobilized from adipose tissue. 3,13,15 After weight has stabilized, cholesterol saturation of bile is usually lower than before weight reduction. One of the main purposes of this study was to elucidate possible mechanisms for the formation of cholesterol crystals during weight loss. In the gallbladder bile of patients developing crystals, the CDT was shortened from 24 days to 7 days. This effect was apparently not directly related to the cholesterol saturation index, because it was observed in both groups. Several studies have shown that besides cholesterol saturation, biliary proteins also regulate cholesterol crystallization. 2 Both crystallization-promoting and -inhibiting proteins have been described in human bile. 2 Keulemans et al. 26 have shown that the addition of concanavalin-a Sepharose to gallbladder bile from patients with cholesterol gallstones can extract crystallization-promoting proteins and thereby inhibit crystallization. In the present study, we found that extraction of the CABF from gallbladder bile samples obtained after weight reduction increased the mean CDT from 6 days to 10 days. In gallbladder samples obtained from these patients before weight reduction, the CDT averaged 20 days and 26 days before and after extraction of the CABF, respectively. These data are not presented in Results. The

6 HEPATOLOGY, Vol. 41, No. 6, 2005 GUSTAFSSON ET AL fact that the CDT increases after extraction of the CABF strongly suggests that crystallization-promoting proteins are of importance for the formation of cholesterol crystals in the gallbladder bile during weight reduction in obese subjects. Despite a great number of studies, it is still not known which factors in the CABF are most important. We have therefore been unable to establish the success of our extraction procedure, and it cannot be excluded that not all of the activity was removed. This possibility may explain why the increase in CDT after extraction was highest in slower-nucleating samples. In the present study, we also measured the concentration of hexosamine, a marker for mucin, in gallbladder samples from 7 patients before and after weight reduction. The hexosamine concentration was increased by about 100% in 6 of the patients with a short follow-up time and was unchanged in 1 patient with a longer follow-up time. Increased concentration of hexosamine was associated with a shortened CDT in 5 patients and the development of crystals in 3 patients. These results provide further evidence of the importance of mucin in crystal formation during weight reduction in obese subjects. Furthermore, Marks et al. 13 found increased levels ( 50%) of glycoprotein in the duodenal bile of obese subjects during the early phase of weight reduction. Previously, Shiffman et al. 16 reported on the mucin content of the gallbladder bile of obese patients who had developed symptomatic gallstones during weight reduction and had to be cholecystectomized. The concentration of gallbladder mucin obtained at the gastric bypass operation was about the same (mean: 62 g/ml) as that obtained in our patients at the time of the VBG (mean: 71 g/ml). However, at cholecystectomy the mucin concentration had increased manifold (mean: 1,110 g/ml). It cannot be excluded that the gallstone disease had contributed to the very high mucin concentration, because chronic cholecystitis was identified in each case. 32 Another factor that may be of importance for promoting crystallization and gallstone formation during the weight loss period is impaired gallbladder contraction. This factor was not assessed in the present study. However, decreased gallbladder emptying has been observed in people with very low caloric diets who probably did not have enough fat and/or protein to maximally stimulate gallbladder contraction. 10,14,17 In conclusion, the present study has shown that weight reduction in morbidly obese subjects is associated with an increased risk of cholesterol crystal formation and gallstone development. The extraction of the CABF from gallbladder bile obtained after weight reduction prolonged the CDT. The concentration of hexosamine, a marker for mucin, increased approximately two-fold in 6 of 7 patients. 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