BILIARY LIPID SECRETION BEFORE AND AFTER CHOLECYSTECTOMY IN AMERICAN INDIANS WITH CHOLESTEROL GALLSTONES

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1 (; l\~thoe~teholor.y 6(l:1~1 ~ - 1 ~ 1-;. I ~J74 COiJ\Tight l'l,~ I,, TllP Wi ll ia m, &: Wilki ils Co. V(II. 6'3. ~(l. f) Printed ill ( r.sa. BILIARY LIPID SECRETION BEFORE AND AFTER CHOLECYSTECTOMY IN AMERICAN INDIANS WITH CHOLESTEROL GALLSTONES RONALD D. ADLER. M.D.. ALLA~ L. METZGER. M.D.. AND SCOTT M. GRUNDY, M.D.. PH.D. Phoenix Clinical Research Section, National Institute of Arthritis. Metabolism. and Dil?estive Diseases. Phoenix Indian M edical Center. Phoenix. Arizona The effects of cholecystectomy on the hepatic secretion rates of biliary lipids and the relative lipid composition of hepatic bile were examined in 8 American Indian patients with cholesterol gallstones. Using a marker dilution technique, the hepatic secretion rates of cholesterol, bile acids, and phospholipids were measured during continuous duodenal infusion of formula before and several months after cholecystectomy. Before surgery, the Indian patients with gallstones secreted hepatic bile that was more lithogenic than that in a group of white women without stones. When bile was sampled under identical conditions after cholecystectomy. there was no change in biliary lipid composition or in hepatic secretion rates. Thus. cholecystectomy apparently does not correct the defect leading to lithogenic bile in these patients. American Indian women have a high prevalence of c~lolesterol gallstones, 1. 2 and several studies have shown that gallstones in this population are associated with secretion of lithogenic bile by the liver This lithogenic bile results from a combined defect in hepatic secretion of biliary lipids. namely a decreased output of bile acids and an increased output of cholesterol. 4 The enhanced secretion of cholesterol is associated with a high rate of total body cholesterol synthesis,4 and the low bile acid output is associated with Received.July 26, 197:~. Accepted November :3. Address requests for reprints to: Dr. Scott M. Grundy. Section of Metabolism. Department ofmedici ne. Veterang Administration Hospital. San Diego. California The authors are indebted to Dr. Eunice Flock. Mr. Elliott Groszek. Mr. James Hobza. Mr. Robert Collins. Mr. Howard Hughes, Mrs. Marjorie Kennel. Mrs. M argaret Hendrikx. and Miss M arjorie Whelan for excellent assistance. and to the Indian Health Service and the Phoenix Indian Medical Service for cooperation in this study. diminished bile acid pools.5 These findings are consistent with the hypothesis proposed by Small and Rap0 3 that the hepatic secretion of lithogenic bile results from a primary defect in hepatic lipid metabolism. However. there have been recent reports that biliary lipid composition returns to normal after cholecystectomy, and the authors have therefore suggested that abnormalities in hepatic bile acid secretion seen preoperatively may, in fact, be secondary to aberrant function of the gallbladder and/or the enterohepatic circulation Unfortunately, in none of the studies of postcholecystectomy patients thus far reported was it possible to obtain bile under identical physiological conditions pre- and postoperatively. To further elucidate the question of what happens to hepatic biliary lipid secretion after removal of the gallbladder harboring cholesterol stones, we have measured hepatic biliary lipid secretion rates and composition under comparable steady state conditions in 8 Indian patients before and after cholecystectomy. 1212

2 June 1974 BILIARY LIPIDS BEFORE AND AFTER CHOLECYSTECTOMY 1213 Under these conditions we were unable to detect any significant changes in lipid composition or secretion. Methods Patients. Eight southwestern American Indians with gallstone disease were studied for biliary outputs of lipids before and after cholecystectomy. Seven patients were female and 1 was male. Ages ranged from 16 to 6:1 vears (average age 31 years). Patients returned for studies 1 to 12 months after cholecvstectomy (average 5 m onths). Informed conse~t was obtained from all patients before study. The preoperative results for several of the patients have previously been reported. 4 All patients had normal liver function tests before both studies and were taking no medication. None of the patients had hyperlipidemia, but 1 had mild diabetes mellitus. Five of the 8 patients had nonvisualizing gallbladders on oral cholecvstograms. Cholesterol gallstones were obt;ined from all patients a t the time of cholecvstectomy. None had common duct T -tubes aft'er the operation, and the postoperative course was uneventful in all patients. There was no change in the weight of patients after surgery. For the purpose of comparison. results are presented on a group of Indian w omen with gallstones who had visualizing gallbladders on cholecystograms and on white women without stones; the findings in these subjects have previously been presented The Indian women who served as control subjects were comparable in age and obesity to the patients in this study. The white women were not obese and were somewhat younger. Hourly outputs of biliary lipids were measured by the method of Grundy and Metzger. 10 A three-lumen tube was positioned by X-ray in the duodenum such that two proximal outlets were adjacent to the ampulla of Vater, and the third outlet was 10 em distal just beyond the ligament of Treitz. Liquid formula containing fat, carbohydrate, and protein 1 0 was infused t hrough one proximal outlet at a rate calculated to provide the subject's daily caloric requirement. Hourly outputs of biliary cholesterol were determined by marker dilution principles from the samples withdrawn distally. Using {1-sitosterol as a marker, the output of cholesterol was calculated according to the following equation: Cholesterol output (mg/hr ~ {1-sitosterol input (mg/hr) / [cholesterol withdrawn (mg/hr)/{1-sitosterol withdrawn (mg/hr) I (1) Calculation of bile acid and phospholipid outputs was as foll ows: Bile ac id (or phospholipid) output (mg/hr) ~ cholesterol output (mg/hr) " (bile acid or phospholipid to cholesterol ratio) (2) where cholesterol output was that determined from the samples obtained distally (equation 1) and ratios of bile acids (or phospholipids) to cholesterol were those found on samples from the proximal a spiration site. Values for biliary output obtained during the first 4 to 6 hr of studv were not included in the results; we have pre~iously shown that during this period hepatic bile is contaminated with gallbladder bile. 10 After initial gallbladder con traction, hourly outputs become reasonablv constant. and this constancy is maintained f~r up to 20 hr of formula infusion. It seems unlikely that this constancy could have been achieved if the gallbladder had continued to empty significant amounts of residual bile after the first 6 hr or if appreciable intermittent fillin g and emptying of the gallbladder had occurred during the subsequent hours of constant formula in fusion. Biliary lipid outputs in both pre- and postcholecystectomy studies were determined hourly for 6 to 11 hr during this steady state period. Multiple determinations should reduce the errors that would be introduced by transitory inconstancies in gallbladder function, mixing of markers, etc. Biliary lipid composition. Duodenal bile that was aspirated continuously from the proximal aspiration site adjacent to the ampulla of Vater was divided into hourly samples and analyzed for cholesterol bile acids, and phospholipids, as described previously. 1 0 Relative bile acid composition was determined before and after cholecystectomy in samples of bile aspirated during steady state formula infusion. The relative masses of cholic. chenodeoxycholic, and deoxycholic acids were determined by gas-liquid chromatography on a l 'k Hi-Eff 8BP column after conversion of their methyl esters to trimethylsilyl ethers, as previously described Results The mean composition of biliary lipids during constant infusion of liquid formula in 8 Indian patients before and after cholecystectomy is shown in figure 1. Results previously presented for white women

3 1214 ADLER ETAL. Vol. 66, No.6 20 Percent Lecithin o ~ 00 e ~ ~ k Normol Percent Bile Salts Pre-Op. Pl:>st-Op FIG. 1. Mean biliary lipid composition of hepatic bile aspirated from the duodenum during continuous formula infusion in 8 Indian patients before and after cholecystectomy and in 20 white women previously reported. 8 The lines passing through the means represent ± SE for each component. The dashed line represents the maximum solubility of cholesterol as determined by Admirand and Small," and the solid line is the equilibrium solubility as reported by Holzbach et al. J3 without gallstones are given for comparison. 8 These results are plotted on triangular coordinates, according to the method of Admirand and Small. '2 This plot demonstrates that hepatic bile in gallstone patients before surgery was much more lithogenic than that of white control subjects. After surgery, there was no significant change in lithogenicity for the Indian group as a whole. Only 1 patient had significantly improved composition after cholecystectomy. Thus, in most Indian patients with cholesterol gallstones, cholecystectomy did not convert lithogenic bile into normal bile. The mean hepatic secretion rates of cholesterol bile acids, and phospholipids in these same Indian patients before and after operation are shown in figure 2. Secretion rates for white women are also shown for control values. Before surgery, Indian patients had an increased secretion of cholesterol and decreased outputs of bile acids in comparison to white control subjects, as previously reported. 4 After cholecystectomy, no significant changes in secretion rates were found in Indian patients (according to analysis of the paired differences by the t-test). In table 1 are shown average outputs of JO 300 I~ rji w ~.c: "- E '" ~Io ~I Cholesterol Bile Salts Phospholipids Pre 0 Post. Normal 0* FIG. 2. Mean biliary outputs of 8 Indian patients before and after cholecystectomy. Analysis of the paired differences by t-test showed no significant changes. Normal values (asterisk) are those previously reported for 14 white women without biliary tract disease.' biliary lipids for the Indian patients of this study compared with two other groups: (a) Indian women with gallstones who had visualization of gallbladders on oral cholecystograms; and (b) white women without stones. The results for all groups have been normalized to 70 kg ideal weight in order to correct for variations in body weight and structure; the rationale for the normalization has previously been discussed in detail. 4, 9 Since several of these patients had gallbladders that did not visualize before cholecystectomy, comparison with a similar group of Indian patients with gallstones who had visualizing gallbladders would seem particularly important; this comparison is necessary to rule out the possibility that nonvisualization alone may have altered biliary lipid outputs in the preoperative state so as to invalidate comparison with the postoperative state. When these patients were compared with other Indian gallstone patients with visualizing gallbladders, no significant differences were found for either their pre- or postcholecystectomy values. The results also show that abnormalities in lipid secretion leading to increased lithogenicity, as compared with white control subjects, persisted after the operation. Although cholecystectomy produced no change in hepatic secretion of lipids for the period of formula infusion, the diurnal variation in bile lipid composition that is normally seen in subjects with functioning gallbladders was largely obliterated after

4 June 1974 BILIARY LIPIDS BEFORE AND AFTER CHOLECYS TECTOMY 1215 T ABLE 1. Comparison of bliary i lipid outputs Subjects No. Average age Cholesterol Bile acids Phospho lipids mg/70kgiw"/hr ± SEM Indians with gallstones (after cholecystec ± ± 71' 332 ± 28 tomy ) Indians wit h gallstones (before cholecystec ± ± ± 26 tomy) (NS)' (NS) (NS) Indian w omen with gallstones (visualizing gall ± ± ± 29 bladders ) (NS ) (NS) (NS) White women without gallstones ± ± ± 54 (1' < O.Ol)d (P < 0.01) (NS) " IW. ideal weight. /, Values for hourly outputs of bile acids are expressed for conjugated bile acids assuming an average molecular weight of 500. In a previous study,' values were expressed in terms of free bile acids., NS, not significantly different from Indians with gallstones after cholecystectomy. " S ignificantly different from Indians with gallstones at level shown, according to the t test. T ABLE 2. M olar perc entage of choles terol in fa sting and mean infusion h epatic bile Subjects No. Fasting bile" Mean infus ion bile" molar Vc.. ± S EM" Indians with gallstones (a fter cholecvstec tomy). Indian women with gall ± ± 1.1 stones (visualizing gallbladders)c.. ' ± ± 0.7 White women without gallstones" ± ± 0.3 " Fasting bile and mean infusion bile were collected as described previously Mean infusion bile repre sents the average value obtained thro ughout the steady state period of constant formula infusion. In both fas ting and feeding states. duodenal bile should represent hepatic bile without admixture o f gallblad der contents. I> Molar percentage of cholesterol = [moles choles terol moles (cholesterol + bil e acids + phos pholipids) j, Results in these two groups o f patients have previously been presented! cholecystectomy (table 2). Table 2 compares the molar percentage of cholesterol in bile during fasting and feeding in our three groups of subjects. Both white women without gallstones and Indian women with stones who had visualizing gallbladders showed a marked increase in lithogenicity during fasting. In contrast, this diurnal TABLE 3. Relative composition of the three m ajor bile acids in the hepatic bile of 8 gallstone patients before and after cholecystectomy Bile acid Percentage ± SE Pre operatively Post operatively Cholic ± ± 3.0 Deoxycholic 31.6 ± ± 5.3 Chenodeoxycholic ± ± 3.5 fluctuat ion disappeared in Indian patients after cholecystectomy. The relative proportions of cholic, chenodeoxycholic, and deoxycholic acids in samples obtained pre- and postcholecystectomy are shown in table 3. There were no significant differences in bile acid composition; no tendency was noted for an increase in proportion of deoxycholic acid after cholecystectomy, as reported by Malagelada et al. 14 Discussion This study shows that hepatic secretion of lithogenic bile is not corrected by cholecystectomy in American Indian patients with cholesterol gallstones. The fact that these patients continue to secrete an abnormal bile after the operation suggests that the underlying defects leading to lithogenic bile are not altered by removal of the gallbladder. This finding is in contrast to those reported by Simmons et al. 6 and

5 1216 ADLER ETAL. Vol. 66, No.6 Shaffer et al. 7 in which a return to normal bile composition was observed after cholecystectomy. Our failure to observe an improvement in bile composition in Indian gallstone patients thus requires an explanation. Our previous studies have shown that lithogenic bile in Indian patients is at least partly due to an increased hepatic secretion of cholesterol. 4 The persistence of elevated cholesterol outputs after cholecystectomy is not surprising, because this abnormality seems to be related to an increase in total body synthesis of cholesterol. Factors contributing to increased cholesterol synthesis, such as obesity, would not be expected to change with the removal of the gallbladder. The second major factor associated with lithogenic bile in Indian patients is a reduced hepatic secretion of bile acids,4 which is apparently due to decrease in the size of the bile acid pool. 5 Our failure to find an increase in hepatic secretion of bile acids after cholecystectomy suggests that the bile acid pool also did not increase. At present, there is conflicting evidence as to whether bile acid pools increase in white patients after cholecystectomy. Studies carried out by Hepner et al. 15 showed that a group of white patients had essentially normal pools of bile acids after cholecystectomy for gallstones. Although it was not shown that these patients had reduced pools before surgery, these workers studied a comparable group of gallstone patients who clearly demonstrated a reduction in pool sizes. The implications of their findings are that the physiology of bile salts is altered by the removal of the gallbladder, leading to an increase in pool sizes. However, in contrast to these findings, Bell et al. 16 were unable to confirm that bile acid pools are increased after cholecystectomy. In 10 white patients with cholesterol stones, bile acid pools continued to be reduced several months after cholecystectomy. Therefore, in our view, additional evidence must be obtained before the conclusion can be reached that cholecystectomy produces significant alterations in bile acid kinetics. Since hepatic secretion of cholesterol and possibly pool sizes of bile acids are not altered by cholecystectomy, how can the changes in bile acid composition noted by previous workers be explained? We must consider whether these changes may be partly related to different methods of bile collection before and after the operation. Indeed, it is not surprising that hepatic bile collected from the common duct at the time of cholecystectomy is significantly more lithogenic than hepatic bile collected from the same patients postoperatively. In the first instance, the bulk of the bile acid pool is stored in the gallbladder, and hepatic bile becomes increasingly lithogenic throughout the fasting period. 8 In the postoperative state, the pool is more or less continuously circulating, 14 and the diurnal variation in bile composition should be partly obliterated, as demonstrated in this study. Although the composition of fasting bile may be improved after cholecystectomy, this does not imply that the total bile acid pool has expanded or that the underlying homeostatic defect has been corrected. To assure comparability of preand postcholecystectomy studies in a given patient, we have measured biliary lipid composition and secretion rates during stimulation of the enterohepatic circulation of bile acids by continuous infusion of liquid formula; during this procedure, the gallbladder is not contributing significantly to bile flow, so that both pre- and postoperative results should reflect steady state hepatic bile secretion. Under these conditions, cholecystectomy did not favorably alter the composition of hepatic bile. Although we cannot exclude the possibility that results in some groups of non-indian patients might be different, we nevertheless suggest that reports of a return to normal in bile acid composition be viewed with caution until additional studies have been carried out. REFERE~CES 1. Sampliner RE, Bennett PH, Comess LJ, et al: Gallbladder disease in Pima Indians. N Engl J Med 283: , Thistle,JL, Eckhart KL, Nensel RE, et al: Preva-

6 June 1974 BILIARY LIPIDS BEFORE AND AFTER CHOLECYSTECTOMY 1217 lence of gallbladder disease among Chippewa Indians. Mayo Clin Proc 46: , Small DM, Rapo S: Source of abnormal bile in patients with cholesterol gallstones. N Engl J Med 28:i:.S3, Grundy SM, Metzger AL, Adler RD: Mechanisms of lithogenic bile formation in American Indian women with cholesterol gallstones. J Clin Invest 51:3026-:3043, Vlahcevic ZR, Bell CC,]r. Gregory DB, et al: Relationship of bile acid pool size to the formation of lithogenic bile in female Indians of the Southwest. Gastroenterology 62:73-8:3, Simmons F, Ross AP,], Bouchier lad: Alterations in hepatic bile composition after cholecystectomy. Gastroenterology 63: , Shaffer EA, Braasch JW, Small DM: Bile composition at and after surgery in normal persons and patients with gallstones. N Engl J Med 287: ,197:l 8. Metzger AL, Adler RD, Heymsfield S, et al: Diurnal variation in biliary lipid composition. l\' Engl J Med 288:333-:3:36, Grundy SM, Duane We. Adler RD, et al: Biliary lipid outputs in young women with cholesterol gallstones. Metabolism (in press) 10. Grundy SM, Metzger AL: A physiologic method for estimation of hepatic secretion of biliary lipids in man. Gastroenterology 62:1200, Makita M, Wells, WW: Quantitative analysis of fecal bile acids by gas-liquid chromatography, Anal Biochem,5:5:23, , Admirand WH, Small DM: The physicochemical basis of cholesterol gallstone formation in man, J Clin Invest 47: , :3. Holzbach RT, Marsh M, Olszewski M, et al: Cholesterol solubility in bile,,] Clin Invest 52: , 197:3 14. Malagelada,]R, Go VLW, Summerskill WH,], et al: Bile acid secretion and biliary bile acid composition altered by cholecystectomy. Dig Dis 18:455-4;';9, , Hepner GW, Hofmann AF, Klein PD: Altered bile acid metabolism in cholecystectomized patients (abstr). Gastroenterology 64:165, 197:3 16. Bell CC,Jr, Almond HR, Vlahcevic ZR, et al: The effect of cholecystectomy on bile acid pool size, kinetics, and biliary lipid composition in patients with cholesterol gallstones (abstr). Gastroenterology 64:879, 1973