1760 Gut 1994; 35: 1760-1764

Rapid cholesterol nucleation time and cholesterol gall stone formation after subtotal or total colectomy in humans Gut: first published as 10.1136/gut.35.12.1760 on 1 December 1994. Downloaded from

I Makino, K Chijiiwa, H Higashijima, S Nakahara, M Kishinaka, S Kuroki, R Mibu

Abstract Total or subtotal colectomy are the surgical Changes in biliary lipid composition, pH, treatments of choice for patients with ulcera- ionised calcium, total and unconjugated tive or familial adenomatosis coli. The bilirubin, and cholesterol nucleation colon is the site where the primary bile acids time of gall bladder bile samples were are deconjugated and dehydroxylated by examined in six patients who had under- enzymes released from colonic bacteria and gone subtotal or total colectomy between the secondary bile acids that are formed, five months and seven years previously, deoxycholic acid and lithocholic acid, are and values were compared with those mainly absorbed.1-3 Thus, the colon as well as in control patients with no gall stones. The the plays an important role in colectomy group mainly comprised the enterohepatic circulation of bile acids. patients with and familial One of the annoying complications after adenomatosis coli, in whom only a short colectomy is gall stone disease.4 Changes in length of the terminal ileum (mean (SEM) biliary lipids and bile acid composition after 2*25 (0-57) cm) had been resected. The colectomy have been investigated in an animal reconstruction procedures were ileoanal model and in a human one. The increase in the in two patients, terminal cholesterol saturation index of gall bladder bile in two, ileorectal anastomosis in after total with interposed one, and J shaped ileal pouch-anal anasto- jejunal segment as neorectum was reported in mosis in one patient. The distributions of the canine model.5 Harvey et al 6 recently age, sex, and relative body weight were showed an increased cholesterol saturation similar in the two groups. The gall bladder index and rapid cholesterol nucleation time of bile was lithogenic in the post colectomy gall bladder bile similar to values from patients group - these patients had a significantly with cholesterol gall stones7 8 in patients

increased cholesterol saturation index with ulcerative colitis who had undergone http://gut.bmj.com/ (p<001) and rapid cholesterol nucleation colectomy. There is, however, a lack of time (p<0.05) compared with the control detailed information about the kind of gall Departnent of 1, Kyushu group. A significant increase in the molar stones formed and changes in other bile University Faculty of percentage of cholesterol and a decrease components after total or subtotal colectomy Medicine, Fukuoka, in that of total bile acid associated in humans. Japan I Makino with significantly decreased secondary bile This study aimed to examine changes in

K Chijiiwa acids (p<005) were observed in the post biliary lipid composition, bilirubin, ionised on September 27, 2021 by guest. Protected copyright. H Higashijima colectomy group. Gall stones formed in calcium, and the nucleation time of gall S Nakahara bile after human In M Kishinaka two of six patients after colectomy were bladder colectomy. S Kuroki cholesterol stones containing more than addition, gall stones formed after colectomy R Mibu 80% cholesterol by dry weight. Total and were chemically analysed. Correspondence to: unconjugated bilirubin, pH, and ionised Dr Chijiiwa, Department of calcium values were similar in the two Surgery 1, Kyushu University Faculty of groups. The results indicate that after total Methods Medicine, 3-1-1 Maidashi, or subtotal colectomy the composition of Higashi-ku, Fukuoka 812, Japan. gall bladder bile increases the risk of PATIENTS Accepted for publication cholesterol gall stone formation. 23 March 1994 (Gut 1994; 35: 1760-1764) Post colectomy group Six patients who had undergone elective colectomy participated in the study (Table I). There were four women and two men with TABLE I Clinical profile ofthe patients who had undergone colectomy a mean (SEM) age of 41-8 (7 4) years. Age Relative body Primary Time after Total colectomy had been performed in Patient (y) Sex weight (%o)* disease Colectomy Reconstruction colectomy Remarks three patients and subtotal colectomy in the mean time since 1 19 F 134-2 FAC Total IAA 4 y 3 mth Gall stone remainder. The (range) 2 56 F 90 0 UC Total JIPA 4 y 9 mth Gall stone colectomy was 3-7 years (5 months to 7 years). 3 26 M 89-2 UC Subtotal Ileostomy 1 y had ulcerative two 4 46 F 84-5 Multiple Subtotal IRA 4 y 6 mth Three patients colitis, ulcer familial adenomatosis coli, and one patient had 5 67 F 79-5 UC Subtotal Ileostomy 5 mth Gall bladder sludge multiple colonic ulcer. In all six patients only 6 37 M 110-2 FAC Total IAA 7 y very short lengths of the terminal ileum (less than 5 cm, mean (SEM) 2-25 (057) cm) were *Calculated as body weight (kg)/{height (cm) -100} x 100. FAC=familial adenomatosis coli; UC=ulcerative colitis; IAA=ileoanal anastomosis; IRA=ileo- resected. Reconstruction procedures after rectal anastomosis; JIPA=J shaped ileal pouch-anal anastomosis. colectomy were ileoanal anastomosis in two; Rapid cholesterol nucleation time and cholesterol gall stoneformation after subtotal or total colectomy in humans 1761

TABLE ii Composition ofgall bladder bile in patients with hepato-biliary system and their function colectomy and control subjects tests were within the normal range. No signifi- Control patients Post colectomy cant differences were found in age, sex, and the (n= 11) patients (n= 6) relative body weight (colectomy v control, Gut: first published as 10.1136/gut.35.12.1760 on 1 December 1994. Downloaded from Total lipid (g/dl) 12-03 (2-16) 11-27 (1-47) 97.9% V 88.2%) between the two groups. Cholesterol: Informed consent was obtained from all (mM) 15-64 (3 60) 23-75 (4-13) (molar %) (6-27 (0 63)) (11-48 (1-33)*) patients before surgery. The study was Total bile acid: approved by the senior staff committee of the (mM) 167-56 (28 22) 138-35 (18-67) (molar %) (76-8 (1-48)) (66-57 (2.54)*) department. Phospholipid: (Mm) 41-17 (8-45) 45 95 (6-88) (molar %) (16-93 (0-99)) (21 95 (1-38)*) Cholesterol saturation BILE SAMPLES index 1-04 (0-08) 1-56 (0-16)* At the time of surgery and after an overnight pH 7-22 (0-16) 7 30 (0-13) Ionised Ca (mM) 0-95 (0.08) 1-17 (0-17) fast, gall bladder bile was completely aspirated Total bilirubin (mg/ml) 3-12 (0-54) 3-01 (0-71) by needle to avoid stratification of bile, and Unconjugated bilirubin (%) 0-89 (0 05) 1-46 (0-48) care was taken to avoid contamination with *Significantly different from the control (p<001). blood. Fresh gall bladder bile samples thus obtained were kept in sterile test tubes at 37°C in the dark and were immediately subjected to ileostomy in two, ileorectal anastomosis in one; determinations of pH, ionised calcium, and and J shaped ileal pouch-anal anastomosis in total and free bilirubin. Bile samples (3 ml) one patient. None of the patients had gall were ultracentrifuged at 370C for two hours at bladder disease at the time of colectomy: 105 g (55 P-72, Hitachi, Tokyo, Japan) and the this had been confirmed by preoperative isotropic bile samples obtained were used for ultrasonography and intraoperative palpation the nucleation time study as described below. of the gall bladder. Two of the six patients Part ofthe bile sample was stored at - 20°C for (patients 1 and 2) developed gall stone disease chemical analyses. four months and 57 months respectively after colectomy and another patient (patient 5) had gall bladder sludge five months after colectomy BILE ANALYSIS (Table I). These patients underwent chole- The ionised calcium and pH of fresh gall cystectomy. The other three patients under- bladder bile were determined using an went a second operation for closure of automated analyser (CAI 101, Shimadzu, ileostomy with ileoanal anastomosis (patient Kyoto, Japan). Concentrations of individual 3), resection of minor ileocutaneous fistula bile acids and cholesterol were simultaneously after subtotal colectomy with ileorectal determined using gas-liquid chromatography anastomosis (patient 4), and resection of (GC 15 A, Shimadzu, Kyoto, Japan, equipped http://gut.bmj.com/ duodenal tumour (patient 6). with a fused silica column - HiCap-CBPl, 20 mX0 2 mm ID, Shimadzu, Kyoto, Japan) as previously reported.9 The total bile acid Gall stonefree control group concentration was determined as the sum of This group consisted of six men and five the individual bile acids. Phospholipid was women with a mean (SEM) age of 54-6 (4 0) quantitated by the method of Bartlett.'0 The years. There were four patients with colon cholesterol saturation index was calculated on September 27, 2021 by guest. Protected copyright. , four with gastric cancer, two with pan- according to the critical table provided creatic cancer, and one with pheochromo- by Carey.1' Total bilirubin was determined cytoma of the adrenal gland. The absence as described by Michaelsson'2 and the of gall stone disease was confirmed by pre- bilirubin fractions were analysed using high operative ultrasonography and intraoperative performance liquid chromatography (ALC/GP palpation of the gall bladder. C 202, Waters, Milford, MA) according to the All the patients studied were eating a normal method of Spivak and Carey. 13 diet. They had no diseases affecting the Gall stone analysis TABLE iii Gall bladder bile acid composition (%o) in patients with colectomy and controls Gall stones obtained from two patients who had undergone colectomy were dried, weighed, Control patients Post colectomy crushed, and extracted with dimethylsulph- (n= 11) patients (n= 6) oxide-acetone-1 N HCl (90:9:1 v/v/v) and were CA 41-55 (2-71) 61-22 (3-92)* subjected to the determination of cholesterol as CDCA 40 47 (2 26) 38-37 (3 73) DCA 13-10 (3 40) 0 03 (0 03)t previously reported.'4 UDCA 4-46 (1-51) 0.37 (0-22) LCA 0-45 (0-24) ND CA+DCA 54-63 (2 52) 61-25 (3 89) CDCA+LCA+UDCA 45-37 (2 52) 38-75 (3 89) NUCLEATION TIME Primary bile acid 82-01 (2 82) 99-61 (0-24)* The earliest time that cholesterol monohydrate Secondary bile acid 13-54 (3 54) 0 03 (0 03)t crystals appeared in gall bladder bile was determined as previously described,7 8 15 CA=cholic acid; CDCA=chenodeoxycholic acid; DCA=deoxycholic acid; UDCA=ursodeoxycholic acid; based on the method of Holan et al.'6 After LCA=lithocholic acid; ND=not detected; primary bile confirming the absence of cholesterol mono- acid=CA+CDCA; secondary bile acid=DCA+LCA. *Significantly different from the control (p<0-01). hydrate crystals, isotropic bile samples were tSignificantly different from the control (p<005). kept in sterile brown tubes under nitrogen at 1762 Makino, Chijiiwa, Higashijima, Nakahara, Kishinaka, Kuroki, Mibu

group (p<0-0 1). The total lipid concentration was similar in the two groups. The 2.o* concentration of biliary cholesterol was

higher in the colectomy group than in the Gut: first published as 10.1136/gut.35.12.1760 on 1 December 1994. Downloaded from controls, although the difference was not a statistically significant. The molar percentage of cholesterol, however, was significantly (p<001) higher in the colectomy group. The 1. * *o- total bile acid concentration was lower in the colectomy group but this was not statistically significant. The molar percentage of total bile acid was significantly (p<0 01) decreased in the colectomy group. The molar percentage of o- phospholipid was significantly higher in the 20 40 6 80 100 colectomy group. The ionised calcium, pH, and total bilirubin concentration were similar the two groups. The proportion of uncon- Figure 1 Cholesterol saturation index and time after . . . colectomry. No significant correlation was observed between jugated bilirubin was higher in the colectomy the chok esterol saturation index and the time after colectomy group than the controls, but there was no by the method ofleast squares (r= 057, p= 0-24). statistically significant difference. The gall bladder bile acid composition is 370C in the dark without shaking and shown in Table III. The cholic acid level were (examined daily over 21 days for the was significantly higher (p<0-01) and the appeaxrance of cholesterol monohydrate deoxycholic acid value was significantly crystalUs using a polarised microscope (Nikon lower (p<005) (in molar percentage) in the XTP-22, Tokyo, Japan). Where cholesterol colectomy group. The molar percentage of crystal[s did not appear during the observation chenodeoxycholic acid was similar in the two period the nucleation time was recorded as 21 groups. The sum of cholic acid plus its days. metabolite (deoxycholic acid) and that of chenodeoxycholic acid plus its metabolites (lithocholic acid and ursodeoxycholic acid) STATIS;TICAL ANALYSIS were not significantly different between the All daita are given as mean (SEM). Statistical two groups. Primary bile acid was significantly differeinces between the two groups were higher in the controls (p<001). Secondary analys4ed by unpaired Student's t test. The bile acids were absent or only present as traces incidexnce of cholesterol monohydrate crystal in the colectomy group.

nucleation was calculated by the Kaplan-Meier http://gut.bmj.com/ method and compared by the Mantel-Cox test. Correlation between the cholesterol saturation CHOLESTEROL SATURATION INDEX AND TIME index and time after colectomy was examined AFTER COLECTOMY (FIG 1) by the method of least squares. An obviously high cholesterol saturation index (2 03) was observed as early as five months after colectomy (patient 5). Most patients in

Results the post colectomy group showed increased on September 27, 2021 by guest. Protected copyright. cholesterol saturation index within 5 to 57 BILE COMPOSITION months after colectomy. One patient with The biliary lipid composition and cholesterol familial adenomatosis coli and ileoanal saturation index are shown in Table II. The anastomosis (patient 6) showed a relatively low cholesterol saturation index of the gall bladder cholesterol saturation index. No significant bile in the colectomy group was significantly correlation was obsetved between the higher than that in the gall stone free control cholesterol saturation index and the time after colectomy (r=0.57, p=024). 100*

0 Post-colectomy group CHOLESTEROL NUCLEATION TIME (FIG 2) 80- 4 0 In the colectomy group, cholesterol mono- 0- hydrate crystals appeared within three days in 60 0 four of five patients. In contrast, cholesterol

U) crystals did not appear during the observation 40 s ~~~~~~Controls period in four of five control patients. The ~0 colectomy patients had a significantly faster C1 20- nucleation time than the controls (6-4 (3 8) v 18-8 (2.2) days, p<005). U I lb 20 Time (d) ANALYSIS OF GALL STONES OF THE COLECTOMY Figure 2: The incidence ofcholesterol monohydrate crystal GROUP nucleation bladder bile in the stone ofgaUl gall free control Gall stones were cholesterol stones by gross (n=5) and post colectomy (n= 5) groups caleulated by the Kaplan-Mieer method. *Significantly different (p<005) inspection. Chemical analysis showed that from the control group by the Mantel-Cox test. gall stones from patients 1 and 2 contained Rapid cholesterol nucleation time and cholesterol gall stoneformation after subtotal or total colectomy in humans 1763

100% and 80% cholesterol by dry weight, of cholesterol in the present study is probably respectively. secondary to the decreased bile acid secretion as described above. Thus, we consider that the bile acid malabsorption resulted in the altered Gut: first published as 10.1136/gut.35.12.1760 on 1 December 1994. Downloaded from Discussion biliary lipid composition and the increased The current study showed that the biliary lipid lithogenecity in patients with total or subtotal composition, cholesterol saturation index, and colectomy. nucleation time in patients after colectomy Gall bladder bile acid composition was also resembled values in patients with cholesterol changed in patients with colectomy. The appre- gall stones.7 8 15 16 These changes are in good ciable decrease in deoxycholic acid may be the agreement with the fact that the gall stones direct effect of colectomy because secondary formed in the colectomy group were confirmed bile acids are formed and absorbed in the by chemical analysis to be cholesterol gall colon. -3 This change is in good agreement with stones. previous studies showing a reduced excretion of The present results are consistent with those secondary bile acids in the ileal effluent in of Harvey et al6 who reported an increased patients with ileostomy. 8 1930 The significantly cholesterol saturation index and rapid increased cholic acid concentration in gall blad- nucleation time after colectomy in patients der bile also suggests that less cholic acid is with ulcerative colitis. Nearly half of their metabolised further to deoxycholic acid in the control subjects had a nucleation time of less colectomy group. It is thus probable that the than six days, whereas cholesterol crystals did increased proportion of biliary cholic acid not appear within 21 days in most of our and the decreased proportion of deoxycholic control patients. The difference may be due to acid may result from reductions in both the the fact that patients with ulcerative colitis 7a-dehydroxylation of cholic acid and the served as the controls in the study of Harvey passive absorption of deoxycholic acid in et al. Increased excretion of bile acids in the patients with colectomy. A shorter intes- ulcerative colitis patients has been reported, tinal transit time19 and changes in the intestinal even in remission,17 so the bile acid meta- microflora may be responsible for the reduc- bolism might have been changed in their tions. controls. The other important finding in this study is In the present study, the molar percentage of the chemical analysis of gall stones obtained bile acid decreased while cholesterol and from patients with colectomy, since there is no phospholipids increased. The increased published report of this. The gall stones cholesterol saturation index after colectomy formed were cholesterol stones. Lithogenic was a result of changes in the biliary lipids bile appeared soon after colectomy and no cor- (Table II). Bile acid metabolism after colec- relation was found between the cholesterol sat- tomy has been reported by several authors. 18-22 uration index and the time after colectomy http://gut.bmj.com/ An increased faecal bile acid excretion has (Fig 1). The results suggest that the increased been reported after colectomy with continent risk of cholesterol gall stone formation occurs ileostomy, conventional ileostomy, or ileal shortly after colectomy and persists. pouch-anal anastomosis.'9-2' Salemans et al The number of patients with colectomy is recently investigated postprandial serum bile small and they were a heterogeneous group acid levels in colectomy patients with ileal with regard to the original disease and the pouch-anal anastomosis and suggested that reconstruction procedure. Most of these on September 27, 2021 by guest. Protected copyright. bile acid malabsorption occurs after colec- patients had a high cholesterol saturation index tomy.22 Thus, the colectomy patients in the and a rapid nucleation time of gall bladder bile present study may have had malabsorption of irrespective of their original disease or the bile acids. reconstruction procedure. The biliary bile acid Biliary bile acid secretion is regulated by its composition was also very similar in these pool size23 24 and the turnover rate of the patients. These results suggest that colectomy enterohepatic circulation.25 Rutgeerts et a126 itself changes the bile acid metabolism, leading showed that the supersaturated bile is formed to the increased lithogenecity of gall bladder as a result of the more reduced biliary output bile. ofbile acids than of cholesterol in patients who Subtotal or total colectomy had no effect underwent partial ileocolonic resection. They on the pH, ionised calcium, and total and suggested that the partial interruption of the unconjugated bilirubin of gall bladder bile, enterohepatic circulation of bile acids reduces which are responsible for pigment gall stone the size of the bile acid pool and decreases formation. Gall stones formed in the colec- bile acid secretion. In animal experiments, tomy patients were shown by chemical analysis relatively decreased bile acid secretion has also to consist mainly of cholesterol. The results been reported when enterohepatic circulation again support the suggestion that gall bladder has been interrupted.27 28 Although a very bile after colectomy is in a condition to form limited length of terminal ileum was resected cholesterol gall stones. in our patients with colectomy, partial In conclusion, cholesterol gall stones and interruption of the enterohepatic circulation of lithogenic gall bladder bile characterised by an bile acids might be present. increased cholesterol saturation index and Cholesterol absorption is carried out in rapid nucleation time were formed in patients the upper third of the small intestine and is after subtotal or total colectomy. Prophylactic disturbed only when bile acid absorption is or the administration of severely affected.29 The increased proportion cholelitholytic agents may prevent cholesterol 1764 Makino, Chijiiwa, Higashijima, Nakahara, Kishinaka, Kuroki, Mibu

gall stone disease after subtotal or total Claffey WJ. Nucleation time: a key factor in the pathogen- esis of cholesterol gallstone disease. Gastroenterology 1979; colectomy. 77: 611-7. 17 Ejederhamn J, Rafter JJ, Strandvik B. Faecal bile acid This work was supported in part by a grant in aid (no 03670633 excretion in children with inflammatory bowel disease. K Chijiiwa) from the Ministry of Education, Science and Gut 1991; 32: 1346-51. Gut: first published as 10.1136/gut.35.12.1760 on 1 December 1994. Downloaded from Culture, Japan. 18 Kay RM, Cohen Z, Siu KP, Petrunka CN, Strasberg SM. Ileal excretion and bacterial modification ofbile acids and cholesterol in patients with continent ileostomy. Gut 1 Marcus SN, Heaton KW. Deoxycholic acid and the 1979; 21: 128-32. pathogenesis of gall stones. Gut 1988; 29: 522-33. 19 Pedersen BH, Simonsen L, Hansen LK, Giese B, Justesen 2 Samuel P, Saypol GM, Meilman E, Mosbach EH, T, Tougaard L, Binder V. Bile acid malabsorption in Chafizadeh M. Absorption of bile acids from the large patients with an ileum reservoir with a long efferent leg to bowel in man. J Clin Invest 1968; 47: 2070-8. an anal anastomosis. Scand Jf Gastroenterol 1985; 20: 3 Fujii T, Yanagisawa J, Nakayama F. Absorption ofbile acids 995-1000. in dogs as determined by portal blood sampling: evidence 20 Hylander E, Ladefoged K, Nielsen ML, Nielsen OV, Thale for colonic absorption of bile acid conjugates. Digestion M, Jarnum S. Excretion, deconjugation, and absorption 1988; 41: 207-14. of bile acids after colectomy for ulcerative colitis. Scand J 4 Kurchin A, Ray JE, Bluth EI, Merritt CRB, Gathright JB, Gastroenterol 1986; 21: 1137-43. Pehrsson BF, Ferrari BT. Cholelithiasis in ileostomy 21 Natori H, Utsunomiya J, Yamamura T, Benno Y, Uchida patients. Dis Colon 1984; 27: 585-8. K. Fecal and stomal bile acid composition after ileostomy 5 Itoh H, Nakahara S, Nakamura K, Ikeda S, Mibu R, Osato or ileoanal anastomosis in patients with chronic ulcerative K, Nakayama F. Bile composition after total procto- colitis and adenomatosis coli. Gastroenterology 1992; 102: colectomy with interposed jejunal segment as neorectum. 1278-88. Dis Colon Rectum 1989; 32: 711-5. 22 Salemans JMJI, Nagengast FM, Tagerman A, Van Schaik 6 Harvey PRC, McLeod RS, Cohen Z, Strasberg SM. Effect A, De Haan AFJ, Jansen JBMJ. Postprandial conjugated of colectomy on bile composition, cholesterol crystal and unconjugated serum bile acid levels after proctocolec- formation, and gallstones in patients with ulcerative tomy with ileal pouch-anal anastomosis. Scand Jf colitis. Ann Surg 1991; 214: 396-402. Gastroenterol 1993; 28: 786-90. 7 Chijiiwa K, Hirota I, Noshiro H. High vesicular cholesterol 23 Shaffer EA, Small DM. Biliary lipid secretion in cholesterol and protein in bile are associated with formation of gallstone disease: the effect of cholecystectomy and cholesterol but not pigment gallstones. Dig Dis Sci 1993; obesity. _7 Clin Invest 1977; 59: 828-40. 38: 161-6. 24 Rutgeerts P, Ghoos Y, Vantrappen G. The enterohepatic 8 Hirota I, Chijiiwa K, Noshiro H, Nakayama F. Effect of circulation of bile acids during continuous liquid formula chenodeoxycholate and ursodeoxycholate on nucleation perfusion of the duodenum. Jf Lipid Res 1983; 24: 614-9. time in human gallbladder bile. Gastroenterology 1992; 25 Northfield TC, Hofmann AF. Biliary lipid output during 102: 1668-74. three meals and an overnight fast. I Relationship to bile 9 Chijiiwa K, Nakayama F. Simultaneous microanalysis of acid pool size and cholesterol saturation of bile in bile acids and cholesterol in bile by glass capillary column gallstone and control subjects. Gut 1975; 16: 1-17. gas chromatography. Jf Chromatogr 1988; 431: 17-25. 26 Rutgeerts P, Ghoos Y, Vantrappen G. Effects of partial ileo- 10 Barlett GR. Phosphorus assay in column chromatography. colectomy and Crohn's disease on biliary lipid secretion. JBiol Chem 1959; 234: 466-8. Dig Dis Sci 1987; 32: 1231-8. 11 Carey MC. Critical tables for calculating the cholesterol 27 Dowling RH, Mack E, Small DM. Effects of controlled saturation of native bile. J Lipid Res 1978; 19: 945-55. interruption of the enterohepatic circulation of bile salts 12 Michaelsson M. Bilirubin determination in serum and by biliary diversion and by ileal resection on bile salt urine. ScandJ7 Clin Lab Invest 1961; 13 (suppl 56): 1-80. secretion, synthesis, and pool size in the rhesus monkey. 13 Spivak W, Carey MC. Reverse-phase h.p.l.c. separation, Jf Clin Invest 1970; 49: 232-42. quantification and preparation of bilirubin and its 28 Dowling RH, Mack E, Small DM. Biliary lipid secretion conjugates from native bile. 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