Studies on Bile Salt Deconjugation Following Small Bowel Bypass Procedures LESLIE WISE, M.D., HARRY MARGRAF, D.Sc., THEODORE STEIN, M.S.
Using the glycine-1-14C-cholic acid (14C-GCA) test, bile salt deconjugation and excretion were studied in 26 subjects with morbid exogenous obesity before and at selected intervals after jejuno-ileal bypass. In the preoperative group there was no malabsorption or intestinal bacterial deconjugation of the bile salts. In the immediate postoperative period (with relative sterilization of the bowel) there was only a trace of 14C in the breath, but the fecal 14C was highly elevated, indicating severe malabsorption without bacterial activity. In the 8 to 10 day postoperative period both the breath and fecal 14C content were highly elevated, indicating malabsorption and normal bacterial activity. Five to 8 months postoperatively both the breath and fecal 14C content showed only moderate elevation, indicating adaptation. It is suggested that the 14C-GCA test is useful in evaluating the adequacy of small bowel bypass procedures and also in following the adaptive response after the bypass. A significant increase in the breath and fecal 14C soon after surgery followed by rapid decrease suggests an adequate bypass and unusually fast adaptation. If the increase in the breath and fecal 14C soon after the bypass is only moderate, then that suggests an inadequate bypass. T HE MAJOR BILE ACIDS synthesized in the human liver
are cholic acid and chenodeoxycholic acid (primary bile salts); they are present in bile as the sodium and potassium salts, conjugated to glycine or taurine. In response to meals, these conjugated bile acids are secreted into the duodenum. Conjugated bile salts play a vital role in the emulsification, hydrolysis, and absorption of lipids. Under normal circumstances most of the conjugated bile salts are absorbed by active transport in the terminal ileum; then they are reexcreted in bile and are again reabsorbed, and this continuous cycle is termed the enterohepatic circulation of bile salts.4 A small fraction of the conjugated bile salts passes into the colon, where it is deconjugated by bacterial enzymes and it is either absorbed or it is excreted as free acid in the stool. In normal subjects the bile salt pool remains fairly constant, the Submitted for publication September 12, 1974. Partly supported by N.I.H. Grant #AM12333 and #RR00036.
From the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
small amount of fecal loss being balanced by hepatic synthesis from cholesterol.3'8 The purpose of this study was to evaluate the amount of bile salt deconjugation and malabsorption following small bowel bypass procedures, and to assess the role of the 14C-GCA test in the evaluation of the intestinal adaptive response and the adequacy of the bypass. Materials and Methods The studies were performed at the Clinical Research Center in Barnes Hospital. Twenty-six patients with morbid exogenous obesity were studied before and at selected intervals after jejuno-ileal bypass. The type of bypass performed is represented diagrammatically in Fig. 1. In this procedure, 35 cm of proximal jejunum is anastomosed end-to-end to 10 cm of terminal ileum. The intervening segment of small bowel is excluded; the proximal end of this segment is then closed and the distal end is anastomosed end-to-side to the cecum. The preoperative 14C-GCA (Glycine- -114C-cholate) studies were performed about a week prior to operation; the postoperative studies were performed 3 to 4 days following operation (during a period when these patients were receiving parenteral Keflin), 8 to 10 days following operation (when the patients were not receiving antibiotic therapy), and finally, 5 to 11 months postoperatively. Glycine-1-_4C-cholate was given orally to these subjects and the "4CO2 specific activity of expired air was measured by a breath-analysis technique. The amount of "4CO2 in the expired air is an index of the bacterial deconjugation of 14C-GCA.15 The 14C content of a 48 hour stool specimen following the ingestion of 14C-GCA was also measured, and this is an index of malabsorption.
397
398
Ann. Surg. * April 1975
WISE, MARGRAF AND STEIN
FIG. 1. Small bowel bypass procedure
as
performed by the authors.
Results were expressed as a percentage of administered dose per mmole of C02, with the assumption that the average CO2 in expired air is 9 mmoles per kg body weight per hour.5'9 Such correction for weight is important, since the greater the endogenous CO2 production the lower the specific activity.' Fecal 14C was measured in 48 hour stool collections. Samples were homogenized with a blender (Lourdes Instrument Co.); additional water was added, when required, to liquify the fecal matter. Three aliquots were removed from the homogenized sample, were remixed with a Vortex mixer (Scientific Products Co.) and were applied on a 4 x 4 cm desiccated filter paper, Whatman No. 42 (W & R Balston Limited, England), and weighed before and after vacuum desiccation. The three aliquots varied less than l1o (dry weight/wet weight) gravimetrically. Sample 14C was collected by the oxygen flask combustion method (Packard 305 Sample Oxidizer, Packard Instrument Co., U.S.A.), and estimated by a liquid scintillation counter (Packard 3380, Packard Instrument Co.) using external standardization. The radiation exposure associated with the 14C-GCA breath test is less than 0.4 m rad on the date of test as compared to a 5 m rad gonadal exposure from a chest roentgenogram.9
Glycine- -14C-cholic acid, sodium salt, specific activity of approximately 19 mCi/mmole was purchased in 250 ,uCi quantities (from Radiochemical Centre, Amersham). It was dissolved in absolute ethanol so that 10 ,uCi was contained in 0.5 ml solution. A control breath analysis was performed in each patient prior to the administration of 10 ,uCi of the conjugated bile acid which was given in the fasting state at 8:00 am. Four further breath analyses were then performed at two hour intervals, and a final analysis was done 24 hours after the Results administration of the bile salt. The patients received no 1 course of 14CO2 excretion in the In the time Table to had breakfast noon but the food until after prior test, of 14C-GCA is recorded. after the administration breath the test the 8:00 am test the following morning.5 During control period the subjects exthe to exhale instructed preoperative During normally periods the patients were and even then in to 6 hours little 14CO2 up creted very containand then blow the residual air over a drying tube rise above 0.1% did not the excretion most subjects ing silica gel into a counting vial containing the trapping x In the immediate kg body weight. solution. The trapping solution consisted of 4 ml of a 0.5 dose/mmole CO2 molar solution of hyamine hydroxide in methanol:ethanol postoperative period (while on antibiotic therapy) there (1: 1) and thymolphthalein as the indicator of the comple- was no measurable 14CO2 in the breath. Eight to 10 days tion of the neutralization and the trapping of 2 mmoles postoperatively, however, there was a significant inCO2. The exact quantity of CO2 collected was determined crease in the amount of 14CO2 in the breath. All patients by titration of the hyamine stock solution with hy- had very high excretion within 2 hours and this increased drochloric acid. The specific activity of the 14CO2 in the excretion persisted for about 8 hours; by 24 hours there vial was measured by liquid scintillation spectrometry was only minimal excretion of 14CO2. Five to 10 months after the addition of a toluene based scintillation cocktail. after operation again there was a significant increase in TABLE 1. Time Course of "4CO2 in Breath After the Administration of 14C-GCA
Group Preoperative Immediate Postoperative (IV Keflin) 8 to 10 days postoperative 5 to 12 months postoperative
No. of Patients
At 2 hours
26 5
0.01 x 0.004 Trace
0.03 x 0.004 Trace
11
0.51 x 0.12
20
0.24 x 0.04
(% dose/mmole C02 x Kg (Mean + S.E.M.) At 8 hours At 6 hours At 4 hours
At 10 hours
0.10 x 0.01 Trace
0.12 x 0.01 Trace
0.13 x 0.08 Trace
0.59 x 0.10
0.40 x 0.04
0.24 x 0.02
0.04 x 0.02
0.37 x 0.05
0.35 x 0.04
0.27 x 0.04
0.06 x 0.04
Vol. 181 * No. 4
BILE SALT DECONJUGATION
399
0.70 0.60
Msells
-
Preoperative 8-10 days postop. 5-12 months postop.
0.50 FIG. 2. 14CO2 excretion in the breath after the administration of 14C-GCA.
%
Dose 0.40
(per m mole C02 x Kg) 0.30 0.20 0.10
Hours the early 14CO, output, although it was less than in the early postoperative period. Figure 2 depicts the respiratory 14C excretion in a diagrammatic form. During the first 6 hours there was an approximately 20 fold increase in CO2 production in the early postoperative group and a 10 fold increase in the late postoperative group. During the 8-24 hour period the differences in CO2 excretion between the various groups were not significant. In Table 2 the 14CO2 respiratory excretion is expressed as the cumulative percentage of the administered dose. The mean 14C respiratory loss during the first 6 hours in the preoperative group was 2.2%, in the 8 to 10 day postoperative group it was 25.4%, and in the 5 to 12 month postoperative group it was 15.9%. In both postoperative groups the per cent loss was significantly increased (p
Using the glycine-1-14C-cholic acid (14C-GCA) test, bile salt deconjugation and excretion were studied in 26 subjects with morbid exogenous obesity before and at selected intervals after jejuno-ileal bypass. In the preoperative group there was no malabsorption or intestinal bacterial deconjugation of the bile salts. In the immediate postoperative period (with relative sterilization of the bowel) there was only a trace of 14C in the breath, but the fecal 14C was highly elevated, indicating severe malabsorption without bacterial activity. In the 8 to 10 day postoperative period both the breath and fecal 14C content were highly elevated, indicating malabsorption and normal bacterial activity. Five to 8 months postoperatively both the breath and fecal 14C content showed only moderate elevation, indicating adaptation. It is suggested that the 14C-GCA test is useful in evaluating the adequacy of small bowel bypass procedures and also in following the adaptive response after the bypass. A significant increase in the breath and fecal 14C soon after surgery followed by rapid decrease suggests an adequate bypass and unusually fast adaptation. If the increase in the breath and fecal 14C soon after the bypass is only moderate, then that suggests an inadequate bypass. T HE MAJOR BILE ACIDS synthesized in the human liver
are cholic acid and chenodeoxycholic acid (primary bile salts); they are present in bile as the sodium and potassium salts, conjugated to glycine or taurine. In response to meals, these conjugated bile acids are secreted into the duodenum. Conjugated bile salts play a vital role in the emulsification, hydrolysis, and absorption of lipids. Under normal circumstances most of the conjugated bile salts are absorbed by active transport in the terminal ileum; then they are reexcreted in bile and are again reabsorbed, and this continuous cycle is termed the enterohepatic circulation of bile salts.4 A small fraction of the conjugated bile salts passes into the colon, where it is deconjugated by bacterial enzymes and it is either absorbed or it is excreted as free acid in the stool. In normal subjects the bile salt pool remains fairly constant, the Submitted for publication September 12, 1974. Partly supported by N.I.H. Grant #AM12333 and #RR00036.
From the Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110
small amount of fecal loss being balanced by hepatic synthesis from cholesterol.3'8 The purpose of this study was to evaluate the amount of bile salt deconjugation and malabsorption following small bowel bypass procedures, and to assess the role of the 14C-GCA test in the evaluation of the intestinal adaptive response and the adequacy of the bypass. Materials and Methods The studies were performed at the Clinical Research Center in Barnes Hospital. Twenty-six patients with morbid exogenous obesity were studied before and at selected intervals after jejuno-ileal bypass. The type of bypass performed is represented diagrammatically in Fig. 1. In this procedure, 35 cm of proximal jejunum is anastomosed end-to-end to 10 cm of terminal ileum. The intervening segment of small bowel is excluded; the proximal end of this segment is then closed and the distal end is anastomosed end-to-side to the cecum. The preoperative 14C-GCA (Glycine- -114C-cholate) studies were performed about a week prior to operation; the postoperative studies were performed 3 to 4 days following operation (during a period when these patients were receiving parenteral Keflin), 8 to 10 days following operation (when the patients were not receiving antibiotic therapy), and finally, 5 to 11 months postoperatively. Glycine-1-_4C-cholate was given orally to these subjects and the "4CO2 specific activity of expired air was measured by a breath-analysis technique. The amount of "4CO2 in the expired air is an index of the bacterial deconjugation of 14C-GCA.15 The 14C content of a 48 hour stool specimen following the ingestion of 14C-GCA was also measured, and this is an index of malabsorption.
397
398
Ann. Surg. * April 1975
WISE, MARGRAF AND STEIN
FIG. 1. Small bowel bypass procedure
as
performed by the authors.
Results were expressed as a percentage of administered dose per mmole of C02, with the assumption that the average CO2 in expired air is 9 mmoles per kg body weight per hour.5'9 Such correction for weight is important, since the greater the endogenous CO2 production the lower the specific activity.' Fecal 14C was measured in 48 hour stool collections. Samples were homogenized with a blender (Lourdes Instrument Co.); additional water was added, when required, to liquify the fecal matter. Three aliquots were removed from the homogenized sample, were remixed with a Vortex mixer (Scientific Products Co.) and were applied on a 4 x 4 cm desiccated filter paper, Whatman No. 42 (W & R Balston Limited, England), and weighed before and after vacuum desiccation. The three aliquots varied less than l1o (dry weight/wet weight) gravimetrically. Sample 14C was collected by the oxygen flask combustion method (Packard 305 Sample Oxidizer, Packard Instrument Co., U.S.A.), and estimated by a liquid scintillation counter (Packard 3380, Packard Instrument Co.) using external standardization. The radiation exposure associated with the 14C-GCA breath test is less than 0.4 m rad on the date of test as compared to a 5 m rad gonadal exposure from a chest roentgenogram.9
Glycine- -14C-cholic acid, sodium salt, specific activity of approximately 19 mCi/mmole was purchased in 250 ,uCi quantities (from Radiochemical Centre, Amersham). It was dissolved in absolute ethanol so that 10 ,uCi was contained in 0.5 ml solution. A control breath analysis was performed in each patient prior to the administration of 10 ,uCi of the conjugated bile acid which was given in the fasting state at 8:00 am. Four further breath analyses were then performed at two hour intervals, and a final analysis was done 24 hours after the Results administration of the bile salt. The patients received no 1 course of 14CO2 excretion in the In the time Table to had breakfast noon but the food until after prior test, of 14C-GCA is recorded. after the administration breath the test the 8:00 am test the following morning.5 During control period the subjects exthe to exhale instructed preoperative During normally periods the patients were and even then in to 6 hours little 14CO2 up creted very containand then blow the residual air over a drying tube rise above 0.1% did not the excretion most subjects ing silica gel into a counting vial containing the trapping x In the immediate kg body weight. solution. The trapping solution consisted of 4 ml of a 0.5 dose/mmole CO2 molar solution of hyamine hydroxide in methanol:ethanol postoperative period (while on antibiotic therapy) there (1: 1) and thymolphthalein as the indicator of the comple- was no measurable 14CO2 in the breath. Eight to 10 days tion of the neutralization and the trapping of 2 mmoles postoperatively, however, there was a significant inCO2. The exact quantity of CO2 collected was determined crease in the amount of 14CO2 in the breath. All patients by titration of the hyamine stock solution with hy- had very high excretion within 2 hours and this increased drochloric acid. The specific activity of the 14CO2 in the excretion persisted for about 8 hours; by 24 hours there vial was measured by liquid scintillation spectrometry was only minimal excretion of 14CO2. Five to 10 months after the addition of a toluene based scintillation cocktail. after operation again there was a significant increase in TABLE 1. Time Course of "4CO2 in Breath After the Administration of 14C-GCA
Group Preoperative Immediate Postoperative (IV Keflin) 8 to 10 days postoperative 5 to 12 months postoperative
No. of Patients
At 2 hours
26 5
0.01 x 0.004 Trace
0.03 x 0.004 Trace
11
0.51 x 0.12
20
0.24 x 0.04
(% dose/mmole C02 x Kg (Mean + S.E.M.) At 8 hours At 6 hours At 4 hours
At 10 hours
0.10 x 0.01 Trace
0.12 x 0.01 Trace
0.13 x 0.08 Trace
0.59 x 0.10
0.40 x 0.04
0.24 x 0.02
0.04 x 0.02
0.37 x 0.05
0.35 x 0.04
0.27 x 0.04
0.06 x 0.04
Vol. 181 * No. 4
BILE SALT DECONJUGATION
399
0.70 0.60
Msells
-
Preoperative 8-10 days postop. 5-12 months postop.
0.50 FIG. 2. 14CO2 excretion in the breath after the administration of 14C-GCA.
%
Dose 0.40
(per m mole C02 x Kg) 0.30 0.20 0.10
Hours the early 14CO, output, although it was less than in the early postoperative period. Figure 2 depicts the respiratory 14C excretion in a diagrammatic form. During the first 6 hours there was an approximately 20 fold increase in CO2 production in the early postoperative group and a 10 fold increase in the late postoperative group. During the 8-24 hour period the differences in CO2 excretion between the various groups were not significant. In Table 2 the 14CO2 respiratory excretion is expressed as the cumulative percentage of the administered dose. The mean 14C respiratory loss during the first 6 hours in the preoperative group was 2.2%, in the 8 to 10 day postoperative group it was 25.4%, and in the 5 to 12 month postoperative group it was 15.9%. In both postoperative groups the per cent loss was significantly increased (p
