Archives of Disease in Childhood, 1979, 54, 19-24
Intestinal bile salts in cystic fibrosis Studies in the patient and experimental animal J. T. HARRIES, D. P. R. MULLER, J. P. K. McCOLLUM, A. LIPSON, E. ROMA, AND A. P. NORMAN The Institute of Child Health, and The Hospital for Sick Children, Great Ormond Street, London SUMMARY The quantitative and qualitative distribution of bile salts in the duodenal juice of 13 patients with cystic fibrosis (CF) was studied after a test meal. The effects of triolein (TO), bovine serum albumin (BSA), and ricinoleic acid (RA) on the absorption of taurocholate (TCA) in the distal ileum of the rat in vivo was also studied. The mean (and ranges) of total bile salt concentrations, glycine: taurine conjugate ratios, and percentage of dihydroxy bile salts in the patients with CF and pancreatic insufficiency were 3 5 (1 3-6-6) mmol/l, 8-6 (>10-3 1), and 37 (10-60) compared with control values of 7 4 (3 0-16-0) mmol/l, 3 0 (1 -34- 5), and 61 (52-70) respectively. The differences between the control and CF values were statistically significant (P85 %.
Animal experiments. The animal experiments were performed using an in vivo closed-loop model as previously described (Harries and Sladen, 1972; Sladen and Harries, 1972). A 20 cm segment of terminal ileum was used in male Wistar rats weighing 250-300 g after an overnight fast, water being allowed ad libitum. All solutions were prepared on the day of the experiment. The solutions were mixed by ultrasound and gassed with 95% 02 and 5% CO2 to pH 7.1 immediately before instilling approximately 2 ml into the loop. The control solutions contained 10 mmol sodium taurocholate (TCA) (Koch Light)/l], 1 mmol oleate/I and 0 5 mmol monolein/l (Sigma Chemical Company Ltd), 4 mmol KC1/1, 25 mmol NaHCO3/l, and were made up to 280 mosmol/l with NaCI. 14C labelled taurocholate (Radiochemical Centre, Amersham) was added to give an activity of 1 VCi/ml. The test solutions were of a similar composition to the control solutions but, in addition, contained 10 or 30 mmol triolein (Sigma Chemical Company Ltd)/l, 3 % bovine serum albumin (fraction V from Armour Chemical Company), or 5 mmol sodium ricinoleate (Eastman
Chemical Company)/l. The stock lipid solutions were made up in organic solvents, and appropriate aliquots were evaporated to dryness under nitrogen
before mixing with the bile salt-buffer solution. After the absorptive period a 100 ,u aliquot of the fluid remaining in the loop was counted in 10 ml of a dioxan-based scintillant: naphthalene 70 g, PPO 7 g, POPOP 0 0526 g/l dioxan (Hopkin and Williams Ltd). The solution was counted in a LKB Wallac beta counter with external standardisation.
Statistical analysis. The significance of differences between mean values was assessed by the Student's t test.
Results
Duodenal bile salts. Figs 1, 2, and 3 show the values for total bile salt concentrations, the molar ratios of glycine to taurine conjugates, and the percentage of dihydroxy bile salts in the duodenal juice of patients with CF and pancreatic insufficiency compared with controls. Unconjugated bile salts were not detected. In the controls, the mean (and ranges) of total bile salt concentrations, glycine: taurine conjugate ratios, and percentage dihydroxy bile salts were
16 1
0
o 14,
E E
c C 0
12
._o g0
-
0
u
c
°
8
concentrations in o 0
24 2 0
0
duodenal juice of CF patients with pancreatic
0 0
0 = Controls; 0 =CF patients.
insufficiency.
0
6
04
Fig. 1 Total bile salt
00
0 00
S .0 0 0
Intestinal bile salts in cystic fibrosis 21 10 or >10 r
**eeO
9 U) _-.
_.
8
U)
l7
*0
-D
0) 6 0
Fig. 2 Molar ratio of glycine: taurine conjugated bile salts in duodenal juice of CF patients with pancreatic insufficiency.
c .
.
04
0
0):3 -_se_-
.
0
0
02
0 = Controls; 0=CF patients.
0
0
'-1 0
Animal studies. Preliminary experiments were performed in order to establish the relationship between TCA absorption and the absorptive period, and the results are shown in Fig. 4. Luminal disappearance of TCA was very rapid during the first 30 min. By 30 min the mean percentage absorption of TCA was 89% compared with 77% for the 20-min absorptive period. When expressed as Fmol/g wet weight of ileum corresponding mean values for the 1-, 5-, 10-, 20-, 30-, and 40-min absorptive periods were 0-86, 5.77, 9.30, 10-41, 13-74, and 11-20
O L
80 I 0
(A
a
U1)
60
F
0 0 0
.
.0
0 0
'C
8
100
*..
._
0
*.
Fig. 3 Percentage of dihydroxy bile salts in duodenal juice of CF patients with pancreatic
insufficiency.
06
01
0 =Controls; 0 = CF patients.
20 I
L
..
(7)
i
_C,o-
60
75 u c0c0 -at
a a ,._4 ) ua
20
0 v
.
0.
(4)
.c 80
S
40 I-
(4)
at 0
0*
x
0 I-
7.4 (3.0-16.0) mmol/l, 3-0 (1*3-4X5), and 61 (52-70) respectively. Pronounced differences were found in the CF patients with pancreatic insufficiency, corresponding values being 3 5 (1 3-6-6) mmol/l, 8-6 (>10-3 1), and 37 (10-60); P values were
Intestinal bile salts in cystic fibrosis Studies in the patient and experimental animal J. T. HARRIES, D. P. R. MULLER, J. P. K. McCOLLUM, A. LIPSON, E. ROMA, AND A. P. NORMAN The Institute of Child Health, and The Hospital for Sick Children, Great Ormond Street, London SUMMARY The quantitative and qualitative distribution of bile salts in the duodenal juice of 13 patients with cystic fibrosis (CF) was studied after a test meal. The effects of triolein (TO), bovine serum albumin (BSA), and ricinoleic acid (RA) on the absorption of taurocholate (TCA) in the distal ileum of the rat in vivo was also studied. The mean (and ranges) of total bile salt concentrations, glycine: taurine conjugate ratios, and percentage of dihydroxy bile salts in the patients with CF and pancreatic insufficiency were 3 5 (1 3-6-6) mmol/l, 8-6 (>10-3 1), and 37 (10-60) compared with control values of 7 4 (3 0-16-0) mmol/l, 3 0 (1 -34- 5), and 61 (52-70) respectively. The differences between the control and CF values were statistically significant (P85 %.
Animal experiments. The animal experiments were performed using an in vivo closed-loop model as previously described (Harries and Sladen, 1972; Sladen and Harries, 1972). A 20 cm segment of terminal ileum was used in male Wistar rats weighing 250-300 g after an overnight fast, water being allowed ad libitum. All solutions were prepared on the day of the experiment. The solutions were mixed by ultrasound and gassed with 95% 02 and 5% CO2 to pH 7.1 immediately before instilling approximately 2 ml into the loop. The control solutions contained 10 mmol sodium taurocholate (TCA) (Koch Light)/l], 1 mmol oleate/I and 0 5 mmol monolein/l (Sigma Chemical Company Ltd), 4 mmol KC1/1, 25 mmol NaHCO3/l, and were made up to 280 mosmol/l with NaCI. 14C labelled taurocholate (Radiochemical Centre, Amersham) was added to give an activity of 1 VCi/ml. The test solutions were of a similar composition to the control solutions but, in addition, contained 10 or 30 mmol triolein (Sigma Chemical Company Ltd)/l, 3 % bovine serum albumin (fraction V from Armour Chemical Company), or 5 mmol sodium ricinoleate (Eastman
Chemical Company)/l. The stock lipid solutions were made up in organic solvents, and appropriate aliquots were evaporated to dryness under nitrogen
before mixing with the bile salt-buffer solution. After the absorptive period a 100 ,u aliquot of the fluid remaining in the loop was counted in 10 ml of a dioxan-based scintillant: naphthalene 70 g, PPO 7 g, POPOP 0 0526 g/l dioxan (Hopkin and Williams Ltd). The solution was counted in a LKB Wallac beta counter with external standardisation.
Statistical analysis. The significance of differences between mean values was assessed by the Student's t test.
Results
Duodenal bile salts. Figs 1, 2, and 3 show the values for total bile salt concentrations, the molar ratios of glycine to taurine conjugates, and the percentage of dihydroxy bile salts in the duodenal juice of patients with CF and pancreatic insufficiency compared with controls. Unconjugated bile salts were not detected. In the controls, the mean (and ranges) of total bile salt concentrations, glycine: taurine conjugate ratios, and percentage dihydroxy bile salts were
16 1
0
o 14,
E E
c C 0
12
._o g0
-
0
u
c
°
8
concentrations in o 0
24 2 0
0
duodenal juice of CF patients with pancreatic
0 0
0 = Controls; 0 =CF patients.
insufficiency.
0
6
04
Fig. 1 Total bile salt
00
0 00
S .0 0 0
Intestinal bile salts in cystic fibrosis 21 10 or >10 r
**eeO
9 U) _-.
_.
8
U)
l7
*0
-D
0) 6 0
Fig. 2 Molar ratio of glycine: taurine conjugated bile salts in duodenal juice of CF patients with pancreatic insufficiency.
c .
.
04
0
0):3 -_se_-
.
0
0
02
0 = Controls; 0=CF patients.
0
0
'-1 0
Animal studies. Preliminary experiments were performed in order to establish the relationship between TCA absorption and the absorptive period, and the results are shown in Fig. 4. Luminal disappearance of TCA was very rapid during the first 30 min. By 30 min the mean percentage absorption of TCA was 89% compared with 77% for the 20-min absorptive period. When expressed as Fmol/g wet weight of ileum corresponding mean values for the 1-, 5-, 10-, 20-, 30-, and 40-min absorptive periods were 0-86, 5.77, 9.30, 10-41, 13-74, and 11-20
O L
80 I 0
(A
a
U1)
60
F
0 0 0
.
.0
0 0
'C
8
100
*..
._
0
*.
Fig. 3 Percentage of dihydroxy bile salts in duodenal juice of CF patients with pancreatic
insufficiency.
06
01
0 =Controls; 0 = CF patients.
20 I
L
..
(7)
i
_C,o-
60
75 u c0c0 -at
a a ,._4 ) ua
20
0 v
.
0.
(4)
.c 80
S
40 I-
(4)
at 0
0*
x
0 I-
7.4 (3.0-16.0) mmol/l, 3-0 (1*3-4X5), and 61 (52-70) respectively. Pronounced differences were found in the CF patients with pancreatic insufficiency, corresponding values being 3 5 (1 3-6-6) mmol/l, 8-6 (>10-3 1), and 37 (10-60); P values were
