Clinical Science (1979) 56,273-281
Synthetic detergents in bile-salt-deficient steatorrhoea
R. F. G. J. K I N G , P. D. H O W D L E , J . K E L L E H E R
AND
M. S . L O S O W S K Y
Leeds University, Department of Medicine, St James's Hospital, Leeds, U.K.
(Received 17 July 1978; accepted 23 October 1978)
Summary
1. Fat malabsorption caused by bile-salt deficiency is not easily corrected by standard therapy. It has been shown in rats with bile-salt deficiency that non-ionic detergents can replace bile salts and improve fat absorption. A careful assessment of the place of synthetic detergents such as Tween in human therapy has not been reported. 2. The effects of Tween were studied in 13 patients with bile-salt-deficient steatorrhoea, eight of whom received Tween 80 and five Tween 20. 3. Tween therapy reduced steatorrhoea in 10/13 patients; a mean decrease in faecal fat of 33% was achieved with best effect seen in patients with severe steatorrhoea. 4. In the five patients given Tween 20, duodenal/jejunal intubation was conducted using meals with or without added Tween. Jejunal bile salt concentrations were low in all subjects and Tween had no effect on bile-salt output. 5. With Tween addition micellar free fatty acids and phospholipids were increased in four out of the five patients (mean increases 100% and 260% respectively). The mean ratio of fatty acid to bile salt increased from 1.26 to 3-53 and of phospholipid to bile salt from 0.09 to 0.21 in the presence of Tween. 6. The results suggest that in steatorrhoea due to bile-salt deficiency Tween causes increased micellization of ingested fat and improvement in fat absorption. 7. Tween and other non-ionic detergents may, therefore, find a place in therapy of bile-salt deficiency. Correspondence: Dr J. Kelleher, Department of Medicine, St James's Hospital, Leeds LS9 7TF, U.K.
Key words: bile-salt deficiency, steatorrhoea, synthetic detergents.
Introduction
Bile-salt deficiency has been implicated as the major cause of fat and fat-soluble vitamin malabsorption in biliary obstruction, chronic liver disease and after ileal resection (Poley & Hofmann, 1976; Hofmann & Poley, 1972; Badley, Murphy, Bouchier & Sherlock, 1970; Atkinson, Nordin & Sherlock, 1956). Relief by the administration of bile salts is impracticable but an alternative way may be the administration of synthetic non-ionic detergents, which are of extremely low toxicity and are widely used in the pharmaceutical and food industries as dispersing agents. In initial studies we have shown that certain of the Tweens and Pluronic F68 (Sheltawy & Losowsky, 1975a, b) and Myrj 52 (M. Sheltawy & M. S. Losowsky, unpublished work) improved fat absorption in bile-salt deficiency under experimental conditions and we have, therefore, been encouraged to assess whether they benefit human subjects with bile-salt deficiency. Tween 80 has been shown to have a beneficial effect on fat absorption in a small number of patients with steatorrhoea due to various causes (Boyd & Helfrick, 1951; Dubois, Holt, Kuron, Hashim & Van Itallie, 1964; Jones, Culver, Drummey & Ryan, 1948). We only know of its use in a single case of steatorrhoea due to bile-salt deficiency after ileal resection and cholestyramine administration (Shuster, Spoto & Jacobs, 1970). The effects of Tween 80 on the luminal phase of fat absorption in man are not known. We have already 273
R . F. G. J. King et al.
274
shown that Tween 20 improves the absorption in dietary fat in rats with cholestyramine-induced bile salt deficiency (M. Sheltawy 8c M. S. Losowky, unpublished work). We have, therefore, studied the effect of oral Tweens on the intraluminal phase of fat digestion and on the steatorrhoea in patients with intraluminal bile-salt deficiency. In part this paper was presented to the British Society of Gastroenterology Spring Meeting in April 1977.
Methods Patients
Thirteen patients with suspected bile-salt deficiency of different aetiologies were studied (Table 1). All had steatorrhoea and at the time of study were in hospital and in a stable clinical state as assessed by clinical and laboratory findings.
Informed consent was obtained from each patient and the experimental protocol was approved by the local ethical committee. Experimental design
Each patient was studied over 2 weeks while being fed a constant fat diet, varying between patients from 59 to 115 g of fatlday, mean 80.3 g. Samples were collected for faecal fat estimation on the last 2 days of each week. During the second week polyoxyethylene sorbitan monolaurate (Tween 20, Koch-Light Laboratories Ltd, Colnbrook, Bucks, U.K.) was given to patients 1-5 (Table 1) in a dose of 2 g three times daily with meals at 08.00, 12.00 and 18.00 hours. On days 7 and 14, after an overnight fast, these five patients were intubated with a double-lumen polyvinylchloride tube which was positioned fluoroscopically just proximal to the ligament of Treitz. In each case the tube was positioned by midmorning and the appropriate test meal was given
TABLE1. Relevant clinical details of 13 patients with suspected bile-salt ddciency ~
Patient
no.
Age (years)
Sex
Diagnosis
~
Further clinical details
Faecal fat (% dietary fat excreted) Without Tween With Tween
1
31
F
Crohn’s disease
2
49
F
3
48
F
4
33
F
5
72
F
6
14
M
Chronic active hepatitis Primary biliary cirrhosis Primary biliary cirrhosis Primary biliary cirrhosis Mesenteric infarction
1
59
F
8
46
M
9
36
F
10
48
F
11
51
M
12
52
M
13
53
F
Proctocolectomy, resection 200 cm distal ileum, ileostomy ;cholecystectomy Cholecystectomy 16 years previously. On steroids
84
50
11
19
Cholecystectomy 5 months previously. Diabetic on insulin
31
25
34
30
14
14
38
32
15
42
34
15
34
25
9
16
31
30
47
29
52
38
Ileal resection and right hemicolectomy with end-to-end anastomosis, leaving 300 cm of small bowel Systemic sclerosis Involvement of majority of gastrointestinal tract. Colostomy. Bacterial overgrowth of smallbowel Previous ulcer Partial gastrectomy, vagotomy and Roux-en-Y surgery anastomosis. Bacterial overgrowth small bowel, exocrine pancreatic insufficiency Chronic active Previous vagotomy, pyloroplasty and cholecystohepatitis jejunostomy with Roux-en-Y anastomosis On steroids Chronic active hepatitis On steroids and cholestyramine Chronic active hepatitis Malignant bile Intrahepatic obstruction at level of portaduct obstruction hepatis Malignant bile Intrahepatic obstruction at level of portaduct obstruction hepatis
Bile-salt-deficient steatorrhoea 275 immediately afterwards. Intestinal contents were from the first two periods were prepared in aspirated continuously, every 5 rnin for the next duplicate. The oil phase and pellet were not investi120 min. The volume of each 5 min fraction was gated further. measured and the sample divided into two, one Duplicate 2 ml samples of whole contents and sample being pooled into 30 rnin collections and of the micellar phase were extracted twice stored in ice for subsequent measurement of with ether/heptane/ethanol (1: 1 :1, by vol.) pancreatic enzymes, the second being heated for 10 (Blankenhorn & Ahrens, 1955). The extracts were rnin at 70°C,cooled for 5 min and then pooled into transferred to vials and the solvent evaporated. To 30 rnin collections and maintained at 37OC. This each vial was added 1 ml of ethanol and two drops heat treatment effectively denatures most digestive of Bromothymol Blue (1 g/l) as indicator; fatty enzymes (Hofmann & Borgstrom, 1964) with the acids were then titrated with tetra-n-butylexception of phospholipases and will prevent ammonium hydroxide (1 0 mmol/l). further lipolysis (Porter, Saunders, Tytgat, Brunser Total bile acids of whole contents and micellar & Rubin, 1971). The pH of each pooled sample phase were determined in duplicate by the enzymic was recorded and adjusted to pH 5-7 where it was method of Sheltawy & Losowsky (197%). not already within this range. The samples were Phospholipids were extracted by the method of then shaken by hand for 20 s and portions taken Bligh & Dyer (1959); 2 ml samples of extracts were for ultracentrifugation, bile-salt analysis, phosphoevaporated under N, and digested with 1-3 ml of lipid content, tracer recovery and fatty acid 11.7 M-perchloric acid to liberate phosphate analysis. (Marinetti, 1962), which was estimated by the Patients 6-13 (Table 1) were studied without method of Fiske & Subbarrow (1935). Standards intubation and received polyoxyethylene sorbitan of phosphatidylcholine and inorganic phosphate monoleate (Tween 80, Koch-Light Laboratories were taken through the same procedure. For the estimation of individual phospholipids further 2 ml Ltd) during the second week in a dose of 2 g three samples of the extracts were dried under N,, times daily with meals. Patients 7 and 9 (Table 1) were studied for the following third week, after redissolved in a small volume of chloroform and stopping the Tween. applied to t.1.c. plates of silica gel H (thickness 0.5 mm) with standards of phosphatidylcholine and lysophosphatidylcholine. Plates were developed in Test meal chloroform/methanol/water (65:25 :4, by vol.) The test meal used on day 7 consisted of a (Nelson, 1972), spots visualized by exposure to mixture of 20 g of corn oil (Brown and Polson, iodine vapour and the amounts of phospholipids U.K. Ltd.), 10 g of locasol (Cow and Gate Baby estimated by comparative densitometry with reference to standards. Foods, Guildford, Surrey, U.K.), 10 g of limePancreatic function was estimated on the flavoured Hycal (Beecham Products, Brentford, original samples of whole contents, by measureMiddlesex, U.K.), 250 ml of water and 2 pCi of ment of tryptic activity by using N-benzoyl-L[ 14Clpolyethyleneglycol. The test meal given on arginine ethylester as substrate (Wiggins, 1967). day 14 was identical except for the addition of 6 g of Tween 20. Each meal was thoroughly homo[ 14C]Polyethyleneglycol markers in the meals, genized for 2 min in a domestic liquidizer, whole contents and derived micellar phases immediately before consumption. were counted in duplicate 200 pl samples, using a toluene/Triton (2 :1, v/v) scintillator containing 0.1 g/l p-bis-(5-phenyloxazoyl-2-yl)benzene Analysis (POPOP) and 4 g/12,5-diphenyloxazole (PPO) in a 12 ml of the whole contents from each 30 rnin Packard Tri-Carb model 3375 liquid-scintillation period were centrifuged at 30000 rev./min in an counter. Recovery was calculated from the d.p.m. MSE Superspeed 65 centrifuge (average 110000 and juice volumes. g) for 20 h (Hofmann & Borgstrom, 1962). All the Faecal fat was determined on collections from the last 2 days of each experimental period by tubes contained three distinct phases: a top oily phase, a central micellar phase and a pellet. Each the method of Van de Kamer, Huinink & Weyers (1949) using a continuous chromium sesquioxide tube was carefully split in a tube slicer so that the marker (Walker, Kelleher, Davies & Losowsky, micellar phase could be aspirated into a clean tube, which was kept at 37°C before the removal of 1971). Statistical significance between the faecal fat samples for analyses. Micellar-phase solutions
276
R.F. G.J. King et al.
excretion of patients on control or Tween-supplemented diets was assessed by use of the paired ttest and the Wilcoxon Rank test. Other results were assessed by using the paired t-test. Values are given as mean & 1 SD. Results Ten meals were given to five patients. The recovery by aspiration averaged 66% f 15%, only two recoveries being less than 60% (41% each). Of the recovered volume, most (79% -t 22%) was aspirated in the first hour. In two subjects, extending the collection period for a further 30 min (120-150 min) gave little additional recovery (5% and 10%). Although the pH varied between 3.69 and 8.81, 85% of specimens were within the pH range 5-8. All patients had normal pancreatic function and Tween 20 had no significant effect on the output of trypsin. The mean output of trypsin without Tween 20 was 10.36 f 2.5 units/ml and with Tween 20 11.60 rf: 2.9 units/ml; these results were not significantly different (P > 0.1). During Tween administration there was a significant (P < 0.02) decrease in faecal fat (Fig. 1). Ten of the 13 patients had a decreased faecal fat while on Tween therapy; the greatest decrease occurred in those patients with the most severe steatorrhoea. Three of the five patients taking Tween 20 showed a decrease in faecal fat as did all but one of the eight patients taking Tween 80. Two patients (7 and 9, Table 1) had a further faecal fat measurement after Tween therapy was stopped. In both the fat excretion returned to pretreatment values, 80% and 33% of dietary fat intake respectively. Bile-salt concentrations were usually lower in the micellar phase than in total juice (78% f 19% of total juice) and only the micellar results are presented (Table 2). Three patients (2,4 and 5) had an extremely poor bile-salt response to the meal. In each, less than 350 prnol was recovered in the 2 h. In patients 1 and 3, total bile-salt output was only at the lower limit of normal. The total bile-salt output from the ten intubations averaged 820 f 780 ymol with a range of 114-2291 pmol. Bile-salt concentrations were less than 7 mmol/l in every half-hour period (Table 2); in patient no. 2 concentrations never exceeded 2 mmol/l, the probable critical micellar concentration in viva Although patients 1 , 3 , 4 and 5 had isolated periods where values were higher than 2 mmoV1, the concentrations reached were relatively low compared with Mansbach, Cohen & Leff (1975). In
only two instances did a half-hour collection have a bile-salt concentration greater than 5 mmol/l. Tween 20 had no effect on bile-salt output. The fatty acid concentrations and total fatty acid aspirated during 2 h, for the micellar phase and the whole contents, are shown in Table 3. The normal micellar fatty acid concentration after a test meal had been reported as 8-12 mmol/l by Mansbach et al. (1975) and 7.5-20 mmol/l by Badley et al. (1970). In general the concentration of fatty acid in the micellar phase after the first test meal was low. Only two values were greater than 7 mmol/l (Table 3), and only four out of 20 samples were greater than 4 mmol/l. There was a correlation between bile-salt and fatty acid concentrations ( r = 0.79 P (0.05) and there was always a large reserve of fatty acid in the oil phase. After a meal containing Tween 20 the concentrations of fatty acids recovered from the micellar phase in three patients (nos. 1, 2 and 4) were substantially greater than after the first meal. In the other two (3 and 5) they were similar, and in patient no. 5 the micellar fatty acid concentration was maintained despite a marked decrease in bile-salt concentration (Tables 2 and 3). P < 0.02
80
'\
70
-e
P
60
50
d
-t
.B 40 c -u
6(
30
20 2
5
10
C
10
(4Tween
(+)Tween
F I ~1.. Effect of Tween 20 ( 8 )or Tween 80 (H) on faecal fat in 13 patients with suspected bile-salt deficiency. The l i e s connect results from individual patients; the numbers refer to patients described in Table 1.
Bile-salt-dejicient steatorrhoea
277
TABLE2. Micellar bile-salt concentration in individual half-hour periods, and total bile output (2 h) in five subjects after test meals given without (a) or with (b) Tween 20 Bile salt concn. (mmol/l)
Patient
no.
l 2 3 4 5
a b a b a b a b a b
0-30 min
3C-60 min
60-90 min
1.9 3.2 1.6 0.3 6.5 1.3 0.2 0.3 2.0 0.2
2.0 1.6 1.0 1.8 1.4 3.8 0.5 2.4 0.9 0.3
3.3 2.3 0.6 0.8 1.5 0.9 0.6 0.6 3.2 1.0
9C-120 min
Total bilesalt output ijmol) C-120 min
3.9 5.9 1.5 1.1 0.8 0.3 0.3 0.7 1.3 Not analysed
1500 1750 250 250 2300 1150 300 300 300 100
TABLE3. Duodenal juice fatty acid concentration in the micellar phase and in whole contents of individual half-hour periods and the total fatty acid output (2 h) in five patients with bile-salt deficiency after test meals given without (a) and with (b) Tween 20 Fatty acid concn. (mmol/l)
Patient no.
a 1 b
a 2
b a 3 b
a 4 b
a 5 b
Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents
0-30 min
30-60 min
60-90 min
90-120 min
4.6 10.5 16.5 44.7 0.2 62.4 2.7 16.0 10.8 34.7 2.8 21.9 0.4 46.4 0.3 27.5 0.8 45.8 0.3 1.0
0.5 5.4 10.9 20.8 1.7 48.1 1.2 51.0 1.9 14.9 13.7 17.8 0.2 21.2 12.1 25.3 0.3 40.3 0.3 2.0
3.9 4.3 14.2 54.2 0.2 24.3 2.8 37.8 2.2 16.2 0.4 5.6 0.2 30.7 4.1 17.4 6.8 32.7 1.6 8.4
16.4 25.8 36.3 35.7 2.6 93,6 3.4 43.0 3.3 14.1 0.4 8.8 0.2 14.3 1.8 11.8 0.2 2.9
Phospholipids were recovered in the whole contents mainly as lysophosphatidylcholine. In general the micellar phospholipid concentrations in all patients were lower than the range given by Mansbach et al. (1975) in healthy controls (0.761.12 mmol/l) although in patients 1 and 3 (Table 4) two of the micellar samples were within this range. After the control test meal there was no detectable phospholipid in any of the samples of micellar phase from patients 2 and 4 (Table 4). After a Tween-supplemented test meal an increase in 19
-
Total fatty acid output (umol) 0-120 min 2350 5150 9400 23 000 300 12 750 750 8550 3300 13 100 2000 11 600 150 19 100 950 5000 250 3700 150 650
micellar phospholipid was found. The total micellar phospholipid increased after Tween treatment in four of the five patients. The molar ratio of fatty acid and of phospholipid to bile salt in the micellar phase in the presence and absence of Tween 20 is shown in Fig. 2. The ratio of fatty acid to bile salt increased in all five subjects in the presence of Tween 20 (P < 0.05). The mean ratio increased from 1.3 f 0.6 to 3-5 & 1.8. Similarly an increase in the phospholipid to bile salt ratio occurred in four out of five subjects in
R . F. G.J. King et al.
278
TABLE4. Duodenal juice phospholipid concentration in the micellar phase and in whole contents of individual half-hour periods and the total phospholipid output ( 2 h) in five patients with bile-salt deficiency after test meals given without (a)and with (b) Tween 20 Phospholipid concn. (mmol/l)
Patient no.
u
1
b u 2
b u
3
b u
4
b u
5
b
0-30 min
30-60 min
60-90 min
0.2 0.4 0.5
0.1 0.4 0.4 0.5
0.4 0.5 0.4 0.9
0
0
0
0
0.4 0.1 0.9 0.2
0.2 0.1 0.4
0.7
1.0 0.1 0.4
0.7 0.1 0.5 0.3 0.6 0.1 0.2
100 25 100 450 750 200 400
Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents
0.8 0 0.4 0.1 0.2 1.6 2.2 0.3 0.5 0 0.1
0.1 0.2 0.2 0.5 0.1 0.2
0 6
(6) 005< P < O 1
05 04
02
0
1
(-)Tween
(+)Tween
Total phospholipid output 90-120 min (umol) 0-120 min
4
I (-)Tween
0.1
1.4 1.5
0.9 1.2 1.4 1.7
150 250 300 450
0
0
0
0
0.1 0.3 0.6 0.1 0.6 0.1 0.4
0.2 0.1 0.3 0.5 0.7 0.3 0.4
0.2 0.1 0.4
100 25 100 25 50 50
0 0.2
No sample No sample
100
belonging to a three component system. If the molar concentrations are expressed as percentages, as used for analysis of the constituents of bile (Admirand & Small, 1968) then it is possible to construct a graph with triangular co-ordinates. A significantly greater proportion (P< 0.01; by chi-squared) of the points from Tween-supplemented meals (11 out of 19) than from the nonsupplemented (2 out of 20) lie above the 'normal' region calculated from the data of Mansbach et al. (1975) (Fig. 3).
(+)Tween
FIG.2. Duodenal micellar fatty acid/bile salt molar ratio (a), phospholipid/bile salt molar ratio (b) after standard test meals with ( 0 ) or without ( 0 ) Tween 20 in five patients with bile-salt deficiency. The lines connect the means of all determinations from individual patients; the numbers refer to patients described in Table 1.
the presence of Tween 20 (P < 0.06), the mean ratio increasing from 0.1 f 0.1 to 0.2 f 0.1. The one subject in whom the ratio remained constant was the one with by far the highest bile-salt concentration and the only one which was initially above the critical micellar concentration. The micellar concentrations of bile salts, phospholipids and fatty acids can be considered as
Discussion
Available evidence clearly implicates the importance of the micellar phase for normal fat digestion and absorption. Although non-micellar absorption of long-chain fatty acids can occur (Blomstrand, Carlberger & Forsgren, 1969), the severity of steatorrhoea in the presence of reduced concentrations of bile salts implies that this is not quantitatively important. Thus the use of artificial detergents to alter the aqueous solubilization of fatty acids towards the micellar phase would seem to have potential for use in patients with fat malabsorption due to low bile-salt concentrations. The results obtained from faecal fat studies of our patient group show that therapy with either
Bile-salt-deficient steatorrhoea 279 five of these patients with steatorrhoea was characterized by low concentrations of fatty acid due to a deficiency in bile salts. The total bile-salt concentration in aspirated duodenal fluid was lower than the optimum of 8-12 mmol/l (Poley & Hofmann, 1976). The aqueous concentration was even lower, probably due to non-specific adsorption of bile salt to suspended food particles, as has been shown to occur in normal adults (Mansbach et al., 1975) and in patients with intestinal resection (Poley & Hofmann, 1976), or liver disease (Badley et al., 1970). In many samples from our subjects the bile-salt concentrations were lower than the criticaI micell& concentration of 2 mmolfl (Poley & Hofmann, 1976). Even in those patients in whom some bile-salt concentrations FIG.3. Molar concentrations of micellar bile salts, were at or near the critical micellar concentration phospholipids and fatty acids, after test meals in five patients with bile salt deficiency plotted on triangular co(patients 1, 3 and 5, Table 2) the quantity of ordinates. Points represent individual half-hour periods micelles may be insufficient throughout the day to with ( 0 ) or without ( 0 )Tween 20 administration. The solubilize a standard dietary lipid load. This may shaded area represents the normal range calculated from result in steatorrhoea and confirms the work of the data of Mansbach et al. (1975). Badley et al. (1970), who suggested that a mean conjugated bile-salt concentration of greater than 4 mmol/l is required to provide an adequate number of micelles. Tween 80 or Tween 20 confers a significant degree Tween as a non-ionic detergent would be of improvement of steatorrhoea (Fig. 1). Although expected to disperse in aqueous solution to form the detergent-supplemented period always followed micelles, which could be expanded by lipid the control period by design, two patients were molecules. In four of the five patients Tween 20 taken into a third control period where a supplementation caused a substantial increase in corresponding worsening of steatorrhoea was seen. both micellar fatty acid and phospholipid concenIt was encouraging that the patients deserving the trations. The effect was particularly manifested at greatest reduction in their output of faecal fat were low bile-salt concentrations. It cannot be assumed, precisely those in whom Tween therapy was most however, that Tween micelles, presumably different successful. in size and shape from bile-salt micelles, would The therapeutic dose of Tween used in this necessarily function to improve fat absorption. present study was based on previous reports of its However, steatorrhoea did improve in 10 out of 13 use in human subjects (Boyd & Helfrick, 1951; patients and in those with the severest steatorrhoea Dubois et al., 1964; Jones et al., 1948). It is not the improvement was greatest. This suggests that known if this dose of 6 g/day is sufficient to Tween 20 micelles can substitute, to some extent, produce a maximal reduction in faecal fat or if for the physiological bile-salt molecules in fat larger doses of the detergent would have improved absorption. From these studies it is not clear the steatorrhoea still further. Both Tween 20 and whether mixed micelles containing both Tween and Tween 80 are completely hydrolysed in the bile salts can be formed. intestine, the polyoxyethylene component remainThe fatty acid concentration in the oil phase was ing unabsorbed and excreted in the faeces while the always in excess of that in the micellar phase and it fatty acid moiety is available for absorption along therefore seems unlikely that the low concenwith dietary fat (Elworthy & Treon, 1967). trations of fatty acids were due to previous Released fatty acid may contribute to faecal fat if it absorption, or that availability of fatty acid was a is unabsorbed but at the Tween dose used here factor limiting the micellar concentration of fatty would add only approx. 1% to that derived from acids. Tween 20 and Tween 80 have been reported dietary intake. Thus the fatty acid content of the to inhibit pancreatic lipase (Minnard, 1953) in administered detergent will not influence the intervitro, but the inhibition is reversed by the presence pretation of fat absorption. of bile salts. It is not known if such inhibition The aqueous phase of the duodenal contents of
280
R.F. G.J. King et al.
occurs in vim, though the fact that total free fatty acid concenGation is increased in the presence of Tween 20 (Fig. 3) would suggest that such inhibition, if present, did not significantly influence the results of the present study. There is evidence that phospholipid depletion in the gut of experimental animals causes changes in the mucosal cells and impairs fat absorption (O'Doherty, Yousef & Kukis, 1974). These experiments showed that biliary diversion in the rat lowers the activity of acylglycerol transferases and causes retention of dietary triglyceride in the intestinal mucosa. Although the results of Rodgers (1975) conflict with this view, this may be because the experiments were of too short a duration to detect the effect. The micellar phase in our untreated patients usually contained a low concentration of phospholipids and in some untreated patients intestinal fluid contained no micellar phospholipid despite a reserve in the oil phase. It has not been previously recognized that patients with bile-salt depletion may have a deficiency of micellar phospholipid supplied to the mucosa and this may be a factor in decreasing fat absorption. The redistribution of phospholipid from oil to the aqueous phase seen in patients on Tween (Fig. 3) may thus be of importance. It may give even more benefit to supplement some patients with both Tween and phosphatidylcholine. The high ratio of lysophosphatidylcholine in the micellar phase is in agreement with that found by Mansbach et al. (1975) in normal volunteers. Phosphatidylcholine would be digested in the lumen by pancreatic phospholipase. The question of whether phospholipids penetrate the mucosal cells during absorption has not been resolved. Our evidence suggests that lysophosphatidylcholine would predominate if both the fatty acid and phospholipid contents of micelles entered simultaneously. In a scheme put forward by Harrison & Leat (197S), however, phosphatidylcholine directly enters the mucosal cells alone. Despite the difficulty of extrapolating to the practical clinical situation, it seems possible that the increased fat absorption obtained by the use of artificial detergents in patients with bile-salt deficiency may prove of therapeutic value. Our data do not permit an evaluation of the effects of Tween on fat-soluble vitamin absorption but improvement in fat absorption might well improve this too and furthermore may provide a solution to the problem of maintaining a micellar supply of the essential long-chain fatty acids, liioleic and arachidonic, in such patients.
Acknowledgments
This work was supported by a grant from the Medical Research Council. The skilled technical assistance of Mrs W. J. Kennedy is greatly appreciated. References ADMIRAND, W.H. & SMALL,D.M. (1968) The physicochemical basis of cholesterol gallstone formation in man. Journal of Clinical Investigation, 47,1043-1052. ATKINSON, M., NORDIN,B.F.C. & SHERLOCK, S. (1956) Malabsorption and bone disease in prolonged obstructive jaundice. Quarterly Journal of Medicine, 25,299-3 12. BADLEY, B.W.D., MURPHY, G.M., BOUCHIER,I.A.D. & SHERLOCK,S. (1970) Diminished micellar phase lipid in patients with chronic non-alcoholic liver disease and steatorrhoea. Gastroenterology, 58,78 1-789. BLANKENHORN, D.H. & AHRENS,JR., E.H. (1955) Extraction, isolation' and identification of hydrolytic products of triglyceride digestion in man. Journal of Biological Chemistry, 212,69-81. BLIGH,E.G. & DYER,W.J. (1959) A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37,9 1 1-9 17. BLOMSTRAND, R., CARLBERGER, G. & FORSGREN L. (1969) intestinal absorption and metabolism of ''CC-labelledfatty acids in the absence of bile in man. Acta Chirurgica Scandinavica, 135,329-339. BOYD,H. & HELFRICK,F. (195 1) Coeliac disease treated with polyoxyethylene sorbitan monoleate. Journal of Pediatrics, 38,493-497. DUBOIS,J.J., HOLT,P.R., KURON,G.W., HASHIM,S.A. & VAN ITALLIE,T.B. (1964) Effect of Tween 80 on cholestyramineindwed malabsorption. Proccedings of the Society f o r Experimental Biology andMedicine ( N .Y.),117,226-229. ELWORTHY, P.H. & TREON,J.F. (1967) Physiological activity of non-ionic surfactants In: Non-Ionic Sufactants, p. 923-970. Ed. Schick, MJ. Edward Arnold, London. Y. (1935) Colourimetric determinFISKE,C.H. & SUBBARROW, ation of phosphorus. Journal of Biological Chemistry, 66, 375-400. HARRISON,F.A. & LEAT, W.M.F. (1975) Digestion and absorption of lipids in non-ruminant and ruminant animals: A comparison. Proceedings of the Nutrition Society, 34, 203209. HOFMANN,A.F. & BORGSTROM, B. (1962) Physico-chemical state of lipids in intestinal content during their digestion and absorption. Federation Proceedings, 21,43-50. HOFMANN,A.F. & BORGSTROM, B. (1964) The intraluminal phase of fat digestion in man. Journal of Clinical Investigation, 43,247-257. HOFMANN,A. F. & POLEY,J.R. (1972) Role of bile acid malabsorption in pathogenesis of diarrhea and steatorrhea in patients with ileal resection. Gastroenterology, 62.9 18-934. JONES,C.M., CULVER,P.J., DRUMMEY, G.D. & RYAN,A.E. (1948) Modification of fat absorption in the digestive tract by the use of an emulsifying agent. Annals of Internal Medicine, 29, 1-10. MANSBACH, C.M., COHEN,R.S. & LEFF, P.B. (1975) Isolation and properties and the mixed lipid micelles present in intestinal content during fat digestion in man. Journal of Clinical Investigation, 56,781-791. MARMETTI,G.V. (1962) Chromatographic separation, identification and analysis of phosphatides. .lournal of Lipid Research, 3,l-20. MINNARD, F.N. (1953) The inhibition of the action of pancreatic lipase by esters of polyoxyethylene sorbitan. Journal of Biologicai Chemistry, 657-660. NELSON, G.J. (1972) Blood Lipids and Lipoproteins: Quantitation, Composition and Metabolism, p. 55. Wiley Interscience, New York.
Bile-salt-deficient steatorrhoea O’DOHERTY,P.J.A., YOUSEF,I.M. & KUKIS,A. (1974) Effect of phosphatidylcholine on triacylglycerol synthesis in rat intestinal mucosa. Canadian Journal of Biochemistry, 52, 726-733. POLEY,J.R. & HOFMANN, A.F. (1976) Role of fat maldigestion in pathogenesis of steatorrhoea in ileal resection. Gastroenterology, 11,3844. PORTER,H.P., SAUNDERS,D.R., TYTGAT,G., BRUNSER,0. & RUBIN,C.E. (1971) Fat absorption in bile fistula man - a morphological and biochemical study. Gastroenterology, 60, 1008- 1019. RODGERS,J.B. (1975) Lipid absorption in bile fistula rats, lack of a requirement for biliary lecithin. Biochimica et Biophysica Acta, 398,92-100. SHELTAWY,M.J. & LOSOWSKY,M.S. (1975a) Effect of nonionic detergents on the absorption of oleic acid in the anaesthetised rat. Biomedicine, 22 (6),468-472.
281
SHELTAWY,M.J. & LOSOWSKY, M.S. (1975b) Effect of nonionic detergents on the absorption of fat and 6-tocopherol in the rat. Nutrition and Metabolism, 18,265-27 1. SHELTAWY, M.J. & LOSOWSKY, M.S. (1975~)Determination of faecal bile acids by an enzymic method. Clinica Chimica Acta, 64,127-132. SHUSTER, F., SPOTO, R.G. & JACOBS, M.N. (1970) Cholestyramine and Polysorbate-80 in the treatment of cholerheic enteropathy. American Journal of Digestive Diseases, 15,353-358. VAN DE KAMER,J.H., HUININK,H. TEN B. & WEYERS,H.A. (1949) Rapid method for the determination of fat in faeces. Journal of Biological Chemistry, 111,347-355. WALKER,B.E., KELLEHER, J., DAVIES, T. & LOSOWSKY, M.S. (1971) Chemical faecal fat using single stools. Scandinavian Journal of Gastroenterology, 6,277-280. WIGGINS,H.S. (1967) Simple method for estimating trypsin. Gut, 8,415-416.
Synthetic detergents in bile-salt-deficient steatorrhoea
R. F. G. J. K I N G , P. D. H O W D L E , J . K E L L E H E R
AND
M. S . L O S O W S K Y
Leeds University, Department of Medicine, St James's Hospital, Leeds, U.K.
(Received 17 July 1978; accepted 23 October 1978)
Summary
1. Fat malabsorption caused by bile-salt deficiency is not easily corrected by standard therapy. It has been shown in rats with bile-salt deficiency that non-ionic detergents can replace bile salts and improve fat absorption. A careful assessment of the place of synthetic detergents such as Tween in human therapy has not been reported. 2. The effects of Tween were studied in 13 patients with bile-salt-deficient steatorrhoea, eight of whom received Tween 80 and five Tween 20. 3. Tween therapy reduced steatorrhoea in 10/13 patients; a mean decrease in faecal fat of 33% was achieved with best effect seen in patients with severe steatorrhoea. 4. In the five patients given Tween 20, duodenal/jejunal intubation was conducted using meals with or without added Tween. Jejunal bile salt concentrations were low in all subjects and Tween had no effect on bile-salt output. 5. With Tween addition micellar free fatty acids and phospholipids were increased in four out of the five patients (mean increases 100% and 260% respectively). The mean ratio of fatty acid to bile salt increased from 1.26 to 3-53 and of phospholipid to bile salt from 0.09 to 0.21 in the presence of Tween. 6. The results suggest that in steatorrhoea due to bile-salt deficiency Tween causes increased micellization of ingested fat and improvement in fat absorption. 7. Tween and other non-ionic detergents may, therefore, find a place in therapy of bile-salt deficiency. Correspondence: Dr J. Kelleher, Department of Medicine, St James's Hospital, Leeds LS9 7TF, U.K.
Key words: bile-salt deficiency, steatorrhoea, synthetic detergents.
Introduction
Bile-salt deficiency has been implicated as the major cause of fat and fat-soluble vitamin malabsorption in biliary obstruction, chronic liver disease and after ileal resection (Poley & Hofmann, 1976; Hofmann & Poley, 1972; Badley, Murphy, Bouchier & Sherlock, 1970; Atkinson, Nordin & Sherlock, 1956). Relief by the administration of bile salts is impracticable but an alternative way may be the administration of synthetic non-ionic detergents, which are of extremely low toxicity and are widely used in the pharmaceutical and food industries as dispersing agents. In initial studies we have shown that certain of the Tweens and Pluronic F68 (Sheltawy & Losowsky, 1975a, b) and Myrj 52 (M. Sheltawy & M. S. Losowsky, unpublished work) improved fat absorption in bile-salt deficiency under experimental conditions and we have, therefore, been encouraged to assess whether they benefit human subjects with bile-salt deficiency. Tween 80 has been shown to have a beneficial effect on fat absorption in a small number of patients with steatorrhoea due to various causes (Boyd & Helfrick, 1951; Dubois, Holt, Kuron, Hashim & Van Itallie, 1964; Jones, Culver, Drummey & Ryan, 1948). We only know of its use in a single case of steatorrhoea due to bile-salt deficiency after ileal resection and cholestyramine administration (Shuster, Spoto & Jacobs, 1970). The effects of Tween 80 on the luminal phase of fat absorption in man are not known. We have already 273
R . F. G. J. King et al.
274
shown that Tween 20 improves the absorption in dietary fat in rats with cholestyramine-induced bile salt deficiency (M. Sheltawy 8c M. S. Losowky, unpublished work). We have, therefore, studied the effect of oral Tweens on the intraluminal phase of fat digestion and on the steatorrhoea in patients with intraluminal bile-salt deficiency. In part this paper was presented to the British Society of Gastroenterology Spring Meeting in April 1977.
Methods Patients
Thirteen patients with suspected bile-salt deficiency of different aetiologies were studied (Table 1). All had steatorrhoea and at the time of study were in hospital and in a stable clinical state as assessed by clinical and laboratory findings.
Informed consent was obtained from each patient and the experimental protocol was approved by the local ethical committee. Experimental design
Each patient was studied over 2 weeks while being fed a constant fat diet, varying between patients from 59 to 115 g of fatlday, mean 80.3 g. Samples were collected for faecal fat estimation on the last 2 days of each week. During the second week polyoxyethylene sorbitan monolaurate (Tween 20, Koch-Light Laboratories Ltd, Colnbrook, Bucks, U.K.) was given to patients 1-5 (Table 1) in a dose of 2 g three times daily with meals at 08.00, 12.00 and 18.00 hours. On days 7 and 14, after an overnight fast, these five patients were intubated with a double-lumen polyvinylchloride tube which was positioned fluoroscopically just proximal to the ligament of Treitz. In each case the tube was positioned by midmorning and the appropriate test meal was given
TABLE1. Relevant clinical details of 13 patients with suspected bile-salt ddciency ~
Patient
no.
Age (years)
Sex
Diagnosis
~
Further clinical details
Faecal fat (% dietary fat excreted) Without Tween With Tween
1
31
F
Crohn’s disease
2
49
F
3
48
F
4
33
F
5
72
F
6
14
M
Chronic active hepatitis Primary biliary cirrhosis Primary biliary cirrhosis Primary biliary cirrhosis Mesenteric infarction
1
59
F
8
46
M
9
36
F
10
48
F
11
51
M
12
52
M
13
53
F
Proctocolectomy, resection 200 cm distal ileum, ileostomy ;cholecystectomy Cholecystectomy 16 years previously. On steroids
84
50
11
19
Cholecystectomy 5 months previously. Diabetic on insulin
31
25
34
30
14
14
38
32
15
42
34
15
34
25
9
16
31
30
47
29
52
38
Ileal resection and right hemicolectomy with end-to-end anastomosis, leaving 300 cm of small bowel Systemic sclerosis Involvement of majority of gastrointestinal tract. Colostomy. Bacterial overgrowth of smallbowel Previous ulcer Partial gastrectomy, vagotomy and Roux-en-Y surgery anastomosis. Bacterial overgrowth small bowel, exocrine pancreatic insufficiency Chronic active Previous vagotomy, pyloroplasty and cholecystohepatitis jejunostomy with Roux-en-Y anastomosis On steroids Chronic active hepatitis On steroids and cholestyramine Chronic active hepatitis Malignant bile Intrahepatic obstruction at level of portaduct obstruction hepatis Malignant bile Intrahepatic obstruction at level of portaduct obstruction hepatis
Bile-salt-deficient steatorrhoea 275 immediately afterwards. Intestinal contents were from the first two periods were prepared in aspirated continuously, every 5 rnin for the next duplicate. The oil phase and pellet were not investi120 min. The volume of each 5 min fraction was gated further. measured and the sample divided into two, one Duplicate 2 ml samples of whole contents and sample being pooled into 30 rnin collections and of the micellar phase were extracted twice stored in ice for subsequent measurement of with ether/heptane/ethanol (1: 1 :1, by vol.) pancreatic enzymes, the second being heated for 10 (Blankenhorn & Ahrens, 1955). The extracts were rnin at 70°C,cooled for 5 min and then pooled into transferred to vials and the solvent evaporated. To 30 rnin collections and maintained at 37OC. This each vial was added 1 ml of ethanol and two drops heat treatment effectively denatures most digestive of Bromothymol Blue (1 g/l) as indicator; fatty enzymes (Hofmann & Borgstrom, 1964) with the acids were then titrated with tetra-n-butylexception of phospholipases and will prevent ammonium hydroxide (1 0 mmol/l). further lipolysis (Porter, Saunders, Tytgat, Brunser Total bile acids of whole contents and micellar & Rubin, 1971). The pH of each pooled sample phase were determined in duplicate by the enzymic was recorded and adjusted to pH 5-7 where it was method of Sheltawy & Losowsky (197%). not already within this range. The samples were Phospholipids were extracted by the method of then shaken by hand for 20 s and portions taken Bligh & Dyer (1959); 2 ml samples of extracts were for ultracentrifugation, bile-salt analysis, phosphoevaporated under N, and digested with 1-3 ml of lipid content, tracer recovery and fatty acid 11.7 M-perchloric acid to liberate phosphate analysis. (Marinetti, 1962), which was estimated by the Patients 6-13 (Table 1) were studied without method of Fiske & Subbarrow (1935). Standards intubation and received polyoxyethylene sorbitan of phosphatidylcholine and inorganic phosphate monoleate (Tween 80, Koch-Light Laboratories were taken through the same procedure. For the estimation of individual phospholipids further 2 ml Ltd) during the second week in a dose of 2 g three samples of the extracts were dried under N,, times daily with meals. Patients 7 and 9 (Table 1) were studied for the following third week, after redissolved in a small volume of chloroform and stopping the Tween. applied to t.1.c. plates of silica gel H (thickness 0.5 mm) with standards of phosphatidylcholine and lysophosphatidylcholine. Plates were developed in Test meal chloroform/methanol/water (65:25 :4, by vol.) The test meal used on day 7 consisted of a (Nelson, 1972), spots visualized by exposure to mixture of 20 g of corn oil (Brown and Polson, iodine vapour and the amounts of phospholipids U.K. Ltd.), 10 g of locasol (Cow and Gate Baby estimated by comparative densitometry with reference to standards. Foods, Guildford, Surrey, U.K.), 10 g of limePancreatic function was estimated on the flavoured Hycal (Beecham Products, Brentford, original samples of whole contents, by measureMiddlesex, U.K.), 250 ml of water and 2 pCi of ment of tryptic activity by using N-benzoyl-L[ 14Clpolyethyleneglycol. The test meal given on arginine ethylester as substrate (Wiggins, 1967). day 14 was identical except for the addition of 6 g of Tween 20. Each meal was thoroughly homo[ 14C]Polyethyleneglycol markers in the meals, genized for 2 min in a domestic liquidizer, whole contents and derived micellar phases immediately before consumption. were counted in duplicate 200 pl samples, using a toluene/Triton (2 :1, v/v) scintillator containing 0.1 g/l p-bis-(5-phenyloxazoyl-2-yl)benzene Analysis (POPOP) and 4 g/12,5-diphenyloxazole (PPO) in a 12 ml of the whole contents from each 30 rnin Packard Tri-Carb model 3375 liquid-scintillation period were centrifuged at 30000 rev./min in an counter. Recovery was calculated from the d.p.m. MSE Superspeed 65 centrifuge (average 110000 and juice volumes. g) for 20 h (Hofmann & Borgstrom, 1962). All the Faecal fat was determined on collections from the last 2 days of each experimental period by tubes contained three distinct phases: a top oily phase, a central micellar phase and a pellet. Each the method of Van de Kamer, Huinink & Weyers (1949) using a continuous chromium sesquioxide tube was carefully split in a tube slicer so that the marker (Walker, Kelleher, Davies & Losowsky, micellar phase could be aspirated into a clean tube, which was kept at 37°C before the removal of 1971). Statistical significance between the faecal fat samples for analyses. Micellar-phase solutions
276
R.F. G.J. King et al.
excretion of patients on control or Tween-supplemented diets was assessed by use of the paired ttest and the Wilcoxon Rank test. Other results were assessed by using the paired t-test. Values are given as mean & 1 SD. Results Ten meals were given to five patients. The recovery by aspiration averaged 66% f 15%, only two recoveries being less than 60% (41% each). Of the recovered volume, most (79% -t 22%) was aspirated in the first hour. In two subjects, extending the collection period for a further 30 min (120-150 min) gave little additional recovery (5% and 10%). Although the pH varied between 3.69 and 8.81, 85% of specimens were within the pH range 5-8. All patients had normal pancreatic function and Tween 20 had no significant effect on the output of trypsin. The mean output of trypsin without Tween 20 was 10.36 f 2.5 units/ml and with Tween 20 11.60 rf: 2.9 units/ml; these results were not significantly different (P > 0.1). During Tween administration there was a significant (P < 0.02) decrease in faecal fat (Fig. 1). Ten of the 13 patients had a decreased faecal fat while on Tween therapy; the greatest decrease occurred in those patients with the most severe steatorrhoea. Three of the five patients taking Tween 20 showed a decrease in faecal fat as did all but one of the eight patients taking Tween 80. Two patients (7 and 9, Table 1) had a further faecal fat measurement after Tween therapy was stopped. In both the fat excretion returned to pretreatment values, 80% and 33% of dietary fat intake respectively. Bile-salt concentrations were usually lower in the micellar phase than in total juice (78% f 19% of total juice) and only the micellar results are presented (Table 2). Three patients (2,4 and 5) had an extremely poor bile-salt response to the meal. In each, less than 350 prnol was recovered in the 2 h. In patients 1 and 3, total bile-salt output was only at the lower limit of normal. The total bile-salt output from the ten intubations averaged 820 f 780 ymol with a range of 114-2291 pmol. Bile-salt concentrations were less than 7 mmol/l in every half-hour period (Table 2); in patient no. 2 concentrations never exceeded 2 mmol/l, the probable critical micellar concentration in viva Although patients 1 , 3 , 4 and 5 had isolated periods where values were higher than 2 mmoV1, the concentrations reached were relatively low compared with Mansbach, Cohen & Leff (1975). In
only two instances did a half-hour collection have a bile-salt concentration greater than 5 mmol/l. Tween 20 had no effect on bile-salt output. The fatty acid concentrations and total fatty acid aspirated during 2 h, for the micellar phase and the whole contents, are shown in Table 3. The normal micellar fatty acid concentration after a test meal had been reported as 8-12 mmol/l by Mansbach et al. (1975) and 7.5-20 mmol/l by Badley et al. (1970). In general the concentration of fatty acid in the micellar phase after the first test meal was low. Only two values were greater than 7 mmol/l (Table 3), and only four out of 20 samples were greater than 4 mmol/l. There was a correlation between bile-salt and fatty acid concentrations ( r = 0.79 P (0.05) and there was always a large reserve of fatty acid in the oil phase. After a meal containing Tween 20 the concentrations of fatty acids recovered from the micellar phase in three patients (nos. 1, 2 and 4) were substantially greater than after the first meal. In the other two (3 and 5) they were similar, and in patient no. 5 the micellar fatty acid concentration was maintained despite a marked decrease in bile-salt concentration (Tables 2 and 3). P < 0.02
80
'\
70
-e
P
60
50
d
-t
.B 40 c -u
6(
30
20 2
5
10
C
10
(4Tween
(+)Tween
F I ~1.. Effect of Tween 20 ( 8 )or Tween 80 (H) on faecal fat in 13 patients with suspected bile-salt deficiency. The l i e s connect results from individual patients; the numbers refer to patients described in Table 1.
Bile-salt-dejicient steatorrhoea
277
TABLE2. Micellar bile-salt concentration in individual half-hour periods, and total bile output (2 h) in five subjects after test meals given without (a) or with (b) Tween 20 Bile salt concn. (mmol/l)
Patient
no.
l 2 3 4 5
a b a b a b a b a b
0-30 min
3C-60 min
60-90 min
1.9 3.2 1.6 0.3 6.5 1.3 0.2 0.3 2.0 0.2
2.0 1.6 1.0 1.8 1.4 3.8 0.5 2.4 0.9 0.3
3.3 2.3 0.6 0.8 1.5 0.9 0.6 0.6 3.2 1.0
9C-120 min
Total bilesalt output ijmol) C-120 min
3.9 5.9 1.5 1.1 0.8 0.3 0.3 0.7 1.3 Not analysed
1500 1750 250 250 2300 1150 300 300 300 100
TABLE3. Duodenal juice fatty acid concentration in the micellar phase and in whole contents of individual half-hour periods and the total fatty acid output (2 h) in five patients with bile-salt deficiency after test meals given without (a) and with (b) Tween 20 Fatty acid concn. (mmol/l)
Patient no.
a 1 b
a 2
b a 3 b
a 4 b
a 5 b
Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents
0-30 min
30-60 min
60-90 min
90-120 min
4.6 10.5 16.5 44.7 0.2 62.4 2.7 16.0 10.8 34.7 2.8 21.9 0.4 46.4 0.3 27.5 0.8 45.8 0.3 1.0
0.5 5.4 10.9 20.8 1.7 48.1 1.2 51.0 1.9 14.9 13.7 17.8 0.2 21.2 12.1 25.3 0.3 40.3 0.3 2.0
3.9 4.3 14.2 54.2 0.2 24.3 2.8 37.8 2.2 16.2 0.4 5.6 0.2 30.7 4.1 17.4 6.8 32.7 1.6 8.4
16.4 25.8 36.3 35.7 2.6 93,6 3.4 43.0 3.3 14.1 0.4 8.8 0.2 14.3 1.8 11.8 0.2 2.9
Phospholipids were recovered in the whole contents mainly as lysophosphatidylcholine. In general the micellar phospholipid concentrations in all patients were lower than the range given by Mansbach et al. (1975) in healthy controls (0.761.12 mmol/l) although in patients 1 and 3 (Table 4) two of the micellar samples were within this range. After the control test meal there was no detectable phospholipid in any of the samples of micellar phase from patients 2 and 4 (Table 4). After a Tween-supplemented test meal an increase in 19
-
Total fatty acid output (umol) 0-120 min 2350 5150 9400 23 000 300 12 750 750 8550 3300 13 100 2000 11 600 150 19 100 950 5000 250 3700 150 650
micellar phospholipid was found. The total micellar phospholipid increased after Tween treatment in four of the five patients. The molar ratio of fatty acid and of phospholipid to bile salt in the micellar phase in the presence and absence of Tween 20 is shown in Fig. 2. The ratio of fatty acid to bile salt increased in all five subjects in the presence of Tween 20 (P < 0.05). The mean ratio increased from 1.3 f 0.6 to 3-5 & 1.8. Similarly an increase in the phospholipid to bile salt ratio occurred in four out of five subjects in
R . F. G.J. King et al.
278
TABLE4. Duodenal juice phospholipid concentration in the micellar phase and in whole contents of individual half-hour periods and the total phospholipid output ( 2 h) in five patients with bile-salt deficiency after test meals given without (a)and with (b) Tween 20 Phospholipid concn. (mmol/l)
Patient no.
u
1
b u 2
b u
3
b u
4
b u
5
b
0-30 min
30-60 min
60-90 min
0.2 0.4 0.5
0.1 0.4 0.4 0.5
0.4 0.5 0.4 0.9
0
0
0
0
0.4 0.1 0.9 0.2
0.2 0.1 0.4
0.7
1.0 0.1 0.4
0.7 0.1 0.5 0.3 0.6 0.1 0.2
100 25 100 450 750 200 400
Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents Micellar phase Whole contents
0.8 0 0.4 0.1 0.2 1.6 2.2 0.3 0.5 0 0.1
0.1 0.2 0.2 0.5 0.1 0.2
0 6
(6) 005< P < O 1
05 04
02
0
1
(-)Tween
(+)Tween
Total phospholipid output 90-120 min (umol) 0-120 min
4
I (-)Tween
0.1
1.4 1.5
0.9 1.2 1.4 1.7
150 250 300 450
0
0
0
0
0.1 0.3 0.6 0.1 0.6 0.1 0.4
0.2 0.1 0.3 0.5 0.7 0.3 0.4
0.2 0.1 0.4
100 25 100 25 50 50
0 0.2
No sample No sample
100
belonging to a three component system. If the molar concentrations are expressed as percentages, as used for analysis of the constituents of bile (Admirand & Small, 1968) then it is possible to construct a graph with triangular co-ordinates. A significantly greater proportion (P< 0.01; by chi-squared) of the points from Tween-supplemented meals (11 out of 19) than from the nonsupplemented (2 out of 20) lie above the 'normal' region calculated from the data of Mansbach et al. (1975) (Fig. 3).
(+)Tween
FIG.2. Duodenal micellar fatty acid/bile salt molar ratio (a), phospholipid/bile salt molar ratio (b) after standard test meals with ( 0 ) or without ( 0 ) Tween 20 in five patients with bile-salt deficiency. The lines connect the means of all determinations from individual patients; the numbers refer to patients described in Table 1.
the presence of Tween 20 (P < 0.06), the mean ratio increasing from 0.1 f 0.1 to 0.2 f 0.1. The one subject in whom the ratio remained constant was the one with by far the highest bile-salt concentration and the only one which was initially above the critical micellar concentration. The micellar concentrations of bile salts, phospholipids and fatty acids can be considered as
Discussion
Available evidence clearly implicates the importance of the micellar phase for normal fat digestion and absorption. Although non-micellar absorption of long-chain fatty acids can occur (Blomstrand, Carlberger & Forsgren, 1969), the severity of steatorrhoea in the presence of reduced concentrations of bile salts implies that this is not quantitatively important. Thus the use of artificial detergents to alter the aqueous solubilization of fatty acids towards the micellar phase would seem to have potential for use in patients with fat malabsorption due to low bile-salt concentrations. The results obtained from faecal fat studies of our patient group show that therapy with either
Bile-salt-deficient steatorrhoea 279 five of these patients with steatorrhoea was characterized by low concentrations of fatty acid due to a deficiency in bile salts. The total bile-salt concentration in aspirated duodenal fluid was lower than the optimum of 8-12 mmol/l (Poley & Hofmann, 1976). The aqueous concentration was even lower, probably due to non-specific adsorption of bile salt to suspended food particles, as has been shown to occur in normal adults (Mansbach et al., 1975) and in patients with intestinal resection (Poley & Hofmann, 1976), or liver disease (Badley et al., 1970). In many samples from our subjects the bile-salt concentrations were lower than the criticaI micell& concentration of 2 mmolfl (Poley & Hofmann, 1976). Even in those patients in whom some bile-salt concentrations FIG.3. Molar concentrations of micellar bile salts, were at or near the critical micellar concentration phospholipids and fatty acids, after test meals in five patients with bile salt deficiency plotted on triangular co(patients 1, 3 and 5, Table 2) the quantity of ordinates. Points represent individual half-hour periods micelles may be insufficient throughout the day to with ( 0 ) or without ( 0 )Tween 20 administration. The solubilize a standard dietary lipid load. This may shaded area represents the normal range calculated from result in steatorrhoea and confirms the work of the data of Mansbach et al. (1975). Badley et al. (1970), who suggested that a mean conjugated bile-salt concentration of greater than 4 mmol/l is required to provide an adequate number of micelles. Tween 80 or Tween 20 confers a significant degree Tween as a non-ionic detergent would be of improvement of steatorrhoea (Fig. 1). Although expected to disperse in aqueous solution to form the detergent-supplemented period always followed micelles, which could be expanded by lipid the control period by design, two patients were molecules. In four of the five patients Tween 20 taken into a third control period where a supplementation caused a substantial increase in corresponding worsening of steatorrhoea was seen. both micellar fatty acid and phospholipid concenIt was encouraging that the patients deserving the trations. The effect was particularly manifested at greatest reduction in their output of faecal fat were low bile-salt concentrations. It cannot be assumed, precisely those in whom Tween therapy was most however, that Tween micelles, presumably different successful. in size and shape from bile-salt micelles, would The therapeutic dose of Tween used in this necessarily function to improve fat absorption. present study was based on previous reports of its However, steatorrhoea did improve in 10 out of 13 use in human subjects (Boyd & Helfrick, 1951; patients and in those with the severest steatorrhoea Dubois et al., 1964; Jones et al., 1948). It is not the improvement was greatest. This suggests that known if this dose of 6 g/day is sufficient to Tween 20 micelles can substitute, to some extent, produce a maximal reduction in faecal fat or if for the physiological bile-salt molecules in fat larger doses of the detergent would have improved absorption. From these studies it is not clear the steatorrhoea still further. Both Tween 20 and whether mixed micelles containing both Tween and Tween 80 are completely hydrolysed in the bile salts can be formed. intestine, the polyoxyethylene component remainThe fatty acid concentration in the oil phase was ing unabsorbed and excreted in the faeces while the always in excess of that in the micellar phase and it fatty acid moiety is available for absorption along therefore seems unlikely that the low concenwith dietary fat (Elworthy & Treon, 1967). trations of fatty acids were due to previous Released fatty acid may contribute to faecal fat if it absorption, or that availability of fatty acid was a is unabsorbed but at the Tween dose used here factor limiting the micellar concentration of fatty would add only approx. 1% to that derived from acids. Tween 20 and Tween 80 have been reported dietary intake. Thus the fatty acid content of the to inhibit pancreatic lipase (Minnard, 1953) in administered detergent will not influence the intervitro, but the inhibition is reversed by the presence pretation of fat absorption. of bile salts. It is not known if such inhibition The aqueous phase of the duodenal contents of
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occurs in vim, though the fact that total free fatty acid concenGation is increased in the presence of Tween 20 (Fig. 3) would suggest that such inhibition, if present, did not significantly influence the results of the present study. There is evidence that phospholipid depletion in the gut of experimental animals causes changes in the mucosal cells and impairs fat absorption (O'Doherty, Yousef & Kukis, 1974). These experiments showed that biliary diversion in the rat lowers the activity of acylglycerol transferases and causes retention of dietary triglyceride in the intestinal mucosa. Although the results of Rodgers (1975) conflict with this view, this may be because the experiments were of too short a duration to detect the effect. The micellar phase in our untreated patients usually contained a low concentration of phospholipids and in some untreated patients intestinal fluid contained no micellar phospholipid despite a reserve in the oil phase. It has not been previously recognized that patients with bile-salt depletion may have a deficiency of micellar phospholipid supplied to the mucosa and this may be a factor in decreasing fat absorption. The redistribution of phospholipid from oil to the aqueous phase seen in patients on Tween (Fig. 3) may thus be of importance. It may give even more benefit to supplement some patients with both Tween and phosphatidylcholine. The high ratio of lysophosphatidylcholine in the micellar phase is in agreement with that found by Mansbach et al. (1975) in normal volunteers. Phosphatidylcholine would be digested in the lumen by pancreatic phospholipase. The question of whether phospholipids penetrate the mucosal cells during absorption has not been resolved. Our evidence suggests that lysophosphatidylcholine would predominate if both the fatty acid and phospholipid contents of micelles entered simultaneously. In a scheme put forward by Harrison & Leat (197S), however, phosphatidylcholine directly enters the mucosal cells alone. Despite the difficulty of extrapolating to the practical clinical situation, it seems possible that the increased fat absorption obtained by the use of artificial detergents in patients with bile-salt deficiency may prove of therapeutic value. Our data do not permit an evaluation of the effects of Tween on fat-soluble vitamin absorption but improvement in fat absorption might well improve this too and furthermore may provide a solution to the problem of maintaining a micellar supply of the essential long-chain fatty acids, liioleic and arachidonic, in such patients.
Acknowledgments
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