Absorption of short-chain fatty acids in human stomach and rectum

NUTRITION RESEARCH, Vol. 11, pp. 841-847,1991 0271-5317/91 $3.00 + .00 Printed in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.

ABSORPTION OF SHORT-CHAIN FATTY ACIDS IN HUMAN STOMACH AND RECTUM

David R. Saunders, M.D., F.R.C.P.(C). Department of Medicine, University of Washington, Seattle, USA, and Dunn Clinical Nutrition Unit, Cambridge, UK

ABSTRACT

Increased interest in the metabolism and therapeutic potential of short-chain fatty acids (SCFA) prompted this study of the absorption rates of acetate, propionate, and butyrate in the human rectosigmoid, and in the human stomach. In human rectosigmoid, all SCFA were absorbed from neutral infusates, but propionate and butyrate absorption exceeded that of acetate. In human stomach, acetate was absorbed in the protonated form only, and butyrate was absorbed at a faster rate than acetate. These observations are compatible with both popular models of SCFA absorption: non-ionic diffusion, and carrier-mediated anionic exchange. KEY WORDS - SCFAAbsorption Human Stomach and Rectum INTRODUCTION

Short-chain fatty acids (SCFA), mainly acetic, propionic, and butyric are the principal metabolites of anaerobic microbial fermentation of carbohydrate in the lumen of the large intestine. SCFAare the major anions in fecal water. There is increasing interest in the absorption and metabolism of SCFA especially since they are an important energy source for colonocytes (I,2). They may restore inflamed colonic epithelium which has been deprived of luminal SCFA by being surgically separated from the fecal stream (3). In the proximal colon of the guinea pig, transport rates for the 3 SCFA are similar: about half of SCFAare transported in the ionized form. On the other hand, in the distal guinea pig colon, transport rates increase with chain length and transport of SCFA is mainly as unionized acids (4). Data on the relative rates of SCFA absorption in humans are conflicting. Dawsonand his colleagues described a rank order of butyrate > propionate > acetate for absorption rates when a mixture of SCFAwas infused into a surgically isolated segment of colon (5). An experiment in which the entire human colon was infused, however, indicated that absorption of acetate, propionate, and butyrate were similar (6) as did an experiment in which SCFA in dialysis bags were presented to the human rectum (7). The aims of the present investigation were to assess relative rates of SCFA absorption in two readily accessible sites in the human gut: rectum and stomach. In the stomach, one might expect that SCFA, whose pKas are 4-5, would be better absorbed in their unionized form. Butyric acid should be absorbed more rapidly than acetic because unionized butyric would be more l i p o p h i l i c than unionized acetic, and i t would be, therefore, more readily transported into l i p i d membranes of gastric epithelium. 841

842

D.R. SAUNDERS MATERIALAND METHODS

Protocols were approved by the Human Subjects Review Committee of the University of Washington in May of 1989, and again in May of 1990. Patients who were being screened for colonic neoplasia were invited to participate and to sign consent before their colonoscopy. They had been prepared for colonoscopy by being purged with magnesium citrate 2 d before, by ingesting only clear liquids I d before, and by drinking 4 L of a poorly-absorbed electrolyte solution (6olytely, Braintree Laboratories, Braintree, MA 02184) 18 h before. The colonoscopist examined the large intestine to the cecum. Visualization of the mucosa was maximized by washing and by aspirating residual luminal contents. SCFA absorption was undertaken in those patients who had a normal rectosigmoid, and who continued to consent to the experiment. Experiments in the rectosigmoid were conducted by inserting a plastic tube (OD 5mm) for 10 cm into the rectum. Any residual fluid in the rectum was aspirated before i n s t i l l i n g 100 ml of SCFA solution, pH 7 (4 mmols each of sodium acetate, sodium propionate, and sodium butyrate plus 4 mg of phenol red). Rectal contents were mixed every min by withdrawing and reinfusing 20 ml of fluid for 30 min when the rectum was emptied. Two sets of experiments were conducted in the human stomach. In the f i r s t set, the absorption of a single SCFA, acetate, was measured when i t was presented in its ionized, or unionized form. An H2-receptor antagonist was used to block gastric acid secretion so that meals of sodium acetate would not be acidified; ranitidine, 150 mg (Glaxo Pharmaceutical NC 27709) was ingested at 06:30. Two hours later, a tube, 3 mm OD, with a tungsten weighted tip (Superior Feeding Tube, Healthcare Group, Inc., Cumber]and, RI 02864) was swallowed. The end of the tube was positioned in a dependent portion of the stomach by using.a water recovery test (8). One of the two test meals was selected by flipping a coln, and i t was infused over 5 min. Thereafter, gastric contents were mixed each min by withdrawing and reinfusing 50 ml portions. Twenty min later, gastric contents were aspirated. The second test meal was infused after a respite of 30 min. The acidic test meal, pH 2.8, consisted of 50 mmols of acetic acid in 500 ml of distilled water. The neutral test meal consisted of 50 mmols of sodium acetate in 500 ml of water. Both meals contained 20 mg of phenol red as a poorly absorbed, water-soluble marker, and sodium chloride so that calculated osmolality was 300 mosmols per Kg. In the second set of experiments, rates of gastric absorption of acetic, propionic, and butyric acids were compared. Gastric acid secretion was not blocked with ranitidine because only acidic test meals were infused (20 rmols of acetic acid, 20 mmols of propionic acid, and 20 mmols of butyric acid in 500 ml of distilled water containing 4 mg of phenol red). A single meal was given on each of 4 days. Gastric contents were mixed for 20 min before being withdrawn. Analytic Techniques Acetic, propionic, and butyric acids were measured in colonic infusates and effluents, and in gastric meals and aspirates by using isobutyric acid as the internal standard. 0.2 ml of sample were mixed with 5 ml of 1.6 mM isobutyric acid in water: portions of this mixture were acidified with 10 mM HCl. Then 1 ul of the acidified mixtures was injected onto a c a p i l l a r y column (15 m x 0.53 mm coated with DB wax, J and W S c i e n t i f i c , Folsom, CA 95630) in a Hewlett-Packard 5890 gas chromatogram with flame ionization detector. The coefficients of variation of

FATTY ACID ABSORPTION

843

measuring the 3 SCFA by ratios of peak height were less than 5%. Acetate, in those experiments in which only acetate was infused into the stomach, was measured by mixing 0.2 ml of test meals and of gastric aspirates with 3 ml of an internal standard (2.7 mM propionic acid in 0.25 N sodium hydroxide). Propionic acid was an appropriate internal standard because i t was present only in trace amount in gastric aspirates. Portions of the mixture were acidified with equal volumes of 1M phosphoric acid so that acetic and propionic could be quantified by gas liquid chromatography on a 1.8 m glass column, ID 2 mm, packed with Tenax (Pye Unicam, Cambridge, UK) in a Pye Unicam Series 204 with flame ionization detector. Column temperature was 110 C with a flow rate of nitrogen of 40 ml per min. The coefficient of variation of measuring acetate in 6 samples of 100 mH acetic acid was 2.8% using ratios of peak height. Phenol red in alkalinized mixtures of SCFAwas measured spectrophotometrically at 560 hR. Calculations The fraction of SCFAabsorbed is given by i -

[SCFA] out

x

[SCFA] in

[PR] in [PR] out

were [PR~ in is the concentration of phenol red in the test meal, or infusate, and [P~ out is the concentration of phenol red in the gastric aspirates, or rectal effluents.

Statistical Methods Paired t tests were performed with a Hewlett-Packard 41CV c a l c u l a t o r . P values of < 0.01 were accepted as i n d i c a t i n g a s i g n i f i c a n t difference when 3 groups of data were being compared with each other. Results are expressed as means • SD. RESULTS Absorption from Human Rectum SCFA remained ionized in t h i s experiment where i n i t i a l and f i n a l pH of infusates and e f f l u e n t s was 7. Butyrate was absorbed more r a p i d l y than acetate ( t = 6.g3; p < 0,01), and propionate was absorbed more r a p i d l y than acetate ( t = 5.81~ p < 0.01) when a mixture of these three SCFA were i n s t i l l e d into the rectum (Table 1). The comparison between proptonate and butyrate did not reach s t a t i s t i c a l significance ( t = 3.37: p ~ 0.01).

844

D.R. SAUNDERS TABLE I Absorption of SCFA by Human Rectum * Subject

Ac (% of

Pr administered

Bu dose)

I

17

21

24

II

29

37

40

III

51

56

58

IV

12

14

17

V

22

26

34

VI

18

23

24

* 100 ml, pH 7.4, of 40 mM sodium acetate, 40 mM sodium propionate, and 40 mM sodium butyrate were i n s t i l l e d into the rectum after colonoscopyt rectal contents were aspirated 30 min l a t e r . Subject I did not have a colonoscopy, but he was cleansed with 4 L of a poorly absorbed electrolyte solution. Absorption from Human Stomach

Only protonated acetate was absorbed from the stomach when acetate alone was fed. Ranitidine prevented the a c i d i f i c a t i o n of meals of sodium acetate so that v i r t u a l l y a l l of the acetate remained ionized (Table 2). No absorption of acetate was detected ( -2 + 2%) when sodium acetate was fed (Table 2) whereas 16 + 2% was absorbed when acetic acid was fed ( t = 18.51: p < 0.01).

FATTY ACID ABSORPTION

845

TABLE I I Effect of pH on Acetate Absorption by Human Stomach*

Experiment

Meal

p_H

Acetate Absorbed

initial

final

(% of

dose)

1

7.5 2.9

7.0 3.2

3 17

2

7.6 2.9

6.9 3.2

3 12

3

7.5 2.8

7.2 3.3

4 13

4

7.5 2.8

6.8 3.1

3 18

5

7.2 2.8

6.9 3.0

1 16

6

7.4 2.7

6.7 3.0

1 17

* Six experiments were performed in Subject I. The order of the two meals, I00 mM sodium acetate, pH 7, or 100 mM acetic acid, pH 3, was selected by flipping a coin. Meals were infused over 5 min and gastric contents were withdrawn ZO min later. Butyric was absorbed at a faster rate than acetic ( t = 10.83: p < 0.01) when a mixture of acetic, propionic, and butyric was infused at a pH of 2.8 (Table 3). Propionic may have been absorbed more rapidly than acetic, but the comparison failed to reach s t a t i s t i c a l significance ( t = 4.16: p > 0.01).

846

D.R. SAUNDERS TABLE I I l Absorption of SCFA by Human Stomach* Experiment

Ac Pr Bu (% of administered dose)

1

10

13

19

2

15

24

22

3

16

20

26

4

14

20

25

* Meals of 500 ml, pH 2.8, 40 mM acetic, 40 mM propionic, and 40 mM butyric were infused over 5 min on 4 occasions into Subject I. Gastric contents were removed 20 min later. DISCUSSION The human rectosigmoid, after colonoscopy, was thought to be ideal for examining the relative rates of SCFA absorption because endogenous SCFA had been eliminated. Patients undergo intensive purging, and the colonoscopist aspirates much of the remaining luminal fluid as the colonic mucosa is inspected. We found that concentrations of SCFA in residual rectal fluid were less than 3 mMcompared with more than 100 mM in normal fecal water (g). The major drawback of this experimental model of absorption is that the absorptive surface area is unknown. Absolute absorption rates, therefore, are unknown, but relative rates of absorption of mixtures of probe molecules should be accurate. SCFAwere introduced at pH 7 into the rectosigmoid so that they existed in their ionized forms in lumen contents. The fact that butyrate and propionate were absorbed more rapidly than acetate is compatible with both popular models of SCFA absorption. Non-ionic diffusion, which occurs in guinea pig distal colon (4) would favor more lipophilic butyrate over acetate. A carrier-mediated bicarbonate exchange process, which has been described in apical membrane vesicles of rat distal colon (10) might favor butyrate i f the carrier had greater a f f i n i t y for but~rcate than acetate. The acidic environment of the human stomach offered a unique opportunity to study the absorption of unionized SCFA. Pilot experiments revealed that acetate absorption increased as gastric pH decreased when sodium acetate, pH 7, was i n s t i l l e d into the fasting stomach and serial samples of gastric contents were withdrawn over 10 min. Theseobservations indicated that the stomach might be impermeable to acetic acid (pKa 4.76) in i t s ionized form. Ranitidine, 150 mg, prevented acidification of the test meal and under these conditions no gastric absorption of acetate was detected (Table 2). Butyric acid was absorbed more rapidly than acetic acid when the relative rates of absorption of acetic, propionic, and butyric acids were compared (Table 3). This result is consistent with the idea that the rapidity of SCFA absorption is related to the length of the carbon chain which determines s o l u b i l i t y in l i p i d epithelial membranes (5).

FATTY ACID ABSORPTION

847

We did not contemplate infusing acidic solutions (pH 2.8) into the human rectum. Future experiments should examine interrelationships among absorption rates of SCFA in human colonic absorptive cell membranes. ACKNOWLEDGEMENTS

The author thanks Dr. Oohn Cummings of the Dunn Clinical Nutrition Unit for his hospitality and help during a short sabbatical leave, Dr. Irena King for coaxing the N-P 5890 to behave, and Dr. Ed Lipkin for criticizing the manuscript.

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2)

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