Experimental Physiology (1991), 76, 615-618 Printed in Great Britain
EFFECT OF SHORT-CHAIN FATTY ACIDS ON CALCIUM ABSORPTION BY THE RAT COLON T. LUTZ AND E. SCHARRER Institute of Veterinary Physiology, University of Zurich, Winterthurerstrasse 260, 8057 ZIurich, Switzerland (MANUSCRIPT RECEIVED 5 APRIL 1991, ACCEPTED 8 APRIL 1991) SUMMARY
An in vivo luminal perfusion technique was used to investigate whether short-chain fatty acids influence the absorption of Ca by the rat colon. Na and water absorption were also determined. In the distal colon, acetate and butyrate caused a significant increase in Ca absorption, while the absorption of Na and water were not affected. In the proximal colon, butyrate did not influence Ca absorption, but significantly enhanced Na and water absorption. These results are in part consistent with the presence of a Ca-H exchanger in the apical membrane of the distal colon mediating Ca uptake into the epithelial cell. INTRODUCTION
Calcium is actively absorbed by the proximal small intestine and by the large intestine (Karbach & Rummel, 1987; van Os, 1987). Active Ca absorption at both intestinal sites is stimulated by vitamin D (Favus & Angeid-Backman, 1985; van Os, 1987; Karbach, 1989), but whether the mechanism of absorption is the same in the small and large intestine is not known. Ca enters the epithelial cell during absorption along a steep electrochemical gradient across the luminal membrane of the epithelial cell and is extruded across the antiluminal membrane by a Ca-ATPase and a Ca-Na exchanger which is driven by the Na gradient (van Os, 1987). Since short-chain fatty acids (SCFAs; acetate, propionate and butyrate), derived from microbial fermentation of carbohydrates in the large intestine, constitute the major anions in hindgut contents, we investigated whether SCFAs affect Ca absorption by the colon. Na and water absorption were also studied. An in vivo luminal perfusion technique was used. METHODS
The distal colon and the proximal colon of adult male rats (mean body wt, 250 g) were luminally perfused (perfusion rate 0-5 ml min-1) under anaesthesia (xylazine-HCl, 10 mg kg-1 and ketamine-HCl, 50 mg kg-1, I.M.) and luminal disappearance of Ca was determined. The buffer solutions contained Phenol Red as an unabsorbable marker for measuring water absorption (Petith & Schedl, 1978). The composition of the buffers (pH 6-0) was as follows (mmol 1-1): CaCl2, 1; KCl, 5; NaHCO3, 20; NaH2PO4, 30; NaCl, 90; lactic acid, 5. In the experimental solution, 60 or 30 mmol 1-1 NaCl were replaced by 60 or 30 mmol 1-1 sodium acetate or sodium butyrate. Absorption rates were determined following a 30 min equilibration period. Ca was determined either photometrically with a test kit (No. 0714534, Hoffmann-La Roche, Basel) or by measuring 45Ca activity. In this case, the buffer solutions contained 20 pCi 1-1 45Ca (45CaC12, Amersham International, England); 45Ca was measured in a,-counter (Kontron Instruments).
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T. LUTZ AND E. SCHARRER
The concentration of Na was determined with a flame photometer. The results are presented as means_S.E.M. Differences between means were statistically evaluated using Student's t test and when more than two means were compared, an ANOVA with the post hoc test of Bonferroni was used for statistical evaluation of differences between means. RESULTS
In the distal colon (Table 1) absorption of Ca calculated from disappearance of 45Ca was enhanced to the same extent by 60 mmol 1-1 acetate or butyrate. At a concentration of 30 mmol 1-1, only butyrate stimulated Ca absorption significantly. The stimulating effect of 60 mmol 1-1 SCFA was somewhat greater than that of 30 mmol 1-1 SCFA. Na and water absorption in the distal colon were not affected by acetate or butyrate. In an additional experiment, in which Ca was determined photometrically, 60 mmol 1-1 butyrate produced a stimulation of Ca absorption (P < 0-05) similar to that in the previous experiment (control vs. experimental value, 136±0*50 vs. 2*77±0-48 jumol (100 mg dry wt)-l (30 min)-'; n= 1 3). In the proximal colon (Table 2) 60 mmol 1-1 butyrate did not affect Ca absorption under identical conditions, but caused a significant enhancement of Na and water absorption. In view of the lack of effect of 60 mmol l-l butyrate on Ca absorption, the effect of 30 mmol 1-1 butyrate was not tested in the proximal colon.
Table 1. Influence of different concentrations of acetate or butyrate on electrolyte absorption in the distal colon Absorption expressed as ,mol (100 mg dry wt)-1 (30 min)-I Plus 60 mmol 1-1 SCFA
Control (n=6)
Calcium Sodium Water t
1*09±0*12 98±11 740±83
Acetate (n=7)
2.12+0-22** 122±15 870±119
Butyrate (n=7) 2-13±0.11** 112±8 853±44
Plus 30 mmol 1-1 SCFA
Control
(n=9) Calcium Sodium Water t
1-04±0*12 98±11 682±75
Acetate
(n=8)
1*60±0*17 107±15 766±111
Significantly different from control, ** P < 0*01 .
t gl (100 mg dry wt)-I (30 min)-I.
Butyrate
(n=9) 1-82±0-22** 108±8 734±53
617
CALCIUM ABSORPTION BY THE COLON
Table 2. Influence of 60 mmol 1-1 butyrate on electrolyte absorption in the proximal colon Absorption expressed as gmol (100 mg dry wt)-1 (30 min)-1
Calcium Sodium Water t
Control (n=10)
Plus 60 mmol 1-I butyrate (n=10)
1*49±O016 153±7 977±56
1*48±0-37 228±14*** 1230±87*
Significantly different from control, *** P < 0 *0001; * P < 0.05. t pi (100 mg dry wt)-1 (30 min)-1. DISCUSSION
The results presented show for the first time that acetate and butyrate stimulate Ca absorption in the distal colon without influencing Na and water absorption. In the proximal colon, Na and water absorption, but not Ca absorption, were enhanced by butyrate. A stimulatory effect of SCFAs on Na absorption in the proximal colon has been observed previously (Lutz, Wiirmli & Scharrer, 1991). SCFAs seem to stimulate Na absorption by their effect on the Na-H exchanger located in the apical membrane of the colon (Argenzio, Miller & Engelhardt, 1975; Lutz et al. 1991). SCFAs are mainly present in their dissociated form in the colonic lumen (Argenzio et al. 1975) and have to be protonated prior to absorption since they are predominantly absorbed in their undissociated form (Rechkemmer, Ronnau & Engelhardt, 1988). After diffusion into the epithelial cell, the SCFAs dissociate immediately because of the prevailing intracellular pH of about 7-0 (pKa of SCFAs-4.8). Thus, protons are liberated and can be secreted in exchange for Na so stimulating Na absorption in the proximal colon (Scharrer & Lutz, 1990; Lutz et al. 1991). Since Na absorption in the distal colon was not stimulated by SCFAs, a Na-H exchanger seems not to be operative under the present experimental conditions. By analogy with Na absorption by a Na-H exchanger in the proximal colon, a Ca-H exchanger located in the apical membrane of the epithelium might be involved in Ca absorption by the distal colon. Since, due to its higher lipid solubility, butyric acid enters the epithelial cell faster than acetic acid (Rechkemmer et al. 1988) and therefore provides more protons, butyrate should stimulate Ca absorption more than acetate if a Ca-H exchanger is involved. Such a trend occurred when the perfusion fluid contained 30 mmol 1-1 SCFA but not when it contained 60 mmol 1-1. At first glance this poor relationship between chain length of SCFAs and their stimulatory effect on Ca absorption argues against a Ca-H exchanger being operative in Ca absorption. It is however possible that the protons, released by SCFAs, in addition reduce the affinity of the Ca-ATPase of the antiluminal membrane for intracellular Ca and thus inhibit Ca extrusion. Such an effect was shown in red blood cells (Gassner, Luterbacher, Schatzmann & Wuthrich, 1988). This inhibition might partially counteract the stimulating effect of SCFAs on Ca absorption by their impact on
618
T. LUTZ AND E. SCHARRER
the apical Ca-H exchanger. With this model, the almost equipotent stimulating effect of acetate and butyrate could be explained: butyrate as the more efficient proton donator would be more potent than acetate in stimulating the Ca-H exchanger. The inhibitory effect of butyrate on the basolateral Ca-ATPase would also be greater than that of acetate. The net effect could be a nearly equipotent stimulation of Ca absorption by butyrate and acetate. A Ca-H exchanger could also be involved in Ca absorption in the proximal colon. A lower activity of this exchanger in the proximal colon compared with that in the distal colon might just be sufficient to overcome the inhibitory effect of intracellular protons delivered by butyric acid on the antiluminal Ca-ATPase, resulting in no net effect of SCFAs on Ca absorption. It seems unlikely that the effect of SCFAs on Ca absorption is due to an increase in the energy supply to colonocytes, since there was no stimulation of Na absorption by SCFAs in the distal colon, a process which is known to be similarly energy-dependent. REFERENCES ARGENZIO, R. A., MILLER, N. & ENGELHARDT, W. VON (1975). Effect of volatile fatty acids on water and ion absorption from the goat colon. American Journal of Physiology 229, 997-1002. FAVUS, M. J. & ANGEID-BACKMAN, E. (1985). Effects of 1,25(OH)2D3 and calcium channel blockers on cecal calcium transport in the rat. American Journal of Physiology 248, G676-681. GASSNER, B., LUTERBACHER, S., SCHATZMANN, H. J. & WUTHRICH, A. (1988). Dependence of the red cell calcium pump on the membrane potential. Cell Calcium 9, 95-103. KARBACH, U. (1989). Cellular-mediated and diffusive magnesium transport across the descending colon of the rat. Gastroenterology 96, 1282-1289. KARBACH, U. & RUMMEL, W. (1987). Calcium transport across the colon ascendens and the influence of 1,25-dihydroxyvitamin D3 and dexamethasone. European Journal of Clinical Investigation 17, 368-374. LUTZ, T., WURMLI, R. & SCHARRER, E. (1991). Short chain fatty acids stimulate magnesium absorption by the colon. In Magnesium - a Relevant Ion? Proceedings of the 3rd European Congress on Magnesium, ed. LASSERRE, B. & DURLACH, J., pp. 131-137. John Libbey, London. PETITH, M. M. & SCHEDL, H. P. (1978). Effects of magnesium deficiency on duodenal and ileal magnesium absorption and secretion. American Journal of Digestive Diseases 23, 1-5. RECHKEMMER, G., RONNAU, K. & ENGELHARDT, W. V. (1988). Fermentation of polysaccharides and absorption of short chain fatty acids in the mammalian hindgut. Comparative Biochemistry and Physiology 90A, 563-568. SCHARRER, E. & LUTZ, T. (1990). Effects of short chain fatty acids and K on absorption of Mg and other cations by the colon and caecum. Zeitschriftfar Erndhrungswissenschaft 29, 162-168. VAN OS, C. H. (1987). Transcellular calcium transport in intestinal and renal epithelial cells. Biochimica et Biophysica Acta 906, 195-222.
EFFECT OF SHORT-CHAIN FATTY ACIDS ON CALCIUM ABSORPTION BY THE RAT COLON T. LUTZ AND E. SCHARRER Institute of Veterinary Physiology, University of Zurich, Winterthurerstrasse 260, 8057 ZIurich, Switzerland (MANUSCRIPT RECEIVED 5 APRIL 1991, ACCEPTED 8 APRIL 1991) SUMMARY
An in vivo luminal perfusion technique was used to investigate whether short-chain fatty acids influence the absorption of Ca by the rat colon. Na and water absorption were also determined. In the distal colon, acetate and butyrate caused a significant increase in Ca absorption, while the absorption of Na and water were not affected. In the proximal colon, butyrate did not influence Ca absorption, but significantly enhanced Na and water absorption. These results are in part consistent with the presence of a Ca-H exchanger in the apical membrane of the distal colon mediating Ca uptake into the epithelial cell. INTRODUCTION
Calcium is actively absorbed by the proximal small intestine and by the large intestine (Karbach & Rummel, 1987; van Os, 1987). Active Ca absorption at both intestinal sites is stimulated by vitamin D (Favus & Angeid-Backman, 1985; van Os, 1987; Karbach, 1989), but whether the mechanism of absorption is the same in the small and large intestine is not known. Ca enters the epithelial cell during absorption along a steep electrochemical gradient across the luminal membrane of the epithelial cell and is extruded across the antiluminal membrane by a Ca-ATPase and a Ca-Na exchanger which is driven by the Na gradient (van Os, 1987). Since short-chain fatty acids (SCFAs; acetate, propionate and butyrate), derived from microbial fermentation of carbohydrates in the large intestine, constitute the major anions in hindgut contents, we investigated whether SCFAs affect Ca absorption by the colon. Na and water absorption were also studied. An in vivo luminal perfusion technique was used. METHODS
The distal colon and the proximal colon of adult male rats (mean body wt, 250 g) were luminally perfused (perfusion rate 0-5 ml min-1) under anaesthesia (xylazine-HCl, 10 mg kg-1 and ketamine-HCl, 50 mg kg-1, I.M.) and luminal disappearance of Ca was determined. The buffer solutions contained Phenol Red as an unabsorbable marker for measuring water absorption (Petith & Schedl, 1978). The composition of the buffers (pH 6-0) was as follows (mmol 1-1): CaCl2, 1; KCl, 5; NaHCO3, 20; NaH2PO4, 30; NaCl, 90; lactic acid, 5. In the experimental solution, 60 or 30 mmol 1-1 NaCl were replaced by 60 or 30 mmol 1-1 sodium acetate or sodium butyrate. Absorption rates were determined following a 30 min equilibration period. Ca was determined either photometrically with a test kit (No. 0714534, Hoffmann-La Roche, Basel) or by measuring 45Ca activity. In this case, the buffer solutions contained 20 pCi 1-1 45Ca (45CaC12, Amersham International, England); 45Ca was measured in a,-counter (Kontron Instruments).
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T. LUTZ AND E. SCHARRER
The concentration of Na was determined with a flame photometer. The results are presented as means_S.E.M. Differences between means were statistically evaluated using Student's t test and when more than two means were compared, an ANOVA with the post hoc test of Bonferroni was used for statistical evaluation of differences between means. RESULTS
In the distal colon (Table 1) absorption of Ca calculated from disappearance of 45Ca was enhanced to the same extent by 60 mmol 1-1 acetate or butyrate. At a concentration of 30 mmol 1-1, only butyrate stimulated Ca absorption significantly. The stimulating effect of 60 mmol 1-1 SCFA was somewhat greater than that of 30 mmol 1-1 SCFA. Na and water absorption in the distal colon were not affected by acetate or butyrate. In an additional experiment, in which Ca was determined photometrically, 60 mmol 1-1 butyrate produced a stimulation of Ca absorption (P < 0-05) similar to that in the previous experiment (control vs. experimental value, 136±0*50 vs. 2*77±0-48 jumol (100 mg dry wt)-l (30 min)-'; n= 1 3). In the proximal colon (Table 2) 60 mmol 1-1 butyrate did not affect Ca absorption under identical conditions, but caused a significant enhancement of Na and water absorption. In view of the lack of effect of 60 mmol l-l butyrate on Ca absorption, the effect of 30 mmol 1-1 butyrate was not tested in the proximal colon.
Table 1. Influence of different concentrations of acetate or butyrate on electrolyte absorption in the distal colon Absorption expressed as ,mol (100 mg dry wt)-1 (30 min)-I Plus 60 mmol 1-1 SCFA
Control (n=6)
Calcium Sodium Water t
1*09±0*12 98±11 740±83
Acetate (n=7)
2.12+0-22** 122±15 870±119
Butyrate (n=7) 2-13±0.11** 112±8 853±44
Plus 30 mmol 1-1 SCFA
Control
(n=9) Calcium Sodium Water t
1-04±0*12 98±11 682±75
Acetate
(n=8)
1*60±0*17 107±15 766±111
Significantly different from control, ** P < 0*01 .
t gl (100 mg dry wt)-I (30 min)-I.
Butyrate
(n=9) 1-82±0-22** 108±8 734±53
617
CALCIUM ABSORPTION BY THE COLON
Table 2. Influence of 60 mmol 1-1 butyrate on electrolyte absorption in the proximal colon Absorption expressed as gmol (100 mg dry wt)-1 (30 min)-1
Calcium Sodium Water t
Control (n=10)
Plus 60 mmol 1-I butyrate (n=10)
1*49±O016 153±7 977±56
1*48±0-37 228±14*** 1230±87*
Significantly different from control, *** P < 0 *0001; * P < 0.05. t pi (100 mg dry wt)-1 (30 min)-1. DISCUSSION
The results presented show for the first time that acetate and butyrate stimulate Ca absorption in the distal colon without influencing Na and water absorption. In the proximal colon, Na and water absorption, but not Ca absorption, were enhanced by butyrate. A stimulatory effect of SCFAs on Na absorption in the proximal colon has been observed previously (Lutz, Wiirmli & Scharrer, 1991). SCFAs seem to stimulate Na absorption by their effect on the Na-H exchanger located in the apical membrane of the colon (Argenzio, Miller & Engelhardt, 1975; Lutz et al. 1991). SCFAs are mainly present in their dissociated form in the colonic lumen (Argenzio et al. 1975) and have to be protonated prior to absorption since they are predominantly absorbed in their undissociated form (Rechkemmer, Ronnau & Engelhardt, 1988). After diffusion into the epithelial cell, the SCFAs dissociate immediately because of the prevailing intracellular pH of about 7-0 (pKa of SCFAs-4.8). Thus, protons are liberated and can be secreted in exchange for Na so stimulating Na absorption in the proximal colon (Scharrer & Lutz, 1990; Lutz et al. 1991). Since Na absorption in the distal colon was not stimulated by SCFAs, a Na-H exchanger seems not to be operative under the present experimental conditions. By analogy with Na absorption by a Na-H exchanger in the proximal colon, a Ca-H exchanger located in the apical membrane of the epithelium might be involved in Ca absorption by the distal colon. Since, due to its higher lipid solubility, butyric acid enters the epithelial cell faster than acetic acid (Rechkemmer et al. 1988) and therefore provides more protons, butyrate should stimulate Ca absorption more than acetate if a Ca-H exchanger is involved. Such a trend occurred when the perfusion fluid contained 30 mmol 1-1 SCFA but not when it contained 60 mmol 1-1. At first glance this poor relationship between chain length of SCFAs and their stimulatory effect on Ca absorption argues against a Ca-H exchanger being operative in Ca absorption. It is however possible that the protons, released by SCFAs, in addition reduce the affinity of the Ca-ATPase of the antiluminal membrane for intracellular Ca and thus inhibit Ca extrusion. Such an effect was shown in red blood cells (Gassner, Luterbacher, Schatzmann & Wuthrich, 1988). This inhibition might partially counteract the stimulating effect of SCFAs on Ca absorption by their impact on
618
T. LUTZ AND E. SCHARRER
the apical Ca-H exchanger. With this model, the almost equipotent stimulating effect of acetate and butyrate could be explained: butyrate as the more efficient proton donator would be more potent than acetate in stimulating the Ca-H exchanger. The inhibitory effect of butyrate on the basolateral Ca-ATPase would also be greater than that of acetate. The net effect could be a nearly equipotent stimulation of Ca absorption by butyrate and acetate. A Ca-H exchanger could also be involved in Ca absorption in the proximal colon. A lower activity of this exchanger in the proximal colon compared with that in the distal colon might just be sufficient to overcome the inhibitory effect of intracellular protons delivered by butyric acid on the antiluminal Ca-ATPase, resulting in no net effect of SCFAs on Ca absorption. It seems unlikely that the effect of SCFAs on Ca absorption is due to an increase in the energy supply to colonocytes, since there was no stimulation of Na absorption by SCFAs in the distal colon, a process which is known to be similarly energy-dependent. REFERENCES ARGENZIO, R. A., MILLER, N. & ENGELHARDT, W. VON (1975). Effect of volatile fatty acids on water and ion absorption from the goat colon. American Journal of Physiology 229, 997-1002. FAVUS, M. J. & ANGEID-BACKMAN, E. (1985). Effects of 1,25(OH)2D3 and calcium channel blockers on cecal calcium transport in the rat. American Journal of Physiology 248, G676-681. GASSNER, B., LUTERBACHER, S., SCHATZMANN, H. J. & WUTHRICH, A. (1988). Dependence of the red cell calcium pump on the membrane potential. Cell Calcium 9, 95-103. KARBACH, U. (1989). Cellular-mediated and diffusive magnesium transport across the descending colon of the rat. Gastroenterology 96, 1282-1289. KARBACH, U. & RUMMEL, W. (1987). Calcium transport across the colon ascendens and the influence of 1,25-dihydroxyvitamin D3 and dexamethasone. European Journal of Clinical Investigation 17, 368-374. LUTZ, T., WURMLI, R. & SCHARRER, E. (1991). Short chain fatty acids stimulate magnesium absorption by the colon. In Magnesium - a Relevant Ion? Proceedings of the 3rd European Congress on Magnesium, ed. LASSERRE, B. & DURLACH, J., pp. 131-137. John Libbey, London. PETITH, M. M. & SCHEDL, H. P. (1978). Effects of magnesium deficiency on duodenal and ileal magnesium absorption and secretion. American Journal of Digestive Diseases 23, 1-5. RECHKEMMER, G., RONNAU, K. & ENGELHARDT, W. V. (1988). Fermentation of polysaccharides and absorption of short chain fatty acids in the mammalian hindgut. Comparative Biochemistry and Physiology 90A, 563-568. SCHARRER, E. & LUTZ, T. (1990). Effects of short chain fatty acids and K on absorption of Mg and other cations by the colon and caecum. Zeitschriftfar Erndhrungswissenschaft 29, 162-168. VAN OS, C. H. (1987). Transcellular calcium transport in intestinal and renal epithelial cells. Biochimica et Biophysica Acta 906, 195-222.
