p-carotene intestinal absorption: bile, fatty acid, pH, and flow rate effects on transport DANIEL Division Detroit,
HOLLANDER
AND
of Gastroenterolugy, Michigan
PAUL
E. RUBLE,
and Harper-Grace
Hospitals,
48201
HOLLANDERJIANIEL~AND PAUL E. RUBLEJR. ~Carotene intestinal absorption: bile, fatty acid, pH, and fZow rate effects on transport. Am. J. Physiol. 235(6): E686-E691, 1978 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 4(6): E686-E691,1978. -Beta carotene absorption in the unanesthetized rat was investigated by recirculating a micellar perfusate that contained p-carotene through jejunal and ileal intestinal loops. Radioautography revealed extensive distribution of the provitamin throughout the layers of the small bowel. A linear relationship was found between the concentration of p-carotene in the perfusate and its rate of absorption at perfusate concentrations of 0.541 PM. Increases in the perfusate hydrogen ion concentrations, additions of fatty acids of varied chain lengths and degrees of saturation, and an increase in the perfusate flow rate caused higher rates of p-carotene absorption. Increase in the perfusate sodium taurocholate concentration above 2.5 mM did not change the absorption rate of /%carotene. These experiments indicate that pcarotene absorption takes place by passive dfision. The process of diffusion can be modulated by intraluminal factors that change the physical characteristics of perfusate or stimulate the intracellular cleavage reaction of carotene to retinal.
absorption in the rat; lipid transport; micellar
JR.
Wayne State University
provitamin components
A absorption;
intestinal
Following column purification of p-carotene (12>, its purity was found by thin-layer chromatography to be greater than 97% (24). If more than 6% impurities were detected, repurification was performed. The p-carotene stick solution was stored in benzene in the dark under a nitrogen atmosphere at - 10°C. Purified grade sodium taurocholate (K & K Laboratories, Plainview, NY) was found by thin-layer chromatography (8) to have less than 2% impurities, consisting primarily of cholic acid. Octanoic, stearic, oleic, linoleic, and linolenic acids (Sigma Chemical, St. Louis, MO) were more than 97% pure when purchased. The standard perfusate consisted of a micellar solution prepared in a Krebs phosphate buffer at pH 7.4 (25). The solution contained 123 mM NaCl, 4.9 mM KCl, and 1.2 mM MgSO,*7 H,O in the phosphate buffer base* Bile salts, fatty acids, monoglycerides, and p-carotene were added in concentrations as specified. More than 97% of p-carotene was found by methods described previously (9, 10) to be contained within the micellar particles. To prevent p-carotene degradation by ultraviolet radiation, all purification and experimental work was performed in a darkened room. METHODS
Male Sprague-Dawley rats weighing between 200 is a major dietary source of vitamin A and 250 g were fed regular chow (Check-R-Board, Novi, in man. Vitamin A is a lipid-soluble micronutrient MI) and tap water ad libitum and were not fasted prior necessary for support of growth, differentiation of epi- 1 to experimentation. Surgical preparation of the proxithelial tissues, and preservation of vision (3). Vitamin mal jejunal and distal ileal, isolated, intestinal loops A has also been shown t;o counteract the effect of some and their cannulation has been previously described carcinogen .ic agents (23), enhance antitumor chemo- (9). The rat was allowed to awaken from the ether therapy (2), and decrease tumor incidence following anesthesia and was placed in a restraint cage. The exposure to tumorogenic viruses (20). cannulas from each intestinal segment were connected The purpose of the present series of experiments was to separate X-ml reservoirs. A totally occlusive roller to investigate the kinetics and mechanisms of p-caro- pump (Buchler Instruments, Fort Lee, NJ) controlled tene absorption by perfusing intact proximal and distal the flow rate of perfusate from the reservoirs into the small intestinal loops in the unanesthetized rat. The inflow cannulas. The outfl .ow cannulas were al lowed to influences of the addition of fatty acids and variation in drain into the reservoirs by gravity. The animal’s body the perfusate pH, p-carotene concentration, bile salt temperature was monitored by a rectal probe connected concentration, and flow rate on the absorption rate of to a thermostatic temperature controller (Thermistemp P-carotene by the small bowel were also assessed. model 74, Yellow Springs Instruments, Yellow Springs, OH), which intermittently activated a forced-air heatMATERIALS ing device. The animal’s body temperature was main[15-15’-14C]P-carotene (sp act of 32 &i/mg) and non- tained at 38°C. Each reservoir was- filled with 10 ml of radioactive p-carotene were obtained through the gen- perfusate solution which contained ,&carotene and other compontints as specifi.ed. Just prior to perfu .sion, erosity of the F. Hoffman LaRoche, Basel, Switzerland.
BETA CAROTENE
EM6
0363-6119/78/0000-0000$01.25
Copyright
0 1978 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
P-CAROTENE
INTESTINAL
E687
ABSORPTION
three lOO+l samples were taken from each reservoir in order to measure the initial specific activity of p-carotene. After a 30-min equilibration period, disappearance of p-carotene from the perfusate was monitored by removing loo-p1 samples of the perfusate every 5 min over a 25min period. The absorption rate of pcarotene was calculated by linear regression analysis of absorption of the compound versus tim .e and by using the value of the slope as the mean value for absorption (4). In preliminary -experiments without radioactive @ carotene, [ Wllabeled inulin was added to the perfusate and was used as a nonabsorbable indicator to evaluate possible fluid shifts that could have taken place during the perfusion period (15). In both the proximal and distal intestinal segments Z-5% net fluid absorption was found to take place during the 55-min perfusion period. Net fluid absorption remained in the same range in subsequent experiments that explored the influence of changes in the pH, bile salt concentration, and presence of fatty acids. The final perfusate volume at the conclusion of the experiments varied between 8.8-9.1 ml. The decrease in perfusate volume was primarily due to sampling volumes during the experiments. Therefore, no correction of the absorption rate for fluid shifts was undertaken in this communication. At the end of each experiment, the rat was killed by an overdose of ether, the remaining perfusate solution volume was measured, and the perfused intestinal segments were gently disengaged- and removed. To ensure a constant degree of intestinal stretch, the segments were suspended with a 10-g wt attached to their most dependent portion and were dried at room temperature for 24 h, after which their lengths were measured. The final stretched length of the small bowel segments varied from 9.0-10.8 cm. The absorption rate of p-carotene in this communication was expressed as a function of bowel length. Radioactive determinations. Aliquots (100 ~1) of the intestinal perfusate were placed directly into scintillation counting vials containing a dioxane-based scintillator (11). Radioactivity was assayed in a Beckman LS 250 liquid scintillation counter with automatic quench calibration at ambient temperature. Radioactivity measurements were carried to a counting error of -+ 170. Morphological assessment of intestinal tissue and radioautography. During preliminary experiments, O.&cm sections of perfused bowel, as well as bowel from adjacent segments that had not been perfused, were fixed in 10% buffered formaldehyde and then examined by light microscopy. Intestinal cross sections were also processed for radioautography (18) to ascertain visually the site of accumulation and distribution of p-carotene in the intestinal tissue or the cell’s luminal surface. All the coded microscopic tissue sections were examined by two independent morphologists who did not have information regarding the section’s source or length of perfusion. Stutistical calculations. The data were analyzed by the use of the Student t test (22), regression analysis (4), and analysis of variance (F test) (22).
RESULTS
Adsorption of @carotene to the reservoirs and tubing. Lipid compounds can adsorb to or be absorbed into the tubing and reservoirs. The resultant loss of the compound introduces artifacts into the apparent disappearance rates of these compounds from the perfusate. In preliminary studies, the stan .dard micellar solution with 1 PM ,&carotene was circulated through the tubing and reservoirs only for a 55-min period. The range of carotene removal from the perfusate in four such experiments was 2-5%. Because the rate of disappearance was minimal, data presented in this communication were not corrected for this artifact. Radioautography of @carotene absorption. Tissue sections were examined after processing for radioautography (18) to obtain a visual -estimate-as to the distribution of the radioactivity on or within the absorptive cells. Some accumulation of radioactivity was seen in the brush-border area of the enterocytes. Visual estimation of multiple sections revealed that most of the radioactivity accumulated in the enterocytes (Fig. 1) and in the lamina propria in and around the lymphatic vessels (Fig. 2). We did not find evidence for excessive adsorption of ‘p-carotene to the luminal surface of the enterocytes. The effect of p- carotene perfusate concentration on its absorption rate. Tracer amounts of radioactive /3-carotene and nonradioactive p-carotene were added to the micellar solution, which was composed of 10 mM sodium taurocholate, 2.5 mM oleic acid, and 2.5 mM monoolein in the standard Krebs phosphate buffer at pH 7.4. Beta carotene concentrations were varied from 0.5-11 PM. A separate group of 3-6 animals was studied at each concentration and five data points were obtained from each animal experiment. The mean rate of p-carotene absorption at each concentration was plotted against the concentration (Fig. 3). The plot revealed a linear relationship between the two parameters. Analysis of variance of the two slopes disclosed no difference between p-carotene absorption by the two intestinal regions. The influence of sodium taurocholate concentration on pcarotene absorption. The perfusate solution consisted of the standard Krebs phosphate buffer at pH 7.4, p-carotene at a constant concentration of 1.0 PM, and sodium taurocholate at concentrations of 2.5-15 mM. Incremental increases in the perfusate’s sodium taurocholate concentration did not change p-carotene’s absorption rate by the jejunal and ileal loops (Table 1). Microscopic examinations of the tissue sections disclosed no structural changes at any of the bile salt concentrations studied. The effect of hydrogen ion concentration on pear+ tene absorption. The hydrogen ion concentration of the perfusate was varied by changing the ratio of monobasic and dibasic forms of phosphate in the perfusate. The perfusate contained i0 mM sodium taurocholate, 2.5 mM oleic acid, 2.5 mM monoolein, and 1.0 PM pcarotene. An increase (P < 0.01) in the rate of pcarotene absorption by both the proximal and distal small intestinal segments was noted as the hydrogen
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
E688
1. Radioautograph of intestinal perfusion.
FIG.
min
D. HOLLANDER
ion concentration (Table 2).
(731x) of jejunal Intestinal lumen
was increased
cross section after 55 is at top. Absorptive
in a stepwise fashion
The effect ofperfusate flow rate on pcarotene absorption. The thickness of the unstirred water layer at the
absorptive cell luminal membrane was gradually decreased by increasing the perfusate flow rate (14). The perfusate consisted of 10 mM sodium taurocholate, 2.5 mM oleic acid, 2.5 mM monoolein, and 1.0 PM pcarotene. A separate group of animals was studied at each flow rate. A progressive increase in p-carotene absorption by both the proximal and distal intestinal segments was found as the perfusate flow rate was increased (Table 3). Despite the increase in perfusate flow rate (0.5-15 mUmin), net fluid absorption remained constant (3-5%) throughout the 55-min period of perfusion. This observation indicated that the small intestinal surface area did not change at higher perfusion rates. The effect of fatty acid addition on pcarotene absorption. Absorption rate of p-carotene in the presence of
2.5 mM fatty acids and 10 mM sodium taurocholate in the per&sate was compared to base-line absorption rates of p-carotene in the presence of sodium taurocholate only. The p-carotene concentration was kept constant at 1.0 PM. Both the proximal and distal small bowel segments absorbed p-carotene at a higher rate V’ < 0.01) after the addition of any of the fatty acids (Table 4). The absorption rate was the highest when oleic acid (C,,:,) was present in the per&sate. The separate additions of linoleic (C!& and linolenic (C&
cells are loaded of radioactively
AND
P. E. RUBLE,
with dark dots, each representing labeled p-carotene. Hematoxylin-eosin
JR.
an accumulation stain.
acids caused @carotene to be absorbed at a rate lower than that in the presence of oleic acid (C8:,) (Table 4). DISCUSSION
In this report, we investigated the mechanism and kinetics of p-carotene absorption by the small bowel in the unanesthetized rat. Intact vascular and lymphatic circulation to the intestine was preserved while a micellar perfusate that contained p-carotene was circulated through proximal and distal intestinal loops. The relationship between the concentration of p-carotene in the perfusate and its absorption rate remained linear as the concentration of p-carotene was varied from 0.5-11.0 PM (Fig. 3). The linearity of the relationship suggests that absorption of the compound in the physiological range of concentrations takes place by passive diffusion. The present experimental data in vivo and their interpretation are supported by the in vitro studies of El-Corab and co-workers (51, who found that the addition of metabolic inhibitors did not retard p-carotene uptake by everted gut sacs and that there was no evidence for saturation kinetics. Bile salts are necessary for micellar solubilization of p-carotene, which possesses virtually no aqueous solubility. Bile salts are also required for the active conversion of pcarotene to retinal by the rat intestinal mucosa (6, 7). In this series of experiments, the rate of p-carotene absorption by the small intestine did not change as the intraluminal sodium taurocholate con-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
P-CAROTENE
INTESTINAL
E689
ABSORPTION
j ’
centration was varied from 2.5-15 mM (Table 1). The lack of significant influence of sodium taurocholate’s concentration on /?-carotene absorption by the small intestine would suggest that once the detergent properties of bile salts are fulfilled by achieving the critical micellar concentration, further increase in their concentration does not facilitate p-carotene’s absorption rate. The influence of the hydrogen ion concentration on p-carotene absorption was studied by modifying the perfusate pH while the concentrations of p-carotene and bile salts were kept constant. An increase in the hydrogen ion concentration was paralleled by a significant increase in the rate of p-carotene absorption by the small intestine (Table 2). The increased absorption rate is probably secondary to pH-induced changes in the surface charges of the cell membrane. Because both the luminal absorptive cell membrane (19) and the micellar surface (1) are negatively charged, electro-
FIG. 2. Radioautograph (731x) of jejunal lamina propria. Base of a crypt, upper left corner; muscularis mucosa, lower right corner. Accumulation of radioactivity is seen in and around lymphatic vessel in center of field. Hematoxylin-eosin stain.
static forces would decrease the rate of micellar diffusion toward the intestinal cells as they come into close approximation (13). An increase in the hydrogen ion concentration would diminish the negative surface charges of the cell membrane and would, therefore, enhance the rate of diffusion of the p-carotene-carrying micelles toward the cell membrane. The effect of changes in the solution’s pH from 5.3 to 8.3 is not caused by changes in the solubility of p-carotene or sodium taurocholate: the aqueous solubility of p-carotene is negligible despite its somewhat higher solubility in alkaline solutions, and sodium taurocholate has a pK, of 1.8 (1). The unstirred water layer could be a barrier to the diffusion of pcarotene if the compound’s rate of transfer across the lipid cell membrane is faster than its rate of diffusion through the unstirred water layer (26). In order to explore the relationship between the thickness of the unstirred water layer and the absorption rate of
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
D. HOLLANDER p-CAROTENE
ABSORPTION IN-V tv0
PfWXlMAL 57&43.9X = .
AND
3. Influence ofperfusate on @carotene absorption
flow
TABLE
Flow Rate, mllmin
No, of Animals
0.5 2.5 5.0 10.0 15.0
18 9 9 9 9
Absorption, Proximal
79.3 113.5 133.4 145.4 157.8
2 t k * *
P value
4.1 3.1 2.8 1.7 3.7
co.01 CO.01 *CO.01 co.01
P. E. RUBLE,
JR.
rate
(pmol/min)/lO
cm
Distal
63,3 77.6 107.7 116.6 131.0
-t 2 -tk *
P value
2.0 2.5 2.9 3.3 5.8
HOLLANDER
AND
of Gastroenterolugy, Michigan
PAUL
E. RUBLE,
and Harper-Grace
Hospitals,
48201
HOLLANDERJIANIEL~AND PAUL E. RUBLEJR. ~Carotene intestinal absorption: bile, fatty acid, pH, and fZow rate effects on transport. Am. J. Physiol. 235(6): E686-E691, 1978 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 4(6): E686-E691,1978. -Beta carotene absorption in the unanesthetized rat was investigated by recirculating a micellar perfusate that contained p-carotene through jejunal and ileal intestinal loops. Radioautography revealed extensive distribution of the provitamin throughout the layers of the small bowel. A linear relationship was found between the concentration of p-carotene in the perfusate and its rate of absorption at perfusate concentrations of 0.541 PM. Increases in the perfusate hydrogen ion concentrations, additions of fatty acids of varied chain lengths and degrees of saturation, and an increase in the perfusate flow rate caused higher rates of p-carotene absorption. Increase in the perfusate sodium taurocholate concentration above 2.5 mM did not change the absorption rate of /%carotene. These experiments indicate that pcarotene absorption takes place by passive dfision. The process of diffusion can be modulated by intraluminal factors that change the physical characteristics of perfusate or stimulate the intracellular cleavage reaction of carotene to retinal.
absorption in the rat; lipid transport; micellar
JR.
Wayne State University
provitamin components
A absorption;
intestinal
Following column purification of p-carotene (12>, its purity was found by thin-layer chromatography to be greater than 97% (24). If more than 6% impurities were detected, repurification was performed. The p-carotene stick solution was stored in benzene in the dark under a nitrogen atmosphere at - 10°C. Purified grade sodium taurocholate (K & K Laboratories, Plainview, NY) was found by thin-layer chromatography (8) to have less than 2% impurities, consisting primarily of cholic acid. Octanoic, stearic, oleic, linoleic, and linolenic acids (Sigma Chemical, St. Louis, MO) were more than 97% pure when purchased. The standard perfusate consisted of a micellar solution prepared in a Krebs phosphate buffer at pH 7.4 (25). The solution contained 123 mM NaCl, 4.9 mM KCl, and 1.2 mM MgSO,*7 H,O in the phosphate buffer base* Bile salts, fatty acids, monoglycerides, and p-carotene were added in concentrations as specified. More than 97% of p-carotene was found by methods described previously (9, 10) to be contained within the micellar particles. To prevent p-carotene degradation by ultraviolet radiation, all purification and experimental work was performed in a darkened room. METHODS
Male Sprague-Dawley rats weighing between 200 is a major dietary source of vitamin A and 250 g were fed regular chow (Check-R-Board, Novi, in man. Vitamin A is a lipid-soluble micronutrient MI) and tap water ad libitum and were not fasted prior necessary for support of growth, differentiation of epi- 1 to experimentation. Surgical preparation of the proxithelial tissues, and preservation of vision (3). Vitamin mal jejunal and distal ileal, isolated, intestinal loops A has also been shown t;o counteract the effect of some and their cannulation has been previously described carcinogen .ic agents (23), enhance antitumor chemo- (9). The rat was allowed to awaken from the ether therapy (2), and decrease tumor incidence following anesthesia and was placed in a restraint cage. The exposure to tumorogenic viruses (20). cannulas from each intestinal segment were connected The purpose of the present series of experiments was to separate X-ml reservoirs. A totally occlusive roller to investigate the kinetics and mechanisms of p-caro- pump (Buchler Instruments, Fort Lee, NJ) controlled tene absorption by perfusing intact proximal and distal the flow rate of perfusate from the reservoirs into the small intestinal loops in the unanesthetized rat. The inflow cannulas. The outfl .ow cannulas were al lowed to influences of the addition of fatty acids and variation in drain into the reservoirs by gravity. The animal’s body the perfusate pH, p-carotene concentration, bile salt temperature was monitored by a rectal probe connected concentration, and flow rate on the absorption rate of to a thermostatic temperature controller (Thermistemp P-carotene by the small bowel were also assessed. model 74, Yellow Springs Instruments, Yellow Springs, OH), which intermittently activated a forced-air heatMATERIALS ing device. The animal’s body temperature was main[15-15’-14C]P-carotene (sp act of 32 &i/mg) and non- tained at 38°C. Each reservoir was- filled with 10 ml of radioactive p-carotene were obtained through the gen- perfusate solution which contained ,&carotene and other compontints as specifi.ed. Just prior to perfu .sion, erosity of the F. Hoffman LaRoche, Basel, Switzerland.
BETA CAROTENE
EM6
0363-6119/78/0000-0000$01.25
Copyright
0 1978 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
P-CAROTENE
INTESTINAL
E687
ABSORPTION
three lOO+l samples were taken from each reservoir in order to measure the initial specific activity of p-carotene. After a 30-min equilibration period, disappearance of p-carotene from the perfusate was monitored by removing loo-p1 samples of the perfusate every 5 min over a 25min period. The absorption rate of pcarotene was calculated by linear regression analysis of absorption of the compound versus tim .e and by using the value of the slope as the mean value for absorption (4). In preliminary -experiments without radioactive @ carotene, [ Wllabeled inulin was added to the perfusate and was used as a nonabsorbable indicator to evaluate possible fluid shifts that could have taken place during the perfusion period (15). In both the proximal and distal intestinal segments Z-5% net fluid absorption was found to take place during the 55-min perfusion period. Net fluid absorption remained in the same range in subsequent experiments that explored the influence of changes in the pH, bile salt concentration, and presence of fatty acids. The final perfusate volume at the conclusion of the experiments varied between 8.8-9.1 ml. The decrease in perfusate volume was primarily due to sampling volumes during the experiments. Therefore, no correction of the absorption rate for fluid shifts was undertaken in this communication. At the end of each experiment, the rat was killed by an overdose of ether, the remaining perfusate solution volume was measured, and the perfused intestinal segments were gently disengaged- and removed. To ensure a constant degree of intestinal stretch, the segments were suspended with a 10-g wt attached to their most dependent portion and were dried at room temperature for 24 h, after which their lengths were measured. The final stretched length of the small bowel segments varied from 9.0-10.8 cm. The absorption rate of p-carotene in this communication was expressed as a function of bowel length. Radioactive determinations. Aliquots (100 ~1) of the intestinal perfusate were placed directly into scintillation counting vials containing a dioxane-based scintillator (11). Radioactivity was assayed in a Beckman LS 250 liquid scintillation counter with automatic quench calibration at ambient temperature. Radioactivity measurements were carried to a counting error of -+ 170. Morphological assessment of intestinal tissue and radioautography. During preliminary experiments, O.&cm sections of perfused bowel, as well as bowel from adjacent segments that had not been perfused, were fixed in 10% buffered formaldehyde and then examined by light microscopy. Intestinal cross sections were also processed for radioautography (18) to ascertain visually the site of accumulation and distribution of p-carotene in the intestinal tissue or the cell’s luminal surface. All the coded microscopic tissue sections were examined by two independent morphologists who did not have information regarding the section’s source or length of perfusion. Stutistical calculations. The data were analyzed by the use of the Student t test (22), regression analysis (4), and analysis of variance (F test) (22).
RESULTS
Adsorption of @carotene to the reservoirs and tubing. Lipid compounds can adsorb to or be absorbed into the tubing and reservoirs. The resultant loss of the compound introduces artifacts into the apparent disappearance rates of these compounds from the perfusate. In preliminary studies, the stan .dard micellar solution with 1 PM ,&carotene was circulated through the tubing and reservoirs only for a 55-min period. The range of carotene removal from the perfusate in four such experiments was 2-5%. Because the rate of disappearance was minimal, data presented in this communication were not corrected for this artifact. Radioautography of @carotene absorption. Tissue sections were examined after processing for radioautography (18) to obtain a visual -estimate-as to the distribution of the radioactivity on or within the absorptive cells. Some accumulation of radioactivity was seen in the brush-border area of the enterocytes. Visual estimation of multiple sections revealed that most of the radioactivity accumulated in the enterocytes (Fig. 1) and in the lamina propria in and around the lymphatic vessels (Fig. 2). We did not find evidence for excessive adsorption of ‘p-carotene to the luminal surface of the enterocytes. The effect of p- carotene perfusate concentration on its absorption rate. Tracer amounts of radioactive /3-carotene and nonradioactive p-carotene were added to the micellar solution, which was composed of 10 mM sodium taurocholate, 2.5 mM oleic acid, and 2.5 mM monoolein in the standard Krebs phosphate buffer at pH 7.4. Beta carotene concentrations were varied from 0.5-11 PM. A separate group of 3-6 animals was studied at each concentration and five data points were obtained from each animal experiment. The mean rate of p-carotene absorption at each concentration was plotted against the concentration (Fig. 3). The plot revealed a linear relationship between the two parameters. Analysis of variance of the two slopes disclosed no difference between p-carotene absorption by the two intestinal regions. The influence of sodium taurocholate concentration on pcarotene absorption. The perfusate solution consisted of the standard Krebs phosphate buffer at pH 7.4, p-carotene at a constant concentration of 1.0 PM, and sodium taurocholate at concentrations of 2.5-15 mM. Incremental increases in the perfusate’s sodium taurocholate concentration did not change p-carotene’s absorption rate by the jejunal and ileal loops (Table 1). Microscopic examinations of the tissue sections disclosed no structural changes at any of the bile salt concentrations studied. The effect of hydrogen ion concentration on pear+ tene absorption. The hydrogen ion concentration of the perfusate was varied by changing the ratio of monobasic and dibasic forms of phosphate in the perfusate. The perfusate contained i0 mM sodium taurocholate, 2.5 mM oleic acid, 2.5 mM monoolein, and 1.0 PM pcarotene. An increase (P < 0.01) in the rate of pcarotene absorption by both the proximal and distal small intestinal segments was noted as the hydrogen
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
E688
1. Radioautograph of intestinal perfusion.
FIG.
min
D. HOLLANDER
ion concentration (Table 2).
(731x) of jejunal Intestinal lumen
was increased
cross section after 55 is at top. Absorptive
in a stepwise fashion
The effect ofperfusate flow rate on pcarotene absorption. The thickness of the unstirred water layer at the
absorptive cell luminal membrane was gradually decreased by increasing the perfusate flow rate (14). The perfusate consisted of 10 mM sodium taurocholate, 2.5 mM oleic acid, 2.5 mM monoolein, and 1.0 PM pcarotene. A separate group of animals was studied at each flow rate. A progressive increase in p-carotene absorption by both the proximal and distal intestinal segments was found as the perfusate flow rate was increased (Table 3). Despite the increase in perfusate flow rate (0.5-15 mUmin), net fluid absorption remained constant (3-5%) throughout the 55-min period of perfusion. This observation indicated that the small intestinal surface area did not change at higher perfusion rates. The effect of fatty acid addition on pcarotene absorption. Absorption rate of p-carotene in the presence of
2.5 mM fatty acids and 10 mM sodium taurocholate in the per&sate was compared to base-line absorption rates of p-carotene in the presence of sodium taurocholate only. The p-carotene concentration was kept constant at 1.0 PM. Both the proximal and distal small bowel segments absorbed p-carotene at a higher rate V’ < 0.01) after the addition of any of the fatty acids (Table 4). The absorption rate was the highest when oleic acid (C,,:,) was present in the per&sate. The separate additions of linoleic (C!& and linolenic (C&
cells are loaded of radioactively
AND
P. E. RUBLE,
with dark dots, each representing labeled p-carotene. Hematoxylin-eosin
JR.
an accumulation stain.
acids caused @carotene to be absorbed at a rate lower than that in the presence of oleic acid (C8:,) (Table 4). DISCUSSION
In this report, we investigated the mechanism and kinetics of p-carotene absorption by the small bowel in the unanesthetized rat. Intact vascular and lymphatic circulation to the intestine was preserved while a micellar perfusate that contained p-carotene was circulated through proximal and distal intestinal loops. The relationship between the concentration of p-carotene in the perfusate and its absorption rate remained linear as the concentration of p-carotene was varied from 0.5-11.0 PM (Fig. 3). The linearity of the relationship suggests that absorption of the compound in the physiological range of concentrations takes place by passive diffusion. The present experimental data in vivo and their interpretation are supported by the in vitro studies of El-Corab and co-workers (51, who found that the addition of metabolic inhibitors did not retard p-carotene uptake by everted gut sacs and that there was no evidence for saturation kinetics. Bile salts are necessary for micellar solubilization of p-carotene, which possesses virtually no aqueous solubility. Bile salts are also required for the active conversion of pcarotene to retinal by the rat intestinal mucosa (6, 7). In this series of experiments, the rate of p-carotene absorption by the small intestine did not change as the intraluminal sodium taurocholate con-
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 20, 2018. Copyright © 1978 American Physiological Society. All rights reserved.
P-CAROTENE
INTESTINAL
E689
ABSORPTION
j ’
centration was varied from 2.5-15 mM (Table 1). The lack of significant influence of sodium taurocholate’s concentration on /?-carotene absorption by the small intestine would suggest that once the detergent properties of bile salts are fulfilled by achieving the critical micellar concentration, further increase in their concentration does not facilitate p-carotene’s absorption rate. The influence of the hydrogen ion concentration on p-carotene absorption was studied by modifying the perfusate pH while the concentrations of p-carotene and bile salts were kept constant. An increase in the hydrogen ion concentration was paralleled by a significant increase in the rate of p-carotene absorption by the small intestine (Table 2). The increased absorption rate is probably secondary to pH-induced changes in the surface charges of the cell membrane. Because both the luminal absorptive cell membrane (19) and the micellar surface (1) are negatively charged, electro-
FIG. 2. Radioautograph (731x) of jejunal lamina propria. Base of a crypt, upper left corner; muscularis mucosa, lower right corner. Accumulation of radioactivity is seen in and around lymphatic vessel in center of field. Hematoxylin-eosin stain.
static forces would decrease the rate of micellar diffusion toward the intestinal cells as they come into close approximation (13). An increase in the hydrogen ion concentration would diminish the negative surface charges of the cell membrane and would, therefore, enhance the rate of diffusion of the p-carotene-carrying micelles toward the cell membrane. The effect of changes in the solution’s pH from 5.3 to 8.3 is not caused by changes in the solubility of p-carotene or sodium taurocholate: the aqueous solubility of p-carotene is negligible despite its somewhat higher solubility in alkaline solutions, and sodium taurocholate has a pK, of 1.8 (1). The unstirred water layer could be a barrier to the diffusion of pcarotene if the compound’s rate of transfer across the lipid cell membrane is faster than its rate of diffusion through the unstirred water layer (26). In order to explore the relationship between the thickness of the unstirred water layer and the absorption rate of
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D. HOLLANDER p-CAROTENE
ABSORPTION IN-V tv0
PfWXlMAL 57&43.9X = .
AND
3. Influence ofperfusate on @carotene absorption
flow
TABLE
Flow Rate, mllmin
No, of Animals
0.5 2.5 5.0 10.0 15.0
18 9 9 9 9
Absorption, Proximal
79.3 113.5 133.4 145.4 157.8
2 t k * *
P value
4.1 3.1 2.8 1.7 3.7
co.01 CO.01 *CO.01 co.01
P. E. RUBLE,
JR.
rate
(pmol/min)/lO
cm
Distal
63,3 77.6 107.7 116.6 131.0
-t 2 -tk *
P value
2.0 2.5 2.9 3.3 5.8
