AMERICAN
JOURNAL
o I> PrIYsIoLOGY
Vol. 228, No. 5, May
Uptake
1975.
Pri?&
in U.S.A.
and compartmental
fatty acid by rat small SEYMOUR MISHKIN, MORDECHAI Gaslrointestinal Research Luboratory, McGill Royal Victoria Hqhtal, Montrea!, Canadu
distribution intestine
SEYMOUR, MORDECHAI YALOVSKY, AND JACQUES I, Uptake and compartmental distribution of fatty acid by rat small intestine in Go. Am. J. Physiol. 228(5): 1409-141.4. 1975.The uptake and esterification of micellar [3H]oleate and [‘“Cl palmitate were uniform along the entire length of the small intestine in vivo. Fatty acid (FA) radioactivity taken up by the small intestine could be described in terms of four functionally distinct compartments analogous to those described in vitro. The KRP-extractable compartment (KEC) and albumin-extractable compartment (AEC) contained reversibly adherent unesterified FA radioactivity, while the tissue free and esterifred FA compartments contained irreversibly bound radioactivity. Whereas 27 yO and 63y0 of FA uptake were reversibly bound in the KEC and AEC by the most proximal and most distal regions of the small intestine in vitro (IS), less than 10yO was contained in these compartments in vivo, independent of locationLinear inverse relationships were found between tissue FA esterification and proportion of FA radioactivity present in the KEC, AEC, and the tissue free FA compartment in vivo. These observations allow for the possibility that FA molecules pass through these compartments prior to esterification.
acids; small intestinal and C3H]oleate.
in vivo
YALOVSKY, ANI JACQUES I. KESSLER lhiiversity Clinic, Ijefiartment of Medicine,
MISHKIN, &SSLER.
absorption of long-chain fatty in vivo; micellar [14C]palmitate
of
absorption
IT IS GENERALLY ACCEPTED that the cellular uptake of free fatty acid (FFA) by any cell is a non-energy-dependent process that can be described as a mass-action exchange between the bathing solution and the cell surface (27). Furthermore, it is also accepted that the first step in FFA utilization is the binding of fatty acid in unesterified form to the cell surface. This conclusion was reached independently from studies with ascites tumor cells (28), erythrocytes (9), leukocytes (7), diaphragm (23, 25), heart slices (25), and acanthameba (29). Recent studies in our laboratory have extended these findings to the absorption of FFA by the small intestine (11, 15-l 7). We have demonstrated that micellar FA taken up in vitro was distributed into four functionally distinct compartments : the Krebs-Ringerphosphate (KRP) -extractable compartment (KEG) and albumin-extractable compartment (AEC), which contained reversibly adherent FFA, and the tissue free and esterified fatty acid compartments, which contained irreversibly bound FA. A linear inverse relationship existed between the proportion of FA esterified and the proportion of the FA uptake reversibly bound in the KRP-extractable and albumin-extractable compartments, independent of
small intestinal segment, duration, or temperature bation (15, 17). I n view of the possible implications findings for the pathophysiology of FA absorption intact animal, we have been anxious to determine the above-mentioned compartments are present This paper reports the results of such studies. METHODS
AND
of incuof these int the wheiher in vvo.
MATERIALS
Materials. Palmitic and oleic acid and monolein were purchased from the Hormel Institute, Austin, Minn., and sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC), and sodium glycochenodeoxycholate (NaGCDC) from Maybridge Research Chemicals, Tintagel, Cornwall, U.K. These chemicals were certified as at least 99% pure by the suppliers and confirmatory tests with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were in close agreement with the stated purities. The [ 1-14C]palmitate and [9,10-“Hloleate (New England Nuclear Corp., Boston) were certified to be 98 % pure by the supplier and the stated purities were confirmed by TLC and GLC analysis; [‘“Cl inulin also was obtained from New England Nuclear. All chemicals used were reagent grade and all organic solvents were doubly distilled. Analyticcll procedures. Thin-layer chromatography was carried out on standard glass plates (20 x 20 cm) coated with Silica Gel-G (E. Merck Company, Darmstadt, Germany) For the separation of lipid fractions, 2’,7’dichlorofluorescein was incorporated into the silica gel and the solvent system used consisted of n-hexane-diethyl etheracetic acid-methanol, 90 : 20: 2 :3 (vol/vol) (4). The lipid fractions after separation were identified by comparison with simultaneously chromatographed standards (Hormel were visualized by ultraviolet Institute). The fractions illumination and the corresponding silica gel as well as appropriate blanks were scraped into counting vials containing 12 ml of scintillation solution, made up of 4 g of Omnifluor (New England Nuclear) dissolved in 1 liter of toluene. Total radioactivity of the different incubation and washing solutions as well as the tissue homogenates was assayed in l-ml aliquots added to 12 ml of Bray’s solution (2). All samples were counted in a Tri-Carb liquid-scintillation counter (model 3375, Packard Instrument Company, Inc., Downers Grove, Ill.) with a minimum efficiency of 90 % for 14C in toluene and 75 % for 14C in Bray’s solution. The corresponding counting efficiencies for 3H were 40 % and 26 %, respectively. The samples were corrected for quenchl
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
1410 ing by the channel-ratio method (1) or by internal standardization. General jmmdure. Male Sprague-Dawley rats weighing 200-300 g (Quebec Breeding Laboratories, LaPrairie, Quebec) were fasted overnight and anesthetized with ether. Between the duodenojejunal junction and the ileocecal valve, six equal segments, 6 cm in length, were isolated carefully between double ligatures Pla .ced SO as not to damage blood vessels. Each segment was then l”.l ected w ith 0.8 ml of a micellar solution containing 1.5 mM [3H]oleate (1 .O &i/pmol), 1.5 mM monolein, 7.5 mM sodium taurodeoxycholate, and 10 mM glucose made up in 0.15 M KRP buffer (pH 6.3, 37”C, Ca++ and Mg++ omitted). In some of the experiments equimolar [““Cl pal mitic acid (0.05 &i/pmol) replaced C3H]oleic acid. In preliminary experiments, it was found that 10 mM sodium taurocholate or 7.5 mM sodium glycochenodeoxycholate did not alter the results obtained with 7.5 mM sodium taurodeoxycholate. In some experiments [14C]inulin was incorporated into some of the micellar solu .tions. Duri “g incubation, the in its normal ana tomical position small intestin .e remained and was covered with gauze soaked with normal saline (37°C). After 10 min of incubation each segment was removed after its vascular supply was ligated. Each segment was rinsed in iced saline, drained, and everted with the use of a blunt upholstery needle. The everted segments were rinsed sequentially in a series of unlabeled washing solutions maintained at 4°C to ensure that the release of [3H]oleate or [14C]palmitate was unaffected by the energydependent processes of the tissue. The rinsing procedure was carried out in the following order: a) a single 20-s rinse in an identical but unlabeled micellar solution; b) five separate Z-min rinses in KRP buffer (pH 6.3); c) 10 separate Z-min rinses in 2.5 % solutions of fat-free albumin dissolved in saline (pH 7.4, 0.15 M). Each rinse was carried out in a ZO-ml volume of rinsing solution by gently swirling the tissue every 30 s. The rinsing sequence has been fully described in previous communications (17). Examination of histological sections with light microscopy did not reveal any significant alterations in intestinal morphology by the rinsing procedure used. In preliminary experiments carried out to establish this rinsing sequence, 18 segments obtained from three animals were randomly subjected to one of three rinsing sequences after 10 min of incubation in micellar [aH]oleate and 20 s of rinsing in unlabeled micelle, namely: 1) 15 separate Z-min rinses in KRP buffer (6 sacs), 2) 15 separate Z-min rinses in albumin solution (6 sacs); (3) five separate 2-min rinses in KRP buffer, followed by 10 separate 2-min rinses of albumin (6 sacs). (The results obtained are described in Figs. 1 and 2.) Similar experiments were carried out with 12 segments incubated in micellar rllC]After completion of the rinsing sequence; the palmitate. sacs were weighed and homogenized in 10 ml of methanol (4°C) in an all-glass Potter-Elvehjem homogenizer. onemilliliter aliquots of incubation medium, rinsing solutions, and tissue homogenate were directly counted in Aquasol (New England Nuclear) _ The remaining samples were extracted with chloroform-methanol 2: 1 (vol/vol) (8), and aliquots of t.he lipid extract were taken for TLC to determine the distribution of fatty acid radioactivity among the free and esterified fatty acid fractions.
MISHKIN,
YALOVSKY,
AND
KESSLER
The “total uptake” of fatty acid was calculated by subtracting the radioactivity remaining in the lumen of the sac after incubation from the amount present in the micellar incubation medium injected. The tissue uptake of fatty acid radioactivity (pm01 X 10B3/g wet wt) was calculated by addition of radioactivity released into each of the rinsing solutions to that remaining in the tissue at the end of the rinsing sequence. The radioactivity released from the segments during the 20-s rinse in unlabeled micelle was not included in calculations of tissue uptake and was considered to be part of the radioactivity remaining in the lumen after incubation. The assumptions inherent in these calculations have been dealt with in an earlier communication (17). It was a consistent finding that total uptake and tissue uptake were identical for each segment studied, indicating that during the short incubation period insignificant amounts of radi oactivity had been absorbed into the systemic circulation. This was confirmed by the absence of significant radioactivity in the serum and livers of a number of animals tested. As a result all discussion of FA uptake refers to tissue uptake of fatty acid. Experiments in which the sacs were extracted before the rinsing sequence yielded values for total uptake that were within 1 SD of the mean value obtained by the method described above. The amount of [sH]oleate esterified at the end of the incubation period was 1.11 & 0.10 pmol/g wet wt and that after rinsing for 30 min at 4°C was 1.12 & 0.09 pmol/g wet wt. The corresponding values for 114C]palmitic acid ester&cation were O-68 & 0.09 and 0.68 & 0.15, respectively. These results are consistent with the hypothesis that labeled fatty acid released during the rinsing procedure had not been derived from the lipolysis of esterified fatty acid. Furthermore, the substitution of [9, lo-3HIpalmitic acid for the 14C-labeled acid in the incubation medium did not change the results obtained. This finding is consistent with the conclusion that oxidation of the terminal carboxyl group had not occurred to any significant extent under our experimental conditions. A number of preliminary experiments were carried out 15-cm ct was with a micellar solution containing 1.5 mM [aH]oleate, 1.5 mR/1: monoolein, 7.5 mM sodium taurodeoxycholate, and 10.0 mM glucose (pH 6.3, 37°C) at a rate of 1.0 ml/min by means of a Holter peristaltic pump (model RL-175, Brent Surgical Limited, Toronto). The effluent was collected for 2-min periods into serial tubes with a fraction collector (LKB model 7000). After 20 rnin of perfusion, when absorption had reached a steady state, unlabeled KRP buffer (pH 6.3, 4°C) was infused in place of the radioactive micellar solution. The infusion of KRP buffer was continued for 20 min at which time the radioactivity measured in the effluent was very low, At this time the perfusing medium was changed to 2.5 g/ 100 ml albumin made up in KRP buffer (pH 7.4, 0.15 M). Perfusion with the albumin solution was continued for 20 min. Statistical methods. The Student t test was used for comparing individual differences. Only 1’ values
JOURNAL
o I> PrIYsIoLOGY
Vol. 228, No. 5, May
Uptake
1975.
Pri?&
in U.S.A.
and compartmental
fatty acid by rat small SEYMOUR MISHKIN, MORDECHAI Gaslrointestinal Research Luboratory, McGill Royal Victoria Hqhtal, Montrea!, Canadu
distribution intestine
SEYMOUR, MORDECHAI YALOVSKY, AND JACQUES I, Uptake and compartmental distribution of fatty acid by rat small intestine in Go. Am. J. Physiol. 228(5): 1409-141.4. 1975.The uptake and esterification of micellar [3H]oleate and [‘“Cl palmitate were uniform along the entire length of the small intestine in vivo. Fatty acid (FA) radioactivity taken up by the small intestine could be described in terms of four functionally distinct compartments analogous to those described in vitro. The KRP-extractable compartment (KEC) and albumin-extractable compartment (AEC) contained reversibly adherent unesterified FA radioactivity, while the tissue free and esterifred FA compartments contained irreversibly bound radioactivity. Whereas 27 yO and 63y0 of FA uptake were reversibly bound in the KEC and AEC by the most proximal and most distal regions of the small intestine in vitro (IS), less than 10yO was contained in these compartments in vivo, independent of locationLinear inverse relationships were found between tissue FA esterification and proportion of FA radioactivity present in the KEC, AEC, and the tissue free FA compartment in vivo. These observations allow for the possibility that FA molecules pass through these compartments prior to esterification.
acids; small intestinal and C3H]oleate.
in vivo
YALOVSKY, ANI JACQUES I. KESSLER lhiiversity Clinic, Ijefiartment of Medicine,
MISHKIN, &SSLER.
absorption of long-chain fatty in vivo; micellar [14C]palmitate
of
absorption
IT IS GENERALLY ACCEPTED that the cellular uptake of free fatty acid (FFA) by any cell is a non-energy-dependent process that can be described as a mass-action exchange between the bathing solution and the cell surface (27). Furthermore, it is also accepted that the first step in FFA utilization is the binding of fatty acid in unesterified form to the cell surface. This conclusion was reached independently from studies with ascites tumor cells (28), erythrocytes (9), leukocytes (7), diaphragm (23, 25), heart slices (25), and acanthameba (29). Recent studies in our laboratory have extended these findings to the absorption of FFA by the small intestine (11, 15-l 7). We have demonstrated that micellar FA taken up in vitro was distributed into four functionally distinct compartments : the Krebs-Ringerphosphate (KRP) -extractable compartment (KEG) and albumin-extractable compartment (AEC), which contained reversibly adherent FFA, and the tissue free and esterified fatty acid compartments, which contained irreversibly bound FA. A linear inverse relationship existed between the proportion of FA esterified and the proportion of the FA uptake reversibly bound in the KRP-extractable and albumin-extractable compartments, independent of
small intestinal segment, duration, or temperature bation (15, 17). I n view of the possible implications findings for the pathophysiology of FA absorption intact animal, we have been anxious to determine the above-mentioned compartments are present This paper reports the results of such studies. METHODS
AND
of incuof these int the wheiher in vvo.
MATERIALS
Materials. Palmitic and oleic acid and monolein were purchased from the Hormel Institute, Austin, Minn., and sodium taurodeoxycholate (NaTDC), sodium taurocholate (NaTC), and sodium glycochenodeoxycholate (NaGCDC) from Maybridge Research Chemicals, Tintagel, Cornwall, U.K. These chemicals were certified as at least 99% pure by the suppliers and confirmatory tests with gas-liquid chromatography (GLC) and thin-layer chromatography (TLC) were in close agreement with the stated purities. The [ 1-14C]palmitate and [9,10-“Hloleate (New England Nuclear Corp., Boston) were certified to be 98 % pure by the supplier and the stated purities were confirmed by TLC and GLC analysis; [‘“Cl inulin also was obtained from New England Nuclear. All chemicals used were reagent grade and all organic solvents were doubly distilled. Analyticcll procedures. Thin-layer chromatography was carried out on standard glass plates (20 x 20 cm) coated with Silica Gel-G (E. Merck Company, Darmstadt, Germany) For the separation of lipid fractions, 2’,7’dichlorofluorescein was incorporated into the silica gel and the solvent system used consisted of n-hexane-diethyl etheracetic acid-methanol, 90 : 20: 2 :3 (vol/vol) (4). The lipid fractions after separation were identified by comparison with simultaneously chromatographed standards (Hormel were visualized by ultraviolet Institute). The fractions illumination and the corresponding silica gel as well as appropriate blanks were scraped into counting vials containing 12 ml of scintillation solution, made up of 4 g of Omnifluor (New England Nuclear) dissolved in 1 liter of toluene. Total radioactivity of the different incubation and washing solutions as well as the tissue homogenates was assayed in l-ml aliquots added to 12 ml of Bray’s solution (2). All samples were counted in a Tri-Carb liquid-scintillation counter (model 3375, Packard Instrument Company, Inc., Downers Grove, Ill.) with a minimum efficiency of 90 % for 14C in toluene and 75 % for 14C in Bray’s solution. The corresponding counting efficiencies for 3H were 40 % and 26 %, respectively. The samples were corrected for quenchl
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 14, 2019.
1410 ing by the channel-ratio method (1) or by internal standardization. General jmmdure. Male Sprague-Dawley rats weighing 200-300 g (Quebec Breeding Laboratories, LaPrairie, Quebec) were fasted overnight and anesthetized with ether. Between the duodenojejunal junction and the ileocecal valve, six equal segments, 6 cm in length, were isolated carefully between double ligatures Pla .ced SO as not to damage blood vessels. Each segment was then l”.l ected w ith 0.8 ml of a micellar solution containing 1.5 mM [3H]oleate (1 .O &i/pmol), 1.5 mM monolein, 7.5 mM sodium taurodeoxycholate, and 10 mM glucose made up in 0.15 M KRP buffer (pH 6.3, 37”C, Ca++ and Mg++ omitted). In some of the experiments equimolar [““Cl pal mitic acid (0.05 &i/pmol) replaced C3H]oleic acid. In preliminary experiments, it was found that 10 mM sodium taurocholate or 7.5 mM sodium glycochenodeoxycholate did not alter the results obtained with 7.5 mM sodium taurodeoxycholate. In some experiments [14C]inulin was incorporated into some of the micellar solu .tions. Duri “g incubation, the in its normal ana tomical position small intestin .e remained and was covered with gauze soaked with normal saline (37°C). After 10 min of incubation each segment was removed after its vascular supply was ligated. Each segment was rinsed in iced saline, drained, and everted with the use of a blunt upholstery needle. The everted segments were rinsed sequentially in a series of unlabeled washing solutions maintained at 4°C to ensure that the release of [3H]oleate or [14C]palmitate was unaffected by the energydependent processes of the tissue. The rinsing procedure was carried out in the following order: a) a single 20-s rinse in an identical but unlabeled micellar solution; b) five separate Z-min rinses in KRP buffer (pH 6.3); c) 10 separate Z-min rinses in 2.5 % solutions of fat-free albumin dissolved in saline (pH 7.4, 0.15 M). Each rinse was carried out in a ZO-ml volume of rinsing solution by gently swirling the tissue every 30 s. The rinsing sequence has been fully described in previous communications (17). Examination of histological sections with light microscopy did not reveal any significant alterations in intestinal morphology by the rinsing procedure used. In preliminary experiments carried out to establish this rinsing sequence, 18 segments obtained from three animals were randomly subjected to one of three rinsing sequences after 10 min of incubation in micellar [aH]oleate and 20 s of rinsing in unlabeled micelle, namely: 1) 15 separate Z-min rinses in KRP buffer (6 sacs), 2) 15 separate Z-min rinses in albumin solution (6 sacs); (3) five separate 2-min rinses in KRP buffer, followed by 10 separate 2-min rinses of albumin (6 sacs). (The results obtained are described in Figs. 1 and 2.) Similar experiments were carried out with 12 segments incubated in micellar rllC]After completion of the rinsing sequence; the palmitate. sacs were weighed and homogenized in 10 ml of methanol (4°C) in an all-glass Potter-Elvehjem homogenizer. onemilliliter aliquots of incubation medium, rinsing solutions, and tissue homogenate were directly counted in Aquasol (New England Nuclear) _ The remaining samples were extracted with chloroform-methanol 2: 1 (vol/vol) (8), and aliquots of t.he lipid extract were taken for TLC to determine the distribution of fatty acid radioactivity among the free and esterified fatty acid fractions.
MISHKIN,
YALOVSKY,
AND
KESSLER
The “total uptake” of fatty acid was calculated by subtracting the radioactivity remaining in the lumen of the sac after incubation from the amount present in the micellar incubation medium injected. The tissue uptake of fatty acid radioactivity (pm01 X 10B3/g wet wt) was calculated by addition of radioactivity released into each of the rinsing solutions to that remaining in the tissue at the end of the rinsing sequence. The radioactivity released from the segments during the 20-s rinse in unlabeled micelle was not included in calculations of tissue uptake and was considered to be part of the radioactivity remaining in the lumen after incubation. The assumptions inherent in these calculations have been dealt with in an earlier communication (17). It was a consistent finding that total uptake and tissue uptake were identical for each segment studied, indicating that during the short incubation period insignificant amounts of radi oactivity had been absorbed into the systemic circulation. This was confirmed by the absence of significant radioactivity in the serum and livers of a number of animals tested. As a result all discussion of FA uptake refers to tissue uptake of fatty acid. Experiments in which the sacs were extracted before the rinsing sequence yielded values for total uptake that were within 1 SD of the mean value obtained by the method described above. The amount of [sH]oleate esterified at the end of the incubation period was 1.11 & 0.10 pmol/g wet wt and that after rinsing for 30 min at 4°C was 1.12 & 0.09 pmol/g wet wt. The corresponding values for 114C]palmitic acid ester&cation were O-68 & 0.09 and 0.68 & 0.15, respectively. These results are consistent with the hypothesis that labeled fatty acid released during the rinsing procedure had not been derived from the lipolysis of esterified fatty acid. Furthermore, the substitution of [9, lo-3HIpalmitic acid for the 14C-labeled acid in the incubation medium did not change the results obtained. This finding is consistent with the conclusion that oxidation of the terminal carboxyl group had not occurred to any significant extent under our experimental conditions. A number of preliminary experiments were carried out 15-cm ct was with a micellar solution containing 1.5 mM [aH]oleate, 1.5 mR/1: monoolein, 7.5 mM sodium taurodeoxycholate, and 10.0 mM glucose (pH 6.3, 37°C) at a rate of 1.0 ml/min by means of a Holter peristaltic pump (model RL-175, Brent Surgical Limited, Toronto). The effluent was collected for 2-min periods into serial tubes with a fraction collector (LKB model 7000). After 20 rnin of perfusion, when absorption had reached a steady state, unlabeled KRP buffer (pH 6.3, 4°C) was infused in place of the radioactive micellar solution. The infusion of KRP buffer was continued for 20 min at which time the radioactivity measured in the effluent was very low, At this time the perfusing medium was changed to 2.5 g/ 100 ml albumin made up in KRP buffer (pH 7.4, 0.15 M). Perfusion with the albumin solution was continued for 20 min. Statistical methods. The Student t test was used for comparing individual differences. Only 1’ values
