In vivo absorption of medium-chain fatty acids by the rat colon exceeds that of short-chain fatty acids

GASTROENTEROLOGY 2001;120:1152–1161

In Vivo Absorption of Medium-Chain Fatty Acids by the Rat Colon Exceeds That of Short-Chain Fatty Acids JIMMY R. JØRGENSEN,* MARK D. FITCH,‡ PER B. MORTENSEN,* and SHARON E. FLEMING‡ *Department of Medicine, Section of Gastroenterology, Copenhagen University Hospital, The Rigshospital, Copenhagen, Denmark; and ‡Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California

Background & Aims: Short-chain fatty acids (SCFAs) are main fuels of the colonic epithelium, and are avidly absorbed by the colon of animal and man. The current knowledge on colonic metabolism and absorption of medium-chain fatty acids (MCFAs) is limited. In some clinical situations, colonic absorption of high-energy substances could compensate for reduced absorptive capacity because of a shortened or malfunctioning small bowel. We evaluated and compared colonic absorption and metabolism of MCFAs (octanoate, decanoate, and dodecanoate), SCFAs (acetate and butyrate), and longchain fatty acids (LCFAs) (oleate). Methods: Rats were surgically operated on to cannulate a 7-cm segment of proximal colon, isolate the vasculature, and cannulate the right colic vein draining this segment. The lumen was perfused with 14C-labeled substrates for 100 minutes. Right colic vein blood was analyzed for total 14C, 14CO , and metabolites by scintillation counting and 2 high-performance liquid chromatography. Results: The transport from the colonic lumen to mesenteric blood of substrate carbon from MCFAs exceeded by 2–13-fold that of SCFAs and LCFAs. The CO2 production from the oxidation of MCFAs was as high as or higher than that from SCFAs. CO2 produced from the LCFA, oleate, was lower than from SCFAs or MCFAs. In addition to CO2, ketone bodies were major metabolites of SCFAs and MCFAs. Ketogenesis from butyrate and the MCFAs was significantly higher than from acetate and oleate. A substantial proportion (50%–90%) of all substrates was absorbed without being metabolized. Conclusions: The colonic epithelium serves to absorb and partially metabolize MCFAs. For patients with a compromised smallbowel function, colonic absorption of MCFAs could represent an important way of receiving calories.

hort-chain fatty acids (SCFAs) are produced in the colon of nonruminant animals and humans by fermentation of unabsorbed carbohydrates and dietary fiber. Colonic absorption of SCFAs is well known and is estimated to contribute 5%–10% to total energy requirements in man.1,2 Medium-chain fatty acids (MCFAs) are normally not present in colonic contents. For patients with compromised small bowel function, as is seen in

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short bowel syndrome, pancreatic insufficiency, and extrahepatic biliary obstruction, colonic absorption of these more energy-rich fatty acids (FAs) could represent an important way of retrieving calories. The colon has not often been considered to be a site of fat absorption. Accordingly, the nutritional benefit of treating patients having a diminished absorption of longchain triglycerides using medium-chain triglycerides (MCT) has been explained by a rapid absorption of MCT in the small bowel.3,4 Nevertheless, some studies have suggested that colonic absorption of MCFAs can take place in the rat,5,6 dog,7 and in man.8,9 Also, in small bowel resected patients administered MCT, a preserved colon was associated with a significant improvement in the absorption of MCFAs compared with patients with a jejuno- or ileostomy,10 suggesting colonic absorption of MCFAs. An accurate way to evaluate the extent to which colonic absorption of MCFAs takes place was therefore desired. It seems from in vitro studies that SCFAs (especially butyrate), are important fuels for colonic epithelial cells (isolated colonocytes),11–13 and help maintain mucosal integrity and health.14 Other in vitro studies have shown that MCFAs may be comparable to butyrate as substrates for colonocyte oxidation.15 However, aspects of FA metabolism are not always predicted from data using isolated colonocytes, but require the use of an in vivo model.16 For the purpose of doing comparative studies between SCFAs and MCFAs, we were therefore prompted to use our novel method that permits investigation of rat colonic metabolism and absorption of substrates in vivo.16 To extend our investigation of the effect of increasing chain length on metabolism and absorption of FAs, the Abbreviations used in this paper: FA, fatty acid; HPLC, high-performance liquid chromatography; LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid; MCT, medium-chain triglycerides; SCFA, short-chain fatty acid. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.23259

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long-chain fatty acid (LCFA) oleate was included in experiments. To our knowledge, this is the first model that simultaneously restricts metabolism to a selected colonic segment and permits metabolism and absorption to be studied in vivo.

Materials and Methods In this in vivo model, rats were surgically operated on to cannulate a segment of proximal colon, isolate the vasculature, cannulate the right colic vein draining this segment, and install an infusion catheter in the left saphenous vein. The lumen was then continually perfused with 14C-labeled substrates, and all blood draining the segment was collected from the right colic vein and analyzed for total 14C, 14CO2, and metabolites. The experimental protocols are based on procedures that have been used previously.16

Chemicals [1-14C]-labeled acetate, butyrate, octanoate, decanoate, dodecanoate, and oleate (all sodium salts) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Unlabeled fatty acids (sodium salts), acetylcysteine, and antibiotics were obtained from Sigma Chemical Co., Inc. (St. Louis, MO). Sodium pentobarbital was obtained from Abbot Laboratories, Inc. (North Chicago, IL). Sodium heparin was obtained from Elkins-Sinn, Inc. (Cherry Hill, NJ). All silicone tubing was medical grade (Baxter-Scientific Products, McGaw Park, IL). Cholylsarcosine was a generous gift from Dr. Alan Hofmann, Department of Medicine, University of California, San Diego, CA.

Animals Male Sprague–Dawley rats, 6 months of age, weight 473 ⫾ 7 g (mean ⫾ SE), were obtained from Simonsen Laboratories, Inc. (Gilroy, CA). Whole body blood volume was assumed to be 5.8% of body weight.17 All procedures involving animals were reviewed and approved by the Animal Care and Use Committee, University of California, Berkeley, CA.

Substrates and Solutions All substrates were 10 mmol/L in Krebs–Henseleit buffer without calcium and with antibiotics (amphotericin B, kanamycin monosulfate, penicillin G, and streptomycin sulfate) and acetylcysteine as previously described.16 [1-14C]labeled FAs were added to the corresponding unlabeled solutions to produce a specific activity of approximately 0.05 ␮Ci/␮mol. The bile acid cholylsarcosine was used to dissolve dodecanoate and oleate. Twenty millimoles per liter of cholylsarcosine was needed to make a clear micellar solution (37°C) with dodecanoate, whereas only 7.5 mmol/L was needed with oleate, and the former concentration was chosen throughout experiments. Filtration experiments through 0.22-␮m filters and spectrophotometric analysis of dodecanoate and oleate solutions showed that large molecular aggregates or microcrystals were not present.

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Surgical Procedure and Experimental Protocols On the evening before the experiment, blood was obtained from 6 rats. On the day of the experiment, 1 rat was anesthetized by intraperitoneal injection of sodium pentobarbital. It was placed on a thermostated operating pad with a rectal temperature probe. A nose cone ventilator provided oxygen while the respired gases were continually exhausted through a liquid trap (800 mL of 0.5 mol/L NaOH) to retain carbon dioxide for later 14C analysis. An abdominal midline incision was made and the cecum was exteriorized onto a 37°C platform. After digitally expelling fecal pellets, the colon was cleared of mesenteric connective tissue, lymphatic vessels, and overlying portions of the pancreas by blunt dissection. The middle colic artery and vein were tied off and then cut to allow repositioning of the middle region of the colon within the body cavity. The right colic vein was then exposed. A cannula for infusion of substrate solution was inserted into the lumen of the colon segment at the position of the middle colic vein and secured tightly in place by sutures around the colon at this location. A second cannula for the exit of effluent was inserted into the lumen of the proximal colon at the ceco-colonic junction and also secured tightly with sutures around the colon. The segment was then flushed to clear it of excess mucus and any remaining digesta. In preparation for the installation of 3 vascular cannulae, the animal was heparinized by injecting 200 United States Pharmacopeia (USP) units into the right saphenous vein. The cannula supplying donor blood was installed into the left saphenous vein, and another cannula was installed into the aorta to measure blood pressure. Donor blood infusion rate was adjusted to maintain a systolic pressure of 160 –180 mm Hg. Finally, a 26G cannula was inserted into the right colic vein and secured in place with 2 sutures, establishing a blood flow of approximately 0.2 mL/min.

Lumen Perfusion and Sample Collection Labeled substrate solution was delivered to the colon segment through a 37°C warming loop at 2.0 mL/min for 1 minute to displace the flush solution. The flow rate was then reduced to 1.0 mL/min and blood collections were begun. Blood samples were collected on ice at 10-minute intervals under mineral oil to avoid loss of carbon dioxide. Luminal perfusion continued for 100 minutes. Whole blood was analyzed immediately after collection for 14C and for 14CO2.16 Expired 14CO2 caught in the liquid trap was quantified also. At the end of the experiment, the perfused segment was removed, blotted, measured, and minced partially by hand. Aliquots of tissue (100 –150 mg wet wt) were digested in tissue solubilizer, and radioactivity was determined as previously described.16

Blood Metabolite Analysis Metabolites for high-performance liquid chromatography (HPLC) analysis were extracted from frozen whole blood

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aliquots using a modified Bligh and Dyer methanol:chloroform (2:1, vol/vol) procedure.18 Equal volumes of blood taken from time points 70, 80, and 90 minutes were pooled and spiked with a solution of unlabeled standards containing 10 mmol/L each of lactate, 3-OH butyrate, acetate, propionate, butyrate, octanoate, decanoate, and dodecanoate. The chloroform phase was transferred to a 5 mL Reacti-Vial (PierceChem Co, Rockford, IL), titrated to pH 8 with 0.1% KOH, and evaporated to dryness using nitrogen gas. The aqueous phase was titrated to pH 8, evaporated to one third its original volume using nitrogen gas, transferred to the Reacti-Vial containing the dried chloroform phase, frozen, and lyophylized. The dried deproteinized samples were derivatized for HPLC analysis19 using 200 ␮L acetonitrile, 100 ␮L bromoacetophenone (50 mg/mL in acetonitrile), and 100 ␮L 18crown-6 ether (25 mg/mL in acetonitrile) at 100°C. After 30 minutes, the samples were evaporated to dryness, redissolved in 50 ␮L of 50% ethanol, and evaporated to dryness again. A second derivatization step was done by redissolving the samples in 350 ␮L acetonitrile, adding another 50 ␮L bromoacetophenone reagent, and reacting at 100°C for an additional 30 minutes. The derivatives were then dried and redissolved in 500 ␮L of 85% acetonitrile for injection. Samples were analyzed by reversed phase HPLC using a 25 ⫻ 0.46 – cm, 5␮m particle size octadecylsilane column (Ultrasphere, Beckman Instruments, Fullerton, CA). The elution rate (1.0 mL/min) and gradient conditions (6 minutes at 30% acetonitrile, then a linear increase in acetonitrile concentration of 1.33% per minute for 6 minutes, then a linear increase in acetonitrile concentration of 7.1% per minute for 7 minutes reaching 100% acetonitrile at 19 minutes) were sufficient to resolve lactic, acetoacetic, 3-OH butyric, acetic, propionic, butyric, octanoic, decanoic, dodecanoic, and oleic acids in 36 minutes. The HPLC column effluent fractions were collected and counted in 15 mL of Hionic Fluor cocktail. All scintillation counting was done in a 1600TR liquid scintillation counter (Packard Instruments, Co., Meriden, CT).

Calculations Calculations were based on the specific activities of substrates and the appearance of radioactivity in mesenteric blood. Total substrate absorption was calculated from total 14C appearing in whole blood. The production of CO2 was calculated from 14CO2 trapped from whole blood. The amount of radioactivity in each remaining metabolite was determined, and subsequently converted to its corresponding fraction (percent) of the radioactivity of all metabolites. By multiplying these fractions with the radioactivity expected to be found (i.e., total 14C minus 14CO2), the quantity of substrate carbon converted into each metabolite could be determined. Metabolism was quantified as the rate at which molar substrate and molar substrate carbon (i.e., adjusted for the number of carbon atoms in each substrate molecule) was absorbed into mesenteric blood. Data are reported as ␩moles substrate or substrate carbon per gram wet weight per minute,

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where wet weight refers to the fresh weight of the blotted colonic segment exposed throughout to the substrate.

Experimental Design and Statistical Analyses Twenty-four rats were randomly assigned to 7 different substrate groups so that a total of 3– 4 rats were exposed to each substrate. As the bile acid cholylsarcosine was used to dissolve dodecanoate and oleate, butyrate was tested without and with cholylsarcosine to investigate any unintended effect of cholylsarcosine on transport or metabolism of substrates. Because of unequal variance among substrates, statistical differences among group means were determined on log-transformed data. When comparing total absorption of substrates and CO2 production, group means were based on the marginal mean value of 3 consecutive 10-minute intervals (70-, 80-, and 90-minute time points) at steady-state conditions (see Results), and a 2-way analysis of variance (ANOVA) on repeated measures was used. When comparing data from the HPLC analysis of pooled blood, a 1-way ANOVA was used. Differences were considered to be statistically significant at P ⬍ 0.05. The Tukey HSD procedure was used as a follow-up test for multiple comparisons. The statistical procedures in SPSS20 were used to perform statistical analyses.

Results Validity of the Perfusion Technique The cannulated colonic segments had an average length of 6.9 ⫾ 0.2 cm (mean ⫾ SE) and a wet weight of 0.94 ⫾ 0.04 g and consisted of the proximal third of the entire colon length. Sufficient substrate was perfused through the cannulated segment to prevent substantial changes to substrate concentration or to availability of substrate to the epithelium during experimentation. On average, ⬍2% of the 10 mmol/L substrates was transported into mesenteric blood during the 100-minute perfusion period (acetate: 1.1% ⫾ 0.1%; butyrate: 1.2% ⫾ 0.2%; butyrate with cholylsarcosine: 1.2% ⫾ 0.2%; octanoate: 3.2% ⫾ 0.6%; decanoate: 2.9% ⫾ 0.3%; dodecanoate: 0.9% ⫾ 0.1%; oleate: 0.1% ⫾ 0.0%). Breath CO2 was quantitatively collected throughout experiments. Blood samples were also taken from the aorta at the end of experiments. The sum of radioactivity in breath CO2 and whole body blood taken from all experiments was 3.7% ⫾ 0.6% and 0.5% ⫾ 0.1%, respectively, of the radioactivity transported into the mesenteric blood. Furthermore, significant differences were not seen for any of these 2 measures between substrates (P ⬎ 0.05), indicating that the procedure effectively prevented transport of luminal perfusate into tissues other than the cannulated intestinal segment regardless of substrate. Of the total radioactivity perfused with substrate solutions, 99.3% ⫾ 0.7% was recovered.

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Figure 1. Influence of perfusion time on the rate of molar substrate transport into mesenteric blood. Blood was collected at 10-minute intervals during the 100-minute perfusion period.

Pooling of Blood Collected for HPLC Analysis During Steady-State Absorption The molar absorption of each substrate had apparently stabilized and reached steady-state conditions after 30 – 60 minutes (Figure 1), and the values of 3 consecutive 10-minute intervals, corresponding to the 60 –90-minute perfusion period, were chosen for statistical analysis. For these 10-minute intervals, no substrate by time interaction was seen (P ⫽ 0.93), allowing the statistical comparison among the corresponding mean values for the 7 substrates (two-way ANOVA). Correspondingly, no time effect within substrate was noted (P ⫽ 0.60), showing that steady state absorption of substrates had taken place during the 60 –90-minute perfusion period. This allowed equivolumetric pooling of blood from this period for later HPLC analysis of metabolites. Steady-State Rate of Molar Substrate Absorption Table 1 shows the rate of total molar substrate absorption and the rate of metabolite production from

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Figure 2. Influence of perfusion time on the rate of molar CO2 transport into mesenteric blood. Blood was collected at 10-minute intervals during the 100-minute perfusion period.

absorbed substrates. Expressed in this manner, the presence of cholylsarcosine in the medium did not significantly affect the absorption or metabolism of butyrate except for a slight decrease in lactate production. Among the substrates, the rate of total octanoate absorption was significantly higher (3– 4-fold) than absorption of acetate, butyrate, or dodecanoate. The rate of total oleate absorption was minimal and was lowest of all substrates. The rate of substrate absorbed and transported into the blood without being metabolized also was significantly higher (4-fold) for octanoate than for acetate, butyrate, or dodecanoate. Steady-state conditions were also achieved for the rates of substrates oxidized to CO2 for the 60 –90-minute perfusion period (no time interaction, P ⫽ 0.33, Figure 2). In addition, no substrate by time interaction was seen for this period (P ⫽ 0.97), allowing statistical analysis using a single mean steady-state value. The molar rate of substrate conversion to CO2 was significantly higher for acetate than for octanoate, decanoate, dodecanoate, or oleate. There was a trend toward decreasing molar con-

Table 1. Steady-State Rate of Total Molar Substrate Absorption and Transport Into Mesenteric Blood During the 60 –90-Minute Perfusion Period of Proximal Rat Colon Converted to other metabolitesb Substrate (10 mmol/L)

Total substrate absorption

Unmetabolized substrate

Converted to CO2

Converted to ketone bodiesa

Acetate (3) Butyrate (4) Butyrate ⫹ cs (4) Octanoate (3) Decanoate (4) Dodecanoate ⫹ cs (3) Oleate ⫹ cs (3) P

140.7 ⫾ 138.1 ⫾ 23.8bc 147.3 ⫾ 21.5bc 462.4 ⫾ 68.3d 259.8 ⫾ 35.9cd 117.8 ⫾ 6.4b 21.9 ⫾ 3.9a ⬍0.001

80.0 ⫾ 69.0 ⫾ 14.8b 81.4 ⫾ 16.2b 413.5 ⫾ 65.6d 231.5 ⫾ 34.8cd 97.1 ⫾ 8.4bc 17.6 ⫾ 4.8a ⬍0.001

45.7 ⫾ 30.9 ⫾ 4.8de 27.6 ⫾ 1.5de 22.6 ⫾ 2.1cd 13.8 ⫾ 1.6c 6.6 ⫾ 0.3b 2.3 ⫾ 0.4a ⬍0.001

5.4 ⫾ 24.9 ⫾ 4.7c 28.5 ⫾ 6.4c 14.1 ⫾ 1.4c 5.3 ⫾ 0.8b 4.3 ⫾ 0.4b 0.0 ⫾ 0.0a ⬍0.001

19.7bc

14.7b

3.6e

1.3b

Converted to lactate 5.4 ⫾ 6.0 ⫾ 0.6e 3.8 ⫾ 0.4cd 2.4 ⫾ 0.4bc 1.3 ⫾ 0.1b 1.3 ⫾ 0.1b 0.0 ⫾ 0.0a ⬍0.001

0.5de

Known

Unknown

1.8 ⫾ 0.2 2.4 ⫾ 0.8 2.6 ⫾ 0.6 4.3 ⫾ 1.2 3.5 ⫾ 0.5 5.9 ⫾ 1.3 1.8 ⫾ 1.0 0.08

2.5 ⫾ 0.4b 5.0 ⫾ 1.2b 3.0 ⫾ 0.3b 5.6 ⫾ 0.8b 4.4 ⫾ 0.8b 2.6 ⫾ 0.6b 0.2 ⫾ 0.1a ⬍0.001

NOTE. Values in nmol substrate/g ⫻ min (mean ⫾ SE). Number per group shown in parentheses. Values within columns with different superscripts are significantly different at P ⬍ 0.05 (Tukey HSD procedure). cs, cholylsarcosine. aAcetoacetate ⫹ 3-OH butyrate. b The known fraction of other metabolites consists of acetate, propionate, butyrate, octanoate, decanoate, dodecanoate, and oleate (perfused substrate not included). The unknown fractions were not further characterized.

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version to CO2 with increasing molecular weight of substrate (Table 1). The rate of substrate converted to ketone bodies was significantly higher for butyrate and octanoate than for acetate, decanoate, dodecanoate, and oleate. Low rates of substrate metabolism to lactate and other minor constituents were noted. Steady-State Rate of Molar Substrate Carbon Absorption In terms of energy available to the tissue, the rate of substrate carbon absorption is relevant and was calculated by multiplying the rates of molar substrate absorption by the number of carbon atoms in each corresponding substrate molecule. The rate of total molar substrate carbon absorption was significantly higher for octanoate, decanoate, and dodecanoate than for acetate, butyrate, or oleate (Table 2). Over 13-fold more substrate carbon was transported into the blood with luminal perfusion of octanoate than with acetate. The rate of molar substrate carbon transported into mesenteric blood as unmetabolized substrate was again significantly higher for octanoate, decanoate, and dodecanoate than for acetate, butyrate, and oleate. The rate of CO2 production from substrates was higher for octanoate than for acetate, dodecanoate, or oleate (Table 2). In fact, the CO2 production from octanoate was 2-fold higher than from acetate and dodecanoate and was 4-fold higher than from oleate. The rate of substrate carbon converted to ketone bodies was significantly lower for acetate and oleate than for all the other substrates. Statistical differences between the rates of butyrate carbon absorption or metabolism without and with cholylsarcosine were not seen.

Relative Absorption of Unmetabolized Substrate and Metabolites The relative proportion of substrate transported into mesenteric blood without being metabolized was substantial (Table 3) at 80%–90% for octanoate, decanoate, and dodecanoate, which exceeded the approximate 50%–55% proportions for acetate and butyrate (P ⬍ 0.05). The corresponding proportions of substrate oxidized to CO2 were around 5% of absorbed substrate for the MCFAs (octanoate, decanoate, and dodecanoate), 10% for oleate, and 20%–33% for the SCFAs, acetate, and butyrate (P ⬍ 0.05). Proportions of lactate and other known and unknown metabolites were low for all substrates.

Discussion The aim of developing this in vivo method was to restrict absorption and metabolism of substrates to a selected colonic segment. As also seen in our previous study,16 an insignificant amount of labeled substrates unintentionally escaped from the lumen of the perfused segment. This radioactivity amounted to about 4% of that transported into mesenteric blood, rendering an accurate assessment of the fate of luminal substrates possible. Previously, absorption of FAs has been estimated by measuring their disappearance from the intestinal lumen.9,21–23 In studies using the rat jejunum, appearance of 14C label in portal blood has also been used to quantitate FA absorption.24 In the present study, FAs and metabolites were measured in blood draining a continu-

Table 2. Steady-State Rate of Total Substrate Carbon Absorption and Transport Into Mesenteric Blood During the 60 –90-Minute Perfusion Period of Proximal Rat Colon Converted to other metabolitesb Substrate (10 mmol/L)

Total substrate absorption

Unmetabolized substrate

Acetate Butyrate Butyrate ⫹ cs Octanoate Decanoate Dodecanoate ⫹ cs Oleate ⫹ cs P

281.3 ⫾ 552.5 ⫾ 95.2a 589.2 ⫾ 86.0a 3699 ⫾ 546.4c 2597 ⫾ 358.9bc

159.9 ⫾ 276.1 ⫾ 59.1a 325.6 ⫾ 64.6a 3307 ⫾ 524.9c 2315 ⫾ 347.5bc

1413 ⫾ 76.8b 393.7 ⫾ 70.4a ⬍0.001

1165 ⫾ 101.2b 316.8 ⫾ 87.3a ⬍0.001

39.4a

29.5a

Converted to CO2 91.3 ⫾ 123.4 ⫾ 110.2 ⫾ 180.4 ⫾ 137.6 ⫾

7.2b 19.3bc 6.2bc 17.0c 16.1bc

79.7 ⫾ 3.0b 40.7 ⫾ 7.7a ⬍0.001

Converted to ketone bodiesa

Converted to lactate

Known

Unknown

10.7 ⫾ 99.6 ⫾ 18.7c 115.8 ⫾ 25.6c 112.8 ⫾ 11.1c 52.8 ⫾ 7.8c

⫾ ⫾ ⫾ ⫾ ⫾

3.6 ⫾ 9.7 ⫾ 3.0ab 10.6 ⫾ 2.3ab 34.5 ⫾ 9.6bc 34.9 ⫾ 4.6bc

4.9 ⫾ 0.8ab 19.9 ⫾ 4.6bcd 11.8 ⫾ 1.2bc 44.4 ⫾ 6.5d 44.1 ⫾ 8.1d

70.5 ⫾ 15.7c 31.9 ⫾ 17.7ac ⬍0.001

30.7 ⫾ 7.7cd 3.2 ⫾ 2.0a ⬍0.001

2.6b

51.5 ⫾ 4.9c 0.5 ⫾ 0.5a ⬍0.001

10.9 23.8 15.2 19.0 13.0

1.1b 2.6c 1.5bc 3.5bc 1.5bc

15.8 ⫾ 0.7bc 0.6 ⫾ 0.6a ⬍0.001

0.4a

NOTE. Values in nmol substrate carbon/g ⫻ min (mean ⫾ SE). Values within columns with different superscripts are significantly different at P ⬍ 0.05. (Tukey HSD procedure). cs, cholylsarcosine. aAcetoacetate ⫹ 3-OH butyrate. bThe known fraction of other metabolites consists of acetate, propionate, butyrate, octanoate, decanoate, dodecanoate, and oleate (perfused substrate not included). The unknown fractions were not further characterized.

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Table 3. The Relative Proportions of Substrate Absorbed and Transported Into Mesenteric Blood During the 60 –90-Minute Perfusion Period of Proximal Rat Colon Other metabolitesb Substrate (10 mmol/L) Acetate Butyrate Butyrate ⫹ cs Octanoate Decanoate Dodecanoate ⫹ cs Oleate ⫹ cs P

Total 100 100 100 100 100 100 100

Unmetabolized substrate

CO2

Ketone bodiesa

Lactate

Known

Unknown

56.1 ⫾ 49.4 ⫾ 3.2a 54.1 ⫾ 5.0a 89.1 ⫾ 1.4c 88.7 ⫾ 1.2c 82.1 ⫾ 2.6c 77.7 ⫾ 10.1bc ⬍0.001

33.1 ⫾ 22.6 ⫾ 0.6c 19.7 ⫾ 3.1c 5.0 ⫾ 0.7a 5.4 ⫾ 0.5a 5.7 ⫾ 0.2a 10.7 ⫾ 3.0b ⬍0.001

3.7 ⫾ 18.2 ⫾ 1.7c 19.6 ⫾ 2.3c 3.1 ⫾ 0.4b 2.1 ⫾ 0.4b 3.7 ⫾ 0.5b 0.1 ⫾ 0.1a ⬍0.001

3.9 ⫾ 4.5 ⫾ 0.3c 2.7 ⫾ 0.4b 0.5 ⫾ 0.0a 0.5 ⫾ 0.0a 1.1 ⫾ 0.0a 0.2 ⫾ 0.2a ⬍0.001

1.4 ⫾ 0.4 1.7 ⫾ 0.5 1.8 ⫾ 0.3 1.0 ⫾ 0.4 1.5 ⫾ 0.3 5.1 ⫾ 1.3 10.4 ⫾ 6.7 0.07

1.8 ⫾ 0.4ab 3.6 ⫾ 0.6b 2.1 ⫾ 0.2ab 1.2 ⫾ 0.0ab 1.8 ⫾ 0.5ab 2.2 ⫾ 0.6ab 1.0 ⫾ 0.7a 0.04

3.0ab

2.5c

0.5b

0.2c

NOTE. Values in percent (mean ⫾ SE). Values within columns with different superscripts are significantly different at P ⬍ 0.05 (Tukey HSD procedure). cs, cholylsarcosine. aAcetoacetate ⫹ 3-OH butyrate. b The known fraction of other metabolites consists of acetate, propionate, butyrate, octanoate, decanoate, dodecanoate, and oleate (perfused substrate not included). The unknown fractions were not further characterized. The other known metabolites were primarily octanoate and decanoate (2.1% and 1.4%, respectively) for dodecanoate ⫹ cs substrate and were primarily acetate, decanoate, and dodecanoate (2.3%, 1.7%, and 5.2%, respectively) for oleate ⫹ cs substrate.

ally perfused colonic segment. This method offers the advantage of an equal comparison of the absorption of FAs at steady-state conditions (unchanged concentrations in luminal perfusate, steady-state absorption rates), which is not true for methods measuring disappearance. In addition, our in vivo model enabled us to determine the degree to which the colonic epithelium uses FAs, as well as to characterize and quantify the metabolites that were made available to the rest of the body. Total Absorption Our direct measurements of the rates of MCFA absorption in vivo support previous indications of colonic absorption of MCFAs.5–10 The total molar absorption of octanoate was over 3 times higher than that for either acetate or butyrate (Table 1). Additionally, in terms of the total energy available to colonic or other tissues, the differences between rates of molar absorption of substrate carbon from MCFAs and SCFAs were even more pronounced (Table 2). It is well-recognized that octanoate and decanoate leave the small intestinal mucosa almost exclusively by the portal blood route,4,25–27 whereas some controversy exists as to the preferred route of dodecanoate transport.28 –30 Therefore, it is possible that the absorption of dodecanoate would have been even higher than now reported if our model had allowed for the study of substrate absorption via the lymphatic vessels. Lymphatic absorption of dodecanoate would, however, require the ability of the colonic mucosa to esterify FAs, which may not be fulfilled. In this study, MCFAs provided up to 13 times the level of substrate carbon to the rat colon compared with

equimolar SCFAs (Table 2). If the present data can be extended to the human colon, a diet rich in MCT would be highly favorable in terms of whole body energy supply. Besides small bowel resected patients with a preserved colon, who have been shown to benefit from a partial replacement of long-chain triglycerides with MCT,10 other patients with compromised small-bowel function could gain from dietary MCT as well. This could be patients with pancreatic insufficiency (e.g., chronic pancreatitis) and patients with insufficient biliary secretion (caused by decreased production, extrahepatic biliary obstruction, or decreased reabsorption) because the absorption of MCT (either as MCT or MCFAs) is not dependent on the presence of pancreatic lipase or bile acids.4,31 The bile acid cholylsarcosine was used to solubilize dodecanoate and oleate. Contrary to other bile acids, cholylsarcosine is not deconjugated or dehydroxylated by the colonic flora.32 Hypothetically, it may therefore keep its ability to form mixed micelles in colonic contents without causing or worsening diarrhea, which determined our choice of bile acid.32 Cholylsarcosine improves intestinal absorption of fat33–35 in short bowel patients either with or without a colon who have lost the primary site of bile acid reabsorption through terminal ileum resection. In addition, cholylsarcosine did not alter epithelial transport or metabolism of butyrate (Table 2), suggesting that it has no negative influence on the transport and metabolism of other FAs. Thus, this compound could potentially be used as an adjuvant in colonic fat absorption.

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The molar rate of total oleate absorption was low compared with the rates for both SCFAs and MCFAs (Table 1) despite complete solubilization into micelles with cholylsarcosine. This low transport rate may be expected for a tissue that only rarely is exposed to significant levels of dietary LCFAs. In this study, the total carbon available from oleate in mesenteric blood was equivalent to the level of that from SCFAs (Table 2), suggesting that LCFAs, if available and solubilized in the colonic lumen, may have the potential to provide substantial energy. To our knowledge, only 2 reports have dealt with LCFA absorption by the colon.36,37 Although suggesting that some colonic absorption of LCFAs exists, data from those 2 studies were based on fecal recovery of LCFAs after cecal instillation, i.e., LCFAs were not recovered in either blood or lymph. Our present report is the first, to our knowledge, that shows direct evidence that the colonic mucosa can both transport and metabolize LCFAs. Although further studies would be needed to clarify the extent to which colonic absorption of LCFAs can contribute to the energetic needs of humans with a poorly functioning small intestine, the current data provide an opportunity for performing a predictive test. To determine the extent to which FAs absorbed from the colon could provide energy for the animals used in this study, a theoretical calculation was performed. This calculation assumed that all FA carbon absorbed into the mesenteric blood would eventually be oxidized to provide energy for the animal. It assumed also, based on previous observations, that absorption would be similar along the length of the colon, and that there would be no diurnal variations in FA absorption, assumptions that have been used previously to calculate daily colonic SCFA transport.38 – 40 These calculations indicated that colonic absorption of FAs could provide nearly 25% of the energy needed to meet the basal metabolic needs of the animal (Table 4). The MCFAs, octanoate and decanoate, contributed substantially more energy under our experimental conditions than did either the SCFAs or LCFA evaluated. Although these calculations must be used cautiously, they support the notion that the colon may, under certain circumstances, be exploited as a critical site for absorption of energy-providing substances. Present evidence is not conclusive as to the mechanism of SCFA absorption, and this issue has only been sparsely addressed as regards to MCFAs. Paracellular transport may well be one mechanism by which FAs move from lumen to blood, but it is probably not the sole mechanism because metabolites would then not be present in blood. However, the fact that the absorption of the larger

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Table 4. Theoretical Energy Provided to the Rat by Colonic Absorption of FAs Substrate (10 mmol/L)

Energy to rat (kJ/day)a

Contribution to BMRb (%)

Acetate Butyrate Octanoate Decanoate Dodecanoate Oleate Total

0.6 1.6 12.0 8.6 4.8 1.4 29.0

0.5 1.3 10.0 7.2 4.0 1.1 24.1

aNet

energy theoretically available to an animal from an FA absorbed into mesenteric blood ⫽ [ATP produced from flux of FA to acetyl-CoA ⫺ ATP used in the activation of the FA molecule ⫹ ATP produced from flux of the FA-derived acetyl-CoA units to CO2 via TCA cycle metabolism]. To express this ATP as energy available to an animal (kJ/day), the ATP (nmol/g colon ⫻ min) was multiplied by appropriate factors for average weight of colonic tissue/animal (4 g colon/rat), minutes in a day (1440 min/day), complete conversion of ATP to kJ (74 kJ/mol ATP),41 and conversion of nmol to mol (1/109). Calculations were performed using the following formula: [(FA absorption (nmol/g ⫻ min) ⫻ 5 nmol ATP produced per ␤-oxidation cycle ⫻ number of ␤-oxidation cycles needed to convert FA to acetyl-CoA units) ⫺ (FA absorption (nmol/g ⫻ min) ⫻ 2 nmol ATP used for activating each nmol of FA) ⫹ (FA absorption (nmol/g ⫻ min) ⫻ 12 nmol ATP produced per nmol acetyl-CoA oxidized via the TCA cycle ⫻ number of acetyl-CoA units produced from the FA)] ⫻ 4 g colon/rat ⫻ 1440 min/day ⫻ 74 kJ/ATP ⫻ 1/109. b The BMR of an adult rat was assumed to be 120 kJ.42

molecular weight MCFAs exceeded that of SCFAs goes against the notion of predominantly paracellular absorption of SCFAs and MCFAs. Alternative methods have been used by other investigators to estimate the colonic absorption of FAs. In several studies, the disappearance of FAs from dialysis bags in the rectal lumen of humans has been used to predict colonic transport rates.9,21,22 In those studies, disappearance rates of FAs were calculated as ␮mol 䡠 cm⫺2 䡠 h⫺1. Expressing our data in these units and normalizing for the different FA concentrations used in the experiments (assuming a linear relation between the concentration of FAs and absorption, which seems to apply for SCFAs in vivo16) allows a direct comparison with the data of others. Our method shows absorption rates of 46%, 48%, and 56% for acetate, and 43% and 71% for butyrate when compared with the disappearance method.9,21 For octanoate and decanoate, our in vivo determination yielded 163% and 82% of the values determined in humans using the dialysis bag method.9 When compared with data obtained in vivo by measuring the disappearance of SCFAs from the rat cecum,23 our data had corresponding values of 37% and 35% for cecal absorption of acetate and butyrate, respectively. Some of the observed differences can probably be ascribed to the use of different methodologies or species but also, in part,

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to the difficulty in accurately determining surface areas that are comparable across studies. Production of Metabolites by the Colonic Epithelium The metabolism of SCFAs by the colonic epithelium has attracted much attention because a decreased ability of colonocytes to use butyrate in vitro has been linked to various disease states of the colon, such as ulcerative colitis and the tumorigenic process of colon cancer.43,44 Data on epithelial in vivo metabolism of FAs are few16 and differ substantially from data obtained with the use of isolated colonocytes. Thus, maximum butyrate oxidation to CO2 in vivo seems to occur at a concentration somewhere between 5 and 10 mmol/L,16 several-fold higher than reported with isolated colonocytes,13,15,45 and this prompted the use of a substrate concentration of 10 mmol/L. Production of CO2. Energetic needs of the colonic epithelium are primarily met by the production of CO2 from butyrate oxidation.11,12 In agreement with previous in vitro results,15 the in vivo production of CO2 from octanoate and decanoate in this study was equivalent to that from butyrate (Table 2). Thus, epithelial cells can fulfill their basic energy requirements as easily from octanoate and decanoate as from the SCFAs. All of the substrates used in this study were 14C labeled on the number one carbon and all will generate a 1-14C-acetyl group in the first step of oxidation to CO2. Valid comparisons between substrates can be made regarding the relative rates of substrate use. Because all of the acetyl CoA groups (labeled or unlabeled) released via ␤-oxidation of a FA are expected to enter a common pool, this approach can be used to quantitate total CO2 production. The CO2 production data presumes that each molecule of substrate from which a labeled CO2 is produced is entirely oxidized to acetyl CoA. In addition, our approach assumes that CO2 produced by the colonic epithelium is fully transported to the mesenteric blood. Although these assumptions may be in error to a limited extent, any error would exist equally for all substrates. At first glance, the low rate of CO2 production from oleate is noteworthy (Table 2). This might be attributable to low mitochondrial transport because carnitine was not provided in this experiment. Further observation indicates, however, that CO2 production was in proportion to overall transport (Table 3), suggesting that the mitochondrial membrane is not likely the rate limiting step for oleate oxidation by colonic mucosa in vivo. Production of ketone bodies and lactate. Our in vivo results support previous in vitro findings that ketone bodies are major metabolites of butyrate and oc-

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tanoate, but not of acetate.15 In particular, the ratio of CO2:ketone bodies was 10:1 for acetate, 1:1 for butyrate, and 1.5:1 for octanoate. Although only 3% of the absorbed octanoate was converted to ketone bodies because of its high molar absorption, it was equivalent to butyrate in the molar production of ketone body carbon transported into the bloodstream and made available to other tissues. Using isolated colonocytes, others have reported that acetate and butyrate, in contrast to glucose, also did not stimulate lactate production.12,13,46 In the present study as well, lactate production from the MCFAs was not different from that from the acetate or butyrate. Absorption of unmetabolized substrate. The production of the 2 major metabolites, CO2 and ketone bodies, seem to follow saturation kinetics in vivo,16 and substantial proportions of substrates absorbed by the epithelium were not further metabolized (Table 3). This is particularly evident in the MCFA substrates in which total absorption of substrate carbon was highest. Octanoate and decanoate provided 12-fold and 8-fold increases relative to equimolar butyrate, respectively, in unmetabolized substrate carbon. Once transported into the bloodstream, the fate of these FAs is probably complete oxidation by the liver27 or peripheral tissues. Comparing Colonic Absorption of FAs With That of the Small Intestine Although wide histologic differences prevail between the colon and the small intestine, we have compared our data with similar data obtained by others in the small intestine. Because factors such as perfusion rate,47 composition of perfusion solution,48 and choice of method may influence the absorption of FAs, our data were compared with those of Westergaard et al.47 They investigated the disappearance of SCFAs and MCFAs from solutions perfused through segments of the rat jejunum in vivo, using a perfusion rate (1.5 mL/min) close to ours (1 mL/min). To make a meaningful comparison, the original data of the 2 studies were adjusted for surface area and to a substrate concentration of 1.0 mmol/L. In this manner, our total substrate absorption data corresponded to an apparent permeability of 1.5, 4.3, 2.5, and 1.0 ⫻ 10⫺2 nmol 䡠 cm⫺2 䡠 s⫺1 for butyrate, octanoate, decanoate, and dodecanoate, respectively. The jejunal data presented by Westergaard et al.47 were converted to corresponding values of 9.4, 13.0, 14.8, and 14.1 ⫻ 10⫺2 nmol 䡠 cm⫺2 䡠 s⫺1, respectively. Some of the differences noted can probably be explained by a larger true surface area of the small intestine than colon as well as by a faster perfusion rate used in their study. In comparison to the colon, absorption from the small in-

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testine was 3– 6 times higher for butyrate, octanoate, and decanoate, and 14 times higher for dodecanoate. This is expected because the jejunum is the primary site of LCFA absorption, and lymphatic absorption of FAs was precluded in our model. The lower rate of dodecanoate absorption can be attributed to a lower monomeric concentration in the perfusate and perhaps, in addition, to the inability of the colonic epithelium to esterify fatty acids. In conclusion, by the use of an in vivo rat model, we have shown that the colonic epithelium serves to absorb and partially metabolize MCFAs and that absorption of MCFAs can exceed that of SCFAs in relation to the total carbon absorbed. Octanoate and decanoate are comparable with butyrate in meeting the energy requirement of the colonic epithelium while providing 4 – 6-fold increases in total energy transported into the bloodstream. If these results can be extended to the human colon, dietary replacement of LCFA with MCFA in patients with compromised small bowel absorption may result in increased available energy through colonic absorption of MCFAs.

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15. Jørgensen JR, Clausen MR, Mortensen PB. Oxidation of shortand medium-chain C2-C8 fatty acids in Sprague–Dawley rat colonocytes. Gut 1997;40:400 – 405. 16. Fitch MD, Fleming SE. Metabolism of short-chain fatty acids by rat colonic mucosa in vivo. Am J Physiol 1999;277:G31–G40. 17. Biological values. In Lab chows: the control factor. St. Louis, MO: Ralston-Purina Co., Inc., 1977. 18. Kates M. Techniques of lipidology; isolation, analysis and identification of lipids. In: Work TS, Work E, eds. Laboratory techniques in biochemistry and molecular biology. New York: American Elsevier, 1975;351–352. 19. Bleiberg B, Steinberg JJ, Katz SD, Wexler J, LeJemtel T. Determination of plasma lactic acid concentration and specific activity using high-performance liquid chromatography. J Chromatogr 1991;568:301–308. 20. SPSS. SPSSX User’s guide. 2nd ed. Chicago: SPSS, 1986. 21. McNeil NI, Cummings JH, James WPT. Short chain fatty acid absorption by the human large intestine. Gut 1978;19:819 – 822. 22. McNeil NI, Cummings JH, James WPT. Rectal absorption of short chain fatty acids in the absence of chloride. Gut 1979;20:400 – 403. 23. Fleming SE, Choi SY, Fitch MD. Absorption of short-chain fatty acids from the rat cecum in vivo. J Nutr 1991;121:1787–1797. 24. Bernard A, Carlier H. Absorption and intestinal catabolism of fatty acids in the rat: effect of chain length and saturation. Exp Physiol 1991;76:445– 455. 25. Hashim SA, Krell K, Mao P, Van Itallie TB. Portal venous transport of free pelargonic acid following intestinal instillation of tripelargonin. Nature 1965;207:527–528. 26. Kiyasu JY, Bloom B, Chaikoff IL. The portal transport of absorbed fatty acids. J Biol Chem 1952;199:415– 419. 27. Fernandes J, Van de Kamer JH, Weijers HA. The absorption of fats studied in a child with chylothorax. J Clin Invest 1955;34: 1026 –1036. 28. Bloom B, Chaikoff IL, Reinhardt WO. Intestinal lymph as pathway for transport of absorbed fatty acids of different chain lengths. Am J Physiol 1951;166:451– 455. 29. Isselbacher KJ. Mechanisms of absorption of long and medium chain triglycerides. In: Senior JR, ed. Medium chain triglycerides. Philadelphia: University of Pennsylvania Press, 1968:21. 30. Sigalet DL, Winkelaar GB, Smith LJ. Determination of the route of medium-chain and long-chain fatty acid absorption by direct measurement in the rat. J Parenter Enteral Nutr 1997;21:275–278. 31. Jensen MM, Christensen MS, Høy C-E. Intestinal absorption of octanoic, decanoic, and linoleic acids: effect of triglyceride structure. Ann Nutr Metab 1994;38:104 –116. 32. Lillienau J, Schteingart CD, Hofmann AF. Physicochemical and physiological properties of cholylsarcosine. A potential replacement detergent for bile acid deficiency states in the small intestine. J Clin Invest 1992;89:420 – 431. 33. Gruy-Kapral C, Little KH, Fordtran JS, Meziere TL, Hagey LR, Hofmann AF. Conjugated bile acid replacement therapy for shortbowel syndrome. Gastroenterology 1999;116:15–21. 34. Heydorn S, Jeppesen PB, Mortensen PB. Bile acid replacement therapy with cholylsarcosine for short-bowel syndrome. Scand J Gastroenterol 1999;34:818 – 823. 35. Weinaud I, Hofmann AF, Jordan A, Caspary F, Stein J. Cholylsarcosine use for bile acid replacement in short bowel syndrome (SBS) (abstr). Gastroenterology 1999;116:G0441. 36. Ammon HV, Phillips SF. Inhibition of colonic water and electrolyte absorption by fatty acids in man. Gastroenterology 1973;65: 744 –749. 37. Segal L, Kneip J, Levitt MD. Fate of oleate in the colon of the rat. J Lab Clin Med 1990;115:249 –253. 38. Marty J, Vernay M. Absorption and metabolism of the volatile fatty acids in the hind-gut of the rabbit. Br J Nutr 1984;51:265–277.

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39. Luciano L, Reale E, Rechkemmer G, von Engelhardt W. Structure of zonulae occludentes and the permeability of the epithelium to short-chain fatty acids in the proximal and the distal colon of guinea pig. J Membr Biol 1984;82:145–156. 40. Hoverstad T. Studies of short-chain fatty acid absorption in man. Scand J Gastroenterol 1986;21:257–260. 41. Newsholme EA, Leech AR. Biochemistry for the medical sciences. New York: Wiley, 1983:19. 42. Kleiber M. The fire of life. An introduction to animal energetics. New York: Wiley, 1961:205. 43. Roediger WE. The colonic epithelium in ulcerative colitis: an energy-deficiency disease? Lancet 1980;2:712–715. 44. Zhang J, Wu G, Chapkin RS, Lupton JR. Energy metabolism of rat colonocytes changes during the tumorigenic process and is dependent on diet and carcinogen. J Nutr 1998;128:1262–1269. 45. Kight CE, Fleming SE. Nutrient oxidation by rat intestinal epithelial cells is concentration dependent. J Nutr 1993;123:876 – 882. 46. Ardawi MS, Newsholme EA. Fuel utilization in colonocytes of the rat. Biochem J 1985;231:713–719.

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47. Westergaard H, Holtermu¨ller KH, Dietschy JM. Measurement of resistance of barriers to solute transport in vivo in rat jejunum. Am J Physiol 1986;250:G727–G735. 48. Holtug K, Hove H, Mortensen PB. Stimulation of butyrate absorption in the human rectum in vivo. Scand J Gastroenterol 1995; 30:982–988. Received May 25, 2000. Accepted November 22, 2000. Address requests for reprints to: Sharon E. Fleming, Ph.D., Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA 94720-3104. e-mail: [email protected]; fax: (510) 642-0535. Supported by the following foundations: P. Carl Petersen, Beckett, Codan, Ferring, Jacob Madsen & Olga Madsen, The Danish Foundation for the Advancement of Medical Science, and the Danish Medical Research Council; and by the Agriculture Experiment Station (U.S.). The authors thank Mark Hudes, Ph.D., University of California, Berkeley, California, for statistical consultation, and Klavs Holtug, M.D., Copenhagen University Hospital, The Rigshospital, for helpful discussion regarding the mechanism of fatty acid absorption.