Effects of Calcium and Magnesium Ions Upon Fat Absorption by Sacs of Everted Hamster Intestine

Vol. 73, No.2

73:421-424, 1977 Copyrigh t © 1977 by the American Gastroenterological Association

GAST ROENTEROLOGY

Printed in U.S A.

RAPID COMMUNICATIONS EFFECTS OF CALCIUM AND MAGNESIUM IONS UPON FAT ABSORPTION BY SACS OF EVERTED HAMSTER INTESTINE Elliott W. Strauss, Division of Biolog ical and Medical Sciences Brown University; Providence, Rhode Island 02912 ABSTRACT. Sacs of everted hamster jejunum were incubated with fatty acid and mono g lyceride in bile salt solutions either with or without added salts of calcium and magnesium. At very low concentrations of ca2+ and Mg2+, the intestine produced very few chylomicrons. However, at physiological concentrations of the ions, the g ut-preparation released numerous chylomicron-like particles into the serosal fluid. Previous investi gations of fat absorption employed the everted hamster intestine for incubations with bile salt micellar solutions containin g very low concentrations of calci urn and magnes i urn ions 1, 2, 3, The results provided important information about the uptake of fatty acid and monoglyceride, and the resynthesis of triglyceride, but the gut-preparation translocated very little lipid from the mucosal to the serosal fluid. Completion of the transfer would have been equivalent to the absorption of fat in vivo. It was possible that lipia-uptake and resynthesis occurred normally in vitro, but that deterioration of-rissues durin g isolation prevented chylomicron production, the final step during the absorption of fat. I t also was possible that a deficiency in the incubation medium rather than in the tissues was primarily responsible for the defective chylomicronproduction . In view of the theoretical importance of the experiments with the everted hamster intestine with respect to mechanisms of fat absorption, it seemed important to find out if there were suitable conditions under which the sacs would absorb fat. MATERIALS AND METHODS Fasted hamsters were sacrificed and the jejunum removed. The tissue was everted, washed, and segments (2 - 3 em) were fashioned into sacs 4 . Each everted sac was incubated in 3 ml of the lipid-containing solutions indicated below. During control incubations, the mucosal

fluid contained either Krebs-Rin g er phosphate buffer 5, pH 6.3 or 7.4, or the bicarbonate buffer s, pH 7.4, without added salts of calcium and magnesium. Each ml included 2.4 ~moles of sodium taurodeoxycholate, 0.6 ~moles of [l- 1 '*C]- l1nolenic or oleic acid (1. 6 ~Ci/~mole), 0.3 ~moles of 2-monoolein, and 2 mg of glucose. The experimental, divalent cation incubations used media which were similar to the control media except for added CaCl 2 and Mgso 4 (or Mg Cl 2 ) at concentrations wh~ch were 2.5 and 1.2 mM, respectively. The serosal fluid consisted of the same buffer added to the mucosal side but without added glucose or lipid. Flasks containing phosphate buffer were gassed with 100 % 0 2 ; those containing bicarbonate buffer were gassed with 5 % C0 2 , 95 % Oz. Incubation was at 37° for 60 minutes. Lipids were extracted from acidified fluids and homo genized tissues as described by Johnston and Borg strom 1, Chromato g raphy was performed by the method of Brown and Johnston 6, Samples were dried under nitrogen, dissolved in benzene and portions were counted in duplicate in a liquid scintillation counter. The sodium taurodeoxycholate was a gift from Dr. John M. Johnston, University of Texas, Southwestern Medical School, Dallas, Texas, and Dr. Alan F. Hofmann, Mayo Clinic Foundation, Rochester, Minn. The fatty acids were purchased from Applied Science Laboratories, State Colle g e, Penn. The 2-monoolein was supplied through the generosity of Dr. F. H. Mattson,

Submitted April 22, 1977 421

TABLE 1 Effect of ca2+ and Mg2+ upon translocation of 14 C-labeled total lipids and tri glycerides by sacs of everted hamster jejunum. The segment of jejunum nearest to the duodenum was called #1, the next jejunal segment, #2, and so on. Total lipids entering serosal fluid were expressed as counts/min/mg of intestine wet weight (CPM/mg), and triglycerides as percentage of the total lipids (%TG). The sacs were incubated with 14 C-labeled linolenic (LEN) or oleic (OLEIC) acid in media at pH 6.3 or 7.4 usin~ phosphate (P0 4 ) or bicarbonate (Hco 3 ) buffer. ca2+ and Mg2 were either added to(+), or deleted from (0), the medium. Buffer

Seg #

ca2+ + Mg2+

6.3 6.3 7.4 7.4

P04 P04 P04 P0 4

1 2 1 2

0 + 0 +

LEN LEN

7.4 7.4

HC03 HC03

1 2

0 +

LEN

7.4

HC0 3

1

OLEIC OLEIC OLEIC

7.4 7.4 7.4

PO 4 P0 4 P04

OLEIC OLEIC

7.4 7.4

OLEIC OLEIC

7.4 7.4

Fatty Acid

pH

LEN LEN LEN LEN

cpm/mg (± SE) 17 988 8 1340

-n 1

%TG

1 1

49 96 18 94

25 (± 2) 820 (± 320)

3 3

12 (± 3) 87 (± 5)

+

2008 (± 354)

5

1 2 3

+ + +

1470 1750 1570

2 1 1

P04 Po 4

1 2

0 0

64 47

2 1

HC0 3 HC0 3

1 2

+ 0

658 26

1 2

Procter and Gamble Co., Cincinnati, Ohio. The chemical and radiochemical purities of the lipids were confirmed by means of qualitative and quantitative thin-layer and gas-liquid chromatography. Electrolytes were of analytical grade and water was distilled in glass.

1

90 43

from mucosal to serosal solution, the tissue took up a large amount of lipid from the control medium. However, durin g the divalent cation incubations, the sacs translocated considerable lipid into the serosal fluid which became turbid and contained as much as 10% of tracer which had been present originally in the mucosal fluid. Triglycerides comprised 78-96% of the total translocated lipid. The presence of divalent cations markedly stimulated the appearance of radioactive-triglyceride on the serosal side (Table 1) During the incubations with added ca2+ and Mg2+, the serosal fluid contained numerous particles having Brownian motion as seen by means of dark field microscopy. The serosal fluid was centrifuged in a

RESULTS The experiments in Table 1 demonstrate that very little radioactive lipid was translocated from mucosal to serosal solution during incubations in media without added ca2+ and Mg2+ , and that triglycerides were only a small part of the total radioactive lipid. Most of the radioactivity (99.5%) on the serosal side was extracted with acid ether-heptane. In spite of the low net transfer of lipid 422

The total lipid and the triglyceride content in a sac at each of these four time intervals are given in Fig. 2, indicating that triglyceride was released increasingly with time. The values indicating lipid translocation in Table 1 have no apparent relationship to the kind of fatty acid, pH, type of buffer, or the location of the intestinal segment in vivo.

Beckman 40.3 rotor under a force of 106 G min. Particles containing 60% of the total radioactive lipid were found in the upper layer of fluid comprising 33% of the total volume. These properties are consistent with those described for chylomicrons 7,8. Electron micrographs of the centrifuged serosal fluid indicated that the fluid contained chylomicronlike particles having a diameter of about 0.1 microns (Fig. 1).

800 w

::>

(/') (/')

..... (.!)

400

::E

'::E

CL

u

0-15

15-30

30-45 45-60

MINUTES

Fig. 2 The average time course for lipid secretion in two experiments. The total height of a bar indicates total 14 C-lipids entering serosal fluid during each 15 min period. The height of the stippled bar indicates the released triglycerides.

DISCUSSION The addition of ca2+ and Mg2+ to lipid-containing media permitted considerable net translocation of lipid including the release of chylomicron-like particles by sacs of everted hamster intestine. The translocation of lipid from mucosal to serosal solution by this preparation of the gut resembled rat absorption in the living animal. The impaired translocation at very low concentrations of ca2+ and Mg2+ resulted evidently from the composition of the media rather than an originally defective tissue preparation. According to the present results, the necessary concentrations of electrolytes in a medium for fat absorption in vitro included those of ca2+ or Mg2+ or ca2+ and Mg2+. The influence of pH or type of buffer upon lipid secretion were not apparent in the

Fig. 1 Transmission electron micrograph of chylomicron-like particles.

The time course for the release of radioactive triglyceride and total lipids into the serosal fluid was measured. For the experiments a small glass cannula was tied to one end of the sac, and 1 ml of fluid introduced. At the end of 15 minutes incubation the entire contents were removed and replaced by fresh buffer. This process was repeated at the end of 30, 45 and 60 minutes-. 423

present study although such effects were not ruled out. The reasons were not entirely clear for effects of external ca2+ and Mg2+ upon the production of chylomicron-like particles. In additional experiments involving morphological study of briefly incubated tissue, supplements of ca2+ and Mg2+ affected the intracellular mechanisms for lipid transfer 9. Results of prolonged incubations such as 60 min suggested that additions of ca2+ and Mg2+ also protected the viability of the tissues. Whatever the mechanisms, the present results demonstrated that incubations of everted hamster intestine in media with added ca2+ and Mg2+ may be used advantageously for studying the intestinal secretion of triglyceride.

6. Brown JL, Johnston JM: Radioassay of lipid components separated by thin-layer chromatography. J Lipid Res 3: 480-481. 1962 7. Dole VP. Hamlin JT III: Particulate fat in lymph and blood. Physio Rev 42: 674-701, 1962 8. HatchET, Lees RS: Practical methods for plasma lipoprotein analysis. In Advances in Lipid Research. Edited by R Paoletti and D Kritchevsky. New York, London, Academic Press, 1968 p 1-68 9. Strauss EW: Importance of Ca2+ and Mg2+ for fat absorption in vitro (abstract). Gastroenterology 68: 992' 1975

Supported in part by a grant, 2 ROl AM13186 from the National Institutes of Health, and by a grant from the American Cancer Society, Rhode Island Division, Inc.

REFERENCES 1. Johnston JM, Borgstrom B: The intestinal absorption and metabolism of micellar solutions of lipids. Biochim Biophys Acta 84: 412-423, 1964 2. Kern F Jr, Borgstrom B: Quantitative study of the pathways of triglyceride synthesis by hamster intestinal mucosa. Biochim Biophys Acta 98: 520-531, 1965 3. Strauss EW: Electron microscopic study of intestinal fat absorption in vitro from mixed micelles containing linolenic acid monoolein and bile salt. J Lipid Res 7: 307-323, 1966 4. Wilson TH, Wiseman , G: The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J Physiol London 123: 116-125, 1954 5. Luca HF de, Cohen PP: Suspending media for animal tissues. In Manometric Techniques. Edited by WW Umbreit, RH Burris and JF Stauffer. Minneapolis, Burgess Publishing Co, 1964, p 131-133 424