INTESTINAL CALCIUM TRANSPORT: COMPARISON OF DUODENUM AND ILEUM IN VIVO IN THE RAT

Transcription

1 GASTROENTEROLOGY Copyright 1972 by The Williams & Wilkins Co. Vol. 62, No.4 Printed in U.S.A. INTESTINAL CALCIUM TRANSPORT: COMPARISON OF DUODENUM AND ILEUM IN VIVO IN THE RAT M. K. YOUNOSZAI, M.D. AND HAROLD P. SCHEDL, M.D., PH.D. Gastroenterology Research Laboratories, Departments of Pediatrics and Medicine, University of Iowa College of Medicine, Iowa City, Iowa Rats were loaded parenterally with,sca and the lumen of the duodenum and ileum were perfused with either a calcium-free solution (NaCI) or a solution containing 3.4 mm 'calcium (NaCI + Ca). Net movements of,sca and.dca and their intestinal tissue and plasma concentrations were measured. In the NaCI study the net ileal calcium secretion was 2 to 3 times that in the duodenum. In the NaCI + Ca studies.dca was absorbed, 45Ca was secreted, and the four fluxes across the mucosa could be calculated. The ileal mucosa to lumen flux was 1.5 times that in the duodenum. Lumen to mucosa fluxes were several times greater than mucosa to lumen fluxes. The duodenallumen to mucosa flux was 1.7 times that in the ileum. Fluxes at the base of the cell were much greater than those at the brush border in both intestinal segments. In man 1 and in animals 2 ' s a calcium load presented to the intestinal mucosa is absorbed more rapidly in the duodenum than the ileum. In previous studies' we perfused the lumen of the in situ rat intestine with solutions containing calcium and 45Ca and demonstrated that the greater net absorption in the duodenum was the result of a higher lumen to plasma flux (LP) than in the ileum. We estimated the plasma to lumen (PL) calcium flux indirectly as the difference between LP flux and net calcium absorption. PL fluxes were of approximately the same magnitude in both duodenum and ileum. At low luminal calcium concentrations Received June 2, Accepted October 22, Address requests for reprints to: Dr. M. K. Younoszai, Gastroenterology Research Laboratories, Departments of Medicine and Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa This work was supported by Public Health Ser vice Training Grant T 1 AM 5390 and Public Health Service Research Grants AM and HD The technical assistance of Margaret Young is gratefully acknowledged. (0.2 to 0.4 mm), LP flux was about double PL flux in the duodenum, but LP and PL fluxes were approximately equal in the ileum. These findings on ileal calcium transport have important implications regarding the role of the small intestine in calcium homeostasis. Because PL flux is crucial for calcium conservation, we measured PL flux directly in these studies. We loaded rats parenterally with 4SCa and measured appearance of 4SCa in the intestinal lumen during steady state luminal perfusion. When the luminal solution. contained 4DCa the two calcium isotopes moved in opposite directions: luminal 4DCa was absorbed and parenteral 4SCa was secreted. The four fluxes across the mucosal cell could be calculated directly from luminal, mucosa, and plasma 4DCa and 4SCa concentrations and net movements. Findings were consistent with our previous indirect estimates. In the absence of luminal calcium there was net intestinal calcium secretion, which was 2 to 3 times greater m the ileum than in the duodenum. 565

2 566 YOUNOSZAI AND SCHEDL Vol. 62. No.4 Materials and Methods Male albino rats (Carworth Laboratories) weighing 150 to 230 g were fed a regular Wayne Lab Blox. Twenty-four hours before perfusion they were injected intramuscularly with 25 /LC of "Ca per 100 g of body weight (initial specific activity, 12 mc per mg; New England Nuclear, Boston, Mass.) in 0.1 N HCl (100 /Lc per mi). All rats were fasted, with distilled water ad libitum for 24 hr after 45Ca injection. Pilot studies demonstrated constancy of plasma and intestinal tissue concentration of 45Ca (and 40Ca) during the 2-hr period 24 hr after injection of 45Ca. The experimental procedure was similar to that described previously.6 Ten- to 15-cm segments of the duodenojejunum and terminal ileum were cannulated and perfused in situ by the single pass technique for 2 hr at the rate 0.3 ml per min (model no. 206 AutoAnalyzer pump). The pump rate was constant and was measured both before and after each experiment. The solutions perfused had initial osmolalities of 294 to 300 milliosmoles per kg and consisted of either 165 mm sodium chloride (9.30 g per liter) (NaC!) or 159 mm sodium chloride and 3.4 mm calcium chloride (NaCI + Ca). All solutions contained polyethylene glycol (PEG, Carbowax 4000, 2 g per liter) as nonabsorbed indicator. Effluents from each segment in each rat were collected separately at 0 to 45, 45 to 60, 60 to 75, and 75 to 90 min after the start of perfusion. The entry of 45Ca into the intestinal lumen was determined by measuring the radioactivity in each of the effluent solutions. One milliliter of the initial solution or effluents was added to plastic counting vials containing 1 ml of a 6 mm solution of the disodium salt of ethylenediaminetetraacetic acid (Endrate, 1: 20 dilution) and 0.2 ml of 3 M trichloroacetic acid. Ten milliliters of a modified Brays solution 7 were added and the samples were counted in a liquid scintillation spectrometer (Beckman LS 250). Since the "Ca concentration (counts per minute per milliliter) in the effluents obtained during the last two collection periods was constant within ± 5%, the rate of "Ca secretion into the lumen was based on the combined values calculated for these two periods and expressed on the basis of V2 hr. After termination of the perfusion, blood was obtained from the inferior vena cava and anticoagulated with Endrate solution. Radioactivity in O.l-ml aliquots of plasma was counted after addition of the scintillation solution as above and 0.2 ml of 3 M trichloroacetic acid. The perfused intestinal segments were stripped from the mesentery and cut at the inlet and outlet cannulas. Length was measured by placing the segment straight and under no tension on absorbent paper. Segments were then weighed and cut longitudin~lly. The luminal surface was scraped firmly With the edge of a microscope slide. The mucosal scrapings, designated as mucosa, and the underlying tissues were separated and their wet and dry weights were obtained. After incinerating the two layers at 570 C for 24 hr, the ash was dissolved in 0.5 ml of a hot solution composed of equal volumes of 3 M trichloroacetic acid and glacial acetic acid. The contents of the crucibles were transferred quantitatively into 5-ml volumetric flasks for determination of calcium and 45Ca. Calcium was measured using a model 303 Perkin Elmer Atomic Absorption Spectrometer. 45Ca was determined as above. Recovery of calcium and "Ca from the blood, perfusion solution, and tissue is 97 to 100% by the methods described. PEG was determined by the method of Hyden." Calculations The rates per 1/2 hr given in "Results" are the sums from the last two 15-min periods calculated as follows: Secretion. Net <sca secretion (counts per minute per 15 min) = V. [45Ca] Net calcium secretion (/Lmoles per 15 min) = V. [40Ca] where V is the volume of the effluent in milliliters, [45Ca] is 45Ca concentration in the effluent, counts per minute per milliliter, and [,oca] is the concentration of calcium in the effluent, micromoles per milliliter. V is calculated as follows: V = PR-PEGR where PR is the pump rate, milliliters per 15 min, and PEGR is the ratio of the initial to the final PEG concentration. Absorption (in the NaCI + Ca studies). Net calcium absorption (/Lmoles per 15 min) = PR[40Catl - PR[40Ca, JPEGR The subscripts i and f refer to initial and final values, and the other symbols have the same meaning as above.

3 April 1972 DUODENAL AND ILEAL CALCIUM TRANSPORT 567 Unidirectional fluxes. Calculation of the calcium fluxes and rate constants are described in "Appendix." Results Data on intestinal segments, plasma, and luminal contents and their 40Ca and 45Ca concentrations and specific activities are shown in table 1 and figure 1. The lengths of the perfused duodenal and ileal segments were similar. The wet weight of the duodenal mucosa per unit length was significantly greater than that in the ileum for both groups. In both NaCl and NaCl + Ca studies, at the end of the perfusion period the mean concentrations of calcium (fig. 1) and 45Ca (table 1) in the ileal lumen were higher than those in the corresponding duodenal lumen. Ileal tissue calcium and 45Ca concentrations were usually twice those in the duodenal tissues and plasma. These differences were statistically significant and present in each individual rat. Calcium concentrations in the duodenal mucosa tended to parallel those in the plasma. However, although duodenal mucosa and plasma calcium concentrations did not differ (fig. 1), 45Ca concentrations in duodenal mucosa were significantly lower than those in the plasma (table 1). Duodenal tissue specific activities were also significantly lower than ileal tissue specific activities. In the NaCl study 45Ca specific activities were the same in the lumen (duodenum and ileum), ileal tissue, and plasma. In the NaCl + Ca study the tissue and lumen specific activities were lower than plasma because of the calcium in the perfusion solution. Calcium concentrations (fig. 1) were significantly lower in the mucosa than in the corresponding underlying tissue in the duodenum but not in the ileum, in both the NaCl and NaCl + Ca studies. Table 2 shows the net calcium movements in the perfused segments. There was net secretion of 45Ca in both intestinal segments in each individual rat. In both TABLE 1. Intestinal segments, "Ca concentrations and specific activities (mean and SE) Perfusion solution NaCl' NaCI + Ca' Segment Duodenum Ileum P valuet> Duodenum Ileum Length " ± ± ± ± 0.9 Mucosa, mg/cm ± ± 0.9 < ± ± 1.4 Underlying tissue, mg/cm...,' ± ± 1.2 < ± ± 1.3 "ea concentrations, counts per min x 10-3 /ml or g wet weight Lumen ± ± 0.04 < ± ± 0.03 Mucosa..., ' ± 0.9 c 44.9 ± 1.1 < ± O.4 c 45.3 ± 0.9< Underlying tissue ± 1.7 c 46.9 ± 2.1 < O ± 1.3a 55.2 ± 2.3'" Plasma " ± ± 1.1 Specific activities, counts per min x 10-3/ /Lmole Lumen..... " ± 0.8" 12.4 ± 1.0 NS ± ± 0.0 Mucosa....., ' ± ± 0.7 < ± ± 0.5 Underlying tissue 9.5 ± ± 0.4 < ± ± 0.7 Plasma ± ± 0.4 P value b < 0.05 < 0.05 < 0.05 < < NS < 0.01 < 0.01 a Eleven animals in each group. b P values are for differences between duodenum and ileum. c These two fractions of the same segment differ significantly, P < " Significantly greater than corresponding tissue values, P < 0.01.

4 568 YOUNOSZAI AND SCHEDL Vol. 62. No. 4 NaCl and NaCl + Ca studies the mean ileal 45Ca secretion rates were significantly greater than those in the duodenum (P < 0.01) on both weight and length bases. In the NaCl perfusion there was NaGI Perfusion NaCI + Co Perfusion t MUCOSA t PLASMA LUMEN UNDERLYI NG TISSUE FIG. 1. Calcium concentrations at the end of perfusion (mean ± SE). Mean ileal values are shown as open circles and duodenal values as closed circles. All ileal luminal and tissue concentrations are significantly greater than corresponding duodenal values (P < 0.01). net calcium secretion into both segments. Depending on how data were expressed, the mean ileal secretion rates were about 2 to 3 times those in the duodenum. In the NaCl + Ca study there was net calcium absorption from the intestinal lumen. The duodenal calcium absorption rate was more than double that in the ileum, and the difference was statistically significant (P < 0.05). Figure 2 shows the unidirectional calcium fluxes and rate constants for the duodenal and ileal segments perfused with NaCl + Ca. These were calculated as described in "Appendix." The estimated entry and exit fluxes at both the brush border and the basal-lateral membranes of the mucosal cell are given. All data were calculated from mean values given in table 2 and figure 1. In both segments fluxes at the base of the cell were more than double those at the brush border. Mucosa to lumen (ML) flux was greater in the ileum than in the duodenum, but the reverse or lumen to mucosa (LM) flux was greater in the duodenum than the ileum. The mucosa to plasma (MP) flux was greater than the plasma to mucosa (PM) flux in both segments, and duodenal TABLE 2. Net movements of " Ca and calcium (mean and SE) Perfusion solution NaCl" NaCl '+ Ca" Segments Duodenumb Ileum /) Duodenumb Ileum/> Secretion " Ca, counts per min x 10- '/ 0.5 hr per g mucosal dry weight..., ± ± ± ± g mucosal wet weight " 6. 3 ± ± ± ± 1.4 g segment wet weight.... " ± ± ± ± 0.6 cm length , ' ± ± ± ± 0.03 Calcium, ILmoles/0.5 hr per g mucosal dry weight ± ± 1.6 g mucosal wet weight ± ± 0.12 g segment wet weight.... " " 0.22 ± ± 0.03 cm length ± ± Absorption Calcium, ILmoles/0.5 hr per g mucosal dry weight ± ± 5.0 g muc9sal wet weight ± ± 1.2 g segment wet weight ± ± 0. 5 cm length ± ± 0.02 a Eleven animals in each group. b All comparisons between corresponding duodenal and ileal data differ significantly (P < 0.05).

5 April 1972 DUODENAL AND ILEAL CALCIUM TRANSPORT 569 DUODENUM LUMEN MUCOSA E.C.F. (PLASMA) ~:.:~mt~t: ILEUM ~:~,:~::: :::.:L~l:~::: FIG. 2. Calcium fluxes and their rate constants, K. The four fluxes across the mucosa are shown (/Lmoles per 0.5 hr per g of mucosal wet weight) : LM, lumen to mucosa; ML, mucosa to lumen; MP, mucosa to plasma; and PM, plasma to mucosa. The lumen of the duodenum and ileum perfused with a solution of 3.4 mm calcium chloride in 159 mm sodium chloride. E.C.F., extracellular fluid. MP and PM fluxes were greater than the corresponding ileal fluxes. The rate constants for the fluxes showed relationships that were similar to the fluxes. All duodenal rate constants were higher than the corresponding ileal rate constants. Discussion Most previous studies of intestinal calcium transport in the rat have focused on the duodenum and have measured calcium absorption. Absorption in other segments of the small intestine and secretion of calcium into the lumen have not received much attention. However, both absorption and secretion are important for calcium homeostasis, and the contribution of the duodenum to calcium transport must be considered in relation to the more distal segments of the small intestine. In this in vivo study of calcium transport in the rat small intestine, both luminal secretion of parenterally injected 45Ca, and absorption of luminal 40Ca were measured directly. Net secretion and absorption rates were compared in the duodenum and terminal ileum. From these data and the concentrations of 40Ca and 45Ca in the intestinal lumen, mucosa, and plasma we calculated calcium fluxes across the brush border and basal-lateral membrane of the intestinal mucosal cell (see "Appendix"). Net 45Ca secretion (table 2) was significantly greater in the ileum than the duodenum regardless of the luminal calcium concentration or the basis for expressing the data. In the absence of luminal calcium (NaCI perfusion) there was net calcium secretion in both segments. Secretion rates (~moles per 0.5 hr per g of mucosal wet weight) were significantly greater in the ileum (1.41) than in the duodenum (0.50). A similar relationship was seen for 45Ca secretion rates in the two segments. In the NaCI + Ca perfusion net 45Ca secretion is proportional to the ML flux since the probability that secreted 45Ca will be reabsorbed is very low in the presence of large amounts of exogenous 40Ca in the lumen. In fact, the ratio of duodenal to ileal net 45Ca secretions 11.4: 18.3 (table 2) was the same as the ratio of the ML calcium fluxes: 1.2: 1.8 (fig. 2). The ML calcium flux (~moles per 0.5 hr per g of mucosal wet weight) was greater in the ileum (1.8) than the duodenum (1.2). The NaCI and NaCI + Ca studies show the contrasting responses of duodenum and ileum to the low and the high (3.4 mm) luminal calcium concentrations. In the ileum at low luminal calcium concentration, the net calcium secretion (~moles per 0.5 hr per g of mucosal wet weight) approximated the ML flux: 1.4 (table 2) as compared with 1.8 (fig. 2). The net ileal 45Ca secretion rates were also approximately the same at both low and high luminal calcium concentration. This contrasts with the duodenal response: at the low luminal calcium concentration net calcium secretion (~moles per 0.5 hr per g of mucosal wet weight) was 0.50 (table 2) less than half the ML flux 1.2 (fig. 2). The net duodenal 45Ca secretion rates were also comparably lower at the low as compared with the high luminal calcium concentration. The much greater reabsorption of secreted calcium and 45Ca in the duodenum than in the ileum explains this difference. At the low luminal calcium concentration reabsorption is low and secretion is high in the ileum. At the high luminal calcium concentration duodenal absorption was also greater than ileal (table 2), in accordance with the

6 570 YOUNOSZAI AND SCHEDL Vol. 62, No.4 LM and ML flux difference shown in figure 2. The LM: ML flux ratio at the brush border is about 7 in the duodenum and 3 in the ileum. The absolute values of fluxes are much lower at the brush border than at the basal cell membrane (fig. 2). Thus the transport mechanisms operating at the brush border and base of the cell show different characteristics. At the brush border transport is more unidirectional with the LM: ML flux ratio of about 7 in the duodenum and 3 in the ileum. At the base of the cell, transport is more bidirectional with MP: PM flux ratios of about 1.4 in both segments. The relation between absorptive fluxes is the same in the duodenum and ileum: the MP flux is about 3 times the LM flux. For the secretory fluxes, the PM flux is 14 times the ML flux in the duodenum but only about 6 times greater in the ileum. The ileal tissue calcium concentration was almost double that in the duodenum (fig. 1). In addition all of the ileal calcium exchanged with the transport pool, since tissue and plasma 45Ca specific activities were identical in the NaCl studies (table 1). Duodenal calcium specific activity was 80% of that of plasma, and the 45Ca concentration in the duodenal mucosal transport pool was corrected for the nonexchangeable calcium using data from the NaCl perfusion ("Appendix"). Since flux is the product of a concentration term and a permeability factor (K, fig. 2), permeabilities are greater in the duodenum to give higher fluxes at lower concentrations. Our findings regarding ileal calcium secretion are important in understanding calcium homeostasis. Net secretion of calcium into the lumen, i.e., loss of endogenous calcium, occurred during perfusion of the calcium-free NaCl solution. Thus, at least during acute restriction of calcium intake, the ileum is unable to conserve calcium. Since the colon does not appear to conserve calcium,9 acute calcium restriction should give a negative calcium balance. Animals adapt to a low calcium intake by increasing the efficiency of calcium absorption, but nevertheless continue to lose endogenous calcium in the stool. Our data suggest the limiting factor in this adaptation may be ileal function. Previous studies in the rat directed toward determining the site of the ratelimiting step in mucosal calcium absorption have implicated the basal-lateral cell membrane rather than the brush border.5, 10 In these studies we have contrasted the behavior of duodenum and ileum and found absorptive activity greatly enhanced in duodenum relative to ileum. The most striking difference between these two sites is in fluxes at the base of the cell (fig. 2). At a duodenal mucosal calcium concentration of half that in the ileum, basal fluxes were twice as great. Assuming that the greater duodenal absorptive capacity is related to this most striking difference between the duodenum and ileum, it may be speculated that the rate-limiting step is at the base of the cell. Appendix The following assumptions were made for calculating the calcium and 45Ca fluxes: (1) calcium exchanged between three well mixed compartments consisting of the intestinal lumen, mucosal epithelium, and extracellular fluid (ECF); (2) plasma calcium and 45Ca concentrations approximated those in the ECF, and those in the mucosal scraping approximated calcium concentrations in mucosal epithelial cells; (3) steady state conditions prevailed in the last liz hr of perfusion, during which the sum of calcium fluxes into the intestinal mucosa were equal to the sum of calcium fluxes out of the mucosa; rates of entry and removal of calcium and 45Ca were not changing appreciably; (4) the average of the initial and final luminal calcium concentrations represented calcium concentration in the perfusion fluid along the length of perfused segment; (5) the quantity of calcium moving out of a compartment per unit time is determined by the concentration of calcium in that compartment and the rate constant for the flux. Figure 2 shows the three compartments

7 April 1972 DUODENAL AND ILEAL CALCIUM TRANSPORT 571 and the directions of calcium movement; lumen to mucosa (LM), mucosa to lumen (ML), mucosa to ECF (MP), and ECF to mucosa (PM); K I, K 2, K 3, and K 4 are the corresponding rate constants. Rates of calcium absorption and secretion in the NaCI + Ca perfusion are then as follows: Net calcium LM flux = K1[Cah - K 2 [Ca]M (1) Net 45Ca ML flux = K 2 [45Ca]M - K 1[45Ca]L (2) where [Ca] is the total calcium concentration in micromoles per gram of mucosal wet weight or milliliter, [HCa] is the concentration of 45Ca in counts per minute per gram of mucosal wet weight or per milliliter, and the subscripts Land M designate lumen and mucosa. Since steady state conditions exist, the net amount of calcium and 45Ca being transported at the mucosal brush border should equal that being transported at the basal-lateral cell membrane. This makes it possible to calculate calcium fluxes between intestinal mucosa and plasma. Rates of calcium transport at the basal-lateral membrane are then as follows: Net calcium MP flux = K 3 [Ca]M - K4[Ca]p (3) Net 45Ca PM flux = K4[45Ca]p - K 3 [45Ca]M (4) where subscripts M and P designate mucosa and ECF. Equations 1 and 2, and 3 and 4 can be solved in the usual way as two simultaneous equations with the pair of rate constants as unknowns (K 1 and K 2 for equations 1 and 2 and K 3 and K 4 for equations 3 and 4). The values for calcium and 45Ca concentrations and net transport rates are measured directly and substituted in the equations. Individual fluxes are obtained as the products of the rate constants (K) and calcium concentrations in the compartments. In the NaCI perfusion the duodenal mucosal 45Ca concentration is only 80% of that in plasma (table 1). This indicates that a fraction of the duodenal mucosal calcium is nonexchangeable and does not participate in the transport process. In calculating fluxes the mucosal calcium concentration in the NaCI + Ca studies is corrected for this nonexchangeable calcium. In the ileum no correction is necessary because mucosal and plasma specific activities were the same. REFERENCES 1. Wensel RH, Rich C, Brown AC, et al: Absorption of calcium measured by intubation and perfusion of the intact human small intestine. J Clin Invest 48: , Cramer CF: Sites of calcium absorption and the calcium concentration of gut contents in the dog. Can J Physiol Pharmacol 43:75-78, Kimberg DV, Schachter D, Schenker H: Active transport of calcium by intestine: effects of dietary calcium. Am J Physiol 200: , Krawitt EL, Schedl HP: In vivo calcium transport by rat small intestine. Am J Physiol 214: , Schachter D, Kowarski JD, Finkelstein RW: Tissue concentration differences during active transport of calcium by intestine. Am J Physiol 211: , Urban E, Schedl HP: Comparison of in vivo and in vitro effects of vitamin D on calcium transport in the rat. Am J Physiol 217: , Beckman Instructions: LS-233, LS-250 Liquid Scintillation Systems. Fullerton, Calif: Beckman Instruments, Inc, 1967, p 6 8. Hyden SA: A turbidometric method for the determination of higher polyethylene glycols in biological materials. Kgl Lantbruks-Hogskol Ann 22: , Urban E: Calcium transport by the rat colon in vivo. Clin Res 14:73, Urban E, Schedl HP: Vitamin D, tissue calcium and calcium transport in the in vivo rat small intestine. Am J Physiol 219: , 1970