AMERICAN JOURNAL OF PHYSIOLOGY Vol. 230, No. 5, May 1976. Printed in U.S.A.
Intestinal calcium absorption: ifferences in transport between duode urn and ileum JOSE BEHAR AND MORRIS D. KERSTEIN Departments of Medicine and Surgery, Veterans Administration School of Medicine, West Haven, Connecticut 06516
diet; vitamin
D; sodium;
sugars;
solvent
and Yale University
out according to the method of Urban and Schedl (17). lbino male rats of the same strain weighing 90-120 g ere selected at random and fed with a vitamin Ddeficient diet (USP vitamin D-free rachitogenic test diet, General Biochemicals, Chagrin Falls, Ohio) for at least 12 wk before the experiments were carried out. these animals were then repleted with 50,000 U > of vitamin Dz (calciferol, Kremer-Urban Co., injection 48 and 24 waukee, Wise.) by intramuscular h before the experiments were performed. Repleted anire immediately separated from the deficient
BEHAR,JOSE, AND MORRIS D. KERSTEIN. Intestinal calcium absorption: differences in transport between duodenum and ileum. Am. J. Physiol. 230(5): 1255-1260. 1976. -The interaction of calcium with sugar, sodium, and water absorption was studied in rats fed laboratory chow and in vitamin D-deficient and -repleted rats with the use of intestinal loops in vivo. Calcium absorption in the duodenum was enhanced by Dglucose only in the vitamin D-deficient state. In both vitamin D-deficient and -repleted ileum, calcium absorption decreased when NaCl was replaced by mannitol; however, it remained unchanged when NaC’l -was replaced by choline chloride or urea. Calcium absorption was enhanced by actively transported sugars and by increasing net water generated by differences in osmotic pressure and urea. er all experimental conditions there was a strong association between net water and calcium absorption in the ileum. These findings suggest that calcium absorption is enhanced by solvent, drag, although the role of sodium cannot be excluded entirely.
rachitogenic
Hospital
m intestinal loop of duodenum-jejunurn (duodenal loop) or of terminal ileum (ileal loop) was perfused in vivo by a method previously reported (1, 11). All test solu prior to perfusion were isstonic (290-310 m0s nd their composition consisted of ‘I. mM CaCl,, 150 NaCl, and 5 mM KC1 unless specifically indicated otherwise. The pH of the isotonic NaCl solution was 7.4 and that of all other solutions varied. fram 5.3 to 5.7. In the experiments with vitamin D-deficient and -rep1 animals the concentratisn of CaCl, was 3 mM; this urn concentration has been suggested to demonstrate the vitamin D effect in the small intestine in viva (17). The tracer used was g7Ca; it was added ta the test solutions immediately before the experiments. (PD) measurements agar bridges (PE-10 d through the distal ge was placed on the serosal surfaces; the tip was in contact with the ser0sa opposite to that where the tip of the mucosal bridge was ed to a calomel eleclocated. The bridges were con eaker in the Ringer trode, each one immersed in 0m the calomel halfsolution. The resulting voltage cells was determined in a differential high-input impedance amplifier (Orion Research). The symbol Of the PD elative mucosal potential with respect to e. Before each measurement, the system was shart-circuited by connecting a Ringer-agar bridge between the two Ringer beakers to determine cumulative drift m Calculation. Polyethylene glycol (Carbowax 4000) was used as a marker (5) and was determined by the method of Malawer and Powell (9). Lumen-to-plasma (L-P) transport of calcium and net absorption of calcium, sodium, and water were calculated by the equa-
drag
THE DUODENAL TR NSPORT OF CALCIUM appears to take place in a two-step transport process. Calcium enters the epithelial cells by passive or facilitated transport and is extruded from the cell against an electrochemical gradient by active transport (6, 14, 21). The latter trans-dependent process port step appears to be a vitamin (15, 17, 21). The transport of calcium in the ileum, however, has not been clearly defined. Further, the rates of calcium absorption in the small intestine are also influenced by a variety of chemical agents, some of them of physiological significance (21). Martin and DeLuca (10) have shown in vitro that calcium transport in the duodenum is ependent on sodium transport. Lactose and other sugars appear to stimulate calcium absorption, exerting a greater effect in the ileum (7, 19). The present studies were designed to investigate the interaction of calcium transport with sodium, sugars, and water absorption in rats fed Purina laboratory chow and in vitamin D-deficient and -repleted rats in vivo. METHODS
Albino male rats of the Charles River strain weighing X0-200 g and fed Purina laboratory chow (Mg 0.19%, Ca 0.94%, and vitamin D 3.3 IU/g) were used. The experiments with vitamin D-deficient animals were carried 1255
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1256
J.
tions of Wasserman, Kallfelz, and Comar (23). The assumption of these calculations is that the backflow of 47Ca from plasma to lumen is small. Serum specific activity at the end of 1 h of perfusion was less than 250 counts/min per pmol of calcium, in contrast to the perfusate, which averaged 5,000 counts/min per pmol of calcium, so that back diffusion of the tracer was small. The results of calcium and sodium transport are expressed as millimicromoles and micromoles per centimeter per hour, respectively, and microliters per centimeter per hour for net water absorption” The unpaired Student t test was applied to the experimental data obtained to determine statistical significance. The following equations were used: Net water absorption = PR (1 - PEG,/PE&). (1) Lumen-to-plasma [47Ca] = 47Ca - (47CaF x PEG/PEGF) PR I (SA1 + SA,/2) Net absorption
[40Ca] = PR [4*Car - (4*Cal: (3) x PEGJPEG,)] where SA = specific activity (counts/min per pm01 ‘“‘Ca), PR = pump rate, I = initial solution, F = final solution, and PEG = polyethylene glycol. Net sodium absorption was calculated with equation 3. The 47Ca was obtained as calcium chloride from Cambridge Nuclear Corp. Radioactivity of initial and final samples was determined with the Packard gamma well counter. Because of the relatively short half-life of 47Ca (4.5 days) all counts to be compared were corrected to a standard reference time. Chemical measurements of total calcium (40Ca) were determined by atomic absorption spectroscopy (model 290, Perkin-Elmer Corp., Norwalk, Conn.). Sodium was measured with the flame photometer and osmolarities of the solutions were determined with an osmometer (Advanced Instruments Inc., Boston, Mass.). All samples were determined in duplicate and the coefficient of variation was less than 3%. RESULTS
Effect of sodium replacement on calcium absorption (Table 1). Lumen-to-plasma transport of calcium with isotonic solutions of NaCl (150 mM) was greater in the duodenum than in the ileum (P < 0.001). The L-P transport of calcium in the duodenum was not affected by the isosmotic replacement of NaCl with mannitol and choline chloride (P > 0.05). The transmural PD under these conditions became markedly positive (P < 0.001) and the final concentration of sodium in the perfusate was 28 mM or less. In the ileum, replacement of NaCl with mannitol resulted in a decrease in L-P transport of calcium (P c O.OOl>, but remained unchanged when replaced with choline chloride (P > 0.05). Both solutes increased the positivity of the transmural PD (P < 0.001) with a final concentration of sodium in the perfusate of less than 18 mM. The isosmotic increase in the intraluminal concentration of NaCl from 0 to 150 mM caused a progressive increase in L-P transport of calcium in the ileum (Table
AND
M.
D.
KERSTEIN
TABLE I. Effect of sodium replacement by mannitol choline on calcium absorption and transmural PD in duodenum and ileum
--~Test
Solution,*
NaCl
Duodenum 150 50 0 0
Ileum 150 50 0
(2)
BEHAR
0
mM Other
i
L-P Transport ‘:Ca, mtt.rnoll cm per h
0 Mannitol 190 Manni tolj 290 Choline Cl+ 120
0 Mannitol 190 Mannitol$ 290 Choline Cl$ 120
Transmural PD, mV
P 0.05) and only moderate stimulatory effect on net sodium absorption (P < 0.05) in the ileum and duodenum. Effect of water movement on calcium absorption. Manipulation of net water movement was accompanied by
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CALCIUM
TRANSPORT
IN
DUODENUM
AND
1257
ILEUM
marked changes in net calcium absorption in the ileum with strong association between the magnitude of net water absorption and net calcium absorption. There was a significant correlation between net water and calcium absorption when NaCl was replaced with mannitol and choline chloride (r = 0.88, P < 0.001; Fig. 1) and between net water absorption generated by increasing the intraluminal sodium concentration (O-150 mM) and net calcium absorption (r = 0.88, P < 0.001; Fig. 2). 400
J
t
= 67.4 r = 0.96
I 0
0 -50 L
I 50
II
+0.47X
duodenum (open (a> was replaced
400
(pl/cm/hr)
I
SECRETION
I
I
I
ABSORPTION
(pl/cm/hr)
3. Correlation between net water and calcium duodenum (open symbols) and ileum (closed symbols) mannitol (0) and urea (a) solutions. FIG.
v
absorption in with isosmotic
0
V
0
A
0
300
V
A
V A
250
r” =i E
V
A
200
z) 0 150
a 100
SO
0
-50
0
50
100
150
200
ABSORPTION
SECRETION
A WATER
(pl/cm/hr)
2. Relationship between net water and calcium transport in duodenum (open symbols) and ileum (closed symbols) when concentration of NaCl was increased in test solution from OmM (V), to 50 mM (0) and to 150 mM (A). FIG.
I
A WATER
Y, = 310 -0.09x I = -0.13
V
i
I
between net water and calcium transport in symbols) and ileum (closed symbols) when NaCl with choline chloride (U) and mannitol (0).
350
;
I 150
ABSORPTION
A WATER
1. Correlation
I 100
I
SECRETION
‘z r \ E 0 1
A A
0
YC
FIG.
+ 0.24X
YC = 304 -0.1x r = -0.18
0
-I
Y, = 307 I = 0.43
Furthermore, the isosmotic replacement of mannitol with urea resulted in an increase in net water and calcium absorption with a significant correlation between these two measurements (r = 0.96, P < 0.001; Fig. 3). Mannitol, a poorly absorbable substance, caused mild net water secretion, whereas urea generated net water flow. Urea also increased the positivity of the PD (+ 3.8 t 0.4 mV). Hypotonic solutions (105 mosM) with a NaCl concentration of 50 mM were able to increase net water and calcium absorption above the levels observed with isotonic solution of equal NaCl concentration (P c 0.001). Under these experimental conditions, there was a positive association between net water and calcium absorption (r = 0.77, P < 0.01; Fig. 4). A strong correlation between net water and calcium absorption was also observed with the addition of 25 mM of sugars, of which all but fructose caused an increase in net water and calcium absorption (r = 0.93, P < 0.001; Fig. 5). A poor association between net water and calcium absorption was observed in the duodenum (Figs. l-5). Marked changes in the magnitude of net water absorption did not influence net calcium absorption, which remained independent of the composition of intraluminal solutions. Effect of vitamin D on calcium absorption (Table 3). Neither vitamin D deficiency nor repletion affected the L-P transport of calcium in the ileum with isotonic NaCl solutions (P > 0.05). The effect of mannitol, urea, and D-
Downloaded from www.physiology.org/journal/ajplegacy at Macquarie Univ (137.111.162.020) on February 13, 2019.
1258
J. BEHAR
Y, = 328 + 0.03x r = 0.03
400
A
350
5
M.
II.
MERSTEIN
glucose on L-P transport of calcium was also not influenced by vitamin D. D-Glucose stimulated net calcium absorption despite vitamin D deficiency (P < 0.05). A similar strong correlation between net water and calcium absorption was observed in the deficient and repleted states (r = 0.93, P < 8.001 and P” = 0.89, P +C .OOl, respectively; Fig. 6). The L-P transport of calcium in the duodenum, however, was reduced by vitamin D deficiency and was restored by vitamin D repletion (P < 0.001). The effect of urea and n-glucose on L-P transport of calcium, which
A
L < E 300 \" 0 2 =c 250 Is z 3
AND
TABLE 3. Effect of sodium, mannitol, urea, and o-glucose on calcium absorption in vitamin Ddeficient and -repleted duodenum and ileum ---
200
z: 0 Q 150
Y, = 70 + 0.36X ? = 0.77
Vitamin Test Solution*
100
50
1
25
1
50
I
75
100
A WATER
I
I
I
125
150
175
(pl/em/hr)
FIG. 4. Effect of net water flow on net calcium absorption in duodenum (open symbols) and ileum (closed symbols). Water flow was induced by varying osmolarity of perfusate from 105 mosM (hypotonic solutions, A) to 300 mosM (isotonic solutions, 0) with a constant NaCl concentration of 50 mM.
400
Y, 8243
Vitamin
D Repleted
L-P transport ‘“Ca, nip/cm per h
P-3
---------T
Duodenum NaCl Manni to1 LJrea D-GlUcoSe, 25 mM
516.7 501.7 580.0 578a3
-t 7.6* rt 4.0 I? 5.8 t 7.9
i
ileum NaCl Mannitoll Urea D-GlUCOSe, 25 mM
203.3 146.7 285.0 395.0
+ 7.2 -r- 4.9 +_ 6.19 -t 8.47
:
Values CaCl, in comparing D-glucose.
+ 0.54X
D Deficient
1
NS 0.001 0.001
0.001 0.001 1 0.001
PI/--
821.7 810.0 823.3 818.3
5 I I -t
11.4 9.3 12.6 13.0
201.7 123.3 266.7 366.7
zt 6.0 -+ 7.6 XL4.9 -+ 14.1
NS NS NS
t 0.001 0.001
0.001.
------- A----
are means -t- SE of 6 experiments * Concentration of test solution was 3 mM. t Unpaired Student t test calcium transport between NaCl and mannitol, urea and
300
350 & \ E 300 \u 0 ZE E* 250 V z 3 3
250 h 2 \ fj 200 \ 5 z =t
E V
200
E 3 i3
i Q
150
v
DEFICIENT--Yc = 78 + 0.69X r = 0.93
ov 0
REPLETEDYc = 76 + 0.74X I = 0.09
Ip
150
IO0
< (3 a SO
100 0
-50
SO 0
50
100
A
WATER
150
200
250
1
I
1
I
SO
100
150
200
WATER in test (O),
1
250
ABSORPTION
SECRETION
(pl/em/hr)
FIG. 5. Correlation between net water and calcium absorption duodenum (open symbols) and ileum (closed symbols) with solutions of NaCl 150 mM alone (Cl) and with 25 mM of fructose o-glucose (P’), 3-0-methylglucose (A), and n-galactose (0).
I
0
(pi/cm/Iv)
FIG. 6. Relation between net water and calcium absorption in ileum of vitamin D-deficient (closed symbols) and vitamin D-repleted (open symbols) animals. Calcium was perfused in isosmotic solutions ofmannitol (0). NaCl (A). urea (Cl). and n-glucose (0).
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CALCIUM
TRANSPORT
IN
DUODENUM
AND
1259
ILEUM
was negligible in the vitamin D-repleted state, became evident in the vitamin D-deficient duodenum (P c 0.001). Furthermore, the effect of the magnitude of water flow on calcium absorption was also apparent in the duodenum of vitamin D-deficient animals. A higher association was observed between net water and calcium absorption in the vitamin D-deficient state (r = 0.70, P c 0.001; Fig. 7) that was significantly different (P < 0.05, t tests of the slopes) from the association between these two measurements in the repleted state (r = 0.34, P > 0.05). DISCUSSION
In agreement with previous studies (6, 14) our results indicate that the rat duodenum and ileum are able to bring about net calcium transport against an electrochemical gradient, with greater rates of absorption in the duodenum. These results also point out significant additional differences in the absorption of calcium between the duodenum and ileum. These differences become evident when calcium interacts with water, sodium, and sugar absorption. Calcium absorption in the duodenum in vivo appears to be independent of the composition of the intraluminal fluid, whereas in the ileum it is influenced by the concentration of sodium, by actively transported sugars, and by the rates of net water absorption. Manipulation of the composition of intraluminal solutions, however, caused similar changes in the transmural PD and in the rates of net sodium and water absorption in both duodenum and ileum. 900
REPLETE0 Y, = 727 + 0.16X r = 0.34 0
800
_
*
Cl
O# 0
CJ0
0
A
DEFICIENT Y, = 382 + 0.43X r = 0.70
300
200 -50 SECRETlON
I
1
0
50
I
1
100
1
I
150
200
I
250 J
ABSORPTION
A WATER (pl/cm/hr) 7. Relationship between net water and calcium absorption in duodenum of vitamin D-deficient (closed symbols) and vitamin Drepleted (open symbols) animals. Calcium was perfused in isosmotic solutions of mannitol (O), NaCl (A), urea (Cl), and D-glucose (0). FIG.
Changes in the transmural PD do not seem to have any effect on calcium absorption in the ileum. Choline chloride solutions caused PD changes of a magnitude similar to those with mannitol solutions without affecting calcium absorption. Although the increase in the positivity of the transmural PD observed with mannitol and choline chloride should enhance lumen-to-cell movement of calcium (12), it is possible that this flux was not of sufficient magnitude to alter net calcium absorption significantly (1>. Previous work has presented conflicting evidence on the effect of sodium on calcium absorption in vitro (10, 24). Our studies in intestinal loops in vivo cannot resolve this controversy since the interaction of sodi urn and calcium absorption under these experimental conditions appears more complex th .an in vitro systems. First, calcium absorption was affected by sodium in the ileum exclusively and only when sodium chloride was replaced by mannitol or when greater rates of sodium transport were induced by actively transported sugars. Second, sodium diffused back in the perfusate of sodium-free solutions and we should assume that its concentration was even higher in the unstirred layer (13). Nevertheless, it is theoretically possible that sodium might contribute to the ileal absorption of calcium, which could be dependent on the rates of lumen-to-plasma movement of sodium, and the sodium found in the perfusate could simply be the result of plasma-to-lumen secretion. This hypothesis fails to explain the finding that calcium absorption remained unaffected when sodium chloride was replaced by choline chloride and urea. It is also conceivable, although highly speculative, that choline chloride might increase the concentration of sodium ions in the unstirred layer by increasing the density of negative charges at the luminal surface of the brushborder membrane (2, 4). This higher concentration of sodium ions at the membrane could then become available to the transport of calcium despite its low intraluminal concentration. However, the effect of urea on calcium absorption cannot be reconciled with either hypothesis. The finding that stimulation of net water flow by differences in osmotic pressure and by passively transported urea enhanced net calcium absorption in the ileum suggests that, in addition to an active transport process, calcium may be transported by solvent drag. Previous studies have sho wn that net w ,ater flow across biological membranes can stimulate the net transport of charged solutes against an electrochemical gradient (1, 18). This hypothesis is supported by the strong association between net water and calcium absorption under a variety of experimental conditions. Solvent drag could explain the discrepancy in the rates of calcium absorption between mannitol and choline chloride or urea solutions. Actively transported sugars could also increase net calcium absorption by generating net water flow. Water movement could drag calcium across the ileal mucosa through the tight junction of the epithelial cells, as has been suggested for magnesium absorption (1) In accord with the observations of Urban and Schedl
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J.
1260 (17), calcium absorption in the ileum was not affected by vitamin D. Furthermore, vitamin D deficiency did not inhibit the stimulatory effect of n-glucose on calcium absorption. However, it decreased calcium absorption in the d uodenum, whi ch w ‘as restored by vitamin D repletion. Moreover, in the vitamin D- deficient state, calcium absorption was more responsive to the composition of’ the intraluminal fluid with greater correlation between net water and calcium absorption. These findings would suggest that net water flow exerts an effect under conditions of impaired or less efficient calcium transport in the vitamin D-deficient duodenum. These findings support and could also explain previous observations (7, 19) that lactose stimulation of calcium absorption was greater in the rat ileum in vivo and that this lac tose effect was independent of the action of vitamin D @I . It is possibl e that lactose increa ses the net absorption of calcium by generating net water flow during the acti ve transport of i ts intraluminal end products, D- glucose and n-galactose (22). The contribution of this additional transport process to calcium homeostasis may become physiologically significant in
BEHAR
AND
M.
D.
KERSTEIN
malabsorption syndromes affecting the proximal small intestine or in vitamin D-deficient stat!es. Finally, the results of this study do not elucidate the structural or physiological factors responsible for the differences in calcium absorption between the duodenum and ileum. The former, with a larger pore radius, should be more permeable than the ileum to the transport of most ions (3). It is possib le, however, that the ileum is more permeable to the passive transpor t of calcium. Calcium appears more readily secreted in the ileum and colon (16, 20, 25) with inefficient calcium transport systems. The impermeability of the duodenum could be the result of a highly efficient active transport process, which becomes more permeable with an impaired calcium transport in the vitamin D-deficient state. We are indebted to Ann able technical assistance, vice, and to Judy Johnson manuscript. Received
for publication
Bartiss and Allyn Moore for their invaluto Emanuel Lerner for his statistical adand Mary Murray for preparation of the 11 July
1975.
REFERENCES 1. BEHAR, J. Magnesium absorption by the rat ileum and colon. Am. J. Physiol. 227: 334-340, 1974. 2. BIEBERDORF, F. A., S. MORANSKJ, AND J. S. FORDTRAN. Effect of sodium, mannitol, and magnesium on glucose, galactose, 3-0methylglucose and fructose absorption in the human ileum. Gastroenterology 68: 58-66, 1975. 3. CLARKSON, T. W., AND A. ROTHSTEIN. Transport of monovalent cations by the isolated small intestine of the rat. Am. J. PhysioZ. 199: 898-906, 1960. 4. HOGBEN, C. A. M., D. J. Tocco, B. B. BRODIE, AND L. S. SCHANKER. On the mechanism of intestinal absorption of drugs. J. Pharmacol. Exptl. Therap. 125: 275-282, 1959. 5. JACOBSON, E. D., D. C. BONDY, S. A. BROITMAN, AND J. S. FORDTRAN. Validity of polyethylene glycol in estimating water volume. Gastroenterology 44: 761-767, 1963. 6. KRAWITT, E. L., AND H. P. SCHEDL. In vivo calcium transport by rat small intestine. Am. J. Physiol. 214: 232-236, 1968. 7. LENGEMANN, F. W. The site of action of lactose in the enhancement of calcium utilization. J. Nutr. 69: 23-27, 1959. 8. LENGEMANN, F. W., R. H. WASSERMAN, AND C. L. COMAR. Studies on the enhancement of radiocalcium and radiostrontium absorption by lactose in the rat. J. Nutr. 68: 443-456, 1959. 9. MALAWER, S. J., AND D. W. POWELL. An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifer. GastroenteroZogy 53: 250-256, 1967. 10. MARTIN, D. L., AND H. F. DELUCA. Influence of sodium on calcium transport by the rat small intestine. Am. J. Physiol. 216: 1351-1359, 1969. II. POWELL, D. W., AND S. J. MALAWER. The relationship between water and solute transport from isosmotic solutions by rat intestine in vivo. Am. J. PhysioL. 215: 49-55, 1968. 12. ROSE, R. C., AND S. G. SCHULTZ. Studies on the electrical potential profile across the rabbit ileum. Effect of sugars and amino acids on transmural and mucosal electrical potential differences. J. Gen. Physiol. 57: 639-663, 1971. 13. SALTZMAN, D. A., F. C. RECTOR, AND J. S. FORDTRAN. The role of intraluminal sodium in glucose absorption in uiuo. J. CLin. In-
vest. 51: 876-885, 1972. 14. SCHACHTER, D., E. B. DOWDLE, AND H. SCHENKER. Active transport of calcium by the small intestine of the rat. An2. J. Physiol. 198: 263-268, 1960. 15. SCHACHTER, D., D. V. KIMBERG, AND H. SCHENKER. Active transport of calcium by intestine; action and bioassay of vitamin D. Am. J. Physiol. 200: 1263-1271, 1961. 16. URBAN, E. Calcium transport by the rat colon in uiuo. CLin. Res. 14: 73, 1971. 17. URBAN, E., AND H. P. SCHEDL. Comparison of in vivo and in vitro effects of vitamin D on calcium transport in the rat. Am. J. Physiol. 217: 126-130, 1969. 18. USSING, H. H., AND B. ANDERSON. The relation between solvent drag and active transport of ions. Proc. In tern. Congr. Biochem., 3rd) Brussels, 1955. 19. VAUGHAN, 0. W., AND L. J. FILER, JR. The enhancing action of certain carbohydrates on the intestinal absorption of calcium in the rat. J. Nutr. 71: 10-14, 1960. 20. WALLING, M. W., AND D. V. KIMBERG. Active secretion of calcium by adult rat ileum and jejunum in vitro. Am. J. PhysioZ. 225: 415-422, 1973. 21. WASSERMAN, R. H. The Transfer of Calcium and Strontium Across the Biological Membranes. New York: Academic, 1963, p. 127-182. 22. WASSERMAN, R. H., AND C. L. COMAR. Carbohydrates and gastrointestinal absorption of radiostrontium and radiocalcium in the rat. Proc. Sot. Exptl. Biol. Med. 101: 314-317, 1959. 23. WASSERMAN, R. H., V. A. KALLFELZ, AND C. L. COMAR. Active transport of calcium by rat duodenum in uiuo. Science 133: 883884, 1961. 24. WASSERMAN, R. H., AND A. N. TAYLOR. The non-essentiality of sodium ions for intestinal calcium transport. Proc. Sot. Exptl. BioZ. Med. 114: 479-482, 1963. 25. YOUNOSZAI, M. K., AND H. P. SCHEDL. Intestinal calcium transport: comparison of duodenum and ileum in uivo in the rat. Gastroenterology 62: 565-571, 1972.
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Intestinal calcium absorption: ifferences in transport between duode urn and ileum JOSE BEHAR AND MORRIS D. KERSTEIN Departments of Medicine and Surgery, Veterans Administration School of Medicine, West Haven, Connecticut 06516
diet; vitamin
D; sodium;
sugars;
solvent
and Yale University
out according to the method of Urban and Schedl (17). lbino male rats of the same strain weighing 90-120 g ere selected at random and fed with a vitamin Ddeficient diet (USP vitamin D-free rachitogenic test diet, General Biochemicals, Chagrin Falls, Ohio) for at least 12 wk before the experiments were carried out. these animals were then repleted with 50,000 U > of vitamin Dz (calciferol, Kremer-Urban Co., injection 48 and 24 waukee, Wise.) by intramuscular h before the experiments were performed. Repleted anire immediately separated from the deficient
BEHAR,JOSE, AND MORRIS D. KERSTEIN. Intestinal calcium absorption: differences in transport between duodenum and ileum. Am. J. Physiol. 230(5): 1255-1260. 1976. -The interaction of calcium with sugar, sodium, and water absorption was studied in rats fed laboratory chow and in vitamin D-deficient and -repleted rats with the use of intestinal loops in vivo. Calcium absorption in the duodenum was enhanced by Dglucose only in the vitamin D-deficient state. In both vitamin D-deficient and -repleted ileum, calcium absorption decreased when NaCl was replaced by mannitol; however, it remained unchanged when NaC’l -was replaced by choline chloride or urea. Calcium absorption was enhanced by actively transported sugars and by increasing net water generated by differences in osmotic pressure and urea. er all experimental conditions there was a strong association between net water and calcium absorption in the ileum. These findings suggest that calcium absorption is enhanced by solvent, drag, although the role of sodium cannot be excluded entirely.
rachitogenic
Hospital
m intestinal loop of duodenum-jejunurn (duodenal loop) or of terminal ileum (ileal loop) was perfused in vivo by a method previously reported (1, 11). All test solu prior to perfusion were isstonic (290-310 m0s nd their composition consisted of ‘I. mM CaCl,, 150 NaCl, and 5 mM KC1 unless specifically indicated otherwise. The pH of the isotonic NaCl solution was 7.4 and that of all other solutions varied. fram 5.3 to 5.7. In the experiments with vitamin D-deficient and -rep1 animals the concentratisn of CaCl, was 3 mM; this urn concentration has been suggested to demonstrate the vitamin D effect in the small intestine in viva (17). The tracer used was g7Ca; it was added ta the test solutions immediately before the experiments. (PD) measurements agar bridges (PE-10 d through the distal ge was placed on the serosal surfaces; the tip was in contact with the ser0sa opposite to that where the tip of the mucosal bridge was ed to a calomel eleclocated. The bridges were con eaker in the Ringer trode, each one immersed in 0m the calomel halfsolution. The resulting voltage cells was determined in a differential high-input impedance amplifier (Orion Research). The symbol Of the PD elative mucosal potential with respect to e. Before each measurement, the system was shart-circuited by connecting a Ringer-agar bridge between the two Ringer beakers to determine cumulative drift m Calculation. Polyethylene glycol (Carbowax 4000) was used as a marker (5) and was determined by the method of Malawer and Powell (9). Lumen-to-plasma (L-P) transport of calcium and net absorption of calcium, sodium, and water were calculated by the equa-
drag
THE DUODENAL TR NSPORT OF CALCIUM appears to take place in a two-step transport process. Calcium enters the epithelial cells by passive or facilitated transport and is extruded from the cell against an electrochemical gradient by active transport (6, 14, 21). The latter trans-dependent process port step appears to be a vitamin (15, 17, 21). The transport of calcium in the ileum, however, has not been clearly defined. Further, the rates of calcium absorption in the small intestine are also influenced by a variety of chemical agents, some of them of physiological significance (21). Martin and DeLuca (10) have shown in vitro that calcium transport in the duodenum is ependent on sodium transport. Lactose and other sugars appear to stimulate calcium absorption, exerting a greater effect in the ileum (7, 19). The present studies were designed to investigate the interaction of calcium transport with sodium, sugars, and water absorption in rats fed Purina laboratory chow and in vitamin D-deficient and -repleted rats in vivo. METHODS
Albino male rats of the Charles River strain weighing X0-200 g and fed Purina laboratory chow (Mg 0.19%, Ca 0.94%, and vitamin D 3.3 IU/g) were used. The experiments with vitamin D-deficient animals were carried 1255
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1256
J.
tions of Wasserman, Kallfelz, and Comar (23). The assumption of these calculations is that the backflow of 47Ca from plasma to lumen is small. Serum specific activity at the end of 1 h of perfusion was less than 250 counts/min per pmol of calcium, in contrast to the perfusate, which averaged 5,000 counts/min per pmol of calcium, so that back diffusion of the tracer was small. The results of calcium and sodium transport are expressed as millimicromoles and micromoles per centimeter per hour, respectively, and microliters per centimeter per hour for net water absorption” The unpaired Student t test was applied to the experimental data obtained to determine statistical significance. The following equations were used: Net water absorption = PR (1 - PEG,/PE&). (1) Lumen-to-plasma [47Ca] = 47Ca - (47CaF x PEG/PEGF) PR I (SA1 + SA,/2) Net absorption
[40Ca] = PR [4*Car - (4*Cal: (3) x PEGJPEG,)] where SA = specific activity (counts/min per pm01 ‘“‘Ca), PR = pump rate, I = initial solution, F = final solution, and PEG = polyethylene glycol. Net sodium absorption was calculated with equation 3. The 47Ca was obtained as calcium chloride from Cambridge Nuclear Corp. Radioactivity of initial and final samples was determined with the Packard gamma well counter. Because of the relatively short half-life of 47Ca (4.5 days) all counts to be compared were corrected to a standard reference time. Chemical measurements of total calcium (40Ca) were determined by atomic absorption spectroscopy (model 290, Perkin-Elmer Corp., Norwalk, Conn.). Sodium was measured with the flame photometer and osmolarities of the solutions were determined with an osmometer (Advanced Instruments Inc., Boston, Mass.). All samples were determined in duplicate and the coefficient of variation was less than 3%. RESULTS
Effect of sodium replacement on calcium absorption (Table 1). Lumen-to-plasma transport of calcium with isotonic solutions of NaCl (150 mM) was greater in the duodenum than in the ileum (P < 0.001). The L-P transport of calcium in the duodenum was not affected by the isosmotic replacement of NaCl with mannitol and choline chloride (P > 0.05). The transmural PD under these conditions became markedly positive (P < 0.001) and the final concentration of sodium in the perfusate was 28 mM or less. In the ileum, replacement of NaCl with mannitol resulted in a decrease in L-P transport of calcium (P c O.OOl>, but remained unchanged when replaced with choline chloride (P > 0.05). Both solutes increased the positivity of the transmural PD (P < 0.001) with a final concentration of sodium in the perfusate of less than 18 mM. The isosmotic increase in the intraluminal concentration of NaCl from 0 to 150 mM caused a progressive increase in L-P transport of calcium in the ileum (Table
AND
M.
D.
KERSTEIN
TABLE I. Effect of sodium replacement by mannitol choline on calcium absorption and transmural PD in duodenum and ileum
--~Test
Solution,*
NaCl
Duodenum 150 50 0 0
Ileum 150 50 0
(2)
BEHAR
0
mM Other
i
L-P Transport ‘:Ca, mtt.rnoll cm per h
0 Mannitol 190 Manni tolj 290 Choline Cl+ 120
0 Mannitol 190 Mannitol$ 290 Choline Cl$ 120
Transmural PD, mV
P 0.05) and only moderate stimulatory effect on net sodium absorption (P < 0.05) in the ileum and duodenum. Effect of water movement on calcium absorption. Manipulation of net water movement was accompanied by
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CALCIUM
TRANSPORT
IN
DUODENUM
AND
1257
ILEUM
marked changes in net calcium absorption in the ileum with strong association between the magnitude of net water absorption and net calcium absorption. There was a significant correlation between net water and calcium absorption when NaCl was replaced with mannitol and choline chloride (r = 0.88, P < 0.001; Fig. 1) and between net water absorption generated by increasing the intraluminal sodium concentration (O-150 mM) and net calcium absorption (r = 0.88, P < 0.001; Fig. 2). 400
J
t
= 67.4 r = 0.96
I 0
0 -50 L
I 50
II
+0.47X
duodenum (open (a> was replaced
400
(pl/cm/hr)
I
SECRETION
I
I
I
ABSORPTION
(pl/cm/hr)
3. Correlation between net water and calcium duodenum (open symbols) and ileum (closed symbols) mannitol (0) and urea (a) solutions. FIG.
v
absorption in with isosmotic
0
V
0
A
0
300
V
A
V A
250
r” =i E
V
A
200
z) 0 150
a 100
SO
0
-50
0
50
100
150
200
ABSORPTION
SECRETION
A WATER
(pl/cm/hr)
2. Relationship between net water and calcium transport in duodenum (open symbols) and ileum (closed symbols) when concentration of NaCl was increased in test solution from OmM (V), to 50 mM (0) and to 150 mM (A). FIG.
I
A WATER
Y, = 310 -0.09x I = -0.13
V
i
I
between net water and calcium transport in symbols) and ileum (closed symbols) when NaCl with choline chloride (U) and mannitol (0).
350
;
I 150
ABSORPTION
A WATER
1. Correlation
I 100
I
SECRETION
‘z r \ E 0 1
A A
0
YC
FIG.
+ 0.24X
YC = 304 -0.1x r = -0.18
0
-I
Y, = 307 I = 0.43
Furthermore, the isosmotic replacement of mannitol with urea resulted in an increase in net water and calcium absorption with a significant correlation between these two measurements (r = 0.96, P < 0.001; Fig. 3). Mannitol, a poorly absorbable substance, caused mild net water secretion, whereas urea generated net water flow. Urea also increased the positivity of the PD (+ 3.8 t 0.4 mV). Hypotonic solutions (105 mosM) with a NaCl concentration of 50 mM were able to increase net water and calcium absorption above the levels observed with isotonic solution of equal NaCl concentration (P c 0.001). Under these experimental conditions, there was a positive association between net water and calcium absorption (r = 0.77, P < 0.01; Fig. 4). A strong correlation between net water and calcium absorption was also observed with the addition of 25 mM of sugars, of which all but fructose caused an increase in net water and calcium absorption (r = 0.93, P < 0.001; Fig. 5). A poor association between net water and calcium absorption was observed in the duodenum (Figs. l-5). Marked changes in the magnitude of net water absorption did not influence net calcium absorption, which remained independent of the composition of intraluminal solutions. Effect of vitamin D on calcium absorption (Table 3). Neither vitamin D deficiency nor repletion affected the L-P transport of calcium in the ileum with isotonic NaCl solutions (P > 0.05). The effect of mannitol, urea, and D-
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1258
J. BEHAR
Y, = 328 + 0.03x r = 0.03
400
A
350
5
M.
II.
MERSTEIN
glucose on L-P transport of calcium was also not influenced by vitamin D. D-Glucose stimulated net calcium absorption despite vitamin D deficiency (P < 0.05). A similar strong correlation between net water and calcium absorption was observed in the deficient and repleted states (r = 0.93, P < 8.001 and P” = 0.89, P +C .OOl, respectively; Fig. 6). The L-P transport of calcium in the duodenum, however, was reduced by vitamin D deficiency and was restored by vitamin D repletion (P < 0.001). The effect of urea and n-glucose on L-P transport of calcium, which
A
L < E 300 \" 0 2 =c 250 Is z 3
AND
TABLE 3. Effect of sodium, mannitol, urea, and o-glucose on calcium absorption in vitamin Ddeficient and -repleted duodenum and ileum ---
200
z: 0 Q 150
Y, = 70 + 0.36X ? = 0.77
Vitamin Test Solution*
100
50
1
25
1
50
I
75
100
A WATER
I
I
I
125
150
175
(pl/em/hr)
FIG. 4. Effect of net water flow on net calcium absorption in duodenum (open symbols) and ileum (closed symbols). Water flow was induced by varying osmolarity of perfusate from 105 mosM (hypotonic solutions, A) to 300 mosM (isotonic solutions, 0) with a constant NaCl concentration of 50 mM.
400
Y, 8243
Vitamin
D Repleted
L-P transport ‘“Ca, nip/cm per h
P-3
---------T
Duodenum NaCl Manni to1 LJrea D-GlUcoSe, 25 mM
516.7 501.7 580.0 578a3
-t 7.6* rt 4.0 I? 5.8 t 7.9
i
ileum NaCl Mannitoll Urea D-GlUCOSe, 25 mM
203.3 146.7 285.0 395.0
+ 7.2 -r- 4.9 +_ 6.19 -t 8.47
:
Values CaCl, in comparing D-glucose.
+ 0.54X
D Deficient
1
NS 0.001 0.001
0.001 0.001 1 0.001
PI/--
821.7 810.0 823.3 818.3
5 I I -t
11.4 9.3 12.6 13.0
201.7 123.3 266.7 366.7
zt 6.0 -+ 7.6 XL4.9 -+ 14.1
NS NS NS
t 0.001 0.001
0.001.
------- A----
are means -t- SE of 6 experiments * Concentration of test solution was 3 mM. t Unpaired Student t test calcium transport between NaCl and mannitol, urea and
300
350 & \ E 300 \u 0 ZE E* 250 V z 3 3
250 h 2 \ fj 200 \ 5 z =t
E V
200
E 3 i3
i Q
150
v
DEFICIENT--Yc = 78 + 0.69X r = 0.93
ov 0
REPLETEDYc = 76 + 0.74X I = 0.09
Ip
150
IO0
< (3 a SO
100 0
-50
SO 0
50
100
A
WATER
150
200
250
1
I
1
I
SO
100
150
200
WATER in test (O),
1
250
ABSORPTION
SECRETION
(pl/em/hr)
FIG. 5. Correlation between net water and calcium absorption duodenum (open symbols) and ileum (closed symbols) with solutions of NaCl 150 mM alone (Cl) and with 25 mM of fructose o-glucose (P’), 3-0-methylglucose (A), and n-galactose (0).
I
0
(pi/cm/Iv)
FIG. 6. Relation between net water and calcium absorption in ileum of vitamin D-deficient (closed symbols) and vitamin D-repleted (open symbols) animals. Calcium was perfused in isosmotic solutions ofmannitol (0). NaCl (A). urea (Cl). and n-glucose (0).
Downloaded from www.physiology.org/journal/ajplegacy at Macquarie Univ (137.111.162.020) on February 13, 2019.
CALCIUM
TRANSPORT
IN
DUODENUM
AND
1259
ILEUM
was negligible in the vitamin D-repleted state, became evident in the vitamin D-deficient duodenum (P c 0.001). Furthermore, the effect of the magnitude of water flow on calcium absorption was also apparent in the duodenum of vitamin D-deficient animals. A higher association was observed between net water and calcium absorption in the vitamin D-deficient state (r = 0.70, P c 0.001; Fig. 7) that was significantly different (P < 0.05, t tests of the slopes) from the association between these two measurements in the repleted state (r = 0.34, P > 0.05). DISCUSSION
In agreement with previous studies (6, 14) our results indicate that the rat duodenum and ileum are able to bring about net calcium transport against an electrochemical gradient, with greater rates of absorption in the duodenum. These results also point out significant additional differences in the absorption of calcium between the duodenum and ileum. These differences become evident when calcium interacts with water, sodium, and sugar absorption. Calcium absorption in the duodenum in vivo appears to be independent of the composition of the intraluminal fluid, whereas in the ileum it is influenced by the concentration of sodium, by actively transported sugars, and by the rates of net water absorption. Manipulation of the composition of intraluminal solutions, however, caused similar changes in the transmural PD and in the rates of net sodium and water absorption in both duodenum and ileum. 900
REPLETE0 Y, = 727 + 0.16X r = 0.34 0
800
_
*
Cl
O# 0
CJ0
0
A
DEFICIENT Y, = 382 + 0.43X r = 0.70
300
200 -50 SECRETlON
I
1
0
50
I
1
100
1
I
150
200
I
250 J
ABSORPTION
A WATER (pl/cm/hr) 7. Relationship between net water and calcium absorption in duodenum of vitamin D-deficient (closed symbols) and vitamin Drepleted (open symbols) animals. Calcium was perfused in isosmotic solutions of mannitol (O), NaCl (A), urea (Cl), and D-glucose (0). FIG.
Changes in the transmural PD do not seem to have any effect on calcium absorption in the ileum. Choline chloride solutions caused PD changes of a magnitude similar to those with mannitol solutions without affecting calcium absorption. Although the increase in the positivity of the transmural PD observed with mannitol and choline chloride should enhance lumen-to-cell movement of calcium (12), it is possible that this flux was not of sufficient magnitude to alter net calcium absorption significantly (1>. Previous work has presented conflicting evidence on the effect of sodium on calcium absorption in vitro (10, 24). Our studies in intestinal loops in vivo cannot resolve this controversy since the interaction of sodi urn and calcium absorption under these experimental conditions appears more complex th .an in vitro systems. First, calcium absorption was affected by sodium in the ileum exclusively and only when sodium chloride was replaced by mannitol or when greater rates of sodium transport were induced by actively transported sugars. Second, sodium diffused back in the perfusate of sodium-free solutions and we should assume that its concentration was even higher in the unstirred layer (13). Nevertheless, it is theoretically possible that sodium might contribute to the ileal absorption of calcium, which could be dependent on the rates of lumen-to-plasma movement of sodium, and the sodium found in the perfusate could simply be the result of plasma-to-lumen secretion. This hypothesis fails to explain the finding that calcium absorption remained unaffected when sodium chloride was replaced by choline chloride and urea. It is also conceivable, although highly speculative, that choline chloride might increase the concentration of sodium ions in the unstirred layer by increasing the density of negative charges at the luminal surface of the brushborder membrane (2, 4). This higher concentration of sodium ions at the membrane could then become available to the transport of calcium despite its low intraluminal concentration. However, the effect of urea on calcium absorption cannot be reconciled with either hypothesis. The finding that stimulation of net water flow by differences in osmotic pressure and by passively transported urea enhanced net calcium absorption in the ileum suggests that, in addition to an active transport process, calcium may be transported by solvent drag. Previous studies have sho wn that net w ,ater flow across biological membranes can stimulate the net transport of charged solutes against an electrochemical gradient (1, 18). This hypothesis is supported by the strong association between net water and calcium absorption under a variety of experimental conditions. Solvent drag could explain the discrepancy in the rates of calcium absorption between mannitol and choline chloride or urea solutions. Actively transported sugars could also increase net calcium absorption by generating net water flow. Water movement could drag calcium across the ileal mucosa through the tight junction of the epithelial cells, as has been suggested for magnesium absorption (1) In accord with the observations of Urban and Schedl
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J.
1260 (17), calcium absorption in the ileum was not affected by vitamin D. Furthermore, vitamin D deficiency did not inhibit the stimulatory effect of n-glucose on calcium absorption. However, it decreased calcium absorption in the d uodenum, whi ch w ‘as restored by vitamin D repletion. Moreover, in the vitamin D- deficient state, calcium absorption was more responsive to the composition of’ the intraluminal fluid with greater correlation between net water and calcium absorption. These findings would suggest that net water flow exerts an effect under conditions of impaired or less efficient calcium transport in the vitamin D-deficient duodenum. These findings support and could also explain previous observations (7, 19) that lactose stimulation of calcium absorption was greater in the rat ileum in vivo and that this lac tose effect was independent of the action of vitamin D @I . It is possibl e that lactose increa ses the net absorption of calcium by generating net water flow during the acti ve transport of i ts intraluminal end products, D- glucose and n-galactose (22). The contribution of this additional transport process to calcium homeostasis may become physiologically significant in
BEHAR
AND
M.
D.
KERSTEIN
malabsorption syndromes affecting the proximal small intestine or in vitamin D-deficient stat!es. Finally, the results of this study do not elucidate the structural or physiological factors responsible for the differences in calcium absorption between the duodenum and ileum. The former, with a larger pore radius, should be more permeable than the ileum to the transport of most ions (3). It is possib le, however, that the ileum is more permeable to the passive transpor t of calcium. Calcium appears more readily secreted in the ileum and colon (16, 20, 25) with inefficient calcium transport systems. The impermeability of the duodenum could be the result of a highly efficient active transport process, which becomes more permeable with an impaired calcium transport in the vitamin D-deficient state. We are indebted to Ann able technical assistance, vice, and to Judy Johnson manuscript. Received
for publication
Bartiss and Allyn Moore for their invaluto Emanuel Lerner for his statistical adand Mary Murray for preparation of the 11 July
1975.
REFERENCES 1. BEHAR, J. Magnesium absorption by the rat ileum and colon. Am. J. Physiol. 227: 334-340, 1974. 2. BIEBERDORF, F. A., S. MORANSKJ, AND J. S. FORDTRAN. Effect of sodium, mannitol, and magnesium on glucose, galactose, 3-0methylglucose and fructose absorption in the human ileum. Gastroenterology 68: 58-66, 1975. 3. CLARKSON, T. W., AND A. ROTHSTEIN. Transport of monovalent cations by the isolated small intestine of the rat. Am. J. PhysioZ. 199: 898-906, 1960. 4. HOGBEN, C. A. M., D. J. Tocco, B. B. BRODIE, AND L. S. SCHANKER. On the mechanism of intestinal absorption of drugs. J. Pharmacol. Exptl. Therap. 125: 275-282, 1959. 5. JACOBSON, E. D., D. C. BONDY, S. A. BROITMAN, AND J. S. FORDTRAN. Validity of polyethylene glycol in estimating water volume. Gastroenterology 44: 761-767, 1963. 6. KRAWITT, E. L., AND H. P. SCHEDL. In vivo calcium transport by rat small intestine. Am. J. Physiol. 214: 232-236, 1968. 7. LENGEMANN, F. W. The site of action of lactose in the enhancement of calcium utilization. J. Nutr. 69: 23-27, 1959. 8. LENGEMANN, F. W., R. H. WASSERMAN, AND C. L. COMAR. Studies on the enhancement of radiocalcium and radiostrontium absorption by lactose in the rat. J. Nutr. 68: 443-456, 1959. 9. MALAWER, S. J., AND D. W. POWELL. An improved turbidimetric analysis of polyethylene glycol utilizing an emulsifer. GastroenteroZogy 53: 250-256, 1967. 10. MARTIN, D. L., AND H. F. DELUCA. Influence of sodium on calcium transport by the rat small intestine. Am. J. Physiol. 216: 1351-1359, 1969. II. POWELL, D. W., AND S. J. MALAWER. The relationship between water and solute transport from isosmotic solutions by rat intestine in vivo. Am. J. PhysioL. 215: 49-55, 1968. 12. ROSE, R. C., AND S. G. SCHULTZ. Studies on the electrical potential profile across the rabbit ileum. Effect of sugars and amino acids on transmural and mucosal electrical potential differences. J. Gen. Physiol. 57: 639-663, 1971. 13. SALTZMAN, D. A., F. C. RECTOR, AND J. S. FORDTRAN. The role of intraluminal sodium in glucose absorption in uiuo. J. CLin. In-
vest. 51: 876-885, 1972. 14. SCHACHTER, D., E. B. DOWDLE, AND H. SCHENKER. Active transport of calcium by the small intestine of the rat. An2. J. Physiol. 198: 263-268, 1960. 15. SCHACHTER, D., D. V. KIMBERG, AND H. SCHENKER. Active transport of calcium by intestine; action and bioassay of vitamin D. Am. J. Physiol. 200: 1263-1271, 1961. 16. URBAN, E. Calcium transport by the rat colon in uiuo. CLin. Res. 14: 73, 1971. 17. URBAN, E., AND H. P. SCHEDL. Comparison of in vivo and in vitro effects of vitamin D on calcium transport in the rat. Am. J. Physiol. 217: 126-130, 1969. 18. USSING, H. H., AND B. ANDERSON. The relation between solvent drag and active transport of ions. Proc. In tern. Congr. Biochem., 3rd) Brussels, 1955. 19. VAUGHAN, 0. W., AND L. J. FILER, JR. The enhancing action of certain carbohydrates on the intestinal absorption of calcium in the rat. J. Nutr. 71: 10-14, 1960. 20. WALLING, M. W., AND D. V. KIMBERG. Active secretion of calcium by adult rat ileum and jejunum in vitro. Am. J. PhysioZ. 225: 415-422, 1973. 21. WASSERMAN, R. H. The Transfer of Calcium and Strontium Across the Biological Membranes. New York: Academic, 1963, p. 127-182. 22. WASSERMAN, R. H., AND C. L. COMAR. Carbohydrates and gastrointestinal absorption of radiostrontium and radiocalcium in the rat. Proc. Sot. Exptl. Biol. Med. 101: 314-317, 1959. 23. WASSERMAN, R. H., V. A. KALLFELZ, AND C. L. COMAR. Active transport of calcium by rat duodenum in uiuo. Science 133: 883884, 1961. 24. WASSERMAN, R. H., AND A. N. TAYLOR. The non-essentiality of sodium ions for intestinal calcium transport. Proc. Sot. Exptl. BioZ. Med. 114: 479-482, 1963. 25. YOUNOSZAI, M. K., AND H. P. SCHEDL. Intestinal calcium transport: comparison of duodenum and ileum in uivo in the rat. Gastroenterology 62: 565-571, 1972.
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