Regulation of Intestinal Calcium Absorption in the ...

Regulation of Intestinal Calcium Absorption in the Laying Quail: Independent of Kidney Vitamin D Hydroxylation1 ARIE BAR, URI EISNER, GIULIA MONTECUCCOLI2 ANDSHMUEL HURWITZ Institute of Animal Science, Agricultural Research Organization, The Vulcani Center, Bet Dagan, Israel

Changes in calcium absorption under different nutritional or physiological condÃ¬tions were demonstrated previously in the laying fowl (1). Two types of regulatory mechanisms have been suggested: (a) an increase in calcium absorption associated with an increase in calcium binding protein (CaBP), observed either under conditions of calcium depletion or at the onset of egg production (1, 2); and (b) a higher calcium absorption during eggshell calcification than during shell gland (uterine) inactivity. The latter does not involve any significant changes in CaBP level (2, 3). The changes produced by dietary calcium restriction (4-6) or by the onset of

sponse to calcium restriction manifested by increased intestinal CaBP and calcium absorption (1, 9-11) does not occur when the only sources of vitamin D activity in the diet are compounds which do not have to undergo further hydroxylation in the kidney, such as dihydrotachysterol (DHT3) (10), 1,25(OH)2-CC (4) or 1 a-OH-cholecalciferol (l Â«-OH-CC) (11). The mechanism for the second type of regulation of calcium absorption which operates in laying birds during the laying cycle, is still unknown. The purpose of this study was to examine the possibility that this mechanism also involves modulaâ€”

egg production are probably mediated fhrniicrli rnrrpsnnnrlino1 pJiantrpÂ« in thp fortnrOUgn corresponding cnanges in ine lor mation OÃ•1,25 ( OH )2-cholecalcifCrol (1,25(OH)2-CC) by the kidney3 and its aCCU-

Received for publication February 17, 1976. * Contribution from the Agricultural Research Orpanlzatlon, The Volcanl Center, Bet Dafian, Israel. 1Â»75 Series, No. 289-E. Supported by the Poultry M^^D^T^LÂ¡sfeiuStltute of Human Physl-

mulation in the mucosal cell * (7) .where

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ABSTRACT Two experiments were carried out with laying quail (Coturnix coturnix japÃ³nica) fed either cholecalciferol (40 /Â¿g/kg) or 1 a-hydroxycholecalciferol (8 /Â¿g/kg). Recovery following vitamin D deple tion, as indicated by egg production and shell deposition, was faster in quail fed 1 a-hydroxycholecalciferol than in those fed cholecalciferol. Overall intestinal calcium absorption was higher in the 1 a-hydroxycholecalciferol-fed quail due to a higher absorption in the middle parts of the small intestine. This was associated with corresponding differences in the concentration of calcium binding protein. Intestinal calcium absorption was markedly higher during periods of shell formation than during periods of uterine inactivity in quail fed either cholecalciferol or 1 a-hydroxycholecal ciferol. It is suggested, therefore, that the mechanism responsible for this difference is independent of vitamin D hydroxylation in the kidney. J. Nutr. 106: 1336-1342, 1976. INDEXING KEY WORDS calcium absorption â€¢calcium binding protein â€¢cholecalciferol â€¢1 a-hydroxycholecalciferol â€¢laying quau

REGULATION

OF Ca ABSORPTION

IN LAYING QUAIL

1337

tion of 1,25(OH)2-CC synthesis. The study was carried out using laying quail (Coturnix coturnix japÃ³nica) characterized by their high rate of egg production, their short generation interval, their low body weight, and by their similarity to the do mestic laying hen with regard to the pres ence of the same intestinal or uterine CaBP and their similar regulatory mech anism of calcium absorption (12). These quails were fed continuously either cholecalciferol (CC) or 1 Â«-OH-CC,5a cholecalciferol derivative which does not have to undergo further hydroxylation in the kidney.

uterine inactivity (105-120 minutes after oviposition). Their intestines were exposed and immediately divided into five segments each.6 The content of each segment was removed by gentle manipulation, dried overnight at 105Â°,ashed for 6 hours at 650Â°,and dissolved in hydrochloric acid. The samples were then analyzed for cal cium and 91Ycontent. Cumulative net cal cium absorption at the level of any intesti nal segment was calculated by:

METHODS

where (Ca/Y)f and (Ca/Y)( are the ratios of calcium to 01Yin the feed and the in testinal segment, respectively. Chemical and radioisotope assays. Cal cium in plasma and in dry-ashed samples of intestinal content was determined by direct EDTA titration under ultraviolet light using Fluorescein complexon as an indicator. Inorganic phosphorus was deter mined by the method of Gomori (15) in filtrates of plasma treated with 20% trichloric acetic acid. Bones were cleaned of adhering tissues, dried at 105Â°,and ashed at 650Â°for 6 hours. Eggshells were weighed, dried, ashed and dissolved in hydrochloric acid similar to the intestinal contents. Eggshell calcium was determined by direct EDTA titration using hydroxy naphthol blue 7 as indicator (16). Shell weight per unit area (shell density) was calculated using the formula of Mueller and Scott (17). The 91Yof the dissolved ash samples of intestinal content was counted in a liquid Scintillation spectrometer8 using Bray solution. CaBP measurements. The uterus and the different intestinal segments 6 were rinsed in ice cold 154 mM sodium chloride, cut into small pieces, diluted (1:4, w/v) with buffer pH 7.4 (in mM: Tris HC1, 13.7; NaCl, 119; KCL 4.5; glucose, 0.09), hoe Synthesized by Prof. Y. Mazor of the Weizmann Institute of Science, Rehovot, Israel. "Duodenum, upper jejunum, lower Jejunum, upper ileum and lower ileum. Entry of the bile ducts was taken as the end of the duodenum and the Intestine between this point and the entry of ceca was divided into four equal segments. The upper segments are defined as the jejunum and the two lower ones as the ileum. 7 Malllnckrodt Chemical Works, St. Louis, Mo. 8 Packard Instrument Co. Inc., Downers Grove, 111.


Animals and diet. Laying Japanese quail were kept in individual laying cages placed in a windowless room. The room was il luminated by fluorescent light with no ultraviolet spectrum. The birds were fed ad libitum a commercial diet containing (by chemical analysis) 3.4% calcium and 0.7% phosphorus. At the age of 10 weeks, after laying for 4 to 5 weeks, 50 quail were chosen on the basis of their egg produc tion rate (0.8-0.9 eggs/day) and were fed a vitamin D-deficient, semipurified diet composed of (in %): soybean oil meal (45% protein), 50.0; glucose, 37.5; soy bean oil, refined, 3.0; sodium chloride, 0.25; DL-methionine, 0.15; vitamin mix (13), 0.5; trace mineral mix (13), 0.1; dicalcium phosphate, 2.5; and calcium carbonate, 6.0. After 16 days of consuming this diet some of the quails stopped laying, while others laid eggs without shells or with very thin ones. They were then divided into two groups and were fed for 5 additional weeks similar semipurified diets containing either 40 /tg/kg of cholecalciferol or 8 /xg/kg of 1 Â«-OH-CC.5An additional similar experi ment was performed 2 months later, using laying quail of the same hatch. Intestinal absorption. Calcium absorp tion by intact birds was measured using yttrium-91 (91Y) as a nonabsorbed refer ence substance, as described previously ( 14 ). For 4 days prior to killing, the quail were fed the same diets but labeled with 80 /xC/kg of 91Y.Birds were bled and then killed by an overdose of sodium pentobarbital either while forming an eggsnell ( 1217 hours after oviposition) or during

% absorption of calcium (Ca/Y), - (Ca/Y)i X 100 (Ca/Y),

1338

BAR, EISNER, MONTECUCCOLI AND HURWITZ

mogenized at 4Â°,and centrifuged for 20 minutes at 35,000 X g in a refrigerated OJÃŽOai centrifuge (18). Supernatant CaBP concentration was ^ co o>8 NIO! â€¢u measured by a radial immunodiffusion as Â«^â€¢^N say using a specific antiserum 9 for purified Ã³Ã¡|ZlÃ-Ã-cogIIIÃ¢me chick intestinal CaBP (19). Supernatants were diluted to attain a final concentration of 10 to 30 /xg CaBP/ml. Agars were stained with tannic acid (4%) in order to visualize the precipitation rings (20). cholecalcifelIjjjâ€” Â±0.06Â» quail. of 20 Iu le diet supp Statistical procedures. Means, standard Â±0.2Â§N00â€¢Â«â€¢HCOt10.6 errors, analyses of variance or Student's o00 -H f-tests were calculated according to stan ^ai o>M dard procedures (21). S2 RESULTS Feeding the laying quail a vitamin-Deâ€¢u >.lieIIIfS 2 gcol> S deficient diet for 16 days resulted in a sig COdoOrolÃ¬Sui0052 nificant ( P < 0.01 ) reduction in egg pro *i?CO 00O)i duction (from 0.8-0.9 eggs/day to 0.3 and m 0.2 egg/day in the first and second experi _J|fios ment, respectively), and the few eggs laid were poorly calcified (table 1). The re oÂ§*eÂ» 5 covery from the vitamin-D-deficient con dition was faster in quail fed la-OH-CC ='S' divi' were than in those fed CC (table 1). Egg pro Val diet. DÃŒ peÂ«t*t 35-day > n1Ã¯nd Â±0.05 duction of the la-OH-CC-fed quail Â±0.4oo0.53 .6 OooT' reached a high constant level within 7 days, while the CC-fed quail reached this SO6 level only after 14 days. Eggshell quality (eggshell density or the calcium content of ). respectively, ] q.Â§'S16,Ã€K)Ã eggshell NÃ®r*.to1".2â€¢ogfWla-OH-CC8la-OH-CC88VQi58la-OH-CC89Â§ZÂ£Qae>0.74 wÂ¿ 814to eggshells) of la-OH-CC-fed quail reached Â±0.11Â» diet; day on Â»the Â±0.22MooHH-HÂ«M00â€”Ã¯Ã¯SodeienS 10.4 ud a high constant level within 3 days, while o * -H g the CC-fed quail reached this level only co â€¢"â€¢C4IH after 7 or more days. Plasma calcium or inorganic phosphorus (table 2) was not significantly different in tsTJc"2Â°Ã¡|â€¢S_ quail fed either vitamin D supplement OSob-Â«5 during the period of shell formation or of *â€¢*S|Ã¯ uterine inactivity. The slight increase in COCO CO-LI bone ash of the la-OH-CC-treated quail S"I'll'â€¢Â«iÃ-r was not significant. Duodenal or uterine CaBP level (table 3) was not significantly affected by dietary source of vitamin D or COtÂ»SgÃ¬ by the stage of egg shell formation. Intestinal absorption of calcium was higher ( P < 0.01 ) during egg shell forma tion than during periods of uterine inactiv Â®â€” Ãœ ity (fig. 1, table 3) in both CC- and lasfifiW Â§a s OS'T)&Â£l U SIlUlliw .Ã¤. OH-CC-fed quail. This difference between the quail forming an eggshell and not = Â«t.S-s-s1111jforming one, found mainly in the duodeof 8Calculated"*S or ne W( period not are'

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â€¢ The specific antiserum for chick CaBP was gen erously supplied by Dr. R. H. Wasserman of the Veterinary College, Cornell University. Ithaca, N.T.


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REGULATION OF Ca ABSORPTION IN LAYING QUAIL

1339

TABLE 2 Plasma calcium and inorganic phosphorus and bone ash as influenced by dietary source of vitamin D activity" concentrationformationplasma Vitamin D sourceCholecalciferol ml+ la-OH-CholecalciferolBlood

ashmg

phosphorusmg/100 calcium ml

mg/100

26.8Â±0.7 27.5Â±0.8 27.0Â±0.6 25.2Â±0.7

+

0 Values given in the table are meansisEM of the parameters.

matter183Â±

7.6Â±1.1 5.8Â±0.4 7.9Â±1.0 6.5Â±0.3Tibia

7 198Â±10 191Â±8 208Â±10

% dry

56.0Â±0.8 57.4Ãœ.4 57.0Â±0.6 60.0Â±1.2

of 20 laying quail. Groups did not differ significantly

than the duodenum, where the difference between the two dietary treatments occurred. Results of intestinal calcium absorption and the recovery from the vitamin D-deficient state obtained in this experiment were similar to those of the first, and therefore are not given in detail, As in the first experiment, duodenal CaBP level during shell formation was not significantly different between the CC- and the la-OH-CC-fed quail (table 4). However, in the lower parts of the intestine, CaBP level was found to be higher in the la-OH-CC-fed quail than in the CC-fed quail, DISCUSSION At the concentration used in this study, la-OH-CC proved to be as effective as a five-times-higher concentration of CC, in supporting egg production, eggshell qual-

TABLE 3 The effect of dietary source of vitamin D activity on intestinal calcium absorption and on duodenal and uterine CaBP"

Vitamin D sourceCholecalciferol

Eggshell absorption formation6 calcium"'"1+of

la-OH-CholecalciferolIntestinal

+

49.1Â±3.7 22.1Â±2.8 66.9Â±4.5 23.1Â±6.3CaBP

concentrationDuodenummg/g2.33Â±0.12

2.70Â±0.24 2.88Â±0.13 2.46Â±0.33 2.76Â±0.23 2.32Â±0.22Uterusmg/g2.24Â±0.15 2.62Â±0.13

" Values given in the table are meansÂ±SEM of 6 to 8 laying quail. * The eggshell-forming quail (+) were killed 12 to 17 hours after oviposition. Quail not forming eggshells ( â€”)were killed 105 to 120 minutes after oviposition, when a new egg was found in the magnum. ' Intestinal overall net calcium absorption was calculated from the calcium-to-yttrium-91 ratio of the lower ileum and expressed as a percentage of feed intake. d Factorial analysis indicated a significant effect (P < 0.01) of the vitamin D source, eggshell formation status and a significant interaction between both factors.


num and the jejunum, was significant (P < 0.01) for each of the tested segments. Intestinal calcium absorption was higher ( P < 0.01 ) in la-OH-CC-fed quail than in those fed CC, during periods of shell formation ( table 3, fig. 1 ). This difference was the result mainly of jejunal absorption. No significant difference in calcium absorption between the two dietary groups was found during periods of uterine inactivity. A second experiment was conducted (a) to confirm the results of the first with respect to intestinal calcium absorption and the recovery from the vitamin-deficient state; and ( b ) to test the possible relation ship between the higher calcium absorption in la-OH-CC-fed quail during periods of eggshell formation, and a possible corresponding change in the level of CaBP in the parts of the intestinal tract lower

in any

1340

BAR, EISNER, MONTECUCCOLI AND HURWITZ

10

20

INTESTINAL

30

Â¿0

50

LENGTH, cm

ity, bone ash, plasma calcium or inorganic phosphorus. Similar to the known effect of la-OH-CC in vitamin D-deficient chicks (22-24), the recovery of laying quail from vitamin D deficiency was faster when the quail were fed la-OH-CC than when fed CC. In a previous study (11), we showed that 8 fig la-OH-CC/kg diet, promotes a higher calcium absorption ability and

TABLE 4 CaBP level along the intestine of cholecalciferol- or la-OH-cholecalciferol-fed laying quail"

Intestinal6 CaBP Vitamin D sourceCholecalciferol

jejunummg/g0.76Â±0.27

la-OH-CholecalciferolDuodenummg/g2.84Â±0.18 1.04Â±0.21 2.96Â±0.08Upperjejunummg/g2.11Â±0.13 2.52Â±0.18Lower2.33Â±0.09Ileum0.19Â±0.03 0 Values given in the table are meansÂ±SEM of 5 to 6 laying quail killed during periods of eggshell formation. Factorial analysis of variance indicated significant effects (P < 0.01) of vitamin D sources and intestinal segments. 6Entry of the bile duct was taken as the end of the duodenum and the intestine between this point and the entry of ceca was divided into four equal segments. The upper two segments were defined as the jejunum and the lower two segments as the ileum.


Fig. 1 Cumulative percentage of net calcium absorption in laying quail fed cholecalciferol or la-OH-cholecalciferol during periods of eggshell formation or uterine inactivity. Cholecalciferolfed, eggshell forming, O; cholecalciferol-fed, uterine inactivity, â€¢; la-OH-CC-fed, eggshell forming, fj; la-OH-CC-fed, uterine inactivity, â€¢. SE of the means were 6.5, 4.8, 4.9 and 5.5 for the upper jejunum, lower jejunum, upper ileum and lower ileum, respectively.

CaBP concentration in the duodenum of chicks, than 40 /Â¿gCC/kg diet. In the present study with laying quail using the same respective concentrations of vitamin D supplements, an increase in calcium ab sorption and CaBP occurred in the jeju num, rather than in the duodenum of quail during the period of eggshell formation (fig. 1). The reason for this difference in the site of effect of la-OH-CC between chicks and laying quail is not clear. It could be related either to a species dif ference or to the possibility that CaBP in the laying quail, unlike in the chick, had reached maximal concentrations. It is puzzling however, that an increase in cal cium absorption due to la-OH-CC did not occur also during uterine inactivity. In the ileum, a large increase in CaBP occurred in response to la-OH-CC without a concomitant increase in calcium absorp tion. It has been shown (25) that the frac tion of active calcium in the ileal contents of the laying hen is much lower than that of the jejunal contents. This may be the limiting factor for calcium absorption in the ileum. Since in vivo net calcium ab sorption, rather than absorption capacity, has been measured in the present work, the lack of response of calcium absorption in the ileum to la-OH-CC does not neces sarily reflect on its absorption capacity. Laying quail, during periods of eggshell formation, absorbed more calcium than during periods of uterine inactivity, when fed either CC or la-OH-CC. This increase in calcium absorption was not associated with a corresponding increase in the con-

REGULATION OF Ca ABSORPTION IN LAYING QUAIL

6. 7.

8.

9.

10.

11.

12.

13.

ACKNOWLEDGMENTS

The authors thank Professor R. H. Wasserman of Cornell University, Ithaca, New York, for the generous supply of the specific antiserum, and Professor Y. Mazor of the VVeizmann Institute of Science, Rehovot, Israel, for the synthetic la-hydroxycholecalciferol. The technical assist ance of Mrs. M. Cotter is gratefully ac knowledged. LITERATURE

14.

15.

16.

CITED

1. Hurwitz, S., Bar, A. & Cohen, I. (1973) Regulation of calcium absorption by fowl intestine. Am. J. Physiol. 225, 150-154. 2. Bar, A. & Hurwitz, S. (1972) Relationship of duodenal calcium-binding protein to cal cium absorption in the laying fowl. Comp. Biochem. Physiol. 41B, 735-744. 3. Bar, A. & Hurwitz, S. (1975) Intestinal and uterine calcium-binding protein in laying hens during different stages of egg formation. Poultry Sci. 54, 1325-1327. 4. Deluca, H. F. ( 1974 ) Regulation of func tional vitamin D metabolism: A new endo crine system involved in calcium homeostasis. Biochem. Soc. Spec. Pubi. 3, 5-26. Ed. P. R. Fraser. 5. Norman, A. W. & Henry, H. (1974) 1,25Dihydroxycholecalciferolâ€”an hormonally ac

17. 18.

19.

20. 21.

tive form of vitamin Da. Recent Progr. Hor mone Res. 30, 431-480. Kodicek, E. (1974) The story of vitamin D from vitamin to hormone. Lancet 1, 325-329. Edelstein, S., Harell, A., Bar, A. & Hurwitz, S. (1975) The functional metabolism of vitamin D in chicks fed low-calcium and lowphosphorus diets. Biochem. Biophys. Acta 385, 438-442. Emtage, J. C., Lawson, D. E. M. & Kodicek, E. ( 1974 ) The response of the small in testine to vitamin D. Isolation and properties of chick intestinal polyribosomes. Biochem. J. 140, 239-247. Morrissey, R. L. & Wasserman, R. H. (1971) Calcium absorption and calcium-binding pro tein in chicks on differing calcium and phos phorus intakes. Am. J. Physiol. 220, 15091515. Bar, A. & Wasserman, R. H. (1973) Con trol of calcium absorption and intestinal cal cium-binding protein synthesis. Biochem. Bio phys. Res. Commun. 54, 191-196. Bar, A., Hurwitz, S. & Edelstein, S. (1975) Response of renal calcium-binding protein: Independence of kidney vitamin D hydroxyla tion. Biochim. Biophys. Acta 411, 106-112. Bar, A., Dubrov, D., Eisner, U. & Hurwitz, S. (1976) Calcium-binding protein and cal cium absorption in the laying quail (Coturnix coturnix japÃ³nica). Poult. Sci. 55, 622-628. Hurwitz, S., Harrison, H. C. & Harrison, H. E. (1967) Effect of vitamin D3 on the in vitro transport of calcium by the chick in testine. J. Nutr. 91, 319-323. Hurwitz, S. & Bar, A. (1965) Absorption of calcium and phosphorus along the gastro intestinal tract of the laying fowl as influenced by dietary calcium and shell formation. J. Nutr. 86, 433-438. Gomori, G. (1942) A modification of the colorimetrie phosphorus determination for use with photometric colorimeter. J. Lab. Clin. Med. 27, 955-960. Hurwitz, S. & Bar, A. (1966) Calcium de pletion and repletion in laying hens. 1. Ef fect on calcium in various bone segments, in egg shells and in blood plasma, and on cal cium balance. Poultry Sci. 45, 345-352. Mueller, C. D. & Scott, H. M. (1940) The porosity of the egg shell in relation to hatchability. Poultry Sci. 19, 163-166. Wasserman, R. H. & Taylor, A. N. (1966) Vitamin D3-induced calcium-binding protein in chick intestinal mucosa. Science 152, 791793. Corradino, R. A. (1973) Embryonic chick intestine in organ culture. A unique system for the study of the intestinal calcium ab sorptive mechanism. J. Cell Biol. 58, 64-78. Simmons, P. (1971) Quantitation of plasma proteins in low concentration using RID. Clin. Chim. Acta 35, 53-57. Snedecor, G. W. & Cochran, W. G. (1968) Statistical Methods. 6th ed., Iowa State Uni versity Press, Ames, Iowa.

22. Holick, M. F.. Semmler. E. T-, Schnoes, H. K.


centration of duodenal CaBP (table 3) (1-3). Thus, the la-OH-CC-fed quail were capable of regulating their intestinal ab sorption of calcium at least as efficiently as those fed CC. Since la-OH-CC does not have to undergo any hydroxylation in the kidney (22), it may be concluded that the increased calcium absorption as a response to the stimulus of shell formation is inde pendent of changes in vitamin D hydroxyl ation in the kidney. This conclusion is further supported by the facts that (a) the increase in calcium absorption during periods of eggshell for mation was not associated with a corre sponding increase in CaBP (table 3); (fo) kidney 1-hydroxylase activity was the same during periods of eggshell formation and of uterine inactivity;3 and ( c ) distribution of vitamin D metabolites in the intestinal mucosa was found to be rather indepen dent of the uterine activity in quail (65% and 53% 1.25(OH)2-CC of total metab olites in the quail forming and not forming eggshell, respectively) during eggshell for mation or uterine inactivity.

1341

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MONTECUCCOLI

& DeLuca, H. F. (1973) la-Hydroxy deriv ative of vitamin D3: A highly potent analog of lot,25-dihydroxyvitamin D3. Science 180, 190191. 23. Okomura, W. H., Mitra, M. N., Wing, R. M. & Norman, A. W. ( 1974 ) Chemical synthe sis and biological activity of 3-deoxy-lahydroxyvitamin D3, an analog of la,25(OH)Â»Da, the active form of vitamin D3. Biochem. Biophys. Res. Commun. 60, 179-185.

AND HURWITZ

24. Zerwekh, J. E., Brumbaugh, P. F., Haussler, D. H., Cork, D. J. & Haussler, M. R. (1974) la-Hydroxyvitamin D3. An analog of vitamin D which apparently acts by metabolism to la,25-dihydroxyvitamin D3. Biochemistry 13, 4097-4102. 25. Hurwitz, S. & Bar, A. ( 1968 ) Activity, con centration, and lumen-blood electrochemical potential difference of calcium in the intestine of the laying hen. J. Nutr. 95, 647-654.
