EFFECTS OF 25-HYDROXYVITAMIN D, ON RAT DUODENUM, JEJUNUM, AND

Vol.

THE JOURNAL 249, No. 4, Issue

OF BIOLOGICAL

CHEMISTRY

of February 25, pp. 1156-1161, Printed in U.S.A.

1974

Effects of 25-Hydroxyvitamin Duodenum, Jejunum, and CORRELATION Ds METABOLITES*

Oli’

CALCIUM

ACTIVE

D, on Rat Ileum TRANSPORT

WITH

TISSUE

LEVELS

OF VITAMIN

(Received for publication, MARLIN

W.

WALLING,~

RlmmAY J.

FAVUS,$

AND

DANIEL

V.

August 17, 1973)

KIMBERGT[

From the Depa&nelrt of Medicine, Harvard Medical School, and the Gastrointestinal Unit of the Department of Medicine, Beth Israel Hospital, Boston, Massachusetts02215

Jejunum and ileum from vitamin D-deficient rats actively secreted calcium when studied in vitro, while duodenum from these animals actively absorbed calcium. Acute repletion with a single physiologic dose of 25-hydroxyvitamin D3 (25-OH D3) (15 i.u.) increased calcium absorption in duodenum and ileum but had no effect on jejunum. Studies of the distribution of 25-OH D3 and its metabolites in these tissues demonstrated that levels of 1,25-(OH)* Da per cell were highest in duodenum, much lower in ileum and lower still in jejunum. The increased calcium absorption in duodenum and ileum was proportional to the levels of 1,25(OH)% Ds in these tissues. This was not the case for jejunum however, because while 1,25-(OH)* D3 was present, calcium transport rates were unchanged. Another finding was a much higher percentage of water-soluble metabolites of 25-OH Da in jejunum and ileum than in duodenum. This observation may relate to an enterohepatic circulation of these compounds. These experiments suggest that previous reports of decreased calcium absorptive responses by lower small bowel (relative to proximal duodenum) to either treatment with vitamin D or prolonged dietary calcium deprivation, can be explained by the differential cellular localization of 1,25(OH):! DB (duodenum > ileum > jejunum).

of the vitamin, transfer of 45Ca into the serosal compartment of everted intestinal sacs was greatly increased in proximal duodenum, while the small intestine 20 cm or more distal to the pylorus responded only slightly. Similarly, Urban and Schedl (2) reported that vitamin D-repletion resulted in greater increases in calcium absorption from in viva perfused loops of rat duodenum than comparable preparations of ileum. Kimberg et al. (3) also showed that vitamin D-repletion produced much larger serosal 45Ca/mucosal 45Ca (I :0) ratios in everted gut sacs from rat duodenum than in gut sacs from ileum. These investigators also found that the effect of dietary calcium deprivation on I : 0 ratios was greater in duodenum than in ileum (3). We have recently confirmed this latter observation in the study of transmural calcium fluxes across rat duodenum and ileum in. vitro (4). Since the ca 1clum . transport enhancement which occurs in response to dietary calcium deprivation appears to be mediated through changes in the metabolism of vitamin D (5, 6), the differential response of duodenum and ileum to both vitamin D-repletion and calcium deprivation may be manifestations of the same phenomenon. The purpose of the present study was to examine the effects of repletion with a physiologic dose of 25-011 Dzl (15 i.u.) on calcium transport by rat duodenum, jejunum, and ileum and to attempt to correlate changes in calcium transport with levels of 1 ,25-(OH)2 D3 and other metabolites of the vitamin in these intestinal segments. The results indicate that the differential calcium transport response between duodenum and ileum can apparently be explained by differences in tissue 1 ,25-(0H)2 D3 levels, while the response of jejunum cannot.

Studies by Schnchter et al. (1) dcmonstratrd that when vitamin Ddeficient rats were repleted with a pharmacological dose * This work was supported by Grants AM-13696, AM-05114, and CA-051G7 from the National Institlltes of Health of the United States Public Health Service. $ To whom reprint requests should be addressed. § Present address, Department of Medicine, Michael Reese Hospital and Medical Center and the University of Chicago l’ritzkcr School of Medicine, Chicago, Illinois. In receipt of United States Pltblic Health Service Special Postdoctoral Ilesearch Fellowship AM-53375. f Recipient of a liesearch Career Development Award from the National Institute of Arthritis, Metabolism, and IXgestive Diseases (AM-19377).

MATICRIALS

AKD

ME,THODS

Malt Sherman rats (Camm Research Inst., Wayne, N.J.) obtained at a weight of 90 to 100 g were raised in the dark for 6 weeks on a vitamin D-deficient diet which contained 0.8% cal1 The abbreviations used are: 25.OH-113, 25-hydroxyvitamin 113; 1,25-(OH)2 113, l,Z&dihydroxyvitamin 113; 24,%5-(OH)2 D3, 24,25-dihydroxy-vitamin l)r; 25,X(OH)* 1)3, 25,2Gdihydroxyvitamin 113; JMB, unidirectional flux from the mucosal to serosal surface of intestine; JBM, serosal to mucosal flux; JP~‘<,*, net flux derived from Jus-JSM; XC, short-circuit current; fZ1, electrical resistance of intestine; PI), transmural electrical potential difference.

1156

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SUMMARY

1157 by Lund and DeLuca (14). The aqueous phases were re-extracted with an additional volume of chloroform to assure maximum recovery. The material in the chloroform phases was concentrated by evaporation under a stream of NP and then dissolved in a small volume of chloroform-hexane, 65:35 (v/v) for subsequent chromatographic separation. Radioactivity in portions of the lipid-soluble and aqueous phases was determined as previously described (9) with an Intertechnique model SL-40 liquid scintillation spectrometer (Intertechnique Division, Teledyne Isotopes, Westwood, N.J.). Sephadex LH-20 Column Chromatography-The lipid-soluble metabolites from mucosa and plasma were eluted from columns containing 20 g of Sephadex LH-20 resin (Pharmacia Fine Chemicals Inc., Piscataway, N.J.). The columns were eluted with 345 ml of chloroform-hexane, 65:35 (v/v) then stripped of more polar fractions with 150 ml of chloroform-hexane, 70:30 (v/v) as described by Holick and DeLuca (15). Radioactivity was measured on aliquots removed from each collecting tube and fractions containing the peaks of radioactivity were combined. The pooled fractions for each peak were dried under a stream of Nz and dissolved in 3.0 ml of methanol for subsequent reaction with paraperiodic acid as we have previously described (16). To test the reproducibility of the separation techniques, rat plasma containing tritiated 25-OH D3 and its metabolites was extracted and chromatographed in triplicate with the following results: per cent recovery of total tritium following extraction procedures = 82.6, 85.4, and 86.6; per cent of recovered material in the lipid phase = 94.5, 95.1, and 93.8; per cent in aqueous phase = 5.5, 4.9, and 6.2; per cent of lipid phase eluting as 25.OH D) = 80.4, 84.6, and 85.4; per cent as 24,25-(OH)2 D3 = 15.5, 14.2, and 13.8; per cent as 1 ,25-(OH)z D3 = 0.4, 3.1, and 0.8. These data indicate that the method of chromatographic separation is quite reproducible and that differences between the metabolite peaks of experimental groups in excess of SC/o of the total radioactivity probably represent biological differences rather than variance due to methodology. Plasma Calcium and Phosphorus illeasurements-Plasma calcium concentration was determined with a Perkin-Elmer atomic absorption spectrometer (model 290 13) in the presence of 1.070 lanthanum chloride. Inorganic phosphorus was determined by the method of Fiske and SubbaRow (17). Iron Deferminations-Non-heme iron was measured in both whole blood and mucosa by the method of Briickmann and Zondek (18) as modified by Foy et al. (19). Total iron concentration in these tissues was determined by the method of Lee and Stumm (20). RESULTS

Intestinal Calcium Transport-Duodenum from vitamin D-deficient animals actively absorbed calcium while both jejunum and ileum actively secreted calcium toward the luminal side of the tissue (Table I). The 937-pmole (15 i.u.) dose of 25-OH Ds increased active calcium absorption by duodenum, had little effect on jejunum and increased ileal absorption sufficiently to produce no net flux across this tissue. All changes were due to increases in mucosal to serosal fluxes (Jhls) and serosal to mucosal flux (JsM) was unaffected by 25-OH D3 treatment. The short circuit current (SCC) and intestinal resistance (Rr) for duodenum, jejunum and ileum were unchanged by 25-OH Da treatment; however, the SCC was much greater in ileum and jejunum than in duodenum (no treatment, ileal SCC = 104.8 ~amps~cm-2 f 6.6 SE., n = 7; +25-OH Da, ileal SCC =


cium, 0.4’% phosphorus, and 0.24% magnesium by weight (test diet 69280, General Biochemicals Div., Mogul Corp., Chagrin Falls, Ohio). After 5 weeks on this diet, animals were randomized by weight into two groups (mean weight, 213 g). The first group received no treatment and was used to establish vitamin D-deficient calcium transport levels. Animals in the second group received a single dose of 937 pmoles (15 i.u.) of 25.OH Da intrajugularly under light ether anesthesia 24 hours before sacrifice. Animals used in the calcium transport studies received unlabeled 25-OH D3 (generously provided by Dr. John C. Babcock, Upjohn Co., Kalamazoo, Mich.), while animals used in the study of vitamin D metabolites received a similar dose of 25-0H-[26,27-3H] D3 (New England Nuclear Corp., Boston, Mass.) diluted to a specific activity of 600 mCi per mmole with unlabeled material. All of the repleted animals received the 25.OH D3 dissolved in 95% ethanol as a 50.~1 dose. Calcium Transport Studies-Animals were fasted overnight and sacrificed by cerebral concussion followed by exsanguination. The experimental technique was the same as we have previously described (modified Ussing-type apparatus) (4, 7). The incubation medium was a bicarbonate-buffered KrebsRinger solution gassed with 95% 02-5% COz, containing 1.25 mM calcium, no phosphorus and 11 mM n-glucose (4). The transmural potential difference (PD) was nulled using the method of correcting for fluid resistance described by Field et al. (8). All electrical parameters are reported for time periods when transmural calcium fluxes were at steady state. Net calcium fluxes were measured across paired pieces of intestine from the same animal, therefore, paired t tests were used to evaluate differences between mucosal to serosal fluxes (J& and serosal to mucosal fluxes (JSM) within each group. The segments of duodenum were obtained from the most proximal 5 to 6 cm of small intestine, jejunum from the middle 20 cm and ileum from the distal 15 to 20 cm. The vitamin D-deficient animals were studied during a a-day period immediately prior to the experiments on 25.OH Do-repleted animals. Tissue Preparation-Animals injected with 25.OH-[26,273H] Ds for the study of vitamin D metabolites were sacrificed as described above following an overnight fast. Blood was collected in heparinized tubes on ice and aliquots of whole blood were removed for subsequent determinations of non-heme and total iron. Following centrifugation of the remaining blood, aliquots of plasma were taken for the measurement of calcium and phosphorus concentrations and the remainder was pooled and stored under Nz at -70”. The entire small intestine was removed, rinsed with ice-cold isotonic saline, and gently blotted. The mucosal layer from the proximal 8 cm (duodenum), middle 30 cm (jejunum), and distal 20 cm (ileum) was scraped from the underlying coats and Portions of the mucosal scrapings from each pooled by region. region were removed for non-heme and total iron determinations and the remainder was stored under NB at -70”. Extraction of Radioactivity-Homogenates of intestinal mucosa (10%; w/v) were prepared in glass-distilled water using a Waring Blendor at medium speed for 20 s. Portions of mucosal homogenates and plasma were digested with NCS tissue solubilizer (Amersham-Searle Corp., Arlington Heights, Ill.) and total tritium content was then determined as previously described (9). Portions of mucosal homogenates were also used for measurement of total protein and DNA content as previously reported (10-12). The lipid phases of mucosal homogenates and plasma were extracted using the method of Bligh and Dyer (13) as modified

1158 necessarily also unaffected by 25-OH D3 treatment. However, due to differences in SCC and RI from tissue to tissue the rejrom vitamin sultant 1’D values were the same (mean I’D values for the six groups ranged from 3.1 to 3.8 mv). Treated animals received 937 pmoles of 25-OH L)s intrajugularly 24 hours before sacrifice. Calcium fluxes were studied with the 25-08-[26,g7-3H]DZ Metaboliles-Plasma contained almost transmural PD nulled by a short-circuit current using the ~TL vitro entirely 25-OH D3 and lipid-soluble products of its metabolism technique described under “Materials and Methods.” Net fluxes (Table II). Although there were comparable concentrations were obtained by measuring unidirectional fluxes across paired of total radioactivity in all levels of small intestine, there was a pieces of intestine from each level of the intestine. JN~,~is net much higher percentage of lipid-soluble material in the duoflux and was determined from J,~s-Jsu (mucosal to serosal flux denum, more nearly equal amounts of lipid-soluble and watcrminus serosal to mucosal flux) so that positive values of JN~,~indisoluble material in ileum and predominantly water-soluble cate active calcium absorption; negative values indicate active metabolites in jejunum. secretion. Since Peak V elutcd from Scphades LII-20 columns may conTransmural calcium fluxes tain 25,26-(OH), DZ in addition to 1 ,25-(011)2 D3 (15, al), Tissue and experimental periodate oxidation was performed to test the homogeneity of condition INet JXS JSM the material in this peak. lieaction with paraperiodic acid I I consistently produced less than a 20% loss in radioactivity in both Peaks IV and V, and there wcrc no tliffcrenccs in the results Duodenum No treat9.6 zk 1.4 +26.1 rk 5.2h of this reaction with mctabolitrs obtained from the three lcvcls 35.7 zk 5.9a ment of small intestine. The effects of periodation, together with the +25-OH Da 53.9 f 3.2c,’ 11.5 ?t 1.oe +42.4 f 3.0’ elution volumes indicate that Peak IV is indcctl predominantly Jejunum 25-OH Da and Peak V is predominantly 1 ,25-(011)2 DS. Thcrc -15.1 f 1.9i No treat15.8 f 1.1 30.9 f 1.69,’ were insufficient quantities of Peak Va mctabolitcs to test by ment this method, however, in a prior study, reaction of a peak with +25-OH Da 18.3 f l.Oi 28.6 f 1.5’” -10.3 f 1.5 the identical elution volume confirmed the assumption that this Ileum metabolite is predominantly 24,25-(OH)* D3 (16). No treat17.7 f 1.2 33.2 f O.gl -15.5 f 0.8’” As shown in Table I I, the major lipid-soluble radioactive comment pound in both plasma and intestine was unaltered 25-011 Da, -4.3 f 2.2 +25-OH Da 29.4 f 1.5” 32.7 f 1.4O and the predominant metabolite of 25-011 D3 in all tissues was a No treatment duodenal Jars > no treatment duodenal JEM, 1 ,25-(OH)n Da; only small amounts of 24,25-(OH)Z D3 were p < 0.001. found in these tissues. Much more 1 ,25-(OII)Z Ds localized in * All values + S.lS. duodenum with progressively lower quantities in ileum and c +25-OH UI duodenal JMS > +25-OH DB duodenal JSM, p < jejunum; this was found to be the case whether the data wcrc 0.001. d +25-OH D3 duodenal Jaas > no treatment duodenal JMS, expressed on the basis of protein content or, as shown in Table II, on the more meaningful basis of DNA content. There was p < 0.02. 61.5 pg of DNA per mg of protein in duodenum, 54.2 pg of 8 +25-OH I), duodenal Js~n not > no treatment duodenal JSM, DNA per mg of protein in ileum and 49.5 pg of DNA per mg of p > 0.05. The conccnt.ration of 24,25-(OH)z D3 was f $25OH Da duodenal ~~~~ > no treatment duodenal JN(,$, protein in jejunum. p < 0.02. higher in ileum than in duodenum and much lower in jejunum g No treatment jejunal Jsu > no treatment jejnnal Jars, p < (Table II). 0.001. A portion of the radioactivity measured in mucosal scrapings h No treatment jejunal JIM not > +25-OH I& jejunal Jw, p > is contributed by blood cntrappcd in the submucosa at the time 0.30. of sacrifice. An estimation of the degree of such contamination i No treatment jejunal JN~~ not > +25-OH Dt jejunal JN<.~,p > was made by measuring the non-hcmc and total iron content of 0.05. blood and mucosa. The iron in blood is primarily hcmoglobinj +25&H D3 jejunal Jr,fs not > no treatment jejunal JMS, p < iron together with a very small amount of transferrin-bound 0.20. iron. In intestinal mucosa, the quantity of total iron less the k +25-OH DS jejunal Jen > +25-OH Da jejunal Jae, p < 0.001. amount of non-hcme iron would provide an estimate of thehemo2 No treatment ileal JIM > no treatment ileal JYE, p < 0.001. m No treatment ileal Jxet > +25-OH DS iIeaI JN~~, p < 0.061. globin-iron, the latter reflecting the volume of entrapped blood. n +25-OH Ds ileal JMS > no treatment ileal J~IE, p < 0.001. The quantities of blood contaminating the mucosa were similar 0 +25-OH Da ileal JIM not > +25-OH Da ileal JMS, p > 0.70. at all three levels of intestine; about 0.9 ~1 per ml of 10% homogenate. Based on these determinations, the maximal contribution of blood-borne vitamin D metabolites to scraped 120.2 f 7.9, n = 7; no treatment, jejunal SCC = 93.3 f 12.0, n = 8, +25-OH DB, jejunal SCC = 89.6 f 9.0, n = 8; no mucosal metabolite content would be 1 pmolc of 25.OH Da per treatment, duodenal SCC = 41.4 =t 3.3, n = 7; +25-OH D3 g of DNA and 0.14 pmolcs of 1,25-(011)~ D3 per g of DNA; duodenal SCC = 42.5 =t 2.6, n = 8). lntestinal resistance these quantities represent negligible proportions of the total mucosal content of these metabolites (Table II). (Rr) was highest in duodenum, much lower in jejunum, and Plasma Calcium and Phosphorus Levels-Plasma calcium was least in ileum (no treatment, duodenal RI = 85.9 ohms.cm2 f unaffected by the acute dose of 25-OH D3 (no treatment plasma 4.5 S.E., n = 7; +25-OH Da duodenal RI = 81.2 f 4.7, n = calcium = 2.40 mM + 0.03 S.E., n = 16; +25-OH D, plasma 8; no treatment, jejunal RI = 48.8 ZIZ 3.4, n = 8; +25-OH D,, jejunal RI = 43.4 •t 2.6, n = 8; no treatment, ileal RI = 32.3 f calcium = 2.39 + 0.03, n = 23). Plasma phosphorus was decreased by 25-OH Da treatment (no treatment plasma phos1.3, n = 7; +25-OH Da, ileal Rr = 27.9 f 1.7, n = 7). Bephorus = 2.91 InM f 0.07 S.E., n = 16, +25-OH D3 plasma cause the transmural I’D is a function of SCC and RI, it was TABLE

Steady state calcium

I

jluxes across duodenum, jejunum, D-dejicient and 25-OH Da-treated

and rats

ileuwL


1159 II

TABLE

Distributio?l

of 25-OH

[26,2YJH]

Da a)ld metabolic

products

The tissues were obtained 24 hours after a 937.pmole dose of 25OH [26,27-“H] D3. Tissues were extracted as described under “Materials and Methods.” The results for each tissue represent Peaks are labeled according to pooled material from 30 animals. the nomenclature of Holick and DeLuca (15) : Peaks I and Ia are esters of 25-OH D3, Peak IV is 25.OH D3, Peak Va is predomi-

Tissue

Total

tissue

radioactivity

mentag lipidsoluble radioactivity

water-

soluble radioactivity

Peaks I and Ia

18,299 dpm/ml

95.1

4.9

(1.6)Q

Duodenum.

626 dpm/g

of DNA

85.4

14.6

(3 .O)

Jejunum..

736 dpm/g of DNA

36.1

63.9

(5.0)

Ileum.

710 dpm/g

59.3

40.7

(7.4)

Q Numbers in parentheses

represent

3H-label

in plasma

and small

intestine

: I‘mentag :e

Plasma....

of DNA

containing

nantly 24,25-(OH)2 Ds, Peak V is predominantly 1,25-(OH), D3. The concentrations of metabolites in Peaks IV, Va, and V are based on the assumption that the specific activity of the material in each of these peaks is identical to that of the injected 25.OH126,27-3H] 1)s. No concentrations are estimated for Peaks I, Ia, and the strip since the molecular composition is unknown.

the percentage,

Peak IV 25OH Dx

11.6 pmoles/ml (89.2)a 204.5 pmoles/g DNA (47.6) 117.2 pmoles/g I>NA (484) 171.5 pmoles/g DNA (46.5)

Peak V 1,25-(OH)2 Da

Peak Va 24.25.(0H)r Ds

of of of

0.1 pmoles/ml (l.l)a 22.6 pmoles/g DNA (5.3) 5.3 pmoles/g DNA (2.2) 38.3 pmoles/g DNA (10.4)

of of of

1.1 pmoles/ml (8.1)a 189.3 pmoles/g DNA (44.0) 106.9 pmolesjg DNA (44.3) 131.4 pmoles/g DNA (35.6)

Strip

@)a

of (0.1)

of (0.1)

of (0.1)

for each tissue, of the total dpm values eluted from the column in each peak.

7

p zo-

DISCUSSION

There is considerable evidence which indicates that I ,25(OH), D3 is the endogenously produced form of vitamin D3 that acts on intestine to increase calcium absorption in growing animals (22-28). Moreover, the calcium transport response in chick ileum has clearly been shown to be proportional to the amount of 1,25-(OH)* Da localized in the small intestine (27). It has been known for some time that the calcium absorptive process in rat duodenum is more responsive to vitamin D-repletion than the process in the more distal small intestine (l-3). The mechanisms underlying the differential responses of various segments of small intestine to vitamin D have remained unclear. This responsiveness has generally been reported as absorption per cm* or per cm of intestinal length. Either parameter involves the assumption that different segments of the small bowel have similar effective surface areas for transport. Expressing transport data on the basis of intestinal mucosal wet or dry weight requires an assumption that all of the tissue being studied has equivalent transport function. Neither of these methods for expressing such data may be entirely valid. More importantly, since the mucosa is heterogeneous, in neither case can the surface area of the cells actually involved in calcium transport be quantified. Since vitamin D repletion causes an increase in villus height (29), it may be exceedingly difficult to define conditions where changes in effective surface area can be eliminated as a potential source of any differences observed in transport responses. This regional specialization could also be due to either the localization of differing amounts of metabolites of the vitamin (particularly 1,25-(OH)* D3) in the cells of each segment, or inherent differences in tissue sensitivity to this metabolite. The fact that 25.OH D, administration produced increases in active calcium absorption that were directly proportional to the tissue 1,25-(OH)2 Ds levels in duodenum and ileum (Fig. l), is consistent with the former of these two possibilities. Since

50

100 pmoles

1.25~(OH)rD,

150 per g DNA

FIG. 1. Changes in intestinal calcium active transport following 25.OH D3 administration versus intestinal 1,25-(OH), Da content. Values for picomoles of 1,25-(OH)2 Da per g of DNA are those in Table II. Jhls is movement from the mucosal to the serosal side of the tissue in vitro and net flux (JN=~) is Jllls minus serosal to mucosal flux (JIM). Since JIM in all tissues was unchanged by 25.OH Da treatment (Table I), only changes in JM~ and JN~~ are plotted. Calcium fluxes are derived from Table I with the numbers representing absolute differences between fluxes of vitamin D-deficient and 25.OH Dt-repleted animals for each tissue. All changes in the fluxes were in a positive or absorptive direction. Both JUS and JN~~ for duodenum and ileum increase proportionally with increasing mucosal 1,25-(OH)2 Da content. No acute response to 25-OH Da treatment was observed in jejunum (Table I).

specific and saturable binding of 1,25-(OH)2 D, to intestinal mucosal chromatin has been demonstrated (27), and since a recent report indicates the existence of specific cytoplasmic receptor molecules as well (30), differences in cellular uptake at various levels of gut may largely reflect differences in the numbers of receptor sites or their affinity for 1 ,25-(OH)2 Da. The lack of an acute jejunal response in terms of changes in calcium transport relative to the observed tissue levels of 1,25(OH)2 D3 (Table II, Fig. I), as well as the prevalence of watersoluble metabolites in the mid and distal small intestine (Table II), deserve special comment. \Vhile the jejunal calcium absorptive process may indeed be less responsive to 1,25-(OH), D3, it may simply respond more slowly than duodenum or ileum.


phosphorus = 2.43 f 0.09; p < 0.001). Since the animals were fasted for at least one-half of the period following the administration of 25.OH D8, changes in the plasma levels of animals receiving 25-011 D, cannot be attributed cntircly to effects on either intestinal absorption or on bone and kidney.

1160 denum or chick ileum, studies correlating intestinal mucosal vitamin D3 metabolite patterns with levels of intestinal calcium transport have generally used mucosa obtained from either long segments or the entire small intestine. Our results indicate that, at least in the rat, it is undesirable to correlate transport rates from segments where this parameter is maximal with metabolite data derived from several regions of the small intestine. For example, pooling of mucosa from the entire rat small intestine would result in substantially lower concentrations of 1 ,25-(0H)2 D3 per mg of protein or per g of DNA than would be found in material obtained only from duodenum. Conclusions drawn from studies in which comparisons are made between calcium absorptive responses and vitamin D metabolite levels from different segments of the small intestine should be qualified accordingly. Acknowledgment-We would like to thank Gail Millar Fishbein for her expert assistance in the chromatographic separation of vitamin D1 metabolites. REFERENCES 1. SCHACHTER, D., KIMBERG, D. V., AND SCHENKER, H. (1961) Amer. J. Physiol. 200, 1263 2. URBAN, E., AND SCHEDL, H. P. (1970) Amer. J. Physiol. 219, 944 D. V., SCHACHTER, D., AND SCHENKER, H. (1961) 3. KIMBERG, Amer. J. Physiol. 200, 1250 M. W., AND KIMBERG, D. V. (1973) Amer. J. Physiol. 4. WALLING, 226, 414 I. T., GRAY, R. W., AND DELUCA, H. F. (1971) Proc. 5. BOYLE, Nat. Acad. Sci. U. S. A. 68, 2131 J. L., GRAY, R. W., BOYLX, I. T., KNUTSON, J., AND 6. OMDAHL, DELUCA, H. F. (1972) Nature New Biol. 237, 63 M. W., AND ROTHMAN, S. S. (1969) Amer. J. Physiol. 7. WALLING, 217, 1144 M., FROMM, D., AND MCCOLL, I. (1971) Amer. J. 8. FIELD, Physiol. 220, 1388 R. D., KIMBERG, D. V., AND GERSHON, E. (1970) J. 9. BAERG, Clin. Invest. 49, 1288 N. J., FARR, A. L., AND RANDALL, 10. LOUTRY, 0. H., ROSEBROUGH, R. J. (1951) J. Biol. Chem. 193, 265 11. SCHMIDT, G., AND THANNHAUSER, S. J. (1945) J. Biol. Chem. 161, 83 K. W., AND MYERS, A. (1965) Nature 206, 93 12. GILE~, 13. BLIGH. E. G.. AND DYER. W. J. (1959) Canad. J. Biochem. Phykol. 37,‘911 ’ \ ’ 14. LUND, J., AND DELUCA, H. F. (1971) J. Lipid Res. 7,739 15. HOLICK, M. F., AND DELUCA, H. F. (1971) J. Lipid Res. 12, 460 16. FAVUS, M. J., KIMBERG, D. V., MILLAR, G. N., AND GERSHON, E. (1973) J. Clin. Invest. 62, 1328 17. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol. Chem. 66, 375 18. BRUCKMAN, G., AND ZONDEK, S. G. (1940) J. Biol. Chem. 136, 23 19. FOY, A. L., WILLIAMS, H. L., CORTELL, S., AND CONRAD, M. E. (1967) Anal. Biochem. 18, 559 20. LEE, G. F., AND STUMM, W. (1960) J. Amer. Water Works Ass. 62, 1567 21. SUDA, T., DELUCA, H. F., SCHNOES, H. K., TANAKA, Y., AND HOLICK, M. F. (1970) Biochemistry 9, 4776 22. NORMAN, A. W., MYRTLE, J. F., MIDGETT, R. J., NOWICKI, H. G., WILLIAMS, V., AND POPJ~K, G. (1971) Science (Wash.) 173, 51 J., HOLICK, M., SUDA, T., TANAKA, Y., AND DELUCA, 23. OMDAHL, H. F. (1971) Biochemistry 10, 2935 J. F., AND NORMAN, A. W. (1971) Science (Wash. 24. MYRTLE, D.C.) 171,79 M. R., BOYCE, D. W., LITTLEDIKE, E. T., AND 25. HAUSSLER, RASMUSSEN, H. (1971) Proc. Nat. Acad. Sci. U. S. A. 68, 177 C. A., AND DELUCA, H. F. (1972) J. Clin. Invest. 51, 26. FROLIK, 2900


There is no available data which can be brought to bear on this latter possibility. However, it is quite possible that the metabolites of vitamin D3 localizing in jejunum may only be related in part to calcium absorption, because the jejunum appears to be the major site of vitamin D-enhanced (31, 32), calcium-independent (32), phosphate absorption in the rat. Finally, one must also consider the potential role of an enterohepatic circulation of vitamin D metabolites in relation to the results of the present study. The absorption of vitamin D3 is dependent on its interaction with bile salts and significant absorption occurs in rat jejunum (33). Furthermore, there is evidence suggesting that both lipid-soluble metabolites of vitamin D3 and watersoluble conjugates of metabolites undergo biliary excretion (34). Although this has not been the subject of careful investigation, it may not be unreasonable to presume that both lipid and water-soluble metabolites undergo an enterohepatic circulation, in which case a major part of the enteric uptake phase for lipidsoluble vitamin D metabolites might also occur in the jejunum. If indeed, 1,25-(OH)2 Da were to undergo biliary excretion and enteric uptake, then some undefined portion of this metabolite in jejunal mucosa would be present as a part of a transport pool, and as such its presence might be unrelated to target organ effects on calcium or phosphate transport. Such a phenomenon would contribute to the apparently aberrant calcium transport response of jejunum to 1,25-(OH)2 Da. No matter what the absorptive fate of lipid-soluble vitamin D metabolites may be, the high concentrations of water-soluble metabolites in the distal small intestine probably do reflect the enterohepatic circulation of these compounds. In any event, with the apparent exception of jejunum, previous reports of diminished calcium absorptive responses to vitamin D treatment by distal as opposed to proximal small intestine seem to be best explained by differences in the quantities of the biologically active form of the vitamin localizing in the mucosa at these levels. The present data also provide the first demonstration of active calcium secretion by jejunum and ileum from vitamin D-deficient rats. In a previous study, we observed active calcium secretion by both ileum and jejunum from adult rats and reported that rats raised to adulthood on a vitamin D-free diet in the dark (14 weeks) were no longer capable of active ileal calcium secretion (4). These adult animals were very nearly in an avitaIminotic rather vitamin D-deficient state which probably accounts for the differing observations. Subsequently, we have been able to demonstrate active calcium secretion by the ileum of growing rats when they are raised on a high calcium diet (35). It therefore appears likely that both rat jejunum and ileum actively secrete calcium into the gut lumen either when vitamin D3 intake is restricted or when conversion of the vitamin to 1 ,25-(OH)2 DB is suppressed by prolonged feeding of a high calcium diet (5). In accordance with the results of our earlier studies on the effects of dietary calcium deprivation on rat duodenum (7, 36) and ileum (4, 35), the changes in either the magnitude or direction of net calcium fluxes observed in the present study apparently occurred through increases in JMs rather than alterations in JsM. The contribution of calcium secretion by the lower small bowel to the negative calcium balance found in vitamin D-deficient animals deserves further investigation. Since duodenum from vitamin D-deficient animals actively absorbed calcium while the larger portion of the small bowel actively secreted calcium, use of duodenal absorption as a criterion for the transport status (absorptive versus secretory) of the entire small intestine could be misleading. While calcium absorption has been studied chiefly in rat duo-

1161 27. Ts.11, H. C., WONG, R. G., >ZND NORMAN, A. W. (1972) J. Biol. Chem. 247, 5511 28. BOYLE, I. T., MIR~VICT, L., GRAY, 11. W., HOLICK, M. F., AND DI,;Luc.\, H. F. (1972) Endocrinology 90, GO5 29. BIIXGE, 8. F., AND ALPERS, L). H. (1973) Gastroenlerology 64, 982 30. ~~~~~~~~~~~~~ M. It., AND BRUMUAUGH, P. F. (1973) Fed. Proc. 32, 918 31. HARRISON, H. IX., AND HARRISON, H. C. (1961) Amer. J. Physiol. 201, 1007

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S., 11~~ SCHACHTICR,

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(19G9)

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244,

211 33. SCHACHTER, J. Clin. 34. AVIOLI, L. D~Luca, 35. WALLING, 423 36. WALLING, 246, 5007

I)., FINICJCLSTEIN, J. D., AND KO~~I~SICI, S. (1964) Invest. 43, 787 V., LP;E, S. W., MCI)ONN,D, J. E., LUND, J., AND H. F. (1967) J. C&n. Invest. 46, 983 M. W., AND KIMI~ERG, 11. V. (1973) Fed. Proc. 32,

M.

W.,

AND

ROTHMAN,

S. S. (1970)

J.

Biol.

Chem.


Effects of 25-Hydroxyvitamin D3 on Rat Duodenum, Jejunum, and Ileum : CORRELATION OF CALCIUM ACTIVE TRANSPORT WITH TISSUE LEVELS OF VITAMIN D3 METABOLITES Marlin W. Walling, Murray J. Favus and Daniel V. Kimberg J. Biol. Chem. 1974, 249:1156-1161.

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EFFECTS OF 25-HYDROXYVITAMIN D, ON RAT DUODENUM, JEJUNUM, AND

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