Ar~rrrv~s Vol.

OF BIOCHEMISTRY AND BIOPHYSICS 188, No. 1, May, pp. 145-156, 197X

Effect of Dietary Calcium and Phosphorus on Intestinal Absorption and Vitamin D Metabolism’ M.

Department

of Biochemistry,

L.

RIBOVICH

AND

H.

F.

DELUCA’

College of Agricultural and Life Sciences, 1Jniversity Madison, Wisconsin, .537i% Received

August

2, 1977; revised

Calcium

January

of Wisconsin-Madfson,

23, 1978

To understand better dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin D:, and diets differing in calcium and phosphorus content was made. All known vitamin D metabolites were ultimately identified by high-pressure liquid chromatography. In addition to the known metabolites (25.hydroxyvitamin D,, 24,25dihydroxyvitamin D:x, 1,25dihydroxyvitamin D.1, 25,2G-dihydroxyvitamin I&, and 1,24,25-trihydroxyvitamin D,,), several new and unidentified metabolites were found. In addition to 1,25dihydroxyvit.amin D:, and 1,24,25-trihydroxyvitamin D.,, the levels of some of the unknown metabolites could be correlated with intestinal calcium transport. However, whether or not of intestinal calcium absorption by any of these metabolites plays a role in the stimulation low dietary calcium or low dietary phosphorus remains unknown.

Both low dietary calcium and phosphorus stimulate intestinal calcium absorption in rats when supplemented with vitamin D (1,2) The mechanism of dietary regulation of intestinal calcium transport was unknown until after the metabolism of vitamin D was elucidated. It is now well established that vitamin Da is first converted in the liver to 25hydroxyvitamin D,3 (25-OH-Da)” (3-5). This metabolite is then hydroxylated to either 24,25-dihydroxyvitamin Dz (24,25-(OH)zDs) (6, 7) or 1,25dihydroxyvitamin D:, (1,25-(0H)zDa) (8-12). ’ This research work was supported by a programproject grant from the National Institutes of Health (No. AM-148811, U. S. Energy and Research Development Administration Contract EY-7G-S-O2-lGG8, and the Harry Steenbock Research Fund. ’ To whom all correspondence should be sent. ” Abbreviations used: 25OH-D,, 25hydroxyvitamin Da; 24,25-(OH12Da, 24,25-dihydroxyvitamin D.,; 1,25-(OH)zD.,, 1,25dihydroxyvitamin D.,; LC, low calcium; HC, high calcium; LP, low phosphorus; NP, normal phosphorus; HAPS, hydroxyalkoxypropyl Sephadex; hplc, high-pressure liquid chromatography; 1,24,25-(OH)~& 1,24,25trihydroxyvitamin D:,; 25,2G(OH)&, 25,2G-dihydroxyvitamin D:,; I-, correlation coefficient; uv, ultraviolet.

Since 1,25-(OH)zDa is the metabolically active form of the vitamin in intestinal calcium transport (13), it appeared that the effect of low dietary calcium and phosphorus in the intestine may be mediated by a stimulation of 1,25-(OH)zDz synthesis. Boyle et al. (14) showed that rats on a low calcium diet given small amounts of vitamin D accumulate mainly 1,25-(OH)zDJ1 in the intestine, whereas animals on a high calcium diet accumulate predominantly 24,25-(OH)zD,?. Similarly, Tanaka et al. (15, 16) demonstrated that rats on a low phosphorus diet accumulate more 1,25-(OH)& in the intestine than rats fed a diet containing adequate phosphorus. Therefore, if an animal can regulate 1,25-(OH)zD:< synthesis in response to either dietary calcium or phosphorus, then providing the animal with 1,25-(OH)zD:j should bypass the regulation point and eliminate the animal’s ability to alter its intestinal calcium absorption in response to changes in dietary calcium and phosphorus. As expected, rats supplemented with vitamin D:l or 25-OH-D, are able to adapt their intestinal calcium transport to a low calcium diet, while rats administered 1,25-(OH)zD:j show a high effi-

145 C@G3-98Gl/78/lSSl-0145$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.

146

RIBOVICH

AND

ciency of calcium absorption independent of dietary calcium levels (17). However, rats fed a low phosphorus diet transport calcium more efficiently than those fed a normal phosphorus diet when supplemented with vitamin Ds, 25OH-Da, or 1,25-(OH)zDs (17).

These results suggest that stimulation of intestinal calcium absorption by low dietary calcium is the direct consequence of 1,25(OH)2D3 synthesis. They also suggest that in addition to stimulating I,25(OH)zDs synthesis, low phosphorus diets have some other unknown effect on intestinal calcium transport. It may be that dietary and/or serum phosphorus may modulate directly the intestinal calcium absorption system, the metabolism of 1,25-(OH)zDa, the uptake of 1,25-(OH)zDs by the intestine, or the elaboration of some unknown humoral agent. To help elucidate the mechanism of dietary regulation of intestinal calcium absorption, a quantitative assessment of the metabolites in plasma and duodenum of rats given daily doses of radioactive vitamin DB and diets differing in calcium and phosphorus content was made. In addition to the known metabolites of vitamin D3, several new and unidentified metabolites were discovered following extensive chromatography. Some of the unknown metabolites could be correlated with intestinal calcium absorption. MATERIALS

AND

METHODS

Animals. Male weanling rats (50-60 g) were purchased from the Holtzman Co. (Madison, Wise.). They were individually housed in hanging wire cages with free access to distilled water and a vitamin D-deficient diet (18). The diets used were a low calcium diet (LC, 0.02% Ca, 0.3% P), a high calcium diet (HC, 2.0% Ca, 0.3% P), a low phosphorus diet (LP, 1.2% Ca, 0.1% P), and a normal phosphorus diet (NP, 1.2% Ca, 0.3% P) (14, 19). Approximately 42 rats were maintained on each diet for 3.5 weeks. After 10 days, 30 rats from each group were administered daily 250 ng of [1,2‘HIvitamin Da in 0.05 ml of propylene glycol by gastric tube. This supplementation was continued for 14 days. The last dose of [“HIvitamin DB was given 24 h before sacrifice. The remaining rats in each dietary group were treated similarly with either nonradioact,ive vitamin DS or the control vehicle. Active calcium transport was studied in the rats given the nonradioactive vitamin Da. The method used was the everted intes-

DE LUCA tinal sac technique of Schachter and Rosen (20) as modified by Martin and DeLuca (21). The animals given the [1,2-3H]vitamin DB were killed, blood was collected in heparinized tubes, and the proximal 10 cm of duodenum was removed and rinsed with ice-cold 0.9% (w/v) saline. The mucosa was separated from the underlying coats by scraping with a glass slide. Both the plasma and duodenal mucosa were frozen and stored under nitrogen until extraction. Radioactive vitamin Da. [1,2-“HIVitamin DB was prepared in this laboratory according to the method of Neville and DeLuca (22). To check its radiochemical purity, the compound was applied to a Sephadex LH20 column (1 X 60 cm) equilibrated and developed with chloroform:Skellysolve B (1:1, v/v). The peak tubes, which gave the expected uv absorption spectra bLl,X 264,Am 230), were pooled and an aliquot was applied to a hydroxyalkoxypropyl Sephadex (HAPS) column (1 X 80 cm) equilibrated with chloroform: methanol:water (20:70:10). A single peak was obtained with a recovery of 92.6%. An aliquot was applied to a high-pressure liquid chromatography (hplc) system utilizing a pBondapak CIH (Waters) column (4 mm x 30 cm) equilibrated with methanokwater (955) and cochromatographed with synthetic crystalline vitamin Da. A single peak of radioactivity was found which exactly cochromatographed with vitamin Da, indicating a purity of >95%. The specific activity was determined as 51,000 dpm/25 ng. Extraction of radioactivity. The duodenal mucosa and plasma were each pooled for each dietary group. Homogenates of mucosa (20%) were prepared in water using a Potter-Elvehjem homogenizer. Mucosal homogenates and plasma were subjected to a lipid extraction according to the method of Bligh and Dyer (23) as modified by Lund and DeLuca (24). The chloroform phase was removed and the aqueous phase reextracted twice with chloroform. The combined chloroform phases were evaporated to dryness with a flash evaporator. Residual water was removed as an azeotrope by addition of 100% ethanol. The remaining lipid was analyzed for vitamin D metabolites by extensive chromatography. Chromatography. The ultimate goal of the chromatography was to take known vitamin D metabolites from the initial columns and, after removing residual lipid by various chromatographic systems, cochromatograph the metabolites with authentic vitamin D metabolites by high-pressure liquid chromatography. The chloroform-soluble metabolites in the plasma and duodenum were applied sequentially to a series of chromatographic systems. A description of these chromatographic systems is shown in Table I, including column packings, dimensions, volumes, and composition of eluting solvents, etc. The figure number that displays the resultant column profile for each system is also indicated. Each chromatogram was determined by counting a small aliquot of each individual column fraction.

CALCIUM,

PHOSPHORUS,

AND

380rnS

Column packing

1

Sephadex LH-20

2X57cm

2

4mmX30cm

3

hplc PPorasil (Waters) Sephadex LH-20

4

Sephadex LH-20

2X60cm

5

HAPS

lX80cm

6

hplc PBondapak (Waters) HAPS

7 8 9

.Sephadex

700

900

lHC lX18cm LH-20

4mmx30cm

11

Sephadex LH-20

12

hplc two Zorbax-SIL (DuPont) in series

lX57cm lX18cm 2.1 mm x 25 cm

2.1 mm X 25 cm

hallmr

160rnl

Frocllonr

SYSTEMS

Eluting solvents (v/v) 75:25 CHC&/Skellysolve B 15:85 Isopropanol/SkellysoIve 1:l CHC&/Skellysolve B 65:35 CHCla/Skellysblve B 20:70:10 CHClJMeOH/H20 95:5 MeOH/H20

Figure

1 2 B 3 4,8 5 6

3709

14:53:33 CHCb/MeOH/H20 90:10:5 Skellysolve B/CHCL/MeOH 2.5:97.5 Isopropanol/SkellysoIve B 7.5:92.5

3000

Isopropanol/SkellysoIve B 9O:lo:lO Skellysolve B/CHClJMeOH 1090

lX18cm

hplc pPsorasil (Waters) hplc two Zorbax-SIL (DuPont) in series

10

Operating pressure (psi)

3X58cm

4mmx30cm

T&n

I

OF CHROMATOGRAPHIC

Column dimensions

FmnlMI

FIG. 1. Chromatogram of high dietary calcium plasma extract on a Sephadex LH-20 column (2 x 57 cm) developed in chloroform:Skellysolve B (75:25).

TABLE Column

147

D METABOLISM

peaks were eluted (II, III, Ar, and IV) (Fig. 4). The relative elution ratios of the peaks to peak III (vitamin Da) were: II, 0.61 ? 0.04; AT, 1.34 f 0.07; and IV, 1.64 + 0.04. Peak III was then applied to column 5 and two peaks were obtained, peak B and peak III (Fig. 5). The relative elution ratio of peak B to peak III (vitamin D:,) was 1.33 f .02. Peak III was finally applied to column 6 and two peaks were eluted: peak C and a peak which cochromatographed with synthetic vi-

Chloroform-soluble metabolites from the plasma or duodenum were applied to column 1 (Fig. 1). In addition to metabolites varying in polarity from vitamin D3 to 1,25-(OH)zDz, some of which were tentatively identified by elution of authentic vitamin D metabolites on this column system, four metabolites more polar than 1,25-(OH)zDa were found (VI, VII, VIII,, and VIII). The relative elution ratios of these peaks to peak V, (1,25-(OH)pDa) were: VI, 1.37 + 0.09; (mean + S.D.); VII, 1.80 r?~0.07; VIII,, 2.17 + 0.09; and VIII, 2.54 f 0.04. Peak VIII was tentatively identified by its elution ratio relative to 1,25-(OH)zD:j as reported by Kleiner-Bossaller and DeLuca (25). It was ultimately identified as 1,24,25-(OH)zDa by cochromatography with the authentic metabolite on column 2 (Fig. 2). In plasma, the part of the original chromatogram (Fig. 1) from the void volume up to and including peak V, was applied to column 3 (Fig. 3). Seven peaks varying in polarity from peak II to peak VI were found (II, III, A, A’, IV, V,, and V,,). The relative elution ratios of these peaks to peak III (vitamin Da) were: II, 0.56 f 0.02; A, 1.54 + 0.02; A’, 1.87 + 0.03; IV, 2.22 f 0.06; V,,, 2.83 + 0.07; and, V,,, 3.69 f 0.06. In the duodenum, the part of the original chromatogram (Fig. 1) from the void volume to peak IV was applied to column 4. Four

DESCRIPTION

VITAMIN

806

Isopropanol/Skellysolve

B

7 9

10, 11 12

148

RIBOVICH 1,24,25-(0W30S

AND

DE LUCA

I

1400

5

III

4000

-vitamin

D3

IV - 25-OH-D3 3000

0

2

4

6

8

I8 ml

IO

12

14

16

18

20

Fractions

FIG. 2. Cochromatography of high dietary calcium plasma peak VIII with 1,24(R),25-(OH)&, on hplc. The system consisted of a PPorasil column (4 mm x 30 cm) (700 psi) developed in isopropanol:Skellysolve B (1585).

FIG. 3. Chromatogram of low dietary calcium plasma sample on a Sephadex LH-20 column (3 X 58 cm) developed in chloroform:Skellysolve B (1:l).

tamin D:, (Fig. 6). The elution ratio of peak C to vitamin Da was 0.66 + 0.02. Peak IV was successively applied to columns 7, 8, and 9 (Fig. 7). A single peak was noted in each case for columns 7 and 8 (Table I). Two peaks were eluted from column 9: peak D and a peak which cochromatographed with synthetic 25 OH-D:,. The elution ratio of peak D to 25-OH-D? was 1.33 f 0.00 (Fig. 7). From column I (Fig. I), the region of the chromatogram from peak V, to peak V, was applied to column 4. Three peaks were eluted from this column: peaks V,, Vb, and V, (Fig. 8). Peak V, was successively passed through columns 8 and 10 (Fig. 9). From the last column, peak V, was identified as 24,25-(OH)pD:,. Peak V, (Fig. 8) from plasma was applied to column 11 and two peaks were eluted: peak V,. (1,25-(OH)zDa) and V,., (Fig. 10). The elution ratio of peak V,, to peak V, was 1.42 -t 0.02. Peak V, from duodenum was applied to a smaller column 11 (1 x 18 cm) and two peaks were eluted: peak V, (1,25-OH)2Da) and peak E (Fig. 11). The elution ratio of peak E to peak V, was 0.44 f 0.03. Peak V,. was finally applied to column 12 and two peaks were found: 1,25-(OHhDa and a peak which migrates near 25,26-(OH)zDn (Fig. 12).

465 ml fractions

FIG. 4. Chromatogram of low dietary calcium duodenum sample on a Sephadex LH-20 column (55 g, 2 x 60 cm) developed in chloroform:Skellysolve B (65:35).

5.6 ml

fractions

FIG. 5. Chromatogram of low dietary phosphorus duodenum peak III on a hydroxyalkoxypropyl Sephadex (HAPS) column (1 x 80 cm) developed in chloroform:methanol:water (20:70:10).

3000

r

024

c

6

8

IO

12

14

16

18

20

FIG. 6. Cochromatography of low dietary calcium plasma peak III with crystalline vitamin D:, on hplc. The system consisted of a PBondapak CIH column (4 mm x 30 cm) (900 psi) developed in methanol:water (95:5). Sephadex LH-20 was purchased from Pharmacia Fine Chemicals, Inc., Piscataway, N. J. The hydroxyalkoxypropyl Sephadex was made according to the procedure of Ellingboe et al. (26). The final product had a C,5-C,n chain length, and the weight increase

CALCIUM,

PHOSPHORUS,

AND

VITAMIN

149

D METABOLISM "c

6000r

4000 I a n

I "c

2000

.i, 0

1.8 ml

Fractions

FIG. 7. Cochromatography of high dietary calcium duodenum peak IV with synthetic 25OH-Da and [‘%]25-OH-Da on hplc. The system consisted of a PPorasil column (4 mm x 30 cm) (800 psi) developed in isopropanol:SkellysoIve B (2.5:97.5).

20 A0 60 5.0 ml Fractions FIG. 10. Chromatogram of low dietary calcium plasma peak V,. on a Sephadex LH-20 column (1 X 57 cm) developed in Skellysolve B:chloroform:methanol (90:10:10).

FIG. 8. Chromatogram of low dietary phosphorus plasma sample on a Sephadex LH-20 column (55 g, 2 x 60 cm) developed in chloroform:Skellysolve B (65:35).

6000

4000

z D

: x b 2-a

2OmJ

2

4

a3

l,2%0H)2

m

r

-(OH):, D3

I E 5 ‘;

0

40 30 3.45 ml Fractions

FIG. 11. Chromatogram of low dietary phosphorus duodenum peak V,. on a Sephadex LH-20 column (1 x 18 cm) developed in Skellysolve B:chloroform: methanol (9O:lO:lO).

6000 24,25

20

IO

0

6

6

09

lo 12 14 16 ml Fractions

18 20

FIG. 9. Cochromatography of low dietary phosphorus plasma peak V, (Fig. 8) with 24(R),25-(OH)2D:g on hplc. The system consisted of two Zorbax-Sil columns (2.1 mm x 25 cm) (3700 psi) in series developed in isopropanol:Skellysolve B (7.5:92.5). corresponded to a hydroxyalkyl group content of 50% (w/w). Radioactivity determination. Radioactivity in the chloroform-soluble plasma, mucosal homogenate, phase, aqueous phase, and protein interphase was

I EZI dpm k

z0 4000

0

dpm ‘%

f : x b :: 4

2000

0

2

4

6

8

IO

12

14

16

IS

20

IO ml Fractions

FIG. 12. Cochromatograph of low dietary phosphorus plasma peak V, with 25,26-(OH)zDs, 1,25-(OH)~DX, and “C-1,25-(OH)aD.1 on hplc. The system consisted of two Zorbax-Sil columns (2.1 mm x 25 cm) (3ooO psi) in series developed in isopropanol:Skellysolve B (1090). determined by counting duplicate samples. For the chloroform-soluble phase, the methanol strip, and the column effluents, the solvent was evaporated to dryness and the residue dissolved in a toluene counting solution as described previously (27). The mucosal homogenate was analyzed for radioactivity after solubilization in Protosol (New England Nuclear) at 50°C. The dissolved sample was then mixed with the count-

150

RIBOVICH

AND

ing solution. The radioactivity in the plasma and aqueous phase was determined by addition of an aliquot to 13.5 ml of Aquasol (New England Nuclear). Radioactivity in the protein interphase was determined after combustion in a Packard Tri-Carb Model 305 sample oxidizer. All samples were counted in a Packard Tri-Carb Model 3375 liquid scintillation counter equipped with an automatic external standardization system. Disintegrations per minute were calculated for each sample using an internal standard of [:‘H]toluene. For the protein interphase, the disintegrations per minute were calculated using [“HIwater as the internal standard. For the column effluents, the disintegrations per minute were calculated using the automatic external standard. Compounds. Vitamin DS was purchased from Philips-Duphar (The Netherlands). The 25-OH-D, was a gift from Dr. John Babcock of the Upjohn Company (Kalamazoo, Mich.). The 1,25-(OH)& 24(R),25-(OH)pD:i, and 1,24(R),25-(OH):jD:, were generously supplied by Dr. M. Uskokovic of the Hoffmann-LaRoche Company (Nutley, N.J.). The 25,26(OHhDcr was synthesized by the method of Lam et al. (28). 25-OH-[26,27-.‘H]D:, (9.2 Ci/mmol) was synthesized in this laboratory by the method of Suda et al. (29). 1,25-(OH),-[26,27-“H] (9.2 Ci/mmol) was synthesized enzymatically from 25-OH-[26,27-“HID:, (9.2 Ci/mmol) by the method of Frolik and DeLuca (30). 24(R),25-(OH),-[26,27”H]T):, (9.2 Ci/mmol) was preTABLE

DE LUCA pared enzymatically from 25-OH-[26,27”H]D:, by the procedure of Knutson and DeLuca (31). 1,24(R),25(OH)J[26,27-“H]D:j was prepared as described by Tanaka et al. (32). The [26,27-“‘Cl25-OH-D:] was synthesized by the method of Suda et al. (29). The [26,27“‘C]1,25-(OH)& was synthesized enzymatically as described by Gray et al. (33). All compounds were checked for purity by Sephadex LH-20 chromatography (34) and by high-pressure liquid chromatography (35). RESULTS

Tables II and III give the final concentration of metabolites in the plasma and duodenal mucosa. The data are expressed as either picomoles or disintegrations per minute per milliliter of plasma or gram of mucosa. All values were corrected for counting, transfer, and radioactive decay losses, and the known vitamin D metabolites were also corrected for recovery losses from chromatography. This latter correction was made by determining the recovery of authentic vitamin D metabolites from each chromatographic system (Table IV). The recovery of authentic vitamin D metabolites ranged from 75 to 98%. Unknown metaboII

CONCENTRATION OF VITAMIN D METAROLITES IN PLASMA“ Dietary

Peaks LC (0.02% Ca, 0.3% P) II D:, C B A A’ 25-OH-D,3 VI, VI, Vb 24,25-(OH)zD:1 1,25-(OHhD:, (??25 ’26.(OHhD,,) v,, VI VII VIII,, 1,24,25-(OH):,D:g

464.3 178.1 (0.22)h 305.0 126.2 79.2 183.7 2516.0 (3.08) 139.4 123.2 363.5 120.1 (0.14) 547.0 (1.30) 56.1 (0.070) 239.2 582.1 145.9 -’ 247.6 (0.56)

(2.0% c::o.,, 763.0 440.9 (0.55) 168.8 75.3 226.4 632.4 6466.4 (7.93) 351.6 47.3 418.8 2614.0 (3.20) 128.8 (0.29) 42.4 (0.053) 132.1 330.3 144.3 55.1 78.3 (0.18)

P)

groups (1.2% CL,S.l% P) 1417.6 1311.2 (1.67) 399.3 133.4 287.7 632.4 7739.0 (9.63) 382.5 176.3 337.3 851.9 (1.04) 567.9 (1.34) 81.2 (0.099) 163.8 571.8 175.4 264.6 (0.59)

n Data are expressed as disintegrations per minute per milliliter of plasma. h Data in parentheses are expressed as picomoles per milliliter of plasma. ’ -, no detectable radioactivity.

NP (1.2% Ca, 0.3% P) 284.4 (0.35) 442.1 48.8 229.8 835.9 7282.5 (8.93) 363.9 252.2 472.2 2810.3 (3.44) 69.4 (0.16) 85.0 (0.103) 234.8 250.0 139.2 53.2 57.9 (0.13)

CALCIUM,

PHOSPHORUS,

AND

151

D METABOLISM

olite found in highest concentration in the plasma. Rats on the high calcium, low phosphorus, or normal phosphorus diet had levels greater than 7.0 pmol/ml of plasma. Rats on the low calcium diet had a much lower level (-3.1 pmol/ml). Rats on the high calcium or normal phosphorus diet had the highest levels of 24,25-(OH)2D3 (>3.0 pmol/ml). This metabolite was also present in the low phosphorus diet fed rats but was very low in the low calcium diet fed rats (0.14 pmol/ml). Almost identical levels of 1,25-(OH)zDs were found in rats on either the low calcium or low phosphorus diet

lites which corn&rated with known metabolites on specific chromatographic systems were assumed to have similar recoveries. The reproducibility of each system is expressed as the coefficient of variation (standard deviation f mean x lOO), as a percentage, and is also shown in Table IV. The steady-state levels of vitamin D metabolites in the plasma are shown in Table II. Six known vitamin D metabolites were identified by hplc: vitamin Dg, 25OH-Da, 24,25-(OH)zDa, 1,25-(OH)nDs, (?? 25,26(OH)zD3), and 1,24,25-(OH)3D3. As expected, 25-OH-D3 is the vitamin D metabTABLE Peaks

VITAMIN

III

CONCENTRATION OF VITAMIN D METAROLITES IN DLJO~ENALMUCOSA" Dietary Groups

II Vitamin Da B C 25-OH-D:, D AT V, 24,25-(OH)aDa Vb 1,25-(OHhD.3 E VI VIII

(O.OE Ca, 0.3% P)

(2.ZcCa, 0.3% P)

625.7 290.7 (0.39)h 663.1 -1 286.5 (0.36) 16.0 115.5 63.3 192.9 (0.47) 79.7 21.4 59.6

265.3 241.7 (0.29) 325.7 105.3 715.5 (0.87) 48.1 60.2 30.5 350.2 (0.44) 28.2 58.6 (0.14) 25.3 8.53 27.0

(1.2YCa 0.1% P) ’ 466.0 1217.8 618.4 43.9 516.3 29.3 51.2 34.2 78.4 50.8 151.8 55.9 22.1 34.4

(l.&‘Ca, 0.3%P) 328.0 369.0 305.0 32.4 687.5 56.7 59.8 139.0 456.7 86.4 79.7 17.6 -

(1.55)

(0.63)

(0.11) (0.37)

(0.47)

(0.85)

(0.54) (0.18)

-

n Data are expressed as disintegrations per minute per gram of mucosa. ’ Data in parentheses are expressed as picomoles per gram of mucosa. ’ -, no detectable radioactivity.

RECOVERYOFAIJTHENTICVITAMIN

TABLE IV D METABOLITESFROMCORRESPONDINC;

CHROMATOGRAPHICSYSTEMS"

Chromatographic systems (column No.)

Vitamin D:, 25OH-D:, 24,25-(OH)zDa 1,25-(OH)zDa 1,24,25-(OH)&

1

3

(8.1)’ 90.4 97.6 84.8 79.5 88.9

(5.0) 93.4 87.4

4

(f3.6) 92.3 98.5 89.9 92.2

5 (10.5) 87.9

7

8

(3.8)

(1.0)

92.0

82.6 75.2

” The recoveries of known vitamin D metabolites are expressed as percentages. ’ The hplc system for each known vitamin D metabolite is described under Materials ’ The reproducibility of each system is expressed as coefficient of variation (%).

11

hplc?

(2.3)

94.3

93.3 94.4 84.2 84.4 81.3

and Methods.

(3.3) (1.0) (19.7) (5.5) (0.2)

152

RIBOVICH

AND

(-1.3 pmol/ml). This level was much greater than that found in high calcium or normal phosphorus diet fed rats. The amount of metabolite which migrated near 25,26-(OH)zD3 on hplc was very low regardless of dietary conditions (SO.1 pmol/ml). The highest amount of 1,24,25-(OH)sD3 was found in rats on either a low calcium or low phosphorus diet (0.5-0.6 pmol/ml). Rats on high calcium or normal phosphorus diets had lower levels of 1,24,25-(OH)zDs (0.1-0.2 pmol/ml). The majority of unknown metabolites in the plasma were found in low amounts with the possible exception of peaks II, A’, and VI. Table V shows the intestinal transport data for the different dietary groups. As expected, rats on the low calcium and low phosphorus diets had significantly higher intestinal calcium transport ratios than rats on the high calcium and normal phosphorus diets, respectively. Correlation coefficients (r) were determined between the amount of vitamin D metabolite in the plasma and the increase in calcium transport ratios due to vitamin D in the four dietary groups. Thus four points were used for the calculation of correlation coefficients. Table VI shows that the levels of three metabolites had a statistically significant positive correlation with intestinal calcium transport: 1,25-(OH)zD3, peak VI, and 1,24,25(OH)SDS. Peak B gave a high positive correlation coefficient (+0.94) although statistical significance was not achieved. Two metabolites demonstrated a statistically significant negative correlation: 24,25TABLE

DE LUCA

(OH)zD3 and peak VIII,. Another metabolite which showed a high negative correlation was peak Vb (-0.89). Table III shows the steady-state levels of vitamin D metabolites in the duodenal mucosa. Four known vitamin D metabolites were identified by hplc: vitamin D,?, 25-OHDs, 24,25-(OH)zD:j, and 1,25-(OH)&. No 25,26-(OH)zD3 was detected in the mucosa. Although peak VIII was found in the duodenum, too little radioactivity was obtained for cochromatography with authentic l,24,25-(OH)ZDB on hplc. The relative elution ratio of peak VIII to 1,25-(OH)iD:, was TABLE VI COKHEI,ATION DATA HETWRENAMWJNT OF

VITAMIN D METAROLITE IN PLASMA AND INTESTINAL CALCIIJMTHANSPORT Metabolite

r value

II D:g C B A A’ 25-OH-Da

+0.42 +0.46 +0.31 +0.94 -0.25 -0.63 -0.36 -0.45 0.09 -0.89 -0.95 +0.99 +0.25 +0.24 +0.96 +0.68 -1.00 +0.99

VI, VI, Vb 24,25-(OH)yD:, 1,25-(OH)zD:g (?? 25,26-(OH)zD:j) v