Biological activity of a vitamin D metabolite

ARCHIVES

OF

BIOCHEMISTRY

AND

Biological H. MORII,

BIOPHYSICS

Activity J. LUND,

503-512 (1967)

120,

of a Vitamin P. F. NEVILLE,2

D Metabolite’ AND

H. F. DELUCA

Departmentof Biochemistry, University of Wisconsin, Madison, Received November 28, 1966

Wisconsin 6%%’

A metabolite of vitamin D which is obtained from the carcassesof rats given aHvitamin D1 has been isolated by silicic acid column chromatography and tested for its effectiveness in curing rickets, stimulation of calcium transport by everted intestinal sacs, and in the elevation of serum calcium concentration. The metabolite was as effective as vitamin D in all three systems. In addition it stimulated intestinal transport of calcium within S-10 hours after its oral administration to vitamin D-deficient rats, whereas vitamin D itself required a 20-hour lag for its action. The physiologic significance of these findings is discussed.

The idea that vitamin D may be converted to a metabolically active form has recently

mineral

calcium

and the gastrointestinal tract was removed and discarded. The carcass was skinned, freed of most the muscle, ground in a meat grinder, and extracted with methanol and chloroform as described earlier (2). The blood was also extracted by these solvents as previously described (2). The chloroform extracts to which 1 mg a-tocopherol was added as an antioxidant were dried in a flash evap-

mobilization.

Furthermore,

this

metabolite(s) of vitamin D acts much more gained momentum with the isolation of vitarapidly than does vitamin D itself in stimumin D metabolites which are capable of lating calcium transport by everted incuring rickets in rats (1, 2). One of these testinal sacs. metabolites is found in large quantities in MATERIALS AND METHODS bone, liver and blood, and is at least as active in curing rickets as is the parent vitamin (2). Preparation of rats. Male weanling rats were obFurther experiments demonstrated that as tained from the Holtzman Co. of Madison, Wisthe dosage of vitamin D was decreasedto a consin. They were maintained in hanging wire truly physiologic dose, the proportion of this cages and fed ad lib&m a purified, vitamin Dmetabolite increased to the point where it is deficient diet described earlier (3). This diet, the major form of the vitamin present (2). which contains 0.47% Ca and 0.3% P, does not Kinetics of its formation in vivo shows that induce rickets in rats but produces a severe vitait appears in quantity before an observable min D deficiency in 34 weeks characterized by low physiologic effect of the vitamin is obvious serum Ca++ and reduced growth (4). When the rats reached this degree of deficiency they were used in (Neville, Prees, and DeLuca, in prep- the following experimenta. aration). Although this metabolite has not Production of metabolite fraction. The vitamin yet been identified, its very interesting in D-deficient rats were given 8000 IU each of either vivo characteristics have prompted our fur- 1,2-SH-vitamin D3 (5) or randomly labeled ‘Hther investigation of its physiologic effects. vitamin D3 (6), both of which were purified just beIt will be demonstrated in this manuscript foreuse and were judgedradiochemicallypure. The that this polar metabolite(s) of vitamin D radioactive vitamin was dissolved in cottonseed not only cures rickets but activates calcium oil and given orally in 0.1 ml. Seventeen hours transport in the intestine and elevates serum later the rats were killed, their blood was collected presumably

by

activating

bone

1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported by grant AMO-5800-05 NTN from the U.S. Public Health Service. e Postdoctorate fellow of the U.S. Public Health Service, No. 5-F2 AM-25,634-02.

508

VITAMIN

4000

s

3000

6 v) z 3

2000

2 -e 1000

20

30 40 IO ml FRACTIONS

50

509

D METABOLITE

6C

FIU. 1. A silicic acid chromatographic profile of a chloroform extract of the carcass of a rat given 10,009 IU of aH-vitamin D, 17 hours previously. 0, Radioactivity.

orator under a stream of Ns. The residue was dissolved in Skellysolve B (a petroleum fraction boiling at 6567”) and chromatographed on silicic acid columns using gradient elution already described (1,2). Four components were usually obtained (Fig. 1). The peak 4 obtained from these chromatograms was once again rechromatographed before use. The peak 4 fraction was then dissolved in cottonseed oil (Wesson). The dosages administered were calculated on the assumption that the specific activity of the metabolite is the same as that of the original radioactive vitamin. For example, if the original 3H-vitamin Da contained 800 dpm/IU, then the number of units of metabolite was calculated by dividing its total dpm by 800. Line test assay or rickets cure test. Weanling rats of either sex were obtained from the Holtzman Co. and were fed the rachitogenic diet of Steenbock and Black (7) for 21 days. The diet was modified by the substitution of egg white protein for wheat gluten and by the addition of water-soluble vitamin at the levels described earlier (3). After the al-day depletion period, a single 4 IU dose of either standardvitamin D or metabolite was administered. Controls received the oil vehicle alone. Seven days later the rats were killed and the line test was performed on sectioned radii and ulnae of individual rats. The biological activity was ascertained as described in the U.S. Pharmacopeia (8). Serum calcium response. Vitamin D-deficient rats fed the purified diet as described above were used in all experiments. When their serum calcium concentrations fell to 4-5 mg ‘% they were used in the following experiments. The deficient rats received 5 IU of peak 4 me-

tabolite every day for 4 days or a total of 20 IU. At the end of this period, the rats and others which had not received the metabolite fraction were killed and their serum calcium concentrations were determined by the method of Webster (9). Everted intestinal sacs. The vitamin D-deficient rats were given a single 5 IU dose of metabolite orally in oil solution while the controls received the oil vehicle alone. After 20 or 24 hours the rats were killed and calcium transport by everted intestinal sacs as described earlier was carried out (9). The data are expressed as a ratio of the 4%a concentration on the serosal side over that on the mucosal side. In another experiment vitamin D-deficient rats were given a single dose of 10 IU of metabolite or vitamin Da in oil solution. The rats were then killed at the times indicated for the determination of calcium transport by everted intestinal sacs and serum calcium concentration. RESULTS

It was once again confirmed that biologically active metabolites of vitamin D do exist in the tissues of rats given vitamin D. Figure 1 shows a typical silicic acid column chromatogram profile of a chloroform exTABLE

I

RELATIVE ABILITY OF METABOLITES FROM VARIOUS TISSUES~ TO CURE RICKETS IN RATS Biological activitP TiSSUfZ

Kidney Intestinal Serum Liver Bone

mucosa

1

2

3

4

1.7 1 0 1.2 -

2.5 1.9 0.5 -

4.0 4.0 4.5 4.0 3.8

2.0 2.5 3.7 3.8 3.5

0 One to four represent the peaks eluted as shown in Fig. 1 from silicic acid column chromatography of chloroform extracts of tissues of rats which had been maintained for 3-4 weeks on a vitamin D-free diet and sacrificed 24 hours after administration of 500 IU of 3H-vitamin D,. 6 Test animals received amounts of metabolites corresponding to 4 IU of parent vitamin, according to radioactivity. Animals which received 4 IU of standard vitamin Da gave an average value of 4. Each value in the table is the average of eight assay rats. Peak 1 fractions from kidney and intestine were pooled for assay. The assays were carried out according to the line test method described in the U.S. Pharmocopeia (8).

510

MORII, TABLE

SERUM

CALCIUM

ET AL.

II

TABLE

RISE IN RESPONSE TO PEAK 4 METABOLITE

Vitamin D-deficient rats received 0.1 ml of cottonseed oil containing 5 IU of peak 4 or oil only at 0,24,48, and 72 hours. After 96 hours, the animals were sacrificed and the serum calcium levels were determined. Control animals received the oil vehicle alone. The numbers in parentheses represent the number of animals in each group.

IV

EFFECT OF ORAL ADMINISTRATION OF 10 IU VITAMIN Da AND AN EQUIVALENT AMOUNT OF METABOLITE (PE,ZK 4) OF VITAMIN D, ON CA TRaNSPORT BY EVERTED INTESTINAL SACS

The experiments were carried out as described in Table III except that a 10 IU dose of either vitamin Da or peak 4 metabolite was used. Hoursafter administration

““Ca Sero~al/‘~Ca Mucosal 10 IU vitamin Da

Peak 4 metabolite

Control (mg %) Peak 4 (mg %)

Control Serum Ca (mg/lOO ml) 4.1 f 0.2”~ b 6.5 f (5)

0.3b

(5)

0 f Standard error of the mean. bp < 901 TABLE

III

STIMULATION OF CALCIUM TRANSPORTIN EVERTED INTESTINAL SACS BY A VITAMIN D METABOLITE 2:

dosage

20 24 24

Deficient

control; ratio5

Vitamin Dg; ratioa

Peak 4 metabolite; ratio9

1.44 i 0.10” 3.49 f O.llbs c 2.53 f O.Ogb*c

(6) (4) 1.57 f 0.11 2.58 f 0.13” (4) (3) 1.64 f 0.08 3.48 f 0.12c (3) (4)

(4) 2.44 f O.llC (5) 2.00 f 0.13” (4)

a Vitamin D-deficient rats were given either 5 IU of vitamin DI or 5 IU of metabolite orally in 0.1 ml of cottonseed oil, and the controls received the oil vehicle alone. At the times indicated, the rats were killed and everted sacs were prepared from their duodenum. Calcium-45 transport expressed as a ratio of Wa (serosal side)/Wa (mucosal) was measured as described in the text. b Standard deviation. The figures in parentheses represent the number of animals in each group. “p < .OOl

tract of a whole rat which had received 3Hvitamin D, 17 hours before it had been killed. Again the four radioactive fractions described previously (1, 2) appeared. Peak 1 has already been identified as an ester of vitamin D and long-chain fatty acids (10). Peak 3 is unaltered vitamin Da (1) while peaks 2 and 4 are as yet unidentified. Table I summarizes the results of line test

0.98 f

0.03

(31)”

-

1.09 f 1.09 f

0.79 f 1.17 f

0.03 (4) 0.13 (4)

0.97 f 1.80 f

1.13 f

0.09 (9)

1.86 f

0.99 f

0.03

0.04 (3) 0.23 (3)

2 4 6 8

10 12

(5)

1.71 f

0.15

1.09 f

0.14 (5)

1.78 f

0.11 (3)

1.78 f

16 20 24 48 72 96

1.73 1.53 2.23 2.61 1.69

(16)d

0.15

(3)

0.11 (3)b 0.19 (7)c

f

0.13

f

0.19 (3)C

-

f f f

0.12 (3)’ 0.07 (3)d 0.27 (3)”

-

0.15

(3)d (10)

a & Standard error of the mean. Numbers in parentheses show the numbers of animals in each group. b p < .025 above control. c p < .005 above control. d p < .OOl above control.

assays of these fractions prepared from different tissues of rats given 3H-vitamin DD. Clearly all four fractions were able to cure rickets to some extent. Most important is the fact that peak 4 was in some cases as effective as was the parent vitamin (peak 3). The effectiveness of peak 4 metabolite in curing rickets by the line test method prompted our examination of its ability to carry out the other two well-known physiologic actions of the vitamin, namely the elevation of serum calcium concentration by its bone mobilization action and the increased calcium transport by intestine. Table II demonstrates that the peak 4 metabolite is capable of bringing about the rise in serum calcium concentration, and Table III shows that it is effective in stimulating the transport of calcium in everted small intestinal sacs. These effects of the metabolite have been repeated many times. Thus two additional parameters have been measured which

VITAMIN

D METABOLITE

demonstrate the biological activity of the peak 4 metabolite. Perhaps most interesting is that the peak 4 metabolite acts much more rapidly than does the parent vitamin in stimulating calcium transport by small intestine (Table IV). A statistically significant effect of peak 4 metabolite on calcium transport can be observed as early as 8 hours after its oral administration, whereas 20 hours is required for comparable action by the parent vitamin DE. DISCUSSION

The present communication establishes firmly by three independent criteria the biological activity of peak 4 metabolibe formed in viva from vitamin Da. There is little doubt that this metabolite can (a) cure rickets, (b) stimulate the transport of calcium across small intestine, and (c) increase serum calcium concentration presumably by stimulating bone mineral mobilization. Thus its action most certainly mimics those of the parent vitamin. Of greatest significance is the fact that the metabolite acts much more rapidly than does the parent vitamin in initiating calcium transport in intestine. Clearly the metabolite fraction is effective within 8 hours after oral administration, whereas vitamin D itself is effective only after 20 hours. This in itself certainly constitutes evidence to support the idea that this fraction is or contains the metabolically active form of the vitamin. It has already been demonstrated that as the dose of vitamin D given per animal approaches the level considered truly physiological, the proportion of the radioactivity in the tissues that exists as peak 4 becomes greater, approaching 80 % (Neville, Press, and DeLuca, unpublished results; 2). Additional experiments demonstrate that the peak 4 metabolite is formed rapidly and appears in maximal quantity long before the first biochemical effects of the vitamin occur including the stimulation of 3H-erotic acid incorporation into RNA (11). Despite the evidence cited, it is not possible as yet to conclude that the peak 4 metabolite is in fact the metabolically active form of the vitamin. This can only be established when the bio-

511

chemical systems responsible for vit’amin D actions are identified and shown t’o utilize this metabolite to the exclusion of vitamin D itself. The nature of the peak 4 metabolite is not known at the present time. That it is in fact a metabolite and not an artifact has been clearly established (2). Recent work with new chromatographic systems has revealed that the peak 4 fraction is not homogenous but contains more than one component (Neville and DeLuca, unpublished results). Preparative scale production of the components of the peak 4 region is currently in progress with the aim of identification of those which are biologically active. Only two components which appear in the silicic acid chromatogram profile have as yet been identified. Peak 1 has been shown to be an ester of vitamin D and a long-chain fatty acid (usually palmitic) (Lund, Horsting, and DeLuca, submitted for publication). Peak 3 has been shown to be primarily the unaltered vitamin D (l), although it may contain an additional metabolite. Peak 2 is as yet unidentified although the possibility that it might be pre-vitamin Dg has been ruled out (12). Peak 4 metabolite is a normal component of human serum and has been shown to be formed from 3H-vitamin Ds (DeLuca, Lund, Rosenblum, and Lobeck, submitted for publication). It is also formed by rachitic chicks much as in the case of rats (Imrie, Drescher, and DeLuca, unpublished results). Thus the metabolite 4 is not confined to a single species but is of general occurrence. Although it is not possible to conclude that metabolite(s) 4 is the metabolically active form of the vitamin, it is nevertheless highly significant that a metabolite of vitamin D has been shown to be biologically active by three distinct parameters covering all known physiologic actions of the vitamin. Regardless of the final position the metabolite holds in the sequence of events resulting in the physiologic expression of vitaminD action, its identification should at least add to our knowledge of modifications of the vitamin D molecule which are possible to permit expression of the vitamin’s action.

512

MORII, ACKNOWLEDGMENT

We are grateful to Roberta lent technical assistance.

Prees for her excel-

REFERENCES 1. NORMAN, A. W., LUND, J., AND DELUCA, H. F., Arch. Biochem. Biophys. 108.12 (1964). 2. LUND: J., AND DELUCA, H. F., J. Lipid Res. 7, 739 (1966). 3. DELUCA, H. F., GUROFF, G., STEENBOCK, H. REISER, S., AND MANATT, M. R., J. Nutr. 76, 175 (1961). 4. STEENBOCK, H., AND HERTING, D. C., J. Nutr. 67, 449 (1955).

ET AL. 5. NEVILLE, P. F., AND DELUCA, H. F., Biochemistry 6, 2201 (1966). 6. NORMAN, A. W., AND DELuc.~, H. F., Biochemistry 2, 1160 (1963). 7. STEENBOCK, H., AND BLACK, A., J. Biol. Chem. 64, 263 (1925). 8. U.S. PHARMACOPEIA, 14th revision. Mack Publishing Co., Easton, Pa. (1955). 9. WEBSTER, W. W., Am. J. Clin. Path01 131, 330 (1960). 10. LUND, J., HORSTING, M., AND D~Luc.4, H. F., Arch. Y48. 11. ZULL, J. E., STOHS, S. J., AND DELUCA, H. F., Federation Proc. 26, 545 (1966). 12. LUND, J., Ph.D. Thesis, University of Wisconsin (1966).