THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, 14, July 25 ... · THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 14, Issue of July 25, p.8261-8271, 1982 Printed in U.S.A Identification

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 14, Issue o f July 25, p. 8261-8271, 1982 Printed in U.S.A

Identification of a New C-23 Oxidation Pathway of Metabolism for 1,25=Dihydroxyvitamin D3 Present in Intestine and Kidney*

(Received for publication, December 23, 1981)

Norio Ohnuma and Anthony W. Normanf From the Department of Biochemistry, University of California, Riverside, California 92521

Evidence is presented for the existence of a new C-23 oxidation pathway for the metabolism of the hormon- ally active form of vitamin D3, namely lu,25-dihydrox- yvitamin D3 (1,26(OH)zD3). Homogenates of intestinal mucosa or kidney, but not liver, from rats and chicks convert 1,25(0H)z[26,27-3H]D3 into two new metabo- lites, la,25-dihydroxy-23-oxo-vitamin DS (1,25(OH)z-23- OXO-D~) and lcu,25,26-trihydroxy-23-oxo-vitamin D3 (1,25,26(OH)3-23-oxo-D3), and an unknown metabo- lite(s) which has been possibly subjected to side chain modification/cleavage so that the tritium of the sub- strate has been converted into a form which is volatile. The isolation and chemical characterization of 1,25(OH)z-23-oxo-D3 and 1,25,26(OH)3-23-oxo-D3 from homogenates of chick intestinal mucosa has recently been described (Ohnuma, N., Kruse, J., Popjak, G., and

Based on kinetic studies with chick intestinal homoge- nates, the proposed C-23 oxidation pathway is:

oxo-D3 J unknown metabolite with altered side chain. The relative enzyme activities of the C-23 pathway in

tissues from vitamin D-replete chicks are: intestine, kidney, liver, 18:3:less than 1. The activity of the C-23 pathway in homogenates of chick intestinal mucosa can be enhanced 1 0 ~ by prior priming of the birds with a single intravenous dose of 500 ng (1.3 nmol) of 1,25(OH)zD3; the induction of the enzyme activity is maximal by 3-6 h and returns to basal levels by 12 h. A comparison was made in chick and rat intestinal homogenates of this C-23 pathway and the previ- ously known C-24 oxidation pathway, which con- verts 1,25(OH)zD3 into 2,24,25-trihydroxyvitamin D3 (1,24,25(OH)3D3); it was calculated that under these conditions 72% of the 1,25joH)z& was metabolized by the C-23 oxidation pathway, 13% by the C-24 pathway, and only 14% by other as yet unspecified pathways. It is proposed that the newly discovered C-23 pathway for metabolism of 1,25(OH)zD3 by the target intestinal mucosa and kidney may play a prominent role under physiological conditions of controlling the tissue levels of this hormonally active form of vitamin D3.

N o ~ ~ B z ~ , A. W. (1982) J. BioC. Chem 257, 5097-5102).

1,25(OH)zD3 -+ 1,25(OH)z-23-0~0-D3 + 1,25,26(OH)3-23-

In the past decade, significant progress has been made in our understanding of the metabolism and action of vitamin D (2). For the expression of its biological activity, vitamin D is

* This work was supported by United States Public Health Service Grant AM-09012-017. This is Paper XX in a series entitled “Studies on the Metabolism of Calciferol”; the previous paper in this series is Ref. 1. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

# To whom aIl inquiries should be addressed.

first metabolized in the liver to 25-hydroxyvitamin Ds. 25(OH)D3’ is then subjected to la-hydroxylation to yield la,25-dihydroxyvitamin D3 (3,4) or 24-hydroxylation to yield 24R,25-dihydroxyvitamin Ds (5 , 6 ) ; both of these dihydroxy- lated metabolites are believed to be the principal agents which generate the complete spectrum of vitamin D-mediated bio- logical responses (7,8). 25(OH)D3 also has been shown to be 23-hydroxylated to yield 23,25-dihydroxyvitamin DB (9) and 26-hydroxylated to yield 25S,26-dihydroxyvitamin D3 (10); the biological functions of these metabolites are not yet known.

While it is well established from data obtained in the chick (11-13), rat (14), and man (15, 16) that 1,25(OH)2& is the dominant active form of vitamin D in the intestine, mediating calcium and phosphorus absorption, very little is known con- cerning the detailed pathways of further metabolism of this seco-steroid? The work of Kumar et at. (la, 19) suggested that the intestine or liver was involved in the side chain oxidation of 1,25(OH)2D3. In as little as 6 h after dosing rats (18) or chicks (19) with physiological doses of 1,25(0H)2[26,27- l4C]-D3, between 50 and 70% of the administered carbon 14 could be detected as 14C02. In a follow-up study, Esvelt et ai. (20) isolated and purified from extracts of rat liver and intes- tine a new metabolite which was chemically characterized to be a side chain-shortened metabolite of 1,25(OH)2D3-namely la- hydroxy-24,25,26,27-tetranor-C-23-carboxyI-vitamin D,. Recently, we reported the existence of another metabolite of 1,25(OH)& (21-23) in the plasma of animals dosed with 1,25(OH)zD3; this substance was isolated in pure form and its structure was determined to be 1,25-dihydroxyvitamin D:<- 26,23-lactone (24). In the course o€ studying the biological activity (23) and pathway(s) of biosynthesis (25) of the 1,25(OH)2Da-26,23-lactone (in rat intestinal homogenates) we discovered the existence of two additional metabolites of 1,25(OH)~D3. In an accompanying paper (I) , we have de- scribed their isolation from homogenates of chick intestinal mucosa with 1,25(0H)?D3 as substrate and their chemical characterization. Their structures were reported to be 1,25- dihydroxy-23-oxo-vitamin D, and 1,25,26-trihydroxy-23-0~0-

’ The abbreviations used are: 25(OH)Ds, 25-hydroxyvitmin D,?; 1,25(OH)~D3, la,25-dihydroxyvitamin D,?; 24,25(OH)~D3, 24R,25-di- hydroxyvitamin Ds; 25,26(0H)*D3, 25S,26-dihydroxyvitamin Ds; 1,- 24,25(OH)~D3, Ia,24R,25-trihydroxyvitamin Da; 1,25(0H)~D~-26,23- lactone, In,25-dihydroxy-26,23-lactone-vitamin D;j; calcitroic acid, la- hydroxy-24,25,26,27-tetranor-C-23-carboxyl-vitmin D3; 23,25(OH)~D:{, 23,25-dihydroxyvitmin Da; 1,25,26(OH)aDs, la,25,26-trihydroxyi- tamin D3; 1,25(OH)~-23-oxo-Ds or 1,25-Prime, 10,25-dihydroxy-23- oxo-vitamin Da; 1,25,26(0H)3-23-0xo-D3 or Peak-X and Peak-X- Prime, 1,25,26-trihydroxy-23-oxo-vitamin D3; HPLC, high perform- ance liquid chromatography; TLCK, N-a-p-tosyl-L-lysine chloro- methyl ketone; PMSF, phenylmethylsulfonyl fluoride; TPCK, L-1- tosylamide-2-phenylethyl chloromethyl ketone methyl ester.

According to the International Union of Pure and Applied Chem- istry Committee on the Nomenclature of Biological Chemistry, vi- tamin DS (cholecalciferol) i s defined as a steroid (17). The chemical name is 9,10-seco-cholesta-5,7,10(19)-triene-3-,B-o1.

8261

8262 C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin 0 3

vitamin D3. This paper reports results of a study of the physiological and enzymological conditions which optimize the intestinal conversion of 1,25(OH)2D3 by a new C-23 oxi- dation pathway into these two new metabolites.

MATERIALS AND METHODS4

RESULTS

Metabolism in Vivo of 1,25(OH)2D3 in the Rat-A preceding paper in this series (25) reported studies on the metabolism of 1,25(OH)2D3 in the rat under both in vitro and in vivo condi- tions. A polar fraction termed Peak I11 (migrating in the region of 1,24R,25(OH),D3) obtained from Sephadex LH-20 column chromatography of an intestinal mucosal extract was subsequently resolved by two-step HPLC (p-Porasil column; elution solvent isopropyl alcoholhexane, 11:89 (v/v) or iso- propyl alcohol:dichloromethane, 1O:lOO (v/v)) into three sep- arate vitamin D metabolites. They were identified as authen- tic 1,24R,25(OH)SDa, 1,25(OH)2D3-26,23-lactone, and a new metabolite Peak X (subsequently determined (1) to be 1,- 25,26(OH)3-23-oxo-D3). It then was of interest to ascertain the relative concentrations in blood and the intestine of these metabolites of 1,25(OH)& and to determine their metabolic relationship to one another. The tissue concentration of these metabolites determined in the plasma or small intestine are presented in Table I. In plasma, 1,25(OH)2[”H]D3, 1,25(OH)z[”H]D3-26,23-lactone, and 1,24,25(OH)3[”H]D3 were determined to be approximately 0.1 pmol, 0.04 pmol, and 0.006 pmol per ml, respectively, while 1,25,26(OH)3-[3H]23-~~~-D3 could not be detected. In the intestinal mucosa, 1,25(OH)2[3H] D3 was determined to be the predominant radioactive metab- olite (approximately 0.3 pmol per g of wet tissue). Other metabolites, 1,25,26(0H)3-[3H]23-oxo-D3, 1,25(OH)2[3H]D3- 26,23-lactone, and 1,24,25(OH)3[3H]D3, were approximately 1.5% or less of the radioactive 1,25(OH)zD3. In spite of their low concentration, it should be noted that these three metab- olites were produced and are present in the intestine under physiological conditions. It also is noteworthy that 1,25,26(OH)3-23-oxo-D3 was only found in the intestine and could not be detected in the plasma. In contrast, 1,25(OH)zD3- 26,23-lactone was determined to be the major metabolite of 1,25(OH)zD3 in plasma and this is consistent with our previous observation that the maximum level of this metabolite was determined to be approximately 0.4-0.7 pmol/ml at 8 h after administration of 400 ng/kg doses of 1,25(OH)2D3 to vitamin D-replete rats (25).

Metabolism in Vitro of 1,25(oH)~D3 by Rat Small Intes- tine-Since 1,25,26(OH)3-23-oxo-D3 was undetectable in plasma extracts, the metabolite was postulated to be produced in the small intestine. Supportive evidence for this idea was provided by an incubation of 1,25(OH)2[3H]D3 with a homog- enate of intestinal mucosa obtained from vitamin D-replete rats which had received 1.25 pg/kg of 1,25(OH)2D3 intrave- nously, 8 h before death. As shown in Fig. 1, the HPLC profile of the intestinal metabolites produced in vitro revealed 1,- 25,26(OH)3-23-oxo-D3 to be the major metabolite of 1,25(OH)2D3 when 1,25(0H)@6,27-3H]D3 was used as the substrate at a concentration of 3 X M.

The time course of production of these polar metabolites in

Portions of this paper (including “Materials and Methods,” por- tions of ‘‘Results,” Figs. 10-14, and Footnote 3 ) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Be- thesda, MD 20814. Request Document No. 81M-3153, cite the au- thors, and include a check or money order for $8.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the journal that is available from Waverly Press.

an intestinal homogenate with a substrate concentration of 1,25(OH)zD3 of 30 IIM is shown in Fig. 2. It can be seen that 1,25,26(OH)3-23-oxo-D3 is a major metabolite produced by the intestinal mucosa and that its maximal production under these conditions was achieved by 5-10 min.

Further evidence for the possible physiological significance of the production of 1,25,26(0H)3-23-oxo-D3 was provided by determination of the 1,25(OH)~D3 substrate dependence for the production in vitro of 1,25,26(OH)3-23-oxo-D3 and 1,24,25(OH)3D3 by the homogenate of rat small intestinal mucosa (Fig. 3). Since the production of the 1,25,26(0H)3-23- 0xo-D~ and 1,24,25(OH)3D3 showed a sigmoid curve for the substrate concentration dependence, neither an apparent K,,, nor apparent Vmax could be obtained precisely for the produc- tion of these metabolites. However, the difference in the estimated kinetic parameters for these two metabolites is striking. The estimated K,,, (-5 X lo-’ M) and V,,, (-80 pmol/ 150 mg of mucosa/l5 min) for the 1,25,26(OH)3-23-oxo-D3 production is clearly smaller than the K, (22 X lo” M) and V,,, (-500 pmol/l50 mg of mucosa/l5 min) for the reaction responsible for the production of 1,24,25(OH)3Ds. When the substrate concentration was higher than M, ~ , ~ E I ( O H ) ~ D ~ was metabolized predominantly to 1,24,25(OH)3D,3. In con- trast, when the substrate concentration was lower than -4 X lo-’ M, 1,25,26(0H)3-23-oxo-D3 was produced as the dominant metabolite of 1,25(OH)zD3. Accordingly, it is reasonable to consider that 1,25,26(OH)3-23-oxo-D3 is the major metabolite produced by small intestine from 1,25(OH)2D3 under physio-

TABLE I Metabolism in vivo of 1,25(OH)~[26,27-”HJD:~ in ruts

Rats were administered an oral 60.5-pmol (126 ng/kg body weight) dose of 1,25(OH)s[26,27-3HH]D,, (14 Ci/mmol) 8 h before death. U.D. indicates that the metabolite was undetectable. Values in parentheses are per cent of total metabolites present.

Tissue concentration -

Radioactive metabolite Plasma Small intestinal

mucosa

fmol/g

1,25(OH)2D;3 99.8 271 1,25,26(OH):~-23-OXO-D;~ U.D. 4.2 (1.58) 1,25(0H)~D:~-26,23-1actone 43.0 (28.98) 3.6 (1.3%) 1,24,25(OH)sDs 6.2 (4.2%) 2.2 (0.8%)

y 2 0 I /

”

[L , , , , , / / , 1 , 1 , 1 1 1 , , , / 1 1 , 1

0 10 20 30 ELUTION VOLUME ( m i l

40

FIG. 1. Production in uitm of 1,25,26(OH)3-23-oxo-D~ from 1,25(OH),D3 by homogenates of rat small intestine. 1,25(OH)2[26,27-3H]D3 was incubated with the homogenate of small intestinal mucosa from rats primed with 1.25 pg/kg of 1,25(OH)*Ds 8 h before death. The polar fraction (Peak 111) from Sephadex LH-20 column chromatography (data not presented) was pooled and then co-chromatographed with authentic 1,25(OH)~D:3 (1 yg) and 1,24R,25(OH)3D3 ( 2 pg) on a HPLC column of y-Porasil eluted with isopropyl alcohol:hexane, 11:89 (v/v). The incubation was carried out as described under “Materials and Methods.” The absorbance at 254 nm of authentic 1,25(OH)~Ds, and 1,24R,25(OH)nD:3, is indicated by the solid line.

C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin D3 8263

1 1 1 1 I l l

30 nM

d

0 5 10 15 20 25 30

INCU8ATION T I M E (mm)

FIG. 2. Time course of 1,25(OH),D3 metabolism (30 11~) by a homogenate of rat small intestinal mucosa. Incubation condi- tions and metabolite separation are described under “Materials and Methods.” o “ 0 , radioactivity present in Peak I11 obtained from Sephadex LH-20 (data not shown, see “Materials and Methods”). The Peak 111 was rechromatographed on HPLC to generate the data points shown by the closed symbols. M, Peak X; A-A, 1,24,25(OH)3D3 fraction. Note that the 1,24,25(OH)3Da fraction may contain a small amount of 1,25(OH)2D3-26,23-lactone since the HPLC elution system employed does not separate these two metabolites.

Y+ PEAK - X

0 250 500 750 1000 1250 1500 1750 200

1

MI 0 50 100 150 200 250 300 350 400 nM

0 PC

SUBSTRATE CONCENTRATION

FIG. 3. Substrate concentration dependency of the produc- tion of 1,25,26(OH)3-23-oxo-D3 and 1,24,25(0H)& from 1,25(OH),D3 by a homogenate of rat small intestinal mucosa. Vitamin D-replete rats received 1.25 pg/kg of 1,25(OH)2D3 intrave- nously 8 h before death. The homogenates were incubated with varying concentrations of 1,25(0H),[26,27-’H]D3 for 15 min and the metabolites were separated by HPLC. Results are expressed as pico- moles of each metabolite produced per incubation. Incubation con- ditions are described under “Materials and Methods.” -, Peak X; m, 1,24,25(OH)sD~.

logical conditions. This conclusion is also consistent with the results presented in Figs. 4, 5, and 7 which present similar data for homogenates of chick intestinal mucosa.

Metabolism in Vitro of 1,25(oH)2D3 by Homogenates from Chick Small Intestinal Mucosa-To investigate the nature of this in vitro metabolism of 1,25(OH)2D3 in homogenates of chick intestinal mucosa, time course experiments were per- formed using chicks of varying vitamin D status (also see Fig. 11 in the miniprint). When the intestinal homogenate obtained from vitamin D-deficient (-D) chicks which had been primed before death with l,25(OH)2D3 was incubated for 30 min at a 1,25(OH)2D3 substrate concentration of 2.25 m, a marked decrease of the substrate (Peak 11) was observed by 5 min and it was accompanied with an increase of Peak 111. Peak I11 gradually decreased with time (Fig. 4, B and C). Radioactiv- ity recovered in the water:methanol layer following chloro- form:methanol extraction also increased with time, and the maximal level was achieved within approximately 10 min of the incubation (Fig. 4B). In contrast, the homogenate from

vitamin D-deficient chicks which received only vehicle alone (Fig. 4) could not metabolize in sigmfkant amounts 1,25(OH)2[3H]D3 to either Peak I11 or the metabolite(s) re- covered in the water:methanol layer. These results are con- sistent with the results obtained from the study of 1,25(OH)2D3 metabolism in vitro by rat small intestine (25). An essentially identical result was obtained from the chicks which had received daily oral doses (1.3 nmol) of 1,25(OH)2Ds for 2 weeks (Fig. 4, C and D). The decrease of Peak I1 was again accompanied with an increase of radioactivity in the water:methanol layer, as well as an increase of Peak I11 which reached a maximum by 5 min and was then followed by a gradual decrease with time. This transient increase of Peak I11 was considered to be due to a consumption of the substrate Peak I1 (1,25(OH)2De) and due to degradation of Peak 111. In fact, when the incubation was performed at a 10 times higher substrate (1,25(OH)~D3) concentration (23.9 nM), the radio- activity recovered in Peak I1 decreased gradually from 34,000 dpm to 12,000 dpm with time (Fig. 4 0 ) and the radioactivity recovered in Peak I11 increased up to 15,000 dpm by 30 min. In every case, the more polar metabolite Peak IV was ob- served. However, the amount was not significant. Further- more, the radioactivity recovered in the water:methanol layer

i 7 1 1 I I I I I I I 1

A -0; (-1,251 B -0; (+1.251 2.25 nM 2.25 nM -

i 1 1 1 1 1 1 1 1 1 1

0 5 IO 15 30 0 5 IO 15 30 INCUBATION TIME (mm)

1 1 1 1 I 1 1 1 1 1 - C. +1,25;(+1.25) D. +1.25;(+1,25) - 2.25nM

5 4 - 2 3 . 9 n M

” - n

a a a 0

b i - t a

1 1 1 I I I I

0 5 IO 15 30 0 5 IO 15 30

INCUBATION TIME ( m i n )

FIG. 4. Time course of 1,25(OH)~[26,27-’~D, metabolism in homogenates of chick small intestinal mucosa. A represents the kinetics of 1,25(OH)2D3 metabolism in the homogenate prepared from vitamin D-deficient chicks given 0.1 ml of the vehicle alone. B represents the kinetics obtained from vitamin D-deficient chicks given 500 ng (1.3 nmol) of 1,25(OH)2D3 6 h previously. C and D are the results obtained from 1,25(0H)2D3-replete animals primed with 500 ng of 1,25(OH)xD~ 6 h before death. The substrate concentration of 1,25(OH)xD3 is 2.25 nM (for A, B, and C) or 23.9 nM (for D). Peak I, W, Peak 11, D”Q Peak 111, A”-& Peak IV, V”-V; water:methanol layer, O ” - O . Peaks I, 11, 111, and IV are shown in Fig. 11. The water:methanol layer represents the total radioactivity recovered following ch1oroform:methanol extraction of the incubation mixture by a modified procedure of Bligh and Dyer (29).

8264 C-23 Pathway of Metabolism of 1,25-Dihydroxyuitamin D.7

Water-MeOH Layer ( x ld’dpm )

FIG. 5. Linear regression analysis of relationships between Peak XI and water:MeOH layer (lef3 panel), between Peak 1x1 and water:MeOH layer (centerpanel), and between the radio- activity “unrecovered” in the CHCb layer and the radioactiv- ity “recovered” in the water:MeOH layer (right panel). Each point on the left-hand panel corresponds to that in Fig. 4 (A-V).

I 7 0 1 r 1 1 I 1 0 1 I

v g f i 0 +1.25; 23.9nM +1,25; 2.25 nM

15 ~

10 -

5 -

0 -

t

I l l 1 I I I I I 1

0 5 IO 15 30 0 5 10 15 30 INCUBATION TIME (mm)

FIG. 6. Time course of 1,25(OH)zD3 metabolism in homoge- nates of chick small intestinal mucosa. 1,25(0H)2D~-replete chicks (+1,,25) were used; their duodenal segments were used; their duodenal segments were removed at 6 h after intravenous injection of 500 ng of 1,25(OH)2D3; and the chicks and then the mucosal homog- enate was prepared as described under “Materials and Methods.” The 1,25(OH)~D3 substrate concentrations used here are 2.25 nM (A) or 23.9 nM (B) . Details of the experiment are described under “Materials and Methods.” W, polar fraction (Peak 111) from Sephadex LH-20 column; M, 1,25,26(OH)3-23-oxo-Ds; A-A, 1,24,25(OH)sD3.

was considered to be 3H20 and/or a volatile 3H-fragment of the side chain of the 1,25(OH)2[26,27-3H]D3 which was used as substrate, since most of the radioactivity (>95%) in the water:methanol layer was lost by evaporation under a flow of forced air. Thus, it was postulated that 1,25(OH)2[3H]D3 could be metabolized via metabolite(s) eluted in the fraction of Peak I11 from the Sephadex LH-20 column to yield an as yet unidentified 1,25(OHhD3 metabolite(s) which had lost tritium from C-26 and C-27, possibly by side chain cleavage.

This idea was studied by examining the relationship be- tween the radioactivity determined in Peak I1 (1,25(OH)2D3) and Peak I11 (1,25,26(OH)3-23-oxo-D3) and radioactivity “unrecovered” in the chloroform layer with the radioactivity “recovered” in the water:methanol layer (Fig. 5). It can be seen that: (a) the decrease of Peak I1 (1,25(OH)&) is pro- portional to the increase of tritium in the water:methanol layer (4 = 0.96, for n = 8); ( b ) the increase of Peak I11 (1,25,26(OH)3-23-oxo-D3) is proportional to the increase of tritium in the water:methanol layer (9 = 0.96, n = 5); and ( c ) the increase of tritium “recovered” in the water:methanol layer is proportional to the tritium “unrecovered” in the chloroform layer (3 = 0.94, n = 10). “Unrecovered” radioac-

Each point on the center panel corresponds to that in Fig. 4 0 . Points in the rzght-hand panel correspond to those in C and D of Fig. 4. Linear regression was performed with a least square method; ? represents the square of the correlation coefficients. A definition of “recovered” and “unrecovered” radioactivity is given in the text.

tivity = ((initial substrate radioactivity) - (radioactivity re- covered in chloroform layer)).

The radioactivity in the water:methanol layer was deter- mined to contain approximately 45% of the “unrecovered radioactivity.” Since almost all of the radioactivity in the HzO/methanol layer is lost by evaporation and since, on the other hand, solvent evaporation is also required for measure- ment of the tritium in the chloroform layer, it can be postu- lated that approximately 55% of the “unrecovered” radioac- tivity in the chloroform layer is present as a 3H-containing metabolite(s) which is readily lost by evaporation and thus cannot be determined. The relationships presented in Fig. 5 enable us to presume either that: (i) the metabolite(s) present in the Peak I11 fraction must be an intermediateb) of 1,25(OH)zD3 leading to a metabolite having a shortened side- chain or (ii) that the metabolite(s) has been modified in the

I 1 I 7 I I I

25 -

- E 20- p.

W 15-

0 J

m 2 I O L W I

SUBSTRATE CONCENTRATION

FIG. 7. Substrate concentration dependence of the metabo- lism of 1,25(0H)2D3 in homogenates of small intestinal mucosa or kidney tissue obtained from 1,25(OH)2D3-replete chicks. The homogenates were prepared from animals which had received 500 ng of 1,25(OH)2D3 intravenously 6 h before death and were incubated with varying concentrations of 1,25(OH)a[26,27-”H]D:3 for 5 min (for the intestinal mucosa homogenate) or 10 min (for the kidney homog- enate). Results are expressed as picomoles of each metabolite pro- duced per incubation. .”--., 1,25,26(0H):3-23-oxo-D3; m, 1,24,25(OH)3D3. Circles are data obtained from intestinal homoge- nates and bores are data from kidney homogenates.

C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin D3 8265

side chain in a manner that results in partial loss of the radioactivity on C-26 and C-27.

Fig. 6 presents the time courses of the in vitro production of the Peak X and 1,24,25(OH)3D3 by chick intestinal homog- enates. When the reaction was performed at a substrate concentration of 2.25 n ~ , the 1,25,26(OH)3-23-oxo-D3 in- creased to the maximum (5500 dpm/ml of incubation) by 5

1,24,25(OH)3D3 showed a maximum production by 1 min. When the substrate (1,25(OH)&) concentration was

20

I 5 min and then gradually decreased with time, while

z 2.25 nM, the metabolite 1,25,26(OH)3-23-oxo-D3 was present 0 in a larger amount than 1,24,25(OH)3D3, while the amount a of 1,25,26(OH)3-23-oxo-D3 was smaller than that of rn 3 1,24,25(OH)3D3 when the substrate concentration was 23.9 0 I O nM. 5 Fig. 7 presents data concerning the 1,25(OH)& sub-

E was determined to be the major metabolite. In contrast, when

l-

\ v) w

strate concentration dependence for the production of 1,- -1 24,25(OH)3D3 and peak X. When the 1,25(OH)2D3 substrate 0 concentration was higher than -2 X M, 1,24,25(OH)aDa

5 employed, Peak X was observed to be the major product. a lower 1,25(OH)zD3 substrate concentration (<lo-* M) was

Although neither the apparent K , nor apparent Vmax could be determined precisely, the difference in the kinetic parameters for the production of the metabolites was clear. The apparent K , and V,,, for the production of 1,25,26(OH)a-23-oxo-D3 was estimated to be -1.5 X lo-@ M and -12 pmol/5 min/100 mg of mucosa, while the apparent K,,, and V,,, for the 1,- 24,25(OH)3D3 production was estimated to be >8 X M and >40 pmol/5 min/100 mg of mucosa, respectively. Accord-

24 36 ingly, the metabolite 1,25,26(OH)3-23-oxo-D3 is demonstrated HOURS AFTER I.V. DOSING OF to be the predominant metabolite of 1,25(OH)& under cer-

tain in vitro conditions which closely approximate physiolog- ical circumstances.

0

0 3 6 12

20 I-U. OF 1,25(OH)~D3

FIG. 8. Time course of induction and decay of 1,25(OH)zD3 Induction Of the Intestinal Catabolism of 1,25(ow2[26,27- metabolizing activity in homogenates of small intestinal mu- 3H1D3 following single Intmvenous Injection O f 1,25(off.)zD3 cosa. Vitamin D-deficient chicks (three/group) were induced with a to Vitamin D-deficient Chicks-Further evidence for the

animals were killed at 0 (received vehicle only, 1,3, 6, 12, 24, or 35 h the intestine was provided by a t h e cowse study of the seco- after dosing. Incubations were carried out at 37 "C for 5 min at a steroid metabolism induced by the with 1,25(OH)zD3 substrate concentration of 6.5 nM. Each metabolite was

with a solvent system of isopropyl alcohol:hexane, 11:89 (v/v). Details activity in Vitro, it Was necessary to employ a short time are described under "Materials and Methods." Total radioactivity incubation (5 min) and relatively high substrate Concentration

single intravenous injection of 500 ng (20 units) of 1,25(OH)zDa. The physiological significance of the 1,25(OH)JJ3 metabolism in

separated by HPLC equipped with a p-porsil column and eluted 1,25(OH)ZD3 prior to death (Fig* 8). To measwe the enzymatic

recovered in chloroform layer after ch1oroform:methanol extraction, (6.5 nM) to avoid substrate depletion during incubation since M, 1,25(OH)zD3 &action, 1,25,26(OH)3-23-oxo-Da, the metabolism of 1,25(OH)2[26,27-3H]Da proceeds very rap- - 1,24,25(OW3D3, A"+; &I represents the mean f s. E. of i dy (see for instance ~ i ~ . 4 ~ ) . Under the incubation conditions contains another metabolite designated 1,25-Prime besides Used here, approximately 98% of the added substrate was 1,25(OH)zD3 recovered without incubation. The 1,25(OH)2D3 fraction

1,25(OH)2D3 as is shown in Fig. 14.4. The 1,24,25(OH)3D3 peak also consumed during the 5-min incubation with the intestinal contains a trace amount of 1,25(OH)~D3-26,23-lactone as shown in homogenate prepared from the vitamin D-deficient chicks Fig. 140. primed with 1,25(OH)~& 3 h previously. As presented in Fig,

TABLE I1 Oxidative degradation of 1,25(0H)& by chick small intestinal mucosa homogenate

The data represent the mean * S.E. from three separate incubations. One incubation of 5 min was c-ed out for each bird, except for the first listed sample which was a "zero time control." The initial substrate concentration of 1 , 2 5 ( 0 ~ ) ~ ~ ~ is 19.5 pmol/incubation.

Time after injection 1,25(OH)*Ds substrate C-23-oxidized metabolites C-21-oxidized metabolite of 1,25(OH)zD:I rernamng 1,25(OH)?-23-oxo-D3 +

1,25,26(OH)3-23-oxo-D3 1,24,25(OH)aDs Total

pmol pnol pmol p n o l 0 0

0.23 f 0.08 0.26 f 0.07

18.4 f 1.68 16.9 rt 0.78

1 13.2 rt 2.59 2.56 f 1.54 0.86 f 0.73 3 0.37 f 0.03".' 13.2 f 1.33",h

16.6 f 0.67

6 2.42 f O.lOn,' 15.9 f 1.39

12 13.1 f 0.12".h 2.62 & 0.33".' 15.0 rt 1.01

24 0.75 & 0.18 15.7 rC_ 0.30

36 14.4 f 0.34 14.4 f 0.35",'

16.5 f 1.23 1.68 -C 0.46 15.0 f 0.44 1.62 f 0.37

0.56 f 0.36".' 11.8 f 0.76".h 1.85 f 0.09

12.9 f 0.40b 1.31 rt 0.07 0.27 * 0.00 12.2 f 0.33".h 1.68 rC_ 0.07 0.50 f 0.03".'

Statistically significant from the incubations, 0 h, 0 min. p < 0.05. ' Statistically significant from the incubations of 0 h, 5 min. p < 0.05.

8266 c-23 Pathway of Metabolism of 1,25-Dihydroxyuitamin D3

8, the induction of 1,25(OH)zD3 metabolism in the intestine of vitamin D-deficient chicks reaches its maximum at 3-6 h after intravenous 1,25(OH)2D3 injection. The induction is very short lived and has returned almost to base-line levels 12 h after dosing. Although studies with protein synthesis inhibitors have not been undertaken, the possibility of protein synthesis cannot be eliminated from this induction process because it has been suggested that protein synthesis is involved in the renal 25(OH)D3-24-hydroxylase induction (32-34).

As described under “Materials and Methods,” the con- version of 1,25(OH)2[%]D3 to 1,25(OH)z-23-oxo-Dn and 1,- 25,26(OH)j-23-0xo-D2 was accompanied by a loss of tritiated metabolites due to volatilization. By suitable calculation it was possible to evaluate the 1,25(0H)*D3 substrate depend- ence for the loss of tritium (a measure of C-23 oxidation) and compare it to the amount of 1,24,25(OH)3D3 formed (a meas- ure of C-24 oxidation). These recalculated results are shown in Table 11. The markedly enhanced catabolism of 1,25(OH)2D3 was observed at 3-6 h following intravenous injection of 1,25(OH)2D3 to vitamin D-deficient chicks. The small intestinal mucosa from chicks primed with 1,25(OH)zD3 3 h before death could metabolize 72% of 1,25(OH)zD3 to 1,25(OH)2-23-oxo-D:~ or 1,25,26(OH)3-23-oxo-D3 and 13% of the substrate to 1,24,25(OH)&. The participation of other metabolic pathways utilizing 1,25(OH)2D3 is likely less than 14%. From the results described above, it can be concluded that 1,25(OH)2D3 undergoes further metabolism by the small intestine principally via the C-23 oxidation pathway.

DISCUSSION

This communication and a recent paper (1) report the discovery and evaluation of the possible physiological signifi- cance of a new (3-23 oxidation pathway for the further metab- olism of 1,25(OH)2D3. The previous paper (1) describes the isolation and chemical characterization of two C-23-oxidized metabolites-namely 1,25(OH)2-23-oxo-D3 and 1,25,26(0H)3- 23-0XO-D3. This paper deals with the induction in vivo by 1,25(OH)zD3 of the metabolism in vitro of 1,25(OH)zD3 in tissues of vitamin D-deficient chicks as well as vitamin D- replete chicks or rats. The results of this study demonstrate that the small intestine and kidney metabolize 1,25(OH)*D,? to 1,25(OH)z-23-oxo-D3, 1,25,26(OH)3-23-oxo-D3, and 1,- 24,25(OH)3D3 and that this further metabolism is markedly induced by priming the animals with 1,25(OH)zD3 3-6 h before death.

When 1,25(OH)~[Z6,27-’H]D~ is used as the in uztro sub- strate, the production of the two new metabolites is accom- panied with a loss of radioactivity into the water:methanol layer following Bligh and Dyer extraction (29). Furthermore, this H20:methanol-extractable radioactivity is readily lost by evaporation, indicating that 1,25(OH)~[26,27-~H]Ds undergoes oxidative side chain cleavage (18, 19) to give a metabolite having a shortened side chain. The exact chemical nature of this putative metabolite has not yet been determined however it may be calcitroic acid.5 The production of this metabolite from 1,25(0H)z/26,27-3H]D3 would necessarily result in loss of the side chain tritium.

The kinetic relationships between the remaining substrate 1,25(OH)#H]D3 and its radioactive metabolites determined after in uitro incubation of 1,25(OH)2[26,27-3H]D,3 with the small intestinal homogenate from chicks primed with 1,%(OH)zD3 (see Fig. 6) indicate that 1,25(OH)2D3 is princi- pally metabolized via 1,25,26(OH)3-23-oxo-D3. Thus, it can be concluded that 1,25,26(OH)3-23-oxo-D3 is an intermediate product in the catabolism of 1,25(OH)zD3.

ted for publication. S. Ishizuka, S. Ishimoto, and A. W. Norman, manuscript submit-

VITAMIN 0 3

z 1a,25(;H)pD, *

la.24.25(OH)3D,

I v lq25(OH)2-23-oxo-D, [ la.23.25.26(OH)4D3]

I I“ [la.23.25(OH),D3]

7 J. 3. la,25.26(OH13-23-oxo-D3 la.25(OH)~-26.23-lactone-D~

la(OH)-24.25.26.27-letranor-23-COOH-D~ (Calctlrotc Actd)

FIG. 9. Pathways of metabolism of 1,25(OH)zD,. The com- pounds listed in brackets have not yet been chemically characterized; there is evidence for the existence of 23,25,26(OH)~D:$ (41).

The other newly isolated metabolite, 1,25(OH)2-23-oxo-D3 (l) , is considered to be the sole precursor of 1,25,26(0H)’-23- oxo-Ds on the basis of the following observations. (a) When 1,23(OH)2[26,27-3H]D3 was incubated with homogenate of small intestinal mucosa from chicks primed with 1,25(OH)& 1,25,26(OH)3[3H]Dj could not be detected. (6) 1,25,26(0H)3- 23-oxo-& has been prepared from 1,25(OH)& by in vitro incubation with chick intestinal ,mucosa homogenate (1). However, it was not produced when l~x,25S,26(OH)~D~ was utilized as the substrate for the in vitro preparation (1). ( e ) When 1,25(0H)~-[26,27-~H]23-oxo-D~ which had been pre- pared enzymatically was incubated with the chick intestinal mucosa homogenate, considerable amounts of radioactivity migrated in the fraction corresponding to the elution of 1,25,26(OH)3-23-oxo-D3 on a HPLC p-Porasil c ~ l u m n . ~

Thus, it is reasonable to propose that 1,25(OH)2D3 is fist metabolized to 1,25(OH)2-23-oxo-D3, presumably via forma- tion of 1,23,25(OH)3D3, although this compound has not yet been found? 1,25(OH)~-23-oxo-D~ is then metabolized to 1,25,26(OH)&23-oxo-D3 followed by a further degradation to a metabolite having a shortened side chain, presumably cal- citroic acid (20). An alternative possibility that 1,25(OH)zD3 is metabolized to 1,25,26(OH)3-23-oxo-D3 via 1,25S,26(OH)aD3 seems to be unlikely since 1,25,26(OH)3D3 is not an effective substrate for the production of 1,25,26(OH)3-23-oxo-D3.5 From these considerations, it can be proposed that 1,25(OH)zD~ undergoes metabolism by two alternative pathways; one path- way includes (2-23 oxidation as the fist step and the second pathway includes C-24 oxidation as the first step. These relationships are summarized in Fig. 9.

A highly correlated linear regression equation (see “Materials and Methods”) which described the remaining 1,25(OH)zD3 as a linear function of observed C-23-oxidized metabolites and C-24-oxidized metabolite has been obtained following a 5-min incubation of 1,25(OH)2D,7 with the homog- enates. By using this relationship, the actual amounts of the C-23- or C-24-oxidized metabolites produced during the incu- bation has been calculated to estimate the participation of the C-23 or (2-24 oxidation on the 1,25(OH)2D3 metabolism occur- ring in the small intestinal mucosa. In the small intestinal mucosa from chicks primed with 1,25(OH)~D3 h before death, the participation of the C-23, the C-24 oxidation, and other unknown metabolic alterations is estimated to be 72%, 13% and less than 14%, respectively.

Additional evidence in support of the physiological signifi- cance of the C-23 oxidation pathway for the further metabo- lism of 1,25(OH)2D3 has been obtained from a study of the substrate concentration dependence on the production of

However, the non-la-hydroxylated seco-steroid 23,25(OH)?D3 has been isolated and chemically characterized by Ikekawa et al. (40).

C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin DB 8267

1,25,26(OH)3-23-oxo-D3 and 1,24,25(OH)& from 1,25(OH)~D3 by the small intestinal mucosa homogenate. The relative pro- duction rates of 1,25,26(OH)3-23-oxo-D3 and 1,24,25(OH)~D3 vary strikingly depending upon the substrate concentration as assessed in both the rat intestinal homogenate (Fig. 2) and chick intestinal homogenate (Figs. 4, 6, and 7). 1,25,26(0H)3- 23-oxo-D3 is the major metabolite of 1,25(OH)~D3 when the substrate concentration is lower than lo-* M and the relative production of 1,25,26(OH)3-oxo-D3 and 1,24,25(OH)~D3 is con- stant when the substrate concentration is lower than 6.5 X IO-' M. Therefore, the participation of the C-23 oxidation pathway for 1 , 2 f ~ ( o H ) ~ D ~ seems likely to play an important role under conditions of physiological concentrations of

The enzymatic activities necessary for the new C-23 path- way of metabolism of 1,25(OH)& for the production of both 1,25,26(OH)3-23-oxo-D3 and 1,24,25(OH)3D~ are present in homogenates prepared from either rat small intestinal mucosa (Fig. 2) or chick kidney (Fig. 14), as well as chick small intestinal mucosa (Figs. 4, 6, and 7). Also, both metabolites have been detected in the intestine of rats which received oral doses of 1,25(OH)2[3H]De (126 ng/kg) in the physiological range 8 h previously (Table I). The marked difference in the rate of metabolism of 1,25(OH)& in intestinal homogenates from vitamin D-deficient chicks primed and unprimed with 1,25(oH)zD3 (Fig. 4) suggests that significant levels of the 1,25(0H)2D3-metabolizing enzymes are present in the normal animal.

Kumar et al. (38) have previously reported that there is almost no difference between intact and nephrectomized vi- tamin D-deficient rats in 14COz expiration when the animals are given an intravenous dose of 1,25(OH)~[26,27-'~CC]D~. This observation suggested that extrarenal tissue must participate in the metabolite transformations of 1,25(OH)zD3 which led to l4CO2 formation. Subsequently, calcitroic acid, a side chain- oxidized metabolite of 1,25(OH)zD~ was isolated, identified, and shown to be quantitatively the most prominent metabolite of 1,25(OH)2D3 in liver and intestine after the administration of the seco-steroid to vitamin D-deficient rats (20). The site of this side chain metabolism was unclear, although an entero- hepatic location was proposed (35). Recently, in a study on the distribution of calcitroic acid following 1,25(0H)2[26,27-

C]D3 administration to vitamin D-deficient rats, it was shown that liver and small intestine possess the capability for the side chain oxidation of this steroid (31). Our experiments described in this paper have clearly demonstrated that this side chain oxidation takes place in the small intestine as well as the kidney, but not in the liver. Also, our observation that 1,25(OH)2D3 is metabolized almost exclusively via 1,- 25,26(OH)3-23-oxo-D:1 to the presumed calcitroic acid would be consistent with the previous observation (13) that 1,24,25(0H),3[26,27-'4C]Da is not a good substrate for the pro- duction of expired I4CO2.

A fundamental question raised by this study is whether the participation of the small intestine and kidney in the metab- olism of 1,25(OH)z& plays a significant role in regulation of calcium and phosphorus homeostasis. The biological activities of the newly isolated metabolites 1,25(OH)2-23-oxo-D3 and 1,25,26(OH)3-23-0xo-D3 have not yet been fully assessed; pre- liminary data indicate that both metabolites compete effec- tively with 1,25(OH)& for the chick intestinal receptor meas- ured under in vitro conditions according to Wecksler and Norman (39) and also have some modest capability to stimu- late intestinal calcium ab~orption.~ If the rapid metabolism of 1,25(OH)2DS in the target intestine and kidney is related to

' E. Mayer, N. Ohnuma, and A. W. Norman, manuscript submitted

1,25(OH)zD3.

14

for publication.

some as yet undefined biological activity, then both 1,25(0H)2- 23-OXO-D3 and 1,25,26(OH)3-23-oxo-D3 are prime candidates for consideration. The structural relationship between vitamin D analogs and vitamin D activity has been intensively inves- tigated (36,37) under both in vivo and in vitro conditions. It is well established that the la-hydroxyl and C-25-hydroxyl functionalities are essential for vitamin D3 to exert their biological activities; also, an intact 8-carbon side chain with a C-25-hydroxyl provides optimum binding to the intestinal receptor (37). Thus, 1,25(OH)2-23-oxo-D3 satisfies these struc- tural requirements. The second new metabolite 1,25,26(0H)3- 23-0xO-D~ may also satisfy the structural requirements even though the C-26 position is hydroxylated. This latter com- pound is present in the form of C-26,23-hemiketal (1) and the C-26-hydroxyl group does not exhibit hydroxyl functionality. One attractive possibility is that these new metabolites which are produced only in target tissues and which are not detect- able in the blood may act as local or paracrine hormonal agents.

Besides 1,24,25(OH):jD:~, 1,25(OH)2-23-oxo-D3, and 1,- 25,26(OH)3-23-oxo-D,3, another metabolite, namely 1,25- (OH)2D:3-26,23-lactone, was detected in the lipid extracts of the intestinal homogenate incubated with 1,25(0H)2[26,27-:1H] DB; however, the 1,25(OH)2D3-26,23-lactone was not present in significant amounts in comparison to other metabolites. Although it may be possible to postulate that 1,25(OH)2D.1- 26,234actone is an intermediate between 1,25,26(OH):j-23-oxo- D3 and calcitroic acid, no direct evidence supporting this possibility is available at the present time. Of interest is the fact that the 1,25(0H)2D:1-26,23-1actone has been determined to be a prominent metabolite of 1,25(OH)& in the plasma of rats given 1,25(OH)zD:$ previously; however, neither 1,25(OH)z-23-0xo-D3 nor 1,25,26(0H):1-23-oxo-D., have been detected in the plasma.

The detailed evaluation of this new C-23 oxidation pathway for the metabolism of 1,25(OH)zD3 and the biological activities of the newly isolated metabolites still remain to be elucidated. A further investigation of these problems is now in progress in our laboratory.

Acknowledgments-We gratefully acknowledge many helpful dis- cussions with Professor W. H. Oksmura and Dr. Eberhard Mayer.

REFERENCES 1. Ohnuma, N., Kruse, J., Popjak, G., and Norman, A. W. (1982) J.

2. Norman, A. W. (1979) Vitamin D The Calcium Homeostatic

3. Norman, A. W., Myrtle, J. F., Midgett, R. J., Nowicki, H. G.,

4. Holick, M. F., Schnoes, H. K., DeLuca, H. F., Suda, T., and

5. Holick, M. F., Schnoes, H. K., DeLuca, H. F., Gray, R. W., Boyle,

6. Henry, H. L., Taylor, A. N., and Norman, A. W. (1977) J. Nutr.

7. Henry, H., and Norman, A. W. (1978) Science 201, 835-837 8. Norman, A. W., Henry, H. L., and Malluche, H. (1980) Life Sci.

9. Tanaka, Y., Wichmann, J. K., Schnoes, H. K., and DeLuca, H. F.

10. Suda, T., DeLuca, H. F., Schnoes, H. K., and Blunt, J. W. (1969)

11. Brumbaugh, P. F., and Haussler, M. R. (1974) J. Biol. Chem. 249,

12. Wecksler, W. R., and Norman, A. W. (1980) J. Biol. Chem. 255,

13. Brumbaugh, P. F., and Haussler, M. R. (1975) J. Biol. Chem. 250,

14. Wecksler, W. R., Ross, F. P., and Norman, A. W. (1979) J. Biol.

Biol. Chem. 257, 5097-5102

Steroid Hormone, Academic Press, New York

Williams, V., and Popjak, G. (1971) Science 173, 51-54

Cousins, R. J. (1971) Biochemistry 10, 2799-2804

I. T., and Suda, T. (1972) Biochemistry 11,4251-4255

107, 1918-1926

27,229-237

(1981) Biochemistry 20,3875-3879

Biochemistry 8,3515

1258-1262

3571-3574

1588-1594

Chem. 254,9488-9491

8268 C-23 Pathway of Metabolism of 1,25-Dihydroxyvitarnin D,

15. Wecksler, W. R., Ross, F. P., Mason, R. S., and Norman, A. W.

16. Brickman, A. S., Coburn, J. W., Massry, S. G., and Norman, A.

17. IUPAC-IUB Commission on Biochemical Nomenclature (1966)

18. Kurnar, R., Harnden, D., and DeLuca, H. F. (1976) Biochemistry

19. Kumar, R., and DeLuca, H. F. (1976) Biochem. Biophys. Res. Commun. 69, 197-200

20. Esvelt, R. P., Schnoes, H. K., and DeLuca, H. F. (1979) Biochem- istry 18,3977-3983

21. Ohnuma, N., Bannai, K., Ishizuka, S., Kiyoki, M., Fukushima, H., Naruchi, T., Hashimoto, Y., and Noguchi, T. (1978) Pharma- cometrics 16, 1103-1121

22. Kiyoki, M., Bannai, K., Ohnuma, N., Fukushirna, H., Naruchi, T., Hashimoto, Y., and Noguchi, T. (1978) Pharmacometrics 16,

23. Ohnuma, N., Kiyoki, M., Bannai, K., Naruchi, T., Hashimoto, Y.,

24. Ohnuma, N., Bannai, K., Yarnaguchi, H., Hashimoto, Y., and

25. Ohnuma, N., and Norman A. W. (1982) Arch. Biochem. Biophys.

26. Norman, A. W., and Bishop, J. E. (1980) Methods Enzymol. 67,

27. Norman, A. W., and Wong, R. G. (1972) J. Nutr. 102, 1709-1718

(1980) J. Clin. Endocrinol. Metab. 50, 152-157

W. (1974) Ann. Intern. Med. SO, 161-168

J. Biol. Chem. 241,2987-2991

15,2420-2423

1145-1151

Noguchi, T., and Norman, A. W. (1980) Steroids 36, 27-39

Norman, A. W. (1980) Arch. Biochem. Biophys. 204,387-391

213, 139-147

424-426

28. Norman, A. W. (1972) J. Nut?. 102, 1243-1245 29. Bligh, E. G., and Dyer, W. Y. (1959) Can. J. Biochem. Physwl.

30. Friedlander, E. J., and Norman, A. W. (1975) Arch. Biochem.

31. Esvelt, R. P., and DeLuca, H. F. (1981) Arch. Biochem. Biophys.

32. Henry, H. L. (1979) J. Biol. Chem. 254,2722-2729 33. Tanaka, Y., Castillo, L., and DeLuca, H. F. (1977) J. Biol. Chem.

34. Castillo, L., Tanaka, Y., DeLuca, H. F., and Ikekawa, N. (1978)

35. Kumar, R., and DeLuca, H. F. (1977) Biochem. Biophys. Res.

36. Procsal, D. A., Okamura, W. H., and Norman, A. W. (1975) J.

37. Wecksler, W. R., Okamura, W. H., and Norman, A. W. (1978) J.

38. Kumar, R., Schnoes, H. K., and DeLuca, H. F. (1978) J. Biol.

39. Wecksler, W. R., and Norman, A. W. (1980) Methods Enzymol.

40. Ikekawa, N., Eguchi, T., Hirano, Y., Tanaka, Y., and DeLuca, H.

41. Ishizuka, S., Ishimoto, S., and Norman, A. W. (1982) Arch. Bio-

37,911-917

Biophys. 170, 731-738

206.403-413

252, 1421-1424

Min. Elec. Metab. 1, 198-207

Commun. 76,253-258

Biol. Chem. 250,8382-8388

Steroid Bwchem. 9,929-937

Chem. 253,3804-3809

67,494-500

F. (1981) Folia. Endocrinol. Jpn. 57,675-681

chem. Biophys., in press

C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin D3

One international unit (IU) of vitamin D3 (cholecalciferol) is defined by the World Health Organization as being 25 ng which is equivalent to 65 pmol. There is no formal international unit definition for 1,25(OH)zD3; however 1.0 unit (U) has been defmed (28) as being 65 pmol.

C-23 Pathway of Metabolism of 1,25-Dihydroxyvitamin 0 3

( L C l . 41

1,25[UH) 0 . p o l = 15.6 pmol - 1.13 x f1,25-Pnme.pmol + Peak-X.po l1 2 3 - 1.48 x [1,24.25(DH)303.pnoll. r = 0.92

P P I

0 15 30 45 60 85 110 t-5

ELUTION VOLUME (ml) . ."

m. Sephadex LH-ZD co lumn ch rmatog raph ic p ro f l i e of l,25[DH)~-[26.27-3H]-D3 meta-

g iven 500 ng (20 U) of 1.25(OH)2D3 in t ravenour ly , 6 h p r e r i a u r l y . The haopenate "dl

b o l l t e r p r d u c e d b y hamagenates O f m a l l i n t e s t i n a l mucosa frm vltdmin 0-def ic ient Chicks

incubated wi th 1.25(DH)2-[26.27-3H]-D3 a t a concent ra t ion O f 2.25 nM f o r 30 .in. Peak I IS a n a r t i f a c t o f 1,25(DH)2-[26.27-3H]-0~ [see reference 25). The subs t ra te L.25(OH)2- [26.77-3H]-D3 e l u t e s i n t h e Peak I? region. Peaks Ill and IV are po lar and more po lar

metabol i tes o f 1,25[OH)~Og. The c o l m n vas e l u t e d w t h d solvent o f ch1orofom:hexane:

methdnol, 75:23:2. ("/VI.

Next t h e c o i p a s i t l a n o f t h e P e a k - I l l frm t h e Sephadex LH-2D (see i l g . 11) de rwed

frm the ch i ck m tes t i na l hmogena te MI analyzed by HPLC. As s h o w ~n Flg. 12. Peak I 1 1

was resolved i n t o two major c l a r r e r o f r a d i o a c t l v e peaks on t h e HPLC y-Poras l l colmn * i c h

was eluted w i t h a s o l v e n t I y s t m of l ~ ~ p r ~ p d n ~ l : d , ~ h l o r m e t h a n e . 10:lOo. I v / v ) . They were i d e n t i f i e d t o be 1.24.25(0H)303 and t h e newly iso la ted metabolite 1,25,26(DH))-23-01o-D)

[Peak XI.

ELUTION VOLUME ( m 0

C-23 Pathway of Metabolism of 1,25-Dihydroxyuitamin D3

FRACTION NUMBER ilr. 1-40; l.5mlllr., Ir 41-50: 2.5nlflr.l

*, induced by pr iming chicks w i t h 1,25(OH)203 e. The i n d u c t i o n Of t h e 1.25- m. Vltarnln 0 s ta tus and t i s s u e s p e c i f i c i t y on the 1,25[0X)~03 degmdat icm 2

(OH1203 metabolIrm was examined i n t h e I n t e s t i n e , k i d n e y and l i v e r O f t h e c h i c k s w i t h

d i f f e r l n g v i t a m i n 0 s ta tus . The animal preparat ion i s descr ibed under Mater ia ls and

Methods. The m a l l i n t e s t i n e . k i d n e y and l i v e r *ere m o v e d fm t h e sane animals and 101 homogenater *ere prepared i n 0.25 M I U C ~ O S ~ . Af te r incubat ion an a l i q u o t o f t h e c h l o r o f o m e x t r a c t was chrml tographed on i Sephddex LH-20 column e l u t e d w i t h a so lvent of Chloroform:

hexane:methanol, 75:23:2, ( V l v ) . The 1,25(OH)203 Substrate COnCentrPtion i s 2.25 nH or 23.9 nu. Peak I 1 1 an a r t i f a c t produced from the 1,25(0H)~-[~H]-O3 (see re fe rence 25).

Peak I 1 1 1 t h e s u b s t r a t e 1,25(OH)2-[3H]-03. Peaks 111 and I V are p o l i l ~ m e t a b a l l t e s o f

1,25(0H)2-[3H]-o3. The s o l i d l i n e r e p r e s e n t s t h e m e t a b o l i c p r o f i l e o b t a i n e d f m t h e

animal glven vehic le a lone 6 h before sacrifice, w h i l e t h e dashed l i n e r e p r e s e n t s t h e

p ro f i l e ob tz lned frm the animal glven 500 ng of 1.25(0H)~D3 6 h be fo re sac r i f i ce ( t op

panels -0 b l rdS ; m idd le pane ls v i t am in 03 - rep le te b i rds ; bo t tm pane ls 1,25(0H)203

r e p l e t e b i r d s ) .

As i nd i ca ted i n F ig . 14, essen t ia l l y i den t l ca l me tabo l i c p ro f i l es were ob ta ined f p r

The HPLC ana lys is was car r ied ou t under the exac t ly iden t lca l Condition for these sampler.

vl tarni i l 0-deficient In te r t l ne , v i t am in 0 -de f l c len t k ldney and 1,25(OH)203-replete kidney.

The major metabo l i tes are Peak-X [l ,25.26(011)3-23-oxa-03, 1.24,25(011)303 and 1,ZL:Pnme

8271

Figure. HPLC p r o f i l e s O f 1,25(0H)203 metabo l i tes fomed frm the hanopenate O f v i t a -

m i n O d e f i c i e n t C h i c k m a l l i n t e s t i n a l 1111cosa (panel A aod 0). w t h e k i d n e y h m q e n a t e

frm vitamin 0-replete (panel B and E) o r 1,25(OH)~D3-replete chicks (panel C and F). "t vehic le" represents b i rds that received vehic le a lone. "t 1,25(OH)~O3" represents

b i rds g i ven 500 ng O f 1,25(OH)~D3 i n t r a v e n o u r l y 6 h be fore sacr i f i ce . Incubat ion t ime

(5 or 30 m i " ) i s i n d i c a t e d i n t h e panel. The l i p i d e x t r a c t s of t h e incubation m i x t u r n

[I ml) were d i r e c t l y a p p l i e d on a HPLC u-Poras l l colmn developed With a so lvent o f i so-

f i l e s o f t h e 1,24,25(OH)303 f rac t ions shown on panels A , B and C, respec t ive ly . The propyl alcohol:hexane, 11:89 (panels A , B and C ) . Panels 0, E and F l e p m s e n t HPLC p m -

~ 0 1 m n s ( u - P o r a r i l ) were e l u t e d w t h i s o p r o p y l l 1 C ~ h o l : d i c h l a r D m e t h l n e 10:100, ("1") . The l,Z5(OH)2D3 subs t ra te concent ra t ions are: 6.5 (51,000 dpn lml ) (ma11 In tes t ine)

~r 2.25 nn (48,600 d p l m l ) ( k i d n e y homogenate).

~1,Z5(OH)2-Z3-oxo-03] which g ives 1 shoulder on t h e e l u t e d peak of 1,25(OH)203 on the

prof l le . Trace amounts of 1,25(0Hl~03-Z6,Z3-lat~"~ were also resolved from 1.24.25.

(0H)qOg on t h e u - P o r a n l column developed w i t h isopropyl alcoho1:dichloramethane (10:100,

" I " ) .