Metabolism of 25-hydroxyvitamin D3 in kidney homogenates of chicks supplemented with vitamin D3

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

November 29,1978

Pages

601-607

METABOLISM OF 25-HYDROXYVITAMIN D3 IN KIDNEY HOMOGENATES OF CHICKS SUPPLEMENTED WITH VITAMIN D3 YOSHIKO TAKASAKI,

NOBORU HORIUCHI,

and TATSUO SUDA*

Department of Biochemistry, Showa University l-5-8, Hatanodai, Shinagawa-ku, Tokyo 142, Received

October

Dental Japan

School,

4,1978

Kidney homogenates from chicks fed a vitamin D-deficient diet Summary: for 10 days and supplemented with 6.5 nmol of vitamin D3 48 hr prior to sacrifice metabolized [3H]-25-hydroxyvitamin D3 (25-OH-D31 to 24,25-dihy--in vitro droxyvitamin D3 [24,25-(OH)2-D3] and 3 other metabolites (peaks A, C and E). When the homogenates were incubated with purified L3H]-24,25-(OH)2-D3, 3 simiOn high pressure liquid lar metabolites (peaks A', C' and E') were produced. chromatography, peaks A, C and E migrated to exactly the same respective pOSitions as peaks A', C' and E'. Kidney homogenates from D-deficient chicks failed to produce these metabolites from [3H]-2%OH-D3 or L3H]-24,25-(OH)2-D3. These results strongly suggest that the new metabolites reported here are synthesized via 24,25-(OH)2-D3 in the kidney of chicks supplemented with vitamin D3. INTRODUCTION It

is now well

hydroxyvitamin

established

D3 (25-OH-D3)

la,25-dihydroxyvitamin 25-(OH)2-D3] this

(1).

has not been the ly-

selective More

phosphate,

or during

kidney

--in vitro

of these

vitamin

situation

(3H]-25-OH-D3

thought

in

to 25-

the kidney

in

the

levels

form,

of plasma

has been were

since

calcium

of 24,25-(OH)2-D3, circumstances

to

D3 (24,

to be an active

when animals

D depletion,

however,

associated

investigated

with extensive-

in need of calcium

and more 24,25-(OH)2-D3

or

was produced

(2-6).

of investigating

homogenates

* To whom all Abbreviations dihydroxyvitamin

was produced

metabolized

or 24,25-dihydroxyvitamin

significance

metabolites

first

and subsequently

change

The physiological

la,25-(OH)2-D3

In the course that

The biological

formation

in the opposite

has been

a remarkable

established.

D3 is

in the liver,

la,25-(OHj2-D3 induces

and phosphate

vitamin

D3 [lu,25~-(OH)~-D~]

(1).

metabolite

that

from chicks to 3 other

renal

24-hydroxylase

supplemented metabolites

with besides

activity, vitamin

D3

we found metabolize

24,25-(OHj2-D3.

The

correspondence should be addressed. used: 25-OH-D3, 25-hydroxyvitamin D3; la,25-(OH)2-D3, 24,25-dihydroxyvitamin D3. D3; 24,25-(OHj2-D3,

la,25-

0006-291X/78/0852-0601$01.00/0 601

Copyright All rights

0 1978 by Academic Press. Inc. of reproduction in any form reserved

Vol. 85, No. 2, 1978

present

report

OH-D3 via

BIOCHEMICAL

describes

that

24,25-(OH)2-D3

in

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

these

new metabolites

the kidney

of chicks

MATERIALS Animals: One-day-old White days on a vitamin D-deficient calcium and 0.45% phosphorus. 6.5 nmol of vitamin D3 48 hr minced with removed, rinsed, 0.2 M sucrose containing 15 5 mM sodium succinate.

are

synthesized

supplemented

from 25-

with

vitamin

D3.

AND METHODS

Leghorn cockerel chicks were maintained for 10 purified soy protein diet (7) containing 3% Most of the animals were orally predosed with prior to sacrifice. The kidneys were quickly a garlic press, and homogenized in 4 volumes of mM Tris-Cl (pH 7.4), 2 mM MgC12, 0.4 mM NADP, and

Incubation: The homogenates (1.5 ml) were incubated in 20 ml-Erlenmeyer flasks with 10 pmol (0.1 uCi) of either [26,27-3H]-25-OH-D3 (Radiochemical Centre, Amersham) or [26,27-3H]-24,25-(OH)2-D3. The iucubation was carried out at 37OC for 5-60 min under 100% oxygen. The reactions were terminated by the addition of 9 ml of methanol-chloroform (2:l v/v). Extraction was performed as reported by Gray et al. (8). Chromatography: Chromatography of the extracts was carried out on a 1 x 30 cm column of Sephadex LH-20 (10 g) using a solvent of 65% chloroform - 35% hexane High pressure liquid chromatography(HPLC) according to Holick and DeLuca (9). was performed on a Waters HPLC system (Model 440) equipped with a Zorbax-Sil column (2.1 mm x 25 cm, Du Pant) . The solvent system was either 0.67% or 2% Thirtymethanol in dichloromethane according to Ikekawa and Koizumi (13). Determifour 0.25 ml fractions were collected at a flow rate of 0.5 ml/min. nation of radioactivity was carried out with a Packard liquid scintillation counter (Model 3255) as previously reported (10). Preparation of [26,27-3~1-24,25-(OH)~-~~i Kidney homogenates of chicks predosed intravenously with 312.5 pmol of la-hydroxyvitamin D3 24 hr prior to sacrifice were incubated with r3H]-25-OH-D3 at 37OC for 20 min. [3~]-24,25(OH)2-D3 was purified on the Sephadex LH-20 and HPLC column described above. Crystalline vitamin D3 was purchased from Tokyo Chemical Industry, and 25OH-D3 from Phillips-Dupher Co., Amsterdam. Crystalline la,25-(OH)2-D3 and 24R,25-(OH)2-D3 were gifts from Dr. M. R. Uskokovie, Hoffman-La Roche Inc., New Jersey. la-hydroxyvitamin D3 was a gift from Dr. I. Matsunaga, Chugai Pharmaceutical Co., Tokyo. RESULTS AND DISCUSSION Administration

of 6.5

ly suppressed

renal

tivity

I).

In the course

that

the

ity,

(Table we found

from Sephadex

LH-20

The heterogeneity

25-OH-D3-la-hydroxylase

columns of the

by Garabedian

OH-D3 and the

24,25-(OHj2-D3

were

individually

and the

were

both

heterogeneous fraction

applied

fractions

(11)

fractions

on Sephadex

to HPIC column,

602

the

LH-20 former

by HPLC.

LH-20

(12).

activ-

isolated

when purified

Sephadex

ac-

24-hydroxylase

and Gray et al. from

complete-

24-hydroxylase

renal

24,25-(OH)2-D3

24,25-(OHj2-D3 et al. --

chicks

and enhanced

of investigating

25-OH-D3

been reported

(a))

D3 to D-deficient

nmol of vitamin

column

When the

columns

(Fig

was separated

has

251. into

~~-

Failure

100% N2*** 24,25-(OHj2-D3

Boiled** 24,25-(OH),-0,

24,25-(OHj2-D3

Control 25-OH-D3

___...

24,25-(OH),-D3

Control 25-OH-D3

Substrate

Incubation*

I.

of

-.---.

0 0

0 0

0

0

0

0

15.4 13.9

0

22.2

15.9

0

39.6

0 ~- ___.-_._.

Trace -15.8

0

Trace

24.8

0

%

0

0

0

0

47.6

45.2

25.8

23.3

0

0

0

0

Peak

new metabolites

25-OH-D3

the

17.6

%

A

of

0

Peak

synthesis

%

C

91.7

92.0

98.5

97.7

27.3

25.9

23.1

9.5

89.6

87.2

0

0

24,25-(OHj2-D3

by kidney

%

were

homogenates

The 25-OH-D3 and the 24,25-(OH)2-D3 fractions from Sephadex LH-20 columns Figures in Table I are expressed as percentage of the sum of the radioactivity on HPLC. * All incubations were performed for 60 min. l * boiled homogenates for 5 min at 100°C prior to incubation. *** incubated for 60 min under 100% N2.

6.5

0

nmol

Predose of D3

Table

%

E

separately recovered

0

0

0

0

8.8

8.4

8.0

5.1

Trace

Trace

0

0

Peak

from

in

%

to HPLC columns. l-34 fractions

8.3 applied each

8.0 0

1.5

0

2.3

2.4

5.1

5.0

6.7

3.2

5.4

12.0

8.8

Others

0

%

chicks.

0

0

0

0

0

0

0

63.2

73.6

la,25-(OH)2-D3

D-deficient

Vol. 85, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL

4 (b) t

25-OH-D3 Fraction

.-;;E 3

RESEARCH COMMUNICATIONS

s .-z3

24,25-(OH)>-D,

4 2

e

24.25~(OH),-03 Fract!on

,

‘: 0

2-

; e

Fraction

. 25-OH-D, Fractmn

l-

Number

( 3rd)

Fig 1. Sephadex LH-20 chromatographic profiles of extracts of kidney homogenates from chicks orally dosed with 6.5 nmol of vitamin D3. The reaction conditions are described under MATERIALS AND METHODS. The columns containing 10 g of Sephadex LH-20 were eluted with chloroform : n-hexane (65:35, v/v) and 3 ml fractions were collected. Panel (a) represents a chromatographic profile of the extracts when the homogenates were incubated for 60 min with 10 pmol (0.1 PCi) of [3H]-25-OH-D3 and panel (b) with 10 pmol (0.1 UCi) of 13H]-24,25(OH) 2-D3.

2 peaks: with

authentic

3 peaks: tic

an unknown

peak referred

25-OH-D3

2 unidentified

24R,25-(OH)2-D3

that

from

metabolites

of vitamin in this

radioactive 1.

might

was referred

3 peaks:

failed

were

2 unidentified

reported

that

2.

was

by Gray et al.

604

produced

new metabolites

(12).

[3H]-24,25-(OH)2-D3, on Sephadex

LH-20

not

fraction and peak

which

produce

25-OH-D3

to exactly

the

was also D' which

a columns

peak was homogeneous, did

similar

Thus,

Peak A' migrated

C' and E',

However,

(12).

fraction

The homogenates

resemble

administration

purified

radioactive

authen-

whereas

by Gray et al.

25-OH-D3

with

into

here,

from

with

separated

(12).

of lc~,25-(OH]~-D~

those

coincided

coincided

them

The 24,25-(OHj2-D3 peaks

latter

to produce

the

cc)).

B which

new metabolites

reported

incubated

in the

to as peak A'.

as peak A on HPLC.

Some of these

to those

be different

(Fig

On HPLC the

administration

HPLC showed

[3H]-24,25-(OH)2-D3 tion

prior

peak appeared (b)).

(b)).

recently

D3 or 25-OH-D3 study

(a)).

A, and peak

C and E, and peak D which

r3H]-25-OH-D,

When the homogenates

(Fig

2.

those

demonstrated

2.

peaks (Fig

chromatographically they

(Fig

to as peak

from

same posi-

separated

coincided

into with

BIOCHEMICAL

Vol. 85, No. 2, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

i.

10

5 Time

( mln

15

10

5

Time

1

15

(min)

Fig 2. HPLC profiles of the 25-OH-D3 and the 24,25-(OH)2-D3 fractions colHPLC Panels (a) and (c) represent lected from the Sephadex LH-20 columns. (tubes 6-13) shown on Fig 1. (al and (b), profiles of the 25-OH-D3 fraction respectively. Panels (b) and (d) indicate those of the 24,25-(OH)2-D3 fraction (tubes 16-35) on Fig 1. (a) and (b), respectively. Before applying to 500 pmol of authentic 25-OH-D3 (in panels (a) and (c)) or 24R, HPLC columns, The columns 25-(OH)2-D3 (in panels (b) and (d)) were added to each extract. were eluted with a solvent of either 0.67% methanol (in panels (a) and (c)) or 2% methanol (in panels (b) and (d)) in dichloromethane.

authentic

24R,25-(OH12-D3

exactly were

(Fig

the same respective preheated

at 100°C

and E' were

produced,

matically.

Production

under

100% nitrogen Figure

D3 and peaks D3 for in 5-10

5-60

2.

positions for

5 min,

indicating

(Table

3 indicates

as peaks neither

that

of these

On HPLC peaks

(a)).

metabolites

time

Amounts

min and decreased

C and E gradually

increased

A, C and E nor

metabolites

are

was also

suppressed

peaks

A',

synthesized

C' enzy-

completely

I). course

of change

A, C and E when the homogenates min.

to

When the homogenates

C and E.

peaks

these

C' and E' migrated

of 24,25-(OHj2-D3 thereafter. during

in

the amounts-of

were produced

On the other the incubation

605

incubated

with

attained hand,

period

24,25-(OHJ2F3H]-25-OH-

a maximal

amounts of 60 min.

level

of peaks

A,

When the

Vol. 85, No. 2, 1978

BIOCHEMICAL

0

AND BIOPHYSICAL

30

15 Incubation

Time

RESEARCH COMMUNICATIONS

45 ( min

60

)

proFig 3. Time course of change in the amounts of the 25-OH-D3 metabolites duced by kidney homogenates from chicks dosed with 6.5 nmol of vitamin D3. Incubations were carried out with 10 pmol (0.1 uCi) of [3H]-25-OH-D3 as described under MATERIALS AND METHODS. Results are expressed as percentage of the radioactivity recovered as peak A (--A- ), peak C (-0 ), and peak E (--&-). The vertical bar 24,25-(OH),-D3 ( -), indicates standard error of the mean of 3-4 experiments.

homogenates major

were

incubated

metabolite

relatively

was peak

minor

failed

ones

to produce The results

(peaks

behaviors fied

with

reported

here

D-deficient the time

course

A, C and E fits Biological in view under

of the investigation

Second,

25-OH-D3

from

peaks

or 24,25-(OHj2-D3

of change

in the

these amounts

in the kidney

metabolites

(Table

I).

Third, kidney

of chicks

from puri-

A, C and E migrate

Fourth,

chicks

chromatographic

are produced

C' and E'.

produced

were

of 25-OH-D3

C' and E' with

on HPLC peaks A',

and E (or E')

the metabolites

A',

the

from D-deficient

24,25-(OH)2-D3

24,25-(OHj2-D3.

to produce

to exactly

the homogenates homogenates

from 25-OH-D3. of 24,25-(OH),-D3

from

Finally, and peaks

the above hypothesis.

significance control

25-OH-D3 that

as peaks

fail

with

A (or A')

A, C and E, respectively,

positions

chicks

First,

D3.

to peaks

the same respective

indicate via

[3H]-24,25-(OH)2-D3,

homogenates

from

synthesized

or

and peaks

Kidney

metabolites

[3H]-24,25-(OH)2-D3.

do not produce

I).

these

vitamin

similar

13H]-25-OH-D3

C (or C'),

(Table

A, C and E) are

supplemented

with

of

mechanism in our

these

metabolites

of 25-OH-D3

laboratory.

606

is metabolism,

of considerable which

is

interest currently

Vol. 85, No. 2, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1.

DeLuca,

H. F.,

2.

Boyle, I. T., Gray, USA 68, 2131-2134.

3.

Tanaka, 574.

4.

Horiuchi, N., Suda, (1974) FEBS Letters

5.

Tanaka,

Y.,

6.

Hughes, Baylink,

M. R., Brumbaugh, P. F., Haussler, D. J. (1975) Science 190, 578-580.

-7.

Gmdahl, J., Biochemistry

8.

Gray, R. W., Gmdahl, J. L., Ghazarian, J. Biol. Chem. 247, 7528-7532.

9.

Holick,

Y.,

and Schnoes, R. W.,

and DeLuca, T., 43,

and DeLuca,

and DeLuca,

H. F.

H. F.

and DeLuca,

S.,

Ezawa,

(1974)

Suda,

H. F.

11.

Garabedian, M., Bailly Du Bois, Balsan, S. (1978) Endocrinology

12.

Gray, R. W., Caldas, A. E., Weber, Biochem. Biophys, Res. Commun. 82,

13.

Ikekawa,

Proc.

M. R., Y.,

Y.,

607

J. Chrmatogr.

154,

Wergedal,

Sci.

566-

J. E.,

and DeLuca,

Res.

12,

Pezant,

J. L., and Ghazarian, 121-128.

N. (1976)

Acad.

E.

1198-1200.

Ogata, E., Ezawa, I., Biophys. 171, 540-548.

M., Corvol, M. T., 102, 1262-1268.

Nat.

631-666.

and Ogata,

and DeLuca,

J. Lipid

e,

Biophys.

Sano. 183,

J. G.,

N., Suda, T., Sasaki, S., E. (1975) Arch. Biochem.

(1971)

I.,

Tanaka,

(1971)

Rev. Biochem.

Biochem.

Science

T.,

Horiuchi, Shimazawa,

and Koizumi,

H. F. Arch.

10.

N.,

Ann.

(1973)

Sasaki, 353-356.

Holick, M. F., lo, 2935-2940.

M. F.,

H. K. (1976)

-119,

H. F.

H. F.

and (1971)

(1972)

460-465. SanO, Y., E.,

and

J. G. (1978) 227-232.

and