Quantitative changes of bone collagen crosslinks and precursors in vitamin D deficiency

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

QUANTITATIVE CHANGES OF BONE COLLAGEN CROSSLINKS AND PRECURSORS IN VITAMIN D DEFICIENCY Gerald

L. Mechanic,

Dental

Received

Svein

U. Toverud

and Warren

K. Ramp

Research

Center and the Departments of Biochemistry and of Pharmacology University of North Carolina Chapel Hill, North Carolina 27514

April

12, 1972

SUMMARY The crosslinks from NaB3H4-reduced bone collagen of 4-week-old rachitic and normal chicks were compared. The ratio of the reduced crosslink 6,6'-dihydroxylysinonorleucine (DHLNL) to 6-hydroxylysinonorleucine (HLNL) was significantly increased in vitamin D deficiency. The ratio of s-hydroxynorleucine (HNL) (reduced crosslink precursor of HLNL) to the reduced unknown peak 1 was decreased while that of HLNL to HNL was unaffected. The data indicate that vitamin D affects the quantitative relationships between crosslinks and aldehydic precursors that are present in mineralizing macromolecular matrices. INTRODUCTION The time-dependent their

respective

crosslinks

macromolecular tein

soon

formation

matrix

after

aggregation

its

into

and relative

of collagen synthesis

abundance having

maturation

(6,7,8).

The major

has been

different

reducible

bases

labelling

base

crosslinking

compounds

architectures

crosslinks

in bovine

studies

indicate

that

in calf

is bone

In bovine

quantitatively collagen foetal

bone,

in dentine

of both)

which

is

AcademicPress,lnc.

in types

and in various

states

tissues (7).

reduction

are

of

6,6'-

Furthermore, of the Schiff

(24% of the

former)

and

(7).

comparatively 760

Copyright01972,by

when

and A6y7 dehydrohydroxylysino-

important

(25%-50%

ensues

demonstrated

mineralized

--in vivo

in the pro-

have been

and 6-hydroxylysinonorleucine

Do,7 dehydrodihydroxylysinonorleucine

norleucine

formed

Differences

(2,5).

and

the fibrillar are

occurs

collagen

precursors

Aldehydes

Schiff

matrix

crosslink

to stabilize

of NaB3H4-reducible

dihydroxylysinonorleucine deuterium

shown

(1,2).

(3,4);

a polymeric

in tissues

of aldehydic

less

mineralized

than

Vol. 47, No. 4, 1972

mature

bone,

activity

in

leucine

(7).

precursors

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6,6'-dihydroxylysinonorleucine the elution

profile

while

Simultaneously, are

possesses

and a lower

ratio

of

57% of the radio-

11% is due to &-hydroxylysinonor-

markedly

in evidence.

more mineral,

represents

smaller

However,

amounts

more mature

much larger

quantities

of reduced

bone,

of the

6,6'-dihydroxylysinonorleucine

which

aldehydic

contains

crosslink

precursors

to b-hydroxylysinonor-

leucine. This links

report

in

concerns

the macromolecular

that

the same relationships

also

exist

between

MATERIALS

rachitogenic

diet 2.2

diet

$1 170650).

D on the reducible

collagen.

are present

vitamin with

The data

between

and normal

foetal

chick

crossindicate

and mature

bone

in a windowless

bone

collagen.

room and fed a

1.4% Ca and 1.1% P (General of the chicks

(+D group)

vitamin

D3, dissolved

in corn

g of diet.

corn

The remaining

oil

added.

were weighed

twice

a week.

groups

were

at random

the purpose

housed

One half

D per only

were

containing

was added

The chicks selected

of bone

D deficient

male chicks

to which

the diet

that

vitamin

chick

Diet

I.U.

matrix

of vitamin

AND METHODS

One-day-old

Inc.,

the effect

from both

received

oil,

chicks

At the sacrificed

Biochemicals, this

to provide (-D group)

received

end of 4 weeks,

8 chicks

by decapitation

for

of removing: (1)

a blood

sample

for

serum

Ca determination

by atomic

absorption

spectrometry. (2)

one tibia

for

(3)

the other

tibia

tion The tibias The diaphysis

described

and staining

from

for

and ash

in neutral

(6OOOC) determinations formalin,

of sections

with

hematoxylin

chicks

from both

groups

of marrow

and analytical

(105'C)

fixation

3 additional

was cleaned

NaB3H4 reduction,

dry weight

and subjected

chromatography

(1,6,7). 761

decalcificaand eosin.

were

removed.

to demineralization, of the bone

collagen

as

Vol.47,No.4,1972

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE 1 EFFECT ON CHICKS AFTER 4 WEEKS

TIBIA Body Weight 9

Serum Calcium mg/lOO ml

Dry Weight 9

% Ash of Dry Weight

- Vitamin

D

MEAN

237

5.5

0.572

37.1

+ Vitamin

D

MEAN

328

10.1

1.029

43.2

SE

8.6

P


0.12

0.03

0.63




RESULTS AND DISCUSSION Table features severe

1 shows

that

of vitamin

sections

epiphyseal

deficient

decreased

bone weight

chromatographic Note,

of the

in rachitic

increase

to remain peak.

constant

showed

bone

the 3 bones

the smaller relative

in both

groups

obtained

because

amounts to unknown

in Figure

peak

bone. of other

also

1.

The quantitative were

1, while

concordant. and 6-

there

Unknown

aldehydic

data

substances

762

(10).

D

&hydroxynorleucine

crosslink

seems to be slightly

Quantitative unknown

trabeculae

and vitamin

each group of

of the

shortened

normal

gain,

The histo-

is an peak

and was used as a quantitative

the reduced

6,6'-dihydroxylysinonorleucine, D deficient

from

ash.

widening

6,6'-dihydroxylysinonorleucine.

Dihydroxynorleucine,

vitamin

NaB3H4-reduced

the usual body weight

and percentage

and markedly

are presented

bone,

in

wide

collagen of

diminished

characteristic

of

patterns

demonstrated

(9):

profiles

hydroxylysinonorleucine apparent

tibias

and irregular,

elution chick

of chicks

rickets

cartilage

Typical

-D group

D deficiency

hypocalcemia,

logical

the

1 seems

reference precursor

more

on this

compound

present

in this

of

abundant could

not

section

of

in be

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

8 75 0 : “, %I ; P e 25. 00

50

200

150

300

LEGEND TO FIGURE 1 Typical radioactive elution profiles of NaB3Hq-reduced normal (upper) and vitamin D deficient (lower) chick bone collagens. Dihydroxynorleucine elutes at fractions 70-73 (upper) and 68-71 (lower). Unknown peak 1 elutes at fractions 88-91 (upper) and 80-83 (lower). Equal amounts of radioactivity are present in both unknown peak 1's. s-Hydroxynorleucine (HNL) elutes at fractions 108-112 (upper) and 99-104 (lower). Dihydroxylysinonorleucine (DIUNL) elutes at fractions 320-323 and hydroxylysinonorleucine (HLNL) elutes at fractions 331-333 for both profiles.

the

chromatogram.

the radioactivity areas

under

under the 4 peaks.

The pattern to that resembles

for

The data

foetal

exhibited bovine

an intermediate

were

expressed

the curves These

are

and calculating presented

by the vitamin bone while stage

quantitatively

that

in development

763

the ratios in Table

D deficient for

by integrating between

the

2. collagen

the normal as compared

chick

is

similar

bone

collagen

to mature

bovine

Vol. 47, No. 4, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 2 Ratios of NaB3H4-Reduced Intermolecular Crosslinks and Aldehydic Precursors in Vitamin D Deficient and Normal Chick Bone Collagen

RATIOS

-D

+D

-D

1.40

.696

1.71

3.81

1.52

.728

1.88

4.08

1.51

.771

2.48

3.15

1.48

.731

2.02

3.68

SE

0.031

P

Unk.

bone

(

-D Hi: DHNL HLNL Peak 1 -

(7).

the of that

insufficient

a normal Electron

)

0.02

the similar

of the

ratios

s-hydroxynorleucine

base,

groups

that

in vitamin

involving

is being

to unknown

both

peak

bone

to satisfy

cohesive

mineralizing indicate

the

crosslinking

collagen

matrix.

that

rachitic

764

rat

D

formed.

However,

1 and the high

ratio

suggests

of C!-amino-adipic-6-semialdehyde

in rachitic

of 6-

cr-amino-adipic-6-

to 6-hydroxylysinonorleucine

quantities

micrographs

for

indicate

Schiff

0.4

bone

HNL) and hydroxylysine,

6,6'-dihydroxylysinonorleucine

synthesized for

proportion

(unreduced of

study,

0.132

to e-hydroxynorleucine

a normal

low ratio

(

Vitamin D deficient rickets Normal bone c-hydroxynorleucine s,b'-dihydroxylysinonorleucine 6'-hydroxylysinonorleucine See Figure 1

In the present

semialdehyde

0.255

0.001

hydroxylysinonorleucine deficiency

HLNL HNL

+D

Treatment

Mean

DHLNL HLNL

HNL Peak 1

Unk.

are being requirements

collagen

needed

has a normal

Vol. 47, No. 4, 1972

appearance graphic

BIOCHEMICAL

and does not localization

concerned normal

with

of [la-3H]

matrix

of Norman

as an affector may stimulate

aldehydic

crosslink strongly

stabilize

in collagen

processes On the basis

of lysyl precursor suggest

that

(11)

in order

an important

between

mineralizing role

vitamin

data that

we propose

in the collagen

a relationship

Autoradio-

to the elaboration

vitamin collagen

in the formation

of a

vitamin

D acts

vitamin

to facilitate molecule

D is

and the

that

a-amino-adipic-&i-semialdehyde.

the macromolecular may play

suggests

and Taylor

oxidase

residues

(13).

of the present

or enzyme synthesis, of lysyl

collagen

leading

and Wasserman

the synthesis conversion

that

(14)

bone

cholecalciferol

(12).

of protein

increased

the data

from normal

the biochemical

collagen

hypotheses

differ

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

D the

to the In any event,

D and the crosslinks matrix.

Crosslinking

of a normal

tissue

of Sam Weddington,

Unni

architecture. ACKNOWLEDGEMENTS We are Kristoffersen, was supported

grateful

for

the

Judy Knight, by USPHS Grant

technical Carol

assistance

Ellison

and Catherine

Lane.

This

research

DE-02668.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14.

Tanzer, M. L. J. Biol. Chem. 243, 4045 (1968). Tanzer, M. L. and Mechanic, G. Biochem. Biophys. g. Commun. 32, 885 (1968). Pinnell, S. R. and Martin, G. R. -----_ Proc. Nat. Acad. Sci. U. S. 6l, 708 (1968). Siegal, R. C. and Martin, G. R. J. E. Chem. 245, 1953 (1970). Kang, A. H., Faris, B. and Franzblau, C. Them. Biophys. --Res -*Commun 3!3, 175 (1970). Mechanic, G. and Tanzer, M. L. Biochem. Biophys. Res --Commun. ffl, 1597 (1970). Mechanic, G., Gallop, P. M. and Tanzer, M. L. Biochem. Biophys. -*Res -*Commun 45, 644 (1971). Mechanic, G. --Israel J. -Med. -Sci. 7, 458 (1971). Migicovsky, B. B. and Belanger, L. F. Radioisotopes and Bone (F. C. McLean, P. Lacroix and A. M. Budy, eds.), Blackwell Sci. Publ., Oxford and Edinburgh, 1962. Yuile, C. L., Chen, P. S. and Bossman, A. B. Arch. Pathol. 83, 241 (1967). -Wasserman, R. H. and Taylor, A. N. Science 152, 791 (1966). Kodiecek, E. and Thompson, G. A. In Structure and Function of Connective and Skeletal Tissue, Butterworth, London, pg. 369, 1965. Robinson, R. A. and Sheldon, H. In Calcification in Biological Systems (R. Sognnes, ed.), AAAS, Washington, D. C., pg. 261, 1960. Norman, A. W. Biolopical Reviews 43, 97 (1968). 765