jejunal and ileal calcium-binding protein and on plasma calcium

Effect of vitamin

D

or

calcium

deficiency

on

_duodenal,

jejunal

and

ileal

_{calcium-binding protein}

and

on

_plasma

calcium

and

_{25-hydroxycholecalciferol}

levels

in

the

_{growing pig}

Monique

THOMASSET,

A. POINTILLART Paulette

CUISINIER-GLEIZES,

L. GUÉGUEN

N. GOUHIER A. SUBRENAT C. COLIN A. PORTEU

INSERM U. 120 (Pr H. Mathieu), 44, chemin de Ronde, 78110 Le Vésinet

* _{Station de Recherches de Nutrition LN.R.A. 78350}

Jouy en _Josas.

Summary.

In vitamin D-deficient pigs the amount of intestinal

_{calcium-binding}

protein

(CaBP)

decreased

_by

33 p. 100 in the

_proximal

duodenum and

by

70 p. 100 in the

mid-jejunum ; it was unmeasurable in the distal ileum. Plasma calcium levels declined

signi-ficantly

and

_{plasma 25-hydroxycholecalciferol}

level was undetectable. The _pigs

_adapted

to a calcium-deficient diet _by

considerably

_increasing

jejunal

and ileal CaBP.

In chicks and rats,

25-hydroxycholecalciferol

(25-OHD,)

is the

_major

_circulating

metabolite of vitamin

_D

₃

_,

whereas

_{1,25-dihydroxycholecalciferol (1,25(OH)!Dg)}

is the

_major

_hormonally

active metabolite of vitamin

_D

₃

_increasing

intestinal

_absorption

of calcium and

_inducing

the

_synthesis

of intestinal

_{calcium-binding}

_protein

_(CaBP)

(Spencer

et

_al.,

_1976).

In these

_species

evidence is now

_accumulating

that CaBP is

probably

involved in the

_complex

_{process of intestinal calcium}

_transport

_(Wasserman,

Taylor

and

Fullmer,

_1974).

On the other

hand,

little is known

_concerning

vitamin D

metabolism and its action mechanism in

_pigs.

The

_present

_study

_reports

the effect of vitamin D or calcium

_deficiency

on CaBP isolated from the

proximal

duodenum,

mid-jejunum

and distal ileum, and on

_plasma

calcium and

_25-OHD,

levels.

Material and methods.

- Animals and Diets.

Vitamin-D

_deficiency.

- Two

groups of

Large-White

pigs

were used. In the first

group, vitamin D-deficient

pigs

were

_produced

as

_previously

described

_{(Pointillart,}

Garel and

_Guéguen,

_1978).

From

_weaning

at 5 weeks _{of age, the}

_piglets

received a

semi-synthetic

vitamin D-deficient diet

_containing

_{0.9 p. 100 Ca and 0.6}

_{p. 100}

P. In the second group, control

pigs

were

_produced

from sows

_receiving

the standard

_pig

diet

with the usual vitamin D content. After

_weaning

at 5

weeks,

_they

received a

semi-synthetic

diet

_containing

_{0.8 p. 100} Ca, _{0.6 p. 100 P and 2 500 IU of} vitamin

_D,/kg

of diet. The animals were

_slaughtered

7 to 8 weeks later. 13

_pigs

_{of each group} were used.

Calcium

_deficiency.

- Five-week old L. W.

piglets

received either a low-calcium

diet

₍₀

_{Ca) (0.1}

_{p. 100}

Ca,

0.6 p. 100

P)

or a control diet

_(NCa)

(0.8

_{p. 100}

Ca,

0.6 p. 100

P)

containing

2 5001U of vitamin

_D,/kg

diet. Seven

_pigs

in _{each group} were

_slaughtered

4 weeks later.

-

Calcium-binding

protein

(C

A

BP)

analysis

(Freund

and

Bronner,

_1975).

20 cm of

the

_proximal

duodenum,

_mid-jejunum

and distal ileum of each animal were

_excised,

everted and the mucosal tissue

_homogenized

in 4 x its volume of a modified

Tris-saline

buffer ;

the

_homogenate

was

_centrifuged

at _{100 000 g and} the

_supernatant

lyo-philized (S100).

A low molecular

_weight

fraction was isolated from this material

_by

Sephadex

G-75 column

_{chromatography}

_{(dimensions :}

1.6 x 100

_cm).

This material

(peak

B,

v,/v,

=

2.0)

was

_pooled

and defined as CaBP. The CaBP content was

evaluat-ed

_by

its

_{calcium-binding}

_activity

determined as a function of the assay calcium

concen-tration ; the amount of added calcium was 0-100

_ygjml

and the final free calcium

concentration varied from 0.2 to 15

₅

_yM.

The

_binding

data were

_analyzed

_by

saturation

kinetics. The

_{calcium-binding}

_capacity

was calculated

_by

the method of Scatchard and

expressed

as nmol Ca

_bound/mg

_protein

in

_peak

B.

_Equal

amounts

₍₁₇₅

_mg)

of S100

_protein

were

_applied

to the column for

_quantitative

_comparison.

All

_procedures

were carried out at 4 !C. Protein was monitored at 280 nm. 45Ca was

_{analyzed by}

liquid

scintillation.

- Plasma

analysis.

« Vitamin D status » was evaluated

_by

the

_plasma

concentra-tion

_{of 25-OHD}

₃

measured

_by

a

_competitive

_{protein-binding}

_assay

_(Belsey,

De Luca

and

Potts,

1974).

Plasma calcium was determined

_by

atomic

_absorption

spectropho-tometry.

Results.

Vitamin

_{D-deficiency.}

- Table 1 shows that in the vitamin D-deficient

_pigs

the mucosal CaBP levels decreased

_{significantly}

_by

_{about 33 p. 100} in the

_proximal

duode-num and

_by

_{71 p. 100} in the

_{mid-jejunum ;}

_they

remained undetectable in the distal ileum. Table 1 also shows that

_jejunal

CaBP levels

_represented

_only

_{17 p. 100 and 9 p.} 100 of the duodenal CaBP levels in control and vitamin D-deficient

_pigs,

_{respectively.}

The

_{plasma 25-OHD,}

concentration

_dropped

to an undetectable value

_(<

4

_fL9/

₁₎

in the vitamin D-deficient

_pigs.

There was a

_{statistically significant}

_positive

correlation between duodenal CaBP levels and

_plasma

_25-OHD,

levels :

where y is the duodenal CaBP content

_expressed

as nmol Ca

_bound/mg

_protein

(peak

B)

and x is the

_{plasma 25-OHD}

₃

level

_expressed

as

_fL9/

_1.

The

_plasma

calcium levels of the vitamin D-deficient animals were lower

_(p

!

_0.02)

than in the controls.

Calcium

_deficiency.

- Table 2 shows that when the

pigs

were

placed

on a

low-calcium diet for 4 weeks the mucosal CaBP

_appeared

in the distal

ileum,

rose

_by

about

170 p. 100 in the

_mid-jejunum,

but was not

_{significantly changed}

in the

_proximal

duodenum. Table 2 also shows that in calcium-deficient

_pigs

the CaBP levels of the

jejunal

segment

approached

those of the duodenal

_segment

and that the CaBP content

of the ileal

_segment

_represented

_{about 20 p. 100 of both other}

_segments.

In calcium-deficient

_pigs

the

_{plasma 25-OHD,}

concentrations

_dropped

to 5

_!

0.8

_fL9/

_’

and the

plasma

calcium concentrations

_markedly

decreased to

_{76:::!: 5 mg/I.}

Discussion.

The

_present

_study

confirms that in

_pigs

fed a normal

calcium,

phosphorus

and vitamin

_D

₃

_{-supplemented}

diet the CaBP levels decrease

_along

the small intestine to become very low or unmeasurable in _{the ileum. These data agree with}

_previous

reports

(Arnold

et

_{al., 1975 ;}

Fox et

_al.,

_1977).

In addition our results

clearly

demonstrate that intestinal CaBP is sensitive to

dietary

vitamin

_D,.

_{This appears from both the}

_significant

decrease in the intestinal CaBP levels in vitamin D-deficient

_pigs

and the

_positive

_relationship

between duodenal CaBP and

_{plasma 25-OHD,}

levels. The

_jejunal

_segment

_appears more

_dependent

on

dietary

vitamin

_D

₃

than the duodenal

_segment.

An essential

_point

is that in 7 or 8-week

vitamin D-deficient

_pigs

a

_large

amount of duodenal CaBP is associated with an

unde-tectable

_{plasma 25-OHD,}

concentration.

_Harrison,

Hitchman and Brown

₍₁₉₇₅₎

noted

no

_significant

decrease in the duodenal CaBP in 3 vitamin D-deficient

_pigs

as

_compared

to 3 control

_pigs.

Our

_{previously reported}

_experiments

in the rat indicated that in 6-week vitamin D-deficient rats duodenal

CaBP,

isolated and measured

_by

the same

method,

decreased

_by

about _{80 p. 100}

₍₂₂

to 5 nmol.Ca

_bound/mg

_protein),

serum

calcium levels fell to 59

_{mgjl (Thomasset,}

Cuisinier-Gleizes and

Mathieu,

1977)

and

serum

_25-OHD,

concentrations

_dropped

to undetectable values

_(Thomasset

unpublish-ed

_data).

This

_emphasizes

the differences between intestinal CaBP values in control rats and

_pigs.

On the other

hand,

the vitamin D

_dependency

of intestinal CaBP is more

marked in rats than in

_pigs.

The latter

difference, however,

may be due to the insuffi-cient duration of the vitamin D

_deficiency

in our

_{pig experiments.}

The

_present

_study

also indicates that

_pigs

can

adapt

to a 4-week calcium

depriva-tion

_by

_increasing

the concentration of the CaBP in the

_mid-jejunum

and

_by

_promoting

the CaBP

_synthesis

in the _{distal ileum without any}

_change

in CaBP levels in the duodenal

segment.

Such data have

_already

been noted in two 2-week calcium-deficient

_pigs

as

compared

to two control

_pigs

_(Arnold

et al.,

1975).

It is

_important

to note that when

_pigs

were fed a low-calcium diet for 7

weeks,

the duodenal CaBP levels rose

_by

about

100 p. 100

(144

vs.

76) (Pointillart, unpublished data). Consequently

the response of the

duodenal

_segments

seems

_dependent

on the duration of the calcium

_deficiency

and

posterior

to the

_adaptation

of the

_mid-jejunum

or distal ileum. Thus,

_pigs

as well as rats

(Thomasset,

Cuisinier-Gleizes and

Mathieu,

₁₉₇₇₎

can

_adapt

to calcium

_deprivation

_by

increasing

intestinal CaBP content.

The rise in CaBP levels in the distal ileum of calcium-deficient

_pigs

is

_pertinent,

since _up to now the duodenum rather than the ileum has been considered as a

_primary

target

organ for vitamin

D,.

This distal

adaptive

capacity

to increase CaBP

synthesis

has been

_reported

after massive small bowel resection in

_pigs

_(Margolis

et al.,

_1977).

In

our

_study

calcium

_deficiency

is associated with a

_drop

in

_{plasma 25-OHD,}

levels. This decrease may be due to a more

rapid

conversion

of 25-OHD

3

into

1,25(OH)

2

D

3

.

Indeed

it has been

_reported

that renal

_25-ODH

₃

_{1-hydroxylase}

_activity

_(Boyle

et

_al.,

_1972),

as well as

_{plasma 1,25(OH),D,}

levels

(Hughes

et

al.,

1975),

increased in calcium-deficient rats.

The

_possible

association between the increase in CaBP levels and the increase in calcium

_{absorption ability}

in the different intestinal

_segments

of calcium-deficient

_pigs

CaBP level in the distal

_{three-quarters}

of the small intestine and noted an increase in duodenal calcium

_absorption

_{without any}

_{corresponding}

increase in the CaBP levels

in the same

_segment.

Thus,

intestinal CaBP in

_pigs

is

_dependent

on

_dietary

vitamin

_D

₃

or

_dietary

cal-cium. The

_jejunum

_appears to

_rely

more on

_dietary

vitamin

_D

₃

than the duodenum.

Adaptation

to a low-calcium diet

_by

_promoting

CaBP

_synthesis

was marked in the

jejunum

and the ileum. This

_study

_suggests

that a low-calcium diet

_promotes

intestinal

CaBP

_synthesis,

an

_expression

of the hormonal action of

_1,25(OH),D,

in the distal

ileum,

which is not

_usually

considered as an essential

_target

tissue for vitamin D.

Commission CNERNA

_{Digestion-Absorption/Association}

des _{Physiologistes,} Paris 5-6 octobre 1978.

Acknowledgments.

- This

study

was

_supported

in

_part

_{by C.R.L.}

76.5.201.4 of INSERM and

_by

ATP 2427 of C.N.R.S.

Résumé. Chez le porc carencé en vitamine D la _quantité de « CaBP » décroît de 33 _p.

100 dans le duodénum

proximal,

de 70 p. 100 dans le _jéjunum médian et reste non

mesu-rable dans l’iléon distal. La calcémie chute

significativement

et le taux

plasmatique

de

25-OHD,

est indosable. Le porc

s’adapte

à un

_régime

carencé en calcium en _augmentant

considérablement son taux de CaBP

_jéjunale

et iléale.

References

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