Zinc transport by transferrin in rat portal blood plasma

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ZINC TRANSPORT BY TRANSFERRIN IN RAT PORTAL BLOOD PLASMA

G.W. Evans'

and T.W. Winter'

United States Department of Agriculture Agricultural Research Service Human Nutrition Laboratory Grand

Received

July

Forks,

North

Dakota,

58201

18, 1975 Sumary

Zinc transport in mesenteric lymph and zinc distribution in portal plasma and venous plasma were examined in rats that had been given an oral dose of 65Zn. Less than 1% of an oral dose of 65Zn appeared in the mesenteric lymph over a period of 8 hr. In portal plasma, approximately 70% of the isotope recovered after gel-filtration chromatography was bound to a protein that was identified as transferrin on the basis of molecular weight and electrophoretic properties. In venous plasma, the major fraction of 'j5Zn was bound to albumin while the remainder of the isotope was associated with higher molecular weight proteins including transferrin and ae-macroglobulins. These results demonstrate that zinc is transported from the intestine to the liver via the portal blood, and the results demonstrate that zinc is transported in portal plasma bound to transferrin.

Introduction Several of zinc

in venous

remainder Until of zinc

investigations plasma

of the zinc

recently,

associated

normal

the possible

that

or serum is bound

when Boyett

in serum from

had considered

is

have demonstrated

with

and Sullivan

to albumin other (4)

and cirrhotic role

the major while

protein examined

humans,

of transferrin

very

in zinc

fraction the

species

(l-5).

the distribution few investigators metabolism.

'Research Chemist, United States Department of Agriculture, Human Nutrition Laboratory, Grand Agricultural Research Service, North Dakota. 2 Department of Medicine, Grand

of Biochemistry, University Forks, North Dakota. 1218

of North

Dakota,

Forks, School

BIOCHEMICAL

Vol. 66, No. 4,1975

During

the

course

transport,

of our

we examined

in the portal

blood

of the isotope following

report

investigations

the

into

distribution

of the

in the

AND BIOPHYSICAL

rat

the

absorption

of orally

plasma

describes

zinc

and discovered

portal

RESEARCH COMMUNICATIONS

is

results

65Zn

administered

that

bound

and

approximately

70% The

to transferrin.

of our experiments.

Methods In Experiment was examined. with

65Zn transport

1,

A 500-g

sodium

male,

pentobarbital

and the mesenteric

Sprague-Dawley

after

lymph

duct

Carrier-free

65 Zn (International

California)'

was diluted

NaCl,

which

in a graduated

restraining

cage and given

collected

8 hr.

and monitored Thereafter,

was removed, Packard

for

the rat

and the

Whole

carcass

over

the entire for

Irvine,

in 0.85% the via

the

in a

Lymph samples

hourly

was monitored

2

65

Zn distribution

a period

were of

intestinal

radioactivity

in portal

Sprague-Dawley

rats

had been fed

was made,

into

was kept

to water.

was decapitated,

and 3 Long-Evans

were

the animal

radioactivity

rats

The rats

Corp.,

pCi/ml

was injected

access

Sprague-Dawley

the

and Nuclear

tubing.

tract in a

Body Counter.

In Experiment

days while

was made

and lymph was collected

while

free

of one rat

polyethelene

of 0.1

solution

cylinder

incision with

Chemical

was closed

lymph

was anesthetized

a midline

to a concentration

The incision

cannula

rat

was cannulated

and 1.0 ml of the isotope

duodenum.

in mesenteric

other

animals

anesthetized

and carrier-free

rats

were

and venous examined.

a zinc-deficient

diet

had been fed a diet with

sodium

65Zn (0.1

pentobarbital, @i

in

blood,

of 2

One of the (6)

of Purina

for

30

Lab Chow.

a midline

1.0 ml 0.85% NaCl)

incision was

'Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.

1219

Vol. 66, No. 4, 1975

injected

BIOCHEMICAL

into

portal

vein

the duodenum. and blood

approximately sample samples plasma

were

was drawn

was inserted

a heparinized from

from the

syringe.

the portal

inferior

in a table-top

into After

vein,

a second

vena cava.

centrifuge

after

the

The blood which

the

was removed.

packed

samples

with

that

collected

G-150

in 3-ml

gamma-well

were

Ion exchange with

Each sample

DEAE-Sephadex 0.02

through

gradient

(limit

Aliquots

of 1.2 ml were

buffer

Electrophoresis

Piscataway,

M Tris

the sample

- 0.05 which

was monitored

radioactivity

was carried

out on a 0.9-

M Tris

M HCl,

of the sample,

the column

after

- 0.5 M HCl,

collected

and monitored

x 15-cm

Chemicals) pH 8.6,

that

had

as starting

25 ml of the starting

which

= 2 M Tris

Chicago

10 min.

Fine

- 0.05

was

in a Nuclear for

(Pharmacia

M HCl,

in a LKB Uvicord

was counted

was performed Samples

System.

membranes

inersed

strips

were

with were

a loo-ml pH 8.6) for

linear

was applied.

radioactivity

scanned

a Beckman Model applied

in barbital

Following

18 min at 250 volts. the acetate

0.02

x 90-cm column

in

counter.

Electrophoresis

acetate

Chemicals,

with

for

application

was passed

the gamma-well

Fine

Absorbance

monitored

with

Following

to a 1.5-

was used to elute

chromatography

been equilibrated buffer.

(Pharmacia

aliquots.

counter.

packed

individually

had been equilibrated

The samples

buffer

applied

The same buffer

pH 8.6.

column

were

Sephadex

New Jersey)

Model

into

needle

was drawn

was withdrawn

centrifuged

Plasma

II.

A 27-gauge

3 ml of blood

of blood

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with

Microzone

to Beckman cellulose

buffer,

staining

R-101

pH 8.6,

and subsequent

a Beckman Microzone

and run for destaining, Desitometer,

R-110. Results When 65 Zn transport

(Experiment

l),

and Discussion

in the mesenteric

85% of the administered

1220

lymph was examined dose was detected

in the blood

BIOCHEMICAL

Vol. 66, No. 4, 1975

and carcass

(minus

intraduodenal

in the

that

zinc

gastrointestinal

of ?!n.

injection

was detected indicate

the entire

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

lymph is

Only

collected

not

tract)

0.13%

over

of the

8 hr after administered

the 8 hr period.

dose

These

transported

through

the mesenteric

the results

obtained

in Experiment

the

results

lymph

system. Figure

1 illustrates

samples

of portal

Sephadex

G-150.

radioactivity with

that

recovered

of proteins

and venous

In the portal was eluted

a molecular

of the

plasma

weight

plasma,

at a volume

WRTAL

PL

VENOUS

PLASMA

ELUTION

Figure

1.

corresponding

was eluted

a molecular

were

chromatographed

70% + 5% (S.D.)

of approximately

radioactivity with

plasma

weight

90,000

2 when

with daltons,

on

of the

recovered

that

of proteins

and 10% 2 3%

in a peak corresponding of approximately

70,000

with daltons.

VOLUME(ml)

Elution of 65Zn and protein from portal and venous plasma on Sephadex G-150. The results illustrated were taken from one experiment but are representative of the results obtained with 1 Sprague-Dawley rat and 3 Long-Evans rats fed a stock diet and 1 Sprague-Dawley rat fed a zinc-deficient diet. The inset illustrates the elution volumes of 3 proteins run as standards: T = transferrin; A = albumin; H = hemoglobin.

1221

Vol.66,No.4,1975

BIOCHEMICAL

In venous volume

plasma

> 300,000

daltons),

75 ml (90,000

daltons)

deficiency

had no apparent

plasma.

plasma

Fraction

When portal by electrophoresis,

2 protein

One of the protein

bands

second

band migrated

purify

Fraction

Sephadex

the

with

with

remainder

three-fourth

90 ml).

When the

analyzed

by electrophoresis

starting

buffer

protein

band that

migrated

of portal

plasma

albumin

the presence (Figure

by gel-filtration

plasma

and smaller Following the

fractions

The fraction

fraction

fraction

is bound

eluted

contained

buffer

(80 ml -

with

isotope

the

based

G-150 with

weight properties,

from

the

to transferrin

intestine while

of a

2).

migrated

on molecular

fractions

consisted

(Figure

band that

DEAE were the

II from Sephadex

protein

peak

from

However,

while

determination indicate via

a second

to albumin.

chromatography that

eluted

transferrin

Fraction

bound

10% of the

the gradient

that

transported

is

to DEAE-

in a single

and electrophoretic

of zinc

of the rat

applied

65Zn eluted

the

To further

the starting

protein.

results,

chromatography

the major

the portal

These

2).

with

contained

with

while

Approximately

of the eluted

of a single

observed.

were

was eluted

no stainable portion

indicated

that

isotope

the major

was analyzed

B-globulins.

samples

the fractions

contained

Electrophoresis

and the

gradient.

that

G-150

the a2-macroglobulins

of the way through

fractions

of 65Zn in either

consistently

was eluted

contained

single

with

G-150,

column

in

Zinc-

distribution

were

transferrin

of the recovered

approximately

that

bands

a linear

from the

daltons).

I from Sephadex

migrated

void

of the 65Zn was eluted

- 70,000

on the

I from Sephadex

and eluted

65Zn recovered

effect

or venous

at the

and 40% + 10% of the radioactivity

at 95 ml - 105 ml (60,000

plasma

Zn was eluted

15% 55%

was eluted

portal

65

20% + 2% of the recovered

(proteins

a peak around

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

65Zn were

at the void

volume

1222

venous

plasma

on Sephadex

analyzed

by electrophoresis.

contained

al-macroglobulins,

G-150,

Vol.66,No.4,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PORTAL PLASMA Fraction I otter OEAE

PORTAL Fraction

PLASMA II

n

TRANSFERRIN

n

PLASMA

n

CONTROL

ALBUMIN

Figure

2.

Illustration electrophoresis.

02-macroglobulins, that

eluted

B-globulins,

fraction

of

results

agree

other

zinc

65

and the

those

in venous

protein

species

The experiments from

the

Moreover, portal in portal zinc carried

intestine

plasma

of other

plasma

described liver

demonstrate

plasma

bound

to transferrin.

blood

by transferrin

investigators is

above

results

in venous

while that

following

the

fraction

migrated

with

and largest

only

albumin.

These

who have demonstrated

distributed

among albumin

and

(l-5).

to the

is

obtained

The third contained

or serum

our

plasma

proteins

B-globulins.

Zn in the venous with

recordings

and gamma globulins

at 70 ml - 80 ml contained

the o2-macroglobulins

that

of the densitometer

bound

indicate

exclusively

via

that

is

zinc Since

to transferrin

is bound to the

that

dietary

zinc

portal

blood

the

transported

the major while

to albumin,

the metal

liver

to becoming

prior

1223

is

system.

in rat

portion

the major

passes

of zinc

portion

of

apparently bound

to albumin

Vol.66,No.4,

BIOCHEMICAL

1975

or incorporated Boyett

into

fraction

(4)

fraction

authors

may be involved

suggest

previously

that

the

exchange.

in the transferrin-

extremely

The results in

may play

role

the zinc

remains

observations

transferrin

unrecognized

that

constant,

the transferrin-o2-macroglobulin

in zinc

with

that

demonstrated

of human plasma

suggested

human serum combined paper

o2-macroglobulin.

and Sullivan

a2-macroglobulin and these

zinc

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in zinc

rats

observed

described

an extremely

important

in

in this but

metabolism.

Acknowledgments The authors their technical USDA Cooperative

thank Carrie Grace, Linda Hintgen, and Joan Rude for This work was supported in part by the assistance. Agreement No. 12-14-100-11, 176 (61) Amend. 1.

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Giroux,

E.L.,

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