Immunoreactive atrial natriuretic factor (IR-ANF) in human plasma

Vol. 128, No. 3, 1985

BlOCHEMlCAL

AND BlOPHYSlCAL

RESEARCH COMMUNlCATlONS Pages 1350-1357

May16.1985

IMMUNOREACTIVE ATRIAL J.

Gutkowska*,

M. Bourassa**, M. Cantin

110 Pine Received

March

22,

NATRIURETIC

Clinical Avenue

FACTOR (IR-ANF)

IN HUMAN PLASMA

D. Roy**, G. Thibault, and J. Genest

R. Garcia,

Research Institute of Montreal West, Montreal, Quebec, Canada

H2W lR7

1985

A direct radioimmunoassay for ANF in human plasma was synthetic a-human atria1 peptide (Ser 99-Tyr 126) was used for the iodinated tracer and the standards. The sensitivity of 1.9 pg/ml. Concentration of immunoreactive ANF (IR-ANF) in clinically normal subjects was 65.3 + 2.5 pg/ml (mean f SE). who underwent atria1 pacing an increase of about 100 percent IR-ANF was observed. IR-ANF was extracted from human plasma and purified by HPLC. The main immunoreactive isolated peak 0 1985 Academic Press, Inc. molecular weight peptide.

The in the

presence

atria

of secretory

of mammals (1,

natriuretic

factors,

Kangawa

and Matsuo

(IO)

(Ser

diuretic

and natriuretic atria1

MATERIALS Blood

2,

3,

9%Tyr

purified,

126)

natriuretic

a

factor

from

active rat

human atria1

potent

atria extracts,

vasorelaxant

We now report

activity.

has been

Biologically

isolated

from

with

in cardiocytes

4).

ANF) have been

peptide

tive

granules

developed. A preparation of the method is plasma of 59 In two patients in circulating by Vycor glass contained 'a low

the

demonstrated

peptides (5-9).

Recently,

a 2%amino

activity presence

(atria1

as of

acid

well

as

immunoreac-

in human plasma.

AND METHODS

withdrawal

Blood was drawn into chilled tubes containing EDTA and (per 1 ml blood) Blood was centrifuged at pepstatin 10 ~1 (0.5 mM) and 10 ~1 PMSF (1 mM). 4000 rpm for 20 min at 4°C. Plasma was assayed immediately or stored at -70°C for only a few days. Preparation Inc.) was

of iodinated

Iodination of human atria1 natriuretic was performed using the chloramine-T purified by HPLC chromatography

* To whom correspondence ** Institut de Cardiologie 0006-291X/85 Copyrght All rights

ANF peptide (Peninsula Laboratories method (11). The iodinated tracer on a Cl8 PBondapak column. The

should be addressed. de Montrbal.

$1.50

63 I985 by Academic Press, Inc. of reproduction in any form resewed.

1350

BIOCHEMICAL

Vol. 128, No. 3, 1985

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

To 5 ug of a-human ANF radioactive peaks were tested for immunoreactivity. dissolved in 10 ~1 sodium phosphate buffer, pH 7.4, kept in ice, 1 mCi of Na-1251 (40 ~1) was added and mixed in a Vortex. Then, 20 ~1 of a 0.5 mg/ml was added solution of chloramine-T in 0.1 M sodium phosphate buffer, pH 7.4, and again mixed. After 30 set, 20 ~1 of 1 mg/ml of sodium metabisulfide in 0.1 M buffer, pH 7.4, was added. The iodination mixture was applied to a C1e uBondapak column (0.39 x 30 cm) and the radioactive material was eluted with a linear gradient of 20% to 50% acetonitrile with 0.1% trifluoroacetic acid with One milliliter eluates were a slope of 0.5%/min and a flow rate of 1 ml/min. collected into tubes containing 100 ~1 of 1% BSA. Aliquots of IO ~1 from each After testing the immunoreactivity fraction were counted in a gamma counter. of the radioactive peaks, the tracer was stored in 0.1 M acetic acid (- 6 x 10 cpm in 250 1~1 0.1 M acetic acid) at -20°C. ANF standards The synthetic a-human atria1 peptide (Ser 39-Tyr 126) was used (Peninsuia Laboratories Inc.), taking into consideration that this commercially available preparation contains 66% of pure peptide. A stock solution of 1 mg/ml in 0.1 M acetic acid was fractionated into 50-~1 aliquots and stored at -20°C. The stock solution was diluted in RIA buffer to a final concentration ranging One hundred microliters of standards were used from 1.9 pglml to 488 pglml. for preparation of the standard curve. Radioimmunoassay

procedure

All reagents The entire procedure was done at 4°C in polystyrene tubes. were diluted in RIA buffer, containing 0.1% BSA and 0.1% Triton, pH 7.4. The duplicate 25 ~1, 50 ~1 or 100 ul plasma samples or standards were mixed with 100 ~1 of antiserum (Peninsula Laboratories Inc.) in a total volume of 200 ~1 and incubated overnight at 4°C. Then, 100 ~1 of iodinated tracer (- 6000 cpm) were added, mixed and incubated overnight at 4°C. Free and bound fractions were separated by goat anti-rabbit gamma globulin (100 ~1, I:501 in the presence of a pool of normal rabbit serum (100 ~1, 1:25). After adding 1 ml of polyethylene glycol 6000 (6.25% solution), the tubes were centrifuged at 4000 rpm at 4°C for 20 min. The precipitate was washed with 1 ml 0.1 M sodium phosphate buffer, pH 7.4, and recentrifuged. The supernatant was aspirated in vacuum and the radioactivity in precipitates was measured in a LKB gamma Rack counter. Extraction

of

IR-ANF

from

human plasma

To 10 ml of Human IR-ANF has been extracted from plasma by Vycor glass. fresh plasma 5 ml of Vycor glass (Corning Glassware, NY) suspension (50 mg activated glass powder in 1 ml deionized water) was added and rotated during 30 min at 4°C. After 2 min centrifugation at 3000 rpm the supernatant was then aspirated and the glass powder was washed with 30 ml of deionized water, with 20 ml 0.1 M HCI. The adsorbed IR-ANF was eluted from the glass powder with 10 ml of a purified acetone:water mixture (60:40) during 30 min rotation at 4°C. After a short centrifugation, acetone was evaporated under a nitrogen stream and the aqueous solution lyophilized in a Speed-Vat and further purified by HPLC on a C,, in-Bondapak column (0.39 x 30 cm) (Waters Inc., Milford, Mass.). The column was eluted with a linear gradient of 15% to 45% acetonitrile with 0.1% trifluoroacetic acid with a slope of 0.5%/min and a flow rate of 1 ml/min. Two-milliliter fractions were collected, 200 1-11 aliquots were lyophilized in a Speed-Vat, then dissolved in 0.1 M phosphate buffer, pH 7.4, containing 0.1% BSA. ANF content was determined by radioimmunoassay. 1351

Vol.

128,

No. 3, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

The recovery of IR-ANF-during the extraction procedure has been studied using the radioactive tracer (- 20,000 cpm) in 6 individual plasma samples and measuring the radioactivity of the supernatants at every step. RESULTS Assay

conditions Various

ture,

conditions

dilution

of the

investigated

curves

Data

(done

with

the

obtained

(Fig.

1).

included

subtraction average

radioligand

binding

coefficient

control

determined software

paralleled

antibody final

zero

temperaserum were

reproducible

binding

counts

from

and calculation

of

B/B0

The concentrations

of

IR-ANF

of the

gamma counter).

dose was inhibited

standard

rabbit

conditions,

transformation

of 7.8% (n = 9). the

assay

of duplicates

in the

time,

and normal

of non-specific

logit-log

available at

such as incubation

or sample.

from

of variation

100 ~1)

assay

and second

of each tube,

were

the

Under

dose of standard,

samples

or

were

reduction

counts

each

first

and optimized.

standard

the

affecting

Fifty by 114.8

Dilutions

standard percent

pg/ml

of plasma

ANF samples

for in

curve of with

the a

(50 ~1

curve.

Sensitivity The with

sensitivity

95% of confidence,

of the

assay,

was found

the

least

to be 0.19

-. . o-

amount

distinguishable

from

pg/tube.

3.0 2.4

1.8

z Ig4

l .

1.2 0.6

-0.6 -1.2

-

-1.8

,-

.

. . .

1::L-L 1.6

Figure

1:

Calibration

curve

of

Ser

3.2

4.8

In [ANFI

(pg/mlI

99-Tyr

126

from 1.9 to 488 pg/ml.

1352

6.4

with two-fold serial

dilutions

0

Vol. 128, No. 3, 1985

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

Precision The

within-assay

10 replicates (16.7%) for

of

precision 3 plasma

was assessed

two

consecutive

Cross-reactivity The

of the

samples

from

assay

was assessed

was 10.3%;

duplicate

the

measurements

from

results

between-assay

of 5 plasma

in

precision two

assays,

days.

of the antiserum

antiserum

has

fragments

of ANF,

natriuretic

factor

been

tested

some of which (13,

14)

for

obtained

cross-reactivity during

with

purification

of

different rat

I).

(Table

TABLE I CROSS-REACTIVITY OF ANF-RELATED PEPTIDES WITH ANTISERUM OF ATRIALNATRIURETIC FACTOR

Cross-reactivity

Peptide (Ser 99-Tyr 126) *

r-ANF

(Arg 101-Tyr

126)

*

90

r-ANF

(Asp Ill-Tyr

126)

*

50

r-ANF

(Ser 103-Tyr (Atriopeptin

III)

126)

*

r-ANF

(Ser 103-Tyr (Atriopeptin

II)

100

125)

*

27

126) -

57

-ANF

(pGlu 116-Tyr

r-ANF

(Ser 103-Ser (Atriopeptin

r-ANF

(Arg IO?-Cys 121) **

0.001

r-ANF

(Arg lOl-Ser

123) **

0.001

r-ANF

(Arg 102-Tyr

126) **

33

r-ANF

(Ser 103-Tyr

126) **

22

r-ANF

(Ser 104-Tyr

126) **

21

r-ANF

(Glu

126) ***

8

h:

Human

-

This

54-Tyr

123) I)

3

*

r: sequence

(X)

100

h-ANF

* **

for

Rat

is common to both human and rat ANF (Gln of

original sequence has been substituted by PGlu). Specifications given by Peninsula Laboratories Inc.

Sequences obtained by proteolytic digestion Tyr 126) (13). *** Sequence obtained by isolation and purification

1353

of

the

ANF (Arg lOlof rat ANF (14).

atria1

Vol. 128, No. 3, 1985

Analytical

analytical

of

cold

recovery

(n

buffer

has been found

Normal

values Blood

samples

studied. -

to be 95.3

from

pg/ml).

from

The recovery

The control

119.6

ranged

22).

q

by adding

and 100 pg/ml)

Recovery

+ 4.8%

has been tested

ANF (50 pg/ml

concentration. 72.4

RESEARCH COMMUNICATIONS

recovery

The ties

8lOCHEMlCALAND8lOPHYSlCAL

values

to 93.6%

of the

of values

a

mean + SE

of ANF added

normotens

ive

(mean f SE) 65.3

were

the

to

of RIA

(mean + SE 1.

normal,

IR-ANF were

quanti-

and measuring

with

same amount

+ 1.9% (n = 10)

of

The majority

to human plasma

43%

59 clinically

two different

situated

subjects + 2.5

between

were

(9.6

pg/ml

50 pg/ml

and

70 pg/ml. The stability stored

at

of

-2O'C

at -?O"C,

during

2 to 4 days. at -20°C

in spite

are

Isolation The is shown 31%

to

in table of IR-ANF results

pool

of

101.0

pg/ml.

presence

atria1

beginning

from

samples

the

samples

In

of samples

was observed

degradation

of IR-ANF

enzyme

when

inhibitors.

content

(13).

of

IR-ANF

The plasma

of treatment

in two

samples

and 24 hours

patients were

who

obtained The results

later.

human plasma

These low molecular

human

majority

plasma

IR-ANF

plasmatic pacing

time.

of proteolytic

of human plasma

2. The IR-ANF was eluted

plasma After

agreement

with

detected,

considering

radioactive

the

of

in some we observed

of HPLC purification

in figure

a

in the

by assaying

II.

acetonitrile.

contains

in IR-ANF

of the

20 min after

shown

intervals

pacing on circulating

subjected

before,

was assessed

various

However,

We have determined were

for

no changes

of atria1

Effect

in plasma

and -70°C

stored

kept

IR-ANF

results weight

extraction

theoretical

from

the

C,,

that

the

main

suggest peptide.

used for

determined

and HPLC,

a 62% recovery

of

the 62.6

(62.0

tracer. 1354

IR-ANF pg/ml

f 5.3%;

glass

column

immunoreactive of by

content which

by Vycor

PBondapak

The concentration

extraction

value

extracts

peak

IR-ANF

direct

with

in

a

RIA

was

was 57 pg/ml,

in

should

have

n = 6) as determined

been using

Vol.

128,

No. 3, 1985

BIOCHEMICAL

AND

TABLE IR-ANF

IN PATIENTS

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

II

SUBJECTED

PACING

TO ATRIAL

IR-ANF (pg/ml) Patient

later

before

after

24 hours

153262-O

77.2

139.9

106.1

134643-1

91.3

174.4

81.5

DISCUSSION A

method

allowing

importance

in assessing

biologically

potent

when

large

ANF

measurement

the

physiological

peptide.

number

radioimmunoassay

the

This

of clinical is a simple

in human plasma

0

using

4

of ANF in body fluids and physiopathological 1125-ANF

samples

2:

Chromatography of UBondapak column.

16

Vycor

for

available

20

FRACTION

Figure

assayed.

technique

a commercially

12

be

24

is

conven

ent

The

descr

bed

determination

antiserum.

32

36

of this

The

of

extract

of

human

40

plasma

IR-

labe 1 ing

NUMBER

glass

1355

3

increasing

roles

radioimmunoassay

are to

and sensitive

8

is of

on Cl8

Vol. 128, No. 3, 1985

yields

procedure

tion

BIOCHEMICAL

curves

variations

using

method

using this

are very

as an endocrine

Nothing Our results

increase

method,

presumably of atria1

human plasma,

4 weeks.

The cal ibra-

with very

reproducible

small

however,

suggest that

presence

origin.

about

immunoreactive

of

The human heart

the nature

it is a low moIecuIar

in the patients

of IR-ANF

the

ANF in

can thus

be con-

organ.

is known,

may be applied

method

this

for at least

in ID50 (cv = 7.8%; n = 9).

We have shown,

sidered

which is stable

a tracer

obtained

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

weight peptide.

who underwent

for physiological

of the circulating

atria1

peptide.

The observed

pacing shows that this

and physiopathological

studies.

ACKNOWLEDGEMENTS We wish to thank

critical technical

Doctor E.L. Schiffrin for his useful discussions review of this manuscript. We thank Mrs. L. Martin-Falstrault assistance and Ms. F. De Coste for secretarial help.

and the for

This work was supported by a Medical Research Council of Canada grant to the Multidisciplinary Research Group on Hypertension, and by grants given by the Quebec Heart Foundation, the National Research Council of Canada and the “Minister-e de la science et de la technologie du Quebec".

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de Bold: A:J. (1979) Proc, Soc.‘Exp.-Bioi. Med. 161, 508-511. Cantin, M., Gutkowska, J., Thibault, G., Milne, R.W., Ledoux, S., Min Li, S Chapeau, C., Garcia, R., Hamet, P., and Genest, J. (1984) Hiitochemistry 80, 113-127. de Bold, A.J., Borenstein, H.B., Veress, A.T., and Sonnenberg, H. (1981) Life Sci. 28, 89-94. Garcia, R., Cantin, M., Thibault, G., Ong, H., and Genest, J. (1982) Experientia 38, 1071-1073. Flynn, T.G., de Bold, M.L., and de Bold, A.J. (1983) Biochem. Biophys. Res. Comm. 117, 859-865. Seidah, N.G., Lazure, C., Chretien, M., Thibault, G., Garcia, R., Cantin, M Genest, J., Nutt, R.F., Brady, S-F., Lyle, T.A., Paleveda, W.J., Coiton, C.D., Ciccarone, T.M., and Veber, D.F. (1984) Proc. Natl. Acad.

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Gutkowska, J., Horky', K., Thibault, G., Januszewicz, P., Can-tin, M., and Genest, J. (1984) Biochem. Biophys. Res. Comm. 125, 315-323. J.F. (1980) Cardiac Pacing, Weliens, H.J.J., Bdr, F.W., and Muncharaz, 2nd Edition, pp. 317-340, Grune & Stratton Inc. Thibault, G., Garcia, R., Carrier, F., Seidah, N.G., Lazure, C., Biophys. Res. Chretien, M., Cantin, M. and Genest, J. (1984) Biochem. Commun. 125, 938-946. Thibault, G., Garcia, R., Cantin, M., Genest, J., Lazure, C., Seidah, N.G. and Chretien, M. (1984) FEBS Letter 167, 352-356.

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