Cortisol and cortisone levels in umbilical cord plasma and maternal plasma of normal pregnancies

497

CORTISOL

AND CORTISONE AND MATERNAL Robyn

LEVELS PLASMA

Postgraduate

A. Dormer

IN UMBILICAL

OF NORMAL

and John

CORD

PLASMA

PREGNANCIES

T. France

School of Obstetrics and Gynaecology, University of Auckland National Women's Hospital, Auckland, 3, Nets Zealand

Received: 10/30/72

ABSTRACT A method for assaying cortisol and cortisone using chromatography on either paper or Sephadex LH-20 columns for isolation, followed by competitive protein binding, has been applied to umbilical cord and maternal plasma samples. In mixed cor$ plasma the mean cortisol concentration was 6.0 - 0.8 pg/lOO ml $n = 9) and the mean cortisone concentration was 13.5 - 2.9 pg/lOO ml In cord artefial plasma the mean cortisol (n = 9). concentration was 6.3 - 2.9 ~g~100 ml (n = 6) and the mean cortisone level was 10.1 - 2.5 ~g/lOO ml (n = 6). For Ford venous plasma, the mean level of cortisol was 5.6 - 1.5 pg/'lOO ml (n = 6) and of cortisone was 13.5 2 Ma+ternal plasma gave a mean 2.4 pg/lOO ml (n = 6). value of cortisol of+42.3 - 4.5 lJ-g/100ml (n = 6) and of cortisone of 6.2 - 0.9 EJ-g/100ml. The results of this study suggest that the fetus at term-gestation The significance of this production produces cortisol. compared with placental transfer of maternal cortisol into the fetal circulation however is uncertain. INTRODUCTION The factors metabolism Although across

in the human the transfer

the placenta

demonstrated if any,

influencing

from mother

and

on a supply

and

understood.

its metabolites

to fetus

it is not known

relies

production

are poorly

of cortisol

(1,2,3),

the fetus

fetus

cortisol

has been

to urhat extent, of maternal

21:4

STEROIDS

498

cortisol.

The human newborn appears able to produce an

adequate supply of cortisol for its own needs (4). Cortisol production

rates during the first few days of life, as

measured by isotopic dilution methods, are in the range observed in adults when allowance

is made for differences

in body surface area (5). It has yet to be established if the autonomy in cortisol production

of the newborn is

acquired at birth, or earlier in gestation. Knowledge of the concentrations

of cortisol and

cortisone in umbilical cord artery and umbilical cord vein in normal and abnormal pregnancies in defining the relative contributions

may be helpful of maternal and

fetal cortisol to the fetal circulation.

Values of

cortisol and cortisone in mixed and separated arterial and venous cord plasma and in maternal plasma of normal pregnancies

are reported in this paper.

concentrations

The steroid

were measured by competitive protein

binding methods. MATERIALS

AND METHODS

Radioactive steroids: 1, 2-H3-c rtisol (specific activity 44 Ci/mmole) and 1, Z-H 9- cortisone (specific activity 52 Ci-mmole) uere purchased from New England Nuclear Corporation, Boston, Massachusetts, and were repurified by paper chromatography in the solvent system benzene:methanol:water (2:l:l). Authentic steroids for Non-radioactive steroids: reference standards were obtained from fkapharm, RamatGan, Israel.

April 1973

STEROIDS

499

Solvents: All reagent grade solvents u1ere redistilled before use. Solvents of higher quality were used without further purification. Sephadex I-H-20 (2S-lOLIp,particle size) was obtained from Pharmacia, Uppsala. Paper chromatoqraphy: Ulhatman No.1 chromatography paper was washed with methanol by elution in a chromatography tank for 24 hours prior to use, Fullers earth was used in the form of 15 mg pellets. Florisil {60-100 mesh) was purchased from Sigma Chemical Co., St.Louis, Missouri, Fines were removed by suspending the Florisil in distilled water and then decanting the supernatant, the procedure being repeated six times. After drying, IO gm Florisil was mechanically shaken overnight with IO gm dextran (N.W. 60,000-90,000) in 60 ml distilled mater. The dextran-coat,ed Florisil was filtered on a Buchner without mashing and oven-dried. Plasma samples: Samples were collected from uncomplicated pregnancies terminating in vaginal delivery of a normal baby. Umbilical cord mixed arterial and venous blood was collected immediately after the cord was cut at delivery. The blood was allowed to drain from the cord into a heparinired container while the placenta still remained within the uterus. Samples of cord venous and arterial blood were collected separately into heparinized tubes from a clamped segment of the umbilical cord immediately after delivery. Maternal blood samples mere taken from an antecubital arm vein within a few minutes of delivery, The blood samples were centrifuged and the plasma stored at -2OOC until taken for analysis. Method for determination

of cortisol and cortisone

Duplicate 0.1 ml samples of plasma were added to tracer amounts (1000 cpm) of 1, 2-Hz-cortisol and 1, Z-Hz-cortisone from which solvent had been evaporated under nitrogen. After allowing 15 minutes at room temperature for equilibration of the steroids with the plasma, the samples were extracted in 1 ml dichloromethane by shaking for 15 minutes. Following centrifuqation, the plasma was frozen in an acetone/dry ice mixture and the organic phase decanted off into a test-tube. The dichloromethane extracts were taken to near dryness under a stream of nitrogen in a water bath and the residues were applied to No. 1 Uihatman chromatography

500

STEROIDS

21:4

The papers were equilibrated overnight and run for 5-7 hours in the Bush B5 solvent system benzene: methanol: water (2:l:l). The regions corresponding to cortisol and cortisone were identified by scanning in a Nuclear Chicago Actigraph III Radiochromatogram Scanner, separately cut out and eluted with 2.5 ml of 95% ethanol. The eluates were blown down, taken up in 1 ml of ethanol and 1/5th removed for estimating recovery. The remaining portion of each of the steroid extracts was taken to dryness and assayed by a competitive protein binding method as outlined in Table I. paper.

Latterly, paper chromatography of the extracts was replaced by chromatography on 0.5 cm x 13 cm Sephadex LH 20 columns in benzene:methanol,(95:5). The cortisone and cortisol fractions were blown down and treated in the same manner as the eluates from the paper chromatograms. The competitive protein binding assays were carried out according to techniques originally described by Murphy (6). The plasma source of binding protein was stripped of endogenous steroid by'addition of an equal volume of charcoal solution (containing 4 mg charcoal/ml borate buffer pli 7.8). The solutions of plasma and charcoal were first heated to 45OC, mixed and kept at 45OC for five minutes. After centrifuging to sediment the charcoal, the supernatant was decanted and filtered through filter paper (Ufhatman No. 542). The resultant 50% plasma solution ufas diluted with distilled water to give the required concentration in t%e presence of H3-steroid. A small volume of standard cortisol or cortisone was pipetted into a series of tubes, and an equal volume of 25% ethanol added to the sample tubes. To each sample and standard tube was added 1 ml of tritiated plasma binding solution. The tubes were vortexed, incubated at 4OoC for seven minutes and transferred to an ice bath at O°C for ten minutes. In the assays for cortisone, the tubes were kept in the ice bath while 80 mg of dextran-coated Florisil was added and each tube wortexed slowly for 30 seconds. After twenty-five minutes, 0.5 ml of the supernatant was pipetted from each tube into a counting vial for measurement of radio-activity. For the cortisol assays, the test tubes and rack were transferred to a horizontal shaking machine kept in a 4oC cold room. One pellet of Eullers earth was added to each tube and the rack shaken for 2 minutes. After centrifuging for five minutes at rt°C, 0.5 ml af

Cortisol

Cortisol

Cortisone

Cortisone

Cord

Maternal

Cord

Maternal

protein

assays

plasma

plasma

TABLE

2

_H3_ cortisone

4% chicken

_H3 -cortisone

8% chicken

_H3 -cortisol

2.5% human pregnancy plasma (3rd trimester)

-H3-cortisol

1% human pregnancy plasma (3rd trimester)

CEIG Source - labelled steroid employed

bindinq

I

Steroid

Cortisone

Cortisone

Cortisol

Cortisol

Plasma sample

Cord

Maternal

Cord

Maternal 44.5

9.6

14.6

27.7

(1.1q/lOOml)

Value before chromatographic purification

39.6

5.9

7.3

13.3

Value - paper chromatography method (i_1q/lOOml)

Specificity of the method as shown by agreement of results after by paper chromatography or Sephadex LH-20 column chromatoqraphy

Steroid to be assayed

for competitive

Plasma for analysis

Conditions

TABLE

39.4

6.1

7.4

13.4

column

of steroids Value - LH-20 chromatography method (~g/lOOml)

isolation

80 mg dextrancoated florisil

80 mg dextrancoated florisil

15 mg Fullers earth pellet

15 mg Fullers earth pellet

Adsorbent

STEROIDS

502

the supernatant was removed for measurement of radioactivity. Radioactivity counts were measured in a Packard Tri-Carb Liquid Scintillation Spectrometer Model 3365 in IO ml Bray's scintillation fluid. Calculations The steroid concentration (in EJ-g/100ml plasma) was calculated by the following formula (for a 0.1 ml plasma sample): pg/lOO Where

ml

=

x x 1.25 x 100 R

X = ng determined in the assay R = $ recovery of tritiated reference

EVALUATION

steroid

OF THE METHOD

This has been determined with samples separated by paper chromatography unless otherwise mentioned. Specificity The specificity of the method rests on the chromatographic separation of cortisol and cortisone from endogenous steroid that would otherwise compete for binding sites on the corticosteroid binding Levels of cortisol and cortisone prior to globulin. separation and after separation on paper and Sephadex The results are tabulated LH-20 columns were measured. The close agreement of the results obtained in Table 2. using the two different chromatographic systems indicates that in both of these methods cortisol and cortisone are measured free from interfering substances. Assay without chromatographic purification gives higher values because of the presence of cross-reacting substances. Precision The precision of the total method was estimated by calculation of the standard deviation of the mean of replicate determinations on aliquots of pooled cord and pooled maternal plasma samples. Assay of 11 samples of a pool of cord blood gave mean cortisol level of 5.9 pg/lOO ml with a standard deviation of 0.4 pg/'lOO ml and a mean cortisone level

a

ApriI 1973

of 13.3 100 ml.

STEROIDS

ii,g/lOO

ml with

503

a standard

deviation

of

1.8

j_~g/

Assays of 6 samples of a pool of maternal plasma gave a mean cortisol level of 39.6 Pg/lOO ml with a standard deviation of 3.0 ~g/lOO ml, and a mean cortisone level of 7.3 ii.g/lOO ml with a standard deviation of 1.6 pg/lOO ml. Accuracy

and

recovery

The accuracy of each measuring the concentrations

method was determined by of cortisol and cortisone in pooled maternal plasma and pooled cord plasma before and after the addition of cold cortisol and cortisone at 3 dose levels of concentrations ranging from 2 to The mean values of the levels of steroid 30 ~g/~OO ml. found expressed as a percentage of the levels expectyd mere for cortisof+and cortisone respectively lOl.l$ - 0.+9 plasma, and 98.4% (SD)% and 104.3% - 2.5% for maternal 1.4% and 100.3% + 2.7% for cord plasma. These values mere all corrected for experimental losses. Mean recoveries of individual radioactive steroids from IO determinations after extraction and separation, each for both the paper chromatography and Sephadex LH-20 column chromatography methods were: Paper

Chromatoqraphy

Cor tisol

76.7%

2 8.8

Cortisone

73.1%

2 10.3

(SD) (SD}

Column

ChromatoqraPhy

66.4%

+ 11.2

(SD}

68.5%

,f 10.2

(SD)

Blanks Method blanks mere obtained from chromatography paper separation by eluting portions of blank paper chromatograms equivalent to the regions of cortisol and and from Sephadex LH-20 column separation by cortisone, collecting equivalent elution volumes from blank columns. There was no difference in the blank values obtained from paper or from Sephadex LH-20 columns, The blanks for maternal cortisone had a mean level 0.13 pg/lOU ml with a standard deviation of 0.10 pg/lClU and for cord cortisone a mean level of 0.23 yg/lOO ml and a standard deviation of 0.2 pg/lOO ml. F-or cortisol, of

ml

21:4

STEROIDS

504

the blanks mere 0;2 2 0.35 yg/lOO ml for maternal samples and 0.26 - 0.43 for cord samples (n = 6 in each case). RESULTS The concentrations series

of mixed

are listed standard

of cortisol

cord plasma

in Table

deviation

are in agreement

3.

and cortisone

from

9 normal

pregnancies

The mean

cortisol

level

was 6.0 2 0.8 pg/lOO with

in a

thosereported

ml.

urith

These

by other

values

workers

(4,7,&g). In Table cortisone

4 are presented

in arterial

and in maternal

plasma

deviations

6.3 + 2.9 pg/lOO pg/100

ml, and

4.5 rJ_g/100ml. with

standard

for maternal The

mean

deviations

2.4 iJ-g/100ml, and The

0.9 yg/lOO

ml.

tuith those

reported

of 2 pools

of cord

value

for maternal cord plasma

arterial

From

plasma

results

f

cord

plasma

13.5 +

plasma

6.2 2

are comparable from analysis

obtained

ml and a mean

analysis

42.3

of cortisone

peripheral

(IO), who

with

5.6 2 1.5

cord plasma

plasma

plasma,

plasma

plasma

for arterial

for venous

of 7 pg/lOO

of 10.5 i_Lg/lOOml.

cord

cord

concentrations

by James

cord

of cortisol

peripheral

mere

and

for 6 normal

for arterial

for venous

ml,

venous

concentrations

were,

10.1 2 2.5 tJ_g/100ml,

cortisol

plasma,

of cortisol

at delivery,

The mean

pregnancies. standard

cord

levels

a mean

cortisone

of 2 pools

value

of cord

and

Cortisol

40.4 36.1 38.7 43.6 49.9 45.2

42.3

4.5

Mean

S.D.

separate

2 SD

0.9

6.2

7.3 6.0 6.6 4.4 6.6 6.1

Cortisone

in

Mean

cortisone

CHA COO KAN McC PAR McD

normal

and

venous

+ 0.8 4

9.9 8.0 8.6 7.4 14.2 12.6 10.1 2.5

5.0 5.5 5.6 6.3 9.5 5.6 6.3 2.9

cord

13.5

14.9 13.6 13.5 17.4 10.7 10.2 17.0 13.7 10.6

11.3 14.8 10.2 13.2 17.5 14.0 13.5 2.4

5.1 4.7 5.1 6.2 8.5 3.8 5.6 1.5

Plasma Cortisone

in maternal

Cord Venous Cortisol

and

2 2.9

Cortisone

plasma,

plasmas

i~,g/lOO ml)

mixed

Cord Arterial Plasma Cortisol Cortisone (~q/lOO ml)

arterial

TABLE

6.0

3 card

6.6 6.4 6.3 5.9 4.9 4.3 6.1 5.4 5.9

of

UIAR ADM OAK DAN LET HAR MID EDM MIS

a series Cortisol

in

Subject

cortisone

Maternal Plasma Subject Cortisol

plasma

and

Cortisol

TABLE

21:4

STEROIDS

506

venous

plasma

he obtained

100 ml and a mean levels

cortisone

of cortisol

peripheral workers

a mean

and

plasma

are

cortisol

value

cortisone similar

value

of 14 pg/lOO determined

to levels

of 7 kg/ ml.

The

in maternal

found

by other

(4,7,8).

DISCUSSION The high

concentration

cortisol

in cord

plasma

studies

(4,7,8,9)

to cortisone

unit

The reason

so active

as reported

reflects

of cortisol (11).

why the fetus cortisol

from

excessive Unlike

corticosteroid. are high

level

is low in the fetus

concentration blood

of free

is relatively

difference levels cortisol

in total

in fetal levels

on transcortin Giroud

levels

neurborn some controlling

and placenta

to cortisone

cortisol

circulating

(12). despite

also

through

would

tend

(12,13).

(4), there

the reduction

in fetal

the large

of cortisol

progesterone

to increase

for binding

free

sites

If, as Hillman

is in the fetus

'illdefinedimmaturity'

where

the

The high

levels.

competition

and albumin

protecting

the transcortin

consequently

cortisol

has yet

in the mother,

and

higher

are

of biologically-active

of transcortin,

plasma

have proposed

feto-placental

the situation

there

metabolism

it is a mechanism

levels

to

and in similar

the extensive

but probably

to be resolved,

relative

here

in the human

in converting

the fetus

of cortisone

and

and

of the enzymes and

cortisone

to

April 1973

their

tetrahydro

cortisol

to

disposing that

derivatives, could

be

cortisol.

It

has

conversion

in

the

of

in

mechanism

regulating

levels of

the

fetal

The

contribution

in

little

led or

be

Other

of

contribution

is

through

the

placenta

compartment.

cortisol have to with the

and

in shown

cortisol

to (15).

prewiable adrenals

have

of

that

of

progesterone

conclude

with the

adrenal in

by

the

fetus.

the

vitro

perfusion demonstrated and

to

fetal

transfer

maternal there is

is

no

producing

techniques

metabolizing

(16)

that

secretion

fetal

results

admini-

that

although

of

The

produced

from

addition, have

to

hand,

the

cortisol

labelled

compared

capable

fetuses

C I4

cortisol

amounts,

In

to

suggested

cortisol

other

be

to

cortisol

maternally

(2)

were

significant it

of

insignificant

evidence

not

cortisol

maternal

and

adrenal

On the

of

uncertain.

co-workers

also

levels

cut

is

transport

fetal

does

cortisol

cortisol

plasma

clear

maternal

corticosteroids

of

Pasqualini

important

of

circulation

(7,8)

of

(14).

placental

workers

an

transfer

by

cortisol

may

Migeon

no

to

of

means

shourn

placenta

non-radioactive

cortisol

been

conversion

the

conversion

important

The

fetal

of

an

cortisone

circulation

the

studies

stered

the

the

(11).

fetus

cortisone

into

then

cortisone

of

the

occur

507

STEROIDS

progesterone experiments conversion

A~-C~,,

steroids

by

(17)

to cortisol.

passing

through

to cortisone

maintain

fetal

concentration cortisol

for the fetus. could

production

plasma

because

cortisol

resulting

hand,

cortisol exceed

venous

cortisol

levels.

was measured

and arterial between

plasma

the venous

not therefore

origin

of cortisol

is not a cortisol venous term,

blood does

then

and arterial

arterial

difference

with

the placenta

on the metabolic

venous

finding

explanation

of the

Since

favouring

the fetus,

though

should

was found

This

gradient

there cord

at least

the significance

transfer

of

study

circulation.

that

On the

source

of cord

levels.

in the fetal

of

levels

samples

of

(1).

in this

a conclusive

cortisol,

levels

was the major

no significant

appear

in concentration

of labor

Houtever, when

transfer

at the time

maternal

cord

in

of cortisol

the difference

the stress

compared

through

of information

source

the major

concentration

produce

this production cortisol

was

in paired

it would

cortisol

if placental

offer

(14,

to

than arterial

production

in the fetus

circulation

contain

of the raised

if fetal

converted

levels.

should

from

in

be necessary

to be at a maximum

delivery

other

the fetal may

Furthermore,

be expected

cortisol

may be largely

entering

cortisol

venous

of maternal

maternal

the placenta

adequate

Cord higher

Moreover,

before

18) so that

does

21:4

STEROIDS

508

at of

of maternal

is uncertain clearance

in the absence rate

of

April 1973

cortisol

in the feto-placental

Cortisone

venous

levels

plasma

than

venous-arterial of 3.4 pg/lOO therefore fetal

found

in cord

that

to be higher

arterial

as has been

ments

by Pasqualini

It

would

in the

transplacental to cortisone

The relatively in cortisone

as

high

concentration

of cortisone

previously

mean

concentration

converted

metabolism

shown

The

of cortisone

from

difference

appreciable

fetus

mainly

the placenta.

venous-arterial

in cord

at P
level

cortisol

through

plasma.

in cortisone

the high

arises

of maternal

represents

unit.

ml uIas significant

seem

it passes

were

difference

circulation

passage

cord

509

STEROIDS

by the

by perfusion

experi-

(11).

FOOTNOTE The following trivial names have been used: cortisol Z~-tr~hydroxypregn-4-en-3, 20 dione), 018, 17% cortisone (17a, Zl-dihydroxypregn-4-en-3, 11, 20-trione). REFERENCES 1.

Migeon, C.J., Prystotusky, H., Grumbach, M.M. J.CLIN.INVEST. 35, 488 (1956) Byron, M.C.,

2.

Migeon, INVEST.

3.

Migeon, C.J., Bertrand, 3. and PRO~.HORM.~ES., l7, 207 (1961)

4.

Hillman, D.A. and Giroud, 25, 243 (1965)

5.

Kenny, J.M., Malvaux, PEDIATRICS, 31, 360

C.J., Eertrand, 36, 1350 (1957)

J. and Wall,

Gemzell,

C.J.P.,

P. and (1963)

p-E.,

and

J.CLIN.

C.A.,

REC.

J.CLIN.ENDOCRINOL,

Migeon,

C.J.P.,

21:4

STEROIDS

510

6.

Murphy, (1967)

7.

Bro-Rasmussen, ENDOCR. 40,

B.E.P.

J.CLIN.ENDOCRINOL.METAB.

579

F., Buus, (1962)

0.

8.

Schweitzer, STEROIDS,

M., Branchaud, l4, 519 (1969).

9.

Klein, G.P., de Levis, M. STEROIDS, l9, 275 (1972)

10.

James,

11.

Pasqualini, Diczfalusy,

12.

Sandberg, A.A., EXCERPTA %11967).

13.

Rosenthal, H.E., Slaunmhite, A.A. J.CLIN.ENDOCRINOL. 29,

14.

Osinski,

15.

Villee, D.B. and INTERN.CONGR.SER.

16.

Bird, C.E., Uliqvist, S ., J.CLIN.ENDOCRINOL.

17.

Jackanicz, T.M., BIOCHIM,BIOPHYS.ACTA.

18.

C.St-G. and Hall, 49, 1384, (1971).

V.H.T.

EUROPEAN J.R. E.,

P.A.,

C.

and

D.,

Giroud,

Giroud,

1,

777

ACTA.

C.J.P.,

5

(1966) U.N. and 209 (1970).

H. and Slaunwhite INTERN.CONGR.SER.

Jr. lU.R. 352 (1969)

973

C.J.P.,

B.L., Wiqvist, BIOCHEM. 1,

Rosenthal, MED.(AMST)

187,

Trolle,

and

J.STEROIDS,

Nguyen, J.STEROID

NATURE

and

27,

and

Jr. 132,

Sandberg,

(1960)

EXCERPTA Villee, C.A. 83, 709 (1964) N., Diczfalusy, E. 26, 1144 (1966)

MED.(AMST)

and

Solomon,

Wiqvist, N. and Diczfalusy, 176, 883 (1969) Giroud,

C.J.P.

CANADIAN

E.

J.

BIOCHEM