On the processing of saliva samples for progesterone assay

539

3193

ON THE PROCESSING

OF SALIVA

Sila

Banerjee

Department New York

Received

SAMPLES FOR PROGESTERONE ASSAY and Mortimer

Levitz

of Obstetrics and Gynecology University School of Medicine New York, New York 10016

U-28-83

ABSTRACT

Reports from several laboratories indicate that the concentration of progesterone in the saliva provides a valid indicator of corpus luteum function. However; optimal conditions for the treatment and stprage of saliva specimens prior to analysis was not addressed in these papers. We have found that 1) sonication of saliva in brief bursts produces a homogeneous sample from which progesterone is removed quantitatively by a single extraction with hexane in a vortex mixer, and 2) prompt freezing of saliva is important since storage of samples at room temperature for 48 hours results in a significant decrease in radioimmunoassayable progesterone. Four normal women provided daily saliva specimens throughout one menstrual cycle and Excellent serum samples every 3 days during the luteal phase. correlations between the progesterone profiles in the two fluids were obtained. INTRODUCTION Recent

reports

from

of the

measurement

of

assess

luteal

function

progesterone

levels

hyperprolactinemia, been

shown

to

insufficiency The

the

concentrations in

of

women

attest

progesterone (l-4).

have

been

found

whereas

the

duration

be significantly

advantages

of

to

the

in the

in

low

anovulatory

of the

validity saliva

Abnormally

curtailed

salivary

or 24 hours adequately

heterogeneous

aggravated

laboratories

to saliva

cycles

progesterone

in women with

and peak

luteal

has phase

(5).

venipunctures discussed

several

on

urine

(5). nature

thawing

Volume 42, Number 5

But of

frozen

S

sampling collections too saliva. samples

=FIDEOXD=

little

regimens are

obvious

attention Moreover, whereby

over

repeated

and have has this

filamentous

been

been paid

problem

to is

material

November 1983

interferes

with in

progesterone Prolonged observation values

pipetting. saliva

stability

samples

would

cited showed

Another

in

permit

reference

little

or

concern maintained

mailing

of

5 suggested

no change

is

the

stability

at

room

specimens. that

on storage

temperature. An unpublished

salivary at

of

room

progesterone temperature

for

48 hours. The conditions and

to

purposes for

of the

this

investigation

pretreatment

reinvestigate

the

of

consequences

were saliva

to

samples of

storing

determine prior

to

samples

optimum analysis at

room

temperature. SUBJECTS AND METHODS Four women (ages 25-32) with no history of menstrual problems and no hormonal preparations volunteered for the study. Eath receiving In addition, blood samples contributed a daily 4 ml saliva sample. were obtaind at three day intervals starting at the estimated time of The saliva samples were promptly divided into two equal ovulation. portions. One aliquot was stored at -20“ C, wheres the other was maintained at room temperature for 48 hours and then frozen at -20" C. The sera were separated and frozen until analyzed. Preliminary studies were conducted to assess the effect of various pretreatments of saliva on the recovery of tritium after 160 Ci/mmol (New England Nuclear, Boston, addin (3H) progesterone, MA) ? 0 saliva. Each procedure was conducted with 0.4 ml saliva and where indicated, 3 ml of organic solvent. Following extraction of the (3H) progesterone in a vortex mixer, the tube was centrifuged and The organic phase was quickly rozen in a dry ice-acetone slurry. poured into a 6 ml plastic counting vial and evaporated under nitrogen. The radioactivty was determined in a Packard Tri-Carb scintillation counter, Model 3255 (Packard Instrument Co., Downers Grove, IL), after the addition of 0.2'ml ethanol and 4 ml Hydrofluor (National Diagnostics, Somerville, NJ). The regimens with saliva were as follows: 1. vortex, without extraction with organic solvent; 2. vortex after adding isopentane; 3. centrifuge at 1000 x g for 5 min and counting the supernatant fraction; 4. centrifuge and extra&ion of the supernatant with isopentane; 5. vortex in the presence of hexane intermittent ultrasonication (Model W 140, Branson Sonic Power and 6. co., Plainview, NY) at setting 6 for a total of 20-30 set, vortex extraction with hexane. Progesterone radioimmunoassay with 3 ml hexane. minimize emulsion

in (6).

saliva and serum was quantified by a standard Saliva (400 ul) was sonicated and extracted The mixture was vortexed in three short spurts to formation, centrifuged at 1000 x g for 5 min and

S

541

-X-REOXD-

quickly frozen. The hexane was poured into a 12 x 75 mm glass tube and -evaporated under nitrogen. With mixing, there were added sequentially, 10 ul ethanol, 100 ul (3H) progesterone (2000 cpm) in 0.9% NaCl and 0.1% each phosphate buffer (0.033 M, pH 7.0) containing of sodium azide and gelatin, and 100 ul of monoclonal antibody to kindly provided by Drs. R.D. Bulbrook and D.Y. progesterone (11 P 27, Imperial Cancer Research Fund Laboratories) diluted to a final Wang, The tube was incubated first at 37' C for concentration of 1:250,000. 45 min and then in an ice-bath for 45 min. Dextran-coated charcoal, 200 ul (0.5% washed Norit A containing 0.0005% dextran in phosphate buffer) was added and after vortexing, the mixture was returned to the The samples were centrifuged in ice-bath for an additional 30 min. the cold at 1250 x g for 10 min and 0.3 ml of the supernatant fraction was counted for at least 5 min in Hydrofluor. The counts per minute were converted to pg/ml saliva in the usual way by comparisons incremental O-150 pg progesterone standards. Sera were with quantified in a similar way with minor modifications. A mixture of 20'0 ul serum 200 ul hosphate buffer and 700 cpm (3H) progesterone (to correct for losses P was extracted with hexane. The hexane was One evaporated and the residue was dissolved in 50 ul ethanol. milliliter of buffer was added and the tube was incubated 30 min at 37” c. .To a plastic 12 x 75 mm tube were added 100 ul serum extract, 100 ul 'antibody (final concentration 1:120,000) and 100 ul (3Hb After incubation for 20 min at 37 progesterone (5000 cpm) in buffer. C and30min at4'C, 250 ul of dextran-coated charcoal were added. the mixture was Following further 20 min incubation in ice, centrifuged at 1250 x g for 10 min and 300 ul of the supernatant fraction was counted. RESULTS (3H) then

Progesterone treated

examined

was added

in

a variety

It

1).

(Table

heterogenous

mixtures

to

Simple

saliva.

samples

afforded were

diminished

recovery

Several results (1). activity

The

data is

prevail

ways

after

average

still

from

the

the

addition

with

by heterogeneity columns

accumulated

of

of

tritium

were

column

of

or room

temperature

particulate

the

isopentane

that

deviations

by

that

in

(11%). spurious

centrifugation

significant matter

standard

resulted

suggested

be obviated

that

progesterone

however,

have

data

(3H)

standard

saliva could

the

were

of

frozen

with

3 and 4 indicate by

first

recoveries;

unacceptable

working

The samples

and recoveries

Extraction

with

samples.

and pipetting

large.

in

saliva

can be seen

vortex

investigators caused

of

acceptable

deviations

to

derived

radiofrom

Tritiun

recovered

from

----------------------------------------------------------

frozen

Vortex

wa

saliva

samples

Vortex, extract

(14)

TABLE 1 mixed with regimens.

___-_--------_--_-------------------------------89.2 a13.5 78.8 k11.5

Centrifuge

(3H)

77.5 k3.6 (11)

progesterone

and then

submitted

Sonicate

to

99.5 ,t2.5b (11)

78.4 +lO.O 67.9 +19.6b (5) (10) ----------------------------

79.6 k21.2 (13)

--------------------------

Vortex

Extraction with hexane after -----------------------------

samples

-------------__-----------------------saliva

ex-

96.7 217.0 (4)

70.0 k13.6 (12)

Centrifuge, tract

Treatment of stored ---------------------------------Storage of samples Direct assay, or assay after extraction with isopentane --------------------------------------------------------------------------

Freshly

99.0 +5.6 (4)

percent kS.D. of added tritium recovered. samples shown in parentheses. were done on separate saliva specimens except for this category where an additional divided in half and one was frozen immediately, whereas the other was maintained for 48 h prior to freezing. The respective values after sonication and extraction and 54.1 k6.3.

Room temperature 98.0 k16.7 81.3 k11.1 48 h (5) (5) -----------------------------------------------------------------------Values indicate a. Number of b. All assays 5 samples were room temperature were 97.8 k4.6

various

---me-

at

freshly

frozen

indicate room

saliva.

Moreover,

considerable temperature in

samples

cannot

that

standard

of

eier,

we

geneous

that

consistently been

temperature but

brief

sonication

easy

to

pipet.

Specimens days

the

also

of

the

of

were

more

followed

con-

by

hexane How-

advantage. produces

a

100% of

phase

in samples

that

had

had been

maintained

at

room

homogeneous

were

extracted

the

homo-

Moreover,

rendered

tritium

indicated

gave

specimens

hexane that

unextracted

serum

isopentane

no particular

frozen.

two-thirds

than

of

The incon-

and

with

offered

is

two

extracted

samples

into

for

only

rather

4 samples

same way.

Our experiences

partitioned

freshly

the

deviations

Only

the

vortexing

that

mixture

in

Nevertheless, saliva

found

standard

samples.

examined between

hexane

results.

extraction

were

be explained. with

large

among

deviations

extraction

sistent

differences specimens

sistency

the

by

with

tritium

sonication

hexane

(Table

1). Sonication the

study.

saliva were

Figure

of

in

quickly

temperature profiles the

and

the

kept

at

sample samples assays

temperature

that

had been

obtained shown

in

in Figure

for

luteal

IC did

not

follow

in

which

the

of

one was

in the to

the

apparent

the

cycle.

this

pattern.

at

room

progesterone

feature

is

in the

standing

important found

for

Analyses

of

This

phase

half

of

compared

quickly.

cycle.

concentrations

progesterone 48 hours,

frozen the

the

adopted

progesterone

after

feature

Another of

in

was frozen

between

method

menstrual

divided

A striking

levels

of

a single

saliva.

basic

concentrations

other

48 hours.

lower room

the

and

was the

on samples

parallelism

serum

consistently

the

throughout

parallel

for is

extraction

I presents

4 volunteers

performed

frozen

in

and hexane

the

hormone is

saliva

the samples

corresponding only

in

the

Most

of

the

S

TREOXD-

FRESH FROZEN

6

2

10

14

16

22

26 2 6 DAY OF CYCLE

10

14

16

22

Figure 1. Daily concentrations of progesterone in saliva in the menstrual cycle and in the serum every third day during the presumed Saliva samples were luteal phase in normal women aged 25-33 years. one was frozen quickly and the other was divided into two parts; Each pair was frozen after standing at room temperature for 2 days. The subject shown in panel D estimated her analyzed simultaneously. luteal phase incorrectly. DISCUSSION The

data

throughout levels

the in

of

sparse

in corpus

progesterone

phase

in

progesterone

and the

confirm

and accurate

(5),

but function

saliva.

preliminary are

reflected

The

validation

have

been

saliva

the

that of

serum salivary

normal

luteal

fragmentary

indications in the of

in

with

findings

indicator

states the

profiles

comparisons

previous

pathological

luteum in the

on the cycle

a sensitive

Studies

reports

defects

luteal is

function.

here

menstrual

the

progesterone

the

reported

these

are concentrations preliminary

and that

?CXlEOX’D6

:s

findings

has

important

been

implicated

breast

disease

the

luteal

yield

as

attracting

technical,

relates

pipetting

glass

losses

wool of

centrifugation view.

or

provides

stability the

inexpensive. luteal concentration 48

hours.

population

of

deserve

to

for

a few

mixture by

relates at

room

to

exhibited on exposing Parenthetically,

the

the

mail

the

the

To

of

significant

"the

background

this factor

is

hexane.

from

saliva

of time. the

donor

convenient

samples

obtained in

noise"

in

period

both

room

removed

with

specimens

to

poor

following

progesterone

decline samples

The

troublesome

an extended

saliva

result

support

extraction

of the

filtering

could

progesterone

a process bulk

facilitate

obtained

this

of

the

purely

or

samples

which

for

service,

a

frozen

stability

transfer

first,

matter. we

single

and

procedures

obviates

temperature

permit via

a

The

saliva.

Both

from

to

volunteers.

particulate

seconds

the

personnel

centrifugation

freshly

to

convenience

of

(8).

may sample

delivered

comment.

to

saliva

trained

deviations of

Unfortunately, phase

cost,

standard

would

laboratory

of

during

study,

daily

requiring

bound

vortexing

feature

maintained

a

solids

quantitatively

second

of

until

malignant

sampling

advantage

resorted

remove

a homogeneous

essentially

to

to

large

Sonication

samples

have

daily

and stored

has

of

a prospective

heterogeneity

progesterone

or

recoveries

Such

the

investigators

through

The

to

study

development

a linkage,

in terms

our

insuffficiency

in

a large

of

the

venipunctures

apparent

phase

preferably

volunteer

daily

features

in such

The

by the

is

of

factor

cycle,

data.

serum

Two

and

the

over

capability 4.

in

of

frozen

laboratory,

Luteal

To confirm

definitive

obtain

a risk

(7).

phase

immediately

ramifications.

and in

the

progesterone

temperature of

the

for assay

precludes

making

nature the

of

the

was not

coincidental

not

that

samples

possible unextractable results room

by us is

highly

products

of

stand

at

products

with room appear

contradiction dictates

that

analyzed.

exists.

saliva

samples

kept

of frozen

of

for

the

to

2 days

was

to

the frozen

origin

difference

are

stored

the

monoclonal

antibodies

on allowing

saliva

so that appears

been

as g months

to these

a

puzzling

that

caution

frozen

has

at

ma;

However,

the

our

saliva

that

in

into

between

in

hexane,

values

added

progesterone

any ever&it

progesterone

than

freshly

periods.

be stored

more

bacterial

formed

with

as long

be

Non-specific

extended

best

for

to

is

progesterone

In

with

corresponds

progesterone

specific.

for

still

the

explanation

be unextractable

Constancy samples

One

temperature to

of

cross-react

temperature

enzymes

is

The

tritium

converting

stability

used react

or

phase.

may

the

fraction

for

(5).

cross

room

Unresolved

reported

of

as compared

agents

temperature

antibody

saliva

This

saliva

products.

and the

at

progesterone

causative

not it

one-third

maintained

in the

did

However,

hexane.

Enzymes

proliterative

that

investigated.

assayable

the

product(s)

were

with

specimens.

in

approximately

that

extracted

diminished

comparisons

transformation

antibody

saliva

similar

state

until

recorded

for

and by a

grant

(5).

ACKNOWLEDGEMENTS

from

This work the Guttman

was supported by USPHS Grant Breast Diagnostic Institute.

CA-02071

REFERENCES

1. Sorgo, (1983).

W.,

2. Connor, PHARMACOL. 3. Luisi,

M.L., Sanford, 60, 410 (1982). M., Franchi, F.,

Manella,

B.

and

Zachmann,

L.M.,

Howland,

B.E.,

P.M.,

Silvestri,

Kicovic,

M.,

HORMONE RES. I.7,

153

CAN. J. PHYSIOL. D.,

Cossu,

G.,

Catarsi,

A.L.,

Barletta,

D.,

and Gasperi,

M.,

J.

STEROID

BIOCHEM.

I4,

1069 (1981). 4.

Walker,

R.F.,

Read,

G.F.

and Riad-Fahmy, G.F., F.Z.

Walker,

D.,

CLIN. CHEM. 25, R.F.and

and Goebelsmann,

Griffiths, U.,

2030 K.,

STEROIDS 24,

Mauvais-Jarvis, P., Sitruk-Ware, R., Kuttenn, F. and Sterkers, N. RESEARCH IN BREAST DISEASE, Vol.1, R.D. Bulbrook and D.J. Taylor, Editors. Alan R. Liss, New York (1979), p. 25. 8. Cedard, L., Janssens, Y., MC Domough, P.G., Nessmann, C.,and Zorn, J.R. 30th ANNUAL MEETING OF THE SOCIETY FOR GYNECOLOGICINVESTIGATION, Washington, D.C., (1983), p. 158.

In: