On the stability of salivary progesterone under various conditions of storage

967

ON THE STABILITY OF SALIVARYPROGESTERONE UNDERVARIOUS CONDITIONS OF STORAGE Sila Banerjee and Mortimer Levitz Department of Obstetrics and Gynecology New York University School of Medicine New York, New York 10016 and

Car] R. Rosenberg Department of Environmental Medicine New York University School of Medicine New York, New York 10016 Received 12-18-85 Revised 3-4-86

ABSTRACT

The concentrations of progesterone in saliva of women exhibited significant decreases when the fluid was stored in plastic vials for 3 days at room temperature or 37 C. The addition of antibiotics or a variety of metabolic poisons to the saliva prior to storage did not prevent the progesterone decrement. However,the addition of albumin (2 g/dl) was protective, suggesting that the protein impeded adsorption of salivary progesterone by the plastic container. Saliva could be maintained at 31 C for 3 days in glass vials or at -20 C in plastic containers for indefinite periods without loss of progesterone t i t e r s . These data indicate that a patient under ]uteal function assessment may collect saliva samples in glass vials at regular intervals during the latter half of her cycle and store them in the freezer compartment of the refrigerator until shipment by mail to the laboratory for progesterone assay. With special care, plastic vials charged with albumin may also be used. INTRODUCTION I t has been documented that the concentrations of progesterone in the saliva may serve as a valid indicator of luteal function in (1-5).

women

The advantages of salivary sampling regimens over repeated

venipunctures

or

24- hour

urine

collections

are obvious.

But

inadequate attention has been paid to the elucidation of conditions by December 1985

Steroids

Volume 46, Number 6

968

~

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I ~I

which the patient could collect and store specimens and send them to the

laboratory

without

for

loss of

assay with

minimum inconvenience and expense

progesterone t i t e r ,

Expediencyand c o s t can be

accommodated by mail shipment, but there are no reports of systematic studies designed to determine whether saliva progesterone is stable to the

conditions

unpublished

normally encountered in

observation cited

in

mailing.

a

However in

recent

review,

it

an was

reported that salivary progesterone values undergo l i t t l e or no change on standing at room temperature for 48 hours (6). neither

d a t a nor

specific details

Unfortunately,

were presented.

The present

report expands on this observation and presents evidence that saliva progesterone t i t e r s days,

are remarkably stable at 37 C at

provided the

samples are stored in

glass,

least not

for

3

plastic.

However, progesterone s t a b i l i t y similar to that observed in glass was achieved

when saliva

was stored in plastic

in

the

presence of

albumin. MATERIALS AND METHODS Vessels: Two plastic containers and one glass container were tested as short- term storage vessels for saliva. The plastic vials were polyethylene (40-217 Mini Vials, Nuclear Associates, Carle Place, NY) and polypropylene (60.542, Sarstedt Inc., Princeton, NJ). The glass vials (986492) were from Wheaton Scientific, M i l l v i l l e , NJ. They were chosen because of convenient volume (6-7 ml) and cost, which is relatively low since they are used extensively as mini-vials in s c i n t i l l a t i o n counting. In addition, we are continuing to test the s t a b i l i t y of salivary progesterone on long-term storage at -20 C in 2 ml vials (72.694, Sarstedt, Inc.). Saliva samples and progesterone assays: Specimens were obtained from normally cycling women in their mid-luteal phase. The samples were frozen for 2-72 hours and submitted to ultrasonication as described previously (5). One-milliliter aliquots were exposed to a variety of conditions designed to inhibit degradation of progesterone. Progesterone was quantified by RIA (5,7). Treatments

of

saliva

stored in

plastic:

Significant

losses in

~

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x "~ i

969

progesterone t i t e r s were observed with saliva samples stored in polyethylene for 3 days at room temperature (5). The samples were divided into two groups of two. One group was treated according to one of the regimens cited below (see A to E) and the other was untreated. Then, within each group, one sample was kept at room temperature and the other at -20 C. After 3 days the samples were assayed for progesterone and the values were compared. The treatments were as follows: A-Antibiotics. A mixture of penicillin-streptomycin and 100 ~g/ml, respectively) was added.

(100 units

B-Chemical preservatives. In each study, one of the following compounds was added to the final concentration indicated: sodium azide (0.1%); EDTA (2 mg/ml); sodium b i s u l f i t e (5 mg/ml); cacodylic acid (32 mg/ml); sodium arsenate (33 mg/ml). C-pH modification. Sulfuric acid (0.05-0.1%) to pH 3-6. D-Sterilization. The sample was immersed in boiling water for 30 min. E-Albumin. The essentially f a t t y Co., St. Louis, MO) as described in the

saliva sample was added to the vial containing acid-free bovine albumin (A-6003, Sigma Chemical to a final concentration of 2 g/dl, then processed text.

Stability o f salivary progesterone i__nnglass vials - comparison with : Saliva samples were sequentially frozen, thawed and icated. One-milliliter aliquots were placed in glass and stored for 3 days either at -20 C, room temperature or 37 C. Two additional aliquots w e r e placed in either polyethylene or polypropylene vials and stored at -20 C or room temperature for 3 days. Then the samples were assayed for progesterone. RESULTS AND DISCUSSION In a previous study (5) we showed that storage of saliva in

polyethylene

vials at

samples

room temperature for 3 days resulted in

about a 30% decrease in progesterone t i t e r s .

Exploring the possibility

that microorganisms in the saliva were responsible for this phenomenon, antibiotics, metabolic poisons, s t e r i l i z a t i o n and lowering the pH were examined as possible means of preserving progesterone t i t e r s .

None of

these

results

approaches was successful.

which are not shown: 1.

To b r i e f l y summarize the

The decrease in all specimens exposed to room

temperature for 3 days was in the 30% range.

2.

None of these agents

8 T ~ 1 o i ~ s

970

per se caused the decrease. These data posed the possibility that a component of the plastic, perhaps

a plasticizer,

immunogenic. decrease

modified the progesterone, rendering i t less

An alternative, perhaps more likely, explanation for the

is that progesterone was partially adsorbed by the

I t has been recognized for several years that the addition of

plastic. albumin

to aqueous solutions containing nonpolar steroids such as progesterone impedes or even prevents uptake of the solute by the wails of vessels or tubing (8). in

Parenthetically,

we had found that 1-56% of 3H added

the form of 3H-progesterone to saliva was unextracted with organic

solvents after storing the fluid in plastic vials at room temperature for

2 days (5).

Table I

shows the

comparison of

the

salivary

progesterone t i t e r s when the samples were stored in polyethylene vials in the

presence and absence of albumin, at temperatures likely to be

encountered

by samples that

are mailed. The values are similar for TABLE 1

Salivary progesterone concentrations following storage in plastic vials with and without albumin Storage Conditions Sample

Glass -20 C

1 2 3 4 5 6

194 208 189 101 242 85

Plastic -20 C Rm Temp 37 C 72 h 200 188 74 225 75

115 102 26 104 33

33 58 13 50 13

Albumin in Plastic -20 C Rm Temp 37 C 72 h 167 192 230 90 176 103

169 163 156 78 165 73

157 154 188 64 179 112

Values in pg/ml are the means of assays done in duplicate. The deviations were less than 10% except f o r samples 2 and 4, column 5 in which the deviations were 25%. The p l a s t i c v i a l s were of polyethylene. Blank spaces denote that samples were not analyzed.

T

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z ~

971

samples maintained in the frozen state, whether in glass or plastic in the

presence or absence of albumin. For samples stored in

exposure for

3 days at 25-37 C resulted in a large

progesterone t i t e r s .

The addition of 2 g/dl

sample stored in plastic generally

of

decline

immunoassayable progesterone. However, there are

at

25-37 C,

considerable

diminution

albumin

prevented the

albumin mode of preventing progesterone loss.

plastic,

to

of the

in radio-

drawbacks to the

Upon storage for 3 days

the sample develops an offensive c a r e and labor must go into the

odor.

Furthermore

regulation

of

final

albumin concentrations witin reasonable limits in vials that would be mailed bidirectionally samples containing necessitating

between laboratory

and patient.

albumin formed gels on extraction

with

a second extraction with solvent - a step not

Finally, hexane, required

with samples containing no protein supplements. Although glass

is

fragile and slightly

more expensive than

plastic, i t is s t i l l acceptable in a large-scale screening program for detecting

abnormalities

in ]utea] function.

Greater precaution

prevent breakage during mailing would be dictated. in

Table 2 demonstrate the remarkable s t a b i l i t y of

saliva stored in glass vials. or

the

temperature

to

storage or

at

-20 C in

1,

glass,

37 C for 3 days in

radioimmunoassayable progesterone. -20 C in plastic.

The data presented progesterone in

From analysis of the individual results

normalized averages shown in Table 2,

compared

to

glass

it

saliva

is

evident that

stored

at

room

showed no decrease in

This was also true for storage at

On the other hand,

as in the study shown in Table

the progesterone concentrations diminished significantly (p< 0.001)

when the

specimens were stored in plastic at room temperature.

We

8

972

~m~m~aoi~s

TABLE 2 Salivary progesterone concentrations following storage in glass and plastic vials under various conditions Storage Conditions Sample -20 C

Glass Vials Rm Temp 72 h

Plastic Vials -20 C Rm Temp 72 h

37 C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

115 145 96 120 114 142 133 128 154 94 133 125 165 128 250 205

100 135 93 108 116 144 130 124 156 78

113 145 101 120 112 146 140 127 156 86

Mean

100

94.9•

100•

109 160 88 125 93 142 130 125 123 67 125 130 160 131 230 206 94.9•

47 78 35 59 54 121 90 76 112 39 103 123 148 110 165 165 65.6•

Values in pg/ml are the averages of assays done in duplicate. The deviations were genera]|y in the 10% range. Blank spaces denote that samples were not analyzed. Means• are relative to values for glass at -20 C normalized to 100. Comparing the mean decline in radioimmunoassayable progesterone from -20 C to room temperature between glass and plastic vials s t a t i s t i c a l l y : t = 4.32 (p=0.00012). Samples 1-10 were stored in polyethylene (Nuclear Associates) 11-16 were in polypropylene (Sarstedt, Inc.). have a study under way to test the in

s t a b i l i t y of progesterone stored

polypropylene vials over the long term.

progesterone

titer

Thus far no diminution in

has been observed over 9 months in

samp|e maintained at -20 C.

and

a

pooled

9g " m

It

is

emphasized that

progesterone. of

=to

9

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973

the present study focused only on

No conclusions may be drawn concerning the s t a b i l i t y

other salivary steroids stored in either

For example, the concentrations of

glass or plastic

estrio] in the saliva of pregnant

women have been reported to be stable after storage in vials

for

vials.

10 days at room temperature (9).

In

polyethylene

that

s t u d y the

authors rendered the saliva 10% with respect to sodium chloride

upon

receipt of the samples. We have adopted the luteal function in women. samples

of

her

specimens

Starting

saliva are collected

menstruation. in

following regimen for the

with day 10 of the cycle, 1-2 ml in glass vials every 2 days until

The subject stores the

samples in the frozen state

home refrigerator until the cycle is are

mailed

to

the

assessment of

completed.

laboratory

for

Then

the

analysis,

Parenthetically, no change in progesterone values has been observed in saliva

specimens stored upside down in

glass vials

aluminum-lined

polypropylene screw caps.

In

under way, 11

women have thus far complied with

Considering progesterone profiles as the indicator,

a

covered by pilot

study

this

schedule.

luteal

status in

each subject could be assessed.

ACKNOWLED(~MENTS This work was supported by USPHSgrants CA-34588 and CA-02071 and by a grant from the Guttman Breast Diagnostic Institute. The monoclona] antibody to progesterone, 11P27 (10), was kindly provided by Drs. R.D. Bu]brook and D.Y. Wang, Imperial Cancer Research Fund Laboratories.

974

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mm

REFERENCES I. 2.

Sorgo, W., Manella, B.

(1983).

and Zachmann, M., HORMONERES. i__7, 153

Connor, M.L., Sanford, L.M. and HowIand, B.E., CAN. J. PHYSIOL. PHARMACOL. 60, 410 (1982). 3. Luisi, M., Franchi, F., Kicovic, P.M., SiIvestri, D., Cossu, G., Catarsi, A.L., Barletta, D. and Gasperi, M., J. STEROIDBIOCH~. L4, 1069 (1981). 4. Walker, R.F., Wilson, D.W., Truran, P.L., Read, G.F., Richards, G., Walker, S.M. and Riad-Fahmy, D., J. ENDOCRINOL.104, 441 (1985). 5. Banerjee, S. and Levitz, M., STEROIDS42, 539 (1983). 6. Riad-Fahnly, D., Read, G.F., Walker, R.F. and Griffiths, K., ENDOCRINE REV. 3, 367 (1982). 7. Katagari, H., Stanczyk, F.Z. and Goebe]smann, U., STEROIDS24, 225 (1974). 8. Levin,J., Friedrich, E.H. and Lobotsky, J., J. CLIN. ENDOCRINOL. METAB. 25, 1519 (1965). 9. Besch, N.F., Huang, N.H., Nguyen, H., Wait, R.B. and Besch, P.K., in:Immunoassa~s of Steroids in Saliva (Read, G.F., Riad-Fahmy, O., Walker, R.F. and Griffiths, K., Editors), Alpha Omega Publishing Ltd., Cardiff, Wales, UK (1982), p 10. 10. Fant], V.E., Wang, D.Y. and Knyba, R.E., J. STEROIDBIOCHEM. i__77, 125 (1982).