Saliva as a Matrix for Measuring free Androgens: Comparison with Serum Androgens in Polycystic Ovarian Disease*

r

~

•

...

FERTILITY AND STERILITY Copyright © 1979 The American Fertility Society

Vol. 31, No.5, May 1979 Printed in U.S.A.

SALIVA AS A MATRIX FOR MEASURING FREE ANDROGENS: COMPARISON WITH SERUM ANDROGENS IN POLYCYSTIC OVARIAN DISEASE*

ROY G. SMITH, PH.D.t PAlIGE K. BESCH, PH.D. BUENA DILL, M.T. (A.S.C.P.) VEASY C. BUTTRAM, JR., M.D. Reproductive Research Laboratory, St. Luke's Episcopal Hospital, and Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, Texas 77030.

We report a simple and direct procedure for the measurement of circulating free testosterone concentrations by using saliva as a matrix rather than serum. There is a close correlation between saliva testosterone values measured by radioimmunoassay, calculated values of free testosterone, and free testosterone estimated by equilibrium dialysis. Our method is direct and has the advantage that the biologic fluid can be obtained routinely by noninvasive techniques outside the clinic during a course of therapy. We also show that a single saliva value is of greater diagnostic use than any of the currently used androgen assays. Testosterone was found to be elevated in the saliva of 17 infertility patients diagnosed as having polycystic ovarian syndrome, 14 of these patients were hirsute. Fertil SteriI31:513, 1979

It is now generally accepted that physiologically effective levels of steroids are reflected by the concentration that is not bound to plasma proteins. It is the "free" steroid which interacts with specific receptors in the target cells to elicit normal, or in certain cases abnormal, responses. For this reason it is the concentraton of "free" hormone which is most important diagnostically when a hormonally related abnormality is evident. Thus a reliable and simple method for the measurement of free steroids would prove invaluable for clinical evaluations. In our initial work we have decided to develop a method for the measurement of free circulating levels of testosterone. Although our methods will probably prove to be applicable for other steroids,

testosterone was chosen because of its obvious relevance to hirsutism and infertility in females. All of the methods currently used to estimate free testosterone, with the exception of equilibrium dialysis, are indirect ones. To estimate free testosterone indirectly, the concentraton of sex steroid-binding protein must be quantitated together with the total testosterone in serum. Serum testosterone is measured by radioimmunoassay and testosterone/estradiol-binding globulin (TEEG) is measured by any of a number of methods which include polyacrylamide gel electrophoresis, 1 gel filtration,2. a ammonium sulfate precipitation,4.5 charcoal adsorption,a. 6. 7 and diethylaminoethyl (DEAE) cellulose chromatography.7.s In addition to measuring total testosterone and TEBG, the equilibrium association constant for testosterone binding to TEBG must be known together with the amount of albumin in the serum and the equilibrium association constant for testosterone binding to albumin. Using all of these parameters it is possible to calculate free testosterone. 7 However, since this measurement is an indirect one it may prove to be inaccurate, as it has been shown that

Received April 3, 1978; revised December 22, 1978; accepted January 8, 1979. *Presented at the Thirty-Fourth Annual Meeting of The American Fertility Society, March 29 to April 1, 1978, New Orleans, La. tReprint requests: Dr. Roy G. Smith, Department ofObstetrics and Gynecology, Baylor College of Medicine, 1200 Moursund Avenue, Houston, Tex. 77030.

513

May 1979

SMITHETAL.

514

certainly in the case of drugs the ratio of free to protein-bound drug varies with age and disease states. This may be caused by such factors as altered albumin concentrations and the presence of compounds which displace the drug from the plasma proteins. 9 The determination of free testosterone by dialysis is frought with analytical and methodical problems since it is time-consuming and to determine losses accurately it is necessary to use a doubleisotope assay. Thus the procedure is too cumbersome for routine assays which may have to be repeated several times during diagnosis or during a course of therapy. Robin et al. 10 have recently developed a routine assay for the measurement of free cortisol in plasma by ultrafiltration. Their procedure is not suitable for measuring free testosterone, since the membranes used in the ultrafiltration adsorb testosterone. We have developed an assay for the direct measurement of free testosterone by utilizing saliva rather than serum. If the transfer of a compound from plasma to saliva is a passive process, then the saliva levels should correlate with the concentration of the non-protein bound compound. l l A good correlation of salivary levels reflecting the free level of digoxin, 12 cortisol,13 theophylline,14 and many other drugs has now been established. 15. 16 We have shown that a good correlation exists between the concentration of testosterone in saliva, as measured by radioimmunoassay, and free testosterone as determined indirectly by calculation and free testosterone as measured directly using equilibrium dialysis. 17 To establish such correlations, testosterone and TEBG were measured in 13 normal subjects and 17 subjects with diagnosed polycystic ovarian syndrome, of whom 14 were hirsute. MATERIALS AND METHODS

Saliva was collected by expectoration followed by centrifugation to remove debris. Blood was obtained by venipuncture, and the serum was collected after centrifugation. Both saliva and serum (1.0 ml) were extracted by agitating with ether (5.0 ml) for 10 seconds. After snap freezing at -800 C the organic layer was removed and evaporated under a gentle stream of nitrogen. Recovery from either saliva or serum was 90 ± 2%. Testosterone values were measured by usual radioimmunoassay (RIA) procedures with a specific testosterone antibody purchased from Wien Laboratories, Succasunna, N. J. Incubation was at 4 0 C for 15 hours,

and separation of bound from free testosterone was achieved with dextran-charcoal. The blanks determined by using either charcoal-stripped serum or charcoal-stripped saliva were <1 ng/dl and 1.3 ng/dl, respectively. A corresponding correlation was made in the saliva measurements. The measured values of testosterone in saliva are very low, and in the case of normal females the values approach the limit of sensitivity of the assay. For this reason each determimition was carried out twice in duplicate. The measured value was considered to be valid only when the interassay standard deviation was <10%. All calculations were performed with a Hewlett-Packard Mini-Computer 9815A, using an RIA program designed to give a linear regression analysis of a log-logit plot with recording of quality control parameters. The College of American Pathologists' Assurance Program was utilized as a reference basis with other laboratories for testosterone values. TEBG was determined by the procedure described by Rosner. 5 Equilibrium dialysis was performed by the method described by Forest et al.,17 using either undiluted serum or 5:1 diluted serum. When diluted serum was used, a factor correcting for the dilution of serum was applied. The dilution factor was redetermined for each assay. Radioimmunoassays for dehydroepiandrosterone (DHEA), androstenedione, and dihydrotestosterone were performed using highly specific antisera obtained from Radioassay Systems, Carson, Calif.; Miles Yeda, Elkhart, Ind.; and Dr. Rao of the Southwest Foundation, San Antonio, Tex., respectively. The concentration offree testosterone was calculated by substituting in the following equationsIS:

+ X2KA where KT = equilibrium association constant for TEBG, 3.5 x lOs M-l (reference 19); Xl = molar concentration of TEBG binding sites; b = molar concentration of testosterone in the serum; X2 = molar concentration of albumin binding sites, 5 x 10-4 M (reference 20); KA = equilibrium association constant of testosterone for albumin, 4.2 x 104 M-l (reference 18); and free = 111 + (bound/free) . Bound/free =

KT (xl - b)

b. RESULTS

It was first necessary to establish that a linear relationship exists between serum testosterone and the calculated value for free testosterone in the group of 13 normal females and the 17 females

f

l

SALIVA AS A MATRIX FOR MEASURING FREE ANDROGENS

Vol. 31, No.5

4.0 0.91 ~

"!i1

"-

3.0

... ~ ~

00

2.0

... ,

~ ~

:5

1.0

'"

30

•

110

90

70

50

SEPLIM T

NG/DL

FIG. 1. Correlation of total serum testosterone as measured by radioimmunoassay and free testosterone as calculated by the formula under "Materials and Methods": comparison of normal patients (.) with patients with POS (0) (P < 0.001).

diagnosed as having polycystic ovarian syndrome (POS) by laparoscopy. Figure 1 shows that there is a very good correlation (r = 0.91; P < 0.001) between calculated free testosterone and serum testosterone in both groups of subjects, although there does appear to be a greater spread of values among the subjects with POS. Figure 2 shows a plot of calculated free testosterone versus testosterone as determined in saliva. There is a very significant correlation in both groups of subjects (r = 0.83; P < 0.001), but again there was a greater deviation in the POS subjects. Interestingly, the slope of the curve did not equal 1, rather the slope was 0.47. This difference in slope from the predicted value is perhaps indicative of either active transport of testosterone from serum to saliva or a nonlinearity in the measurement of the low values of testosterone in saliva as

compared, with the higher serum values. The addition of known amounts of testosterone to saliva and the reconstitution of charcoal-stripped saliva with testosterone, however, showed that the RIA was indeed linear at low testosterone concentrations. In addition, Scatchard analysis 21 of saliva showed that no binding proteins with a high affinity for testosterone were present in saliva. Thus we do not have a simple explanation for this apparent anomaly other than some form of active transport phenomenon. This anomaly is not necessarily important, since Figure 2 clearly shows that the saliva value is directly proportional to the calculated value. The significance of the saliva assay was also confirmed when free testosterone as measured by equilibrium dialysis was compared with testosterone values. It can be seen from Figure 3 that when free testosterone was estimated a good correlation existed between the two methods. A theoreticalline has been drawn through the points (slope = 1) to illustrate the close correlation. In our hands, however, it was much more difficult to obtain reproducible values by equilibrium dialysis, and each specimen had to be assayed in duplicate at least three times. The utility of salivary testosterone values is clearly shown in Table 1, which represents the androgen profiles of 15 patients with POS. If one considers the DHEA, androstendione, 5a-dihydro-

~ 0\ c:

6

•

NOR~ALS

5

Z

R

/

/ /

4

IrJ'l

3

0

/ /0

0

0 0

...

E1! Q

>-'"'

~ ~

:5

0

0

I./'l l.&J

0

•

2 ./

•

.-

-- -

/

0

2.0

P

0

/

I-

0

2.0

0

: "-.

I-

~

"z '"

"-

/

/ /00

0: W

0.83

=

0

/

o POS

l.&J

4.0

515

••,

0

J/ •

/

0 0

00 Li.O

6.0

SALIVA T NG/DL

FIG. 2. Correlation of saliva testosterone as measured by radioimmunoassay and free testosterone as calculated by the formula under "Materials and Methods": comparison of normal patients (.) with patients with POS (0) (J' < 0.001).

3 SALIVA FREE TESTOSTERONE., ng/dl

FIG. 3. Correlation of "free" testosterone as determined by equilibrium dialysis '7 and testosterone as measured in saliva by radioimmunoassay: comparison of normal patients (.) with patients with POS (0) (R = 0.82; slope = 0.78; P < 0.001). The theoreticalline (---) is drawn with a slope = 1, which would be perfect correlation.

SMITHETAL.

516

May 1979

TABLE 1. Androgen Profiles of Fifteen Patients with POSa DHEA

DHT

(SID-3380)

Androstenedione (0.7-3.1)

(135-280)

(1.1>-2.1)

TEBG

T (28-55)

SalivaT (0.4-2.4)

nglmJ

nglml

pglmJ

pg DHT boundl
ngl
ngl
2734 1094 3388 2171 2968 4125 1334 12661' 4744 2035 3513 2876 3399 781 c 1714c

3.7 2.7 3.4 4.3 3.4 4.8 3.4 2.3 3.6 3.6 4.8 4.1 5.2 1.9 3.1

421 370 392 434 120 520 317 226 362 388 273 371 393 202 282

0.9

97 83 73 95 108 101 90 42 65 75 85 78 86 42 55

5.6 3.1 4.0 4.9 5.9 5.1 2.4 2.6 3.4 3.0 4.2 3.0 4.9 3.1 3.2

1.1

0.7 1.1

0.7 0.9 1.0 0.8 0.4 0.6 1.0 1.5 1.4 1.3 1.7

Saliva/total T (1-3.S)

X

100

5.8 3.7 5.5 5.2 5.5 5.1 2.7 6.2 5.2 4.0 4.9 3.9 5.7 7.4 2.7

aSteroids in the serum of each patient were determined by radioimmunoassay using antisera highly specific for each hormone. TEBG was measured by the ammonium sulfate precipitation method described by Rosner. 5 The normal ranges are given in parentheses. bNot hirsute. cNot hirsute but on prednisone therapy.

testosterone (DHT), TEBG, and testosterone values in serum there is no single parameter which could be used diagnostically. However, a comparison of testosterone saliva and the percentage of saliva testosterone as compared with serum testosterone does appear to be diagnostic. Saliva testosterone was also measured in a patient with amenorrhea and ovarian hyperthecosis. Prior to wedge resection of both ovaries, her saliva concentration of testosterone was 16.7 ng/dl and the calculated free concentration was 14.2 ng/dl. After surgery the saliva value decreased to 7 ng/dl, and within 6 weeks the patient resumed menses. DISCUSSION

We have shown a clear correlation between free testosterone and testosterone as measured in saliva by RIA. The saliva values are directly proportional to serum free testosterone; thus by a single assay we can determine very simply the physiologically important concentration ofthis hormone. Furthermore, in patients with polycystic ovaries, free testosterone appears to be diagnostic since all of the patients had elevated testosterone concentrations in their saliva. The assay is potentially more meaningful than calculated values for free testosterone. The calculations 7 assume a normal TEBG molecule, a normal albumin molecule, a normal concentration of albumin, the absence of any agents which might affect the equilibrium association constants for testosterone, and the absence of any agents which

might displace testosterone from the serum binding proteins. A major significance of this report is that, by using saliva as a matrix, multiple sampling by noninvasive techniques can be carried out by nonclinical personnel. Furthermore, Goldzieher et aJ.22 have demonstrated that to achieve adequate sampling of blood for the accurate measurement of testosterone it is necessary to pool three samples drawn at 6- to 16-minute intervals. This sampling problem is overcome by using saliva, which is generally collected over a 30-minute period, and the results from the saliva assay are more physiologically relevant than the serum values. The results confirm our preliminary observations of the relevance of steroid concentrations in saliva. 23 Acknowledgments. We wish to acknowledge the research support of St. Luke's Episcopal Hospital, Texas Medical Center, Houston, Texas, to the Reproductive Research Laboratory, and Dr. A. N. Poindexter for his collaboration.

REFERENCES 1. Corvol PL, Chramback A, Rodbard D, Bardin CW: Physical properties and binding capacity of testosterone-estradiolbinding globulin in human plasma determined by polyacrylamide gel electrophoresis. J BioI Chern 246:3435, 1971 2. Vermeulen A, Verdonck L, Van der Straeten M, Orie N: Capacity of the testosterone-estradiol-binding globulin in human plasma and influence of specific binding of testosterone on its metabolic clearance rate. J Clin Endocrinol Metab 29:1470, 1969 3. Kato T, Horton R: Studies oftestosterone binding globulin. J Clin Endocrinol Metab 28:1160, 1968

Vol. 31, No.5

SALIVA AS A MATRIX FOR MEASURING FREE ANDROGENS

4. O'Connor S, Baker HWG, Dulmanis A, Hudson B: The measurement of sex steroid binding globulin by differential ammonium sulphate precipitation. J Steroid Biochem 4:331,1973 5. Rosner W: A simplified method for the quantitative determination of testosterone-estradiol binding globulin in human plasma. J Clin Endocrinol Metab 34:983, 1972 6. Rosenfeld RL: Plasma testosterone binding globulin and indexes of the concentration of unbound plasma androgens in normal and hirsute subjects. J Clin Endocrinol Metab 32:717, 1971 7. Weist WG, Paulson JD, Keller DW, Warren JC: Free testosterone.concentration in serum: a method for determination. Am J Obstet Gynecol 130:321, 1978 8. Mickelson KE, Petra PH: A filter assay for the sex steroid binding protein (SBP) of human serum. FEBS Lett 44:34, 1974 9. Dvorchik BH, Vesell ES: Pharmokinetic interpretation of data gathered during therapeutic drug monitoring. Clin Chern 22:868, 1976 10. Robin P, Predine J, Milgrom E: Assay of unbound cortisol in plasma. J Clin Endocrinol Metab 46:277, 1978 11. Kien P: In Concepts in Biochemical Pharmacology. Berlin, Springer-Verlag, Part 1, p 213 12. Huffman DH: Relationship between digoxin concentrations in serum and saliva. Clin Pharmacol Ther 17:310, 1975 13. Walker RF, Riad-Fahmy D, Read F: Adrenal status assessed by direct radioimmunoassay of cortisol in whole saliva or parotid saliva. Clin Chern 24:1460, 1978 14. Koysooko R, Ellis EF, Levy G: Relationship between theophylline concentration in plasma and saliva of man. Clin Pharmacol Ther 15:454, 1974

517

15. Cook CE, Amerson E, Poole WK, Lesser P, O'Tuama L: Phenyltoin and phenobarbital concentrations in saliva and plasma measured by radioimmunoassay. Clin Pharmacol Ther 18:742, 1975 16. Horning MG, Brown L, Nowlin J, Lertratanangkoon K, Kellaway P, Zion TE: Use of saliva in therapeutic drug monitoring. Clin Chern 23:157, 1977 17. Forest MG, Rivarola MA, Migeon CJ: Percentage binding of testosterone, androstenedione and dehydroisoandrosterone in human plasma. Steroids 12:323, 1968 18. Pearlman WH: Measurement of testosterone binding sites. In Karolinska Symposia on Research Methods in Reproductive Endocrinology, 2nd Symposium: Steroid Assay by Protein Binding, Edited by E Diczfalusy. Stockholm, Karolinska Institute, 1970, p 225 19. Rosner W, Smith RN: Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma. Use of a novel affinity column. Biochemistry 14:4813, 1975 20. Diem K, Lentner C (Editors): Documenta Geigy Scientific Tables, Seventh Edition. Basle, Ciba-Geigy Ltd, 1970, P 583 21. Scatchard G: The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660, 1949 22. Goldzieher JW, Dozier TS, Smith KD, Steinberger E: Improving the diagnostic reliability of rapidly fluctuating plasma hormone levels by optimized multiple-sampling techniques. J Clin Endocrinol Metab 43:824, 1976 23. Besch PK, Smith RG, Besch NF, Buoy ME: Saliva as a matrix for determining circulating levels of free steroids and drugs. Physiologist 20:9, 1977