0021-972X/92/7403-0698$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

UTILITY QUANTIFICATION

Vol. 74, No. 3 Printed

OF AN ORAL OF SALIVA

DIFFUSION

SINK (ODS) DEVICE

CORTICOSTEROIDS

IN HUMAN

FOR SUBJECTS

k2.3

James E. Shipley t, Norman E. Alessi t , Stephen E. Wade $, Albert D. Haegele $, and Beth Helmbold t t Department of Psychiatry, Box 0840, University of Michigan, 1500 East Medical Center Dr., Ann Arbor, MI 48109-0840 $ Hammersmith Laboratories, PO Box 774324, Steamboat Springs, CO 80477 ABSTRACT: Measurement of cortisol by assay of single blood or saliva samples is inherently imprecise due to the episodic secretion of cortisol. In addition, assay of blood usually quantifies total cortisol, rather than separating free hormone, which is proportionately the much smaller fraction. Furthermore, the free fraction may be disproportionately higher in hypercortisolism. Urinary free cortisol is one measure that provides both a time integral and a focus on the free fraction, but it is inconvenient and prone to collection error in unsupervised ambulatory subjects. The Oral Diffusion Sink (ODS) apparatus takes up corticosteroids from saliva according to first-order kinetics and may provide a practical alternative. We assessed the utility of the ODS in a study of seven healthy volunteers admitted to the CRC for three days. Data on day two from 0700 - 1100 h and 1100 - 1500 h were compared between the ODS and three other means of assessing cortisol: urinary free cortisol (UFC), blood, and saliva. The subjects all tolerated wearing the ODS device without any complaint. High correlations were observed between ODS values vs. data for UFC, plasma, and saliva determinations. In summary, the ODS device was well tolerated and collected reliable corticosteroid data, and thus provides a new, non-invasive methodology for studies of I-lPA function in health and disease.

Cortisol is the subject of investigations in normal physiology and various disease states. It may be measured simply and inexpensively by subjecting blood samples to analysis for total cortisol. The result does not distinguish the influence of bound (approximately 95%) versus free (5%) sources of hormone. Thus, despite the greater physiologic importance of free hormone, the test result is mainly representative of the bound fraction. Furthermore, in states of hypercortisolism, the increase in free fraction is disproportionately higher than the increase in the bound hormone [l], which only compounds the inaccuracy. A further issue with a single blood sample is that it provides only incomplete information about either secretory bursts or the cumulative time-integrated exposure Single saliva sample of tissue to the hormone. measurements are useful in that they correspond to the free fraction [2], but due to the presence of 11-hydroxysteroid dehydrogenase enzyme in the parotid gland, the relative quantity of cortisol (F) vs. its 11-dehydro metabolite cortisone (E) may be influenced by differential activity of this enzyme from individual to individual. Another measure of cortisol, urinary free cortisol (UFC), provides information about cumulative clearance of free cortisol, but it is cumbersome to obtain and prone to collection error in unsupervised ambulatory subjects. 1 Supported by MH42031, AA07378, and MOl-RR00042 and the facilities and staff of the Kughn Clinical Research Center of the University of Michigan Hospitals 2 submitted July 11,199l 3 reprint requests to the first author

One of us (S.E.W.) has developed a diffusion sink method for assessment of hormones in biological fluids [3, 41. This method has been applied to measurement of corticosteroids both in interstitial fluid of rats [3] and in saliva of humans [5]. In humans, measurements are made by use of an oral diffusion sink (ODS) device worn in the lateral gutter of the mouth, loosely attached to a molar by a length of dental floss. The device is cylindrical in form (2 mm x 15 mm) and is constructed with an outer tubular shell of polycarbonate plastic, perforations in which give access to a membrane of cellulose acetate formed on the inside of the polycarbonate tube. Inside this membrane is contained a quantity of anticorticosteroid serum. When the device is placed in a medium containing corticosteroids, these hormones are accumulated by the antiserum at a rate determined by the properties of the cellulose acetate this membrane (according to first order kinetics); The membrane excludes protein-bound steroids. accumulation of corticosteroids by an ODS device provides a direct measurement of the time integral of free corticosteroid concentration in the medium, under conditions like those in saliva and interstitial fluid [5]. Our initial investigation documented a high correlation (r = 0.78, p < 0.005) between total (F + E) corticosteroids in saliva pooled from frequent samplings vs. corticosteroids measured over the same time interval using the ODS [5]. The aims of the present study were to replicate this fmding and to determine the extent to which the ODS results would be correlated with two other types of corticosteroid assessments, namely UFC and the average of frequent (Q 20 min) plasma samples.

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METHOD Protocol: Seven male volunteers with a mean age of 27.6 * 5.3 and without evidence of medical or psychiatric illness or abnormal physical examination or screening laboratory were admitted to the CRC for three days. Beginning at 0700 h on day 2, an ODS device was left in place in each side of the mouth in the lateral gutter by affixing it to a molar. Pilot studies have demonstrated a high correlation between the results of the two sides (S. Wade, unpublished). Subjects could drink only water and no food was consumed while the ODS devices were in place. At 1100 h the ODS devices were removed, the subject ate lunch, and new devices were installed until 1500 h. During this 8 h study period, subjects had 2 ml of blood removed every 20 min via a heparin lock for determination in the clinical laboratory of the University of Michigan Department of Psychiatry of total cortisol by HPLC. On the same 420 min schedule, subjects provided 2 ml of saliva which was shipped on dry ice to Hammersmith Laboratories and pooled over respective 4 hr time blocks for analysis of total corticosteroids by HPLC [6]. Urine was collected over the respective 4 h time blocks and UFC was determined by HPLC in the Psychiatry Department clinical laboratory. ODS devices were refrigerated before use, and after use were shipped at ambient temperature in individual tubes containing 400 ul of 70% methanol to Hammersmith Laboratories for analysis [7]. From the quantity of E and F Data Analysis: accumulated by each pair of ODS devices and determined by HPLC [71, and the duration of exposure of the devices, we calculated the time-averaged total corticosteroid concentration of saliva, using the calibration previously established for these ODS devices [5]. This time-averaged total saliva corticosteroid concentration (hereafter ODS corticosteroids) was compared by Pearson correlation to the UFC, to the average of 420 min plasma cortisols, and to the total corticosteroid concentration in pooled Q20 min saliva samples, for each 4 h period (0700-1100 h and 1100-1500 h) in all subjects. Missing data occurred for one subject in the 0700 h-l 100 h time block and a different subject in the 1100 - 1500 h time block, making the sample size 6 subjects for each time block.

PEARSON CORRELATIONS FOR ODS VALUES VS. THE SAME TIME BLOCK FOR PLASMA, SALIVA, AND UFC CORTICOSTEROIDS TIME BLOCK 0700 h-1100 h SOURCE Mean Plasma Pooled Saliva UFC

1100 h-1500 h

.77*

.81*

.E**

.88**

.96**

* p-c.05

** p-c .Ol

.lJ - .20 22

. . . . * 24 26 28

. - .30 32

. . . 34 36

- - 1 38 40

ODSCorticosteroids(nmol/L)0700-11OOh Figure 1. Pearson correlation between the ODS corticosteroid determinationand UFC from 0700 -1100 h.

RESULTS The subjects all tolerated wearing the ODS device without any complaint. Total corticosteroids for the ODS device in general were highly correlated with corresponding determinations from mean plasma, pooled In the case of the saliva, and UFC (table below). correlation with plasma for the 1100 -1500 h time block, there was one outlier who had the lowest average plasma concentration (4.6 ug/dl). With this outlier removed, the correlation was r = 0.95 (p< 0.05). Scattergrams for the correlations between ODS corticosteroids and UFC from the respective time blocks are shown in figures 1 and 2.

ODSCorticosteroids(nmol/L) llOO-15OOh Figure 2. Pearsoncorrelationbetweenthe ODS corticosteroid determinationand UFC from 1100-1500 h.

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DISCUSSION The ODS device reliably documented corticosteroids in saliva. There was a high correlation between the ODS values and those from pooled saliva, UFC, or the mean of frequent plasma samples. This was true despite the fact that four different assays in two different laboratories were utilized for these four different types of specimens. We did not test the limits of detection of the ODS, since this study was conducted near the peak of circadian secretory activity, but pilot work (S. Wade, unpublished) has shown the device to be capable of detecting concentrations as low as 25% of the values observed in the present study. Thus, the device may be able to detect the lower levels expected during evening or night in circadian studies of corticosteroid secretion. The ODS device was well tolerated, and in fact no subject complained of irritation or other discomfort. The ODS is constructed of plastic and is radiopaque, so that in the unlikely event of a subject swallowing it, the device may be documented radiographically to have passed through the GI tract. The device is disposable, obviating any risk of disease transmission. The ODS device appears robust in clinical laboratory use. We were conservative and return-shipped the ODS devices using methanol as a preservative. Subsequent studies have shown that devices may be shipped for analysis at ambient temperature without methanol. The manufacturing procedure is reliable and permits batchwise calibration of the rate of uptake by the membrane [5]. The device and assay service are available commercially (Hammersmith Laboratories, Steamboat Springs, CO) at rates similar to other assays of cortisol. Potential applications of the ODS device include those situations in which the measure of interest is a time-integral of free cortisol secretion. This translates most closely to UFC determinations. Given the simplicity of storage and use of the ODS, it could someday provide a complementary or alternate method to UFC determinations. To further assess this potential, we are currently examining in a much larger group of subjects the correlation between UFC and ODS values for 3-4 hr blocks across the day. This may suggest during which circadian time period the use of the ODS will most reliably correlate with the level of cortisol secretion obtained by a 24 hr UFC.

The ODS may also have utility when corticosteroid secretion in saliva is specifically of interest, as in the recent studies of such abnormalities in psychiatric patients [8]. Dysregulation of cortisol secretion may be studied readily by means of saliva samples [9, 101, thus obviating the need for either cumbersome collection of urine or a stressinducing venipuncture procedure. REFERENCES 1. Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab. 1981;81:58-68. 2. Vining RF, McGinley RA. The measurement of hormones in saliva: possibilities and pitfalls. J Steroid Biochem. 1987;27:81-94. 3. Wade SE. A direct method for determination of the time integral of corticosterone availability in interstitial fluid of rats. Analyt Biochem. 1984;138:365-371. 4. Wade SE. Time-integrated measurement for determining substances in biological fluids. U.S. patent 4594,326; 1986. 5. Wade SE, Haegele AD. Time-integrated measurement of corticosteroids in human saliva by oral diffusion sink technology. Clin Chem. 1991;37:1166-1172. 6. Wade SE, Haegele AD. Differential measurement of cortisol and cortisone in human saliva by HPLC with uv detection. J Liq Chrom. 1991;14:1813-1827. 7. Haegele AD, Wade SE. Ultrasensitive differential measurement of cords01 and cortisone in biological samples using fluorescent ester derivatives in normal phase HPLC. J Liq Chrom. 1991;14:1133-1148. 8. Cook N, Harris B, Walker R. Clinical utility of the dexamethsone suppression test assessed by plasma and salivary cortisol determinations. Psychiatry Res. 1986;18:143-150. 9. Galard R, Gallart JM, Catalan R, Schwartz S, Argue110 JM, Castellanos JM. Salivary cortisol levels and their correlation with plasma ACTH levels in depressed patients before and after the DST. Am J Psychiatry. 1991;148:505508. 10. Guechot J, Lepine JP, Cohen C, Fiet J, Lemperiere T, Dreux C. Simple laboratory test of neuroendocrine disturbance in depression. Neuropsychobiol. 1987;18: 1-4.

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Utility of an oral diffusion sink (ODS) device for quantification of saliva corticosteroids in human subjects.

Measurement of cortisol by assay of single blood or saliva samples is inherently imprecise due to the episodic secretion of cortisol. In addition, ass...
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