Quantification of the Salivary Steroid Hormones

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Current Pharmaceutical Analysis, 2010, 6, 182-197

Quantification of the Salivary Steroid Hormones Considered Bio-markers in Clinical Research Studies and Sports Medicine

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Alina Plenis* and Tomasz Baczek Department of Pharmaceutical Chemistry, Medical University of Gdask, Gdask, Poland Abstract: Steroid hormones are important in controlling human body functions as a part of the endocrine system together with neuronal systems and the immune system. Application of the assay of the steroid hormones treated as biomarkers was recently illustrated in certain cases, for example in clinical diagnosis of stress, the Cushing syndrome, congenital adrenal hyperplasia, and infertility, as well as in the field of sports medicine. The assessment of the steroid hormones in the body fluids has so far been typically based on serum and urine. However, the use of saliva as the diagnostic medium has recently grown in popularity among the scientists and clinicians because of sample collection, which is quick, uncomplicated, and non-invasive. Moreover, steroid hormones are not bound to protein in saliva. Therefore, salivary determination is an excellent approach for evaluation of free steroid hormones. The present study provides an overview of the analytical methods applied for salivary steroid measurements in the current clinical laboratory practice. It describes and thoroughly discusses the recent achievements associated with optimisation of the analytical conditions for the steroid assay, obtained through application of the modern separation techniques such as liquid chromatography, mass spectrometry, versus non-separation techniques such as the immunological methods. Moreover, the issues associated with optimization of the extraction procedures are presented, since sample pre-treatment is the most limiting and crucial step in analyses of biological fluids. In addition, the study evaluates the consequences of any pre-analytical variation preceding the application of the assay methodologies, stemming from the collection strategy and the subsequent storage conditions. It further provides several examples of application in diverse fields of interest such as psychology, pharmacology, clinical endocrinology, or sports medicine.

Keywords: Saliva, Biomarkers, Steroid hormones, Clinical research studies, Sports medicine, Separation and non-separation techniques. 1. INTRODUCTION Saliva attracted the researchers’ attention many years ago, when sensitive and selective radio- [1-3] and enzymeimmunoassays [4-6] became widely available. Over the following decade researchers explored the characteristics of saliva as a biological fluid in terms of steroid hormone measurements. The results of those studies were described in reports, also in a number of reviews [7-9]. In the function of a diagnostic fluid for determination of the steroid hormones, saliva represents an alternative matrix collectible in a non-invasive and more convenient way than blood or even urine. The collection procedure of the salivary samples is neither painful nor traumatic and, the oral fluid sampling is safe for the operator and the patient, and no special training or equipment is necessary. The storage of the salivary samples is easy and low-cost. In addition, if required, the patients can conveniently collect the samples themselves. These characteristics enable monitoring several biomarkers in special populations (e.g., infants, children, elderly and non-collaborative subjects), as well as in many circumstances when sampling the serum, plasma or urine is impossible. This type of testing may serve determination of *Address correspondence to this author at the Department of Pharmaceutical Chemistry, Medical University of Gdask, Hallera 107, 80-416 Gdask, Poland, Tel: +48 58 3493131; Fax: +48 58 3493130; E-mail: [email protected] 1573-4129/10 $55.00+.00

the concentrations of the endogenous steroid compounds that may signal abnormal physiological functioning. Moreover, salivary fluid testing may be used in diagnosing disease, following the progress, remission and reoccurrence of the disease, monitoring the hormones in therapy, and detecting any illegal steroid hormones in doping treatment. The salivary fluid contains many steroids of interest also found in plasma, and in many cases reflects the free fraction of the circulating steroid hormones [7,10]. Therefore, it has become a popular sampling fluid in psychobiology, pharmacology, and sports medicine. However, using saliva as diagnostic fluid has some disadvantages. For most biomarkers the levels of the salivary steroid hormones are about 10–100 times lower than their concentrations in the blood, hence very sensitive analytical methods are required. Other disadvantages of the salivary steroid assays include limited specimen volume, interferences from other exogenous steroids, food and/or beverages, and no standardised testing procedures for the collection and storage of the saliva samples. This study provides an overview of the current status and future applications of the salivary steroid hormones. The structures of these hormones are shown in Fig. (1). It offers a detailed description of the well-established procedures, the recent progress in the analytical methods applied for salivary steroid assays, and the numerous applications of the methods in the clinical laboratory practice and sports medicine. Fig. (2) shows the statistics data on the percent of publica© 2010 Bentham Science Publishers Ltd.

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[14]. Similar effects accompany e.g. the host factors (hereditary inclinations and oral hygiene), the use of medication, physical exercise [15], the method of sampling, and the time of the day. Consequently, the composition and consistency of saliva can vary both quantitatively and qualitatively [16,17]. The components of saliva further include steroid hormones which pass through the cell membranes into the salivary glands by diffusion [10,11]. Unconjugated steroids, being highly lipid-soluble molecules, are transferred due to their solubility in the lipid-rich cell membranes [18]. This type of passive intracellular diffusion remains unaffected by the saliva flow rate. In many cases high correlation in terms of the hormone concentration levels has been confirmed for saliva and blood [7,10,19-21]. On the other hand, polar molecules enter saliva together with water in the process of ultrafiltration. Because the molecule size is a major factor in the process, the level of larger lipid-insoluble molecules such as the conjugated steroid hormones is highly dependent on the salivary flow rate. For example, the level of the insoluble lipid of dehydroepiandrosterone sulphate (DHEA-S) reflects less than 1% of the non-protein-bound plasma concentration [10].

Fig. (1). Chemical structures of steroid hormones.

tion devoted to the subject of steroid hormones considered as biomarkers in clinical research studies and sports medicine since 1990. Moreover, salivary steroid level measurements and their corresponding biomarker diagnostic implications in psychology, clinical endocrinology, stress research studies, and sports medicine are critically discussed. 1.1. Hormone Transport to Saliva In humans, saliva fulfils several functions involved in maintaining oral health and homeostasis. It plays an actively protective role in lubrication, tissue coating, digestion, and interacts with the microorganisms found in it [11]. Saliva is a complex fluid comprising secretions of the major salivary glands (parotis, submandibularis and sublingualis) and a large number of minor salivary glands. The glandula parotis produces a serous fluid, the glandula submandibularis a seromucous secrete, the glandula sublingualis specialises in secreting nothing more but mucous saliva, whereas the minor glands secrete viscous secrete [12]. Other saliva components are of non-glandular origin, to name but bronchial and nasal mucosal secretions, gingival crevicular fluid, microorganisms, and food particulates [13]. Healthy adult subjects normally produce 500–1500 ml of saliva per day at a rate of approximately 0.5 ml/min, however certain physiological conditions (e.g. anger or fright) can alter saliva production

Moreover, the levels of the steroid hormones of cortisol and cortisone are dependent not only on the transport into saliva, but also on the activity of two isoforms of 11 hydroxysteroid dehydrogenase (11 -HSD). One of the two isomers, 11 -HSD type 1, acts predominantly as an 11oxo-reductase (i.e. converts cortisone to cortisol), while 11 -HSD type 2 catalyzes 11 -dehydrogenation, that is the opposite reaction (i.e. inactivation of cortisol to cortisone) [22]. This enzymatic conversion may alter the proportions between salivary and plasma free cortisol, and as such may explain certain diversity in the results obtained in different assays. This problem is of particular significance in the case of data obtained under the immunological methods which are based on cross-reactivity between the antibodies. In these cases we frequently face the situation that the results not only reflect the cortisol concentration, but produce falsely elevated values because both glucocorticoids are measured. 1.2. Saliva Collection and Storage Used as a biological matrix saliva has several advantages, mainly at the collection and storage steps. However, the salivary flux and composition may be affected by several factors which can influence the concentration of some steroids and modify their levels. For this reason, standardisation of the salivary sample collection and storage processes is of great importance in saliva analysis. Even though it is possible to obtain samples of glandularduct saliva, crevicular fluid, and mucosal transudate under specially designed collecting methods, obtaining whole saliva remains most feasible. This particular method is widely used in the laboratory practice. Currently, there are different modalities facilitating the production/stimulation and collection of saliva. Both the advantages and disadvantages of each modality should be taken into account. Many studies have used unstimulated whole saliva collected by passive drooling or spitting directly into a collector vial [17]. However, such direct collection method can not be applied for research with infants [23,24], geriatric [25,26] and older patients with

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Fig. (2). The statistic data presented the percent of publication devoted to the subject of steroid hormones considered as biomarkers in clinical research studies [a] and sports medicine [b] since 1990.

xerostomia [27,28]. Moreover, the procedure is less effective than application of an adsorbent device [29-31]. Stimulated whole saliva can be obtained using citric acid, but the acid may affect the immunoassay analysis results by interfering with the antibody binding [29,32-34]. Recently, several commercially available devices have been used to facilitate saliva collection. The most commonly used are the Salivette® (Sarsted) and Quantisal® (Immunalysis) pads of cotton, polyester, polyethylene, or cellulose used to absorb saliva from the subjects’ mouths. The usefulness of the described devices and other saliva collection systems has been evaluated in many publications [35-38]. The results have shown that the recovery of steroids from saliva can depend on the applied collection system. Moreover, cottonbased absorbent materials can potentially affect the results of immunoassays for salivary steroids to a profound degree. Sampling with cotton wool may significantly increase the salivary testosterone [31,32,39], dehydroepiandrosterone (DHEA) [29,32,38], estradiol and progesterone [32], as well as 17-hydroxyprogesterone (17OHP) [40] concentrations in immunoassays. In contrast, the results for the salivary cortisol, DHEA–S, and cotinine are not affected under the cotton collection methods [32,35]. The storage procedure and the lapse of time from the collection affect mainly the analysis of the biochemical variables characterised by temperature instability and bacterial growth. For this reason, the choice of the storage procedure preceding the analysis depends on the type of steroid and takes into account its stability. Nevertheless, this particular analytical problem is not serious in research because the hormones are stable in saliva. For example, the concentration of the saliva progesterone has been found stable over 3 months at room temperature [20]. No degradation of the DHEA in saliva samples has been observed at room temperature at 4˚C [41,42]. Aldosterone has been found stable with saliva kept at both the room temperature, and 4˚C for up to 7 days [43]. No loss of cortisol has been detected at -85˚C, or at the refrigerator temperature of 4˚C for at least 19 and 18 days, respectively. Moreover, blood contamination in saliva

samples is rare and its effects on the measurement of salivary hormones is small [44]. In contrast, the average levels of the salivary testosterone and cortisol have been found significantly lower in samples stored for 10 days at room temperature than in samples frozen on the day of collection [41]. In addition, cortisol decreases significantly after the third thawing [45]. The concentrations of cortisol, 17OHP, and progesterone decrease significantly over 21 consecutive days in native saliva samples, both at room temperature and after storage in the refrigerator. The decrease continues from day 5 onward. Centrifuging the saliva before storage increases the stability of 17OHP and progesterone up to day 9. The method, however, does not work for cortisol, even though the decrease rate is lower in centrifuged saliva. To prevent degradation of the salivary steroids in effect of bacterial growth, sodium azide (NaN3) is added to the saliva samples as the preservative. It has been confirmed that stability of the salivary steroids after addition of NaN3 improves, however one needs to take into account the possible interference of NaN3 with horseradish peroxydase, a common component of enzyme immunoassays [46]. 1.3. Saliva Sample Pretreatment Routine clinical laboratories should aim at subjecting samples to rapid pre-analytical processing, making sure that manual sample manipulation is reduced to the minimum and the desired sensitivity and high specificity are ensured. Sample pretreatment is of great importance in salivary steroid analysis, especially because the steroid concentration levels are very low. The specific steps in the sample pretreatment process depend on the analytical method selected for the measurement of the salivary steroids. The advantage of the immunological methods consists in simple and rapid sample pretreatment. In many cases the RIA or ELISA analysis requires no more but centrifugation to remove the particulate matter [47-51]. This is sometimes supplemented with a freeze–thaw cycle to break down the mucopolysaccharides [52,53]. Homogenisation of the saliva sample through sonication can improve the recovery of the salivary steroids such as progesterone, cortisone, 17OHP, testosterone, or estradiol,

Salivary Steroid Hormones as Biomarkers

however no improvement has been observed for cortisol and androstenedione [54]. Several studies have also reported steroid extraction from saliva samples under the liquid-liquid procedure (LLE). For example, cortisol has been extracted from a saliva sample using ethanol [23], dichloromethane [55], and ether diethyl [56]. However, the recovery of cortisol from the extracted samples has proved lower than the level obtained from unextracted samples when dichloromethane was used as the organic solvent (83.1 and 98.6%, respectively) [55]. Testosterone [57-59], progesterone [60], and estradiol [61] have been extracted from saliva with ether diethyl, whereas a mixture of dichloromethane and polyethylene glucose PGF 10000 has been used to extract aldosterone [43]. However, the solvent-extraction step with diethyl ether has, in case of testosterone, not increased the level of the steroid recovered [59]. Whenever chromatography is applied to determine the salivary steroids, sample preparation is the critical aspect. Analysis under the chromatographic methods has always required prior employment of the LLE or solid phase extraction (SPE), normally with a protein precipitation step beforehand. The LLE requires no special equipment, but may call for large volumes of organic solvents. Moreover, the procedure may be time-consuming and its automation is difficult. In contrast, the on-line SPE technique proves highly sensitive due to the preconcentration factor, high speed, and easy automation, but the extraction cost per sample is high. Both the LLE and SPE extraction procedures have been used in preparation to the chromatographic analysis of the salivary steroid hormones. In the first successful applications of HPLC for determination of the salivary cortisol the HPLC analysis was preceded by dichloromethane deprotenisation and concentration [62]. In presented methods, 2 ml of saliva was sufficient to improve the detection limit. Okurama et al. [63] have developed a more sensitive, fully automated column–switching HPLC system for the assay of the salivary cortisol. The system consists of a polymer-coated, mixed-functional silica (PCMF) column for deprotenisation, and a CN column for frontal concentration and separation and requires using only 0.1 ml of saliva samples. Jönsson et al. [64] were the first to develop and validate the method of analysing the salivary cortisol using liquid chromatography–tandem mass spectrometry (LC–MS/MS). In the work-up procedure the saliva samples were proteinprecipitated using acetonitrile of the acidic pH. The supernatant was evaporated in nitrogen flow and analysed under the LC–MS/MS technique using ESI in the positive mode. The disadvantage of the reported LC-MS/MS method came down to low recovery of cortisol from the saliva samples, identified at 64%. Moreover, concentrating solvents in nitrogen flow can be laborious and time consuming when processing numerous samples. Nelson et al. [65] investigated other evaporation techniques in preparation for the salivary cortisol LC-MS/MS assay. They compared three concentration techniques, i.e. the nitrogen flow, freeze drying, and centrifugal vacuum concentration, in the saliva sample work-up procedure before determining the salivary cortisol under the LC–MS/MS method

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described by Jönsson et al. [64]. The obtained results revealed there was no significant difference between the cortisol levels detected irrespective of the evaporation methods. Evaporation through freeze drying or centrifugal vacuum concentration did not improve the recovery of cortisol from saliva samples. De Palo et al. [66] have reported using the solid phase extraction procedure for simultaneous determination of the salivary cortisol and cortisone. They subjected a saliva sample (500 l) to the SPE pre-treatment to eliminate any interferences and achieve a ten-fold increase in the salivary cortisol and cortisone concentrations. The extraction procedure was performed on the SPE Discovery DSC-18 columns and the analytes were eluted with diethyl ether. The recovery levels under the SPE extraction procedure were 85 and 82% for cortisol and cortisone, respectively. Kataoka et al. [67] have applied automated in-tube solidphase microextraction (SPEM) in the determination of cortisol in human saliva. The optimum in-tube SPME conditions were: 20 draw/eject cycles, samples sized 40 l, and the Supel Q PLOT capillary column as the extraction device. The extracted compounds were desorbed easily from the capillary into the mobile phase. Cortisol recovery from a saliva sample was higher than 95.5%. In the sensitive liquid chromatography–electrospray ionisation-tandem mass spectrometric (LC–ESI-MS/MS) method of quantification of the salivary dehydroepiandrosterone (DHEA), as described by Higashi et al. [42], the saliva sample (200 l) was deproteinised with acetonitrile and purified using a Strata-X cartridge with a reversed-phase polymeric sorbent. The analytes were eluted with ethyl acetate. The mean recovery of the DHEA from the saliva samples was 85.4%. The same extraction procedure as the one described above was used by Shibayama et al. [68] in their LC– MS/MS method of simultaneous determination of the salivary testosterone and DHEA. The technique enabled quantification of the salivary testosterone and DHEA in a saliva sample of 500 l. The steroid recovery rates at the pretreatment stage were about 90%. Turpeinen et al. [69] used liquid-liquid extraction with dichloromethane before conducting an LC-MS/MS analysis of the salivary cortisol. In that case a saliva sample sized 0.1 ml was required. The mean recovery of the steroid added to the saliva samples ranged from 95% to 106%. Liquid-liquid extraction of testosterone from saliva samples (1 ml) was also reported by Sakaguchi et al. [70] and Yasuda et al. [71], as the sample pretreatment step before the LC-MS/MS analysis. In those methods ethyl acetate was used as the organic solvent. Frasconi et al. [48] and Baid et al. [72] have reported employing the LC-MS/MS as the reference method in determination of cortisol [48,72] and cortisone [48] in saliva. However, in their papers they gave no details of the data validation, except mentioning that saliva samples were analysed without any pre-treatment [48] and that the value of LOD was 4 ng/ml [72].

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2. ANALYTICAL METHODS FOR QUANTIFICATION OF THE SALIVARY STEROID HORMONES 2.1. Immunological Methods The immunological techniques are currently the most commonly reported methods employed in assays of the steroid hormones because of their simplicity and sensitivity above the LC–MS/MS levels. Moreover, immunoassays involve no or minor extraction steps, can be easily automated and applied to deal with large numbers of samples. The most common immunological methods of determining the salivary steroids are based on radioimmunoassay (RIA) [2,3,20,33,72,73], enzyme-linked immunosorbent assay (ELISA) [4,5,6,59,71], luminescence immunoassay (LIA) [47,49] and a time-resolved fluorescence immunoassay (TRFIA) [43,74]. Several commercial kits for determination of the salivary steroids have recently been marketed by different manufacturers, all available and widely used in research. Some of them represent adaptations of the methods designed for the serum assays. Modifications of the procedures/techniques have led to obtaining methods suitable for saliva. The adaptations found in commercial kits include adjustment of the protein content in the standard buffer and dilution of the samples, which improves precision and accuracy of the salivary steroid hormone measurements. For example, Yao et al. [55] and Höferl et al. [75] reported an adaptation of the DELFIA TM cortisol kit for the salivary cortisol quantification. Modifications of the Diagnostic Systems Laboratories’ 125l double antibody test kit for quantification of the DHEA and estradiol in the serum have been presented by Shritcliff et al. [32] and Granger et al. [33] respectively. Other adaptations of the commercial test kits for determination of cortisol [49,76,77], estradiol [61] and testosterone [31] in saliva have also been reported. Other reports present the application of new immunoassays for the measurement of the salivary steroids, offering higher selectivity and sensitivity than RIAs and ELISAs, such as the surfaceplasmon resonance-based immunosensor assay (SPR immunosensor) [48,78,79], the immunoelectrochemical sensor assay [51], and the magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) [53]. Actually, RIAs are replaced with non-radioactive ELISAs because there is no need to train the personnel in handling radioactive reagents. However, the reliability of the steroid immunoassays has been shown to be dubious because of the lack of specificity caused by interference from the cross-reacting steroids and other substances similar in structure. This lack of specificity may lead to incorrect result interpretation or too high steroid concentrations obtained. For example, cortisol may interfere with prednisolone (57%), 11deoxycortisol (12%), and other steroids (