Saliva Diagnostics

IBL INTERNATIONAL GMBH Flughafenstrasse 52a Phone: +49 (0)40-53 28 91-0 [email protected] D-22335 Hamburg, Germany Fax: +49 (0)40-53 28 91-11 www.IBL-International.com

Introduction ...... 4 Physiology of the Salivary Glands...... 4 Free, Bound, and Conjugated Steroids...... 6 The Importance of the Flow Rate ...... 6 Preanalytical Aspects...... 9 The Saliva Sampling Device...... 9 Validation of saliva sampling devices ...... 9 Influence of contamination ...... 10 Limits for blood tolerance in saliva diagnostics...... 10 Useful Hints for Saliva Testing ...... 13 Measurement of Steroids in Saliva...... 14 Chemiluminescence Technique...... 14 ELISA Technique...... 15 General Aspects of Immunoassays...... 15 Advantages of the salivary Steroid Immunoassays of IBL-International...... 17 ...... 18 Physiology and Time Dependent Concentration ...... 18 Normal ranges...... 22 Saliva Collection for Cortisol Assessment ...... 22 Indications...... 23 Stress research...... 23 Occupational Medicine ...... 24 Sports medicine...... 26 Jet lag-effects due to overseas travel ...... 27 Veterinary Medicine...... 28 Advantages of the IBL-International Cortisol Saliva Immunoassays.... 28 Test characteristics of the IBL-International Cortisol Luminescence IA...... 29 Test characteristics of the IBL-International Cortisol ELISA...... 30 Literature...... 31 ...... 32 Physiology and Time Dependent Concentration ...... 32 Testosterone level in males...... 32 Testosterone level in females ...... 33 Seasonal changes of Testosterone concentrations ...... 36 Normal Ranges ...... 38 Indications...... 38 Gynaecology...... 39 Andrology ...... 40 Paediatrics...... 40 Sports medicine – Overtraining syndrome...... 41 Veterinary Medicine...... 42 Advantages of the IBL-International Testosterone Saliva Luminescence IA 43 Test characteristics of the IBL-International Testosterone Luminescence IA44 Test characteristics of the IBL-International Testosterone ELISA ...... 45 Application for serum samples ...... 46 Standardisation of the IBL-Testosterone Luminescence Immunoassay by GCMS ...... 48

Page 2 ...... 50 Physiology and Time Dependent Concentration ...... 50 Normal Ranges ...... 53 Indications...... 54 Corpus Luteum Insufficiency ...... 54 Dysregulation...... 54 Application for serum samples ...... 55 Veterinary Medicine...... 55 Advantages of the IBL-International Progesterone Saliva Luminescence IA 56 Test characteristics of the IBL-International Progesterone Lum IA ...... 57 Literature...... 58 17- β - ...... 60 Physiology and Time Dependent Concentration ...... 60 Normal ranges...... 61 Indications...... 62 Precocious puberty (Pubertas praecox) in girls ...... 62 Gynecomastia in males ...... 62 In-vitro fertilization and embryo transfer treatment...... 62 Veterinary Medicine...... 64 Application for Serum Samples...... 64 Advantages of the IBL-International Estradiol Saliva Immunoassays ...... 65 Test Characteristics of the IBL-International 17-ß-Estradiol Lum IA...... 66 Test Characteristics of the IBL-International 17-ß-Estradiol ELISA...... 67 Literature...... 68 DHEA...... 69 Physiology and Time Dependent Concentration ...... 69 Normal ranges...... 73 Indications...... 74 Endocrinology...... 74 Anti-Aging Medicine...... 74 Psychology ...... 76 Neuroendocrinology...... 76 Sports Medicine...... 77 DHEA in Veterinary Medicine ...... 77 Advantages of the IBL- International DHEA Luminescence IA...... 78 Test Characteristics of the IBL-International DHEA Luminescence IA...... 79 Literature...... 80 Other Websites ...... 82 Anti-Aging Medicine ...... 82 Information About Salivadiagnostics ...... 82

This brochure discusses the determination of steroids in saliva. It presents both published results and unpublished data gathered at IBL-International. One of the goals of this brochure is to provide stimulation to the scientific community for additional investigative work moving forward in time.

Page 3 Introduction

For many years saliva has been used as a biological fluid for the detection of different such as electrolytes, hormones, drugs and in human and veterinary medicine. Sample collection is non-invasive, painless and very convenient especially for the patients. Collecting saliva samples is possible any time, day or night because of the overall convenience of collection. Saliva collection can be accomplished under circumstances where blood collection is difficult or inadvisable.

Saliva is the specimen of choice in a variety of the subsets of traditional medical care as well as emerging areas of health measurement and monitoring. With the ability to evaluate a pooled saliva sample from multiple collections it is possible to get a reliable assessment of hormone concentration even if distinct diurnal fluctuations occur.

Steroid hormone assessment from saliva also allows the specific determination of biologically active or “free” fraction of target hormone.

Assays for salivary steroid determination must be extremely sensitive as the concentrations of biologically active, free fractions are significantly lower than the concentrations of total analyte in serum. The required sensitivity can be achieved with chemiluminescence, a technology used in the saliva assays from IBL-International.

Physiology of the Salivary Glands

More than one mechanism exists for blood components to pass the membrane barrier into the salivary ducts.

1. The first mechanism is the passing through the space between the acinar cells. Because of barriers in the intercellular space, called tight junctions, only molecules with a relatively small molecular weight (MW < 1900) may pass through (MW H 2O = 18; MW Na = 23; MW steroids ca. 300; MW = 66,000; MW CBG = 49,500; MW SHBG = 115,000).

2. The second mechanism is the filtration through pores of the cell membranes. This transfer is only possible for substances of a MW < 400 (e.g. water, electrolytes).

3. The third mechanism is the selected transport through the cell membrane: a) Passive diffusion of lipophilic molecules (e.g. steroids) b) Active transport through protein channels (e.g. peptides) c) Pinocytosis: passing into the cell by taking along a part of the cell membrane and forming a vacuole in the cell; on the other side of the cell the vacuole membrane is reintegrated into the cell membrane and the contents of the vacuole is released in the duct of the glands (e.g. larger proteins such as ).

Page 4 The following table summarizes the concentration of analytes other than hormones, in saliva and blood according to different literature sources. Unfortunately, a precise description and evaluation of the analytical method is often not mentioned and therefore, in some cases a measurement of artifacts should be assumed (i.e. the level of SHBG or LDH in saliva may derive from a contamination with blood or crevicular fluid).

Table 1: Analyte levels in saliva and in plasma (according to several literature sources).

Analyte Mixed Saliva Plasma Unit In General Water 97 – 99.5 90 - 93 % pH (5.6)6.4 – 7.4 (7.9) 7.4 Substrates Albumin 246 – 344 34 000 – 48 000 mg/L Cholesterol 3 – 15 150 – 300 mg/dL Creatinine 0.07 – 0.2 < 1.1 mg/dL Glucose < 2 55 – 115 mg/dL Protein 1.1 – 1.8 (6.4) 66 – 87 g/L Urea 17 – 41 < 50 mg/dL Uric Acid 0.7 – 6.0 < 7.0 mg/dL Enzymes (37 °C) α-Amylase 11 900 – 305 000 < 220 U/L AP < 11 < 270 U/L SGOT (ASAT) < 43 < 38 U/L SGPT (ALAT) < 11 < 41 U/L LDH 113 - 609 < 480 U/L Lysozyme 6 - 12 3 – 9 mg/L Electrolytes / Minerals Calcium 0.88 – 2.05 2.20 – 2.55 mmol/L Chloride 5 – 40 96 – 108 mmol/L Magnesium 0.08 – 0.56 0.70 – 1.05 mmol/L Phosphate 1.4 – 13.2 0.87 – 1.45 mmol/L Potassium 6.4 – 37 3.3 – 5.1 mmol/L Sodium 2 – 21 133 – 145 mmol/L Other IgA 42 – 174 850 – 4 000 mg/L CBG, male 38 ± 18 39 700 ± 6 300 µg/L CBG, female 72 ± 71 42 200 ± 5 600 µg/L SHBG, male 19 ± 10 ?? 15 – 100 nmol/L SHBG, female 63 ± 60 ?? 15 - 120 nmol/L < 0.5 250 - 350 mg/dL

Page 5 Free, Bound, and Conjugated Steroids

Steroids are found in blood in conjugated and in unconjugated form. The conjugated form is excreted by the kidneys, and the steroids may be linked with Sulfates, Glucuronides etc. The unconjugated form of the steroids is mostly bound to various binding proteins like CBG, SHBG etc. This bound fraction represents the vast majority of the unconjugated steroid fraction (between 95% and 99% of the total hormone) present in plasma or serum. The bound fraction is biologically inactive and it is sometimes described as a biological inactive reservoir of the . The biologically active hormone is exclusively the small fraction of free steroid hormone and represents between 1 and 5% of the total concentration of the steroid in serum. Therefore any measurement of steroid hormones in serum or plasma will be mainly a reflection of the inactive hormone. Only a small and indeterminate percentage of the resultant measurement will be the biologically active hormone. Currently there is no reliable immunoassay available for the measurement of the free hormone fraction in serum. The only reliable method available is equilibrium dialysis. Equilibrium between the free fraction of hormones in serum and saliva has been observed, but is directly related to the size, polarity and ionicity of the hormone.

The Importance of the Saliva Flow Rate

The flow rate of saliva may influence the concentration of salivary hormones. This is the case if the hormone has a high molecular weight or if the molecule is polar or even ionic. Under these circumstances the concentration of the hormone in saliva will be influenced by the flow rate. On the other hand, the concentration of small and non-polar molecules in saliva has not been shown to be dependent on the flow rate. This is a very important aspect in a discussion of the utility of salivary diagnostics. If the concentration of the target analyte is dependent on the flow rate it is difficult to accurately determine hormone levels. A classical example is the measurement of DHEA and DHEA-S in saliva. DHEA is a non-polar and small molecule. Therefore the free fraction will pass from the vascular system to the salivary system. The salivary DHEA concentration reflects specifically the free fraction found in blood and consequently the hormone activity. DHEA-S is highly polar due to the ionic composition and thus cannot pass the membrane. As a result, most of the DHEA-S present in saliva originates from micro bleeding, and testing for DHEA-S in saliva is only useful as a tool for measuring levels of blood contamination in the sample.

Page 6 Diffusion of free steroid hormones from blood into saliva:

Blood Saliva

bound + free only free Membrane

Membrane

Figure 1: Free and protein bounded steroid hormones in blood and free hormones in saliva. The concentration of free hormones is the same on both sides of the membrane.

Page 7 The following table summarizes steroid hormone levels in saliva and blood in some physiological situations described in literature. It can be seen that the saliva/plasma ratio of the steroids ranges from approximately 1:10 (Cortisol) up to more than 1:100 (Testosterone). For some hormones such as Estradiol, very small concentrations are found in saliva, requiring a highly sensitive method for measurement. Blood contamination can cause false positive results in some salivary diagnostic applications, and should be taken into consideration when strategizing testing and reporting.

Table 2: Steroid hormone levels in saliva and in plasma (according to several literature sources)

Mixed Analyte Remark Plasma Unit Saliva Aldosterone non pregnant female 29 – 118 80 – 790 pmol/L adult men 140 – 630 1200 – 11000 pmol/L Androstene-dione adult women 62 – 482 1400 – 11900 pmol/L Cortisol peak in adults 13.8 – 48.9 190 – 690 nmol/L Cortisol 8 hours. after peak 1.4 – 8.6 55 – 250 nmol/L premenop. women 0.3 – 1.0 4.5 – 34.5 nmol/L DHEA adult men 0.3 – 1.7 6.2 – 43.3 nmol/L 2 – 18 26 – 650 pmol/L Estradiol middle cycle “peak” 9 – 29 180 – 1420 pmol/L mmol/L Estriol 40 weeks gestation 4.5 – 9.8 330 – 1596

adult women 10 – 21 92 – 1294 pmol/L Estrone adult men 10 – 21 92 – 555 pmol/L adult men 50 – 360 150 – 4900 pmol/L 17-OH-Progesterone 140 – 320 600 – 8800 pmol/L follicular phase < 160 500 – 3500 pmol/L Progesterone luteal phase 200 – 1600 4900 – 72000 pmol/L premenop. women 10 – 52 200 – 2860 pmol/L Testosterone adult men 95 – 205 9900 – 27800 pmol/L premenop. women 10 – 26 80 – 1270 pmol/L 5α-Dihydrotesto-sterone adult men 34 – 172 860 - 3410 pmol/L

Page 8 Preanalytical Aspects

The Saliva Sampling Device

It is important to use a sampling device which does not interfere with the salivary analyte under investigation. In contrast to serum or plasma, there is a very low analyte level in saliva. Therefore interactions between the surface of the sampling device and the analyte are much more likely. As previously mentioned, the analytes in saliva testing are small non-polar molecules. Such molecules show a tendency to stick to plastic material. The most critical of these analytes seems to be Progesterone which is highly non-polar. Therefore any saliva sampling device should be validated by measuring the recovery of Progesterone in saliva. It has been demonstrated that Polyethylene is highly absorptive for Progesterone, while glass for instance is not. Glass seems to be completely absorption free even for Progesterone, but has significant disadvantages. Glass is fragile and prone to breakage, and typically the stoppers are made of polyethylene which is highly absorptive. Because of these disadvantages, glass collection devices are not optimal for salivary applications.

The best plastic material seems to be Polypropylene, although the absorption characteristics are highly dependent on the purity of the material. Recycled polypropylene, for instance, shows significant absorption activity. Therefore IBL has introduced a special ultra-pure polypropylene sampling device named SaliCap which demonstrates an absorption characteristic of less than 5%, even in low concentrations of Progesterone in saliva. In case that the SaliCaps from IBL are not used for saliva sample collection and transport, each individual laboratory should alternatively validate their own collection device with critical attention to analyte absorption rates. With the IBL collection system, the saliva is transferred by the patient into the SaliCap by means of a polypropylene straw. Due to the short contact time of saliva with the surface of the straw the influence on measurable analyte is negligible. Use of the Salivettes® is not recommended, as these devices show significant interference even for Cortisol. The observed levels of interference are even greater if swabs are used together with the Salivettes®.

Validation of saliva sampling devices

For quantifying the adsorption properties of potential saliva collecting devices it is recommended to perform absorption studies. For these studies, 3H labeled Progesterone in physiological concentrations in pooled saliva can be used. Marked Progesterone is stored in glass and in the studied collecting device. After an overnight incubation on a laboratory shaker, aliquots from both samples are measured in a beta counter. The adsorption can be assessed as the difference between the values obtained from the collecting device and that from the reference (glass).

Alternatively, validation studies also can be performed by using the Progesterone Luminescence Immunoassay test kit Cat.-No. RE62021. Again physiological concentrations should be applied and incubated overnight on a laboratory shaker.

Page 9 Influence of blood contamination

It is of utmost importance to avoid any significant blood contamination, as the total steroid hormone concentration in blood can be up to 100 times higher than in saliva. Blood contamination is saliva is a critical issue. Therefore, if quantitative measurements of blood contamination are necessary, it is recommended to measure either DHEA-S or Transferrin. This is not necessary for routine purposes when there is not any visible blood contamination. Still, every patient should be aware that they must discard any sample which shows even a slightly red color. In this case the patient should rinse the sampling device 2 times with tap water, wait for another 10 minutes and sample again. The laboratory should deep freeze all samples for at least for 3 hours or overnight. Then the samples should be thawed again, mixed and centrifuged for 5 minutes, in order to obtain a clear supernatant. If the supernatant still is not completely clear, the freeze/thaw cycle has to be repeated as many times as needed and the centrifugation time should be extended to 15 minutes. Then the clear supernatant should be visually inspected in front of a white background. Any samples with slightly red color should be discarded.

Limits for blood tolerance in saliva diagnostics

The visual inspection of saliva samples in the SaliCap device (in front of a white background) will result in the following detection limits: 1. Non pretreated Saliva samples in the SaliCap: Detection limit 0.2% v/v. 2. Treated Saliva samples (Freeze/thaw cycle(s), centrifugation; transference of clear into a clean glass tube): Detection limit 0.1% v/v.

The influence of blood also has been investigated by spiking saliva with the blood of the same individual. The following figure shows this effect in case of Cortisol.

700

600

500

400 652 300

200 423.5 255.1 100 168.4 of (%) Cortisol Increase 100 102.3 97.7 94.3 119.4 0 0 0.06 0.125 0.25 0.3 0.625 1.25 2.5 5

Concentration of Blood (% v/v)

Figure 2: Influence of blood contamination on the salivary Cortisol level. Blood (Cortisol level 22µg/dL) has been added in increasing concentration to saliva (Cortisol level 0.86ng/mL). The total Cortisol concentration in saliva has been measured by Cortisol Luminescence Immunoassay and was plotted at the respective blood concentration level. The starting sample has been blood free saliva.

Page 10 The following table shows the positive bias of blood contamination at the visibility limit of 0.1% v/v. When the visibility limit was 0.2% the bias was twice as much.

Table 3: Bias caused by blood contamination

Analyte Ratio blood vs. saliva conc. Bias at visibility limit Cortisol 24 2% Testosterone 84 8% Progesterone luteal 56 6% Estradiol 91 9%

The following figure shows the results of blood contamination studies. These studies are similar to those described in Figure 2.

Influence of blood contamination on Testosterone in saliva (Sample of a female with the low Testosterone concentration of 7.2pg/mol) 200 180 160 140 120 100 80 60

Recovery (%) 40 20 0 0.000 0.015 0.031 0.063 0.125 0.250 0.500

Blood contamination in % (v/v)

Visible red color

Figure 3: Influence of blood contamination on the salivary testosterone level.

Page 11 Influence of blood contamination on Estradiol in saliva (Sample of a female with the Estradiol concentration of 3.7pg/mL)

200

180

160

140

120

100

80 Recovery (%) 60

40

20

0 0.000 0.015 0.031 0.063 0.125 0.250 0.500 Blood contamination in % (v/v)

Visible red color

Figure 4: Influence of blood contamination on the salivary Estradiol level.

Page 12 Useful Hints for Saliva Testing

Some hints for the collection of saliva are summarized in the following list.

Table 4: Instructions for saliva testing

− The use of the collection devices SaliCap from IBL International is highly recommended for the assessment of all steroid hormones in saliva. − Do not use cotton or polyester rolls for saliva collection. − Do not use Salivettes ® or any other non-validated devices. − Do not collect saliva samples within 15 minutes after brushing teeth or use of dental floss. − Do not collect saliva within 30 minutes after eating or drinking or gum chewing. − In case of doubt, rinse the oral cavity with pure mineral water or tap water before collecting saliva. − Do not use swabs with citric acid for stimulation of saliva flow. − If stimulation of saliva flow is really needed, chew inert material such as Parafilm ®. − Saliva samples exhibiting any slight reddish tinge must be discarded and a new sample must be taken when there is no oral bleeding. − Saliva samples for steroid measurement may be stored: − 7 days at room temperature. − 4 weeks at 2 - 8°C. − for longer periods at <-20°C.

Page 13 Measurement of Steroids in Saliva

Chemiluminescence Technique

Chemiluminescence assays were considered to be approximately ten times more sensitive than immunoassays for some years. For this reason IBL-International developed first saliva assays using glow luminescence in microtiter format. Chemiluminescence is a specific term describing light emission as a release of energy created during chemical reaction. This technology allows exact measurements below the picogram range. Instrumentation for measuring glow luminescence is relatively inexpensive (compared to flash luminescence) as no injectors are needed. There are many manufacturers of luminometers on the market. The table below is a list of well known manufacturers.

Table 5: Manufacturers of luminometer Manufacturer / Luminometer Multifunctional Reader Web page Anthos Lucy 1, 2, 3, AutoLucy

http://www.anthos-labtec.com Zenyth 1100 and 3100 Berthold-Detection Systems MPL1 & 2

http://www.berthold-ds.com Orion Centro LB960 Berthold Technologies Mithras LB940 MicroLumatPlus http://www.bertholdtech.com (luminescence, fluorescence) LB96V Bio-Rad Laboratories Lumimark Mikroplate

http://www.bio-rad.com (luminescence, absorbance) FLX800 (luminescence; fluorescence) Bio-Tek Instruments GmbH Synergy; FL600 http://www.biotek.com (luminescence ; fluorescence ; absorbance) BMG Labtechnologies LUMIstar Galaxy FLUOstar Galaxy http://www.bmg-labtechnologies.com (luminescence, fluorescence ; absorbance) HARTA Corporation MicroLumi 96 http://www.hartacorporation.com Hidex Oy Plate Chameleon

http://www.hidex.com (luminescence ; fluorescence ; absorbance) Molecular Devices Corporation Analyst AD/ Analyst HT, Acquest Lmax http://www.moleculardevices.com (luminescence; fluorescence; absorbance) Packard Fusion Universal Microplate Analyzer LumiCount http://www.packardbioscience.com (luminescence ;fluorescence ; absorbance) Perkin Elmer/Wallac 1420 Victor

http://www.perkinelmer.com (luminescence ;fluorescence ; absorbance) SFRI Laboratoire Lumax2

http://www.sfri.com Lumax3 GENios Tecan Schweiz AG Spectra FluorPlus, Safire, Ultra http://www.tecan.ch (luminescence,fluorescence, absorbance) Manufacturer / web page Luminometer Multifunctional Reader ThermoLabsystems Luminoskan Fluoroskan Ascent FL http://www.labsystems.fi Ascent (luminescence and fluorescence) Turner Designs Inc. The Reporter

http://www.turnerdesigns.com UltraSource, Inc. Stripwell

http://www.ultrasourceinc.com Luminometer

Page 14 ELISA Technique

The ELISA technique remains one of the diagnostic methods of choice for the majority of laboratories. Although the luminescence method offers high sensitivity, the appropriate material for working with such assays is quite expensive and for this reason not well installed in the laboratories.

In order to offer our customers reliable salivary ELISA kits, IBL-International developed assays by use of an enzymatic signal amplification system. This new technology make possible the achievement a very good sensitivity also with an ELISA test. The composition and performance of the ELISA and luminescence immunoassays are very similar. The main difference is the substrate reaction which is responsible for the colorimetric or luminescence signal generation.

General Aspects of Immunoassays

For the evaluation of the overall quality and utility of any laboratory immunoassay, the following criteria play a significant role.

Sensitivity : It is common to mention the analytical sensitivity of any quantitative immunoassay. Analytical sensitivity is determined by measuring replicates of the zero standard, calculating the mean value and the standard deviation (SD) of the signal, and identifying a concentration of analyte which corresponds to the mean value +/- 2 SD. This parameter can be influenced by the matrix of the zero standard. If it is not a saliva matrix but a highly diluted serum matrix or only a buffer solution, the calculated analytical sensitivity will be artificially and erroneously low, and therefore a non accurate reflection of the true sensitivity of that particular assay. Therefore, it is important to evaluate functional sensitivity by using saliva samples with low concentrations of analyte and calculating the coefficient of variation of replicate measurements at these low concentrations.

Range of linearity of the assay (shape of the standard curve): The concentrations of analyte in physiological, pathological and eventually therapeutic situations should be within the linear range of the standard curve of the assay. Typically the standard curves generated by immunoassays (designed for serum applications and later modified for saliva diagnostics) do not have linear slope characteristics where the majority of the saliva samples are found.

Cross reactivity of the to hormones similar to the analyte: In some salivary steroid assays a very low cross reactivity is important in order to be able to distinguish the target analyte from other hormones, which are common in saliva in high concentrations (e.g. the concentration of Cortisone in saliva is 2 to 5 times higher than the concentration of Cortisol, subsequently a low cross reactivity to Cortisone in a Cortisol assay is required).

Serum measurements: Most of the saliva assays from IBL-International can also be used for serum analysis. In order to achieve this, the samples have to be diluted between 1:20 to 1:50 with buffer. Results of the serum application

Page 15 represent the total steroid concentration in serum. Serum applications can be done with IBL-International Cortisol, Testosterone, and DHEA Saliva test.

Veterinary applications: As saliva from many species is very similar, the matrix effect can be neglected. Therefore, steroid saliva assays in general can be applied in veterinary medicine without any special precaution. Diluted veterinary serum samples can also be used in salivary test systems (see above). In this case, very tiny amounts of serum have to be sampled, which is very advantageous if small animals have to be investigated (mice, birds, etc).

The following graphics outline the determination of steroids in saliva in IBL- International luminescence assays.

Page 16

Figure 5: Scheme of the IBL-International Luminescence Immunoassays for steroid hormones in saliva

Advantages of the salivary Steroid Immunoassays of IBL- International

The following table summarizes the advantages of the IBL International Immunoassay assays.

− FDA cleared 510(k). − Applicable for saliva and serum samples. − High sensitivity. − Good precision at low concentrations. − Extended range of linearity. − Low cross reactivity to relevant metabolites. − Low sample volume (20µl or 50µl). − Easy test procedure, no extraction. − Almost all kit reagents ready to use. − 2 kit controls (normal and elevated). − Long acting, stable glow luminescence signal or ELISA technique − Applicable to automatic systems.

Page 17 Cortisol

Physiology and Time Dependent Concentration

Cortisol is produced in the cortex of the adrenal glands. The lipophilic steroid hormone is released into circulation and bound to proteins: 90% to corticoid binding globulin (CBG) and 8% to albumin. Only about 4% of the total Cortisol in the blood is free. Only this free hormone fraction in blood is actively available for the target cells. These facts have to be taken into account when assessing correlation studies of Cortisol in blood and in saliva. At 145 - 180ng/mL (400 – 500nmol/L) of total Cortisol plasma levels the CBG is saturated. Above this concentration the percentage of free Cortisol increases. Therefore the plasma level of total Cortisol depends from the CBG concentration. Any increased CBG level leads to an elevated Cortisol plasma level, whereas the free Cortisol concentration in plasma and saliva still is normal. The CBG concentration is affected by various conditions such as , liver disease, , polycystic ovary syndrome and application of different drugs (e.g. contraceptives).

Figure 6: Correlation of salivary Cortisol to Cortisol in serum (Vining et al., 1983). On the left: Relationship between salivary and total serum Cortisol in time-matched samples. On the right: Relationship between salivary and unbound serum Cortisol in time-matched samples.

Page 18 16

14

12

10

8

6

4 Salivary Cortisol (ng/ml) 2

0 4 6 8 10 12 14 16 18 Time of day [h]

Figure 7: Diurnal rhythm of salivary Cortisol, different wake-up times.

The release of Cortisol is regulated by the Corticotropin Releasing Hormone (CRH) from the hypothalamus and the Adrenocorticotrope Hormone (ACTH) of the pituitary gland via a negative feedback mechanism. This release is influenced also by other factors such as stress and physical exercise mainly because of the positive feedback of higher adrenaline levels on ACTH release.

Normal Cortisol concentration in human saliva during the day is highly dynamic. In the graph above, diurnal profiles for three individuals are shown with the typical Cortisol peak in the early morning. The time of the Cortisol peak is not dependent upon the absolute time and also is not influenced by daylight. It is dependent on wake-up timing of each individual.

Page 19 The mean diurnal cycle of salivary Cortisol in 110 healthy adult people is shown in the following graph and table (Westermann et al., 2004):

100

80

60

40

20 Peak maximum [%]

0 2 4 6 8 10 12 14 Time after awakening [h]

Figure 8: Mean diurnal Cortisol in saliva profile of 110 healthy adults. The graph shows the mean value +/- 2 standard deviations. Samples were measured with the IBL Cortisol Luminescence Immunoassay.

Table 7: Day time dependent normal range periods of Cortisol in saliva of 110 adult persons. Samples were measured with the IBL Cortisol Luminescence Immunoassay.

Hours after Saliva Cortisol Values (nmol/L) 5% 95% Awakening Percentile Median Percentile 0 – 1.5 5.1 18.9 40.2 1.5 – 3 3.6 11.8 28.4 3 – 6 2.1 6.7 15.7 6 – 9 1.8 5.5 12.1 9 – 15 0.9 3.3 9.2

Page 20 The highest values are found 30 to 90 minutes after the average wake up time:

45 Normal 40

35

30

25

20 Number 15

10

5

0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 Peak delay (h) 28.02.2006 14:21:18 DAYSORT.WST Figure 9: Time intervals of maximum Cortisol peak of 110 healthy adults after waking up. Samples were measured with the IBL Cortisol Luminescence Immunoassay.

Table 8: Maximum daily saliva Cortisol values in relation to sex and age. Samples were measured with the IBL Cortisol Luminescence Immunoassay.

Saliva Cortisol Peak Tested Individuals (nmol/L) Minimum Median Maximum 39 Males (17 – 59 years) 10.3 24.3 62.0 61 Females (17 – 61 years) 6.8 28.1 78.4 10 Seniors (62 – 77 years) 14.0 31.2 53.7

Page 21 Normal ranges

Table 9: Guideline for the expected saliva Cortisol values.

Values (ng/mL) Wake up time 1.4 – 8.9 Peak (within 90 min. after average wake up time) 5.0 – 17.7 3.5 – 4.5 hours after cortisol peak 1.0 – 2.8 7.5 – 8.5 hours after cortisol peak 0.5 – 3.1 Dexamethason suppression test* Cut off (normal/Cushing disease) 1.0 Borderline 0.7 – 1.6 Addison disease < 0.4 Cushing disease 7.2

*Application of 1mg Dexamethason orally 11pm; saliva collection next morning immediately after awakening.

The table below provides detailed instructions for saliva Cortisol determinations. Alternative sampling strategies may be applied as well, depending on the specific medical needs.

Table 10: Saliva Collection strategies for Cortisol.

Saliva Collection for Cortisol Assessment

Day Profile - 5 samples in the morning. The first sample immediately after wake up, 4 more samples at intervals of 30 minutes. - 1 sample around noon (e.g. 12am) - 1 sample in the afternoon (e.g. 4pm) - 1 sample in the evening (e.g. 10pm)

Short profile - 2 samples: at 8am and at 10pm

Morning Peak - 5 samples in the morning. The first sample immediately after wake up, 4 more samples at intervals of 30 minutes.

Page 22 Indications

Cortisol determination is useful in endocrinology, psychology, sports medicine, pediatrics, anti-aging medicine, veterinary medicine and others.

The classical indication of Cortisol determination is the Cushing Syndrome. In this case the saliva sampling should be done in the late evening or even at midnight. The Cortisol concentration should be low (< 3ng/mL). In the following some specific areas for Cortisol determinations are described in more detail.

Stress research

In many studies the endocrinological response of individuals to different kinds of stress is tested. In order to differentiate the variable nature of individual reaction to stress, there has to be a standardized test which can be applied to all participants. The Trier Social Stress Test (TSST) is generally accepted as such a method. Students performing the TSST every day at the same time for a week can be divided into two groups regarding their Cortisol levels during the study. The “high responders” demonstrate a strong increase of salivary Cortisol levels at the same times on following days, whereas the “low responders” don’t show this increase.

Figure 10: Cortisol response on standardized stress situations. Students were exposed to the Trier Social Stress Test (TSST) between 4pm and 7pm on 5 days (C. Kirschbaum et al., 1995).

Page 23 There are suggestions that other factors can influence the level of Cortisol in response to stress in humans: Gender and seem to have an impact on this hormone profile and the application of drugs, such as oral contraceptives, seems to decrease the response of Cortisol to stress according to Kirschbaum et al. (1999).

14 50 Stress Stress 45 12 40 10 35 30 8 25 6 20 4 15 10 2 Total Plasma Cortisol (nmol/l : 10) Salivary Free Cortisol (nmol/l) 5 0 0 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 Time (min.) Time (min.)

Luteal phase (n = 21) Men (n = 20) Follicular phase (n = 19) OC user (n = 21)

Figure 11: Impact of gender, menstrual cycle and oral contraceptives during stress on salivary free Cortisol and total plasma Cortisol. Healthy men and women run through the Trier Social Stress Test (TSST). Basal Cortisol level are taken the day before (C. Kirschbaum et al., 1999).

Occupational Medicine

A common issue in some work environments is the adaptation of people to shift work. There are some people who seem to adapt quickly to night-shift work without any problems, while other workers show signs of psychological difficulties during the adaptation time. In a documented study (Hennig et al. 1998) it is suggested that it is possible to differentiate the adapters and non- adapters by means of diurnal salivary Cortisol profiling.

In this study 24 nurses were involved. Saliva samples were collected for two days at the early working shift (beginning at 6:00am; ending at 9:00pm) and in the following 7 nights of the night shift (beginning at 9:00pm; ending at 6:00am). Salivary Cortisol was assessed in each saliva sample.

In the next figure (page 24) it is shown that the Cortisol morning peak decreased during the night-shift, whereas there developed a “night Cortisol peak” at 9:00pm. On the fifth night the Cortisol of the night peak exceeded that of the morning peak. By looking at the individual Cortisol profiles, two different “types” of nurses can be identified:

6 of the 24 nurses involved in the study showed Cortisol profiles even in the 7 th night whereas the night Cortisol peak didn’t exceed the morning peak. These people are called “non-adapters” (see Figure 12).

Page 24 In addition these 6 nurses also experienced psychological problems during the adaptation period of the night-shift. It is not clear if concurrent treatment with could convert these “non-adapters” into “adapters”.

Figure 12: Diurnal salivary Cortisol profile of 24 nurses during two days of the early shift (beginning at 6:00 am; ending at 9:00 pm) and 7 following days of the night-shift (beginning at 9:00 pm; ending at 6:00 am; J. Hennig et al., 1998).

Figure 13: Difference of the salivary Cortisol concentration of the morning peak and the night peak of 24 nurses during 7 days of the night shift (beginning at 9:00pm; ending at 6:00am). In 18 nurses (called “adapter”) after the fifth night the night Cortisol peak exceeded the morning peak, whereas in 6 nurses (called “non-adapter”) this didn’t happen even in the seventh night (J. Hennig et al., 1998).

Page 25 Sports medicine

During physical exercise the free salivary Cortisol concentration increases with the intensity of the exercise.

20

18

16

14

12 Physical 10 Exercise

8

Salivary Cortisol (ng/mL) 6

4

2

0 -20 -10 0 10 20 30 40 50 60 70 M inutes

Day 2: 10 min./100 rpm/113 kJ Day 3: 10 min./75 rpm/84kJ Day 4: 8 min./100 rpm/91 kJ Day 5: 10 min./75 rpm/84 kJ Day 7: 10 min./50 rpm/55 kJ

Figure 14: Salivary Cortisol dependent on physical exercise. Male on different days with increasing exercise (from 55 kJ up to 113 kJ); Measured with the IBL Luminescence Immunoassay.

This graph suggests that there may be a measurable difference in the Cortisol peak between well trained people and so called “non runners”.

9 Runners (n = 13) Non-runners (n = 13) 8

7

6

5

4

3 SalivaryCortisol (ng/ml) 2

1

0 Pre-exercise 25 min. of exercise 10 min. post 30 min. post exercise exercise

Figure 15: Salivary Cortisol response during physical exercise of trained and non-trained people. (D. L. Rudolph and E. McAuley, 1998). Runners = cross-country runners (ca. 100 km per week for 2 years); non-runners = students; exercise = 30 min. treadmill run (60% of max. heart rate and max. oxygen consumption).

Page 26 Jet lag-effects due to overseas travel

When traveling from the United States to Europe by airplane, people have during their first week in Europe two Cortisol peaks, one (decreasing) peak related to the “normal” wake up time in the US, and the other (increasing) peak coming up related with the European wake up time. During the first week in Europe, the USA morning peak vanishes and the new European peak increases to normal level. It seems that these findings are related to the jet lag phenomenon as the subjective jet lag feeling seems to be limited to the time when both peaks are to be seen.

2nd day 4th day 6th day

12

10

8

6 Cortisol (pg/mL) Cortisol

4

2

0 12:00 AM 3:00 AM 6:00 AM 9:00 AM 12:00 PM 3:00 PM 6:00 PM 9:00 PM USA time

US wake-up time Europe wake-up time

Figure 17: Salivary Cortisol levels after return to Germany. Data obtained after a trip from the USA to Germany. Samples measured with the Cortisol Luminescence Immunoassay from IBL- International.

Page 27 Veterinary Medicine

The IBL saliva test kits are able to measure saliva samples from many species without pretreatment or precaution. This level of species compatibility is not applicable with a serum assay. There are two reasons why animal saliva can be used in the IBL chemiluminescence’s assays.

- The Cortisol molecule is absolutely identical in any mammals.

- There is no significant difference in the protein content of saliva among different species. That is why no extraction is necessary.

The Cortisol Luminescence Immunoassay from IBL also offers the option for testing serum samples in human and veterinary testing. Especially the use in veterinary applications is to mention. This is an attractive feature since the dilution of serum samples (e.g. 1:50) essentially removes any potential protein matrix effect. Therefore no extraction of animal serum samples is necessary. Due to the high dilution, only extremely small serum sample volumes (< 1 µl) are needed. Therefore serum assessment can easily be performed on very small vertebrates.

Advantages of the IBL-International Cortisol Saliva Immunoassays

- The only FDA cleared saliva and serum/plasma assay. - High analytical sensitivity. - Good specificity (specifically regarding cortisone). - Good precision (functional sensitivity). - Good linearity in the clinically relevant concentration range. - Two levels of controls included in the kit. - Standards, controls and conjugate ready to use. - Adaptable to automated instruments.

Page 28 Test characteristics of the IBL-International Cortisol Luminescence Immunoassay

Principle: Competitive Chemiluminescence Immunoassay.

Intended use: Quantitative determination of free Cortisol in saliva and total Cortisol in Calibrator A diluted serum.

Regulatory status: CE, FDA-cleared 510(k).

Format: 12 microtiter strips with 8 wells each.

Sample: 20µL or 50 µl saliva or 50µL diluted serum (human or animal).

Standards: 7 standards ready to use; Concentrations 0.0/ 0.3/ 0.6/ 2.0/ 6.0/ 15.0/ 40.0ng/mL

Incubation: 1st incubation: 3 hours (room temperature). 2nd incubation: 10 min. (room temperature).

Substrate: Luminol, ready to use.

Expected values: Hours after Normal range (ng/mL) wake up upper limit lower limit 0.0 – 1.5 1.85 14.5 1.5 – 3.0 1.3 10.2 3.0 – 6.0 0.76 5.68 6.0 – 9.0 0.65 4.68 9.0 – 12.0 0.33 3.33

Sensitivity: 0.05 ng/mL

Precision: Intraassay: 3.1 – 3.4 % at 1.25 – 29.17 ng/mL Interassay 4.1 – 2.1 % at 2.60 – 29.69 ng/mL

Specifity: Cross reactivity (Abraham Method) Cortisone 4.5 % Prednisone 2.1 % Corticosterone 2.0 %

Controls: 2 kit controls

Automation: Applicable on various automated instruments (e.g. Triturus).

Cat. No.: RE62011 (96 Tests) or RE62019 (960 Tests)

Page 29 Test characteristics of the IBL-International Cortisol ELISA

Principle: Competitive Enzyme Immunoassay.

Intended use: Quantitative determination of free Cortisol in saliva and total cortisol in Calibrator A diluted serum.

Regulatory status: CE, FDA-cleared 510(k).

Format: 12 microtiter strips with 8 wells each.

Sample: 50 µl saliva or diluted serum (human or animal).

Standards: 7 standards ready for use Concenrations: 0/0.3/0.6/2.0/6.0/15.0/40.0 ng/mL

Incubation: 1st incubation: 2 hours (room temperature, plate shaker) 2nd incubation: 30 min (room temperature, plate shaker).

Substrate: TMB, ready for use.

Expected values: Hours after Normal range (ng/mL) wake up upper limit lower limit 0.0 – 1.5 1.85 14.5 1.5 – 3.0 1.3 10.2 3.0 – 6.0 0.76 5.68 6.0 – 9.0 0.65 4.68 9.0 – 12.0 0.33 3.33

Sensitivity: 0.05 ng/mL

Precision: Intraassay: 3.1 – 7.3 % at 2.7 – 23.4 ng/mL Interassay 6.4 – 9.3 % at 5.4 – 23.5 ng/mL

Specifity: Cross reactivity (Abraham Method) Cortisone 4.2 % Corticosterone 1.4 % Prednisone 2.5 %

Controls: 2 kit controls.

Automation: Applicable on various automated instruments.

Cat. No.: RE52611

Page 30 Literature

- Hennig, J. et al. Upright posture influences salivary Cortisol. Psychoneuroendocrinology 25: 69 – 83, 2000. - Kirschbaum, C. et al. Impact of Gender, Menstrual Cycle Phase, and Oral Contraceptives on the Activity of the Hypothalamus-Pituitary- Adrenal Axis. Psychosomatic Medicine 61: 154 – 162, 1999. - Hennig, J. et al. Changes in Cortisol Secretion During Shiftwork: Implications for Tolerance to Shiftwork? Ergonomics 41: 610 – 621, 1998. - Rudolph, D.L. et E. McAuley. Cortisol and Affective Responses to Exercise.Journal of Sports Sciences 16 : 121 – 128, 1998. - Kirschbaum, C. et al. Stress- and Treatment-induced Elevations of Cortisol Levels Associated with impaired Declarative Memory in Healthy Adults. Life Sciences 58: 1475 – 1483, 1996. - Kirschbaum, c. et al. Short Term Estradiol Treatment Enhances Pituitary- Adrenal Axis and Sympathetic Responses to Psychosocial Stress in Healthy Young Men. J Clin Endocrinol Metab 81: 3639 – 3643, 1996. - Kirschbaum, C. et al. Sex-Specific Effects of Social Support on Cortisol and Subjective Responses to Acute Psychological Stress. Psychosomatic Medicine 57: 23 – 31, 1995. - Kirschbaum, C. et al. Persistent High Cortisol Responses to Repeated Psychological Stress in a Subpopulation of Healthy Men Psychosomatic Medicine 57: 468 – 474, 1995. - Kirschbaum, C. et al. Preliminary Evidence for Reduced Cortisol Responsitivity to Psychological Stress in Women Using Oral Contraceptive Medication. Psychoneuroendocrinology 20: 509 – 514, 1995. - Kirschbaum, C. et D. H. Hellhammer Salivary Cortisol in Psychoneuro- endocrine Research: Recent Developments and Applications. Psychoneuroendocrinology 19:313 – 333, 1994. - Vining R. F., et. al. The measurement of hormones in saliva: possibilities and pitfalls. J. steroid Biochem. 27:81 - 94, 1987. - Westermann, J. et al. Determination of Cortisol in Saliva and Serum by a Luminescence-Enhanced Enzyme Immunoassay. Clin. Lab. 50, 11-24, 2004.

Page 31 Testosterone

Physiology and Time Dependent Concentration

Testosterone level in males

In males almost all Testosterone is produced in the Leydig cells of the testicles. The release of Testosterone is regulated by LH () from the anterior part of the pituitary gland. The FSH (follicle stimulating hormone), also from anterior part of the pituitary gland, has a positive effect on the Sertoli cells of the testicles. They produce the binding protein which activates the development of sperm cells mediated by Testosterone in the cells. Testosterone itself has a negative feedback on the release of GNRH (gonadotropin releasing hormone) in the hypothalamic region and FSH and LH in the pituitary gland.

The concentration of Testosterone in males is decreasing with increasing age.

120

100

80

60

Concentration in pg/ml 40

20

18 - 29 years 30 - 39 years 40 - 49 years 50 - 59 years 60 - 69 years Age Dependence of Saliva Testosterone of Males

Figure 18: Age dependence of the saliva Testosterone values of 287 men. The saliva samples were collected in the afternoon (The graph shows the median, the 25–75% and the 5–95% percentiles). Samples were measured with the IBL Testosterone Luminescence Immunoassay.

The age dependence is especially seen in men who live in the western hemisphere. Investigations regarding saliva Testosterone of men who live in the USA, Congo, Nepal and Paraguay show that the age dependent differences decrease in the described range (Ellison et al., 2002).

Page 32 Testosterone level in females

In females almost half of the Testosterone is produced from the cortex of the adrenal glands. The other half is produced in the ovaries. The release of Testosterone from the ovaries is regulated via GNRH (Hypothalamus) by FSH and LH (pituitary gland). The production of Testosterone from the adrenal cortex is regulated via CRH (corticotropin releasing hormone, Hypothalamus) by ACTH (adrenocorticotrope hormone, pituitary gland).

In females the blood Testosterone level is 10 to 20 times lower than in males. This makes the determination of Testosterone with conventional immunoassays very difficult, especially in females and children. Recently published articles have stated that results when measured by fully automated systems have mostly been incorrect (Taieb et al., 2003, Herold et al., 2003).

In saliva the concentration of free Testosterone in females is 3 to 5 times lower when compared with male saliva. The concentration of Testosterone seems to be nearly independent of the age.

50

40

30

20

10 Concentration in pg/ml

0 18 - 29 years 30 - 39 years 40 - 49 years 50 - 59 years 60 - 69 years Age Dependence of Saliva Testosterone of Femals

Figure 19: Age dependence of the saliva Testosterone values of 249 women. The saliva samples were collected in the afternoon (The graph shows the median, the 25–75% and the 5– 95% percentiles). Samples were measured with the IBL Testosterone Luminescence Immunoassay.

Page 33 The following figure 20 shows the statistics of an evaluation of the Testosterone day profile of obviously healthy males.

140

120

100

80

60 Testosterone (pg/ml) Testosterone

40

20

0 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Daytime (h) Figure 20: Mean diurnal Testosterone concentration in pg/ml (mean value +/- 1 standard deviation) of 18 males (age 25–59). Samples were collected every 30 minutes starting after awakening. Samples were measured with the IBL Testosterone Luminescence Immunoassay.

Similar to Cortisol, testing has shown that there is a morning peak of Testosterone in males. Statistically this morning peak is difficult to see as it is highly dependent on sleeping habits and disappears rather quickly.

The Testosterone level in men clearly fluctuates in short periods of time (e.g. 90 minutes) because of the fluctuating release of GNRH and LH in the hypothalamic region and the pituitary gland. It is assumed that the pituitary gland releases 8 to 16 pulses per day. These fluctuations are very high especially in young men.

The following graph shows the individual Testosterone morning peak and the short time variations of the free fraction as measured in saliva of healthy male individuals. The age of these individuals has been between 20 and 51 years.

Page 34 Diurnal Fine Profile: Testosterone in Saliva of Males

160 51 years 140 26 years 28 years 120 22 years 20 years 100

80

60 Concentrationinpg/ml

40

20

Day Time in Hours from 6 a.m to 6 p.m.

Figure 21: Diurnal fine profile of the saliva Testosterone concentration in pg/mL of 5 men (age 20, 22, 26, 28 and 51 years). The saliva samples were collected every 10 to 15 minutes. Variation coefficient: around 30%. Samples were measured with the IBL Testosterone Luminescence Immunoassay.

The high dynamics of Testosterone concentration requires a special sampling strategy for routine determinations. Single samples in most cases will give arbitrary results, which are difficult to reproduce. Therefore we strongly recommend collection of at least 5 samples over a period of 2 hours. The laboratory has to mix aliquots before measuring. The mixture will give an average free Testosterone value which in fact represents the hormone activity over the period of sampling.

Page 35 Seasonal changes of Testosterone concentrations

Initially it was assumed that the male Testosterone concentration was somehow influenced by the seasons. Interestingly this assumption was confirmed after a long term study. The highest Testosterone values were measured during the months of May and June.

Testosterone in Saliva: Annual Profile

160 (Man 35 Years)

140

120

100

80

60

40

20

0 Dez-02 Jan-03 Mrz-03 Mai-03 Jun-03 Aug-03 Okt-03 Nov-03 Jan-04

Figure 22: Annual profile of the saliva Testosterone concentration in pg/mL of a man (age 35 years). The saliva samples were collected daily in the morning and measured as a weekly saliva pool. Samples were measured with the IBL Testosterone Luminescence Immunoassay.

In blood only 1–2% of Testosterone is not bound to proteins. Only this free fraction of Testosterone has endocrine effects on the target cells. 50 to 60 % of the circulating Testosterone is strongly bound to SHBG (sex hormone binding globulin) while the remaining part is less strongly fixed to albumin. As with other steroid hormones the levels of the binding globulins, and therefore the concentration of bound Testosterone, is dependent on many physiological and pathological situations in humans. SHBG values may be increased in hyperthyroidism, applications, higher age and cirrhosis of the liver. SHBG values may be decreased in hypothyroidism, applications, obesity and nephrotic syndrome. This means that Testosterone values in serum and free Testosterone in saliva will not ideally correlate.

Page 36 Saliva-Serum Comparison IBL Luminescence Immunoassay

9000 y = 67.5x + 165; N=75; R2 = 0.73 8000 7000 6000 5000 4000

Serum (pg/mL) Serum 3000 2000 1000 0 0,0 20,0 40,0 60,0 80,0 100,0 120,0 Saliva (pg/mL)

Figure 23: Saliva and serum Testosterone concentrations in pg/ml. The samples were collected at the same time. Measured with IBL Luminescence Immunoassay. The serum samples were diluted 1:40.

The fraction of free Testosterone in blood or serum can be calculated either as free androgen index by the values of the whole Testosterone concentration and that of SHBG or as biological disposal Testosterone by the values of total Testosterone, SHBG and albumin (http://www.issam.ch/freetesto.htm). These calculated values have a wide variability, which implicates the addition of the variability of these two or three parameters. Alternatively the direct measurement of free Testosterone in serum is possible by equilibrium dialysis. There are several publications available showing that none of the commercial immunoassays for the measurement of free Testosterone in serum gives any useful results (A.Vermeulen 1999). Concerning these assays there is a large gap between the claims and reality. The only routine method for direct measurement of free Testosterone is the salivary assays.

Page 37 Normal Ranges

Saliva Testosterone concentrations:

Age Male Female (years) (pg/mL) (pg/mL) 20–29 41 – 143 5 - 49 30–39 32 – 100 5 - 49 40–49 30 – 98 4 - 49 50–59 30 – 92 4 - 49 60–69 23 – 87 3 - 39

Woman:Hirsutism 15 - 90 PCO:Syndrome 25 - 145

As already described the concentrations of Testosterone in saliva are dependent on sex, age and the time of day. These are clearly shown over a specific time period in both males and in females. Therefore we strongly recommend collection of at least 5 samples over a period of 2 hours. The laboratory then has to mix equal aliquots of each single sample. The mixture will give an average free Testosterone value which in fact represents the hormone activity over the period of sampling.

In case the morning peak is to be investigated sampling should start immediately after the first wake up and continue every 15 minutes for approximately 2 hours.

Indications

Besides the main indications of Testosterone measurement in endocrinology, i.e. diagnosis of hirsutism in women, evaluation of hormone dysregulation in children and differential diagnosis in hypoandrogenism in men, there are many other questions in psychology, sports medicine, anti-aging medicine and veterinary medicine which call for the assessment of Testosterone levels in situations where taking blood samples can be very difficult if not impossible (see also chapter Cortisol: indications).

Some interesting clinical aspects of salivary Testosterone assessment by various publications and our own research are shown in the following chapters:

Page 38 Gynaecology

The comparison of concentrations of various in serum and salivary Testosterone levels in healthy women and patients with Hirsutism indicates that saliva shows the best discrimination between the two groups.

18 Healthy persons (n = 19) Hirsutism cases (n = 50) 16 14

12

10 8

6

4 2

0 nmol/l µmol/l nmol/l pmol/l pmol/l : 10 Testosterone, serum DHEA-S, serum Androstendione, serum Free Testosterone, Free Testosterone, serum saliva

Figure 24: Various androgen levels during hirsutism (J. Osredkar et al., 1989).

But the variation of Testosterone levels during the day in women is significant. Therefore taking multiple samples during the day is recommended. The success of antiandrogen therapy can also be monitored by the measurement of salivary Testosterone levels. There is a significant decrease of Testosterone concentrations in the menstrual cycles of hirsute patients following the therapy.

0,3 23 Hirsutism cases 0,25

0,2

0,15

0,1 Normal

Salivary Testosterone (nmol/l) Testosterone Salivary 0,05

0 -1 0 1 2 3 4 Menstrual Cycle

Figure 25: Salivary Testosterone level during antiandrogen therapy in hirsutism. Therapy: 100 mg cyproteronacetate daily from day 5–15; 50µg Ethinyl-Estradiol, 150µg levonorgestrol daily from day 5–26 (J. M. Gomez et al., 1992).

Page 39 Andrology

For insufficient functions of the gonads () the concentration of Testosterone is found below the normal range. Low concentrations of free Testosterone may result in disorders of psychological, somatovegetative and sexual nature. Besides , states of exhaustion, disturbed potency and infertility, loss of muscle and bone density also may occur. In the past, determinations of Testosterone have been mostly done in serum. The determination of salivary free Testosterone is much superior especially in the low concentration range. In this range fully automated routine methods for measuring total Testosterone in serum fail. Clinically it is the lower values of Testosterone which are important to correctly quantify. This is not possible in case of serum Testosterone (see Taieb et al., 2003).

Paediatrics

Butler et al. (1989) described in a study with 84 boys (age 7.3–16.2 years) that salivary Testosterone concentrations correctly reflect the gonad function in this age group. They compared the mean daily salivary Testosterone level of 6 samples with the clinical pubertal stages classified according to the TANNER schedule (G1–G5; next figure).

Figure 26: Correlation of the mean salivary Testosterone level (6 samples; mean ±95% confidence interval) to the pubertal stage according to the TANNER schedule (G1 – G5) of 84 boys (age 7.3 – 16.2 years).white= genital maturity; grey= pubic hair (Butler, G.E. et al., 1989)

These results potentially correlate to other age classes as well, including the possibility that in older men salivary Testosterone levels are reflected in the gonad function.

Page 40 Sports medicine – Overtraining syndrome

During intensive, physical exercise, such as running, it is known that the free salivary Cortisol concentration increases followed by an increase of the free Testosterone. However there appears to be a difference in the Cortisol peak between the well-trained marathon runner and the occasional runner.

Figure 27: Salivary Cortisol and Testosterone level during a marathon run (N. J. Cook et al., 1992); During Marathon run: • Cortisol level (nmol/L); ▪ Testosterone level (pmol/L) Control Day: ◦ Cortisol level (nmol/L); ▫ Testosterone level (pmol/L)

18 180 Cortisol in Saliva Testosterone in Saliva 16 160

14 140

12 120 Begin of 10 Jogging 100

8 80

6 60 Cortisol in Saliva (ng/ml) Saliva in Cortisol 4 40 Testosterone in Saliva (pg/ml) Saliva in Testosterone End of 2 20 Jogging 0 0 5:40 PM 6:20 PM 7:00 PM 7:40 PM

Figure 28: Salivary Cortisol and Testosterone level during a 5 km run measured with IBL- International Luminescence Immunoassay.

Page 41 The relation of anabolic active Testosterone to catabolic active Cortisol is discussed in connection with overtraining syndrome. Overtraining syndrome is a situation where, in spite of regeneration and without clear recognizable physiological reason, persistent decrease in power and efficiency can be observed over several weeks. The ratio of Testosterone/Cortisol should be a valuable indicator of physical strain during sports training and should be applied as a training control tool under hormonal aspects. In practice almost no clear, significant results can be seen when hormonal investigations are conducted in serum, especially since the blood samples are collected under poorly standardized conditions and in the state of rest (Urhausen et al.).

The diagnosis of “overtraining syndrome” is therefore a challenge in sports medicine. The systematic capture of the actual state of health is done with the help of standardized questionnaires, and this method is the most sensitive diagnostic criterion in studies of overtraining syndrome up to now.

The use of saliva as a diagnostic, sampling material could improve this situation because saliva has several advantages. In exercise situations as well as in states of rest, the sample material can be collected without the help of specialized medicinal personnel. Diurnal profiles can replace monthly or weekly profiles. There are a number of good reasons why saliva should be the sample material of the future if the overtraining syndrome needs to be investigated.

Veterinary Medicine

Saliva from nearly any animal can be measured using salivary IBL test kits and is possible without any further pre-treatment or precaution. This is very different when compared to serum assays. There are two reasons why animal saliva can be used without any problems.

− The Testosterone molecule is absolutely identical in any animal − There is no significant difference in the protein content of saliva from different species. That is why no extraction is necessary.

The IBL Testosterone Luminescence Immunoassay also offers the option for the investigation of serum samples in human and veterinary testing and especially in veterinary medicine this is an attractive option. The dilution of serum samples (e.g. 1:20) practically removes the protein matrix. Therefore no extraction of animal serum samples is necessary if tested by the Luminescence Immunoassay methodology. Due to the high dilution only extremely small serum sample volumes (2µl) are needed, so that such investigations easily can be performed in very small vertebrates.

Page 42 Advantages of the IBL-International Testosterone Saliva Luminescence Immunoassay

- The first FDA cleared test kit for saliva and serum testing - Excellent analytical and functional sensitivity - Good specificity (especially with regard to 5-alpha-DHT) - Excellent precision - Good linearity in the clinical relevant concentration range - Excellent correlation with GCMS (y = 1.03x-0.014; R = 0.999) - 2 controls provided with the kit - Standards, controls and conjugate are ready for use - applicable for automated instruments

The IBL Testosterone Luminescence Immunoassay is not only the first FDA cleared Testosterone salivary assay but it has also been cleared by the FDA for application in serum samples. Therefore this assay can be used in clinical purposes in cases of salivary samples as well as serum samples.

Page 43 Test characteristics of the IBL-International Testosterone Luminescence Immunoassay

Principle: Competitive Chemiluminescence Immunoassays

Intended use: Quantitative determinations of active free Testosterone in saliva and total Testosterone in serum.

Format: 12 microtiter strips with 8 wells each.

Sample: 50 µl Saliva (or 1:20 or higher diluted serum).

Standards: 7 standards ready to use; 0.0/6.4/16/40/100/240/760 pg/mL

Incubation: 4 hours. (room temperature, plate shaker) 10 min. (room temperature)

Substrate: Acridan based substrate

Expected values: Age Male Female (Saliva) (years) (pg/mL) (pg/mL) 20–29 41 – 143 5 - 49 30–39 32 – 100 5 - 49 40–49 30 – 98 4 - 49 50–59 30 – 92 4 - 49 60–69 23 – 87 3 - 39

Woman:Hirsutism 15 - 90 PCO:Syndrome 25 - 145

Sensitivity: 1.8 pg/mL

Precision: Intra-assay: 1.5 – 3.0% at 38 – 541pg/mL Inter-assay 7.0 – 4.0% at 21 – 557pg/mL

Specificity: Cross reactivity (Abraham method) 5α-DihydroTestosterone 1.6% Androstenedione 0.7% Methyl-Testosterone 0.3%

Controls: 2 kit controls

Automation: Applicable on various automated instruments (e.g. Triturus)

Cat.-No.: RE62031

Page 44 Test characteristics of the IBL Testosterone ELISA

Principle: Competitive Enzyme Immunoassay

Intended use: Quantitative determinations of active free Testosterone in saliva and total Testosterone in serum.

Format: 12 microtiter strips with 8 wells each.

Sample: 50 µl Saliva (or 1:20 or higher diluted serum).

Standards: 7 standards ready to use; 0.0/6.4/16/40/100/240/760 pg/mL

Incubation: 2 hours. (room temperature, plate shaker) 15 min. (room temperature, plate shaker)

Substrate: TMB, ready to use

Expected values: Age Male Female (Saliva) (years) (pg/mL) (pg/mL) 20–29 41 – 143 5 - 49 30–39 32 – 100 5 - 49 40–49 30 – 98 4 - 49 50–59 30 – 92 4 - 49 60–69 23 – 87 3 - 39

Woman:Hirsutism 15 - 90 PCO:Syndrome 25 - 145

Sensitivity: 2.0 pg/mL

Precision: Intraassay: 4.2 – 15.1 % at 9.0 – 508.7 pg/mL Interassay 5.5 – 6.0 % at 48.7 – 502.6 pg/mL

Specificity: Cross reactivity (Abraham method) 11 β-OH-Testosterone 4.2 11 α-OH-Testosterone 3.6 Dihydrotestosterone 2.5

Controls: 2 kit controls

Automation: Applicable on various automated instruments (e.g. Triturus)

Cat.-No.: RE52631

Page 45 Application for serum samples

The reliable measurement of low serum Testosterone concentrations in women and children by routine methods is very difficult if not impossible as evidenced by Taieb at all 2003.

- Automated systems usually give results in the low concentration range which show a bias of up to the factor of 5 if compared with the reference method GCMS. - There are no commercial control sera available at a concentration of <0.6ng/mL. - There are no external quality trials using samples in concentration ranges <0.6ng/mL. - Methods using extraction procedures seem to be reliable also in the low concentration range.

In case of serum samples with low concentrations of Testosterone the IBL salivary Luminescence Immunoassay may offer a reliable method for the Testosterone measurement:

- Serum should be diluted 1:20 or higher and tested without extraction step (like a saliva sample). In this case of course the result will reflect the total Testosterone concentration in serum. - The better alternative would be to switch to saliva sampling and to directly measure the free Testosterone fraction.

Table 14: The following table shows the results of correlation studies using serum samples by RIA, GCMS, LCMS and Luminescence Immunoassay (samples have been diluted).

Reference Sex Serum N Linear Regression Correlation Measured Method Dilution Luminescence Coefficient Range (ng/mL) Immunoassay= RIA Male 1:40 71 =1.26RIA+0.15 R=0.97 Up to 8.0 GC/MS Male 1:40 30 =0.96GCMS+0.25 R=0.98 Up to 8.6 GC/MS Female 1:40 15 =0.91GCMS+0.05 R=0.98 Up to 1.1 LC/MS Female 1:20 77 =0.99LCMS+0.003 R=0.97 Up to 1.5

These results show that the IBL Testosterone Luminescence Immunoassay can also be used successfully in the quantitative measurement of serum samples, especially in the low concentration range.

Page 46 The following graph shows the result of a correlation study using female sera. In this study the IBL Luminescence Immunoassay has been correlated with the LCMS method. Comparison of Methods: Serum Testosterone of Females

3

2

1

Y = 1.02*X - 0.056; N=99; R=0,89 0 95,0% Confidential Interval (Line) 95,0% Confidential Interval (Data) Assay1:20 Dilution,Serum nmol/l in 0,5 1,0 1,5 2,0 2,5 IBL- LC/MS Reference Method, in nmol/l

Figure 29: Comparison of serum Testosterone values in the normal range of females (lower than 1ng/mL). The sera were diluted 1:20 with zero standard for the measurement with the IBL Luminescence Immunoassay.

Sera of children should be diluted 1:5. Samples in the concentration range of about 0.05ng/mL can also be reliably measured between the 2nd and 3rd standard of the standard curve.

Table 15: Measurement of children sera by IBL Luminescence Immunoassay

Standard curve Serum Values of children Concentration RLU Child No.: Pre-values RLU Testosterone (ng/mL) (ng/mL) 1:5 Dilution (ng/mL) 0 107,262 6.4 99,870 1 < 0.1 102,022 0.02 16 84,578 2 < 0.1 90,414 0.06 40 61,234 3 < 0.1 90,225 0.07 100 37,950 4 < 0.1 82,890 0.09 250 20,626 5 < 0.1 78,690 0.11 760 6,714

Conclusion: The Testosterone Luminescence Immunoassay method from IBL can be successfully applied for the measurement of free Testosterone in saliva and also for the measurement of total Testosterone in sera of low concentrations. Such low concentration sera typically originate from females, children, and hypogonadic males.

Page 47 Standardisation of the IBL-Testosterone Luminescence Immunoassay by GCMS

In order to correctly calibrate the Testosterone assay a reference laboratory (Prof. Siekmann, University of Bonn) measured 30 saliva pools by GCMS method.

The figure below shows a comparison between GCMS method of Prof. Dr. Siekmann and the IBL Testosterone Luminescence Immunoassay.

160.0

140.0 y = 0.9505x + 7.7704 R = 0.9853 120.0

100.0

80.0 60.0

40.0

20.0

IBL Assay Testosterone (pg/mL) 0.0 0 20 40 60 80 100 120 140 160 GC-MS (pg/mL)

Figure 30: Calibration of the IBL Testosterone saliva assay by GCMS.

Page 48 Literature

- Cook N. J. et. al. Salivary Cortisol and Testosterone as markers of stress in normal subjects in abnormal situations. In Kirschbaum C. et al. (eds.): Assessment of hormones and drugs in saliva in biobehavioral research. Hofgrefe & Huber Publishers, Seattle, 1992 - Gomez, J. M. et al. Salivary Testosterone as an Index of Antiandrogen Therapy in Hirsutism. Recenti Progressi in Medicina 83: 672 – 674, 1992 - Dabbs, J. M. Salivary Testosterone Measurements: Collecting, Storing, and Mailing Saliva Samples Physiology & Behaviour 49: 815 – 817, 1991 - Dabbs, J. M. Salivary Testosterone Measurements: Reliability Across Hours, Days, and Weeks. Physiology & Behaviour 48: 83 – 86, 1990 - Butler, G. E. et al. Salivary Testosterone Levels and the Progress of Puberty in the Normal Boy. Clinical Endocrinology 30: 587 – 596, 1989 - Osredkar, J. et al. Salivary free Testosterone in Hirsutism. Ann Clin Biochem 26: 522 – 526, 1989 - Ellison, P.T., et al. Poplulation variation in age-related decline in male salivary Testosterone. Human Reproduction 17(12): 3251 – 3253, 2002 - Taieb, J., et al. Testosterone Measured by 10 Immunoassays and by Isotope-Dilution Gas Chromatography-Mass Spectrometry in Sera from 116 Men, Women and Children. Clinical Chemistry 49(8): 1381 – 1395, 2003 - Urhausen, A., Kindermann, W., Übertraining. Deutsche Zeitschrift für Sportmedizin, 53: 121 – 122, 2002 - Urhausen, A., Kindermann, W., Current markers fort he diagnosis of overtraining syndrome in the practice of training. Deutsche Zeitschrift für Sportmedizin, 51: 226 – 233, 2000 - A. Vermeulen et al., A Critical Evaluation of Simple Methods for the Estimation of Free Testosterone in Serum. The Journal of Clinical Endocrinology and Metabolism, 1999, 84:3666-3672

Page 49 Progesterone

Physiology and Time Dependent Concentration

Progesterone is produced in the cortex of the adrenal glands in both sexes as a precursor of the other steroid hormones synthesized at this site. Besides, small amounts of Progesterone are produced in the testicles of males and also in the brain of both sexes. The majority of Progesterone in females is produced by the Corpus luteum in the ovaries and during pregnancy in the placenta and can be measured in serum (total concentration) and in saliva (free fraction).

Progesterone concentration changes significantly during pregnancy. After delivery Progesterone falls rapidly into the normal range of the menstrual cycle (next figure).

Pregnancy (last 2 month))

2500

2000

1500

1000

Birth 500 Progesterone in Saliva Progesterone /(pg mL)

0

1 1 1 1 1 1 1 1 1 1 1 1 1 200 200 200 200 200 200 200 200 200 200 200 200 200

22.08. 29.08. 05.09. 12.09. 19.09. 26.09. 03.10. 10.10. 17.10. 24.10. 31.10. 07.11. 14.11.

Figure 31: Progesterone concentration of a female in the last weeks of pregnancy und directly after the birth, values measured with the IBL Luminescence Immunoassay

Page 50 Progesterone during the Menstrual Cycle without Contraceptives (N=27, age 19-43)

250

200

150

100 Saliva (pg/ml) Progesterone

50

0 Bleeding 3 5 7 9 11 13 15 17 19 21 23 25 27 29 Day of the Menstrual Cycle

Figure 32: Salivary Progesterone of the menstrual cycle of 27 female, measured with the IBL Luminescence Immunoassay.

Like Testosterone, Progesterone in saliva, at least in females, shows clear short time dynamic changes of concentration. This is especially obvious during the luteal phase of the menstrual cycle (Figure 33).

Progesterone in Saliva, Diurnal Fine Profile

700 Female 1 Female 2 600 Female 3 500

400

300

200

Concentration (pg/mL) Concentration 100

0 0 1 2 3 4 5 Time (h)

Figure 33: Salivary Progesterone diurnal fine profiles of 3 females during the luteal phase or the beginning of the luteal phase; saliva sample collection every 10 minutes; measured with the IBL Luminescence Immunoassay.

Page 51 The diurnal short time fluctuation is found with a wavelength of about 90 to 300 minutes. This fluctuation, most importantly caused by GNRH (gonadotropin releasing hormone) from the hypothalamic region, is essential for the receptors on the target cells (of the pituitary glands). The fluctuation of the GNRH level affects the release of FSH and LH. The secondary effect most probably is the short time fluctuation as shown in Figure 33.

The fluctuation of Progesterone values is found in saliva as well as in serum (Fig. 34). Therefore in case of Progesterone measurements multiple sampling is highly recommended in order to avoid arbitrary results.

30 1.8

1.6 25 1.4

20 1.2

1 15 0.8

10 0.6

0.4 Progesterone in Saliva (ng/mL) Saliva in Progesterone Progesterone in Plasma (ng/mL) in Progesterone 5 0.2

0 0 8:00 AM 12:00 AM 4:00 PM 8:00 PM 12:00 PM 4:00 AM

Progesterone in Plasma Progesterone in Saliva Figure 34: Diurnal fluctuation of the Progesterone level in a woman during the 21 st day of the cycle (T. M. Delfs et al., 1994).

The high dynamics of Progesterone concentration requires a special sampling strategy at least for routine determinations. Single samples in most cases will give arbitrary results which are difficult to reproduce. Therefore we strongly recommend collecting at least 5 samples during a period of 2 hours. The laboratory mixes the aliquots. The mixture will give an average free Progesterone value which in fact represents the hormone activity during the period of sampling.

Page 52 Normal Ranges

The following is our recommendation for the collection of saliva samples in order to check regular and irregular female menstrual cycles:

Table 16: Collection of saliva samples

Collection of saliva samples Menstrual cycle Between day 20 and 23 after begin of menstrual bleeding 3–5 samples in the morning (about 8am) in intervals of 30 minutes. In disturbed menstrual cycles 3–5 saliva samples in the morning in intervals of 30 minutes at day 2, 10, 12, 14, 16, 18 and 24 after begin of the menstrual bleeding.

Meno Pause Hormone replacement therapy 8–12 hours after application. Hormone replacement therapy with continued transdermal use 24–48 hours after application.

In special applications (e.g. Corpus luteum insufficiency) or in studies, alternative timing of saliva collection may be useful.

Expected values for salivary free Progesterone: * Measurement 8–12 hours after application

Table 17: Normal values Progesterone in saliva Values (pg/mL) Females: Premenopausal Follicular phase 28-82 Luteal phase 127-446 Postmenopausal 18-51 Oral hormone replacement therapy* 100–500 Transdermal application (cream)* 1000-10000 Males < 59

Page 53 Indications

The two main indications for Progesterone measurements are the Corpus Luteum insufficiency as well as the Dysregulation in females. In anti-aging medicine it might be necessary to evaluate the Progesterone level before and during the replacement therapy.

Corpus Luteum Insufficiency

If multiple sampling is applied the salivary determination of free Progesterone is useful for detecting this insufficiency.

Dysregulation

Anovulatorical menstrual cycles can result in absolute decreased saliva Progesterone and saliva Estradiol values (next figure). Anovulational Menstrual Profile

70 Progesterone in pg/ml Estradiol in pg/ml x 0.1 60

50

40

30 Concentration 20

10

0 5 10 15 20 25 30 Days of the Menstrual Cycle

Figure 35: Disturbed menstrual cycle of a female, 24 years old, with pituitary adenoma and increased prolactin values, measured with IBL Luminescence Immunoassay.

Page 54 The following figure illustrates the possibilities of abnormal Progesterone profiles in women.

900 800 700 600 500 400 300 200 100 Salivary Progesterone (pmol/l) 0 -14-12-10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 Day of menstrual cycle 5th percentile of the normal values 95th percentile of normal values mean of normal values profile with premature luteinisation profile with a short luteal phase profile with a poor progesterone surge

Fig. 36: Salivary Progesterone profiles during the menstrual cycle of women. Day 0 is the day of ; day –14 is the day of bleeding (Bolaji et al., 1992).

Application for serum samples

The use of serum samples is not possible with the Progesterone IBL Luminescence Immunoassay assay. This is only possible in the case of Cortisol, Testosterone, and Estradiol.

Veterinary Medicine

The measurement of veterinary saliva samples is possible in any animal using the IBL Luminescence Immunoassay without any further pretreatment or precaution. This is very different compared to serum assays. There are two reasons why animal saliva can be used without any problems.

- The Progesterone molecule is absolutely identical in any animal. - There is no significant difference in the protein content of saliva from different species. That is why no extraction is necessary.

The Progesterone Luminescence Immunoassay from IBL does not offer the option for the investigation of animal serum samples.

Page 55 Advantages of the IBL-International Progesterone Saliva Luminescence Immunoassay

− The first FDA cleared test kit for saliva measurements. − Excellent analytical and functional sensitivity. − Good specificity. − Excellent precision. − Good linearity in the clinical relevant concentration range. − Excellent sensitivity of 2.6 pg/mL. − 2 controls provided with the kit. − Standards, controls and conjugate are ready to use. − Applicable for automated instruments.

The IBL Progesterone Luminescence Immunoassay is the first salivary assay 510(k) cleared by the FDA in the USA which means it can be used for clinical applications as well as research applications.

Page 56 Test characteristics of the IBL-International Progesterone Luminescence Immunoassay

Principle: Competitive Chemiluminescence Immunoassay.

Intended use : Quantitative determination of free Progesterone in saliva.

Regulatory status: CE/ FDA cleared according to 510(k).

Format: 12 microtiter strips with 8 wells each.

Sample: 20 µl saliva.

Standards: 7 standards ready to use; Concentrations 0/ 10/ 25/ 50/ 100/ 300/ 1000 pg/mL.

Incubation: 4 hours (room temperature, plate shaker) 10 min. (room temperature)

Substrate: Acridan based, ready to use.

Expected values: Female: Premenopausal Follicular phase 28-82 pg/mL Luteal phase 127-446 pg/mL Postmenopausal 18-51 pg/mL Male: < 59 pg/mL

Sensitivity: 2.6pg/mL

Precision: Intra-assay: 6.0–0.7% at 11.6–822pg/mL Inter-assay 18.8–5.0% at 10.6–817.1pg/mL

Specificity: Cross reactivity (Abraham Method) 17 α-OH-Progesterone 1.8% 6α-Methyl-17 α-OH-Progesterone 1.4% Pregnenolone 0.4%

Controls: 2 kit controls.

Automation: Applicable on various automated instruments (e.g. Triturus).

Cat. No.: RE62021

Page 57 Test characteristics of the IBL Progesterone ELISA

Principle: Competitive Enzyme Immunoassay.

Intended use : Quantitative determination of free Progesterone in saliva.

Regulatory status: CE

Format: 12 microtiter strips with 8 wells each.

Sample: 50 µl saliva.

Standards: 6 standards ready to use; Concentrations 0/ 20/ 100/ 500/ 2000/ 5000 pg/mL.

Pretreatment: Heating the samples at 60°C for 1 hour

Incubation: 1 hour (room temperature, plate shaker) 10-20 min. (room temperature, plate shaker)

Substrate: TMB based, ready to use.

Expected values: Female, Premenopausal: Follicular phase < 100 pg/mL Luteal phase 100-500 pg/mL Postmenopausal: < 50 pg/mL

Sensitivity: 20 pg/mL

Precision: Intra-assay: 13.3–7.4% at 32.9–302.7pg/mL Inter-assay 12.7–19.9% at 30.8–241.1pg/mL

Specificity: Cross reactivity (Abraham Method) Deoxycorticosterone 1.7 % 17 α-OH-Progesterone 0.4 % 5α-Androstan-3β,17 β-diol 0.3 % Pregnenolone 0.2 %

Controls: 2 kit controls.

Automation: Applicable on various automated instruments (e.g. Triturus).

Cat. No.: DB52621

Page 58 Literature

− Delfs, T. M. et al. 24-Hours Profiles of Salivary Progesterone Fertil Steril 62: 960 – 966, 1994 − Lenton E. A. et al. Measurement of Progesterone in Saliva : Assessment of the Normal Fertile Range Using Spontaneous Conception Cycles. Clinical Endocrinology 28: 637 – 646, 1988 − Luisi, M. et al. Radio-Immunoassay of Salivary Progesterone for Monitoring Ovarian Function in Female Infertility. Ann. Biol clin. 45: 449 – 452, 1987

Page 59 17- β - Estradiol

Physiology and Time Dependent Concentration

Estradiol is secreted into circulation by the ovaries, placenta, adrenal gland, and testes or is produced by extra-glandular conversion of secreted androgen precursors. Estradiol is biologically the most active of the naturally produced human . In the postmenopausal women, Estradiol originates from extra-glandular conversion of androgens and circulates in low, non-cyclic concentrations.

In prepubertal children and in males salivary Estradiol concentrations are low and non-cyclic. The changes in the hormone levels are registered during the different the phases of the menstrual cycle (Figure. 37). Similar to Testosterone and Progesterone, Estradiol also shows short time pulsating dynamics especially in females (Figure 38).

Estradiol During Menstrual Cycle (n=18 women age 19-43)

18

16

14

12

10

8

6 Estradiol (pg/mL) Estradiol 4

2

0 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 Day of Menstrual Cycle

Figure 37: Monthly saliva Estradiol profile during the menstrual cycle of 18 women, measured with the IBL-Luminescence Immunoassay.

Page 60 Estradiol in Saliva, Diurnal Fine Profile

6 Female 1 Female 2 5

4

3

2 Concentration (pg/mL) Concentration 1

0 0 1 2 3 4 Time (h)

Figure 38: Salivary Estradiol of the diurnal fine profile of 2 females during the luteal phase; saliva sample collection every 10 minutes; measured with the IBL Luminescence Immunoassay.

In Figure 38 can be seen that salivary Estradiol (like Progesterone and Testosterone) shows a considerable pulsating dynamic, in short cycles of about 60 to 90 minutes. Therefore single saliva determinations will result in arbitrary values. Therefore, multiple sampling for Estradiol determination is highly recommended. We advise the collection of at least 5 samples during a period of 2 hours. The laboratory should mix equal aliquots of each single sample. The mixture would give a mean free Estradiol value, which in fact represents the hormone activity during the period of sampling.

Normal ranges

The following table summarizes the expected values for 17-beta-Estradiol in saliva.

Table 19: Normal values of Estradiol in Saliva.

Estradiol in saliva (pg/ml) Female Premenopausal Follicular phase 0.2 – 10.4 Midcycle peak 5.8 – 21.2 Luteal phase 0.8 – 10.8 Postmenopausal < 3.2 Male < 2.5

Page 61 Indications

Precocious puberty (Pubertas praecox) in girls

Estradiol measurements may be utilized in the assessment of precocious puberty in girls. The determination of free Estradiol concentrations in saliva in children is non-invasive and therefore very convenient.

Gynecomastia in males

Free Estradiol measurements in saliva may be helpful in the assessment of unexplained Gynecomastia, especially in cases with slightly or non-elevated serum Estradiol values. Such dysregulation can be seen much clearer when measuring the hormone activity (free hormone fraction) in saliva.

In-vitro fertilization and embryo transfer treatment

The concentration of salivary Estradiol in stimulated cycles of female patients with in-vitro fertilization (IVF) and embryo transfer programs can be easily followed by salivary measurements. Such a diagnostic strategy is much more convenient for the patient even in repetitive sampling within short time intervals.

Gandia et al. also described that salivary Estradiol values can be used as markers for the biological response in the case of IVF program. In such a program Estradiol provides valuable information on follicular growth and in the monitoring of induction of ovulation (Figure. 39). They concluded that saliva might substitute serum to determine Estradiol especially in the IVF programs in which daily blood samples are required.

Figure 39: Plasma and saliva Estradiol in the induction of ovulation Mean value of the daily Estradiol concentration of 22 females of the IVF program with 4 different stimulation protocols (hormonal therapy from the third or fourth day of the cycle up to the ovulation induced with 5000 IU hCG = day 0), (Gandia et al. 1992).

Page 62 Increased Estradiol values under stimulation treatment have also been found with the IBL-Luminescence Immunoassay (Figure 40). However, the larger variability in the saliva results must be noted and considered by establishing certain conditions in the technique of saliva sampling. Repeated saliva collection within a short period of time (e.g. 30 minutes) is highly recommended.

Figure 40: Estradiol in saliva of 22 females of the IVF program.OCP = oral contraception; Stim = ovarian stimulation, different types of stimulation; A = 6-12 days, B = 4-9 days, C = 2-5 days before retrieval; retrieval = day of ovum retrieval; Pregnancy 14-19 days later; measured with the IBL-Luminescence Immunoassay.

The success of the IVF and embryo transfer program can be confirmed with the determination of Progesterone in saliva (Figure 41). Repeated saliva collection within short period of time (e.g. 30 minutes) is again recommended.

Page 63 Figure 41: Progesterone in saliva of 22 females of the IVF program. OCP = oral contraception; Stim = ovarian stimulation, different types of stimulation; A = 6-12 days, B = 4-9 days, C = 2-5 days before retrieval; retrieval = day of ovum retrieval; Pregnancy 14-19 days later; measured with the IBL-Luminescence Immunoassay.

Veterinary Medicine

The measurement of veterinary saliva samples with IBL salivary test kits is possible in many animals without any further pretreatment or precaution. This is very different compared to serum assays. There are two reasons why animal saliva can be used without any problems: the Estradiol molecule is absolutely identical in any animal and there is no significant difference in the protein content of saliva from different species. That is why no extraction is necessary. The Estradiol Luminescence Immunoassay from IBL also offers the option of testing animal serum samples. This is an attractive option in veterinary medicine. The dilution of serum samples (e.g. 1:20) practically removes the protein matrix. Therefore no extraction of animal serum samples is necessary if tested by the Estradiol Luminescence Immunoassay. Due to this dilution only extremely small serum sample volumes (2-5 µl) are needed, so that such investigations easily can be performed in very small vertebrates

Application for Serum Samples

The reliable measurement of low Estradiol concentrations in serum of males and children by routine immunoassay methods is very difficult up to impossible. This has been published several times (e.g. Stanczyk at al 2003). In low concentration range, only methods using extraction procedures seem to give reliable results.

Also with our IBL-International Estradiol Luminescence Immunoassay we recommend an extraction step before analyzing the serum samples. In this case the result reflects the total Estradiol concentration in serum. The best alternative would be to switch to saliva sampling and to measure the free Estradiol fraction directly.

Page 64 Advantages of the IBL-International Estradiol Saliva Immunoassay

− FDA cleared kit for saliva (free).

− high analytical sensitivity (0.3 pg/mL).

− very good precision.

− good linearity in the clinical relevant concentration range.

− two levels of controls included in the kit.

− standards, controls and conjugate ready to use.

− applicable for automated instruments.

The IBL Estradiol Luminescence Immunoassay is the first salivary assay 510(k) cleared by the FDA in the USA which means it can be used for clinical applications as well as research applications.

Page 65 Test Characteristics of the IBL-International 17-ß-Estradiol Luminescence Immunoassay

Principle Competitive Chemiluminescence Immunoassay

Intended use Quantitative determination of free 17 β-Estradiol in saliva.

Format 12 microtiter strips with 8 wells.

Sample 50µl saliva.

Standards 7 standards ready to use. 0 / 0.9 / 2.0 / 4.0 / 8.0 / 16 / 64pg/mL

Incubation 4 hours. (room temperature, plate shaker) 10 min. (room temperature)

Substrate Acridan based, ready to use

Expected values Female Premenopausal Follicular phase 0.8 – 7.7 pg/mL Midcycle phase 3.4 – 14.3 pg/mL Luteal phase 1.1 – 7.8 pg/mL Postmenopausal < 4.3 pg/ml Male 0.4 - 3.3 pg/ml

Sensitivity 0.3 pg/ml

Precision Intraassay: 13.3 – 7.2 % at 1.6 – 39.6 pg/mL Interassay 14.8 – 11.2 % at 2.4 – 33.6 pg/mL

Specificity Cross reactivity (Abraham Method) Estrone 14% Estriol 0.5%

Controls 2 kit controls

Automation Assay tested on different microtiter plate instruments.

Cat. No. RE 62041

Page 66 Test Characteristics of the IBL 17-ß-Estradiol ELISA

Principle Competitive Enzyme Immunoassay

Intended use Quantitative determination of free 17 β-Estradiol in saliva.

Format 12 microtiter strips with 8 wells.

Sample 100µL saliva.

Standards 6 standards ready to use. 0 / 1 / 5 / 10 / 50 / 100 pg/mL

Incubation 2 hours. (room temperature) 30 min. (room temperature)

Substrate TMB, ready to use

Expected values Female Premenopausal Follicular phase 1.3 – 7.8 pg/mL Midcycle phase 3.8 – 16.0 pg/mL Luteal phase 1.2 – 8.4 pg/mL Postmenopausal 0.6 – 4.4 pg/mL Male < 4.7 pg/mL

Sensitivity 0.4 pg/ml

Precision Intraassay: 9.9 – 1.0 % at 2.1 – 17.8 pg/mL Interassay 11.1 – 5.7 % at 2.8 – 13.1 pg/mL

Specificity Cross reactivity (Abraham Method) Estrone 0.2 % Estriol 0.05 %

Controls 2 kit controls

Automation Assay tested on different microtiter plate instruments.

Cat. No. RE52601

Page 67 Literature

− Gandia, A. et al., Salivary Estradiol as a Marker of the Biological Response to Induction of Ovulation, In Kirschbaum C. et al. (eds.): Assessment of hormones and drugs in saliva in biobehavioral research. Hofgrefe & Huber Publishers, Seattle, 1992. − Stanczyk F.Z., Cho M.M., Endres D.B., Morrison J.L., Patel St., Paulson R.J., Limitations of direct estradiol and testosterone immunoassay kits. Steroids 68, 1173-12178 (2003).

Page 68 DHEA

Physiology and Time Dependent Concentration

Dehydroepiandrosterone (DHEA) and its sulphate ester dehydroepi- androsterone sulphate (DHEA-S), as C-19 steroid hormones, belong to the group of androgens. As they are almost exclusively (96%) produced in the adrenal cortex, they are the most important adrenal androgens. Additionally, they are also produced in the gonads and in the brain (Wolkowitz et al., 2000).

Due to quantity produced, they can be considered as the main products of human steroid biosynthesis. DHEA-S circulates in blood in 20-fold higher concentrations than any other hormones. The concentration of DHEA-S exceeds the DHEA levels by approximately 300 to 500 times (Orentreich et al. 1984; Ebeling et al., 1994). It serves as a kind of repository form of DHEA. The conversion of DHEA-S into DHEA takes place very quickly and in almost all body tissues because the corresponding enzymes are available ubiquitously.

While the hydrophilic DHEA-S represents the inactive pre-hormone, the lipophilic DHEA can be reabsorbed by the cells of the peripheral tissues, converted into androgens and estrogens and then released into circulation. Only free, non-protein-bound, DHEA can enter the cells and be converted. This is also the case in saliva, where only the free DHEA is available. Therefore, the concentration of biologically active DHEA can be measured easily and directly in saliva.

When no gonadal steroid production is possible, as during or after a total gynecological operation, sexual steroids are available in a physiological concentrations. They are the result of low extragonadal synthesis and the peripheral conversion of DHEA.

The following graph shows an example of a daily profile of steroids in saliva for a menopausal woman. All steroids are in normal post-menopausal range.

Page 69 Cortisol Diurnal profile of Steroids in Saliva: Female, 60 years old. DHEA Progesterone 10000 Testosterone Estradiol 1000

100

10 Conc. Conc. (pg/mL)

1

0 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 Daytime (h)

Figure 42: Daily profile of steroid in saliva of a menopausal woman (60 years). Samples are measured with the IBL Luminescence Immunoassays.

In blood, DHEA-S and DHEA are loosely bounded to albumin (Dunn et al., 1981; Longkope, 1986). In saliva, only the free active hormone is found. While the concentration of DHEA in saliva is around 1/30 of those in sera, the concentration of DHEA-S in saliva is even lower (1/500 to 1/1000) and is dependent on the saliva flow rate. As opposed to the free steroid molecule, the highly hydrophilic sulphated molecule prevents it from passing into the saliva. As a result, high DHEA-S concentrations are primarily a result of oral micro bleeding.

In comparison to DHEA-S, the free DHEA in saliva is a relevant parameter for research and routine diagnostics. The level of DHEA in sera and in saliva is slightly higher in men than in woman (see Tab. 21).

Page 70 Total DHEA in sera and free DHEA in saliva correlate, as shown in the following graphic (Swinkels et al., 1990). Due to the weak binding of the proteins, the correlation is not always ideal.

Comparison of DHEA in Serum and Saliva

Y = 3428 + 109*X; N=19; R=0,79 95,0% Confidential Interval (Line) 95,0% Confidential Interval (Data) 15000

10000

5000 DHEA in Serum (in pg/mL) (in Serum in DHEA

0

20 40 60 80 100 DHEA in Saliva (in pg/mL)

Figure 43: Correlation of values of DHEA in sera and saliva, according to T. Fischer.

The normal DHEA concentration alters significantly with age. In adults, peak concentrations of DHEA are expected between the 25th and the 35th year. This peak is followed by a slow but continuous decline until the concentration finally reaches the minimum of 10-20% of the peak values in advanced age. This age- related decline of DHEA secretion is known as “Adrenopause” and is the result of a reduction of the production of DHEA in the adrenal cortex. The following graphic and table show the age-dependency of DHEA concentration in saliva in men.

600

500

400

300

200 Concentration in pg/mL in Concentration

100

19-29 30-39 40-49 50-59 60-80 Years DHEA in Saliva of Male, Age Dependence

Figure 44: Age dependency of DHEA in saliva for 78 men. Graphics shows the median and the 25 - 75% and 5 - 95% percentile. Values measured by IBL-Luminescence Immunoassay.

Page 71 In children, no circadian concentration differences are present. At birth the DHEA values are very high, dropping sharply to barely measurable values, and remain at low levels until the 7th to 10th year of life, when the adrenal cortex matures and DHEA values begin to rise again (De Peretti et al., 1978).

500

400

300

200 Concentration in pg/ml in Concentration

100

0 5-7 years 9 - 13 years 15 - 20 years DHEA in Saliva of Children and Adolescents

Figure 46: DHEA in saliva, age dependency in children and teenagers, 37 volunteers, with median, 25 - 75% percentile and 10 - 90% percentile, measured with IBL-Luminescence Immunoassay.

In saliva DHEA values are observed to be pulsatile as with other steroid hormone with clear oscillation range around the middle value. The following illustration shows the pulsatile occurrence of DHEA in saliva, which is attributed to the pituitary pulses.

DHEA in Saliva, Female, 61 Years Old

800 700 600 500 400 300 Conc.(pg/mL) 200 100 0 09:15 10:45 12:15 13:45 15:15 17:15 18:45 20:15 22:00 23:30 1:00 Daytime

Figure 47: Daytime related fluctuations of the DHEA concentration in saliva, measured with IBL Luminescence Immunoassay.

Page 72 Normal ranges

Tab. 21: Expected values for DHEA-S- and DHEA

Sera Saliva DHEA-S 1) DHEA 2) DHEA-S 3) DHEA 4) (ng/mL) (ng/mL) (ng/mL) (ng/mL) Men 1000 – 4200 1.8 – 12.5 0.2 – 2.7 0.05 – 0.65 Women 800 – 3900 1.3 – 9.8 0.2 – 2.5 0.04 – 0.60

1) Reference range for IBL-International DHEA-S ELISA; Cat. No.: RE52181. 2) Reference range for IBL-International DHEA ELISA; Cat. No.: RE52221. 3) Data for Diametra DHEA-S ELISA. 4) Reference range for IBL-International DHEA Luminescence Immunoassay; Cat. No.: RE62051.

Tab. 22: Age Dependency of DHEA in saliva values, measured with IBL- Luminescence Immunoassay

Concentration of DHEA in saliva (pg/mL) Age Males (Years) N Median 10%-Percentile 90%-Percentile 19-30 14 334 154 620 31-40 21 252 139 573 41-50 23 249 133 490 51-60 16 183 99 462 61-80 4 91 50 180

During the day, DHEA values have the highest level in the morning and decrease over the day to the lowest level during night time. This pattern is somewhat similar to Cortisol. The rhythm of the daily secretion declines after the 40th to 50th year of life.

Due to the pulsatile dynamics of DHEA secretion repeated saliva sampling is recommended. We recommend collecting 5 saliva samples within 2 hours. In the laboratory, equal volumes of the individual saliva samples can be mixed. This mixed sample results in a mean free DHEA value, which represents the active hormone concentration in a reproducible way.

Page 73 Indications

Due to the ubiquitous occurrence of DHEA, its determination is useful in many fields of the medicinal diagnostics and in research.

Following some DHEA application areas:

− Effect of androgen and estrogen in the gynecology and andrology. − Cardio and vasoprotective effects: Low levels of DHEA are associated with high rates of cardiovascular illness. − Antiproliferative and antioxidative effect of DHEA turns into a retardation in the formation of tumors. − Effect in growth and on the glucose metabolism. − Influence on bone metabolism and reduction of risk of osteoporosis. − Neurosteroidal effects. − Correlation between DHEA and wellness or lifetime. − Some of the effects of DHEA should be looked in detail.

Endocrinology

The necessity of determination of DHEA is related to the suspicion of adrenal insufficiency or presence of tumours in the adrenal gland, to adrenogenital syndrome and in the context of differential diagnosis of hirsutisms and virilisms. The substitution of DHEA in women with insufficiency in adrenal cortex show a detectable improvement of the wellness and mood (Arlt et al. 1999).

Anti-Aging Medicine

Therapeutic replacement of DHEA is possible, targeting levels that corresponding to those typically found in young adults. DHEA replacement is a concept based on studies that document the age-related reduction of DHEA and DHEA-S in sera and the corresponding decline in physiological capacity and biological processes in the human body. The application of daily doses of DHEA, between 20-50mg, may manifest feelings of youthful vigor and vitality in some individuals, and allow them to recover more quickly from physical disorders. Administration of therapeutic DHEA may be done either in the mornings or afternoons (Cherniske, 1998). Patient monitoring is recommended.

The diagnosis may require the DHEA values to be measured on mixed saliva samples or on a daily profile.

The following graph shows the concentration of DHEA in saliva after a single application of 50 mg of DHEA:

Page 74 DHEA in Saliva; after substitution of 50mg DHEA

6000

5000

4000

3000

2000 Conc.(pg/mL)

1000

0 10:00 10:20 10:40 11:00 11:20 11:40 12:00 12:20 12:40 13:00 13:20 13:40 14:00 14:20 14:40 16:30 Daytime Figure 48: Concentration of DHEA in saliva of a young healthy man (27 years) after application of 50mg DHEA, measured in 10 minute time spans with the IBL DHEA Luminescence Immunoassay.

Immediately after the ingestion of the DHEA pill, the level of DHEA increases notably reaching a peak and decreases rapidly thereafter. After one hour the level of DHEA reaches a level which represents a 3 to 4 fold increase of the original concentration. After 2 hours as a consequence of the peripheral conversion of DHEA, the testosterone and estradiol concentrations in saliva increase as well, achieving 2 to 3 times of the original value (see following graph). Because the DHEA was applied to a young man with normal DHEA values, both steroid values are physiologically rather high.

Testosterone and Estradiol in Saliva, after Application of DHEA

350

300

250

200

150

100 Estradiol (pg/mL) Testosteron Testosterone (pg/mL) 50 Estradiol 0 10:00 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00 14:30 16:30 Daytime

Figure 49: Concentration of testosterone and estradiol in saliva of a young healthy man (27 years) after application of 50mg DHEA, measured in 10 minutes time span with IBL- Luminescence Immunoassay.

Page 75 Determination of hormone levels is an extremely practicable method for the examination of the pharma-kinetic and monitoring of therapy in the context of Anti-Aging-Medicine, and it allows the detection of overdoses.

Psychology

DHEA is known as anti-stress hormone and as the opposite of cortisol. McCraty et al. indicates an improvement of emotional wellness and a reduction of negative reception in the context of a one-month self management program. While the concentration of cortisol in saliva diminishes to a quarter, the concentration of DHEA in saliva of the testing group doubles compared to that of the reference group (McCraty et al., 1998).

Neuroendocrinology

While the circulating DHEA has its effects indirectly on the peripheral tissue, through the intracellular conversion to androgen and/ or estrogen without cell receptors, it works directly on the brain by the interaction with neurotransmitter receptors. Based on this it can be established that DHEA is produced in the brain and furthermore that DHEA can be seen as a neurosteroid which is related to different aspects of the brain activity such as thought, sleep, fear, aggression or also dementia (Swe-Bom et al., 2003).

DHEA concentrations in cerebrospinal fluid are comparable to those measurable in saliva (see the following table). The IBL Luminescence Immunoassay kit allows the direct measurement without any pretreatment as pre-concentration for cerebrospinal fluid samples in comparison with other traditional test methods.

Tab. 23: DHEA in cerebrospinal fluid of elderly patients with or without mental illness, measured with IBL-Luminescence Immunoassay.

DHEA in Cerebrospinal Fluid Control group (N=10) 15 – 131 pg/mL 26 Patients with different kind of 18 – 144 pg/mL dementia 3 Patients with confirmed Alzheimer 189 – 293 pg/mL disease

The DHEA Luminescence Immunoassay assay can also be used to quantify analyte in cerebrospinal fluid, although such samples constitute far greater risk to the patient than a simple saliva sample and would need to be medically justified.

Page 76 Sports Medicine

Besides cortisol and testosterone DHEA has also been recommended as a good marker of training stress (Flynn et al., 1997). Indeed, the balance between catabolic (Cortisol) and anabolic hormones (Testosterone, DHEA) may have important implications for performance and recovery processes. However, the testosterone-cortisol ratio has mainly been used by researchers in training and overtraining investigations (Flynn et al., 1997).

Considering the very low concentrations of testosterone in women, particularly in saliva, the measurements of DHEA is suggested. Thus the DHEA determination in saliva has served as a substitute for testosterone measurements to study training responses in elite female handball players (Filaire et al., 2000).

Therefore it is considered appropriate to use salivary DHEA to evaluate training processes in women athletes.

DHEA in Veterinary Medicine

The measurement of veterinary saliva samples with IBL salivary test kits is possible in many animals without any further pre-treatment or precaution. This is very different when compared to serum assays. There are two reasons why animal saliva can be used without any problems.

− The DHEA molecule is absolutely identical in any animal − There is no significant difference in the protein content of saliva from different species.

The DHEA Luminescence Immunoassay from IBL also offers the option for the investigation of serum samples in human and veterinary testing. Especially in veterinary medicine this is an attractive option. The dilution of serum samples (e.g. 1:20) practically removes the protein matrix. Therefore no extraction of animal serum samples is necessary if tested by the DHEA Luminescence Immunoassay. For this reason only extremely small serum sample volumes (2-5 µL) are needed and such investigations can easily be performed in very small vertebrates.

It must be considered though that the level of DHEA may be not detectable in all kind of animals; this is especially true in the case of rodents. Regardless, in each case, it should be established whether or not the measurement of DHEA is reasonable.

Page 77 Advantages of the IBL- International DHEA Luminescence Immunoassay

The IBL DHEA Luminescence Immunoassay has the following characteristics:

− Applicable for saliva and for diluted sera. − High analytic Sensitivity. − Good Specificity. − Very good Precision. − Good Linearity in the relevant clinical range. − 2 kit controls at different concentrations. − Ready to use Standards, Controls and Conjugate. − Applicable to devices.

DHEA Luminescence Immunoassay can be used for research and for both saliva and sera samples.

Page 78 Test Characteristics of the IBL-International DHEA Luminescence Immunoassay

Principle: Competitive Chemiluminiscence-Immunoassay.

Intended use: Quantitative measurement of free DHEA in saliva and total DHEA in diluted sera.

Regulatory status: CE

Format: 12 microtiter strips with 8 wells each

Sample: 50 µl saliva or diluted serum

Standards: 7 standards ready for use 0; 12.3; 37; 111; 333; 1000; 3000 pg/mL

Incubation: 4 h. (room temperature, plate shaker) 10 min. (room temperature)

Substrate: Acridan based, ready for use

Expected values: Male 19-30 years 154 - 620 pg/mL 31-40 years 139 - 574 pg/mL 41-50 years 133 - 490 pg/mL 51-60 years 99 - 463 pg/mL 61-80 years 50 - 180 pg/mL

Sensitivity: Analytical: 3 pg/mL Functional: 10 pg/mL

Linearity: 83 – 118% for 161 – 2572 pg/mL, dilution until 1:32l Recovery: 83 – 120%

Specificity: % Cross reactivity (Abraham-Method) Estrogen 0.32 Androstendion 0.26 17a-Hydroxypregnenolone 0.24 Androsterone 0.23 Pregnenolone 0.20 DHEA-S 0.025

Controls: Two kit controls. Automation: Applicable to different devices. Cat.-Nr.: RE 62051.

Page 79 Test Characteristics of the IBL-International DHEA ELISA

Principle: Competitive Enzyme Immunoassay.

Intended use: Quantitative measurement of free DHEA in saliva

Regulatory status: CE

Format: 12 microtiter strips with 8 wells each

Sample: 50 µl saliva

Standards: 6 standards ready for use 0; 10; 60; 120; 480; 1440 pg/mL

Incubation: 1 h. (room temperature) 20 min. (room temperature)

Substrate: TMB based, ready for use

Expected values: Male 19-30 years 103 - 578 pg/mL 31-40 years 116 - 472 pg/mL 41-50 years 110 - 475 pg/mL 51-60 years 86 - 488 pg/mL 61-80 years 42 - 184 pg/mL

Sensitivity: Analytical: 2.2 pg/mL Functional: 5.6 pg/mL

Precision Intraassay: 4.7 – 3.4 % at 48.9 – 239.9 pg/mL Interassay: 5.6 – 12.2 % at 56.9 – 241.6 pg/mL

Specificity: % Cross reactivity (Abraham-Method) Progesterone 0.23 Androsterone 0.06 17a-Hydroxypregnenolone 0.07 Androsterone 0.06 Pregnenolone 0.01 DHEA-S 0.004

Controls: Two kit controls.

Automation: Applicable to different devices.

Cat.-Nr.: RE 52651.

Page 80 Literature

− Wolkowitz, O. M. et al., Neuropsychiatric Effects of (DHEA) in Kalimi, M., Regelson, W. (eds.): Dehydroepiandrosterone (DHEA) – Biochemical, Physiological and Clinical Aspects. Walter de Gruyter Berlin, New York, 2000, 271-298. − Orentreich, N., et al., Age changes and sex differences in serum dehydroepiandrosterone sulphate concentrations throuout adulthood. J.Clin.Endocrinol.Metabol. 59: 551-555, 1984. − Dunn, J., F. et al. Transport of steroid hormones: Binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J.Clin.Endocrinol. Metabol. 53: 58-68, 1981. − Longkope, C. Adrenal and gonadal androgen secretionin normal females. J.Clin.Endocrinol.Metabol. 15: 213-228, 1986. − Swinkels, L.M.H.W et al. Concentrations of total and free dehydroepiandrosterone in saliva of normal and hirsute women under basal conditions and during administration of dexamethasone/synthetic corticotrophin. Clin.Chem. 36/12: 2042-2046, 1990. − De Peretti, E. et al., Pattern of plasma dehydroepiandrosterone sulphate levels in humans from birth to adhulthood: evidence for testicular production. J.Clin.Endocronol.Metabol. 47: 572-577, 1978. − Swe-Bom, Kim et al., Neurosteroids : Cerebrospinal Fluid Level for Alzheimer’s Disease and Vascular Dementia Diagnosis., J.Clin.Endocrinol.Metabol. 88: 5199-5208, 2003. − Arlt, W. et al., Dehydroepiandrostendione replacement in womaen with adrenal insufficiency. N.Engl.J.Med. 341/14: 1013-1020, 1999. − Fischer, Tobias. Ein Radioimmunoassay (RIA) für die Messung von Dehydroepiandrosteron (DHEA) im Speichel. Inaugural-Dissertation der Bayrischen Julius-Maximilains-Universität Würzburg, Januar 2004 − Cherniske, St,. DHEA: Die Pille für ein langes Leben, Rowohlt 1998. − McCraty, R. et al. The impact of a new emotional self-management program on stress, emotions, heard rate variability, DHEA and Cortisol. Integr.Physiol.Behav.Sci 33(2): 151-170, 1998. − Flynn, M.,G.. et al. Hormonal responses to excessive training: Influence of cross training. Int.J.Sports Med. 18: 191-196, 1997. − Fry, A.C. et al. Resistance exercise overtraining and overreaching. − Filaire, E. et al. DHEA rather than testosterone show saliva androgen responses to exercise in elite femal handball players. Int.J.SportsMed.; 21: 17-20, 2000.

Page 81 Other Websites

Anti-Aging Medicine http://www.worldhealth.net http://www.drhuber.at http://www.healthDiagnostics.de http://www.hormoneundantiaging.de

Information About Salivadiagnostics http://www.criminology.fsu.edu/journal/hold.html http://www.uni-duesseldorf.de/~ck/index.html

Version 5; 06/2009

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