SalivaA reliable sample matrix in bioanalytics?

Dr. Michael Gröschl

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Classification of saliva by glandular origin

watery secretion: contributes between 20% (basal) to 50% (stimulated)

mixed serous and mucous saliva: 65%.

Only mucous saliva: 4%.

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Transport mechanisms from blood into saliva

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Commercial Collection Devices

Gröschl M; Current status of salivary hormone analysis; Clin Chem. 54(11):1759-69 2008

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Easy and non-invasive collection

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Advantages and Disadvantages

• Non invasive: very suitable for children

• Rapid and simple: useful for profiles in short time intervals

• No sterile precautions

• At-home collection possible

• Intimacy of the patient not disturbed

• Salivary levels reflect the non protein bound and active concentration of hormones or drugs

• Stickiness (Mucins)

• Sensitive Methods are often needed

• Commercial Kits are often not available

• Interfering factors (Contamination with blood and food) must be avoided

• Non-standardised collection procedure

• Diurnal variation of salivary secretion

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Applications for Saliva DiagnosticsExample 1: Assessment of hormones in saliva

Persistent high cortisol responses to repeated psychological stress in a subpopulation of healthy menKirschbaum et alPsychosom Med. 1995;57(5):468-74.

Relationship between salivary progesterone, 17-hydroxyprogesterone, and cortisol levels throughout the normal menstrual cycle of healthy postmenarcheal girlsGröschl et alFertil Steril. 2001 Sep;76(3):615-7.

Caution for unreliable testing procedures offered in the internet !!!

Example from theinternet (vendornot shown).

These assessments ofsingle time points(no profile, nostimulus) areworthless swizzleand discreditscientific salivaanalysis

Applications for Saliva DiagnosticsExample 1: Assessment of hormones in saliva

Source of DNA analysis in forensic settings.Saliva can be easily obtained without violating privacy.All drugs of abuse presently investigated in screening programmes can be detected in saliva.

Applications for Saliva DiagnosticsExample 2: Saliva in forensic medicine

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Metha

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Midazolam

Temazep

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Simple LC-MS/MS Screening and Quantification Methods for the Analysis of 41 Common Pain Drugs in an Oral Fluid MatrixX. Chen, et alAB SCIEX, Foster City, CA 94404, USA

Applications for Saliva DiagnosticsExample 3: Suitability of saliva in drug monitoring

Quantification of busulfan in saliva and plasma in bone marrow transplantation in childrenusing liquid chromatography mass spectrometryRauh M, Stachel D, Kuhlen M, Gröschl M, Holter W, Rascher WClin Pharmacokin. 2006 45(3):305-16.

a) Pharmacokinetics of busulfan in plasma and saliva in a child after 1 mg/kg busulfan (• plasma, osaliva). b) Correlation between saliva and plasma busulfan, r = 0.958, p < 0.001, n = 69.

Applications for Saliva DiagnosticsExample 4: Saliva in sports medicine and doping control

The relationship between serum and saliva erythropoietin concentrations in adults, full-term and premature infantsHung GY, Jeng MJ, Lin CY, Soong WJ, Hwang B.Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi. 1998 Nov-Dec;39(6):380-5.

Measurement of salivary steroids (Cortisol and testosterone) during long term physical labor (Sportive running)

Changes in adrenal and testicular activity monitored by salivary sampling in males throughout marathon runsCook NJ, Read GF, Walker RF, Harris B, Riad-Fahmy D. Eur J Appl Physiol Occup Physiol. 1986;55(6):634-8.

Saliva collection for EPO testing at marathon race

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Applications for Saliva DiagnosticsExample 5: Therapy control in systemic metabolic diseases

Sputum endothelin-1 is increased in cystic fibrosis and chronic obstructive pulmonary diseaseChalmers GW, Macleod KJ, Sriram S, Thomson LJ, McSharry C, Stack BH, Thomson NC.Eur Respir J. 1999 Jun;13(6):1288-92.

Log endothelin (ET)-1 concentrations in normal subjects and patients with cystic fibrosis (CF)). Vertical barsrepresent median and interquartile range. *: p<0.05 versus normal subjects;.

IgA antibodies against MenC in different vaccination groups before (pre) and after (post) vaccination at 4 or 5 years of age

Salivary antibody response to vaccination with meningococcal A/C polysaccharide vaccine in childrenNurkka A, et alVaccine. 2000 19(4-5):547-56.

Example 6: Specific immune response in saliva

Vaccination groups

IgA titer against MnC

Example 7: Salivary markers for gastrointestinal diseases

Parameters of non-specific immune response in the oral cavity• a,b-Defensins bind to bacteria surfaces, cause chemotaxis of immune cells, increase expression of interleucines and cytocines (Gallo et al, J Allergy

Clin Immunol. 2002 10(6):823-31.)• Cathelicidin inhibit bacterial enzymes, growth inhibition of gram+ and gram- bacteria (Murakami et al, J Dent Res. 2002 81(12):845-50.) • Adrenomedullin inhibits growth of gram+ and gram- bacteria (Gröschl M, et al, Regulatory Peptides 2009 154: 16–22)

Salivary growth factors and oral wound healing• Effect of oral epidermal growth factor (EGF) on mucosal healing in rats with duodenal ulcer. (Chao JC, et al. World J Gastroenterol. 2003 9:2261-5)• Vascular endothelial growth factor (VEGF) in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis.

(Taichman N, et al. Lab Invest. 1998 78:869-75)• Salivary leptin induces increased expression of growth factors in oral keratinocytes. (Gröschl M, et al J Mol Endocrinol. 2005 34:353-66)

Salivary biomarkers in tumor diagnostics• Detection of telomerase activity in saliva from oral squamous cell carcinoma patients . (Zhong LP, et al Int J Oral Maxillofac Surg. 2005 34:566-70)• Salivary soluble CD44: a potential molecular marker for head and neck cancer (Franzmann EJ, et al Cancer Epidemiol Biomarkers Prev. 2005

14:735-9)• Salivary leptin as a candidate diagnostic marker in salivary gland tumors (Schapher M, et al Clin Chem. 2009 May;55(5):914-22) • Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma (St John MA, Arch Otolaryngol

Head Neck Surg. 2004 Aug;130(8):929-35)

Conclusion

A growing number of analytes from various fields of research can reliably be monitored in saliva.• collection is easy, non invasive, non stressful.• particularly useful when repeated collections are required

but drawing blood is impractical or unethical.• particularly useful for „Yes-No“ answers e.g. for screening

and epidemiologic applications. • Salivary concentrations of drugs and hormones represent

the free fractions of serum.• Further standardising of saliva collection and sensitive

methods are required.

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