Thermo Scientific B · R · A · H · M · S CT-proAVP LIA for use in endocrinology

1. Thermo Scientific B·R·A·H·M·S CT-proAVP LIA for use in endocrinology February 2011

2. Vasopressin & CT-proAVP - FAQs • What is Vasopressin (CT-proAVP) and where is it produced? • What is the physiological role of Vasopressin? • Why not simply measure Vasopressin? • Is CT-proAVP produced together with Vasopressin? • Do both analytes show the same kinetics? • Which CT-proAVP levels should be expected in normals? • Thermo Scientific B·R·A·H·M·S CT-proAVP LIA in the Differential Diagnosis of Diabetes insipidus • What about the performance of the Thermo Scientific B·R·A·H·M·S CT-proAVP LIA Assay?

3. What is Vasopressin and where is it produced?

4. Structure of Vasopressin O NH2 O NH2 -C • Arginine-Vasopressin (AVP) • synonym: Vasopressin or antidiuretic hormone (ADH) • peptide hormone • 9 amino acids • Disulfide bridge between two cysteine amino acids • C-terminal amidation NH2-

5. Synthesis of Vasopressin • Synthesis as a precursor hormone (pre-pro-vasopressin) in the hypothalamus • Cleavage and transport in granules • down the axons • Storage in granules in the posterior pituitary • Release into nearby capillaries upon • appropriate stimulation Figures adapted from: Golenhofen, Basislehrbuch Physiologie, Urban & Fischer; and Morgenthaler NG et al.: Clin Chem 2006 Information: Russel IC and Glover PJ: Critical Care and Resuscitation 2002; Ranger GS: IJCP 2002; Oghlakian G and Klapholz M: Cardiology in Review 2009

6. What is the physiological role of Vasopressin?

7. Vasopressin - physiological role Main role: Regulation of water balance - Increased plasma osmolality - Decreased arterial circulating volume AVP: acts via V2-receptors in the kidney -> water retention AVP: Synthesis in the Hypothalamus Figure adapted from: Knoers NV N Engl J Med. 2005 May 5;352(18):1847-50

8. Vasopressin (AVP) effects • Effects of AVP dependent on concentration : • maximal antidiuretic effect: below 15 pg/ml • vasoconstrictor effect at higher concentrations • very little effect on blood pressure at physiological levels! Singh Ranger G, Int J Clin Pract 2002; 56(10):777-782

9. Vasopressin in stress situation Myocardialinfarction STRESS AVP ACTH Cortisol

10. Why not simply measure Vasopressin?

11. Vasopressin Quantification of Vasopressin is difficult

12. Vasopressin Receptor Vasopressin Quantification of Vasopressin is difficult

13. Vasopressin Receptor Vasopressin Vasopressin Platelets Quantification of Vasopressin is difficult

14. Protease Vasopressin Vasopressin Receptor Vasopressin Vasopressin Platelets Quantification of Vasopressin is difficult

15. Protease Vasopressin Vasopressin Receptor Vasopressin Vasopressin Platelets Quantification of Vasopressin is difficult Further problem: very unstable ex vivo (even frozen)

16. Protease Vasopressin Vasopressin Receptor Vasopressin Vasopressin Platelets Quantification of Vasopressin is difficult Further problem: very unstable ex vivo (even frozen) Only specialized labs measure AVP (time to results several days) Not a single FDA approved AVP assay on the market LIMITED CLINICAL USE

17. Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Morgenthaler NG et al., Clin Chem. 2006

18. Signal Peptidase Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Vasopressin CT-proAVP Neurophysin II Morgenthaler NG et al., Clin Chem. 2006

19. Signal Peptidase ProhormoneConvertase Vasopressin Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Vasopressin CT-proAVP Neurophysin II Neurophysin II CT-proAVP Morgenthaler NG et al., Clin Chem. 2006

20. Signal Peptidase ProhormoneConvertase Vasopressin Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Vasopressin CT-proAVP Neurophysin II Neurophysin II CT-proAVP Morgenthaler NG et al., Clin Chem. 2006

21. Signal Peptidase ProhormoneConvertase Vasopressin Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Vasopressin CT-proAVP Neurophysin II Neurophysin II CT-proAVP very stable ex vivo CT-proAVP Morgenthaler NG et al., Clin Chem. 2006

22. Signal Peptidase ProhormoneConvertase Vasopressin Prohormone processing and assay Signal Vasopressin CT-proAVP Neurophysin II Vasopressin CT-proAVP Neurophysin II Neurophysin II CT-proAVP very stable ex vivo CT-proAVP Morgenthaler NG et al., Clin Chem. 2006

23. Is CT-proAVP produced together with Vasopressin? Do both analytes show the same kinetics in vivo?

24. Correlation of Vasopressin and CT-proAVP LIA Assay r = 0.78 Jochberger S et al., Schock 2009 31: 132-138 Validation in: Jochberger S et al., Intensive Care Med 2009 35:489-497 Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9.

25. CT-proAVP – like Vasopressin – is rapidly degraded in vivo t1/2: few minutes Morgenthaler et al. Clin Chem 2006

26. CT-proAVP – like Vasopressin – is rapidly degraded in vivo t1/2: few minutes Morgenthaler et al. Clin Chem 2006

27. CT-proAVP – like Vasopressin – is rapidly degraded in vivo 97.5 % percentile normals: t1/2: few minutes Morgenthaler et al. Clin Chem 2006

28. CT-proAVP – Stimulation via osmoreceptors CT-proAVP behaves like AVP Hypertonic saline infusion / thirsting Hypotonic saline infusion Control n=8 Szinnai et al. JCEM (2007)

29. CT-proAVP correlates better with osmolality than Vasopressin Balanescu S. et.al. JCEM 2011 in press

30. CT-proAVP- stimulation via baroreceptors/ hemorrhagic shock, model CT-proAVP behaves like AVP Morgenthaler et al. Shock 2007

31. Which CT-proAVP levels should be expected in normals?

32. CT-proAVP is not age-related Normal distribution Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

33. CT-proAVP levels dependent on gender Significantly higher levels in males 706 healthy volunteers Bhandari SS et al, Clinical Science (2009) 116, 257–263

34. CT-proAVP: Influence of exercise Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

35. CT-proAVP: Influence of exercise 97.5 % percentile normals: Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

36. CT-proAVP LIA in the differential diagnosis of Diabetes insipidus

37. What is Diabetes insipidus ? • Diabetes Insipidus (DI) is a disorder in which there is an abnormal increase in urine output, fluid intake and often thirst (polyuria-polydipsia-syndrome). • Urine output is increased because it is not concentrated normally -> the urine is not yellow but pale, colorless or watery. • Diabetes Insipidus is divided into three types, each of which has a different cause and must be treated differently. 

38. Types of Diabetes insipidus • Central Diabetes Insipidus(also known as neurogenic DI): The most common type of DI is caused by a lack of vasopressin. • Treatment: various drugs including a modified vasopressin known as desmopressin or DDAVP • Nephrogenic Diabetes insipidus(also known as renalDI): is caused by an inability of the kidneys to respond to the "antidiuretic effect" of normal amounts of vasopressin. • Treatment: It cannot be treated with DDAVP and, depending on the cause, may or may not be curable by eliminating the offending drug or disease. 

39. Types of Diabetes insipidus • Central Diabetes Insipidus(also known as neurogenic DI): The most common type of DI is caused by a lack of vasopressin. • Treatment: various drugs including a modified vasopressin known as desmopressin or DDAVP • Nephrogenic Diabetes insipidus(also known as renalDI): is caused by an inability of the kidneys to respond to the "antidiuretic effect" of normal amounts of vasopressin.  • Treatment: It cannot be treated with DDAVP and, depending on the cause, may or may not be curable by eliminating the offending drug or disease.  Diagnostic Challenge: All types of Diabetes insipidus also as partial forms existing!

40. Types of Diabetes insipidus (II) • primarypolydipsia : occurs when vasopressin is suppressed by excessive intake of fluids. • most common type of polyuria-polydipsia-syndrome • most often caused by an abnormality in the part of the brain that regulates thirst or by psychogenic illnesses (psychogenic polydipsia) • difficult to differentiate from central DI because it mimics DI.   Interested in more?: http://www.diabetesinsipidus.org Also in French and Spanish language

41. Differential Diagnosis of Diabetes insipidus • Clinical Challenges: Differential diagnosis of patients with polyuria-polydipsia syndrome • State-of-the art diagnosis: • 1. Stimulation of AVP release via a Water deprivation test • 2.Indirect measurement of AVP release by monitoring of urine osmolality and - volume during water deprivation • (ability to concentrate urine). • 3. Additional Desmopressin administration to differentiate nephrogenic DI from central DI. • Direct AVP measurement becomes not the diagnostic reference standard because of its methological limitations (instability of analyte and uncomfortable assay handling)

42. CT-proAVP for Differential diagnosis of Diabetes insipidus ability to concentrate urine during water deprivation , indirect measurement via urine- volume and – osmolality ability to respond to desmopressin intake

43. Differential diagnosis of Diabetes insipidus Diagnosis without water deprivation and Desmopressin stimulation possible! ability to concentrate urine during water deprivation , indirect measurement via urine- volume and - osmolality

44. Differential diagnosis of Diabetes insipidus Diagnosis without water deprivation possible! Differential diagnosis of partial DI possible ability to concentrate urine during water deprivation , indirect measurement via urine- volume and - osmolality

45. CT-proAVP course during water deprivation Mean value of CT-proAVP in primarypolydipsia Mean value of CT-proAVP in central DI

46. Conclusion:Current state-of -the art - method WDT gives no reliable results in the differential diagnosis of polyuria-polydipsia syndrome!CT-proAVP is superior to the current method of choice and revives the concept of the direct test in the polyuria- polydipsia syndrome. Superiority of CT-proAVP in diagnosing Diabetes insipidus Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome – revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011

47. CT-proAVP: Diagnosis of central DI totalis and nephrogenic DI in the 1st blood draw basal CT-proAVP [pmol/l] (fasting, in the morning after 8h dehydration) < 2.6 >20 Sensitivity (%) 95 100 Specificity (%) 100 100 Central Diabetes nephrogenic insipidustotalis Diabetes insipidus Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome – revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011

48. Best separation of primary polydipsia and partial central DI (in contrast to current methods including AVP measurements)specificity 100%sensitivity 86% Differential diagnosis of unclear cases after water deprivation erum -Na+ Poster: Fenske W: 14th Annual meeting of the neuroendocrinology section of the DGE October 15, 2010 (Munich) Paper accepted at JCEM Jan. 2011

49. 2nd blood draw: Stimulated CT-proAVP differentiates safe between central DI partialis and Primary Polydipsia Index Δ CT-proAVP [8h-16h] x 1000 [pmol/L/mmol/L] S-Na+ [16h] <20 >20 Sensitivity (%) 10086 Specificity (%) 86100 central Diabetes primary insipiduspartialispolydipsia Fenske W. et.al. Copeptin in the differential diagnosis of the polyuria- polydipsia syndrome – revisiting the direct and indirect water deprivation tests. JCEM accepted January 2011

50. Suspicion of Diabetes insipidus with Polyurie-Polydipsie-Syndrome CT-proAVP basal (in the morning, fasting, after 8h dehydration) CT-proAVP >=2,6 - 20 pmol/L CT-proAVP <2,6 pmol/L CT-proAVP >20 pmol/L • Ratio of CT-proAVP-Delta (8 to16h) and Serum-Na+ (16h) = CT-proAVP-Index Central Diabetes insipidus totalis Renal Diabetes insipidus CT-proAVP-Index <20 CT-proAVP-Index >=20 Primary Polydipsia Central Diabetes insipidus partalis Reliable differential diagnosis of DI with the help of CT-proAVP CT-proAVP stimulated and Serum-Na+ (after 16 hours dehydration)