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

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?

What is Vasopressin and where is it produced?

Structure of Vasopressin NH2 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

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 Vasopressin (AVP/ADH) is synthesized as a large precursor hormone mainly in the supraoptic nuclei (N. supraopticus) and to a lesser extent in the paraventricular nuclei (N. paraventricularis) located in the hypothalamus. The initial biologically inactive precursor macromolecule, pre-pro-vasopressin, is a 164 AS protein, which is sequentially cleaved to provasopressin and then to the biologically active peptide vasopressin. Vasopressin, along with its binding protein, neurophysin II, and the glycoprotein CT-proAVP are transported in neurosecretory granules down the axons, into the posterior pituitary, where it is stored. The entire process takes around two hours. From here the granules are released into the nearby capillaries upon receipt of various stimuli to the posterior pituitary. Neurophysin II, which dissociates from the vasopressin molecule prior to secretion, does not appear to have any significant physiological role. The role of CT-proAVP is unclear. It may have a role during the intracellular processing of provasopressin, possibly contributing to correct structural formation of the precursor, which leads to efficient proteolytic maturation. 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

What is the physiological role of Vasopressin?

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

Vasopressin (AVP) effects receptor location effect kidney water retention V1a vascular smooth muscle cells strong vasoconstriction V1b endocrine cells (e.g. pituitary) regulation of ACTH secretion during stress 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! There are three receptors for AVP: the V2, the V1a and the V1b receptor. All AVP receptors are G-protein coupled. The V2 receptor is best known for mediating vasopressin´s antidiuretic effects in the kidney. The V1a receptor, located in vascular smooth muscle cells, is associated with AVPs vasoconstrictor effect. ADH has maximal antidiuretic action at concentrations below 15 pg/mL. Above this level, it has no antidiuretic activity, and even create the opposite effect. Potent vasoconstrictor effects occur at higher concentrations The V1b receptor is mainly present in the pituitary. Its activation plays a major role in the regulation of ACTH (adrenocorticotropic hormone) secretion during physiologic stress. Singh Ranger G, Int J Clin Pract 2002; 56(10):777-782

Vasopressin in stress situation Myocardial infarction STRESS AVP ACTH Vasopressin is released upon physiologic stress. In response to vasopressin, the anterior pituitary releases adrenocorticotropic hormone (ACTH) into the blood system. ACTH is transported to the remotely located adrenal gland. ACTH stimulates the production of cortisol in the adrenal cortex. Cortisol is released into the blood stream. Cortisol affects the body's reaction to stress. Cortisol is a corticosteroid hormone or glucocorticoid produced by the adrenal cortex, that is part of the adrenal gland (in the zona fasciculata and the zona reticularis of the adrenal cortex). It is usually referred to as the "stress hormone" as it is involved in response to stress and anxiety. It increases blood pressure and blood sugar, and reduces immune responses. Cortisol

Why not simply measure Vasopressin?

Quantification of Vasopressin is difficult Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours

Quantification of Vasopressin is difficult Receptor Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours

Quantification of Vasopressin is difficult Receptor Vasopressin Platelets Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours

Quantification of Vasopressin is difficult Receptor Vasopressin Protease Vasopressin Platelets Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours

Further problem: very unstable ex vivo (even frozen) Quantification of Vasopressin is difficult Vasopressin Vasopressin Receptor Vasopressin Protease Vasopressin Platelets Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours Further problem: very unstable ex vivo (even frozen)

Quantification of Vasopressin is difficult Receptor Vasopressin Protease Vasopressin Platelets Problems associated with Vasopressin measurement ca. 90% are associated with platelets Metabolized by vasopressiases from liver and kidney – short plasma half life time (rapidly cleared from plasma) unstable in isolated plasma (even at –20°C) measurement only possible by competitive immunoassay (limitations in sensitivity) measurement requires sample extraction large sample volume required (1-4 ml serum/plasma) time to result: up to 72 hours 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

Prohormone processing and assay Signal Vasopressin Neurophysin II CT-proAVP CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

Prohormone processing and assay Signal Peptidase Vasopressin Neurophysin II CT-proAVP Signal Vasopressin Neurophysin II CT-proAVP CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

Prohormone Convertase Prohormone processing and assay Signal Peptidase Vasopressin Neurophysin II CT-proAVP Signal Vasopressin Neurophysin II CT-proAVP Vasopressin Prohormone Convertase CT-proAVP Neurophysin II CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

Prohormone Convertase Prohormone processing and assay Signal Peptidase Vasopressin Neurophysin II CT-proAVP Signal Vasopressin Neurophysin II CT-proAVP Vasopressin Prohormone Convertase CT-proAVP Neurophysin II CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

Prohormone Convertase Prohormone processing and assay Signal Peptidase Vasopressin Neurophysin II CT-proAVP Signal Vasopressin Neurophysin II CT-proAVP Vasopressin Prohormone Convertase CT-proAVP Neurophysin II CT-proAVP very stable ex vivo CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

Prohormone Convertase Prohormone processing and assay Signal Peptidase Vasopressin Neurophysin II CT-proAVP Signal Vasopressin Neurophysin II CT-proAVP Vasopressin Prohormone Convertase CT-proAVP Neurophysin II CT-proAVP very stable ex vivo CT-proAVP is very stable in whole blood and plasma/serum. Because of the lager molecule the measurement with a more sensitive sandwich-immunoassay is possible (BRAHMS CT-proAVP LIA and BRAHMS CT-proAVP Kryptor) Morgenthaler NG et al., Clin Chem. 2006

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

Correlation of Vasopressin and CT-proAVP LIA Assay The correlation between CT-proAVP and vasopressin is highly significant (see above: different independent studies). Because of its stoichiometric generation, CT-proAVP offers a simple and readily method for vasopressin determination. The good correlation found between CT-proAVP and vasopressin supports this reasoning. CAVE: AVP values are significantly lower than CT-proAVP values due to the binding of AVP to platelets. Because of the limited sensitivity of the AVP assays the correlation is weaker in the lower concentration range. 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.

CT-proAVP – like Vasopressin – is rapidly degraded in vivo Healthy volunteers fasted overnight, consumed a 1 l water load the next morning and had a standardized lunch of 1200 kcal. The individual curves for 2 participants are shown. After an initial high value of the postfasting sample, CT-proAVP decreased rapidly in the male volunteer after he drank 1 l of water and increased again later during the day and after food intake, whereas the values in the woman were constantly low, and initially detectable values decreased to below the detection limit. Again CT-proAVP shows the same pattern as vasopressin. Changes are within the normal range. t1/2: few minutes Morgenthaler et al. Clin Chem 2006

CT-proAVP – like Vasopressin – is rapidly degraded in vivo Healthy volunteers fasted overnight, consumed a 1 l water load the next morning and had a standardized lunch of 1200 kcal. The individual curves for 2 participants are shown. After an initial high value of the postfasting sample, CT-proAVP decreased rapidly in the male volunteer after he drank 1 l of water and increased again later during the day and after food intake, whereas the values in the woman were constantly low, and initially detectable values decreased to below the detection limit. Again CT-proAVP shows the same pattern as vasopressin. Changes are within the normal range. t1/2: few minutes Morgenthaler et al. Clin Chem 2006

CT-proAVP – like Vasopressin – is rapidly degraded in vivo 97.5 % percentile normals: Healthy volunteers fasted overnight, consumed a 1 l water load the next morning and had a standardized lunch of 1200 kcal. The individual curves for 2 participants are shown. After an initial high value of the postfasting sample, CT-proAVP decreased rapidly in the male volunteer after he drank 1 l of water and increased again later during the day and after food intake, whereas the values in the woman were constantly low, and initially detectable values decreased to below the detection limit. Again CT-proAVP shows the same pattern as vasopressin. Changes are within the normal range. t1/2: few minutes Morgenthaler et al. Clin Chem 2006

Hypertonic saline infusion / thirsting CT-proAVP – Stimulation via osmoreceptors CT-proAVP behaves like AVP 8 healthy adults were included in this study. Hypotonic saline infusion lead to CT-proAVP decrease. A 17 hour period of simultaneous thirsting and hypertonic saline infusion induced a CT-proAVP increase. This study shows that CT-proAVP behaves like Vasopressin and therefore strongly correlates to changes in serum osmolality. Control Hypotonic saline infusion Hypertonic saline infusion / thirsting n=8 Szinnai et al. JCEM (2007)

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

CT-proAVP behaves like AVP CT-proAVP- stimulation via baroreceptors/ hemorrhagic shock, model CT-proAVP behaves like AVP Copeptin levels and mean arterial pressure (MAP) in four baboons before, during, and after hemorrhagic shock. Before the experiment, animals were fasted overnight. All the animals were anesthetized and placed on a ventilator. The animals were then bled down to a MAP of 40 mmHg in two stages. Over the first 30 min, the MAP was reduced to 60 mmHg. The MAP was then reduced from 60 to 40 mmHg over the next 30 min. During the first hour of resuscitation, the MAP was brought to 100 mmHg by the infusion of 25% of the shed blood plus Ringer solution, and during the third hour of resuscitation, an additional 25% of the shed blood plus Ringer solution was administered to raise the MAP to baseline levels. The total duration in hours and the time points of blood drawn during bleeding and reperfusion are indicated. R0 is the time point after 3 h of bleeding and immediately before reperfusion was started. Copeptin concentrations in hemorrhagic shock increased markedly from a median of 7.5 to 269 pmol/L. After reperfusion, Copeptin levels dropped. The MAP followed an inverse kinetic in all animals. Copeptin behaves like vasopressin (dependent on blood volume!) Morgenthaler et al. Shock 2007

Which CT-proAVP levels should be expected in normals?

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

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

CT-proAVP: Influence of exercise 6 male and 6 female volunteer blood donors performed a bicycle exercise test, starting with 50 W and increasing in 50 W steps every 3 minutes until physical exhaustion or maximum exercise activity. After exercise the median CT-proAVP concentration increased significantly. In two individuals, CT-proAVP concentrations were monitored after exercise. In both individuals, the CT-proAVP concentration had returned to the original pre-exercise value 1h after exercise completion. Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

CT-proAVP: Influence of exercise 97.5 % percentile normals: 6 male and 6 female volunteer blood donors performed a bicycle exercise test, starting with 50 W and increasing in 50 W steps every 3 minutes until physical exhaustion or maximum exercise activity. After exercise the median CT-proAVP concentration increased significantly. In two individuals, CT-proAVP concentrations were monitored after exercise. In both individuals, the CT-proAVP concentration had returned to the original pre-exercise value 1h after exercise completion. Morgenthaler NG et al., Clin Chem. 2006 Jan;52(1):112-9

CT-proAVP LIA in the differential diagnosis of Diabetes insipidus

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. 

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 renal DI): 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. 

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 renal DI): 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!

Types of Diabetes insipidus (II) primary polydipsia : 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

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)

CT-proAVP for Differential diagnosis of Diabetes insipidus central DI primary Polidipsia Nephrogenic Urine Volume/ fluid intake Excessive Urine- Osmolality low CT-proAVP basal low (< 2.6 pmol/l) (~3 pmol/l) high (>20 pmol/l) CT-proAVP increase after thirsting no yes small State-of-the-art diagnosis ability to concentrate urine during water deprivation , indirect measurement via urine- volume and – osmolality ability to respond to desmopressin intake

Differential diagnosis of Diabetes insipidus central DI primary Polidipsia Nephrogenic Urine Volume/ fluid intake Excessive Urine- Osmolality low CT-proAVP basal low (< 2.6 pmol/l) (~3 pmol/l) high (>20 pmol/l) CT-proAVP increase after thirsting no yes small State-of-the-art diagnosis Diagnosis without water deprivation and Desmopressin stimulation possible! ability to concentrate urine during water deprivation , indirect measurement via urine- volume and - osmolality

Differential diagnosis of Diabetes insipidus central DI primary Polidipsia Nephrogenic Urine Volume/ fluid intake Excessive Urine- Osmolality low CT-proAVP basal low (< 2.6 pmol/l) (~3 pmol/l) high (>20 pmol/l) CT-proAVP increase after thirsting no yes small State-of-the-art diagnosis 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

CT-proAVP course during water deprivation Mean value of CT-proAVP in primary polydipsia in central DI

Superiority of CT-proAVP in diagnosing Diabetes insipidus 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. 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

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 insipidus totalis 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

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

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 (%) 100 86 Specificity (%) 86 100 central Diabetes primary insipidus partialis polydipsia 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

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

Advantages for the diagnostic routine Significantly higher diagnostic accuracy for all variations of Diabetes insipidus and primary Polydipsia Considerably eased differential diagnosis of Polyuria-Polydipsia-Syndrome Reduced physical and psychical exposure of the patient due to simplified WDT and redundancy of desmopressin stimulation Support of safe therapeutic decisions with highly sensitive measurement values Overall cost reduction due to reduced complexity, less lab consulting and no prescription of desmopressin

What about the performance of the LIA assay? 52

Reminder: Why not measure AVP directly? AVP is very unstable in plasma even at -20 °C storage (sample transport frozen or blood collection directly in the lab) AVP is largely attached to platelets AVP assays performed with the required accuracy are available in only a few selected laboratories (non of them FDA cleared) Sample extraction necessary Time to result up to 72 hours Sample volume 1-4 ml plasma No reliable clinical results

Advantages Thermo Scientific B·R·A·H·M·S CT-proAVP LIA sample volume only 50µl for plasma and serum one-step procedure (time to result 3 hours) stable analyte (at room temperature) highest sensitivity sandwich-immunoassay clinical superiority shown

CT-proAVP LIA assay parameters Sample type serum, plasma Volume 50 µl Incubation time 2 hours Stability at RT minimum 8 hours Stability at 2-8°C 14 days Freezing and thawing No influence up to 3 cycles Analytical assay sensitivity < 0,4 pmol/L FAS (20% CV) < 1 pmol/L Direct measuring range 0,4 - 1250 pmol/L Data taken from IFU (instructions for use) 55