Measurement of Renin Activity using Tandem Mass Spectrometry Ravinder J Singh, PhD, DABCC Mayo Clinic, Rochester, MN
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1 Measurement of Renin Activity using Tandem Mass Spectrometry Ravinder J Singh, PhD, DABCC Mayo Clinic, Rochester, MN
2 Renin Angiotension System Weber, et al. NEJM 2001; 345:1690.
3 Renin Angiotension System Angiotensinogen Renin Ag I ACE Ag II Vascular AT1 receptor Vasoconstriction
4 Renin Angiotension System Ag II Adrenal AT1 receptor Aldosterone Aldosterone Receptor Activity Na + reabsorption via Na + -Cl - cotransporter Epithelial Na + channel
5
6 PAC-PRA ratio and Diagnosis Plasma Renin Activity (PRA) Plasma Aldosterone Concentration (PAC) PRA PAC (PAC-PRA ratio 20) and PAC 15 Primary Aldosteronism
7 Feedback Loop Renin + Aldosterone PAC/PRA = 10
8 Feedback Loop Renin Aldosterone +
9 Feedback Loop Renin + Aldosterone
10 Feedback Loop Renin + Aldosterone PAC/PRA = 10
11 Feedback Loop Renin Aldosterone +
12 Feedback Loop Renin + Aldosterone
13 Feedback Loop Renin Aldosterone + PAC/PRA = 10
14 Feedback Loop a Renin Aldosterone +
15 Feedback Loop a Aldosterone + Renin PAC/PRA > 20
16 Primary Aldosteronism May result from: 1) a single adenoma, 2) multilple adenomata 3) primary hyperplasia 4) adrenal cortical carcinoma
17 Vasculature of Adrenal Gland
18 Laboratory studies Vein Aldosterone Cortisol Aldosterone/Cortisol (ng/dl) (µg/dl) ratio Right adrenal Left adrenal Inferior vena cava
19 Adrenalocetomy
20 Plasma Renin Activity Angiotensinogen Renin Ag I ACE Ag II
21 Methodologies for PRA RIA for Ang-I Competitive-binding HPLC-RIA
22 Revolution LC-MS/MS
23 Collision-assisted dissociation spectra of Ang1 Intensity, cps A B m/z m/z Asp-Arg-Val-Tyr-Ile-His- Pro-Phe-His-Leu Asp-Arg-Val-Tyr-Val-His- Pro-Phe-His-Leu 1278 Clinical Chemistry 1999;45:659
24 LC-MS/MS Ag I-peptide Asp Arg Val Tyr Ile His Pro Phe His Leu MW 1278 Fragmentation Parent ion= Ag I+ H 2 O + 2H + = 1298/2=649 Asp Arg Val Tyr Ile His MW Daughter ion= fragment+1h + = 784/1
25 Precision of the HPLC-ESI-MS/MS assay %CV PRA Mean Within- Between- (ng Ang1/mL/h) concentration day day (ng Ang1/mL)
26 Features of the MS/MS assay Sensitivity= 0.14 ng/ml/h same as RIA Linearity= vs ng/ml/h Precision= 11% for 0.78 ng/ml/h ESI interface-positive ionization mode
27 Multiplexing and Throughput Sensitive and Rapid Determination of Angiotensin I Utilizing Online Extraction and LC-MS/MS
28 Ang I Applied Biosystems API 5000 Tandem Mass Spectrometer with APCI Source Aria TX-4 HTLC System (Thermo-Fisher) Analytical Column: Targa C18, 33 x 4.6 mm, 5 microns HTLC Column: Cyclone, 50 x 1.0 mm Flowrates: HTLC Column 5.0 ml/min, Analytical Column 0.25 ml/min Injection Volume: 30 μl Run Time: 6.5 minutes with a 1.5 minute Analysis Window
29 Parent Ion and daughter ion Ag I-peptide Asp Arg Val Tyr Ile His Pro Phe His Leu MW 1278 Fragmentation Parent ion= Ag I+ H 2 O + 3H + = 1299/3=433 Asp Arg Val Tyr Ile MW Daughter ion= fragment+1h + = 647/1
30 Ang-I MRM Transitions AngioI_ to m/z AngioI_ to m/z AngioIS_1 d to m/z AngioIS_2 d to m/z
31 High Throughput Front-End Device (Cohesive/Thermo) Cohesive TLX4 System
32 Load- No Tee...Loop out Transfer- Tee...Loop in Wash-Regenerate No Tee...Loop in
33
34 High Density MS/MS The parking lot
35 Ang-I Calibration Curve y = x R 2 = AngI Area / IS Area Ang I (ng/ml)
36 Trend and Variability Ang-I IS Intensity 2.00E+05 IS Area 1.50E E E E Injection #
37 CV as a function of Ang-I 18% 16% 14% CV 12% 10% 8% 6% 4% 2% 0% Ang-I ng/ml
38 Ang-I -0 ng/ml
39 Ang-I -0.1 ng/ml
40 Ang-I Patient
41 Method Comparison Bias RIA/PRA (ng Ang1/ml/h) LC-MS/MS PRA (Ang1 ng/ml/h) Clinical Chemistry 1999;45:659
42 Ang-I Method Comparison Angio I (ng/ml) LC-MS/MS Y = x R= 0.96 N = 59 Identity line Y = X Angio I (ng/ml) RIA
43 Prevalence of Primary Aldosteronism Conn had predicted 20% (Conn s Syndrome) 12 cases per year (presenting at Mayo) until 1980, when PAC/PRA was proposed increase in number of cases to 150 per year in 1990 and is continuously increasing The frequency of primary aldosteronism is 10 %
44
45 The Cardiorenal Axis: A Balance between the NPS and RAAS ANP, BNP Natriuretic Diuretic Renin and aldosterone inhibiting Vasodilating Renin-angiotensin angiotensin- aldosterone system Sodium retaining Antidiuretic Vasoconstricting CP
46 ANG II's Role in Cardiovascular Pathology Mehta, P. K. et al. Am J Physiol Cell Physiol 292: C82-C ; doi: /ajpcell Copyright 2007 American Physiological Society
47 Survival Ang-II and Mortality
48 A Robust Method for the Quantification of Angiotensin II in Serum and Plasma using Turboflow-LC-MS/MS
49 Ang-II Transitions Asp Arg Val Tyr Ile His Pro Phe Parent ion= Ag II+ H 2 O + 2H + = 1048/2=524 Parent ion= Ag II+ H 2 O + 3H + = 1049/3=349.7 MW 1028
50 Ang-II MRM Transitions Q /784.5 Native Angiotensin II 527.0/784.5 Labeled Angiotensin II Q /136.2 Native Angiotensin II 351.8/136.2 Labeled Angiotensin II
51
52 Validation Linearity y = x R 2 = Measured (pg/ml) Expected (pg/ml)
53 Collection Tube EDTA Plasma Heparin Plasma ACD Plasma Serum Spec. ID Pink K2 EDTA Green Sodium Heparin % Difference between EDTA & Heparin Yellow ACD (soln B) % Difference between EDTA and ACD Red % Difference between EDTA & Serum % % % % % % % % % % % % % % % Mean 130% 110% 461%
54 PRA-Ang II Ang II pg/ml PRA ng/ml/hr
55 The Role of ANG II in Vascular Smooth Muscle Cell Contraction Mehta, P. K. et al. Am J Physiol Cell Physiol 292: C82-C ; doi: /ajpcell Copyright 2007 American Physiological Society
56 Thanks for your Attention!
57
58 Mineralocorticoid hyperfunction Hyperaldosteronism Primary Conn s Syndrome Aldosterone-producing adrenal adenomas, bilateral idiopathic hyperplasia of the zona glomerulosa or adrenal carcinoma Secondary Renal artery stenosis, chronic renal failure, reninsecreting tumor, CHF, nephrotic syndrome, cirrhosis and malignant hypertension Other Ectopic Cushing s Syndrome or congenital adrenal hyperplasia due to enzyme deficiency
59 Primary Aldosteronism May result from: 1) a single adenoma, 2) multilple adenomata 3) primary hyperplasia 4) adrenal cortical carcinoma
60 Conn s clinical features Hypertension Cause of 1-2% of hypertension cases Muscle weakness and fatigue
61 Methodologies for Steroids Analysis Corticosteroids RIA ELISA GC/MS GC/MS/MS HPLC/MS HPLC/MS/MS Aldosterone Immunoassaychemiluminescence RIA ELISA GC/MS GC/MS/MS
62
63 PAC-PRA ratio and Diagnosis Plasma Renin Activity (PRA) Plasma Aldosterone Concentration (PAC) PRA PAC (PAC-PRA ratio 10) Secondary hyperaldosteronism
64 PAC-PRA ratio and Diagnosis Plasma Renin Activity (PRA) Plasma Aldosterone Concentration (PAC) PRA PAC Congenital adrenal hyperplasia Exogenous mineralo-corticoid Cushing s syndrome 11-β OHSD deficiency
65 Why Is the Kidney Important in CHF? The Kidney I: regulates cardiac loading by control of intravascular volume II: modulates the RAAS which possesses direct actions upon myocardial structure CP
66 Adrenal gland Adrenaline Adrenal Cortex (outer) Adrenal Medulla (center)
67
68 Plasma Renin Activity Angiotensinogen Renin Ag I ACE Ag II Aldosterone
69 Laboratory findings in primary hyperaldosteronism Elevated serum and urine aldosterone Suppressed plasma renin activity Hypokalemia Hyperkaluria Urine K > 30 mmol/d Metabolic alkalosis High normal serum sodium (very rarely hypernatremia is present)
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