William O. Slade (Presenter)
LabCorp
Bio: After completing a Ph.D. at Virginia Tech where he developed targeted and global methods for proteomics in plants, Dr. William Slade moved to the University of North Carolina at Chapel Hill where he developed novel methods for the analysis of post-translational modifications, specifically phosphorylation and redox modifications. During this time he also developed software for the analysis of large phosphoproteomics data sets and the discovery of novel antimicrobial peptides from medicinal plants. Dr. Slade is currently a Researcher in the Mass Spectrometry Research & Development group with Dr. Russ Grant at LabCorp where he is working to bring mass spectrometry to the forefront of clinical proteomics.
Authorship: William O. Slade, Christopher M. Shuford, and Russell P. Grant
(1) Laboratory Corporation of America Holdings
| Short Abstract Plasma Renin Activity (PRA) measures the capacity of circulating Renin and Angiotensinogen to generate Angiotensin 2 (Ang2) using the quantity of its precursor, Angiotensin 1 (Ang1), which is linearly correlated with Ang2 abundance and, thus, predicts hypertension (1,2). Historically, EDTA plasma is the required specimen type for measurement of renin activity due to the chelation of divalent cations from the plasma, which reduces degradative loss of Ang1 to Ang2 during the “generation” procedure by conversion from the metalloprotease, Angiotensin Converting Enzyme (3). As a reference laboratory that receives a many incorrect specimen types, we tested the stabilizing effect of supplemental EDTA on Ang1 in EDTA and non-EDTA samples types. Our preliminary results indicate that supplemental EDTA is required to prevent degradation of Ang1 regardless of sample type. |
Long Abstract
Plasma Renin Activity (PRA) measures the capacity of circulating Renin and Angiotensinogen to generate Angiotensin 2 (Ang2) using the quantity of its precursor, Angiotensin 1 (Ang1), which is linearly correlated with Ang2 abundance and, thus, predicts hypertension (1,2). Historically, EDTA plasma is the required specimen type for measurement of renin activity due to the chelation of divalent cations from the plasma, which reduces degradative loss of Ang1 to Ang2 during the “generation” procedure by conversion from the metalloprotease, Angiotensin Converting Enzyme (3). As a reference laboratory that often receives a many incorrect specimen types, we studied the effect of supplementing specimens (EDTA and non-EDTA) with additional EDTA in order to observe the stabilizing effect on Ang1 during the generation.
Methods
To evaluate the recovery of Ang1 in the generation by sample type and EDTA concentration, we employed two stable isotope-labeled forms of Ang1 as internal standards. Internal standard 1 (IS1), which was labeled at three amino acids, was added to each specimen prior to generation, while internal standard 2 (IS2), which was labeled at two amino acids, was added to each specimen after generation. For generation, samples were transferred to a 37°C water bath and aliquots were collected at 0, 15, 45, 90, 120, and 180 minutes, fortified using IS2 to 400ng/mL and precipitated using methanol before LC-MS/MS. Thus, the measured IS1/IS2 ratio after 0 minutes represented 100% recovery of IS1 and, thus, a decrease in this ratio over the time course demonstrates degradation of IS1 (i.e., degradation of Ang1). This study was performed using matched draws of EDTA-Plasma, Heparin-Plasma, and Serum from 2 volunteers. Generation of the samples was induced by addition of 100mM Tris-Acetate, pH 5.7 containing 0, 3, 7.5, or 15mg/mL K2-EDTA (final concentration in sample) and 167µM PMSF.
Results
In EDTA-Plasma samples, recovery of IS1 after 15 minutes ranged from 63 to 80%, 28 to 43% after 45 minutes, 11-22% after 90 minutes, 7-18% after 120 minutes, and 3-10% after 180 minutes. Including 15 mg/mL EDTA increased recovery to 89-92% after 15 minutes, 88-90% after 45 minutes, 85-86% after 90 minutes, 68-83% after 120 minutes, and 68-86% after 180 minutes. Per patient, Li-Heparin plasma displayed similar trends and ranges with the exception that without supplemental EDTA recovery of IS1 dropped to 27-43% after 15 minutes and subsequently decreased to <5%. Without EDTA, recovery of IS1 in serum decreases to 29-51% after 15 minutes and subsequently decreases < 11%. Including 15 mg/mL supplemental EDTA increases recovery in serum: 83-87% after 15 minutes, 71% after 45 minutes, 53-56% after 45 minutes, 44-48% after 90 minutes, 36-33% after 120 minutes, and 33-36% after 180 minutes.
Discussion
Our preliminary results indicate that supplemental EDTA is required to prevent degradation of Ang1 not only in non-EDTA specimens, but also in EDTA-plasma, which has ~1.8 mg/mL K2-EDTA after full collection. For example, degradation of IS1 during generation ranged from 57-98% after 45min and 80-99% after 3h without supplemental EDTA in both EDTA-plasma specimens tested. In contrast, supplementation using 15mg/mL EDTA yields 68-86% recovery of IS1. Further, heparinized plasma displayed similar recoveries of IS1 when using supplemental EDTA, suggesting IS1 and, thus, Ang1 can be recovered accurately during generation of Heparinized-Plasma. Unfortunately, serum degrades ≥30% or ≥ 67% of IS1 after 45 min or 3h generation even with supplemental EDTA. Given that the clinical utility of PRA measurements requires optimal recovery of Ang1, our results indicate supplemental EDTA is required regardless of sample type. This and additional studies with a greater number of specimens will be presented confirming the optimal concentration of supplemental EDTA required to fully stabilize Ang1 during the generation procedure. Additionally, the stabilizing impact of PMSF and AEBSF on various specimen types and supplemental EDTA concentrations will be explored and contrasted. We will also present a facile colorimetric test that differentiates K2-EDTA plasma from other sample types.
References & Acknowledgements:
(1) Christen Y, et al. Circulation. (1991) 83:1333-42.
(2) Bragat AC, et al. J Hypertens (1997) 15:459-65.
(3) Rehan M, et al. Clinical Biochemistry (2015) 48:377-387.
| Description | Y/N | Source |
| Grants | yes | LabCorp |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |