MSACL 2017 US Abstract

Development and Validation of a LC-MS/MS Method for L-Arginine (ARG) and Asymmetric/Symmetric Dimethylarginine (ADMA/SDMA)

Xander van Wijk (Presenter)
University of California, San Francisco

Bio: I received a BSc in Molecular Life Sciences from Maastricht University, The Netherlands and a MSc in Biomedical Sciences from Hasselt University, Belgium. I obtained My PhD in Medical Sciences at the Department of Biochemistry from Radboud University Nijmegen, The Netherlands. After that I was a postdoctoral fellow at the University of California, San Diego. I am currently a Clinical Chemistry Fellow at the University of California, San Francisco.

Authorship: Xander M. R. van Wijk, Alan H. B. Wu, Kara L. Lynch
Department of Laboratory Medicine, University of California, San Francisco and Zuckerberg San Francisco General, San Francisco, CA, USA

Short Abstract

Asymmetric and symmetric dimethylarginine (ADMA and SDMA) are involved in endothelial function via inhibition of endothelial nitric oxide synthase. Elevated levels are associated with cardiovascular risk and renal failure. The latter is particularly true for SDMA as renal excretion is the primary route of its elimination. ADMA and SDMA levels are also increased in critically ill and septic patients. In this regard, the L-arginine (ARG)/ADMA ratio is decreased as well. In light of a greater effort of clinically validating new markers for sepsis, we developed and validated a method for ARG, ADMA, and SDMA. The method we describe here is a relatively simple, reversed phase method, without the need for derivatization. We are currently investigating the clinical utility of ARG, ADMA, and SDMA as markers for sepsis.

Long Abstract

Introduction

Asymmetric and symmetric dimethylarginine (ADMA and SDMA) are formed by methylation of L-arginine (ARG) residues in proteins and are released into the circulation after protein hydrolysis. ADMA is a potent inhibitor of endothelial nitric oxide synthase (eNOS), which generates nitric oxide (NO) from ARG. NO is a protective molecule for the vasculature by dilating blood vessels and inhibiting platelet aggregation and leukocyte adhesion. Via eNOS inhibition, ADMA is an important mediator of endothelial function. SDMA can inhibit eNOS by limiting the ARG uptake via inhibition of a cationic amino acid transporter (hCAT2b). ADMA is mainly eliminated (~80%) by means of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which converts it into dimethylamine and L-citrulline. Cardiovascular risk factors such as oxidized LDL, hyperglycemia, and smoking, decrease DDAH activity and thereby elevate ADMA levels. The remaining 20% is excreted via the kidneys. There is a strong link between ADMA levels and atherosclerosis and cardiovascular events, whereas the link with renal disease ranges from good to absent. As for SDMA, renal excretion is the primary route of elimination and SDMA is a good marker for renal function. SDMA is also increasingly valued as a predictor of cardiovascular events. ADMA and SDMA levels are also increased in critically ill and septic patients. In this regard, the ARG/ADMA ratio is decreased. In light of a greater effort of clinically validating new markers for sepsis, we set out to develop and validate a method for ARG, ADMA, and SDMA.

Methods

Calibrators, controls, and internal standards. The following standards were purchased from Sigma-Aldrich: ARG (cat# 90538 Fluka), ADMA (cat# D4268), both as hydrochloride salts, and SDMA (cat# D0390), as di(p-hydroxyazobenzene-p’-sulfonate) salt. The following internal standards were purchased as hydrochloride salts from Cambridge Isotope Laboratories: ARG-13C6 (cat# CLM-2265-H-0.05) and ADMA-D7 (cat# DLM-7476-PK). SDMA-D6 (Cat# D463582) was purchased from Toronto Research Chemicals.

A calibrator stock solution containing 25 mM ARG, 250 µM ADMA, and 250 µM SDMA was prepared in water. 5 μL of this calibrator stock was added to 245 μL PBS (or in certain cases to plasma or serum) to obtain a 500/5/5 µM (ARG/ADMA/SDMA) calibrator. A total of 10 calibrators were prepared in PBS, ranging from 500/5/5 µM to 500/5/5 nM. An internal standard (IS) stock solution containing 1.25 mM ARG-13C6, 25 µM ADMA-D7, and 25 µM SDMA-D6 was prepared in water.

For controls, serum and plasma pooled from 21-25 donors were used. The target values for serum were 122, 0.515, 0.735 µM; the target values for plasma were 111, 0.664, 0.824 µM, for ARG, ADMA, and SDMA respectively.

Sample preparation. 250 μL sample was mixed with 5 uL IS solution and 750 uL PBS. An Oasis 30 mg MCX 96 well solid phase extraction (SPE) plate (Cat# 186000248) was used for sample preparation. Conditioning of the SPE plate was done with 600 μL MeOH/H2O/NH4OH (50/45/5, v/v/v) and equilibration was done with 600 μL PBS, after which the samples (1 mL) were loaded. The plate was washed with 600 μL 0.1 M HCl and 600 μL MeOH. Elution was performed with 400 μL MeOH/H2O/NH4OH (50/45/5, v/v/v). Samples were dried under nitrogen at 37 °C and dissolved in 100 μL mobile phase A (see below for composition).

LC-MS/MS conditions. Mobile Phase A (MPA) was 5 mM ammonium formate and 0.05% formic acid in H2O, pH ~3.1-3.3. Mobile Phase B was 0.05% formic acid in acetonitrile. A Kinetex 2.6 μM F5 100A, 150 x 4.6 mm column (Phenomenex 00F-4723-E0) was used with a PFP security guard ULTRA column and holder (Phenomenex AJO-8773 and AJO-9000, respectively).Run conditions were as follows: 100% A for 8 min, 95% B for 2 min, and 100% A for 4 min. The flow rate was 0.35 mL/min.

The following transitions were monitored using a 3200 QTRAP (AB Sciex) in multiple reaction monitoring (MRM), positive, ESI mode: m/z 175.0 → 60.0 for ARG, m/z 203.1→ 46.1 for ADMA, m/z 203.1→ 172.0 for SDMA, m/z 181.0 → 61.1 for ARG-13C6, m/z 210.1 → 46.0 for ADMA-D7, and m/z 209.1→ 175.1 for SDMA-D6. Curtain gas was set to 20, collision gas to medium, ionspray voltage to 5500, temperature to 750 ºC, ion source gas 1 to 50, ion source gas 2 to 40, and interface heater to on.

Results

Retention times. The retention time for ARG and ARG-13C6 was 4.13±0.01 min (mean ± SD), for both. The RT for ADMA and ADMA-D7 was 5.54±0.04 min and 5.53±0.03 min, respectively. The RT for SDMA and SDMA-D6 was 5.44±0.02 min and 5.42±0.02 min, respectively.

Linearity. Linearity was achieved for ARG from 0.5 μM to 500 μM (y = 0.0386x + 0.0246, R2 = 0.9981). Linearity for ADMA and SDMA was achieved from 0.0125 μM to 5 μM (ADMA: y = 4.0508x – 0.0557, R2 = 0.995; SDMA: 2.1441x + 0.0067, R2 = 0.9987).

Imprecision. Imprecision was determined by running 5 replicates of pooled serum and plasma samples. The CV for ARG was 2.7% and 4.4% for serum and plasma, respectively. For ADMA this was 6.5% and 2.2% and for this SDMA was 5.3% and 9.4%. Between-run imprecision is currently being determined.

Matrix effect. The matrix effect was determined in two ways. First, the slope of the calibration curve in PBS was compared to the slope of the curve in pooled serum and plasma. The slope of the curve of ARG spiked in serum and plasma was similar to PBS, i.e. 90.9% and 90.2% of the slope in PBS, respectively (0.0351x, R2 = 0.9985 and 0.0348x, R2 = 0.9986 respectively). For ADMA this was 98.0% and 99.4%, respectively (3.9705x, R2 = 0.9974 and 4.028x, R2 = 0.9966, respectively). For SDMA this was 102.4% and 101.2%, respectively (2.1956x, R2 = 0.9933 and 2.1705x, R2 = 0.993, respectively). Second, pre-extraction vs. post-extraction spiking experiments were performed with pooled serum and plasma. For ARG the uncorrected matrix effect was 61.0% for serum, which was corrected by the IS to 116.2%. For plasma, the uncorrected matrix effect was 79.2% and the IS-corrected value was 103.4%. The uncorrected and IS-corrected matrix effect values for ADMA are as follows: serum: 86.0% and 104.4%; plasma: 84.1% and 94.3%. The uncorrected and IS-corrected matrix effect values for SDMA are: serum: 75.7% and 120.0%; plasma: 79.9% and 106.3%.

Recovery. Recovery was determined by pre-extraction vs. post-extraction spiking experiments. Uncorrected and IS-corrected recovery values for ARG were 64.7% and 81.8%, respectively, for serum, and 46.9% and 98.0%, respectively, for plasma. For ADMA these values were 70.7% and 97.9%, respectively, for serum, and 60.2% and 113.2%, respectively, for plasma. For SDMA these values were 72.3% and 97.4%, respectively, for serum and 44.3% and 102.7%, respectively, for plasma.

Interferences. Possible interferences of hemolysis and lipemia were determined by diluting the hemolyzed or lipemic sample 1:1 with PBS containing 125 μM ARG/1.25 μM ADMA/1.25 μM SDMA. This value was compared to a calculated average by running the hemolyzed or lipemic sample and the spiked PBS sample separately. For ARG in serum, the % actual of calculated values were 92.6% and 94.5% for hemolysis and lipemia, respectively. For plasma these values were 100.3% and 105.7%. For ADMA in serum, these values were 108.6% and 111.8% for hemolysis and lipemia, respectively, and for plasma this was 115.3% and 102.0%. For SDMA in serum, the % actual of calculated values were 111.3% and 100.1% for hemolysis and lipemia, respectively. For plasma these values were 101.2% and 90.1%.

Carry-over. Carry-over was determined by injecting mobile phase A after increasing concentrations of analyte. For ARG, carry-over was 7.5% after 0.5 μM, 2.7% after 1.25 μM and this further decreased to 0.3% after 50-500 μM. For ADMA, carry-over was 0.4% at 0.0125 μM and 0.0% after 0.125-5 μM. For SDMA, carry-over was 0.5% at 0.0125 μM and 0.0-0.1% after 0.05-5 μM.

Stability. IS-corrected post-extraction stability for ARG at 4 ºC was 91.8% and 98.1% after 46h for serum and plasma, respectively. After 107-110h, this was 102.0% and 106.4%, respectively. For ADMA, post-extraction stability was 114.0% and 102.3% after 46h for serum and plasma, respectively. After 107-110h, this was 84.0% and 93.9%, respectively. For SDMA, post-extraction stability was 96.4% and 98.9% after 46h for serum and plasma respectively. After 107-110h, this was 114.2% and 96.3%, respectively.

Discussion

Most methods for ARG, ADMA, and SDMA that are described in the literature are based on HILIC separation or reversed-phase separation after butanol derivatization. The method we describe here is a relatively simple, reversed phase method, without the need for derivatization. The reportable range of the method covers the whole range of values expected in control subjects and critically ill and septic patients. We are currently measuring control and patient samples to investigate the utility of ARG, ADMA, and SDMA as markers for sepsis. These samples are divided into the following groups according to the criteria proposed by the American College of Chest Physicians and Society of Critical Care medicine: 1) infection without sepsis, 2) sepsis, 3) severe sepsis, and 4) septic shock.


References & Acknowledgements:


Financial Disclosure

DescriptionY/NSource
GrantsyesAmerican Heart Association and AACC
Salaryno
Board Memberno
Stockno
Expensesno

IP Royalty: no

Planning to mention or discuss specific products or technology of the company(ies) listed above:

no