AACC 2019 -- Symmetric and asymmetric dimethylarginine (SDMA and ADMA) are metabolites of the post-translational modification of arginine residues in histones. SDMA is produced by all body tissues and is cleared almost exclusively by the kidneys, making it an attractive biomarker for the evaluation of renal function. In this study, the development of an LC-MS/MS method for the measurement of SDMA is described and its performance reviewed against traditional renal markers in individuals with microalbuminuria or abnormal thyroid function.

1. Presented at AACC 2019
Accuracy
SDMA, Creatinine and Cystatin C in Normal Patients versus
Patients with Albuminuria
Thyroid status and renal marker measurement
Conclusions
Creatinine measurement serves as a staple tool in the
assessment of renal disease, importantly for the calculation of
estimated glomerular filtration rate. Creatinine measurement is
impacted by some pathological conditions, including hyper- and
hypothyroidism.
SDMA by LC-MS/MS measurement offers an accurate and
precise method for the assessment of renal function. SDMA
levels rise as expected in subjects with known renal dysfunction,
demonstrated by comparing normal subjects to subjects with
albuminuria. Unlike creatinine, SDMA values are not affected by
gender, as shown through a unisex reference interval. Likewise,
SDMA is not affected by thyroid status. Based on these
observations, SDMA may serve as an alternative strategy to
assess renal function in certain patient populations.
Introduction
Creatinine measurement has served as a staple in the
assessment of renal function. While it serves as an inexpensive
and effective renal marker in some populations, creatinine values
can be confounded by some diseases. SDMA measurement by
liquid chromatography-tandem mass spectrometry (LC-MS/MS)
provides an alternative assessment of renal function in such
scenarios.
Materials and Methods
Serum and K2-EDTA plasma were collected from consented
normal donors. Remnant specimens from individuals with
microalbuminuria or abnormal TSH values were de-identified per
standard operating procedures. SDMA, arginine, ADMA, and
internal standard SDMA-d6 were extracted from specimens by
protein precipitation and dilution. Calibrators were prepared from
spiked charcoal-stripped K2-EDTA human plasma with SDMA.
SDMA and its internal standard were analyzed using Waters
UPLC system (Milford, MA) coupled to an AB Sciex QTRAP 5500
mass spectrometer (Washington, D.C.) in multiple reaction
monitoring (MRM) mode. Chromatographic separation was
performed using Phenomenex normal phase column (Torrance,
CA). The mobile phases consisted of 1% formic acid in water
(phase A) and 50:50 methanol:acetonitrile (phase B). Phase A was
adjusted 15% to 60% and phase B from 85% to 40% from 4.1 to
4.8 minutes before returning to initial conditions. SDMA and its
internal standard were detected by positive electrospray ionization
with the following transitions: m/z 203→172 and internal standard
m/z 209→175.
Cystatin C was measured on a Siemens Behring Nephelometer II
(Malvern, PA). Thyroid stimulating hormone (TSH) was measured
on the Beckman Coulter Access DxI (Brea, CA). Creatinine was
measured on the Roche cobas 8000 (Indianapolis, IN) using
enzymatic and Jaffé reagent systems. Quality control (QC)
samples were assayed prior to SDMA, cystatin C, creatinine and
TSH measurements.
Results
Linearity
Precision
Reference Interval
Abstract
Background
Symmetric and asymmetric dimethylarginine (SDMA and
ADMA) are metabolites of the post-translational modification of
arginine residues in histones. SDMA is produced by all body
tissues and is cleared almost exclusively by the kidneys,
making it an attractive biomarker for the evaluation of renal
function. In this study, the development of an LC-MS/MS
method for the measurement of SDMA is described and its
performance reviewed against traditional renal markers in
individuals with microalbuminuria or abnormal thyroid function.
Methods
Serum and K2-EDTA plasma were collected from consented
normal donors. Remnant specimens from individuals with
microalbuminuria or abnormal TSH values were de-identified
per standard operating procedures. SDMA, arginine, ADMA and
internal standard SDMA-d6 were extracted from specimens by
protein precipitation and dilution. Calibrators were prepared
from spiked charcoal-stripped K2-EDTA human plasma with
SDMA. SDMA and its internal standard were analyzed using
Waters UPLC system (Milford, MA) coupled to an AB Sciex
QTRAP 5500 mass spectrometer (Washington, D.C.) in
multiple reaction monitoring (MRM) mode. Chromatographic
separation was performed using Phenomenex normal phase
column (Torrance, CA). The mobile phases consisted of 1%
formic acid in water (phase A) and 50:50 methanol: acetonitrile
(phase B). Phase A was adjusted 15% to 60% and phase B
from 85% to 40% from 4.1 to 4.8 minutes before returning to
initial conditions. SDMA and its internal standard were detected
by positive electrospray ionization with the following transitions:
m/z 203→172 and internal standard m/z 209→175.
Cystatin C was measured on a Siemens Behring Nephelometer
II (Malvern, PA). Thyroid stimulating hormone (TSH) was
measured on the Beckman Coulter Access DxI (Brea, CA).
Creatinine was measured on the Roche cobas 8000
(Indianapolis, IN) using enzymatic and Jaffé reagent systems.
Quality control (QC) samples were assayed prior to SDMA,
cystatin C, creatinine and TSH measurements.
Results
The LC-MS/MS method for SDMA is linear from 10 to
1000 ng/mL. Intra-day and inter-day precision is <3.2% and
<6%, respectively. Accuracy studies by spike-and-recovery
yielded recoveries from 89-99%. Specimen dilution was verified
up to eight-fold. The reference interval was verified at 73.0 -
125.1 ng/mL for adult males and females. Specimen stability
was established for up to 7 days ambient (20-25°C) and
refrigerated (2-8°C) temperatures and up to 12 months for
-70°C. Freeze-thaw stability was established for 5 cycles at
-70°C and 3 cycles at -20°C. No interference was observed for
hemolyzed, icteric or lipemic samples.
Mean creatinine, cystatin C, and SDMA values were statistically
significantly different (p<0.0001) between normal donors and
individuals with microalbuminuria. Mean creatinine values were
statistically significantly different (p<0.01) between hyperthyroid
(TSH<0.1 μIU/mL) and hypothyroid (TSH >10 μIU/mL)
individuals. Cystatin C and SDMA showed no statistically
significant difference in these individuals (p>0.05).
Conclusions
SDMA measurement by LC-MS/MS provides an analytically
robust alternative to creatinine for the evaluation of renal
function. Like creatinine and cystatin C, SDMA was elevated in
individuals with microalbuminuria. Unlike creatinine, SDMA
measurement was unaffected by thyroid status.
Measurement of Symmetric Dimethylarginine by LC-MS/MS for the
Evaluation of Renal Function
E. Wagner, D. Chu, K. Mercado, D. Fattore, C. Zipperle, E. Fix, S. Fischer and M. Militello; Covance Central Laboratory Services, Indianapolis, IN
0
20
40
60
80
100
120
140
160
180
Creatinine,
Enzymatic
(μmol/L)
Creatinine, Jaffe
(μmol/L)
Cystatin C
(μg/dL)
SDMA (ng/mL)
Meananalytevalue(unitsbelow)
TSH <0.1 μIU/mL
TSH >10 μIU/mL
0
50
100
150
200
250
Creatinine,
Enzymatic
(μmol/L)
Creatinine, Jaffe
(μmol/L)
Cystatin C
(μg/dL)
SDMA (ng/mL)
Meananalytevalue(unitsbelow)
Normal
Albuminuria
Precision Type Level 1 Level 2 Level 3
Target Value 30 ng/mL 150 ng/mL 775 ng/mL
Within run CV% 3.1% 2.7% 2.3%
Between run CV% 3.3% 1.5% 2.3%
Sample Recovery Sample Recovery Sample Recovery
ACC75 101.6 ACC18 99.44 ACC565 101.7
ACC66 99.39 ACC15 106 ACC465 102.65
ACC57 96.32 ACC12 99.17 ACC365 94.58
ACC45 98 ACC965 104.97 ACC265 100.91
ACC36 88.89 ACC865 103.27 ACC165 99.21
ACC30 102 ACC765 103.32 ACC140 97.29
ACC24 102.5 ACC665 95.74