MSACL 2024 Abstract

Introduction
Adipocytes (fat cells) express multiple proteins that function in the homeostatic regulation of glucose and lipid metabolism. Insulin regulates the translocation and secretion of many of these proteins in response to changes in energy balance. One of these proteins, adiponectin, serves as a biomarker of metabolic dysregulation, where decreased expression is associated with hyperinsulinemia, high liver lipid content, and obesity, all of which are significant risk-factors for cardiovascular disease. Currently, enzyme-linked immunosorbent assays (ELISA) are commonly used for the quantification of adiponectin. However, this approach is expensive, labor-intensive, and not ideally precise, presenting an opportunity for improvement for which a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach is well-suited.

Here, we describe a validated LC-MS/MS method for quantifying adiponectin concentration without prior immunoenrichment in human serum and compare its performance to quantitation by ELISA.

Methods

LC-MS/MS:
Human serum specimens utilized during assay development were fully de-identified random specimen discards from our clinical laboratory. Calibration standards, quality controls (QC), and internal standards (IS) were made by reconstituting recombinant human adiponectin or peptides in MilliQ water and further diluting in stripped serum. Concentrations of calibrator and IS stock solutions were established using amino acid analysis (AAA) by an independent laboratory. Calibration standards, QCs, and patient specimens were thermally denatured and digested with TPCK trypsin after addition of IS in sodium deoxycholate (DOC) solution. Digestion was halted and DOC precipitated with formic acid. After centrifugation, sample supernatants were transferred to a 96-well plate for injection onto an Agilent StreamSelect LC system coupled to an Agilent 6495C QqQ MS. Adiponectin was chromatographically resolved using Kinetex 2.6 µm C18 columns (Phenomenex), and gradients of water and acetonitrile (both containing 0.1% formic acid). Analyte and IS were monitored using electrospray ionization in positive-ion mode with multiple reaction monitoring (MRM).'

ELISA:
Calibration standards, detection antibody solution, 3,3’,5,5’-tetramethylbenzidine (TMB) substrate, stopping solution, and dilution schemes were provided in kit from manufacturer (Otsuka, Human Adiponectin ELISA kit). Serum specimens were diluted using a pretreatment buffer, denatured on a heat block, vortexed, centrifuged, and diluted with an in-house sample diluent. Diluted specimens, calibrators, and QCs were then placed on a Dynex DSX automated ELISA platform for plating and subsequent execution of the immunoassay protocol. Adiponectin levels were calculated using the color intensity measured at 450 nm.

Results
The ELISA and LC-MS/MS methods were compared using the results obtained for 47 serum specimens. Deming regression analysis showed a R2 = 0.9671 correlation between methods with a slope of 0.957.
The sensitivity for the LC-MS/MS method was assessed using 20 serum blanks and 20 low concentration samples (0.78 µg/mL) to assess precision near the lower limit of quantitation. The limit of quantitation for LC-MS-MS was found to be 0.74 µg/mL, 2.56 times more sensitive than validated ELISA method (2 µg/mL).

Precision of the LC-MS/MS was conducted using 3 different concentrations of adiponectin which span both low and high range of detection. Each of the 3 samples were run 4 separate times with 5 replicates (N=20) where each level showed <10% CV.

Discussion
We have developed and validated an accurate, precise, and (most importantly) sensitive LC-MS/MS approach to measuring adiponectin in serum samples. Quantifying adiponectin via LC-MS/MS has high potential for use in reference laboratories; clinical decision-points are at the lower end of the measurement range (3-5 µg/mL). This approach demonstrated over 2-fold greater sensitivity compared with the ELISA method. Additionally, the LC-MS/MS approach requires no preliminary enrichment of the target using expensive monoclonal antibodies, thus improving assay cost. Using a traceable AAA-quantified calibrator also facilitates future standardization of adiponectin quantification, empowering standardized results across multiple laboratories.