Oxidized LDL is stable in human serum under extended thawed-state conditions ranging from -20 °C to room temperature

Abstract

Introduction: Oxidized LDL (oxLDL) is formed by the spontaneous reaction between aldehyde byproducts of lipid peroxidation and lysine residues of apolipoprotein B within LDL. Clinically, oxLDL is used as a marker of coronary artery disease and predictor of metabolic syndrome risk. Despite its popularity as a clinical marker, no systematic studies of oxLDL stability, in which serum or plasma has been pre-analytically exposed to an array of different time and temperature conditions, have been carried out.

Objective: To systematically evaluate the stability of oxLDL in human serum samples exposed to thawed conditions (> -30 °C) for varying periods of time while monitoring a second protein/small molecule redox system as a positive control for non-enzymatic biomolecular activity.

Methods: OxLDL was measured in serum samples, from 24 different humans, that had been pre-exposed to three different time courses at 23 °C, 4 °C and -20 °C using ELISA kits from Mercodia that employ the 4E6 mouse monoclonal antibody. A liquid chromatography/mass spectrometry-based marker of serum exposure to thawed conditions known as ΔS-Cys-Albumin was employed as a positive control.

Results: OxLDL was stable in serum exposed to 23 °C for up to 48 h, 4 °C for 21 days, or -20 °C for 65 days. ΔS-Cys-Albumin changed dramatically during these time courses (p < 0.001).

Conclusions: OxLDL is remarkably stable ex vivo in human serum samples exposed to thawed conditions.

Keywords: CAD, coronary artery disease; CHD, coronary heart disease; OxLDL, oxidized low-density lipoprotein; Oxidized LDL; Serum; Stability.

Fig. 1

Stability of oxLDL in serum over time at thawed conditions of A) 23 °C, B) 4 °C, and C) −20 °C. Serum aliquots from 12 GI cancer patients and 12 cancer-free donors were monitored at each temperature. No significant differences were observed between any thawed-state exposure time point and a single set of control aliquots kept continuously at −80 °C.

Fig. 2

ΔS-Cys-Albumin inversely reflects the oxidation of albumin to S-cysteinylated albumin . Here it serves as a positive control for ex vivo changes that occur in a different protein/small molecule redox system present in serum when it is exposed to thawed conditions at A) 23 °C, B) 4 °C, and C) −20 °C. Serum aliquots from the same 12 GI cancer patients and 12 cancer-free donors shown in Fig. 1 were monitored at each temperature. Data points represent the average of all 24 individuals and error bars represent inter-donor standard deviation.