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

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.

Tracking the Stability of Clinically Relevant Blood Plasma Proteins with Delta-S-Cys-Albumin-A Dilute-and-Shoot LC/MS-Based Marker of Specimen Exposure to Thawed Conditions.

Biomolecular integrity can be compromised when blood plasma/serum (P/S) specimens are improperly handled. Compromised analytes can subsequently produce erroneous results-without any indication of having done so. We recently introduced an LC/MS-based marker of P/S exposure to thawed conditions called ΔS-Cys-Albumin which, aided by an established rate law, quantitatively tracks exposure of P/S to temperatures greater than their freezing point of -30 °C. The purposes of this study were to (1) evaluate ΔS-Cys-Albumin baseline values in gastrointestinal cancer patients and cancer-free control donors, (2) empirically assess the kinetic profiles of ΔS-Cys-Albumin at 23 °C, 4 °C, and -20 °C, and (3) empirically link ΔS-Cys-Albumin to the stability of clinically relevant proteins. ΔS-Cys-Albumin was measured at ≥ 9 different time points per exposure temperature in serum and K2EDTA plasma samples from 24 separate donors in aliquots kept separately at 23 °C, 4 °C, and -20 °C. Twenty-one clinically relevant plasma proteins were measured at four time points per temperature via a multiplexed immunoassay on the Luminex platform. Protein stability was assessed by mixed effects models. Coordinated shifts in stability between ΔS-Cys-Albumin and the unstable proteins were documented by repeated measures and Pearson correlations. Plasma ΔS-Cys-Albumin dropped from approximately 20% to under 5% within 96 h at 23 °C, 28 days at 4 °C, and 65 days at -20 °C. On average, 22% of the 21 proteins significantly changed in apparent concentration at each exposure temperature (p < 0.0008 with >10% shift). A linear inverse relationship was found between the percentage of proteins destabilized and ΔS-Cys-Albumin (r = -0.61; p < 0.0001)-regardless of the specific time/temperature of exposure. ΔS-Cys-Albumin tracks cumulative thawed-state exposure. These results now enable ΔS-Cys-Albumin to approximate the percentage of clinically relevant proteins that have been compromised by incidental plasma exposure to thawed-state conditions.

Insight into the molecular function and transcriptional regulation of activator protein 1 (AP-1) components c-Jun/c-Fos ortholog in red lip mullet (Liza haematocheila).

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.

Delta-S-Cys-Albumin: A Lab Test that Quantifies Cumulative Exposure of Archived Human Blood Plasma and Serum Samples to Thawed Conditions.

Exposure of blood plasma/serum (P/S) to thawed conditions (> -30 °C) can produce biomolecular changes that skew measurements of biomarkers within archived patient samples, potentially rendering them unfit for molecular analysis. Because freeze-thaw histories are often poorly documented, objective methods for assessing molecular fitness before analysis are needed. We report a 10-µl, dilute-and-shoot, intact-protein mass spectrometric assay of albumin proteoforms called "ΔS-Cys-Albumin" that quantifies cumulative exposure of archived P/S samples to thawed conditions. The relative abundance of S-cysteinylated (oxidized) albumin in P/S increases inexorably but to a maximum value under 100% when samples are exposed to temperatures > -30 °C. The difference in the relative abundance of S-cysteinylated albumin (S-Cys-Alb) before and after an intentional incubation period that drives this proteoform to its maximum level is denoted as ΔS-Cys-Albumin. ΔS-Cys-Albumin in fully expired samples is zero. The range (mean ± 95% CI) observed for ΔS-Cys-Albumin in fresh cardiac patient P/S (n = 97) was, for plasma 12-29% (20.9 ± 0.75%) and for serum 10-24% (15.5 ± 0.64%). The multireaction rate law that governs S-Cys-Alb formation in P/S was determined and shown to predict the rate of formation of S-Cys-Alb in plasma and serum samples-a step that enables back-calculation of the time at which unknown P/S specimens have been exposed to room temperature. A blind challenge demonstrated that ΔS-Cys-Albumin can detect exposure of groups (n = 6 each) of P/S samples to 23 °C for 2 h, 4 °C for 16 h, or -20 °C for 24 h-and exposure of individual specimens for modestly increased times. An unplanned case study of nominally pristine serum samples collected under NIH-sponsorship demonstrated that empirical evidence is required to ensure accurate knowledge of archived P/S biospecimen storage history.