Optimizing Sensitivity of Methods for Detecting Analytes Released During Inflammation

During inflammation, there are microparticles that are released from the formation of blood clots. From these microparticles, bioactive lipids such as eicosanoids, which can be further categorized as prostaglandin, thromboxanes, and leukotrienes, are released. There are also proteins such as LOX-1, known to participate in the pathogenesis of atherosclerotic cardiovascular disease and lung cancer and is expressed by neutrophils in patients with COVID-19. Soluble LOX-1 protein (sLOX-1) was found to become elevated in blood plasma of patients with diabetes, atherosclerosis, non-small cell lung cancer and acute myocardial infarction. The clinical challenge is that these biomarkers are difficult to detect because they are at low concentrations in the bloodstream. The goal of my project is to develop more sensitive methods to detect these biomarkers to aid in the diagnosis, monitoring, and progression of diseases.

This project involved creating a lipidomic and proteomic profile of human blood plasma and serum samples. All samples were from de-identified healthy donors under IRB-approved protocols with written informed consent. Liquid Chromatography – Mass Spectrometry (LCMS) is the ideal method of creating lipidomic profiles of lipid analytes due to its high specificity and sensitivity. Plasma samples’ lipids contents were extracted using 2:1:1 v/v chloroform/methanol/water or 3:2 v/v hexane/isopropanol. These extraction methods were compared for their sensitivity in detecting certain analytes, and it was found that 3:2 v/v hexane/isopropanol was the better extraction method. Nanotraps, made of various types of nanoparticles, were utilized to bind to sLOX-1 in sample and deplete them for analysis using ELISA, where it was discovered that the majority of sLOX-1 fraction was depleted by the Nanotraps in citrated plasma/serum with recombinant sLox-1 but not much in human blood serum alone. Future sample preparation modifications are needed before LC-MS analyses of Nanotrap-associated sLOX-1.

Hello my name is Joelle Nguyen and this is my project on “Optimizing the Sensitivity of Methods for Detecting Analytes Released During Inflammation.” To start with an introduction on the problem I’m trying to address with my project. The biomarkers involved in inflammation and found in the blood are difficult to detect at low concentrations so it’s necessary to develop assays and lab techniques that have enough sensitivity to detect these biomarkers. These biomarkers can include bioactive lipid mediators such as thromboxanes, prostaglandin, and leukotrienes which form from the oxidation of arachidonic acid, and there are biomarkers categorized as proteins such as the sLox-1 receptor. Specific eicosanoids as seen in this diagram here including thromboxane and HETE species are pro-inflammatory mediators that can play a role in platelet aggregation or inflammatory responses. sLox-1 receptors are elevated in blood plasma of patients with diabetes, atherosclerosis, non-small cell lung cancer and acute myocardial infarction. Detecting these biomarkers can aid in the diagnosis and monitoring of diseases as well as their progression. The hypothesis tested was that methods can be developed to improve the sensitivity of inflammatory biomarker detection by LCMS for lipids and proteins.

So to move on to the methodology of my project, there were two approaches. One was comparing two different extraction methods for the lipid analytes found in blood plasma samples for a lipidomic mass spectrometry analysis, and the other was examining the extent of depletion using nanotraps on the sLox-1 protein receptor in plasma/serum samples for a proteomic mass spectrometry analysis. To go into more detail on the lipidomic experiment, I had compared 2:1:1 v/v chloroform/methanol/water with 3:2 v/v hexane/isopropanol as potential extraction solvents. To compare these methods, I analyzed the peak areas of lipid analytes from duplicate citrated plasma samples and used JMP to provide a descriptive statistical analysis. For the proteomic experiment, there were six dyed nanoparticles used as Nanotraps to deplete sLox-1 in plasma/serum samples and an ELISA test was done to analyze the concentration of depleted sLox-1. The samples were not analyzed with mass spectrometry quite yet but the ELISA results provided future directions for sample preparation and LCMS analysis.

Here are the results for the lipidomic profile made using LCMS. The experiment had two different blood donors, and for this donor here you can see that there is a higher peak area for lipid analytes extracted by hexane on the JMP graph to the left and visually to the right you see that 3:2 v/v hexane/isopropanol has a higher peak area for the specific lipid analytes called 4-HNE-H2O. The same can be said for the second donor in my experiment where again the hexane extracted analytes had a higher peak area both on the JMP plot and the mass spectra graph for 4 HNE-H2O. Now for the mass spectrometry proteomics experiment, there was the ELISA data collected which showed that not a lot of sLox-1 depleted from the nanotraps, only around 20% or less for serum with free sLox-1. The figure to the right shows that citrated blood plasma combined with recombinant sLox-1 protein had a much higher depletion of sLox-1 with 90-100% depleted.

Therefore based on the results of my experiments, the 3:2 v/v hexane/isopropanol is the better solvent to use for extracting lipids from citrated blood plasma samples for a lipidomic profile. For the proteomic experiment, the nanotraps shown in the excel bar graphs showed that they did not interfere with ELISA detection of sLox-1 in control samples. The nanotraps only depleted 0 to 20% of free sLox-1 in serum and depleted 40-100% of recombinant sLox-1 in citrated plasma. The next steps for my project would include doing more experimental trials with both extraction methods to see that 3:2 v/v hexane/isopropanol produces reproducible data. There could also be future modifications to plasma/serum sample preparation to improve sLox-1 binding to the nanotraps.

Thank you for listening and I’d like to acknowledge Dr. Hoemann, Dr. Girgis, Dr. Karen Lee, Dr. Luchini, Rayan Ibrahim Alhammad, and Julia Leonard for their help throughout this project.