A cell‐based approach to quantifying endotoxin in human plasma samples

Abstract

Poor dietary choices are thought to contribute to the risk of developing a variety of chronic diseases, in part, through promoting a state of low‐grade inflammation. For example, there are multiple research reports indicating that high‐fat meals promote a transient post‐prandial inflammatory state. Several investigators have hypothesized that high fat meals promote the absorption of microbial endotoxin across the gut wall and that once in the circulation this promotes inflammation through the activation of TLR4 signaling pathway on circulating innate immune cells (e.g., monocytes). Direct assessment of endotoxin in human blood samples is problematic. Reliance on the limulus amebocyte lysate (LAL) test for quantifying endotoxin in a protein‐rich matrix, such as human plasma, requires that samples be heated prior to the analyses. Recovery of endotoxin spiked into plasma samples prior to heating is remarkably low, typically less than 1%. This phenomena, commonly referred to as neutralization, raises grave doubts about the suitability of the LAL test for quantifying endotoxin in the human circulation. Many researchers have long recognized the need for a more robust and accurate method for quantifying low levels of endotoxin in human circulation. In 1995, a cell‐based bioassay approach referred to as monocyte activation test (MAT) was developed and marketed as an alternative to LAL test. Unfortunately, the MAT requires that blood samples are processed within a few hours of collection, which greatly limits the utility of this alternative approach. We have developed and tested a cell‐based bioassay (CBA) for quantifying endotoxin in human plasma following storage at −80°C by using THP‐1 cells (i.e., a human monocytic cell line) stably‐transfected with a luciferase reporter driven by multiple NF‐kB promoters. Our CBA provides reliable and reproducible quantitation of endotoxin in unheated human plasma samples with a limit of detection approaching 0.05 endotoxin units (EU)/mL of plasma. Sensitivity and precision of our CBA compares well with the performance of a recently released commercial cell line (InvivoGen). In conclusion, both of these CBAs appear to provide the research community with a reliable alternative to the LAL test for quantifying endotoxin in human plasma.Support or Funding InformationThis research was supported in part by Grant Number P50AT006273 from the National Center for Complementary and Integrative Health (NCCIH), the Office of Dietary Supplements(ODS), and the National Cancer Institute (NCI). Additional funding was provided by the Food‐for‐the‐21st Century Program ‐Nutrition Cluster, which is part of the College of Agriculture, Food and Natural Resources at the University of Missouri ‐ Columbia. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NCCIH, ODS, NCI, or the National Institutes of Health.