Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

Abstract

Systemic infl ammatory response syndrome (SIRS) is defi ned by a cluster of clinical signs that include tachycardia, leukocytosis, tachypnoea, and pyrexia ( 1 ). Although SIRS is a major consequence of sepsis (i.e., a deleterious, nonresolving infl ammatory response to infection that can lead to multiple organ dysfunction and septic shock), it can be caused by many pathological events that are not associated with the bacterial infection ( 1 ). It is of major importance to distinguish between noninfectious SIRS, sepsis, and septic shock in critically ill patients because these groups of patients are markedly different in terms of care and clinical outcome. Positive microbiological identifi cation tests [using conventional microbiological tests or bacterial DNA detection ( 2 ) as well as a number of infl ammatory and infectious biomarkers, including cytokines, procalcitonin, immune cell markers or a combination of these ( 3 )] have been proposed, but at this stage, none has proved to be reliable enough to ascertain the occurrence and extent of infection in high-risk patients with SIRS. Interestingly, and as documented further by our group in the prospective multicenter cohort EPISS study ( 4 ), Gramnegative bacilli are the most frequently identifi ed pathogens in patients with septic shock. In this context, the direct quantitation of the culprit component of the Gram-negative bacterium [i.e., lipopolysaccharide (LPS), which triggers SIRS by interacting with the CD14/Toll-like receptor 4 /myeloid differentiation factor 2 receptor complex at the surface of leukocytes ( 1 ) Abbreviations: 3HM, 3-hydroxymyristic acid or 3-hydroxymyristate; LAL, limulus amebocyte lysate; LOD, limit of detection; LOQ, limit of quantifi cation; LPS, lipopolysaccharide; PLTP, phospholipid transfer protein; SIRS, systemic infl ammatory response syndrome; SOFA, sequential organ failure assessment .

Characterization of endotoxins on orthopaedic fixation screws, using physicochemical surface analyses

“…The FDA guidelines have received some criticism due to the fact, for example, that it is not easy to elute LPS from the test item. One study showed that only 40–70% of inoculated LPS was recovered from devices following a very stringent extraction protocol in ethanol …”

“…However, in this study, the authors used only small implants, due to the extremely costly LAL assay reagents. Lipscomb et al tested 260 surgical instruments, from nine primary care units, and used 4,6‐diamidino‐2‐phenylindole dihydrochloride to detect LPS and proteinaceous contaminations. Their results were negative for LPS, but 60% showed a high degree of protein soiling (0.4–4.2 μg protein/mm 2 ).…”

Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry

“…Alternatively, a wide variety of mass spectrometry (MS)-based techniques has been applied for the detection of complex LPS or lipid A [27–30]. For example, Caroff et al reported direct micro-extraction and analysis of intact LPS by combined thin-layer chromatography and matrix-assisted laser desorption ionization (MALDI) MS [27].…”

“…Instead, other approaches for the measurement of LPS using MS are to fractionate intact LPS, extract specific fatty acids and derivatize them prior to analysis. Gas chromatography (GC)- or liquid chromatography (LC) coupled to MS is then applied for the separation, detection and quantitation of targeted fatty acid [30–32]. These methods can also be applied to carbohydrate-containing samples, which normally interfere with endotoxin biological assays but still suffer from multiple sample pre-treatment steps and tedious, time-consuming method development.…”

Dietary lipid emulsions and endotoxemia

“…It is increasingly used in metabolic studies and therefore was adapted to the plasma of different species to this aim (Laugerette et al, 2015). Of note, emerging methods aim at quantifying LPS molecules via the analysis of 3-hydroxymuristic acid from their lipid A in plasma (Pais de Barros et al, 2015).…”