Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy.

Abstract

We have compared gas chromatography and mass spectrometry (GC-MS) analysis with the Limulus amebocyte lysate (LAL) assay to quantify native meningococcal lipopolysaccharides (LPS) in five patient plasmas containing > 5 ,gg/liter by LAL. 3-Hydroxy launric acid (3-OH-12:0) was used as a specific lipid A marker of neisserial LPS. The quantitative LAL results were confirmed by GC-MS (r = 0.98, P = 0.006). Seven patient plasmas were centrifuged at 103,000 g and the sedimentation behavior of native LPS compared with reference plasma proteins and with apo Al and apo B100 representing high and low density lipoproteins. After 15 min ofcentrifugation, 84±2% (mean±SE) of the recovered LPS were found in the lower onethird of the centrifuged volume, whereas 6±1% remained in the upper one-third volume, indicating that meningococcal endotoxin circulates as complexes with high sedimentation coefficients. Bacterial outer membrane fragments were detected in the bottom fractions of three patient plasmas examined by means of electron microscopy. In three patient plasmas ultracentrifuged for 60 min at 103,000 g, the levels of apo Al and apo B100 revealed minor changes, whereas only 1±1% of the recovered LPS remained in the upper one-third and 91±2% were found in the lower one-third volume. Few bioreactive LPS appear to be complexed with high and low density lipoproteins in meningococcal septic shock plasma. (J. Clin. Invest. 1992.

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

“…They may thus produce false positive or false negative results ( 9 ). Quantitation of 3HM by GC/MS or GC/MS/MS corresponds to the direct measurement of LPS mass ( 8,10,11 ), but the detection of small amounts of 3HM in complex media is restricted by the limited amounts of lipid-rich samples that can be loaded on GC columns.…”

Distribution of 3-Hydroxy Fatty Acids in Tissues after Intraperitoneal Injection of Endotoxin

“…Lipopolysaccharide (LPS), 4 a constituent of the outer membrane of gram-negative bacteria, is the endotoxin responsible for a variety of pathologic inflammatory reactions that can lead to septic shock and death. Little is known about the translocation of endotoxin into the bloodstream and about its tissue distribution and degradation, mainly because of problems encountered in measuring this potent and multifunctional compound in body fluids and tissues.…”

“…The 3-OH FAs (usually of 10-to 18-carbon chain lengths) have been suggested as chemical markers for LPS in mammalian samples. Low concentrations of 3-OH FAs have been found in normal human sera and plasma (3,4 ). Recently, the same 3-OH FAs were identified in rat blood, and because the concentrations were approximately the same in germ-free as in conventional rats, they were assumed to originate mainly from mitochondrial ␤-oxidation of endogenous FAs (5 ).…”

“…Many sepsis patients with endotoxemia fail to show positive blood cultures for gram-negative bacteria, suggesting the persistence of endotoxin after clearance of the bacteria, leakage of endotoxin from the gut, or deficiencies in blood culture technology. Analysis of plasma and cerebrospinal fluid for meningococcal LPS by measurement of 3-OH 12:0, the specific 3-OH FA of Neisseria (9 ), was suggested as a possible alternative to the LAL activity assay (4,14 ). Interestingly, Limulus activity in plasma from patients with meningococcal septicemia showed a close quantitative association with the 3-OH FA results.…”

Significances of OMV and Extracellular Vesicle Proteomics

“…Proteomic profiling of OMV of Campylobacter jejuni using high resolution LTQ-orbitrap spectroscopy identified 134 vesicular proteins which have the significant role in the bacterial infection and communication [112]. Virulence role of the OMV can also be understood from the proteomic study of the samples obtained from the septic human or rat serum [113,114]. Proteomic analyses of extracellular proteins in the Acinetobacter baumannii have revealed their role in defense machinery against the macrophagic attack and also in the state of oxidative stress [115].…”