Meningococcal endotoxin in lethal septic shock plasma studied by gas chromatography, mass-spectrometry, ultracentrifugation, and electron microscopy.
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 greater than 5 micrograms/liter by LAL. 3-Hydroxy lauric 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 A1 and apo B100 representing high and low density lipoproteins. After 15 min of centrifugation, 84 +/- 2% (mean +/- SE) of the recovered LPS were found in the lower one-third 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 A1 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.
Brandtzaeg
P
P
Department of Pediatrics, Ullevål University Hospital, Oslo, Norway.
Bryn
K
K
Kierulf
P
P
Ovstebø
R
R
Namork
E
E
Aase
B
B
Jantzen
E
E
D004731:Endotoxins / Q000097:blood*; D004926:Escherichia coli / Q000472:pathogenicity; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000097:blood*; D008854:Microscopy, Electron; D009345:Neisseria meningitidis / Q000378:metabolism*; D012772:Shock, Septic / Q000097:blood*; D014461:Ultracentrifugation
D004731:Endotoxins; D008070:Lipopolysaccharides