Performance characteristics of the limulus amebocyte lysate assay and gas chromatography-mass spectrum analysis of lipopolysaccharides relative to nitric oxide production by peritoneal exudates of cells.
Limulus amebocyte lysate (LAL) assay and gas chromatography-mass spectrum (GC-MS) analysis are usually used for the quantification of lipopolysaccharides (LPS) from the environment. The LAL assay measures endotoxin units to represent the LPS biological function but GC-MS analysis measures decanoic (C(10:0)) and dodecanoic (C(12:0)) 3-hydorxy fatty acids (3-OH FA) concentration to represent the LPS chemical composition. A study was carried out using these methods to evaluate their degree of correlation. Using the culture supernatants of 30 independent strains of Pseudomonas aeruginosa, the bacterial supernatants gave of 0.53+/-0.45 and 2.49+/-1.75mg/l of C(10:0) and C(12:0) 3-OH FA, respectively, compared to 17.96+/-13.28mg/l of LPS with the LAL assay (1ng/ml of LPS congruent with0.78EU/ml). The 3-OH FA concentration relative to the endotoxin unit dose in the supernatants exhibited a positive correlation (r(2)=0.5182, C(10:0); r(2)=0.4359, C(12:0)). When supernatants having a high level of endotoxin were used to treat peritoneal exudates cells, nitric oxide (NO) was generated in a dose-dependent manner (r(2)=0.6174). To determine if either C(10:0) or C(12:0) 3-OH FA can act as an indictor of LPS quantity was correlated with this immunostimulatory effect, the correlation of these 3-OH FA concentrations against the produced NO levels was evaluated. This also exhibited a positive correlation; however, the two indicators of 3-OH FA gave different dose-responsible performances (r(2)=0.3211, C(10:0); r(2)=0.4527, C(12:0)).
Lin
Hsi-Hsun
HH
Department of Infectious Disease, E-DA Hospital/I-Shou University, Kaoshiung, Taiwan.
Huang
Shiao-Ping
SP
Hsieh
Hui-Chun
HC
Chen
Colin S
CS
Chen
Ya-Lei
YL
D000818:Animals; D005227:Fatty Acids / Q000032:analysis*; D008401:Gas Chromatography-Mass Spectrometry; D008033:Limulus Test / Q000379:methods*; D008070:Lipopolysaccharides / Q000032:analysis* / Q000633:toxicity; D051379:Mice; D008807:Mice, Inbred BALB C; D010537:Peritoneum / Q000166:cytology; D011550:Pseudomonas aeruginosa / Q000502:physiology*; D012680:Sensitivity and Specificity
D005227:Fatty Acids; D008070:Lipopolysaccharides