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

Abstract

Background: 3-Hydroxy fatty acids (3-OH FAs) with 10-to 18-carbon chain lengths are constituents of the endotoxin [lipopolysaccharide (LPS)] of gram-negative bacteria. We investigated whether these FAs may be used as chemical markers in measuring endotoxin concentrations in mammalian tissue samples. Methods: We used gas-liquid chromatography-tandem mass spectrometry to measure 3-OH FAs in serum and tissues (heart, liver, and skeletal muscles) of rats after intraperitoneal injection of Escherichia coli LPS. One group of rats (group I) received a single LPS dose of 20 mg/kg of body weight; group II rats received the same total dose but over the course of 10 days (2 mg/kg each day). Rats receiving saline (group III) were used as controls. Results: 3-OH FAs with chain lengths of 10, 12, 14, 16, and 18 carbons were detected in all studied types of samples. Group I rats had 50-fold and group II rats had 3-fold higher serum concentrations of 3-hydoxytetradecanoic acid (3-OH 14:0, the predominant 3-OH FA of E. coli LPS) than group III rats. Concentrations of 3-OH 14:0 in livers from group I and II rats were similar and fourfold higher than in group III rats, whereas concentrations of the same acid in skeletal and heart tissues did not differ among the three groups of rats. 3-OH 14:0 dominated in heart and liver of group III rats, whereas 3-OH 16:0 (followed by 3-OH 14:0) dominated in skeletal muscles and blood. Conclusions: 3-OH FAs 10 -18 carbons in length, probably originating from endotoxin and mitochondrial ␤-oxidation, are abundant in rat liver, skeletal muscles,

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

“…In earlier studies, the direct quantitative assay of 3HM was conducted by using GC/MS ( 8,10,11,21,22 ). In the present study, a highly signifi cant correlation was observed between GC/MS and HPLC/MS/MS methods.…”

“…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.…”

Routine clinical laboratory tests correspond to increased serum levels of 3-hydroxy fatty acids, markers of endotoxins, in cardiosurgery patients

“…Earlier studies [15,16] have shown that the distribution of 3-OH FAs in mammalian tissues and in blood can be detected by GLC-MS ion trap instrumentation. An experimental model of germ-free rats indicated a dual origin of these compounds in the blood and soft tissues: because of the unique structure of LPS it is believed that 3-OH FAs are chemical markers of Gram--negative bacteria.…”

“…We previously used 3-hydroxy fatty acids (3-OH FAs) for monitoring endotoxin levels in the blood and sera of rats [15]. 3-OH FAs, structural constituents of the lipid A part of LPS, are chemical markers of endotoxin and are quantified by an analytic method [13].…”

Determination of endotoxin by the measurement of the acetylated methyl glycoside derivative of Kdo with gas–liquid chromatography-mass spectrometry

“…Lipopolysaccharides were prepared with phenol-water extraction method (Westphal and Jann, 1965) and purified on Sepharose 2B column in 0.01 M NH 4 HCO 3 (Romanowska, 1967) as detailed previously (Rybka et al, 2003). Samples of rat serum from previous investigation (Szponar et al, 2003) were used. Samples of the phage T4 preparations cultured on wild type strain E. coli B were obtained from Bacteriophage Laboratory in our Institute.…”