Determination of lipid A and endotoxin in serum by mass spectroscopy

Abstract

ABSTRACrA quantitative technique for determining lipid A content of endotoxin added to serum by combined gas chromatography-mass spectrometry is described. This MATERIALS AND METHODS Serum Samples. New Zealand white rabbits weighing approximately 2 kg, and not fed overnight, were used. Blood was obtained by heart puncture under sterile conditions. Blood was also obtained aseptically from human volunteers by venipuncture. Serum was separated by centrifugation and stored in pyrogen-free tubes at -200.Solvents. All solvents were either freshly distilled or were liquid chromatography grade and purchased from Burdick and Jackson Co. Determination of #(OH) Myristic Acid Content of LipidA. Varying amounts of Salmonella minnesota R595-lipid A or Escherichia coli 0127:B8 endotoxin in 5 ml of pyrogen-free 0.85% wt/vol NaCl or in 3-5 ml of rabbit or human serum were hydrolyzed with 5 ml of 8 M HCI for 4.5 hr at 1100. The free fatty acids of the hydrolysate were extracted three times with 10 ml of diethyl ether. The ether extracts were pooled, washed with 5 ml of pyrogen-free water, and dried under nitrogen. The extracted free fatty acids were methylated by the procedure of Schlenk and Gellerman (27), dried under nitrogen, dissolved in 0.5 ml of pentane, and applied to a dry silica gel column 60-200 mesh (0.5 X 3 cm). The column was eluted with 3% ether in pentane to remove saturated and unsaturated methyl esters of fatty acids (28). The remaining polar fatty acids, including hydroxy fatty acid methyl esters, were eluted quantitatively with 20% ether in pentane and dried under nitrogen. After addition of 0.25 ml of trimethylsilyl reagent (MesSi imidazole, Applied Science), the samples were shaken on a Vortex mixer. After 2 min, 1 ml of water was added. The Me3Si ethers of hydroxy fatty acid methyl esters were extracted three times with 1 ml of pentane. The pentane extracts were pooled and dried carefully under a nitrogen stream at 300. The dried Me3Si ethers were dissolved in 0.1 ml of acetonitrile, mixed on a Vortex, and filtered through pasteur pipettes plugged with silanized glass wool.Two to five microliters of the acetonitrile solution were injected into a gas chromatograph-mass spectrometer (model 5992 A, Hewlett-Packard, Palo Alto) equipped with a membrane separator. The relative abundance at 315.4 atomic mass unit was determined by using a selected ion monitoring program in conjunction with a six-ion data acquisition program available from Hewlett-Packard. The glass column (2 mm inner diameter X 1.8 m) was packed with 3% Silar-lOC on 100/120 Abbreviations: #(OH), fl-hydroxy; Me3Si, trimethylsilyl; Me3Si-Mefl(OH)C14, trimethylsilyl ether of ,B(OH) methyl myristate.

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

“…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 ).…”

“…Interestingly, Limulus activity in plasma from patients with meningococcal septicemia showed a close quantitative association with the 3-OH FA results. In earlier studies (3,15 ), convincing dose-response relationships were found between serum concentrations of 3-OH FAs and added amounts of LPS. However, it should be noted that these studies were performed using GLC-MS analysis in the selected-ion monitoring mode, a method that provides high sensitivity but may lack specificity in sample matrices that are chemically complex.…”

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.…”

Chemical markers for bacteria in extraterrestrial samples

“…However, viable bacteria were not detected (using classical microbiological culture) in the lunar material when it was first brought by the Apollo missions (Oyama et al, 1970). Unfortunately, chemical methods that detect dead bacterial remnants, or non-culturable bacteria, were in a primitive stage of development at that time (Maitra et al, 1978;Fox et al, 1980Fox et al, , 1993Fox et al, , 1995Fox et al, , 1996Findlay et al, 1983;Saraf and Larsson, 1996;Saraf et al, 1999;Bal and Larsson, 2000;Kozar et al, 2000).…”

“…However, most environmental organisms are non-culturable and remain undetected. Analytical microbiology techniques (primarily GC-MS) detect not only viable bacteria, but also their nonviable cell envelope remnants when present in complex biological matrices (Maitra et al, 1978;Fox et al, 1980;Findlay et al, 1983;). More recently, gas chromatography tandem mass spectrometry (GC-MS/MS) has been proven to be capable of detecting terrestrial bacterial markers at minute concentrations in environmental and clinical samples (Fox et al, 1995(Fox et al, , 1996Saraf and Larsson, 1996;Saraf et al, 1999;Bal and Larsson, 2000;Kozar et al, 2000).…”