William N. Howald

Porphyromonas gingivalis Lipopolysaccharide Contains Multiple Lipid A Species That Functionally Interact with Both Toll-Like Receptors 2 and 4

ABSTRACT The innate host response to lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis is unusual in that different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) as well as an antagonist or agonist for TLR4. In this report it is shown that P. gingivalis LPS is highly heterogeneous, containing more lipid A species than previously described. In addition, purification of LPS can preferentially fractionate these lipid A species. It is shown that an LPS preparation enriched for lipid A species at m/z 1,435 and 1,450 activates human and mouse TLR2, TLR2 plus TLR1, and TLR4 in transiently transfected HEK 293 cells coexpressing membrane-associated CD14. The HEK cell experiments further demonstrated that cofactor MD-2 was required for functional engagement of TLR4 but not of TLR2 nor TLR2 plus TLR1. In addition, serum-soluble CD14 effectively transferred P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4. Importantly, bone marrow cells obtained from TLR2−/− and TLR4−/− mice also responded to P. gingivalis LPS in a manor consistent with the HEK results, demonstrating that P. gingivalis LPS can utilize both TLR2 and TLR4. No response was observed from bone marrow cells obtained from TLR2 and TLR4 double-knockout mice, demonstrating that P. gingivalis LPS activation occurred exclusively through either TLR2 or TLR4. Although the biological significance of the different lipid A species found in P. gingivalis LPS preparations is not currently understood, it is proposed that the presence of multiple lipid A species contributes to cell activation through both TLR2 and TLR4.

Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity: differential activities of tetra‐ and penta‐acylated lipid A structures on E‐selectin expression and TLR4 recognition

Porphyromonas gingivalis is a Gram‐negative bacterium strongly associated with periodontitis, a chronic inflammatory disease of the tissue surrounding the tooth root surface. Lipopolysaccharide (LPS) obtained from P. gingivalis is unusual in that it has been shown to display an unusual amount of lipid A heterogeneity containing both tetra‐ and penta‐acylated lipid A structures. In this report, it is shown that penta‐acylated lipid A structures facilitate E‐selectin expression whereas tetra‐acylated lipid A structures do not. Furthermore, it is shown that tetra‐acylated lipid A structures are potent antagonists for E‐selectin expression. Both tetra‐ and penta‐acylated lipid A structures interact with TLR4 although experiments utilizing human, mouse and human/mouse chimeric TLR4 proteins demonstrated that they interact differentially with the TLR4 signalling complexes. The presence of two different structural types of lipid A in P. gingivalis LPS, with opposing effects on the E‐selectin response suggests that this organism is able to modulate innate host responses by alterations in the relative amount of these lipid A structures.

Identification of the Lipophilic Factor Produced by Macrophages That Stimulates Steroidogenesis1

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC column...

Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.

Validation of a soy food frequency questionnaire with plasma concentrations of isoflavones in US adults

OBJECTIVE To validate assessment of soy intake using food frequency questionnaires (FFQs) compared with plasma isoflavone (genistein and daidzein) concentrations. DESIGN Cross-sectional analysis of soy isoflavone intake and plasma analysis of isoflavones. SUBJECTS 77 men and women, age range 20 to 40 years, recruited from the Seattle metropolitan area. MAIN OUTCOME MEASURES Isoflavone intake was determined from responses to a 40-item soy FFQ and from tofu and soymilk intake assessed as part of a comprehensive FFQ used for the Womens Health Initiative (WHI FFQ). Isoflavone concentrations in fasting blood samples were determined by liquid chromatography-mass spectrometry. STATISTICAL ANALYSES Correlation coefficients were calculated for: a) isoflavone intake assessed by the soy FFQ and the WHI FFQ, b) intake assessed by the soy FFQ and plasma isoflavone concentrations, and c) intake assessed by the WHI FFQ and plasma isoflavone concentrations. RESULTS Isoflavone intake was highly correlated between the soy FFQ and the WHI FFQ (r = 0.84). Genistein and daidzein intakes determined by the soy FFQ were significantly correlated with plasma concentrations (r = 0.53 and 0.45, respectively). Isoflavone intake assessed from the WHI FFQ was also correlated with plasma concentration (r = 0.46 and 0.45). Soymilk and tofu were the two major contributors to isoflavone intake (38.6%). CONCLUSIONS A soy-specific, 40-item FFQ assessed isoflavone intake with good validity. Isoflavone intake assessed by the WHI FFQ (tofu and soymilk) had lower correlations with plasma concentrations compared with the soy FFQ. Nonetheless, assessment of the two foods is a reasonably good marker for soy food consumption in this sample.

Analysis of cyclophosphamide and five metabolites from human plasma using liquid chromatography-mass spectrometry and gas chromatography-nitrogen-phosphorus detection.

An assay method for the quantification of cyclophosphamide (CY) and five metabolites from human plasma is presented. The procedure is adapted to the chemical properties of the compounds of interest: non-polar compounds are extracted into methylene chloride, concentrated and analyzed by GC-NPD after derivatization, and the remaining aqueous fraction is deproteinated with acetonitrile-methanol prior to separation via reversed-phase HPLC and detection using atmospheric pressure ionization (API)-MS. Standard curves are linear over the required range and reproducible over five months. Plasma concentration-time profiles of CY and metabolites from a patient receiving CY by intravenous infusion (60 mg/kg, once a day for two days) are presented.

Structural characterization of prenyl groups attached to proteins

Certain mammalian proteins are modified at a carboxyl-terminal cysteine by a thioether-linked prenyl group, the 15-carbon farnesyl or the 20-carbon geranylgeranyl moiety. Here, we describe analytical methods to determine the presence of a prenyl modification, the structure of the prenyl group, and the lipid: protein molar ratio for candidate proteins or their proteolytic fragments. Methods for the synthesis of prenyl standards are also presented. Methyl iodide or Raney nickel treatment is used to release the prenyl group from the protein for further analysis. When the prenyl group has been radiolabeled biosynthetically, two analytical techniques are available. Methyl iodide-released material, primarily the prenyl alcohol, can be cochromatographed with known standards using reverse-phase high-performance liquid chromatography to provide indirect structural data. Raney nickel-released material, primarily the unsubstituted hydrocarbon, can be analyzed by radiometric gas chromatography, which offers more precise structural information. However, unequivocal determination of the structure and quantitation of the mass of the released prenyl compound requires the use of gas chromatography-coupled mass spectrometry.

Interaction of secobarbital with warfarin pseudoracemates

To evaluate the interaction of secobarbital with racemic warfarin or R,S(±)‐warfarin, S(−)‐warfarin was synthesized with 13C‐label in the 2‐position of the coumarin nucleus and added to 12C‐R(+)‐warfarin to form a 12C‐/13C‐warfarin pseudoracemate. Six normal subjects received 1.5 mg/kg of this “cold‐labeled” pseudoracemate. It was given with and without a daily oral dose of secobarbital, 100 mg, beginning 7 days before the warfarin and continuing throughout the hypoprothrombinemia. Plasma samples were obtained daily and analyzed for warfarin and for one‐stage prothrombin activity. Unchanged warfarin in plasma was fractionated by forward‐phase high‐pressure liquid chromatography, and enantiomorphic ratios were determined by chemical ionization–mass spectrometry with pentadeuterio‐warfarin as the internal standard. There was a reduction of the hypoprothrombinemia of the pseudoracemate during the secobarbital regimen over that on warfarin alone (p < 0.001). There was an increase in plasma clearance of R‐warfarin (p < 0.05) and an increase in plasma clearance of S‐warfarin (p < 0.003) during the secobarbital regimen over that on warfarin alone. It was concluded that secobarbital diminished the hypoprothrombinemia of pseudoracemic warfarin by increasing plasma clearance of the more hypoprothrombinemic S‐warfarin and by increasing plasma clearance of the less hypoprothrombinemic R‐warfarin.

The use of mass spectrometry in the study of chemically-reactive drug metabolises. Application of MS/MS and LC/MS to the analysis of glutathione- and related S-linked conjugates of N-methylformamide

The S-(N-methylcarbamoyl) derivatives of glutathione, cysteine and N-acetylcysteine, the S-linked conjugates derived from a reactive metabolite of N-methylformamide (NMF), were studied in mice dosed with an equimolar mixture of NMF and deuterium-labelled NMF. Following preparation of N-benzyloxycarbonyl derivatives in aqueous media, the title conjugates were isolated, purified as their methyl esters and subjected to analysis by fast atom bombardment mass spectrometry (FAB/MS), fast atom bombardment tandem mass spectrometry (FAB/MS/MS) or thermospray liquid chromatography/mass spectrometry (TSP LC/MS). Characteristic isotope clusters in the FAB or TSP mass spectra facilitated recognition of drug metabolites, while constant neutral loss (89 u) and daughter ion scanning tandem mass spectrometry (MS/MS) experiments provided unique structural information on the conjugates of interest. It is concluded that the combined use of stable isotopes, aqueous-phase derivatization and contemporary mass spectrometric techniques represents a powerful approach for the analysis of glutathione adducts and related S-linked conjugates of chemically-reactive drug metabolites.

Subnanomolar quantification of caffeine's in vitro metabolites by stable isotope dilution gas chromatography-mass spectrometry

A method for the quantification of subnanomolar levels of in vitro metabolites of caffeine by an isotope dilution gas chromatographic-mass spectrometric (GC-MS) assay has been developed and applied. Trideuteromethylated analogs of each primary metabolite were synthesized and added after incubations of caffeine with human liver microsomes high in cytochrome P4501A2. HPLC separation of the metabolites prior to GC-MS quantification allowed the isolation of theobromine and paraxanthine which coeluted by GC and enabled quantification over a larger dynamic range. Quantitative analysis was performed on the n-propylated derivatives by selected-ion monitoring of either the M+. ions for the dimethylxanthines or [M-C3H6]+. ions for 1,3,7-trimethyluric acid. For the least abundant metabolite (1,3,7-trimethyluric acid), the detection level on column was 200 pg. Replicate analyses exhibited intra- and inter-day variability of 4.2 and 7.9%, respectively. This assay has been successfully used in the quantification of caffeines primary metabolites in more than 180 incubations, at varying substrate concentrations and with multiple enzyme sources.