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Alvin Fox - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the methyl ester o methyl acetate derivative of Muramic Acid for the determination of peptidoglycan in environmental samples by ion trap gc ms ms
2004Co-Authors: Aleksandra Sebastian, Alvin Fox, William M Harley, Lennart LarssonAbstract:Muramic Acid (MA) is a unique amino sugar that is a constituent of the peptidoglycan (PG) present in prokaryotic cell walls. MA can serve as a marker for quantifying bacterial load, e.g. in indoor environments, by using gas chromatography–tandem mass spectrometry (GC-MS-MS). We demonstrated recently that the methyl ester O-methyl acetate (MMA) derivative can be used to detect MA in house dust by ion-trap GC-MS-MS. However, since the MMA derivative is not formed from free MA quantification was not optimal. Here we report 1) significant improvements in sample preparation of the MMA derivative and 2) an evaluation of the performance of derivative, using for comparison the alditol acetate derivative, the gold standard in quantitative trace analysis of MA in complex matrices. The MMA derivative was analysed using an MS instrument with internal ionization and the alditol acetate derivative was analysed using an instrument with external ionization. 13C-labelled cyanobacteria, containing MA in their PG, were used as the internal standard. A linear relationship was found between the two methods in studies on 27 parallel samples of airborne dust from school classes collected on filters. Although the analytical sensitivity of the MMA derivatives was somewhat slightly lower than of the alditol acetate derivative, this may be due to differences in yield of derivative, sample clean-up efficiency, or different performance of the GC columns or MS instruments. However preparation of the MMA derivative is quick and compatible with preparation of methyl esters of 3-hydroxy fatty Acids (used as markers of Gram negative endotoxin) allowing the levels of both markers to be determined in the same dust sample. In conclusion, the MMA procedure can be used to determine MA in environmental samples with good reproducibility provided the concentration of the 13C-labelled MA internal standard in the cyanobacteria is first determined with an alternative method.
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trace analysis of Muramic Acid in indoor air using an automated derivatization instrument and gc ms2 or gc ms3
2002Co-Authors: William M Harley, Michael P Kozar, Alvin FoxAbstract:Abstract An automated derivatization instrument has been developed for the preparation of alditol acetates from bacterial hydrolysates for analysis by gas chromatography–mass spectrometry (GC–MS). The current report demonstrates the utility of the automated instrument for the more demanding task of trace analysis of Muramic Acid (Mur) in airborne dust using gas chromatography-tandem mass spectrometry (GC–MS2). Conditions for efficient derivatization of Mur, vital for trace analysis, are rigorous including lactam and imido group formation under anhydrous conditions. Furthermore, as the detection limit is lowered, possible contamination or carry-over of samples becomes an increasingly greater consideration and must not occur. The instrument meets these criteria and was successfully used for assaying the levels of Mur in laboratory air, which were found to be much lower than in the previous studies of heavily occupied schools and agricultural environments. The potential for GC–MS3 in further lowering the detection limit was also demonstrated.
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analysis of a stable halogenated derivative of Muramic Acid by gas chromatography negative ion chemical ionization tandem mass spectrometry
2002Co-Authors: Michael P Kozar, Alvin FoxAbstract:Muramic Acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography-tandem mass spectrometry (GC-MS-MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC-MS-MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC-MS-MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4-5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC-NICI-MS-MS or as alditol acetates by electron impact GC-electron impact ionization MS-MS serve as complementary approaches for trace detection in complex matrices.
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Muramic Acid is not generally present in the human spleen as determined by gas chromatography tandem mass spectrometry
2002Co-Authors: Michael P Kozar, Jon D Laman, Alvin FoxAbstract:It has been hypothesized that bacterial debris may accumulate in tissues of the reticuloendothelial system (RES) serving as an inflammatory stimulus for human disease. In support of this hypothesis, Muramic Acid (Mur), a component of bacterial peptidoglycan (PG), has previously been reported to be present in culture-negative human spleen. High-performance liquid chromatography (HPLC) was employed in these analyses, and a peak was detected at the retention time of Mur. However, HPLC is best used as a screening technique, and it is vital that these tentative observations be reexamined by the state-of-the-art approach (gas chromatography-tandem mass spectrometry [GC-MS(2)]). Indeed, in the present work using GC-MS(2), Mur was not detected in six out of seven human spleens previously examined by HPLC. However, Mur was categorically detected at minute concentrations, 50 ppb, in one spleen. In conclusion, since Mur is not generally found in culture-negative human spleen, in future studies, these tissues can serve as negative controls. The study of Mur levels in inflammation (e.g., reactive arthritis) could prove important in testing the hypothesis that bacterial debris persisting in tissues could serve as a depot inciting diseases of unknown etiology.
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failure to detect Muramic Acid in normal rat tissues but detection in cerebrospinal fluids from patients with pneumococcal meningitis
2000Co-Authors: Michael P Kozar, Alvin Fox, Mark T Krahmer, Barry M GrayAbstract:Muramic Acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of Muramic Acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatography-tandem mass spectrometry, to identify and quantify the levels of Muramic Acid in tissues. Muramic Acid was not found in normal rat brain or spleen. However, when tissues were spiked with Muramic Acid, it was readily identified. The detection limit was
Lennart Larsson - One of the best experts on this subject based on the ideXlab platform.
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endotoxin ergosterol Muramic Acid and fungal dna in dust from schools in johor bahru malaysia associations with rhinitis and sick building syndrome sbs in junior high school students
2016Co-Authors: Dan Norback, Jamal Hisham Hashim, Pawel Markowicz, Gui Hong Cai, Zailina Hashim, Faridah Ali, Lennart LarssonAbstract:This paper studied associations between ocular symptoms, rhinitis, throat and dermal symptoms, headache and fatigue in students by ethnicity and in relation to exposure to chemical microbial markers and fungal DNA in vacuumed dust in schools in Malaysia. A total of 462 students from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated (96% response rate). Dust was vacuumed from 32 classrooms and analysed for levels of five types of endotoxin as 3-hydroxy fatty Acids (C10, C12, C14, C16 and C18 3-OH), Muramic Acid, ergosterol and five sequences of fungal DNA. Multiple logistic regression was applied. Totally 11.9% reported weekly ocular symptoms, 18.8% rhinitis, 15.6% throat and 11.1% dermal symptoms, 20.6% headache and 22.1% tiredness. Totally 21.1% reported pollen or furry pet allergy (atopy) and 22.0% parental asthma or allergy. Chinese students had less headache than Malay and Indian had less rhinitis and less tiredness than Malay. Parental asthma/allergy was a risk factor for ocular (odds ratio = 3.79) and rhinitis symptoms (OR = 3.48). Atopy was a risk factor for throat symptoms (OR = 2.66), headache (OR = 2.13) and tiredness (OR = 2.02). There were positive associations between amount of fine dust in the dust samples and ocular symptoms (p < 0.001) and rhinitis (p = 0.006). There were positive associations between C14 3-OH and rhinitis (p < 0.001) and between C18 3-OH and dermal symptoms (p = 0.007). There were negative (protective) associations between levels of total endotoxin (LPS) (p = 0.004) and levels of ergosterol (p = 0.03) and rhinitis and between C12 3-OH and throat symptoms (p = 0.004). In conclusion, the amount of fine dust in the classroom was associated with rhinitis and other SBS symptoms and improved cleaning of the schools is important. Endotoxin in the school dust seems to be mainly protective for rhinitis and throat symptoms but different types of endotoxin could have different effects. The ethnic differences in symptoms among the students deserve further attention.
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evaluation of the methyl ester o methyl acetate derivative of Muramic Acid for the determination of peptidoglycan in environmental samples by ion trap gc ms ms
2004Co-Authors: Aleksandra Sebastian, Alvin Fox, William M Harley, Lennart LarssonAbstract:Muramic Acid (MA) is a unique amino sugar that is a constituent of the peptidoglycan (PG) present in prokaryotic cell walls. MA can serve as a marker for quantifying bacterial load, e.g. in indoor environments, by using gas chromatography–tandem mass spectrometry (GC-MS-MS). We demonstrated recently that the methyl ester O-methyl acetate (MMA) derivative can be used to detect MA in house dust by ion-trap GC-MS-MS. However, since the MMA derivative is not formed from free MA quantification was not optimal. Here we report 1) significant improvements in sample preparation of the MMA derivative and 2) an evaluation of the performance of derivative, using for comparison the alditol acetate derivative, the gold standard in quantitative trace analysis of MA in complex matrices. The MMA derivative was analysed using an MS instrument with internal ionization and the alditol acetate derivative was analysed using an instrument with external ionization. 13C-labelled cyanobacteria, containing MA in their PG, were used as the internal standard. A linear relationship was found between the two methods in studies on 27 parallel samples of airborne dust from school classes collected on filters. Although the analytical sensitivity of the MMA derivatives was somewhat slightly lower than of the alditol acetate derivative, this may be due to differences in yield of derivative, sample clean-up efficiency, or different performance of the GC columns or MS instruments. However preparation of the MMA derivative is quick and compatible with preparation of methyl esters of 3-hydroxy fatty Acids (used as markers of Gram negative endotoxin) allowing the levels of both markers to be determined in the same dust sample. In conclusion, the MMA procedure can be used to determine MA in environmental samples with good reproducibility provided the concentration of the 13C-labelled MA internal standard in the cyanobacteria is first determined with an alternative method.
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structure of Muramic Acid tms derivative mass spectrum s base ion m z 185 used for quantification of bacterial peptidoglycan
2002Co-Authors: Karol Bal, Lennart Larsson, Eugenia Mielniczuk, Zbigniew MielniczukAbstract:The use of trimethylsilyl (TMS)-derivatisation for determining Muramic Acid in environmental and clinical samples by gas chromatography-mass spectrometry provides high detection sensitivity; however, questions have been raised as concerns the chemical structure of the entity giving the strong signal of m/z 185. In the present communication we present evidence that this entity results from the formation of a lactam structure of Muramic Acid upon derivatisation.
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new and simple procedure for the determination of Muramic Acid in chemically complex environments by gas chromatography ion trap tandem mass spectrometry
2000Co-Authors: Karol Bal, Lennart LarssonAbstract:A gas chromatographic-ion trap tandem mass spectrometric method was developed for the quantification of Muramic Acid, a marker of bacterial peptidoglycan, in environmental and clinical specimens. Samples (bacteria, house dust and urine) were heated in methanolic hydrochloric Acid overnight and extracted with hexane for removal of hydrophobic compounds. The aqueous phase was evaporated and heated in acetic anhydride and pyridine after which the product, the acetate derivative, was washed with dilute hydrochloric Acid and water. The described method is both rapid and simple to apply, and produces a stable derivative. It should become widely used for measuring peptidoglycan in chemically complex environments.
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determination of Muramic Acid in organic dust by gas chromatography mass spectrometry
1995Co-Authors: Zbigniew Mielniczuk, Eugenia Mielniczuk, Lennart LarssonAbstract:A method is described for the quantitation of Muramic Acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric Acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography--mass spectrometry. The Muramic Acid derivative gave two peaks upon injection into the gas chromatograph--mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5-1500 ng of Muramic Acid. In a house dust sample, 40 ng of Muramic Acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.
Karen F Fox - One of the best experts on this subject based on the ideXlab platform.
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comparison of ion trap and triple quadrupole gas chromatography tandem mass spectrometry in the quantitative and qualitative trace analysis of Muramic Acid in complex matrices
1999Co-Authors: Mark T Krahmer, Karen F Fox, Alvin FoxAbstract:Abstract During the past few years we helped introduce the use of gas chromatography-tandem mass spectrometry (GS-MS/MS) for trace detection of chemical markers for bacteria and their constituents in complex clinical and environmental matrices. A particular marker of interest is Muramic Acid found in bacteria but not elsewhere in nature. Absolute identification is achieved using the product ion spectrum and levels of chemical markers for bacteria determined by multiple reaction monitoring. Work directly comparing, the utility of ion trap and triple quadrupole GC-MS/MS instrumentation, in the quantitative and qualitative analysis of Muramic Acid, is presented here.
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absolute identification of Muramic Acid at trace levels in human septic synovial fluids in vivo and absence in aseptic fluids
1996Co-Authors: Alvin Fox, Karen F Fox, B Christensson, D Harrelson, Mark T KrahmerAbstract:This is the first report of a study employing the state-of-the-art technique of gas chromatography-tandem mass spectrometry for absolute identification of Muramic Acid (a marker for peptidoglycan) at trace levels in a human or animal body fluid or tissue. Daughter mass spectra of synovial fluid Muramic Acid peaks (> or = 30 ng/ml) were identical to those of pure Muramic Acid. Absolute chemical identification at this level represents a 1,000-fold increase in sensitivity over previous gas chromatography-mass spectrometry identifications. Muramic Acid was positively identified in synovial fluids during infection and was eliminated over time but was absent from aseptic fluids.
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gas chromatography tandem mass spectrometry for trace detection of Muramic Acid a peptidoglycan chemical marker in organic dust
1995Co-Authors: Alvin Fox, Lloyd Wright, Karen F FoxAbstract:Abstract Muramic Acid (MA) is a chemical marker for bacterial peptidoglycan. MA was analyzed (as its alditol acetate) at trace levels in organic dust using electron impact gas chromatography-tandem mass spectrometry (GC-MS-MS) and gas chromatography-mass spectrometry (GC-MS). GC-MS-MS in the multiple ion reaction mode (MRM) was demonstrated to exhibit considerably decreased background over GC-MS in the selected ion mode (SIM). MA was readily detected in air conditioning and house dust and extraneous peaks were not observed in chromatograms of plants or fungi in the MA region. GC-MS-MS has considerable potential as a tool for trace detection of Muramic Acid in organic dust and other complex matrices.
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electrospray tandem mass spectrometry for analysis of native Muramic Acid in whole bacterial cell hydrolysates
1994Co-Authors: Gavin E Black, Alvin Fox, Karen F Fox, Peter A Snyder, Philip B SmithAbstract:Muramic Acid is an amino sugar found in eubacterial cell walls and not elsewhere in nature. This study explored the use of electrospray tandem mass spectrometry (ESI MS/MS) in analysis of underivatized Muramic Acid in bacterial hydrolysates. Fungal hydrolysates were used as negative controls. The only processing used was hydrolysis in sulfuric Acid followed by extraction with an organic base (N,N-dioctylmethylamine) to remove the Acid prior to ESI MS/MS analysis. Compared with pure Muramic Acid, bacterial hydrolysates produced more complex ESI mass spectra, such that the protonated molecular ion at m/z 252 was barely detectable. In contrast, product ion spectra of m/z 252 were identical among pure Muramic Acid, Gram positive bacteria, and Gram negative bacteria. However, no characteristic product ion spectrum was manifested from m/z 252 in fungal samples. This allowed ready, visual differentiation of bacteria and fungi. Multiple reaction monitoring (MRM) following Muramic Acid fragmentations (m/z 252-->144 and m/z 252-->126) increased sensitivity and allowed quantitative differentiation when compared with the MRM of the internal standard N-methyl-D-glucamine (m/z 196-->44). ESI MS/MS required minimal sample preparation and allowed rapid sample throughput for analysis of Muramic Acid in whole bacterial cell hydrolysates.
Mark T Krahmer - One of the best experts on this subject based on the ideXlab platform.
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failure to detect Muramic Acid in normal rat tissues but detection in cerebrospinal fluids from patients with pneumococcal meningitis
2000Co-Authors: Michael P Kozar, Alvin Fox, Mark T Krahmer, Barry M GrayAbstract:Muramic Acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of Muramic Acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatography-tandem mass spectrometry, to identify and quantify the levels of Muramic Acid in tissues. Muramic Acid was not found in normal rat brain or spleen. However, when tissues were spiked with Muramic Acid, it was readily identified. The detection limit was
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comparison of ion trap and triple quadrupole gas chromatography tandem mass spectrometry in the quantitative and qualitative trace analysis of Muramic Acid in complex matrices
1999Co-Authors: Mark T Krahmer, Karen F Fox, Alvin FoxAbstract:Abstract During the past few years we helped introduce the use of gas chromatography-tandem mass spectrometry (GS-MS/MS) for trace detection of chemical markers for bacteria and their constituents in complex clinical and environmental matrices. A particular marker of interest is Muramic Acid found in bacteria but not elsewhere in nature. Absolute identification is achieved using the product ion spectrum and levels of chemical markers for bacteria determined by multiple reaction monitoring. Work directly comparing, the utility of ion trap and triple quadrupole GC-MS/MS instrumentation, in the quantitative and qualitative analysis of Muramic Acid, is presented here.
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monitoring Muramic Acid in air after alditol acetate derivatization using a gas chromatograph ion trap tandem mass spectrometer
1996Co-Authors: Alvin Fox, Mark T Krahmer, Davis HarrelsonAbstract:Abstract Muramic Acid is a chemical marker for bacterial peptidoglycan. Air monitoring was performed in a stable and a dairy. Airborne dust was collected for 68–95 h on Teflon filters for Muramic Acid analysis. Filters were cut in two and the replicates analyzed separately. The filters and associated dust were heated in sulfuric Acid to release Muramic Acid. Muramic Acid was converted to Muramicitol pentaacetate lactam and analyzed by gas chromatography-tandem mass spectrometry using a non-polar GC column (DB-5MS). An ion trap tandem mass spectrometer was used for detection. The precursor mass spectrum of Muramic Acid was dominated by one major high mass ion m z 403 (loss of ketene, m z 42, from the molecule, MW 445) and m z 198 was the most abundant ion in daughter spectra. Both ions retained the unique lactam ring structure. Using multiple reaction monitoring (MRM), the transition m z 403 to m z 198 was used in quantitation. 13C-labeled Muramic Acid was used as an internal standard (monitoring the corresponding ion transition m z 412 to m z 205). MRM chromatograms were free of extraneous background peaks. Excellent sensitivity was also observed with daughter spectra for m z 403 allowing absolute identification of Muramic Acid in airborne dust.
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absolute identification of Muramic Acid at trace levels in human septic synovial fluids in vivo and absence in aseptic fluids
1996Co-Authors: Alvin Fox, Karen F Fox, B Christensson, D Harrelson, Mark T KrahmerAbstract:This is the first report of a study employing the state-of-the-art technique of gas chromatography-tandem mass spectrometry for absolute identification of Muramic Acid (a marker for peptidoglycan) at trace levels in a human or animal body fluid or tissue. Daughter mass spectra of synovial fluid Muramic Acid peaks (> or = 30 ng/ml) were identical to those of pure Muramic Acid. Absolute chemical identification at this level represents a 1,000-fold increase in sensitivity over previous gas chromatography-mass spectrometry identifications. Muramic Acid was positively identified in synovial fluids during infection and was eliminated over time but was absent from aseptic fluids.
Michael P Kozar - One of the best experts on this subject based on the ideXlab platform.
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trace analysis of Muramic Acid in indoor air using an automated derivatization instrument and gc ms2 or gc ms3
2002Co-Authors: William M Harley, Michael P Kozar, Alvin FoxAbstract:Abstract An automated derivatization instrument has been developed for the preparation of alditol acetates from bacterial hydrolysates for analysis by gas chromatography–mass spectrometry (GC–MS). The current report demonstrates the utility of the automated instrument for the more demanding task of trace analysis of Muramic Acid (Mur) in airborne dust using gas chromatography-tandem mass spectrometry (GC–MS2). Conditions for efficient derivatization of Mur, vital for trace analysis, are rigorous including lactam and imido group formation under anhydrous conditions. Furthermore, as the detection limit is lowered, possible contamination or carry-over of samples becomes an increasingly greater consideration and must not occur. The instrument meets these criteria and was successfully used for assaying the levels of Mur in laboratory air, which were found to be much lower than in the previous studies of heavily occupied schools and agricultural environments. The potential for GC–MS3 in further lowering the detection limit was also demonstrated.
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analysis of a stable halogenated derivative of Muramic Acid by gas chromatography negative ion chemical ionization tandem mass spectrometry
2002Co-Authors: Michael P Kozar, Alvin FoxAbstract:Muramic Acid (Mur) is present in the cell wall of Eubacteria and serves as a chemical marker for the trace detection of bacteria and bacterial cell wall debris in complex matrices. There have been numerous studies using a variety of derivatives of Mur, particularly in combination with gas chromatography-tandem mass spectrometry (GC-MS-MS) where the detection limit has been steadily lowered. A stable, halogenated derivative, the pentafluorobenzyl oxime (PFBO) acetate of Mur, has been developed by others and successfully used for GC with electron-capture detection. The current report is the first use of this derivative for GC-MS-MS analysis of Mur, or indeed any other carbohydrate, using negative ion chemical ionization (NICI) with GC-MS-MS. Mur was readily detected in settled surface dust (166 ng/mg), as well as dust collected from indoor air (1.4-5.9 ng/mg). Analyses of Mur as a PFBO acetate by GC-NICI-MS-MS or as alditol acetates by electron impact GC-electron impact ionization MS-MS serve as complementary approaches for trace detection in complex matrices.
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Muramic Acid is not generally present in the human spleen as determined by gas chromatography tandem mass spectrometry
2002Co-Authors: Michael P Kozar, Jon D Laman, Alvin FoxAbstract:It has been hypothesized that bacterial debris may accumulate in tissues of the reticuloendothelial system (RES) serving as an inflammatory stimulus for human disease. In support of this hypothesis, Muramic Acid (Mur), a component of bacterial peptidoglycan (PG), has previously been reported to be present in culture-negative human spleen. High-performance liquid chromatography (HPLC) was employed in these analyses, and a peak was detected at the retention time of Mur. However, HPLC is best used as a screening technique, and it is vital that these tentative observations be reexamined by the state-of-the-art approach (gas chromatography-tandem mass spectrometry [GC-MS(2)]). Indeed, in the present work using GC-MS(2), Mur was not detected in six out of seven human spleens previously examined by HPLC. However, Mur was categorically detected at minute concentrations, 50 ppb, in one spleen. In conclusion, since Mur is not generally found in culture-negative human spleen, in future studies, these tissues can serve as negative controls. The study of Mur levels in inflammation (e.g., reactive arthritis) could prove important in testing the hypothesis that bacterial debris persisting in tissues could serve as a depot inciting diseases of unknown etiology.
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failure to detect Muramic Acid in normal rat tissues but detection in cerebrospinal fluids from patients with pneumococcal meningitis
2000Co-Authors: Michael P Kozar, Alvin Fox, Mark T Krahmer, Barry M GrayAbstract:Muramic Acid serves as a marker for the presence of bacterial cell wall debris in mammalian tissues. There have been a number of controversial and sometimes conflicting results on assessing the levels of Muramic Acid in health and disease. The present report is the first to use the state-of-the art technique, gas chromatography-tandem mass spectrometry, to identify and quantify the levels of Muramic Acid in tissues. Muramic Acid was not found in normal rat brain or spleen. However, when tissues were spiked with Muramic Acid, it was readily identified. The detection limit was
