The presence of acidic polysaccharides and muramic acid phosphate in the walls of Corynebacterium poinsettiae and Corynebacterium betae.
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Cell Wall↗
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Determination of diaminopimelic acid, ornithine, and muramic acid by gas chromatography.
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Bacteria↗
Electrospray tandem mass spectrometry for analysis of native muramic acid in whole bacterial cell hydrolysates.
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.
Cell Wall↗
Synovial fluid muramic acid in acute inflammatory arthritis.
Presence of muramic acid, a bacterial cell wall component, was analysed by gas chromatography-mass spectrometry in synovial fluid (SF) of 40 patients with acute inflammatory arthritis. SF muramic acid was observed in 4/14 patients with acute, culture negative inflammatory arthritis of unclear origin. Each of these four patients had a history of a recent bacterial disease (pansinuitis, purulent leg ulcer, erysipelas, unexplained fever with suspicion of cholecystitis and urinary tract infection). In the bacterial arthritis, SF muramic acid was detected in 6/12 patients (in 2/6 culture negative cases). In the reactive arthritis due to Salmonella or Yersinia, the rate of positivity was 2/14. Nineteen samples of traumatic SF effusion were muramic acid negative. These findings indicate that several cases of undefined acute inflammatory arthritis are of bacterial origin.
Acute Disease↗
Failure To detect muramic acid in normal rat tissues but detection in cerebrospinal fluids from patients with Pneumococcal meningitis.
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 <1 ng of muramic acid/100 mg (wet weight) of tissue. The levels of muramic acid reported in diseased human spleen and spleen of arthritic rats, previously injected with bacterial cell walls, were 100- to 1,000-fold higher. In the present study, muramic acid was also readily detected in the cerebrospinal fluid of patients with pneumococcal meningitis (6.8 to 3,900 ng of muramic acid/ml of cerebrospinal fluid). In summary, there can be an enormous difference in the levels of muramic acid found in different mammalian tissues and body fluids in health and disease. This report could have great impact in future studies assessing the role of bacterial cell wall remnants in the pathogenesis of certain human inflammatory diseases.
Animals↗
Muramic acid as a measure of microbial biomass in estuarine and marine samples.
Muramic acid, a component of the muramyl peptide found only in the cell walls of bacteria and blue-green algae, furnishes a measure of detrital or sedimentary procaryotic biomass. A reproducible assay involving acid hydrolysis, preparative thin-layer chromatographic purification, and colorimetric analysis of lactate released from muramic acid by alkaline hydrolysis is described. Comparison of semitropical estuarine detritus, estuarine muds, and sediments from anaerobic Black Sea cores showed muramic acid levels of 100 to 700 microng/g (dry weight), 34 microng/g, and 1.5 to 14.9 microng/g, respectively. Enzymatic assays of lactate from muramic acid gave results 10- to 20-fold higher. Radioactive pulse-labeling studies showed that [14C]acetate is rapidly incorporated into muramic acid by the detrital microflora. Subsequent loss of 14C, accompanied by nearly constant levels of total muramic acid, indicated active metabolism in procaryotic cell walls.
Acetates↗
Mass spectrometric quantitation of muramic acid, a bacterial cell wall component, in septic synovial fluids.
This is the first report describing the use of gas chromatography-mass spectrometry for detection of muramic acid in infected synovial fluid (SF). Muramic acid is a ubiquitous component of bacterial cell walls, and it has been proposed that it could serve as a chemical marker for the presence of live bacteria or bacterial debris in rheumatoid joints. Our goal was to determine whether muramic acid was present at detectable levels in septic SF, since this would serve as a positive control for studies of reactive and rheumatoid arthritis. Muramic acid was found to be present at levels of less than 250-1,700 ng/ml in 12 septic SF samples (10 of which were culture positive for Staphylococcus aureus and 1 each for Escherichia coli and Streptococcus pneumoniae). Among these samples, those containing low bacterial colony counts did not contain detectable muramic acid. Muramic acid was also not detected in any SF samples from 20 control patients. We conclude that muramic acid can be used as a marker for the presence of bacterial peptidoglycan in SF. With further lowering of gas chromatography-mass spectrometry detection limits, determination of the quantities of bacterial debris present in joints of patients with rheumatoid or reactive arthritis will be attainable.
Cell Wall↗
An improved gas chromatographic method for measuring glucosamine and muramic acid concentrations.
A method of simultaneously measuring glucosamine and muramic acid concentrations in marsh grass litter was developed. Spartina alterniflora samples were preextracted with acetone to remove lipids containing amino sugars and then hydrolyzed in 6 N HCl (100 degrees C, 4.5 h). Amino sugars in the hydrolysates were isolated by ion-exchange chromatography, which gave good recoveries (greater than 90%) and reproducibility (CV less than 5%). Isolated amino sugars were converted to O-methyloxime acetates. beta-Phenylglucose and N-methylglucamine were added as internal standards. Sample derivatives were quantified by capillary column gas chromatography. OV-101 and SE-54 capillary columns completely separated glucosamine and muramic acid from other amino sugars. The detection limit of glucosamine and muramic acid during gas chromatographic analysis was below 30 pmol using splitless-mode injection (SE-54 column). Filamentous fungal and procaryotic biomasses may be estimated simultaneously by using glucosamine and muramic acid biomass conversion factors in conjunction with this method.
Animals↗
Amperometric detection of muramic acid in high-performance liquid chromatography with a post-column reaction.
Highly sensitive detection of muramic acid has been developed by using an amperiometric detector in high-performance liquid chromatography. The reducing ability of muramic acid was coupled with the redox reaction of bis(1,10-phenanthroline)copper(II), CBP, as mediator. CBP, reduced by muramic acid in a post-column reaction, was reoxidized with the amperometric detector, resulting in a highly sensitive and selective detection. A doublet peak was obtained, due to the alpha- and beta-anomers of muramic acid. This was merged into a single peak by adding phosphoric acid to the sample solution. Addition of phosphoric acid to serum, spiked with muramic acid, also clarified the chromatogram. The detection limit was 0.05 ppm (4 pmol of muramic acid injected) in serum samples.
Carbohydrate Metabolism↗
Muramic acid in human peripheral blood leucocytes in different age groups.
Presence of muramic acid (as a marker for bacterial cell wall peptidoglycan) was analysed by gas chromatography and mass spectrometry in the peripheral blood leucocytes of subjects from a range of ages (9-80 years) and groups (healthy individuals, patients with rheumatoid arthritis, osteoarthrosis, essential hypertension or multiple sclerosis). Sixty per cent of the sample from the youngest subjects contained detectable muramic acid. The percentage of people with circulating leucocytes containing muramic acid decreased gradually with age, being less than 5% in all groups over 40 years. No clear correlation between the presence of muramic acid and the disease was observed.
Adolescent↗
CD11a/CD18 and CD11b/18 modulation by lipoteichoic acid, N-acetyl-muramyl-alpha-alanyl-D-isoglutamine, muramic acid and protein A from Staphylococcus aureus.
This study investigates the effect of some components of the Staphylococcus aureus cell wall [lipoteichoic acid (LTA), N-acetyl-muramyl-alanyl-D-isoglutamine (MD), muramic acid (MA) and protein A (PA)] in modulating expression of cell-surface adhesion molecules CD11a/CD18, CD11b/C18 on monocytes qualitatively and quantitatively. Monocytes incubated with bacterial components presented different CD11b/CD18 expressions which were dose-dependent in contrast to controls. The results obtained demonstrated that lymphocytes incubated with bacterial components also increased the expression of CD11a/CD18. The modifications in activation of CD11a/CD18 and CD11b/CD18 expression are probably correlated with modifications of membrane fluidity measured as polarisation fluorescence (P).
Acetylmuramyl-Alanyl-Isoglutamine↗
Determination of muramic acid in organic dust by gas chromatography-mass spectrometry.
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.
Air Microbiology↗
Muramic acid detection in mammalian tissues by gas-liquid chromatography-mass spectrometry.
Muramic acid is a component of the peptidoglycan moiety of cell walls of all bacteria and blue-green algae and is not found elsewhere in nature. A gas-liquid chromatography-mass spectrometry assay for muramic acid in tissue is described. The application of the method to the detection of muramic acid in tissues of rats with streptococcal cell wall-induced polyarthritis is demonstrated. Because the method has the potential to measure total bacterial biomass in tissue, it should prove to be an important assay in elucidating the etiological role of bacterial debris in chronic inflammatory diseases in humans.
Animals↗
Legume lectins interact with muramic acid and N-acetylmuramic acid.
The inhibitory potency of both muramic acid (MurAc) and N-acetylmuramic acid (MurNAc) on various legume lectins, including Glc/Man- and Gal/GalNAc-specific lectins, was investigated by a haemagglutination inhibition technique. Data indicated that many lectins, especially those specific for Glc/Man, specifically interact with MurAc and MurNAc often to a greater extent than with other monosaccharides and their derivatives, such as N-acetylglucosamine (GlcNAc) and sialic acid. Glc/Man-specific lectins were also shown to interact with the muramyl-dipeptide MurNAc-D-Ala-D-isoGln. These interactions could explain why various lectins readily agglutinate some bacterial strains of which cell walls contain peptidoglycans with high amounts of MurNAc.
Carbohydrate Sequence↗
Absolute identification of muramic acid, at trace levels, in human septic synovial fluids in vivo and absence in aseptic fluids.
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.
Arthritis, Infectious↗
Detection of muramic acid in a carbohydrate fraction of human spleen.
In previous studies, we showed that peptidoglycan polysaccharides from anaerobic bacteria normally present in the human gut induced severe chronic joint inflammation in rats. Our hypothesis is that peptidoglycan from the gut flora is involved in perpetuation of idiopathic inflammation. However, in the literature, the presence of peptidoglycan or subunits like muramyl peptides in blood or tissues is still a matter of debate. We were able to stain red pulp macrophages in all six available human spleens by immunohistochemical techniques using a monoclonal antibody against gut flora-derived antigens. Therefore, these human spleens were extracted, and after removal of most of the protein, the carbohydrate fraction was investigated for the presence of muramic acid, an amino sugar characteristic for peptidoglycan. Using three different methods for detection of muramic acid, we found a mean of 3.3 mumol of muramic acid with high-pressure liquid chromatography, 1.9 mumol with a colorimetric method for detection of lactate, and 0.8 mumol with an enzymatic method for detection of D-lactate per spleen (D-lactate is a specific group of the muramic acid molecule). It is concluded that peptidoglycan is present in human spleen not as small muramyl peptides as were previously searched for by other investigators but as larger macromolecules probably stored in spleen macrophages.
Chromatography, High Pressure Liquid↗
Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring.
Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.
Acetylation↗
Biosynthesis of streptococcal cell walls: N-acetyl-D-muramic acid.
Glucose-1-(14)C and acetylglucosamine-1-(14)C were added singly and together with equal amounts of the unlabeled reciprocal to Brain Heart Infusion and used for the culture of Streptococcus pyogenes. The labeling pattern of the rhamnose, glucosamine, and muramic acid in the cell wall supported an intermediary role for acetylglucosamine in providing the C1-C6 moiety of muramic acid. Although radioactivity in the C2-C9 portion of muramic acid suggested that some of the lactyl group (C7-C9) came from glycolytic products, there was also considerable contribution to it from noncarbohydrate sources. Using cell-free extracts, we were unable to demonstrate biosynthesis of acetylmuramic acid, either free or nucleotide-bound, while glycolysis was occurring. The formation of uridine diphosphoacetylmuramic acid has been reported by others who used uridine diphospho-N-acetyl-d-glucosamine, phosphoenolpyruvate, reduced nicotinamide adenine dinucleotide, and reduced nicotinamide adenine dinucleotide phosphate. However, we did not detect the formation of this compound.
Acids↗