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PubMed1996

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↗
PubMed1995

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↗
PubMed1985

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↗
PubMed1967

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↗
PubMed1995

Muramic acid in peripheral blood leukocytes of healthy human subjects.

Peptidoglycan, a specific marker for all bacterial cell walls, was studied in peripheral blood of healthy human subjects by mass spectrometric analysis of muramic acid. Peripheral blood mononuclear and polymorphonuclear cells from 98 healthy adults were hydrolyzed and analyzed by gas chromatography-mass spectrometry as alditol acetate derivatives using selective ion monitoring. Muramic acid was observed in cell samples from 21 of 98 subjects. Blood cultures were done simultaneously and remained negative. As a control, mononuclear and polymorphonuclear cells separated from umbilical vein blood of 41 healthy newborns were studied; all were muramic acid-negative. Since newborns lack gut flora, intestinal absorption of bacteria or of their degradation products appears to be the most likely explanation for the finding.

Adult↗
PubMed2002

Structure of muramic acid TMS derivative mass spectrum's base ion (m/z=185) used for quantification of bacterial peptidoglycan.

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.

Bacteria↗
PubMed1969

Structure of the peptidoglycan of bacterial spores: occurrence of the lactam of muramic acid.

Six major oligosaccharides were released from the peptidoglycan of spores of Bacillus subtilis by lysozyme treatment. They were isolated and characterized as a disaccharide, tetrasaccharide, and hexasaccharide composed of equal amounts of muramic acid and glucosamine and containing two, three, and four acetyl groups, respectively. Three of the compounds were substituted by a single L-alanine residue, and the other three by a single tetrapeptide substituent on the acetylmuramic acid residue at the reducing end of each compound. The other muramic acid residue in the tetrasaccharides (and two of the three in the hexasaccharides) were shown to be present as muramic lactams, a sugar not previously found in nature and, hence, a unique spore constituent. Other features of the structure of spore peptidoglycan are discussed.

Bacillus subtilis↗
PubMed2002

Analysis of a stable halogenated derivative of muramic acid by gas chromatography-negative ion chemical ionization tandem mass spectrometry.

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.

Gas Chromatography-Mass Spectrometry↗
PubMed2000

New and simple procedure for the determination of muramic acid in chemically complex environments by gas chromatography-ion trap tandem mass spectrometry.

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.

Acetic Anhydrides↗
PubMed1968

Structure of the cell wall of Lactobacilli. Role of muramic acid phosphate in Lactobacillus fermenti.

1. The polysaccharide and mucopeptide components of the cell wall of Lactobacillus fermenti, serological group F, were separated by mild conditions of acid hydrolysis; the polysaccharide was composed of glucose and galactose. 2. Soluble cell-wall products were isolated from cell wall lysed by lysozyme and a Streptomyces enzyme preparation. The lysozyme-dissolved fraction contained a greater proportion of mucopeptide. 3. The soluble preparations were heated in dilute acid to hydrolyse the linkage between the polysaccharide and mucopeptide components and then incubated with acid phosphatase. 4. Inorganic phosphate was released from products of Streptomyces enzyme action but not from products of lysozyme action. 5. The phosphate was shown to be present in the mucopeptide as muramic acid phosphate. It is concluded that in the intact wall polysaccharide is joined to muramic acid by a phosphodiester linkage.

Acid Phosphatase↗
PubMed1979

Muramic Acid assay in sediments.

An improved chromatographic assay for muramic acid which is sufficiently sensitive for marine sandy sediments is described; it involves acid hydrolysis, thin-layer chromatography, and gas-liquid chromatography.

Journal Article↗
PubMed2004

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.

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.

Acetates↗
PubMed1992

Assay for N-acetylmuramyl-L-alanine amidase in serum by determination of muramic acid released from the peptidoglycan of Brevibacterium divaricatum.

A method is reported for the determination of N-acetylmuramyl-L-alanine amidase in serum. Muramic acid, released from the interpeptide bridges of Brevibacterium divaricatum peptidoglycan, is measured by a modified colorimetric method. Using this procedure, it was possible to determine N-acetylmuramyl-L-alanine amidase in aliquots of less than 10 microliters human serum with an incubation time of 10 min. Amidase activity was found in all the sera tested (n = 11). The relevance of this simple and fast assay is discussed.

Adult↗
PubMed1993

Synthesis and conformational analysis of muramic acid delta-lactam structures and their 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) derivatives, characteristic of bacterial spore peptidoglycan.

1,6-Anhydro-4-O-benzyl-beta-muramic acid 1',2-lactam (2) was prepared by reduction of 1,6-anhydro-2-azido-4-O-benzyl-2-deoxy-3-O-[(R)-1- methoxycarbonylethyl]-beta-D-glucopyranose (1) followed by cyclisation. Debenzylation of 2 (-->3) and glycosylation of HO-4 with 3,4,6-tri-O-acetyl-2- deoxy-2-phthalimido-beta-D-glucopyranosyl chloride afforded 75% of a beta-(1-->4)-linked disaccharide derivative (7). Removal of the Phth group from 7, then acetylation, and O-deacetylation yielded 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-amino-1,6-anhydro-3-O- [(R)- 1-carboxyethyl]-2-deoxy-beta-D-glucopyranose 1',2-lactam (10) Acetolysis of the 1,6-anhydro ring in the 4-acetate (4) of 3 and the 3',4',6'-triacetate (9) of 10, with saponification of the products 5 and 11, afforded 2-amino-3-O- [(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose 1',2-lactam (6) and 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-amino-3- O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranose 1',2-lactam (12), respectively. The structure of 12 corresponds to that of the disaccharide unit characteristic of the glycan chains of bacterial spore peptidoglycan. 1H NMR spectroscopy indicated that the beta-D-glucopyranose ring in the 1,6-anhydro 1',2-lactam derivatives adopts the BO,3 conformation. On cleavage of the 1,6-anhydro ring by acetolysis, the D-glucopyranose ring adopts the 4C1 conformation. X-ray analysis of 2, 4, and 5 confirmed the proposed structures. Molecular mechanics and molecular dynamics simulations were used to follow the transformation of the BO,3 conformation of the D-glucopyranose ring via transition states to the 4C1 form.

Acetylglucosamine↗
PubMed1990

Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry.

By using the powerful separation technique of capillary gas chromatography combined with the selectivity of mass spectrometric detection, muramic acid was not detectable in purified elementary bodies of Chlamydia psittaci Cal 10 (less than or equal to 0.006%) or C. trachomatis serovar E (less than or equal to 0.02%). This confirms previous reports which suggested the absence of a typical peptidoglycan in Chlamydia spp.

Chlamydia trachomatis↗
PubMed1968

Determination of amino sugars in mixtures containing glucosamine, galactosamine and muramic acid.

A colorimetric method is described whereby the direct quantitative determination of glucosamine, galactosamine and muramic acid can be achieved without previous treatment of the cell-wall hydrolysate, for example by column chromatography. Molar ratios of hexosamines in cell-wall preparations, from a number of bacterial species, determined by this method were found to be in general agreement with previously published results.

Amino Sugars↗
PubMed2000

A pyrophosphate bridge links the pyruvate-containing secondary cell wall polymer of Paenibacillus alvei CCM 2051 to muramic acid.

The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed: [(Pyr4,6)-beta-D-ManpNAc-(1-->4)-beta-D-GlcpNAc-(1-->3)]n-11-(Pyr4,6)-beta-D-ManpNAc-(1-->4)-alpha-D-GlcpNAc-(1-->O)-PO2-O-PO2-(O-->6)-MurNAc- Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. 31P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex. In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.

Bacillus↗