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Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

Fyrestam J, Bjurshammar N, Paulsson E, Johannsen A, Östman C - Anal Bioanal Chem (2015)

Bottom Line: In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry.Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae.Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science and Analytical Chemistry, Analytical and Toxicological Chemistry, 106 91, Stockholm, Sweden.

ABSTRACT
Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus

HPLC-MS/MS chromatogram of porphyrins extracted from A. actinomycetemcomitans cultivated at 7 d. Parts of chromatogram in dashed boxes are enlarged for better visual effect. Analytes present in the sample: UP (184 ng/g), 7P (28 ng/g), CPI (6 ng/g), CPIII (20 ng/g), and PPIX (286 ng/g)
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Fig4: HPLC-MS/MS chromatogram of porphyrins extracted from A. actinomycetemcomitans cultivated at 7 d. Parts of chromatogram in dashed boxes are enlarged for better visual effect. Analytes present in the sample: UP (184 ng/g), 7P (28 ng/g), CPI (6 ng/g), CPIII (20 ng/g), and PPIX (286 ng/g)

Mentions: A. actinomycetemcomitans was cultivated on five agar plates and pooled to get an average of endogenously produced porphyrins. Extracts of A. actinomycetemcomitans were analyzed the same day using the described method and porphyrins were identified through retention time and three characteristic SRM transitions in the MS/MS analysis. A typical chromatogram from bacterial sample analysis is shown in Fig. 4, identifying UP, 7P, CPI, CPIII, and PPIX. MPIX is not an intermediate in biosynthesis of heme and could not be detected in any of the analyzed bacteria extracts. This makes it suitable for use as an internal standard. Highest concentrations were PPIX and UP, with concentrations of 286 and 184 ng/g respectively; 7P, CPI, and CPIII were of lower concentrations with 28, 6, and 20 ng/g, respectively.Fig. 4


Determination of porphyrins in oral bacteria by liquid chromatography electrospray ionization tandem mass spectrometry.

Fyrestam J, Bjurshammar N, Paulsson E, Johannsen A, Östman C - Anal Bioanal Chem (2015)

HPLC-MS/MS chromatogram of porphyrins extracted from A. actinomycetemcomitans cultivated at 7 d. Parts of chromatogram in dashed boxes are enlarged for better visual effect. Analytes present in the sample: UP (184 ng/g), 7P (28 ng/g), CPI (6 ng/g), CPIII (20 ng/g), and PPIX (286 ng/g)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4551553&req=5

Fig4: HPLC-MS/MS chromatogram of porphyrins extracted from A. actinomycetemcomitans cultivated at 7 d. Parts of chromatogram in dashed boxes are enlarged for better visual effect. Analytes present in the sample: UP (184 ng/g), 7P (28 ng/g), CPI (6 ng/g), CPIII (20 ng/g), and PPIX (286 ng/g)
Mentions: A. actinomycetemcomitans was cultivated on five agar plates and pooled to get an average of endogenously produced porphyrins. Extracts of A. actinomycetemcomitans were analyzed the same day using the described method and porphyrins were identified through retention time and three characteristic SRM transitions in the MS/MS analysis. A typical chromatogram from bacterial sample analysis is shown in Fig. 4, identifying UP, 7P, CPI, CPIII, and PPIX. MPIX is not an intermediate in biosynthesis of heme and could not be detected in any of the analyzed bacteria extracts. This makes it suitable for use as an internal standard. Highest concentrations were PPIX and UP, with concentrations of 286 and 184 ng/g respectively; 7P, CPI, and CPIII were of lower concentrations with 28, 6, and 20 ng/g, respectively.Fig. 4

Bottom Line: In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry.Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae.Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Science and Analytical Chemistry, Analytical and Toxicological Chemistry, 106 91, Stockholm, Sweden.

ABSTRACT
Biofilms in the oral cavity can be visualized by fluorescence and a common assumption is that the endogenously produced porphyrins in certain bacteria give rise to this fluorescence. Porphyrin content in oral bacteria has been sparingly investigated, and non-selective detection techniques such as utilizing the Soret fluorescence band of porphyrins are often used. In the present study, a quantitative and selective method for the determination of porphyrins in oral bacteria has been developed and validated using high performance liquid chromatography-tandem mass spectrometry. Lysis of bacteria using Tris-EDTA buffer together with ultrasonication showed high microbial killing efficiency ≥99.98%, and sample clean-up using C18-solid phase extraction resulted in low matrix effects ≤14% for all analytes. Using this method, the porphyrin content was determined in the two oral pathogens Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, as well as for baker's yeast, Saccharomyces cerevisiae. Uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, coproporphyrin, and protoporphyrin IX were identified in the investigated microorganisms, and it was shown that the porphyrin profile differs between the two bacteria, as well as for S. cerevisiae. To our knowledge, this is the first time the porphyrin profile has been determined for the bacterium A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus