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Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography.
Anal Biochem. 2006 Dec 01; 359(1):132-40.AB

Abstract

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.

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Authors+Show Affiliations

Ganzera M
Institute of Pharmacy, Department of Pharmacognosy, University of Innsbruck, 6020 Innsbruck, Austria. markus.ganzera@uibk.ac.at
Vrabl P
No affiliation info available
Wörle E
No affiliation info available
Burgstaller W
No affiliation info available
Stuppner H
No affiliation info available

MeSH

Adenine NucleotidesChromatography, High Pressure LiquidCulture MediaEthanolGlucoseHyphaeNADPenicilliumReproducibility of Results

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17054897

Citation

Ganzera, Markus, et al. "Determination of Adenine and Pyridine Nucleotides in Glucose-limited Chemostat Cultures of Penicillium Simplicissimum By One-step Ethanol Extraction and Ion-pairing Liquid Chromatography." Analytical Biochemistry, vol. 359, no. 1, 2006, pp. 132-40.
Ganzera M, Vrabl P, Wörle E, et al. Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography. Anal Biochem. 2006;359(1):132-40.
Ganzera, M., Vrabl, P., Wörle, E., Burgstaller, W., & Stuppner, H. (2006). Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography. Analytical Biochemistry, 359(1), 132-40.
Ganzera M, et al. Determination of Adenine and Pyridine Nucleotides in Glucose-limited Chemostat Cultures of Penicillium Simplicissimum By One-step Ethanol Extraction and Ion-pairing Liquid Chromatography. Anal Biochem. 2006 Dec 1;359(1):132-40. PubMed PMID: 17054897.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography. AU - Ganzera,Markus, AU - Vrabl,Pamela, AU - Wörle,Elisabeth, AU - Burgstaller,Wolfgang, AU - Stuppner,Hermann, Y1 - 2006/10/04/ PY - 2006/08/29/received PY - 2006/09/14/revised PY - 2006/09/14/accepted PY - 2006/10/24/pubmed PY - 2007/1/11/medline PY - 2006/10/24/entrez SP - 132 EP - 40 JF - Analytical biochemistry JO - Anal Biochem VL - 359 IS - 1 N2 - Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined. SN - 0003-2697 UR - https://www.unboundmedicine.com/medline/citation/17054897/Determination_of_adenine_and_pyridine_nucleotides_in_glucose_limited_chemostat_cultures_of_Penicillium_simplicissimum_by_one_step_ethanol_extraction_and_ion_pairing_liquid_chromatography_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-2697(06)00677-4 DB - PRIME DP - Unbound Medicine ER -