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Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography

Michael Kron1, Joseph Leykam, Jessica Kopaczewski

  • 1Department of Medicine, Medical College of Wisconsin, Milwaukee, WI 53226, United States. mkron@mcw.edu

Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
|July 17, 2007
PubMed

Insights

Researchers developed a new method to detect diadenosine oligophosphates (ApnA) in pathogens. This technique successfully identified diadenosine triphosphate (Ap3A) in Brugia malayi parasites, aiding future research on ApnA signaling.

Area of Science:

  • Biochemistry
  • Parasitology
  • Analytical Chemistry

Background:

  • Diadenosine oligophosphates (ApnA) are crucial signaling molecules, but their assessment in eukaryotic pathogens is challenging.
  • Existing mass spectrometry methods for nucleotide phosphate analysis are limited by high salt content and sample loss.
  • The role of ApnA in pathogens like Brugia malayi remains understudied due to technical difficulties.

Purpose of the Study:

  • To develop a simplified and effective method for analyzing ApnA in biological samples.
  • To overcome the limitations of existing techniques for nucleotide phosphate quantification.
  • To investigate the presence of ApnA in the human filarial parasite Brugia malayi.

Main Methods:

  • A novel reverse-phase HPLC (RP-HPLC) method was established using volatile organic buffers at low pH.
  • This method generates distinct elution profiles for adenosine and diadenosine phosphates.
  • The technique was validated by analyzing extracts from Brugia malayi.

Main Results:

  • The developed RP-HPLC method successfully separated and visualized nucleotide phosphates.
  • A major peak at 10.4 min was identified as diadenosine triphosphate (Ap3A) via direct mass spectrometry analysis.
  • Significant quantities of Ap3A were detected in Brugia malayi samples.

Conclusions:

  • The simplified RP-HPLC method effectively detects ApnA in small biological samples, overcoming previous technical hurdles.
  • This technique facilitates the study of ApnA in pathogens, including Brugia malayi.
  • Further research into the physiological and pathophysiological roles of ApnA is now more accessible.

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