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Renal interstitial cAMP and AMP are converted to adenosine: application of a mass spectrometric ion trap assay for purines
Author(s) -
Zacharia Lefteris C.,
Mi Zaichuan,
Jackson Edwin K.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a765
Subject(s) - chemistry , adenosine , hypoxanthine , microdialysis , detection limit , inosine , adenosine deaminase , chromatography , adenosine monophosphate , renal cortex , inosine monophosphate , high performance liquid chromatography , mass spectrometry , extracellular , kidney , medicine , nucleotide , biochemistry , enzyme , gene
cAMP and AMP are delivered to the renal interstitial space via a variety of mechanisms. The goal of this study was to test the hypothesis that renal interstitial cAMP and AMP are a source of adenosine. We developed a rapid (10 min/sample), convenient (no sample preparation), accurate (low bias, high precision) and sensitive (<25 pg detection limit) assay to measure adenosine, AMP, inosine and hypoxanthine in biological fluids using a Thermofinnigan HPLC‐LCQ Duo ion trap mass spectrometer equipped with an electrospray ionization source operated in the positive ion mode. The assay uses a C‐18 Eclipse Zorbax XDB column (4.6 mm x 150 mm), the mobile phase consists of inexpensive solvents (water, methanol), and the flow rate is low (0.5 ml/min). Analytes are well separated and demonstrate a linear relationship between concentration and signal. Addition of cAMP (30 μM) to the perfusates of microdialysis probes inserted into the rat renal cortex significantly increased exit dialysate levels of AMP (from 18 ± 5 to 54 ± 21 ng/ml, mean ± SEM, n=12, p<0.05) and adenosine (from 20 ± 4 to 64 ± 29 ng/ml, p<0.001). Similar results were obtained when the perfusate also contained an adenosine deaminase inhibitor (AMP from 18 ± 6 to 191 ± 134 ng/ml, p<0.05; adenosine from 46 ± 22 to 133 ± 88 ng/ml, p<0.05). AMP (30 μM) in the perfuate also increased exit dialysate levels of adenosine (from 22 ± 5 to 358 ± 59 ng/ml, p<0.001 and from 123 ± 23 to 525 ± 105 ng/ml, p<0.001, without and with inhibition of adenosine deaminase, respectively. We conclude that interstitial cAMP and AMP is a viable source of interstitial adenosine in the kidney This work was supported by DK68575