Simultaneous determination of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in biological samples using solid-phase extraction and high-performance liquid chromatography.
A sensitive and rapid method for measuring simultaneously adenosine, S-adenosylhomocysteine and S-adenosylmethionine in renal tissue, and for the analysis of adenosine and S-adenosylhomocysteine concentrations in the urine is presented. Separation and quantification of the nucleosides are performed following solid-phase extraction by reversed-phase ion-pair high-performance liquid chromatography with a binary gradient system. N6-Methyladenosine is used as the internal standard. This method is characterized by an absolute recovery of over 90% of the nucleosides plus the following limits of quantification: 0.25-1.0 nmol/g wet weight for renal tissue and 0.25-0.5 microM for urine. The relative recovery (corrected for internal standard) of the three nucleosides ranges between 98.1 +/- 2.6% and 102.5 +/- 4.0% for renal tissue and urine, respectively (mean +/- S.D., n = 3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop freezing technique is mandatory to observe the tissue levels of the nucleosides under normoxic conditions. The resulting tissue contents of adenosine, S-adenosylhomocysteine and S-adenosylmethionine in normoxic rat kidney are 5.64 +/- 2.2, 0.67 +/- 0.18 and 46.2 +/- 1.9 nmol/g wet weight, respectively (mean +/- S.D., n = 6). Urine concentrations of adenosine and S-adenosylhomocysteine of man and rat are in the low microM range and are negatively correlated with urine flow-rate.
Delabar
U
U
Department of Pharmacology, Medical Faculty, University of Tübingen, Germany.
Kloor
D
D
Luippold
G
G
Mühlbauer
B
B
D000241:Adenosine / Q000378:metabolism / Q000652:urine*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012435:S-Adenosylhomocysteine / Q000378:metabolism / Q000652:urine; D012436:S-Adenosylmethionine / Q000378:metabolism / Q000652:urine*; D013056:Spectrophotometry, Ultraviolet
D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D000241:Adenosine