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-aden osylhomocysteine and S-adenosylmethionine in renal tissue, and for the anal ysis 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-performanc e liquid chromatography with a binary gradient system. N-6-Methyladenosine is used as the internal standard. This method is characterized by an absolu te recovery of over 90% of the nucleosides plus the following limits of qua ntification: 0.25-1.0 nmol/g wet weight for renal tissue and 0.25-0.5 mu M for urine. The relative recovery (corrected for internal standard) of the t hree nucleosides ranges between 98.1+/-2.6% and 102.5+/-4.0% for renal tiss ue and urine, respectively (mean+/-S.D., n=3). Since the adenosine content in kidney tissue increases instantly after the onset of ischemia, a stop fr eezing technique is mandatory to observe the tissue levels of the nucleosid es 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 mu M range and are negatively correlated wit h urine flow-rate. (C) 1999 Elsevier Science B.V. All rights reserved.