Determinants of the S-Adenosylhomocysteine (SAH) Technique for the Local Assessment of Cardiac Free Cytosolic Adenosine

Abstract

The S-adenosylhomocysteine (SAH) technique allows the estimation of the free cytosolic adenosine concentration using the kinetic properties of the enzyme SAH-hydrolase (adenosine+homocysteine→SAH+H2O). Besides the cytosolic adenosine concentration, the local SAH signal may also depend on the local homocysteine availability, the continuous production of SAH from S-adenosylmethionine (SAM→SAH+CH3) and the activity of the enzyme SAH-hydrolase. These variables were studied with high spatial resolution (sample dry mass 25 mg) in left ventricular myocardium from 26 anesthetized open-chest dogs in which heart rate averaged 86±14 beats/min and mean aortic pressure 96±17 mmHg. Homocysteine infusion (48 mg/kg i.v.) increased the normal plasma homocysteine concentration from 5.0± 0.8 to 586±40μmafter 30 min when the average tissue concentration was 94% of the plasma concentration and similar in low and high flow areas (flow range 0.04 to 1.91 ml/min/g). Local SAH content was 1.18±0.48 nmol/g under control conditions and increased to 4.33±0.59 nmol/g within 60 min following competitive blockage of the SAH-hydrolase by adenosine dialdehyde (10μmol/kg i.v.). This increase of the SAH content was slightly more in high than in low-flow areas (P<0.01). Regional SAH-hydrolase activity (9.0±0.5 nmol/min/g) was comparable in high and low flow areas. All three variables exhibited an observed variability which was larger than the methodical variability suggesting significant spatial heterogeneity in the myocardium. A regrouping analysis indicated that between four and five samples taken from distant sites should be averaged to obtain a robust estimate of the above metabolic parameters. Reconciling the measurements with a mathematical model of cardiac adenosine metabolism and fitting of the measured SAH tissue levels gave an estimate of 72 pmol/min/g for the mean transmethylation rate. Estimates of the cytosolic adenosine concentration of cardiomyocytes and endothelial cells under control physiological conditions were 24 and 7μm, respectively. Thus, the present measurements provide a basis for the quantitative assessment of the local cytosolic adenosine concentration in relation to blood flow.