Absolute quantification and NMR visibility of glycogen in the isolated, perfused rat heart using 13C NMR spectroscopy

NMR spectroscopy, possibly, does not detect 100% of large molecules such as glycogen (mol.wt = 10(7)-10(9)). Using both NMR and chemical quantification methods, we have, therefore, determined the NMR visibility of cardiac glycogen (defined as the ratio of the NMR value to the chemical value, expressed as a percentage) in the isolated, perfused heart. Rats (n = 7) were pretreated for 60 min with 0.2 mg/kg isoproterenol (s.c.) to deplete their endogenous myocardial glycogen stores (mainly 12C). The hearts were then aerobically perfused (65 cm H2O, at 37 degrees C) in a double-walled chamber (the annulus contained a standard), for 70 min with Krebs buffer plus 3.5 mM [13C]1-glucose and 5 mM sodium acetate (natural abundance). From 70 to 175 min the sole substrate was natural abundance acetate (5 mM). 13C NMR spectra for glycogen quantification were acquired in two different ways; by applying 896, 90 degree pulses at 0.33 s intervals with 1H decoupling ('fast', practical spectra) and by applying 896, 90 degree pulses at 5 s intervals ('slow', impractical spectra). Hearts were then removed from the magnet, freeze-clamped (-196 degrees C) and analysed chemically. Cardiac glycogen, quantified from the 'fast' spectra (using conversion factors) and the 'slow' spectra was 16.8 +/- 1.1 and 16.1 +/- 1.8 (mean +/- SEM) mumol glucosyl units/heart, respectively. After correction of the chemical value for the residual [12C]glycogen (determined from 1H NMR spectra of the extracted glycogen after hydrolysis), the NMR-visibilities were calculated to be 101 +/- 6 and 109 +/- 7%, for the 'fast' and 'slow' spectra, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)