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Absolute quantification and NMR visibility of glycogen in the isolated, perfused rat heart using 13 C NMR spectroscopy
Author(s) -
Garlick Pamela B.,
Pritchard Ronald D.
Publication year - 1993
Publication title -
nmr in biomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.278
H-Index - 114
eISSN - 1099-1492
pISSN - 0952-3480
DOI - 10.1002/nbm.1940060113
Subject(s) - glycogen , chemistry , nuclear magnetic resonance spectroscopy , nmr spectra database , analytical chemistry (journal) , carbon 13 nmr , nuclear magnetic resonance , chromatography , spectral line , biochemistry , stereochemistry , physics , astronomy
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 0.2mg/kg isoproterenol (s.c.) to deplete their endogenous myocardial glycogen stores (mainly 12 C). The hearts were then aerobically perfused (65 cm H 2 O, at 37 °C) in a double‐walled chamber (the annulus contained a standard), for 70 min with Krebs buffer plus 3.5 mM [ 13 C]1‐glucose and 5 mM sodium acetate (natural abundance). From 70 to 175 min the sole substrate was natural abundance acetate (5 mM). 13 C NMR spectra for glycogen quantification were acquired in two different ways; by applying 896, 90° pulses at 0.33 s intervals with 1 H decoupling (‘fast’, practical spectra) and by applying 896, 90° pulses at 5 s intervals (‘slow’, impractical spectra). Hearts were then removed from the magnet, freeze‐clamped ( −196 °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) μmol glucosyl units/heart, respectively. After correction of the chemical value for the residual [ 12 C]glycogen (determined from 1 H 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. We conclude that, despite its size, cardiac glycogen is 100% visible in NMR spectra of isolated rat hearts.