Open AccessPhosphorus‐31 nuclear‐magnetic‐resonance study of phosphorylated metabolites compartmentation, intracellular pH and phosphorylation state during normoxia, hypoxia and ethanol perfusion, in the perfused rat liver
Author(s) -
DESMOULIN Franck,
COZZONE Patrick J.,
CANIONI Paul
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb10555.x
Subject(s) - phosphomonoesters , cytosol , chemistry , ethanol , perfusion , pi , intracellular ph , adenine nucleotide , metabolism , intracellular , biochemistry , endocrinology , nucleotide , medicine , phosphate , biology , enzyme , inorganic phosphate , gene
A quantitative analysis of the phosphorus‐31 NMR spectra of excised perfused rat liver has been carried out at 80.9 MHz using a 30‐mm sample cell.1 The results indicate that in liver from fed rats, all intracellular ATP is detected by NMR. In contrast, only the cytosolic fractions of P i and ADP can be observed as indicated by careful analysis of spectra obtained from perchloric acid liver extracts and intact liver under valinomycin perfusion. In well‐oxygenated perfused liver the ATP concentration is 7.4 mM. Values of 5.3 mM and 0.9 mM are found respectively for P i and ADP concentrations in the cytosolic compartment. Cytosolic pH value (pH i ) is 7.25±0.05 and free magnesium concentration 0.5 mM. 2 Addition of 70 mM (0.4%) ethanol to the perfusate of a fed rat liver induces 25% and 38% reduction of ATP and P i levels, respectively. A large amount of sn ‐glycerol 3‐phosphate is synthesized (up to 11 mM) in the cytosol. After ethanol withdrawal, a large overshoot in cytosolic P i is observed, which is indicative of a net uptake of P i across the plasma membrane that occured during ethanol oxidation. No significant pH variation is observed during ethanol infusion. 3 In perfused liver ofrats subjected to 48‐h fasts, the concentrations of cytosolic phosphorylated metabolities are 5.3 mM, 0.8 mM and 11.5 mM for ATP, ADP and P i , respectively. The perfusion of the liver with 70 mM ethanol does not change the adenine nucleotide levels, while the P i content is decreased by 10%. 4 During a 4‐min hypoxia, induced by reducing the perfusion flow rate from 12 ml to 3 ml min −1 (100 g body weight) −1 , ATP concentration decreases to 5.8 mM in the fed rat liver. Cytosolic P i and ADP increase to 8.7 mM and 1.6 mM, respectively. The cytosolic pH evolves to more acidic values and reaches 7.02±0.05 at the end of the 4‐min hypoxic period.