The novel phosphorylated pyrrolidine diethyl(2-methylpyrrolidin-2-yl)phosphonate (DEPMPH) was evaluated as a 31 P NMR probe of the pH changes associated with ischemia/reperfusion of rat isolated hearts and livers. In vitro titration curves indicated that DEPMPH exhibited a 4-fold larger amplitude of chemical shift variation than inorganic phosphate yielding an enhanced NMR sensitivity in the pH range of 5.0 -7.5 that allowed us to assess pH variations of less than 0.1 pH units. At the non-toxic concentration of 5 mM, DEPMPH distributed into external and cytosolic compartments in both normoxic organs, as assessed by the appearance of two resonance peaks. An additional peak was observed in normoxic and ischemic livers, assigned to DEPMPH in acidic vesicles (pH 5.3-5.6). During severe myocardial ischemia, a third peak corresponding to DEPMPH located in ventricular and atrial cavities appeared (pH 6.9). Mass spectrometry and NMR analyses of perchloric extracts showed that no significant metabolism of DEPMPH occurred in the ischemic liver. Reperfusion with plain buffer resulted in a rapid washout of DEPMPH from both organs. It was concluded that the highly pH-sensitive DEPMPH could be of great interest in noninvasive ex vivo studies of pH gradients that may be involved in many pathological processes.Because of the dependence of the chemical shift of phosphates on pH (1-3), 31 P NMR spectroscopy has progressively become the standard method for the measurement of intracellular pH (pH i ) 1 in biological systems, mostly using inorganic orthophosphate (P i ) as a naturally occurring pH probe (4 -6). Although this technique was first applied to measure pH in a variety of biological fluids, the development of pulsed Fourier transform NMR and wide-bore supraconducting magnets has soon allowed the noninvasive study of cell cultures and isolated perfused organs under physiologic or pathologic conditions. In particular, the use of 31 P NMR has considerably increased our understanding of the dynamics of pH i changes during ischemia-induced acidosis in the heart (7-9) and liver (10, 11). Although P i resonance has been successfully used to describe the main pH i -regulatory systems in these organs (12, 13), more subtle trans-sarcolemmal proton movements that could occur in different pathologies may escape investigation if they are related to extra-and intracellular pH values different by less than 0.2-0.3 pH units (4). In addition to this relative lack of resolution, P i levels vary with cell metabolism, and the chemical shift of P i has been demonstrated to be affected by ionic strength (4 -6).The search for improved exogenous 31 P NMR pH indicators as alternatives to P i yielded a variety of alkyl-and aminoalkylphosphonic acids having their resonance peak distinct from that of phosphorylated metabolites, and an NMR sensitivity ⌬␦ ab (defined as the mean difference between the chemical shifts of the protonated ␦ a and the unprotonated ␦ b forms) in the range of P i (i.e. 2-3 ppm). There have been a number of studies on the in v...
The purpose of this study was to test the hypothesis that mitochondrial permeability transition might be implicated in mitochondrial and intact organ dysfunctions associated with damage induced by reperfusion after cold ischaemia. Energetic metabolism was assessed continuously by 31P-NMR on a model system of isolated perfused rat liver; mitochondria were extracted from the livers and studied by using top-down control analysis. During the temperature transition from hypothermic to normothermic perfusion (from 4 to 37 degrees C) the ATP content of the perfused organ fell rapidly, and top-down metabolic control analysis of damaged mitochondria revealed a specific control pattern characterized by a dysfunction of the phosphorylation subsystem leading to a decreased response to cellular ATP demand. Both dysfunctions were fully prevented by cyclosporin A, a specific inhibitor of the mitochondrial transition pore (MTP). These results strongly suggest the involvement of the opening of MTP in vivo during the transition to normothermia on rat liver mitochondrial function and organ energetics.
This study was designed to test the effects of short-chain fatty acids (SCFA) with an even number of carbon atoms on hepatic energy metabolism. The effect of the SCFA was evaluated by measuring liver ATP content and oxygen consumption. The ATP content was evaluated using (31)P nuclear magnetic resonance in isolated liver from fed rats. In addition, respiratory activity (VO(2)) was assessed using Clark electrodes. The livers were perfused with acetate, butyrate or a medium chain length fatty acid, octanoate, at a concentration of 0.05--5.0 mmol/L. The addition of each substrate enhanced the rate of the net ATP consumption (V(i)), establishing a new ATP steady state that required a perfusion time of > or = 20 min, dependent on the chain length and concentration of the fatty acid (FA). The initial V(i) was unchanged for acetate and the ATP level stabilized at 58% of the initial level. Both butyrate and octanoate induced a dose-dependent increase in V(i). This may reflect an ATP-consuming process for the intracellular pH regulation observed during the acidosis associated with the beta-oxidation pathway. At the new steady state, the ATP concentration was approximately 45% of the initial level for both FA. VO(2) was both rapidly and reversibly increased, and the change was a function of butyrate or octanoate concentration and of the chain length. K(m) values were similar for butyrate and octanoate. Because all of the effects were similar for butyrate and octanoate, in contrast to acetate, we suggest that the impairment of the energy metabolism by butyrate resulted from an increase in the FADH(2)/NADH ratio due to beta-oxidation. In conclusion, the difference in the hepatic oxidation pathways of two products of intestinal fermentation (acetate and butyrate) explains their different actions on energy metabolism.
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