Simple cation-exchange high-performance liquid chromatography optimized to the measurement of metabolites in the effluents from perfused rat livers using refractive index and ultraviolet detectors.

A high-performance liquid chromatographic method designed to analyse effluents from perfused organs is described. In the case of rat liver, compounds released by the liver are readily separated and quantitated, using a strong cation exchanger (Aminex HPX 87H), two detectors connected in series (ultraviolet detector at 210 nm and refractive index detector), and by optimizing the concentration of sulphuric acid in the mobile phase. Chromatographic conditions described in the present work enable the quantitation, in a single run, of metabolites derived from the tricarboxylic acid cycle, glycolysis, ketogenesis, adenine nucleotides catabolism and ethanol oxidation. The advantage of this method stems from its ease of implementation, sensitivity and flexibility.

Variations of plasma sialic acid and N-acetylglucosamine levels in cancer, inflammatory diseases and bone marrow transplantation: a proton NMR spectroscopy study.

Proton NMR spectroscopy allows the detection in plasma of resonances arising from N-acetyl-glucosamine (NAG) and N-acetyl-neuraminic acid (NANA) which have been shown to be borne by acute phase glycoproteins. These resonances can be identified using 2 different protocols of spectrum acquisition detecting different physical states in the global pool of glycoproteins, ie mobile and less mobile moieties of glycosylated chains. In this study we demonstrate that NMR spectroscopy allows a precise monitoring of the variations of glycosylated residues in cancers, inflammatory processes and bone marrow transplantation. The most important findings are that: i), the distribution of glycosylated residues varies with the origin of the cancerous tissue; ii), the level of these residues is a function of tumor development; iii), the concentrations in NAG and NANA are well correlated with the standard biological parameters of acute phase and leucocyte activation. Proton NMR spectroscopy of glycosylated residues in plasma may offer a new means of monitoring sialic acid in cancer and other pathological conditions.

[Modification of the relative plasma concentrations of methyl and methylene groups in cancer: a study using proton NMR spectroscopy]. / Modification des concentrations relatives des groupements méthyle et méthylène plasmatiques dans la pathologie cancéreuse: une étude par spectroscopie RMN du proton.

Fossel et al. have recently proposed the proton NMR examination of plasmatic lipoproteins--and more precisely the determination of an index obtained from the averaged linewidth of the CH2 and CH3 resonances--as a possible tool for detection of cancer. Many evaluations conducted on an international basis have demonstrated that initial expectations were not met and that the test lacked sensitivity, specificity, and predictive value to be accepted as a screening and diagnostic tool. In our evaluation we have collected plasma from healthy subjects, from patients with various kinds of cancer at different stages of evolution and therapy, and from patients suffering from a variety of pathologies, including benign tumors. In accordance with Chmurny et al., we observed that the linewidth index (LWI) is precise and reproducible when care is taken in the handling and storage of samples and in the fasting of subjects. After finding no predictive value to the test, we have reanalyzed the spectra and studied the variations of the ratio defined by the methylene signal area over the methyl signal area. This ratio is significantly increased in cancer. Furthermore, it offers a better separation of statistical populations permitting a more precise discrimination between cancer, other pathologies and controls. We have also found that malignant tumors arising from mesenchyma (sarcoma, leukemia, lymphoma) induce less important variations in the CH2/CH3 ratio than adenocarcinoma or glioma, when such differences cannot be documented using the LWI. These observations are particularly interesting since they might bring new information on the metabolic modifications of the LWI and the CH2/CH3 ratio might reflect the embryologic origin of the tumors and raise the issue of the heterogeneity of cancer disease.

Carbon-13 and phosphorus-31 NMR study of hepatic metabolism in the perfused rat liver.

Phosphorus-31 nuclear magnetic resonance (NMR) has been used to determine non-invasively absolute concentrations of phosphorylated metabolites in the perfused rat liver. It has been shown that the NMR method does detect cytoplasmic ATP and ADP (ATP:ADP ratio of 15 +/- 3) with no contribution from mitochondrial adenine nucleotides. The concentration of ATP was 7.2 +/- 0.3 mM in the cytosol of well-oxygenated liver, after two hours of perfusion with a Krebs-Ringer buffer. Other phosphorylated metabolites were detected, mainly inorganic phosphate (1.1 mumol/g liver wet weight), phosphorylcholine (1.0 mumol/g wet weight), glycerophosphorylethanolamine (0.34 mumol/g wet weight) and glycerophosphorylcholine (0.30 mumol/g wet weight). The intracellular pH measured from the position of the Pi resonance has a value of 7.2 +/- 0.1. It is likely that the detectable Pi originates from the cytosolic compartment since a pH value of 7.4-7.6 would be expected for the mitochondrial matrix. Natural abundance carbon-13 NMR has also been used to follow the glycogen breakdown in situ by measuring the intensity of the glycogen C-1 resonance in the perfused liver spectrum as a function of the perfusion time. The glycogenolytic process has been studied as a function of the glucose content of the perfusate. Rate of glycogenolysis from 2.7 to 0.16 muEq glycosyl units g wet weight-1 min-1 were found when glucose concentration in the perfusate was varied from 0 to 50 mM. The fate of 90% enriched [2-13C] acetate has been studied in the perfused rat liver by 13C-NMR in order to investigate the mitochondrial metabolism and the interrelations between cytosolic and mitochondrial pools of metabolites. Some compounds of the intermediary metabolism where found to be extensively labelled, e.g. glutamate, glutamine, acetoacetate and beta-hydroxybutyrate. Under our experimental conditions, labelling of glutamate reached a steady-state within 30 min after the onset of perfusion of 20 mM acetate. In addition, the observed incorporation of carbon-13 isotope into glutamine can be linked to the operation of the glutamate-glutamine antiporter and to the high activity of the cytosolic glutamate synthetase. The finding of both active glutaminase and glutamine synthetase activity in the same liver cells is an evidence of the existence of an active glutamine-glutamate futile cycle.

31P-NMR study of high-energy phosphorylated compounds metabolism and intracellular pH in the perfused rat heart.

Using 31P-NMR and haemodynamical measurements, this work assesses different aspects of myocardial preservation improvement during a global ischaemia, based on a simultaneous and correlated study of high-energy phosphorylated compounds, intracellular pH and left ventricular function. Isolated perfused working rat hearts were subjected to 2 or 3 h of hypothermic ischaemia followed by 30 or 45 min of reperfusion. A study of the influence of pH and buffer used in cardioplegic solutions has demonstrated a better preservation of high-energy phosphates and an improved functional recovery when using a pH 7.0, glutamate - containing solution. Protection provided by cardioplegia can be enhanced by the appropriate use of a fluorocarbon-oxygenated cardioplegic reperfusate. The use of nifedipine, a calcium antagonist, in the cardioplegic solutions, does not provide any additional protection under hypothermic conditions.

Effect of nifedipine in hypothermic cardioplegia: a phosphorus-31 nuclear magnetic resonance study.

The ability of nifedipine to enhance myocardial protection was assessed on isolated perfused rat hearts subjected to 180 min of hypothermic (20 degrees C), global ischemia, followed by 45 min of normothermic reperfusion. Intracellular pH, ATP, Pi and phosphocreatine content were serially measured at 4 min intervals by phosphorus-31 nuclear magnetic resonance spectroscopy and correlated with simultaneously recorded hemodynamic parameters. Addition of nifedipine (0.075 mumol/l and 0.5 mumol/l) to Saint Thomas' cardioplegic solution reduced Pi accumulation during ischemic arrest and increased phosphocreatine levels during reperfusion. Post-ischemic functional recovery was not improved at a drug concentration of 0.075 mumol/l and was depressed at 0.5 mumol/l. These results clearly show that the presence of nifedipine in Saint Thomas' cardioplegic solution does not provide significant additional myocardial protection under hypothermic conditions.

Enhanced cardioplegic protection by a fluorocarbon-oxygenated reperfusate: a phosphorus-31 nuclear magnetic resonance study.

Prolonged global ischemia results in a defect in oxygen extraction during early reperfusion. This study was thus undertaken to assess the effects of maintaining cardioplegia at the onset of reoxygenation in view of channeling available energy toward reparative cell processes rather than mechanical activity. Twenty-four isolated perfused rat hearts were subjected to 120 min of 15 degrees C ischemia. Group I (control) was reperfused with the standard Krebs perfusion medium whereas in groups II and III the initial reperfusate consisted of an oxygenated alkaline cardioplegic solution prior to the resumption of Krebs perfusion. Oxygenation of the cardioplegic reperfusate was ensured by fluorocarbons at a concentration of 10% (O2 content: 5.5 vol %; group II) or 20% (O2 content: 9 vol %; group III). In addition to hemodynamical determinations, high-energy phosphates and intracellular pH were monitored serially by phosphorus-31 nuclear magnetic resonance spectroscopy. After 30 min of reperfusion postischemic recovery of aortic flow was better in group II (74.0 +/- 5.9% of control) than in group I (59.1 +/- 5.4% of control, P less than 0.05). This functional improvement correlated with a higher postischemic increase in phosphocreatine levels (103.21 +/- 11.21% vs 74.12 +/- 3.59%, at 3 min of reperfusion, P less than 0.05) without significant differences in total ATP content. Group III hearts exhibited a slow recovery as evidenced by a severe depression in aortic flow, coronary arteriovenous difference, and total phosphate content during the 15 initial minutes of reperfusion. These results show that the protection provided by cardioplegia can be improved by a fluorocarbon-oxygenated cardioplegic reperfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the pH of cardioplegic solutions on intracellular pH, high-energy phosphates, and postarrest performance. Protective effects of acidotic, glutamate-containing cardioplegic perfusates.

The common practice of using alkalotic cardioplegic solutions is not supported by experimental evidence. The present study was conducted to assess the effects of varying the pH (7.00, 7.40, and 7.70 at 20 degrees C) of a glutamate-containing cardioplegic solution on intracellular pH, high-energy phosphate content, and postarrest functional recovery and to compare the effects of various buffers (glutamate, bicarbonate, TRIS, and histidine) at a given pH (7.00 and 7.40). Isolated perfused rat hearts were subjected to 2 hours of cardioplegic arrest at 15 degrees C followed by 30 minutes of reperfusion. Intracellular pH and high-energy phosphate content were measured at 4 minute intervals by phosphorus 31 nuclear magnetic resonance spectroscopy. These data were correlated with postischemic recovery of function. There was no significant difference between the intracellular pH values recorded at the end of arrest in the three glutamate-containing groups. However, the acidotic solution (pH 7.00) resulted in better preservation than the alkalotic solution (pH 7.70), as evidenced by a higher creatine phosphate content at the end of arrest (61% +/- 9% of control values versus 30% +/- 9% [mean +/- standard error of the mean], p less than 0.05), a higher adenosine triphosphate content at the end of reperfusion (102% +/- 5% versus 82% +/- 6%, p less than 0.05), and a faster recovery of aortic flow (at 3 minutes of reperfusion, 91% +/- 11% versus 51% +/- 11%, p less than 0.05). Subsequent comparison of buffers showed that bicarbonate, TRIS, and histidine were equally effective in maintaining intracellular pH close to control values during arrest. Conversely, the use of glutamate resulted in a more pronounced fall in intracellular pH, which correlated with a better preservation of adenosine triphosphate and a better functional recovery than in the other groups. Overall, the greatest extent of preservation was provided by the pH 7.00 glutamate-containing cardioplegic solution. We conclude that additional protection can be conferred to the cold, chemically arrested heart by combining mild intracellular acidosis, which lowers metabolic needs during arrest, most likely through a limitation of calcium overload, and provision of glutamate, which may act as a substrate for anaerobic energy production while allowing intracellular pH to be kept within the appropriate range.