Preservation of amino acids during long term ischemia and subsequent reflow with supplementation of L-arginine, the nitric oxide precursor, in the rat heart.

We investigated whether L-arginine, used in heart preservation to limit endothelial damage, may influence the pool of amino acids during long term ischemia and reflow. Isolated isovolumic rat hearts (n = 23) were submitted to 8 h of hypothermic ischemia after cardioplegic arrest with the Centre de Résonance Magnétique Biologique et Médicale (CRMBM) solution with or without L-arginine (Arg and No Arg groups respectively). Hearts were freeze-clamped after ischemia (n = 11) or submitted to 60 min of reflow (n = 12) and freeze-clamped. Eight hearts were perfused aerobically for 20 min and freeze-clamped (No ischemia group). Addition of L-arginine to the CRMBM solution limited aspartate depletion and decreased lysine level at the end of ischemia. After reflow, L-arginine supplementation increased the pool of glutamate and arginine and limited the depletion of serine, asparagine, glycine and taurine. We conclude that adding L-arginine to the CRMBM cardioplegic solution during long term ischemia preserved the amino acids pool.

L-arginine during long-term ischemia: effects on cardiac function, energetic metabolism and endothelial damage.

BACKGROUND: We have evaluated the addition of L-arginine, a precursor of nitric oxide, to a cardioplegic solution (named CRMBM) designed for long-term heart preservation. METHODS: Isolated isovolumic-perfused rat hearts (n = 22) were arrested with the CRMBM solution either with (Arg) or without L-arginine (2 mmol/L) (Arg group, n = 12, vs control group n = 10), submitted to 8 hours of cold storage (4 degrees C) in the solution, and then reperfused for 60 minutes at 37 degrees C. In 11 hearts, we evaluated the quality of cardiac preservation with P-31 magnetic resonance spectroscopy and the measure of function and cellular integrity. Endothelium-dependent and independent vasodilatations were measured in 11 other hearts, using 5-hydroxytryptamine and papaverine to assess endothelial and smooth muscle function. RESULTS: Adding L-arginine to the cardioplegic solution improved functional recovery during reflow, as shown by the rate pressure product (31% +/- 3% for control vs 47% +/- 3% for Arg, p = 0.003) together with higher coronary flow and diminished contracture. Purine release in coronary effluents during reperfusion was lower in the Arg group. During ischemia and reflow kinetics of intracellular pH and high-energy phosphates were similar in both groups. Coronary endothelium-dependent vasodilatation was similarly impaired in both groups, but smooth muscle was less altered with L-arginine. CONCLUSIONS: As an additive to the CRMBM cardioplegic solution, L-arginine provides a protective effect for long-term heart preservation. Our data do not show coronary endothelial protection as the prominent mechanism.

Metabolic and functional effects of low-potassium cardioplegic solutions for long-term heart preservation.

Cardioplegic solutions used to arrest the heart during open heart surgery and cardiac transplantation are based on potassium as a cardioplegic agent in a concentration range of 15-35 mM. However, high to moderate K+ concentrations increase Ca2+ influx and impair endothelial function. We have therefore evaluated the possible advantage of a lower potassium concentration in a new cardioplegic solution (named CRMBM solution) designed for long-term heart preservation. Nine isolated perfused rat hearts were submitted to 8 h of hypothermic ischemia after cardioplegic arrest, followed by 60 min of reflow at 37 degrees C. Two cardioplegic solutions were compared: (1) the CRMBM solution with 10 mM potassium (K-10 group), and (2) the CRMBM solution with 4 mM potassium (K-4 group). The quality of heart preservation was assessed by a metabolic study using P-31 magnetic resonance spectroscopy (energy metabolism and intracellular pH) combined to a functional evaluation and a measure of cellular integrity (biochemical assays in effluents and tissues). Decreasing the potassium concentration to 4 mM improved heart preservation, as shown by a higher functional post-ischemic recovery represented by the rate pressure product and a better preservation of cellular integrity. The evolutions of intracellular pH and high energy phosphate levels during ischemia and reflow were similar in both groups.

Optimized cardiac graft preservation: a comparative experimental study using P-31 magnetic resonance spectroscopy and biochemical analyses.

BACKGROUND: The University of Wisconsin (UW), St. Thomas (ST) and Broussais (B) solutions were compared to the CRMBM solution, that we developed for long term heart preservation. METHODS: Isolated isovolumic rat hearts were arrested with each cardioplegic solution (n = 5) to 8 hearts in each group), submitted to 12 hours of cold storage (4 degrees C) in the same solution and then reperfused for 60 minutes at 37 degrees C. Function was measured during control and reflow. High energy phosphates and intracellular pH were monitored by P-31 magnetic resonance spectroscopy. Analyses were performed by biochemical assays and HPLC in coronary effluents (CK, Pi, lactate, purines) and in freeze-clamped hearts (amino acids, nucleotides, CK, LDH) at the end of reperfusion. RESULTS: Functional recovery was significantly improved with the new cardioplegic solution (50+/-12% recovery for the rate pressure product at the end of reflow vs 8+/-3% with UW, 0% with B and with ST). This result was correlated with the best metabolic and cellular protection as assessed in particular by higher PCr levels during reflow (30+/-3% vs 10+/-3% with UW, 8+/-4% with B, and 7+/-1% with ST) as well as reduced creatine kinase leakage during reflow (110+/-15 IU/60 minute vs 270 +/- 57 IU/60 minute with UW, 323+/-36 IU/60 minute with Broussais solution and 237+/-18 IU/60 minute with ST). CONCLUSION: This new solution is more effective in prolonged myocardial protection than the three most widely used solutions.

The influence of temperature on metabolic and cellular protection of the heart during long-term ischemia: a study using P-31 magnetic resonance spectroscopy and biochemical analyses.

We have compared the influence of two different cold temperatures (below 10 degreesC) for cardiac ischemia by measuring a large variety of hemodynamic and metabolic parameters during ischemia and reflow. Isolated isovolumic rat hearts were arrested with a preservation solution which was developed in our laboratory and then submitted to 5 h of cold storage (4 degreesC, group I; and 7.5 degreesC, group II) in the same solution. After an additional period of 50 min of ischemia at 15 degreesC with intermittent cardioplegic infusion, hearts were reperfused for 60 min at 37 degreesC. Function was assessed during the control period and reflow. High-energy phosphates and intracellular pH were followed by 31P magnetic resonance spectroscopy. Analyses of metabolites and enzymes were performed by biochemical assays and HPLC in coronary effluents and in freeze-clamped hearts to assess cellular integrity. The energetic pool was better preserved at 4 degreesC during ischemia (ATP at the end of 4 degreesC ischemia, 59 +/- 7% in group I vs 31 +/- 5% in group II, P < 0.01) and reflow (P < 0.05) but membrane protection was higher when increasing the temperature to 7.5 degreesC (reduction of creatine kinase leakage, 89 +/- 16 IU/min in group I vs 51 +/- 5 IU/min in group II, P < 0.05). As a result, functional recovery, represented by the rate pressure product, was higher in hearts preserved at 7.5 degreesC (52 +/- 6% recovery in group I vs 77 +/- 7% in group II at the end of reflow, P < 0.05). Altogether, cold storage at 7.5 degreesC provides a better protection than storage at 4 degreesC.

Analysis of plasma lipids by NMR spectroscopy: application to modifications induced by malignant tumors.

Lipid extracts of plasma were studied by 31P and 1H NMR spectroscopy at 9.4 T. Signals recorded on lipid mixtures were assigned to different lipid classes using a data base built with two-dimensional 1H COSY spectra of seven standard lipids. Signals unique to glycerophospholipids, sphingolipids, and triacylglycerols were identified. 31P and 1H NMR spectroscopy was used to study qualitative and quantitative modifications induced in plasma by malignant tumors. The results show a significant increase in triglyceride/phospholipid ratio and a concomitant decrease of total phospholipids in patients with cancer. In order to check for the possible presence of particular lipids such as glycolipids in these patients, 1H COSY spectra were recorded on the intact plasma and on extracts of plasma lipids in patients with cancer and in healthy subjects. Only in one case of ovarian cancer, a cross-peak at 1.35 and 4.15 ppm, corresponding to fucose residue in glycolipids, was detected.

CSF and serum metabolic profile of patients with Huntington's chorea: a study by high resolution proton NMR spectroscopy and HPLC.

We studied both cerebrospinal fluid (CSF) and serum of 11 patients suffering from Huntington's disease (HD) and 12 control subjects by combining high resolution proton NMR spectroscopy and HPLC. NMR spectroscopy analysis of the CSF shows a significant increase (60%) in pyruvate concentration in HD patients. No unexpected molecules were detected. Glutamate, glutamine, aspartate, proline and GABA levels were found unchanged in the CSF of HD patients, using HPLC analysis. Conversely, a significant increase (30%) in the CSF level of glycine was detected. These observations are in agreement with the metabolic hypothesis of HD physiopathogenesis. In addition, the protocol combining NMR spectroscopy and HPLC provides a straightforward evaluation of brain metabolic status and blood-brain-barrier function.

Catabolism of adenine nucleotides and its relation with intracellular phosphorylated metabolite concentration during ethanol oxidation in perfused rat liver.

Ethanol-induced perturbations in the energy metabolism and in the catabolism of adenine nucleotides were investigated by 31P NMR spectroscopy and HPLC analyses in perfused rat liver. Ethanol oxidation reduced the redox potential of the hepatocyte, leading to an intracellular accumulation of sn-glycerol 3-phosphate. This accumulation, in turn, led to a cytosolic P(i) depletion with stoichiometric relationship close to 1/1 for an initial period of 2 min. The concentration of nucleoside 5'-triphosphates (83 +/- 4% of ATP) was decreased during ethanol oxidation, reaching about 66% of its control value [2.88 +/- 0.02 mumol.(g of liver wet wt)-1] at high ethanol doses (10 and 70 mM). The depletion of P(i) relieved the inhibition exerted by this compound on AMP deaminase, key enzyme in the catabolism of adenine nucleotides. The degradation of AMP was monitored by HPLC analyses of the adenine nucleosides and bases released in the effluents. Integration over time of the total release of these metabolites accounted for the depletion of ATP recorded in the same time by 31P NMR spectroscopy. This result suggests that ATP depletion occurring during ethanol oxidation originated from an enhanced degradation of adenine nucleotides. There was a strong linear correlation (r2 = 0.92) between cytosolic P(i) level and allantoin release rate during ethanol perfusion. Cytosolic P(i) and allantoin release exhibited biphasic behavior, the recovery toward the initial levels being related to the release of P(i) in the cytoplasm during the complete catabolism of adenine nucleotides. Finally, the depletion of P(i) affected the glycogenolysis pathway, with a maximal inhibition of ca. 19% of the initial level.

Graphic-aided study of metabolic modifications of plasma in cancer using proton magnetic resonance spectroscopy.

Proton high-resolution MRS of human plasma allows the rapid detection, on the same spectrum, of many compounds originating from different metabolic pathways. In this paper, we illustrate the modifications of the plasma metabolic profiles recorded by proton NMR spectroscopy in different classes of cancers. These modifications can be easily monitored with graphic aids such as 'star plots' which define for each type of cancer a particular pattern describing the most altered metabolic pathways. By using 'star plots' three types of metabolic patterns have been distinguished: (i) the 'inflammatory' pattern characterized by an increase of glycosylated moieties of glycoproteins; (ii) a 'lipid modified' pattern, characterized by various modifications occurring mainly in the lipid moieties detected by MRS; and (iii) a pattern which is often observed in sarcomas and mainly characterized by an alteration in the N-acetyl glucosamine/N-acetyl neuraminic acid ratio. This study demonstrates the ability of proton MRS of plasma to rapidly detect the occurrence of metabolic modifications brought about by cancer evolution or therapy.

[Plasma glycosylated residues demonstrated by proton NMR spectroscopy. Value in the detection of heart graft rejection]. / Résidus glycosylés plasmatiques mis en évidence par spectroscopie de RMN du proton. Intérêt dans la détection du rejet de greffe cardiaque.

In conjunction with biopsy and Doppler studies, we analysed by high resolution proton NMR spectroscopy the blood plasma of 22 heart transplant recipients. There was a significant variation in the glycosylated residues of proteins with the development of acute cardiac rejection. A more extensive study is underway to assess the sensitivity and specificity of this approach for the early diagnosis of acute cardiac rejection.

High resolution NMR spectroscopy of physiological fluids: from metabolism to physiology.

High resolution NMR spectroscopy of physiological fluids provides quantitative, qualitative and dynamic information on the metabolic status of the interstitial and plasma compartments under a variety of pathophysiological conditions. The simultaneous detection and quantitation by NMR spectroscopy of numerous compounds of the intermediary metabolism offers a new insight in the understanding of the milieu intérieur. NMR spectroscopy of physiological fluids offers a unique way to define and monitor the global metabolic homeostasis in humans. The development of this analytical approach is still limited by the scarcity of pluridisciplinary teams able to fully exploit the wealth of information present on the NMR spectrum of a fluid. While application in pharmacology and toxicology is already established, the main areas of current development are cancer, hereditary metabolic disorders, organ transplantation and neurological diseases.

Quantitation of metabolites in human blood serum by proton magnetic resonance spectroscopy. A comparative study of the use of formate and TSP as concentration standards.

Formate has been evaluated as an alternative standard to quantitate human serum metabolites in 1H NMR spin-echo spectra. The comparison between added formate and 3-(trimethylsilyl) 3,3,3,3-tetradeutero-propionic acid (TSP) shows that, unlike TSP, formate does not interact with serum macromolecules. Transverse and longitudinal proton relaxation times have been measured on several serum metabolites, in the presence of ammonium chloride. With the exception of glucose, values of metabolite concentrations derived from Hahn spin-echo spectra recorded on serum containing 15.4 mM exogenous formate as a standard, are in excellent agreement with the results of biochemical and chromatographic assays, after correction for differential relaxation effects. This approach can be readily used for quantitation of metabolites from blood serum (and eventually other physiological fluids) in normal and in pathological situations not involving disorders of endogenous formate metabolism.

The effects of ethanol concentration on glycero-3-phosphate accumulation in the perfused rat liver. A reassessment of ethanol-induced inhibition of glycolysis using 31P-NMR spectroscopy and HPLC.

The dose-dependent effect of ethanol on the hepatic metabolism of the perfused rat liver has been investigated by (a) 31P-NMR spectroscopy for the follow-up of intracellular phosphorylated metabolites and (b) HPLC for compounds released in the effluents. Perfusion of livers from fed rats with ethanol induced an increase in the level of sn-glycerol 3-phosphate and net accumulations of 3.30 +/- 0.33 and 0.69 +/- 0.15 mumol x g-1 wet liver were reached after 20 min, for 70 mM and 0.5 mM ethanol, respectively. sn-Glycerol-3-phosphate accumulation was fully detected by 31P NMR as indicated by comparing quantitations based on NMR and biochemical assays. Ethanol administration up to a concentration of 10 mM induced a dose-dependent decrease in the release of lactate + pyruvate by the liver. Lactate release decreased from 1129 +/- 39 to 674 +/- 84 nmol x min-1 x g-1, while pyruvate decreased from 230 +/- 9 to 6.2 +/- 0.4 nmol x min-1 x g-1, after 20 min of perfusion with 10 mM ethanol. Nevertheless, the flux through 6-phosphofructo-1-kinase, as measured by both the accumulation of sn-glycerol 3-phosphate and release of lactate + pyruvate, was not affected in the early phase of ethanol oxidation. Finally, data obtained from oxygen consumption, the release of acetate and the accumulation of sn-glycerol 3-phosphate do not support the involvement of the microsomal ethanol-oxidizing system in the catalysis of ethanol oxidation, even at high doses of alcohol.

Early detection of heart transplant rejection using cardiac echography combined with the assay of glycosylated residues in plasma by proton NMR spectroscopy.

Early diagnosis of acute cardiac graft rejection by non-invasive methods is required for medical, organizational, psychological and economic reasons. We have monitored 18 heart recipients over a period of 2.5 years using endomyocardial biopsies (EMB), cardiac Doppler-echography (CDE) and proton NMR spectroscopy assay of plasma glycosylated residues. Diastolic parameters of CDE and assay of the glycosylated residues by NMR spectroscopy respectively detect 42 and 45% of the acute low grade (mild or moderate) histological rejections. The combination of the two methods allows the detection of 65% of rejections. The strategy combining plasma NMR spectroscopy and echography is pertinent to the non-invasive detection of acute cardiac rejections with low histological grade.

[Study of biological fluids by nuclear magnetic resonance spectroscopy]. / Etude des liquides biologiques par spectroscopie de résonance magnétique nucléaire.

The use of nuclear magnetic resonance (NMR) spectroscopy in the study of biofluids is rapidly developing and might soon constitute a new major medical application of this technique which benefits from technological and methodological progress such as higher magnetic fields, new probe design, solvent suppression sequences and advanced data processing routines. In this overview, the clinical and pharmacological impact of this new approach is examined, with emphasis on the NMR spectroscopy of plasma, cerebrospinal fluid and urine. Applications to pharmacokinetics and toxicology are illustrated. Interestingly, a number of biochemical components of fluids which are not usually assayed by conventional biochemical methods are readily detected by NMR spectroscopy which is clearly a new competitive entrant among the techniques used in clinical biology. Its ease-of-use, cost effectiveness and high informational content might turn it into a major diagnostic tool in the years to come.