Laurence Le Moyec

Proton Nuclear Magnetic Resonance Spectroscopy of Urine and Plasma in Renal Transplantation Follow-Up

Urine and plasma samples from 39 patients who underwent renal transplantation were analyzed by proton nuclear magnetic resonance (NMR) spectroscopy. The most relevant resonances for evaluating renal function after transplantation were those arising from citrate, trimethylamine-N-oxide (TMAO), alanine, and lactate when compared to creatinine. A resonance at 3.7 ppm was related to cyclosporine toxicity when associated with elevated levels of TMAO. The respective variations in these metabolites in urine could contribute to prognosis and diagnosis of renal function impairment related to cyclosporine toxicity or overdosage, or to rejection. Thus, NMR spectroscopy should improve the follow-up and management of renal transplantation patients.

Trimetazidine prevents renal injury in the isolated perfused pig kidney exposed to prolonged cold ischemia.

BACKGROUND Ischemia caused by cold storage (CS) and reperfusion of the kidney is often responsible for delayed graft function after transplantation. Significant attention has been focused on the cascade of events involved in ischemia-reperfusion injury, with the objective of identifying drugs to ameliorate the functional damage that occurs. METHODS The purpose of this study was to evaluate the renal function of isolated perfused pig kidneys after 48 hr of CS with Euro-Collins (EC) solution plus trimetazidine (EC+TMZ), standard EC solution, or University of Wisconsin (UW) solution. Normothermic isolated perfused pig kidneys were randomized into five experimental groups: (A) control group (cold flush with cold heparinized saline and immediately reperfused; n=6); (B) cold flush with cold heparinized saline with TMZ (10(-6) M), n=6; (C) 48 hr of CS with EC and reperfusion (n=8); (D) 48 hr of CS with EC+TMZ alone and reperfusion (n=8); (E) 48 hr of CS with UW and reperfusion (n=8). Proton nuclear magnetic resonance spectroscopy and biochemical studies were performed for the functional evaluation during reperfusion. Lipid peroxidation was also determined. Histological examination (optical and electron microscopy) was performed after CS and reperfusion. RESULTS Using TMZ, the renal perfusate flow rate as well as the glomerular filtration rate and proximal tubular function were significantly improved. This improvement of renal function during reperfusion was correlated with a less significant cellular and interstitial edema. In addition, tubular injury markers were significantly lower in the group preserved with EC+TMZ, and TMZ reduced lipid peroxidation dramatically during reperfusion. CONCLUSIONS The addition of TMZ to the EC solution increased the preservation quality and renal tubular function, and gave protection from reperfusion injury better than EC alone or UW. These results strongly suggest that TMZ has a cytoprotective effect and may therefore be useful for kidney preservation.

Evaluation of postnatal renal function in fetuses with bilateral obstructive uropathies by proton nuclear magnetic resonance spectroscopy

OBJECTIVES To precisely evaluate fetal renal function in cases of bilateral fetal urinary tract obstruction, we analyzed fetal urine metabolites by nuclear magnetic resonance spectroscopy. STUDY DESIGN Fifty-six specimens of fetal urine samples in utero were retrospectively analyzed by proton nuclear magnetic resonance. Group 1 (n = 21) consisted of survivors for > or = 1 year with normal serum creatinine (< 50 mumol/L), for group 2 (n = 17) of survivors with abnormal serum creatinine (> 50 mumol/L), and group 3 (n = 18) of those with bilateral histologic dysplasia associated with fetal (termination of pregnancy) or neonatal death. RESULTS A specific nuclear magnetic resonance profile was obtained for each of the three groups of renal function. A two-dimensional representation of alanine-valine and valine-threonine concentrations allows clear differentiation between the three degrees of renal function, with a sensitivity and specificity of 88% and 86%, respectively. CONCLUSIONS In fetal bilateral urinary tract obstruction a fetal urine nuclear magnetic resonance profile allows an accurate identification of renal function at birth.

Aminoglycoside and glycopeptide renal toxicity in intensive care patients studied by proton magnetic resonance spectroscopy of urine.

ObjectiveAminoglycoside and glycopeptide antibiotics are responsible for renal toxicity. In most cases, the nephrotoxicity is limited to a reversible tubular injury, but an acute and sustained renal failure may occur. The aim of our study was to explore the renal function of patients given these antimicrobial agents with proton magnetic resonance spectroscopy of urine. This technique is able to detect, in urine samples, a wide range of metabolites reflecting renal tubular function. The variables assessed by magnetic resonance spectroscopy were compared with the routine markers of renal function: creatinine, urea, and 24-hr urine volume. DesignProspective clinical study. SettingIntensive care unit. PatientsAll patients in an intensive care unit receiving an aminoglycoside and/or a glycopeptide were included in the study if they presented with signs of renal dysfunction. All experiments were performed on urine samples collected for the routine follow-up of these patients. InterventionProton spectra were acquired with water suppression, and the peak intensity of each metabolite was reported in relationship to the intensity of the creatinine peak. Measurements and Main ResultsThe ratio values obtained by magnetic resonance spectroscopy were compared with the values of creatininemia and blood urea obtained routinely by biochemistry and with the value of the 24-hr urine volume by logistic regression and general linear models. This statistical analysis showed that the ratio of dimethylamine to creatinine was highly correlated with creatininemia. ConclusionsDimethylamine is an osmolyte released from the medullar region of the kidney. Thus, our study demonstrated that nephrotoxicity from aminoglycosides and glycopeptides is not limited to proximal tubular toxicity but also may involve the medullar region (Henle loop and collecting duct) of the nephron.

Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion

The proton NMR spectra of K562 cells contain resonances of lipids. When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed. The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy. Therefore, the K562 drug‐sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug‐free medium, were used in this study. The P‐glycoprotein (P‐gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy. The phenotype reversion is due to the decrease of the P‐gp function and an increased entrance of anthracycline drug when compared with the resistant cells. The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content. A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids. Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance. These results are in favour of a different lipid organization or of localization within the cell. Copyright

pH regulation during ischaemia-reperfusion of isolated rat hearts, and metabolic effects of 2,3-butanedione monoxime

We investigated changes in pHi during ischaemia-reperfusion of isolated rat hearts using phosphorus nuclear magnetic resonance spectroscopy (31P NMR). Hearts were separated into three groups according to the perfusion buffer: bicarbonate-buffered Krebs solution, HEPES-buffered Krebs solution, or bicarbonate-buffered Krebs solution plus 10(-6) M 5-(N-ethyl-N-isopropyl) amiloride (EIPA). In HEPES buffer and in bicarbonate buffer plus EIPA, pH at the end of 30 min of ischaemia and pH oscillations observed during early reperfusion were lower than in bicarbonate buffer. Thus, the presence of two pH regulation mechanisms (Na(+)-H+ antiport and Na(+)-HCO3- symport) was confirmed in the isolated rat heart, while in HEPES buffer, pH was regulated by Na(+)-H+ antiport, and in bicarbonate buffer plus EIPA, by Na(+)-HCO3- symport. When cardiac contraction was inhibited by 10 mM 2, 3-butanedione 2-monoxime (BDM), we observed, in all cases, a less pronounced decrease in pHi at the end of ischaemia, and in pHi oscillations at the onset of reperfusion. These effects were similar to those observed with 150 x 10(-8) M verapamil and might thus be related to a decrease in intracellular calcium. However, with BDM, a greater reduction in the pH recovery rate was observed only in HEPES buffer, suggesting a possible phosphatase-like effect affecting the Na(+)-H+ exchange. Whatever the buffer used, the protective effect of BDM was reflected by an increase in the rate pressure product, which was not observed with verapamil.

Proton magnetic resonance spectroscopy of human amniotic fluids sampled at 17–18 weeks of pregnancy in cases of decreased digestive enzyme activities and detected cystic fibrosis

Low digestive enzyme activities in human amniotic fluid can be observed in normal and disease-affected pregnancies: cystic fibrosis, trisomy 21, intestinal atresia. Amniotic fluids were analyzed by proton nuclear magnetic resonance (NMR) spectroscopy in order to specify prenatally the etiology of low digestive enzyme activities observed at 17-18 weeks of amenorrhea. A total of 114 amniotic fluid samples were collected at 17-18 weeks of amenorrhea. Karyotyping and assays of digestive enzyme activities were performed in all cases. Samples were divided into six groups according to enzyme activities and pathology. Proton spectra were retrospectively recorded. Many compounds, such as amino acids and carboxylic acids, were detected by NMR. The same resonance intensities (normalized to creatinine) were observed in the six groups. Nevertheless, an unidentified resonance at 1.05 ppm was detected in seven out of 13 cases of cystic fibrosis affected fetuses. The NMR spectra demonstrated the stability of the amniotic fluid composition at 17-18 weeks of amenorrhea, even when the fetus was affected by a disease such as trisomy 21 or intestinal atresia. The resonance associated with most cases of cystic fibrosis should be further investigated.

Energetics of endurance exercise in young horses determined by nuclear magnetic resonance metabolomics.

Long-term endurance exercise severely affects metabolism in both human and animal athletes resulting in serious risk of metabolic disorders during or after competition. Young horses (up to 6 years old) can compete in races up to 90 km despite limited scientific knowledge of energetic metabolism responses to long distance exercise in these animals. The hypothesis of this study was that there would be a strong effect of endurance exercise on the metabolomic profiles of young horses and that the energetic metabolism response in young horses would be different from that of more experienced horses. Metabolomic profiling is a powerful method that combines Nuclear Magnetic Resonance (NMR) spectrometry with supervised Orthogonal Projection on Latent Structure (OPLS) statistical analysis. 1H-NMR spectra were obtained from plasma samples drawn from young horses (before and after competition). The spectra obtained before and after the race from the same horse (92 samples) were compared using OPLS. The statistical parameters showed the robustness of the model (R2Y = 0.947, Q2Y = 0.856 and cros-validated ANOVA p < 0.001). For confirmation of the predictive value of the model, a test set of 104 sample spectra were projected by the model, which provided perfect predictions as the area under the receiving-operator curve was 1. The metabolomic profile determined with the OPLS model showed that glycemia after the race was lower than glycemia before the race, despite the involvement of lipid and protein catabolism. An OPLS model was calculated to compare spectra obtained on plasma taken after the race from 6-year-old horses and from experienced horses (cross-validated ANOVA p < 0.001). The comparison of metabolomic profiles in young horses to those from experienced horses showed that experienced horses maintained their glycemia with higher levels of lactate and a decrease of plasma lipids after the race.

Metabolomics with Nuclear Magnetic Resonance Spectroscopy in a Drosophila melanogaster Model of Surviving Sepsis

Patients surviving sepsis demonstrate sustained inflammation, which has been associated with long-term complications. One of the main mechanisms behind sustained inflammation is a metabolic switch in parenchymal and immune cells, thus understanding metabolic alterations after sepsis may provide important insights to the pathophysiology of sepsis recovery. In this study, we explored metabolomics in a novel Drosophila melanogaster model of surviving sepsis using Nuclear Magnetic Resonance (NMR), to determine metabolite profiles. We used a model of percutaneous infection in Drosophila melanogaster to mimic sepsis. We had three experimental groups: sepsis survivors (infected with Staphylococcus aureus and treated with oral linezolid), sham (pricked with an aseptic needle), and unmanipulated (positive control). We performed metabolic measurements seven days after sepsis. We then implemented metabolites detected in NMR spectra into the MetExplore web server in order to identify the metabolic pathway alterations in sepsis surviving Drosophila. Our NMR metabolomic approach in a Drosophila model of recovery from sepsis clearly distinguished between all three groups and showed two different metabolomic signatures of inflammation. Sham flies had decreased levels of maltose, alanine, and glutamine, while their level of choline was increased. Sepsis survivors had a metabolic signature characterized by decreased glucose, maltose, tyrosine, beta-alanine, acetate, glutamine, and succinate.

Detection and follow-up, after partial liver resection, of the urinary paracetamol metabolites by proton NMR spectroscopy.

Background: Combination drug therapy is often used to achieve optimal analgesia in surgery. Paracetamol can be used as one component of an analgesic regime following hepatic resection. Objective: This study was designed to investigate paracetamol and its metabolites by proton NMR spectroscopy in patient urine and to assess whether N-acetyl-p-benzoquinone imine (NAPQI, a hepatotoxic metabolite) formation is increased after liver resection. Method: We studied the excretion of acetaminophen and its metabolites by 5 patients who were operated on for partial liver resection by proton NMR spectroscopy. As an intravenous infusion 1 g of paracetamol was given over 15 min every 6 h during 48 h. The first injection was given in the operating theatre after liver resection was completed. Urine samples were collected before injection (T1) and 24 and 48 h after the first injection (T2 and T3); the samples were frozen and kept at -20°C up to the analysis by NMR spectroscopy. Results: Metabolites of the paracetamol were detected for all patients. Among the discerned metabolites, 4 were identified as metabolites of paracetamol: paracetamol glucuronide, paracetamol sulfate, N-acetyl-L-cysteinyl paracetamol (metabolite of NAPQI) and paracetamol. Their ratios, respectively, were: 46-82.9, 12.6-30.0, 0.5-5.5 and 1.43-3.54%. Conclusion: This study showed that there was no increase in the formation of toxic metabolite (NAPQI) after treatment with paracetamol in these few cases of liver resections. A larger study is necessary to confirm these results.