F. Seguin

Early Energy Deficit in Huntington Disease: Identification of a Plasma Biomarker Traceable during Disease Progression

Huntington disease (HD) is a fatal neurodegenerative disorder, with no effective treatment. The pathogenic mechanisms underlying HD have not been elucidated, but weight loss, associated with chorea and cognitive decline, is a characteristic feature of the disease that is accessible to investigation. We, therefore, performed a multiparametric study exploring body weight and the mechanisms of its loss in 32 presymptomatic carriers and HD patients in the early stages of the disease, compared to 21 controls. We combined this study with a multivariate statistical analysis of plasma components quantified by proton nuclear magnetic resonance (1H NMR) spectroscopy. We report evidence of an early hypermetabolic state in HD. Weight loss was observed in the HD group even in presymptomatic carriers, although their caloric intake was higher than that of controls. Inflammatory processes and primary hormonal dysfunction were excluded. 1H NMR spectroscopy on plasma did, however, distinguish HD patients at different stages of the disease and presymptomatic carriers from controls. This distinction was attributable to low levels of the branched chain amino acids (BCAA), valine, leucine and isoleucine. BCAA levels were correlated with weight loss and, importantly, with disease progression and abnormal triplet repeat expansion size in the HD1 gene. Levels of IGF1, which is regulated by BCAA, were also significantly lower in the HD group. Therefore, early weight loss in HD is associated with a systemic metabolic defect, and BCAA levels may be used as a biomarker, indicative of disease onset and early progression. The decreased plasma levels of BCAA may correspond to a critical need for Krebs cycle energy substrates in the brain that increased metabolism in the periphery is trying to provide.

Radiofrequency map of an NMR coil by imaging

We propose a new imaging method to obtain a map of the radiofrequency (RF) field amplitude over a sample. The sequence contains three RF pulses (alpha, 2 alpha, and alpha) and produces two images by a classical spin echo and a stimulated echo. A third image is computed and gives the distribution of the flip angle alpha, and so the RF amplitude, over the sample. The accuracy of the flip angle determination is verified on an homogeneous sample and results show a good correlation between experimental and theoretical flip angles in the range of 50 degrees to 130 degrees. Experiments with a surface coil and a resonator show the method is available in an inhomogeneous RF field. Images obtained on the calf of a volunteer confirms the independence of the computed RF distribution from proton density, T1, or T2 contrast.

Nucleotide content, oxydative phosphorylation, morphology, and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight‐line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/108 cells and from 0.6 to 6.0 nmole/108 cells, respectively. Moreover, 31P‐NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/109 spermatozoa/min. FCCP, an uncoupler of oxydative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3–, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3– altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxydative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo‐osmotic medium, mitochondrial oxydative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece. Mol. Reprod. Dev. 53:230–243, 1999.

1H-NMR and 31P-NMR analysis of energy metabolism of quiescent and motile turbot (Psetta maxima) spermatozoa.

31P-NMR and (1)H-NMR were used to monitor changes of several compounds with high-energy bonds and metabolites prior to and after the initiation of motility of turbot spermatozoa (Psetta maxima). The obtained (31)P-NMR spectra revealed the presence of phosphomonoesters, phosphodiester, intracellular inorganic phosphate (Pi), phosphocreatine (PCr), and free nucleotide triphosphate. Following the activation of motility, the di- and tri-phosphate nucleotides, PCr, phosphomonoesters levels dropped while Pi levels increased. A significant increase of lactate was also seen at the end of the swimming phase. The compositions of seminal fluid and urine were also determined. Lipoproteins, formic acid, amino acid, and citric acid were detected in seminal fluid. Dimethyl amine, trimethylamine, and trimethylamine oxyde were found in urine. These data suggest that at least a part of the energy required during the swimming phase results from anaerobic fermentation and oxidative phosphorylation. J. Exp. Zool. 286:513-522, 2000.

A pair analysis of the delayed graft function in kidney recipient: the critical role of the donor.

PURPOSE The aim of this study was to analyze the importance of donor factors and especially the potential role of hemodynamic management in regard to delayed graft function in paired kidney recipient patients after renal transplantation and to analyze the urine of organ donors by proton-nuclear magnetic resonance spectroscopy to identify urine markers potentially correlated with delayed graft function in recipient patients. METHODS A prospective multicenter epidemiologic study was conducted. A logistic regression model taking into account paired data was used. RESULTS Data from 72 donors and the 144 corresponding paired recipients were analyzed. Univariate analysis showed that age of donor, previous history of tobacco, ischemic cause of brain death, norepinephrine infusion, and recipient age were the risk factors for delayed graft function. After adjusting for correlated outcome data and controlling for other potential prognostic factors, 3 variables remained significantly associated with outcome: donor age (odds ratio [OR], 10.7), hemodynamic status (OR, 0.167), and hydroxyl-ethyl starch infusion (OR, 0.135). Proton-nuclear magnetic resonance analysis evidenced 3 metabolites of interest in donors (trimethylamine-N-oxide, citrate, and lactate). However, these peaks were not correlated the clinical parameters in donors. CONCLUSIONS Paired analysis of kidney transplantation emphasizes the important role of factor donor associated with delayed graft function in recipient. Thus, a particular attention should be paid to the hemodynamic management of donor.

Elevated CSF N-acetylaspartylglutamate in patients with free sialic acid storage diseases

Objective: To investigate body fluids of patients with undiagnosed leukodystrophies using in vitro 1H-NMR spectroscopy (H-NMRS). Methods: We conducted a cross-sectional study using high-resolution in vitro H-NMRS on CSF and urine samples. Results: We found a significant increase of free sialic acid in CSF or urine in 6 of 41 patients presenting with hypomyelination of unknown etiology. Molecular genetic testing revealed pathogenic mutations in the SLC17A5 gene in all 6 patients. H-NMRS revealed an increase of N-acetylaspartylglutamate in the CSF of all patients with SLC17A5 mutation (range 13–114 μmol/L, reference <12 μmol/L). Conclusion: In patients with undiagnosed leukodystrophies, increased free sialic acid in CSF or urine is a marker for free sialic acid storage disorder and facilitates the identification of the underlying genetic defect. Because increase of N-acetylaspartylglutamate in CSF has been observed in other hypomyelinating disorders, it can be viewed as a marker of a subgroup of hypomyelinating disorders.

Free sialic acid storage disease without sialuria.

We performed high‐resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria. Ann Neurol 2009;65:753–757

Acquisition of spin echo and stimulated echo by a single sequence: Application to MRI of diffusion

A new method is described to measure the restricted diffusion coefficient with magnetic resonance imaging. The two images necessary to calculate the diffusion image are obtained with a simultaneous acquisition of a spin-echo and a stimulated echo, and so, in half the time needed by usual spin-echo or stimulated echo method. A different diffusion contrast is created on each echo. A map of an estimate of the diffusion coefficient and an estimation of T1 value are obtained with only one experiment. The accuracy of the method has been evaluated on phantom and results are in agreement with values found in previous papers and with measurements performed with a usual spin-echo method. Furthermore, in vivo measurements have shown that this method can be used without electrocardiogram triggering.

31P NMR study of intracellular pH during the respiratory burst of macrophages

The metabolic events occurring during the respiratory burst of macrophages previously primed in vivo with lipopolysaccharide were studied by 31P nuclear magnetic resonance, using the P388D1 cell line as a model of the mature macrophages. Using perchloric acid extracts, the presence of phosphocreatine was shown in the primed cells, indicating that in control P388 D1 macrophages, in which no phosphocreatine was seen, in vivo maturation was incomplete. The cells primed in vivo exhibited greater maturation than the control cells, as well as greater creatine kinase activity. Perfusion of gel-embedded macrophages allowed the monitoring of phosphorylated metabolite peak intensities and of the intracellular pH. After the respiratory burst of the primed macrophages had been triggered by concanavalin A, these intensities did not alter significantly, but the intracellular pH decreased. 31P NMR spectra reflected transient acidification in the primed cells, possibly due to the formation of endocytic vesicles and their fusion with lysosomes.

Apports de la spectroscopie par résonance magnétique nucléaire des fluides dans l’étude de maladies métaboliques et neurodégénératives

Resume La spectroscopie par resonance magnetique nucleaire — ou SRM — in vitro est une methode d’analyse biochimique permettant l’analyse metabolique des fluides biologiques tels que le liquide cephalorachidien ou les urines. Les principaux avantages de la SRM in vitro sont la preparation minimale des echantillons avant leur analyse, la possibilite d’analyser simultanement des composes de nature differente, et les informations structurales apportees sur les metabolites presents dans l’echantillon. Au cours des dix dernieres annees, la SRM in vitro a ainsi permis l’identification de maladies metaboliques nouvelles, dont certaines sont accessibles a une therapie. La recherche d’anomalies metaboliques, par SRM in vitro , chez des patients presentant une maladie neurologique inexpliquee represente donc une nouvelle approche physiopathologique, en particulier en association avec des analyses genetiques de type analyse de liaison ou recherche de gene candidat. Grâce a l’utilisation d’outils statistiques multiparametriques, permettant de comparer l’ensemble du profil metabolique entre differents groupes, la SRM in vitro est egalement une methode de choix face au defi que represente la recherche de biomarqueurs dans des maladies neurologiques connues.