Physiology, Pathophysiology and Clinical Relevance of D-Amino Acids Dynamics: From Neurochemistry to Pharmacotherapy.

Over three decades ago, two independent groups of investigators identified free D-aspartic and later D-serine in specific brain nuclei and endocrine glands. This finding revealed a novel, non-proteinogenic role of these molecules. Moreover, the finding that aged proteins from the human eye crystallin, teeth, bone, blood vessels or the brain incorporate D-aspartic acids to specific primary protein sequences fostered the hypothesis that aging might be related to D-amino acid isomerization of body proteins. The experimental confirmation that schizophrenia and neurodegenerative diseases modify plasma free D-amino acids or tissue levelsnurtured the opportunity of using D-amino acids as therapeutic agents for several disease treatments, a strategy that prompted the successful current application of D-amino acids to human medicine.

Age-Associated Upregulation of Glutamate Transporters and Glutamine Synthetase in Senescent Astrocytes In Vitro and in the Mouse and Human Hippocampus.

Aging is marked by complex and progressive physiological changes, including in the glutamatergic system, that lead to a decline of brain function. Increased content of senescent cells in the brain, such as glial cells, has been reported to impact cognition both in animal models and human tissue during normal aging and in the context of neurodegenerative disease. Changes in the glutamatergic synaptic activity rely on the glutamate-glutamine cycle, in which astrocytes handle glutamate taken up from synapses and provide glutamine for neurons, thus maintaining excitatory neurotransmission. However, the mechanisms of glutamate homeostasis in brain aging are still poorly understood. Herein, we showed that mouse senescent astrocytes in vitro undergo upregulation of GLT-1, GLAST, and glutamine synthetase (GS), along with the increased enzymatic activity of GS and [3H]-D-aspartate uptake. Furthermore, we observed higher levels of GS and increased [3H]-D-aspartate uptake in the hippocampus of aged mice, although the activity of GS was similar between young and old mice. Analysis of a previously available RNAseq dataset of mice at different ages revealed upregulation of GLAST and GS mRNA levels in hippocampal astrocytes during aging. Corroborating these rodent data, we showed an increased number of GS + cells, and GS and GLT-1 levels/intensity in the hippocampus of elderly humans. Our data suggest that aged astrocytes undergo molecular and functional changes that control glutamate-glutamine homeostasis upon brain aging.

SRL pathogenicity island contributes to the metabolism of D-aspartate via an aspartate racemase in Shigella flexneri YSH6000.

In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.

Contenido de glutamato, aspartato y glicinato en diversos preparados de la comida peruana / Content of glutamate, aspartate and glycinate in different preparations of Peruvian food

Objetivo: Evaluar la concentración de glutamato, aspartato y glicinato en alimentos elaborados en Lima, Perú. Material y Métodos: Se analizó el contenido de glutamato, aspartato y glicinato, aminoácidos no esenciales presentes en diez comidas peruanas: lomo saltado, arroz con pollo, arroz chaufa, rocoto relleno, seco de carne, ceviche de caballa, escabeche de pollo, tallar¡n saltado de carne, causa de conserva de pescado, ají de pollo. Se realizó el análisis del contenido de aminoácidos en cada una de los preparados por cromatografía líquida HPLC según Quattrocchi y Laba por el método de derivatización pre-columna. Resultados: Se encontró mayor contenido de glutamato: 2463 mg/100g en tallarín saltado de carne. El aspartato y glicinato fueron más abundantes en ceviche de caballa con 1707,95 mg/100g y 893,73 mg/100g respectivamente. Conclusiones: En las comidas analizadas se halló mayor contenido de glutámico lo cual se relaciona al buen sabor y aceptabilidad de las comidas elaboradas en Lima, PerúAU)

Objective: to evaluate the concentration of glutamate, aspartite and glycinate in foods made in Lima, Peru. Material and Methods: The present study analyzed the content of non-essential amino acids glutamate, aspartite and glycinate, present in ten Peruvian foods. Those selected were “lomo saltado, arroz con pollo, arroz chaufa, rocoto relleno, seco de carne, ceviche de caballa, escabeche de pollo, tallar¡n saltado de carne, causa de conserva de pescado, aj¡ de pollo”. The analysis of the content of amino acids on the ten Peruvian Creole food was done by liquid chromatography HPLC according to Quattrocchi and Laba by pre-columna derivatization method. Results: Highest content of glutamate was found in “tallarin saltado de carne”: 2463 mg / 100g. Aspartite and glycinate were more abundant in ceviche with 1707,95 mg / 100g and 893,73 mg / 100g respectively. Conclusions: in the analyzed meals we found high content of glutamate which relates to the good taste and acceptability of meals prepared in Lima, Peru

Prospective analysis of 44 consecutive liver transplants performed at a university hospital

PURPOSE: To analyze the intraoperative and immediate postoperative biochemical parameters of patients submitted to orthotopic liver transplantation. METHODS: Forty four consecutive orthotopic liver transplants performed from October 2009 to December 2010 were analyzed. The patients (38 male and eight female) were divided into two groups: group A, survivors, and group B, non-survivors. Fifty percent of group A patients were Chid-Pugh C, 40% Chid-Pugh B and 10% Chid-Pugh A. In group B, 52% of the patients were Chid-Pugh C, 41% Chid-Pugh B, and 17% Chid-Pugh A. All orthotopic liver transplants were performed by the piggy-back technique without a portacaval shunt in an anhepatic phase. ALT, AST, LDH and lactate levels were determined preoperatively, at five, 60 minutes after arterial revascularization of the graft and 24 and 48 hours after the end of the surgery.( or: after the surgery was finished). RESULTS: There were no preoperative clinical differences (Child and Meld) between the two groups. The times of warm and hypothermal ischemia were similar for both groups (p>0.05). Serum aminotransferases levels at five and 60 minutes after arterial revascularization of the graft were similar (p>0.05) for both groups, as also were lactate levels at the time points studied. There was no significant difference in Δ lactate between groups at any time point studied (p>0.05). No significant difference was observed between groups during the first 24 and 48 hours after surgery (p>0.05). CONCLUSION: No significant difference in any of the parameters studied was observed between groups. Under the conditions of the present study and considering the parameters evaluated, no direct relationship was detected between the intraoperative situation and the type of evolution of the patients of the two groups studied.(AU)

Exchange of extracellular L-glutamate by intracellular D-aspartate: the main mechanism of D-aspartate release in the avian retina.

D-aspartate is present in significant concentrations throughout the nervous tissue but its physiological role is still under discussion. Here, we report the process of d-aspartate release in retinal cells. [(3)H]-d-aspartate release occurs through a glutamate/aspartate exchange mechanism using excitatory amino acid transporters. This process is sodium-dependent and it is not prevented by glutamate receptor antagonists such as MK-801, DNQX or AIDA nor mimicked by glutamatergic agonists like kainate, NMDA or trans-ACPD. In vitro experiments indicate that the great majority of d-aspartate release is performed by neuronal cells and to a much lower extent by glial cells. This glutamate-mediated release process is mimicked by the competitive glutamate transporter antagonist l-trans-PDC and inhibited by the non-competitive transporter antagonist TBOA. Instead of the classical calcium-dependent exocytosis or transporter-reversal mediated neuronal release, d-aspartate efflux in the retina occurs mostly, if not exclusively, via an exchange of external l-glutamate by d-aspartate predominantly present in the cytoplasmatic compartment of neurons. These data also suggest that this process narrows down the specificity of excitatory signaling in the microenvironment of the synapses, reinforcing NMDA receptor activation by d-aspartate at the cost of reduction in the overall activation of excitatory amino acid receptors promoted by l-glutamate.

Effect of the L- or D-aspartate on ecto-5'nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A(2A) receptors.

Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5'-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5'-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5'-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A(2A) receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A(1) receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5'-nucleotidase and on cellular damage. The adenosine formed from ecto-5'-nucleotidase stimulation preferentially acted on adenosine A(2A) receptor which is probably co-operating with the neurotoxicity induced by amino acids.

Niveles extracelulares de glutamato y aspartato en el fluido gingival en la periodontitis / Extracellular glutamate and aspartate levels in the gingival crevicular fluid of periodontitis patients

El objetivo de este trabajo fue comparar los niveles extracelulares de glutamato y aspartato en el fluido del surco gingival (GCF) de personas adultas, en la periodontitis crónica localizada inducida por placa (PCIP) y la gingivitis inducida por placa (GIP). La enfermedad periodontal produce cambios inflamatorios en los tejidos de sostén de las piezas dentales afectadas. El análisis químico del GCF, con diferentes métodos de colección y análisis, ha sido usado para determinar la presencia de algunos elementos inflamatorios que aparecen en la enfermedad periodontal, tales como diversas enzimas, aminoácidos, etc.Las muestras del GCF se tomaron con la técnica de microdiálisis en las zonas dentales con PCIP con una profundidad del surco > 3 mm; pérdida de soporte > 2mm y en las zonas dentales con GIP en el mismo paciente (n=10) Total de muestras: 100. Para medir el glutamato y aspartato en el GCF se usó la técnica de electroforesis capilar acoplada a laser con detección inducida por fluorescencia (CZE-LIFD). Los resultados mostraron que en los dientes con PCIP el glutamato disminuyó (p<0.05) y el aspartato aumentó (p< 0.02) en comparación con los dientes con GIP.

The objective of this work was to compare glutamate and aspartate levels in periodontal chronic localized disease (PCIP) and dental zones with gingivitis (GIP) in the gingival crevicular fluid (GCF). Periodontal inflammation produces histological changes, increase of blood irrigation and also increase of subgingival fluid. GCF was recognized as an inflammatory exudes derived from the periodontal tissue. Different methods to collect and analyze GCF samples had been used to identify some substances in the GCF, such as the proteinglycans metabolite, to be a possible marker of active periodontal disease. A combination of microdialysis in situ in dental zones with PCIP (probing depth > 3 mm; attachment level > 2 mm) and dental zones with GIP (n=10), total samples: 100, and capillary zone electrophoresis coupled to a laser induced fluorescence detection (CZE-LIFD) was used to measure extracellular concentrations of amino acids: glutamate and aspartate in the GCF in adult patients The results showed that glutamate decrease (p<0.05) and aspartate increase (p<0.02) in PCIP disease zones compared with dental zones with GIP. We observed chemical in vivo evidence that differentiate the GIP zones and PCIP zones.

Aspectos clave del ciclo de la úrea con relacion al metabolismo energético y proteico en vacas lactantes / Key aspects of the urea cicle, related to energetic and proteic metabolism in lactating dairy cows

El uso de altas cantidades de fertilizantes nitrogenados en las lecherías especializadas, ha conducido a cambios importantes en las características nutricionales de los forrajes, incrementando el contenido de nitrogeno total (proteina cruda) y su fracción soluble (fracción) a expensas de la proteína verdadera. Este hecho ha generado un aumento exagerado del contenido de nitrogeno fermentable que aparece como amonio el cual no alcanza a ser utilizado por la flora ruminal y pasacon relativa facilidad al torrente circulatorio; posteriormente debe ser transformado en el higado a area y eliminado en la orina o en la leche. Las vacas lactantes utilizan sus reservas proteicas (ademas del propionato ruminal), a favor de la sintesis de glucosa. Los aminoacidos producto del catabolismo proteico, sufren un proceso de transaminación para confluir, la mayoria de ellos, en el glutamato a partir de Á-cetoglutarato como cetoacido receptor. En el interior de la mitocondria, en presencia dela glutamato deshidrogenasa es liberado el amonio y este, en presencia de la carbamoil C fosfato sintetasa, bicarbonato y dos moles de ATP, forma el carbamoil fosfato (CaP), el que más adelante liberarón área. El amonio libre procedente de la absorción ruminal puede formar también Carbamoil Fosfato (CbP), sin embargo, esta reacción tiene baja afinidad a diferencia de la formación del glutamato a partir del mismo amonio y Á- cetoglutarato. La unión del glutamato con otra molécula de amonio produce glutamina la cual es un vehículo muy empleado por el organismo para deshacerse del amonio a nivel renal. La retención del amonio como glutamato constituye un gasto del Á- cetoglutarato, el cual es un importante precursor de la glucosa con lo que la gluconeogenesis a partir de este metabolito se puede ver disminuida. Paralelamente, el aumento en la actividad ureogênica conlleva al incremento en las necesidades de ciertos aminoácidos, como la metionina, para la...

The effect of D-aspartate on luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, GABA and dopamine release.

Since D-aspartate stimulates prolactin and LH release, our objective was to determine whether D-aspartate modifies the release of hypothalamic and posterior pituitary factors involved in the control of their secretion and whether its effects on these tissues are exerted through NMDA receptors and mediated by nitric oxide. In the hypothalamus, D-aspartate stimulated luteinizing hormone-releasing hormone (LHRH), alpha-melanocyte-stimulating hormone (alpha-MSH) and GABA release and inhibited dopamine release through interaction with NMDA receptors. It increased nitric oxide synthase (NOS) activity, and its effects on LHRH and hypothalamic GABA release were blunted when NOS was inhibited. In the posterior pituitary gland, D-aspartate inhibited GABA release but had no effect on dopamine or alpha-MSH release. We report that D-aspartate differentially affects the release of hypothalamic and posterior pituitary factors involved in the regulation of pituitary hormone secretion.

Insulin-like growth factor-1 increases activity and surface levels of the GLAST subtype of glutamate transporter.

Glutamate uptake systems are the primary mechanisms involved in excitatory amino acids clearance, their regulation is extremely important for proper neuronal function. Using cultured chick cerebellar Bergmann glia cells, the involvement of receptor tyrosine kinases in glutamate uptake was studied. Treatment of the cells with insulin-like growth factor-1 but not epidermal growth factor or neuronal growth factor, induces a dose and time dependent increase in [(3)H]-D-aspartate uptake that is sensitive to wortmannin, an inhibitor of phosphatidylinositol 3-kinase. Saturation experiments show a significant increase in V(max), suggesting that the amount of transporter molecules at the cell membrane under insulin-like growth factor-1 treatment is augmented. This interpretation was strengthen by equilibrium-binding experiments and by the fact that the increase in [(3)H]-D-aspartate uptake was not dependent on protein synthesis. The present studies suggest that insulin-like growth factor-1 signaling is involved in modulation of glutamate transporter cell surface expression.