L-carnitine protects DNA oxidative damage induced by phenylalanine and its keto acid derivatives in neural cells: a possible pathomechanism and adjuvant therapy for brain injury in phenylketonuria.

Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.

Pharmacokinetics of low molecular weight phenolic compounds in gerbil plasma after the consumption of calafate berry (Berberis microphylla) extract.

Calafate is a berry with high concentration of anthocyanins and hydroxycinnamic acids that grows in South Patagonia. To date, no metabolism studies of phenolic compounds using calafate have been carried out. A calafate extract was characterized by HPLC-DAD-ESI-MS/MS. After extract administration (300 mg/kg), a pharmacokinetic study of phenolic compounds in gerbil plasma was performed by GC-MS/MS. Sixteen phenolic acids increased after intake. Phenylacetic acid derivatives exhibit the highest concentration, while main increase of phenolic catabolites was observed 2 h post-intake. 3-hydroxyphenylacetic and phenylacetic acids increased at 4-8 h post-intake. All catabolites found in gerbil plasma exhibit concentration peaks between 0.1 and 1 µM, however no parental anthocyanins were detected. Establish in vivo plasmatic concentration ranges of phenolic compounds derived from polyphenol consumption following WHO recommendations, plays a key role to carry out future in vitro assays in order to correctly assign biological benefits of calafate berry consumption.

Conformational equilibrium of phenylacetic acid and its halogenated analogues through theoretical studies, NMR and IR spectroscopy.

This paper presents a study on the conformational preferences of phenylacetic acid (PA) and its halogenated analogues (FPA, CPA, BPA). To clarify the effects that rule these molecules' behaviour, theoretical calculations were used, for both the isolated phase and solution, combined with nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy. Most conformations of phenylacetic acid and its halogenated derivatives are stabilized through the hyperconjugative effect, which rules the conformational preference. NMR analyses showed that even with the variation in medium polarity, there was no significant change in the conformation population. Infrared spectroscopy showed similar results for all compounds under study. In most spectra, two bands were found through the carbonyl deconvolution, which is in accordance with the theoretical data. It was possible to prove that variation in the nature of the substituent in the ortho position had no significant influence on the conformational equilibrium.

Determination of the binding properties of the uremic toxin phenylacetic acid to human serum albumin.

Uremic toxins are compounds normally excreted in urine that accumulate in patients with chronic kidney disease as a result of decreased renal clearance. Phenylacetic acid (PAA) has been identified as a new protein bound uremic toxin. The purpose of this study was to investigate in vitro the interaction between PAA and human serum albumin (HSA) at physiological and pathological concentrations. We used ultrafiltration to show that there is a single high-affinity binding site for PAA on HSA, with a binding constant on the order of 3.4 × 10(4) M(-1) and a maximal stoichiometry of 1.61 mol per mole. The PAA, at the concentration reported in end-stage renal patients, was 26% bound to albumin. Fluorescent probe competition experiments demonstrated that PAA did not bind to Sudlow's site I (in subdomain IIA) and only weakly bind to Sudlow's site II (in subdomain IIIA). The PAA showed no competition with other protein-bound uremic toxins such as p-cresyl-sulfate or indoxyl sulfate for binding to serum albumin. Our results provide evidence that human serum albumin can act as carrier protein for phenylacetic acid.

Identification, isolation and optimization of antifungal metabolites from the Streptomyces Malachitofuscus ctf9

An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.

Identification, isolation and optimization of antifungal metabolites from the Streptomyces Malachitofuscus ctf9.

An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.

Identification, isolation and optimization of antifungal metabolites from the Streptomyces Malachitofuscus ctf9

An indigenous Streptomyces isolate CTF9, exhibiting promising antifungal activity against Mucor miehei and Candida albicans in pre-screening studies, was investigated by cultivation in a 50-L fermenter and by subsequent isolation, purification, and structure elucidation of the active metabolites. Based on the morphological, biochemical, and physiological characterization, as well as the 16S rRNA gene sequence, the isolate CTF9 was identified as Streptomyces malachitofuscus. Using a series of chromatographic techniques, two pure compounds were isolated from the obtained extracts after the fermentation of the isolate CTF9. The isolated compounds were identified as phenylacetic acid and indolyl-3-lactic acid by mass spectrometry (MS) and NMR analysis. The culture optimization studies revealed that the isolate CTF9 can use a variety of low-cost carbon and nitrogen sources to generate the maximum quantity of industrially important metabolites at an elevated temperature of 35°C and at a pH 7.8.

Bio-synthesis and hydrolysis of ethyl phenylacetate and ethyl 2-phenylpropionate in organic solvent by lyophilized mycelia

To select the best biocatalysts for ethanol acylations with phenylacetic and 2-phenylpropionic acids, lyophilized mycelia of Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 and R. oryzae CBS 32847 were tested in this study. The carboxylesterase activities of A. oryzae MIM and R. oryzae 11207, which revealed to be the best biocatalysts, were investigated either in 0.1 M phosphate buffer or in n-heptane to catalyze the hydrolysis or the synthesis of ethyl esters of these acids, respectively. A. oryzae proved more effective than R. oryzae, probably due to more favorable microenvironment conditions and thermodynamic scenario. The results in terms of product formation and substrate consumption versus time were used to estimate the maximum conversion yields, the equilibrium constants and the times needed to reach half maximum conversion, thus providing sufficient information about these equilibria.

Micélios liofilizados de Aspergillus oryzae CBS 10207, A. oryzae MIM, Rhizopus oryzae CBS 11207, R. oryzae CBS 39134, R. oryzae CBS 26028 e R. oryzae CBS 32847 foram testados neste estudo com vista à seleção do melhor biocatalisador para efetuar a acilação de etanol com ácidos fenilacético e 2-fenilpropiônico. As atividades carboxilesterásicas de A. oryzae MIM e R. oryzae 11207, que resultaram ser os melhores biocatalisadores, foram investigadas tanto em tampão fosfato 0,1 M como em n-heptano para catalisar a hidrólise ou a síntese dos ésteres etílicos destes ácidos. A. oryzae pareceu ser mais eficaz que R. oryzae, provavelmente devido a condições micro-ambientais e a um cenário termodinâmico mais favoráveis. Os resultados obtidos em termos de formação do produto e consumo dos substratos em função do tempo foram usados para a estimativa dos rendimentos de conversão máximos, as constantes de equilíbrio e os tempos necessários para alcançar metade da conversão máxima, fornecendo desta forma suficientes informações sobre esses equilíbrios.