Domingo Blanco

Melatonin uptake in prostate cancer cells: intracellular transport versus simple passive diffusion

Abstract:  Melatonin, an indole mainly synthesized in the pineal gland during the dark phase, plays a role as an endogenous antioxidant and an anticancer agent in many tumors. Melatonin, at pharmacological concentrations, inhibits cell growth and induces neuroendocrine differentiation in prostate cancer cells. Classically it has been considered that melatonin enters freely into most of cells by passive diffusion through the cell membrane; however, there are few studies examining how melatonin is taken up by cancer cells. The aim of the present paper was to study the uptake of melatonin into human androgen‐dependent LNCaP and androgen‐independent PC‐3 prostate cancer cells. Increased concentrations of melatonin induced a rapid and transitory rise in intracellular melatonin content in both cell types, with a peak of maximal content at 6 hr after melatonin addition, following a rhythmic uptake; melatonin was found in both cytoplasm and nuclear fractions. Inhibition of protein or RNA synthesis partially blocked melatonin uptake in both cell lines. Interestingly, melatonin pulse incubation led to a higher uptake after four cycles of pulse incubation. Neither extracellular Ca2+/K+ alterations nor the presence of bovine serum albumin or charcoal‐stripped serum modified the profile of melatonin uptake. On the contrary, chemical binding of melatonin to BSA totally prevented melatonin from entering into cells. The present data support the hypothesis that a facilitated diffusion or an active process rather than simple passive diffusion through the cell membrane is the major mechanism in melatonin uptake by prostate cancer cells and it accounts for its intracellular concentration (350 nm–3.3 μm), which is related to its anti‐tumor actions.

Micellar electrokinetic capillary chromatography analysis of diuretics in pharmaceutical formulations

This paper describes the development of an electrophoretic method used to analyse diuretics, and its use in the analysis of pharmaceutical formulations. Chlorthalidone, furosemide, hydrochlorothiazide and triamterene were separated in 5 min by micellar electrokinetic capillary chromatography using a carrier containing sodium dodecylsulphate as surfactant, and were subsequently detected spectrophotometrically using a diode-array detector. The limits of detection (S/N=3) were in all cases less than 1.2 mugml(-1) for all compounds. Satisfactory repeatability was obtained for migration times (<1.6%) and corrected peak areas (<5.3%) in the analysis of pharmaceutical formulations. The reproducibility was less than 4% for all samples tested. The average recoveries obtained, which ranged between 97 and 100%, testify to the accuracy of the proposed method.

Solid-phase extraction and determination of trace aroma and flavour components in cider by GC-MS

SummaryMinor volatile compounds are responsible for the aromas of cider. A simple technique for the analysis of these components is described based on solid-liquid phase extraction followed by quantitation by gas chromatography-mass spectrometry (GC-MS). The method is quantitative for analysis of alcohols, esters, lactones, phenols, and medium and long chain-length fatty acid.

Determination of Organic Acids in Apple Juice by Capillary Liquid Chromatography

Abstract The organic acids quinic, malic, shikimic and citric were separated on a packed reverse phase fused-silica capillary column using 0.01 M K2HPO4/H3PO4 at pH 2.7 as eluent at a flow rate of 2 μL/min, and determined with UV detection. Conventional liquid chromatographicequipment was adapted for such purposes. The organic acids were able to be separated with detection limits of 2.4, 2.1, 0.04 and 2.9 ng, respectively. Application of the proposed method to the quantification of organic acids in apple juice is reported.

Determination of Water Soluble Vitamins By Liquid Chromatography with Ordinary and Narrow-Bore Columns

Abstract A reversed phase high performance liquid chromatographic method is described for the simultaneous determination of nicotinic acid, nicotinamide, pyridoxine, thiamine, folic acid and riboflavin. The vitamins were separated on a C8 bonded phase column and eluted with a dihidrogen phosfate buffer, hexanesulphonate and methanol as the eluent. A rapid spectral detector Diode Array was used to optimise the detection of each vitamin. All vitamins were separated in less than 12 minutes. This method was applied to the determination all the vitamins in multivitamin Pharmaceuticals. Recovery studies showed good results for all solutes (93.4%–106.5%) and some day coefficients of variation ranging from 0.91% to 4.95%. Narrow bore columns were recommended because this alternative provided a good separation efficiency, plus greater economy and sensibility. Finally, the performance of an ordinary UV detector and that of a rapid spectral detector in this type of determination were critically compared.

Biochemical study of the ripening of cider apple varieties

ZusammenfassungDie Änderung der Zusammensetzung der wichtigsten Zucker und organischen Säuren in fünf asturianischen Apfelsorten wurden während der Endphase des Reifens durch Hochleistungsflüssigchromatographie untersucht. Ebenfalls wurden im gleichen Zeitraum die Veränderungen im Stickstoff-, Polyphenol-, Zucker- und Stärkegehalt dieser Sorten bestimmt. Unterschiede ergaben sich bei den wichtigsten Zuckerarten (Fructose, Saccharose und Glucose) und Sorbitol der fünf Apfelsorten. Die größte Fructosemenge wurde in der Endphase des Reifens angereichert. Mit fortschreitendem Reifen nahm der Gesamtzuckergehalt zu, wohingegen der Gehalt an Apfelsäure, Stärke, Stickstoff und Polyphenolen abnahm.SummaryChanges in the major sugars and organic acids in five Asturian apple varieties during the final stage of ripening were studied using HPLC. The changes in total nitrogen, polyphenols, sugars and starch in these varieties were also studied over the same period. Differences were noted among the major sugars (fructose, sucrose und glucose) and sorbitol for the five varieties studied. Fructose accumulated in a greater quantity in fruit during the final stage of ripening. The overall sugar content increased, while the malic acid and starch contents decreased in the final stage, which was preceded by a minimum in the nitrogen and polyphenol contents.

Development and validation of new methods for the determination of melatonin and its oxidative metabolites by high performance liquid chromatography and capillary electrophoresis, using multivariate optimization.

Melatonin (N-acetyl-5-metoxytriptamine, MEL) has focused a lot of attention as consequence of its multiple functions. MEL is a potent endogenous antioxidant and a free radical scavenger that reacts with several sort of radicals generating various metabolites. Two of them are N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK) and N1-acetyl-5-methoxykynurenine (AMK). These compounds are important because they have also antioxidant actions as well as other important biological properties. In the present work, we develop two methods to detect and quantify these compounds (MEL, AFMK and AMK) in the same sample. For this purpose we used an experimental design, and utilized high performance liquid chromatography (HPLC-DAD) and micellar electrokinetic chromatography (MEKC) techniques with diode array detector in both of them. The limit of detection/quantification for MEL, AFMK and AMK were respectively 44/94, 18/38 and 23/51 ng mL(-1) by using HPLC and 13/44, 37/124 and 47/156 ng mL(-1) by using MEKC. This is the first time that these compounds have been separated in the same chromatogram or electroferogram. The time of analysis was faster using MEKC. Furthermore, this technique showed better resolution but HPLC offered better limit of detection and quantification for metabolites. Both methods were validated and correlation coefficients were higher than 0.999 and the range of recovery of those methods were 99.6-103.7%. Precision was evaluated as repeatability and intermediate precision with relative standard derivation <5%. When a 5 microg mL(-1) solution of these compounds were analyzed with both methods we do not observed any statistically significance differences. Moreover, we analyzed 3COHM (cyclic-3-hydroximelatonin), another known metabolite of melatonin, by using the same methods. The employment of these methods will offer a useful tool to contribute to answer the role of MEL, AFMK and AMK in biological system and both methods can be used in routine analysis for these compounds.

Study of various treatments to isolate low levels of cider proteins to be analyzed by capillary sieving electrophoresis

Abstract Four different sample treatments (ethanol precipitation, dialysis, ultrafiltration, and gel filtration) to isolate cider proteins were tested in this work. Purified cider proteins were analyzed by capillary sieving electrophoresis (CSE) using linear polyacrylamide as a sieving medium under optimized conditions; with this technique, separation, and molecular weight determination of proteins are possible. The electropherograms obtained in the protein analysis with each treatment were compared to choose the one that led to the best results. This was found to be ultrafiltration; many peaks were obtained in the electrophoretic profile and their spectra corresponded to a protein. Bradford analysis confirmed this choice. These results were compared with those obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE), the molecular weights of the protein bands agreeing with the molecular weights of the electrophoretic peaks obtained with ultrafiltration.

Determination of Fat-Soluble Vitamins by Liquid Chromatography in Pediatric Parenteral Nutritions

Abstract A reversed phase high performance liquid chromatographic method using narrow-bore columns packed with 3 μm particles is described for the simultaneous determination of transretinol, ergocalciferol, DL-α-tocopherol and phytomenadione. The fat-soluble vitamins were separated in a C18 bonded phase column and eluted with methanol as the eluent pumped at a flow rate of 0.2 mL/min. A spectral detector was used and the wavelenghts were set at 325, 265, 284 and 250 nm for vitamin A, D2, E and K1 respectively. All vitamins were separated in less than 13 minutes. This method was applied to the determination of fat-soluble vitamins in the pediatric parenteral nutritions. The effect of the nutrition composition, daylight and the plastic infusion tubing on the stability of the vitamins was studied. Recovery studies showed good results for all solutes (191.0% −110.5%) and the coefficients of variation were always less than 3%.

Influence of cider-making technology on low-boiling-point volatile compounds

ZusammenfassungDie hauptsächlichen niedrigsiedenden Aromakomponenten aus zwölf Apfelweinen, die mit verschiedenen technischen Methoden (verschiedene Preßgeschwindigkeiten und Klärungssysteme) erhalten wurden, wurden durch Gaschromatographie analysiert. Die chromatographischen Daten zeigten, daß der Anteil an Ethylacetat, Methanol, Propanol,Isobutanol, 2-Me-thylbutanol und 3-Methylbutanol von der Preßgeschwindigkeit abhängt, ebenso wie der Anteil an Methanol,Isobutanol, 2-Methylbutanol und 3-Methylbutanol vom Klärungssystem. Ein Einfluß beider Faktoren wurde nur fürIsobutanol und 3-Methylbutanol beaobachtet, während der Butanolanteil von der angewandten Technologie unabhängig ist.AbstractMajor low-boiling-point aroma components present in twelve ciders obtained by different technological methods involving different pressing speeds and clarification systems were evaluated by gas chromatography. Variance analysis of chromatographic data show that ethyl acetate, methanol, propanol, isobutanol, 2-methyl butanol and 3-methyl butanol contents depend on the pressing speed. In the same way, the metanol, isobutanol, 2-methylbutanol and 3-methylbutanol contents depend on the clarification system employed. The interactive effect of both factors was observed only forIsobutanol and 3-methylbutanol whereas the butanol content was independent of the technology used.