Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC.

Melatonin (N-acetyl-5-methoxytryptamine) is a potent endogenous antioxidant and free radical scavenger that has attracted much attention as a consequence of its multiple biological functions. In addition to other physiological properties, it has clear antiproliferative activity in several types of cancer cell. The concentration of melatonin necessary to inhibit cell growth is much higher than its blood physiological concentrations in some tumor types. For years its indolic nature has impeded proper monitoring, by molecular or immunological techniques, of its uptake by cancer cells. In this work we developed a simple, rapid, and validated analytical method for detection and quantification of MEL inside normal and cancer cells. For this purpose we performed high-performance liquid chromatographic analysis after liquid-liquid extraction of the indole from biological samples. The method was validated, and the correlation coefficient for amounts from 0.125 to 1.25 microg was higher than 0.999, with a range of recovery near 100%. Precision was evaluated as repeatability, and for intermediate precision, the relative standard deviation was less than 5%. The method was used to study the stability of the indole in solution and to determine intracellular melatonin concentrations in normal (PNT1A) and several cancer (LNCaP, DU-145, PC-3) prostate cell lines. Intracellular LOQ/LOD were 7.23/2.83, 23.17/9.07, 4.03/1.83, and 6.51/2.53 nmol L(-1), or 1.82/4.66, 0.56/1.45, 3.26/8.34, and 2.02/5.17 attogram in each cell in PNT1A, LNCaP, DU145, and PC-3 cells, respectively. Because there was no information about intracellular levels of melatonin inside normal or tumor prostate cells after treatment with the indole, nor a relationship between its antiproliferative activity and its intracellular concentration, this is the first time that, by using an analytical method combined with measurement of cellular volume by flow cytometry, the intracellular concentration of MEL has been estimated. Also, data obtained here explain why the antiproliferative properties of MEL vary in different cell types. This is, moreover, the first time that by increasing the intracellular concentration of melatonin, its antitumor properties have been promoted in prostate cancer cells. This process can be monitored by the method developed here.

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.

Polyphenols and antioxidant properties of almond skins: influence of industrial processing.

Almond (Prunus dulcis[Mill.] D.A. Webb) skins have been proposed as a source of bioactive polyphenols. In this article, the phenolic composition and antioxidant activity of almond skins obtained from different processes (blanching [freeze-drying], blanching + drying, and roasting) were studied. A total of 31 phenolic compounds corresponding to flavan-3-ols (33% to 56% of the total of phenolic compounds identified), flavonol glycosides (9% to 36%), hydroxybenzoic acids and aldehydes (6% to 26%), flavonol aglycones (1.7% to 18%), flavanone glycosides (3% to 7.7%), flavanone aglycones (0.69% to 5.4%), hydroxycinnamic acids (0.65% to 2.6%), and dihydroflavonol aglycones (0% to 2.8%) were determined in the skins from 3 different varieties of almonds. The total contents of phenolic compounds identified were significantly (P < 0.05) higher (around 2-fold) in the roasted samples than in the blanched almonds (freeze-dried). Industrial drying (oven drying) of the blanched almond skins produced an increase (< 2-fold) in the contents of phenolic compounds, although the results were only statistically significant (P < 0.05) for some samples. The antioxidant activity (ORAC values) was higher for the roasted samples (0.803 to 1.08 mmol Trolox/g), followed by the samples subjected to blanching + drying (0.398 to 0.575 mmol Trolox/g) and then the blanched (freeze-dried) samples (0.331 to 0.451 mmol Trolox/g). Roasting is the most suitable type of industrial processing of almonds to obtain almond skin extracts with the greatest antioxidant capacity.

Evolution of red wine anthocyanins during malolactic fermentation, postfermentative treatments and ageing with lees.

A comparative study was conducted on nine batches of wine, from the same initial wine, subjected to malolactic fermentation and ageing in barrels, under different technological conditions: Malolactic fermentation in barrel or in tank, with or without wine clarification, ageing with or without lees and stirring or no stirring of the lees. Samples were taken of the initial wine, of the wine at the end of malolactic fermentation, of the wines after clarifying treatments, and after 3, 6, 9, 12 and 14 months of ageing in the barrel, making a total of 48 wines. As a result of the anthocyanin analysis of all the wines studied, a total of 21 different anthocyanin compounds were detected, which can be classified into four groups: simple glucosides, acetyl glucosides, cinnamoyl glucosides and pyroanthocyanins. During MLF, it was shown that the effect of the container used seems to be more important than the metabolic activity of the bacteria responsible for the process. From application of the LSD test, significant differences were found in the concentrations of all the anthocyanin compounds identified due to ageing time and significant differences were also revealed for most anthocyanin compounds in relation to the manufacturing method, especially the presence or absence of lees.

Hydroxycinnamic acids and ferulic acid dehydrodimers in barley and processed barley.

Hydroxycinnamic acid content and ferulic acid dehydrodimer content were determined in 11 barley varieties after alkaline hydrolysis. Ferulic acid (FA) was the most abundant hydroxycinnamate with concentrations ranging from 359 to 624 microg/g dry weight. p-Coumaric acid (PCA) levels ranged from 79 to 260 microg/g dry weight, and caffeic acid was present at concentrations of <19 microg/g dry weight. Among the ferulic acid dehydrodimers that were identified, 8-O-4'-diFA was the most abundant (73-118 microg/g dry weight), followed by 5,5'-diFA (26-47 microg/g dry weight), the 8,5'-diFA benzofuran form (22-45 microg/g dry weight), and the 8,5'-diFA open form (10-23 microg/g dry weight). Significant variations (p < 0.05) among the different barley varieties were observed for all the compounds that were quantified. Barley grains were mechanically fractionated into three fractions: F1, fraction consisting mainly of the husk and outer layers; F2, intermediate fraction; and F3, fraction consisting mainly of the endosperm. Fraction F1 contained the highest concentration for ferulic acid (from 77.7 to 82.3% of the total amount in barley grain), p-coumaric acid (from 78.0 to 86.3%), and ferulic acid dehydrodimers (from 79.2 to 86.8%). Lower contents were found in fraction F2, whereas fraction F3 exhibited the lowest percentages (from 1.2 to 1.9% for ferulic acid, from 0.9 to 1.7% for p-coumaric acid, and <0.02% for ferulic acid dehydrodimers). The solid barley residue from the brewing process (brewer's spent grain) was approximately 5-fold richer in ferulic acid, p-coumaric acid, and ferulic acid dehydrodimers than barley grains.

Fast determination of procyanidins and other phenolic compounds in food samples by micellar electrokinetic chromatography using acidic buffers.

Procyanidins are phenolic oligomers, mainly composed of (+)-catechin and (-)-epicatechin units that exhibit certain sensorial and physiological properties of interest (e.g., astringency and bitterness of food, antioxidant activity, etc.). This paper shows the development of a micellar electrokinetic chromatography (MEKC) method for the separation of three procyanidin dimers (B1, B2, and B3), their monomers ((+)-catechin and (-)-epicatechin), and the cis- and trans-forms of p-coumaric acid. Separation conditions are optimized in terms of buffer pH, SDS concentration, and washing routine between injections. The best results in terms of peak resolution and reproducibility between separations were obtained with a MEKC running buffer at pH 5 with 100 mM SDS and a washing routine that includes a rinse step with 0.1 M sodium hydroxide. Using this new MEKC method it is possible to separate in less than 5 min the seven substances. More interestingly, it is demonstrated that the low pH used in this MEKC method allows one to obtain clean electropherograms when samples are injected. The method is shown to be reproducible between different days with relative standard deviation (RSD) values lower than 1% for migration times and lower than 7% for peak areas (3 days, 24 injections). The usefulness of this procedure to determine these compounds in effluents from food processing (i.e., soaking water from lentils, white beans and black beans) and in food by-products (i.e., almond peels) considered as potential procyanidin sources is demonstrated. To our knowledge, this is the first report of separation and determination of procyanidins in food samples done by capillary electrophoresis.

Effects of wine phenolics and sorghum tannins on tyrosinase activity and growth of melanoma cells.

In this study, three different phenolic (anthocyanin, other flavonoid, and phenolic acid) fractions from wine and a condensed tannin preparation from sorghum were tested for their effects on melanogenesis of normal cells and growth of human melanoma cells. The wine phenolic fractions decreased melanogenic activity (tyrosinase activity) at concentrations that resulted in a slight variation in melanocyte viability. Sorghum tannins, however, increased melanogenic activity, although no increase was found in total melanin at the concentrations that least affect melanocyte viability. Incubation of human melanoma cells with the wine fractions and sorghum tannins resulted in a decrease in colony formation, although the effect was not dose dependent in all cases. These results suggest that all of these phenolic fractions have potential as therapeutic agents in the treatments of human melanoma, although the mechanisms by which cellular toxicity is effected seem to be different among the fractions.

Behavior of monosaccharides, phenolic compounds, and color of red wines aged in used oak barrels and in the bottle.

A red wine with appropriate basic quality characteristics for aging was stored in oak barrels for 12 months and then bottled and aged for a further 6 months. The same ambient conditions of temperature and humidity were maintained throughout the entire aging process. The barrels used were made from three different species of oak by four different cooperages and had been used for at least two years. Analysis of variance and principal component analysis were run on the values for hexoses, pentoses, total anthocyanins, ortho-diphenols, low- and high-polymer polyphenols, and color parameters to study the behavior of the monosaccharides and polyphenols in response to the factors of aging time, the oak variety employed, and the source cooperage where the barrels had been made. Time trends for all the phenolic components were directly related to aging time, with low-polymeric polyphenols (LPPs) being the most affected by wood type and source cooperage. Wine color was defined by a basic red color which decreased with aging time in the barrel and was altered by yellowish pigment components differing for each of the barrels in which oxidative aging took place and by increased stability of the blue copigments. Principal component analysis showed that samples of the same source wine aged in different barrels tended to be grouped together according to each of the aging intervals considered.

Natural occurrence of alternariol and alternariol methyl ether in Spanish apple juice concentrates.

A limited survey of 32 samples of apple juice concentrates, destined for the production of commercial juices, was carried out in order to evaluate the natural occurrence of the Alternaria metabolites alternariol and alternariol methyl ether. A high-performance liquid chromatographic method based on solid-phase extraction columns for extraction and purification of the toxins was used. Both mycotoxins were found as natural contaminants in 50% of the samples analyzed. Levels of alternariol were in the range 1.35-5.42 ng/ml. Alternariol methyl ether was present in most cases only at trace levels, and the highest amount detected was 1.71 ng/ml in one sample.

Determination of alternariol and alternariol methyl ether in apple juice using solid-phase extraction and high performance liquid chromatography.

The present work describes a new method for determination of alternariol (AOH) and alternariol methyl ether (AME) in apple juice using solid-phase extraction (SPE) columns for extraction and cleanup of samples for high-performance liquid chromatography (HPLC). Chromatograms of spiked samples show that both toxins can be easily detected without interferences, and good recoveries for AOH (82.8 +/- 7.4%) and AME (91.9 +/- 6.1%) with detection limits as low as 1.6 and 0.7 micrograms/l, respectively, were obtained.

Determination of patulin in apple juice by high-performance liquid chromatography with diode-array detection.

A method is described for the detection of patulin in apple juice and the simultaneous determination of the phenolic composition. Spectral data obtained with diode-array detection showed that patulin can be easily distinguished from compounds eluting under the same conditions. The detection limit for patulin was 8.96 micrograms/l.