RESUMEN
Understanding the metabolic and pharmacokinetic fate of a drug in humans is a key factor in its development and registration, as well as in the elaboration of new therapeutic agents. To carry out these studies, stable isotope labeling techniques have been effectively used by drug metabolism scientists and toxicologists in order to gain better understanding of the drugs' disposition, bioavailability and toxicity in in vivo studies. Among the different analytical techniques used, mass spectrometry (MS) coupled to separation techniques has become the detection method of choice due to its high sensitivity and selectivity. In vitro quantification of metabolite levels in biofluids by MS is often difficult if a proper internal standard is not available due to the inherent problems associated with the technique (e.g. chromatographic coelutions, ion suppression, low reproducibility etc.). Stable isotope coding approaches alleviate these drawbacks and allow comparative drug metabolomics studies similarly to the differential proteomic techniques developed in the last decade. This review describes a selection of methodological improvements in the use of stable isotopes labeling in combination with MS to detect drug metabolites. In the first part of the paper, the application of labeled compounds to study the absorption, distribution, metabolism, excretion and toxicology of drugs (ADMET) in addition to the elucidation of metabolic pathways is presented. In the second part, recent developments of stable isotope coded tags for the in vitro relative metabolite quantification in biofluids are presented.
Asunto(s)
Marcaje Isotópico/métodos , Preparaciones Farmacéuticas/metabolismo , Animales , Descubrimiento de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Metabolómica , Pruebas de Toxicidad/métodosRESUMEN
The present study investigates the antioxidant mechanism of grape procyanidins and, in particular, their aptitude to establish redox interactions with two important components of the endogenous antioxidant system of muscle tissues, α-tocopherol (α-TOH) and ascorbic acid (AA). To this end, the progress of lipid oxidation was monitored in fish muscle supplemented with grape procyanidins at the concentrations usually employed in antioxidant food applications, and then related to the redox stability of the endogenous α-TOH and AA. In addition to the lipid oxidation protective effect, the incorporation of procyanidins also provided an improvement of the redox stability of the endogenous components in a straight procyanidinic concentration-dependent manner. Results showed the capacity of procyanidins to repair oxidised α-TOH at medium-long term, and to delay the AA depletion. Therefore, such cooperative redox interaction of exogenous procyanidins adequately complements the natural α-TOH regenerative system supplied by AA that is efficient at the early post mortem stages.
Asunto(s)
Ácido Ascórbico/química , Conservación de Alimentos/métodos , Conservantes de Alimentos/química , Carne/análisis , Músculo Esquelético/química , Proantocianidinas/química , Vitis/química , alfa-Tocoferol/química , Animales , Peces , Peroxidación de LípidoRESUMEN
Microcystins (MC) are a family of hepatotoxic cyclic heptapeptides produced by a number of different cyanobacterial species. Considering the recent advances in the characterization of deprotonated peptides by mass spectrometry, the fragmentation behavior of four structurally related microcystin compounds was investigated using collision-induced dissociation (CID) experiments on an orbitrap mass spectrometer. It is demonstrated in this study that significant structural information can be obtained from the CID spectra of deprotonated microcystins. A predominant ring-opening reaction at the isoMeAsp residue, as well as two major complementary fragmentation pathways, was observed, reducing the complexity of the product ion spectra in comparison with spectra observed from protonated species. This proposed fragmentation behavior was applied to characterize [Leu(1)]MC-LR from a cyanobacterial cell extract. In conclusion, CID spectra of microcystins in the negative ion mode provide rich structurally informative mass spectra which greatly enhance confidence in structural assignments, in particular when combined with complementary positive ion CID spectra.
Asunto(s)
Microcistinas/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Iones/química , Microcystis/química , Modelos Moleculares , ProtonesRESUMEN
The influence of polymerization (number of monomers) and galloylation (content of esterified gallates) of oligomeric catechins (proanthocyanidins) on their effectiveness to prevent lipid oxidation in pelagic fish muscle was evaluated. Non-galloylated oligomers of catechin with diverse mean polymerization (1.9-3.4 monomeric units) were extracted from pine (Pinus pinaster) bark. Homologous fractions with galloylation ranging from 0.25 to <1 gallate group per molecule were obtained from grape (Vitis vinifera) and witch hazel (Hamamelis virginiana). The results showed the convenience of proanthocyanidins with medium size (2-3 monomeric units) and low galloylation degree (0.15-0.25 gallate group/molecule) to inhibit lipid oxidation in pelagic fish muscle. These optimal structural characteristics of proanthocyanidins were similar to those lately reported in fish oil-in-water emulsions using phosphatidylcholine as emulsifier. This finding suggests that the antioxidant behavior of polyphenols in muscle-based foods can be mimicked in emulsions prepared with phospholipids as emulsifier agents. The present data give relevant information to achieve an optimum use of polyphenols in pelagic fish muscle.
Asunto(s)
Conservación de Alimentos/métodos , Peroxidación de Lípido/efectos de los fármacos , Músculo Esquelético/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Polifenoles/química , Polifenoles/farmacología , Alimentos Marinos/análisis , Animales , Peces , Conservantes de Alimentos/química , Conservantes de Alimentos/farmacología , Hamamelis/química , Músculo Esquelético/efectos de los fármacos , Pinus/química , Corteza de la Planta/química , Relación Estructura-Actividad , Vitis/químicaRESUMEN
Polyphenolic fractions extracted from pine (Pinus pinaster) bark, grape (Vitis vinifera) pomace, and witch hazel (Hamamelis virginiana) bark were selected for investigating the influence of the number of phenolic units, polymerization, and the content of esterified galloyl residues (galloylation) on their efficacy for inhibiting lipid oxidation in fish lipid enriched foodstuffs. Experiments carried out with nongalloylated pine bark fractions with different polymerization degrees demonstrated that the number of catechin residues per molecule modulates their reducing and chelating properties in solution. In real food systems such as bulk fish oil and fish oil-in-water emulsions, the efficacy against lipid oxidation was highly dependent on the physical location of the antioxidant at the oxidative sensitive sites. The lowest polymerized fractions were the most efficient in bulk fish oil samples, whereas proanthocyanidins with an intermediate polymerization degree showed the highest activity in fish oil-in-water emulsions. Galloylation did not influence the antioxidant effectiveness of proanthocyanidins in bulk fish oils. The presence of galloyl groups favored the antioxidant activity of the polyphenols in emulsions, although results indicated that a high degree of galloylation did not improve significantly the activity found with medium galloylated proanthocyanidins. The results obtained in this research provide useful information about the relationship between structure and antioxidant activity in order to design antioxidant additives with application in fish oil-enriched functional foods.
Asunto(s)
Antioxidantes/química , Aceites de Pescado/química , Flavonoides/química , Fenoles/química , Corteza de la Planta/química , Extractos Vegetales/química , Animales , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Flavonoides/aislamiento & purificación , Flavonoides/farmacología , Gadus morhua , Hamamelis/química , Humanos , Oxidación-Reducción/efectos de los fármacos , Fenoles/aislamiento & purificación , Fenoles/farmacología , Pinus/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Polifenoles , Proantocianidinas/química , Gusto , Vitis/químicaRESUMEN
The efficiency of different reductants (reduced glutathione, ascorbic acid, and catalase) and metal chelators [ethylenediaminetetraacetic acid (EDTA), citric acid, sodium tripolyphosphate (STPP), and adenosine-5'-triphosphate (ATP)] to inhibit lipid oxidation promoted by fish hemoglobin was investigated. The inhibitory activity on hemoglobin-catalyzed lipid oxidation was also evaluated for grape oligomeric catechins (proanthocyanidins), which have both reducing and chelating properties. The antioxidant activity was studied in two different lipid oxidation models, liposomes and washed minced fish muscle. Grape proanthocyanidins were found to be significantly more effective than other reductants to prevent hemoglobin-mediated lipid oxidation in both liposomes and washed fish muscle. Reduced glutathione was also efficient to retard lipid oxidation at the same molarity in washed fish muscle, whereas catalase and ascorbic acid showed a lower antioxidant activity. Metal chelators were less active than reductants, and consequently, the former were necessarily evaluated at much higher concentration than grape proanthocyanidins and reducing compounds. STPP was found to be the iron chelator with the strongest efficiency to delay hemoglobin-mediated lipid oxidation followed by EDTA. Citric acid and ATP were ineffective in retarding lipid oxidation in both systems. Grape proanthocyanidins provided the most extensive protection to preserve hemoglobin at ferrous state in washed fish muscle. Our results draw attention to the greater capacity of reducing compounds to prevent fish hemoglobin-mediated lipid oxidation in comparison with iron chelators, suggesting that the free radical scavenging and/or reduction of ferrylHb species are crucial actions to avoid the pro-oxidant capacity of fish hemoglobin.
Asunto(s)
Quelantes/farmacología , Peces/sangre , Hemoglobinas/química , Peroxidación de Lípido/efectos de los fármacos , Sustancias Reductoras/farmacología , Animales , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Catalasa/farmacología , Glutatión/farmacología , Músculos/química , Oxidación-Reducción , Proantocianidinas/farmacología , Vitis/químicaRESUMEN
In an emulsion of corn oil in water with the addition of caffeic acid (Caf-OH) and alpha-tocopherol (alpha-TOH), Caf-OH was found to be very active in delaying lipid oxidation without affecting significantly the kinetics for alpha-TOH degradation. In contrast, Caf-OH addition to fish muscle retarded both the degradation of endogenous alpha-TOH and the propagation of lipid oxidation, measured by peroxide value (PV) and thiobarbituric acid reactive substances (TBARS), with increasing effect with increasing Caf-OH addition (55.5-555.1 micromol/kg). Electron spin resonance (ESR) spectroscopy confirmed a higher capacity of Caf-OH to regenerate alpha-TOH via reduction of the alpha-tocopheroxyl radical compared to other cinnamic acid derivatives (o-coumaric, ferulic, and chlorogenic acids). Degradation of endogenous ascorbate (AscH(-)) was accelerated at higher concentration of Caf-OH in fish tissue, suggesting a role of AscH(-) in the regeneration of Caf-OH. These results indicate that the antioxidant mechanism of Caf-OH implies the protection of endogenous alpha-TOH localized in tissue membranes where lipid oxidation is initiated and, at the same time, Caf-OH regeneration by the endogenous AscH(-). These combined effects result in a stronger antioxidant protection against lipid oxidation by favoring, as a final point, the protection of alpha-TOH, which is suggested as the last defense of fish muscle against lipid oxidation.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/metabolismo , Ácidos Cafeicos/farmacología , Peces/metabolismo , Músculo Esquelético/metabolismo , alfa-Tocoferol/metabolismo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Ácido Ascórbico/química , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Sinergismo Farmacológico , Peroxidación de Lípido/efectos de los fármacos , Músculo Esquelético/química , Músculo Esquelético/efectos de los fármacos , Oxidación-Reducción , alfa-Tocoferol/químicaRESUMEN
A procedure for the determination of volatile compounds derived from lipid oxidation of fish muscle samples is presented. Analytes are concentrated on a solid-phase microextraction fiber employed in the headspace mode (HS-SPME), and selectively determined using gas chromatography in combination with mass spectrometry (GC-MS). The influence of several parameters on the efficiency of microextraction such as type of fiber, volume of sample, time, temperature, salting-out effect and stirring was systematically investigated. A saline extraction of fish muscle followed by incubation on a Carboxen-polydimethylsiloxane fiber during 30min at 60 degrees C gave the most effective and accurate extraction of the analytes. Quantification of them was performed by MS in the selected ion monitoring mode and by the internal standard method. Satisfactory linearity, repeatability and quantification limits were achieved under these conditions. The method was applied to the determination of the volatile compounds associated to oxidation of Atlantic horse mackerel (Trachurus trachurus) minced muscle and excellent correlations were obtained with chemical indexes for monitoring lipid oxidation as peroxide value and thiobarbituric acid reactive substances. This combined technique is fast, simple, sensitive, inexpensive and useful to monitor target compounds associated to fish rancidity as 1-penten-3-ol, 2,3-pentanedione or 1-octen-3-ol.
Asunto(s)
Aldehídos/análisis , Ácidos Grasos/análisis , Músculos/química , Microextracción en Fase Sólida/métodos , Conservación de Alimentos , Cromatografía de Gases y Espectrometría de Masas , Metabolismo de los Lípidos , Oxidación-Reducción , VolatilizaciónRESUMEN
Headspace solid-phase microextraction (HS-SPME) is proposed for isolating and determining the headspace volatiles formed during oxidation of fish-oil-in-water emulsions. Three different fiber coatings were tested and compared for sensitivity and reproducibility. A carboxen/polydimethylsiloxane (CAR-PDMS) fiber coating was found to be the most suitable for the analysis of emulsion volatiles. The main factors affecting the microextraction process on CAR-PDMS were then evaluated by a factorial design: amount of sample, time and temperature of extraction and stirring. The incubation of 0.5 g of emulsion at 60 degrees C during 30 min leads to the most effective extraction of volatiles associated with lipid oxidation of fish oil emulsions. The HS-SPME method coupled with GC-MS allowed the qualitative and quantitative analysis of the volatiles derived from oxidation of real fish oil enriched foods such as milk and mayonnaise. The method here proposed is very fast and simple and yields high sensitivity, with good repeatability for all target compounds.