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RATIONALE: Natural variations in the abundance of the stable isotopes of nitrogen (δ15N) and carbon (δ13C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ15N and δ13C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions. METHODS: Prostate cancer cells (PC3) were cultured in different conditions and their δ15N and δ13C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia. RESULTS: Successive cell culture passages generally resulted in reproducible δ15N and δ13C values. The impact of culture medium composition on δ15N and δ13C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ13C in bulk cell samples, with only the former affecting δ15N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided. CONCLUSIONS: Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ13C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach.
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Isótopos de Carbono , Medios de Cultivo , Espectrometría de Masas , Isótopos de Nitrógeno , Humanos , Isótopos de Carbono/análisis , Isótopos de Carbono/metabolismo , Isótopos de Nitrógeno/análisis , Isótopos de Nitrógeno/metabolismo , Espectrometría de Masas/métodos , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Glutamina/metabolismo , Glutamina/análisis , Neoplasias de la Próstata/metabolismo , Masculino , Células PC-3 , Línea Celular Tumoral , Técnicas de Cultivo de Célula/métodosRESUMEN
Carbon-13 isotopomics of triacylglycerol (TAG) fatty acids or free fatty acids in biological matrices holds considerable potential in food authentication, forensic investigations, metabolic studies, and medical research. However, challenges arise in the isotopic analysis of short- and medium-chain (C4 to C10) fatty acid methyl esters (SMCFAMEs) through gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). The high volatility of these esters results in losses during their preparation, leading to isotopic fractionation. Moreover, the methoxy group added to acyl chains requires the correction of δ13C values, thereby increasing the uncertainty of the final results. Analyzing free fatty acids (FFAs) addresses both issues encountered with SMCFAMEs. To achieve this objective, we have developed a new protocol enabling the isotopomics of individual fatty acids (FAs) by GC-C-IRMS. The same experiment also provides the FA profile, i.e., the relative percentage of each FA in the TAG hydrolysate or its concentration in the studied matrix. The method exhibited high precision, as evidenced by the repeatability and within-lab reproducibility of results when tested on TAGs from both animal and vegetal origins. Compared to the analysis of FAMEs by GC-C-IRMS, the current procedure also brings several improvements in alignment with the principles of green analytical chemistry and green sample preparation. Thus, we present a two-in-one method for 13C-isotopomic and metabolomic biomarker quantitation within quasi-universal TAG compounds, encompassing the short- and medium-acyl chains.
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While clinical evidence remains limited, an extensive amount of research suggests a beneficial role of n-3 polyunsaturated fatty acid supplementation in cancer treatment. One potential benefit is an improvement of protein homeostasis, but how protein metabolism depends on proinflammatory cytokines in this context remains unclear. Here, using the natural abundance of the stable isotopes of nitrogen as a marker of changes in protein metabolism during a randomized, double-blind, controlled clinical trial, we show that protein homeostasis is affected way faster than proinflammatory cytokines in metastatic breast cancer patients supplemented with n-3 polyunsaturated fatty acids. We provide some evidence that this response is unrelated to major changes in whole-body substrate oxidation. In addition, we demonstrate that more fatty acids were impacted by metabolic regulations than by differences in their intake levels during the supplementation. This study documents that the percentage of patients that complied with the supplementation decreased with time, making compliance assessment crucial for the kinetic analysis of the metabolic and inflammatory responses. Our results highlight the time-dependent nature of metabolic and inflammatory changes during long-chain n-3 fatty acid supplementation.
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The natural abundance of heavy stable isotopes (13C, 15N, 18O, etc.) is now of considerable importance in many research fields, including human physiology. In fact, it varies between tissues and metabolites due to isotope effects in biological processes, that is, isotope discriminations between heavy and light isotopic forms during enzyme or transporter activity. The metabolic deregulation associated with many diseases leads to alterations in metabolic fluxes, resulting in changes in isotope abundance that can be identified easily with current isotope ratio technologies. In this review, we summarize the current knowledge on changes in natural isotope composition in samples (including various tissues, hair, plasma, saliva) found in patients compared to controls, caused by human diseases. We discuss the metabolic origin of such isotope fractionations and highlight the potential of using isotopes at natural abundance for medical diagnosis and/or prognostic.
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15N natural isotope abundance (NIA) is systematically higher in infants' hair than in that of their mothers at birth. This study aimed to investigate this difference in plasma pools. We compared 15N NIA values for plasma amino acid (AA) pools (free + protein-bound) in the umbilical cord artery (UCA) and vein (UCV) and in the maternal vein (MV) at birth. This preliminary study included 7 mother-infant dyads. Whole plasma was treated (HCl) to hydrolyze protein. Following derivatization, AAs were separated using gas chromatography and compound-specific 15N NIA values were measured on-line using an isotope ratio monitoring mass spectrometer. 15N NIA plasma AA pools in the UCA and UCV were highly correlated to the MV, r 2 > 0.89 and r 2 > 0.88 (both P < 10-4) respectively. The full model found a significant effect of sampling compartment (P = 0.02) and AA type (P < 0.0001) on 15N NIA plasma AA values. 15N NIA plasma AA was 0.74 higher (P = 0.01) in the MV than in the UCA. This study indicates that a decrease in 15N NIA for plasma AA pools occurs in the fetal-placental unit. Trial registration: ClinicalTrials.gov identifier: NCT00607061.
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Aminoácidos/sangre , Cabello/química , Intercambio Materno-Fetal , Isótopos de Nitrógeno/análisis , Arterias Umbilicales/química , Femenino , Retardo del Crecimiento Fetal/sangre , Feto/irrigación sanguínea , Humanos , Lactante , Espectrometría de Masas , Fenómenos Fisiologicos Nutricionales Maternos , Embarazo , Venas Umbilicales/químicaRESUMEN
Dichloroacetate (DCA) is an investigational drug targeting the glycolytic hallmark of cancer by inhibiting pyruvate dehydrogenase kinases (PDK). It is metabolized by GSTZ1, which has common polymorphisms altering enzyme or promoter activity. GSTZ1 is also irreversibly inactivated by DCA. In the first clinical trial of DCA in a hematological malignancy, DiCAM (DiChloroAcetate in Myeloma), we have examined the relationship between DCA concentrations, GSTZ1 genotype, side effects, and patient response. DiCAM recruited seven myeloma patients in partial remission. DCA was administered orally for 3 months with a loading dose. Pharmacokinetics were performed on day 1 and 8. Trough and peak concentrations of DCA were measured monthly. GSTZ1 genotypes were correlated with drug concentrations, tolerability, and disease outcomes. One patient responded and two patients showed a partial response after one month of DCA treatment, which included the loading dose. The initial half-life of DCA was shorter in two patients, correlating with heterozygosity for GSTZ1*A genotype, a high enzyme activity variant. Over 3 months, one patient maintained DCA trough concentrations approximately threefold higher than other patients, which correlated with a low activity promoter genotype (-1002A, rs7160195) for GSTZ1. This patient displayed the strongest response, but also the strongest neuropathy. Overall, serum concentrations of DCA were sufficient to inhibit the constitutive target PDK2, but unlikely to inhibit targets induced in cancer. Promoter GSTZ1 polymorphisms may be important determinants of DCA concentrations and neuropathy during chronic treatment. Novel dosing regimens may be necessary to achieve effective DCA concentrations in most cancer patients while avoiding neuropathy.
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Ácido Dicloroacético/farmacocinética , Resistencia a Antineoplásicos/genética , Glutatión Transferasa/genética , Mieloma Múltiple/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/genética , Administración Oral , Anciano , Ácido Dicloroacético/administración & dosificación , Ácido Dicloroacético/efectos adversos , Drogas en Investigación/administración & dosificación , Drogas en Investigación/efectos adversos , Drogas en Investigación/farmacocinética , Femenino , Genotipo , Glutatión Transferasa/metabolismo , Semivida , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/sangre , Mieloma Múltiple/genética , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Estudios Prospectivos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/antagonistas & inhibidores , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismoRESUMEN
In the article (Romek et al. 2013) we reported the values of δ15N () and δ13C () obtained by.
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The natural isotope abundance in bulk organic matter or tissues is not a sufficient base to investigate physiological properties, biosynthetic mechanisms, and nutrition sources of biological systems. In fact, isotope effects in metabolism lead to a heterogeneous distribution of 2H, 18O, 13C, and 15N isotopes in metabolites. Therefore, compound-specific isotopic analysis (CSIA) is crucial to biological and medical applications of stable isotopes. Here, we review methods to implement CSIA for 15N and 13C from plant, animal, and human samples and discuss technical solutions that have been used for the conversion to CO2 and N2 for IRMS analysis, derivatization and isotope effect measurements. It appears that despite the flexibility of instruments used for CSIA, there is no universal method simply because the chemical nature of metabolites of interest varies considerably. Also, CSIA methods are often limited by isotope effects in sample preparation or the addition of atoms from the derivatizing reagents, and this implies that corrections must be made to calculate a proper δ-value. Therefore, CSIA has an enormous potential for biomedical applications, but its utilization requires precautions for its successful application.
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Métodos Analíticos de la Preparación de la Muestra/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Isótopos/análisis , Métodos Analíticos de la Preparación de la Muestra/instrumentación , Animales , Cromatografía de Gases y Espectrometría de Masas/instrumentación , Humanos , Isótopos/química , Cinética , Plantas/metabolismoRESUMEN
Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13C and 15N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes.
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Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the production of glutamine from glutamate, ammonium and ATP. Although being essential in plants for N assimilation and recycling, kinetic commitments and transition states of the reaction have not been clearly established yet. Here, we examined 12C/13C, 14N/15N and H2O/D2O isotope effects in Arabidopsis GS1 catalysis and compared to the prokaryotic (Escherichia coli) enzyme. A14N/15N isotope effect (15V/K ≈ 1.015, with respect to substrate NH4+) was observed in the prokaryotic enzyme, indicating that ammonium utilization (deprotonation and/or amidation) was partially rate-limiting. In the plant enzyme, the isotope effect was inverse (15V/K = 0.965), suggesting that the reaction intermediate is involved in an amidation-deamidation equilibrium favoring 15N. There was no 12C/13C kinetic isotope effect (13V/K = 1.000), suggesting that the amidation step of the catalytic cycle involves a transition state with minimal alteration of overall force constants at the C-5 carbon. Surprisingly, the solvent isotope effect was found to be inverse, that is, with a higher turn-over rate in heavy water (DV ≈ 0.5), showing that restructuration of the active site due to displacement of H2O by D2O facilitates the processing of intermediates.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/biosíntesis , Proteínas de Arabidopsis/química , Isótopos de Carbono/química , Glutamato-Amoníaco Ligasa/química , Glutamina/metabolismo , Cinética , Isótopos de Nitrógeno/análisis , Solventes/químicaRESUMEN
Natural (15)N abundance (δ(15)N) varies between individual amino acids (AAs). We hypothesized that δ(15)N of nontransaminating and essential AAs ("source" AAs, such as phenylalanine) present in animal tissues could be used as a marker of dietary origin, whereas δ(15)N of transaminating AAs ("trophic" AAs, such as glutamic acid) could give more detailed insights into animal feed efficiency. Two diets based on dehydrated Lucerne pellets were tested in growing lambs, which promoted different feed efficiencies. No dietary effects were noted on δ(15)N of any AAs analyzed in lamb muscle. In addition, δ(15)N of phenylalanine was unexpectedly similar to that of glutamic acid, suggesting that δ(15)N of AAs is significantly derived from the metabolism of the rumen microbiota and, thus, are not suited for diet authentication in ruminants. In contrast, the δ(15)N of transaminating AAs facilitates an improved prediction of animal feed efficiency compared to the classical isotopic bulk N analysis.
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Aminoácidos/análisis , Alimentación Animal/análisis , Carne/análisis , Músculo Esquelético/química , Isótopos de Nitrógeno/análisis , Ovinos/metabolismo , Aminoácidos/metabolismo , Animales , Músculo Esquelético/metabolismo , Isótopos de Nitrógeno/metabolismoRESUMEN
The seeds of Carapa procera are exploited extensively in West African ethnopharmacy for the treatment of several pathologies, including inflammation. They also are effective as insect antifeedants and as a mosquito repellent. With the aim of identifying bioactive principles, an ethyl acetate extract of the defatted seeds was made and fractionated. Two principle compounds were isolated. One of these, 5,6-dehydro-7-deacetoxy-7-oxogedunin (1), while known from another genus of the Meliaceae, is newly identified from the genus Carapa and its X-ray structure is described for the first time. In addition, 1 displayed strong anti-clonogenic activity at 10 µM. The other compound, mexicanolide (2), is known from this species and showed neither cytotoxicity nor anti-clonogenicity. These differences in efficacy are discussed in relation to known structure-activity relationships of limonoids.
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Antineoplásicos Fitogénicos/aislamiento & purificación , Limoninas/aislamiento & purificación , Meliaceae/química , Extractos Vegetales/aislamiento & purificación , Triterpenos/aislamiento & purificación , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Limoninas/química , Limoninas/farmacología , Estructura Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Semillas/química , Triterpenos/química , Triterpenos/farmacologíaRESUMEN
PURPOSE: A better understanding of the control of body fat mass and distribution is required for both human health and animal production. The current study investigates plasma parameters in response to changes in body fat mass. METHODS: Pigs from two lines divergently selected for residual feed intake were fed diets contrasted in energy sources and nutrients. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received isoproteic and isoenergetic diets, either rich in starch (LF) or in lipids and fibres (HF). At the end of the feeding trial, plasma samples were analysed by (1)H NMR spectroscopy and standard hormonal and biochemical assays. RESULTS: Pigs fed the HF diet had lower (P < 0.01) perirenal and subcutaneous adipose tissue relative masses than pigs fed the LF diet. Metabolomic approach showed a clear discrimination between diets, with lower (P < 0.05) plasma levels of creatinine-lysine, creatine, tyrosine, proline, histidine, lysine, phenylalanine and formate but higher (P < 0.001) plasma VLDL-LDL levels in HF pigs than in LF pigs. Plasma concentrations of triglycerides were higher (P < 0.001), while plasma concentrations of ß-hydroxybutyrate, leptin, glucose, insulin and urea were lower (P ≤ 0.05) in HF pigs than in LF pigs. Plasma levels of leptin, creatine and urea were positively correlated (r = 0.3, P < 0.05) with relative adipose tissue masses. CONCLUSION: These data indicate that metabolites associated with energy and protein metabolism were involved in the response to a high-fat, high-fibre diet. Relevant plasma indicators of metabolic flexibility related to changes in body adiposity were then proposed.
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Adiposidad , Biomarcadores/sangre , Dieta/veterinaria , Metabolómica , Ácido 3-Hidroxibutírico/sangre , Tejido Adiposo/metabolismo , Alimentación Animal , Animales , Glucemia/metabolismo , Peso Corporal , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Creatina/sangre , Creatinina/sangre , Grasas de la Dieta/administración & dosificación , Fibras de la Dieta/administración & dosificación , Histidina/sangre , Insulina/sangre , Leptina/sangre , Modelos Lineales , Metabolismo de los Lípidos , Lisina/sangre , Espectroscopía de Resonancia Magnética , Masculino , Fenilalanina/sangre , Prolina/sangre , Porcinos , Triglicéridos/sangre , Tirosina/sangreRESUMEN
Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.
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Productos Biológicos/química , Espectroscopía de Resonancia Magnética , Plantas/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Isótopos de Carbono/química , Línea Celular Tumoral , Femenino , Humanos , Solanum lycopersicum/química , Solanum lycopersicum/metabolismo , Resonancia Magnética Nuclear Biomolecular , Plantas/metabolismoRESUMEN
The natural xanthines caffeine, theobromine, and theophylline are of major commercial importance as flavor constituents in coffee, cocoa, tea, and a number of other beverages. However, their exploitation for authenticity, a requirement in these commodities that have a large origin-based price-range, by the standard method of isotope ratio monitoring by mass spectrometry (irm-MS) is limited. We have now developed a methodology that overcomes this deficit that exploits the power of isotopic quantitative (13)C nuclear magnetic resonance (NMR) spectrometry combined with chemical modification of the xanthines to enable the determination of positional intramolecular (13)C/(12)C ratios (δ(13)Ci) with high precision. However, only caffeine is amenable to analysis: theobromine and theophylline present substantial difficulties due to their poor solubility. However, their N-methylation to caffeine makes spectral acquisition feasible. The method is confirmed as robust, with good repeatability of the δ(13)Ci values in caffeine appropriate for isotope fractionation measurements at natural abundance. It is shown that there is negligible isotope fractionation during the chemical N-methylation procedure. Thus, the method preserves the original positional δ(13)Ci values. The method has been applied to measure the position-specific variation of the (13)C/(12)C distribution in caffeine. Not only is a clear difference between caffeine isolated from different sources observed, but theobromine from cocoa is found to show a (13)C pattern distinct from that of caffeine.
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Espectroscopía de Resonancia Magnética con Carbono-13/métodos , Xantinas/química , MetilaciónRESUMEN
Multi-element stable isotope ratios have been assessed as a means to distinguish between fermented cocoa beans from different geographical and varietal origins. Isotope ratios and percentage composition for C and N were measured in different tissues (cotyledons, shells) and extracts (pure theobromine, defatted cocoa solids, protein, lipids) obtained from fermented cocoa bean samples. Sixty-one samples from 24 different geographical origins covering all four continental areas producing cocoa were analyzed. Treatment of the data with unsupervised (Principal Component Analysis) and supervised (Partial Least Squares Discriminant Analysis) multiparametric statistical methods allowed the cocoa beans from different origins to be distinguished. The most discriminant variables identified as responsible for geographical and varietal differences were the δ(15)N and δ(13)C values of cocoa beans and some extracts and tissues. It can be shown that the isotope ratios are correlated with the altitude and precipitation conditions found in the different cocoa-growing regions.
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Cacao/química , Isótopos/química , Espectrometría de Masas/métodos , Fermentación , GeografíaRESUMEN
This review aims at providing an analysis of the various available reference methods in nuclear magnetic resonance, based on recent literature. This paper will provide a tool to help users in choosing the appropriate method depending on the NMR experiment, sample and targeted application. For each method, the crucial parameters that should be particularly considered to reach the accuracy objectives of quantitative NMR are described. After defining the main concepts, the principle, the analytical performance and the main recent applications of each method are described, and a critical discussion is finally provided.
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Espectroscopía de Resonancia Magnética/métodos , Calibración , Humanos , Espectroscopía de Resonancia Magnética/normas , Estándares de ReferenciaRESUMEN
Phenacetin is known to be metabolized to N-AcetylParaBenzoQuinoneImine (NAPQI), which is a common metabolite of paracetamol (also called acetaminophen or APAP). The electrochemical conversion of APAP to NAPQI was shown in 1989 by Getek and co-workers, thus demonstrating the capacity of electrochemistry to mimic the formation of NAPQI from APAP as well as from phenacetin. This study focuses on a preparative electrochemical electrolysis associated with quantitative (1)H NMR. On one hand, this method is able to synthesize reactive metabolites in sufficient concentrations and amounts for NMR analysis. On the other hand, NMR allows the simultaneous detection and quantification of all chemical species, in contrast to mass spectrometry. The combination of electrochemistry with quantitative NMR is thus presented as a relevant method for elucidating the degradation of reactive metabolites and may be considered a valuable complementary tool to EC-MS.
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Acetaminofén/análisis , Benzoquinonas/análisis , Iminas/análisis , Resonancia Magnética Nuclear Biomolecular/métodos , Fenacetina/análisis , Acetaminofén/química , Benzoquinonas/química , Biotransformación , Electrólisis , Humanos , Iminas/química , Fenacetina/química , SolucionesRESUMEN
Since exclusively breast-suckled infants obtain their nutrient only from their mother's milk, it might be anticipated that a correlation will exist between the (15)N/(14)N isotope ratios of amino acids of protein of young infants and those supplied by their mother. The work presented here aimed to determine whether amino nitrogen transfer from human milk to infant hair protein synthesized within the first month of life conserves the maternal isotopic signature or whether post-ingestion fractionation dominates the nitrogen isotope spectrum. The study was conducted at 1 month post-birth on 100 mother-infant pairs. Isotope ratios (15)N/(14)N and (13)C/(12)C were measured using isotope ratio measurement by Mass Spectrometry (irm-MS) for whole maternal milk, and infant hair and (15)N/(14)N ratios were also measured by GC-irm-MS for the N-pivaloyl-O-isopropyl esters of amino acids obtained from the hydrolysis of milk and hair proteins. The δ(15)N and δ(13)C () were found to be significantly higher in infant hair than in breast milk (δ(15)N, P < 0.001; δ(13)C, P < 0.001). Furthermore, the δ(15)N () of individual amino acids in infant hair was also significantly higher than that in maternal milk (P < 0.001). By calculation, the observed shift in isotope ratio was shown not to be accounted for by the amino acid composition of hair and milk proteins, indicating that it is not simply due to differences in the composition in the proteins present. Rather, it would appear that each pool-mother and infant-turns over independently, and that fractionation in infant N-metabolism even in the first month of life dominates over the nutrient N-content.
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Aminoácidos/análisis , Cabello/química , Proteínas de la Leche/química , Leche Humana/química , Adulto , Femenino , Humanos , Lactante , Isótopos de Nitrógeno/análisisRESUMEN
A comparative study of the electrochemical conversion and the biotransformation performed by the cytochrome P450 (CYP450) obtained by rat liver microsomes has been achieved to elucidate the oxidation mechanism of both acebutolol and alprenolol. For this purpose, a wide range of reactions such as N-dealkylation, O-dealkoxylation, aromatic hydroxylation, benzyl hydroxylation, alkyl hydroxylation, and aromatic hydroxylation have been examined in this study, and their mechanisms have been compared. Most of the results of the electrochemical oxidation have been found to be in accordance with those obtained by incubating acebutolol and alprenolol in the presence of CYP450, i.e., N-dealkylation, benzyl hydroxylation, and O-dealkoxylation reactions catalyzed by liver microsomes were found to be predicted by the electrochemical oxidation. The difficulty for the electrochemical process to mimic both aromatic and alkyl hydroxylation reactions has also been discussed, and the hypothesis for the absence of aromatic hydroxylated and alkyl hydroxylated products, respectively, for alprenolol and acebutolol, under the anodic oxidation has been supported by theoretical calculation. The present study highlights the potential and limitation of coupling of electrochemistry-liquid chromatography-high-resolution mass spectrometry for the study of phase I and phase II reactions of acebutolol and alprenolol.