RESUMEN

OBJECTIVES: Inadequate resection margins of less than 5 mm impair local tumor control. This weak point in oncological safety is exacerbated in bone-infiltrating tumors because rapid bone analysis procedures do not exist. This study aims to assess the bony resection margin status of bone-invasive oral cancer using laser-induced breakdown spectroscopy (LIBS). MATERIALS AND METHODS: LIBS experiments were performed on natively lasered, tumor-infiltrated mandibular cross-sections from 10 patients. In total, 5,336 spectra were recorded at defined distances from the tumor border. Resection margins < 1 mm were defined as very close, from 1-5 mm as close, and > 5 mm as clear. The spectra were histologically validated. Based on the LIBS spectra, the discriminatory power of potassium (K) and soluble calcium (Ca) between bone-infiltrating tumor tissue and very close, close, and clear resection margins was determined. RESULTS: LIBS-derived electrolyte emission values of K and soluble Ca as well as histological parameters for bone neogenesis/fibrosis and lymphocyte/macrophage infiltrates differ significantly between bone-infiltrating tumor tissue spectra and healthy bone spectra from very close, close, and clear resection margins (p < 0.0001). Using LIBS, the transition from very close resection margins to bone-infiltrating tumor tissue can be determined with a sensitivity of 95.0%, and the transition from clear to close resection margins can be determined with a sensitivity of 85.3%. CONCLUSIONS: LIBS can reliably determine the boundary of bone-infiltrating tumors and might provide an orientation for determining a clear resection margin. CLINICAL RELEVANCE: LIBS could facilitate intraoperative decision-making and avoid inadequate resection margins in bone-invasive oral cancer.

Asunto(s)

Márgenes de Escisión , Neoplasias de la Boca , Análisis Espectral , Humanos , Neoplasias de la Boca/cirugía , Neoplasias de la Boca/patología , Análisis Espectral/métodos , Masculino , Femenino , Persona de Mediana Edad , Anciano , Invasividad Neoplásica , Calcio/análisis , Potasio/análisis , Mandíbula/cirugía , Mandíbula/patología , Rayos Láser

RESUMEN

PURPOSE: Microscopic tumor spread beyond the macroscopically visible tumor mass in bone represents a major risk in surgical oncology, where the spatial complexity of bony resection margins cannot be countered with rapid bone analysis techniques. Laser-induced breakdown spectroscopy (LIBS) has recently been introduced as a promising option for rapid bone analysis. The present study aimed to use LIBS-based depth profiling based on electrolyte disturbance tracking to evaluate the detection of microscopic tumor spread in bone. METHODS: After en bloc resection, the tumor-infiltrated mandible section of a patient's segmental mandibulectomy specimen was natively investigated using LIBS. Spectral and electrolytic depth profiles were analyzed across 30 laser shots per laser spot position in healthy bone and at the tumor border. For the histological validation of the lasered positions, the mandibular section was marked with a thin separating disc. RESULTS: Solid calcium (Ca) from hydroxyapatite and soluble Ca from dissolved Ca can be reliably differentiated using LIBS and reflect the natural heterogeneity of healthy bone. Increased potassium (K) emission values in otherwise typically healthy bone spectra are the first spectral signs of tumorous bone invasion. LIBS-based depth profiles at the tumor border region can be used to track tumor-associated changes within the bone with shot accuracy based on the distribution of K. CONCLUSION: Depth profiling using LIBS might enable the detection of microscopic tumor spread in bone. In the future, direct electrolyte tracking using LIBS should be applied to other intraoperative challenges in surgical oncology to advance rapid bone analysis by spectroscopic-optical techniques.

Asunto(s)

Neoplasias de la Boca , Potasio , Humanos , Potasio/análisis , Proyectos Piloto , Análisis Espectral/métodos , Calcio/análisis , Rayos Láser , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/cirugía , Electrólitos

RESUMEN

Laser-induced breakdown spectroscopy (LIBS) enables the direct measurement of cell electrolyte concentrations. The utility of LIBS spectra in biomarker studies is limited because these studies rarely consider basic physical principles. The aim of this study was to test the suitability of LIBS spectra as an analytical method for biomarker assays and to evaluate the composition of electrolyte elements in human biomaterial. LIBS as an analytical method was evaluated by establishing KCl calibration curves to demonstrate linearity, by the correct identification of emission lines with corresponding reference spectra, and by the feasibility to use LIBS in human biomaterial, analyzing striated muscle tissues from the oral regions of two patients. Lorentzian peak fit and peak area calculations resulted in better linearity and reduced shot-to-shot variance. Correct quantitative measurement allowed for differentiation of human biomaterial between patients, and determination of the concentration ratios of main electrolytes within human tissue. The clinical significance of LIBS spectra should be evaluated using peak area rather than peak intensity. LIBS might be a promising tool for analyzing a small group of living cells. Due to linearity, specificity and robustness of the proposed analytical method, LIBS could be a component of future biomarker studies.

Asunto(s)

Electrólitos , Rayos Láser , Materiales Biocompatibles , Calibración , Humanos , Análisis Espectral/métodos

RESUMEN

Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials.

Asunto(s)

Atrofia Bulboespinal Ligada al X/sangre , Cromatografía Liquida , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Biomarcadores/sangre , Atrofia Bulboespinal Ligada al X/diagnóstico , Calibración , Cromatografía Liquida/normas , Ensayos Clínicos Fase II como Asunto , Humanos , Masculino , Valor Predictivo de las Pruebas , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray/normas , Espectrometría de Masas en Tándem/normas

RESUMEN

Stearoyl-CoA desaturase 1 (SCD1) plays a role in the development of obesity and related conditions, such as insulin resistance, and potentially also in neurological and heart diseases. The activity of SCD1 can be monitored using the desaturation index (DI), the ratio of product (16:1n-7 and 18:1n-9) to precursor (16:0 and 18:0) fatty acids. Here, different analytical strategies were applied to identify the method which best supports SCD1 biology. A novel effective approach was the use of the SCD1-independent fatty acid (16:1n-10) as a negative control. The first approach was based on a simple extraction followed by neutral loss triglyceride fatty acid analysis. The second approach was based on the saponification of triglycerides followed by fatty acid analysis (specific for the position of the double bond within monounsaturated fatty acids (MUFAs)). In addition to the analytical LC-MS assays, different matrices (plasma total triglyceride fraction and the very low-density lipoprotein (VLDL) fraction) were investigated to identify the best for studying changes in SCD1 activity. Samples from volunteers on a high-carbohydrate diet were analyzed. Both ultra HPLC (UHPLC)-MS-based assays showed acceptable accuracies (75-125% of nominal) and precisions (<20%) for the analysis of DI-specific fatty acids in VLDL and plasma. The most specific assay for the analysis of the liver SCD activity was then validated for specificity and selectivity, intra- and interday accuracy and precision, matrix effects, dilution effects, and analyte stability. After 3 days of high-carbohydrate diet, only the specific fatty acids in human plasma VLDL showed a significant increase in DI and associated SCD1 activity.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Ácidos Grasos/sangre , Espectrometría de Masas/métodos , Dieta , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Humanos , Lipoproteínas VLDL/sangre , Estructura Molecular , Estearoil-CoA Desaturasa/metabolismo

RESUMEN

Deferasirox (Exjade, ICL670, CGP72670) is an iron-chelating drug for p.o. treatment of transfusional iron overload in patients with beta-thalassemia or sickle cell disease. The pharmacokinetics and disposition of deferasirox were investigated in rats. The animals received single intravenous (10 mg/kg) or p.o. (10 or 100 mg/kg) doses of 14C-radiolabeled deferasirox. Biological samples were analyzed for radioactivity (liquid scintillation counting, quantitative whole-body autoradioluminography), for deferasirox and its iron complex [high-performance liquid chromatography (HPLC)/UV], and for metabolites (HPLC with radiodetection, liquid chromatography/mass spectrometry, 1H and 13C NMR, and two-dimensional NMR techniques). At least 75% of p.o.-dosed deferasirox was absorbed. The p.o. bioavailability was 26% at the 10 mg/kg dose and showed an overproportional increase at the 100 mg/kg dose, probably because of saturation of elimination processes. Deferasirox-related radioactivity was distributed mainly to blood, excretory organs, and gastrointestinal tract. Enterohepatic recirculation of deferasirox was observed. No retention occurred in any tissue. The placental barrier was passed to a low extent. Approximately 3% of the dose was transferred into the breast milk. Excretion of deferasirox and metabolites was rapid and complete within 7 days. Key clearance processes were hepatic metabolism and biliary elimination via multidrug resistance protein 2. Deferasirox, iron complex, and metabolites were excreted largely via bile and feces (total > or = 90%). Metabolism included glucuronidation at the carboxylate group (acyl glucuronide M3) and at phenolic hydroxy groups, as well as, to a lower degree, cytochrome P450-catalyzed hydroxylations. Two hydroxylated metabolites (M1 and M2) were administered to rats and were shown not to contribute substantially to iron elimination in vivo.

Asunto(s)

Benzoatos/metabolismo , Benzoatos/farmacocinética , Hierro/metabolismo , Triazoles/metabolismo , Triazoles/farmacocinética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Animales Lactantes/metabolismo , Benzoatos/administración & dosificación , Bilis/química , Análisis Químico de la Sangre , Deferasirox , Circulación Enterohepática , Heces/química , Femenino , Feto/metabolismo , Hierro/análisis , Quelantes del Hierro/administración & dosificación , Quelantes del Hierro/metabolismo , Quelantes del Hierro/farmacología , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Leche Humana/química , Leche Humana/metabolismo , Modelos Biológicos , Estructura Molecular , Placenta/metabolismo , Embarazo , Ratas , Ratas Long-Evans , Ratas Transgénicas , Ratas Wistar , Distribución Tisular , Triazoles/administración & dosificación , Orina/química

RESUMEN

Everolimus (40-O-(2-hydroxyethyl)rapamycin, Certican) is a 31-membered macrolide lactone. In lymphocytes, it inhibits the mammalian target of rapamycin (mTOR) and is used as an immunosuppressant after organ transplantation. Due to its instability in pure organic solvents and insufficient HPLC separation, NMR spectroscopy analysis of its metabolite structures is nearly impossible. Therefore, structural identification based on tandem mass spectrometry (MS/MS) and MS(n) fragmentation patterns is critical. Here, we have systematically assessed the fragmentation pattern of everolimus during liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS and validated the fragment structures by (1) comparison with structurally identified derivatives (sirolimus), (2) high-resolution mass spectrometry, (3) elucidation of fragmentation pathways using ion trap mass spectrometry (up to MS(5)) and (4) H/D exchange. In comparison with the structurally related immunosuppressants tacrolimus and sirolimus, our study was complicated by the low ionization efficiency of everolimus. Detection of positive ions gave the best sensitivity, and everolimus and its fragments were mainly detected as sodium adducts. LC-ESI-MS/MS of everolimus in combination with collision-induced dissociation (CID) resulted in a complex fragmentation pattern and the structures of 53 fragments were identified. These detailed fragmentation pathways of everolimus provided the basis for structural elucidation of all everolimus metabolites generated in vivo und in vitro.

Asunto(s)

Inmunosupresores/química , Sirolimus/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión , Everolimus , Estructura Molecular , Sirolimus/química

RESUMEN

Sensitive radioactivity detection following high performance liquid chromatography (HPLC) separation remains a challenge in many drug metabolism studies with radiolabeled compounds. In this work, solid scintillation counting (SSC) after fraction collection into 96-well plates was evaluated as an off-line radioactivity detection method, in comparison with conventional liquid scintillation counting (LSC). The impact of counting time and biological matrix on the quantification of radiolabeled metabolites and parent drug in samples from animal and human absorption, distribution, metabolism and excretion (ADME) studies was investigated. Three different approaches were used to test whether reliable quantification by off-line SSC detection, which requires an approximately constant counting yield during the entire chromatographic run, can be realized: (i) the measurement of radioactivity-spiked biological blank samples without HPLC separation as an extreme case of biological background, (ii) the measurement of radioactivity-spiked HPLC fractions of biological blank samples and (iii) the comparison of radiochromatograms obtained by off-line SSC and LSC of real samples from ADME studies with radiolabeled compounds. Situations in which variations in SSC yield during an HPLC run are likely to lead to significant errors in quantitation were identified and are discussed. However, examples from a number of animal or human ADME studies showed that in the majority of cases off-line SSC provides very similar quantitative data, compared with the reference method of off-line LSC radioactivity detection. Approaches for validation of the off-line SSC approach in critical cases are discussed. The main advantages of off-line SSC, compared with off-line LSC, are lower detection limits and a substantially higher throughput. Several applications of off-line SSC detection in ADME studies are shown.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Preparaciones Farmacéuticas/análisis , Radioisótopos/análisis , Conteo por Cintilación/métodos , Animales , Preparaciones Farmacéuticas/química , Radioisótopos/química , Ratas , Reproducibilidad de los Resultados

RESUMEN

Liquid chromatography-mass spectrometry (LC-MS) is a common method for profiling biological samples in metabolomics. However, LC-MS data of metabolomic studies are often affected by high noise levels, retention time shifts, and high variability in signal intensities. With a new chip-based nanoelectrospray source it becomes possible to directly infuse complex biological samples such as plasma without any chromatographic separation beforehand. In combination with highly diluted samples and long data acquisition times, the parallel analysis of hundreds of compounds is now possible. In a proof-of-concept study, 10 human plasma samples from females and males were analyzed with the intention to separate the two groups by their different metabolomes. The reproducibility was so high that statistical analysis of the data could be performed without prior normalization. Two groups of female and male samples were separated by a supervised machine learning algorithm, principal component analysis, and hierarchical clustering. Peaks contributing to the group separation were characterized by accurate mass measurement and MS-MS fragmentation and by spiking experiments. The feasibility of direct sample infusion using the new chip-based nanoelectrospray device opens a new dimension for the rapid parallel analysis of complex biological mixtures.

Asunto(s)

Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Espectrometría de Masas/métodos , Nanopartículas/química , Análisis por Matrices de Proteínas/métodos , Proteómica/métodos , Adulto , Precipitación Química , Cromatografía Liquida , Femenino , Humanos , Masculino , Persona de Mediana Edad