RESUMEN
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.
Asunto(s)
COVID-19/virología , Pulmón/citología , Modelos Biológicos , Organoides/citología , Organoides/virología , SARS-CoV-2/fisiología , Técnicas de Cultivo de Tejidos , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/metabolismo , Células Epiteliales Alveolares/virología , COVID-19/metabolismo , COVID-19/patología , Diferenciación Celular , División Celular , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/virología , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/crecimiento & desarrollo , Subtipo H1N1 del Virus de la Influenza A/fisiología , Integrina alfa6/análisis , Integrina beta4/análisis , Queratina-5/análisis , Organoides/metabolismo , Neumonía Viral/metabolismo , Neumonía Viral/patología , Neumonía Viral/virología , SARS-CoV-2/crecimiento & desarrollo , Análisis de la Célula Individual , Receptor de TWEAK/análisisRESUMEN
The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5 + basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5 + cells contained a distinct ITGA6 + ITGB4 + mitotic population whose proliferation segregated to a TNFRSF12A hi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12A hi subset of FACS-purified ITGA6 + ITGB4 + basal cells from human lung or derivative organoids. In vivo, TNFRSF12A + cells comprised ~10% of KRT5 + basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 apical-out organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.
RESUMEN
Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Guía de Kinetoplastida/genética , Regulación de la Expresión Génica , Marcación de Gen , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de la Célula Individual , TranscriptomaRESUMEN
Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Células Enteroendocrinas/metabolismo , Mucosa Intestinal/lesiones , Mucosa Intestinal/metabolismo , Yeyuno/lesiones , Yeyuno/metabolismo , Células Madre/metabolismo , Animales , Antígenos de Diferenciación/genética , Células Enteroendocrinas/patología , Regulación de la Expresión Génica , Mucosa Intestinal/patología , Yeyuno/patología , Ratones , Ratones Transgénicos , Células Madre/patologíaRESUMEN
The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.
Asunto(s)
Autorrenovación de las Células , Intestinos/citología , Receptores Acoplados a Proteínas G/metabolismo , Células Madre/citología , Células Madre/metabolismo , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Femenino , Humanos , Ligandos , Masculino , Ratones , Organoides/citología , Organoides/crecimiento & desarrollo , Análisis de la Célula Individual , Nicho de Células Madre , Transcriptoma , Ubiquitina-Proteína Ligasas/metabolismo , beta Catenina/metabolismoRESUMEN
Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in â¼6 min, with â¼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from â¼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Transcriptoma , Línea Celular , Femenino , Humanos , Leucocitos Mononucleares/química , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.
Asunto(s)
Haplotipos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , ADN/genética , Genoma Humano , Variación Estructural del Genoma , Células Germinativas , Humanos , Conformación de Ácido Nucleico , Proteínas de Fusión Oncogénica/genética , Polimorfismo de Nucleótido SimpleRESUMEN
Tris(2,2'-bipyridine)ruthenium(II) chemiluminescence was investigated for the detection of 3,4-methylenedioxymethamphetamine (MDMA) and several related compounds in street drug samples. Optimization using flow injection analysis showed that the selectivity of the reagent can be targeted towards the detection of secondary amines by altering the pH of the reaction environment. The greater selectivity of this mode of detection, compared to UV-absorbance, reduces the probability of false positive results from interfering compounds. The detection limit for MDMA under these conditions was 0.48 µM. A HPLC method incorporating post-column tris(2,2'-bipyridine)ruthenium(II) chemiluminescence detection was applied to the determination of MDMA in five street drug samples. The results obtained were in good agreement with quantification performed using traditional UV-absorbance detection, which demonstrates the viability of this method for confirmatory analysis of drug samples. This is the first report of tris(2,2'-bipyridine)ruthenium(II) chemiluminescence for the detection of MDMA and related amphetamine derivatives.
Asunto(s)
Drogas Ilícitas/química , Mediciones Luminiscentes/métodos , N-Metil-3,4-metilenodioxianfetamina/análisis , Compuestos Organometálicos/química , Humanos , Límite de Detección , Detección de Abuso de Sustancias/métodosRESUMEN
Previous applications of manganese(IV) as a chemiluminescence reagent have required the use of formaldehyde to enhance the emission intensity to analytically useful levels. However, this known human carcinogen (by inhalation) is not ideal for routine application. A wide range of alternative enhancers have been examined but to date none have been found to provide the dramatic increase in chemiluminescence intensities obtained using formaldehyde. Herein, we demonstrate that ethanol offers a simple, safe and inexpensive alternative to the use of formaldehyde for manganese(IV) chemiluminescence detection, without compromising signal intensity or sensitivity. For example, chemiluminescence signals for opiate alkaloids using 50-100% ethanol were 0.8-1.6-fold those using 2M formaldehyde. This innocuous alternative enhancer is shown to be a particularly effective for the direct detection of thiols and disulfides by manganese(IV) chemiluminescence, which we have applied to a simple HPLC procedure to determine a series of biomarkers of oxidative stress.
Asunto(s)
Alcaloides/análisis , Analgésicos Opioides/análisis , Disulfuros/análisis , Etanol/química , Formaldehído/química , Mediciones Luminiscentes/métodos , Manganeso/química , Compuestos de Sulfhidrilo/análisis , Análisis de Inyección de Flujo , HumanosRESUMEN
Herein we explore modern fabrication techniques for the development of chemiluminescence detection flow-cells with features not attainable using the traditional coiled tubing approach. This includes the first 3D-printed chemiluminescence flow-cells, and a milled flow-cell designed to split the analyte stream into two separate detection zones within the same polymer chip. The flow-cells are compared to conventional detection systems using flow injection analysis (FIA) and high performance liquid chromatography (HPLC), with the fast chemiluminescence reactions of an acidic potassium permanganate reagent with morphine and a series of adrenergic phenolic amines.
Asunto(s)
Aminas/análisis , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodos , Fenol/análisis , Impresión Tridimensional , Ácidos/química , Resinas Acrílicas/química , Aminas/química , Cromatografía Líquida de Alta Presión , Análisis de Inyección de Flujo/métodos , Morfina/análisis , Morfina/química , Octopamina/análisis , Octopamina/química , Fenol/química , Permanganato de Potasio/química , Reproducibilidad de los Resultados , Sinefrina/análisis , Sinefrina/química , Tiramina/análogos & derivados , Tiramina/análisis , Tiramina/químicaRESUMEN
Acidic potassium permanganate chemiluminescence enables direct post-column detection of glutathione, but its application to assess the redox state of a wider range of biological fluids and tissues is limited by its sensitivity. Herein we show that the simple on-line addition of an aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two orders of magnitude, but also provides a remarkable improvement in the selectivity of the reagent towards thiols such as glutathione (compared to phenols and amino acids that do not possess a thiol group). This enhanced mode of detection was applied to the determination of glutathione and its corresponding disulfide species in homogenised striatum samples taken from both wild type mice and the R6/1 transgenic mouse model of Huntington's disease, at both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease progression had not significantly altered the intracellular redox environment.
Asunto(s)
Disulfuro de Glutatión/análisis , Glutatión/análisis , Luminiscencia , Permanganato de Potasio/química , Animales , Cromatografía Líquida de Alta Presión , Cuerpo Estriado/química , Masculino , Ratones , Ratones TransgénicosRESUMEN
We describe a new approach to multiplex detection for HPLC, exploiting parallel segmented outlet flow - a new column technology that provides pressure-regulated control of eluate flow through multiple outlet channels, which minimises the additional dead volume associated with conventional post-column flow splitting. Using three detectors: one UV-absorbance and two chemiluminescence systems (tris(2,2'-bipyridine)ruthenium(III) and permanganate), we examine the relative responses for six opium poppy (Papaver somniferum) alkaloids under conventional and multiplexed conditions, where approximately 30% of the eluate was distributed to each detector and the remaining solution directed to a collection vessel. The parallel segmented outlet flow mode of operation offers advantages in terms of solvent consumption, waste generation, total analysis time and solute band volume when applying multiple detectors to HPLC, but the manner in which each detection system is influenced by changes in solute concentration and solution flow rates must be carefully considered.
Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación , Alcaloides Opiáceos/análisis , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Diseño de Equipo , Límite de Detección , Mediciones Luminiscentes , Compuestos de Manganeso/química , Compuestos Organometálicos/química , Óxidos/química , Papaver/química , Espectrofotometría UltravioletaRESUMEN
Herein we assess the separation space offered by a liquid chromatography system with an optimised uni-dimensional separation for the determination of the key chemical entities in the highly complex matrix of a tobacco leaf extract. Multiple modes of detection, including UV-visible absorbance, chemiluminescence (acidic potassium permanganate, manganese(IV), and tris(2,2'-bipyridine)ruthenium(III)), mass spectrometry and DPPH radical scavenging were used in an attempt to systematically reduce the data complexity of the sample whilst obtaining a greater degree of molecule-specific information. A large amount of chemical data was obtained, but several limitations in the ability to assign detector responses to particular compounds, even with the aid of complementary detection systems, were observed. Thirty-three compounds were detected via MS on the tobacco extract and 12 out of 32 compounds gave a peak height ratio (PHR) greater than 0.33 on one or more detectors. This paper serves as a case study of these limitations, illustrating why multidimensional chromatography is an important consideration when developing a comprehensive chemical detection system.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Nicotiana/química , Extractos Vegetales/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Alcaloides/análisis , Compuestos de Bifenilo/química , Mediciones Luminiscentes , Manganeso/química , Espectrometría de Masas , Compuestos Organometálicos/química , Picratos/química , Permanganato de Potasio/química , Espectrofotometría UltravioletaRESUMEN
Heroin (3,6-diacetylmorphine) and several important extraction and synthesis impurities (morphine, 6-monoacetylmorphine, codeine and 6-acetylcodeine) were determined in illicit drug samples, using high performance liquid chromatography with 'parallel segmented flow', which enabled the simultaneous use of three complementary modes of detection (UV-absorbance, tris(2,2'-bipyridine)ruthenium(III) chemiluminescence and permanganate chemiluminescence). This rapid and sensitive approach for the analysis of street heroin was used to explore the chemistry of a proposed heroin screening test that is based on the relative response with these two chemiluminescence reagents using flow injection analysis. Although heroin was the major constituent of the six drug samples (between 16% and 67% by mass), the synthetic by-product 6-acetylcodeine (2.5-8.3%) made a greater contribution to the total [Ru(bipy)3](3+) chemiluminescence response of the screening test. The signal with permanganate was primarily due to the presence of 6-monoacetylmorphine (0.9-29%), and was therefore indicative of the degree of sample degradation during clandestine manufacture or poor storage conditions prior to the drug seizure. In the second part of the screening test, the sample is treated with sodium hydroxide, which results in a large increase in the signal with permanganate, due to the rapid hydrolysis of heroin to 6-monoacetylmorphine. As the emission of these two reagents with morphinan-alkaloids and their derivatives largely depends on the substituent at the O(3) position, the slower hydrolysis of 6-monoacetylmorphine to morphine, and 6-acetylcodeine to codeine, did not have a major impact on the characteristic pattern of responses in the screening test.
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Codeína/análogos & derivados , Codeína/análisis , Heroína/análisis , Drogas Ilícitas/química , Mediciones Luminiscentes/métodos , Derivados de la Morfina/análisis , Morfina/análisis , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/química , Cromatografía Líquida de Alta Presión , Análisis de Inyección de Flujo , Humanos , Mediciones Luminiscentes/instrumentación , Compuestos de Manganeso/química , Compuestos Organometálicos/química , Óxidos/química , Hidróxido de Sodio/químicaRESUMEN
The selective increase in the oxidation rate of certain organic compounds with permanganate in the presence of environmental "ligands" and reduced species has been ascribed to the different reactivity of the target compounds toward Mn(III), which bears striking similarities to recent independent investigations into the use of permanganate as a chemiluminescence reagent. In spite of the importance of Mn(III) in the light-producing pathway, the dependence of the oxidation mechanism for any given compound on this intermediate could not be determined solely through the emission intensity. However, target compounds susceptible to single-electron oxidation by Mn(III) (such as bisphenol A and triclosan) can be easily distinguished by the dramatic increase in chemiluminescence intensity when a permanganate reagent containing high, stable concentrations of Mn(III) is used. The differences are accentuated under the low pH conditions that favor the chemiluminescence emission due to the greater reactivity of Mn(III) and the greater influence of complexing agents. This study supports the previously postulated selective role of ligands and reducing agents in permanganate oxidations and demonstrates a new approach to explore the chemistry of environmental manganese redox processes.
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Compuestos de Bencidrilo/química , Contaminantes Ambientales/química , Compuestos de Manganeso/química , Manganeso/química , Óxidos/química , Fenoles/química , Triclosán/química , Concentración de Iones de Hidrógeno , Ligandos , Luminiscencia , Oxidación-ReducciónRESUMEN
We have explored the chemiluminescence response of amino acids and related compounds (including the tripeptide glutathione, and disulfides: cystine, homocystine and glutathione disulfide) with several new adaptations of the permanganate and tris(2,2'-bipyridine)ruthenium(III) ([Ru(bipy)(3)](3+)) reagents and a recently developed colloidal manganese(IV) system. The selectivity of the permanganate reagent can be directed towards tyrosine or thiol compounds like cysteine, homocysteine and glutathione by manipulating reaction conditions (providing limits of detection of 4 nM tyrosine and 5 nM glutathione). Colloidal Mn(IV) produced measureable responses with all analytes, but was most suitable for tryptophan, tyrosine, thiols and disulfides, including α-lipoic acid and dihydrolipoic acid, for which the limits of detection were 30 nM and 20 nM, respectively. A stabilised form of [Ru(bipy)(3)](3+), prepared by oxidising [Ru(bipy)(3)](ClO(4))(2) in acetonitrile, exhibited similar selectivity to that of the conventional aqueous reagent. The [Ru(bipy)(3)](3+) reagent was effective for the detection of secondary amino acids such as proline and hydroxyproline, as well as the disulfide, homocystine.
Asunto(s)
2,2'-Dipiridil/análogos & derivados , Aminoácidos/química , Mediciones Luminiscentes , Manganeso/química , Compuestos Organometálicos/química , Permanganato de Potasio/química , 2,2'-Dipiridil/química , Concentración de Iones de HidrógenoRESUMEN
We have examined a range of new and previously described flow cells for chemiluminescence detection. The reactions of acidic potassium permanganate with morphine and amoxicillin were used as model systems representing the many fast chemiluminescence reactions between oxidising agents and organic analytes, and the preliminary partial reduction of the reagent was exploited to further increase the rates of reaction. The comparison was then extended to high-performance liquid chromatography separations of α- and ß-adrenergic agonists, with permanganate chemiluminescence detection. Flow cells constructed by machining novel channel designs into white polymer materials (sealed with transparent films or plates) have enabled improvements in mixing efficiency and overall transmission of light to the photodetector.
RESUMEN
The quantification of low-molecular mass thiols and disulfides involved in cellular redox processes is hindered by oxidation or degradation of analytes during conventional sample preparation steps (including deproteinization and derivatization). Researchers therefore seek techniques that minimize sample handling and permit direct detection of thiols and disulfides within a single chromatographic separation. We demonstrate a new HPLC procedure for these biologically important analytes that incorporates direct chemiluminescence detection with a manganese(IV) reagent. A mixture of seven thiols and disulfides (cysteine, N-acetylcysteine, homocysteine, glutathione (GSH), glutathione disulfide (GSSG), cystine, and homocystine) in their native forms were separated using a C18 column within 20 min. Detection limits for these analytes ranged from 5 × 10(-8) to 1 × 10(-7) M, and the precision for retention times and peak areas was excellent, with relative standard deviations of less than 0.3% and 2%, respectively. This approach was employed to determine two key biomarkers of oxidative stress, GSH and GSSG, in whole blood taken from 12 healthy volunteers. Samples were deproteinized, centrifuged, and diluted prior to analysis using a simple procedure that was shown to avoid significant artificial oxidation of GSH.
Asunto(s)
Disulfuros/análisis , Mediciones Luminiscentes/métodos , Manganeso/química , Compuestos de Sulfhidrilo/análisis , Acetilcisteína/análisis , Adulto , Biomarcadores/análisis , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión/métodos , Cisteína/sangre , Cistina/análisis , Disulfuros/sangre , Femenino , Glutatión/sangre , Disulfuro de Glutatión/sangre , Homocisteína/análisis , Homocistina/análisis , Humanos , Masculino , Estrés Oxidativo , Compuestos de Sulfhidrilo/sangre , Adulto JovenRESUMEN
In this tutorial we describe the construction of chemiluminescence detectors for high performance liquid chromatography (HPLC), comprising the components required to deliver the chemiluminescence reagent, a coiled-tubing flow cell, photomultiplier tube and detector housing, and various options for data acquisition. We also discuss two state-of-the-art commercially available chemiluminescence detectors for HPLC and other flow analysis methodology.
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Cromatografía Líquida de Alta Presión/instrumentación , Mediciones Luminiscentes/instrumentación , Diseño de Equipo , Indicadores y ReactivosRESUMEN
The significant enhancement of acidic potassium permanganate chemiluminescence by Mn(II) results from the concomitant presence of permanganate and Mn(III) in the reagent solution, which enables rapid production of the excited Mn(II) emitter with a wide range of analytes. Furthermore, the key Mn(III) co-reactant can be quickly generated by reducing permanganate with sodium thiosulfate, instead of the slow (~24 h) equilibration required when Mn(ii) is used. The emission from reactions with analytes such as tyrosine and fenoterol was over two orders of magnitude more intense than with the traditional permanganate reagent.