RESUMO
We developed and validated a high-performance liquid chromatographic (HPLC) method for quantifying the bone-specific collagen crosslink, lysylpyridinoline (LP), in urine, LP was purified from cortical bone and characterized by spectrophotometry, HPLC, and 1H NMR spectroscopy. Our HPLC detected urinary LP independently of the sample volume and the range of quantification was between 63 nM and 1 microM. Average recovery of added LP standard to human urine samples was 106 +/- 21%. Mean inter- and intraassay CVs, respectively, for urines containing low, medium, and high concentrations of the crosslink LP were 7.4 and 6.3%. This analytical method is more efficient than previously published HPLC assays for LP because of the significant 24-h reduction in urinary sample preparation time. There was agreement between urinary LP concentrations measured with this method and the Metra Pyrilinks-D enzyme immunoassay (r2 = 0.714). These results emphasize the importance of using a thoroughly standardized HPLC assay as the "gold standard" for comparison of results with newly developed immunoassays.
Assuntos
Aminoácidos/urina , Reabsorção Óssea/urina , Cromatografia Líquida de Alta Pressão/métodos , Idoso , Biomarcadores/urina , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Técnicas de Química Analítica/estatística & dados numéricos , Cromatografia Líquida de Alta Pressão/normas , Cromatografia Líquida de Alta Pressão/estatística & dados numéricos , Feminino , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
A nonextraction, competitive, solid-phase enzyme immunoassay with a monoclonal antibody was developed and validated for measuring progesterone in saliva. The antibody was raised against 11 alpha-hydroxyprogesterone hemisuccinate-bovine serum albumin conjugate and was indirectly immobilized to the walls of microtiter wells. The labeled analyte (progesterone-horseradish peroxidase conjugate) was homologous with the immunogen. The lower detection limit (concentration equivalent to B0-3 SD) was 38 pmol/L of saliva sample. We validated the assay with studies to establish the independence of the concentration determined from the volume of saliva assayed, quantitative recovery of progesterone added to saliva, interference from possible cross-reactants, and agreement with a similar assay that incorporated an extraction step. In addition, we determined luteal-phase concentrations of salivary progesterone in normal women and compared the results with those of an older assay involving a polyclonal antibody.
Assuntos
Anticorpos Monoclonais , Técnicas Imunoenzimáticas , Progesterona/análise , Saliva/química , Feminino , Humanos , Técnicas Imunoenzimáticas/estatística & dados numéricos , Fase Luteal , Microquímica , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Extracts of solid-state cultures of Penicillium capsulatum grown on beet pulp exhibit cellulolytic, hemicellulolytic, and pectinolytic activities. Such extracts catalyzed extensive solubilization of untreated beet pulp. The effects of pH, temperature, and endproducts on the saccharification process were investigated.
RESUMO
Culture filtrates of Talaromyces emersonii UCG 208 grown on beet pulp can convert the polysaccharide components of this agricultural waste to soluble sugars. The saccharification process is facilitated if the pulp is milled or incubated with alkali or peracetic acid before addition of enzyme. However, treatment of unmilled pulp with commercial pectinase prior to incubation with Talaromyces filtrate is also very effective; under suitable conditions, complete hydrolysis of total polysaccharides has been achieved.
