A Quantitative Assay for the Determination of Proline and Hydroxyproline by Capillary Electrophoresis

Abstract

Journal of Liquid ChromatographyPublication details, including instructions for authors and subscription information: ABSTRACTA quantitative spectrophotometric assay for the determination of proline and hydroxyproline is described. The assay is based on the derivatization of the imino acids with the fluorogenic chromophore fluorescamine and the separation of the derivatized analytes by capillary electrophoresis.Fluorescamine reacts efficiently with secondary amino acids to form nonfluorescent aminoenone type chromophores which are easily detected at the low UV region. The assay is simple, rapid, and offers a significant improvement in detection sensitivity when compared with standard colorimetric methods for determining these imino acids. Derivatization with fluorescamine also enhances the degree of resolution of the two imino acids, enabling baseline separation of the derivatized analytes. 1163

Role of Proinflammatory Cytokines IL-18 and IL-1β in Bleomycin-Induced Lung Injury in Humans and Mice

“…The hydroxyproline assay was performed by capillary electrophoresis, as described previously (30). Briefly, lung tissues were freeze dried for 24 to 48 hours.…”

“…An aliquot (500 ml) of the hydrolysate was incubated with 1 ml of 25 mM trisodium phosphate at 608C for 20 minutes, and then 1 ml of 25 M sodium borate was added. A 280-ml aliquot of the sample was conjugated with 40 ml of fluorescamine solution (3 mg/ml fluorescamine in acetone, containing 20 ml of pyridine) for 2 minutes, and analyzed for hydroxyproline at 190-350 nm by capillary electrophoresis (P/ACE System MDQ Capillary Electrophoresis System; Beckman Coulter), as described previously (30). Hydroxy-L-proline (1 mg/ml) and L-proline (1 mg/ml) were dissolved in 0.1 M sodium tetraborate buffer (pH 9.0) and used as a control.…”

Capillary electromigration methods for the study of collagen

“…Fluorescamine reacts readily with secondary amino acids to form nonfluorescent aminoenone-type chromophores which are easily detected in the low UV region [40].…”

Early Postoperative Oral Intake Accelerates Upper Gastrointestinal Anastomotic Healing in the Rat Model

“…After the bursting of the anastomosis, a 1 cm anastomotic segment, i.e., 0.5 cm proximal and distal from the anastomosis, was excised and dissected free of mesenteric fat and adherent omentum. The concentration of hydroxyproline was measured as described by Guzmá n et al [17]. First, the freeze-drying of the specimen and complete hydrolysis with 6 N hydrochloric acid were performed.…”