RESUMO
C-reactive protein (CRP) is a clinical biomarker of inflammation, and high levels of CRP correlate with cardiovascular disease. The objectives of this study were to test our hypothesis that oxidized low-density lipoprotein (ox-LDL) induces the release of CRP from human aortic endothelial cells (HAECs) and to optimize several analytical methods to identify CRP released from cultured cells in a model of atherogenic stress. HAECs were incubated with copper-oxidized LDL, and the supernatant was subsequently purified by diethylaminoethyl chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified an optimal buffer for the elution of CRP, which contained 0.05 M sodium phosphate and 2.0 M NaCl (pH 4.5). Purified CRP was digested with trypsin and subjected to high-performance LC with an optimal mobile phase of acetonitrile-water containing 0.1% formic acid (50:50, v/v) and an optimal mobile phase flow rate of 0.2 mL/min. We identified optimal parameters for MS/MS analysis of CRP, including sheath gas pressure (80 psi), capillary temperature (275 °C), collision energy (25%), tube lens offset (-5 V), auxiliary gas pressure (0 psi), and isolation width of parent ion (m/z value = 3). Characterization of CRP was based on the extracted ion chromatograms and selected multiple-reaction monitoring spectra of three peptides (peptide-1, -2, and -3) derived from trypsin-digested intact CRP standard. CRP peptide-2 and peptide-3 were identified in the supernatant of ox-LDL-treated HAECs. Confirmation of CRP was based on LC-MS/MS and enzyme-linked immunosorbent assay analysis of CRP in purified HAEC supernatant, as well as real-time PCR analysis of CRP mRNA levels in HAECs.
Assuntos
Aorta/citologia , Proteína C-Reativa/biossíntese , Proteína C-Reativa/química , Células Endoteliais/metabolismo , Estresse Oxidativo , Sequência de Aminoácidos , Proteína C-Reativa/genética , Proteína C-Reativa/isolamento & purificação , Cromatografia , Células Endoteliais/efeitos dos fármacos , Humanos , Lipoproteínas LDL/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Between May 2002 and October 2006, 19 patients (17 men and 2 women; average age 57.2; range 47-71 years) received anterior corpectomy and fusion for severe ossification of the posterior longitudinal ligament (OPLL) in our department. Preoperative radiological evaluation showed the narrowing by the OPLL exceeded 50% in all cases, and OPLL extended from one to three vertebrae. We followed-up all patients for 12-36 months (mean 18 months). The Japanese Orthopaedic Association (JOA) score before surgery was 9.3 +/- 1.8 (range 5-12) which significantly increased to 14.2 +/- 1.3 (range 11-16) points at the last follow-up (P < 0.01). The improvement rate (IR) of neurological function ranged from 22.2-87.5%, with a mean of 63.2% +/- 15.2%. The operation also provided a significant increase in the cervical lordosis and the cord flatting rate (P < 0.01). No severe neurological complication developed. We therefore concluded that anterior decompression and fusion was effective and safe in the treatment of the selected patients, although OPLL exceeded 50% diameter of the spinal canal.