Potential plasma biomarkers for progression of knee osteoarthritis using glycoproteomic analysis coupled with a 2D-LC-MALDI system.
BACKGROUND
Although osteoarthritis (OA) is a highly prevalent joint disease, to date, no reliable biomarkers have been found for the disease. In this study, we attempted to identify factors the amounts of which significantly change in association with the progression of knee OA.


METHODS
A total of 68 subjects with primary knee OA were enrolled in the study. These subjects were followed up over an 18-month period, and plasma and serum samples were obtained together with knee radiographs every 6 months, i.e., 0, 6, 12 and 18 months after the enrollment. Progressors and non-progressors were determined from the changes on radiographs, and plasma samples from those subjects were subjected to N-glycoproteomic 2D-LC-MALDI analysis. MS peaks were identified, and intensities for respective peaks were compared between the progressors and non-progressors to find the peak intensities of which differed significantly between the two groups of subjects. Proteins represented by the chosen peaks were identified by MS/MS analysis. Expression of the identified proteins was evaluated in synovial tissues from 10 OA knee joints by in situ hybridization, western blotting analysis and ELISA.


RESULTS
Among the subjects involved in the study, 3 subjects were determined to be progressors, and 6 plasma and serum samples from these subjects were subjected to the analysis together with another 6 samples from the non-progressors. More than 3000 MS peaks were identified by N-glycoproteomic 2D-LC-MALDI analysis. Among them, 4 peaks were found to have significantly different peak intensities between the progressors and non-progressors. MS/MS analysis revealed that these peaks represented clusterin, hemopexin, alpha-1 acid glycoprotein-2, and macrophage stimulating protein, respectively. The expression of these genes in OA synovium was confirmed by in situ hybridization, and for clusterin and hemopexin, by western blotting analysis and ELISA as well.


CONCLUSIONS
In this study, 4 potential biomarkers were identified as potential prognostic markers for knee OA through N-glycoproteomic analysis. To the best of our knowledge, this is the first report for the use of glycoproteomic technology in exploring potential biomarkers for knee OA.
Department of Pathomechanisms, Clinical Research Center, National Hospital Organization Sagamihara Hospital, Sagamihara, Kanagawa, 228-8522, Japan. n-fukui@sagamihara-hosp.gr.jp.
Isao
I
Fukuda
Takeshi
T
Ishihara
Shigeki
S
Ohmachi
Ikue
I
Sakikawa
Atsushi
A
Morita
Minoru
M
Ikeda
Shoji
S
Yamane
Tomoko
T
Toyosaki-Maeda
Yoshihiko
Y
Takinami
Hiroyuki
H
Okamoto
Yoshito
Y
Numata
Naoshi
N
Fukui