Analysis of Electropherogram Profile of Crude Cartilage Oligomeric Matrix Protein for Rheumatoid Arthritis Diagnosis

1 Department of Pharmacochemistry, School of Pharmacy Institut Teknologi Bandung, Jl Ganesha 10, West Java, 40132, Indonesia. 2 Department of Biochemistry, Faculty of Mathematic and Live Sciences, Institut Teknologi Bandung, Jl Ganesha 10, West Java, 40132, Indonesia. 3 Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, Jl Raya Bandung Sumedang km 21, West Java, 45363, Indonesia.


INTRODUCTION
Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by erosive arthritis in symmetric synovial joints.This disease causes pain due to joint damage and dysfunction, decreased quality of life, and even disability (Schuna, 2007).The RA prevalence varies in different populations, generally 0.5-1.0% in the world (Sack et al., 2010) and 0.1-0.3% in Indonesia (Nainggolan, 2009).Epidemiological data suggest that genetic predisposition and exposure to unknown environmental factors play a role in RA triggering (Schuna, 2007).Early diagnosis and therapy in aggressive RA patients is essential to prevent disability due to late treatment (Vittecoq et al., 2003).Cartilage oligomeric matrix protein (COMP) is a secreted glycoprotein compound that found in extracelullar matrix of skeletal tissue (Holden et al., 2005).COMP determines the fiber composition of collagen type II in cartilage and in collaboration with other matrix proteins, to stabilize the collagen tissue (Halasz et al., 2007).The importance of this function was observed from the presence of COMP and its proteolytic fragments which released into synovial fluid and serum during skeletal dysplasia (Holden et al., 2001).So that COMP can be used as a potential biomarker to monitor the development of cartilage damage and During skeletal dysplasia, COMP and its proteolytic fragments were observed in synovial fluid and serum (Holden et al., 2001).This fact was supported the development of COMP as a biomarker to monitor the development of cartilage damage and injury (Vilim et al., 2002) and support the RA diagnosis (Tseng et al., 2009).The most accurate method of determining COMP levels is the enzyme-linked immunosorbent assay (ELISA), but this method is expensive and requires trained analysts.An alternative method to solve this problem is analysis electropherogram profile of crude COMP.The objective of this study was to analyze the electropherogram profile of crude COMP of RA patients and normal individuals, so it can be applied to RA diagnosis.This study was the first study that uses the crude COMP which is isolated from serum of RA patient and normal individuals then analyzed the electropherogram profile of crude COMP.

Subjects
The study was conducted after being approved by Health Research Ethics Committee of Government Hospital of Dr. Hasan Sadikin Bandung, West Java, Indonesia, No. LB.04.01/A05/EC/075III/2016.The RA patients in Rheumatology Clinic and normal individuals were recruited after getting an explanation and signed the informed consent.This study was a follow-up study, all serum of RA patients and individuals normal were collected from previous studies (Saptarini et al., 2017).

Precipitation of Serum COMP
Precipitation of serum COMP was conducted by converting the serum pH to an isoelectric pH of COMP (pI 4.36 ± 0.01) using 0.1 M citric acid solution pH 1.88 ± 0.01.The solution was centrifuged at 12,000 rpm and 4 °C, the precipitated COMP was separated from the supernatant.The precipitated COMP was reconstituted with citrate buffer pH 7.4 ± 0.01 and stored at -80 °C.

Electrophoresis of Crude COMP
Analysis of electropherogram profile of crude COMP was conducted by electrophoresis using sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) with separating gel of 10% polyacrylamide and retaining gel of 5% polyacrylamide (Laemli, 1970).The electropherograms were scanned and analyzed by Gel Analyzer software.The bands on the electropherograms were marked and compared with protein marker.A plot of logarithmic molecular weight versus Rf was generated from the bands of proteins marker to determine the molecular weight of the unknown protein.If the curve is nearly linear, it can be described by the formula y = mx + b, where y is the logarithmic molecular weight, m is the slope, x is the Rf, and b is the y-intercept (http://www.gelanalyzer.com).

Statistical Analysis
Statistical analyzes were conducted by R i386 3.2.0software for Windows under license from the GNU General Public License (http://cran.r-project.org).The electropherogram profile was grouped based on the bands composition, then analysis of variance was performed to the electropherogram profile groups against COMP levels of serum and crude COMP.The statistically significant difference was expressed if p<0.05.

Precipitation of Serum COMP
Eight normal individual serum (code P5 -P12) could not be used for partial purification, because of a little volume of serum.COMP will precipitate at its isoelectric point (pI 4.36 ± 0.01).At isoelectric points, proteins have a zero net charge, so the electrostatic repulsion between protein molecules is gone which lead to aggregates formation (Harrison et al., 2015).Weak and dilute acid (0.1 M citric acid solution) was used to achieve the isoelectric point.It was because weak or dilute acids minimize hydrolysis and denaturation, but still made the aggregates.Protein precipitation is used to concentrate and purify proteins from various contaminants (Harrison et al., 2015).COMP precipitation was accelerated by centrifugation.The colour of crude COMP was whitish bone.After the filtrate was decanted, the crude COMP was reconstituted with phosphate buffer.

Electrophoresis of Crude COMP
Protein and sodium dodecyl sulfate form an anionic complex with a constant negative charge per unit mass.This results in the electrophoresis separation in polyacrylamide gel depending on the protein molecular weight.There is a linear relationship between the logarithm of molecular weight and the relative migration distance of the protein-SDS complex.Trisglycine buffer systems separate proteins with high pH, thus minimize the protein aggregation and make a good separation (Garfin, 2009).The electropherogram profile of RA patients (Fig. 1) and normal individuals (Fig. 2) showed the presence of bands with molecular weights bigger than 200 kDa with various intensities.These bands were predicted as a pentamer form of COMP.The intensity of these band was proportional to COMP level in crude COMP.The higher the COMP level, the darker the band intensity.COMP levels in the crude COMP have been determined (Saptarini et al., 2017).The electropherogram of two RA patients without disease-modifying antirheumatic drugs (DMARDs), i.e.RP23 and RL27, were different from the RA patient electroferogram with prescribed DMARDs.DMARDs, such as methotrexate, was proven to modify the development of joint damage (Lindqvist et al., 2005), which resulting decreased COMP levels along with improved joints condition in RA patients.
The RP23 electropherogram showed that pentamer form more than tetramer form of COMP.The RL27 electropherogram showed that tetramer form more than pentamer form of COMP.The difference in electropherogram profile may be due to disease duration of RP23 (12 months) are longer than RL27 (3 months) and COMP level of RP23 (22.78 g/mL) higher than RL27 (16.53 g/mL).
Image analysis software allows easy and fast protein analysis.Gel Analyzer software was used to create all graphs from electropherogram, then determine the equation (Table 1).Each equation was used to estimate the unknown protein molecular weight on each band.Estimated protein volume can be determined by comparing the band intensity of the sample to the protein marker.All samples (RA patients and normal individuals) had proteins with molecular weights ranging from 54 to 60 kDa which estimated as fragments of the degraded COMP (Fig. 3).COMP levels were estimated to be comparable to the tetramer and pentamer forms of COMP, due to the limitation of capture antibodies in ELISA kits, i.e. mouse anti-COMP monoclonal antibody, which do not react with monomer, dimer, or trimer forms of COMP.Specific electropherogram profiles can be used to differentiate crude COMP of RA patients from normal individuals.Electropherogram of RA patients were dominated by pentamer and dimer bands, whereas electropherograms of normal individuals were dominated by tetramer, trimer, and dimer bands (Table 2).COMP was found in pentamer and its fragments.This was consistent with the literature that COMP and its proteolytic fragments were released into synovial fluid and serum (Holden et al., 2001).The electropherogram profile based on the band composition were categorized into four groups for RA patients and six groups for normal individuals (Table 3).Analysis of variance in each RA patient group showed no significant difference with COMP level of serum (p = 0.56) and crude COMP (p = 0.28).It was the same results for normal individuals, i.e no significant difference with COMP levels of serum (p = 0.59) and crude COMP (p = 0.32).Analysis of variance the electropherogram profile group of RA patients and normal individuals showed significant difference in COMP levels of crude COMP (p = 0.55 x 10 -3 ).It was showed that the electropherogram profile of RA patients can be differentiated from normal individuals, because of the different distribution of COMP oligomers between RA patients and normal individuals.

Table 1 :
Equation of electropherogram of RA patients and normal individuals.

Table 2 :
Distribution of estimated COMP forms in the samples.

Table 3 :
The subject groups based on the bands composition.