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ABSTRAK
TELAAH MARKER ARTRITIS REUMATOID BERDASARKAN
ANALISIS PROFIL ELEKTROFEROGRAM ENDAPAN
CARTILAGE OLIGOMERIC MATRIX PROTEIN SERUM
Oleh
Nyi Mekar Saptarini
NIM: 30713009
(Program Studi Doktor Farmasi)
Artritis reumatoid (AR) merupakan gangguan autoimun sistemik, ditandai dengan adanya artritis erosif pada sendi sinovial yang simetris. Penyakit ini menyebabkan nyeri akibat kerusakan sendi, kegagalan fungsi sendi, dan penurunan kualitas hidup bahkan kecacatan. Diagnosis awal dan pengendalian AR memberikan fungsi pemeliharaan dan mencegah terjadinya kecacatan. Cartilage oligomeric matrix protein (COMP) merupakan biomarker potensial untuk memonitor perkembangan kerusakan dan cedera kartilago. COMP merupakan senyawa glikoprotein yang terutama diekspresikan dalam kartilago, tendon, sinovial, dan fibroblas dermal. Fungsi COMP adalah mengkatalisis fibrilogenesis kolagen. Metode penentuan kadar COMP yang paling akurat adalah enzyme-linked immunosorbent assay (ELISA), tetapi metode ini mahal, memerlukan analis yang terlatih, dan tidak dapat membedakan oligomer COMP. Metode alternatif untuk solusi masalah tersebut adalah analisis profil elektroferogram endapan COMP serum yang dapat memisahkan COMP berdasarkan bobot molekul oligomer. Tujuan penelitian ini adalah menganalisis profil elektroferogram endapan COMP serum pasien AR dan individu normal sehingga dapat diaplikasikan untuk telaah marker AR.
Penelitian ini dilakukan setelah mendapatkan persetujuan etik dan izin penelitian dari Komite Etik Penelitian Kesehatan RSUP Dr Hasan Sadikin Bandung. Pasien AR pada penelitian ini adalah pasien AR yang berkunjung ke Poliklinik Reumatologi RSUP Dr. Hasan Sadikin Bandung pada bulan Maret hingga Mei 2016. Sebanyak 50 pasien AR diekslusi, karena memiliki penyakit kronis lain, seperti hipertensi dan diabetes. Pasien AR yang memenuhi kriteria inklusi hanya 30 orang, maka digunakan 30 individu normal sebagai pembanding. Pasien AR terdiri atas 4 pria (13,3%) dan 26 wanita (86,7%) dengan rasio 1:7. Rata-rata usia adalah 44 ± 11 tahun (rentang 20-64 tahun) untuk pasien AR dan 41 ± 13 tahun (rentang 21-60 tahun) untuk individu normal. Wawancara dilakukan terhadap pasien AR untuk mengetahui informasi usia, durasi kekakuan sendi pada pagi hari, jumlah artritis pada 28 sendi (jari tangan, pergelangan tangan, siku, dan lutut), jumlah artritis pada sendi lengan, artritis simetris, lama sakit, terapi obat, penyuntikan dan/atau operasi yang pernah dilakukan, serta anggota keluarga yang memiliki penyakit sendi. Hasil wawancara diolah untuk menghitung nilai aktivitas penyakit dan menentukan korelasi data hasil wawancara dengan data serologi yang diperoleh melalui penelitian.
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Prevalensi AR tertinggi terjadi pada pasien berusia 46-55 tahun (46,7%). Hasil wawancara menunjukkan bahwa pasien mengunjungi dokter setelah menyadari tubuhnya tidak dapat bergerak secara normal. Hal ini menunjukkan bahwa pasien kurang memiliki pengetahuan atau kesadaran terhadap gejala AR. Rata-rata lama sakit adalah 57,6 ± 48,3 bulan (rentang 3-204 bulan) dengan frekuensi tertinggi selama 10-19 bulan (16,7%). Rentang lama sakit sangat lebar dengan kondisi yang bervariasi, bahkan cenderung membaik karena kepatuhan pasien dalam mengkonsumsi obat. Sebanyak 93,3% pasien diberi terapi DMARD (Disease Modifying Anti-Rheumatic Drugs), AINS (anti inflamasi non steroid), dan analgesik. Pemantauan inflamasi pada pasien AR secara rutin dilakukan melalui penentuan laju endap darah (LED). Nilai LED ditentukan menggunakan metode Westergren. Nilai LED pasien AR adalah 22,0 ± 13,0 mm/jam untuk pria dan 30,8 ± 11,1 mm/jam untuk wanita. Nilai ini lebih tinggi dibandingkan dengan nilai LED individu normal. Nilai LED dan kondisi sendi pasien digunakan untuk menghitung nilai aktivitas penyakit (NAP). Rata-rata NAP adalah 3,4 ± 0,2, yaitu kategori aktivitas artritis yang moderat. Hal ini disebabkan pasien telah mendapatkan terapi obat, bahkan tindakan medis berupa kortikosteroid intramuskular, operasi sendi, ataupun kombinasi keduanya.
Faktor reumatoid (FR) merupakan autoantibodi pertama yang ditemukan berhubungan dengan AR. Penentuan FR dilakukan menggunakan metode aglutinasi lateks. Hasil RF positif ditemukan pada 86,7% pasien AR dan 23,3% individu normal. Individu normal memberikan hasil FR positif karena FR memberikan hasil positif untuk penyakit autoimun lain, selain AR, sedangkan pada penelitian ini, tidak dilakukan pemeriksaaan penyakit autoimun lain.
Pemantauan inflamasi juga dapat dilakukan melalui penentuan kadar protein total, karena sistem imun melepaskan protein fase akut, yang tidak terjadi pada individu normal. Kadar protein total ditentukan dengan metode kolorimetri menggunakan pereaksi Bradford. Kadar protein total adalah 104,7 ± 16,3 mg/mL (rentang 64,95-131,26 mg/mL) pada pasien AR dan 92,3 ± 14,5 mg/mL (rentang 58,11-116,53 mg/mL) pada individu normal. Kadar protein total pasien AR dan individu normal berbeda secara signifikan (p = 0,02).
Pasien AR lebih berisiko kehilangan tulang dan fraktur karena inaktivasi fungsi sendi dan terapi glukokortikoid. Kadar kalsium total ditentukan dengan metode kolorimetri menggunakan pereaksi mureksid. Kadar kalsium total pasien AR (75,3 ± 37,4
µg/mL) lebih rendah dibandingkan dengan individu normal (81,5 ± 39,6 µg/mL),
tetapi tidak ada perbedaan yang signifikan (p = 0,56). Hal ini disebabkan seluruh pasien AR memperoleh suplemen kalsium karbonat untuk mempertahankan kadar kalsium dalam tubuh.
Kadar COMP serum ditentukan dengan metode ELISA. Kadar COMP serum pasien AR (873,2 ± 165,6 ng/mL) lebih rendah dibandingkan dengan individu normal (927,4 ± 90,4 ng/mL), tetapi tidak ada perbedaan yang signifikan (p = 0,15). Hal ini disebabkan kepatuhan pasien dalam mengkonsumsi obat. Alasan lain adalah banyaknya protein lain yang mengganggu interaksi COMP dengan kit ELISA.
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Sehingga diperlukan pemisahan COMP, kemudian dilakukan elektroforesis untuk menentukan bentuk oligomer COMP.
Serum subjek memiliki pH 7,54 ± 0,16 untuk pasien AR dan 7,42 ± 0,12 untuk individu normal. COMP diendapkan pada pH isoelektriknya, yaitu 4,36 ± 0,01. Endapan COMP dilarutkan dan dikarakterisasi meliputi penentuan kadar protein total, kalsium, COMP, dan analisis profil elektroferogram. Kadar kalsium tidak terdeteksi dengan metode kolorimetri. Kadar protein total dalam endapan COMP lebih rendah dibandingkan dalam serum. Hal ini menunjukkan pemisahan berdasarkan pH isoelektrik berhasil memisahkan COMP dari protein lain dalam serum, tetapi pemisahan belum sempurna. Hal ini diamati dari kadar COMP dalam endapan COMP hanya meningkat 26,4 ± 2,6% pada pasien AR dan 17,1 ± 4,1% pada individu normal. Kadar protein total dalam endapan COMP pasien AR dan individu normal tidak berbeda secara signifikan (p = 0,19), sedangkan kadar COMP berbeda secara signifikan (p = 0,52 x 10-5). Hal ini menunjukkan bahwa pengendapan pada pH isoelektrik meningkatkan sensitivitas pengukuran.
Pengendapan pada pH isoelektrik diperlukan untuk mendapatkan profil elektroferogram yang lebih baik, karena memisahkan COMP dari protein lain dalam serum. Metode elektroforesis dapat memisahkan COMP berdasarkan bentuk oligomernya. Hal ini diamati dari profil elektroferogram COMP yang maksimal memberikan lima pita, yaitu pentamer, tetramer, trimer, dimer, dan monomer. Intensitas pita pentamer teramati lebih tinggi dibandingkan dengan pita oligomer lainnya. Profil elektroferogram pasien AR didominasi oleh pita pentamer dan dimer, sedangkan profil elektroferogram individu normal didominasi oleh pita tetramer, trimer, dan dimer. Profil elektroferogram pasien AR dapat dibedakan dari individu normal. Hal ini merupakan keunggulan metode elektroforesis dibandingkan dengan ELISA. Penelitian ini menyimpulkan bahwa profil elektroferogram COMP dapat digunakan untuk telaah marker AR.
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ABSTRACT
STUDY OF RHEUMATOID ARTHRITIS MARKER BASED ON
ANALYSIS OF ELECTROPHEROGRAM PROFILE OF CRUDE SERUM
CARTILAGE OLIGOMERIC MATRIX PROTEIN
By
Nyi Mekar Saptarini
NIM: 30713009
(Doctoral Program in Pharmacy)
Rheumatoid arthritis (RA) is a systemic autoimmune disease, characterized by erosive arthritis in symmetric synovial joints. This disease causes pain due to joint damage, joint dysfunction, and decreased quality of life, even disability. Early diagnosis and controlling of RA proposes maintenance function and prevent disability. Cartilage oligomeric matrix protein (COMP) is a potential biomarker to monitor the development of cartilage damage and injury. COMP is a glycoprotein compound which mainly expressed in cartilage, tendon, synovial, and dermal fibroblasts. The function of COMP is to catalyze collagen fibrilogenesis. The most accurate method for the determination of COMP levels is enzyme-linked immunosorbent assay (ELISA), but this method is expensive, requires trained analysts, and can not distinguish the COMP oligomers. An alternative method to solve this problem is analysis of the electropherogram profile of crude COMP serum which can separate the COMP based on the molecular weight of the oligomer. The objective of this study was to analyze the electropherogram profile of crude COMP serum of RA patients and normal individuals so it can be applied for the study of RA marker.
This study was conducted after obtaining ethical clearance and research approval from the Health Research Ethic Committee of RSUP Dr Hasan Sadikin Bandung. RA patients in this study were RA patients who visited the Polyclinic of Rheumatology RSUP Dr. Hasan Sadikin Bandung from March to May 2016. There was 50 of RA patients were excluded, due to other chronic diseases, such as hypertension and diabetes. Only 30 RA patients who fulfilled the inclusion criteria, so 30 normal individuals were used as a comparison. RA patients consisted of 4 men (13.3%) and 26 women (86.7%) with a ratio of 1:7. The average age was 44 ± 11 years (range 20-64 years) for RA patients and 41 ± 13 years (range 21-60 years) for normal individuals. Interviews were conducted on RA patients for information about age, duration of joint stiffness in the morning, number of arthritis in 28 joints (finger, wrist, elbow, and knee), number of arthritis in arm joints, symmetric arthritis, disease duration, drug therapy, injections and/or surgery, and family members who have joint disease. Interview data are processed to calculate the disease activity score and determine the correlation of interview data with serology data which obtained through research.
The highest RA prevalence was occurred in patients aged 46-55 years (46.7%). The interview data showed that the patient visited the doctor after realizing their body could not move normally. This indicates that the patient lacks knowledge or awareness of RA symptoms. The average of disease duration was 57.6 ± 48.3 months (range 3-204 months) with the highest frequency was 10-19 months (16.7%). The range of disease duration was very wide, but various of RA condition, even tends to improve as patient compliance in consumption of
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prescribed drugs. There were 93.3% of patients were treated with DMARDs (Disease Modifying Anti-Rheumatic Drugs), NSAIDs (non-steroidal anti-inflammatory Drugs), and analgesics.
Inflammation monitoring of RA patients is routinely performed by determination of erythrocyte sedimentation rate (ESR). The ESR values are determined using the Westergren method. The ESR values of RA patient were 22.0 ± 13.0 mm/h for men and 30.8 ± 11.1 mm/h for women. These values were higher than the ESR values of normal individuals. The ESR values and joint conditions of the patients were used to calculate the disease activity score, i.e. 3.4 ± 0.2, which categorized as moderate arthritis activity. These were because the patient have been taking drugs therapy, even the medical treatment, intramuscular corticosteroid, joint surgery, or a combination of both.
The rheumatoid factor (RF) is the first autoantibodies which found to be associated with RA. The RF determination was conducted by latex agglutination method. Positive RF was found in 86.7% of RA patients and 23.3% of normal individuals. Normal individuals were given positive RF, because RF was giving positive results for other autoimmune diseases, other than RA, whereas in this study, the other autoimmune disease was not examined.
Inflammation monitoring of RA patients also can be done through determination of total protein content, because the immune system releases acute phase proteins, which do not occur in normal individuals. Total protein content was determined by colorimetric method using Bradford reagent. Total protein content was 104.7 ± 16.3 mg/mL (range 64.95-131.26 mg/mL) in RA patients and 92.3 ± 14.5 mg/mL (range 58.11 to 116.53 mg/mL) in normal individuals. The total protein content of RA patients and normal individuals was significantly different (p = 0.02).
The RA patients have increased risk of bone loss and fracture due to inactivation of joint function and glucocorticoid therapy. Total calcium content was determined by colorimetric method using murexide reagent. Total calcium content of RA patients (75.3 ± 37.4
µ
g/mL) was lower than normal individuals (81.5 ± 39.6µ
g/mL), but no significant difference (p = 0.56). These were because all the RA patients receive calcium carbonate supplements to maintain calcium levels in the body.Serum COMP levels were determined by ELISA method. The serum COMP levels of RA patients (873.2 ± 165.6 ng/mL) were lower than normal individuals (927.4 ± 90.4 ng/mL), but no significant difference (p = 0.15). These were due to patient compliance in consumption of prescribed drugs. Another reason was other proteins in serum that interfere COMP interactions with ELISA kits. So, that required COMP separation, then electrophoresis was performed to determine the form of COMP oligomer.
The pH serum was 7.54 ± 0.16 for RA patients and 7.42 ± 0.12 for normal individuals. COMP was precipitated at its isoelectric pH, i.e. 4.36 ± 0.01. Crude COMP was dissolved and characterized, involved determine total protein content, calcium, COMP, and analyze the electroferogram profile. Calcium levels were not detected by colorimetric method. Total protein content in crude COMP was lower than serum. These were suggested that separation based on the isoelectric pH successfully separates COMP from other proteins in serum, but the separation was not perfect. It was observed from COMP levels in crude COMP only increased 26.4 ± 2.6% in RA patients and 17.1 ± 4.1% in normal individuals. Total protein content in crude COMP of RA patients and normal individuals was not significantly different
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(p = 0.19), whereas COMP levels were different significantly (p = 0.52 x 10-5). It showed that precipitation on isoelectric pH increase the measurement sensitivity.
Precipitation on isoelectric pH was required to obtain better electropherogram profile, because it separate COMP from other proteins in serum. The electrophoresis method can separate COMP based on its oligomeric form. It was observed from the electroferogram profile of COMP giving five bands, i.e. pentamer, tetramer, trimer, dimer, and monomer. Intensity of pentamer bands was observed higher than other oligomers. The electropherogram profile of RA patients can be distinguished from normal individuals. This is an advantage of electrophoretic method compared to ELISA. This study concluded that the electropherogram profile of COMP can be used for study of RA marker.
