İdrarda total glikozaminoglikan semi-kantitatif ölçümü için hızlı test yöntemi geliştirilmesi
Küçük Resim Yok
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
Giriş: Mukopolisakkaridozlar (MPS'ler), glikozaminoglikanların (GAG'lar) yıkılmasını katalizleyen lizozomal enzimlerdeki kusurlardan kaynaklı kalıtsal, metabolik bir bozukluk olup lizozomal depo hastalıklarının (LSD) bir alt grubudur. MPS organ disfonksiyonuna yol açan dokularda GAG birikimi ile sonuçlanır. Bu nedenle, GAG'lar MPS için önemli biyobelirteçlerdir. Herhangi bir tedavi olmaksızın, şiddetli MPS formları olan hastalar, yaşamın ilk yirmi yılı içinde ölürler. Bu nedenle GAG'ların doğru, hızlı, hassas ve spesifik ölçümlerinin yapılması gereklidir. Birikmiş GAG'lar çeşitli analiz teknikleri kullanılarak saptanırlar. Mevcut en yaygın yöntemlerden biri 1,9 dimetilmetilen mavisi(DMB) katyonik bir boyanın negatif yüklü GAG'lara bağlanarak miktarının ölçülmesine dayanır. Amaç: MPS tanısında kullanılan, idrarda total GAG analizi için mevcut bulunan DMB boya bağlama yöntemiyle bütünleşmiş kağıt tabanlı, kolay ve hızlı semi-kantitatif bir tanı testi geliştirmektir. Yöntem: Ön işleme tabi tutulan ve ardından DMB emdirilmiş kağıtlara, kondroitin sülfatlı(CS) idrar damlatıldığında oluşan mor renk gözlenmiştir. Daha sonra test sistem; Kağıt türü, ön işlem için kullanılan alkol çözeltisi türü, DMB konsantrasyonu , stabilitazör ilavesi, zaman ve sıcaklık, konroitin sülfat konsantrasyonu ve girişim etkisine göre değerlendirilmiştir. Optimal test şeridi seçilerek standart grafik oluşturulmuştur. Bulgular: Optimizasyon çalışmalarından elde edilen sonuçlara göre kağıt seçilmiş ve hızlı test sistemi oluşturulmuştur. Kağıt etanolde inkübe edilmiş ve DMB10x ile boyanmış ve stabilizatör ilavesine gerek duyulmamıştır. Kurutmalar oda sıcaklığında gece boyu olması uygun bulunmuştur. Bilurubin 0,03 mg/ml , askorbik asit 5 mg/ml ve glukoz 20 mg/ml ye kadar test şeritlerinde girişime sebep olmamıştır. CS içeren idrar ve CS içermeyen idrar arasındaki farkın gözlemlendiği en düşük idrarda ki CS konsantrasyonu 0,04 mg/ml'dir. Optimizasyon çalışmalarından sonra yazılıma göre hesaplanan ortalama renk farklarına karşı idrardaki CS konsantrasyonun kalibrasyon eğrisi çizilmiştir ve doğrusal bir grafik(R2=0,9722 ) elde edilmiştir. Sonuç: Total GAG'ın semi-kantitatif olarak belirlenmesi için DMB boyasının renk değişikliklerine dayalı basit, hızlı ve düşük maliyetli bir ilk kağıt tabanlı test geliştirilmiştir. Tasarlanan test kağıdı ile , normal idrarlar ile CS içeren idrarların ayırt edilebileceği gösterilmiştir. Bu çalışma, MPS riski taşıyan hastaları ön teşhisi için bir araç olarak potansiyel faydasını göstermiştir . Gelecekte , daha fazla araştırmayla birlikte MPS tiplerinin ayrılacağı daha spesifik ve rutinde kullanılabilecek bir kağıt tabanlı yöntem geliştirmek mümkündür. Ayrıca akıllı telefonlar ile kombinasyonu halinde MPS için bir tanı aracı olarak kullanılmasına ek olarak, hastaların tedavi sürecini takip etmek için bir yöntem olarak da kullanılabileceği ön görülmüştür. Anahtar Kelimeler; Kağıt ; Hızlı ; Test ; İdrar ; Mukopolisakkaridozlar ; Glikozaminoglikanlar
Development of Rapid Test Method for Semi-Quantitative Measurement of Total Glycosaminoglycan in Urine Introduction: Mucopolysaccharidoses (MPS) is a genetic disease because of defects in lysosomal enzymes that catalyze the breakdown of glycosaminoglycans (GAGs) and are a subset of lysosomal storage diseases (LSD). MPS results in the accumulation of GAG in tissues leading to organ dysfunction. Therefore, GAGs are important biomarkers for MPS. Without any treatment, patients with severe forms of MPS die within the first twenty years of life. For this reason, accurate, fast, sensitive, and specific measurements of GAGs are required. Accumulated GAGs are detected using various analysis techniques. One of the most common methods available is based on the quantification of a 1.9 dimethyl methylene blue (DMB) cationic dye by binding to negatively charged GAGs. Objective: A fast and easy paper-based semi-quantitative diagnostic control integrated with the existing DMB dye-binding method was developed for the analysis of total GAG in urine for use in MPS preliminary analysis. Method: A purple color was observed when urine with chondroitin sulfate (CS) was dripped onto the pretreated and then DMB-impregnated papers. Then the test system; Paper type, type of alcohol solution used for pretreatment, DMB concentration, stabilizer addition, time and temperature, chondroitin sulfate concentration, and interference effect were evaluated. A standard graph was created by choosing the optimal test strip. Results: According to the results obtained from the optimization studies, the paper was selected and a rapid test system was created. The paper was incubated in ethanol and stained with DMB10x and no stabilizer was added. Drying at room temperature overnight was found suitable. Bilirubin did not cause interference in test strips up to 0.03 mg/ml, ascorbic acid 5 mg/ml, and glucose 20 mg/ml. The lowest CS concentration in the urine where the difference between CS-containing and CS-free urine was observed was 0,04 mg/ml. After the optimization studies, the calibration curve of the CS concentration in the urine was drawn against the mean color differences calculated according to the software, and a linear graph (R2=0.9722 ) was obtained. Conclusion: A simple, rapid, and cost-effective first paper-based test based on color changes of DMB dye was developed for the semi-quantitative determination of total GAG. The designed test has shown that normal urines can be distinguished from urines containing CS. This study demonstrated its potential utility as a tool for pre-diagnosing patients at risk for MPS. In the future, with further research, it is possible to develop a more specific and routinely usable paper-based method of separating MPS types. In addition to being used as a diagnostic tool for MPS in combination with smartphones, it has been predicted that it can also be used as a method to follow the treatment process of patients. Keywords; Paper ; Rapid ; Test ; Urine ; Mucopolysaccharidoses ; Glycosaminoglycans.
Development of Rapid Test Method for Semi-Quantitative Measurement of Total Glycosaminoglycan in Urine Introduction: Mucopolysaccharidoses (MPS) is a genetic disease because of defects in lysosomal enzymes that catalyze the breakdown of glycosaminoglycans (GAGs) and are a subset of lysosomal storage diseases (LSD). MPS results in the accumulation of GAG in tissues leading to organ dysfunction. Therefore, GAGs are important biomarkers for MPS. Without any treatment, patients with severe forms of MPS die within the first twenty years of life. For this reason, accurate, fast, sensitive, and specific measurements of GAGs are required. Accumulated GAGs are detected using various analysis techniques. One of the most common methods available is based on the quantification of a 1.9 dimethyl methylene blue (DMB) cationic dye by binding to negatively charged GAGs. Objective: A fast and easy paper-based semi-quantitative diagnostic control integrated with the existing DMB dye-binding method was developed for the analysis of total GAG in urine for use in MPS preliminary analysis. Method: A purple color was observed when urine with chondroitin sulfate (CS) was dripped onto the pretreated and then DMB-impregnated papers. Then the test system; Paper type, type of alcohol solution used for pretreatment, DMB concentration, stabilizer addition, time and temperature, chondroitin sulfate concentration, and interference effect were evaluated. A standard graph was created by choosing the optimal test strip. Results: According to the results obtained from the optimization studies, the paper was selected and a rapid test system was created. The paper was incubated in ethanol and stained with DMB10x and no stabilizer was added. Drying at room temperature overnight was found suitable. Bilirubin did not cause interference in test strips up to 0.03 mg/ml, ascorbic acid 5 mg/ml, and glucose 20 mg/ml. The lowest CS concentration in the urine where the difference between CS-containing and CS-free urine was observed was 0,04 mg/ml. After the optimization studies, the calibration curve of the CS concentration in the urine was drawn against the mean color differences calculated according to the software, and a linear graph (R2=0.9722 ) was obtained. Conclusion: A simple, rapid, and cost-effective first paper-based test based on color changes of DMB dye was developed for the semi-quantitative determination of total GAG. The designed test has shown that normal urines can be distinguished from urines containing CS. This study demonstrated its potential utility as a tool for pre-diagnosing patients at risk for MPS. In the future, with further research, it is possible to develop a more specific and routinely usable paper-based method of separating MPS types. In addition to being used as a diagnostic tool for MPS in combination with smartphones, it has been predicted that it can also be used as a method to follow the treatment process of patients. Keywords; Paper ; Rapid ; Test ; Urine ; Mucopolysaccharidoses ; Glycosaminoglycans.
Açıklama
07.07.2024 tarihine kadar kullanımı yazar tarafından kısıtlanmıştır
Anahtar Kelimeler
Biyokimya, Biochemistry
