Çeşitli dokularda β-glukuronidaz aktivitelerinin radyoaktif iyod-131 ile işaretli fenolftalein-glukuronid kullanılarak ölçülmesi
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Dosyalar
Tarih
2004
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Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
β-Glukuronidaz glukuronid türevi bileşikleri hidroliz eden fakat α-veya β-glukozitlere etki etmeyen bir enzimdir. Bu enzim karaciğer, dalak ve böbreklerde en yüksek seviyede bulunur. Fishman ve grubunun yaptığı çalışmalardan homojenize edilmiş insan tümör dokusunun normal dokulara göre daha yüksek seviyede β-glukuronidaz içerdiği tespit edilmiştir. β-Glukuronidaz aktivitesinin tümörlerde çevresindeki dokulara göre 25 kat kadar yüksek olduğu da rapor edilmiştir. Bazı tümör dokularındaki bu enzimatik aktiviteden dolayı sitotoksik ve ayrıca radyotoksik özelliğe sahip işaretli glukuronid türevi bileşikler kanser araştırmalarında oldukça yüksek teşhis ve tedavi potansiyeline sahiptir. β-Glukuronidaz aktivitesinin klasik ölçüm metodu, genellikle, β-glukuronidaz tarafından fenolftalein-glukuronid'den kopartılan fenolftalein miktarının spektrofotometrik olarak ölçülmesine dayanır. Bu çalışmada, spektrofotometrik teknik kullanılarak fenolftalein'in tespit edilebildiği alt-sınır değeri yaklaşık 0.5 μg / mL olarak bulunmuştur. Bunun anlamı, biyolojik örneklerdeki bu seviyeden daha düşük seviyedeki β- glukuronidaz miktarlarının bu yöntem kullanılarak ölçülemeyeceğidir. Bu düşünceden hareketle, biyolojik örneklerdeki β-glukuronidaz seviyelerini çok daha hassas olarak ölçebilmek için yeni bir yöntem geliştirilmiştir. Bunun için, radyoaktivite sayım tekniği kullanılarak β-glukuronidaz miktarını ölçmek için fenolftalein-glukuronid ve fenil-N-glukuronid iyod-131 ile işaretlenmiş, böylece β-glukuronidaz tarafından koparılan iyod-131 ile işaretli fenolftalein ve anilin miktarları spektrofotometrik tekniğe göre çok daha hassas olarak ölçülebilmiştir. İşaretli fenolftalein ve anilin'in tespit edilebildiği alt-sınır değerleri sırasıyla, yaklaşık olarak 8 pg / mL ve 0.4 pg / mL olarak tespit edilmiştir. Oysa, fenolftalein'in spektrofotometrik olarak tespit edilebildiği alt sınır değeri nükleer tekniğin alt-sınır değerine göre 106 kat daha yüksek bulunmuştur. Bu, çok açık 4 olarak nükleer tekniğin üstünlüğünü göstermektedir. Kuşkusuz ki bu sonuçlar, farklı biyolojik örneklerdeki çok düşük β-glukuronidaz seviyelerinin kolaylıkla ölçülebilmesi anlamını taşımaktadır. Bu da, kanser araştırmalarında büyük önem taşımaktadır. Bazı farklı rat dokularındaki β-glukuronidaz aktivitesinin ölçülmesinden elde edilen sonuçlar, β-glukuronidaz seviyelerinin bu yöntem kullanılarak duyarlı olarak ölçülebildiğini de göstermiştir. Ayrıca bu sonuçlar, bu yöntemin uygulanabilme potansiyelini de ortaya koymuştur. Çalışmanın ikinci kısmında, nükleer tekniğin uygulanabilirlik potansiyeli işaretli fenolftaleinglukuronid yerine işaretli fenil-N-glukuronid kullanılarak benzer biyolojik örnekler üzerinde incelenmiştir. Glukuronid türevi olarak fenil-N-glukuronid kullanılarak bazı farklı rat organları üzerinde yapılan bu çalışmalar, fenolftalein-glukuronid ile yapılan çalışmalardan elde edilen sonuçlarla uyum içerisinde çıkmıştır. Biyodağılım çalışmalarından elde edilen sonuçlar ise, fenolftalein ve fenolftaleinglukuronid'in farklı organlarda birikim yapmış olması, bir bileşik ile onun glukuronid türevinin metabolizmadaki farklılığını ortaya koymaktadır. Sintigrafik çalışmalardan elde edilen sonuçlar fenolftalein ve fenolftalein-glukuronid'in metabolizmadan kolayca temizlenemediğini göstermiştir. Ayrıca, hem fenolftalein'in hem de fenolftalein-glukuronid'in özellikle barsak sistemi için iyi birer radyofarmasötik olarak kullanılabilme potansiyellerini de ortaya koymuştur.
β-Glucuronidase is an enzyme hydrolysing glucuronide conjugates but does not act on α-or β-glucosides. The highest concentrations of this enzyme are found in liver, spleen, and kidney. It is well known from the studies by Fishman et al. that homogenized human tumour tissues contain higher β-glucuronidase activity than adjacent uninvolved tissue. In tumour tissues, β-glucuronidase activity reaching up to about 25 times higher of the surrounding tissues has been also reported. The radiolabelled glucuronide conjugates that possess cytotoxic as well as radiotoxic characteristics have very precious diagnostic and therapeutic potential applications in cancer research because of such enzymatic activity of certain kind of tumour tissues. Conventional method of measuring the β-glucuronidase activity in normal and tumour tissues, thus, is measuring spectrophotometrically the amount of phenolphthalein liberated from its glucuronide by β-glucuronidase activity. The lower detection threshold of phenolphthalein by this technique was found as to be about 0.5 μg / mL in this study. This means that of β-glucuronidase quantity below these units of β-glucuronidase in the biological samples and that this has significant implications on the sensibility of this kind of applications in this field. Starting from this consideration, we tried to develop a new nuclear measurement technique for much lower β-glucuronidase units in biological samples. For this purpose, phenolphthalein-glucuronide and also phenyl-N-glucuronide were radioiodinated with 131I aiming the measurement of β-glucuronidase quantities by using the radioactivity counting technique. So, the quantity of phenolphthalein and aniline radioiodinated with 131I and liberated by β-glucuronidase was aimed to be measured much more sensible than the spectrophotometric measurement technique. The lower detection threshold of radioiodinated phenolphthalein and aniline were found to be about 8 pg / mL and 0.4 pg / mL, respectively. Contrarily, the spectrophotometric detection threshold of phenolphthalein was found as to be about 106 times higher than that of the nuclear detection threshold. This clearly indicates the advantage of the nuclear measurement technique. In addition, these results also promise the potential application of this technique in measuring very low β-glucuronidase units of different biological samples in cancer research. The results obtained from the measurement studies on β-glucuronidase measurement in some different rat tissues have shown that the β-glucuronidase units could reasonably be measured by this technique and have promised the potential application of this technique. At the second step of this study, the applicability potential of this technique was examined in detail on considerably different biological samples using two different glucuronide conjugates such as phenolphthalein-glucuronide and phenyl-N-glucuronide in order to test the type of glucuronide conjugate to be use in β-glucuronidase measurements. The preliminary results obtained on some different rat organs by the means of phenyl-N-glucuronide as an N-glucuronide conjugate have shown that the quantity of β-glucuronidase could reasonably be measured and the results were consistent with those obtained with phenolphthalein-glucuronide. Biodistribution studies showed that phenolphthalein and its glucuronide conjugate in different principal organs of rats have clearly showed a difference between a glucuronide conjugate and its aglycone. The results obtained from scintigraphic studies showed that the phenolphthalein and its glucuronide conjugate couldn’t easily be cleared from the metabolism. Consequently, it should be outlined that phenolphthalein and its glucuronide conjugate promise to be used as radiopharmaceutical agents especially for intestinal system diseases.
β-Glucuronidase is an enzyme hydrolysing glucuronide conjugates but does not act on α-or β-glucosides. The highest concentrations of this enzyme are found in liver, spleen, and kidney. It is well known from the studies by Fishman et al. that homogenized human tumour tissues contain higher β-glucuronidase activity than adjacent uninvolved tissue. In tumour tissues, β-glucuronidase activity reaching up to about 25 times higher of the surrounding tissues has been also reported. The radiolabelled glucuronide conjugates that possess cytotoxic as well as radiotoxic characteristics have very precious diagnostic and therapeutic potential applications in cancer research because of such enzymatic activity of certain kind of tumour tissues. Conventional method of measuring the β-glucuronidase activity in normal and tumour tissues, thus, is measuring spectrophotometrically the amount of phenolphthalein liberated from its glucuronide by β-glucuronidase activity. The lower detection threshold of phenolphthalein by this technique was found as to be about 0.5 μg / mL in this study. This means that of β-glucuronidase quantity below these units of β-glucuronidase in the biological samples and that this has significant implications on the sensibility of this kind of applications in this field. Starting from this consideration, we tried to develop a new nuclear measurement technique for much lower β-glucuronidase units in biological samples. For this purpose, phenolphthalein-glucuronide and also phenyl-N-glucuronide were radioiodinated with 131I aiming the measurement of β-glucuronidase quantities by using the radioactivity counting technique. So, the quantity of phenolphthalein and aniline radioiodinated with 131I and liberated by β-glucuronidase was aimed to be measured much more sensible than the spectrophotometric measurement technique. The lower detection threshold of radioiodinated phenolphthalein and aniline were found to be about 8 pg / mL and 0.4 pg / mL, respectively. Contrarily, the spectrophotometric detection threshold of phenolphthalein was found as to be about 106 times higher than that of the nuclear detection threshold. This clearly indicates the advantage of the nuclear measurement technique. In addition, these results also promise the potential application of this technique in measuring very low β-glucuronidase units of different biological samples in cancer research. The results obtained from the measurement studies on β-glucuronidase measurement in some different rat tissues have shown that the β-glucuronidase units could reasonably be measured by this technique and have promised the potential application of this technique. At the second step of this study, the applicability potential of this technique was examined in detail on considerably different biological samples using two different glucuronide conjugates such as phenolphthalein-glucuronide and phenyl-N-glucuronide in order to test the type of glucuronide conjugate to be use in β-glucuronidase measurements. The preliminary results obtained on some different rat organs by the means of phenyl-N-glucuronide as an N-glucuronide conjugate have shown that the quantity of β-glucuronidase could reasonably be measured and the results were consistent with those obtained with phenolphthalein-glucuronide. Biodistribution studies showed that phenolphthalein and its glucuronide conjugate in different principal organs of rats have clearly showed a difference between a glucuronide conjugate and its aglycone. The results obtained from scintigraphic studies showed that the phenolphthalein and its glucuronide conjugate couldn’t easily be cleared from the metabolism. Consequently, it should be outlined that phenolphthalein and its glucuronide conjugate promise to be used as radiopharmaceutical agents especially for intestinal system diseases.
Açıklama
Anahtar Kelimeler
Fenolftalein-glukuronid, Fenil-N-glukuronid, β-Glukuronidaz, Radyoiyodinasyon, İyod-131, Biyodağılım, Sintigrafi, Phenolphthalein-glucuronide, Phenyl-N-glucuronide, β-Glucuronidase, Radioiodination, Iodine-131, Biodistribution, Scintigraphy.
