Eritrosit galt enzim aktivitesinin belirlenmesi için LC-MS/MS metodu geliştirilmesi, optimizasyonu ve validasyonu
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Tıp Fakültesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaç: Galaktozemi, genellikle galaktoz-1-fosfat üridil transferaz (GALT) eksikliğine bağlı gelişen bir karbonhidrat metabolizması hastalığıdır. GALT eksikliğinin tanısı eritrosit içi enzim aktivitesinin belirlenmesiyle mümkündür. Enzim aktivitesinin belirlenmesine yönelik birçok yöntem tanımlanmıştır. Radyoaktif yöntemler, zahmetli ve tehlikeli olduğu için tercih edilmemektedir. Florometrik yöntemler ise günümüzde tanı ve yenidoğan tarama programlarında en sık kullanılan yöntem olmasına rağmen analitik açıdan yetersiz kalmaktadır. Erken tanı ve tedavinin kritik olduğu bu hastalıkta, enzim aktivitesinin yüksek doğruluk ve güvenirlilik ile belirlenmesi, etkin bir tarama programı ve hastalığın ayırıcı tanısı için oldukça önemlidir. Bu çalışmada, hemolizat ve kuru kan damlası (KKD) örneklerinde GALT aktivitesinin tayinine yönelik, analitik açıdan güçlü, sıvı kromatagrafi-kütle spektrometri (LC-MS/MS) temelli bir yöntem geliştirilmesi; geliştirilen yöntemin valide edilmesi ve florometrik yöntem ile yöntem performansları açısından karşılaştırılması amaçlandı. Ayrıca yöntem için en uygun örnek toplama tüpünün belirlenmesi amacıyla K2EDTA'lı ve lityum heparinli örnek toplama tüplerinin enzim aktivitesine olan etkisi incelendi. Gereç ve Yöntem: Geliştirilen yöntemde izotop işaretli [13C6]-galaktoz-1-fosfat substrat, UDP-N-asetilglikozamin internal standart (İS) olarak kullanıldı. Kromatografik ayrıştırma, Acquity UPLC HSS T3 (1.8 µm, 2.1x50 mm) kolon ile sağlandı. 5 mM amonyum format içeren %50-%50 asetonitril/su çözgen sistemleri mobil faz olarak kullanıldı. Enzimatik reaksiyon sonucunda oluşan izotop işaretli [13C6]-UDP-Gal'un (571/323) kantitasyonu, İS (606/385) pik alanına göre sağlandı ve enzim aktivitesi hesaplandı. Yöntem validasyon parametreleri kapsamında ölçüm limitleri, geri elde, tekrarlanabilirlik, matriks etkisi, seçicilik, taşıma etkisi ve referans aralık çalışmaları yapıldı. Analiz için en uygun örnek tüpünün belirlenmesi ve yöntem performanslarının karşılaştırılması amacıyla, farklı antikoagülan içeren kan tüplerinden elde edilen hemolizat ve KKD örneklerinin enzim aktiviteleri belirlendi ve sonuçlar yorumlandı. Bulgular: Hemolizat örneklerinin GALT aktivitesinin ortalama±SD değeri 12.04±1.88, KKD örneklerinin 3.46±1.06 µmol/grHb/saat olarak belirlendi. Hemolizat yönteminde doğrusal aralık 0.4-100 µM (normalin %1.1-%280'i), KKD yönteminde 0.4-50 µM (normalin %2.4-%310'u) arasındaydı. Gün içi ve günler arası tekrarlanabilirlik çalışmalarında %varyasyon katsayısı (%CV) değerleri <15 bulundu. Geri elde çalışmalarında %90'nın üzerinde geri elde sağlandı. Genel anlamda matriks kaynaklı iyon baskılama tespit edilmedi. KKD örnekleri farklı saklama koşullarında 31 gün boyunca oldukça stabil iken hemolizat örnekleri oda sıcaklığı ve +4 ˚C'de enzim aktivitelerinin yaklaşık %50'sini kaybettiği izlendi. EDTA'lı tüplerden elde edilen KKD örneklerinde ortalama enzim aktivitesi, heparinli tüplerden elde edilen örneklere göre yaklaşık 1.6 kat yüksek tespit edildi. Klasik galaktozemi hastalarının LC-MS/MS ile analizinde herhangi bir enzim aktivitesi saptanmadı. Florometrik yöntem ile <3.5 U/gHb (eşik değer) olarak ölçülen enzim aktiviteleri, çok daha düşük konsantrasyonlarda LC-MS/MS yöntemiyle kantitatif olarak belirlendi. Sonuç: Hemolizat ve KKD örneklerinde, eritrosit içi GALT aktivitesinin yüksek güvenirlilik ve doğruluk ile tayin edilebileceği LC-MS/MS temelli bir yöntem geliştirildi. Yöntem validasyon çalışmaları doğrultusunda ulaşılan performans verileri tatmin edicidir. Çalışmalarda, GALT aktivitesinin tayini için en uygun örneğin K2EDTA içeren tam kan örneği olduğu belirlendi. Geliştirilen yöntem, analitik açıdan karşılaştırılan florometrik yönteme göre üstündür. GALT eksikliğinde enzim aktivitesinin belirlenmesinde LC-MS/MS metodunun kullanılması, hastalığın teşhisinde ve ayırıcı tanısında büyük kolaylıklar sağlayacağı gibi yenidoğan taramalarının etkinliğini artıracağı öngörülmektedir.
Aim: Galactosemia is a carbohydrate metabolism disease that usually develops due to galactose-1-phosphate uridyl transferase (GALT) deficiency. The diagnosis of GALT deficiency is possible by determining the intra-erythrocyte enzyme activity. Many methods for determining enzyme activity have been described. Radioactive methods are not preferred because they are laborious and threatening. Nowadays fluorometric methods are the most frequently used methods in diagnosis and newborn screening programs (NBS) although they are insufficient analytically. As early diagnosis and treatment is critical for galactosemia, determination of enzyme activity with high accuracy and reliability is crucial for an effective screening program and differential diagnosis of the disease. In this study, it was aimed to develop an analytically powerful liquid chromatography-mass spectrometry (LC-MS/MS) based method for the determination of GALT activity in hemolysate and dried blood spot (DBS) samples, to validate the developed method and compare it with the performance of fluorometric method. In addition, the effect of K2EDTA and lithium heparin tubes on enzyme activity was examined in order to determine the most appropriate sample collection tube for the method. Material and Method: In the developed method, the isotope labeled [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine was used as internal standard (IS). Chromatographic separation was achieved with Acquity UPLC HSS T3 (1.8 μm, 2.1 x 50 mm) column. 50%-50% acetonitrile/water solvent systems containing 5 mM ammonium format were used as mobile phase. Quantitation of the isotope labeled [13C6]-UDP-Gal (571/323) formed, as a result of the enzymatic reaction, was achieved according to the peak area of the IS (606/385) and enzyme activity was calculated. In the scope of method validation parameters, measurement limits, recovery, repeatability, matrix effect, selectivity, carryover and reference interval studies were performed. In order to determine the most appropriate sample collection tube for the analysis and to compare the performances of methods, the enzyme activities of both hemolysate and DBS samples obtained from blood tubes containing different anticoagulants were measured and the results were interpreted. Results: The mean ± SD value for GALT activity of hemolysate samples was determined as 12.04±3.68 and 3.46±2.07 μmol/grHb/hour for DBS samples. The linear range was 0.4-100 μM (1.1-280% of normal) in the hemolysate method and 0.4-50 μM (2.4-310% of normal) in the DBS method. The %coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. In recovery studies over 90% recovery was achieved. In general terms, ion suppression from matrix was not detected. While DBS samples were quite stable for 31 days under different storage conditions, hemolysate samples lost approximately 50% of enzyme activities at room temperature and +4 ˚C. Mean enzyme activity for DBS samples obtained from K2EDTA tubes was found approximately 1.6 times higher than samples obtained from heparinized tubes. No enzyme activity was detected with the analysis of classical galactosemia patients’ samples by LC-MS/MS. Enzyme activitiy results reported as <3.5 U/gHb (cut off value) by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method. Conclusion: In the hemolysate and DBS samples a LC-MS/MS based method has been developed in which the intra-erythrocyte GALT activity can be determined with high reliability and accuracy. Performance data obtained in relation with studies of validation method are satisfactory. In the studies, the most appropriate sample for the determination of GALT activity was determined as a whole blood sample containing K2EDTA. The developed method is quite superior to analytical fluorometric methods. It is predicted that the use of LC-MS/MS method in the determining enzyme activity in GALT deficiency will provide great conveniences in diagnosis and differential diagnosis of the disease and increase the effectiveness of newborn screenings.
Aim: Galactosemia is a carbohydrate metabolism disease that usually develops due to galactose-1-phosphate uridyl transferase (GALT) deficiency. The diagnosis of GALT deficiency is possible by determining the intra-erythrocyte enzyme activity. Many methods for determining enzyme activity have been described. Radioactive methods are not preferred because they are laborious and threatening. Nowadays fluorometric methods are the most frequently used methods in diagnosis and newborn screening programs (NBS) although they are insufficient analytically. As early diagnosis and treatment is critical for galactosemia, determination of enzyme activity with high accuracy and reliability is crucial for an effective screening program and differential diagnosis of the disease. In this study, it was aimed to develop an analytically powerful liquid chromatography-mass spectrometry (LC-MS/MS) based method for the determination of GALT activity in hemolysate and dried blood spot (DBS) samples, to validate the developed method and compare it with the performance of fluorometric method. In addition, the effect of K2EDTA and lithium heparin tubes on enzyme activity was examined in order to determine the most appropriate sample collection tube for the method. Material and Method: In the developed method, the isotope labeled [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine was used as internal standard (IS). Chromatographic separation was achieved with Acquity UPLC HSS T3 (1.8 μm, 2.1 x 50 mm) column. 50%-50% acetonitrile/water solvent systems containing 5 mM ammonium format were used as mobile phase. Quantitation of the isotope labeled [13C6]-UDP-Gal (571/323) formed, as a result of the enzymatic reaction, was achieved according to the peak area of the IS (606/385) and enzyme activity was calculated. In the scope of method validation parameters, measurement limits, recovery, repeatability, matrix effect, selectivity, carryover and reference interval studies were performed. In order to determine the most appropriate sample collection tube for the analysis and to compare the performances of methods, the enzyme activities of both hemolysate and DBS samples obtained from blood tubes containing different anticoagulants were measured and the results were interpreted. Results: The mean ± SD value for GALT activity of hemolysate samples was determined as 12.04±3.68 and 3.46±2.07 μmol/grHb/hour for DBS samples. The linear range was 0.4-100 μM (1.1-280% of normal) in the hemolysate method and 0.4-50 μM (2.4-310% of normal) in the DBS method. The %coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. In recovery studies over 90% recovery was achieved. In general terms, ion suppression from matrix was not detected. While DBS samples were quite stable for 31 days under different storage conditions, hemolysate samples lost approximately 50% of enzyme activities at room temperature and +4 ˚C. Mean enzyme activity for DBS samples obtained from K2EDTA tubes was found approximately 1.6 times higher than samples obtained from heparinized tubes. No enzyme activity was detected with the analysis of classical galactosemia patients’ samples by LC-MS/MS. Enzyme activitiy results reported as <3.5 U/gHb (cut off value) by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method. Conclusion: In the hemolysate and DBS samples a LC-MS/MS based method has been developed in which the intra-erythrocyte GALT activity can be determined with high reliability and accuracy. Performance data obtained in relation with studies of validation method are satisfactory. In the studies, the most appropriate sample for the determination of GALT activity was determined as a whole blood sample containing K2EDTA. The developed method is quite superior to analytical fluorometric methods. It is predicted that the use of LC-MS/MS method in the determining enzyme activity in GALT deficiency will provide great conveniences in diagnosis and differential diagnosis of the disease and increase the effectiveness of newborn screenings.
Açıklama
Anahtar Kelimeler
GALT, LC-MS/MS, Yenidoğan Taramaları, Kuru Kan Damlası, Newborn Screening, Dried Blood Spot
