Determination of plasma cytidine deaminase activity by HPLC

Abstract

Cytidine deaminase is an enzyme of nucleic acid metabolism, the measurement of which has been proposed as a useful test for the early detection of pre-eclamptic toxaemia in pregnancy. The enzyme converts the nucleoside cytidine to uridine, with the release of ammonia, and it is the measurement of this latter compound that forms the basis of the conventional methods for the assay of cytidine deaminase. The low activity of the enzyme requires long incubation times, which in turn increase the possibility of contamination by exogenous ammonia. We have developed a new method for determining cytidine deaminase activity, utilising high performance liquid chromatography to measure the production of uridine. This method uses much shorter incubation times and is unaffected by ammonia contamination. This paper describes the development of the method and its comparison with the established assay. The relative merits of each are discussed. Finally, the adaptation of incubation and chromatographic conditions, in order to measure other enzymes of nucleic acid metabolism which are of clinical interest, is briefly mentioned.

CDA Gene Polymorphisms and Enzyme Activity: Genotype–phenotype Relationship in an Italian–Caucasian Population

“…Different analytical methods exist in the literature for measuring CDA activity based either on spectrophotometric determination [3,32] or HPLC assays [7,15,17,33]. Overall, spectrophotometric methods were more cost effective and simpler to execute but provided higher detection limit compared to HPLC.…”

“…Overall, spectrophotometric methods were more cost effective and simpler to execute but provided higher detection limit compared to HPLC. The HPLC method we used has already been validated by other authors, who also used dFdC as substrate [15,17,33]. Based on previous published results [4,6,16], six candidate functional CDA SNPs were genotyped to determine genotype-phenotype correlation.…”

“…The study protocol was approved by the Joint Ethical Committee (JEC) University of Camerino-Azienda ASUR Marche ZT-10 Camerino, in accordance with the Declaration of Helsinki in its revised edition and with international and local regulatory requirements. n CDA activity CDA activity was assessed by a discontinuous direct HPLC previously reported by Richards et al [17] with slight modifications using an Agilent 1100 system. A total of 50 µl of whole blood collected in EDTA-containing vacutainer tubes and stored at -80°C was thawed and diluted 1:10 with water, stored in ice for 20 min and centrifuged for 5 min at 16,000 g. A total of 200 µl of the supernatant was incubated at 37°C in a reaction mixture containing 272 µl of buffer (0.1 M Tris/HCl pH 7.5 and 0.15 M KCl) and 28 µl of cytidine 4 mM.…”

Increased CDA Expression/Activity in Males Contributes to Decreased Cytidine Analog Half-Life and Likely Contributes to Worse Outcomes with 5-Azacytidine or Decitabine Therapy

“…Conversion of cytidine into uridine by plasma at 37°C was measured by high performance liquid chromatography (HPLC) based on published methods(29). Reaction buffer 0.1 M Tris/HCL pH 7.5 (265µl) was added to 25µl of human plasma followed by addition of cytidine to a final concentration of 4.1 mM and 5-flourouridine 0.381 mM (not metabolized by CDA) as an internal control.…”

Novel PET Probes Specific for Deoxycytidine Kinase

“…The deamination assay solution contained purified CDA (0.05 μg/μL) and 140 μM nucleoside analog in 0.125 M phosphate buffer, pH 7.6. The assay was carried out at 37ºC for 1 hour; products were resolved via HPLC using a Waters C18 μBondapak column (3.9 × 300 mm), as previously reported (14). Elution was isocratic using 3% methanol in 0.l M phosphate buffer (pH 5.5) at a flow rate of 1 mL/min.…”