BACKGROUND: Only three inherited metabolic defects have been identified in purine de novo synthesis (PDNS). We present here CE methods for diagnosing defects in the second half of PDNS (from sixth to tenth enzymatic conversion) based on analysis of aminoimidazole ribosides - dephosphorylated intermediates - in urine. METHODS: Assays were performed in an uncoated fused-silica capillary using two electrophoretic separation systems: 60 mmol/l borate - 2-amino-2-methyl-1-propanol-80 mmol/l sodium dodecylsulfate (pH 9.6) and 200 mmol/l phosphate - sodium (pH 1.8). RESULTS: The reported conditions allowed separation of all metabolites from major urinary constituents with analysis time less than 10 min and separation efficiency of 220 and 350 thousands theoretical plates per meter for borate and phosphate system, respectively. The intra- and interday imprecisions were less than 4.4% and 9.9% CV. Potential usefulness of the methods was demonstrated on samples from a patient with adenylosuccinate lyase deficiency and Chinese hamster ovary cell lines defective in PDNS. CONCLUSIONS: CE is a useful and effective tool in the analysis of aminoimidazole ribosides which enables diagnosis of known as well as not so far identified inherited defects of PDNS pathway.
- MeSH
- adenylsukcinátlyasa nedostatek MeSH
- biosyntetické dráhy MeSH
- buněčné linie MeSH
- časové faktory MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- dítě MeSH
- elektroforéza kapilární metody MeSH
- imidazoly moč MeSH
- kojenec MeSH
- křečci praví MeSH
- lidé MeSH
- mladiství MeSH
- molekulární struktura MeSH
- předškolní dítě MeSH
- purinové nukleosidy biosyntéza MeSH
- referenční hodnoty MeSH
- vrozené poruchy metabolismu aminokyselin diagnóza MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- křečci praví MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 2-aminoimidazole MeSH Prohlížeč
- adenylsukcinátlyasa MeSH
- imidazoly MeSH
- purinové nukleosidy MeSH
AICA-ribosiduria is a recently discovered inherited metabolic disease caused by a defect in final steps of purine de novo biosynthesis-5-amino-4-imidazolecarboxamide ribotide (AICAR)-transformylase/inosinemonophosphate (IMP)-cyclohydrolase (ATIC). A rapid and selective capillary electrophoretic method for screening of patients with AICA-ribosiduria is described. The method is based on direct ultraviolet detection of 5-amino-4-imidazolecarboxamide (AICA) and 5-amino-4-imidazolecarboxamide riboside (AICAr) in untreated urine. Background electrolyte consists of 100mM malonic acid adjusted with gamma-aminobutyric acid (pH 2.7). Under the given separation conditions both compounds of interest are well separated from other substances with separation efficiency of 1020000 and 130000 theoretical plates/m for AICA and AICAr, respectively. Total analysis time is 3 min with the limits of detection of 3.6 microM and 4.5 microM for AICA and AICAr, respectively. The usefulness of the presented method for screening of patients with ATIC deficiency is demonstrated on samples of Chinese hamster ovary cell line defective in ATIC activity, spiked urine samples and urine samples from patients treated with high-dose MTX which do not excrete increased amounts of AICA and AICAr compared to untreated controls (p<0.05). The described method is fast and effective enough for diagnostic applications.
- MeSH
- aminoimidazolkarboxamid analogy a deriváty moč MeSH
- dospělí MeSH
- elektroforéza kapilární metody MeSH
- lidé středního věku MeSH
- lidé MeSH
- ribonukleosidy moč MeSH
- vrozené poruchy metabolismu diagnóza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acadesine MeSH Prohlížeč
- aminoimidazolkarboxamid MeSH
- ribonukleosidy MeSH
