Simultaneous quantification of purine and pyrimidine bases, nucleosides and their degradation products in bovine blood plasma by high performance liquid chromatography tandem mass spectrometry.

Improved nitrogen utilization in cattle is important in order to secure a sustainable cattle production. As purines and pyrimidines (PP) constitute an appreciable part of rumen nitrogen, an improved understanding of the absorption and intermediary metabolism of PP is essential. The present work describes the development and validation of a sensitive and specific method for simultaneous determination of 20 purines (adenine, guanine, guanosine, inosine, 2'-deoxyguanosine, 2'-deoxyinosine, xanthine, hypoxanthine), pyrimidines (cytosine, thymine, uracil, cytidine, uridine, thymidine, 2'-deoxyuridine), and their degradation products (uric acid, allantoin, ß-alanine, ß-ureidopropionic acid, ß-aminoisobutyric acid) in blood plasma of dairy cows. The high performance liquid chromatography-based technique coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) was combined with individual matrix-matched calibration standards and stable isotopically labelled reference compounds. The quantitative analysis was preceded by a novel pre-treatment procedure consisting of ethanol precipitation, filtration, evaporation and reconstitution. Parameters for separation and detection during the LC-MS/MS analysis were investigated. It was confirmed that using a log-calibration model rather than a linear calibration model resulted in lower CV% and a lack of fit test demonstrated a satisfying linear regression. The method covers concentration ranges for each metabolite according to that in actual samples, e.g. guanine: 0.10-5.0 µmol/L, and allantoin: 120-500 µmol/L. The CV% for the chosen quantification ranges were below 25%. The method has good repeatability (CV%≤25%) and intermediate precision (CV%≤25%) and excellent recoveries (91-107%). All metabolites demonstrated good long-term stability and good stability within-runs (CV%≤10%). Different degrees of absolute matrix effects were observed in plasma, urine and milk. The determination of relative matrix effects revealed that the method was suitable for almost all examined PP metabolites in plasma drawn from an artery and the portal hepatic, hepatic and gastrosplenic veins and, with a few exceptions, also for other species such as chicken, pig, mink, human and rat.

Separation and identification of purine nucleosides in the urine of patients with malignant cancer by reverse phase liquid chromatography/electrospray tandem mass spectrometry.

Urinary-modified nucleosides have a potential role as cancer biomarkers for a number of malignant diseases. High performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, MS/MS analysis and accurate mass measurements in order to identify purine nucleosides purified from urine. Potential purine nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the mass spectrometric techniques. In this manner, numerous modified purine nucleosides were identified in the urine samples from cancer patients including xanthine, adenosine, N1-methyladenosine, 5'-deoxy-5'-methylthioadenosine, 2-methyladenosine, N6-threonylcarbamoyladenosine, inosine, N1-methylinosine, guanosine, N1-methylguanosine, N7-methylguanine, N2-methylguanosine, N2,N2-dimethyguanosine, N2,N2,N7-trimethylguanosine. Furthermore, a number of novel purine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data including N3-methyladenosine, N7-methyladenine, 5'-dehydro-2'-deoxyinosine, N3-methylguanine, O6-methylguanosine, N1,N2,N7-trimethylguanosine, N1-methyl-N2-ethylguanosine and N7-methyl-N1-ethylguanosine.

Analysis of urinary nucleosides. IV. Identification of urinary purine nucleosides by liquid chromatography/electrospray mass spectrometry.

Modified urinary nucleosides are potentially invaluable in cancer diagnosis. High-performance liquid chromatography (HPLC) was combined with full scan mass spectrometry (MS), tandem mass spectrometry and MSn analysis in order to identify purine nucleosides purified from urine. UV peaks evident in the chromatogram were examined by the various mass spectrometric techniques and adenosine, 1-methyladenosine, xanthosine, N1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, N2,N2,N7-trimethylguanosine, inosine, and 1-methylinosine were each identified in the urine samples from cancer patients. The benefits of the use of LC/MS compared with HPLC alone are discussed.

Rapid screening of high-risk patients for disorders of purine and pyrimidine metabolism using HPLC-electrospray tandem mass spectrometry of liquid urine or urine-soaked filter paper strips.

BACKGROUND: A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips. METHODS: Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available. RESULTS: Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96-107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89-110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency. CONCLUSIONS: HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.

Pharmacokinetics of (-)-beta-D-dioxolane guanine and prodrug (-)-beta-D-2,6-diaminopurine dioxolane in rats and monkeys.

(-)-beta-D-Dioxolane guanine (DXG) is a nucleoside analog possessing potent activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), and hepatitis B virus (HBV) in vitro. Owing to the limited aqueous solubility of DXG, (-)-beta-D-2,6-diaminopurine dioxolane (DAPD), a more water-soluble prodrug of DXG, is being developed for clinical use. The purpose of this study was to characterize the pharmacokinetics of DXG after administration of DXG and DAPD to rats and monkeys. After intravenous administration of DXG, plasma concentrations of the nucleoside declined in a biexponential manner, with a terminal-phase half-life of 0.44 +/- 0.14 hr (mean +/- SD) in rats and 2.3 hr in monkeys. Total clearance of DXG was 4.28 +/- 0.99 liters/hr/kg in rats and 0.72 liters/hr/kg in monkeys. Renal excretion of unchanged DXG accounted for approximately 50% of total clearance in both species. Steady state volume of distribution of DXG was 2.30 liters/kg in rats and 1.19 liters/kg in monkeys. After intravenous administration of DAPD to rats, prodrug concentrations declined with a half-life of 0.37 +/- 0.11 hr. DXG was rapidly generated from DAPD, with approximately 61% of the dose of DAPD being converted to DXG. After administration of DAPD to monkeys, only concentrations of metabolite DXG could be determined owing to rapid conversion of DAPD to DXG during sample collection. The half-lives of DAPD and DXG after intravenous administration determined from urinary excretion data were 0.8 +/- 0.4 and 1.6 +/- 0.2 hr, respectively. Oral bioavailability of DAPD was estimated to be approximately 30%.

Two approaches for determining the urinary excretion patterns of nucleosides--HPLC and CE.

Cancer patients excrete in their urine increased amounts of modified purines and pyrimidines. These modified mucleosides are primary constituents of RNA and are synthesized at the macromolecular level. When RNA is catabolized, most of these modified nucleosides cannot be reutilized; consequently, they are excreted. Due to their increased urinary excretion in conjunction with altered RNA turnover in carcinogenesis, they have been proposed as tumour markers. We developed both reversed-phase high performance liquid chromatographic and capillary electrophoretic approaches to determine normal and modified nucleosides in urine. Data obtained with CE are in good accordance with those from HPLC. The reference excretion levels of urinary nucleosides from healthy volunteers were established, and elevated ones of modified nucleosides from 34 patients with different kinds of cancer were observed. The developed methods are suitable for the analysis of large series of samples for clinical studies.

High-performance liquid chromatographic determination of (-)-beta-D-2,6-diaminopurine dioxolane and its metabolite, dioxolane guanosine, using ultraviolet and on-line radiochemical detection.

(-)-beta-D-2,6-Diaminopurine dioxolane (DAPD) and its metabolite dioxolane guanosine (DXG) have potent activity against hepatitis B virus and HIV, in vitro. A reversed-phase HPLC analytical method using UV and on-line radiochemical detection for the determination of DAPD and DXG in monkey serum and urine is described in this report. Retention times for DXG, DAPD and internal standard (2',3'-didehydro-2'deoxythymidine, D4T) were 5.0, 6.0 and 13.0 min, respectively. The extraction recovery was greater than 97% for DAPD and 94% for DXG. The limit of quantitation for UV detection was 100 ng/ml and 125 ng/ml for DXG and DAPD in monkey serum. The standard curves were linear from 0.1 microgram/ml to 5 micrograms/ml for DXG and 0.125 microgram/ml to 5 micrograms/ml for DAPD. For radiochemical detection, calibration curves of standard solutions of DAPD and DXG were linear in the range of 3500 Bq to 32,000 Bq and 7500 Bq to 60,000 Bq. The intra- and inter-day relative standard deviations were less than 7.2% using UV and less than 8.6% using on-line radiochemical detection. The HPLC method was applied to serum and urine samples collected from a male rhesus monkey that was administered 33.3 mg/kg DAPD with 200 microCi of [3H]DAPD intravenously.

Genetic deficiency of purine nucleoside phosphorylase in the mouse. Characterization of partially and severely enzyme deficient mutants.

Two independent mutations of purine nucleoside phosphorylase were identified in the first-generation progeny of male mice that had been treated with the mutagen N-ethylnitrosourea and mated to untreated females. The common allele in inbred strains is Np-1a and the mutants are assigned the gene symbols Np-1e and Np-1f. Heterozygotes had approximately half normal purine nucleoside phosphorylase activity in erythrocytes and activity of homozygotes was 17 and 5% of NP-1A for NP-1E and NP-1F, respectively. The following properties are consistent with both Np-1e and Np-1f being point mutations: the expression of residual but markedly reduced activity with normal Michaelis constants for inosine and phosphate, altered isoelectric points, and increased thermal lability. The reduction in erythrocyte activity was also evident in other tissues. A metabolic consequence of the mutations was increased purine nucleoside excretion. Inosine and guanosine, total 150 +/- 84 microM, and inosine, deoxyinosine, guanosine, and deoxyguanosine, total 1490 +/- 190 microM, were present in urine of Np-1e/Np-1e and Np-1f/Np-1f mice, respectively, but not in normal urine, less than 10 microM.

Experience with a simple high-performance liquid chromatography method for the analysis of purine and pyrimidine nucleosides and bases in biological fluids.

Purine and pyrimidine nucleosides and bases were analysed using an isocratic reverse-phase HPLC technique and utilising simple sample preparation. The method has been successfully used to separate a large number of biologically occurring nucleic acid products within a 30-minute period and to identify them by their chromatographic and optical properties. A number of metabolic disorders have been identified, including hypoxanthine-guanine phosphoribosyl transferase and ornithine carbamyl transferase deficiencies. The method has been in routine use for 2 years performing both quantitative and qualitative analysis and has proved to be robust and reliable.

[Occurrence of purine deoxyribosides in urine (author's transl)]. / Uber das Vorkommen von Purindesoxyribosiden im Harn.

It was shown that purine deoxyribosides are normal constituents of human urine. Healthy probands excrete about 25 microgram (41% of the total urinary deoxyribosides) per 24 h. Purine deoxyribosides were determined as the acid-labile fraction of the total deoxyribosides by the microbiological assay with Lactobacillus acidophilus R 26.

Abnormal purine metabolism and purine overproduction in a patient deficient in purine nucleoside phosphorylase.

To delineate the normal function of purine nucleoside phosphorylase and to understand the pathogenesis of the immune dysfunction associated with deficiency of this enzyme, we studied purine metabolism in a patient deficient in purine nucleoside phosphorylase, her erythrocytes and cultured fibroblasts. She exhibited severe hypouricemia and hypouricosuria but excreted excessive amounts of purines in her urine, the major components of which were inosine and guanosine. Her urine also contained deoxyinosine, deoxyguanosine and uric acid 9-N riboside. The patient's erythrocytes but not her cultured fibroblasts contained increased concentrations of phosphoribosylpyrophosphate and inosine. The metabolic abnormalities resembled those in the erythrocytes of patients with the Lesch-Nyhan syndrome. Purine nucleoside phosphorylase is a necessary component of the major, if not the sole, pathway for the conversion of purine nucleosides and nucleotides to uric acid. The increased intracellular concentrations of inosine may, by inhibiting adenosine deaminase, be related to the immunologic dysfunction.