Absorption and intermediary metabolism of purines and pyrimidines in lactating dairy cows.

About 20 % of ruminal microbial N in dairy cows derives from purines and pyrimidines; however, their intermediary metabolism and contribution to the overall N metabolism has sparsely been described. In the present study, the postprandial patterns of net portal-drained viscera (PDV) and hepatic metabolism were assessed to evaluate purine and pyrimidine N in dairy cows. Blood was sampled simultaneously from four veins with eight hourly samples from four multi-catheterised Holstein cows. Quantification of twenty purines and pyrimidines was performed with HPLC-MS/MS, and net fluxes were estimated across the PDV, hepatic tissue and total splanchnic tissue (TSP). Concentration differences between veins of fifteen purine and pyrimidine nucleosides (NS), bases (BS) and degradation products (DP) were different from zero (P≤ 0·05), resulting in the net PDV releases of purine NS (0·33-1·3 mmol/h), purine BS (0·0023-0·018 mmol/h), purine DP (7·0-7·8 mmol/h), pyrimidine NS (0·30-2·8 mmol/h) and pyrimidine DP (0·047-0·77 mmol/h). The hepatic removal of purine and pyrimidine was almost equivalent to the net PDV release, resulting in no net TSP release. One exception was uric acid (7·9 mmol/h) from which a large net TSP release originated from the degradation of purine NS and BS. A small net TSP release of the pyrimidine DP ß-alanine and ß-aminoisobutyric acid (-0·032 to 0·37 mmol/h) demonstrated an outlet of N into the circulating N pool. No effect of time relative to feeding was observed (P>0·05). These data indicate that considerable amounts of N are lost in the dairy cow due to prominent intermediary degradation of purines, but that pyrimidine N is reusable to a larger extent.

Phase 2 and pharmacodynamic study of oral forodesine in patients with advanced, fludarabine-treated chronic lymphocytic leukemia.

Forodesine is a new and potent purine nucleoside phosphorylase (PNP) inhibitor. Patients with chronic lymphocytic leukemia (CLL) with primary resistance to fludarabine-based therapy or with progressive disease were eligible for oral forodesine (200 mg/d) for up to 24 weeks. Eight patients with median lymphocyte count of 35.9 x 10(9)/L and median serum beta2 microglobulin level of 6.45 mg/L were treated. Six had Rai stage III to IV and were previously heavily treated (median prior therapy = 5). Two had transient decrease in lymphocyte count to normal, whereas in 5, disease progressed. Adverse events were mild. Steady-state level of forodesine ranged from 200 to 1300 nM and did not reach desired 2 microM level. PNP inhibition ranged from 57% to 89% and steady-state 2'-deoxyguanosine (dGuo) concentration median was 1.8 microM. Intracellular deoxyguanosine triphosphate (dGTP) increase was very modest, from median of 6 microM to 10 microM. Compared with in vivo, in vitro incubations of CLL lymphocytes with 10 or 20 microM dGuo and forodesine (2 microM) resulted in accumulation of higher levels of dGTP (40-250 microM) which resulted in increase in apoptosis. Forodesine has biologic activity in CLL; pharmacodynamic parameters suggest that an alternate dosing schedule and/or higher doses to achieve greater intracellular dGTP may be beneficial in this patient population.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.

Simultaneous determination of metacavir and its metabolites in rat plasma using high-performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS).

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of metacavir and its two metabolites in rat plasma was developed and validated. Tinidazole was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. In addition, these analytes were separated using an isocratic mobile phase on a reverse-phase C18 column and analyzed by MS in the selected reaction monitoring mode. The monitored precursor to product-ion transitions for metacavir, 2',3'-dideoxyguanosine, O-methylguanine and the internal standard were m/z 266.0 → 166.0, m/z 252.0 → 152.0, m/z 166.0 → 149.0 and m/z 248.0 → 202.0, respectively. The standard curves were found to be linear in the range of 1-1000 ng/mL for metacavir, 5-5000 ng/mL for 2',3'-dideoxyguanosine and 1-1000 ng/mL for O-methylguanine in rat plasma. The precision and accuracy for both within- and between-batch determination of all analytes ranged from 2.83 to 9.19% and from 95.86 to 111.27%, respectively. No significant matrix effect was observed. This developed method was successfully applied to an in vivo pharmacokinetic study after a single intravenous dose of 20 mg/kg metacavir in rats.

Acute effect of milk on serum urate concentrations: a randomised controlled crossover trial.

OBJECTIVES: Recent observational studies have highlighted the beneficial role of dairy ingestion in gout prevention. The aims of this study were to determine the acute effects of milk ingestion on serum urate concentrations and examine the mechanisms of these effects. METHODS: This was a short-term randomised controlled crossover trial of milk in 16 healthy male volunteers. The following products were tested (each 80 g protein): soy control, early season skim milk, late season skim milk (containing high concentrations of orotic acid, a naturally occurring uricosuric agent) and ultrafiltrated MPC 85 skim milk. Each participant received a single dose of each product in random order. Serum and urine were obtained immediately before and then hourly over a 3 h period after ingestion of each study product. RESULTS: Ingestion of the soy control led to an increase in serum urate concentrations by approximately 10%. In contrast, ingestion of all milks led to a decrease in serum urate concentrations by approximately 10% (p<0.0001). All products (including soy) rapidly increased the fractional excretion of uric acid (FEUA). Late season milk led to a greater increase in FEUA than MPC 85 (p=0.02) and early season milk (p=0.052). There were no differences over time in serum oxypurines or purine-containing nucleosides. However, all products increased the fractional excretion of xanthine. CONCLUSIONS: Intact milk has an acute urate-lowering effect. These data provide further rationale for long-term intervention studies to determine whether such dietary interventions have an adjunctive role in the management of individuals with hyperuricaemia and gout.

Purines and oxypurines in myocardial ischemia.

OBJECTIVE: To evaluate the plasma levels of purine nucleosides and oxypurines in the presence of other risk factors as additional markers for the diagnosis of myocardial ischemia and severity of myocardial infarction. METHODS: A case control study was conducted on 101 patients with ischemic heart disease (stable angina, n=19: unstable angina, n=29: acute myocardial infarction [AMI]; n=53 patients) admitted to the Cardiology Unit at Al-Kadhimyia Teaching Hospital, Baghdad, Iraq from January to November 2007 in addition to 31 healthy controls. Blood samples were aspirated from those with AMI within the first 12 hours of onset of chest pain. Plasma adenosine (ADO), inosine (INO), hypoxanthine (HYP), and xanthine (XAN) were analyzed by high-performance liquid chromatography. RESULTS: The mean plasma ADO, INO, HYP, and XAN levels were raised in unstable angina over the control values. More increase in all nucleosides and oxypurines was reported in the plasma of patients with AMI as compared to the controls and those of stable angina. The INO (p=0.01) and HYP (p=0.001) values were increased significantly in diabetic men with AMI and at age of < or = 54 years. The mean uric acid values were significantly elevated in hypertensives with unstable angina and smokers with stable angina. CONCLUSION: The levels of purines and their catabolites could be used as additional indices for prior or current ischemia. Pretreatment with such nucleosides, or their oxypurine derivatives, is suggested to improve the regional ventricular function after coronary artery occlusion.

A sensitive and specific liquid chromatography-mass spectrometry method for determination of metacavir in rat plasma.

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the quantification of metacavir in rat plasma using tinidazole as an internal standard (I.S.). Following ethyl acetate extraction, the analytes were separated on a Shim-pack ODS (4.6 microm, 150 mm x 2.0 mm I.D.) column and analyzed in selected ion monitoring (SIM) mode with a positive ESI interface using the respective [M+H](+) ions, 266 for metacavir and 248 for tinidazole. The method was validated over the concentration range of 1-600 ng/mL for metacavir. Between and within-batch precisions (R.S.D.%) were all within 15% and accuracy (%) ranged from 92.2 to 105.8%. The lower limit of quantification (LLOQ) was 1 ng/mL. The extraction recovery was on average 89.8%. The validated method was used for the pharmacokinetic study of metacavir in rats.

Oxypurine and purine nucleoside concentrations in renal vein of allograft are potential markers of energy status of renal tissue.

BACKGROUND: Impairment of organ function derived from ischemia-reperfusion injury is an important problem in solid organ transplantation. Cell alterations induced by ischemia prime the tissue for subsequent damage that occurs during the reperfusion phase. Purine nucleosides and oxypurines are products of adenine nucleotides degradation. Reperfusion and reoxygenation are accompanied by production of reactive oxygen species and free radicals, which lead to damage of graft tissue. The aim of this study was to measure concentrations of adenine nucleotides and their metabolites in renal allograft vein as well as in recipient's peripheral veins during the reperfusion period and to evaluate their usefulness as markers of tissue metabolism in kidney allografts. METHODS: The study enrolled 20 renal transplant recipients. The first blood sample was taken from the recipient's ulnar vein before anastomosing of the kidney graft's vessels with recipient's iliac vessels. Samples were then taken from the renal allograft and ulnar veins 5 min after total graft reperfusion measured with an infrared camera. High-performance liquid chromatography (HPLC) was performed to measure whole blood and plasma concentrations of adenosine triphosphate (ATP), adenosine monophosphate (AMP), guanosine (Guo), inosine (Ino), hypoxanthine (Hyp), xanthine (Xan), uric acid (UA), and uridine (Urd). RESULTS: Hyp and Xan concentrations were significantly increased in renal allograft vein after reperfusion as compared with peripheral vein during the pre- and post-reperfusion periods. CONCLUSIONS: The results of the present study suggest that differences in Hyp and Xan concentrations between renal and peripheral veins reflect metabolic alterations in renal tissue during reperfusion and may be useful for graft function monitoring during reperfusion.

Simultaneous determination of a selective adenosine 2A agonist, BMS-068645, and its acid metabolite in human plasma by liquid chromatography-tandem mass spectrometry--evaluation of the esterase inhibitor, diisopropyl fluorophosphate, in the stabilization of a labile ester-containing drug.

BMS-068645 is a selective adenosine 2A agonist that contains a methyl ester group which undergoes esterase hydrolysis to its acid metabolite. To permit accurate determinations of circulating BMS-068645 and its acid metabolite, blood samples must be rapidly stabilized at the time of collection. A sensitive, rapid and specific liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of BMS-068645 and its acid metabolite in human plasma has been developed and validated using diisopropyl fluorophosphate (DFP) as the esterase inhibitor to prevent BMS-068645 from converting to its acid metabolite. The D(5)-stable isotope labeled analogs of BMS-068645 and its metabolite were used as the internal standards (IS). Analytes and IS in plasma containing 20 mM DFP were acidified and extracted into methyl tert-butyl ether. The liquid-liquid extraction effectively eliminated the strong matrix effect caused by the esterase inhibitor. The chromatographic separation was achieved on a Waters Atlantis C18 column with a run time of 4 min. Detection was performed on a Sciex API 4000 with positive ion electrospray mode (ESI/MS/MS), monitoring the ion transitions m/z 487>314 and 473>300 for BMS-068645 and its acid metabolite, respectively. The method was validated over the range from 0.020 to 10.0 ng/mL for BMS-068645 and 0.050 to 10.0 ng/mL for its acid metabolite. Inter- and intra-run precision for the quality control samples during validation were less than 8.7% and 4.0%, respectively, for the two analytes. The assay accuracy was within +/-5.4% of the nominal values. The esterase inhibitor effectively stabilized BMS-068645 during blood collection and storage. Blood collection tubes containing DFP were easily prepared and used at the clinical sites and could be stored at -30 degrees C for 3 months. This method demonstrated adequate sensitivity, specificity, accuracy, precision, stability and ruggedness to support the analysis of human plasma samples in pharmacokinetic studies.

3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine adducts of workers exposed to asbestos fibers.

Asbestos is the commercial name for a group of silicate minerals naturally occurring in the environment and widely used in the industry. Asbestos exposure has been associated with pulmonary fibrosis, mesothelioma, and malignancies, which may appear after a period of latency of 20-40 years. Mechanisms involved in the carcinogenic effects of asbestos are still not fully elucidated, although the oxidative stress theory suggests that phagocytic cells produce large amounts of reactive oxygen species, due to their inability to digest asbestos fiber. We have conducted a mechanistic study to evaluate the association between 3-(2-deoxy-ß-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) adducts, a biomarker of oxidative stress and lipid peroxidation, and asbestos exposure in the peripheral blood of 327 subjects living in Tuscany and Liguria, Italy, stratified by occupational exposure to asbestos. Adduct frequency was significantly greater into exposed subjects with respect to the controls. M1dG per 108 normal nucleotides were 4.0±0.5 (SE) in 156 asbestos workers, employed in mechanic, naval, petrochemical, building industries, and in pottery and ceramic plants, versus a value of 2.3±0.1 (SE) in 171 controls (p<0.001). After stratification for occupational history, the effects persisted in 54 current asbestos workers, mainly employed in building renovation industry (2.9±0.3 (SE)), and in 102 former asbestos workers (4.5±0.7 (SE)), with p-values of 0.033, and <0.001, respectively. A significant effect of smoking on heavy smokers was found (p=0.005). Our study gives additional support to the oxidative stress theory, where M1dG may reflect an additional potential mechanism of asbestos-induced toxicity.

Short-term safety and pharmacodynamics of amdoxovir in HIV-infected patients.

OBJECTIVES: To evaluate the pharmacodynamics and safety of escalating doses of amdoxovir (DAPD) monotherapy administered to treatment-naive and experienced HIV-1-infected patients over 15 days. DESIGN: Ninety patients with plasma HIV-1 RNA levels between 5000 and 250,000 copies/ml were randomized to DAPD 25, 100, 200, 300 or 500 mg twice daily or 600 mg once daily monotherapy [antiretroviral therapy (ART)-naive and ART-experienced] or to add DAPD 300 or 500 mg twice daily to existing ART. After 15 days of dosing, patients were followed for an additional 7 days. METHODS: Antiviral activity was compared between treatment arms using log10 HIV-1 RNA based on average area under the curve minus baseline to day 15. Safety and tolerability was analyzed by incidence of grade 1 to 4 clinical and laboratory adverse events. RESULTS: In ART-naive patients receiving short-term DAPD monotherapy, a median reduction in plasma HIV-1 RNA of 1.5 log10 copies/ml at the highest doses was observed. In ART-experienced patients, the reduction in viral load observed at each dose was less than that observed in treatment-naive patients (reduction of 0.7 log10 at 500 mg twice daily). The incidence of adverse events was similar across groups with the majority of adverse events reported as mild or moderate in severity. Steady-state plasma concentrations of DAPD and dioxolane guanosine followed linear kinetics. CONCLUSIONS: DAPD was well tolerated and produced antiviral activity in treatment-naive and in some treatment-experienced patients. In ART-experienced patients, the antiviral activity was significant in those with no thymidine-analogue mutations and higher baseline CD4+ cell counts.

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%.

Population pharmacokinetic modeling and model validation of a spicamycin derivative, KRN5500, in phase 1 study.

PURPOSE: KRN5500, a novel spicamycin derivative, shows the greatest activity against a human tumor xenograft model and the highest therapeutic index among spicamycin derivatives. KRN5500 is currently under clinical development in Japan and the United States. The objective of this study was to develop a population pharmacokinetic model that describes the KRN5500 plasma concentration versus time data. METHODS: Data were collected from 18 patients entered in a phase 1 study. These patients received KRN5500 3-21 mg/m2 as a 2-h infusion. A total of 219 concentration measurements were available. The data were analyzed using the nonlinear mixed effect model (NONMEM) program. In addition, the basic and final population pharmacokinetic models were evaluated using bootstrapping resampling. RESULTS: The basic model selected was a two-compartment model with a combination of additive and constant coefficient of variation error models. The basic model fitted well not only the original data, but also 100 bootstrap replicates generated from the original data set. With regard to the effect of covariates selected by generalized additive modeling analysis, gender (SEX) and performance status were found to be possible determinants of the volume of central compartment by NONMEM analysis. The final regression model for V1 was V1 = theta V1 (1--SEX x theta SEX), where V1 is the typical population value of the volume of central compartment, and SEX = 0 if the patient is male, otherwise SEX = 1. The final model was fitted to the 200 bootstrapped samples. The mean parameter estimates were within 15% of those obtained with the original data set. CONCLUSIONS: The KRN5500 plasma concentration versus time data obtained from the phase 1 study were well described by the population pharmacokinetic model. Further evaluation by bootstrapping showed that the population pharmacokinetic model was stable.

The efflux of purine nucleobases and nucleosides from the rat brain.

The efflux of purine nucleobases and their nucleosides from the rat brain was investigated using the brain efflux index (BEI) method. Calculated BEI values showed that purine nucleobases had very rapid initial efflux after the intracerebral injection, which was followed by the slower efflux due to the intracellular trapping of labelled molecules and confirmed by the capillary depletion technique. The efflux of ribonucleosides was much slower than the efflux of nucleobases and the structure of the sugar moiety seemed to be important, since a significant difference in the efflux velocity between ribo- and deoxyribonucleosides was observed. The results of self- and cross-inhibition studies suggested that the efflux of test molecules was saturable and that purines shared the same transport system on the abluminal side of the blood-brain barrier.

Hypoxanthine production by ischemic heart demonstrated by high pressure liquid chromatography of blood purine nucleosides and oxypurines.

An isocratic high pressure liquid chromatographic system was developed for the estimation of purine nucleosides and oxypurines in blood. Use was made of a reversed-phase column. Nucleotides derived from erythrocytes affected the separation; these compounds were removed with A12O3. The recovery of the whole clean-up procedure exceeded 75%, and the lower detection limit of the assay for blood metabolites was 0.1 mumol/l. In 6 healthy volunteers, non-resting, the following blood concentrations (mean values +/- S.D. in mumol/l) were observed: adenosine (less than 0.1), inosine (0.2 +/- 0.1), hypoxanthine (2.2 +/- 1.3) and xanthine (0.2 +/- 0.1). In plasma and serum the total amount of these compounds was 1.9 and 5.4 times higher, respectively, presumably due to nucleotide breakdown during blood processing. The myocardial arterial-venous differences of blood purine nucleosides, oxypurines and lactate were subsequently measured in blood samples from 13 patients with angiographically documented ischemic heart disease, undergoing an atrial pacing stress test. No significant release of adenosine, inosine and xanthine by the heart was detectable in this study. The myocardial arterial-venous difference of lactate changed from 0.01 +/- 0.03 mmol/l (mean +/- SEM) at rest, to -0.10 +/- 0.04 mmol/l during pacing (p less than 0.002). Relatively larger changes were observed for hypoxanthine: pacing increased the arterial-venous difference from -0.01 +/- 0.05 to -0.51 +/- 0.17 mumol/l (p less than 0.02). We conclude that the high pressure liquid chromatographic assay of blood hypoxanthine is a useful tool in the diagnosis of ischemic heart disease.

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.

[Determination of purine and pyrimidine derivatives in acid-soluble fractions of organs and tissues using high-performance reverse-phase ion-pair chromatography]. / Opredelenie purinovykh i pirimidinovykh proizvodnykh v sostave kislotorastvorimykh fraktsii organov i tkanei metodom vysokoéffektivnoi zhidkostnoi obrashchenno-faznoi ion-parnoi khromatografii.

Simultaneous separation of the main ribonucleotides, nucleosides and bases was carried out using tetrabutyl ammonium phosphate (TBA) hetaerons with a reversed-phase (C18) packing material. Chromatographic properties of purine and pyrimidine metabolites in radially compressed NovaPak C18 system was studied at various concentrations of acetonitrile and TBA in the mobile phase. The optimal conditions for gradient separation of nucleotides, nucleosides and bases were evaluated. The procedure was used for analysis of blood from healthy persons and patients with acute myocardial infarction.

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.

Malondialdehyde-deoxyguanosine adducts among workers of a Thai industrial estate and nearby residents.

BACKGROUND: Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. OBJECTIVE: We measured the levels of malondialdehyde-deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources. DESIGN: We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution. RESULTS: The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4-6 years of employment among the petrochemical complexes. CONCLUSIONS: Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution.