Xanthines analysis

In the clinical area, the largest share of analytical methods development and publication has centered on the determination of theophylline in various body fluids, since theophylline is used as a bronchodilator in asthma. Monitoring serum theophylline levels is much more helpful than monitoring dosage levels.44 Interest in the assay of other methylxanthines and their metabolites has been on the increase, as evidenced by the citations in the literature with a focus on the analysis of various xanthines and methylxanthines. 

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

In the early days of meteorite analysis, it was difficult to detect N-heterocycles later, the Murchison meteorite was shown to contain xanthine, hypoxanthine, guanine, adenine and uracil (about 1.3 ppm in total). This meteorite seems to contain various classes of basic and neutral N-heterocycles, as well as isomeric alkyl derivatives. 

K.V. Gobi, Y. Sato, and F. Mizutani, Mediatorless superoxide dismutase sensors using cytochrome c-modified electrodes xanthine oxidase incorporated polyion complex membrane for enhanced activity and in-vivo analysis. Electroanalysis 13, 397-403 (2001). 

Many transition metal complexes have been considered as synzymes for superoxide anion dismutation and activity as SOD mimics. The stability and toxicity of any metal complex intended for pharmaceutical application is of paramount concern, and the complex must also be determined to be truly catalytic for superoxide ion dismutation. Because the catalytic activity of SOD1, for instance, is essentially diffusion-controlled with rates of 2 x 1 () M 1 s 1, fast analytic techniques must be used to directly measure the decay of superoxide anion in testing complexes as SOD mimics. One needs to distinguish between the uncatalyzed stoichiometric decay of the superoxide anion (second-order kinetic behavior) and true catalytic SOD dismutation (first-order behavior with [O ] [synzyme] and many turnovers of SOD mimic catalytic behavior). Indirect detection methods such as those in which a steady-state concentration of superoxide anion is generated from a xanthine/xanthine oxidase system will not measure catalytic synzyme behavior but instead will evaluate the potential SOD mimic as a stoichiometric superoxide scavenger. Two methodologies, stopped-flow kinetic analysis and pulse radiolysis, are fast methods that will measure SOD mimic catalytic behavior. These methods are briefly described in reference 11 and in Section 3.7.2 of Chapter 3. 

Thevis M, Opfermann G, Krug O, Schanzer W. 2004. Electrospray ionization mass spectrometric characteri zation and quantitation of xanthine derivatives using isotopically labeled analogues an application for equine doping control analysis. Rapid Commun Mass Spectrom 18 1553. 

Korman, M., Vindevogel, J., and Sandra, P. (1994). Application of micellar electrokinetic chromatography to the quality-control of pharmaceutical formulations — the analysis of xanthine derivatives. Electrophoresis 15, 1304—1309. 

The first biochemical analysis of a selenium-containing XDH was reported in 1999 by Andreesen s group. This preparation was specific for xanthine and did not hydroxylate nicotinic acid. Moreover, the enzyme contained FAD, acid-labile sulfur, iron, and a dinucleotide molybdenum cofactor. Most intriguing was the near-equimolar presence of tungsten and molybdenum. It should be noted that the culture medium contained nearly equimolar levels of these metals, making one wonder whether the specificity of this enzyme for metal may be relaxed (i.e., can use Mo or W). Selenium was also found in the preparation and could be released by treatment with cyanide indicating it was also a labile cofactor. This further confirmed the chemical nature of the cofactor from the NAH enzyme from the same strain. ... 

Upon purification of the XDH from C. purinolyticum, a separate Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV-visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichlor-oindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ... 

A variety of xanthines including caffeine, theobromine, and theophylline have been found from food materials including.coffee, chocolate, and tea (419-420). Theophylline determination in sera has been much studied. The technique allows the determination of theophylline at serum levels of 1.5-20 mg/liter theophylline with sample sizes ranging from 50 to 10 /xl (42 -425). Hill (426) assayed theophylline using 50 /xl of serum and an analysis time of 8 min with good interbatch precision and accuracy. Alternative methods which allow the determination of as little as 0.1 mg/ml (427) or 20 ng theophylline in 10 ml serum have been described (428). 

Accumulation of Cu(II) complexes with xanthine and xanthosine has also been utilized in stripping analysis [74]. Copper(II) indapamide complex was adsorptively accumulated at a HMDE and used for the determination of the ligand in the cathodic stripping step [75]. 

Fe2S2] clusters are part of the molybdenum containing hydroxylases. Typically, apart from molybdenum and two EPR-distinct iron-sulfur centres there can be FAD as additional cofactor. In Chlostridium purinolyticum a selenium-dependent purine hydroxylase has been characterized as molybdenum hydroxylase. The EPR of the respective desulfo molybdenum (V) signal indicated that the Mo-ligands should differ from those of the well known mammalian corollary xanthine oxidase.197 For the bacterial molybdenum hydroxylase quinoline oxidoreductase from Pseudomonas putida an expression system was developed in order to be able to construct protein mutants for detailed analysis. EPR was used to control the correct insertion of the cofactors, specifically of the two [Fe2S2] clusters.198... 

Upon analysis of the non-flavonoid fraction of wine, pyrimidines have been found (65MI307, 79AJEV98) by chromatography on Sep-hadex 6-25 and phenol assayed by colorimetry. Of the nucleic acids (mainly guanine 96 and xanthine 97) less than 5mg/L can be assumed to be present. 

Richter, T., Shultz-Lockyear, L.L., Oleschuk, R.D., Bilitewski, U., Harrison, D.J., Bi-enzymatic and capillary electrophoretic analysis of non-fluorescent compounds in microfluidic devices Determination of xanthine. Sensors Actuators B 2002, 81, 369-376. 

Incubation of a population of cells with tritiated uridine or hypo-xanthine leads to incorporation of radioactivity into the RNA of all but the mitotic cells. Analysis of the grain counts shows these to fall into a Poisson distribution, the median point of which corresponds... 

An alternative to the terrestrial synthesis of the nucleobases is to invoke interstellar chemistry. Martins has shown, using an analysis of the isotopic abundance of 13C, that a sample of the 4.6 billion year old Murchison meteorite which fell in Australia in 1969 contains traces of uracil and a pyrimidine derivative, xanthine. Samples of soil that surrounded the meteor when it was retrieved were also analyzed. They gave completely different results for uracil, consistent with its expected terrestrial origin, and xanthine was undetectable [48], The isotopic distributions of carbon clearly ruled out terrestrial contamination as a source of the organic compounds present in the meteorite. At 0°C and neutral pH cytosine slowly decomposes to uracil and guanine decomposes to xanthine so both compounds could be the decomposition products of DNA or RNA nucleobases. They must have either travelled with the meteorite from its extraterrestrial origin or been formed from components present in the meteorite and others encountered on its journey to Earth. Either way, delivery of nucleobases to a prebiotic Earth could plausibly have been undertaken by meteors. The conditions that formed the bases need not have been those of an early Earth at all but of a far more hostile environment elsewhere in the Solar System. That environment may have been conducive to the production of individual bases but they may never have been able to form stable DNA or RNA polymers this development may have required the less extreme conditions prevalent on Earth. 

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