Methylxanthines and Methyluric Acids

Caffeine is the most widely consumed central-nervous-system stimulant. It also relaxes smooth muscles, stimulates the cardiac muscle, stimulates diuresis, and appears to be useful in the treatment of some types of headache. Moreover, it has potent antioxidant properties, helps in protecting cells against the UV radiation, and slows down the process of photo-aging of the skin [250]. Theobromine is used as a bronchodilator, as a vasodilator, as a diuretic, and in the treatment of angina pectoris and hypertension [251]. 

The biosynthesis pathway of theobromine and caffeine has been the subject of many studies over the years. Caffeine is produced from the purine nucleotides AMP, GMP, and/or IMP, and theobromine is the immediate precursor of caffeine. 

Various minor routes, dependent upon the broad specificities of the fV-methyltransferases, may also operate in addition to the main caffeine biosynthesis pathway. For instance, caffeine synthase catalyzes the biosynthesis of 3-methylxanthine from xanthine. Paraxanthine is biosynthesized from 7-methylxanthine. 3-Methylxanthine and paraxanthine are immediately converted into caffeine. Paraxanthine is the most active substrate of caffeine synthase, but only limited amounts of paraxanthine accumulate in plant tissues because the N-1 methylation of 7-methylxanthine is very slow [259, 260]. 

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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. 

Georga, K.A. Samanidou, V.F. Papadoyannis, I.N. Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin. J. Chromatogr., B 2001, 759, 209 -218. 

Methylxanthines and methyluric acids are secondary plant metabolites derived from purine nucleotides (Figure 6.59). The most well-known methylxanthines are caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine), which occur in tea (Camellia sinensis. Camellia ptilophylla, and Camellia taliensis) [242,243], coffee (Coffea arabica, Coffea canephora) [244, 245], cacao (Theobroma cacao) [246], and a number of other nonalcoholic beverages of plant origin Ilex paraguariensis, Paullinia cupana. Cola species and Citrus species [247-249]... 

In a narrow sense, purine alkaloids comprise methylxanthines and methyluric acids [1]. This chapter, however, also includes information on cytokinins, which are purine ring-containing phytohormones, and the purine-like neurotoxin alkaloids, saxitoxin and tetrodotoxin. 

Petermann J, Baumann TW (1983) Metabolic relations between methylxanthines and methyluric acids in Cojfea. Plant Physiol 73 961-964... 

Caffeine pharmacokinetics are nonlinear. For example, when comparing a 500 mg dose to a 250 mg dose, the clearance is reduced and elimination half-life is prolonged with the higher dose (Kaplan et al. 1997). Thus, larger doses prolong the action of the drug. Active metabolites of caffeine are paraxanthine, and to a lesser degree, theobromine, and theophylline. Urinary metabolites are I-methylxanthine, l-methyluric acid, and an acetylated uracil derivative. 

The key metabolites of caffeine (a trimethylxanthine) found in plasma, are the dimethylxanthines paraxanthine, theophylline, and theobromine the monomethylxanthine 1-methylxanthine the C-8 oxidized monomethylxanthine 1-methyluric acid and the ring oxidized uracil 5-acetyl-amino-6-amino-3-methyluracil. 

MefaboZ/sm/Excref/on-Xanthines are biotransformed in the liver (85% to 90%) to 1, 3-dimethyluric acid, 3-methylxanthine and 1-methyluric acid 3-methylxanthine accumulates in concentrations approximately 25% of those of theophylline. 

Disposition in the Body. Rapidly absorbed after oral administration bioavailability almost 100%. Metabolic reactions include V-demethylation and oxidation to uric acid derivatives. About 85% of a dose is excreted in the urine in 48 hours with up to 40% of the dose as 1-methyluric acid, 10 to 15% as 1-methylxanthine and up to 35% as 5-acetylamino-6-formylamino-3-methyluracil and 5-acetylamino-6-amino-3-methyluracil other metabolites excreted in the urine include theophylline, 1,7-dimethylxanthine (paraxanthine), 7-methylxanthine, and 1,3-dimethyluric acid. Less than 10% is excreted in the urine as unchanged drug. The extent of V-acetylation is genetically determined. Caffeine, theophylline, theobromine, and paraxanthine are found in plasma from dietary sources especially coffee, tea and cocoa. An average cup of coffee or tea contains approximately 100 mg of caffeine. 

Disposition in the Body. Readily absorbed after oral administration of aminophylline less rapidly absorbed when given as theophylline irregularly absorbed after rectal administration. Rapidly and widely distributed throughout the tissues. Metabolised by V-demethylation to form 3-methylxanthine (which is active but less potent than theophylline), 1,3-dimethyluric acid, and 1-methyluric acid. In adults, about 13% of a dose is excreted in the urine in 24 hours as unchanged drug, with about 15% as... 

Fig. 11.2. Separation of some xanthine derivatives and urinary metabolites Column uBondapak C18 (300x4 mm ID), mobile phase acetonitrile - 0.1 M disodium hydrogen phosphate and 0.1 M sodium dihydrogen phosphate in water (2 38), flow rate 1.5 ml/min, detection UV 254 nm. Peaks 1, uric acid 2, creatinine 3, 1-methyluric acid, 3-methyluric acid and 7-methyluric acid 4, xanthine 5, 7-methylxanthine 6, 1,3-dimethyluric acid 7, 3-methyl-xanthine 8, 1-methylxanthine 9, theobromine 10, 8-chlorotheophyl1ine 11, theophylline and 1,7-dimethylxanthine (paraxanthine) 12, dyphylline 13, caffeine, (reproduced with permission from ref. 80, by courtesy of Clinical Chemistry)...

Precolumn Lichrosorb RP2 10 pm (40x2.1 mm ID), column Ultrasphere ODS 5 pm (250x4.6 mm ID), mobile phase gradient with solvent A 0.01 M sodium acetate and 0.005 M tetrabutylammonium hydrogen sulfate in water (pH 4.9), solvent B same salt concentrations in 50% methanol (pH 4.8). Gradient 0-7.5 min 0 B, 7.5-15 min 0-T5% B, 15-25 min 15-30% B, 25-33 min 30-32% B, 33-38 min 32-45% B and 38-41 min 45-0% 6. Detection UV 280 nm. Peaks 1, xanthine 2, uric acid 3, 3-methyluric acid 4, 7-methyl xanthine 5, 3-methyl xanthine 6, 1-methylxanthine 7, theobromine 8, 3,7-dimethyl uric acid 9, 7-methyluric acid 10, 1-methyluric acid 11, 1,3-dimethyluric acid 12, 1,7-dimethyl xanthine 13, theophylline 14, e-hydroxyethyltheophylline (internal standard) 15, 1,7-dimethyluric acid 16, 1,3,7-trimethyluric acid 17, caffeine, (reproduced with permission from ref. 192, by the courtesy of Journal Chromatographic Science)... 

St-Pierre, M.V. Tesoro, A. Spino, M. MacLeod, S.M. An HPLC method for the determination of theophylline and its metabolites in serum and urine. J.Liq.Chromatogr, 1984, 7, 1593-1608 [extracted dimethyluric acid, methyluric acid, methylxanthine]... 

Hepatic metabolism is predominantly mediated via the cytochrome P-450 (CYP) isoenzymes CYP2E1 and CYP3A4 (8-hydroxylation to 1,3-dimethyluric acid, 40 to 50%), and CYP1A2 (A-demethylation to 3-methylxanthine, 10 to 15%, and 1-methylxanthine). 1-Methylxanthine is further oxidized, by xanthine oxidase, to 1-methyluric acid (15 to 20%). " Caffeine and 3-methylxanthine are the only theophylline metabolites with pharmacological activity, but only... 

However, in two other studies allopurinol 300 mg daily for 7 days did not have any effect on the pharmacokinetics of theophylline, following a single 5-m kg intravenous dose of aminophylline. Similarly, steady-state theophylline levels were not affected by allopurinol 100 mg three times daily in 4 subjects. However, there was an alteration in the proportion of different urinary theophylline metabolites methyluric acid decreased and methylxanthine increased. ... 

Methyluric acids are common decomposition products of methylxanthines such as caffeine. Five such compounds (I- and 7-melhyluric acid, 1,7- and 13-dimethyl-uric acid, and 1,3,7-trimethyluric acid) were extracted from urine and sqjarated on a Cj column (A = 280nm). A two-ramp 95/5— 80/20 (at 8 min)- 70/30 (at 15 min) water (acetate pH 3.5)/methanol gradient generated excellent separation and peak shapes [375]. The linear range was 25 pg/mL to 3 mg/mL with reported detection limits of lOng/mL. 

Theophylline and four of its liver metabolites (1-methyluric acid, 1,3-dimethy-luric acid, 3-methylxanthine, uric acid) were extracted from tissue and baseline resolved on a C,g column (/ = 273nm) using a 13/87 methanol/water (5mM tetrabutylanunonium hydrogensuliate with lOtnM sodium acetate at pH 4.75) mobile phase [588]. Peak shapes were excellent and elution was complete in 20 min. 

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