Theacrine ameliorates experimental liver fibrosis in rats by lowering cholesterol storage via activation of the Sirtuin 3-farnesoid X receptor signaling pathway.

Formulations against liver fibrosis (LF) mitigate the progression of hepatitis to cirrhosis. However, notable toxicity of the currently available anti-LF drugs limits their long-term use. In the study, we aimed to investigate the anti-LF effects of theacrine, a purine alkaloid without obvious toxicity, on high-fat diet-, alcohol-, and carbon tetrachloride-induced LF in rats. The results indicated that 10 and 20 mg/kg of theacrine ameliorated hepatic fibrosis, steatosis, and inflammation in LF rats. Mechanistically, theacrine reduced hepatic stellate cell (HSC)-related α-smooth muscle actin expression, and decreased cholesterol accumulation, followed by decreased expression of transforming growth factor-ß1, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α. In addition, theacrine upregulated the phosphorylation of AMP-activated protein kinase, accompanied by decreased expression of ß-catenin and stearoyl-CoA desaturase 1, and increased the expression of sirtuin 3 (SIRT3). Further investigation revealed that the theacrine-mediated decrease in cholesterol was independent of cholesterol synthesis or low-density lipoprotein (LDL) uptake in hyperlipidemia mice. However, theacrine activated farnesoid X receptor (FXR), a ß-catenin conjugated protein, accompanied with decreased expression of cholesterol 7α-hydroxylase and sterol 12α-hydroxylase. In conclusion, theacrine alleviated experimental LF in rats by lowering cholesterol storage and decreasing cholesterol-related HSC activation. A plausible mechanism of theacrine on cholesterol metabolism may involve activation of SIRT3-FXR signaling pathway followed by decreased intestinal cholesterol absorption.

Attenuation of Tumor Development in Mammary Carcinoma Rats by Theacrine, an Antagonist of Adenosine 2A Receptor.

Caffeine has been reported to induce anti-tumor immunity for attenuating breast cancer by blocking the adenosine 2A receptor. Molecular modeling showed that theacrine, a purine alkaloid structurally similar to caffeine, might be an antagonist of the adenosine 2A receptor equivalent to or more effective than caffeine. Theacrine was further demonstrated to be an effective antagonist of the adenosine 2A receptor as its concurrent supplementation significantly reduced the elevation of AMPK phosphorylation level in MCF-7 human breast cells induced by CGS21680, an agonist of adenosine 2A receptors. In an animal model, the development of mammary carcinoma induced by 7,12-Dimethylbenz[a]anthracene in Sprague-Dawley rats could be attenuated by daily supplement of theacrine of 50 or 100 mg/kg body weight. Both expression levels of cleaved-caspase-3/pro-caspase-3 and granzyme B in tumor tissues were significantly elevated when theacrine was supplemented, indicating the induction of programmed cell death in tumor cells might be involved in the attenuation of mammary carcinoma. Similar to the caffeine, significant elevation of interferon-γ and tumor necrosis factor-α was observed in the serum and tumor tissues of rats after the theacrine supplement of 50 mg/kg body weight. Taken together, theacrine is an effective antagonist of adenosine 2A receptors and possesses great potential to be used to attenuate breast cancer.

Theacrine, a Potent Antidepressant Purine Alkaloid from a Special Chinese Tea, Promotes Adult Hippocampal Neurogenesis in Stressed Mice.

Daily intake of tea has been known to relate to a low risk of depression. In this study, we report that a special variety of tea in China, Camellia assamica var. kucha (kucha), possesses antidepressant effects but with less adverse effects as compared to traditional tea Camellia sinensis. This action of kucha is related to its high amount of theacrine, a purine alkaloid structurally similar to caffeine. We investigated the antidepressant-like effects and mechanisms of theacrine in chronic water immersion restraint stress and chronic unpredictable mild stress mice models. PC12 cells and primary hippocampal neural stem cells were treated with stress hormone corticosterone (CORT) to reveal the potential antidepression mechanism of theacrine from the perspective of adult hippocampus neurogenesis. Results of behavioral and neurotransmitter analysis showed that intragastric administration of theacrine significantly counteracted chronic stress-induced depression-like disorders and abnormal 5-hydroxytryptamine (5-HT) metabolism with less central excitability. Further investigation from both in vivo and in vitro experiments indicated that the antidepressant mechanism of theacrine was associated with promoting adult hippocampal neurogenesis, via the modulation of the phosphodiesterase-4 (PDE4)/cyclic adenosine monophosphate (cAMP)/cAMP response-element binding (CREB)/brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) pathway. Collectively, our findings could promote the prevalence of kucha as a common beverage with uses for health care and contribute to the development of theacrine as a potential novel antidepressant medicine.

A Theacrine-Based Supplement Increases Cellular NAD+ Levels and Affects Biomarkers Related to Sirtuin Activity in C2C12 Muscle Cells In Vitro.

There is evidence in rodents to suggest that theacrine-based supplements modulate tissue sirtuin activity as well as other biological processes associated with aging. Herein, we examined if a theacrine-based supplement (termed NAD3) altered sirtuin activity in vitro while also affecting markers of mitochondrial biogenesis. The murine C2C12 myoblast cell line was used for experimentation. Following 7 days of differentiation, myotubes were treated with 0.45 mg/mL of NAD3 (containing ~2 mM theacrine) for 3 and 24 h (n = 6 treatment wells per time point). Relative to control (CTL)-treated cells, NAD3 treatments increased (p < 0.05) Sirt1 mRNA levels at 3 h, as well as global sirtuin activity at 3 and 24 h. Follow-up experiments comparing 24 h NAD3 or CTL treatments indicated that NAD3 increased nicotinamide phosphoribosyltransferase (NAMPT) and SIRT1 protein levels (p < 0.05). Cellular nicotinamide adenine dinucleotide (NAD+) levels were also elevated nearly two-fold after 24 h of NAD3 versus CTL treatments (p < 0.001). Markers of mitochondrial biogenesis were minimally affected. Although these data are limited to select biomarkers in vitro, these preliminary findings suggest that a theacrine-based supplement can modulate select biomarkers related to NAD+ biogenesis and sirtuin activity. However, these changes did not drive increases in mitochondrial biogenesis. While promising, these data are limited to a rodent cell line and human muscle biopsy studies are needed to validate and elucidate the significance of these findings.

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence.

BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Age-Associated Theophylline Metabolic Activity Corresponds to the Ratio of 1,3-Dimethyluric Acid to Theophylline in Mice.

Age is known as one of influencing factor for theophylline (TP)-metabolizing capacity. In a previous our study, the ratio of TP and its major metabolite 1,3-dimethyluric acid (DMU) in serum (DMU/TP) is a useful index to estimate TP-metabolizing capacity, and this value markedly increased by influencing factor, such as the history of smoking. However, it is unknown whether DMU/TP values in serum reflect age-associated changes of TP-metabolizing capacity. In this study, the effect of age on the DMU/TP values in serum were investigated using mice of different age due to the limited blood sampling in human. The concentrations of TP and its metabolites in mouse serum were simultaneously measured using HPLC. As observed in human serum, serum TP concentrations were closely correlated with DMU concentration in mice, which indicates that the DMU/TP ratio is a good indicator of TP metabolic ability in mice. When TP was administered subcutaneously in 2-28-week-old mice, age-associated changes in the DMU/TP ratio in mice were observed. In conclusion, age-associated changes in TP-metabolizing capacity can be estimated by the DMU/TP ratio in serum.

Theacrine attenuates myocardial fibrosis after myocardial infarction via the SIRT3/ß-catenin/PPARγ pathway in estrogen-deficient mice.

OBJECTIVE: To investigate the role of theacrine in the protection of ventricular remodeling and chronic heart failure after myocardial infarction in the estrogen-deficient mice. MATERIALS AND METHODS: Female C57BL/6 mice aged 8 weeks old were selected and then subjected to bilateral oophorectomy. At 7 days after surgery, the models of the myocardial infarction were established by ligating the anterior descending coronary artery. On the first day after myocardial infarction, Theacrine (20 mg/kg) was administered via gavage for continuous 28 days. Thereafter, the cardiac function in each group of mice was detected via cardiac ultrasonography for small animals at 7, 14, and 28 days after surgery. The mice were sacrificed after 28 days. The infarct size of mice was determined through 2,3,5-triphenyltetrazolium chloride (TTC) and Evan blue double staining assay, while the myocardial fibrosis was assessed via Masson staining assay. The expression levels of collagen-related proteins Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), and the transforming growth factor-ß (TGF-ß) were measured by Western blotting (WB). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining assay was applied to evaluate the myocardial apoptosis, and the WB was employed to detect apoptosis-associated proteins. The expression level of silent information regulator 2 homologue 3 (SIRT3) protein was detected by immunohistochemistry, and the expression levels of SIRT3, ß-catenin and peroxisome proliferator-activated receptor gamma (PPARγ) protein were measured via WB. RESULTS: Compared with those in the Sham group, the ejection fraction (EF) and fractional shortening (FS) in estrogen-deficient mice were significantly lowered, the myocardial fibrosis and myocardial apoptosis were clearly aggravated, and the SIRT3 expression was decreased at 28 days after myocardial infarction. The theacrine could improve the cardiac function after the myocardial infarction in estrogen-deficient mice and relieve both myocardial fibrosis and myocardial apoptosis during chronic remodeling after myocardial infarction in estrogen-deficient mice. After the intervention with theacrine, the estrogen-deficient mice with myocardial infarction had up-regulated SIRT3 and PPARγ levels and a reduced ß-catenin level in the heart. CONCLUSIONS: Theacrine is able to activate SIRT3 and repress myocardial fibrosis and apoptosis after myocardial infarction in ovariectomized mice, thereby improving the cardiac function of ovariectomized mice with myocardial infarction through the possible downstream signal pathway ß-catenin/PPARγ.

Theacrine attenuates epithelial mesenchymal transition in human breast cancer MDA-MB-231 cells.

Theacrine, a purine alkaloid structurally similar to caffeine, has recently become of interest as a potential therapeutic compound. Here, we investigated the antimetastatic potential of theacrine on human breast cancer MDA-MB-231 cells. We observed that theacrine can reverse epithelial-to-mesenchymal transition (EMT), which resulted in a decrease in the levels of mesenchymal markers (Fibronectin, Vimentin, N-cadherin, Twist, and Snail) and an increase in the levels of epithelial markers (Occludin and E-cadherin) in the cells. Additionally, theacrine attenuates TGF-ß-induced EMT, cell adhesion, migration, and invasion in MDA-MB-231 cells. Overall, our results suggest that theacrine may inhibit the breast cancer cell metastasis by reversing the EMT process.

The effects of TeaCrine® and caffeine on endurance and cognitive performance during a simulated match in high-level soccer players.

BACKGROUND: Theacrine (1,3,7,9-tetramethyluric-acid) is a pure alkaloid with a similar structure to caffeine and acts comparably as an adenosine receptor antagonist. Early studies have shown non-habituating effects, including increases in energy and focus in response to Teacrine®, the compound containing pure theacrine. The purpose of this study was to determine and compare the effects of Teacrine® and caffeine on cognitive performance and time-to-exhaustion during a simulated soccer game in high-level male and female athletes. METHODS: Male and female soccer players (N = 24; MAge = 20.96 ± 2.05y, MMaleVO2max = 55.31 ± 3.39 mL/O2/kg, MFemaleVO2max = 50.97 ± 3.90 mL/O2/kg) completed a 90-min simulated treadmill soccer match over four randomized sessions (TeaCrine®, caffeine, TeaCrine® + caffeine, placebo). Cognitive testing at halftime and end-of-game including simple reaction time (SRT), choice RT (CRT), and cognitive-load RT with distraction questions (COGRT/COGRTWrong) was performed, with a run time-to-exhaustion (TTE) at 85% VO2max following end-of-game cognitive testing. Session times and pre-exercise nutrition were controlled. RM-MANOVAs with univariate follow-ups were conducted and significance was set at P < 0.05. RESULTS: TTE trended towards significance in TeaCrine® and TeaCrine® + caffeine conditions compared to placebo (P < 0.052). A condition main effect (P < 0.05) occurred with faster CRT in caffeine and TeaCrine® + caffeine compared to placebo. COGRTWrong showed a significant time main effect, with better accuracy at end-of-game compared to halftime (P < 0.05). A time x condition interaction in SRT (P < 0.05) showed placebo improved from halftime to end-of-game. CONCLUSIONS: The 27-38% improvements in TTE reflect increased performance capacity that may have important implications for overtime scenarios. These findings suggest TeaCrine® favorably impacts endurance and the combination with caffeine provides greater benefits on cognitive function than either supplement independently.

Identification of urate hydroperoxide in neutrophils: A novel pro-oxidant generated in inflammatory conditions.

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid.

Uric acid disrupts hypochlorous acid production and the bactericidal activity of HL-60 cells.

Uric acid is the end product of purine metabolism in humans and is an alternative physiological substrate for myeloperoxidase. Oxidation of uric acid by this enzyme generates uric acid free radical and urate hydroperoxide, a strong oxidant and potentially bactericide agent. In this study, we investigated whether the oxidation of uric acid and production of urate hydroperoxide would affect the killing activity of HL-60 cells differentiated into neutrophil-like cells (dHL-60) against a highly virulent strain (PA14) of the opportunistic pathogen Pseudomonas aeruginosa. While bacterial cell counts decrease due to dHL-60 killing, incubation with uric acid inhibits this activity, also decreasing the release of the inflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF- α). In a myeloperoxidase/Cl-/H2O2 cell-free system, uric acid inhibited the production of HOCl and bacterial killing. Fluorescence microscopy showed that uric acid also decreased the levels of HOCl produced by dHL-60 cells, while significantly increased superoxide production. Uric acid did not alter the overall oxidative status of dHL-60 cells as measured by the ratio of reduced (GSH) and oxidized (GSSG) glutathione. Our data show that uric acid impairs the killing activity of dHL-60 cells likely by competing with chloride by myeloperoxidase catalysis, decreasing HOCl production. Despite diminishing HOCl, uric acid probably stimulates the formation of other oxidants, maintaining the overall oxidative status of the cells. Altogether, our results demonstrated that HOCl is, indeed, the main relevant oxidant against bacteria and deviation of myeloperoxidase activity to produce other oxidants hampers dHL-60 killing activity.

N-Acetyltransferase-2 (NAT2) phenotype is influenced by genotype-environment interaction in Ethiopians.

BACKGROUND AND OBJECTIVES: N-acetyltransferase 2 (NAT2) metabolize several drugs including isoniazid. We investigated the effect of genotype, geographical difference, and smoking habit on NAT2 phenotype in Ethiopians. METHODS: Genotyping for NAT2 191G > A, 341 T > C, 590G > A, and 857G > A was performed in 163 unrelated healthy Ethiopians (85 living in Ethiopia and 78 living in Sweden). The NAT2 phenotype was determined using caffeine as a probe and log AFMU/(AFMU + 1X + 1 U) urinary metabolic ratio (MR) as an index. RESULTS: The frequencies of NAT2*4, *5, *6, *7, and *14 haplotypes were 14.1, 48.5, 30.1, 5.5, and 1.8%, respectively. The frequencies of rapid (NAT2*4/*4), intermediate (heterozygous *4), and slow (no *4 allele) acetylator genotypes were 1.8, 24.6, and 73.6%, respectively. The distribution NAT2 MR was bimodal with 70% being phenotypically slow acetylators. NAT2 genotype (p < 0.0001) and country of residence (p = 0.004) independently predicted NAT2 phenotype. Controlling for the effect of genotype, ethnic Ethiopians living in Ethiopia had significantly higher NAT2 MR than those living in Sweden (p = 0.006). NAT2 genotype-phenotype concordance rate was 75%. Distinct country-of-residence-based genotype-phenotype discordance was observed. The proportion of phenotypically determined rapid acetylators was significantly higher and slow acetylators was lower in Ethiopians living in Ethiopia (39% rapid, 61% slow) than in Sweden (20% rapid, 80% slow). Sex and smoking had no significant effect on NAT2 MR. CONCLUSIONS: We report a high prevalence of NAT 2 slow acetylators in Ethiopians and a conditional NAT2 genotype-phenotype discordance implicating a partial phenotype conversion and metabolic adaptation. Gene-environment interactions regulate NAT2 phenotype.

Theacrine: A purine alkaloid from Camellia assamica var. kucha with a hypnotic property via the adenosine system.

Theacrine (l,3,7,9-tetramethyluric acid), a purine alkaloid from Camellia assamica var. kucha, has diverse pharmacological properties, including sedative and hypnotic activities, anti-inflammatory and analgesic activities, antidepressant effects, and a protective effect against stress-provoked liver damage. The present study aims to investigate the possible mechanism of the hypnotic activity of theacrine. The results revealed that theacrine significantly enhanced pentobarbital-induced sleep at a dose of 3.0mg/kg (i.g.) in mice. Sleep parameter analysis by EEG and EMG showed that theacrine obviously shortened wake time and increased NREM sleep time and that theacrine almost had no effect on REM sleep. Meanwhile, theacrine markedly attenuated caffeine (a nonselective antagonist of adenosine receptor)-induced insomnia. In pretreatment with the adenosine A1 receptor antagonist DPCPX and the A2A receptor antagonist SCH 58261, theacrine significantly reversed the decrease in sleeping time in pentobarbital-treated mice. In addition, theacrine also markedly increased the adenosine content in the hippocampus of rats. These results suggested that theacrine might mediate the adenosine system to augment pentobarbital-induced sleep.

Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2.

Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m-1 s-1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s-1) than Prx1 (k3 = 14.9 s-1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.

A comparative analysis of chemical compositions in Camellia sinensis var. puanensis Kurihara, a novel Chinese tea, by HPLC and UFLC-Q-TOF-MS/MS.

Camellia sinensis var. puanensis Kurihara (Puan tea) is a kind of ancient tea plant newly found in Jiangxipo and the surrounding areas of Puan County (Guizhou, China). People there always believe that drinking Puan tea is beneficial to the promotion of health and prevention of diseases. However, detailed information on its compositions has not been reported. Therefore, in this study, the varieties and contents of purine alkaloids and polyphenols in Puan tea were identified and determined by HPLC and UFLC-Q-TOF-MS/MS. Our results showed that theacrine, but not caffeine, was the dominated purine alkaloid detected in Puan tea. Meanwhile, Puan tea contained B-type procyanidin dimer, trimer and dimer monogallate, which were not detected in Camellia sinensis, Camellia ptilophylla and Camellia assamica var. kucha. The obtained results could support the local uses of Puan tea in health and nutrition and contribute to the research of tea variety.

Cytochrome P450 2A6 Phenotyping Using Dietary Caffeine Salivary Metabolite Ratios and Genotyping Using Blood on Storage Cards in Non-smoking Japanese Volunteers.

BACKGROUND: A simple method of genotyping and phenotyping cytochrome P450 2A6 (CYP2A6) was previously reported using individual blood samples and urinary caffeine metabolite ratios of 1,7-dimethyluric acid (17U) to 1-methylxanthine (1X). OBJECTIVE: Blood spotted onto storage cards and salivary caffeine metabolites were analyzed in 27 healthy non-smoking Japanese volunteers with no prior abstention from dietary caffeine intake. METHODS: 1,7-Dimethylxanthine (17X), 17U, 1X, and caffeine levels in spot saliva samples were determined in Japanese non-smokers by high-performance liquid chromatography under normal dietary caffeine consumption. RESULTS: 17U/17X ratios in saliva were almost constant over time, but those of 17U/1X were variable in two subjects tested before and 1-2.5 h after caffeine treatment (a cup of black tea). In seven subjects, 17U/17X ratios in saliva were highly correlated with those in plasma (r = 0.98, p < 0.01) and well correlated with those in urine samples (r = 0.78, p < 0.05). The average 17U/17X ratios, but not 17U/1X ratios, in saliva under dietary caffeine consumption obtained from subjects with CYP2A6*1/*4 (n=11) and CYP2A6*4/*4 (whole-gene deletion, n=2) genotypes were significantly lower than those from subjects with wild-type CYP2A6*1/*1 (n=14). Genotyping was done by a multiplex real-time polymerase chain reaction method using blood spotted onto storage cards. CONCLUSION: The present results suggest that the decreased CYP2A6 function associated with the whole-gene deletion genotype (determined using blood samples) could be detected using 17U/17X ratios, but not 17U/1X ratios, in spot saliva samples under normal dietary caffeine consumption in Japanese non-smokers, just as it could be detected using urinary 17U/1X ratios.

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.

Polymorphisms of CYP1A2 and CYP2A6 activity: phenotypes and the effect of age and sex in a Nigerian population.

BACKGROUND: CYP1A2 and CYP2A6 are polymorphic enzymes that metabolise several compounds of clinical importance. This study investigated the prevalent phenotypes of these enzymes and the influence of age and sex on enzyme activity in a Nigerian population. METHODS: Caffeine (110 mg) was administered to each of 129 healthy, unrelated subjects (85 males and 44 females) who were non-smokers. Urine voided within 7 h after caffeine administration was collected for a high performance liquid chromatographic assay of caffeine (137X), 1,7-dimethyluric acid (17U) and 1,7-dimethylxanthine (17X). CYP1A2 activity was measured as a ratio of (17U+17X) to 137X, while 17U/17X served as marker for CYP2A6. Transformed data were analysed and the influences of age and sex on activity were also determined. RESULTS: Distribution of CYP1A2 activity in the population was bimodal with a mean±SD of 0.82±0.41, while that of CYP2A6 was trimodal with a mean±SD activity of 0.27±0.42 of the log-transformed urinary molar ratio of metabolites. The influences of age and sex on enzyme activity for both CYP1A2 and CYP2A6 were not significant (p>0.05). CONCLUSIONS: The study established the prevalence of polymorphism in phenotypes of CYP1A2 and CYP2A6 activity in the Nigerian population, but no influence of age and sex on enzyme activity was observed in this population.

In vitro oxidative metabolism of 6-mercaptopurine in human liver: insights into the role of the molybdoflavoenzymes aldehyde oxidase, xanthine oxidase, and xanthine dehydrogenase.

Anticancer agent 6-mercaptopurine (6MP) has been in use since 1953 for the treatment of childhood acute lymphoblastic leukemia (ALL) and inflammatory bowel disease. Despite being available for 60 years, several aspects of 6MP drug metabolism and pharmacokinetics in humans are unknown. Molybdoflavoenzymes such as aldehyde oxidase (AO) and xanthine oxidase (XO) have previously been implicated in the metabolism of this drug. In this study, we investigated the in vitro metabolism of 6MP to 6-thiouric acid (6TUA) in pooled human liver cytosol. We discovered that 6MP is metabolized to 6TUA through sequential metabolism via the 6-thioxanthine (6TX) intermediate. The role of human AO and XO in the metabolism of 6MP was established using the specific inhibitors raloxifene and febuxostat. Both AO and XO were involved in the metabolism of the 6TX intermediate, whereas only XO was responsible for the conversion of 6TX to 6TUA. These findings were further confirmed using purified human AO and Escherichia coli lysate containing expressed recombinant human XO. Xanthine dehydrogenase (XDH), which belongs to the family of xanthine oxidoreductases and preferentially reduces nicotinamide adenine dinucleotide (NAD(+)), was shown to contribute to the overall production of the 6TX intermediate as well as the final product 6TUA in the presence of NAD(+) in human liver cytosol. In conclusion, we present evidence that three enzymes, AO, XO, and XDH, contribute to the production of 6TX intermediate, whereas only XO and XDH are involved in the conversion of 6TX to 6TUA in pooled HLC.

Cytochrome P450 2A6 phenotyping based on dietary caffeine intake in a Japanese population of non-smokers.

Phenotyping of cytochrome P450 2A6 (CYP2A6) was determined by assessing urinary caffeine metabolites in a Japanese population with a high frequency of CYP2A6 whole-gene deletion (CYP2A6*4). The levels of 1,7-dimethyluric acid (17U), 1-methylxanthine (1X), and 1,7-dimethylxanthine (17X) were measured in non-smokers whose CYP2A6 and NAT2 genotypes had been determined. Low 17U/1X ratios were observed in accumulated overnight urine samples of subjects genotyped as CYP2A6*4/*4 after caffeine treatment. The individual 17U/1X ratios in spot urine samples were almost constant before and 2-8 h after caffeine treatment, with or without prior abstention from dietary caffeine. The average 17U/1X ratios obtained from subjects with CYP2A6 *4/ *4 or CYP2A6 *1/ *4 genotypes were significantly lower than those from subjects with wild-type CYP2A6 *1/ *1 under dietary caffeine consumption. The present results suggest that impaired CYP2A6 function associated with CYP2A6 *4/ *4 could be determined using the 17U/1X ratios in spot urine samples under normal dietary caffeine consumption in Japanese non-smokers, without the need for additional caffeine administration or prior abstention from caffeine.