Inhibition study of adenosine deaminase by caffeine using spectroscopy and isothermal titration calorimetry

Kinetic and thermodynamic studies were made on the effect of caffeine on the activity of adenosine deaminase in 50 mM sodium phosphate buffer, pH 7.5, using UV spectrophotometry and isothermal titration calorimetry (ITC). An uncompetitive inhibition was observed for caffeine. A graphical fitting method was used for determination of binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 350 microM by the microcalorimetry method, which agrees well with the value of 342 microM for the inhibition constant that was obtained from the spectroscopy method. Positive dependence of caffeine binding on temperature indicates a hydrophobic interaction.     

Extractionless method for the determination of urinary caffeine metabolites using high-performance liquid chromatography coupled with tandem mass spectrometry

Caffeine is metabolised in humans primarily by cytochromes P450 1A2 and 2A6, xanthine dehydrogenase/oxidase, and N-acetyltransferase 2. The activities of these enzymes show a large variation due to genetic polymorphisms and/or induction by xenobiotics. Ratios of different caffeine metabolites in urine or other body fluids are frequently used to characterise the individual/actual activity of these enzymes. The common analytical method involves extensive sample preparation, followed by HPLC-UV. The presence of numerous other UV-absorbing chemicals in body fluids affects the sensitivity and selectivity of this method. We have developed an HPLC-electrospray-MS-MS method for the determination of 11 caffeine metabolites and two internal standards after a simple, extractionless preparation. Blank urine, obtained after 5 days on a methylxanthine-free diet, contained small amounts of some caffeine metabolites, but no other components producing any confounding signals. Eleven metabolites and internal standards were recovered at 90 to 110% after addition to the blank urine (0.1 to 2.5 micro M in the final sample involving a 20-fold dilution of urine) in the 0.1-2.5 micro M concentration range. Other metabolites, 5-acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU), were detected with similar recovery and precision, but required higher concentrations (3 to 30 micro M). AFMU was completely converted into AAMU by a short alkalisation of urine. The method was explored in six healthy individuals after consuming coffee (4 mg caffeine per kg body mass). These experiments demonstrated the simplicity, high sensitivity and selectivity of the method under conditions used for phenotyping.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

1,3,7-trimethylxanthine (caffeine) may exacerbate acute inflammatory liver injury by weakening the physiological immunosuppressive mechanism

The genetic elimination of A2A adenosine receptors (A2AR) was shown to disengage the critical immunosuppressive mechanism and cause the dramatic exacerbation of acute inflammatory tissue damage by T cells and myeloid cells. This prompted the evaluation of the proinflammatory vs the anti-inflammatory effects of the widely consumed behavioral drug caffeine, as the psychoactive effects of caffeine are mediated largely by its antagonistic action on A2AR in the brain. Because caffeine has other biochemical targets besides A2AR, it was important to test whether the consumption of caffeine during an acute inflammation episode would lead to the exacerbation of immune-mediated tissue damage. We examined acute and chronic treatment with caffeine for its effects on acute liver inflammation. It is shown that caffeine at lower doses (10 and 20 mg/kg) strongly exacerbated acute liver damage and increased levels of proinflammatory cytokines. Because caffeine did not enhance liver damage in A2AR-deficient mice, we suggest that the potentiation of liver inflammation was mediated by interference with the A2AR-mediated tissue-protecting mechanism. In contrast, a high dose of caffeine (100 mg/kg) completely blocked both liver damage and proinflammatory cytokine responses through an A2AR-independent mechanism. Furthermore, caffeine administration exacerbated liver damage even when mice consumed caffeine chronically, although the extent of exacerbation was less than in "naive" mice that did not consume caffeine before. This study suggests an unappreciated "man-made" immunological pathogenesis whereby consumption of the food-, beverage-, and medication-derived adenosine receptor antagonists may modify an individual's inflammatory status and lead to excessive organ damage during acute inflammation.     

Acute and habitual caffeine ingestion and metabolic responses to steady-state exercise.

This study compared the exercise catecholamine and metabolic responses to a caffeine challenge in trained subjects before and after a 6-wk period of increased caffeine ingestion. Trained subjects (n = 6) were challenged with 500 mg of caffeine followed by prolonged exercise before and after 6 wk of increased caffeine ingestion (500 mg ingested before each daily run). A control group (n = 6) of trained subjects followed the same protocol except for caffeine ingestion. Acute caffeine ingestion resulted in increased plasma epinephrine and decreased respiratory exchange ratio (RER) during exercise. After 6 wk of caffeine supplementation, the epinephrine response to exercise or caffeine plus exercise was decreased, although the latter still resulted in a lower RER value compared with exercise without caffeine ingestion. Activity of key metabolic enzymes (hexokinase, citrate synthase, phosphorylase, and 3-hydroxyacyl-coenzyme A dehydrogenase) from biopsies of the gastrocnemius showed no response to 6 wk of this increased adrenergic receptor stimulation and, on the basis of the lower RER, enhanced fat metabolism. This study suggests that caffeine ingestion by trained subjects causes increases in plasma epinephrine and reduces the RER during exercise. However, habitual stimulation results in a general dampening of the epinephrine response to caffeine or exercise. There was no indication that increased adrenergic stimulation and fat oxidation caused any adaptation in the activity of metabolic enzymes.     

Extraction behavior of caffeine and EGCG from green and black tea

A dipping method was developed to extract the catechins (EGCG) and alkaloids (caffeine) from green tea (Korea) and black tea (Sri Lanka). The effects of the solvent composition (water vs. ethanol), extraction time, temperatures, and solvent pH on the amount of catechins (EGCG) and alkaloids (caffeine) extracted from green and black tea were investigated. Reversedphase high-performance liquid chromatography (RP-HPLC) was used to analyze the catechins (EGCG) and alkaloids (caffeine) extracted. The content of EGCG and caffeine in green tea extracts was in the range of 2.04∼0.30 and 10.22∼0.85 mg/g, respectively. The amount of EGCG and caffeine in black tea extracts was in the range of 0.32∼0.24 and 5.26∼1.01 mg/g, respectively. The amount of caffeine extracted from green and black tea was greater than the amount of EGCG. Pure water is the best solvent for extracting EGCG and caffeine from green tea. The amount of caffeine extracted from green and black tea increased as the temperature, extraction time, and hydrogen ion concentration of the solvent increased. Although the amount of EGCG extracted from green tea increased as the temperature increased, the amount of EGCG extracted from black tea was not affected by temperature. The extraction of EGCG from both green and black tea was not affected by the hydrogen ion concentration of the solvent.     

Microdetermination of caffeine in blood by gas chromatography—mass spectrometry

A micromethod for the quantitative analysis of caffeine present in small quantities (100 μl) of whole blood is described. It is based on the gas chromatographic—mass spectrometric analysis of chloroform extracts of biological samples. The method is relatively simple, rapid, specific and sensitive, as little as 20 ng of caffeine can be measured.     

Complete,reversible, drug-specific tolerance to stimulation of locomotor activity by caffeine

The development of tolerance to caffeine-induced stimulation of locomotor activity was evaluated in rats maintained chronically on average daily doses of 160 mg/kg or more of caffeine by the method of scheduled access to drinking water containing the drug. Dose-response curves were determined for caffeine (6.25–100 mg/kg) and $\text{d}$-amphetamine (0.39–6.4 mg/kg) during chronic drug treatment. In addition, the caffeine curve was redetermined 2–3 weeks after removal of the drug from the drinking water. A control group that had scheduled access to drug-free tap water was also tested. Caffeine produced dose-related increases in the locomotor activity of the controls but failed to modify locomotor activity of the chronic caffeine group. In contrast, $\text{d}$-amphetamine increased locomotor activity of both groups comparably. Spontaneous locomotor of the chronic caffeine group was reduced significantly for 4 days after drug-free tap water was substituted for the caffeine solution. The return of spontaneous locomotor activity to baseline values was associated with restored sensitivity to caffeine-induced stimulation of locomotor activity. Thus, chronic administration of caffeine to rats results in the development of tolerance to caffeine-induced stimulation of locomotor activity that is virtually complete, pharmacologically specific, and fully reversible when drug treatment is stopped. Decreases in spontaneous locomotor activity after abrupt termination of chronic caffeine administration follow a time course consistent with a drug withdrawal syndrome.     

Formation of stacking complexes between caffeine (1,2,3-trimethylxanthine) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may attenuate biological effects of this neurotoxin

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin causing symptoms that may resemble those observed in patients suffering from Parkinson's disease. Therefore, MPTP-treated laboratory animals are currently the most favored models to study therapeutic intervention strategies in this disease. It was demonstrated recently that caffeine (1,2,3-trimethylxanthine) intake decreases the risk of Parkinson's disease in various human populations and attenuates MPTP-induced neurological effects in animal models. Since the effects of caffeine on MPTP-treated animals were mimicked by several antagonists of the adenosine A(2A) receptor, it was suggested that caffeine attenuates MPTP toxicity by blocking this receptor. Here, using microcalorimetry and molecular modeling, we demonstrate that caffeine can form stacking (pi-pi) complexes with MPTP. We found that a biological activity of MPTP (induction of mutations in a microbiological mutagenicity assay), which is completely independent on the A(2A) receptor blockade, is significantly reduced by caffeine. Therefore, we suggest that caffeine may attenuate neurotoxicity of MPTP (and possibly other polycyclic aromatic toxins) and reveal its protective effects on the risk of Parkinson's disease not only by blocking the A(2A) receptor but also by sequestering neurotoxin molecules in mixed complexes, especially in stomach.     

Spectrophotometric flow injection determination of caffeine in solid and slurry coffee and tea samples using supported liquid membranes

A method for the determination of caffeine in coffee and tea samples based on the use of supported liquid membranes coupled to a flow system has been developed. The sample may be analysed both as solid and slurry. In the case of solid sample, this is directly placed in the membrane unit, and when the sample is slurry, this is continuously pumped to the membrane unit. In both cases, the caffeine released from the sample passes through the membrane (PTFE/n-undecane:hexyl ether) into an acidic acceptor stream. This stream flows through a spectrophotometric detector allowing the measurement of the absorbance of caffeine at 274 nm. The method shows a linear range between 0.5 and 15 g l⁻¹, with a relative standard deviation of ±3.7% and a sample throughput of 7–8 samples h⁻¹.     

Time effect of rutaecarpine on caffeine pharmacokinetics in rats

Rutaecarpine is reported as a potent inducer of CYP1A2 enzyme in rats. There are natural herbal supplements containing rutaecarpine that are designed to enhance the CYP1A2-dependent removal of caffeine from blood so that people can have coffee later in the day without causing sleep interference. This study aimed to determine the minimum amount of time needed from oral rutaecarpine administration until the observed effect of rutaecarpine on caffeine pharmacokinetics (PK) in 15 male Sprague-Dawley rats. PK parameters for caffeine and its metabolites in the control and rutaecarpine groups were calculated using WinNonlin®. Results showed that orally administered rutaecarpine at 100 mg/kg dose as early as 3 h before oral caffeine administration significantly decreased the oral systemic exposure and mean residence time of caffeine and its metabolites due to decreased caffeine bioavailability (by up to 75%) and increased clearance. The systemic exposure of caffeine and its metabolites were also decreased when caffeine was given intravenously, though this effect was less pronounced than when caffeine was given orally. Although plasma level of rutaecarpine was undetectable (less than 10 ng/mL), rutaecarpine still induced hepatic CYP1A2 activity. Results from 7-methoxyresorufin O-demethylation activity, which is specific to CYP1A2, showed that 3 h after one rutaecarpine oral dose, CYP1A2 activity in rat liver tissue was increased by 3- fold. This finding suggested that rutaecarpine effectively induced CYP1A2 activity in the liver.     

Enhanced degradation of caffeine and caffeine demethylase production by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in bioreactors under fed-batch mode

The growth of Pseudomonas sp. was studied in fed-batch process with an aim to improve the caffeine degradation rate and caffeine demethylase activity. The effects of varying initial caffeine concentrations in the batch mode, increase in the number of feeds, varying feed flow rates, and added nutrients to the feed on the fed-batch process were investigated. A maximum caffeine degradation rate of 0.82 g/L h and maximum caffeine demethylase activity of 2.6 U/mg were achieved using manual intermittent pulse feeds of caffeine with substrate concentration as feedback parameter for the fed batch started with an initial caffeine concentration of 3 g/L. A slight increase in the caffeine degradation rate (0.85 g/L h) and caffeine demethylase activity (3.4 U/mg) was observed when the additional nutrients were added along with caffeine in the feed. This is the first report showing complete degradation of large magnitudes of caffeine amounting to 237 g in 75 h. These results show that the fed-batch conditions achieved in this study using Pseudomonas sp. facilitate the development of a sustainable bioprocess to degrade the high concentrations of caffeine in industrial effluents.     

Caffeine as a novel toxicant for slugs and snails

In this study, caffeine is shown to act as both a repellent and toxicant against slugs and snails. This research is the first to document the potential of caffeine as a molluscicide. A drench treatment using a 1% or 2% solution of caffeine caused 100% of slugs (Veronicella cubensis) to exit treated soil, and the majority of these slugs subsequently died from caffeine poisoning. A 2% solution of caffeine applied to the growing medium of orchids killed 95% of orchid snails, (Zonitoides arboreus), and gave better control than a liquid metaldehyde product representing the standard commercial control for this pest. Using leaf‐dip bioassays, we discovered that slugs tended to avoid feeding on plant material treated with caffeine solutions 0.1%, and caffeine solutions as low as 0.01% significantly reduced overall feeding by slugs. Due to concerns about chemical residues, available molluscicides generally cannot be applied directly to food crops for control of slug and snail pests. Caffeine is a natural product which is approved as a food additive. Therefore, caffeine may prove useful for protecting food crops from slugs and snails.     

Pulmonary stenosis with ventricular septal defect, common aorticopulmonary trunk, and dextroposition of the aorta: morphologic and qualitative physiologic effects in caffeine-treated chick embryos

Effects of caffeine administration to Hamburger-Hamilton stage 19 chick embryos (3 days of incubation) were investigated. A morphologic study of the effect of caffeine on cardiogenesis showed that caffeine produced total cardiac malformations in the chick in a dose-related fashion. A maximum frequency of 70.6% was observed with 4.7 mg caffeine. Major malformations included common aorticopulmonary trunk and dextroposition of the aorta accompanied by ventricular septal defect with/without pulmonary stenosis. Qualitative analysis of cinegraphs following exposure of embryos to a single teratogenic dose of caffeine (3.5 mg/egg) produced marked alterations in cardiac function when compared with chick Ringer's controls. Within 3 minutes after exposure to caffeine, dilation of the common ventricle and weak ventricular contractility were observed and persisted for 1 hour. Dose-response data and microcinematographic observations suggest that caffeine induced cardiac anomalies by a direct toxic effect on the embryo rather than by altering cardiac cell function. Our data also suggest that pathophysiologic changes in cardiac function may play an important role in the pathogenesis of caffeine-induced cardiac anomalies in the chick embryo.     

Determination of plasma theophylline by straight-phase high-performance liquid chromatography: Elimination of interfering caffeine metabolites

Several authors have recently reported interference in theophylline analysis by paraxanthine (1,7-dimethylxanthine), an important metabolite of caffeine. A method for the determination of theophylline in plasma is described, eliminating caffeine and related compounds by means of straight-phase high-performance liquid chromatography. The resulting procedure is sufficiently rapid, accurate and sensitive to be applied in routine monitoring of therapeutic levels in patients as well as for pharmacokinetic purposes. Although only 0.1 ml of sample is required, concentrations as low as 0.2 mg/l can be measured with acceptable precision. A brief comparative evaluation of this procedure with a radioimmunoassay is made.     

Stability studies of selected doping agents in urine: caffeine

The stability of caffeine in urine samples has been studied. A high-performance liquid chromatography (HPLC) method for the quantification of caffeine in urine samples was validated for that purpose. The method consists of a liquid-liquid extraction at alkaline pH with chloroform-2-propanol (9:1, v/v) with a salting out effect. 7-Ethyltheophylline was used as internal standard (ISTD). Analyses were performed with an Ultrasphere ODS C18 column using water/acetonitrile (90:10, v/v) as a mobile phase at a flow rate of 1 ml/min. Ultraviolet absorption at 280 nm was monitored. Extraction recoveries for caffeine and 7-ethyltheophylline were 81.4+/-6.0 and 87.3+/-5.7%, respectively. The calibration curves were demonstrated to be linear in the working range of 6-30 microg/ml (r2>0.990). The limit of detection and the limit of quantitation were estimated as 0.7 and 2.0 microg/ml, respectively. Precisions in the range of 1.5-9.2 and 4.1-5.8% were obtained in intra- and inter-assay studies, respectively, using control samples containing 10, 14 and 26 microg/ml of caffeine. Accuracies ranging from 2.9 to 7.4% for intra-assay experiments, and from 3.9 to 5.4% in inter-assay studies were obtained. Stability of caffeine in urine samples was evaluated after long- and short-term storage at different temperature conditions. The batches of spiked urine were submitted to sterilization by filtration. No adsorption of the analyte on filters was observed. Before starting stability studies, batches of reference materials were tested for homogeneity. For long-term stability testing, caffeine concentration in freeze-dried urine stored at 4 degrees C and in liquid urine samples stored at 4, -20, -40 and -80 degrees C was determined at several time intervals for 18 months. For short-term stability testing, caffeine concentration was evaluated in liquid urine stored at 37 degrees C for 7 days. The effect of repeated freezing (at -20 degrees C) and thawing was also studied for up to three cycles. The stability of caffeine was also evaluated in non-sterile samples stored at -20 degrees C for 18 months. No significant loss of the compound was observed at any of the investigated conditions.     

Incorporation of caffeine into a quantitative model of fatigue and sleep

A recent physiologically based model of human sleep is extended to incorporate the effects of caffeine on sleep-wake timing and fatigue. The model includes the sleep-active neurons of the hypothalamic ventrolateral preoptic area (VLPO), the wake-active monoaminergic brainstem populations (MA), their interactions with cholinergic/orexinergic (ACh/Orx) input to MA, and circadian and homeostatic drives. We model two effects of caffeine on the brain due to competitive antagonism of adenosine (Ad): (i) a reduction in the homestatic drive and (ii) an increase in cholinergic activity. By comparing the model output to experimental data, constraints are determined on the parameters that describe the action of caffeine on the brain. In accord with experiment, the ranges of these parameters imply significant variability in caffeine sensitivity between individuals, with caffeine's effectiveness in reducing fatigue being highly dependent on an individual's tolerance, and past caffeine and sleep history. Although there are wide individual differences in caffeine sensitivity and thus in parameter values, once the model is calibrated for an individual it can be used to make quantitative predictions for that individual. A number of applications of the model are examined, using exemplar parameter values, including: (i) quantitative estimation of the sleep loss and the delay to sleep onset after taking caffeine for various doses and times; (ii) an analysis of the system's stable states showing that the wake state during sleep deprivation is stabilized after taking caffeine; and (iii) comparing model output successfully to experimental values of subjective fatigue reported in a total sleep deprivation study examining the reduction of fatigue with caffeine. This model provides a framework for quantitatively assessing optimal strategies for using caffeine, on an individual basis, to maintain performance during sleep deprivation.     

Central nervous system effects of caffeine and adenosine on fatigue

Caffeine ingestion can delay fatigue during exercise, but the mechanisms remain elusive. This study was designed to test the hypothesis that blockade of central nervous system (CNS) adenosine receptors may explain the beneficial effect of caffeine on fatigue. Initial experiments were done to confirm an effect of CNS caffeine and/or the adenosine A(1)/A(2) receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) on spontaneous locomotor activity. Thirty minutes before measurement of spontaneous activity or treadmill running, male rats received caffeine, NECA, caffeine plus NECA, or vehicle during four sessions separated by approximately 1 wk. CNS caffeine and NECA (intracerebroventricular) were associated with increased and decreased spontaneous activity, respectively, but caffeine plus NECA did not block the reduction induced by NECA. CNS caffeine also increased run time to fatigue by 60% and NECA reduced it by 68% vs. vehicle. However, unlike the effects on spontaneous activity, pretreatment with caffeine was effective in blocking the decrease in run time by NECA. No differences were found after peripheral (intraperitoneal) drug administration. Results suggest that caffeine can delay fatigue through CNS mechanisms, at least in part by blocking adenosine receptors.     

Removal of caffeine in sewage by Pseudomonas putida: Implications for water pollution index

A strain of Pseudomonas putida (biotype A) capable of growing on caffeine (1,3,7-trimethylxanthine) was isolated from a domestic wastewater processing operation. It used caffeine as the sole carbon source with a mean growth rate constant (k) of 0.049 h^-1 (approximately 20 h per generation), whereas k for glucose utilization under similar incubation conditions was 0.31 (3.3 h per generation). The isolate contained at least two plasmids, and the increased expression of a 40 kDa protein was attributable to growth on caffeine. Degradation byproducts of caffeine metabolism by the bacterial isolate included other xanthine derivatives. The slow bacterial catabolism of caffeine in sewage has implications for the effectiveness of wastewater purification, re-use and disposal.The author is with the Laboratory for Molecular Ecology, Department of Environmental Analysis and Design, University of California at Irvine, Irvine, CA 92717-5150 U.S.A.     

CaXMT和TCS1突变体基因串联共表达及体外酶活检测分析

咖啡碱和可可碱是茶叶生物碱的主要组分,且咖啡碱是茶叶重要的滋味物质,随着咖啡碱在食品和药物领域的应用愈发广泛,咖啡碱的生物合成成为新的研究热点.目前市场上的咖啡碱主要靠化学合成,为了探索其生物合成途径,该研究将咖啡黄嘌呤核苷甲基转移酶(coffee xanthosine methyltransferase,CaXMT)基因和茶树咖啡碱合成酶(tea caffeine synthase,TCS1)基因的4个突变体分别串联至同一大肠杆菌表达载体pMAL-c5X,诱导融合蛋白共表达,并进行SDS-PAGE凝胶电泳分析.结果表明:目的蛋白成功表达后,应用超声破碎法制备含有目的蛋白的粗酶液,添加底物黄嘌呤核苷(xanthosine,XR)和甲基供体S-腺苷甲硫氨酸(S-adenosyl-L-methionine,SAM)进行体外酶促反应,将反应产物进行高效液相色谱检测.检测结果显示,pMAL-CaXMT-TM2/3/4的体外酶促反应产物仅有可可碱生成,均未见咖啡碱生成.该研究结果为构建生物合成咖啡碱和可可碱的串联共表达载体奠定了基础,也为进一步研究生物合成咖啡碱和可可碱提供了新思路.