Association between caffeine metabolites in urine and muscle strength in young and older adults: A cross-sectional study from NHANES 2011-2012.

BACKGROUND: Elevated levels of reactive oxygen species may contribute to the gradual decline in muscle strength over time. Although caffeine and its metabolites have antioxidant properties that can mitigate oxidative stress, the association of caffeine and its metabolites with muscle strength remains unknown. AIM: To investigate whether caffeine metabolites in urine are associated with muscle strength in young and older adults. METHODS: A cross-sectional study was conducted with 1145 individuals aged over 20 years (n = 801 < 60 years and n = 344 ≥ 60 years) from the National Health and Nutrition Examination Survey (NHANES) 2011-2012. Muscle strength was assessed using a handgrip dynamometer, and combined grip strength was determined by summing the highest value from each hand. Caffeine and its metabolites in urine were quantified using ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (1-methyluric acid, 3-methyluric acid, 7-methyluric acid, 1,3-dimethyluric acid, 1,7-dimethyluric acid, 3,7-dimethyluric acid, 1,3,7-trimethyluric acid, 1-methylxanthine, 3-methylxanthine, 7-methylxanthine, 1,3-dimethylxanthine, 1,7-dimethylxanthine, 3,7-dimethylxanthine, 1,3,7-trimethylxanthine, 5-acetylamino-6-amino-3-methyluracil). Linear regression analyses were performed to determine the association of caffeine and its metabolites with muscle strength in young and older adults, adjusting for confounders. RESULTS: Positive associations between muscle strength and levels of 7-methyluric acid (ß = 0.029; p = 0.021), 1,3-dimethyluric acid (ß = 0.008; p = 0.004), 3,7-dimethyluric acid (ß = 0.645; p = 0.012), 3-methylxanthine (ß = 0.020; p = 0.002), 7-methylxanthine (ß = 0.020; p = 0.006), 1,3-dimethylxanthine (theophylline) (ß = 0.030; p = 0.004) and 3,7-dimethylxanthine (theobromine) (ß = 0.035; p = 0.029) were observed in older adults. In contrast, no such associations were noted in young adults. CONCLUSION: Our study indicates a positive association between certain caffeine metabolites in urine and muscle strength in older adults, but not in younger individuals. These findings indicate that specific caffeine metabolites may contribute to an antioxidant role especially in older adults.

Role of alanine aminotransferase in the effects of urinary caffeine concentration and its primary metabolite concentration on cognitive function in older adults: Bayesian Kernel Machine regression analysis and mediation analysis.

OBJECTIVE: This study aimed to explore the role of alanine aminotransferase (ALT) in the effects of urinary caffeine and its primary metabolites on cognitive function in elderly people. MATERIALS AND METHODS: In this investigation, we meticulously curated a cohort from the 2011-2014 National Health and Nutrition Examination Survey (NHANES) database. Animal fluency emerged as the pivotal metric for assessing cognitive function within our study population. In order to navigate the intricacies of mixture analysis and circumvent potential complexities, we harnessed the power of Bayesian kernel machine regression (BKMR) models. This method allowed us to dissect the nuanced impacts of caffeine and its primary urinary metabolites on cognitive function. While accounting for caffeine and its metabolites, we analyzed the relationship between ALT and cognitive function through non-linear dynamics. Lastly, employing structural equation modeling, we probed the intriguing question of whether ALT mediates the influence of 3,7-dimethylxanthine on cognitive function. This comprehensive approach has unveiled a deeper understanding of the multifaceted interplay among these variables, offering invaluable insights into the determinants of cognitive function within our cohort. RESULTS: After meticulous adjustment for various covariates, our linear regression analysis unveiled a noteworthy finding: 3,7-dimethylxanthine demonstrated a significant positive correlation with cognitive function (p < 0.05). Importantly, within the BKMR model employed, 3,7-dimethylxanthine emerged as the most influential factor within the compound, with posterior inclusion probabilities of 0.995 and 0.939. Furthermore, our single-exposure effect model confirmed its presence at the 25th, 50th, and 75th percentile concentrations of other components within the compound. Interestingly, bivariate concentration curves indicated no interaction within the compound, underscoring the prominent impact of 3,7-dimethylxanthine on cognitive function. Subsequently, through a test of Restricted Cubic Splines (RCS), we revealed a non-linear relationship between ALT and cognitive function at the 10th, 50th, and 90th percentiles (p < 0.05), indicating a heightened risk of diminished cognitive function in the low ALT group. Employing structural equation modeling, we meticulously examined the mediating role of ALT in relation to 3,7-dimethylxanthine and cognitive function. However, our study results did not yield significant evidence of a mediating effect. This comprehensive analysis elucidates the intricate interplay between these variables, unveiling the subtle mechanisms governing cognitive function. CONCLUSIONS: In this study, a noteworthy positive correlation was observed between 3,7-dimethylxanthine and cognitive function. Additionally, a non-linear relationship was identified between ALT and cognitive function, with lower levels of ALT associated with a decline in cognitive function. The RCS trend suggested that higher levels of ALT may similarly lead to diminished cognitive performance. However, in our pursuit to ascertain potential mediation, we regrettably found no significant evidence supporting mediation among these factors involving ALT. This underscores the need for more comprehensive investigations and expanded clinical explorations into the intricate associations among these three pivotal elements.

Investigating the Relations Between Caffeine-Derived Metabolites and Plasma Lipids in 2 Population-Based Studies.

OBJECTIVE: To investigate the relations between caffeine-derived metabolites (methylxanthines) and plasma lipids by use of population-based data from 2 European countries. METHODS: Families were randomly selected from the general population of northern Belgium (FLEMENGHO), from August 12, 1985, until November 22, 1990, and 3 Swiss cities (SKIPOGH), from November 25, 2009, through April 4, 2013. We measured plasma concentrations (FLEMENGHO, SKIPOGH) and 24-hour urinary excretions (SKIPOGH) of 4 methylxanthines-caffeine, paraxanthine, theobromine, and theophylline-using ultra-high-performance liquid chromatography-tandem mass spectrometry. We used enzymatic methods to estimate total cholesterol, high-density lipoprotein cholesterol, and triglyceride levels and the Friedewald equation for low-density lipoprotein cholesterol levels in plasma. We applied sex-specific mixed models to investigate associations between methylxanthines and plasma lipids, adjusting for major confounders. RESULTS: In both FLEMENGHO (N=1987; 1055 [53%] female participants) and SKIPOGH (N=990; 523 [53%] female participants), total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels increased across quartiles of plasma caffeine, paraxanthine, and theophylline (total cholesterol levels by caffeine quartiles in FLEMENGHO, male participants: 5.01±0.06 mmol/L, 5.05±0.06 mmol/L, 5.27±0.06 mmol/L, 5.62±0.06 mmol/L; female participants: 5.24±0.06 mmol/L, 5.15±0.05 mmol/L, 5.25±0.05 mmol/L, 5.42±0.05 mmol/L). Similar results were observed using urinary methylxanthines in SKIPOGH (total cholesterol levels by caffeine quartiles, male participants: 4.54±0.08 mmol/L, 4.94±0.08 mmol/L, 4.87±0.08 mmol/L, 5.27±0.09 mmol/L; female participants: 5.12±0.07 mmol/L, 5.21±0.07 mmol/L, 5.28±0.05 mmol/L, 5.28±0.07 mmol/L). Furthermore, urinary caffeine and theophylline were positively associated with high-density lipoprotein cholesterol in SKIPOGH male participants. CONCLUSION: Plasma and urinary caffeine, paraxanthine, and theophylline were positively associated with plasma lipids, whereas the associations involving theobromine were less clear. We postulate that the positive association between caffeine intake and plasma lipids may be related to the sympathomimetic function of methylxanthines, mitigating the overall health-beneficial effect of caffeine intake.

Metabolomic Changes after Coffee Consumption: New Paths on the Block.

SCOPE: Several studies suggest that regular coffee consumption may help preventing chronic diseases, but the impact of daily intake and the contribution of coffee metabolites in disease prevention are still unclear. The present study aims at evaluating whether and how different patterns of coffee intake (one cup of espresso coffee/day, three cups of espresso coffee/day, and one cup of espresso coffee/day and two cocoa-based products containing coffee two times per day) may impact endogenous molecular pathways. METHODS AND RESULTS: A three-arm, randomized, crossover trial is performed in 21 healthy volunteers who consumed each treatment for one month. Urine samples are collected to perform untargeted metabolomics based on UHPLC-IMS-HRMS. A total of 153 discriminant metabolites are identified. Several molecular features are associated with coffee consumption, while others are linked with different metabolic pathways, such as phenylalanine, tyrosine, energy metabolism, steroid hormone biosynthesis, and arginine biosynthesis and metabolism. CONCLUSION: This information has provided new insights into the metabolic routes by which coffee and coffee-related metabolites may exert effects on human health.

Exploring the Association between Urine Caffeine Metabolites and Urine Flow Rate: A Cross-Sectional Study.

Examination of urine excretion of caffeine metabolites has been a simple but common way to determine the metabolism and effect of caffeine, but the relationship between urinary metabolites and urine flow rate is less discussed. To explore the association between urinary caffeine metabolite levels and urine flow rate, 1571 participants from the National Health and Nutrition Examination Survey (NHANES) 2011-2012 were enrolled in this study. We examined the association between urinary caffeine metabolites and urine flow rate with linear regression models. Separate models were constructed for males and females and for participants aged <60 and ≥60 years old. A positive association was found between concentrations of several urinary caffeine metabolites and urine flow rate. Three main metabolites, namely, paraxanthine, theobromine, and caffeine, showed significance across all subgroups. The number of caffeine metabolites that revealed flow-dependency was greater in males than in females and was also greater in the young than in the elderly. Nevertheless, the general weakness of NHANES data, a cross-sectional study, is that the collection is made at one single time point rather than a long-term study. In summary, urinary concentrations of several caffeine metabolites showed a positive relationship with the urine flow rate. The trend is more noticeable in males and in young subgroups.

Caffeine and Caffeine Metabolites in Relation to Insulin Resistance and Beta Cell Function in U.S. Adults.

The relationship between caffeine and insulin resistance (IR) has been assessed only in terms of caffeine intake, and the association between caffeine and beta cell function (BCF) remains unclear. This study examines the association between urinary caffeine and its metabolites, IR, and BCF in nondiabetic, noninstitutionalized US adults in order to account for the inter-individual differences in caffeine metabolism. Data on urinary caffeine and its metabolites, IR and BCF from adults aged 20 years and older who participated in the 2009-2010 and 2011-2012 National Health and Nutrition Examination Surveys were analyzed (n for caffeine = 994). IR and BCF were assessed using homeostatic model assessment (HOMA) and urinary caffeine and its metabolites were measured using high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry. After adjusting for all covariates, increases in urinary 1,3-DMU, 1,7-DMU, 1,3,7-TMU, theophylline, paraxanthine, caffeine, and AAMU were significantly associated with increased HOMA-IR and HOMA-ß (HOMA of insulin resistance and beta cell function). Compared with individuals in the lowest quartile of urinary 1,3-DMU, 1,7-DMU, 1,3,7-TMU, theophylline, paraxanthine, caffeine, and AAMU, the regression coefficients for HOMA-IR and HOMA-ß were significantly higher among those in the highest quartile. After stratification by prediabetes status, HOMA-IR and HOMA-ß showed significant positive associations with urinary caffeine and its metabolites among subjects with normal fasting plasma glucose levels. Our cross-sectional study showed that caffeine and its metabolites were positively related to IR and BCF.

On-line in-tube solid phase microextraction coupled to capillary liquid chromatography-diode array detection for the analysis of caffeine and its metabolites in small amounts of biological samples.

In-tube solid phase microextraction (IT-SPME) coupled on-line to capillary liquid chromatography with diode array detection provides a simple and fast analytical methodology for the simultaneous quantitation of caffeine and its three primary metabolites (theobromine, paraxanthine and theophylline) in micro samples of serum, saliva and urine matrices. The sample amount required for one analysis was only 2.5 µL of saliva, 6.25 µL of serum or 40 µL of urine, a requirement for its implementation in a hospital laboratory for preterm newborns, where sample availability is a major problem. In standard conditions, 25 µL of diluted saliva or serum (or 100 µL of urine) were processed by IT-SPME in 30 cm of commercially available capillary GC column coated with ZB-FFAP (100% nitroterephthalic modified polyethylene glycol). The retained compounds were desorbed from the IT-SPME capillary by the mobile phase (a gradient mixture of water and methanol) and the separation was carried out in a C18 column (150 mm × 0.5 mm i.d., 5 µm particle size). Analytes eluted before 14 min, at a flow rate of 15 µL min-1, and were detected by absorbance at 275 nm. The calibration graphs presented good linearity (R2 > 0.99), without the presence of matrix effect, and recoveries between 84 and 112% were obtained. Limits of detection (S/N = 3) were 0.1 µg·mL-1 in serum and 0.5 µg·mL-1 in saliva and urine samples, for all compounds, and the intra- and inter-day variation coefficients (n = 3) were between 3 and 17%. Analytical figures of merit were similar to those proposed by other methodologies, but using lower sample volume and a faster and simpler sample treatment and analysis. Paired samples of serum and saliva from preterm newborns treated with caffeine at the pediatric intensive care unit were analyzed by the method, with statistically equivalent results for caffeine concentrations.

Evaluating Novel Markers for Specimen Validity Testing.

CONTEXT.­: Synthetic urine products are commercially marketed for the purpose of specimen substitution for urine drug screens. These products are widely popular because they yield negative drug screen results, meet criteria for specimen validity testing, and are easily accessible and affordable. Current specimen validity criteria are ineffective for detecting these synthetic products, and new markers of specimen validity are required. OBJECTIVE.­: To develop and evaluate a multicomponent liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for urine specimen validity testing. DESIGN.­: A quantitative LC-MS/MS assay was developed for caffeine, cotinine, theobromine, and urobilin in urine. The assay was applied to known synthetic urine products (n = 10) as well as human specimens received for pre-employment testing (n = 500), for-cause workplace testing (n = 100), and medical pain management monitoring (n = 200). Specimens devoid of all 4 validity markers were subjected to follow-up testing that involved microscopic urinalysis and comprehensive gas chromatography mass spectrometry for drugs, pharmaceuticals, hormones, and lipids. RESULTS.­: Of the experimental groups, 10 of 10 synthetic urine products (100%), 12 of 500 pre-employment specimens (2.4%), and 4 of 200 pain management specimens (2.0%) failed the experimental LC-MS/MS assay. Follow-up testing indicated that each of the failed specimens was nonphysiologic in nature. CONCLUSIONS.­: Simultaneous application of the 4 experimental validity markers appeared to be a robust method for detecting nonphysiologic specimens. New markers of specimen validity must be developed in order to identify commercially available synthetic urine products.

Determination of Urinary Caffeine Metabolites as Biomarkers for Drug Metabolic Enzyme Activities.

Caffeine is commonly taken via the daily dietary consumption of caffeine-containing foods. The absorbed caffeine is metabolized to yield various metabolites by drug-metabolizing enzymes, and measuring the levels of each caffeine metabolite can provide useful information for evaluating the phenotypes of those enzymes. In this study, the urinary concentrations of caffeine and its 13 metabolites were determined, and the phenotypes of drug metabolic enzymes were investigated based on the caffeine metabolite ratios. Human urine samples were pretreated using solid phase extraction, and caffeine and its metabolites were analyzed using liquid chromatography-tandem mass spectrometry. Based on the urinary caffeine metabolite concentrations, the caffeine metabolite ratios were calculated for six human subjects at specified time points after caffeine intake. Variations in urinary metabolite levels among individuals and time points were reported. In addition, the resultant enzyme activities showed different patterns, depending on the metabolite ratio equations applied. However, some data presented a constant metabolite ratio range, irrespective of time points, even at pre-dose. This suggests the possibility of urinary caffeine metabolite analysis for routine clinical examination. These findings show that urinary caffeine and the metabolite analysis would be useful in evaluating metabolic phenotypes for personalized medicine.

Determination of cocaine adulterants in human urine by dispersive liquid-liquid microextraction and high-performance liquid chromatography.

This study aimed to determine simultaneously five major street cocaine adulterants (caffeine, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine by dispersive liquid-liquid microextraction (DLLME) and high-performance liquid chromatography. The chromatographic separation was obtained in gradient elution mode using methanol:water plus trifluoroacetic acid 0.15% (v/v) (pH = 1.9) at 1 mL min-1 as mobile phase, at 25 °C, detection at 235 nm, and analysis time of 20 min. The effect of major DLLME operating parameters on extraction efficiency was explored using the multifactorial experimental design approach. The optimum extraction condition was set as 4 mL human urine sample alkalized with 0.5 M sodium phosphate buffer (pH 12), NaCl (15%, m/v), 300 µL acetonitrile (dispersive solvent), and 800 µL chloroform (extraction solvent). Linear response (r2 ≥ 0.99) was obtained in the range of 180-1500 ng mL-1 with suitable selectivity, quantification limit (180 ng mL-1), mean recoveries (33.43-76.63%), and showing relative standard deviation and error (within and between-day assays) ≤15%. The analytes were stable after a freeze-thaw cycle and a short-term room temperature stability test. This method was successfully applied in real samples of cocaine users, suggesting that our study may contribute to the appropriate treatment of cocaine dependence or with the cases of cocaine acute intoxication.

Urine Caffeine Concentration in Doping Control Samples from 2004 to 2015.

The ergogenic effect of caffeine is well-established, but the extent of its consumption in sport is unknown at the present. The use of caffeine was considered "prohibited" until 2004, but this stimulant was moved from the List of Prohibited Substances to the Monitoring Program of the World Anti-Doping Agency to control its use by monitoring urinary caffeine concentration after competition. However, there is no updated information about the change in the use of caffeine as the result of its inclusion in the Monitoring Program. The aim of this study was to describe the changes in urine caffeine concentration from 2004 to 2015. A total of 7488 urine samples obtained in official competitions held in Spain and corresponding to athletes competing in Olympic sports (2788 in 2004, 2543 in 2008, and 2157 in 2015) were analyzed for urine caffeine concentration. The percentage of samples with detectable caffeine (i.e., >0.1 µg/mL) increased from ~70.1%, in 2004⁻2008 to 75.7% in 2015. The median urine caffeine concentration in 2015 (0.85 µg/mL) was higher when compared to the median value obtained in 2004 (0.70 µg/mL; p < 0.05) and in 2008 (0.70 µg/mL; p < 0.05). The urine caffeine concentration significantly increased from 2004 to 2015 in aquatics, athletics, boxing, judo, football, weightlifting, and rowing (p < 0.05). However, the sports with the highest urine caffeine concentration in 2015 were cycling, athletics, and rowing. In summary, the concentration of caffeine in the urine samples obtained after competition in Olympic sports in Spain increased from 2004 to 2015, particularly in some disciplines. These data indicate that the use of caffeine has slightly increased since its removal from the list of banned substances, but urine caffeine concentrations suggest that the use of caffeine is moderate in most sport specialties. Athletes of individual sports or athletes of sports with an aerobic-like nature are more prone to using caffeine in competition.

Associations of Urinary Caffeine and Caffeine Metabolites With Arterial Stiffness in a Large Population-Based Study.

OBJECTIVE: To assess the influence of caffeine on arterial stiffness by exploring the association of urinary excretion of caffeine and its related metabolites with pulse pressure (PP) and pulse wave velocity (PWV). PARTICIPANTS AND METHODS: Families were randomly selected from the general population of 3 Swiss cities from November 25, 2009, through April 4, 2013. Pulse pressure was defined as the difference between the systolic and diastolic blood pressures obtained by 24-hour ambulatory monitoring. Carotid-femoral PWV was determined by applanation tonometry. Urinary caffeine, paraxanthine, theophylline, and theobromine excretions were measured in 24-hour urine collections. Multivariate linear and logistic mixed models were used to explore the associations of quartiles of urinary caffeine and metabolite excretions with PP, high PP, and PWV. RESULTS: We included 863 participants with a mean ± SD age of 47.1±17.6 years, 24-hour PP of 41.9±9.2 mm Hg, and PWV of 8.0±2.3 m/s. Mean (SE) brachial PP decreased from 43.5 (0.5) to 40.5 (0.6) mm Hg from the lowest to the highest quartiles of 24-hour urinary caffeine excretion (P<.001). The odds ratio (95% CI) of high PP decreased linearly from 1.0 to 0.52 (0.31-0.89), 0.38 (0.22-0.65), and 0.31 (0.18-0.55) from the lowest to the highest quartile of 24-hour urinary caffeine excretion (P<.001). Mean (SE) PWV in the highest caffeine excretion quartile was significantly lower than in the lowest quartile (7.8 [0.1] vs 8.1 [0.1] m/s; P=.03). Similar associations were found for paraxanthine and theophylline, whereas no associations were found with theobromine. CONCLUSION: Urinary caffeine, paraxanthine, and theophylline excretions were associated with decreased parameters of arterial stiffness, suggesting a protective effect of caffeine intake beyond its blood pressure-lowering effect.

A Versatile Probe for Caffeine Detection in Real-Life Samples via Excitation-Triggered Alteration in the Sensing Behavior of Fluorescent Organic Nanoaggregates.

Excitation triggered alteration in the sensing behavior of fluorescent nanoaggregates was explored in water, considering caffeine as the "target analyte". Merely by changing the excitation wavelength, we could specifically excite either the monomeric species or the fluorescent nanoaggregates. The monomer showed highly sensitive interaction with caffeine despite poor selectivity, while the "strongly associated" fluorescent nanoaggregates displayed relatively high selectivity with low sensitivity. In addition, the extent of self-aggregation was also found to be influenced by the micropolarity of the local surroundings and the electronics of the core carbazole unit. Furthermore, the present protocol was utilized for the estimation of caffeine in different beverages and biological fluids with reasonably high accuracy. Inexpensive, portable paper strips were designed for a rapid, on-site detection of caffeine without involving sophisticated instruments or trained technicians.

Direct Analysis of Biofluids by Mass Spectrometry with Microfluidic Voltage-Assisted Liquid Desorption Electrospray Ionization.

Signal suppression by sample matrix in direct electrospray ionization-mass spectrometric (ESI-MS) analysis hampers its clinical and biomedical applications. We report herein the development of a microfluidic voltage-assisted liquid desorption electrospray ionization (VAL-DESI) source to overcome this limitation. Liquid DESI is achieved for the first time in a microfluidic format. Direct analysis of urine, serum, and cell lysate samples by using the proposed microfluidic VAL-DESI-MS/MS method to detect chemical compounds of biomedical interest, including nucleosides, monoamines, amino acids, and peptides is demonstrated. Analyzing a set of urine samples spiked with dihydroxyphenylalanine (DOPA) showed that the assay had a linear calibration curve with r2 value of 0.997 and a limit of detection of 0.055 µM DOPA. The method was applied to simultaneous quantification of nucleosides, that is, cytidine, adenosine, uridine, thymidine, and guanosine in cell lysates using 8-bromoadenosine as internal standard. Adenosine was found most abundant at 26.5 ± 0.57 nmol/106 cells, while thymidine was least at 3.1 ± 0.31 nmol/106 cells. Interestingly, the ratio of adenosine to deoxyadenosine varied significantly from human red blood cells (1.07 ± 0.06) to cancerous cells, including lymphoblast TK6 (0.52 ± 0.02), skin melanoma C32 (0.82 ± 0.04), and promyelocytic leukemia NB4 cells (0.38 ± 0.06). These results suggest that the VAL-DESI-MS/MS technique has a good potential in direct analysis of biofluids. Further, because of the simplicity in its design and operation, the proposed microfluidic liquid DESI source can be fabricated as a disposable device for point-of-care measurements.

Water pipe (Shisha, Hookah, Arghile) Smoking and Secondhand Tobacco Smoke Effects on CYP1A2 and CYP2A6 Phenotypes as Measured by Caffeine Urine Test.

Public policies to stop or reduce cigarette smoking and exposure to secondhand smoke and associated diseases have yielded successful results over the past decade. Yet, the growing worldwide popularity of another form of tobacco consumption, water pipe smoking, has received relatively less attention. To the best of our knowledge, no study to date has evaluated the effects of water pipe smoking on cytochrome P450 (CYP450) activities and drug interaction potential in humans, whereas only limited information is available on the impact of secondhand smoke on drug metabolism. In a sample of 99 healthy volunteers (28 water pipe smokers, 30 secondhand tobacco smoke exposed persons, and 41 controls), we systematically compared CYP1A2 and CYP2A6 enzyme activities in vivo using caffeine urine test. The median self-reported duration of water pipe smoking was 7.5 h/week and 3 years of exposure in total. The secondhand smoke group had a median of 14 h of self-reported weekly exposure to tobacco smoke indoor where a minimum of five cigarettes were smoked/hour for a total of 3.5 years (median). Analysis of variance did not find a significant difference in CYP1A2 and CYP2A6 activities among the three study groups (p > 0.05). Nor was there a significant association between the extent of water pipe or secondhand smoke exposure and the CYP1A2 and CYP2A6 activities (p > 0.05). Further analysis in a subsample with smoke exposure more than the median values also did not reveal a significant difference from the controls. Although we do not rule out an appreciable possible impact of water pipe smoke and secondhand smoke on in vivo activities of these two drug metabolism pathways, variability in smoke constituents from different tobacco consumption methods (e.g., water pipe) might affect drug metabolism in ways that might differ from that of cigarette smoke. Further studies in larger prospective samples are recommended to evaluate water pipe and secondhand tobacco smoke effects on CYP450 function, particularly at higher smoke exposure conditions.

The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6ß-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2.

Chemical and biochemical characterization and in vivo safety evaluation of pharmaceuticals in drinking water.

The water constituents that are currently subject to legal control are only a small fraction of the vast number of chemical substances and microorganisms that may occur in both the environment and water resources. The main objective of the present study was to study the health impact resulting from exposure to a mixture of pharmaceuticals that have been detected in tap water at low doses. Analyses of atenolol, caffeine, erythromycin, carbamazepine, and their metabolites in blood, urine, feces, fat tissue, liver, and kidney after exposure to a mixture of these pharmaceuticals in treated drinking water were performed. The effects of this exposure were assessed in rats by measuring biochemical markers of organ injury or dysfunction. Simultaneously, the selected pharmaceuticals were also quantified in both physiological fluids and organ homogenates by liquid chromatography-tandem mass spectrometry (performed in multiple reaction monitoring mode and full scan mode). Following exposure of rats to a concentration of a pharmaceutical which was 10 times higher than the concentration known to be present in tap water, trace levels of some pharmaceuticals and their metabolites were detected in biological samples. This exposure did, however, not lead to significant organ injury or dysfunction. Thus, the authors report an experimental model that can be used to characterize the safety profile of pharmaceuticals in treated drinking water using a multiorgan toxicity approach. Environ Toxicol Chem 2016;35:2674-2682. © 2016 SETAC.

Fluorescence Based Turn-on Probe for the Determination of Caffeine Using Europium-Tetracycline as Energy Transfer Complex.

The study describes a simple and sensitive fluorometric sensor based on the enhancement of fluorescence intensity of Europium ion (Eu(3+)) - tetracycline (TC) charge transfer complex on addition of caffeine. The Eu(3+)-TC ternary complex has a characteristic emission peak at 615 nm (λex = 375 nm), the intensity of which increases with increase in concentration of caffeine. The caffeine sensor assay was found to be linear in the range of 0.0515 mM to 51.5 mM. The limit of detection and quantification were found to be 0.0515 mM and 0.382 mM, respectively. A caffeine recovery of 90 to 110 % in biological samples (serum and urine) indicated minimal interference by commonly present excipients in the samples. Rosenthal plots to calculate the binding capacity of caffeine with the Eu(3+)- TC complex revealed an association constant (K) of 238 x 10(3) L/mol and binding number (N) of 1.9. Bland-Altman plot comparing the developed assay and HPLC showed good agreement between values obtained by both the methods. The proposed fluorescent chemical sensor is a rapid and convenient method to determine caffeine with excellent recovery and low detection limit. The probable reaction mechanism for the formation of the turn on fluorescent probe enhancer is discussed.

The CYTONOX trial.

INTRODUCTION: In Denmark, it is estimated that 3-5% of children are obese. Obesity is associated with pathophysiological alterations that may lead to alterations in the pharmacokinetics of drugs. In adults, obesity was found to influence important drug-metabolising enzyme pathways. The impact of obesity-related alterations on drug metabolism and its consequences for drug dosing remains largely unknown in both children and adults. An altered drug metabolism may contribute significantly to therapeutic failure or toxicity. The aim of this trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 substrates in obese versus non-obese children. METHODS: The CYTONOX trial is an open-label explorative pharmacokinetic trial. We intend to include 50 obese and 50 non-obese children. The primary end points are: in vivo clearance of CYP3A4, CYP2E1 and CYP1A2 substrates, which will be defined by using well-tested probes; midazolam, chlorzoxazone and caffeine. Each of the probes will be administered as a single dose. Subsequently, blood and urine samples will be collected at pre-specified times. CONCLUSION: The aim of the CYTONOX trial is to investigate the in vivo activity of CYP3A4, CYP2E1 and CYP1A2 in obese and non-obese children. The results are expected to be used in the future as a basis for drug dosing recommendations in obese children. FUNDING: The study was funded by the Danish Regions' "Medicinpuljen". The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. TRIAL REGISTRATION: EudraCT: 2014-004554-34.

Correction.

In the article by Guessous et al (Guessous I, Pruijm M, Ponte B, Ackermann D, Ehret G, Ansermot N, Vuistiner P, Staessen J, Gu Y, Paccaud F, Mohaupt M, Vogt B, Pechère-Bertschi A, Martin PY, Burnier M, Eap CB, Bochud M. Associations of ambulatory blood pressure with urinary caffeine and caffeine metabolite excretions. Hypertension. 2015;65:691­696. doi: 10.1161/HYPERTENSIONAHA.114.04512), which published online ahead of print December 8, 2014, and appeared in the March 2015 issue of the journal, a correction was needed.One of the author surnames was misspelled. Antoinette Pechère-Berstchi has been corrected to read Antoinette Pechère-Bertschi.The authors apologize for this error.