Comparison of caffeine consumption behavior with plasma caffeine levels as exposure measures in drug-target Mendelian randomization.

Mendelian randomization is an epidemiological technique that can explore the potential effect of perturbing a pharmacological target. Plasma caffeine levels can be used as a biomarker to measure the pharmacological effects of caffeine. Alternatively, this can be assessed using a behavioral proxy, such as average number of caffeinated drinks consumed per day. Either variable can be used as the exposure in a Mendelian randomization investigation, and to select which genetic variants to use as instrumental variables. Another possibility is to choose variants in gene regions with known biological relevance to caffeine level regulation. These choices affect the causal question that is being addressed by the analysis, and the validity of the analysis assumptions. Further, even when using the same genetic variants, the sign of Mendelian randomization estimates (positive or negative) can change depending on the choice of exposure. Some genetic variants that decrease caffeine metabolism associate with higher levels of plasma caffeine, but lower levels of caffeine consumption, as individuals with these variants require less caffeine consumption for the same physiological effect. We explore Mendelian randomization estimates for the effect of caffeine on body mass index, and discuss implications for variant and exposure choice in drug target Mendelian randomization investigations.

Metabolomics analysis of urine from patients with alcohol-associated liver disease reveals dysregulated caffeine metabolism.

Excess alcohol intake causes millions of deaths annually worldwide. Asymptomatic early-stage, alcohol-associated liver disease (ALD) is easily overlooked, and ALD is usually only diagnosed in more advanced stages. We explored the possibility of using polar urine metabolites as biomarkers of ALD for early-stage diagnosis and functional assessment of disease severity by quantifying the abundance of polar metabolites in the urine samples of healthy controls (n = 18), patients with mild or moderate liver injury (n = 21), and patients with severe alcohol-associated hepatitis (n = 25). The polar metabolites in human urine were first analyzed by untargeted metabolomics, showing that 209 urine metabolites are significantly changed in patients, and 17 of these are highly correlated with patients' model for end-stage liver disease (MELD) score. Pathway enrichment analysis reveals that the caffeine metabolic pathway is the most affected in ALD. We then developed a targeted metabolomics method and measured the concentration of caffeine and its metabolites in urine using internal and external standard calibration, respectively. The described method can quantify caffeine and its 14 metabolites in 35 min. The results of targeted metabolomics analysis agree with the results of untargeted metabolomics, showing that 13 caffeine metabolites are significantly decreased in patients. In particular, the concentrations of 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil are markedly decreased with increased disease severity. We suggest that these three metabolites could serve as functional biomarkers for differentiating early-stage ALD from more advanced liver injury.NEW & NOTEWORTHY Our study using both untargeted and targeted metabolomics reveals the caffeine metabolic pathway is dysregulated in ALD. Three caffeine metabolites, 1-methylxanthine, paraxanthine, and 5-acetylamino-6-amino-3-methyluracil, can differentiate the severity of early-stage ALD.

Analysis of ADORA2A rs5760423 and CYP1A2 rs762551 Genetic Variants in Patients with Alzheimer's Disease.

Various studies have been conducted, exploring the genetic susceptibility of Alzheimer's disease (AD). Adenosine receptor subtype A2a (ADORA2A) and cytochrome P450 1A2 (CYP1A2) are implicated in pathways such as oxidative stress and caffeine metabolism, which are associated with AD. The aim of this study was to explore for any potential association between the ADORA2A rs5760423 and the CYP1A2 rs762551 genetic variants and AD. A case-control study was performed with a total of 654 subjects (327 healthy controls and 327 patients with AD). Five genetic models were assumed. We also examined the allele-allele combination of both variants. The value of 0.05 was considered as the statistical significance threshold. A statistically significant association was found between ADORA2A rs5760423 and AD, as the "T" allele was associated with increased AD risk in recessive (OR = 1.51 (1.03-2.21)) and log-additive (OR = 1.30 (1.04-1.62)) genetic modes. In the codominant model, the TT genotype was more prevalent compared to the GG genotype (OR = 1.71 (1.09-2.66)). The statistical significance was maintained after adjustment for sex. No association between CYP1A2 rs762551 or allele-allele combination and AD was detected. We provide preliminary indication for a possible association between the ADORA2A rs5760423 genetic polymorphism and AD.

Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC-MS/MS.

Caffeine is a widely consumed psychostimulant with several mechanisms of action and various positive and negative effects on organisms. Caffeine undergoes extensive hepatic metabolism to form main metabolites such as theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid. However, interspecies diversities have been observed in caffeine metabolism. In the present study, we developed a sensitive and straightforward ultra-high-performance liquid chromatography-tandem mass spectrometry method to quantify caffeine and its primary metabolites, namely theobromine, theophylline, paraxanthine, and 1,3,7-trimethyluric acid in rat plasma. After extraction of analytes using micro solid-phase extraction plate, analytes were separated by elution gradient on the Acquity UPLC HSS T3 (50 × 2.1 mm, 1.8 µm) column over 4 min. The detection was done on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring modes using a positive electrospray ionization interface. The method was successfully validated according to the European Medicine Agency guideline over a concentration range of 5-1500 ng/ml for caffeine, 5-1200 ng/mL for theobromine, and 2.5-1200 ng/mL for theophylline, paraxanthine, and 1,3,7-trimethyluric acid. The developed method was applied to analyze samples from animal experiments focusing on the metabolism and effects of caffeine and caffeine-containing beverages.

Rapid identification and quantitative validation of a caffeine-degrading pathway in Pseudomonas sp. CES.

Understanding the genes and enzymes involved in caffeine metabolism can lead to applications such as production of methylxanthines and environmental waste remediation. Pseudomonas sp. CES may provide insights into these applications, since this bacterium degrades caffeine and thrives in concentrations of caffeine that are three times higher (9.0 g L(-1)) than the maximum tolerable levels of other reported bacteria. We took a novel approach toward identifying the enzymatic pathways in Pseudomonas sp. CES that metabolize caffeine, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities. Here we describe an optimized, rapid alternative strategy based on multiplexed LC-MS/MS assays and show its application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison of proteomes from bacteria grown in the absence and presence of caffeine, the latter condition of which was found to have a highly induced capacity for caffeine degradation. Comparisons were made using stable isotope dimethyl labeling, differences in the abundance of particular proteins were substantiated by reciprocal labeling experiments, and the role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Overall, multiple new components of a N-demethylase system were identified that resulted in rapid pathway validation and gene isolation using this new approach.

Metabolomics Unveils Disrupted Pathways in Parkinson's Disease: Toward Biomarker-Based Diagnosis.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by diverse symptoms, where accurate diagnosis remains challenging. Traditional clinical observation methods often result in misdiagnosis, highlighting the need for biomarker-based diagnostic approaches. This study utilizes ultraperformance liquid chromatography coupled to an electrospray ionization source and quadrupole time-of-flight untargeted metabolomics combined with biochemometrics to identify novel serum biomarkers for PD. Analyzing a Brazilian cohort of serum samples from 39 PD patients and 15 healthy controls, we identified 15 metabolites significantly associated with PD, with 11 reported as potential biomarkers for the first time. Key disrupted metabolic pathways include caffeine metabolism, arachidonic acid metabolism, and primary bile acid biosynthesis. Our machine learning model demonstrated high accuracy, with the Rotation Forest boosting model achieving 94.1% accuracy in distinguishing PD patients from controls. It is based on three new PD biomarkers (downregulated: 1-lyso-2-arachidonoyl-phosphatidate and hypoxanthine and upregulated: ferulic acid) and surpasses the general 80% diagnostic accuracy obtained from initial clinical evaluations conducted by specialists. Besides, this machine learning model based on a decision tree allowed for visual and easy interpretability of affected metabolites in PD patients. These findings could improve the detection and monitoring of PD, paving the way for more precise diagnostics and therapeutic interventions. Our research emphasizes the critical role of metabolomics and machine learning in advancing our understanding of the chemical profile of neurodegenerative diseases.

Tea Consumption and New-Onset Acute Kidney Injury: The Effects of Milk or Sweeteners Addition and Caffeine/Coffee.

Aims: To explore the relationship between tea consumption and the risk of incident acute kidney injury (AKI) and examine the effects of coffee consumption, genetic variation in caffeine metabolism, and the use of tea additives (milk and sweeteners) on this association. Methods: Using data from the UK Biobank, 498,621 participants who were free of AKI and had information on tea intake were included. Black tea is the main type consumed in this population. Dietary information was collected from standardized and validated Food-Frequency Questionnaire (FFQ). Outcome was incident AKI, determined via primary care data, hospital inpatient data, death register records, or self-reported data at follow-up visits. Results: After a median follow-up period of 12.0 years, 21,202 participants occurred AKI. Overall, there was a reversed J-shaped relation between tea consumption and incident AKI, with an inflection point at 3.5 cup/d (p for nonlinearity < 0.001). The relation was similar among participants with different genetically predicted caffeine metabolism (p-interaction = 0.684), while a more obvious positive association was found between heavy tea consumption and AKI when more coffee was consumed (p-interaction < 0.001). Meanwhile, there was a reversed J-shaped relationship for drinking tea with neither milk nor sweeteners, and a L-shaped association for drinking tea with milk (with or without sweeteners) with incident AKI. However, no significant association was found between drinking tea with sweeteners only and incident AKI. Conclusions: There was a reversed J-shaped relation between tea consumption and incident AKI, suggesting that light to moderate tea consumption, especially adding milk, can be part of a healthy diet.

Serum Phosphorus, Serum Bicarbonate, and Renal Function in Relation to Liver CYP1A2 Activity.

The liver plays an important role in normal metabolism and physiological functions such as acid-base balance; however, limited epidemiologic studies have investigated how the liver contributes toward acid-base balance using non-invasive biomarkers. We determined associations between serum biomarkers related to acid-base balance and renal function with liver CYP1A2 activity. We used data from 1381 participants of the 2009-2010 National Health and Nutrition Examination Survey (NHANES) with measurements of serum phosphorus, serum bicarbonate, caffeine intake, caffeine metabolites, and estimated glomerular filtration rate (eGFR). Liver CYP1A2 activity was estimated using urine caffeine metabolite indices, which were calculated as the ratio of one of the urine caffeine metabolites (i.e., paraxanthine and 1-methyluric acid) to caffeine intake. We analyzed associations in the whole data set and in different strata of hepatic steatosis index (HSI) based on different cut-points. We found that serum bicarbonate was positively associated with CYP1A2 activity in the whole data set when comparing persons with bicarbonate at Q4 to Q1 (ß = 0.18, p = 0.10 for paraxanthine; ß = 0.20, p = 0.02 for 1-methyluric acid). Furthermore, serum phosphorus was positively associated with CYP1A2 activity only in the stratum of 30 ≤ HSI < 36. Lastly, low eGFR was significantly associated with lower CYP1A2 activity measured with paraxanthine in the whole dataset and in all the strata with HSI < 42; when comparing eGFR < 60 to eGFR > 90, ß estimates ranged from -0.41 to -1.38, p-values ranged from 0.0018 to 0.004. We observed an opposite trend in the highest stratum (HSI ≥ 42). Non-invasive measurements of serum bicarbonate, serum phosphorus, and eGFR have dynamic associations with CYP1A2 activity. These associations depend on the extent of liver damage and the caffeine metabolite used to assess CYP1A2 activity.

Plasma metabolomic characterization of premature ovarian insufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) patients are predisposed to metabolic disturbances, including in lipid metabolism and glucose metabolism, and metabolic disorders appear to be a prerequisite of the typical long-term complications of POI, such as cardiovascular diseases or osteoporosis. However, the metabolic changes underlying the development of POI and its subsequent complications are incompletely understood, and there are few studies characterizing the disturbed metabolome in POI patients. The aim of this study was to characterize the plasma metabolome in POI by using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) metabolomics and to evaluate whether these disturbances identified in the plasma metabolome relate to ovarian reserve and have diagnostic value in POI. METHODS: This observational study recruited 30 POI patients and 30 age- and body mass index (BMI)-matched controls in the Center for Reproductive Medicine, Department of Gynecology and Obstetrics, Nanfang Hospital, Southern Medical University, from January 2018 to October 2020. Fasting venous blood was collected at 9:00 am on days 2-4 of the menstrual cycle and centrifuged for analysis. An untargeted quantitative metabolomic analysis was performed using UHPLC-MS/MS. RESULTS: Our study identified 48 upregulated and 21 downregulated positive metabolites, and 13 upregulated and 48 downregulated negative metabolites in the plasma of POI patients. The differentially regulated metabolites were involved in pathways such as caffeine metabolism and ubiquinone and other terpenoid-quinone biosynthesis. Six metabolites with an AUC value > 0.8, including arachidonoyl amide, 3-hydroxy-3-methylbutanoic acid, dihexyl nonanedioate, 18-HETE, cystine, and PG (16:0/18:1), were correlated with ovarian reserve and thus have the potential to be diagnostic biomarkers of POI. CONCLUSION: This UHPLC-MS/MS untargeted metabolomics study revealed differentially expressed metabolites in the plasma of patients with POI. The differential metabolites may not only be involved in the aetiology of POI but also contribute to its major complications. These findings offer a panoramic view of the plasma metabolite changes caused by POI, which may provide useful diagnostic and therapeutic clues for POI disease.

Metabolic profiling identifies the significance of caffeine metabolism in CKD.

Background: With the development of chronic kidney disease (CKD), there are various changes in metabolites. However, the effect of these metabolites on the etiology, progression and prognosis of CKD remains unclear. Objective: We aimed to identify significant metabolic pathways in CKD progression by screening metabolites through metabolic profiling, thus identifying potential targets for CKD treatment. Methods: Clinical data were collected from 145 CKD participants. GFR (mGFR) was measured by the iohexol method and participants were divided into four groups according to their mGFR. Untargeted metabolomics analysis was performed via UPLC-MS/MSUPLC-MSMS/MS assays. Metabolomic data were analyzed by MetaboAnalyst 5.0, one-way ANOVA, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) to identify differential metabolites for further analysis. The open database sources of MBRole2.0, including KEGG and HMDB, were used to identify significant metabolic pathways in CKD progression. Results: Four metabolic pathways were classified as important in CKD progression, among which the most significant was caffeine metabolism. A total of 12 differential metabolites were enriched in caffeine metabolism, four of which decreased with the deterioration of the CKD stage, and two of which increased with the deterioration of the CKD stage. Of the four decreased metabolites, the most important was caffeine. Conclusion: Caffeine metabolism appears to be the most important pathway in the progression of CKD as identified by metabolic profiling. Caffeine is the most important metabolite that decreases with the deterioration of the CKD stage.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Urate oxidase from tea microbe Colletotrichum camelliae is involved in the caffeine metabolism pathway and plays a role in fungal virulence.

Tea is one of the most well-known, healthy beverages in the world. Tea plants produce caffeine as a secondary metabolite. Colletotrichum camelliae is one of the most important microbes frequently isolated from tea fields, and it causes anthracnose disease in tea plant. In the present work, we performed molecular microbiology and transcriptomic analyses of the C. camelliae - tea plant interaction to investigate the mechanism of fungal virulence and plant defense. Upon infection of tea plant with C. camelliae, we observed alterations in the expression of fungal transcripts, including those of many genes associated with caffeine metabolism, such as those encoding various transporters, xanthine dehydrogenase, and urate oxidase (UOX). In particular, the deletion of C. camelliae urate oxidase (CcUOX), which is involved in the caffeine metabolism pathway, reduced fungal tolerance to caffeine, and impaired fungal virulence. CcUOX is involved in caffeine metabolism by the degradation of uric acid contents. C. camelliaeΔCcUOX mutants impaired uric acid degradation in vivo. The CcUOX gene was cloned from C. camelliae, overexpressed in Escherichia coli, and the recombinant CcUOX protein displayed maximum activity at 30°C and a pH of 4.0. The recombinant CcUOX efficiently reduced uric acid in vitro suggesting a promising application in caffeine-contaminated environment management and in producing food with low purine contents to prevent uric acid related human diseases, such as hyperuricemia and gout.

Functional single-nucleotide polymorphism (rs762551) in CYP1A2 gene affects white coffee intake in healthy 20- to 40-year-old adults.

Coffee is one of the most consumed beverages in the world, but the extent to which it is consumed is affected by both environmental and genetic factors. Genome-wide association studies and candidate date association studies have identified several gene variants associated with increased consumption of coffee. Functional single-nucleotide polymorphisms in rs762551 (cytochrome P450 1A2 [CYP1A2]) and rs5751876 (adenosine receptor A2A [ADORA2A]) has been linked to individual caffeine response. Coffee intake has been shown to affect lipid metabolism. We thus hypothesize that rs762551 (CYP1A2) A allele carriers consume more coffee than C allele carriers and that rs5751876 (ADORA2A) C allele carriers consume less coffee than T allele carriers. Additionally, we hypothesize that CYP1A2 genotype can modulate serum glucose concentrations and lipid profile. A total of 421 participants aged 20 to 40 years were recruited from 2016 to 2018 in Poznan, Poland. Genotyping of CYP1A2 and ADORA2A was performed using TaqMan probes. Individuals with AA CYP1A2 genotype consumed relatively more coffee with milk (72.81 ± 10.15 mL/1000 kcal vs 43.38 ± 6.42 mL/1000 kcal, P = .008) and with milk or cream than did C allele carriers, whereas the rs5751876 ADORA2A polymorphism was not associated with coffee or tea intake. Additionally, subjects with AA CYP1A2 genotype had 10% higher serum triacylglycerol (TG) concentrations than C allele carriers. This study suggests that CYP1A2 rs762551 polymorphism is associated with coffee intake and serum TG concentrations in healthy 20- to 40-year-old adults.

The determination of the Paraxanthine/Caffeine ratio as a metabolic biomarker for CYP1A2 activity in various human matrices by UHPLC-ESIMS/MS.

The Cytochrome P450 CYP1A2 is a central enzyme in the metabolism of drugs and xenobiotics. The overall activity of this enzyme is influenced by a complex array of biochemical, dietary, and genetic factors. One of the simplest ways to probe the overall output of CYP1A2 is to measure the ratio between the concentration of a precursor and a product of its activity. With the growing interest in the Paraxanthine/Caffeine ratio, the need arises to develop improved analytical methods specifically optimized for the rapid and sensitive determination of paraxanthine and caffeine in biological samples. We report a new optimized method for the determination of caffeine and paraxanthine in various human matrices. The method involved direct determination following protein precipitation based on ultra high performance liquid chromatographic separation with tandem mass spectrometric detection (UHPLC-ESIMS/MS). The method offers an improvement in the detection limit over previously published methods by at least 10-fold (0.1 pg), rapid chromatographic separation (ca. 5 min), the utilization of a green chromatographic solvent (5% v/v ethanol), direct determination with little sample preparation, and the employment of isotopically labeled internal standards and qualifier ions to ensure accuracy. Method validation in urine, saliva, and plasma was performed by spiking at various concentration levels where the recovery and repeatability were within ±15% and ±10%, respectively. The method was applied to investigate the levels of caffeine and paraxanthine in volunteers following controlled caffeine administration and to investigate the inter- and intra-individual variability in the paraxanthine/caffeine ratio in volunteers following an unrestricted caffeine diet. In conclusion, the developed UHPLC-ESIMS/MS method is optimized specifically for the simultaneous determination of the paraxanthine/caffeine ratio in multiple biological matrices, offers several advantages over the current methods, and is well suitable for application in large clinical studies.

Tobacco Smoke and CYP1A2 Activity in a US Population with Normal Liver Enzyme Levels.

Non-alcoholic fatty liver disease (NAFLD) is common among 30% of American adults. Former and current smokers are at higher risk for NAFLD compared to never smokers. The ratio of urine caffeine metabolites to caffeine intake-namely, urine caffeine metabolite indices-has previously been used as a proxy for CYP1A2 activity, which is one of the main liver metabolizing enzymes. CYP1A2 activity is associated with NAFLD progression. No studies to our knowledge have examined the associations of liver enzymes, smoking intensity, and secondhand smoke (SES) with CYP1A2 activity (using caffeine metabolite indices) across smoking status. We analyzed national representative samples from the 2009-2010 National Health and Nutrition Examination Survey (NHANES). Interestingly, even within a normal range, several liver enzymes were associated with caffeine metabolite indices, and patterns of many of these associations varied by smoking status. For instance, within a normal range, aspartate aminotransferase (AST) in never smokers and bilirubin in current smokers were inversely associated with 1-methyluric acid and 5-acetylamino-6-amino-3-methyluracil (URXAMU). Furthermore, we observed a common pattern: across all smoking statuses, higher AST/alanine aminotransferase (AST/ALT) was associated with 1-methyluric acid and URXAMU. Moreover, in current smokers, increased lifelong smoking intensity was associated with reduced caffeine metabolite indices, but acute cigarette exposure as measured by SES levels was associated with increased caffeine metabolite indices among never smokers. In summary, commonly used liver enzyme tests can reflect the CYP1A2 activity even within a normal range, but the selection of these enzymes depends on the smoking status; the associations between smoking and the CYP1A2 activity not only depend on the intensity but also the duration of tobacco exposure.

A Thyroid Hormone-Independent Molecular Fingerprint of 3,5-Diiodothyronine Suggests a Strong Relationship with Coffee Metabolism in Humans.

Background: In numerous studies based predominantly on rodent models, administration of 3,5-diiodo-L-thyronine (3,5-T2), a metabolite of the thyroid hormones (TH) thyroxine (T4) and triiodo-L-thyronine (T3), was reported to cause beneficial health effects, including reversal of steatohepatosis and prevention of insulin resistance, in most instances without adverse thyrotoxic side effects. However, the empirical evidence concerning the physiological relevance of endogenously produced 3,5-T2 in humans is comparatively poor. Therefore, to improve the understanding of 3,5-T2-related metabolic processes, we performed a comprehensive metabolomic study relating serum 3,5-T2 concentrations to plasma and urine metabolite levels within a large general population sample. Methods: Serum 3,5-T2 concentrations were determined for 856 participants of the population-based Study of Health in Pomerania-TREND (SHIP-TREND). Plasma and urine metabolome data were generated using mass spectrometry and nuclear magnetic resonance spectroscopy, allowing quantification of 613 and 578 metabolites in plasma and urine, respectively. To detect thyroid function-independent significant 3,5-T2-metabolite associations, linear regression analyses controlling for major confounders, including thyrotropin and free T4, were performed. The same analyses were carried out using a sample of 16 male healthy volunteers treated for 8 weeks with 250 µg/day levothyroxine to induce thyrotoxicosis. Results: The specific molecular fingerprint of 3,5-T2 comprised 15 and 73 significantly associated metabolites in plasma and urine, respectively. Serum 3,5-T2 concentrations were neither associated with classical thyroid function parameters nor altered during experimental thyrotoxicosis. Strikingly, many metabolites related to coffee metabolism, including caffeine and paraxanthine, formed the clearest positively associated molecular signature. Importantly, these associations were replicated in the experimental human thyrotoxicosis model. Conclusion: The molecular fingerprint of 3,5-T2 demonstrates a clear and strong positive association of the serum levels of this TH metabolite with plasma levels of compounds indicating coffee consumption, therefore pointing to the liver as an organ, the metabolism of which is strongly affected by coffee. Furthermore, 3,5-T2 serum concentrations were found not to be directly TH dependent. Considering the beneficial health effects of 3,5-T2 administration observed in animal models and those of coffee consumption demonstrated in large epidemiological studies, one might speculate that coffee-stimulated hepatic 3,5-T2 production or accumulation represents an important molecular link in this connection.

Caffeine Content and Related Gene Expression: Novel Insight into Caffeine Metabolism in Camellia Plants Containing Low, Normal, and High Caffeine Concentrations.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

Long-term coffee consumption, caffeine metabolism genetics, and risk of cardiovascular disease: a prospective analysis of up to 347,077 individuals and 8368 cases.

BACKGROUND: Coffee is one of the most widely consumed stimulants worldwide and is generally considered to be safe or even beneficial for health. However, increased risk of myocardial infarction and hypertension has been suggested for individuals who carry a functional variant at cytochrome P450 1A2 (CYP1A2), which makes them less effective at metabolizing caffeine. OBJECTIVES: The aim of this study was to examine if the CYP1A2 genotype or a genetic score for caffeine metabolism (caffeine-GS) modifies the association between habitual coffee consumption and the risk of cardiovascular disease (CVD). METHODS: Genetic data and information on habitual coffee intake and relevant covariates were available for 347,077 individuals in the UK Biobank, including 8368 incident CVD cases. We used logistic regression to test for the association between coffee intake and CVD risk, and whether the association varies with CYP1A2 genotype or caffeine-GS. RESULTS: The association between habitual coffee intake and CVD risk was nonlinear, and, compared with participants drinking 1-2 cups/day, the risk of CVD was elevated for nondrinkers, drinkers of decaffeinated coffee, and those who reported drinking >6 cups/day (increase in odds by 11%, 7%, and 22%, respectively, P-curvature = 0.013). CYP1A2 genotype and caffeine-GS were not associated with CVD (P ≥ 0.22 for all comparisons). There was no evidence for an interaction between the CYP1A2 genotype or caffeine-GS and coffee intake with respect to risk of CVD (P ≥ 0.53). CONCLUSIONS: Heavy coffee consumption was associated with a modest increase in CVD risk, but this association was unaffected by genetic variants influencing caffeine metabolism.

The Role of Genetics in Moderating the Inter-Individual Differences in the Ergogenicity of Caffeine.

Caffeine use is widespread among athletes following its removal from the World Anti-Doping Agency banned list, with approximately 75% of competitive athletes using caffeine. While literature supports that caffeine has a small positive ergogenic effect for most forms of sports and exercise, there exists a significant amount of inter-individual difference in the response to caffeine ingestion and the subsequent effect on exercise performance. In this narrative review, we discuss some of the potential mechanisms and focus on the role that genetics has in these differences. CYP1A2 and ADORA2A are two of the genes which are thought to have the largest impact on the ergogenicity of caffeine. CYP1A2 is responsible for the majority of the metabolism of caffeine, and ADORA2A has been linked to caffeine-induced anxiety. The effects of CYP1A2 and ADORA2A genes on responses to caffeine will be discussed in detail and an overview of the current literature will be presented. The role of these two genes may explain a large portion of the inter-individual variance reported by studies following caffeine ingestion. Elucidating the extent to which these genes moderate responses to caffeine during exercise will ensure caffeine supplementation programs can be tailored to individual athletes in order to maximize the potential ergogenic effect.

Rapid Proteomics to Prospect and Validate Novel Bacterial Metabolism Induced by Environmental Burden.

Understanding the pathophysiology of genes and enzymes involved in caffeine metabolism can have extracurricular benefits, such as providing distinct methylxanthines as intermediates for pharmaceutical synthesis, and also improve environmental waste remediation. The strains Pseudomonas putida CBB5 and CES may provide insights into these applications because they may both be induced to degrade caffeine, yet the latter thrives in concentrations >8.0gL-1; threefold higher than any other bacteria. We took a novel approach toward identifying the enzymatic pathways in both Pseudomonas sp. CES and a deletion mutation of strain CBB5, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities to determine caffeine response elements. Here, we describe two optimized, rapid alternative strategies based on multiplexed SIL assays and demonstrate their application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison between enriched lysate fractions drawn from bacterial proteomes grown in the absence and presence of caffeine. Comparisons were made using stable isotope dimethyl labeling and expression differences were substantiated by reciprocal labeling experiments. The role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Multiple new components of N-demethylase system were discovered within a fraction of the lysate enriched specifically for this activity. We also describe how to expand the biological context (and reduce systemic bias) by adapting the protocol for total lysate analysis. We combined off-line prefractionation with the speed and resolution advantages of the Orbitrap LUMOS. The global protocol revealed 2406 proteins 1789 of which were quantified between treatments revealing, among other insights, a new antagonistic degradation pathway for vanillin that is completely suppressed by caffeine treatment.