Showing papers on "Theobromine published in 2020"

Theobromine alleviates diet-induced obesity in mice via phosphodiesterase-4 inhibition

Abstract: Modern science has given much attention to the treatment of obesity by activating brown adipose tissue (BAT) and browning of white adipose tissue (WAT). Recent studies have identified theobromine, a derivative of cocoa, as a potent natural component actively browning white fat cells. Here, we aimed to deduce the anti-obesity effect of theobromine involving phosphodiesterase (PDE) dependent-regulatory pathway in obese animal models. For examining activity of theobromine, C57BL/6 mice were fed with high fat diet and treated with theobromine to determine the expression levels of protein markers by immunoblot analysis and gene targets by quantitative real-time PCR. Other methods used include histopathological studies, immunofluorescence and molecular docking approaches. Theobromine alleviated diet-induced obesity in mice by browning of iWAT and activating BAT. Further, theobromine actively interacted with PDE4D and inhibited its activity in adipose tissues and cells potentiating energy expenditure. Moreover, the regulatory action of theobromine via inhibition of PDE4D was mediated by β3-AR signaling pathway. Altogether, the current results signifies critical role of theobromine in reducing obesity by regulation of lipid metabolism through inhibition of PDE4, indicating its potential as a major therapeutic medicinal compound.

Unique Role of Caffeine Compared to Other Methylxanthines (Theobromine, Theophylline, Pentoxifylline, Propentofylline) in Regulation of AD Relevant Genes in Neuroblastoma SH-SY5Y Wild Type Cells.

Abstract: Methylxanthines are a group of substances derived from the purine base xanthine with a methyl group at the nitrogen on position 3 and different residues at the nitrogen on position 1 and 7. They are widely consumed in nutrition and used as pharmaceuticals. Here we investigate the transcriptional regulation of 83 genes linked to Alzheimer’s disease in the presence of five methylxanthines, including the most prominent naturally occurring methylxanthines—caffeine, theophylline and theobromine—and the synthetic methylxanthines pentoxifylline and propentofylline. Methylxanthine-regulated genes were found in pathways involved in processes including oxidative stress, lipid homeostasis, signal transduction, transcriptional regulation, as well as pathways involved in neuronal function. Interestingly, multivariate analysis revealed different or inverse effects on gene regulation for caffeine compared to the other methylxanthines, which was further substantiated by multiple comparison analysis, pointing out a distinct role for caffeine in gene regulation. Our results not only underline the beneficial effects of methylxanthines in the regulation of genes in neuroblastoma wild-type cells linked to neurodegenerative diseases in general, but also demonstrate that individual methylxanthines like caffeine mediate unique or inverse expression patterns. This suggests that the replacement of single methylxanthines by others could result in unexpected effects, which could not be anticipated by the comparison to other substances in this substance class.

Metabolite and Transcriptome Profiling on Xanthine Alkaloids-Fed Tea Plant (Camellia sinensis) Shoot Tips and Roots Reveal the Complex Metabolic Network for Caffeine Biosynthesis and Degradation.

Abstract: While caffeine is one of the most important bioactive metabolites for tea as the most consumed non-alcohol beverage, its biosynthesis and catabolism in tea plants are still not fully understood. Here, we integrated purine alkaloid profiling and transcriptome analysis on shoot tips and roots fed with caffeine, theophylline, or theobromine to gain further understanding of caffeine biosynthesis and degradation. Shoot tips and roots easily took up and accumulated high concentrations of alkaloids, but roots showed much faster caffeine and theophylline degradation rates than shoot tips, which only degraded theophylline significantly but almost did not degrade caffeine. Clearly feedback inhibition on caffeine synthesis or inter-conversion between caffeine, theophylline, and theobromine, and 3-methylxanthine had been observed in alkaloids-fed shoot tips and roots, and these were also evidenced by significant repression of TCS and MXMT genes critical for caffeine biosynthesis. Among these responsively repressed genes, two highly expressed genes TCS-4 and TCS-8 were characterized for their enzyme activity. While we failed to detect TCS-4 activity, TCS-8 displayed N-methyltransferase activities towards multiple substrates, supporting the complex metabolic network in caffeine biosynthesis in tea plants since at least 13 TCS-like N-methyltransferase genes may function redundantly. This study provides new insight into complex metabolic networks of purine alkaloids in tea plants.

Isolation, characterization and application of theophylline-degrading Aspergillus fungi

Abstract: Caffeine, theobromine and theophylline are main purine alkaloid in tea. Theophylline is the downstream metabolite and it remains at a very low level in Camellia sinensis. In our previous study, Aspergillus sydowii could convert caffeine into theophylline in solid-state fermentation of pu-erh tea through N-demethylation. In this study, tea-derived fungi caused theophylline degradation in the solid-state fermentation. The purpose of this study is identify and isolate theophylline-degrading fungi and investigate their application in production of methylxanthines with theophylline as feedstock through microbial conversion. Seven tea-derived fungi were collected and identified by ITS, β-tubulin and calmodulin gene sequences, Aspergillus ustus, Aspergillus tamarii, Aspergillus niger and A. sydowii associated with solid-state fermentation of pu-erh tea have shown ability to degrade theophylline in liquid culture. Particularly, A. ustus and A. tamarii could degrade theophylline highly significantly (p < 0.01). 1,3-dimethyluric acid, 3-methylxanthine, 3-methyluric acid, xanthine and uric acid were detected consecutively by HPLC in A. ustus and A. tamarii, respectively. The data from absolute quantification analysis suggested that 3-methylxanthine and xanthine were the main degraded metabolites in A. ustus and A. tamarii, respectively. 129.48 ± 5.81 mg/L of 3-methylxanthine and 159.11 ± 10.8 mg/L of xanthine were produced by A. ustus and A. tamarii in 300 mg/L of theophylline liquid medium, respectively. For the first time, we confirmed that isolated A. ustus, A. tamarii degrade theophylline through N-demethylation and oxidation. We were able to biologically produce 3-methylxanthine and xanthine efficiently from theophylline through a new microbial synthesis platform with A. ustus and A. tamarii as appropriate starter strains.

Antifungal Activity of Methylxanthines Based on Their Properties

Abstract: Wood materials for construction purposes can be attacked by various wood-destroying fungi. An ideal wood-preserving substance is supposed to be environment- and health-friendly. For this reason, the effects of the most relevant and non-toxic methylxanthines, such as caffeine and its metabolites theobromine and theophylline, on fungal growth, together with their degradability related to their properties were analyzed in this study. Agar tests with four wood-destroying fungal species (Serpula lacrymans, Coniophora puteana, Gleophyllum sepiarium, and Trametes versicolor) were performed after 28 days of substance exposure. Caffeine exhibited a 100% inhibitive effect on fungal growth, contrary to theobromine, which was not effective in that respect. Theophylline exhibited variable effects on the analyzed fungi. The analysis of degradability indicated the persistence of caffeine and theobromine, but theophylline was degraded up to 34%. The relation of toxicity to chemical structure of studied methylxanthines indicated the dipole moment and lipophilicity as important parameters affecting the antifungal properties. Both caffeine and theophylline are suitable potential candidates for antifungal active substances.

Biotransformation of green tea (Camellia sinensis) by wine yeast Saccharomyces cerevisiae.

Abstract: Wine yeast Saccharomyces cerevisiae 71B was used in fermentation of green tea to modulate the volatiles and nonvolatiles. After fermentation, higher alcohols, esters, and acids, such as isoamyl alcohol, isobutanol, ethyl octanoate, ethyl decanoate, octanoic, and decanoic acids were generated. Some key aroma compounds of tea including linalool, hotrienol, dihydroactinidiolide, and 2-phenylethanol increased significantly. Among these compounds, linalool and 2-phenylethanol increased by 1.3- and 10-fold, respectively, which impart floral and fruity notes to fermented green tea. Alkaloids including caffeine, theobromine, and theophylline were reduced significantly after fermentation, while the most important free amino acid in tea, theanine, was not metabolized by S. cerevisiae. Tea catechins decreased whereas gallic and caffeic acids increased significantly, resulting in the unchanged antioxidant capacity of the fermented green tea. Hence, this work highlighted the potential of using S. cerevisiae to modulate green tea aroma and nonvolatiles. PRACTICAL APPLICATION: A novel fermented tea is produced by yeast fermentation. Saccharomyces cerevisiae led to significant changes in tea volatiles and nonvolatiles. Antioxidant capacity remained stable after fermentation.

Comparison of Two Dietary Supplements for Treatment of Uric Acid Renal Lithiasis: Citrate vs. Citrate + Theobromine

Abstract: Background. Uric acid (UA) renal lithiasis has a high rate of recurrence and a prevalence ranging from 10% and 15%, depending on the population. The most important etiological factor is persistence of urinary pH below 5.5 and one of the most common treatments is alkalization with citrate. Recent studies demonstrated that theobromine, which is abundant in chocolate and cocoa, is a potent inhibitor of UA crystallization. Aim. The aim was to compare the efficacy of citrate versus citrate + theobromine as treatment for UA lithiasis. Methods. This randomized cross-over trial investigated the efficacy of two treatments in 47 patients with UA renal lithiasis. Urine volume, pH, UA excretion, theobromine excretion, and risk of UA crystallization (RUAC) at baseline and at the end of each intervention period were measured. Results. Each treatment significantly reduced the risk of UA crystallization compared to basal values. The RUAC after citrate + theobromine was lower than the RUAC after citrate, although this difference was not statistically significant. Conclusion. The combined consumption of citrate and theobromine may be a promising strategy for the prevention of UA kidney stones.

3-Methylxanthine production through biodegradation of theobromine by Aspergillus sydowii PT-2

Abstract: Methylxanthines, including caffeine, theobromine and theophylline, are natural and synthetic compounds in tea, which could be metabolized by certain kinds of bacteria and fungi. Previous studies confirmed that several microbial isolates from Pu-erh tea could degrade and convert caffeine and theophylline. We speculated that these candidate isolates also could degrade and convert theobromine through N-demethylation and oxidation. In this study, seven tea-derived fungal strains were inoculated into various theobromine agar medias and theobromine liquid mediums to assess their capacity in theobromine utilization. Related metabolites with theobromine degradation were detected by using HPLC in the liquid culture to investigate their potential application in the production of 3-methylxanthine. Based on theobromine utilization capacity, Aspergillus niger PT-1, Aspergillus sydowii PT-2, Aspergillus ustus PT-6 and Aspergillus tamarii PT-7 have demonstrated the potential for theobromine biodegradation. Particularly, A. sydowii PT-2 and A. tamarii PT-7 could degrade theobromine significantly (p < 0.05) in all given liquid mediums. 3,7-Dimethyluric acid, 3-methylxanthine, 7-methylxanthine, 3-methyluric acid, xanthine, and uric acid were detected in A. sydowii PT-2 and A. tamarii PT-7 culture, respectively, which confirmed the existence of N-demethylation and oxidation in theobromine catabolism. 3-Methylxanthine was common and main demethylated metabolite of theobromine in the liquid culture. 3-Methylxanthine in A. sydowii PT-2 culture showed a linear relation with initial theobromine concentrations that 177.12 ± 14.06 mg/L 3-methylxanthine was accumulated in TLM-S with 300 mg/L theobromine. Additionally, pH at 5 and metal ion of Fe2+ promoted 3-methylxanthine production significantly (p < 0.05). This study is the first to confirm that A. sydowii PT-2 and A. tamarii PT-7 degrade theobromine through N-demethylation and oxidation, respectively. A. sydowii PT-2 showed the potential application in 3-methylxanthine production with theobromine as feedstock through the N-demethylation at N-7 position.

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

Abstract: 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.

Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1

Abstract: The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62 Mb and 4.53 Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia. • Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1. • The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively. • This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.

Caffeine inhibits Notum activity by binding at the catalytic pocket

Abstract: Notum inhibits Wnt signalling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signalling by small molecule inhibition of Notum may be of therapeutic benefit in a number of pathologies including Alzheimer’s disease. Here we report Notum activity can be inhibited by caffeine (IC50 19 µM), but not by demethylated caffeine metabolites: paraxanthine, theobromine and theophylline. Cellular luciferase assays show Notum-suppressed Wnt3a function can be restored by caffeine with an EC50 of 46 µM. The dissociation constant (Kd) between Notum and caffeine is 85 µM as measured by surface plasmon resonance. High-resolution crystal structures of Notum complexes with caffeine and its minor metabolite theophylline show both compounds bind at the centre of the enzymatic pocket, overlapping the position of the natural substrate palmitoleic lipid, but using different binding modes. The structural information reported here may be of relevance for the design of more potent brain-accessible Notum inhibitors. Zhao et al. show that caffeine restores Wnt signaling activity by suppressing Notum. The high-resolution crystal structure of Notum bound with caffeine suggests that caffeine may compete with Notum’s natural substrate. Structural insights from this study may therapeutically benefit several pathologies including Alzheimer’s disease.

Methylxanthines Inhibit Primary Amine Oxidase and Monoamine Oxidase Activities of Human Adipose Tissue.

Abstract: Background: Methylxanthines including caffeine and theobromine are widely consumed compounds and were recently shown to interact with bovine copper-containing amine oxidase. To the best of our knowledge, no direct demonstration of any interplay between these phytochemicals and human primary amine oxidase (PrAO) has been reported to date. We took advantage of the coexistence of PrAO and monoamine oxidase (MAO) activities in human subcutaneous adipose tissue (hScAT) to test the interaction between several methylxanthines and these enzymes, which are involved in many key pathophysiological processes. Methods: Benzylamine, methylamine, and tyramine were used as substrates for PrAO and MAO in homogenates of subcutaneous adipose depots obtained from overweight women undergoing plastic surgery. Methylxanthines were tested as substrates or inhibitors by fluorimetric determination of hydrogen peroxide, an end-product of amine oxidation. Results: Semicarbazide-sensitive PrAO activity was inhibited by theobromine, caffeine, and isobutylmethylxanthine (IBMX) while theophylline, paraxanthine, and 7-methylxanthine had little effect. Theobromine inhibited PrAO activity by 54% at 2.5 mM. Overall, the relationship between methylxanthine structure and the degree of inhibition was similar to that seen with bovine PrAO, although higher concentrations (mM) were required for inhibition. Theobromine also inhibited oxidation of tyramine by MAO, at the limits of its solubility in a DMSO vehicle. At doses higher than 12 % v/v, DMSO impaired MAO activity. MAO was also inhibited by millimolar doses of IBMX, caffeine and by other methylxanthines to a lesser extent. Conclusions: This preclinical study extrapolates previous findings with bovine PrAO to human tissues. Given that PrAO is a potential target for anti-inflammatory drugs, it indicates that alongside phosphodiesterase inhibition and adenosine receptor antagonism, PrAO and MAO inhibition could contribute to the health benefits of methylxanthines, especially their anti-inflammatory effects.

Effective Recognition of Caffeine by Diaminocarbazolic Receptors.

Abstract: Caffeine is a competitive inhibitor of adenosine receptors and possesses wide pharmacological activity. Artificial receptors recognizing caffeine potentially have a wide range of biomedical and industrial applications. Herein, we describe two structurally related and readily available artificial receptors: 1) a macrocyclic receptor, which binds caffeine with the unprecedented affinity of 9.3 μM, though with poor selectivity; and 2) a tweezers-like structure, showing an affinity of 26 μM and a 4.5-fold and 6-fold selectivity compared to theophylline and theobromine, respectively. Binding affinities were measured by 1 H NMR titrations and were confirmed by isothermal titration calorimetry. The X-ray structure of the complex between caffeine and the acyclic receptor revealed the origin of the recognition, explained the selectivity, and shed light on the role of hydrogen bonding and CH-π/π-π interactions.

Production of 7-methylxanthine from Theobromine by Metabolically Engineered E. coli

Abstract: In this work, a novel biocatalytic process for the production of 7-methylxanthines from theobromine, an economic feedstock has been developed. Bench scale production of 7-methlxanthine has been demonstrated. The biocatalytic process used in this work operates at 30 OC and atmospheric pressure, and is environmentally friendly. The biocatalyst was E. coli BL21(DE3) engineered with ndmB/D genes combinations. These modifications enabled specific N7- demethylation of theobromine to 7-methylxanthine. This production process consists of uniform fermentation conditions with a specific metabolically engineered strain, uniform induction of specific enzymes for 7-methylxanthine production, uniform recovery and preparation of biocatalyst for reaction and uniform recovery of pure 7-methylxanthine. Many E. coli BL21(DE3) strains metabolically engineered with single and/or multiple ndmB/D genes were tested for catalytic activity, and the best strains which had the higher activity were chosen to carry out the N-demethylation reaction of theobromine. Strain pBD2dDB had the highest activity for the production of 7-methylxanthine from theobromine. That strain was used to find the optimum amount of cells required to achieve complete conversion of theobromine to 7-methylxanthine within two hours. It was found that the optimum concentration of pBD2dDB strain to achieve 100% conversion of 0.5 mM theobromine to 7-methylxanthine was 5 mg/mL. The cell growth of pBD2dDB strain was studied using two different growth media, (Luria-Bertani Broth and Super Broth). Super broth was found to be the best medium to produce the highest amount of cell paste (1.5 g). Subsequently, the process was scaled up in which 2 L reaction volume was used to produce 7-methylxanthine (100% conversion) from 0.5 mM theobromine catalyzed by pBD2dDB strain. The reactions was carried out at 30 oC and 250 rpm shaker speed, and the reaction medium was 50 mM potassium phosphate buffer (pH=7). 7-methylxanthines was separated by preparative chromatography with high recovery, and the product solution was collected, purified by drying at 120-140 oC for 4 hours and, recovered (127 mg). Purity of the isolated 7-methylxanthine was comparable to authentic standards with no contaminant peaks, as observed by HPLC, LC-MS, and NMR.

Monitoring caffeine intake in children with a questionnaire and urine collection: a cross-sectional study in a convenience sample in Switzerland

Abstract: The objectives of this study were (1) to estimate caffeine intake and identify the main sources of intake using a dietary questionnaire, (2) to assess 24-h urinary excretion of caffeine and its metabolites, and (3) to assess how self-reported intake estimates correlates with urinary excretion among children in Switzerland. We conducted a cross-sectional study of children between 6 and 16 years of age in one region of Switzerland. The participants filled in a dietary questionnaire and collected a 24-h urine sample. Caffeine intake was estimated with the questionnaire. Caffeine, paraxanthine, theophylline, and theobromine excretions were measured in the urine sample. Correlations between questionnaire-based intake and urinary excretion estimates were assessed using Spearman correlation coefficients. Ninety-one children were included in the analysis (mean age 10.6 years; 43% female). The mean daily caffeine intake estimate derived from the diet questionnaire was 39 mg (range 0–237), corresponding, when related to body weight, to 1.2 mg/kg (range 0.0–6.3). Seven children (8%) had a caffeine intake above the upper recommended level of 3 mg/kg per day. The main sources of caffeine intake were cocoa milk (29%), chocolate (25%), soft drinks (11%), mocha yogurt (10%), tea (8%), and energy drinks (8%). The 24-h urinary excretion of caffeine was 0.3 mg (range 0.0–1.5), paraxanthine 1.4 mg (range 0.0–7.1), theophylline 0.1 mg (range 0.0–0.6), and theobromine 14.8 mg (range 0.3–59.9). The correlations between estimates of caffeine intake and the 24-h urinary excretion of caffeine was modest (ρ = 0.21, p = 0.046) and with the metabolites of caffeine were weak (ρ = 0.09–0.11, p = 0.288–0.423). Caffeine intake in a sample of children in a region of Switzerland was relatively low. The major sources of intake were cocoa milk, chocolate and soft drinks. Self-reported caffeine intake correlated weakly with urinary excretion of caffeine and some of its main metabolites. NCT02900261.

Trapping Acrolein by Theophylline/Caffeine and Their Metabolites from Green Tea and Coffee in Mice and Humans.

Abstract: Acrolein (ACR) is found exogenously as a widespread environmental pollutant and endogenously, where it is thought to be involved as a pathogenic factor in the progression of many pathological conditions. Eliminating ACR by dietary-active substances has been found to be one potential strategy to prevent ACR-associated chronic diseases. This study first compared the scavenging ACR efficacy of four purine alkaloids, theophylline (TP), paraxanthine (PXT), theobromine (TB), and caffeine (CAF), and then, TP, CAF, and their metabolites were investigated for their ability to trap ACR in vivo. Our results indicated that TP, which possesses an -NH moiety at the N-7 position, exhibits the best ACR-trapping capacity in vitro, while CAF has a slight ability to trap ACR due to the substitutions by -CH3 at the N-1, N-3, and N-7 positions. After oral administration of TP or CAF, the ACR adducts of TP and the metabolites of TP or CAF (e.g., mono- and di-ACR-TP, mono-ACR-1,3-DMU, and mono-ACR-1-MU) were detected in urinary samples obtained from both TP- and CAF-treated mouse groups by using ultra-performance liquid chromatography-tandem mass spectrometry. The quantification studies demonstrated that TP and its metabolites significantly trapped ACR in a dose-dependent manner in vivo. Furthermore, we also detected those ACR adducts of TP and TP/CAF's metabolites in human urine after four cups of green tea (2 g tea leaf/cup) or two cups of coffee (4 g coffee/cup) were consumed per day. Those results indicated that dietary TP or CAF has the potential capacity to scavenge ACR in vivo.

Online measurement of dissolved oxygen in shake flask to elucidate its role on caffeine degradation by Pseudomonas sp.

Abstract: Caffeine is a plant alkaloid present in the large ratio over other emerging pollutants and it causes serious health effects on overdosage. Microbial degradation of caffeine produces metabolites tha...

Detection of caffeine and its main metabolites for early diagnosis of Parkinson's disease using micellar electrokinetic capillary chromatography.

Abstract: Caffeine (CA) is a common xanthine alkaloid found in tea leaves, coffee beans, and other natural plants, and is the most widely used psychotropic substance in the world. Accumulating evidence suggests that low plasma levels of CA and its metabolites may serve as reliable diagnostic markers for early Parkinson's disease (PD) patients. In this study, we demonstrated a new MEKC method for determining CA and its three main downstream metabolites, paraxanthine (PX), theobromine (TB), and theophylline (TP), in human plasma. Plasma samples were collected, and analyzed using MEKC, after SPE. The running buffer was composed of 35 mM phosphate, pH of 10.5, and 25 mM SDS. The separation voltage was 15 kV and the detection wavelength was at 210 nm. Under the optimum conditions, four distinct analytes were completely separated and detected in less than 12 min. Method limits of detection were as low as 7.5 ng/mL for CA, 5.0 ng/mL for TB, and 4.0 ng/mL for both PX and TP. The recoveries were between 88.0% and 105.9%. This method was successfully applied to 27 human plasma samples. The results indicate that the plasma concentrations of the four analytes are significantly lower in patients with early PD than in control subjects (p < 0.05). The area under curve was improved to 0.839 when CA and its three main metabolites were included, suggesting that MEKC testing of CA, TP, TB, and PX may serve as a potential method for early diagnosis of PD.

Formulation of chocolate with carob and low caffeine and theobromine content, no added sugar, dairy free, gluten free, soy free, and with or without fibers

Abstract: The patent invention relates to a chocolate formulation with carob, with low caffeine and theobromine contents, animal milk-free, with or without added sugars, gluten-free, soy-free, with or without fiber, using cocoa and carob as the basis, alternatively supplemented with coconut fat, rice derivatives, powdered sweet potato and sugars having a low glycemic index, producing a food product with special nutritional features to meet the requirements of customers with dietary restrictions due to health problems or life style (low-fat or natural diets, body-builders, etc.) with color, odor and flavor attributes which are characteristic of products based on cocoa and carob.

Modulation of CYP2E1 metabolic activity in a cohort of confirmed caffeine ingesting pregnant women with preterm offspring.

Abstract: To ascertain interactions of caffeine ingestion, food, medications, and environmental exposures during preterm human gestation, under informed consent, we studied a cohort of Mexican women with further preterm offspring born at ≤ 34 completed weeks. At birth, blood samples were taken from mothers and umbilical cords to determine caffeine and metabolites concentrations and CYP1A2 (rs762551) and CYP2E1 (rs2031920, rs3813867) polymorphisms involved in caffeine metabolism. In 90 pregnant women who gave birth to 98 preterm neonates, self-informed caffeine ingestion rate was 97%, laboratory confirmed rate was 93 %. Theobromine was the predominant metabolite found. Consumption of acetaminophen correlated significantly with changes in caffeine metabolism (acetaminophen R2 = 0.637, p = 0.01) due to activation of CYP2E1 alternate pathways. The main caffeine source was cola soft drinks. Environmental exposures, especially acetaminophen ingestion during human preterm pregnancy, can modulate CYP2E1 metabolic activity.