Pharmacokinetics and pharmacodynamics of inhaled nicotine salt and free-base using an e-cigarette: A randomized crossover study.

BACKGROUND: Popular "pod-style" e-cigarettes commonly use nicotine salt-based e-liquids that cause less irritation when inhaled and can deliver higher nicotine concentrations than free-base nicotine. We aimed to investigate the pharmacokinetic and pharmacodynamic effects of different nicotine formulations (salt vs. free-base) and concentrations that might influence systemic nicotine absorption and appeal of e-cigarettes. METHODS: In this randomized, double-blind, within-subject crossover study, 20 non nicotine-naïve participants were switched among three e-liquids (free-base nicotine 20mg/mL, nicotine salt 20mg/mL, nicotine salt 40mg/mL) using a refillable pod system and a standardized vaping protocol (one puff every 30 seconds, 10 puffs total). Serum nicotine concentrations and vital signs were assessed over 180 minutes; direct effects, craving, satisfaction, withdrawal, and respiratory symptoms were measured using questionnaires. CYP2A6 genotypes and the nicotine metabolite ratio were also assessed. RESULTS: Eleven (55%) participants were male and the median age was 23.5 years (range 18-67). All three formulations differed significantly in peak serum nicotine concentration (baseline adjusted Cmax, median (range): 12.0ng/mL (1.6-27.3), 5.4ng/mL (1.9-18.7) and 3.0ng/mL (1.3-8.8) for nicotine salt 40mg/mL, nicotine salt 20mg/mL and free-base 20mg/mL, respectively). All groups reached Cmax 2.0-2.5min (median) after their last puff. Differences in subjective effects were not statistically significant. No serious adverse events were observed. CONCLUSION: Free-base 20mg/mL formulations achieved lower blood nicotine concentrations than nicotine salt 20mg/mL, while 40mg/mL nicotine salt yielded concentrations similar to cigarette smoking. The findings can inform regulatory policy regarding e-liquids and their potential use in smoking cessation. IMPLICATIONS: Nicotine salt formulations inhaled by an e-cigarette led to higher nicotine delivery compared to nicotine free-base formulations with the same nicotine concentration. These findings should be considered in future regulatory discussions. The 40mg/mL nicotine salt formulation showed similar nicotine delivery as combustible cigarettes, albeit at concentrations over the maximum limit for e-liquids allowed in the European Union. Nicotine delivery resembling combustible cigarettes might be beneficial for smokers willing to quit to adequately alleviate withdrawal symptoms. However, increased nicotine delivery can also pose a public health risk, raising concerns about abuse liability, especially among youth and non-smokers.

Chronic and postprandial effect of blueberries on cognitive function, alertness, and mood in participants with metabolic syndrome - results from a six-month, double-blind, randomized controlled trial.

BACKGROUND: Anthocyanin and blueberry intakes positively associated with cognitive function in population-based studies and cognitive benefits in randomized controlled trials of adults with self-perceived or clinical cognitive dysfunction. To date, adults with metabolic syndrome (MetS) but without cognitive dysfunction are understudied. OBJECTIVES: Cognitive function, mood, alertness, and sleep quality were assessed as secondary end points in MetS participants, postprandially (>24 h) and following 6-mo blueberry intake. METHODS: A double-blind, randomized controlled trial was conducted, assessing the primary effect of consuming freeze-dried blueberry powder, compared against an isocaloric placebo, on cardiometabolic health >6 mo and a 24 h postprandial period (at baseline). In this secondary analysis of the main study, data from those completing mood, alertness, cognition, and sleep assessments are presented (i.e., n = 115 in the 6 mo study, n = 33 in the postprandial study), using the following: 1) Bond-Lader self-rated scores, 2) electronic cognitive battery (i.e., testing attention, working memory, episodic memory, speed of memory retrieval, executive function, and picture recognition), and 3) the Leeds Sleep Evaluation Questionnaire. Urinary and serum anthocyanin metabolites were quantified, and apolipoprotein E genotype status was determined. RESULTS: Postprandial self-rated calmness significantly improved after 1 cup of blueberries (P = 0.01; q = 0.04; with an 11.6% improvement compared with baseline between 0 and 24 h for the 1 cup group), but all other mood, sleep, and cognitive function parameters were unaffected after postprandial and 6-mo blueberries. Across the ½ and 1 cup groups, microbial metabolites of anthocyanins and chlorogenic acid (i.e., hydroxycinnamic acids, benzoic acids, phenylalanine derivatives, and hippuric acids) and catechin were associated with favorable chronic and postprandial memory, attention, executive function, and calmness. CONCLUSIONS: Although self-rated calmness improved postprandially, and significant cognition-metabolite associations were identified, our data did not support strong cognitive, mood, alertness, or sleep quality improvements in MetS participants after blueberry intervention. This trial was registered at clinicaltrials.gov as NCT02035592.

Blueberry anthocyanin intake attenuates the postprandial cardiometabolic effect of an energy-dense food challenge: Results from a double blind, randomized controlled trial in metabolic syndrome participants.

BACKGROUND & AIMS: Whilst the cardioprotective effects of blueberry intake have been shown in prospective studies and short-term randomized controlled trials (RCTs), it is unknown whether anthocyanin-rich blueberries can attenuate the postprandial, cardiometabolic dysfunction which follows energy-dense food intakes; especially in at-risk populations. We therefore examined whether adding blueberries to a high-fat/high-sugar meal affected the postprandial cardiometabolic response over 24 h. METHODS: A parallel, double-blind RCT (n = 45; age 63.4 ± 7.4 years; 64% male; BMI 31.4 ± 3.1 kg/m2) was conducted in participants with metabolic syndrome. After baseline assessments, an energy-dense drink (969 Kcals, 64.5 g fat, 84.5 g carbohydrate, 17.9 g protein) was consumed with either 26 g (freeze-dried) blueberries (equivalent to 1 cup/150 g fresh blueberries) or 26 g isocaloric matched placebo. Repeat blood samples (30, 60, 90, 120, 180, 360 min and 24 h), a 24 h urine collection and vascular measures (at 3, 6, and 24 h) were performed. Insulin and glucose, lipoprotein levels, endothelial function (flow mediated dilatation (FMD)), aortic and systemic arterial stiffness (pulse wave velocity (PWV), Augmentation Index (AIx) respectively), blood pressure (BP), and anthocyanin metabolism (serum and 24 h urine) were assessed. RESULTS: Blueberries favorably affected postprandial (0-24 h) concentrations of glucose (p < 0.001), insulin (p < 0.01), total cholesterol (p = 0.04), HDL-C, large HDL particles (L-HDL-P) (both p < 0.01), extra-large HDL particles (XL-HDL-P; p = 0.04) and Apo-A1 (p = 0.01), but not LDL-C, TG, or Apo-B. After a transient higher peak glucose concentration at 1 h after blueberry intake ([8.2 mmol/L, 95%CI: 7.7, 8.8] vs placebo [6.9 mmol/L, 95%CI: 6.4, 7.4]; p = 0.001), blueberries significantly attenuated 3 h glucose ([4.3 mmol/L, 95%CI: 3.8, 4.8] vs placebo [5.1 mmol/L, 95%CI: 4.6, 5.6]; p = 0.03) and insulin concentrations (blueberry: [23.4 pmol/L, 95%CI: 15.4, 31.3] vs placebo [52.9 pmol/L, 95%CI: 41.0, 64.8]; p = 0.0001). Blueberries also improved HDL-C ([1.12 mmol/L, 95%CI: 1.06, 1.19] vs placebo [1.08 mmol/L, 95%CI: 1.02, 1.14]; p = 0.04) at 90 min and XL-HDLP levels ([0.38 × 10-6, 95%CI: 0.35, 0.42] vs placebo [0.35 × 10-6, 95%CI: 0.32, 0.39]; p = 0.02) at 3 h. Likewise, significant improvements were observed 6 h after blueberries for HDL-C ([1.17 mmol/L, 95%CI: 1.11, 1.24] vs placebo [1.10 mmol/L, 95%CI: 1.03, 1.16]; p < 0.001), Apo-A1 ([1.37 mmol/L, 95%CI: 1.32, 1.41] vs placebo [1.31 mmol/L, 95%CI: 1.27, 1.35]; p = 0.003), L-HDLP ([0.70 × 10-6, 95%CI: 0.60, 0.81] vs placebo [0.59 × 10-6, 95%CI: 0.50, 0.68]; p = 0.003) and XL-HDLP ([0.44 × 10-6, 95%CI: 0.40, 0.48] vs placebo [0.40 × 10-6, 95%CI: 0.36, 0.44]; p < 0.001). Similarly, total cholesterol levels were significantly lower 24 h after blueberries ([4.9 mmol/L, 95%CI: 4.6, 5.1] vs placebo [5.0 mmol/L, 95%CI: 4.8, 5.3]; p = 0.04). Conversely, no effects were observed for FMD, PWV, AIx and BP. As anticipated, total anthocyanin-derived phenolic acid metabolite concentrations significantly increased in the 24 h after blueberry intake; especially hippuric acid (6-7-fold serum increase, 10-fold urinary increase). In exploratory analysis, a range of serum/urine metabolites were associated with favorable changes in total cholesterol, HDL-C, XL-HDLP and Apo-A1 (R = 0.43 to 0.50). CONCLUSIONS: For the first time, in an at-risk population, we show that single-exposure to the equivalent of 1 cup blueberries (provided as freeze-dried powder) attenuates the deleterious postprandial effects of consuming an energy-dense high-fat/high-sugar meal over 24 h; reducing insulinaemia and glucose levels, lowering cholesterol, and improving HDL-C, fractions of HDL-P and Apo-A1. Consequently, intake of anthocyanin-rich blueberries may reduce the acute cardiometabolic burden of energy-dense meals. CLINICAL TRIAL REGISTRY: NCT02035592 at www.clinicaltrials.gov.

Sex-specific alterations in NAD+ metabolism in 3xTg Alzheimer's disease mouse brain assessed by quantitative targeted LC-MS.

Levels of nicotinamide adenine dinucleotide (NAD+) are known to decline with age and have been associated with impaired mitochondrial function leading to neurodegeneration, a key facet of Alzheimer's disease (AD). NAD+synthesis is sustained via tryptophan-kynurenine (Trp-Kyn) pathway as de novo synthesis route, and salvage pathways dependent on the availability of nicotinic acid and nicotinamide. While being currently investigated as a multifactorial disease with a strong metabolic component, AD remains without curative treatment and important sex differences were reported in relation to disease onset and progression. The aim of this study was to reveal the potential deregulation of NAD+metabolism in AD with the direct analysis of NAD+precursors in the mouse brain tissue (wild type (WT) versus triple transgenic (3xTg) AD), using a sex-balanced design. To this end, we developed a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the measurement of the full spectrum of NAD+precursors and intermediates in all three pathways. In brain tissue of mice with developed AD symptoms, a decrease in kynurenine (Kyn) versus increase in kynurenic acid (KA) levels were observed in both sexes with a significantly higher increment of KA in males. These alterations in Trp-Kyn pathway might be a consequence of neuroinflammation and a compensatory production of neuroprotective kynurenic acid. In the NAD+ salvage pathway, significantly lower levels of nicotinamide mononucleotide (NMN) were measured in the AD brain of males and females. Depletion of NMN implies the deregulation of salvage pathway critical for maintaining optimal NAD+ levels and mitochondrial and neuronal function.

Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome.

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.

Systemic and central nervous system metabolic alterations in Alzheimer's disease.

BACKGROUND: Metabolic alterations, related to cerebral glucose metabolism, brain insulin resistance, and age-induced mitochondrial dysfunction, play an important role in Alzheimer's disease (AD) on both the systemic and central nervous system level. To study the extent and significance of these alterations in AD, quantitative metabolomics was applied to plasma and cerebrospinal fluid (CSF) from clinically well-characterized AD patients and cognitively healthy control subjects. The observed metabolic alterations were associated with core pathological processes of AD to investigate their relation with amyloid pathology and tau-related neurodegeneration. METHODS: In a case-control study of clinical and biomarker-confirmed AD patients (n = 40) and cognitively healthy controls without cerebral AD pathology (n = 34) with paired plasma and CSF samples, we performed metabolic profiling, i.e., untargeted metabolomics and targeted quantification. Targeted quantification focused on identified deregulated pathways highlighted in the untargeted assay, i.e. the TCA cycle, and its anaplerotic pathways, as well as the neuroactive tryptophan and kynurenine pathway. RESULTS: Concentrations of several TCA cycle and beta-oxidation intermediates were higher in plasma of AD patients, whilst amino acid concentrations were significantly lower. Similar alterations in these energy metabolism intermediates were observed in CSF, together with higher concentrations of creatinine, which were strongly correlated with blood-brain barrier permeability. Alterations of several amino acids were associated with CSF Amyloidß1-42. The tryptophan catabolites, kynurenic acid and quinolinic acid, showed significantly higher concentrations in CSF of AD patients, which, together with other tryptophan pathway intermediates, were correlated with either CSF Amyloidß1-42, or tau and phosphorylated Tau-181. CONCLUSIONS: This study revealed AD-associated systemic dysregulation of nutrient sensing and oxidation and CNS-specific alterations in the neuroactive tryptophan pathway and (phospho)creatine degradation. The specific association of amino acids and tryptophan catabolites with AD CSF biomarkers suggests a close relationship with core AD pathology. Our findings warrant validation in independent, larger cohort studies as well as further investigation of factors such as gender and APOE genotype, as well as of other groups, such as preclinical AD, to identify metabolic alterations as potential intervention targets.

Merged Targeted Quantification and Untargeted Profiling for Comprehensive Assessment of Acylcarnitine and Amino Acid Metabolism.

Acylcarnitines and amino acids are key players in energy metabolism; however, analytical methods for comprehensive and straightforward quantitative profiling of these metabolites, without derivatization or use of ion-pairing agents, are lacking. We therefore developed a hydrophilic interaction chromatography (HILIC)-based high-resolution mass spectrometry (HRMS) method for the simultaneous quantification of acylcarnitines and amino acids in a single run, while taking advantage of HRMS data acquired in full-scan mode to screen for additional derivatives and other polar metabolites. A single-step metabolite extraction with internal standard mixture (in methanol) warranted high-throughput sample preparation whose applicability was demonstrated on a panel of human biofluids (i.e., blood plasma, CSF, and urine) and brain tissue. Method accuracy was within 90-106% of validated NIST reference plasma concentrations for the panel of measured amino acids. Amino acid and acylcarnitine extraction recoveries were 87-100% on average, depending on the concentration range spiked. The coefficient of variation (CV) was 1-10% and 1-25% for intra- and interday measurements, respectively, with the highest CVs for the metabolites at the limit of quantification, depending on the biofluid. Acylcarnitine and amino acid signatures or chemical composition barcodes of the different biofluids and human brain tissue were acquired and biofluid- and tissue-associated differences were discussed in the context of their respective physiological roles. Significant differences were observed in the amino acid profiles, whereas acylcarnitine composition did not show biofluid-characteristic or brain region-specific pattern. The retrospective exploration of full-scan all-ion-fragmentation data allowed us to extract the information on unsaturated and hydroxylated acylcarnitine species, amines, and purine and pyrimidine metabolites. This merged targeted and untargeted approach provides an innovative strategy for simultaneous and comprehensive assessment of acylcarnitine and amino acid metabolism in clinical research studies using relevant biofluids and tissue extracts.

Blueberries improve biomarkers of cardiometabolic function in participants with metabolic syndrome-results from a 6-month, double-blind, randomized controlled trial.

BACKGROUND: Anthocyanin-rich blueberry intake is associated with reduced type 2 diabetes and cardiovascular disease (CVD) risk in prospective studies, although long-term randomized controlled trials (RCTs) have not been conducted in at-risk populations. OBJECTIVE: In the longest-duration RCT to date, we examined the effect of 6-mo blueberry intake on insulin resistance and cardiometabolic function in metabolic syndrome. METHODS: A double-blind, parallel RCT (n = 115; age 63 ± 7 y; 68% male; body mass index 31.2 ± 3.0 kg/m2) was conducted, which fed 2 dietarily achievable blueberry intakes [equivalent to 1/2 and 1 cup/d (75/150 g)] compared with matched placebo. Insulin resistance was assessed via the homeostasis model assessment of insulin resistance (primary endpoint) and confirmed by [6-6-2H2]-glucose-labeled, 2-step hyperinsulinemic clamp (n = 20). Clinically relevant cardiometabolic endpoints [including flow-mediated dilatation, augmentation index, lipoprotein status (by nuclear magnetic resonance spectroscopy), and nitric oxide (NO)-related metabolite assay] and anthocyanin metabolism were assessed. RESULTS: A daily intake of 1 cup of blueberries improved endothelial function (flow-mediated dilatation: +1.45%; 95% CI: 0.83%, 2.1%; P = 0.003), systemic arterial stiffness (augmentation index: -2.24%; 95% CI: -3.97%, -0.61%; P = 0.04) and attenuated cyclic guanosine monophosphate concentrations. In statin nonusers (n = 71), elevated high-density lipoprotein cholesterol (+0.08 mmol/L; P = 0.03), high-density lipoprotein particle density (+0.48n, ×10-6; P = 0.002) and apolipoprotein A-I (+0.05 g/L; P = 0.01) concentrations were observed following the 1-cup/d intervention. Treatment compliance was 94.1% (wrapper returns) and total concentrations of anthocyanin-derived phenolic acid metabolites significantly increased, dose-dependently, in serum and 24-h urine (P < 0.01 and P < 0.001, respectively). Insulin resistance, pulse wave velocity, blood pressure, NO, and overall plasma thiol status were unaffected. Likewise, a half cup per day had no effect on any biomarkers. CONCLUSIONS: Despite insulin resistance remaining unchanged we show, to our knowledge, the first sustained improvements in vascular function, lipid status, and underlying NO bioactivity following 1 cup blueberries/d. With effect sizes predictive of 12-15% reductions in CVD risk, blueberries should be included in dietary strategies to reduce individual and population CVD risk. This study was registered at clinicaltrials.gov as NCT02035592.

LC-HRMS data as a result of untargeted metabolomic profiling of human cerebrospinal fluid.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF is analyzed in the context of neurological diseases and is rarely collected from healthy subjects. For this reason, the metabolite variation associated with general phenotypic characteristics such as gender and age have hardly ever been studied. Here we present the hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) data as a result of untargeted metabolomics analysis of a cohort of elderly cognitively healthy volunteers (n = 32). 146 unambiguously identified water soluble metabolites (using accurate mass, retention time and MS/MS matching against spectral libraries) were measured and their abundances across all the subjects depending on their gender are provided in this article. Data tables are available at https://data.mendeley.com/datasets/c73xtsd4s5/1. it's published on mendeley, the DOI is DOI:10.17632/c73xtsd4s5.1. The data presented in this article are related to the research article entitled "A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects" (Gallart-Ayala et al., 2018, In press).

Soy supplementation: Impact on gene expression in different tissues of ovariectomized rats and evaluation of the rat model to predict (post)menopausal health effect.

This toxicogenomic study was conducted to predict (post)menopausal human health effects of commercial soy supplementation using ovariectomized rats as a model. Different target tissues (i.e. breast, uterus and sternum) and non-target tissues (i.e. peripheral blood mononuclear cells (PBMC), adipose and liver) of ovariectomized F344 rats exposed to a commercially available soy supplement for eight weeks, were investigated. Changes in gene expression in these tissues were analysed using whole-genome microarray analysis. No correlation in changes in gene expression were observed among different tissues, indicating tissue specific effects of soy isoflavone supplementation. Out of 87 well-established estrogen responsive genes (ERGs), only 19 were found to be significantly regulated (p < 0.05) in different tissues, particularly in liver, adipose and uterus tissues. Surprisingly, no ERGs were significantly regulated in estrogen sensitive breast and sternum tissues. The changes in gene expression in PBMC and adipose tissue in rats were compared with those in (post)menopausal female volunteers who received the same supplement in a similar oral dose and exposure duration in human intervention studies. No correlation in changes in gene expression between rats and humans was observed. Although receiving a similar dose, in humans the plasma levels expressed as total free aglycones were several folds higher than in the rat. Therefore, the overall results in young ovariectomized female F344 rats indicated that using rat transcriptomic data does not provide a suitable model for human risk or benefit analysis of soy isoflavone supplementation.

Comparative bio-accessibility, bioavailability and bioequivalence of quercetin, apigenin, glucoraphanin and carotenoids from freeze-dried vegetables incorporated into a baked snack versus minimally processed vegetables: Evidence from in vitro models and a human bioavailability study.

The aim was to incorporate vegetables containing the phytochemicals quercetin, apigenin, glucoraphanin and carotenoids into a processed potato-based snack and assess their bioaccessibility and bioavailability. Three different processing routes were tested for incorporation and retention of phytochemicals in snacks using individually quick frozen or freeze-dried vegetables. No significant differences in the uptake or transport of quercetin or apigenin between a vegetable mix or snacks were observed using the CaCo-2 transwell model. Simulated in vitro digestions predicted a substantial release of quercetin and apigenin, some release of glucoraphanin but none for carotenes from either the snack or equivalent steamed vegetables. In humans, there were no significant differences in the bioavailability of quercetin, apigenin or glucoraphanin from the snack or equivalent steamed vegetables. We have shown that significant quantities of freeze-dried vegetables can be incorporated into snacks with good retention of phytochemicals and with similar bioavailability to equivalent steamed vegetables.

A global HILIC-MS approach to measure polar human cerebrospinal fluid metabolome: Exploring gender-associated variation in a cohort of elderly cognitively healthy subjects.

Cerebrospinal fluid (CSF) is a key body fluid that maintains the homeostasis in central nervous system (CNS). As a biofluid whose content reflects the brain metabolic activity, the CSF has been profiled in the context of neurological diseases to provide novel insights into the disease mechanisms. However, a global high-throughput approach to measure a broad diversity of polar metabolites present in CSF is lacking. Although still perceived as challenging and less reproducible, hydrophilic interaction liquid chromatography (HILIC) has recently evolved to offer the unprecedented coverage capacity of water-soluble metabolome. Here, we present a global HILIC high-resolution mass spectrometry-based (HRMS) approach that combines the profiling in acidic pH ESI (+) and basic pH ESI (-) mode to extend the coverage of CSF polar metabolome. This approach allowed us to annotate and measure a broad range of central carbon metabolites (implicated in glycolysis, TCA cycle, nucleotide, amino acid and fatty acid metabolism) in CSF collected from cognitively healthy elderly volunteers (n = 32), using a single extraction method. Metabolite annotation was achieved using the accurate mass, RT and MS/MS criteria, allowing for the characterization of 146 measurable metabolites. Exploration of characterized individual CSF profiles allowed for a discovery of intriguing gender-associated differences, with significantly higher acylcarnitine levels in men and higher taurine levels women. With this case study, we demonstrate the value of combined HILIC ESI ± HRMS profiling to assess CSF metabolome in clinical research studies.

De novo NAD+ synthesis enhances mitochondrial function and improves health.

Nicotinamide adenine dinucleotide (NAD+) is a co-substrate for several enzymes, including the sirtuin family of NAD+-dependent protein deacylases. Beneficial effects of increased NAD+ levels and sirtuin activation on mitochondrial homeostasis, organismal metabolism and lifespan have been established across species. Here we show that α-amino-ß-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the enzyme that limits spontaneous cyclization of α-amino-ß-carboxymuconate-ε-semialdehyde in the de novo NAD+ synthesis pathway, controls cellular NAD+ levels via an evolutionarily conserved mechanism in Caenorhabditis elegans and mouse. Genetic and pharmacological inhibition of ACMSD boosts de novo NAD+ synthesis and sirtuin 1 activity, ultimately enhancing mitochondrial function. We also characterize two potent and selective inhibitors of ACMSD. Because expression of ACMSD is largely restricted to kidney and liver, these inhibitors may have therapeutic potential for protection of these tissues from injury. In summary, we identify ACMSD as a key modulator of cellular NAD+ levels, sirtuin activity and mitochondrial homeostasis in kidney and liver.

A risk assessment-driven quantitative comparison of gene expression profiles in PBMCs and white adipose tissue of humans and rats after isoflavone supplementation.

Quantitative insight into species differences in risk assessment is expected to reduce uncertainty and variability related to extrapolation from animals to humans. This paper explores quantification and comparison of gene expression data between tissues and species from intervention studies with isoflavones. Gene expression data from peripheral blood mononuclear cells (PBMCs) and white adipose tissue (WAT) after 8wk isoflavone interventions in postmenopausal women and ovariectomized F344 rats were used. A multivariate model was applied to quantify gene expression effects, which showed 3-5-fold larger effect sizes in rats compared to humans. For estrogen responsive genes, a 5-fold greater effect size was found in rats than in humans. For these genes, intertissue correlations (r = 0.23 in humans, r = 0.22 in rats) and interspecies correlation in WAT (r = 0.31) were statistically significant. Effect sizes, intertissue and interspecies correlations for some groups of genes within energy metabolism, inflammation and cell cycle processes were significant, but weak. Quantification of gene expression data reveals differences between rats and women in effect magnitude after isoflavone supplementation. For risk assessment, quantification of gene expression data and subsequent calculation of intertissue and interspecies correlations within biological pathways will further strengthen knowledge on comparability between tissues and species.

Flavan-3-ols, theobromine, and the effects of cocoa and chocolate on cardiometabolic risk factors.

PURPOSE OF REVIEW: Although there is growing interest surrounding the potential health benefits of cocoa and chocolate, the relative contribution of bioactive constituents for these effects remains unclear. This review summarizes the recent research on the cardiometabolic effects of cocoa and chocolate with a focus on two key constituents: flavan-3-ols and theobromine. RECENT FINDINGS: Recent meta-analyses suggest beneficial cardiometabolic effects of chocolate following short-term intake, including improvements in flow-mediated dilatation, blood pressure, lipoprotein levels and biomarkers of insulin resistance. Flavan-3-ols may play a role, but it is currently unclear which specific compounds or metabolites are key. Theobromine has also been shown to improve lipoprotein levels in trials, although these findings need verification at habitual intake levels. Longer term dose-response randomized controlled trials are required to determine the sustainability of the short-term effects and the optimal dose. Quantifying levels of bioactives in intervention products and their metabolites in biological samples will facilitate the assessment of their relative impact and the underlying mechanisms of action. SUMMARY: Promising data support the beneficial cardiometabolic effects of cocoa and chocolate intake, with significant interest in the flavan-3-ol and theobromine content. Validated biomarkers of intake together with more relevant mechanistic insights from experimental models using physiologically relevant concentrations and metabolites will continue to inform this research field.

Plasma bioavailability and changes in PBMC gene expression after treatment of ovariectomized rats with a commercial soy supplement.

The health effects of soy supplementation in (post)menopausal women are still a controversial issue. The aim of the present study was to establish the effect of the soy isoflavones (SIF) present in a commercially available supplement on ovariectomized rats and to investigate whether these rats would provide an adequate model to predict effects of SIF in (post)menopausal women. Two dose levels (i.e. 2 and 20 mg/kg b.w.) were used to characterize plasma bioavailability, urinary and fecal concentrations of SIF and changes in gene expression in peripheral blood mononuclear cells (PBMC). Animals were dosed at 0 and 48 h and sacrificed 4 h after the last dose. A clear dose dependent increase of SIF concentrations in plasma, urine and feces was observed, together with a strong correlation in changes in gene expression between the two dose groups. All estrogen responsive genes and related biological pathways (BPs) that were affected by the SIF treatment were regulated in both dose groups in the same direction and indicate beneficial effects. However, in general no correlation was found between the changes in gene expression in rat PBMC with those in PBMC of (post)menopausal women exposed to a comparable dose of the same supplement. The outcome of this short-term study in rats indicates that the rat might not be a suitable model to predict effects of SIF in humans. Although the relative exposure period in this rat study is comparable with that of the human study, longer repetitive administration of rats to SIF may be required to draw a final conclusion on the suitability of the rat a model to predict effects of SIF in humans.

Isoflavone supplement composition and equol producer status affect gene expression in adipose tissue: a double-blind, randomized, placebo-controlled crossover trial in postmenopausal women.

BACKGROUND: Isoflavone supplements, consumed by women experiencing menopausal symptoms, are suggested to have positive effects on menopause-related adiposity and cardiovascular disease risk profile, but discussions about their safety are still ongoing. OBJECTIVE: The objective was to study the effects of an 8-wk consumption of 2 different isoflavone supplements compared with placebo on whole-genome gene expression in the adipose tissue of postmenopausal women. DESIGN: This double-blind, randomized, placebo-controlled crossover intervention consisted of 2 substudies, one with a low-genistein (LG) supplement (56% daidzein + daidzin, 16% genistein + genistin, and 28% glycitein + glycitin) and the other with a high-genistein (HG) supplement (49% daidzein + daidzin, 41% genistein + genistin, and 10% glycitein + glycitin). Both supplements provided ∼ 100 mg isoflavones/d (aglycone equivalents). After the 8-wk isoflavone and placebo period, whole-genome arrays were performed in subcutaneous adipose tissue of postmenopausal women (n = 26 after LG, n = 31 after HG). Participants were randomized by equol-producing phenotype, and data analysis was performed per substudy for equol producers and nonproducers separately. RESULTS: Gene set enrichment analysis showed downregulation of expression of energy metabolism-related genes after LG supplementation (n = 24) in both equol-producing phenotypes and oppositely regulated expression for equol producers (down) and nonproducers (up) after HG supplementation (n = 31). Expression of inflammation-related genes was upregulated in equol producers but downregulated in nonproducers, independent of supplement type. Only 4.4-7.0% of the genes with significantly changed expression were estrogen responsive. Body weight, adipocyte size, and plasma lipid profile were not affected by isoflavone supplementation. CONCLUSIONS: Effects of isoflavones on adipose tissue gene expression were influenced by supplement composition and equol-producing phenotype, whereas estrogen-responsive effects were lacking. LG isoflavone supplementation resulted in a caloric restriction-like gene expression profile for both producer phenotypes and pointed toward a potential beneficial effect, whereas both supplements induced anti-inflammatory gene expression in equol producers. The study was registered at clinicaltrials.gov as NCT01556737.

Estrogen receptor-mediated effects of isoflavone supplementation were not observed in whole-genome gene expression profiles of peripheral blood mononuclear cells in postmenopausal, equol-producing women.

Isoflavones (genistein, daidzein, and glycitein) are suggested to have benefits as well as risks for human health. Approximately one-third of the Western population is able to metabolize daidzein into the more potent metabolite equol. Having little endogenous estradiol, equol-producing postmenopausal women who use isoflavone supplements to relieve their menopausal symptoms could potentially be at high risk of adverse effects of isoflavone supplementation. The current trial aimed to study the effects of intake of an isoflavone supplement rich in daidzein compared with placebo on whole-genome gene expression profiles of peripheral blood mononuclear cells (PBMCs) in equol-producing, postmenopausal women. Thirty participants received an isoflavone supplement or a placebo for 8 wk each in a double-blind, randomized cross-over design. The isoflavone supplement was rich in daidzein (60%) and provided 94 mg isoflavones (aglycone equivalents) daily. Gene expression in PBMCs was significantly changed (P < 0.05) in 357 genes after the isoflavone intervention compared with placebo. Gene set enrichment analysis revealed downregulated clusters of gene sets involved in inflammation, oxidative phosphorylation, and cell cycle. The expression of estrogen receptor (ER) target genes and gene sets related to ER signaling were not significantly altered, which may be explained by the low ERα and ERß expression in PBMCs. The observed downregulated gene sets point toward potential beneficial effects of isoflavone supplementation with respect to prevention of cancer and cardiovascular disease. However, whether ER-related effects of isoflavones are beneficial or harmful should be studied in tissues that express ERs.

Microbiota dynamics and diversity at different stages of industrial processing of cocoa beans into cocoa powder.

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (P < 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and Enterobacteriaceae (1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the Bacillaceae, Pseudomonadaceae, and Enterococcaceae. Eleven species of ThrS were found, but Bacillus licheniformis and the Bacillus subtilis complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing. B. subtilis complex members, particularly B. subtilis subsp. subtilis, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.