Valacyclovir and Acyclovir Are Substrates of the Guanine Deaminase Cytosolic PSD-95 Interactor (Cypin).

Valacyclovir, enzymatically hydrolyzed in the body to acyclovir, is a guanine-based nucleoside analog commonly prescribed as an antiviral therapy. Previous reports suggest that guanosine analogs bind to guanine deaminase; however, it is unclear whether they act as inhibitors or substrates. Data from our laboratory suggest that inhibition of guanine deaminase by small molecules attenuates spinal cord injury-induced neuropathic pain. Here, we examine whether the guanosine analogs valacyclovir and acyclovir are deaminated by cypin (cytosolic PSD-95 interactor), the major guanine deaminase in the body, or if they act as cypin inhibitors. Using purified Rattus norvegicus cypin, we use NADH-coupled assay to confirm deamination of valacyclovir and determined Michaelis-Menten constants. Subsequently, we use tryptophan fluorescence quenching assay to calculate dissociation constants for valacyclovir and acyclovir and find that inclusion of the valine motif in valacyclovir increases affinity for cypin compared to acyclovir. To our knowledge, neither Km nor KD values for cypin has been previously reported for either compound. We use Amplex Red assay and demonstrate that both valacyclovir and acyclovir are cypin substrates and that their metabolites are further processed by xanthine oxidase and uricase. Using molecular dynamics simulations, we demonstrate that an alpha helix near the active site is displaced when valacyclovir binds to cypin. Furthermore, we used LC-MS-based assay to directly confirm deamination of valacyclovir by cypin. Taken together, our results demonstrate a novel role for cypin in deamination of valacyclovir and acyclovir and suggest that therapeutics based on purine structures may be inactivated by cypin, decreasing inhibitory efficacy.

Effects of acute intravenous lipopolysaccharide administration on the plasma lipidome and metabolome in lactating Holstein cows experiencing hyperlipidemia.

INTRODUCTION: The effects of lipopolysaccharides (i.e., endotoxin; LPS) on metabolism are poorly defined in lactating dairy cattle experiencing hyperlipidemia. OBJECTIVES: Our objective was to explore the effects of acute intravenous LPS administration on metabolism in late-lactation Holstein cows experiencing hyperlipidemia induced by intravenous triglyceride infusion and feed restriction. METHODS: Ten non-pregnant lactating Holstein cows (273 ± 35 d in milk) were administered a single bolus of saline (3 mL of saline; n [Formula: see text] 5) or LPS (0.375 [Formula: see text]g of LPS/kg of body weight; n [Formula: see text] 5). Simultaneously, cows were intravenously infused a triglyceride emulsion and feed restricted for 16 h to induce hyperlipidemia in an attempt to model the periparturient period. Blood was sampled at routine intervals. Changes in circulating total fatty acid concentrations and inflammatory parameters were measured. Plasma samples were analyzed using untargeted lipidomics and metabolomics. RESULTS: Endotoxin increased circulating serum amyloid A, LPS-binding protein, and cortisol concentrations. Endotoxin administration decreased plasma lysophosphatidylcholine (LPC) concentrations and increased select plasma ceramide concentrations. These outcomes suggest modulation of the immune response and insulin action. Lipopolysaccharide decreased the ratio of phosphatidylcholine to phosphatidylethanomanine, which potentially indicate a decrease in the hepatic activation of phosphatidylethanolamine N-methyltransferase and triglyceride export. Endotoxin administration also increased plasma concentrations of pyruvic and lactic acids, and decreased plasma citric acid concentrations, which implicate the upregulation of glycolysis and downregulation of the citric acid cycle (i.e., the Warburg effect), potentially in leukocytes. CONCLUSION: Acute intravenous LPS administration decreased circulating LPC concentrations, modified ceramide and glycerophospholipid concentrations, and influenced intermediary metabolism in dairy cows experiencing hyperlipidemia.

Obtainment and characterization of a potential functional ingredient from olive.

Current olive oil production methods generate huge amounts of polluting waste, containing most of the health-related compounds in olive. Here, a new product is obtained from olive after pitting, drying and oil extraction, without generating waste. Its characterization showed the presence, within a single matrix, of more than 90% of the polyphenols present in olive, including hydroyxtyrosols (commonly not transferred to olive oil), dietary fiber, oleic acid and polyalcohols. This product is a potential new functional ingredient, consumption of which may lead to additive and/or synergic activities among its constituents; some of which already have approved health claims. Additionally, the olive oil obtained exhibits profiles of fatty acids and phenolic compounds similar to those of commercial olive oil. The procurement of this potential functional ingredient may represent a new approach to the revalorization of olive that additionally decreases waste.

Contribution of Macromolecular Antioxidants to Dietary Antioxidant Capacity: A Study in the Spanish Mediterranean Diet.

Epidemiological and clinical studies show that diets with a high antioxidant capacity, such us those rich in plant food and beverages, are associated with significant decreases in the overall risk of cardiovascular disease or colorectal cancer. Current studies on dietary antioxidants and dietary antioxidant capacity focus exclusively on low molecular weight or soluble antioxidants (vitamins C and E, phenolic compounds and carotenoids), ignoring macromolecular antioxidants. These are polymeric phenolic compounds or polyphenols and carotenoids linked to plant food macromolecules that yield bioavailable metabolites by the action of the microbiota with significant effects either local and/or systemic after absorption. This study determined the antioxidant capacity of the Spanish Mediterranean diet including for the first time both soluble and macromolecular antioxidants. Antioxidant capacity and consumption data of the 54 most consumed plant foods and beverages were used. Results showed that macromolecular antioxidants are the major dietary antioxidants, contributing a 61% to the diet antioxidant capacity (8000 µmol Trolox, determined by ABTS method). The antioxidant capacity data for foods and beverages provided here may be used to estimate the dietary antioxidant capacity in different populations, where similar contributions of macromolecular antioxidants may be expected, and also to design antioxidant-rich diets. Including macromolecular antioxidants in mechanistic, intervention and observational studies on dietary antioxidants may contribute to a better understanding of the role of antioxidants in nutrition and health.

Regular consumption of an antioxidant-rich juice improves oxidative status and causes metabolome changes in healthy adults.

An improvement in oxidative status is associated with a reduction in the incidence of several chronic diseases. However, daily intake of antioxidants in Western diets is decreasing. This study evaluates the effect of daily consumption of an antioxidant-rich juice (ARJ) on oxidative status, cardiovascular disease risk parameters, and untargeted plasma and urine metabolomes. Twenty-eight healthy young adults participated in an 8-week clinical trial by drinking 200 mL of ARJ (pomegranate and grape) daily. At the end of the study, the subjects showed a significant decrease (-29%) in plasma lipid oxidation (malondialdehyde concentration), and a significant increase (+115%) in plasma antioxidant capacity. Plasma and urine metabolomes were also significantly modified and some ions modified in urine were identified, including metabolites of polyphenols, ascorbic acid and biliary acids. No significant changes were observed in lipid profile, inflammation, blood pressure or glycaemia. These results show that incorporating antioxidant-rich beverages into common diets may improve oxidative status in healthy subjects.

Non-extractable polyphenols, a major dietary antioxidant: occurrence, metabolic fate and health effects.

Current research on dietary antioxidants misses the so-called non-extractable polyphenols (NEPP), which are not significantly released from the food matrix either by mastication, acid pH in the stomach or action of digestive enzymes, reaching the colon nearly intact. NEPP, not detected by the usual analytical procedures, are made up of macromolecules and single phenolic compounds associated with macromolecules. Therefore, NEPP are not included in food and dietary intake data nor in bioavailability, intervention or observational studies. The present paper aims to provide an overview of dietary NEPP - nature, occurrence in diet, metabolic fate and possible health effects. NEPP are a relevant fraction of dietary polyphenols exerting their main biological action in the colon, where they are extensively fermented by the action of microbiota, giving place to absorbable metabolites. NEPP exhibit different potential health-related properties, in particular in relation to gastrointestinal health, such as increases in antioxidant and antiproliferative capacities, reduction of intestinal tumorigenesis and modification of gene expression, as observed in different animal models. Further research into NEPP may provide a better understanding of the health effects of dietary antioxidants.

Changes in Choline Metabolites and Ceramides in Response to a DASH-Style Diet in Older Adults.

This feeding trial evaluated the impact of the Dietary Approaches to Stop Hypertension diet on changes in plasma choline, choline metabolites, and ceramides in obese older adults; 28 adults consumed 3oz (n = 15) or 6oz (n = 13) of beef within a standardized DASH diet for 12 weeks. Plasma choline, betaine, methionine, dimethylglycine (DMG), phosphatidylcholine (PC), lysophosphotidylcholine (LPC), sphingomyelin, trimethylamine-N-oxide (TMAO), L-carnitine, ceramide, and triglycerides were measured in fasted blood samples. Plasma LPC, sphingomyelin, and ceramide species were also quantified. In response to the study diet, with beef intake groups combined, plasma choline decreased by 9.6% (p = 0.012); DMG decreased by 10% (p = 0.042); PC decreased by 51% (p < 0.001); total LPC increased by 281% (p < 0.001); TMAO increased by 26.5% (p < 0.001); total ceramide decreased by 22.1% (p < 0.001); and triglycerides decreased by 18% (p = 0.021). All 20 LPC species measured increased (p < 0.01) with LPC 16:0 having the greatest response. Sphingomyelin 16:0, 18:0, and 18:1 increased (all p < 0.001) by 10.4%, 22.5%, and 24%, respectively. In contrast, we observed that sphingomyelin 24:0 significantly decreased by 10%. Ceramide 22:0 and 24:0 decreased by 27.6% and 10.9% (p < 0.001), respectively, and ceramide 24:1 increased by 36.8% (p = 0.013). Changes in choline and choline metabolites were in association with anthropometric and cardiometabolic outcomes. These findings show the impact of the DASH diet on choline metabolism in older adults and demonstrate the influence of diet to modify circulating LPC, sphingomyelin, and ceramide species.

iPSC-derived retinal pigmented epithelial cells from patients with macular telangiectasia show decreased mitochondrial function.

Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.

Beverages have an appreciable contribution to the intake of soluble dietary fibre: a study in the Spanish diet.

Beverages are generally not taken into account to determine the intakes of dietary fibre (DF) in diets. Soluble dietary fibre (SDF) content was determined in common alcoholic and non-alcoholic beverages - ranging from 0.18 g/l in white wine to 9.01 g/l in instant coffee - and their contribution to the DF intake in the Spanish Mediterranean diet was estimated as 2.13 g/person/day. It is concluded that beverages provide an appreciable amount of SDF in the diet, and the omission of its contribution may lead to underestimate DF intakes.

Dietary fiber and antioxidant capacity in Fucus vesiculosus products.

Several nutraceutical products have been developed from Fucus vesiculosus, a brown edible seaweed, rich in dietary fiber and polyphenolic antioxidants (phlorotannins). The aim of this work was to compare the antioxidant capacity and polysaccharide composition of raw Fucus with those of some common commercial nutraceuticals. All tested products contained a high percentage of dietary fiber (45-59%), raw Fucus powder being the sample with the highest content. Also, raw Fucus powder exhibited significantly higher antioxidant capacity (determined by FRAP, ABTS and ORAC assays) than the commercial fucoidans and commercial antioxidant extracts. Polyphenols (phlorotannins) seem to be the main contributors to Fucus' antioxidant capacity in both raw powder and commercial fucoidans.

Bioavailability of phenolic antioxidants associated with dietary fiber: plasma antioxidant capacity after acute and long-term intake in humans.

Most studies on bioavailability of phenolic antioxidants are focused in foods and beverages in which they may be easily released from the food matrix, reaching a peak in plasma antioxidant capacity 1-2 h after the intake. However, plant foods contain significant amounts of polyphenols associated with dietary fiber. The aim of the present work was to seek the bioavailability of total phenolic antioxidants associated with dietary fiber by measuring plasma antioxidant capacity in human volunteers. An acute intake of 15 g of a dietary fiber rich in associated phenolic antioxidants in healthy volunteers (n = 10) increased antioxidant capacity of plasma in relation to a control group (n = 4), becoming significant 8 h after the intake. This shows that phenolic antioxidants associated with dietary fiber are at least partially bioavailable in humans, although dietary fiber appears to delay their absorption. No significant changes were observed after long-term intake (16 weeks, 34 subjects).

Effects of grape antioxidant dietary fiber in cardiovascular disease risk factors.

OBJECTIVE: The objective of the study was to evaluate the effects of a grape product rich in dietary fiber and natural antioxidants on cardiovascular disease risk factors. METHODS: A randomized, controlled parallel-group trial was carried out. Thirty-four non-smoking (21 normocholesterolemic and 13 hypercholesterolemic) adults were supplemented for 16 wk with 7.5 g/d of grape antioxidant dietary fiber, a natural product containing 5.25 g of dietary fiber and 1400 mg of polyphenols. Nine non-supplemented non-smokers were followed as a control group. Fasting blood samples, blood pressure, and anthropometric readings were obtained at baseline and at week 16. Subjects were allowed to consume their regular diet, which was monitored weekly. RESULTS: Grape antioxidant dietary fiber (7.5 g/d) reduced significantly (P < 0.05) total cholesterol (9%), low-density lipoprotein cholesterol (9%), and systolic and diastolic blood pressures (6% and 5% respectively). Greater reductions in total cholesterol (14.2%) and low-density lipoprotein cholesterol (11.6%, P < 0.05) were observed in hypercholesterolemic subjects. No changes were observed in the control group. There was a reduction of 2.5 points in the Framingham Global Risk Score in the supplemented group. A significant reduction in triacylglycerol concentration took place in the supplemented hypercholesterolemic subjects (18.6%, P < 0.05). CONCLUSION: Grape antioxidant dietary fiber showed significant reducing effects in lipid profile and blood pressure. The effects appear to be higher than the ones caused by other dietary fibers, such as oat fiber or psyllium, probably due to the combined effect of dietary fiber and antioxidants.

Dietary fiber in brewed coffee.

Coffee beans are rich in nondigestible polysaccharides (dietary fiber), which may partially pass into brewed coffee; however, to the authors' knowledge, there is not enough literature on dietary fiber in brewed coffee. A specific method to determine dietary fiber in beverages (enzymatic treatment plus dialysis) was applied to the coffees brewed by the most common methods (espresso, filter, soluble); results showed that brewed coffee contained a significantly higher amount of soluble dietary fiber (0.47-0.75 g/100 mL of coffee) than other common beverages. Coffee dietary fiber contains a large amount of associated antioxidant phenolics (8.7-10.5 mg/100 mL of brewed coffee).