Metabolomic-Based Approach to Identify Biomarkers of Apple Intake.

SCOPE: There is an increased interest in developing biomarkers of food intake to address some of the limitations associated with self-reported data. The objective is to identify biomarkers of apple intake, examine dose-response relationships, and agreement with self-reported data. METHODS AND RESULTS: Metabolomic data from three studies are examined: an acute intervention, a short-term intervention, and a free-living cohort study. Fasting and postprandial urine samples are collected for analysis by 1 H-NMR and liquid chromatography-mass spectrometry (LC-MS). Calibration curves are developed to determine apple intake and classify individuals into categories of intake. Multivariate analysis of data reveals that levels of multiple metabolites increase significantly post-apple consumption, compared to the control food-broccoli. In the dose-response study, urinary xylose, epicatechin sulfate, and 2,6-dimethyl-2-(2-hydroxyethyl)-3,4-dihydro-2H-1-benzopyran increase as apple intake increases. Urinary xylose concentrations in a free-living cohort perform poorly at an individual level but are capable of ranking individuals in categories of intake. CONCLUSION: Urinary xylose exhibits a dose-response relationship with apple intake and performs well as a ranking biomarker in the population study. Other potential biomarkers are identified and future work will combine these with xylose in a biomarker panel which may allow for a more objective determination of individual intake.

Antimalarial Quinoline Drugs Inhibit ß-Hematin and Increase Free Hemin Catalyzing Peroxidative Reactions and Inhibition of Cysteine Proteases.

Malaria caused by Plasmodium affects millions people worldwide. Plasmodium consumes hemoglobin during its intraerythrocytic stage leaving toxic heme. Parasite detoxifies free heme through formation of hemozoin (ß-hematin) pigment. Proteolysis of hemoglobin and formation of hemozoin are two main targets for antimalarial drugs. Quinoline antimarial drugs and analogs (ß-carbolines or nitroindazoles) were studied as inhibitors of ß-hematin formation. The most potent inhibitors were quinacrine, chloroquine, and amodiaquine followed by quinidine, mefloquine and quinine whereas 8-hydroxyquinoline and ß-carbolines had no effect. Compounds that inhibited ß-hematin increased free hemin that promoted peroxidative reactions as determined with TMB and ABTS substrates. Hemin-catalyzed peroxidative reactions were potentiated in presence of proteins (i.e. globin or BSA) while antioxidants and peroxidase inhibitors decreased peroxidation. Free hemin increased by chloroquine action promoted oxidative reactions resulting in inhibition of proteolysis by three cysteine proteases: papain, ficin and cathepsin B. Glutathione reversed inhibition of proteolysis. These results show that active quinolines inhibit hemozoin and increase free hemin which in presence of H2O2 that abounds in parasite digestive vacuole catalyzes peroxidative reactions and inhibition of cysteine proteases. This work suggests a link between the action of quinoline drugs with biochemical processes of peroxidation and inhibition of proteolysis.

Recent Advances in the Application of Metabolomics for Nutrition and Health.

Metabolomics is the study of small molecules called metabolites in biological samples. Application of metabolomics to nutrition research has expanded in recent years, with emerging literature supporting multiple applications. Key examples include applications of metabolomics in the identification and development of objective biomarkers of dietary intake, in developing personalized nutrition strategies, and in large-scale epidemiology studies to understand the link between diet and health. In this review, we provide an overview of the current applications and identify key challenges that need to be addressed for the further development of the field. Successful development of metabolomics for nutrition research has the potential to improve dietary assessment, help deliver personalized nutrition, and enhance our understanding of the link between diet and health.

Role of dietary onion in modifying the faecal bile acid content in rats fed a high-cholesterol diet.

The determination of faecal bile patterns offers new opportunities in the search for non-invasive biomarkers of disease status. The objective of this study was to describe the shifts in faecal bile acid (BA) composition induced by feeding a high-cholesterol/cholic acid diet (HC) over 7 weeks of experimental feeding in Wistar rats, and to evaluate the effect of onion included as a functional ingredient (HCO). A HPLC-MS/MS method allowed the detection of 29 bile acids, 10 of which were tentatively identified and 12 confirmed and quantified by means of standards and calibration curves. The excretion of bile acids revealed a discriminating bile acid profile between the HC and HCO groups compared with the C group. HCO feeding indicated significant changes in specific primary and secondary BA in both the unconjugated and conjugated forms caused by the addition of the onion ingredient to the diet. The results suggest that the induction of microbiome modifications by the HC and HCO diets acts as a critical modifier of the faecal bile acid composition. These modifications might reflect and be linked to changes in the reabsorption of BA at an intestinal level and the process of BA deconjugation in the course of hypercholesterolemia.

Metabolomic Fingerprinting in the Comprehensive Study of Liver Changes Associated with Onion Supplementation in Hypercholesterolemic Wistar Rats.

The consumption of functional ingredients has been suggested to be a complementary tool for the prevention and management of liver disease. In this light, processed onion can be considered as a source of multiple bioactive compounds with hepatoprotective properties. The liver fingerprint of male Wistar rats (n = 24) fed with three experimental diets (control (C), high-cholesterol (HC), and high-cholesterol enriched with onion (HCO) diets) was obtained through a non-targeted, multiplatform metabolomics approach to produce broad metabolite coverage. LC-MS, CE-MS and GC-MS results were subjected to univariate and multivariate analyses, providing a list of significant metabolites. All data were merged in order to figure out the most relevant metabolites that were modified by the onion ingredient. Several relevant metabolic changes and related metabolic pathways were found to be impacted by both HC and HCO diet. The model highlighted several metabolites (such as hydroxybutyryl carnitine and palmitoyl carnitine) modified by the HCO diet. These findings could suggest potential impairments in the energy-lipid metabolism, perturbations in the tricarboxylic acid cycle (TCA) cycle and ß-oxidation modulated by the onion supplementation in the core of hepatic dysfunction. Metabolomics shows to be a valuable tool to evaluate the effects of complementary dietetic approaches directed to hepatic damage amelioration or non-alcoholic fatty liver disease (NAFLD) prevention.

New insights into the effects of onion consumption on lipid mediators using a diet-induced model of hypercholesterolemia.

The levels and roles of lipid mediators can be modified in response to nutritional stimuli. The aim of this study was to investigate shifts in oxylipin and sphingolipid profiles stimulated by a hypercholesterolemic (HC) diet along with the modulating effects of onion introduced as an antioxidant functional ingredient characterized in the diet (HCO). Oxylipin and sphingolipid profiles were determined in plasma and tissues from Wistar rats using LC-MS/MS. Plasma ω-3 and ω-6 PUFA-derived oxylipins decreased in rats after 7 weeks of HC feeding, but did not evidence a further shift with HCO diet. Onion ingredient supplementation modulated the hepatic concentrations of prostaglandins and enhanced ω-3 oxylipins in the liver of HCO-fed rats relative to the HC group. The HC diet induced shifts in plasma sphingolipids, increasing sphingoid bases, dihydroceramides and ceramides, whilst the sphingomyelin, hexosylceramide and lactosylceramide families decreased. The HCO diet modified some HC diet-induced changes in sphingolipids in liver and spleen tissue. Onion supplementation effected changes in lipid mediator levels in diet-induced hypercholesterolemic Wistar rats. The potential of onion as regulator of pro-inflammatory mediators, and possible enhancer of pro-resolution pathways, warrants further study of the interaction of functional ingredients with bioactive lipid mediators and their potential impact on inflammation, oxidative stress and organ dysfunction.

Multiplatform metabolomic fingerprinting as a tool for understanding hypercholesterolemia in Wistar rats.

PURPOSE: The aim was to investigate the impact of hypercholesterolemic diet on the metabolome of male Wistar rats by a multiplatform metabolomic fingerprinting. METHODS: Male Wistar rats were fed with two different diets [control (C) and high-cholesterol diet (HC)-containing 2 % cholesterol and 0.5 % cholic acid]. After 7 weeks of experimental feeding, the rats were euthanized for blood collection and plasma recovery. The metabolite fingerprint was then achieved by applying a multiplatform comprising LC-MS, GC-MS and CE-MS. RESULTS: Multivariate statistical analysis showed a clear separation between the C and HC groups. Individual differences in metabolites were evaluated using univariate statistical analysis, and multiple metabolites were identified and confirmed in the plasma. A global profiling integrates for the first time pathways affected by high-cholesterol diet intake and allowed us to elucidate some of the associated alterations underlying the hypercholesterolemia event in Wistar rats. CONCLUSIONS: HC feeding stimulated the alteration of multiple pathways in Wistar rats, warning of the risk of developing important diseases, which can be modulated by the diet. Further studies are required to investigate the possibilities to revert or ameliorate the negative effects triggered by HC intake.

High-cholesterol diet enriched with onion affects endothelium-dependent relaxation and NADPH oxidase activity in mesenteric microvessels from Wistar rats.

BACKGROUND: The aim of the present study was to examine the effects of onion as functional ingredient on the oxidative status, lipoprotein levels (total cholesterol-TC, HDL-C, LDL-C), triacylglycerides (TAG) and vascular reactivity of mesenteric arteries in hypercholesterolemic Wistar rats. METHODS: Twenty-four animals were fed with three different diets [control, high-cholesterol diet (HC) and high-cholesterol enriched with onion diet (HCO)]. After seven weeks of experimental feeding the rats were euthanized for blood and tissues collection. TC, HDL-C, LDL-C and TAG were measured, and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS(•+)) scavenging capacity and ferric reducing antioxidant power (FRAP) were determined in plasma. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities were assayed in erythrocyte lysates. Endothelium-dependent vasodilation to acetylcholine was evaluated in mesenteric arterial segments. NADPH oxidase (NOX) was also measured by lucigenin-derived chemiluminiscence. RESULTS: The dietary cholesterol content significantly affected plasma lipoprotein levels, increased superoxide generation from NOX, and caused impaired endothelium-dependent vasodilation in the rat mesenteric arteries. Onion ingredient improved antioxidant status in HCO group, as it was evidenced by ABTS(•+) and FRAP values and SOD and GPx enzyme activities compared to the HC-fed group, reduced the increment in NOX activity and reversed endothelial dysfunction promoted by the HC diet. Scavenging of superoxide with TEMPOL or inhibition of NOX with apocynin improved endothelium-dependent vasodilation only in HC-fed rats. CONCLUSIONS: Enrichment of diet with onion as functional ingredient could be proposed as a complementary approach to prevent or partially modulate vascular dysfunction, reducing some of the risk indexes linked to initial development of atherosclerosis.

Effects of hypercholesterolemic diet enriched with onion as functional ingredient on fatty acid metabolism in Wistar rats.

The complex biochemical composition of onions has been studied as a source of biological components with health-related properties. The evolution of hypercholesterolemia is associated with a large range of alterations considered as strong risk factors for many cardiovascular events. The objective of this study was to investigate the effects of onion as functional ingredient on plasma, erythrocyte, liver and adipose tissue fatty acid composition in hypercholesterolemic male Wistar rats. Rats (n=24) were randomly divided into three groups: control (C), high-cholesterol (HC), and high-cholesterol enriched with onion (HCO) groups. At the end of 7weeks, animals were anesthetized and euthanized by extracting blood by cardiac puncture. Plasma, erythrocytes, liver and adipose tissue were collected and immediately stored at -80°C. Fatty acid methyl esters were identified and quantified by GC/MS. Total fatty acid concentration decreased in liver and adipose tissue both in HC and HCO groups. SFA content was significantly higher in plasma, erythrocytes and liver in the C group compared to HC and HCO groups. In contrast, SFAs increased in adipose tissue both in HC and HCO groups compared to the C group. A significant increase in MUFA content in plasma was found in HC and HCO groups compared to the C group; in erythrocytes and liver the increase was lower. In plasma, PUFA content was significantly lower in HC and HCO groups compared to the C group. Interestingly, in liver and adipose tissue, PUFAs increased in HC and HCO groups compared to the C group. Results showed noticeable effects on individual fatty acid composition when assaying high-cholesterol diets in rats, in some cases enhanced by onion enrichment. Further research is needed to deeper understand the involved mechanisms and pathways.

Hyaluronidase inhibiting activity and radical scavenging potential of flavonols in processed onion.

The flavonol content and anti-inflammatory and antioxidant activities of onion treated by high-pressure processing (HPP) and HPP combined with freeze-drying and pulverization (HPP-FD-P) were evaluated. Allium cepa L. var. cepa, 'Recas' was treated at T1 (200 MPa/25 °C/5 min), T2 (400 MPa/25 °C/5 min), and T3 (600 MPa/25 °C/5 min). After treatment, HP-treated and untreated samples were frozen (diced onion, HP-treated). Subsequently, part of the diced samples was freeze-dried and pulverized (pulverized onion, HP-treated and freeze-dried). Flavonol content and anti-inflammatory and antioxidant activities (hyaluronidase inhibiting activity, NO(•), ABTS(•+), and DPPH(•) scavenging capacity, ferric reducing antioxidant power, and antioxidative capacity by photochemiluminescence) were measured in nonhydrolyzed and hydrolyzed extracts. Hydrolysis was carried out in order to evaluate the effect of HPP and HPP-FD-P on both anti-inflammatory and antioxidant activities of extracts mainly containing aglycone forms. HPP-FD-P increased quercetin 3,4'-diglucoside, quercetin 4'-glucoside, quercetin 3-glucoside, and isorhamnetin 3,4'-diglucoside extractability. The present study suggests that HPP (especially treatment at 400 MPa) and HPP-FD-P may be of benefit for obtaining functional ingredients from onion, as suggested by increased NO(•) scavenging capacity and maintenance of the antioxidant activity mainly in hydrolyzed extracts.

Novel approach for the determination of volatile compounds in processed onion by headspace gas chromatography-mass spectrometry (HS GC-MS).

A novel headspace gas chromatography-mass spectrometry (HS GC-MS) method was developed for analysis of volatile compounds in onion (Allium cepa L. var. cepa, 'Recas'). MS was operated using full scan mode and selective ion monitoring (SIM) mode in order to quantify some specific compounds with increased sensitivity relative to full scan mode. The limits of detection and quantitation ranged from 0.01 to 0.10 µg/g and from 0.02 to 3.83 µg/g fresh weight, respectively, for studied compounds. The procedure allowed the identification of eighteen compounds and quantitation of nine compounds in the volatile fraction of onion, belonging mainly to di-, and trisulfides and aldehydes. These methods were applied to evaluate how high-pressure (HP) as a processing technology affects onion volatile compounds, responsible in part of the onion biological activity. Onion samples were treated at T1: 200 MPa/25°C/5 min, T2: 400 MPa/25°C/5 min and T3: 600 MPa/25°C/5 min (treatments). In addition, the difference among diced, freeze-dried and pulverized onions (groups) was studied, in order to select the process more adequate for better preserving volatile compounds. The results obtained in full scan mode showed that both main factors (group and treatment) had a significant effect (P<0.001). There were also significant differences between groups and treatments for all compounds, being the main effect of group more marked by HS GC-MS using selective ion monitoring (SIM) mode. For 2-methyl 2-pentenal, dimethyl trisulfide, and methyl propyl trisulfide it has been observed an increase in freeze-dried and pulverized onion samples compared with diced samples regardless the HP treatment. However, freeze-drying and pulverization processes affected the stability of propionaldehyde, 1-propanethiol, hexanal, dipropyl disulfide, and dipropyl trisulfide, diminishing their content regardless the HP treatment. HP at 200 and 400 MPa/25°C/5 min were the least detrimental treatments to the total fraction of volatile compounds, not affecting or even increasing the levels of some volatile compounds.