Chronic consumption of orange juice modifies urinary excretion of flavanone gut-derived metabolites through gut microbiota modulation.

The metabolism and absorption of citrus flavanones are intrinsically linked to the gut microbiota, creating a bidirectional relationship where these compounds influence the microbiome, and in turn, the microbiota affects their metabolism. This study evaluates the effect of acute and chronic consumption of orange juice (OJ) on the urinary excretion of gut-derived flavanone metabolites and the gut microbiota. Health volunteers ingested 500 mL of OJ for 60 days in a single-arm human intervention study. Blood and feces were collected at baseline and after 60 days, with an additional 24-hour urine collection after a single dose on day 1 and day 63. LC-MS/MS analyzed urinary flavanone metabolites, while 16S rRNA sequencing characterized gut microbiota. Total urinary hesperetin conjugates excretion significantly decreased over 60 days, while gut-derived total phenolic acids, particularly three hydroxybenzoic acids, increased. Moreover, the heterogeneity of the total amount of flavanone conjugates, initially categorizing individuals into high-, medium- and low- urinary excretor profiles, shifted towards medium-excretor, except for five individuals who remained as low-excretors. This alteration was accompanied by a decrease in intestinal ß-glucosidase activity and a shift in the relative abundance of specific genera, such as decreases in Blautia, Eubacterium hallii, Anaerostipes, and Fusicatenibacter, among which, Blautia was associated with higher urinary flavanone conjugates excretion. Conversely, an increase in Prevotella was observed. In summary, chronic OJ consumption induced transient changes in gut microbiota and altered the metabolism of citrus flavanones, leading to distinct urinary excretion profiles of flavanone metabolites.

Interaction between APOE, APOA1, and LPL Gene Polymorphisms and Variability in Changes in Lipid and Blood Pressure following Orange Juice Intake: A Pilot Study.

SCOPE: Chronic orange juice intake is associated with reduced risk of cardiovascular disease, however, a large inter-individual variability in response to orange juice for lipid profile and blood pressure has been observed. This heterogeneity in responsiveness could be associated with single nucleotide polymorphism (SNP), which has not been previously addressed. This study aims to investigate the influence of SNP in apolipoprotein E (APOE), apolipoprotein A1 (APOA1), mevalonate (MVK), and lipase lipoprotein (LPL) genes in the biological response after chronic orange juice intake. METHODS AND RESULTS: Forty-six volunteers ingested 500 mL daily for 60 days and blood pressure and biochemical parameters are measured. Also, SNPs in APOE, APOA1, MVK, and LPL genes are genotyped in the volunteers that are medium/high excretors of flavanone metabolites. Genotypes CC (APOA1), AA, and GG (LPL) are associated with positive health effects of orange juice and the CC (APOE), GG (APOA1), GG, and AA (LPL) genotypes are associated with no effects of orange juice consumption (p < 0.05). CONCLUSION: These results identify for the first-time SNP associated with effects of orange juice on lipid levels and blood pressure, results that may provide bases for future precise nutritional recommendations regarding this flavanone-rich food to lower the risk for cardiovascular disease.

Citrus flavanone metabolites significantly modulate global proteomic profile in pancreatic ß-cells under high-glucose-induced metabolic stress.

Hesperidin and narirutin are the major citrus flavanones. Several studies have associated these compounds with pancreatic ß-cell survival through their capacity to reduce oxidative stress, inflammation, and inhibit apoptosis. However, the molecular mechanisms of action of flavanones in pancreatic ß-cells under high-glycemic stress is still largely unknown. Therefore, this study aimed to decipher molecular mechanisms of flavanone metabolites in pancreatic ß-cells treated with high glucose concentration using untargeted shotgun proteomics. We identified 569 proteins differentially expressed in cells exposed to hesperetin 7-glucuronide (H7G) and 265 in cells exposed to 3-(4'-hydroxyphenyl) propanoic acid (PA). Comparison of global proteomic profiles suggest that these metabolites could counteract changes in protein expression induced by high glucose stress. The bioinformatic analyses suggested that H7G and PA modulated the expression of proteins involved in cell adhesion, cell signaling, metabolism, inflammation, and protein processing in endoplasmic reticulum (ER) pathways. Taken together, this study suggests that H7G and PA can modulate the expression of proteins that may prevent dysfunction of pancreatic ß-cells under stress induced by high glucose.

Citrus flavanone metabolites protect pancreatic ß-cells against cholesterol stress through a multi-proteomic mechanism.

Citrus flavanones may improve oxidative stress and insulin resistance induced by western diets. However, there is a paucity of studies investigating the change in protein expression levels. This study evaluated the protection and the mechanisms of action of citrus flavanone metabolites, hesperetin 7-glucuronide (H7G) and 3-(4'-hydroxyphenyl) propanoic acid (PA), on pancreatic ß-cell function under oxidative stress induced by cholesterol using the global proteomics approach. Cholesterol induced changes in the global proteomic profile in the pancreatic ß-cell line Min6. On the other hand, proteomics analysis identified 254 proteins differentially expressed with H7G and 352 with PA treatments, most of them were opposite to the changes induced by cholesterol. Bioinformatics analysis showed that these proteins are implicated in cell functions like cell signaling (insulin signaling, p30MAPK signaling, and others), metabolism (glucokinase and glutathione metabolisms), and inflammation pathways (TNF-α and NF-κB pathways). Also, the results of molecular docking suggest that H7G and PA could bind to putative transcription factors (PPAR-γ, STAT-3, CREB1, NF-κB, NFYA) and cell signaling proteins (IKK, RAS, Pi3K, ERK), which results in changes in protein expression observed. Altogether, these data suggest that the treatment with H7G and PA protects pancreatic ß-cells against stress induced by cholesterol through multi-proteomic mechanisms of action.

Association between Single Nucleotide Polymorphisms of SULT1A1, SULT1C4, ABCC2 and Phase II Flavanone Metabolites Excretion after Orange Juice Intake.

Citrus fruits and juices are a major source of dietary flavanones, and the regular consumption of these foods is inversely associated with the development of cardiometabolic diseases. However, the biological benefits depend on the bioavailability of these compounds, and previous studies have reported a large interindividual variability in the absorption and excretion of these compounds. Different factors, such as age, gender or genetic polymorphism of genes coding enzymes involved in the metabolism and transport of the flavanones, may explain this heterogeneity. This study aimed to assess the impact of single nucleotide polymorphism of sulfotransferases SULT1A1 and SULT1C4, and ABCC2 transporter genes on excretion of phase II flavanone metabolites in volunteers after 24 h of orange juice intake. Forty-six volunteers ingested a single dose of 500 mL of orange juice and 24-h urine was collected. The hesperetin and naringenin phase II metabolites were quantified in urine, and SNPs in SULT1A1, SULT1C4 and ABCC2 genes were genotyped. A significant (p < 0.05) relationship between the SNPs in these genes and the high excretion of phase II flavanone metabolites were observed. These results identified novel polymorphisms associated with higher absorption of flavanones, which may provide bases for future personalized nutritional guidelines for consuming flavanone-rich foods rich in these nutrients for better benefit from their health properties.

Blood pressure and body fat % reduction is mainly related to flavanone phase II conjugates and minor extension by phenolic acid after long-term intake of orange juice.

Hesperidin and narirutin are the major flavanones present in orange juice, and they are associated with a reduction in risk of cardiometabolic disease. However, there is heterogeneity in their biological responses, which is partly due to the large interindividual variation in these flavonoids' bioavailability. We investigated the relation between interindividual variability in the excretion of phase II conjugates and gut-derived phenolic acids, and cardiometabolic biomarkers response. Seventy-four subjects, both men and women, were included in a single-arm study. Over the 60 days, volunteers consumed 500 mL of orange juice daily. All measurements and blood collections were performed before and after the intervention period. Moreover, 24 h urine collection was performed after first consumption. Individuals were stratified according to the excretion of phase II conjugates and, for the first time, according to phenolic acids in high, medium, and low excretors. Furthermore, for the first time, the ratio between phenolic acids and flavanones-phase II conjugates has shown groups with different metabolization patterns. Groups with a low or intermediate ratio, corresponding to a higher amount of phase II conjugates excreted, showed a significant reduction in body fat % and blood pressure. This finding suggests that these improvements could be associated in a major way to flavanones-phase II conjugates, as well as to phenolic acids and stratification of volunteers according to metabolite excretions could be a good strategy to better understand the effects of orange juice on metabolism and health.

Stratification of Volunteers According to Flavanone Metabolite Excretion and Phase II Metabolism Profile after Single Doses of 'Pera' Orange and 'Moro' Blood Orange Juices.

Large interindividual variations in the biological response to citrus flavanones have been observed, and this could be associated with high variations in their bioavailability. The aim of this study was to identify the main determinants underlying interindividual differences in citrus flavanone metabolism and excretion. In a randomized cross-over study, non-obese and obese volunteers, aged 19-40 years, ingested single doses of Pera and Moro orange juices, and urine was collected for 24 h. A large difference in the recovery of the urinary flavanone phase II metabolites was observed, with hesperetin-sulfate and hesperetin-sulfo-O-glucuronide being the major metabolites. Subjects were stratified according to their total excretion of flavanone metabolites as high, medium, and low excretors, but the expected correlation with the microbiome was not observed at the genus level. A second stratification was proposed according to phase II flavanone metabolism, whereby participants were divided into two excretion groups: Profiles A and B. Profile B individuals showed greater biotransformation of hesperetin-sulfate to hesperetin-sulfo-O-glucuronide, as well as transformation of flavanone-monoglucuronide to the respective diglucuronides, suggestive of an influence of polymorphisms on UDP-glucuronosyltransferase. In conclusion, this study proposes a new stratification of volunteers based on their metabolic profiles. Gut microbiota composition and polymorphisms of phase II enzymes may be related to the interindividual variability of metabolism.