Plasma pharmacokinetics of (poly)phenol metabolites and catabolites after ingestion of orange juice by endurance trained men

Highlights

• Human absorption and metabolism of orange juice (poly)phenols.

• Mammalian and microbiota-mediated conversions.

• Plasma pharmacokinetic of flavanone metabolites and catabolites.

• Unexpected behaviour of 3-hydroxy-3-(phenyl)propanoic acids highlighted.

• Essential data for ex vivo cell studies on the protective effects of flavanones.

Abstract

The health benefits of orange juice (OJ) consumption are attributed in part to the circulating flavanone phase II metabolites and their microbial-derived ring fission phenolic catabolites. The present study investigated these compounds in the bloodstream after acute intake of 500 mL of OJ. Plasma samples obtained at 0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h after OJ intake were analysed by HPLC-HR-MS. Eleven flavanone metabolites and 36 phenolic catabolites were identified and quantified in plasma. The main metabolites were hesperetin-3′-sulfate with a peak plasma concentration (C_max) of 80 nmol/L, followed by hesperetin-7-glucuronide (C_max 24 nmol/L), hesperetin-3′-glucuronide (C_max 18 nmol/L) and naringenin-7-glucuronide (C_max 21 nmol/L). Among the main phenolic catabolites to increase in plasma after OJ consumption were 3′-methoxycinnamic acid-4′-sulfate (C_max 19 nmol/L), 3-hydroxy-3-(3′-hydroxy-4′-methoxyphenyl)propanoic acid (C_max 20 nmol/L), 3-(3′-hydroxy-4′-methoxyphenyl)propanoic acid (C_max 19 nmol/L), 3-(4′-hydroxyphenyl)propanoic acid (C_max 25 nmol/L), and 3-(phenyl)propanoic acid (C_max 19 nmol/L), as well as substantial amounts of phenylacetic and hippuric acids. The comprehensive plasma pharmacokinetic profiles that were obtained are of value to the design of future ex vivo cell studies, aimed at elucidating the mechanisms underlying the potential health benefits of OJ consumption.

Clinical trial registration number

This trial was registered at clinicaltrials.gov as NCT02627547.

Introduction

Orange juice (OJ) flavanones, mainly hesperetin-7-O-rutinoside (hesperidin) and naringenin-7-rutinoside (narirutin), have been reported to have a wide range of effects on human health [1,2]. Epidemiological and clinical studies suggest that a higher intake of citrus flavanones is associated with a 19% decreased risk of stroke [3], and significant improvements in HDL-cholesterol, triacylglycerol, and postprandial microvascular endothelial reactivity, low-density lipoprotein-cholesterol, glucose and insulin sensitivity [4]. Furthermore, flavanone intake has been inversely correlated with postprandial lipid response to a suppressed fatty acid synthesis in the liver, with a consequent reduction in triglyceride production and VLDL secretion in subjects with high cardiovascular risk [5]. A randomised, 12 week, double-blind crossover study has shown that the consumption of OJs containing either normal or a high concentration of flavanones protected against DNA damage and lipid peroxidation, modified the activity of antioxidant enzymes, and reduced body weight in overweight or obese non-smoking adults [6]. Other intervention studies have found that regular consumption of OJ inhibits oxidative stress, inflammatory responses [7] and brings about an improvement in vascular function [8].

The health benefits of OJ consumption are attributed to the absorbable and circulating flavanone metabolites and gut microbial catabolites. Early reports showed that following ingestion of OJ, the sugar moiety of flavanone-O-rutinosides is cleaved in the distal gastrointestinal (GI) tract and the released aglycones are absorbed appearing in the bloodstream as glucuronide and sulfate metabolites. Quantification of metabolites in plasma and urine was based on the amounts of aglycone released by glucuronidase/sulfatase treatment of extracts prior to analysis by HPLC and as a consequence provides only limited information on the identity of the metabolites [[9], [10], [11]].

The advent of HPLC-MS, and the availability of metabolites as reference compounds, facilitated the analysis of samples without recourse to enzyme hydrolysis. Brett et al. [12] were the first to identify flavanone metabolites reporting the presence of the 3′- and 7-glucuronides of hesperetin and the 4′- and 7-glucuronides of naringenin in both plasma and urine after OJ intake. Bredsdorff et al. [13] also identified these four glucuronides in urine, along with hesperetin-3′-sulfate, hesperetin-3′,7-diglucuronide and hesperetin-5,7-diglucuronide, after ingestion of an α-rhamnosidase-treated OJ which contained hesperetin 7-O-glucoside rather than the 7-O-rutinoside.

Traditionally, the bioavailability of OJ flavanones was considered to be low with only relatively small amounts of metabolites entering the systemic circulation [12,14,15]. However, when phenolic catabolites produced by microbiota-mediated ring fission of hesperetin and naringenin were included in estimates of urinary excretion, the overall bioavailability of the flavanones was recognized as being much greater than previously envisaged [1,16].

The volunteers used in the current bioavailability study with OJ were endurance trained men. An earlier publication provided information on urinary excretion of flavanones metabolites and catabolites after OJ consumption and the effect of cessation of training for a period of 7-days [17]. This paper provides comprehensive information on the pharmacokinetics of hesperetin and naringenin metabolites in the blood stream after the consumption of OJ, and also on the appearance of microbiota-derived catabolites including cinnamic, 3-(phenyl)propanoic, phenylacetic and hippuric acids. The information is of value as it will assist the design of future ex vivo cell studies aimed at elucidating the mechanisms underlying the potential health benefits of OJ consumption.