Human O-sulfated metabolites of (−)-epicatechin and methyl-(−)-epicatechin are poor substrates for commercial aryl-sulfatases: Implications for studies concerned with quantifying epicatechin bioavailability

Abstract

Epicatechin is a widely consumed dietary flavonoid and there is substantial evidence that it contributes to the health benefits reported for flavanol-rich cocoa products including dark chocolate. Numerous reports have described the appearance of epicatechin and epicatechin phase-2 conjugates (sulfates and glucuronides of epicatechin and methylepicatechin) in blood and urine samples of subjects following ingestion of epicatechin. The most widely reported method of quantifying total epicatechin in plasma and urine samples involves hydrolysis with a mixture of β-glucuronidase and sulfatase to convert the conjugates to epicatechin aglycone which is subsequently quantified. We observed a lack of hydrolysis of epicatechin sulfates and methylepicatechin sulfates using commercial sulfatases and investigated this further. Samples of urine or plasma from subjects who had consumed epicatechin were subjected to enzyme hydrolysis and then analysed using LC–MS/MS, or analysed without enzyme hydrolysis. Attempts to increase the extent of hydrolysis of epicatechin conjugates were made by increasing the amount of enzyme, hydrolysis pH and length of incubations, and using alternative sources of enzyme. The standard hydrolysis conditions failed to hydrolyse the majority of epicatechin sulfates and methylepicatechin sulfates. Even when the quantity of enzyme and incubation period was increased, the pH optimised, or alternative sources of sulfatases were used, epicatechin monosulfates and methylepicatechin monosulfates remained as major peaks in the chromatograms of the samples. An assessment of literature data strongly suggested that the majority of reports where enzyme hydrolysis was used had significantly underestimated epicatechin bioavailability in humans. Methods for quantifying epicatechin concentrations in blood and urine need to take account of the lack of hydrolysis of (methyl)epicatechin-sulfates, for example by quantifying these directly using LC–MS/MS.

Introduction

Flavonoids are highly bioactive polyphenols that are synthesised by plants and which are widely consumed as part of human diets. They are also the bioactive constituents of a huge range of dietary supplements, including herbal medicines, and have been used in the treatment of various conditions and as health-promoting remedies for centuries. There is considerable epidemiological evidence showing that consumption of certain flavonoids is inversely related to disease risk for cardiovascular diseases, certain cancers and Alzheimer's, among others [1], [2]. In addition, there are now several hundred randomised controlled trials that have investigated the effects of flavonoids or flavonoid-rich foods/beverages on disease risk, particularly for cardiovascular diseases. Meta-analyses of the data from studies concerned with cardiovascular disease have demonstrated that consumption of flavan-3-ols from cocoa and dark chocolate has beneficial effects on flow mediated dilatation (FMD; marker of endothelial function), blood pressure, plasma low-density lipoproteins and insulin resistance [3], [4]. Indeed, these were the outstanding findings of these systematic reviews of all the relevant reported data for flavonoids, and it is arguable that the most robust current evidence of cardiovascular health benefit for any of the flavonoids is for flavan-3-ols and in particular the monomer (−)-epicatechin. Although cocoa and dark chocolate contain a mixture of both monomeric epicatechin and oligomers and polymers of epicatechin, called procyanidins, it has been established that epicatechin alone can induce changes in nitric-oxide (NO) metabolites in blood and NO-dependent FMD [5], [6], and it has been proposed that epicatechin is responsible for the beneficial effects of cocoa [5].

Numerous reports have described the appearance of epicatechin and epicatechin metabolites in blood and urine samples following ingestion of an epicatechin containing meal such as cocoa, chocolate or tea [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17]. In plasma and urine, epicatechin is found as glucuronide and sulfate conjugates of epicatechin and methylepicatechins. Quantitative analysis of these conjugates typically involves treating the biological samples with β-glucuronidase and/or sulfatase in order to hydrolyse the various glucuronidated and sulfated conjugates to obtain a mixture of epicatechin and methylepicatechins that are subsequently quantified against an authentic epicatechin standard [9], [10], [11], [12], [13], [15], [16]. When we reviewed reports of the analysis of epicatechin in blood/urine samples that used enzyme hydrolysis, two main approaches to the enzymatic treatment of biological samples were identified. The first involves treatment of samples with a β-glucuronidase enzyme that also contains some naturally occurring aryl sulfatase, which in turn acts as a secondary activity for the hydrolysis of sulfates. The majority of such reports refer to the method description of Richelle et al. [11]. The other approach is to treat biological samples with a combination of separately prepared β-glucuronidase and sulfatase enzymes. Complete hydrolysis of epicatechin sulfates and glucuronides into the respective aglycone forms is dependent upon a number of factors; the nature of the biological sample, the concentration and sources of enzymes used, and the pH, temperature and length of incubations. Although there is some variation between the methods used, enzyme hydrolyses are typically carried out at pH 5.0 and over about 45 min.

For the purposes of quantifying total epicatechin in samples of urine from a human intervention study in which volunteers consumed chocolate bars containing epicatechin, we treated samples with a mixture of aryl-sulfatase and β-d-glucuronidase with the intention of quantifying the resulting peaks of epicatechin and 3′-methyl/4′-methylepicatechin using LC–MS to monitor parent and fragment ions. However, we noticed that substantial peaks corresponding to epicatechin sulfates and methylepicatechin sulfates were present in sulfatase/glucuronidase-treated samples, and investigated this further.