Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)-epicatechin are poor substrates for commercial aryl-sulfatases

Implications for studies concerned with quantifying epicatechin bioavailability

S. Saha, W. Hollands, P. W. Needs, L. M. Ostertag, B. de Roos, G. G. Duthie, P. A. Kroon

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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 ß-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 has 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 methyl epicatechin 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 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.
Original languageEnglish
Pages (from-to)592-602
Number of pages11
JournalPharmacological Research
Volume65
Issue number6
Early online date25 Feb 2012
DOIs
Publication statusPublished - Jun 2012

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Sulfatases
Catechin
Biological Availability
Hydrolysis
Enzymes
Urine
Glucuronidase
Insurance Benefits
Flavonoids
Sulfates
Eating

Keywords

  • flavonoids
  • polyphenols
  • phenolics
  • dietary bioactives
  • absorption
  • human metabolism
  • phase-2 enzymes

Cite this

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title = "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 {\ss}-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 has 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 methyl epicatechin 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 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.",
keywords = "flavonoids, polyphenols, phenolics, dietary bioactives, absorption, human metabolism, phase-2 enzymes",
author = "S. Saha and W. Hollands and Needs, {P. W.} and Ostertag, {L. M.} and {de Roos}, B. and Duthie, {G. G.} and Kroon, {P. A.}",
note = "Copyright {\^A}{\circledC} 2012. Published by Elsevier Ltd.",
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TY - JOUR

T1 - Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)-epicatechin are poor substrates for commercial aryl-sulfatases

T2 - Implications for studies concerned with quantifying epicatechin bioavailability

AU - Saha, S.

AU - Hollands, W.

AU - Needs, P. W.

AU - Ostertag, L. M.

AU - de Roos, B.

AU - Duthie, G. G.

AU - Kroon, P. A.

N1 - Copyright © 2012. Published by Elsevier Ltd.

PY - 2012/6

Y1 - 2012/6

N2 - 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 ß-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 has 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 methyl epicatechin 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 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.

AB - 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 ß-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 has 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 methyl epicatechin 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 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.

KW - flavonoids

KW - polyphenols

KW - phenolics

KW - dietary bioactives

KW - absorption

KW - human metabolism

KW - phase-2 enzymes

U2 - 10.1016/j.phrs.2012.02.005

DO - 10.1016/j.phrs.2012.02.005

M3 - Article

VL - 65

SP - 592

EP - 602

JO - Pharmacological Research

JF - Pharmacological Research

SN - 1043-6618

IS - 6

ER -