Authors: Robbins, Rebecca J., Leonczak, Jadwiga, Li, Julia, Johnson, J. Christopher, Collins, Tom, Kwik-Uribe, Catherine, Schmitz, Harold H.
Journal of AOAC International
Published: Aug 2012 95
A single-laboratory validation study was performed for an HPLC method to identify and quantify the flavanol enantiomers (+)- and (-)-epicatechin and (+)- and (-)-catechin in cocoa-based ingredients and products. These compounds were eluted isocratically with an ammonium acetate-methanol mobile phase applied to a modified beta-cyclodextrin chiral stationary phase and detected using fluorescence. Spike recovery experiments using appropriate matrix blanks, along with cocoa extract, cocoa powder, and dark chocolate, were used to evaluate accuracy, repeatability, specificity, LOD, LOQ, and linearity of the method as performed by a single analyst on multiple days. In all samples analyzed, (-)-epicatechin was the predominant flavanol and represented 68-91% of the total monomeric flavanols detected. For the cocoa-based products, within-day (intraday) precision for (-)-epicatechin was between 1.46-3.22%, for (+)-catechin between 3.66-6.90%, and for (-)-catechin between 1.69-6.89%; (+)-epicatechin was not detected in these samples. Recoveries for the three sample types investigated ranged from 82.2 to 102.1% at the 50% spiking level, 83.7 to 102.0% at the 100% spiking level, and 80.4 to 101.1% at the 200% spiking level. Based on performance results, this method may be suitable for routine laboratory use in analysis of cocoa-based ingredients and products.
Accumulating data show a causal role for flavanols in the mediation of cardiovascular benefits associated with the consumption of flavanol- and procyanidin-containing foods. Evidence for a direct causal role for procyanidins in this context is far less profound due to the poor absorption of procyanidins. However, it has been proposed that procyanidins may break down in the gastrointestinal tract, resulting in monomeric flavanols, which contribute to the systemic flavanol pool. Verification or rejection of this supposition could significantly affect the interpretation of epidemiologic and dietary intervention data and the design of food-content databases.
We assessed the respective contribution of flavanols and procyanidins to the systemic pool of flavanols and 5-(3,4-dihydroxyphenyl)-γ-valerolactone (γ-VL) in humans.
Test drinks that contained only flavanols (D1), procyanidins with a degree of polymerization that ranged from 2 to 10 (D2-10), or flavanols and procyanidins with a degree of polymerization that ranged from 2 to 10 (D1-10) were consumed by subjects (n = 12) according to a randomized, double-masked, crossover design. Plasma and urine samples were collected postprandially and analyzed.
The ingestion of D1-10 resulted in the systemic presence of flavanols (plasma concentration: 863 ± 77 nmol/L), γ-VLs (24-h urine: 93 ± 18 μmol), and minute concentrations of procyanidin B2. With correction for small residual amounts of flavanols present in D2-10, only negligible concentrations of circulating flavanols were detected after ingestion of the drink, whereas the intake of D1 resulted in circulating flavanol concentrations similar to those detected after D1-10 consumption.
These outcomes show that dietary procyanidins do not contribute to the systemic pool of flavanols in humans. Thus, these data reject the notion that procyanidins, through their breakdown into flavanols and subsequent absorption, causally modulate vascular function.
Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. Although recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn are largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In this study, we developed a method for the analysis of structurally related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally related (-)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (-)-epicatechin-3'-β-D-glucuronide, (-)-epicatechin-3'-sulfate, and a 3'-O-methyl-(-)-epicatechin-5/7-sulfate are the predominant SREM in humans and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting, to a significant extent, of interlaboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic.
Extensive epidemiological and clinical evidence associates diets high in flavanol-containing foods with cardiovascular health benefits in humans. Catechin and epicatechin, the most common flavanols in foods, are present in the diet in different enantiomeric forms. This study investigated the influence of the stereochemical configuration of flavanols on their absorption, metabolism, and biological activity. Healthy adult males were asked to consume equal amounts of the stereochemically pure flavanols (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin (1.5mg/kg bw) in a well-defined cocoa-based, dairy-containing drink matrix, and flavanol levels were subsequently determined in plasma and 24-h urine. The results obtained show that the stereochemical configuration of flavanols has a profound influence on their uptake and metabolism in humans. In addition, we assessed the vasodilatory activity of each flavanol stereoisomer in vivo and found (-)-epicatechin to be the single stereoisomer capable of mediating a significant arterial dilation response. Importantly, this effect was independent of the classic antioxidant properties of flavanols. Overall, these results indicate that the proposed beneficial health effects associated with the consumption of flavanol-containing foods will significantly depend on the stereochemical configuration of the flavanols ingested.
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