Phenolic metabolites of anthocyanins modulate mechanisms of endothelial function

Anthocyanins are reported to have vascular bioactivity, however their mechanisms of action are largely unknown. Evidence suggests that anthocyanins modulate endothelial function, potentially by increasing nitric oxide (NO) synthesis, or enhancing NO bioavailability. This study compared the activity of cyanidin-3-glucoside, its degradation product protocatechuic acid, and phase II metabolite, vanillic acid. Production of NO and superoxide and expression of endothelial NO synthase (eNOS), NADPH oxidase (NOX), and heme oxygenase-1 (HO-1) were established in human vascular cell models. Nitric oxide levels were not modulated by the treatments, although eNOS was upregulated by cyanidin-3-glucoside, and superoxide production was decreased by both phenolic acids. Vanillic acid upregulated p22phox mRNA but did not alter NOX protein expression, although trends were observed for p47phox downregulation and HO-1 upregulation. Anthocyanin metabolites may therefore modulate vascular reactivity by inducing HO-1 and modulating NOX activity, resulting in reduced superoxide production and improved NO bioavailability

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a 13C-tracer study

BACKGROUND: Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown. OBJECTIVE: We investigated the ADME of a (13)C5-labeled anthocyanin in humans. DESIGN: Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-(13)C5-C3G). Biological samples were collected over 48 h, and (13)C and (13)C-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry. RESULTS: The mean +/- SE percentage of (13)C recovered in urine, breath, and feces was 43.9 +/- 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 +/- 1.38% (5.37 +/- 0.67% excreted in urine and 6.91 +/- 1.59% in breath). Maximum rates of (13)C elimination were achieved 30 min after ingestion (32.53 +/- 14.24 mug(13)C/h), whereas (13)C-labeled metabolites peaked (maximum serum concentration: 5.97 +/- 2.14 mumol/L) at 10.25 +/- 4.14 h. The half-life for (13)C-labeled metabolites ranged between 12.44 +/- 4.22 and 51.62 +/- 22.55 h. (13)C elimination was greatest between 0 and 1 h for urine (90.30 +/- 15.28 mug/h), at 6 h for breath (132.87 +/- 32.23 mug/h), and between 6 and 24 h for feces (557.28 +/- 247.88 mug/h), whereas the highest concentrations of (13)C-labeled metabolites were identified in urine (10.77 +/- 4.52 mumol/L) and fecal samples (43.16 +/- 18.00 mumol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids. CONCLUSION: Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation fo

Phenolic Metabolites of Anthocyanins Modulate Mechanisms of Endothelial Function

Anthocyanins are reported to have vascular bioactivity, however their mechanisms of action are largely unknown. Evidence suggests that anthocyanins modulate endothelial function, potentially by increasing nitric oxide (NO) synthesis, or enhancing NO bioavailability. This study compared the activity of cyanidin-3-glucoside, its degradation product protocatechuic acid, and phase II metabolite, vanillic acid. Production of NO and superoxide and expression of endothelial NO synthase (eNOS), NADPH oxidase (NOX), and heme oxygenase-1 (HO-1) were established in human vascular cell models. Nitric oxide levels were not modulated by the treatments, although eNOS was upregulated by cyanidin-3-glucoside, and superoxide production was decreased by both phenolic acids. Vanillic acid upregulated p22^phox mRNA but did not alter NOX protein expression, although trends were observed for p47^phox downregulation and HO-1 upregulation. Anthocyanin metabolites may therefore modulate vascular reactivity by inducing HO-1 and modulating NOX activity, resulting in reduced superoxide production and improved NO bioavailability

Human metabolism and elimination of the anthocyanin, cyanidin-3-glucoside: a C-13-tracer study

Background: Evidence suggests that the consumption of anthocyanin-rich foods beneficially affects cardiovascular health; however, the absorption, distribution, metabolism, and elimination (ADME) of anthocyanin-rich foods are relatively unknown. Objective: We investigated the ADME of a C-13(5)-labeled anthocyanin in humans. Design: Eight male participants consumed 500 mg isotopically labeled cyanidin-3-glucoside (6,8,10,3',5'-C-13(5)-C3G). Biological samples were collected over 48 h, and C-13 and C-13-labeled metabolite concentrations were measured by using isotope-ratio mass spectrometry and liquid chromatography-tandem mass spectrometry. Results: The mean +/- SE percentage of C-13 recovered in urine, breath, and feces was 43.9 +/- 25.9% (range: 15.1-99.3% across participants). The relative bioavailability was 12.38 +/- 1.38% (5.37 +/- 0.67% excreted in urine and 6.91 +/- 1.59% in breath). Maximum rates of C-13 elimination were achieved 30 min after ingestion (32.53 +/- 14.24 mu g C-13/h), whereas C-13-labeled metabolites peaked (maximum serum concentration: 5.97 +/- 2.14 mu mol/L) at 10.25 +/- 4.14 h. The half-life for C-13-labeled metabolites ranged between 12.44 +/- 4.22 and 51.62 +/- 22.55 h. C-13 elimination was greatest between 0 and 1 h for urine (90.30 +/- 15.28 mu g/h), at 6 h for breath (132.87 +/- 32.23 mu g/h), and between 6 and 24 h for feces (557.28 +/- 247.88 mu g/h), whereas the highest concentrations of C-13-labeled metabolites were identified in urine (10.77 +/- 4.52 mu mol/L) and fecal samples (43.16 +/- 18.00 mu mol/L) collected between 6 and 24 h. Metabolites were identified as degradation products, phenolic, hippuric, phenylacetic, and phenylpropenoic acids. Conclusion: Anthocyanins are more bioavailable than previously perceived, and their metabolites are present in the circulation for &lt;= 48 h after ingestion. This trial was registered at clinicaltrials.gov as NCT01106729. Am J Clin Nutr 2013;97:995-1003.</p

Methods for Isolating, Identifying, and Quantifying Anthocyanin Metabolites in Clinical Samples

The metabolic fate of anthocyanins until recently was relatively unknown, primarily as a result of their instability at physiological pH and a lack of published methods for isolating and identifying their metabolites from biological samples. The aim of the present work was to establish methods for the extraction and quantification of anthocyanin metabolites present in urine, serum, and fecal samples. 35 commercial and 10 synthetic analytes, including both known and predicted human and microbial metabolites of anthocyanins, were obtained as reference standards. HPLC and MS/MS conditions were optimized for organic modifier, ionic modifier, mobile phase gradient, flow rate, column type, MS source, and compound dependent parameters. The impact of sorbent, solvent, acid, preservative, elution, and evaporation on solid phase extraction (SPE) efficiency was also explored. The HPLC-MS/MS method validation demonstrated acceptable linearity (R(2), 0.997 +/- 0.002) and sensitivity (limits of detection (LODs): urine, 100 +/- 375 nM; serum, 104 +/- 358 nM; feces 138 +/- 344 nM), and the final SPE methods provided recoveries of 88.3 +/- 17.8% for urine, 86.5 +/- 11.1% for serum, and 80.6 +/- 20.9% for feces. The final methods were applied to clinical samples derived from an anthocyanin intervention study, where 36 of the 45 modeled metabolites were detected within urine, plasma, or fecal samples. The described methods provide suitable versatility for the identification and quantification of an extensive series of anthocyanin metabolites for use in future clinical studies exploring absorption, distribution, metabolism, and elimination.</p