This is an interventional study to investigate formation and pharmacokinetics of reactive carbonyl species adducts of apple polyphenols in human after a single dose of apple consumption.
A crossover design was used. Twelve healthy volunteers were recruited and asked to avoid the consumption of any apple products starting 1 week before intervention and continuing throughout the entire study for a total of 3 weeks. On week 2, each of six participants received 600 g apple blends (seeds removal) in a single dose, and afterwards, urine and blood samples at different timepoints over 24 h, and fecal sample at different timepoints over 48 h were collected. Meanwhile, other six participants received breakfast (without any apple products) only, and urine and blood samples at different timepoints over 24 h, and fecal sample at different timepoints over 48 h were collected. On the contrary, on week 3, six participants who received apple blends on week 2 received breakfast only, and six participants who were used as control on week 2 received apple blends. And urine and blood samples at different timepoints over 24 h, and fecal sample at different timepoints over 48 h for both groups were collected. Samples will be analyzed by LC-MS/MS. Pharmacokinetics of conjugates will be investigated.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
TREATMENT
Masking
DOUBLE
Enrollment
12
Apple blends (600 g) One single dose
Breakfast without any apple-related products
North Carolina Agriculture and Technical State University
Kannapolis, North Carolina, United States
Maximum Urinary Excretion Rates of Major Reactive Carbonyl Species (RCS) Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Urinary excretion rates of major reactive carbonyl species (RCS) conjugates were determined using LC-MS analysis. Urine samples were enzymatically hydrolyzed. Chromatographic separation of RCS-polyphenols (PPs) in human urine was performed on a Gemini column (50 mm × 2.0 mm; i.d. 3 μm) using a Dionex HPLC. Targeted conjugates were identified using an LTQ Velos Pro ion trap mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ion mode. Quantitation for RCS-PPs was carried out by using multiple reaction monitoring (MRM3) transitions (parent → product ion), and data were processed using Xcalibur Quan Browser (Version 4.2.47).
Time frame: Urinary excretion rates (ERs) of ACR-PPs and MGO-PPs at different time intervals (0-2, 2-4, 4-6, 6-9, 9-12, and 12-24 h) after apple consumption were measured. The maximum ERs at 6 h post-dose for ACR-PPs and 9 h post-dose for MGO-PPs were presented.
Change in Plasma Levels of Major RCS Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Levels of RCS adducts of apple polyphenols in plasma will be determined by Liquid chromatography-tandem mass spectrometry analysis.
Time frame: Major RCS conjugates in plasma at different timepoints (0, 0.5, 1, 1.5, 2, 4, 8, and 24 h) after apple consumption were analyzed.
Change in Levels of RCS Adducts of Apple Polyphenols in Feces Over 48 Hours After One Single Dose of Apple Consumption
Liquid chromatography-tandem mass spectrometry will be utilized to assess RCS adducts of apple polyphenols in fecal samples
Time frame: Major RCS adducts of apple polyphenols at different time intervals (0, 0-24, and 24-48 h) after apple consumption were analyzed.
Average Urinary Excretion Rates of Major RCS (e.g. ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
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Average excretion rates of major RCS in urine were determined by Liquid chromatography-tandem mass spectrometry analysis. Urine samples were derivatized with DNPH solution. Chromatographic separation of RCS-DNPH adducts in samples was conducted on an Accucore C8 column. The thermo Q Exactive Plus Orbitrap mass spectrometer equipped with a heated-electrospray ionization (HESI) source was operated in a highly sensitive targeted-single ion monitoring (tSIM) mode, with positive ion polarity from 0 to 4.6 min and negative ion polarity from 4.6 to 13 min, for quantitative analysis.
Time frame: Urinary excretion rates (ERs) of major RCS (e.g. ACR and MGO) at different time intervals (0-2, 2-4, 4-6, 6-9, 9-12, and 12-24 h) post-dose were measured. Average urinary ERs of ACR and MGO within 24 h were presented.
Average Plasma Levels of Major RCS (ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
Plasma levels of major RCS were determined by Liquid chromatography-tandem mass spectrometry analysis. Plasma samples were derivatized with DNPH solution. Chromatographic separation of RCS-DNPH adducts in samples was conducted on an Accucore C8 column. The thermo Q Exactive Plus Orbitrap mass spectrometer equipped with a heated-electrospray ionization (HESI) source was operated in a highly sensitive targeted-single ion monitoring (tSIM) mode, with positive ion polarity from 0 to 4.6 min and negative ion polarity from 4.6 to 13 min, for quantitative analysis.
Time frame: Plasma levels of major RCS (e.g. ACR and MGO) at different time points (0, 0.5, 1.0, 1.5, 2.0, 4.0, 8.0, and 24 h) post-dose were measured. Average plasma levels of ACR and MGO within 24 h were presented.