Trial Outcomes & Findings for Scavenging of Reactive Carbonyl Species by Apple Polyphenols in Human (NCT NCT03911050)
NCT ID: NCT03911050
Last Updated: 2026-07-02
Results Overview
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).
COMPLETED
NA
12 participants
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.
2026-07-02
Participant Flow
Volunteers were recruited through advertisements around the North Carolina Research Campus in Kannapolis, including circular e-mails and flyers. Interested volunteers aged 18-70 y with a BMI (kg/m2) ranging from 17.0 to 30.0 provided written informed consent prior to enrollment. All participants were asked to avoid the consumption of any apple products, medications, and dietary supplements throughout the entire study for a total of 3 weeks.
A one-week wash-in period was implemented.
Participant milestones
| Measure |
Apple Consumption First, Then Control
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and samples (urine and blood and feces) at different timepoints were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control First, Then Apple Consumption
12 subjects took breakfast without any apple-related products in a single dose, and samples (urine and blood and feces) at different timepoints were collected following breakfast.
|
|---|---|---|
|
Wash-in period (1 week)
STARTED
|
6
|
6
|
|
Wash-in period (1 week)
COMPLETED
|
6
|
6
|
|
Wash-in period (1 week)
NOT COMPLETED
|
0
|
0
|
|
First intervention (1 day)
STARTED
|
6
|
6
|
|
First intervention (1 day)
COMPLETED
|
6
|
6
|
|
First intervention (1 day)
NOT COMPLETED
|
0
|
0
|
|
Wash-out period (1 week)
STARTED
|
6
|
6
|
|
Wash-out period (1 week)
COMPLETED
|
6
|
6
|
|
Wash-out period (1 week)
NOT COMPLETED
|
0
|
0
|
|
Second intervention (1 day)
STARTED
|
6
|
6
|
|
Second intervention (1 day)
COMPLETED
|
5
|
6
|
|
Second intervention (1 day)
NOT COMPLETED
|
1
|
0
|
Reasons for withdrawal
| Measure |
Apple Consumption First, Then Control
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and samples (urine and blood and feces) at different timepoints were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control First, Then Apple Consumption
12 subjects took breakfast without any apple-related products in a single dose, and samples (urine and blood and feces) at different timepoints were collected following breakfast.
|
|---|---|---|
|
Second intervention (1 day)
Withdrawal by Subject
|
1
|
0
|
Baseline Characteristics
We did not collect this information.
Baseline characteristics by cohort
| Measure |
All Study Participants
n=12 Participants
12 participants were randomly assigned to took apple blends 600 g (made from two apples with seed removal) or breakfast without any apple-related products in a single dose, and samples (urine and blood and feces) at different timepoints were collected following administration. After wash-out, same 12 participants crossed over to the alternative intervention and followed the same protocol. Samples (urine and blood and feces) at different timepoints were collected.
|
|---|---|
|
Sex: Female, Male
Female
|
5 Participants
n=12 Participants
|
|
Sex: Female, Male
Male
|
7 Participants
n=12 Participants
|
|
Body Mass Index (BMI)
|
22.65 kg/m^2
STANDARD_DEVIATION 3.83 • n=12 Participants
|
PRIMARY outcome
Timeframe: 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.Population: 12 eligible subjects were randomized to the interventions. However, only 11 out 12 participants completed the entire study. Therefore, 11 participants were analyzed in this study.
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).
Outcome measures
| Measure |
Apple Consumption
n=11 Participants
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control
n=11 Participants
12 subjects took breakfast without any apple-related products in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following breakfast.
|
|---|---|---|
|
Maximum Urinary Excretion Rates of Major Reactive Carbonyl Species (RCS) Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Maximum rate of acrolein-polyphenols
|
12.42 nmol/h
Standard Error 4.02
|
0.083 nmol/h
Standard Error 0.033
|
|
Maximum Urinary Excretion Rates of Major Reactive Carbonyl Species (RCS) Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Maximum rate of methylglyoxal-polyphenols
|
1.17 nmol/h
Standard Error 1.06
|
0.019 nmol/h
Standard Error 0.004
|
PRIMARY outcome
Timeframe: 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.Population: Major reactive carbonyl species adducts of apple polyphenols (RCS-PPs), including ACR-PPs and MGO-PPs, in plasma following apple consumption were analyzed. However, only trace levels of ACR-PPs were detected through careful comparison with authentic standards, and these levels were far below the limit of quantification (LOQ) of the current method. Consequently, changes in plasma RCS-PPs after apple consumption could not be reliably assessed.
Levels of RCS adducts of apple polyphenols in plasma will be determined by Liquid chromatography-tandem mass spectrometry analysis.
Outcome measures
| Measure |
Apple Consumption
n=11 Participants
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control
n=11 Participants
12 subjects took breakfast without any apple-related products in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following breakfast.
|
|---|---|---|
|
Change in Plasma Levels of Major RCS Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Levels of methylglyoxal-polyphenols
|
NA nM
Standard Error NA
MGO-PPs in plasma after apple consumption were below the level of detection.
|
NA nM
Standard Error NA
MGO-PPs in plasma from control were below the level of detection.
|
|
Change in Plasma Levels of Major RCS Adducts of Apple Polyphenols Over 24 Hours After One Single Dose of Apple Consumption
Levels of acrolein-polyphenols
|
NA nM
Standard Error NA
Only trace levels of ACR-PPs were detected through careful comparison with authentic standards using an LTQ Velos Pro ion trap mass spectrometer. However, these levels were far below the limit of quantification (LOQ) of the current method. Therefore, changes in plasma ACR-PPs after apple consumption could not be reliably assessed.
|
NA nM
Standard Error NA
ACR-PPs in plasma from control were below the level of detection.
|
PRIMARY outcome
Timeframe: Major RCS adducts of apple polyphenols at different time intervals (0, 0-24, and 24-48 h) after apple consumption were analyzed.Population: Major reactive carbonyl species adducts of apple polyphenols (RCS-PPs), including ACR-PPs and MGO-PPs, in feces following apple consumption were analyzed using an LTQ Velos Pro ion trap mass spectrometer. However, none of RCS-PPs could be detected. Consequently, changes in feces RCS-PPs after apple consumption could not be reported.
Liquid chromatography-tandem mass spectrometry will be utilized to assess RCS adducts of apple polyphenols in fecal samples
Outcome measures
| Measure |
Apple Consumption
n=11 Participants
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control
n=11 Participants
12 subjects took breakfast without any apple-related products in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following breakfast.
|
|---|---|---|
|
Change in Levels of RCS Adducts of Apple Polyphenols in Feces Over 48 Hours After One Single Dose of Apple Consumption
Levels of methylglyoxal-polyphenols
|
NA nmol/g
Standard Error NA
MGO-PPs in feces after apple consumption were below the level of detection.
|
NA nmol/g
Standard Error NA
MGO-PPs in feces from control were below the level of detection.
|
|
Change in Levels of RCS Adducts of Apple Polyphenols in Feces Over 48 Hours After One Single Dose of Apple Consumption
Levels of acrolein-polyphenols
|
NA nmol/g
Standard Error NA
ACR-PPs in feces after apple consumption were below the level of detection.
|
NA nmol/g
Standard Error NA
ACR-PPs in feces from control were below the level of detection.
|
SECONDARY outcome
Timeframe: 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.Population: 12 eligible subjects were randomized to the interventions. However, only 11 out 12 participants completed the entire study. Therefore, 11 participants were analyzed in this study.
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.
Outcome measures
| Measure |
Apple Consumption
n=11 Participants
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control
n=11 Participants
12 subjects took breakfast without any apple-related products in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following breakfast.
|
|---|---|---|
|
Average Urinary Excretion Rates of Major RCS (e.g. ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
Average excretion rate of acrolein
|
2.51 nmol/h
Standard Error 1.19
|
1.89 nmol/h
Standard Error 0.69
|
|
Average Urinary Excretion Rates of Major RCS (e.g. ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
Average excretion rate of methylglyoxal
|
64.17 nmol/h
Standard Error 18.76
|
71.91 nmol/h
Standard Error 22.32
|
SECONDARY outcome
Timeframe: 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.Population: 12 eligible subjects were randomized to the interventions. However, only 11 out 12 participants completed the entire study. Therefore, 11 participants were analyzed in this study.
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.
Outcome measures
| Measure |
Apple Consumption
n=11 Participants
12 subjects took apple blends 600 g (made from two apples with seed removal) in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following administration of apple blends. 11 out of 12 subjects completed the entire study.
|
Control
n=11 Participants
12 subjects took breakfast without any apple-related products in a single dose, and urine samples at different timepoints (0, 0-2, 2-4, 4-6, 6-9, and 9-24 h) were collected following breakfast.
|
|---|---|---|
|
Average Plasma Levels of Major RCS (ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
Average level of MGO
|
256.88 nM
Standard Error 16.71
|
271.23 nM
Standard Error 23.35
|
|
Average Plasma Levels of Major RCS (ACR and MGO) Over 24 Hours After One Single Dose of Apple Consumption
Average level of ACR
|
58.88 nM
Standard Error 7.39
|
55.81 nM
Standard Error 4.16
|
Adverse Events
Apple Consumption
Control
Serious adverse events
Adverse event data not reported
Other adverse events
Adverse event data not reported
Additional Information
Results disclosure agreements
- Principal investigator is a sponsor employee
- Publication restrictions are in place