Clinical Trial Details
— Status: Completed
Administrative data
NCT number |
NCT05840848 |
Other study ID # |
AZ: F-2017-039 |
Secondary ID |
|
Status |
Completed |
Phase |
N/A
|
First received |
|
Last updated |
|
Start date |
November 21, 2017 |
Est. completion date |
June 1, 2018 |
Study information
Verified date |
May 2023 |
Source |
University of Hohenheim |
Contact |
n/a |
Is FDA regulated |
No |
Health authority |
|
Study type |
Interventional
|
Clinical Trial Summary
In vitro studies found supplemental levels of iron and zinc to inhibit the micellization and
cellular uptake of β-carotene. Here, we investigated this in vivo, in a double-blind 3-arm
crossover human trial.
Healthy males (n=6) ingested, with breakfast, a single dose of 15 mg β-carotene in
combination with either a placebo, 25 mg iron or 30 mg zinc capsule. Blood samples were
collected at baseline and hourly for 10 hours. The triacylglycerol-rich fraction (TRF) was
analysed for concentrations of β-carotene and plasma for β-carotene, retinol,
triacylglycerols, LDL- and HDL-cholesterol.
Description:
The study followed a double-blind crossover design with three study arms separated by one
week washout periods. In short, the participants were asked to follow a diet low in
carotenoids by avoiding all orange, yellow, red and green fruits and vegetables for four
days. This was followed by three days of a strictly carotenoid-free diet, which only allowed
foods from a specified list (Supplemental Table A2). On each study day, β-carotene was
administered in the morning after a >10 hour overnight fast (Supplemental Figure A1). All
participants orally ingested, in random order, a single dose of 15 mg β-carotene (BIOVEA)
with either a placebo (empty capsule), 25 mg iron (FeSO4; Woerwag Pharma GmbH & Co. KG,
Boeblingen, Germany) or 30 mg zinc (ZnSO4; Woerwag Pharma) capsule. A standardised dinner was
provided on the evening before the trial and standardised meals were provided during the
entire intervention day (Supplemental Table A3). On the first study day, the amount of food
(weight or volume) consumed by each participant for each meal was recorded and the same
amounts provided during the following two arms to ensure similar food consumption, especially
of fat. Water was available unrestricted for consumption throughout the day. Blood samples
were drawn from an indwelling venous cannula and collected at 0 hours directly before
β-carotene supplementation and then every hour for 10 hours.
For the determination of plasma concentrations of β-carotene, LDL- and HDL-cholesterol, and
triacylglycerols (TAG), blood was collected in tubes containing EDTA (Sarstedt AG & Co,
Nuebrecht, Germany) and immediately centrifuged (3000 × g, 10 min, 4 °C). From the obtained
plasma samples, three aliquots were stored at -80 °C until further analysis and the rest
ultracentrifuged to obtain the triacylglycerol-rich fraction (TRF). For the analyses of liver
and kidney function markers, plasma and serum were obtained from blood sampled at the 0- and
4-hour time points.
The TRF was prepared according to [10]. Briefly, plasma (3.5 mL) was transferred to an
ultracentrifuge tube and carefully overlaid with 8 mL 1.3% sodium chloride and then
ultracentrifuged (Beckman Coulter, OptimaTM L-80 XP Ultracentrifuge) using a swinging bucket
rotor (SW41Ti) at 150 000 x g for 1 hour at 4 °C. Afterwards, the TRF was isolated by
transferring the upper ~6 mL, which was then overlaid with nitrogen gas to minimize oxidation
and stored at -80 °C until extraction.
The plasma samples were randomly extracted and analysed by HPLC according to [15]. Briefly,
40 µL plasma was extracted with an ethanol/n-butanol mixture (50:50) containing
apo-80-carotenal-methyloxime (12µL/100 mL; Fluka Analytical (Merck Group KGaA), Darmstadt,
Germany) as internal standard. After centrifugation, the clear supernatant was analysed by
HPLC.
The TRF was extracted and analysed by HPLC [15]. For the extraction, 100 µl
apo-80-carotenal-methyloxime (12 µL/100 mL) and 2 mL ethanol (for deproteination) were added
to 3 mL of the TRF and vortexed for 30 sec. The solution was extracted twice with 2 mL
hexane. The hexane layers were removed, combined and evaporated in a centrifugal vacuum
concentrator (Christ, RVC 2-25 CD plus) and the dried sample re-dissolved in 100 µL
acetonitrile and immediately analysed by HPLC.
Both the plasma and TRF samples were analysed using a Shimadzu HPLC (LC-10AD) equipped with a
UV-Vis detector (SPD 20A, set at 450 nm). Carotenoids were separated using a ReproSil 80
ODS-2 column (3 µm, 250 x 4.6 mm; Dr. Maisch GmbH, Ammerbuch, Germany) and an eluent in
recirculation mode (82% acetonitrile, 15% 1,4-dioxin, and 3% methanol (vol/vol) containing
100 mM ammonium acetate and 10 mM triethylamine) at a flow rate of 1.5 mL/min [15]. A
β-carotene standard (≥97.0% purity, Sigma-Aldrich) was used to construct a standard curve.
Plasma TAG, HDL- and LDL-cholesterol were analysed by a clinical laboratory (Laborärzte
Sindelfingen, Sindelfingen, Germany).