Effect of Iron and Zinc Supplementation on B-carotene Bioavailability in Healthy Males
- Conditions
- Bioavailability
- Interventions
- Dietary Supplement: Control beta-carotene supplement plus PlaceboDietary Supplement: Control beta-carotene supplement plus iron SupplementDietary Supplement: Control beta-carotene supplement plus zinc Supplement
- Registration Number
- NCT05840848
- Lead Sponsor
- University of Hohenheim
- Brief 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.
- Detailed 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).
Recruitment & Eligibility
- Status
- COMPLETED
- Sex
- Male
- Target Recruitment
- 12
- Male
- aged 18 and 50 years
- overweight (BMI >25 kg/m2),
- metabolic and endocrine diseases,
- drug abuse,
- use of dietary supplements,
- us of any form of medication,
- smoking,
- frequent alcohol consumption (>20 g ethanol/day),
- adherence to a restrictive dietary regimen,
- physical activity of more than 5 h/wk,
- participation in a clinical trial within the past 3 months prior to recruitment,
- a known intolerance against β-carotene, iron and/or zinc supplements.
Study & Design
- Study Type
- INTERVENTIONAL
- Study Design
- CROSSOVER
- Arm && Interventions
Group Intervention Description Control plus Placebo Control beta-carotene supplement plus Placebo B-carotene supplement (15 mg) consumed in the morning after a \>10 hour overnight fast. Control plus iron Supplement Control beta-carotene supplement plus iron Supplement B-carotene supplement (15 mg) and iron sulphate supplement (25 mg) consumed in the morning after a \>10 hour overnight fast. Control plus zinc Supplement Control beta-carotene supplement plus zinc Supplement B-carotene supplement (15 mg) and zinc sulphate supplement (30 mg) consumed in the morning after a \>10 hour overnight fast.
- Primary Outcome Measures
Name Time Method TRF B-carotene 10 hours B-carotene concentration in the triacylglycerol-rich fraction of the plasma
Plasma B-carotene 10 hours B-carotene concentration int he plasma
- Secondary Outcome Measures
Name Time Method Plasma TAG 10 hours triacylglycerol concentration in Plasma
Kidney funtion markers 4 hours Creatinine i.S. and Uric acid measured
Plasma LDL-cholesterol 10 hours LDL-cholesterol concentration in Plasma
Plasma HDL-cholesterol 10 hours HDL-cholesterol concentration in Plasma
liver function markers 4 hours γ-GT, AST, ALT, Alkaline phosphatase and Bilirubin measured
Trial Locations
- Locations (1)
University of Hohenheim
🇩🇪Stuttgart, Baden-Württemberg, Germany