Effect of Iron and Zinc Supplementation on B-carotene Bioavailability in Healthy Males
Condition: Bioavailability
Study type: Interventional

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).
    

Intervention name(s): Control beta-carotene supplement plus Placebo; Control beta-carotene supplement plus iron Supplement; Control beta-carotene supplement plus zinc Supplement
Intervention type(s): Dietary Supplement; Dietary Supplement; Dietary Supplement
Intervention description(s): 15 mg beta-carotene supplement from BIOVEA consumed before breakfast together with an empty capsule.; 15 mg Beta-carotene supplement from BIOVEA consumed before breakfast together with 25 mg iron (FeSO4; Woerwag Pharma GmbH & Co. KG, Boeblingen, Germany); 15 mg Beta-carotene supplement from BIOVEA consumed before breakfast together with 30 mg zinc (ZnSO4; Woerwag Pharma GmbH & Co. KG, Boeblingen, Germany)
Intervention MeSH terms: Beta Carotene; Carotenoids
Allocation: Non-Randomized
Intervention model: Crossover Assignment
Intervention model description: double-blind crossover
Masking: Double (Participant, Investigator)
Primary purpose: Basic Science
Keywords: B-carotene; Iron; zinc; Supplements