Effect of Simultaneously Consuming Iron and Zinc Supplements With That of B-carotene, on the B-carotene Bioavailability in Healthy Males
Phase: NA
Conditions: Bioavailability
Sponsor: University of Hohenheim

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Test plan No. 2017-2  
  
Effects on the bioavailability of β-carotene when taken concomitantly with zinc and 
iron supplements   
  
Table of Contents  
Abstract of the project 4  
1. study objective 4  
2. scientific background 4  
3. hypothesis 5  
3.1 Randomization and blinding 5  
3.2 Target parameters and analysis 5  
4. study population 6  
4.1 Number of subjects 6  
4.2 Duration of the study 6  
5. selection of subjects 7  
5.1 Inclusion criteria 7  
5.2 Exclusion criteria 7  
5.3 Mode of recruitment 7  
5.4 Informed consent of study participants 7  
6. study medication 8  
6.1 Pharmacological-toxicological report 8  
7. medication 9  
7.1 Dosage and mode of administration 9  
8. study procedure 9  
8.1 Screening 9  
8.2 Hospitalization 9  
8.3 Nutrition 9  
8.4 Documentation 10  
8.5 Trial conduct 10  
8.6 Methods 11  
9 Precautionary measures / discontinuation criteria 12  
9.1 Risk-benefit assessment 12  
9.2 Adverse events 12  
9.3 Serious Adverse Events and Serious Unexpected Side Effects 12  
9.4 Discontinuation criteria 13  
9.4.1 Discontinuation of the study in one subject (drop-out) 13  
9.4.2 Discontinuation of the entire study 13  
10 Data collection and analysis 13  
11 Modifications of the study plan 13  
12 Ethical and legal issues 13  
12.1 Legal basis 13  
12.2 Vote of the Ethics Committee 14  
12.3 Documentation, archiving and data protection 14 13 Signatures Error! Bookmark 
not defined.  
14. literature 14  

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List of abbreviations  
  
ADR Adverse Drug Reaction  
AE Adverse Event  
AUC Area-under-the-curve  
BMI Body Mass Index  
FDA Food and Drug Administration, U.S. Food and Drug Administration  
GCP Guidance on Good Clinical Practice  
HPLC High-pressure liquid chromatography  
RDA Recommended Daily Allowances  
SAE Serious Adverse Event  
SOP Standard Operating Procedure  
UE Adverse Event    
Abstract of the project  
  
Title:  
Effects on the bioavailability of β-carotene when taken concomitantly with zinc and iron 
supplements.  
1. study objective  
The objective of this study is to investigate the effect of concurrent intake of zinc or iron 
supplements on the bioavailability of β-carotene. The proposed trial is a purely scientific 
controlled, randomized, double-blind, prospective nutritional study.   
  
2. scientific background  
Iron, zinc, and vitamin A deficiencies are global public health problems and particularly affect 
pregnant and lactating women and children, who are especially vulnerable during the so-called 
1000-day window, the period from conception to the second birthday. According to the Global 
Nutrition Report (2015), 29% of women of childbearing age worldwide are anemic due to iron 
deficiency, 15% of newborns have low birth weight, and 164 million children under the age of 
five are developmentally inhibited, which are signs of zinc deficiency. The situation is worse in 
developing countries, where an estimated 50% of pregnant women and 40% of preschool 
children are anemic (WHO, 2016). A recent summary analysis showed an increase in the 
prevalence of vitamin A deficiency in sub-Saharan populations from 45% in 1991 to 48% in 
2013 (Stevens et al., 2015). These deficiencies are often treated in vulnerable populations 
through administration of supplements, increased dietary intake, and dietary modification. 
While vitamin A deficiency is increasingly treated by switching to a diet rich in β-carotene 
(provitamin A), severe zinc and iron deficiencies are still largely treated with supplements. 
Taking zinc supplements can greatly reduce the frequency and incidence of diarrhea in children 
under five years of age (IOM, 2001), and iron supplementation is still the preferred treatment 
for iron deficiency anemia (IDA) (WHO, 1998).  
  
Since there is usually a simultaneous deficiency of vitamin A, iron and zinc, a combination of 
these micronutrient supplements is often recommended. However, in vitro studies suggest that 
intestinal absorption of β-carotene may be inhibited by iron and zinc (Bengtsson et al., 2009; 
Biehler et al., 2011). Fat-soluble β-carotene, in order to be absorbed in the small intestine, 
must be incorporated into so-called mixed micelles under the action of bile salts. Caco-2 cells 
incubated with micellated β-carotene and different concentrations of iron absorbed on average 

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22% less β-carotene in the presence of iron (Bengtsson et al., 2009). Another study examined 
the effects of different concentrations of magnesium, calcium, iron, and zinc on the ability to 
form micelles and uptake β-carotene in Caco-2 cells (Biehler et al., 2011). The micellation of 
β-carotene and its uptake into Caco-2 cells decreased with increasing concentrations of the 
minerals. Iron and zinc had a much stronger inhibitory effect than calcium and magnesium. In 
both studies, the authors attributed the decreased absorption of β-carotene to an interaction of 
iron with bile salts and a resulting decreased micellation.   
  
Experiments performed in our laboratory with Caco-2 cells confirm the results of the above 
studies, as we also measured a dose-dependent reduction in micellation of β-carotene with 
increasing iron concentration.   
  
To the best of our knowledge, no human studies have been performed to determine the 
influence of iron and zinc supplements on the bioavailability of β-carotene. However, these are 
of great importance and relevance because in vitro systems are not dynamic and cannot 
adequately model the influence of minerals on β-carotene bioavailability. For example, it is 
possible that the continuous secretion of bile salts in vivo may compensate for precipitation by 
minerals and thus the reduction in absorption.  
  
3. Hypothesis  
  
Based on the in vitro experiments described above (see 2), which observed reduced 
micellization of β-carotene in the presence of iron and zinc, we will test the hypothesis that 
when β-carotene is taken simultaneously with iron or zinc, its bioavailability is reduced in 
healthy subjects.  
  
3.1 Randomization and Blinding  
To avoid systematic errors and otherwise influencing the results, the study will be randomized 
and double-blinded. Randomization and blinding will be done before the start of the study and 
will be performed by a staff member who is not actively involved in the study. For this purpose, 
the samples obtained will be randomly numbered and the key to unblinding, i.e., the 
assignment of the randomly generated sample numbers to the specific samples (identified by 
time of sampling and type of intervention), will be stored in sealed envelopes in two different 
locations in locked cabinets.  
Subjects receive the different preparations (15 mg β-carotene and placebo, 15 mg β-carotene 
with 25 mg iron, or 15 mg β-carotene with 30 mg zinc) in capsules in random order. Analysis 
of plasma samples was also randomized and blinded to prevent inadvertent influence of the 
experimenter on the results. Unblinding of the samples for statistical analysis of the data is 
done only after all biochemical analyses have been completed.   
3.1 Target parameters and evaluation  
The bioavailability of β-carotene is determined via the measured plasma and chylomicron 
concentrations by HPLC (see 8.6 Methods). For this purpose, ultracentrifugation is used to 
isolate the chylomicron fraction and extract β-carotene. Pharmacokinetics for plasma and 
chylomicron fraction are calculated using the area under the concentration-time curve. This 
quantity can be used to quantify the bioavailability of β-carotene in comparison between single 
and combined intakes. Statistical analysis is performed using one-factor analysis of variance.   
  
4. study population  

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4.1 Number of subjects  
The required power for the study will be calculated based on the area under the plasma 
concentration-time curve (AUC). Study power is calculated based on the following formula: n 
> F (σ/d)2, where n = number of subjects, σ = standard deviation of the dependent primary 
outcome measure, d = difference between control and treatment classified as significantly 
different in the literature. The corresponding F-values can be found in the table below; for the 
planned study, P < 0.05 and 90% power are assumed, resulting in an F-value of 10.51 (Scales 
& Rubenfeld 2005).  
  
F-value Table:   
  
Power  
 
 
Significance level (P-value)   
80%  
90%  
95%  
99%  
0.100   
6,18  
8,56  
10,82
15,77
0.050   
7,85  
10,51
12,99
18,37
0.025   
9,51  
12,41
15,10
20,86
0.010   
11,68
14,88
17,81
24,03
  
In a comparable study by (Goltz et al. 2013), an AUC of 22.6 ± 7.6 [nmol*L-1*h] could be 
determined after intake of 20-30 mg β-carotene (n=6). This results in a mean value of 22.6 
nmol*L-1*h and a standard deviation of 7.6 nmol*L-1*h. A reduction of bioavailability by more 
than one third would represent a biologically relevant effect, which is why a significant 
difference of rounded up 35%, corresponding to 7.9 nmol*L-1*h, is used for the calculation in 
this study. Thus, n > 10.51 x (7.6/7.9)2 results in a number of at least 9.7, rounded up to 10 
subjects. To allow for a balanced experimental design in which all permutations of the timing 
of the interventions occur in equal numbers, a total of 12 healthy males aged 18-45 years will 
be recruited for this study. Females will be excluded from the study because the menstrual 
cycle affects blood concentrations of carotenoids (Forman et al. 1998).   
  
4.2 Duration of the study  
The duration of the study is approximately 3 weeks per subject and includes 3 interventions, 
each preceded by a 1-week washout period. The three interventions each consist of a single 
oral administration of 15 mg β-carotene with placebo, in combination with 25 mg iron (FeSO4), 
or in combination with 30 mg zinc (ZnSO4).   
   
 5. selection of subjects  
5.1 Inclusion criteria  
Healthy, consenting males aged 18 to 45 years are sought for the study. A prerequisite for 
participation is that the small blood count, parameters of liver and kidney function, lipid 
metabolism, inflammatory markers, blood pressure and body mass index (BMI) are within the 
normal range.   
  
5.2 Exclusion criteria  
Subjects will not be included in the study should at least one of the following exclusion criteria 
be present: Use of medication, previous myocardial infarction, addiction, malignant disease, 
diabetes mellitus, dementia, arterial hypertension, smoking, increased alcohol consumption, 
use of multivitamin tablets or food supplements, extreme physical stress (>5 hours of exercise 

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per week) and in case of a known intolerance/hypersensitivity to the preparation (β-carotene, 
iron, zinc or fillers used).  
Subjects enrolled in another study during the same time period or who have participated in 
another clinical trial within the previous 3 weeks will not be enrolled in the study.  
  
5.3 Mode of recruitment  
Subjects will be recruited by means of posters, flyers, requests on the websites of the 
participating study groups and institutes, and advertisements in local daily newspapers. During 
the initial telephone interview, a short interview is conducted to verify that the subjects meet 
the inclusion criteria of the study. When a sufficiently large number of potential participants has 
been gathered, "screening" takes place. Study participants will receive an expense allowance 
of EUR 250.00 at the end of the study.  
  
5.4 Informed consent of study participants  
A subject can only be included in the study if he/she has given his/her consent to this after 
being informed orally and in writing by an investigator about the nature, significance and scope 
of the study in an appropriate and comprehensible manner.   
At the same time as giving his consent, he must have declared that he agrees to the recording 
of data within the framework of the study and to their review by authorized persons (e.g. 
monitor, auditor). It must be clear to him that he can withdraw his consent at any time and 
without giving reasons, without any disadvantages resulting from this.  
The original of the written consent will be kept in the study folder of the study center. The 
subject will be given a copy of the written patient information and the informed consent form. 
In addition, copies of both documents will be filed in the study folder. Patient information, 
consent form and all other documents received by the participants as well as recruitment 
notifications will be submitted to the responsible ethics committee for approval before use. See 
informed consent forms for both the screening and participation attached at the end of the 
document. 
 
  
6. Test medication  
6.1 Pharmacological-toxicological expert opinion  
Since vitamin A (retinol) can be formed from β-carotene, including in the intestine and liver, it 
enters the reference values for the desirable daily intake of vitamin A (6 mg β-carotene 
corresponds to 1 mg retinol). β-Carotene is part of the daily diet and is absorbed mainly with 
intensely colored vegetables and fruits (spinach, kale, broccoli, lettuce, carrots, tomatoes, 
pumpkin, mango, etc.). Individuals with a high fruit and vegetable intake (5 servings per day) 
consume a mean of 36 mg of β-carotene per day (Salter-Venzon et al. 2016). Because 
βcarotene contributes to vitamin A supply but is not itself an essential nutrient, no adverse 
health effects are expected even with several days of abstinence. In the CARET (Carotene 
and Retinol Efficacy Trial) study, long-term intake (4 years on average) of 30 mg daily of β-
carotene and 25,000 IU retinol (vitamin A) increased the risk of lung cancer in smokers and 
persons with asbestos exposure, but not in nonsmokers (Omenn et al., 1996). However, no 
increased risk is expected with a single intake of β-carotene alone. Nevertheless, to exclude 
any risk, only nonsmokers are included in the study.  
  
The German Nutrition Society (DGE) recommends a daily intake of 10 mg iron for adult men. 
Adults with normal intestinal function have only a very low risk of being exposed to too much 
dietary iron (NIH, 2016). However, ingestion of more than 20 mg/kg body weight (equivalent to 
1.4 g for an average adult of 70 kg body weight) from dietary supplements or medical 

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preparations can cause stomach upset, nausea, constipation, abdominal pain, vomiting, and 
feelings of weakness, especially if taken fasting. Sustained use of iron supplements containing 
25 mg elemental iron or more may result in decreased absorption of zinc and decreased zinc 
plasma levels. In severe cases (for example, a single dose of 60 mg/kg), iron overdose can 
lead to multiple organ failure, fainting, coma, convulsions, and even death ((Papanikolaou & 
Pantopoulos 2005). In the case of a single intake of 25 mg iron (equivalent to the iron content 
of approx. 100 g blood sausage or pork liver), as planned in the present study, no health risks 
are to be expected for the subjects.   
  
The daily intake of zinc recommended by the German Nutrition Society (DGE) for adult men is 
10 mg. The toxicity threshold for zinc is very high. Acute poisoning with 2 g of zinc causes 
gastrointestinal disturbances and fever. Acute poisoning from very high doses of zinc manifests 
primarily as nausea, abdominal cramps, vomiting, tenesmus, and diarrhea (possibly bloody), 
but is very rare (Maret & Sandstead, 2006). One case study reported severe nausea and 
vomiting within 30 min of ingesting 4 g of zinc gluconate (equivalent to 570 mg of elemental 
zinc; NIH, 2016). Daily intakes of 150-450 mg of zinc are associated with low copper status, 
changes in iron function, lowered immune system activity, and reduced HDL concentrations. 
Daily intake of 60 mg zinc for 10 weeks resulted in decreased copper-containing enzymes, 
which are a marker of copper status. The AREDS study with a daily dose of 80 mg zinc in the 
form of zinc oxide for an average of 6.3 years showed increased hospitalizations for urogenital 
complaints. This suggests that chronic intake of zinc has effects on certain aspects in urinary 
tract physiology. Taking 30 mg of zinc once with a commercially available dietary supplement, 
as planned in the present study, does not pose a risk of adverse health effects to the subjects.   
  
In this study, only commercially available low-dose dietary supplements are used and 
administered in single doses. Therefore, there are no health risks associated with either the 
administration of the micronutrients or the one-week washout periods with a low-carotenoid 
diet for the subjects.  
  
7. medication  
7.1 Dosage and mode of administration  
Subjects (n=12) will receive, in random order, a single oral dose of 15 mg β-carotene 
individually plus a placebo capsule (filled with silica and maltodextrin), 15 mg β-carotene plus 
25 mg iron (FeSO4), or 15 mg β-carotene plus 30 mg zinc (ZnSO4) in a double-blinded 
crossover study (interventions separated by one-week washout periods). Commercially 
available dietary supplements are used for this purpose (β-carotene from BIOVEA, 
Ferrogamma (iron) and Zinkit® (zinc) from Wörwag Pharma GmbH & Co. KG).  
  
8. study procedure  
8.1 Screening  
Subjects are considered to be enrolled in the study if they have given their written informed 
consent to participate in the study (see section 5.4).  
First, it is checked that all inclusion criteria are fulfilled and that none of the exclusion criteria 
are present. For this purpose, a medical history is taken regarding the subjects' health status 
and dietary and exercise habits. The subjects are weighed and measured. Furthermore, a 
blood sample will be taken by medical staff (investigator: Dr. Dr. Venturelli). A small blood count 
will be collected from each subject.  
  

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8.2 Hospitalization  
The screening of the subjects and the study will take place at the Institute of Biological 
Chemistry and Nutrition Science. Hospitalization of the subjects is not required.  
  
8.3 Nutrition  
Standardized meals will be served on the study days (from the evening before the intervention 
until the collection of the 10 h blood sample). Subjects will be instructed not to consume foods 
containing β-carotene (see Subject Information and Supplementary Information) during the 
studies. An appropriate list of foods and beverages to be avoided will be given to subjects as 
part of the subject information.  
  
The standardized meals are composed as follows:  
Dinner before the first study day consists of farmhouse bread with butter and camembert, plus 
roasted macadamia nuts and natural yogurt without artificial colors or additives. For breakfast 
on the study day, fresh wheat rolls with cream cheese (natural) and Greek yogurt are served 
in parallel with taking the capsules. Lunch on the study day is served four hours after taking 
the capsules and consists of pasta with cheese sauce, rolls and yogurt ice cream for dessert. 
The snack 6 hours after intervention consists of ricotta cake. For dinner on the study day, 
potato salad, farmhouse bread with butter, almonds, and a yogurt are served. All foods used 
were chosen from the point of view of avoiding additional intake of β-carotene.   
  
8.4 Documentation  
Each subject is assigned an ID number and all study data (which preparation the subject 
received, blood test findings) are recorded under the respective ID number.  
  
8.5 Experimental procedure  
Subjects are considered to be enrolled in the study if all inclusion criteria have been met during 
screening (see section 5.1), none of the exclusion criteria (see section 5.2) are present and 
the subjects have given their written consent to participate in the study.  
  
On the evening before the intervention, subjects will receive a standardized meal and a 
standardized snack for consumption between dinner and 10 pm. Subjects will be asked to drink 
only water and consume no other food after 10 pm and until the intervention on the day of the 
trial. On the morning of the trial day, subjects will receive a single oral dose of 15 mg β-carotene 
plus placebo, 15 mg β-carotene combined with 25 mg iron (FeSO4), or 15 mg β-carotene 
combined with 30 mg zinc (ZnSO4) after breakfast. Standardized meals will be served to study 
participants throughout the day of the intervention. Blood samples will be collected at baseline 
(0 h) and 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 h after ingestion of the study drugs (11 blood samples). 
A total of approximately 120 mL of blood will be collected per intervention.  
  
As the study is a cross-over study with three arms, after each 1-week washout period, the same 
experimental procedure is performed in random order with one of the remaining study 
preparations each time.  
  
8.6 Methods  
The blood test (for screening) is performed by the joint practice for laboratory medicine 
"Laborärzte Sindelfingen" (Nüßstraße 5, 71065 Sindelfingen, phone 07031-79930).  
  

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For the investigation of bioavailability, β-carotene is measured in the blood by means of 
highpressure liquid chromatography (HPLC), which were taken over the entire period. The 
appropriate HPLC analysis for this purpose has been established (Stuetz et al 2016). Isolation 
of the chylomicron fraction is performed according to (Goltz et al 2012).  
  
9. precautionary measures / discontinuation criteria  
9.1 Risk-Benefit Consideration.  
There is no direct benefit to the subjects of the study. They will receive the result of their blood 
values, as well as meals on the day of the trial and the evening before. Should indications of 
health problems of a potential subject come to light in the course of the study or screening, the 
subject in question will be informed of this by the investigator and, if necessary, advised to 
consult a specific specialist. The risks of blood collection are a short-term uncomfortable 
sensation from the needle prick, rarely bruising, and very rarely infection. Participating routine 
physicians will perform the blood draws so that complications or risks to study participants can 
be minimized and virtually eliminated.  
  
9.2 Adverse events  
Since subjects in the planned study will consume only a single oral dose each of 25 mg iron 
(FeSO4) and 30 mg zinc (ZnSO4), and only three times a dose of 15 mg β-carotene, no health 
risks to subjects are expected. As described in 6.1, side effects appeared only with long-term 
intake of very high doses. Furthermore, in this study only commercially available supplements 
in the form of capsules are administered.  
Nevertheless, the principal investigator agrees to document all UE or Adverse Events (AE) as 
well as side effects of the supplements or Adverse Drug Reactions (ADR) that might occur 
during the course of the studies. When AEs or ADRs occur, information on the type, time of 
occurrence, and duration as well as intensity and frequency of the adverse event will be 
documented. AE or ADR are defined as events such as mild nausea, mild dizziness, mild 
reversible change in blood count.  
  
Furthermore, the measure taken as well as a connection with the intake of the preparation or 
whether the symptom was already known before the start of the study is noted. If the event is 
related to the study conduct, the investigator commits to immediately exclude the subject from 
the study.  
  
9.3 Serious Adverse Events and Serious Unexpected Side Effects  
Serious adverse events are defined as events that may lead, for example, to hospitalization, 
persistent disability, life-threatening impairment or even death.  
Suspected Unexpected Adverse Reaction (SUSAR) is an adverse event that is inconsistent in 
nature or severity with the available information on the investigational product, is judged to be 
serious, and for which a relationship to the investigational drug is judged to be at least possible 
by the investigator.  
  
9.4 Discontinuation criteria  
9.4.1 Discontinuation of the study in one subject (drop-out).  
The study is discontinued in an individual subject if the subject withdraws consent to participate 
in the study, the study protocol is violated, an exclusion criterion or disease occurs, or other 
circumstances arise that would jeopardize the subject's health if the subject continued to 
participate in the study.  
  

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9.4.2 Termination of the entire study  
If the suspected case of a serious unexpected adverse reaction occurs during the human study 
after taking 15 mg β-carotene plus placebo, 15 mg β-carotene with 25 mg iron (FeSO4), and 
15 mg β-carotene with 30 mg zinc (ZnSO4), the study will be terminated at any time for the 
benefit and in the interest of the subjects. In this case, the Ethics Committee would be informed 
immediately.  
10 Data collection and analysis  
Statistical analysis will be performed by the study coordinator. Bioavailability will be determined 
by the area under the curve (AUC), maximum plasma concentration (Cmax), and time to reach 
Cmax (Tmax) of β-carotene. Significant differences between interventions will be determined 
using GraphPad Prism 6.0 software using one-factorial analysis of variance for repeated 
measures.  
  
11. amendments to the protocol.  
The applicants are aware that the vote of the Ethics Committee covers only the information 
contained in the application and does not include extensions and changes to the research 
proposal made at a later date. The applicants are aware that in case of changes, a protocol 
annex is required, which must be signed by the study directors, and that all protocol changes 
must be reported to the Ethics Committee. The applicants are aware that a new vote of the 
Ethics Committee must be obtained for protocol changes that are not exclusively formal in 
nature and that contain changes relevant to the study participants. Potential changes in study 
conditions would be brought to the attention of study participants during the informed consent 
process.  
  
12 Ethical and legal issues  
12.1 Legal basis  
The study will be conducted in accordance with the Declaration of Helsinki (as amended) and 
the Guidance on Good Clinical Practice (GCP) Regulations.  
  
12.2 Vote of the Ethics Committee  
The applicants are aware that the prerequisite of the nutritional study is compliance with the 
guidelines and laws listed under item 12.1 and that the initiation and conduct of the study are 
subject to the vote of the Ethics Committee. The Ethics Committee will be informed immediately 
in the event of serious events (section 9.3), study termination (section 9.4) and protocol 
changes (section 11). After completion of the study, the Ethics Committee will be provided with 
a report and, if necessary, a publication.  
  
12.3 Documentation, archiving and data protection  
The recording of the collected findings is done by means of a test sheet. Among other things, 
the test sheet will summarize information about the participating person in pseudonymized 
form (ID number), information about which 15 mg β-carotene with placebo capsule, 15 mg 
βcarotene with 25 mg iron (FeSO4) and 15 mg β-carotene with 30 mg zinc (ZnSO4) preparation 
the person received, findings of the blood test, etc.  
All data collected in the study will be subject to data protection and will be kept strictly 
confidential. Samples will be encrypted and no report or publication will reveal the identity of 
study participants. Blood samples will leave the facility in encrypted form. The transfer of test 
forms and data storage for evaluation will only be done with the anonymized data of the study 
participant. Assignment of personal data to study data will be made only by the study director. 
After completion of the study, the data will be stored for a period of 10 years.  

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Salter-Venzon, D., Kazlova, V., Izzy Ford, S., Intra, J., Klosner, A. E., & Gellenbeck, K. W. 
(2016). Evidence for decreased interaction and improved carotenoid bioavailability by 
sequential delivery of a supplement. Food Science & Nutrition (DOI: 10.1002/fsn3.409)  
  
Scales, D.C. & Rubenfeld, G.D., 2005. Estimating sample size in critical care clinical trials. 
Journal of Critical Care, 20(1), pp.6–11.  
Schiborr, C., Kocher, A., Behnam, D., Jandasek, J., Toelstede, S., & Frank, J. (2014). The oral 
bioavailability of curcumin from micronized powder and liquid micelles is significantly increased 
in healthy humans and differs between sexes. Molecular nutrition & food research, 58(3), 
516527.  
Stevens, G. A., Bennett, J. E., Hennocq, Q., Lu, Y., De-Regil, L. M., Rogers, L., ... & Oehrle, 
S. P. (2015). Trends and mortality effects of vitamin A deficiency in children in 138 low-income 
and middle-income countries between 1991 and 2013: a pooled analysis of population-based 
surveys. The Lancet Global Health, 3(9), e528-e536.  
Stoltzfus, R. J., & Dreyfuss, M. L. (1998). WHO Guidelines for the use of iron supplements to 
prevent and treat iron deficiency anemia (pp. 18-21). Washington DC: Ilsi Press.  
Underwood, B. A. (2000). Dietary approaches to the control of vitamin A deficiency: an 
introduction and overview. Food and Nutrition Bulletin, 21(2), 117-123.  
WHO. 2004. Vitamin and mineral requirements in human nutrition (2nd Ed), Joint expert 
consultation on human vitamin and mineral requirements. Geneva: WHO. Chapter 7. pp. 
87102.  
Stuetz, W., Weber, D., Dollé, M. E., Jansen, E., Grubeck-Loebenstein, B., Fiegl, S., ... & Sikora, 
E. (2016). Plasma Carotenoids, Tocopherols, and Retinol in the Age-Stratified (35–74 Years) 
General Population: A Cross-Sectional Study in Six European Countries. Nutrients, 8(10), 614.  
  
World 
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Organization 
(WHO). 
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“Micronutrient 
Deficiencies.” 
www.who.int/nutrition/topics/ida/en/ (accessed Oct 2016).  
  
  

Kontaktperson: 
Jan Frank 
Universität Hohenheim  
Institut für Biologische Chemie und Ernährungswissenschaft  
Lehrstuhl für Biofunktionalität und Sicherheit der Lebensmittel (140 b)  
Garbenstraße 28; Ökozentrum I, Erdgeschoss, Raum 025  
70599 Stuttgart 
Tel.: 0711-45923609; Email: study.betacarotene@nutres.de 
 
 
Dear Participant, 
 
Thank you for taking part in the screening for the study "The effect of simultaneous oral 
administration of zinc and iron supplements on the bioavailability of β-carotene". 
 
Our study doctor, will now take two tubes of blood (10 ml each) for the determination of the 
following parameters: 
 
 
Bilirubin (total) 
 
Uric acid 
 
Triglycerides 
 
Creatinine 
 
Alkaline phosphotase 
 
Glutamate oxaloacetate transaminase 
 
Gamma-glutamyl transferase 
 
Glutamate pyruvate transferase 
 
Total cholesterol 
 
HDL cholesterol 
 
LDL cholesterol 
 
Blood count 
 
 
In the next step, we will determine your body mass index (height and weight) and your blood 
pressure. The information collected will be kept confidential, but we will inform your laboratory 
(Laborer Sindelfingen), who will perform the blood tests for us, your name and date of birth for 
the correct assignment of the samples. 
You will not be compensated for the screening. However, you are invited to a small breakfast 
after the screening. In addition, you will receive the results of the blood tests by email. 
We will contact you as soon as possible and inform you about the further course of the study 
(the participant information you have already received by e-mail). 
 
Consent 
I hereby agree to participate in the screening for the study "Effects on the bioavailability of β-
carotene during oral administration of zinc and iron preparations" comprising the above-
described tests. 
 
____________________________________ 
 
 
 
____________________________________ 
(Place, Date)  
 
 
 
 
 
(Participant signature) 
 
____________________________________ 
 
 
 
_____________________________________ 
(Place, Date)  
 
 
 
 
 
(Dr. Dr. Sascha Venturelli) 

Seite 1 von 10 
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Participant information 
(October 2017) 
 
"The effect of simultaneous oral administration of zinc and iron supplements on the 
bioavailability of β-carotene" 
 
 
 
 
 
 
 
 
 
 
In the event of any ambiguities, emergencies, unexpected or undesirable occurrences 
during this study, please contact the following contact person at any time: 
 
Universität Hohenheim 
Institut für Biologische Chemie und Ernährungswissenschaft 
Fg. Biofunktionalität und Sicherheit der Lebensmittel (140b) 
Jan Frank 
Garbenstraße 28 
70599 Stuttgart 
Telefon: 0711/45924459 
Email: carotinstudie@nutres.de 

Seite 2 von 10 
Version 4, 2. Oktober 2017 
Dear participant, 
please read this leaflet carefully. You will be given sufficient time and opportunity to clarify all 
questions related to your participation. If you have any further questions or comments, please do 
not hesitate to contact us. After the information you will be asked to sign an agreement to 
participate in the study. With this declaration of consent, you confirm that you have been informed 
in detail about the study, that your participation in the study is voluntary and that you can 
terminate your participation at any time. 
1. What is the purpose of the study? 
1. What is the purpose of this study? 
The aim of this study is to investigate the influence of zinc and iron supplements on the 
bioavailability (intake in the intestine and blood concentrations) of β-carotene (a precursor of 
vitamin A). Iron, zinc and vitamin A deficiency are widespread deficiencies around the world 
that can be caused by sub-optimal nutrition. For prevention and treatment of nutritional 
deficiencies, food supplements are often administered with the appropriate nutrient. In the case 
of simultaneous occurrence of several deficiencies, preparations with a combination of the 
nutrients are also administered. 
In vitro studies (experiments in an artificially produced environment outside a living organism) 
have shown that increased iron and zinc intake due to supplements can reduce the 
bioaccesibility and uptake of β-carotene from food. The current study therefore aims to 
investigate whether the simultaneous consmption of zinc or iron with β-carotene results in a 
reduced absorption and lower blood concentrations compared to β-carotene consumed alone. 
The prospective study is a controlled, randomized, double-blind, study of cross-over design. 
 
2. Study preparations 
Each study day the participant will be given 15 mg of β-carotene (corresponds to a capsule of the 
brand BIOVEA Beta Carotene) per intervention. You will also receive either 25 mg of iron 
(Ferrogamma from Wörwag Pharma GmbH & Co. KG), 30 mg of zinc (zincite from Wörwag 
Pharma GmbH & Co. KG) or capsule-type placebo. The preparations administered are food 
supplements, that are also available in the supermarket and in drugstores. 
 
3. Study execution 
A total of 12 healthy subjects are recruited (men aged 18-45 years). The duration of the study is 
approximately 3-5 weeks for each subject. 
 
There are 3 intervention days (times of administration) which are separated by a minimum of 1-
week washout phase (period in which no test substance is administered). Each intervention is 

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characterized by a one-time oral preparation, so that immediately after ingestion the time 
window of the wash-out phase begins. 
 
The selection period is to take place in October 2017 and the study itself takes place in 
October/November 2017. All examinations and samples are given at the Institute of Biological 
Chemistry and Nutritional Science, Garbenstr. 28-30, 70599 Stuttgart under medical 
supervision. The following investigations are carried out after clarification and voluntary 
consent to the study: 
Screening 
Before entering the study, each candidate is subjected to a preliminary examination. This 
includes a medical survey, measurement of blood pressure, body weight and body size. 
Furthermore, routine blood tests are performed to check general health. In addition to the small 
blood count, the hepatic enzymes used to check the liver function, the creatinine level as a 
parameter for the renal function and the blood fat values are determined as routine parameters. 
The blood sample must be taken after at least 10 hours of dietary fasting, ie before breakfast. 
The time required for this is about 30 minutes. On the basis of this data, it will be decided 
whether the necessary prerequisites for the study participation are fulfilled.  
 
Study day visits 1, 2 and 3 
Time: approx. 12 hours with free time 
After at least 10 hours of fasting, the first blood collection takes place for the determination of 
the baseline. For this purpose, a permanent venous approach in the arm is inserted by the study 
doctor. The cannula remains in your vein for approximately 10 hours for the successive blood 
draws. You will be given a breakfast with which the capsules are taken with β-carotene alone 
and placebo or in combination with iron (FeSO4) or zinc (ZnSO4) under supervision. The 
concentration course of the nutrients in the blood will be determined over a period of 10 hours. 
Blood samples are collected at 11 consecutive times (0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 hours) after 
taking the capsules. After each blood sampling, the cannula is rinsed with saline solution to 
avoid blood clotting. The cannula is then closed until the next blood draw. Four hours after 
breakfast, you will receive a standardized lunch meal, after 6 hours a standardized snack and 
after 9 hours a dinner. It is strictly forbidden to bring or consume your own additional food and 
drinks, as these could change the absorption processes. You may drink water at any time 
without restriction. 
After the 10 h blood sample has been collected on the day of the experiment, this study day is 
cpmpleted and you can leave the study center. 
 

Seite 4 von 10 
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After a one-week wash-out period, the course of the study day with the other preparations is 
repeated in the corresponding dosage form. In order to assess the tolerability of the 
preparations we ask you to complete a short questionnaire, which will be handed out to you on 
the day of the administration. 
 
4. General information 
Nutrition 
During the study period (one week before the first day of study until the end of the third day of 
the study) you should adjust your dietary habits. We ask you to refrain from consuming orange, 
yellow and green foods during the days preceding visits 1, 2 and 3 and during the wash-out 
phases; simply avoid colourful food and replace with white/colouless food. These limitations 
should only be applied to the study period (1 week before the first day of the study to the last 
blood test on the last day of the study) and not as a general recommendation for nutrition. 
Thus, one week before and after the end of the study, β-carotene-rich foods should be avoided 
(see "General information on the β-carotene study") and three days before the respective study 
day, only the food from the list with β- Carotene-poor foods (see list at the end). On this list you 
will also find menu suggestions for the study period. On the evening before visits 1, 2 and 3, we 
ask you to consume a standardized dinner prepared by us, in order to ensure a comparable 
starting conditions between the individual participants and between the visits. You should also 
abstain from consuming alcohol 24 hours before and on the study days. In the wash-out phase 
we ask you to limit your alcohol consumption to a maximum of 20 g / day, which corresponds to 
approx. 0.2 l of wine. At study days 1, 2 and 3 you get a standard breakfast, a standard lunch 
meal, dinner, snacks and water. Food or drinks brought along must not be consumed on the day 
of the study until the final blood sampling after 10 h. 
 
Excercise  
Your usual sports activities can be maintained during the duration of the study. However, we ask 
you to refrain from strenuous physical activities as well as endurance sports in the 24 hours 
before the student days. 
 
Medication 
Intake of all medication must be documented. If the use of medication for medical reasons is 
necessary, please let us know. 
 
5. What adverse events (side effects) or risks can occur? 
The blood draw is done by inserting a cannula for the subsequent blood sampling corresponds 
to routine medical procedures. However, haematomas (bruises), infections and very rarely 

Seite 5 von 10 
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inflammation of the veins, thromboses or nerve injuries can occur. Thrombosis is the occlusion 
of a blood vessel through a blood clot. A total of approximately 380 ml blood will be taken over 
the entire study including the screening (in comparison: in blood donation, 500 ml of blood is 
withdrawn). Please inform the physician about any adverse events occurring during the study. 
The doctor will, for his part, register adverse events and, if necessary, initiate necessary steps. 
The doctor will inform you about any new findings that may affect the benefit or safety of the 
study and thus your consent. 
 
6. Protection of your personal data 
The provisions on medical confidentiality and data protection are complied with in this study. 
During the study, medical records and personal information are collected and collected by you. 
The data important for the clinical examination are entered into a separate documentation sheet 
in a pseudonymized form (that is, encoded by the subject number and without attribution). Only 
these encrypted data is used. The study data are kept for 10 years. 
 
7. Termination of the study 
Your participation in this study is entirely voluntary. You can terminate your participation in the 
study at any time without incurring any disadvantages. It is helpful if you explain to the doctor 
the reason for the termination. However, you are not obliged to state reasons for your decision. 
Your physician may exclude you from this study in the interest of your health. If you terminate 
the study at an early stage, you may request the deletion of the data collected up to that point. 
 
8. Benefits 
There is no guarantee that your participation in this study will give you any personal benefit. If 
desired, you will receive a copy of the results of the routine laboratory parameters of your blood 
tests. 
 
9. Compensation of expenses 
All examinations are free; neither you nor your health insurance company has to pay anything. 
The study supplements and food will be made available to you free of charge. For full 
participation until the last day of the course, you will receive 250 €. Travel expenses are not 
refunded separately. In case of cancellation for personal or technical reasons, you will be 
compensated pro rata. 

Seite 6 von 10 
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These dietary recommendations should only be observed during the study to 
ensure a carotene‐poor diet. It is not a general dietary recommendation. 
 
Carotene‐Poor Foods 
Vegetables: 
Potatoes 
Mushrooms 
Garlic 
White Onions 
Celery Root 
Cauliflower 
White Asparagus 
 
Fruit: 
Lychee 
Coconut 
 
Starch Products: 
White Noodles 
Wheat Flour 
White Rice 
Waffles 
Bread (Except Bread With Cornmeal) 
Noodles 
 
Drinks: 
Coffee 
Water 
Wine 
Beer 
Low-Fat Milk 
 
Meat / Protein‐Rich Foods: 
Pork Meat 
Beef 
Chicken 
Sausage 
Beef Extract 
Soy Products 
White Beans 
 
 
 
 
Other: 
Sunflower Oil 
Olive Oil 
Nuts (Other Than Pistachios) 
Low-Fat Milk And Milk Products 
Soy Milk 
Coconut Milk 
Almond Milk 
Low-Fat Natural Yoghurt 
Low Fat Fresh Cheese 
Low-Fat White Cheese (Feta Cheese, Sheep's 
Cheese, Camembert) 
Mayonnaise 
Pickled White Onions 
Peanut Butter 
Chocolate Spread 
Honey 
Syrup 
Salt 
White Pepper 
Black Pepper 
 
Snacks: 
Chocolate 
Nuts 
Potato Chips, Salted 
 
Use the following foods very sparingly: 
Colored Spices 
Mustard 

Seite 7 von 10 
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Examples of possible meals: 
Breakfast: 
Toast with cottage cheese and low-fat 
natural yoghurt 
Pancakes with honey 
Lychees with natural yoghurt 
Oatmeal with low-fat milk and honey 
Muesli with low-fat milk 
 
Lunch: 
Fleischkäse with potato salad 
Potato salad and sausages 
Mushroom soup with white bread 
Chicken 
mayonnaise 
sandwich 
(white 
bread) with fries 
Fleischkäse sandwich 
Ham and cheese sandwiches (white bread) 
Flammkuchen with onions and bacon 
Sandwich with cottage cheese (white bread) 
Beef burgers (without salad and tomato) 
with feta and bacon and French fries / 
potato chips 
Schnitzel with rice and mushroom sauce 
Chicken Nuggets 
Fish sticks 
 
Dinner: 
White asparagus with ham and butter (no 
hollandaise sauce) 
Rice with chicken and garlic sauce 
Mashed potatoes with pork sausage and 
onion 
Spätzle and pork cutlets 
Grilled beef strips with cauliflower and 
white sauce 
Ricotta stuffed noodles in mushroom sauce 
Fried potatoes or salt potatoes and grilled 
chicken 
Mushroom risotto 
White bean, mushroom and chicken soup 
Deep-fried / baked camembert (without 
cranberries) 
 
snacks: 
Nuts 
Lychees 
Potato Chips

Seite 8 von 10 
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Written declaration of consent to the study 
 
 
"The effect of simultaneous oral administration of zinc and iron supplements on the 
bioavailability of β-carotene" 
 
 
Place of study: Stuttgart, Germany (monocentric) 
Investigator: Dr. med. Gunther Adler 
 
Proband: ____________________________________________ 
                   (Name, Surname)  
 
I was informed verbally and in writing about the aims and the course of the study as well as 
about possible advantages and disadvantages as well as about possible risks. I have received, 
read and understood the written subject information, which belongs to the above study. My 
questions concerning the participation in this study or my questions concerning the medical 
terms used in the subject information have been answered satisfactorily. I can keep the written 
subject information and receive a copy of my written declaration of consent. I had enough time 
to make my decision. I participate voluntarily in the study. I can revoke my consent to the 
participation at any time and without giving reasons, without incurring any disadvantages. I am 
aware that during the study, the requirements and limitations stated in the subject information 
must be observed. In the interest of my health, the study physician can always exclude me from 
the study. In addition, I inform the doctor about the concurrent treatment with another doctor 
as well as about the taking of medication (prescribed by the physician or independently 
acquired). 
 
Reuse of samples taken 
The samples obtained could be of great value at a later stage and beyond the scope of the 
research questioned in this project. Re-use of your samples for further research questions 
requires your consent, which is why we ask you to communicate your wishes in the following. 
 
A prerequisite for the use of biomaterials and data for a specific research project is that the 
research project has been evaluated by an ethics committee. 
 
I agree to the continued use of the blood and urine samples obtained for research purposes 
 

Seite 9 von 10 
Version 4, 2. Oktober 2017 
 I agree. 
 I do not agree 
 
The reuse is carried out in 
 (1) pseudonymized (from the pseudonym only the student can deduce your identity) 
 (2) anonymized (the samples are marked so that an assignment to your person is no 
longer possible) 
 for all study possibilities. 
 for all studies that are in the context of the questionnaire for which the material was 
originally donated. 
 
________________________________  
_________________________________ 
(Place, date) 
 
 
 
(Signiture of participant) 
 
 
Ich bestätige hiermit, dass ich den Probanden mündlich und schriftlich über die Ziele, den Ablauf 
der Studie, über die zu erwartenden Wirkungen, über mögliche Vor- und Nachteile sowie über 
eventuelle Risiken informiert habe. Der Proband hat seine Teilnahme durch seine persönliche 
Unterschrift mit Datum bestätigt. 
 
 
________________________________  
_________________________________ 
(Place, date) 
 
 
 
(Signiture of study doctor: Dr. Gunther Adler) 
 
 
________________________________  
_________________________________ 
(Place, date) 
 
 
 
(Signiture of study leader: Prof. Dr. Jan Frank) 

Seite 10 von 10 
Version 4, 2. Oktober 2017 
 
Data protection consent to the study 
 
"The effect of simultaneous oral administration of zinc and iron supplements on the 
bioavailability of β-carotene" 
 
In the scientific study, personal data and medical findings are collected about you. The 
dissemination, storage and evaluation of these study-related data requires the following 
voluntary consent before participation in the study: 
I agree that data collected in the course of this study are recorded on questionnaires and 
electronic data media and passed on to the client of the study for scientific evaluation in a 
pseudonymized form (ie, no names, initials or the exact date of birth in the encryption code). In 
the case of cancellation of the consent (cancellation of the study participation), you may request 
the deletion of the data collected up to that point. In the case of the publication of the study 
results, the confidentiality of my personal data is also ensured, if at all, the data are used in 
encrypted form. 
 
_______________________________ 
(Place, date) 
 
 
_______________________________ 
(Signiture of participant)