Daily variation of serum hepcidin levels in healthy volunteers during a day of iron-free diet and a day of iron free diet + oral iron supplementation: a cross over study

Hepcidin is a iron regulating hormone, that was first described in 2001.
Recently a serum assay for hepcidin was developed. Hepcidin is produced by
hepatocytes in the liver en is excreted in the plasma Hepcidin is a negative
regulator of iron-absorption. Hepcidin moldulates iron availability by
promoting the internalization and degradation of ferroportin, which is
essential for both iron absorption in the duodenum and recycling of iron/iron
efflux by macrophages. Increased iron stores and inflammation induce hepcidin
expression, whereas suppression occurs during hypoxia and anemia. The serum
hepcidin assay is a promosing assay in the diagnosis of iron metabolims
disorders. Previous research showed a daily variation in serum hepcidin levels.
Following a normal diet, there is a significant increase of serum hepcidin
levels during the day. However, the mechanism of this daily variation is
unknown.

To gain better insights in mechanism of the biological variation of hepcidin.
We investigate the role of iron in the diet, on the biological variation of
hepcidin.

Two weeks before the study starts:
To investigate if a volunteer can be included in the study, a pre-screening
will be performed. 10 mL blood divided in two tubes will be sampled. Using this
blood, hepcidin, CRP, ALAT, Fe, TIBC, ferritine and Hb will be measured.
Further BMI will be calculated and the history of bloodtransfusion en
chemotherapy was investigated.

Day 1:
Patients follow a iron-free diet. This diet is composed by the research-team
using a nevotable. The diet contains the following products:

Drinks:
Black Tea
Coke

Cereal:
Cornflakes

Dairy:
Semi-skimmed milk
cottage cheese
Buttermilk

Other:
Sweetener
Liquorice

During the day, 1x 15mL en 3x 10mL blood will be sampled by this schedule:

8.00AM 10.30AM 1.00PM 4.00PM
Parameter
Hb x
MCV x
CRP x
Fe x x x x
TIBC x x x x
Ferritine x
ALAT x
Hepcidine x x x x
Buis
5ml EDTA (blood) x
10 ml tube (serum) x x x x

TS is calculated by the Fe/TIBC ratio.

The first serum sample (8.00u) should be taken fasted.

The volunteers could choose whether they want a peripheral cannula (blood will
be drawn on the 4 time points) or whether they want four single blood samplings.

In the blood Hb, MCV, CRP, ALAT, Fe, TIBC and ferritin will be measured by
routine measurement, to determine whether the volunteer has a healthy iron
metabolism. Hepcidin levels will be measured by the Matrix Assisted Laser
Desorption/Ionisation Time-of-Flight Massa Spectrometrische (MALDI-TOF MS)
assay.


Day 2 (one week after day 1):
The volunteers follow the same iron-free diet as followed on day one
supplemented with two tablets of 65mg ferrousfumarate. The ferrousfumarate
tablets should be taken half an our before breakfast (65mg) and half an our
before lunch (65 mg).

Bloodsampling and laboratory measurements follow the same protocol as the
protocol on day one.

Day 1: The volunteers follow an iron-free diet.
Day 2: The volunteers follow an iron-free diet, supplemented with tablets of
65mg ferrousfumarate.
9 samples of blood will be taken.

In the pre-screening, the participants will undergo 1 venepunction of 10 mL
blood to test the fasted hepcidin level and iron parameters.
At study day 1, the participants will undergo 4 times a venepunction to draw a
total of 45mL of blood or the participants get a peripheral cannula for one day
to draw a total of 45mL of blood.
At study day 2, the participants will undergo 4 times a venepunction to draw a
total of 45mL of blood or the participants get a peripheral cannula for one
day to draw a total of 45mL of blood.

This puncture can be experienced as irritable for the volunteers.
The participant could develop a hematoma/bruise in the arm, but this risk is
minimal for the information gained by this study about the biological variation
of hepcidin.
If the participant choose a a peripheral cannula, there is a risk the cannula
will clog. A new a peripheral cannula should be placed.

Taking 2 tablets of 65mg ferrufmaraat can, in a few cases, cause
gastro-intestinal symptoms.