3 - Results

↓65

3.1 Plasma Iron Pool

The iron pool in blood plasma, despite its importance as the main iron exchange medium in the vertebrate body, has a small size compared to other pools (1-2 μg Fe per mouse) and is renewed several times per day (table 3.1).

↓66

The table shows that the transferrin-bound plasma iron concentration varies according to the iron status in the body. It tends to be lower under an iron deficient diet and higher in iron loaded conditions. It is in the range of 80-150 μg/dL and is similar to other mammalian species [57-61 ]. In rats, iron deficiency does not seem to lower plasma iron concentration.

TABLE 3.1: 1) Molecular weight of iron 55.847 2) 1.36 ml plasma per 25 mg mouse (see table 2.1) 3) Rounded value of row 4 * 55.847 / 1000 *1.36 ml (body plasma volume)

Fe-Deficient

Fe-Adequate

Fe-Loaded

Plasma iron concentration [μM]

15.2 ± 5.5

19.8 ± 1.4

25.8 ± 1.0

scaled t [μg/dL] 1 

84.0

110.5

144.1

Plasma pool size [μg per mouse] 2

1.15

1.50

1.96

Plasma iron turnover rate [μM/day] (PIT)

139

185

245

Scaled to μg per mouse3

11

14

19

Total clearance rate [d-1]

17.2

18.1

18.8

Plasma half-life time of iron [min]

58

55

53

The Plasma Iron Turnover rate is an indicator of how much iron is cleared from plasma per day. Iron molecules leave the plasma and go to peripheral organs and in our model iron molecules either slowly return to plasma to be again distributed through the organism or are lost due mucosal or skin desquamation. The Plasma Iron Turnover is calculated as follows:

↓67

PIT = 10 [per day] * plasma concentration [μM] – 13 [μM per day].

the slope of 10 and the intercept of -13 were estimated from fig. 5 of [33 ]. Like the plasma iron concentration, the PIT increases in an iron-loaded diet and diminishes under iron-deficient conditions.

The total clearance rate from table 3.1 is calculated by the plasma turnover rate and plasma iron concentration, taken from fig. 5 of [33 ]. In their study turnover was measured at 2 h after administration assuming that the exponential clearance rate constant is given by

↓68

-24/2 * ln {1 - (turnover rate) / (plasma concentration) * 2/ 24} 

This estimate is probably somewhat too low due the influence of tracer reflux. For parameter estimation we therefore rounded this preset value to maximum of 20 per day. To assess the precise total clearance rate would be necessary to perform measurements in groups of mice in a short period right after radioiron injection. This was not done in our experiments, since it is not feasible in mice. Our first measurement was performed at 12h, when most part of injected radioiron was already cleared from plasma. Cavill et al. have summarized the corresponding problems in humans [62 , 63].

With the calculated turnover rate constants it is possible to estimate the half-life of transferrin-bound plasma iron as follows:

↓69

Half-life = ln (2) / turnover rate constant [per day] * 24 * 60 [min])

The half-life of transferrin-bound plasma iron is somewhat slower for the iron deficient diet (58 minutes) and somewhat faster for iron-loaded diet (53 minutes). These values are in the same range from other species like dogs and rats [10 , 59,60 ] and in accordance with other studies [33 , 64].

3.1.1 Tracer uptake into murine Organs

Schümann´s tracer data [27] (see appendix A) allow to estimate clearance parameters of their compartment representation, see table 3.2. These parameters can be used to understand what proportion of the initial injected radioiron dose is absorbed by the organs and later returned to plasma. As mentioned previously, for our optimization purposes it was established that the sum of the fractional rates leaving the plasma was set to 20 per day. Figure 3.1 depicts how many percent of this fixed value is distributed among organs of mice under different dietary conditions.

↓70

Figure 3.1: distribution of radioactive plasma iron between organs.

There are two different scales: one for the organs known to have a high iron storage or demand like liver and bone marrow and a second scale with peripherical organs like the testicles, heart and brain. The percentage values depicted were calculated as follows: The best-fit clearance parameters for flux plasma-organ for the respective diet were divided by the total (=20) and multiplied by 100.

TABLE 3.2: Clearance parameter rates for the best fit and also the upper and lower limits obtained after Monte Carlo perturbations of the measurement values followed by the curve-fit procedure.

 

Fe-Deficient

 

 

Fe-Adequate

 

 

Fe-Loaded

 

Parameter

Best Fit

Lower Limit

Upper Limit

 

Best Fit

Lower Limit

Upper Limit

Best Fit

Lower Limit

Upper Limit

1kp_bon

 

13.22

12.47

13.63

12.67

12.01

13.26

6.92

6.01

7.09 +

2kp_kid

 

0.42

0.28

0.54

0.45

0.36

0.51

1.62

1.18

1.82 +

3kp_int

 

0.98

0.77

1.06

0.9

0.63

1.1

0.93

0.66

1.01

4kp_liv

 

2.27

1.83

2.54

2.61

2.28

2.9

5.25

4.25

5.73 +

5kp_sto

 

0.09

0.06

0.18

0.12

0.08

0.17

0.27

0.21

0.37 +

6kp_intg

 

1.04

0.89

1.32

1.14

0.96

1.35

1.33

1.05

1.50

7kp_fat

 

0.04

0.03

0.05

0.05

0.04

0.05

0.066

0.051

0.075  (+)

8kp_mus

 

0.96

0.8

1.23  -

1.49

1.31

1.8

2.52

2.06

2.75   +

9kp_lun

 

0.79

0.64

1.06 +

0.31

0.22

0.44

0.63

0.56

0.75 +

10kp_duo

 

0.02

0.01

0.15

0.04

0.03

0.06

0.038

0.027

0.050

11kp_bra

 

0.03

0.03

0.03

0.03

0.03

0.04

0.021

0.019

0.022 -

12kp_hea

 

0.11

0.1

0.14

0.13

0.12

0.15

0.36

0.31

0.38 +

13kp_tes

 

0.04

0.04

0.05

0.06

0.05

0.06

0.043

0.037

2.68

14kkid_p

 

0.2

0.11

0.32

0.2

0.14

0.25

0.23

0.16

0.32

15kliv_p

 

0.25

0.21

0.32 +

0.14

0.11

0.16

0.10

0.073

0.12  (-)

16ksto_out

0.18

0.09

1.72

0.37

0.27

0.49

0.29

0.20

0.40

17kfat_p

 

0.1

0.06

0.19

0.13

0.1

0.15

0.099

0.079

0.12

18kmus_p

 

0.03

0.02

0.05 -

0.15

0.12

0.21

0.14

0.11

0.17

19klun_p

 

0.41

0.37

0.52 +

0.19

0.11

0.3

0.086

0.065

0.12  (-)

20kbra_p

 

0.02

0.02

0.03 -

0.06

0.05

0.07

0.028

0.022

0.034 -

21khea_p

 

0.06

0.03

0.08 (-)

0.08

0.06

0.09

0.17

0.14

0.19 +

22ktes_p

 

0.05

0.03

0.07 -

0.09

0.07

0.12

0.067

0.044

7.16

23kspl_p

 

14.61

13.86

15 -

7.29

5.53

9.15

1.91

1.52

2.33 -

24kintg_out

0.03

0.02

0.05

0.04

0.03

0.06

0.072

0.057

0.102  (+)

25kint_out

 

0.3

0.22

0.4

0.36

0.26

0.42

0.22

0.16

0.26 -

26kduo_p

 

0.17

0.12

2.55

0.42

0.32

0.55

0.24

0.18

0.34

27kbon_rbc

1.85

1.74

1.92 +

1.07

0.93

1.26

0.50

0.48

0.57 -

28kbon_spl

0.56

0.4

0.83 +

0.1

0.08

0.13

0.046

0.033

0.058 -

29krbc_spl

0.03

0.02

0.04 -

 

0.06

0.05

0.07

0.032

0.027

0.047 -

fval chi_sqr

0.58

 

 

 

0.72

 

 

1.05

 

 

sq root of mean weighted squared dev

0.07

 

 

 

0.08

 

 

0.07

 

 

This sequence was performed 100 times. The dimension of the data is day-1. The + and – indicate statistically valid change compared to iron-adequate diet (bracketed if only slight).

-

↓71

Considering the two different scales of figure 3.1 (0-2% and 0-70%) we observe that there were organs which did not demonstrate a strong difference in terms of tracer iron uptake between the diets: brain, fat, testicles, intestine and integument. In the case of duodenum, stomach and lungs it is not possible to affirm that there is a representative difference on the iron uptake levels. This can be attributed to technical difficulties associated to the organ size (duodenum, stomach) or the lack of cellular homogeneity of the organ itself (lungs).

On the other hand, it is visible that other organs have a great difference in tracer uptake levels when the mice were held under different diets. The heart and the muscles absorb around 2 times more iron under loading scenarios. In contrast, in the iron-loaded diet the bone marrow absorbs around 30% less in comparison to iron-deficient or -adequate. This difference in absorption in the bone marrow, liver and muscles reflects a “switch” to a storage mode that occurs in the organism when there is iron repletion.

Figures 3.2, 3.3 and 3.4 show the tracer distribution among organs during the study period under the three different diets. We can observe from the figures and numerically assess from tables 1-3 in Appendix A that at the time when the first measurement was performed (12h) the plasma radioiron clearance was already complete. The tracer had been already absorbed by the organs and the tracer level in plasma was very small and remained practically constant.

↓72

Figure 3.2: The best fit of our model to the measured data of mice held under iron adequate diet.

The blue dots with standard deviation are the measurements and the red line of the curve produced by our model.

Figure 3.3: The best fit of our model to the measured data of mice held under iron deficient diet.

The blue dots with standard deviation are the measurements and the red line of the curve produced by our model.

Figure 3.4: The best fit of our model to the measured data of mice held under iron loaded diet.

The blue dots with standard deviation are the measurements and the red line of the curve produced by our model.

↓73

The two components in the plasma curve (first exponential decay and later a small tracer level close to zero) can be attributed to i) the first clearance phase (which happens approximately within one hour [33 ] and ii) to a reflux of iron from the organs to the plasma.

Inspection of the organ curves demonstrate that after 12-24 hours the reflux into plasma starts to take place and follows until the end of the study. In addition, from the same figures it is possible to verify that the organs receive radioiron and store for biosynthetic functions, return it to plasma to be redistributed among other organs or looses it by desquamation, as is the case of intestine and integument.

In addition, the influx and outflux of radioiron from the organs obviously take place simultaneously, in accordance with the steady-state experienced during the development of this study.

↓74

The results presented so far related to iron uptake by the organs were expressed as percent of the injected tracer dose. To have a more precise quantitative description of the amounts exchanged by the organs and present the results in units that can be further used for comparison among species, one should use absolute flux rates.

To calculate the absolute flux rates it is necessary to have the fractional clearance rates (table 3.2) and the iron content of plasma plus the extracellular fluid (table 3.1).

We calculate the flux rates (v plasma_i) as:

↓75

v i_plasmak i_plasma * C plasma/ECF

where k i_plasma  is the fractional clearance rate between plasma into the organ i and C plasma/ECF is the iron content in plasma plus extracellular fluid.

We can observe in figure 3.5 and table 3.3 that three sets of flux rates may be distinguished:

↓76

Figure 3.5: The calculated radioiron fluxes from plasma into organs. Data obtained from

Table 3.3. The calculations were done for the “best-fit” parameters.

Absolute Flux Rates (μg/body/day)

Parameter

Fe-Deficient

Fe-Adequate

Fe-Loaded

1kp_bon

15.19

19.007

13.55

2kp_kid

0.48

0.66

3.17

3kp_int

1.12

1.34

1.83

4kp_liv

2.61

3.92

10.29

5kp_sto

0.105

0.18

0.53

6kp_intg

1.19

1.71

2.602

7kp_fat

0.05

0.067

0.13

8kp_mus

1.106

2.24

4.93

9kp_lun

0.907

0.45

1.22

10kp_duo

0.021

0.064

0.075

11kp_bra

0.032

0.048

0.041

12kp_hea

0.132

0.202

0.71

13kp_tes

0.046

0.082

0.084

14kkid_p

0.48

0.66

3.17

15kliv_p

2.61

3.92

10.29

16ksto_out

0.105

0.182

0.53

17kfat_p

0.05

0.067

0.13

18kmus_p

1.106

2.24

4.93

19klun_p

0.907

0.457

1.22

20kbra_p

0.032

0.048

0.041

21khea_p

0.132

0.202

0.71

22ktes_p

0.046

0.082

0.084

23kspl_p

15.19

19.007

13.55

24kintg_out

1.19

1.71

2.602

25kint_out

1.12

1.34

1.83

26kduo_p

not calculated

not calculated

not calculated

27kbon_rbc

11.66

17.36

12.42

28kbon_spl

3.53

1.63

1.13

29krbc_spl

11.66

17.36

12.42

3.1.2 The Erythropoietic System

↓77

The injected radioiron in plasma is cleared after approximately 1 hour and most part of its molecules is routed to the bone marrow. Due the intensive production of hemoglobin, the erythrocyte production path represents the greatest iron demand [20 , 21]. According to figures 3.1, 3.5 around 63% of iron is taken up by the bone marrow under normal conditions and 66% under iron deficiency. The value obtained here seems to be lower than in other species [6 , 21, 65].

Under iron-loaded diet the uptake by the bone marrow is decreased to just 35%, that is about half of normal uptake. In figures 3.1, 3.5 it is possible to see a switch of the system into a storage mode [66 ]. This increases the amount of iron absorbed initially by the liver and muscles and diminishes the iron to be used in the erythrocyte forming pathway. A corresponding, but slight, decrease in hemoglobin synthesis was observed under this iron loaded condition [27 ].

Some days after injection, for all three conditions, saturation behavior can be observed on the red blood cell curves (figures 3.2, 3.3 and 3.4). In iron-deficient and iron-adequate mice, around 60% of the injected radioiron dose is present as hemoglobin in the red blood cells, and in iron-loaded mice, just 40%.

↓78

This saturation behaviour instead of a continuously increasing curve suggestsrandom elimination of red blood cells (independent of cell age), in addition to the normal lifespan removal. The bone marrow receives daily an influx of 15;19;14 µg iron (table 3.3). Without this random destruction component, radioiron that returned from other organs would be continuously further incorporated into red cells.

Horký et al. [67 ] studied the red cell lifespan and developed an age independent linear component of cell destruction. This linear element in combination with a life-span determined component produced a rate of red cell elimination of 0.012 per day. Our value is higher: between 0.03 and 0.06 per day. However, these values rely on estimated pool sizes, therefore they are not totally reliable.

After about 30 days the red blood cells are senescent or damaged and need to be removed from circulation, so their iron molecules are liberated and used in the synthesis of new healthy erythrocytes. The murine spleen has a dual role: both removing red cells and colloidal iron from circulation [9 , 68] or as an erythropoietic organ [64 ].

↓79

The spleen contains 5% of the total macrophage population and depending on body iron status the spleen either releases or stores iron originated from the red cell destruction. Table 3.2 shows that under iron deficient diet the spleen releases back to plasma approximately 14.6 splenic pools per day while under iron loaded conditions, this amount decreases to just 1.91 pool units per day.

Another source of iron for the spleen is provided by ineffective erythropoiesis, especially under iron deficiency, when the demand for iron increases. Under iron scarcity many erythrocytes are synthesized in the bone marrow receiving less than the appropriate amount of iron to be functional [68 ]. These red cells do not reach the final stage of maturation and are cleaned from blood by the macrophage system. Table 3.2 shows that the rate of transfer between the bone marrow and spleen increases 10 times between iron-deficient and iron-loaded and 5 times between iron-deficient and iron- adequate conditions. This is evidence that the spleen releases iron based on body status or stores it either as ferritin or hemosiderin [69 ].

It is important to mention that in our model there is no distinction between the macrophage populations and therefore RES is mentioned interchangeably with spleen (figure 2.1). To understand the precise role of macrophage population in each organ would be necessary to carry a cell separation, which is known to be technically very difficult [68 ].

↓80

The erythropoietic contribution of the spleen is a minor factor in terms of body iron turnover and erythropoesis. From tables in 1-3 in Appendix A it can be observed that the amount of iron absorbed by the spleen after 12 hours is not high. The ratio between the tracer content of bone marrow and spleen after 12 hours is respectively 50, 60 and 20 in iron-adequate, -loaded and -deficient mice.

3.1.3 Compartment size of Tracer-Accessible Peripheral Pools

The compartment sizes in our model were estimated inorder to calculate the size of the “tracer-accessible pools”. This should reflect the behavior of newly absorbed molecules whichcan be either stored, used for cellular functions of returned to plasma.

Under steady state assumption, to calculate the tracer accessible pool sizes we need to be aware of:

↓81

With these values, we use equation (7).

Comparing the diets, it can be observed in table 3.4 and figure 3.6 that under iron-loaded diet the amount of absorbed and possibly stored radioiron is increased in every organ considered.

↓82

We can see from table 3.4 (and depicted in fig. 3.6) that the pool sizes can be group mainly into three groups:

Figure 3.6: The calculated pool sizes for the different organs under three diets.

Data obtained from table 3.4.

↓83

TABLE 3.4: The calculated pool sizes for the three dietary regimes.

Compartment size

Fe-Deficient

Fe-Adequate

Fe-Loaded

Red blood cells

380

284

390

Integument

42.2

39.2

36.4

Liver

10.4

28.4

99.1

Bone marrow

6.3

16.3

24.7

Muscles

35.5

14.5

35.1

Intestinal tract

3.7

3.8

8.4

Kidneys

2.4

3.3

13.6

Spleen

1.0

2.6

7.1

Heart

2.2

2.6

4.3

Lungs

2.2

2.4

14.3

Brain

1.6

0.8

1.5

Testicles

0.9

0.9

1.3

Fat

0.5

0.5

1.3

Stomach

0.6

0.5

1.9

3.1.4 Hierarchy of Iron Residence Times in Different Organs

To understand how the organs deal with the injected iron molecules – whether they store it for biochemical processes of return it to plasma – is necessary to asses the time scale of the system. In addition, this provides a realistic idea about the stiffness of the system that is being modeled.

Using the fractional clearance rates from table 3.2 we can calculate the residence time of radioiron molecules in different compartments by equation (4), see methods. Table 3.5 shows that there is a rapid circulation between the plasma, bone marrow and spleen (characteristic time below 1 day), intestinal tract (~2-3 days), an average circulation time in liver, fat, muscles and other inner organs (~5-13 days) and a longer residence time in red cells, brain and integument (greater than 16 days).

↓84

Table 3.5: The calculated residence time that iron molecules spend in the organs of mice held under different diets.

Fe-Deficient

Fe-Adequate

Fe-Loaded

expressed in hours

Plasma TF-bound*

0.05

0.05

0.05

1.2

1.2

1.2

Spleen

0.07

0.14

0.52

1.6

3.3

12.6

Bone marrow

0.4

0.9

1.8

9.9

20.5

43.7

Intestinal tract

4.2

2.8

3.9

Stomach

5.7

2.7

3.5

Lungs

2.4

5.2

11.6

Kidneys

5.0

5.0

4.3

Muscles

32.1

6.5

7.1

Liver

4.0

7.3

9.6

Fat

10.3

7.9

10.1

Testicles

20.5

10.8

15.0

Heart

16.8

13.0

6.1

Red blood cells

32.6

16.4

31.4

Brain

49.7

16.4

35.7

Integument

35.4

22.8

14.0

It is also possible to see from table 3.5 that there is a hierarchy of time scales in the system. The following five groups of time periods are in accordance with literature data [23 , [, [] and can be described as:

↓85

It should be noted that the last aspect, the iron excretion, differs from humans. Miceloose around 0.5 % per day of their iron [23 , 24], while humans loose less (only about 0.1% [24,58,72,73]). A characteristic time of 200 days means a considerable part of murine life-expectancy. So the mouse has an iron depot for its whole life, by contrast to humans.

Another fact that can be observed for a number of organs is that for the most part of organs, the time-hierarchy does not change significantly between the three dietary regimes, also in accordance with the conclusion of [33 ]. However, exceptions are bone marrow, liver, spleen and muscles, where the different diets have an impact on the residence time of iron molecules in the organs.

It is also noteworthy from table 3.5 and table 3.4 that there seems to be two kinetically distinct iron pools in mice: one which comprises about 20% of total body iron and has a residence time between 5 and 25 days and the second which comprises the other ~80% and has a residence time of ~200 days. The first pool type resides in liver, integument, fat and muscles, while the second compartment comprises the brain and especially the red cells compartment.

3.1.5 Comparison of Tracer-accessible pools with unlabelled non-heme

↓86

It is useful to understand how well the newly injected tracer iron mixes with the pre-existing iron in tissues. This gives an idea about how the organ's storage and absorption capacity changes under different dietary regimes. It is assumed that the tracer-accessible pool is a fraction of the total non-heme pool of the organs. To identify the pools that become quickly labelled we rely on the dynamics revealed by the tracer molecules. Table 3.6 provides the amount of non-heme iron for some organs under the different diets in this study.

Table 3.6 shows that the amount of non-heme iron increases from 106 to 1600 µg iron (~ 15 fold-changes) in liver of iron-loaded and -adequate diets. On the other hand, under iron-deficiency the pool is reduced to about half of normal non-heme levels (from 106 to 59.5 µg iron).

A dramatic change also happens in spleen, which under iron-loaded diet accumulates more than 120 µg iron (compared to 17.8 µg and 9.9 µg for iron-adequate and -deficient mice - ~ 7 and 12 fold-change, respectively).

↓87

Table 3.6: Non-heme iron content of mouse (C57B6/L) organs in different dietary regimes. 

Diet

Fe-Deficient

Fe-Deficient

Fe-Adequate

Fe-Adequate

Fe-Loaded

Fe-Loaded

Content

Whole organ

Content

Whole organ

Content

Whole organ

Organ

μg/g wwt

μg

μg/ g wwt

μg

μg/g wwt

μg

Liver*

48.8 ± 5.1

59.5 ± 7.9

86.7 ± 14.9

106 ± 20

1310 ± 90

1600 ± 170

Kidneys

36.9 ± 2.3

14.0 ± 2.0

60.8 ± 2.4

23.1 ± 3.2

88.1 ± 7.1

33.5 ± 5.2

Spleen

141 ± 46

9.9 ± 3.5

254 ± 99

17.8 ± 7.4

1760 ± 240

123 ± 24

Heart

71.2 ± 2.4

10.0 ± 1.5

68.2 ± 7.9

9.5 ± 1.8

81.8 ± 10.5

11.5 ± 2.2

Duodenum

2.9 ± 0.3

Lung

9.5 ± 0.8

Brain

13.4 ± 1.7

Muscle

142 ± 29

Frazer et al. [70 give 56 μg/g wwt. Vácha et al. [71 ] report 257 μg/g wwt. he concentration (μg non-heme iron per g of organ) data are unpublished data (K.Schümann for liver, kidneys, spleen and heart) and Vujic-Spasic & Muckenthaler for duodenum, lung, brain and muscle. The whole organ data were calculated as concentration times organ mass. The standard deviation was estimated from that of both sets of measurements (see methods). Note that liver, kidney and spleen store iron (ferritin) with increasing supply, whereas heart does not.

In kidneys there is a decrease of 10 µg iron (~2 fold-change) between non-heme iron in iron-adequate and -deficient mice; and an increase of ~105 µg iron (~7 fold-change) between iron-adequate and -loaded conditions).

Table 3.7 shows how the tracer-accessible pools relate to non-heme iron pools under the three different diets. The calculation used the values of table 3.4, which contain the estimated tracer-accessible pool sizes.

↓88

We can see that the percentage of the liver compartment accessible to the tracer drops from 27 to 6% when comparing iron-adequate with -loaded condition. The spleen also undergoes a reduction from 15 to 6%.

Furthermore, comparing iron-adequate and -loaded conditions, we can see that the kidneys the heart has an increase in the percentage of compartment size occupied by tracer: from 14 to 41% and from 27 to 27% for kidneys and heart, respectively.

Analyzing the difference between iron-adequate and -deficient conditions, we can verify that the percentage occupied by tracer-accessible pools in some organs decrease (liver: 10%, spleen: 5%, heart: 5%) and in kidneys an increase of 3%.

↓89

Table 3.7: Tracer-accessible Iron Pools compared with non-heme Iron in different dietary Regimes.

Diet

Fe-Deficient

Fe-Deficient

Fe-Adequate

Fe-Adequate

Fe-Loaded

Fe-Loaded

Content

% of non-heme iron

Content

% of non-heme iron

Content

% of non-heme iron

Organ

μg per mouse

μg per mouse

μg per mouse

Liver

10.4

17

28.4

27

99.1

6

Kidneys

2.4

17

3.3

14

13.6

41

Spleen

1.0

10

2.6

15

7.1

6

Heart

2.2

22

2.6

27

4.3

37

The tracer-accessible pool sizes were calculated as described in the text. For the “cold” non-heme iron content refer to table 3.6. It may be noted that upon iron loading non-heme iron in liver and spleen is less accessible to tracer uptake, whereas in kidneys and heart the tracer-reachable fraction increases.

3.1.6 Iron Excretion from the body

Iron, as mentioned before, enters the body by the duodenum and to a lesser extent from other parts of the small intestine. Iron is lost from the body via mucosal exfoliation, skin desquamation and also via bile und urine [72 ]. The iron lost by mice is about 2-5 times greater than in other animals and man [24 , 34, 58, 72-74]. Therefore, it may be hard to develop an iron-overload scenario in mice without a severe mutation.

Table 3.3 shows that the losses through skin and intestine desquamation appear to be in the same range. The amount of iron leaving the body through skin desquamation in iron-deficient, adequate and loaded mice are respectively 1.19; 1.71 and 2.6 µg iron per day.

↓90

The iron losses due intestinal mucosal exfoliation (intestine + stomach) are 1.22; 1.5; 2.3 µg iron per day for iron-deficient, -adequate and -loaded, respectively.

3.2 Simulation Studies with the Cellular Model

The kinetic model of iron metabolism that we have sketched in the methodical section needs the assignment of suitable values for the numerous parameters. We achieved this by applying the pool sizes and flux rates as calculated from tracer data in the previous section. The source of these estimated parameters are the tracer data of Schümann et al. (2007) [27 ], complemented by quantitative statements obtained from the physiological literature on iron metabolism. The flux values and pool sizes are stated in fig. 3.5 and 3.6. They define the quantitative range of the major fluxes in all cell types and tissues and set therefore parameters for input, throughput and output of iron, together with the equilibration of ferritin stores and heme synthesis. Other internal events are of minor quantitative importance, and it is therefore possible to set the parameters in a dimensionless form derived from the reference state of an adult, healthy and well-fed (also in terms of iron supply) mouse.

In this part we report findings of simulations performed for several physiological and pathological conditions: i) to see the effect of considerable blood losses by bleeding, ii) to assess the cellular and physiological changes to the organism under iron deficient or loaded diet, iii) to study the role played by hepcidin and IRP system in iron regulation. All results are obtained as parameter portraits (section 2.22). We present extracts here as percentage changes of certain salient parameter jumps.

3.2.1 Simulation of Chronic Blood Loss

↓91

In our simulations we established a daily blood loss with different degrees of severity. In model terms, a leakage of iron was introduced in the RBC pool. Instead of a complete route of RBC to the RES compartment, there is then a parameter characterizing loss of blood iron from the RBC pool to outside the body.

3.2.2 Erythropoiesis

The constant loss of red blood cells has a strong effect on the erythropoietic mechanism of the body (fig. 3.7). The change in the daily blood loss by 1% of the formation rate causes a parallel decrease of serum iron (transferrin saturation) by a factor of 40%. The consequence of lower transferrin saturation levels is that less iron is circulating and available for heme synthesis in the bone marrow. In this organ, the flux v1 (iron uptake by transferrin receptor 1, TFR1, also shares a parallel decrease and for each percent increased of the daily blood loss v1 level is reduced by 14%.

It can also be observed that the heme export (w10) is reduced in parallel under chronic blood loss: 1% increase in the daily loss of iron reduces the export of heme by 12%. It can be observed in the figure 3.7 that the level of RBC is also reduced in parallel and with 1% blood loss per day, it causes a reduction of 23% in the final level of RBC, where a new steady state is reached.

↓92

The reserve of iron in the bone marrow (Phi1 and Lambda) is also reduced under constant blood loss: 1% increase of the daily loss of iron reduces the levels of ferritin and free iron by 12 and 10% respectively.

The presence of the main receptor of iron in the bone marrow (TFR1) is increased when there is a chronic blood loss. For each percent of iron lost in red blood cells, the expression of this receptor increases by 16%. This happens due the stimulatory effect exerted by Erythropoietin (EPO). This hormone is produced at high levels in hypoxia situations, and in our model for each percent of blood lost on a daily basis, there is an increase of 40% in the level of EPO. And due to this increase, the production of TFR1 is also elevated. This is interpretable as a feedback loop in order to bind and internalize as much circulating serum iron as possible.

 

↓93

Figure 3.7: Blood loss and its relation to body iron status indicators.

RBC, hepcidin and transferrin saturation fall in parallel with increases in the daily percentage of red cell loss. On the other hand, EPO increases almost linearly, due the demand for new erythrocytes and the duodenal iron absorption also increases until it reaches a plateau after 1.8% daily loss.

3.2.3 Recycling of iron

Under normal conditions, the major source of iron for the blood plasma is the Reticulo Endothelial System (RES). The iron released from the macrophages consists of molecules that were recycled from senescent red blood cells and ineffective erythropoiesis. In a chronic blood loss scenario, there is a constant decay in the level of the major source of iron molecules (RBC) that would be recycled and released into circulation.

In our simulations we observed that the v7 flux into RES (sum of iron intake from the two above mentioned sources) decreases in parallel with the constant blood loss: 25% less flux for each percent of blood lost daily. Since the system is in a steady-state, even with the constant blood loss, the same happens with the flux w6, which is the export of iron into circulation.

↓94

The iron stores in the macrophages also suffer a decrease (not shown as parameter portrait), For each percent of iron lost daily, the levels of Phi1 (ferritin bound iron) and Lambda (Labile Iron Pool) decrease by 70% and 84% respectively.

3.2.4 Storage

The iron uptake, storage and the eventual export from the liver decreases with blood loss. It can be observed from our simulations that 1% increase in the daily loss of red blood cells implies almost 20% lesser iron uptake by TFR1(v1 flux). As in the bone marrow, in the liver the expression of TFR1 is also increased . However, this increase in liver cells is much less pronounced than in bone marrow. In the liver the expression of this receptor in the cell membrane increases by around 1% for 1% daily blood loss.

The iron stored in ferritin form (Phi 1) and the cellular labile iron pool (Lambda) are reduced aby bout 18% for each percentual increase in the daily blood loss.

↓95

The iron export from the liver cells through ferroportin is also reduced by 20% for one percent loss of red cells. It was also observed that the level of ferroportin loaded with iron molecules (Fpn1) is reduced; even on simultaneous decrease in the expression of hepcidin, which is known to reduce the expression of ferroportin.

A parallel reduction can also be observed in muscle cells. Ferritin iron (Phi 1) and the labile iron pool (LIP) are reduced by 20% for each percentual increase in the daily blood loss. In addition, the absorption through Transferrin receptor 1 (v1) and export through ferroportin (w6) are also reduced as the same proportion as the iron storage pools (figure 3.8).

Figure 3.8: The reserves of iron are linearly depleted with increases in the daily loss of iron.

Both forms, ferritin and free iron (Phi 1 and Lambda) have their levels decreased with increased loss of red cells.

3.2.5 Absorption

↓96

The absorption of dietary iron in the duodenum is mediated through DMT1 (m0 and m1 variables in our model). Its flux, termed v5 as panel in fig. 3.7, increases with a saturation-like curve witzh the daily loss of red cells, reaching a plateau after 1.8% daily blood loss. With 1% daily blood loss, v5 increases 68% and its plateau is achieved after 80% increase in the dietary absorption.

Due this high increase in dietary iron absorption, the iron stores in ferritin form (Phi1) and the labile iron pool (Lambda) also increase correspondingly with increases in blood loss. For a 1% increase in blood loss, we observe an increase of 50% in Phi1 and Lambda. However, they also reach a plateau after about 1.8% daily blood losses.

The iron export to the plasma is also increased in this blood loss scenario. It was observed that with 1% increase in blood loss there is a 68% increase in iron export from the cell.

3.2.6 Excretion

↓97

The excretion of iron from the body happens through 2 distinct mechanisms: by loss of intestinal mucosa cells and by skin desquamation (integument).

From our simulations we observed that with increases in the daily loss of red blood cells (and consequently less iron circulating in the body), the excretion of iron is diminished. This decrease in iron excretion can be observed in equal proportions both in intestine and in the integument. For a percentual increase in blood loss, the flow of iron from the body to outside (flux w13) is reduced by about 20%.

In addition, it can be observed that the iron stores in the intestine and in the skin are also reduced. Both ferritin iron (Phi 1) and the labile iron pool (Lambda) decrease by more than 20% for each percent of daily loss of red cells.

3.2.7 The new steady state

↓98

The new steady state in our simulations, after a slow ultimate drift, would be reached after many days from the beginning of the daily blood loss.

The half-time necessary to achieve the steady state with a 1% blood loss would be around 29 days. With a greater daily loss (2%), the half-time necessary to achieve the steady state would be around 89 days (figure 3.9). This new steady state has different levels of red cells, well below the reference values.

Figure 3.9: Time necessary for the model to establish a new steady state with a daily loss of iron.

As was demonstrated in the previous sections, the dietary uptake to compensate for this new daily loss is increased but does not overcome totally the demand for new red cells. Therefore, a new state exists were there is less circulating red cells than the reference levels.

3.3 Analysis of changes in dietary iron supply

3.3.1 Absorption

↓99

With changes to parameter K5 of our model, the flux v5 (absorption of dietary iron via DMT1) is either increased or decreased. It can be observed that the value of v5 increases exponentially with changes in K5. Increasing or decreasing the value of K5 by 50% causes a change of 20% (figure 3.10).

The iron stores in the cell, either in ferritin form (Phi 1) or in the labile iron pool (LIP) are also increased or decreased according to the amount of dietary iron absorbed by the intestinal cells. Doubling or reducing the amount of available dietary iron causes an increase by 12% of Lambda and Phi 1.

The export of iron from intestinal cells is also increased or decreased by 20% respectively for higher or lower values of K5.

↓100

While the stores and the iron export from the cells increase with greater values of K5, the opposite happens with the amount of active IRP in the cell (Yeff, representing a sum of IRP1 and IRP2). It can be observed that with lower dietary iron supply, the amount of Yeff is more than linearly increased. This happens because with less iron available in the intestinal cells two processes related to IRP are reduced: first, the conversion of IRP1 from its active form into cytosolic aconitase and second, the degradation of IRP2, which happen with iron levels above a certain limit (figure 3.10).

Figure 3.10: Relation of changes in the dietary iron supply (flux v5, controlles by parameter K5) and iron fluxes/storage/regulation.

With increased iron supply, the levels of ferritin iron and iron export through ferroportin are increased. Meanwhile, the activity of the IRP system decreases due degradation of IRP2 and conversion of IRP1 to its aconitase form. Note that the reference level of the model is at K5=5.

3.3.2 Erythropoiesis

The increased transfer of iron from the duodenum into the circulation is accompanied by greater transferrin saturation. The opposite also holds, decrease in dietary iron supply leads to lower transferrin saturation. In quantitative terms, in both directions, either increasing or reducing the dietary availability of iron by 50% changes the ratio tao1 / (tao1 + tao0) by 12% (Figure 3.11).

↓101

It was observed that with decreased iron supply from the diet there is a rise in the EPO levels. This increase results in more TFR1being expressed in the bone marrow. However, even with increased levels of this receptor, the supply of transferrin iron from the circulation is decreased, since the level of transferrin iron is reduced under lower dietary iron supply.

The storage of iron in its ferritin form and in the labile iron pool is also reduced in bone marrow in the case of lower dietary iron supply. This decrease is not drastic, since the reserve of iron in the bone marrow is relatively small by comparison with other organs like liver or spleen (figure 3.11).

Ultimately, the production of heme in the bone marrow is increased or decreased in the case of higher or lower dietary iron supply, respectively. The change leads to higher production of red blood cells, which are increased or reduced in 10% for 50% more or less dietary supply, respectively (figure 3.11). This tendency would be damped by a higher cooperativity (exponent of power law) of the EPO-loop as so far assumed in the model.

↓102

Figure 3.11: With increases in duodenal iron supply, there is an increase in the iron absorption in the bone marrow through TfR1, its ferritin stores also increases and consequently the RBC levels are also increased.

However, the expression level of TfR1 (alpha) at the cell membrane is diminished due the decrease in the EPO levels. In addition, the transferrin saturation is also increased, providing more iron to every organ in the model with exception of RES (which receives more, but indirectly). Note that the reference level of the model is at K5=5.

3.3.3 Recycling

With increased or decreased iron supply by the diet, the absorption of iron by the RES macrophages is also either increased or decreased. The flux v7, which represents the sum of RES iron uptake from senescent red cells and from bone marrow, due toineffective erythropoiesis follows the same trend as the flux v5 from duodenum (figures 3.10 and 3.12).

In addition, the stores in the RES are also reduced in case of lower iron supply in the diet. This reduction in both forms of cellular iron (ferritin and labile iron pool) is very strong: changing the iron supply by 50% causes changes in Phi 1 and Lambda by 66 and 40% respectively.

↓103

It can also be observed that the release of iron from RES is also decreased in case of reduced iron supply by the diet. However, for a change of 50% in the dietary supply, there is a change of just 10% in the level of iron export from the RES.

Figure 3.12: With increased duodenal absorption there is an increase in the iron supply in the RES, due ineffective erythropoiesis.

It can also be observed that the ferritin level in the RES system is also increased. The same happens with the export of iron to blood plasma, it follows the same trend as the two other previous indicators. Note that the reference level of the model is at K5=5.

3.3.4 Storage

In a scenario of reduced iron supply from the diet, the body stores present mainly in the liver will also be depleted. Both forms of iron, in its ferritin form and also as labile iron pool are reduced according to the decrease in iron supply in the intestinal cells. Changing the dietary iron supply in 50% causes a change in lambda and Phi 1 by 10 and 14% respectively.

↓104

In addition, the export of iron from liver cells is also reduced in the case of lower duodenal iron supply as can be observed in figure (figure 3.13).

Figure 3.13: The reserved in the liver and muscles are increased with higher duodenal iron absorption.

Iron in its two forms present in our model, ferritin and the labile iron pool have higher values after changes to the duodenal iron supply. Note that the reference level of the model is at K5=5.

3.3.5 Excretion

Iron losses from the body are also reduced or increased according to the amount of iron absorbed from the diet. In organs like the intestine or the skin (integument), the iron stores in ferritin form or the labile iron pool are either increased or decreased, following the same trend of the flux v5 in the duodenum (figure 3.14).

↓105

In our model, we can observe that the iron excretion from the body, represented by the flux w13, is also increased or decreased on higher or lower dietary iron absorption. Changing by 50% the amount of iron absorbed in the duodenum causes a change of 18 and 14% in the rate of iron loss in the integument and intestine, respectively (figure 3.14).

Figure 3.14: With increased iron levels in the body, the excretion level through skin desquamation and mucosal exfoliation are also raised.

Note that the reference level of the model is at K5=5.

3.4 Hepcidin Studies

3.4.1 Hepcidin seems not to be active in Liver Hepatocytes

Ramey and collaborators [75 ], have published experiments. where hepcidin targeted ferroportin for degradation in liver hepatocytes. If this would be operative in our model, then a hepcidin knock-down should lead to higher expression of liver ferroportin and consequently to lower iron levels in this organ. This would not explain , however, the observed in crease [76-78 ]of iron in hepcidin hemochromatosis. The paper [79], on the other hand, suggests that the hepcidin effect is different dependent on the target organ. In our model it can be shown (fig. 3.15) that with hepcidin acting in liver hepatocytes, there is no increase in stored liver iron, compared with the implementation where hepcidin acts only in RES and duodenum.

↓106

Figure 3.15: Comparison of different model configurations.

If hepcidin also targeted ferroportin in hepatocytes for degradation, there would be not enough iron accumulation to characterize the iron overload reported in several studies [76 - 78 ]. Note that the reference (normal) level of hepcidin expression is at U8=0.3.

3.4.2 DMT1 and ferroportin expression changes

As demonstrated in two different studies [53 , 79], the increase in hepcidin levels leads to a decrease in the expression level of DMT1. It can be observed that with increased levels of hepcidin, the level of DMT1 is reduced by about 10% and with steps toward a hepcidin knock-out mouse there is an increase in the level of DMT1 by up to 50% of its original value (figure 3.16).

The level of ferroportin in the duodenum is also inversely correlated with the level of hepcidin in the organism [53 ]. “Level of hepcidin” in the organism is ambiguous, when related to blood or urine measurements [80 , 81]. It probably refers to the total production in a given time period, and therefore a measure of the turnover rate. Changing the expression levels of hepcidin in our model we obtained the same as their study: reducing hepcidin we observe an increase in the expression level of ferroportin (figure 3.16). This increase in iron transported was also confirmed experimentally by [77 ].

↓107

Reducing the level of hepcidin we observe an increase in the expression of ferroportin, leading to an increase in the iron transport from the basolateral membrane to the plasma (figure 3.17).

Figure 3.16: the increase and decrease in hepcidin expression is inversely followed by expression levels of DMT1 and ferroportin in duodenum.

Near an absolute lack of hepcidin there is an exponential increase in the expression of those two proteins. Note that the normal level of hepcidin expression is at U8=0.3.

3.4.3 Iron in Spleen

According to [78 ] and [77 ] in case of lack of hepcidin it can be observed that the iron level in the spleen is decreased. In our simulations we could observe the same effect (figure 3.17), decreasing the hepcidin levels there is a correlated decrease in the ferritin iron stores in spleen (Phi1).

↓108

This decrease in the iron stores in the RES can be explained by the increase in ferroportin levels and consequently the efflux of iron from the spleen macrophages. This was also confirmed experimentally by [78 ] and [77 ] and can be seen in (figure 3.17). However, these results are in conflict with the findings of [76 ] who observed no changes in the RES iron stores of mice not expressing hepcidin as consequence of the ablation of the HFE gene.

Figure 3.17: with lack of hepcidin there is an increase in the levels of the membrane iron transporter ferroportin.

Consequently the ferritin iron stores in the RES are decreased. Note that the normal level of hepcidin expression is at U8=0.3.

3.4.4 Transferrin Saturation and Erythropoiesis

It was reported [78 ]) [76 ] that the transferrin level of mice lacking hepcidin expression is increased. This could be observed in our simulations where the level of transferrin saturation (Tao1 / ( Tao 0 + Tao 1) in our model) increased according to the reduction of hepcidin in the organism (figure 3.18). This is a characteristic of hemochromatosis and an indicator used to diagnose this disorder.

↓109

However, those same studies reported that there were no changes in the level of RBC in mice lacking hepcidin expression. In our model that there is an increase in the level of RBC in mice lacking hepcidin. However, the increase is not very high until the level of hepcidin is extremely low, a most severe condition where other factors not included here may interfere. Therefore, from (figure 3.18) we may conclude that the problem could be solved in future versions of the models by adapting the cooperativity parameters of EPO signaling or changing the kinetic equations in the expression of TfR1 in bone marrow.

Figure 3.18: at very low values of hepcidin expression we can observe an increase in the erythrocytes and in the transferrin saturation.

These two events are related since with higher serum iron there is more uptake by the bone marrow, which produces an increased number of red cells.

3.5 IRP Studies

According to several studies [44 , 82], IRP 2 dominates the intracellular iron homestasis in mammals. The mentioned studies performed IRP 1 and IRP 2 ablations and concluded that in most part of tissues IRP 2 expression can be increased in order to compensate the lack of IRP 1. However, the opposite is not true: in absence of IRP 2, IRP alone is not able to maintain alone the control of TfR1, DMT1 and ferritin levels  [44 ].

↓110

This feature was implemented and tested in our model. In the reference state IRP1 exerted an effecte that was responsible for 5% of all IRP activity in the cells, while IRP 2 was responsible for the other 95%, in agreement with [56].

With those values we were able to simulate and observe most of the results reported in the literature. Below is a summary of our observations and comparison with the literature.

3.5.1 Transferrin Saturation and Erythropoiesis

The transferrin saturation in our model expressed by (Tao1 / ( Tao 0 + Tao 1)) was not changed when we changed the expression of IRP 1 or IRP 2 to either lower or higher values (figure 3.19). This observation is accordance with the studies of [83 ] and [42 ].

↓111

In addition the RBC levels are reduced when there is a decrease in IRP 2 expression (figure 3.19). This observation was also reported by [83 ] but not by [42 ], who mentioned that there the RBC levels were unchanged in IRP2-/- mice.

Figure 3.19: with lack of IRP2 expression in tissues there is a decrease of RBC despite the fact that no change can be observed in the transferrin saturation.

Please note that the normal level of IRP2 expression is at U2=1.0.

3.5.2 Duodenum

In the study of [83 ] it was observed that the level of ferritin heavy and light chains change when there is a lack of IRP 2 expression. But we do not differentiate in our model the two different isoforms of ferritin, we could observe that there is a decrease of this protein when we simulate an ablation of IRP 2 (figure 3.20).

↓112

Two differences between our model and the experimental results published by [83 ] refer to the levels of DMT1 and ferroportin. In their paper, they report that in the IRP2-/- mice, the level of DMT1 and ferroportin is unchanged. However in our model we observed that the level of DMT1 is decreased with lower expression levels of IRP 2 (figure 3.20). This difference can be due the different isoforms of DMT1 (with or without IRE, alternative splicing. [84 ]). In addition, in a different study where a conditional knock-out targeting only the IRP system in the intestine was made [85 ], it was observed that DMT1 is decreased, in accordance with our simulaion.

The same happens with ferroportin level, where [83 ] report that it is unchanged and in our simulation we found it to be decreased with lower IRP 2 levels (figure 3.20).

These differences confirm the point of view of [44 ] who pointed out that there are important characteristics that are not totally understood regarding the role of IRP1 and IRP2 in different cell types.

↓113

Figure 3.20: with lack of IRP2 expression there is a decrease in ferroportin and DMT1, however, there is an increase in the level of ferritin iron which corresponds to the observations of [83 ]. Note that the normal level of IRP2 expression is at U2=1.0.

3.5.3 Liver

Simulating the ablation of IRP 2 gene in the whole organism we observed in our model an increase in the level of ferritin in the liver (figure 3.21). This observation is in accordance to the experimental studies of [83 ] and [44 ].

In the study of [83 ] it was also reported that the level of TfR1 in liver hepatocytes is reduced under lower expression of IRP2. This characteristic could be also observed in our model (figure 3.21). Since IRP2 exerts a protective role in the mRNA of TfR1, it is expected that there is a decrease in the protein level of this transporter.

↓114

The hepcidin levels in our model and in the experimental model of [83 ] are also in accordance. In their work they reported that there is a slight increase in the expression level of hepcidin, but it was not high enough to be statistically significant. In our model we have the same: there is an increase but not enough to be considered important (figure 3.21).

The only divergence between our model and the experimental work published by the above-mentioned authors refers to the ferroportin level. They report that there is no difference in the expression level of ferroportin in the liver of IRP2 ablated mice, while our theoretical simulations show that the level of ferroportin would be severely reduced under lower expression of IRP 2 (figure 3.21). Since the IRP system has a protective role over the mRNA of ferroportin it would be expected that the level of this protein would be reduced. This discrepancy sugggests the existence of a modified biological mechanism for the regulation of ferroportin in hepatocytes. This mechanism was not yet found. It may involve different preferences for IRP 1 in this molecule and cell-type.

Figure 3.21: with lack of IRP2 expression there is a decrease in ferroportin and TfR1, since the IRP system provides protection of the mRNA of those genes against degradation. However, just the decrease of TfR1 is in accordance with [83 ].

There is an increase in the level of ferritin iron, in accordance to the experimental work of the same author. In addition, there is no change in the hepcidin level. Note that the normal level of IRP2 expression is at U2=1.0.

3.5.4 Spleen

↓115

There is some divergence between the observations in the spleen compartment of our model and the measurements on spleen reported by [83 ]. They reported that the level of ferritin iron in the spleen decreases under IRP 2 ablation. In our model the opposite effect obtained: the level of ferritin increased. In addition, the same authors report that the level of ferroportin is also decreased under this mouse construct, while in our simulations we observed that the level of ferroportin is not changed with lower IRP 2 expression values (figure 3.22).

The only agreement between our model and the experimental data concerns the iron acquisition by the spleen. In our model this originates as “ineffective erythropoiesis”. Galy and collaborators [83 ] report that this acquisition is reduced in IRP 2 ablated animals and the same observation was made in our simulations (figure 3.22).

Again, these differences direct us to the conclusions of [44 ] who pointed out that there are characteristics that are not understood the IRP system in different organs.

↓116

Figure 3.22: with lack of IRP2 expression there is a decrease in ferroportin and in the iron acquisition.

It can also be observed an increase in the ferritin levels in the spleen macrophages which is not in accordance to the literature. Note that the normal level of IRP2 expression is at U2=1.0.

3.5.5 Bone marrow

In the studies of [83 ] and [42 ] there is one point of agreement and one of divergence. Both reported that in erythroid precursors the level of TfR1 is reduced in mice with IRP 2 ablation. This is also in accordance with our simulations and can be explained by the fact that the IRP system prevents the mRNA of TfR1 of getting degraded (figure 3.23).

The observed fact that is different between the two papers deals with the level of ferritin protein: while [42 ] reports that the level of this protein increases, [83 ] observes that its level did not change with IRP 2 ablation. Our simulations are in accordance with [83 ], since we observed no change in the level of this protein (figure 3.23).

↓117

Figure 3.23: In IRP 2 knock-out mice there is no change in the level of ferritin iron in the bone marrow.

However, there is a strong reduction in the level of TfR1. Both observations are in accordance to the study of [83 ]. Note that the normal level of IRP2 expression is at U2=1.0.

3.6 IRP and Hemochromatosis

In the study of [82 ] it was reported some interesting characteristic was reported between the interaction of the IRP system and the lack of hepcidin expression in patients suffering from hereditary hemochromatosis.

The first is that in macrophages and monocytes of HH patients there is an increase in the level of IRP 1 and IRP 2. In our model the effect of both IRPs is summarized in the term Yeff. We can observe in (figure 3.24) that with lower expression of hepcidin there is an increase in the level of effective IRP in spleen macrophages, observation which is in agreement with the experiment.

↓118

The second fact reported by [82 ] is that in the duodenum of both anemia and hemochromatosis patients there is an increase in the level of IRP activity. In our simulations we could also observe the same effect. Reducing the expression of hepcidin or reducing the intake of iron from the diets caused the pathological effects and could explaion the values of IRP activity in duodenum (figure 3.24).

Figure 3.24: In the duodenum of hereditary hemochromatosis or anemic patients there is increased activity of IRP activity.

The same happens in the macrophages of patients with hemochromatosis. Please note that the normal hepcidin level occurs with U8=0.3 and the normal daily food intake is settled at K5 = 5;


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