Abstract 

DEPOT IRON IN THE RAT LIVER AFTER HYPERTRANSFUSION WITH 
RAT ERYTHROCYTES 
 

By F. M. J. ZUYDERHOUDT, J. VAN GOOL, and G. G. A. JORNING.

From the Laboratory of Experimental Medicine, University

 Full Text (PDF)

Author Affiliations

1. Laboratory of Experimental Medicine, University of Amsterdam, Wilhelmina

Gasthuis, Amsterdam, The Netherlands

Abstract 

In the rat liver the deposition of iron was measured after hypertransfusion with rat
erythrocytes. The liver iron fractions were studied during four weeks after the
hypertransfusions. In the first week the haemosiderin iron fraction increased together with the
ferritin iron fraction. Most iron was deposited as ferritin iron. In the last week of the
experiments, while the ferritin iron fraction still increased, the haemosiderin iron fraction
decreased. At the same time plasma iron was utilized when erythropoiesis, which had been
suppressed by the hypertransfusion, recommenced. It is suggested that, under these
experimental conditions, liver haemosiderin iron is used in haemoglobin synthesis.

The authors wish to thank Mr J. G. de Haan and Mr L. Flens for the performing of the
transfusion experiments and Mrs J. Plomp for preparing the histological liver slices.

Footnotes 

  
o Received June 14, 1976. 
Quarterly Journal of Experimental Physiology (1977) 62, 65-72

DEPOT IRON IN THE RAT LIVER AFTER HYPERTRANSFUSION

WITH RAT ERYTHROCYTES. By F. M. J. ZUYDERHOUDT, J. VAN GOOL,
and G. G. A. J6RNING. From the Laboratory of Experimental Medicine, Uni-
versity of Amsterdam, Wilhelmina Gasthuis, Amsterdam, The Netherlands.

(Received for publication 14th June 1976)

In the rat liver the deposition of iron was measured after hypertransfusion with rat erythrocytes.
The liver iron fractions were studied during four weeks after the hypertransfusions. In the first
week the haemosiderin iron fraction increased together with the ferritin iron fraction. Most iron
was deposited as ferritin iron. In the last week of the experiments, while the ferritin iron fraction
still increased, the haemosiderin iron fraction decreased. At the same time plasma iron was utilized
when erythropoiesis, which had been suppressed by the hypertransfusion, recommenced. It is
suggested that, under these experimental conditions, liver haemosiderin iron is used in haemo-
globin synthesis.

The liver is the most important organ in iron-storage [Barry, 1974]. When a
patient needs repeated blood transfusions, while no iron-shortage exists, liver
iron increases [Barry, 1974]. As in the case of young, regularly transfused,
thalassaemia patients iron overload is an important life limiting factor [Letsky,
Miller, Worwood and Flinn, 1974]. So it is of great importance to know the
mechanism by which the iron depot increases after blood transfusions.
In the liver several iron fractions are detectable which can be divided into
three groups: (i) ferritin iron; water soluble protein molecules containing iron
[Crichton, 1971]; (ii) haemosiderin iron; this is more difficult to define, but it is
often characterized as a water insoluble iron fraction [Richter and Bessis,
1965] or as the non-haem, non-ferritin iron fraction [van Wijk, Linder-Horo-
witz and Munro, 1971; Cook, Hershko and Finch, 1973]; (iii) enzyme bound
iron; this fraction can be divided into two groups: the haem-containing en-
zymes like cytochromes and the non haem-containing enzymes like succinate
dehydrogenase.
Whatever the cause of liver iron loading may be, the ferritin and sometimes
also the haemosiderin iron fractions will increase; this is shown in human liver
[Barry, 1974; Morgan and Walters, 1963] as well as in rat liver [van Wijk et al.,
1971; Cook et al., 1973]. The change of these fractions, after blood transfusions
is not well known, especially when studied for long periods. Because of the
difficulty in obtaining human material we used hypertransfused rats as a model
and studied the changes in liver depot iron for four weeks after transfusion
of erythrocytes in normal rats. We measured ferritin, haemosiderin and haem
iron. Some haematological parameters expressing characteristics of iron
metabolism were included.
65
F o

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66 Zuyderhoudt, van Gool and Jorning
METHODS
Overnight fasted male Wistar rats (260-290 g) were hypertransfused, under Nembutal(R)
anaesthesia, via the dorsal penis vein with 6 ml packed rat erythrocytes. Twenty-four hours
after the first transfusion each rat was once more transfused in the same way. The erythro-
cytes to be used were washed three times with 0-15 mol . I 1 NaCl and the cells were packed
by centrifuging at 2000 g for 5 min. With two transfusions each rat received approximately
10,000 pg haemoglobin iron. Control rats received twice the same volume of 0-15 mol .I-
NaCl. The livers were perfused by portal vein in situ with 0 15 mol .I -I NaCl to remove the
blood. A part of every median lobe was used for histological study of iron depot granules, if
present. Staining for iron was with Perls' stain.
Haematocrit, haemoglobin, plasma iron and total iron binding capacity were measured
with standard clinical chemical methods. Ferritin protein, ferritin iron and the percentage
of iron in ferritin were determined as described before [Zuyderhoudt, 1975]. Total liver iron
was measured on liver homogenate using a wet ashing method [Zuyderhoudt, 1975]. Haemo-
siderin was estimated by subtracting the ferritin iron and haem iron from total liver iron.
This haemosiderin value, measured as the non-haem and non-ferritin iron fraction [van
Wijk et al., 1971; Cook et al., 1973] includes the non-haem bound enzyme iron fraction.
There is no indication that this small fraction will be influenced by an enlarged liver depot
iron concentration. Haem iron was estimated by determining the liver haem concentration,
with rat haemoglobin as a standard. This method [Rimington, 1942], used for rat liver homo-
genates by van Wijk et al., [1971], did not yield reproducible results in our hands because of
turbidity. The procedure was extended by adding desoxycholate (end concentration
0 25 mol .I1) to the NaOH in which the liver homogenate has to be dissolved. This was
followed by centrifugation for 10 min at 15,000 g. The extended procedure resulted in
satisfactory recovery of added haemoglobin (deviation less than 4%). The extinction coef-
ficient of rat haemoglobin haem and rat cytochrome c haem differed by less than 5 %. All
tissue concentrations are presented per g liver, wet weight.
The rats received the transfusions on the first and second day and the groups, of 5 rats,
were sacrificed on the 3rd, 6th, 14th, 21st, and 28th-day. Groups receiving erythrocytes are
described as (hyper) transfusion groups and those infused with 0 15 mol .h1 NaCl are
designated as control groups.
Isotope experiments on the 28th day: six hypertransfused rats and three control rats re-
ceived 0 5 ml normal rat plasma containing 5I9Fe. The plasma transferrin was labelled with
59Fe after Hershko, Cook and Finch [1973].
After five days the rats were sacrificed and I 'Fe activity measured in 1 ml samples of blood,
plasma, liver homogenate and in the heated and centrifuged homogenate. The 59Fe in the
heat supernatant (ferritin fraction) was corrected for the ferritin loss in the heat coagulum
[Zuyderhoudt, 1975].
Every point in the figures represents the mean of a group of five observations with the
standard error of the mean (S.E.M.) of the groups. Differences between means were tested
for statistical significance by Student's t test.

RESULTS
The mean weight of the livers in every hypertransfusion group did not change
during the experiment and was equal to that of the control groups. Mean
liver weight in all transfusion rats was 9-11 +±052 g and in the control rats
9.07 ±064 g. Therefore liver iron depot values can be studied and expressed in
tissue concentrations such as pg/g liver.

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68 Zuyderhoudt, van Gool and Joining
Ferritin iron andferritin protein:
Fig. 1 shows that the increase of the total liver iron is mainly caused by
the increase of the ferritin iron depot. Although total liver iron did not change
during the last fourteen days the mean ferritin iron fraction tended to increase
steadily. In accordance with this the highest ferritin protein concentration
was also found in the last week (Fig. 2).

Haem iron:
This fraction remained constant during the experiment in both hypertrans-
fusion and control rats. The mean value in the hypertransfusion rats was
102 +02 pg Fe/g liver, and in the control rats 9-8 +±04 pg Fe/g liver. The
difference is not significant.

Haemosiderin iron:
In the first week after hypertransfusion this fraction increased rapidly and
after reaching its highest value on the 14th day started to decrease (Fig. 3).
The mean value on the 14th day was, compared with the tissue concentration
on the 3rd day, increased by 25 pg Fe/g liver (P < 001); it was 29 pg Fe/g
liver higher than the mean value on the 28th day (P < 0005). Thus haemo-
siderin after an increase in the first two weeks showed a significant decrease
especially in the last week of the experiment.
Haemoglobin and haematocrit:
Both these parameters (Fig. 4) indicated a slow breakdown of the erythro-
cytes and at the 28th day almost normal values had been reached.

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70 Zuyderhoudt, van Gool and Joining
Plasma iron and iron saturation percentage:
The iron binding capacity did not change significantly during this experiment
and showed no difference between hypertransfusion and control groups. Thus
the changes in plasma iron and in the saturation percentage behaved similarly.
Having reached a peak value around the 14th day the plasma iron and the
saturation percentage decreased in the last week by 9 ,umol Fe/l plasma (P <
0'05) and 15% (P < 002) respectively (Fig. 5). We did not expect such a
decrease in a period of constant breakdown of erythrocytes (Fig. 4). It could
be explained by a restart of erythrocyte production. Thus we examined this
decrease of the plasma iron concentration using 59Fe labelled rat plasma (see
below).
Isotope study:
It is well known that erythropoiesis in hypertransfused rats is suppressed
effectively [Hershko et al., 1973]. Although the haematocrit value on the 28th
TABLE I
Percentage of the injected 59Fe dose, at five days after the injection of
labelled normal rat plasma
Hypertransfusion
Control group group Significance of
n=3 n=6 the difference
Plasma <0.1 <0.1
Whole blood 74 + 4 52 + 5 P < 001
Liver 9 1 17 +1 P<0005

day was above the control value it might be possible that erythropoiesis started
again (the haematocrit value almost reached the control level, Fig. 5). This
could explain, in the last week, the fall in plasma iron concentration. To test
this hypothesis we estimated the erythropoiesis by counting the blood 59Fe
activity, five days after transfusion (28th day) of 59Fe labelled rat plasma (see
Methods). Because plasma 9Fe activity was very low (Table I) whole blood
activity was used to measure 5 9Fe incorporation in newly formed erythrocytes.
From Table I it can be concluded that around the 28th day the erythropoiesis
was again 70 % of the control value. This indicates that plasma iron can be used
in haemoglobin synthesis which, very probably, accounts for the decrease in the
plasma iron concentration.
In the hypertransfusion group more labelled iron was incorporated in the liver
as compared with the control rats (Table I), of which 99 ±3 % proved to be
ferritin iron. In the control group 84 +1 % of the liver 5"Fe activity was ferritin
iron. There was no significant difference between those groups in specific
"Fe ferritin activity.-
Histology of the liver:
It is well known that after blood transfusion the iron excess is found especially

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FIG. 6. Representative picture of a liver slice, from the liver of a hypertransfused rat (14th day)
stained for iron. 0
Black spots indicate iron accumulation.

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 Liver depot iron after hypertransfusion 71
in the Kuppfer cells. Haemosiderin is the iron fraction in the tissues that is
often described as coarse, stainable iron granules [Richter and Bessis, 1965].
In histological liver slices these granules can be visualized by staining for iron.
We estimated this fraction as the non-haem and non-ferritin iron fraction and
tried to find a relation between the histological appearance and the measured
iron values.
On the third day no liver iron, in hypertransfusion and control groups,
could be detected by histological methods. The control rat livers remained
negative for iron during the experiment.
On the 6th day in the livers of the hypertransfused animals iron granules
became visible in the cells of the reticuloendothelial system. This probably
corresponds with the increased measured haemosiderin iron (Fig. 3). After the
6th day the livers of these rats remained iron positive. No iron deposition could
be detected in the liver parenchymal cells (Fig. 6). It proved to be impossible
to quantitate the rather low grade of iron deposition in these liver slices. Thus
we could not prove with histological methods a lowering of haemosiderin
content of the liver in the last week of the experiment.

DISCUSSION
As might be expected the plasma iron concentration increased in the hyper-
transfused rats, caused by the suppressed erythropoeisis and the increased
erythrocyte breakdown [Lipshitz, Simon, Lynch, Bothwell and Charlton, 1971;
Cooke et al., 1973]. The large increase of the liver depot iron in the first two
weeks and especially in the first six days might have been caused by rapid
sequestration of the least viable transfused erythrocytes. Most of the deposited
iron appeared in the form of ferritin (Fig. 1, 2). After the 6th day more iron was
incorporated per ferritin molecule: the mean value in the hypertransfused rats
is 1923 atoms of iron/ferritin molecule (n = 15) against 1724 atoms of iron/
ferritin molecule (n = 15) in the control rats (P < 0 01). The haemosiderin
fraction increased, especially in the first six days, and decreased after the 14th
day. It is remarkable that only in the first week, the period of rapid iron depot
forming, did the haemosiderin fraction increase significantly. Because stainable
iron, in the liver sections, is only visible in cells of the reticuloendothelial
system (Fig. 6) it is reasonable to assume that most of the haemosiderin iron
is located in these cells; these cells belong to the cells in the liver which are
active in erythrocyte phagocytosis [Hershko et al., 1973].
The most remarkable data of our experiments concerning liver ferritin and
haemosiderin are those of the last week of observation. During that period
erythropoiesis has started again, as shown by the isotope studies, and this ex-
plains the lowering of plasma iron level. In the liver, however, ferritin increases
while haemosiderin is diminished in this phase. The decrease of the haemo-
siderin iron concentration is in agreement with the observations of Bradford,
Elchlepp, Arstila, Trump and Kinney [1969]; using electron microscopy,
these authors showed that some time after dietary iron loading liver haemo-

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72 Zuyderhoudt, van Gool and Jorning
siderin decreased. They suggested an escape of haemosiderin particles from the
liver into the bile. We do not know if this also occurs in our experiments, but
our observations suggest that during erythropoiesis haemosiderin iron can be
used in preference to ferritin iron. This indicates that haemosiderin can parti-
cipate actively and rather rapidly during iron mobilization.

ACKNOWLEDGMENT
The authors wish to thank Mr J. G. de Haan and Mr L. Flens for the performing of the
transfusion experiments and Mrs J. Plomp for preparing the histological liver slices.

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