European Journal of Clinical Investigation (1993) 23, 32 1-329
REVIEW
Pathogenesis of genetic haemochromatosis
W. STREMMEL, H. D. RIEDEL, C. NIEDERAU & G. STROHMEYER, Department of Medicine,
University Hospital of the Heinrich-Heine University Dusseldorf, Germany
Received 27 August 1992 and in revised form 1 February 1993; accepted 8 February 1993
Abstract. Genetic haemochromatosis is an autosomal
recessive inherited iron overload disease. The genetic
defect and the underlying metabolic error are not
known. Several observations indicate that the 2-4-fold
increase of iron absorption is due to a regulatory defect
of a membrane iron transport system in duodenal
mucosal cells. The key pathophysiologic factor may be
the increase of gut-derived non-transferrin bound iron
liganded to low-molecular mass organic molecules. A
putative membrane carrier protein for non-transferrin
bound iron was identified and preliminary data suggest
its enrichment in plasma membranes of human muco-
sal cells as well as in liver and other organs which are
affected in genetic haemochromatosis. Cellular ac-
cumulation of ionic iron leads to peroxidative decom-
position of organelle membrane phospholipids with
the consequence of cell degeneration and cell death.
Impairment of organ function and structural altera-
tions such as cirrhosis of the liver are clinical manifes-
tations.
Keywords. Haemochromatosis, iron metabolism, lipid
peroxidation, membrane transport.
Introduction
Incidence and genetic defect
Genetic haemochromatosis is a common inherited
disorder of iron metabolism. The mode of inheritance
of the haemochromatosis trait is autosomal recessive.
The gene defect has been localized to chromosome 6 in
proximity to the HLA-A3 histocompatibility locus [ 11.
Based on pedigree analyses and HLA typing carried
out in Europe, USA and Australia it was shown that
HLA-A3 occurred in about 75% of patients with
haemochromatosis compared to 28-30% in controls
[l]. In various studies it was estimated that the gene
frequency is 557% and that about 0.3-0.45% of the
population is homozygous for haemochromatosis
[1,2]. Those studies have in addition permitted defini-
tion of haemochromatosis as homozygosity for the
mutant allele. HLA-typing is also used as diagnostic
Correspondence: Professor Dr W. Stremmel, Medizinische Klinik
der Heinrich-Heine Universitat Diisseldorf, Moorenstrasse 5 , 4000
Diisseldorf, Germany.
tool for screening in families of haemochromatotic
patients [ 1,3].
Despite the success to localize the haemochroma-
tosis gene on the short arm of chromosome 6 in very
close physical proximity (less than 1 centimorgan) to
the HLA-A locus [l], the structural genetic mutation is
still unknown. The resulting biochemical defect is also
not defined. The net pathophysiologic effect of homo-
zygosity for the haemochromatosis allele is iron over-
load of the organism. In advanced symptomatic
disease total body storage iron may be increased to 20-
40 g, compared with approximately 1 g of storage iron
in normal subjects [4].
Physiology of iron metabolism
Under physiologic conditions iron balance is regulated
through control of iron absorption. Absorbed iron is
highly conserved by the organism since no specific
mechanisms for iron excretion are available except the
obligatory losses through exfoliation of gastrointesti-
nal mucosal cells, with smaller amounts being lost
through bile, urine and skin [5]. These losses of 0.5-2
mg per day are balanced by controlled absorption of
0.5-2 mg of iron per day by the upper intestinal
mucosa from a diet containing 10-20 mg of iron [6,7].
Iron stores and iron absorption appear to be recipro-
cally related: as iron stores decline absorption in-
creases and vice versa [7-131. Similarly, increased
plasma iron turnover is associated with increased iron
absorption. Despite these phenomena the mechanisms
of regulation of iron absorption, including signal
transduction and involved mediators, are not known.
Absorption of dietary iron
In Western diets iron is present in equal amounts as
haeme iron (haemoglobin and myoglobin in meat) and
non-haeme iron (vegetables, fruits, cereals). Haeme
iron is taken up by the mucosal cells via endocytosis.
The haeme complex is then degraded by haeme
oxygenase, and Fez+ is released for absorption in the
body [ 14,151. Non-haeme is solubilized in the acid
milieu of the stomach. This ionized iron (predominant-
ly ferric iron) is complexed to small molecules such as
ascorbic acid, citric acid, peptides, amino acids, sugars,
32 1
322 W. STREMMEL et al.
fatty acids, and bile acids serving to prevent iron from
precipitation [5,6,16]. This is of particular importance
in the pH-neutral environment of the duodenum where
the actual absorption process takes place [I 71.
Uptake of iron by mucosal cells involves several
steps: (a) presentation of chelated iron to the cell
surface; (b) binding and translocation across the
microvillous membrane of the mucosal cell; (c) intra-
cellular transport bound to an intracytoplasmic iron
binding factor; (d) release at the basolateral site of the
plasma membrane. In studies with human microvillous
plasma membrane vesicles from duodenum it was
recently shown that the essential step of translocation
across the apical plasma membrane represents a
facilitated, high affinity transport system [ 181. A
glycoprotein composed of three 55 kD subunits was
described as the responsible carrier protein [18]. It was
shown that this protein is regulated by the body iron
status with high expression in iron deficiency anaemia
and downregulation in secondary iron overload dis-
ease [19]. After translocation across the plasma mem-
brane iron has to be complexed to an intracytoplasmic
iron binding factor to prevent cell damage by the
formation of hydroxy radicals. Whether this factor
represents a protein or a suitable organic chelator is
still unclear. The cytoplasmic transit-iron pool is of
significance for intracellular iron metabolism, because
it coordinates the distribution of intracellular iron
according to the cellular needs [20].
Iron storage proteins
Intracellular iron storage proteins are ferritin and
haemosiderin which are of particular importance in
the liver [21,22]. Ferritin is a soluble cytoplasmic
protein serving as pure iron storage protein with the
ability of rapid accumulation and release of iron by a
yet unknown molecular mechanism [23]. Structurally,
ferritin is a spheric complex composed of 24 apoferri-
tin monomers of the isotypes H (heavy, 21 kD) and/or
L (light, 19 kD) with a total apparent molecular weight
of 450.000. To a small extent the complex ferritin
molecule is released in plasma from which it is taken up
by a specific ferritin receptor into certain cell types, e.g.
hepatocytes [24,25]. Haemosiderin is present as an
insoluble complex in secondary lysosomes, particu-
larly in liver, serving as long-term or even final storage
form of iron. It is assumed that haemosiderin is a
proteolytic degradation product of ferritin [26].
Although this amorphous haemosiderin may remain
relatively inert, it may be one major source of the labile
iron thought to be of significance for cellular toxicity
[27]. At the light microscopy level, excess soluble
ferritin appears as a blue homogeneous colouration of
the cytosol on Perl’s Prussian blue staining, while
haemosiderin is identified by the same stain as blue
granular deposits localized most densely around the
biliary canaliculi of hepatocytes.
Transport of iron from gut to liver
The excretion mechanism of iron at the basolateral site
of the mucosal cells is incompletely understood. It is
thought to involve the enzymatic formation of Fe2+
which passes plasma membranes easier than Fe3+[28].
After release from the basolateral site of the plasma
membrane the toxic effects of Fe2+ (initiation of lipid
peroxidation, oxidation of catecholamines) are pre-
vented by several mechanisms: (a) binding to low
molecular weight natural chelating agents (amino
acids, ascorbic acid, etc.); (b) oxidation to Fe3+ by
ceruloplasmin; (c) binding to apo-transferrin which
transports iron via the portal blood to the liver and
other organs [29]. The transferrin bound iron is taken
up by the transferrin receptor endosomal pathway
[22,30]. In addition a non-endosomal pathway for
uptake of transferrin bound iron was suggested [3 1,321.
It was thought to involve reduction of Fe3+-transferrin
at the transferrin receptor site with labilization of Fe2+
binding, translocation across the membrane and rapid
incorporation into cytosolic ferritin. However, there
were methodological problems with this study and the
concept of hepatic iron uptake by an iron reduction
process is questionable [33].
Transferrin is a glycoprotein which like other
plasma glycoproteins can be desialated by liver endo-
thelial cells [34]. This asialotransferrin may then be
taken up into hepatocytes by asialoglycoprotein recep-
tors and enter an endosomal compartment [35]. In
contrast to the pathway of normal (sialylated) trans-
ferrin, asialotransferrin does not recycle to the plasma
membrane but is degraded in the lysosomal compart-
ment [35]. While under physiologic conditions this
pathway is of no major relevance, in alcoholism
asialotransferrin is markedly increased and correlates
with the hepatic iron content and fibrosis as well as
with the amount of alcohol consumed (means for
detection of alcohol abuse) [36,37].
In addition there are endocytotic pathways for
hepatocellular incorporation of haeme. Haeme is
taken up in the form of haemoglobin-haptoglobin
complexes [38], as haeme-haemopexin complexes [39]
and possibly directly from haeme-albumin complexes
[40]. For the uptake of non-transferrin bound iron the
presence of a membrane carrier protein was demon-
strated, similar to the carrier protein identified in
duodenal brush border plasma membranes [ 18,191.
Iron metabolism in haemochromatosis
Definition and classification of haemochromatosis
The term haemochromatosis defines the pathological
deposition of excessive iron in the parenchymal cells of
many organs, leading to cellular toxicity and damage
followed by functional insufficiency. In genetic haemo-
chromatosis the increase in total body iron stores is
due to an inappropriately high level of intestinal iron
 PATHOGENESIS OF GENETIC HAEMOCHROMATOSIS
absorption resulting from an inherited abnormality in
iron metabolism. When the accumulation of excess
iron is due to other conditions, the term secondary iron
overload syndrome is used. Of particular importance
are diseases associated with disordered erythropoiesis,
usually thalassaemia or sideroblastic anaemia. In
addition to the defect in iron utilization and increased
intestinal iron absorption, those conditions are also
complicated by a parenteral iron overload component
due to the requirement of multiple transfusions. This
latter component leads to excessive iron deposition
initially confined to the RE-system. In later stages iron
is transferred also to the parenchymal cells resembling
the organ distribution pattern in genetic haemo-
chromatosis. Due to the autosomal recessive trait of
inheritance the full disease is only expressed in homo-
zygotes. The clinical features of advanced symptoma-
tic haemochromatosis are characteristic. Patients
usually present with lethargy, loss of libido, joint pain,
symptoms related to the onset of diabetes mellitus or
upper abdominal pain. Hepatomegaly, skin pigmen-
tation, testicular atrophy/amenorrhoea, and arthro-
pathy are the most prominent physical signs [41]. The
clinical and biochemical expression of the disease
varies, depending on such factors as age, sex, and
chronic blood loss. The lower prevalence of sympto-
matic disease among females (1 : 10) is attributed to
physiologic iron losses and lower intake of dietary
iron. Young patients, identified by family screening are
often asymptomatic [41]. They rarely present with
advanced haemochromatosis. In these cases cardio-
myopathy is a relatively frequent clinical feature.
Heterozygous carriers of the haemochromatosis
gene may reveal minor biochemical expression of the
disease with a slightly increased level of iron absorp-
tion in 20-32% of the cases (42-44). However,
progressive iron overload with full clinical and bio-
chemical expression is not a feature of heterozygous
subjects, not even in subjects who have an excessive
intake of alcohol [45,46,47].
Speculation about the underlying metabolic defect in
genetic haemochromatosis
The genetic defect in genetic haemochromatosis leads
to an inappropriate 2-4-fold increase in iron absorp-
tion. It indicates a disorder in the regulation of the iron
absorption process.
Regulation of proteins involved in iron metabolism
The first question is whether a defect in the regulation
of transferrin, transferrin receptor and ferritin, as key
proteins of iron metabolism, is responsible for the
increased absorption from gut. The transferrin recep-
tor, although not involved in the uptake of iron at the
luminal brush border plasma membrane [48], was
323
shown to be present at the basolateral site of duodenal
mucosal cells [49-521. At this site the transferrin
receptor facilitates entry of iron into the mucosal cell
from plasma by internalizing plasma derived diferric
transferrin. Transferrin receptors are increased in
anaemia, particularly in iron deficient states [53]. It is
assumed that under these conditions iron is absorbed
in greater amounts by the enterocyte and rapidly
diverted to the blood stream. The reduction of the
intracellular iron pool may activate transferrin recep-
tor gene expression in an attempt to restore cellular
iron balance. Concomitant with this activation of
transferrin receptor gene expression, a decrease in
ferritin mRNAs was observed [53]. On the other hand,
secondary iron overload conditions reveal a down-
regulation of duodenal transferrin receptor and a
significant higher ferritin H- and L-subunit gene
expression [53].Both situations represent the physiolo-
gic response of epithelial cells to iron deficiency and
iron overload, accomplished by the concerted transla-
tional regulation of the mRNAs for ferritin and
transferrin receptor [54]. The genes encoding the
ferritin and the transferrin receptor are regulated in
response to changing levels of iron by alteration in
mRNA translocation efficiency of mRNA stability,
respectively (Fig. 1). Limitation of iron results in an
increase in the expression of the transferrin receptor,
whereas iron excess decreases transferrin receptor
expression. The synthesis of ferritin is regulated in the
opposite direction: it increases in the presence of iron
excess and decreases in iron deficiency. Transferrin
receptor regulation allows for modulation of iron
uptake, and ferritin regulation allows for adequate
sequestration of excess iron and minimizes sequest-
ration when iron is limited. The opposite but coordi-
nate regulation of these two genes is mediated by
similar cis-acting RNA sequence/structure motifs
forming moderately stable stem-loop configurations in
the untranslated regions of both mRNAs (iron respon-
sive elements (IRE’S) [55]. While for ferritin the IRE is
localized in the 5‘ untranslated region of the mRNA
and controls translation initiation, the IRE of the
transferrin receptor is localized at the 3’ untranslated
region of the mRNA [55]. A common trans-acting
RNA-binding protein (IRE-BP, also called iron regu-
latory factor IRF) serves as key participant in the
regulation of both genes [56,57]. This IRE-BP binds
under iron deficient conditions to the IRES of both
mRNAs. This binding is mediated by a complex
mechanism (‘sulfhydryl switch’) controlled by the
cellular iron status [58,59]. Binding of IRE-BP to the
IRE at the 5’ untranslated region of the ferritin mRNA
inhibits translation, while binding to the 3’ IRE of
the transferrin receptor mRNA stabilizes the message.
The net result is that in iron deficiency ferritin
biosynthesis is decreased while the transferrin receptor
expression is increased. The reverse mechanism acts in
secondary iron overload where ferritin expression is
high and transferrin receptor expression is low.
324 W. STREMMEL et al.

Cellular Iron S u m ly

m kw

Cellular Response: IRE-BP aclivily low IRE EP activity high

Regulatory

TIR mRNA Degradalmn TIR

blocked

Ferrilin mRNATranslalion Ferrllln J

eALAS mRNATranslalion sALAS

Homeoslalic Effect reduced iron uptake increased iron uplake

increased bron storage reduced iron storage
increased erylhroid iron ulilizalton reduced erythroid iron ulilizalion

Figure 1. Regulatory feedback mechanisms controlling iron homeostasis. Illustrated is the regulation ofthree key proteins in iron
metabolism: transferrin receptor (TfR), ferritin and erythroid 5-aminolcvulinic acid synthase (eALAS). IRE-BP activity is
modulated by a regulatory intracellular iron pool. Under conditions of low iron supply, this cytoplasmic mRNA-binding protein
becomes active and binds to IRES in the 5’. respectively. 3’ untranslated regions of the mRNA species encoding above proteins of
central importance in iron metabolism. As a consequence. iron uptake, iron storage, and erythroid haeme synthesis are
coordinately regulated in a way to compensate for iron deficiency. At increased iron levels, thc regulatory cascade is invcrsed.
Thus, under physiological steady-state conditions, iron is expected to maintain IRE-BP at an intermediate level of activation,
and thereby to control its own homeostasis. (Reproduced with permission from the publisher, Elsevier Science Publishing
Co., Inc. [<6]).

Pattern of protciin cxpression in genrtic haemochroma-

physiologic conditions hemochromotosis
tosis
High ferritin and low transferrin receptor expression is - mucosal celi
also observed in the liver of patients with untreated
genetic haemochromatosis [60,61]. However, in the
duodenal mucosa of these patients no decrease in
transferrin receptor mRNA was seen, although it was
expected on the basis of the increased body iron stores
[ 5 3 ] .In addition a low level of ferritin transcripts was
detected [53]. Thus, in subjects with genetic haemo-
chromatosis, duodenal ferritin and transferrin recep-
tor mRNAs are still modulated in concert. The lack of
transferrin receptor downregulation as well as reduced
levels of ferritin mRNAs may be secondary to a low Figure 2. Hypothesis to the pathophysiology of iron overload in
mucosal iron content caused by accelerated transfer of genetic haemochromatosis. Compared to physiologic conditions
iron to the blood, similarly as it is observed in anaemic there is an apparent increase in concentration of the membrane iron
carrier protein (MIBP) in mucosal cells and hepatocytes of patients
and sideropenic patients (’paradoxical’ mucosal iron with genetic haemochromatosis. This results in accelerated transfer
deficiency). The mechanism whereby the duodenal of iron from thc gut lumen across the mucosal cells, increased
mucosa is sensed as iron deficient i n genetic haemo- concentration of non-transferrin bound iron in blood, and enhanced
chromatosis is unclear. It is possible that this unknown uptake into hepatocytcs. The transferrin receptor uptake pathway is
downregulated.
mechanism is responsible for the increased absorption
of iron observed in these patients.
Alternatively it is possible that another biochemical
defect. e.g. increased concentration of a membrane
iron carrier protein, may primarily be responsible for that the membrane iron binding protein is physiologi-
the accelerated flux of iron through the duodenal cally regulated by the body iron status with high lcvels
mucosal cell, thus rendering the mucosal cell siderope- in iron deficiency and low levels in secondary iron
nic. Preliminary data on the expression of the mem- overload conditions [19]. These data indicate that a
brane iron binding protein (MIBP), in fact, revealed regulatory defect of its cellular cxpression is present in
that this protein is paradoxically upregulated in genetic haemochromatosis. Whether the unexpected
plasma membranes of the duodenal mucosa and liver high concentration of a normal carrier protein on the
of patients with genetic haemochromatosis [19] (Fig. basis of a genetic abnormality is due to a regulatory
2). In non-haemochromatotic subjects it was shown defect a t the transcriptional or post-transcriptional
 PATHOGENESIS OF GENETIC HAEMOCHROMATOSIS
level is not known. For the latter hypothesis one could
postulate a disordered interaction of a regulatory
RNA binding protein with a secondary RNA motif at
the 3’ untranslated region of the mRNA with the
consequence of enhanced biosynthesis. Thus cellular
iron uptake may be increased and in concert with the
low mucosal ferritin concentration, the translocation
across the enterocyte may be accelerated (Fig. 2). It is
not excluded that the increased expression of this
carrier protein represents the underlying metabolic
defect in haemochromatosis. However, the structure
of the protein and the corresponding mRNA as well as
the localization of the gene on a specific chromosome
are not yet established.
Another hypothesis for the underlying pathogenetic
factor in genetic haemochromatosis suggested a failure
of the RE-system to adequately process and retain iron
with the consequence of increased transfer of dietary
iron to the liver [47]. Although a relative increase of
transferrin receptor expression in relation to the body
iron stores has been observed in peripheral blood
monocytes from patients with genetic haemochroma-
tosis [62], there is no evidence for an intrinsic abnor-
mality in iron metabolism by circulating monocytes or
macrophages [62,63]. However, recent observations
have suggested that the intestinal macrophage, which
may serve a ‘gatekeeper’ function in intestinal iron
absorption, may be defective in genetic haemochroma-
tosis, leading to a decrease in RE-cell iron in the
intestinal mucosa and to an excess in intestinal iron
absorption [64]. However, conclusive data and insights
into the involved metabolic pathway have not been
presented yet.
From a genetic point of view, the hypothesis that the
metabolic defect is due to mutations of the transferrin
or transferrin receptor gene is also unlikely, because
both genes are localized on chromosome 3 [65].
Moreover, the structure of these proteins and their
regulation were shown to be normal in genetic haemo-
chromatosis. The gene encoding the IRE-binding
protein has been localized on chromosome 9 [66], thus
removing this protein as a candidate for the primary
defect in hereditary haemochromatosis. This applies
also to the ferritin genes, because the light (L) and
heavy (H) subunits are localized on chromosome 19
and 11, respectively [67,68,69]. The significance of
ferritin pseudogenes which were identified on chromo-
some 6 is still unclear and at present under investiga-
tion [70].
Puthophysiologic consequences of iron overloud
Increased iron absorption leads to a positive iron
balance and accumulation of excess body iron in
genetic haemochromatosis. It is interesting that only
specific organs are affected by iron loading. Those
include liver and pancreas and to a lesser extent
endocrine glands, particularly the pituitary, as well as
heart and joints.
325
The dark pigmentation of the skin, although a
typical clinical feature, is not alone due to iron
deposits. The epidermis of the skin is thin, and
increased melanin is found in the cells of the basal
layer.
Deposits of iron are observed around the synovial
lining cells of joints and calcium pyrophosphate
crystals may be seen within deposits of calcium
embedded in the synovial tissue. This arthropathy
develops in 25-30% of patients at any stage of the
disease [71]. The small joints of the hands (2nd and 3rd
metacarpophalangeal joints) are the first joints to be
involved. A progressive polyarthritis involving wrists,
hips, and knees may ensue. Roentgenologic manifes-
tations consist of subchondral sclerosis and cystic
changes, loss of articular cartilage, diffuse deminerali-
zation, hypertrophic bone proliferation and calcifica-
tion of the synovium.
The parenchymal deposits of iron in the liver of
patients with genetic haemochromatosis consist of
ferritin and haemosiderin. In early stages, these de-
posits are found in the periportal parenchymal cells,
especially within lysosomes in the pericanalicular
cytoplasm of the hepatocytes. This stage progresses to
perilobular fibrosis and deposition of iron in the bile
duct epithelium, Kupffer cells, and fibrous septa. In
advanced disease the pattern of cirrhosis is character-
ized by broad fibrous septa surrounding large areas of
comparatively normal parenchyma. Hepatocellular
carcinoma without iron deposition in tumour cells is
observed in about 30% of cirrhotic patients, even after
removal of excess body iron stores by phlebotomy
therapy [41].The pathogenesis of hepatocellular carci-
noma is uncertain. One hypothesis suggests a relation
to increased oxidative stress produced by iron with
oxidant damage to the DNA, acting as initiator or
promotor of carcinogenesis [72,74].The recent sugges-
tion that there may be a link to concomitant HBV
infection with integration of HBV-DNA into the
host’s genome is intriguing but not yet conclusively
proven [75].
Although iron deposits in the pancreas are predom-
inantly localized in the exocrine parenchyma with
development of tissue fibrosis, functional impairment
of digestive enzyme secretion is a rare clinical feature.
However, the selective accumulation of iron in B-cells
of the endocrine pancreas in advanced disease contri-
butes to the diabetes often observed in these patients
[41]. Along with the component of impaired insulin
secretion by damaged B-cells, there is an almost
obligatory insulin resistance observed in iron overload
disease [76]. This is due to an insulin receptor/post-
receptor defect in the iron loaded liver, which is-in
contrast to the destruction of the B-cells-reversible
after completion of the phlebotomy therapy [76].
Iron deposition in the heart is observed in 15% of
patients. Those deposits are more extensive in ventri-
cular than in atrial myocardium, resulting in intracel-
lular disruption [77]. The ensuing haemodynamic
derangement is generally designated as dilated cardio-
326 W. STREMMEL et al.
myopathy, but some patients may have a pattern of
myocardial restriction [78].
Another cell type which selectively accumulates iron
in genetic haemochromatosis is the hypogonadotropic
cell of the pituitary gland. Iron induced cell damage
leads to impaired secretion of FSH and LH which
results in the clinical manifestation of hypogonadotro-
pic hypogonadism with impotence in males and amen-
orrhoea in females [76]. Despite secondary low testost-
erone levels in male patients, peripheral sexual
hormone metabolism remains unaltered [76].
Why certain organs and cells are particularly
affected b y excessive iron accumulation remains
unclear. It is not the expression of transferrin or
ferritin receptors which is responsible for the specific
distribution of excessive iron deposition in certain cell
types. Interestingly, in patients with haemochroma-
tosis there is a significant fraction of non-transferrin
bound iron in plasma, which gradually decreases
during the course of phlebotomy treatment [79,80]. It
represents iron ions liganded to low-molecular mass
organic molecules such as citrate which do not
exchange to a significant extent with the free binding
sites on the transferrin molecule [79]. This source of
iron is not obviously correlated with the concentration
of total plasma non-haeme iron, with the exception
that it is usually present when plasma iron is greater
than 40 pmol 1 P ' [79]. Although this non-transferrin
bound iron does not strongly correlate with the
transferrin saturation in plasma, there may be a
correlation to the serum ferritin concentration. It has
been implied that in haemochromatosis, this low-
molecular mass iron complex enters the circulation
from the gut [81] (Fig. 2). Studies in rats and mice have
shown that the liver has an effective uptake system for
these non-transferrin bound iron ions [82,84]. As
candidate carrier protein for cellular uptake of this
non-transferrin bound iron the membrane iron bind-
ing protein (MIBP) could be suggested [ 18,851 (Fig. 2).
In fact, this membrane iron transporter was shown to
be enriched in those organs which are affected in
genetic haemochromatosis [ 191. However, these preli-
minary data need to be confirmed in further studies.
Mechanism of iron induced cellular toxicity
Why does iron overload of cells induce cell damage
and cell death with the consequence of functional
impairment and structural organ damage? This will be
discussed using the example of liver cell injury. Several
mechanisms whereby excess hepatic iron causes cellu-
lar injury with resultant fibrosis and cirrhosis have
been proposed. Iron-induced peroxidative injury to
phospholipids of organelle membranes, in particular
of lysosomes and mitochondria, is the key pathogen-
etic factor [86,87] (Fig. 3). Additional mechanisms may
represent iron-stimulated depletion of vitamin E and C
stores [88,89] and possibly low glutathione levels [90]
as well as a direct stimulation of collagen synthesis by
iron [91]. It is well established that ionic iron forms
(non - transferrin bound iron]
4
(free radicals)
4
lipid peroxidation
1
lability of lysosomes
0..- metabolism of mitochondria 4
microsomal enzymes 4
Figure 3. Mechanism of liver cell damage by iron overload.
highly reactive free radicals, especially from oxygen. In
order to exert potent oxidizing effects, iron must be
present in a reactive and presumably low molecular
weight form because ferritin- or transferrin-bound
iron is relatively unreactive [73,92]. One candidate for
this source of iron may be the non-transferrin bound
iron which is complexed, e.g. to citrate. It is present in
significant concentration in the circulation of hae-
mochromatic patients [79] and readily taken up by
hepatocytes (see above). Alternatively it is possible
that cellular reductants are capable of enhancing the
mobilization of iron from cellular ferritin stores [27].
Moreover, at pH 4.5 (found within lyosomes) iron
from haemosiderin is available for initiation of lipid
peroxidation without addition of reductants [27]. Such
low molecular chelate or free ionic iron may play a
catalytic role in the initiation of peroxidative injury,
either by the Fenton reaction:
Fe2++ 02+Fe3++-02
' 0 2 + 02'23H202
Fe2+ + H 2 0 2 - + F e 3 + + O H - + . 0 H
or by the iron catalyzed Haber-Weiss reaction:
Fe3+- complex+O~-rFe2+-complex+O~
Fe2+- cornplex+H202-~Fe~~
-complex+OH-+ *OH
Net reaction: O ~ + H 2 0 2 = ~ ~ - 0 2 + O H - + *OH
The derived hydroxyl radical (*OH)is highly reac-
tive and capable of initiating numerous free radical
cascades and oxidations. Others have questioned the
involvement of hydroxy radicals and proposed the
initiation of lipid peroxidation by Fe2+ and H202 in a
reaction mediated by an oxidant requiring both Fe3+
and Fe2+ [93].
Regardless of the initiating species the consequence
is a peroxidative decomposition of polyunsaturated
fatty acids of organelle membrane phospholipids. This
damage to cellular organelle membranes causes loss of
fluidity, changes in membrane potential, increased
permeability to ions, and eventual rupture leading to
release of cell and organelle contents [86,87] (Fig. 3).
Increased lability of iron-loaded lysosomes leads to
 PATHOGENESIS OF GENETIC HAEMOCHROMATOSIS
increased autophagy of hepatocytes accounting for the
characteristic changes in liver biopsy specimens from
haemochromatotic patients [94]. In mitochondria
iron-induced lipid peroxidation impairs among other
enzymes the activity of cytochrome oxidase, the termi-
nal enzyme of the mitochondria1 electron transport
chain [56]. Microsomal lipid peroxidation is associated
with a decrease in the concentration and activity of
cytochrome P-450 [90].
In addition, tissue concentrations of vitamin C and
E, two physiologic antioxidants, are often decreased in
haemochromatosis, perhaps due to increased vitamin
oxidation catalyzed by iron [87,89]. Therefore it is
conceivable that the deficiency of these two antioxi-
dants may be of relevance perpetuating the lipid
peroxidation process. Moreover, glutathione, the key
compound in protecting cells against numerous toxins
and oxidative stress was found to be significantly
decreased in experimentally overloaded rats [90].
However, this could not yet be detected in liver biopsy
specimens of patients with genetic haemochromatosis
1951.
Lipid peroxidation induces cell degeneration and
cell death. This continuous process is followed by
permanent regeneration of hepatocytes and prolifera-
tion of fibroblasts with increased synthesis of collagen
leading to cirrhosis. In addition it was proposed that
excess tissue iron may provide a direct stimulus to
collagen biosynthesis leading to the development of
fibrosis without preceding iron-mediated cellular
injury [91].
Conclusion
Progress in the understanding of the molecular mech-
anism of the physiologic regulation of iron protein
metabolism revealed significant insights into the path-
ogenesis of haemochromatosis. Today it seems evident
that the underlying metabolic defect is not related to
alterations of transferrin-, transferrin receptor- or
ferritin-metabolism. The observed high iron absorp-
tion rates may be due to an increased expression of a
membrane iron binding protein functioning as iron
carrier protein in mucosal cells of the upper small
intestine. The same protein seems to be responsible for
cellular uptake of non-transferrin bound iron through-
out the organism. This source of iron derives from the
gut and is significantly increased in genetic haemo-
chromatosis. Interestingly, the membrane iron binding
protein is enriched in those organs which are primarily
affected in this iron overload disease. Cellular accumu-
lation of ionic iron induces lipid peroxidation with the
consequence of cell damage and cell death. The clinical
features of genetic haemochromatosis are the result of
the specific pattern of organ affection by this iron-
induced cellular toxicity.
Acknowledgments
This work was supported by grants STR 9214-3 and
STR 216/4-1 from the Deutsche Forschungsgemeins-
327
chaft. The authors are very much indebted to Mrs Ch.
Scheil for the accurate preparation of the manuscript.
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