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Non-transferrin-bound-iron in serum and low-molecular-weight-iron in the liver of dietary iron-loaded rats
1~. J. Biochem. Vol. 25, No. 2, pp. 223-232, Printed in Great Britain
1993
0020-7
I IX/93 $6.00 + 0.00 Pergamon Press Ltd
NON-TRANSFERRIN-BOUND-IRON IN SERUM AND LOW-MOLECULAR-WEIGHT-IRON IN THE LIVER OF DIETARY IRON-LOADED RATS PETER NIELSEN,’JOCHEND~LLMANN,~ UWE WULFX-IEKEL~ and HELLMUTH C. HEINRICH’ ‘Abtlg Medizinische Biochemie, Physjolo~~h-Chemi~hes Institut and *Anatomisches Institut. Universitltskrankenhaus Eppendorf, Martinistr. 52, 2000 Hamburg 20, Germany [Tel. (49) 4@4717-2389; Fnx (49) 4&4717-1862] ‘Anatomisches Institut, Universitlt Bonn, NuBallee IO, 5300 Bonn, Germany (Received
25 June 1992)
Abstract-l.
The feeding of 0.5% (3,5,5-trimethyIhexanoy~)ferr~ne (TMH-ferrocene) in rats resulted in a severe and progressive liver siderosis (total liver iron, 30mg/g liver wet weight, after 30 weeks). 2. High concentrations of an iron-rich ferritin (up to 250mg/l) were detected in serum of heavily iron-loaded rats forming a large fraction of non-transferrin-bound-iron (5000 pg/dl in maximum). 3. Ferritin and not haemosiderin was the major iron storage protein in the liver. 4. The total liver iron concentration (from 0.4 to > 30mg Fe/g wet wt) but not the cytosolic low-molecular-weight-iron fraction (from 0.5 to 2.5 PM) was extremely increased during iron-loading.
In hereditary (idiopathic) haemochromatosis, increasing amounts of stored iron can induce severe cell and organ damages mainly in liver and pancreas. Despite considerable effort, the mechanism of the iron toxicity in iron-overload is still not known in detail. A recent concept to explain the development and progression of liver fibrosis and cirrhosis in patients with idiopathic haemo~hromatosis is that increasing amounts of intracellular low-molecularweight-iron (LMW-iron) induce the lipid peroxidation in iron-loaded hepatocytes (Britton er al., 1990). However, only few attempts have been made to quantify this iron pool in iron-loaded tissue directly (Mulligan et al., 1986). The research on iron induced cell pathology has thereby been hampered by the fact that no animal model is known to date that can truly simulate the well known clinical symptoms of human iron overload disease. Parenteral administration of iron dextrane results in a siderosis predominantly in reticuloendotheliai cells, thus imitating more the post-transfusional iron overload. Feeding of diets supplemented with iron salts (Richter, 1984), or the elemental iron preparation, “carbonyl” iron, results in a more parenchymal storage of iron in the liver (Park et al., 1987). However, due to a physiologica downregulation of intestinal iron absorption in already iron-loaded animals, the degree of obtainable iron overload is only moderate. This restriction is not valid for diets enriched with the (3,5,Strimethylhexanoyl)ferrocene (TMHferrocene). The short-time feeding (up to 6 weeks) of this compound has shown to increase total liver iron and ferritin iron in rats very efficiently (Kief et al.,
1977; Longueville and Crichton, 1986; Ward et al., 1991). Previous work from our laboratory has demonstrated the outstanding high bioavaiiabiiity of this lipophilic iron compound even in iron-loaded rats (Nielsen et al., 1992, Diillmann et al., 1992). Using this most effective dietary animal model for experimental siderosis hitherto known, the aim of the present investigation study was to study biochemical parameters of iron metabolism in heavily iron-loaded rats. MATERIALS
AND METHODS
Chemicals
(3,5,5-TrimethyihexanoyI)ferr~ene was synthesized from ferrocene and 3,5,S-trimethylhexanoyl chloride as described elsewhere (Nielsen and Heinrich, 1992). Pure rat liver ferritin was prepared from iron-loaded rat liver as described (Osterloh and Aisen, 1989). This procedure includes no heat-denaturation step to avoid possible heat-induced changes in the ferritin molecule. The purity of ferritin was assessed by SDS-poIyacrylamide gel electrophoresis using the Laemmh system with a 12.5% separation gel (Laemmli, 1970). Anti-ferritin was obtained in rabbits against rat liver ferritin and used as antibodies in the radial immunodiffusion according to Mancini et al. (1965). Ten ~1 of serum or liver homogenate-supernatant was applied on the gel containing anti-rat-liver-serum together with the standard solutions of purified ferritin. From the calibration curve the amount of ferritin was calculated. Light and electron microscopy were performed as described (Dullmann et al., 1992). Animals
and diets
Four to six weeks old female Wistar rats were obtained from Wiga (Hannover, Germany). Rats were housed in polyethylene cages with stainless steel wire tops and received 223
PETERNIELSENet al.
224
an iron restricted diet (Altromin C1038, Altromin, Lage, Germany; 6.3 pg Fe/g) and distilled water ud libirum for the next 5 weeks. At the end of this period a mild iron deficiency anaemia was obtained by means of a low haemoglobin concentration (10.2 It 0.4g/dl) and the absence of stainable iron in the liver and bone marrow. From then, rats received Altromin Cl038 in pellet form supplemented with 0.5, w/w TMH-ferrocene. At the end of the respective feeding period, after an overnight fast, rats were sacrificed by exsanguination from the abdominal aorta while under fight ether anaesthesia. The organs were quickly excised and weighed. Serum iron and unsaturated iron binding capacity was measured as described for humans (Gabbe er al., 1982). Written consent was obtained from the local animal research council for the care and use of laboratory animals in all animal experiments.
Acid-washed glass and plastic vials were used throughout. Total tissue iron concentrations were determined as described (Dullmann er al., 1992). Results are expressed as mg of iron per g of liver, wet weight. For the determination of liver iron fractions a modification of the micromethod according to by Zuyderhoudt et al. (1978b) was used. Ten to fifteen mg samples of post-mortem rat liver and 300~1 of water were homogenized in a Potter-Elvejhem glass-glass homogenizer (15 up and down strokes at 5000 rpm). From the homogenates, 10 nl-aliquots were taken for the determination of total liver iron by atomic absorption spectroscopy (AAS). For determination of haem iron, 1 ml of water, I ml of Drabkin solution (50 rig/l KCN, 140 mgil KH?PO,), 0.5 ml I M HCI and 2ml ethyl methyl ketone were added to 30~1 of the homogenate. The mixture was shaken for I min and centrifuged (10 min, 2500 g). To 800 ~1 of the supernatant was added 200~1 of ethanol and the extracted haem iron was quantified by AAS. 200~1 of the remaining homogenates were heated to 72-75’C during IO min. After centrifugation (SOOOg, IOmin), the pellets were washed with 200 pl of water, The supernatants were pooled and iron was determined in aliquots by AAS. The pellets were resuspended in 2 ml of AAS-matrix modifier, homogenized using a teflon coated ultrasonic stick (Sonicator cell disrupter, Ultrasonic Inc.), and the “insoluble” iron fractions (haemosiderin iron and haem iron) were measured by AAS. For sake of simplicity, the always accepted term “haemosiderin” is used for “non-ferritin-storage iron”. The calculated values for total liver iron concentration (ferritin-iron + haemosiderin + haem iron) were in good agreement with the measured values.
protein solutions were used for HPLC or ultrafiltration experiments.
Five hundred pl of the f9Fe-labelled liver cytosol fraction (with or without added EDTA) or serum samples were placed in the upper compartment of an Amicon MPS-I filtration apparatus (YMT-membrane; cut off for proteins, 10,000 Da; Amicon, Diiren, Germany) and centrifuged at IOOOg for 30min. SYFe-activity in the ultrafiltrate was measured by y-~intillation counting, iron was measured by AAS. High
Atomic
~~a~[i~n
qf fiz:er
chromatography
absorption
spectrometry
RESULTS The chronic feeding of (3,5,.5-trinzethylhexanoyl) ferrocene (TMH-ferrocene) resulted in a severe and progressive liver siderosis in rats (Fig. 1, Table I). Liver iron concentrations, as typically found in 35
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.
-
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Iron was quantified by electrothermal atomic absorption spcctrometry (AAS) using a graphite furnace (Perkin Elmer 300 with HGA 72). HPLC-fractions diluted appropriately with triple distilled water were further diluted I: 3 with matrix modifier containing 1% Triton X-100 and 20mM nitric acid. Twenty /II aliquots thereof were transferred into the graphite furnace by an autosampler and the absorbency measured at 248.3 nm.
:
Gove,-molecular -weight -iron
performance
Acid-washed containers and vials were used throughout for collection and processing of liver homogenate samples. Size-exclusions HPLC was performed using an iron-free system typ Ultrochrom GTI (Pha~acia LKB, Freiburg, Germany), equipped with a Superose 12, or a Hiload 16/20-FPLC-size-exclusion column (Pharmacia LKB), respectively. LC effluent was monitored at 280 nm using a variable wavelength detector and at 200-360 or 360-520 nm using a photodiode array detector type L-3000 (E. Merck, Darmstadt, Germany). Thirty mM Tris--HCl. 0.1 M NaCI, t mh4 EDTA, pH 7.4 was used as eluant and fractions of 0.7 (1.5) ml were collected. HPLC-fractions or ultrafiltrates were measured for ‘9Fe-activity in an automated y-scintillation spectrometer (Packard Auto gamma 5360).
25
,/
homogenates
Fresh or thawed liver tissues (0.5-1.0 g) were minced and homogenized in 5 volumes of ice-cold buffer (30 mM TrisHCI, 0.1 M NaCl, pH 7.5) using a Potter-Elvejhem glass-glass homogenizer (6 up and down strokes. I100 rpm). After centrifugation for 30min at 12,000 g, a solution of 59Fe-citrate (80 ~1; 4.5 mM sodium citrate, IO-20 nCi s9FeCI,, 40.-200 ng iron) was added to 1.92ml of the supernatant. After standing at O’C for 24 hr, the 59Felabelled supernatants were filtered through 0.2 /drn disposable filter units (Gelman, acre LC 13). To one part was added EDTA to a final concentration of 2mM. After standing 30min at 37’C. aliquots of the clear
Fig. 1. Liver iron concentration of rats under chronic feeding of TMH-ferrocene. Ten weeks old, iron deficient female Wistar rats were fed a diet enriched with 0.5% TMH-ferrocene up to 79 weeks.
Non-transferrin-bound
serum iron in rats
225
Table I. Serum iron parameters in groups of rats (n = 4-5) under iron enriched diet (0.5% w/w TMHferrocene; mean f SD). Serum iron was measured by spectrophotometry (bathophenanthroline method) and by atomic absorption spectroscopy AAS’
Feeding period (weeks) 0
I 2 4 I1 43 57 79
Liver-Fe concentration (mg Fe/g wet wt) 0.010 * 0.005 2.15+0.50 3.40 + 0.43 5.15rto.54 10.4 * 0.75 28. I i: 7.4 29.9 + 3.S 29.5 i: I.0
Serum-Fe @g/d0 184rt63 143 rt 72 237 it 67 392 rt 103 750 F 220 4108 + 1282 5588 + 914 6557 * 909
S~trophotom~~ Serum-Fe W’dU
TIBCz (pgldl)
TFS)
149 + 50 183 + 14 195*40 346 k 38 696 + 36 913 + 194 1808&432 2289+311
582 f 82 565 * IO 465*77 581 f 62 731 & 41
25&6 34 f 2 42 f 5 60&Q 9s + I -
-
(%)
‘Atomic absorption spectroscopy. *Total iron binding capacity. ‘Transferrin iron saturation.
human
iron overload diseases (3-10 mg Fe/g wet wt) already after 2-6 weeks.
were obtained
Serum iron and serum ferritin in iron-loaded rats The serum iron concentrations were measured both by spectrophotometry using bathophenanthrolinedisulphonic acid as colour reagent (standard procedure for serum iron determination in humans) and by atomic absorption spectroscopy. When analysed spectrophotometrically, the serum iron concentration increased linearly with the feeding time also after the serum transferrin was saturated with iron (Table 1). In severely iron foaded-rats (feeding time > 1I weeks on 0.5% TMH-ferrocene), the determination of serum iron by atomic absorption spectroscopy showed even higher values as compared to the bathophenanthroline method, indicating that substantial amounts of iron did not react with the colour reagent under the experimental conditions. To study the structure of this particular iron fraction in more detail, rat serum was analysed by size-exclusion HPLC (Fig. 2). Most of the serum iron in heavily iron-loaded rats was bound to a protein fraction (peak I, Fig. 2) with higher molecular mass than transferrin (peak II, Fig. Z), whereas no increased amounts of low-molecular-weight-iron (LMW-iron) were found (Fig. 2). The latter result was confirmed by ultrafiltration of rat serum (exclusion limit of the membrane, 10,000 daltons). Only 0.1 I & 0.04% of the serum iron was found in ultrafiltrates from ironloaded rats compared to 0.4% from normal rats. The high-molecular-weight-iron-fraction was unequivocally identified as ferritin because it comigrated with isolated pure rat liver ferritin in size-exclusionchromatography FPLC as well as in SDS-PAGE electrophoresis, and was reactive in a radial immune diffusion assay using anti-rat-liver-ferritin antibodies. In severely iron-loaded rats was found a correlation between serum ferritin and serum iron (measured by AAS), again indicating that ferritin-iron accounts for the considerable fraction of non-transferrin-boundiron (Fig. 3). A protein/iron-ratio of 4.7 : 1 was calculated which is characteristic for iron-rich ferritin. Further proof for the presence of iron rich ferritin
in serum was found by electron microscopy which revealed numerous electron dense particles corresponding to iron cores of ferritin in the lumina of blood vessels (Fig. 4). Liver iron fractions in iran-loaded ruts The total tissue iron, soluble iron (fer~tin), insoluble iron (haemosiderin), and haem iron were determined from lo-20 mg samples of iron-loaded rat liver using a modified method according to Zuyderhoudt et al. (1978b). The total liver iron concentrations measured by this micromethod were in good agreement with the respective values from the wet ashing technique using samples of 0.5-I .Og tissue (data not shown). A linear relation was found between ferritin-iron as well as haemosiderin and the non-haem liver iron (Fig. 5). The ferritin-iron/haemosiderin ratio seemed to be independent from the non-haem liver iron (Fig. 6). This was also true for the haem iron concentration in the liver (0.05-0.3 mg Fe/g wet wt), which besides did not contribute much for the total liver iron content. Low -molecular-weight -iran in the cytosoi fraction iron -loaded rat liver
of
A size-exclusion-chromatography of the liver cytosol fraction from rats with normal and increased iron stores is shown in Fig. 7 and Table 2. EDTA was present in the HPLC eluant, to minimize unspecific binding of free iron to the HPLC-column. In liver homogenates from iron-loaded rats, most of the iron was found within a high-molecular-weight protein fraction (peak I, Fig. 7). As indicated by the absorbancy at 280 nm, this protein fraction was present to a much lower degree in liver homogenates of rats with normal iron stores. This iron-containing protein was identified as ferritin because it comigrated with isolated pure rat liver ferritin in HPLC and was reactive in a radial immuno diffusion assay using anti-rat-liver ferritin antibodies. The absolute values of LMW-iron (Table 2). isolated by size-exclusion-HPLC (Fig. 7, peak II) or by ultrafiltration, were slightly increased in groups of
226
PETER
HORSE
SPLEEN
NIELSEN
el al.
FERRITIN
20 + blue
dextran
2.000.000 i
16
1000000
‘2 2 0
3
12-
P
%
E
.cD 100000
“r 3 2
2
E 0
I
0
10
I
1
20
30 retention
-
10
absorption
IRON-LOADED liver-Fe:
(260
10000 40 time
50
60
70
(min)
nm)
0
calibration curve
RAT
28.4
mg Fe/g
I
w.wt
=8 ._: .r
1000000
56 E 100000
10000 10
20
30 retention
-
absorption
40 time
(280
run)
50
60
70
(min)
~m~~;;;~pt,on spectroscopy)
Fig. 2. Size-exclusion-FPLC of rat serum from an iron-loaded rat (79 weeks on 0.5% TMH-ferrocene). Column Hiload 16/60 Sephadex 200; eluant, 30 mM Tris-HC1 0.15 M NaCI, pH 7.5; flow 1.5 ml/min). Iron content in fractions (dashed area) was measured by atomic absorption spectroscopy. Peak I, ferritin; la, ferritin aggregates in the void volume of the column; peak II, transferrin.
Fig. 3. Serum-ferritin (measured by radical immunodiffusion assay) as a function of serum iron concentration (measured by atomic absorption spectroscopy) in iron-loaded rats.
rats with very severe liver siderosis (142 + 34 pg Fe/g liver) as compared to rats with normal iron stores (23.7 &-8.9 pg/g). However, in relation the protein bound iron, the LMW-iron fraction was similar in rats with normal and increased iron stores. If EDTA was present in the liver homogenate in order to chelate free iron or iron loosely attached to proteins, the amount of ultrafiltrable iron was higher in all groups (Table 2). The distribution of the 59Fe-label did not correspond to the distribution of unlabelled iron in the cytosol fraction because of an incomplete exchange of the label with the protein-bound iron (transferrin, ferritin, haemoglobin). But an almost complete recovery ( > 95%) of the injected 59Fe-activity
Non-transferrin-bound
serum iron in rats
Fig. 4. Numerous iron-containing ferritin molecules within the lumen of a central liver vein of an iron-loaded rat (liver iron, 18.2 mg/g wet wt). E, erythrocytc. Electron micrograph, unstained, x 112,000.
227
PETERNIELSENel al.
Fig. 5. Soluble iron (ferritin iron) and insoluble iron (haemosiderin) in .rat liver cytosol as a‘function of the total liver iron concentration. demonstrated, that the determination of the LMWiron was reliable with the HPLC-technique used.
concentration of > 15 mg iron/g wet wt. As the observed switch from parenchymal to non-parenchyma1 iron storage in the liver during severe TMHferrocene-iron loading goes parallel with the strong increase in serum ferritin concentration, it seems likely that the iron redistribution within the liver is mediated by circulating iron-rich ferritin. No increased amounts of LMW-iron were found in serum of the iron-overloaded rats. This is also different to iron overload diseases in humans. where a significant LMW-iron fraction is found in serum of patients with P-thalassaemia (Hershko rt al., 1978) or hereditary haemochromatosis (Aruoma et al., 1988). This iron fraction, reactive in the bleomycin assay and probably involved in lipid-peroxidation reactions, was shown to be taken up by the liver very efficiently (Wright et al., 1988) and is suspected for being involved in the pathogenesis of cell damage in iron overload (Gutteridge and Halliwell, 1990).
DlSCUSSlON
The chronic feeding of TMH-ferrocene up to 79 weeks resulted in a fast, progressive, and severe liver siderosis in rats (Fig. 1). An similar effective animal model for experimental iron overload is not known in the literature. Serum jkrritin
in iron -loaded
rats
In heavily iron overloaded rats. a large fraction of serum iron was not bound to transferrin and was identified as iron-rich ferritin. The notably high concentrations of serum ferritin (up to 250 mg/l) in iron-loaded rats exceeded more than one order of magnitude the serum ferritin values described in human iron-overload diseases (about lOmg/l in maximum). However, it should be kept in mind, that the liver iron concentration in the iron-loaded rats were also much higher than those found in patients with iron-overload. Human serum ferritin is thought to be secreted from the reticuloendothelial system and to have a low iron content even in ironloaded patients (0.023-0.067 pg Fe/pg of protein, Worwood et al., 1976). The finding of an ironrich serum ferritin (0.21 pg Fe/pg protein) in ironloaded rats suggests that serum ferritin in this case may be released from iron-overloaded, damaged liver cells. This corresponds to results from Ward et al. (1991), who measured an lo-fold increase in total N-acetyl-fl-glucosaminidase activity in the liver homogenate of rats after 6 weeks on TMH-ferrocene-diet, indicating an increase in lysosomal membrane fragility. Using light and electron microscopy, we have recently shown in the same group of iron-loaded rats, that iron storage in the liver starts in hepatocytes (Diillmann et al., 1992). With progression of iron loading iron also accumulated in liver macrophages which finally surpassed the hepatocytes in iron storage. Slight fibrosis was observed above an iron
Lker
iron ,fractions
As expected from earlier studies in moderately iron-loaded rats (Zuyderhoudt et al., 1978a: Longueville et al., 1986, Ward et al., 1991), ferritiniron and haemosiderin increased with the non-haem liver iron concentration in the TMH-ferrocene fed rats. More iron was stored in form of ferritin than in haemosiderin even in heavily iron-loaded rat livers. Even though a considerable variation of the experimental values must be taken into account, the ratio ferritin ironihaemosiderin seemed to be independent from the status of liver siderosis. This is also in agreement with results in hypertransfused rats (Zuyderhoudt et al., 1978a). In human iron overload diseases, controversial results concerning liver iron fractionation are reported. Some authors (Morgan and Walter, 1963; Selden et al., 1980) found the ferritin iron/haemosiderin-ratio gradually falling with increasing iron overload, whereas Zuyderhoudt et al. (1983) described a constant ratio between the two iron storage proteins in patients with primary or secondary iron overload.
.
Y -
-
0
r *
”
0.26
0137
x f
178
.
Fig. 6. The ferritinihaemosiderin-Fe-ratio as a function of liver iron concentration in iron-loaded rats.
Non-transferrin-bound
serum iron in rats
229
NORMAL RAT (Ilver-Fe: 0.4 mg Fe/o w.wt)
terrltln
100
molecular wolght Idalton)
*\
36
1
= 2 80s” g 80‘; 2 E g 400”
6 .= 20 -
20 fractions
IRON-LOADED
RAT
(liver-Fe:
18 mg Fe/g w.wt)
100 ? L CI 80 B s E 8o E “0 40 ?i C 0
.L,
20
20 fractions -
absorptlon
(280 nm)
-+-
69Fe
m
Fe-AAS
Fig. 7. Size-exclusion-FPLC of rat liver homogenate supernatant of a rat with normal (top) or strongly increased (bottom) iron stores (IO weeks on 0.5% TMH-ferrocene). The liver homogenate was lab&d in uitro with tracer dose s9Fe-citrate. Column, Superose 12; eluant, 30mM Tris-HCI 0.15 M NaCl, (PH 7.5; flow 0.7 ml/min). Peak I, ferritin; Ia, ferritin aggregates in the void volume of the column; peak II, LMW-iron. Law -molecular -weight -iron pool in iron -loaded rat Ever cells
Using two ditl‘erent analytical techniques (ultrafiltration and HPLC), a small amount of low-molecular-weight-iron (LMW-iron) was found in liver homogenates from both normal and iron-loaded rats. Whereas the protein bound iron (mainly ferritin) was strongly increased in iron-loaded rats, absolute values of LMW-iron were only slightly higher (up to 2.5 FM) than in rats with normal iron stores (OS PM) (Table 2). This is in agreement with results from Mulligan et al. (1986), who demonstrated the existence of a LMW-iron fraction in various rat tissue independent from body iron stores. A number of investigators have previously reported the presence of
LMW-iron-components in a variety of cells and tissues. However, the chemical structure (iron complex with citrate, Morley and Bezkorovainy, 1983; phosphate, Pollack et al., 1985; amino acids, Bakkeren et al., 1985, or ATP, Weaver and Pollack, 1989) as well as the function (intracellular iron transport, regulation of the biosynthesis of transferrin receptors and ferritin, Fontecave and Pierre, 1991) remains unclear. Using an in vitro assay, Britton et al. (1990) observed an increased prooxidant action of hepatic cytosolic LMW-iron in carbonyl iron overloaded rats. That would support the hypothesis of iron toxicity being the consequence of increased hepatic pool of LMW-iron which is catalytically active in stimulating the lipid peroxidation. In the present
49.8 rt 13.0
61.3 rt 3.9
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23.7 + 8.9 ll.4zt4.3)
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42.8 + 5.2
5.1+
2.5
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8.3 + 2.1 (0.4 + 0. I
4.7 t 0.8 (0.6 I 0.1
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79.6 f 23. I 3.8+ 1.1)
45.2 + 34.5 5.1f 4.4)
32.2 + 8.9 15.5 + 9.7)
Ultrafiltration of rat liver cytosol (cut off > 10,000 Da) AAS3-iron __-s9Fe in vitro label _-______- EDTA f EDTA - EDTA pg iron/g liver + EDTA (% of inject) (% of inject)
overloaded rats
Protein fraction’ LMW-iron2 pg iron/g liver (% of inject)
AAS’-iron
in normal and TMH-ferrocene-iron
WFe-activity (in vim labelling) and iron content (measured by atomic adsorption spectroscopy, AAS) of HPLC-fractions and ultrafiltrates from rat liver cytosol fraction (control, normal rats; group A-C, TMH-ferrocene fed rats, A, liver-Fe, 8.43 I 1.34 mgfp; 8, 16.9 + 2.25 mg/g; C, 30.7 _+0.8 mg Fe/g liver wet wt). Size-exclusion HPLC (column, Supcrose 12; eluant, Tris buffer pH 7.5, I mM EDTA). For ultrafiltration experiments, liver homogenates were preincubated with EDTA (+ EDTA) or without EDTA (- EDTA) (all values, mean f SD). ‘Protein-~und~iron fraction (~,~5~,~ Da, corresponds to peak I in Fig. 7). ‘Low-molecular-weight-iron fraction (< 3000 Da, corresponds to peak II in Fig. 7). ‘AAS, atomic absorption spectroscopy. ‘Supernatant of rat liver homogenate, centrifuged 30 min at 12,OOOg. For details see Materials and Methods.
160.9 + 27.6
17.7 * 9.7
4
Control
42.1 +_ 13.8
n
Protein fraction’ LMW-iron’ (% of inject)
HPLC of rat liver cytosol rPFe in vitro label __~~_.____.
Group
Iron concentration liver cytosol fraction’ (mg/t)
Table 2. Low-mol~ular-weight-iron
Non-transferrin-bound
study, we failed to demonstrate a distinctly increased LMW-iron pool in TMH-ferrocene-iron loaded rats, although these rats had a much higher parenchymal liver siderosis than the carbonyl-iron fed rats used by Britton et al. (1990). It can be speculated that the turnover of catalytically active LMW-iron may be increased during iron-loading more than the absolute amount of this special iron fraction. In patients with idiopathic hemochromatosis, the onset of clinical relevant liver damage (liver fibrosis, liver cirrhosis) is known to occur above a liver iron concentration of 4-5 mg Fe/g wet wt (Bassett et al., 1986). Regarding the severe liver siderosis in the iron-loaded rats of the present study (Fig. 1) on one hand and the absence of severe liver damage on the other hand, it is obvious that iron-loaded rats can tolerate much more iron before developing a severe liver fibrosis than humans. It may well be that the alteration of the LMW-iron pool during ironloading is quite different in patients with idiopathic haemochromatosis. Further in vitro and in viuo studies in experimental iron overload and in human iron overload disease are needed to understand the role of the LMW-iron pools in lipid
pet-oxidation
and
the
development
of liver
fibrosis. A~X-notr,ledgement-We techmcal assistance.
thank
Maria
Fiirtjes
for excellent
REFERENCES
Aruoma 0. I., Bomford A., Polson R. .I. and Halliwell B. (1988) Nontransferrin-bound iron in plasma from hemochromatosis patients: effect of phlebotomy therapy. Blood 12, 14161419. Bakkeren D. L., De Jeu-Jaspars C. M. H., Van der Heul C. and van Eijk H. G. (1985) Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol. Inr. J. Biochem. 17, 9255930. Bassett M. L., Halliday J. W. and Powell L. W. (1986) Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron level associated with fibrosis. Hepatology 6, 24-29. Britton R. S, Ferrali M., Magiera C. J., Recknagel R. 0. and Bacon B. R. (1990) Increased prooxidant action of hepatic cytosolic low-molecular-weight iron in experimental iron overload. Heparology 11, 1038-1043. Diillmann J., Wulfbekel U., Nielsen P. and Heinrich H. C. (1992) Iron overload of the liver by TMH-ferrocene in rats. Acta Anat. 143, 96-108. Fontecave M. and Pierre J. L. (1991) Iron metabolism: the low-molecular-mass iron pool. Biol. Metal. 4, 133-135. Gabbe E. E.. Heinrich H. C. and Icagic F. (1982) Proposal for the standardization of the serum unsaturated iron binding capacity assay, and results in groups with normal iron stores and with prelatent, latent, and manifest iron deficiency. Clin. chim. Acta 119, 5143. Gutteridge J. M. C and Halliwell B. (1990) The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem. Sci. 15, 129-135. Hershko C., Graham Cl., Bates G. W. and Rachmilewitz E. A. (1978) Non-specific serum iron in thalassaemia: an
serum
iron in rats
231
abnormal serum iron fraction of potential toxicity. Br. J. Haemat.
40, 255-263.
Kief H., Crichton R. R., Bahr H., Engelbart K. and Lattrell R. (1977) Induction of ferritin formation in hepatocytes. Proteins of Iron Metabolism, pp. 107-I 14. Grune and Stratton, New York. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680485. Longueville A. and Crichton R. R. (1986) An animal model of iron overload and its application to study hepatic ferritin iron mobilization by chelators. Biochem. Pharmac. 35, 3669-3678. Mancini G., Carbonara 0. and Heremans J. F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2, 235-254. Morgan E. H. and Walters M. N. I. (1963) Iron storage in human disease. J. Clin. Path. 16, 101-107. Morley C. G. D. and Bezkorovainy A. (1983) Identification of the iron chelate in hepatocyte cytosol. IRCS med. Sci. 11, 11061107. Mulligan M., Althaus B. and Linder M. C. (1986) Nonferritin, non-heme iron pools in rat tissues. Inr. J. Biochem. 18, 791-798. Nielsen P. and Heinrich H. C. (1992) Metabolism of iron from (3,5,5-trimethylhexanoyl)ferrocene in the rat. An dietary model for severe iron overload. Biochem. Pharmat. In press. Nielsen P., Engelhardt R., Fischer R., Heinrich H. C., Langkowski L. and Biicheler E. (1992) Non-invasive liver iron quantification by computed tomography in iron overloaded rats. Inrest. Radiol. 27, 312-317. Osterloh K. and Aisen P. (1989) Pathways in the binding and uptake of ferritin by hepatocytes. Biochim. biophys. Acfa 1011,4045. Park C. H., Bacon B. R., Brittenham G. M. and Tavill A. S. (1987) Pathology of dietary carbonyl iron overload in rats. Lab. Invesr. 57, 555-563. Pollack S., Campana T. and Weaver J. (1985) Low molecular weight iron in guinea pig reticulocytes. Am. J. Hemat. 19, 75-84. Richter G. W. (1984) Studies of iron overload. Rat liver siderosome ferritin. Lab. Invest. 50, 26-35. Selden C., Owen M., Hopkins J. M. P. and Peters T. J. (1980) Studies on the concentration and intracellular localization of iron proteins in liver biopsy specimens from patients with iron overload with special reference to their role in lysosomal disruption. Br. J. Haemat. 44, 59343. Ward R. J., Florence A. L., Baldwin D., Abiaka C., Roland F.. Ramsey M. H., Dickson D. P. E., Peters T. J. and Crichton R. R. (1991) Biochemical and biophysical investigations of the ferrocene iron-loaded rat. An animal model of primary haemochromatosis. Eur. J. Biochem. 202, 405410. Weaver J. and Pollack S. (1989) Low-M, iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes. Biochem. J. 261, 787-792. Worwood M.. Dawkins S, Wagstaff M. and Jacobs A. (1976) The purification and properties of ferritin from human serum. Biochem. J. 157, 97-103. Wright T. L., Fitz J. G. and Weisiger R. A. (1988) Nontransferrin-bound iron uptake by rat liver. J. biol. Chem. 263, 1842-l 847. Zuyderhoudt F. M. J., Jiirning G. G. A and Van Go01 J. (1978a) On the non-ferritin depot iron fraction in the rat liver. Biochim. biophys. Acta 543, 5362.
232
PETER NIELSEN er al.
Zuyderhoudt F. M. J., Hengeveld P., Van Go01 J. and Jorning G. G. A. (1978b) A method for measurement of liver iron fractions in needle biopsy specimens and some results in acute liver disease. Clin. c&n. Acra 86, 313-321.
Zuyderhoudt F. M., Sindram J. W., Marx J. M., Jorning G. G. A. and Van Go01 J. (1983) The amount of ferritin and hemosiderin in the livers of patients with iron-loading diseases. Hepatology 3, 232-235.
1993
0020-7
I IX/93 $6.00 + 0.00 Pergamon Press Ltd
NON-TRANSFERRIN-BOUND-IRON IN SERUM AND LOW-MOLECULAR-WEIGHT-IRON IN THE LIVER OF DIETARY IRON-LOADED RATS PETER NIELSEN,’JOCHEND~LLMANN,~ UWE WULFX-IEKEL~ and HELLMUTH C. HEINRICH’ ‘Abtlg Medizinische Biochemie, Physjolo~~h-Chemi~hes Institut and *Anatomisches Institut. Universitltskrankenhaus Eppendorf, Martinistr. 52, 2000 Hamburg 20, Germany [Tel. (49) 4@4717-2389; Fnx (49) 4&4717-1862] ‘Anatomisches Institut, Universitlt Bonn, NuBallee IO, 5300 Bonn, Germany (Received
25 June 1992)
Abstract-l.
The feeding of 0.5% (3,5,5-trimethyIhexanoy~)ferr~ne (TMH-ferrocene) in rats resulted in a severe and progressive liver siderosis (total liver iron, 30mg/g liver wet weight, after 30 weeks). 2. High concentrations of an iron-rich ferritin (up to 250mg/l) were detected in serum of heavily iron-loaded rats forming a large fraction of non-transferrin-bound-iron (5000 pg/dl in maximum). 3. Ferritin and not haemosiderin was the major iron storage protein in the liver. 4. The total liver iron concentration (from 0.4 to > 30mg Fe/g wet wt) but not the cytosolic low-molecular-weight-iron fraction (from 0.5 to 2.5 PM) was extremely increased during iron-loading.
In hereditary (idiopathic) haemochromatosis, increasing amounts of stored iron can induce severe cell and organ damages mainly in liver and pancreas. Despite considerable effort, the mechanism of the iron toxicity in iron-overload is still not known in detail. A recent concept to explain the development and progression of liver fibrosis and cirrhosis in patients with idiopathic haemo~hromatosis is that increasing amounts of intracellular low-molecularweight-iron (LMW-iron) induce the lipid peroxidation in iron-loaded hepatocytes (Britton er al., 1990). However, only few attempts have been made to quantify this iron pool in iron-loaded tissue directly (Mulligan et al., 1986). The research on iron induced cell pathology has thereby been hampered by the fact that no animal model is known to date that can truly simulate the well known clinical symptoms of human iron overload disease. Parenteral administration of iron dextrane results in a siderosis predominantly in reticuloendotheliai cells, thus imitating more the post-transfusional iron overload. Feeding of diets supplemented with iron salts (Richter, 1984), or the elemental iron preparation, “carbonyl” iron, results in a more parenchymal storage of iron in the liver (Park et al., 1987). However, due to a physiologica downregulation of intestinal iron absorption in already iron-loaded animals, the degree of obtainable iron overload is only moderate. This restriction is not valid for diets enriched with the (3,5,Strimethylhexanoyl)ferrocene (TMHferrocene). The short-time feeding (up to 6 weeks) of this compound has shown to increase total liver iron and ferritin iron in rats very efficiently (Kief et al.,
1977; Longueville and Crichton, 1986; Ward et al., 1991). Previous work from our laboratory has demonstrated the outstanding high bioavaiiabiiity of this lipophilic iron compound even in iron-loaded rats (Nielsen et al., 1992, Diillmann et al., 1992). Using this most effective dietary animal model for experimental siderosis hitherto known, the aim of the present investigation study was to study biochemical parameters of iron metabolism in heavily iron-loaded rats. MATERIALS
AND METHODS
Chemicals
(3,5,5-TrimethyihexanoyI)ferr~ene was synthesized from ferrocene and 3,5,S-trimethylhexanoyl chloride as described elsewhere (Nielsen and Heinrich, 1992). Pure rat liver ferritin was prepared from iron-loaded rat liver as described (Osterloh and Aisen, 1989). This procedure includes no heat-denaturation step to avoid possible heat-induced changes in the ferritin molecule. The purity of ferritin was assessed by SDS-poIyacrylamide gel electrophoresis using the Laemmh system with a 12.5% separation gel (Laemmli, 1970). Anti-ferritin was obtained in rabbits against rat liver ferritin and used as antibodies in the radial immunodiffusion according to Mancini et al. (1965). Ten ~1 of serum or liver homogenate-supernatant was applied on the gel containing anti-rat-liver-serum together with the standard solutions of purified ferritin. From the calibration curve the amount of ferritin was calculated. Light and electron microscopy were performed as described (Dullmann et al., 1992). Animals
and diets
Four to six weeks old female Wistar rats were obtained from Wiga (Hannover, Germany). Rats were housed in polyethylene cages with stainless steel wire tops and received 223
PETERNIELSENet al.
224
an iron restricted diet (Altromin C1038, Altromin, Lage, Germany; 6.3 pg Fe/g) and distilled water ud libirum for the next 5 weeks. At the end of this period a mild iron deficiency anaemia was obtained by means of a low haemoglobin concentration (10.2 It 0.4g/dl) and the absence of stainable iron in the liver and bone marrow. From then, rats received Altromin Cl038 in pellet form supplemented with 0.5, w/w TMH-ferrocene. At the end of the respective feeding period, after an overnight fast, rats were sacrificed by exsanguination from the abdominal aorta while under fight ether anaesthesia. The organs were quickly excised and weighed. Serum iron and unsaturated iron binding capacity was measured as described for humans (Gabbe er al., 1982). Written consent was obtained from the local animal research council for the care and use of laboratory animals in all animal experiments.
Acid-washed glass and plastic vials were used throughout. Total tissue iron concentrations were determined as described (Dullmann er al., 1992). Results are expressed as mg of iron per g of liver, wet weight. For the determination of liver iron fractions a modification of the micromethod according to by Zuyderhoudt et al. (1978b) was used. Ten to fifteen mg samples of post-mortem rat liver and 300~1 of water were homogenized in a Potter-Elvejhem glass-glass homogenizer (15 up and down strokes at 5000 rpm). From the homogenates, 10 nl-aliquots were taken for the determination of total liver iron by atomic absorption spectroscopy (AAS). For determination of haem iron, 1 ml of water, I ml of Drabkin solution (50 rig/l KCN, 140 mgil KH?PO,), 0.5 ml I M HCI and 2ml ethyl methyl ketone were added to 30~1 of the homogenate. The mixture was shaken for I min and centrifuged (10 min, 2500 g). To 800 ~1 of the supernatant was added 200~1 of ethanol and the extracted haem iron was quantified by AAS. 200~1 of the remaining homogenates were heated to 72-75’C during IO min. After centrifugation (SOOOg, IOmin), the pellets were washed with 200 pl of water, The supernatants were pooled and iron was determined in aliquots by AAS. The pellets were resuspended in 2 ml of AAS-matrix modifier, homogenized using a teflon coated ultrasonic stick (Sonicator cell disrupter, Ultrasonic Inc.), and the “insoluble” iron fractions (haemosiderin iron and haem iron) were measured by AAS. For sake of simplicity, the always accepted term “haemosiderin” is used for “non-ferritin-storage iron”. The calculated values for total liver iron concentration (ferritin-iron + haemosiderin + haem iron) were in good agreement with the measured values.
protein solutions were used for HPLC or ultrafiltration experiments.
Five hundred pl of the f9Fe-labelled liver cytosol fraction (with or without added EDTA) or serum samples were placed in the upper compartment of an Amicon MPS-I filtration apparatus (YMT-membrane; cut off for proteins, 10,000 Da; Amicon, Diiren, Germany) and centrifuged at IOOOg for 30min. SYFe-activity in the ultrafiltrate was measured by y-~intillation counting, iron was measured by AAS. High
Atomic
~~a~[i~n
qf fiz:er
chromatography
absorption
spectrometry
RESULTS The chronic feeding of (3,5,.5-trinzethylhexanoyl) ferrocene (TMH-ferrocene) resulted in a severe and progressive liver siderosis in rats (Fig. 1, Table I). Liver iron concentrations, as typically found in 35
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Iron was quantified by electrothermal atomic absorption spcctrometry (AAS) using a graphite furnace (Perkin Elmer 300 with HGA 72). HPLC-fractions diluted appropriately with triple distilled water were further diluted I: 3 with matrix modifier containing 1% Triton X-100 and 20mM nitric acid. Twenty /II aliquots thereof were transferred into the graphite furnace by an autosampler and the absorbency measured at 248.3 nm.
:
Gove,-molecular -weight -iron
performance
Acid-washed containers and vials were used throughout for collection and processing of liver homogenate samples. Size-exclusions HPLC was performed using an iron-free system typ Ultrochrom GTI (Pha~acia LKB, Freiburg, Germany), equipped with a Superose 12, or a Hiload 16/20-FPLC-size-exclusion column (Pharmacia LKB), respectively. LC effluent was monitored at 280 nm using a variable wavelength detector and at 200-360 or 360-520 nm using a photodiode array detector type L-3000 (E. Merck, Darmstadt, Germany). Thirty mM Tris--HCl. 0.1 M NaCI, t mh4 EDTA, pH 7.4 was used as eluant and fractions of 0.7 (1.5) ml were collected. HPLC-fractions or ultrafiltrates were measured for ‘9Fe-activity in an automated y-scintillation spectrometer (Packard Auto gamma 5360).
25
,/
homogenates
Fresh or thawed liver tissues (0.5-1.0 g) were minced and homogenized in 5 volumes of ice-cold buffer (30 mM TrisHCI, 0.1 M NaCl, pH 7.5) using a Potter-Elvejhem glass-glass homogenizer (6 up and down strokes. I100 rpm). After centrifugation for 30min at 12,000 g, a solution of 59Fe-citrate (80 ~1; 4.5 mM sodium citrate, IO-20 nCi s9FeCI,, 40.-200 ng iron) was added to 1.92ml of the supernatant. After standing at O’C for 24 hr, the 59Felabelled supernatants were filtered through 0.2 /drn disposable filter units (Gelman, acre LC 13). To one part was added EDTA to a final concentration of 2mM. After standing 30min at 37’C. aliquots of the clear
Fig. 1. Liver iron concentration of rats under chronic feeding of TMH-ferrocene. Ten weeks old, iron deficient female Wistar rats were fed a diet enriched with 0.5% TMH-ferrocene up to 79 weeks.
Non-transferrin-bound
serum iron in rats
225
Table I. Serum iron parameters in groups of rats (n = 4-5) under iron enriched diet (0.5% w/w TMHferrocene; mean f SD). Serum iron was measured by spectrophotometry (bathophenanthroline method) and by atomic absorption spectroscopy AAS’
Feeding period (weeks) 0
I 2 4 I1 43 57 79
Liver-Fe concentration (mg Fe/g wet wt) 0.010 * 0.005 2.15+0.50 3.40 + 0.43 5.15rto.54 10.4 * 0.75 28. I i: 7.4 29.9 + 3.S 29.5 i: I.0
Serum-Fe @g/d0 184rt63 143 rt 72 237 it 67 392 rt 103 750 F 220 4108 + 1282 5588 + 914 6557 * 909
S~trophotom~~ Serum-Fe W’dU
TIBCz (pgldl)
TFS)
149 + 50 183 + 14 195*40 346 k 38 696 + 36 913 + 194 1808&432 2289+311
582 f 82 565 * IO 465*77 581 f 62 731 & 41
25&6 34 f 2 42 f 5 60&Q 9s + I -
-
(%)
‘Atomic absorption spectroscopy. *Total iron binding capacity. ‘Transferrin iron saturation.
human
iron overload diseases (3-10 mg Fe/g wet wt) already after 2-6 weeks.
were obtained
Serum iron and serum ferritin in iron-loaded rats The serum iron concentrations were measured both by spectrophotometry using bathophenanthrolinedisulphonic acid as colour reagent (standard procedure for serum iron determination in humans) and by atomic absorption spectroscopy. When analysed spectrophotometrically, the serum iron concentration increased linearly with the feeding time also after the serum transferrin was saturated with iron (Table 1). In severely iron foaded-rats (feeding time > 1I weeks on 0.5% TMH-ferrocene), the determination of serum iron by atomic absorption spectroscopy showed even higher values as compared to the bathophenanthroline method, indicating that substantial amounts of iron did not react with the colour reagent under the experimental conditions. To study the structure of this particular iron fraction in more detail, rat serum was analysed by size-exclusion HPLC (Fig. 2). Most of the serum iron in heavily iron-loaded rats was bound to a protein fraction (peak I, Fig. 2) with higher molecular mass than transferrin (peak II, Fig. Z), whereas no increased amounts of low-molecular-weight-iron (LMW-iron) were found (Fig. 2). The latter result was confirmed by ultrafiltration of rat serum (exclusion limit of the membrane, 10,000 daltons). Only 0.1 I & 0.04% of the serum iron was found in ultrafiltrates from ironloaded rats compared to 0.4% from normal rats. The high-molecular-weight-iron-fraction was unequivocally identified as ferritin because it comigrated with isolated pure rat liver ferritin in size-exclusionchromatography FPLC as well as in SDS-PAGE electrophoresis, and was reactive in a radial immune diffusion assay using anti-rat-liver-ferritin antibodies. In severely iron-loaded rats was found a correlation between serum ferritin and serum iron (measured by AAS), again indicating that ferritin-iron accounts for the considerable fraction of non-transferrin-boundiron (Fig. 3). A protein/iron-ratio of 4.7 : 1 was calculated which is characteristic for iron-rich ferritin. Further proof for the presence of iron rich ferritin
in serum was found by electron microscopy which revealed numerous electron dense particles corresponding to iron cores of ferritin in the lumina of blood vessels (Fig. 4). Liver iron fractions in iran-loaded ruts The total tissue iron, soluble iron (fer~tin), insoluble iron (haemosiderin), and haem iron were determined from lo-20 mg samples of iron-loaded rat liver using a modified method according to Zuyderhoudt et al. (1978b). The total liver iron concentrations measured by this micromethod were in good agreement with the respective values from the wet ashing technique using samples of 0.5-I .Og tissue (data not shown). A linear relation was found between ferritin-iron as well as haemosiderin and the non-haem liver iron (Fig. 5). The ferritin-iron/haemosiderin ratio seemed to be independent from the non-haem liver iron (Fig. 6). This was also true for the haem iron concentration in the liver (0.05-0.3 mg Fe/g wet wt), which besides did not contribute much for the total liver iron content. Low -molecular-weight -iran in the cytosoi fraction iron -loaded rat liver
of
A size-exclusion-chromatography of the liver cytosol fraction from rats with normal and increased iron stores is shown in Fig. 7 and Table 2. EDTA was present in the HPLC eluant, to minimize unspecific binding of free iron to the HPLC-column. In liver homogenates from iron-loaded rats, most of the iron was found within a high-molecular-weight protein fraction (peak I, Fig. 7). As indicated by the absorbancy at 280 nm, this protein fraction was present to a much lower degree in liver homogenates of rats with normal iron stores. This iron-containing protein was identified as ferritin because it comigrated with isolated pure rat liver ferritin in HPLC and was reactive in a radial immuno diffusion assay using anti-rat-liver ferritin antibodies. The absolute values of LMW-iron (Table 2). isolated by size-exclusion-HPLC (Fig. 7, peak II) or by ultrafiltration, were slightly increased in groups of
226
PETER
HORSE
SPLEEN
NIELSEN
el al.
FERRITIN
20 + blue
dextran
2.000.000 i
16
1000000
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3
12-
P
%
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.cD 100000
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I
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I
1
20
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-
10
absorption
IRON-LOADED liver-Fe:
(260
10000 40 time
50
60
70
(min)
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0
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28.4
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I
w.wt
=8 ._: .r
1000000
56 E 100000
10000 10
20
30 retention
-
absorption
40 time
(280
run)
50
60
70
(min)
~m~~;;;~pt,on spectroscopy)
Fig. 2. Size-exclusion-FPLC of rat serum from an iron-loaded rat (79 weeks on 0.5% TMH-ferrocene). Column Hiload 16/60 Sephadex 200; eluant, 30 mM Tris-HC1 0.15 M NaCI, pH 7.5; flow 1.5 ml/min). Iron content in fractions (dashed area) was measured by atomic absorption spectroscopy. Peak I, ferritin; la, ferritin aggregates in the void volume of the column; peak II, transferrin.
Fig. 3. Serum-ferritin (measured by radical immunodiffusion assay) as a function of serum iron concentration (measured by atomic absorption spectroscopy) in iron-loaded rats.
rats with very severe liver siderosis (142 + 34 pg Fe/g liver) as compared to rats with normal iron stores (23.7 &-8.9 pg/g). However, in relation the protein bound iron, the LMW-iron fraction was similar in rats with normal and increased iron stores. If EDTA was present in the liver homogenate in order to chelate free iron or iron loosely attached to proteins, the amount of ultrafiltrable iron was higher in all groups (Table 2). The distribution of the 59Fe-label did not correspond to the distribution of unlabelled iron in the cytosol fraction because of an incomplete exchange of the label with the protein-bound iron (transferrin, ferritin, haemoglobin). But an almost complete recovery ( > 95%) of the injected 59Fe-activity
Non-transferrin-bound
serum iron in rats
Fig. 4. Numerous iron-containing ferritin molecules within the lumen of a central liver vein of an iron-loaded rat (liver iron, 18.2 mg/g wet wt). E, erythrocytc. Electron micrograph, unstained, x 112,000.
227
PETERNIELSENel al.
Fig. 5. Soluble iron (ferritin iron) and insoluble iron (haemosiderin) in .rat liver cytosol as a‘function of the total liver iron concentration. demonstrated, that the determination of the LMWiron was reliable with the HPLC-technique used.
concentration of > 15 mg iron/g wet wt. As the observed switch from parenchymal to non-parenchyma1 iron storage in the liver during severe TMHferrocene-iron loading goes parallel with the strong increase in serum ferritin concentration, it seems likely that the iron redistribution within the liver is mediated by circulating iron-rich ferritin. No increased amounts of LMW-iron were found in serum of the iron-overloaded rats. This is also different to iron overload diseases in humans. where a significant LMW-iron fraction is found in serum of patients with P-thalassaemia (Hershko rt al., 1978) or hereditary haemochromatosis (Aruoma et al., 1988). This iron fraction, reactive in the bleomycin assay and probably involved in lipid-peroxidation reactions, was shown to be taken up by the liver very efficiently (Wright et al., 1988) and is suspected for being involved in the pathogenesis of cell damage in iron overload (Gutteridge and Halliwell, 1990).
DlSCUSSlON
The chronic feeding of TMH-ferrocene up to 79 weeks resulted in a fast, progressive, and severe liver siderosis in rats (Fig. 1). An similar effective animal model for experimental iron overload is not known in the literature. Serum jkrritin
in iron -loaded
rats
In heavily iron overloaded rats. a large fraction of serum iron was not bound to transferrin and was identified as iron-rich ferritin. The notably high concentrations of serum ferritin (up to 250 mg/l) in iron-loaded rats exceeded more than one order of magnitude the serum ferritin values described in human iron-overload diseases (about lOmg/l in maximum). However, it should be kept in mind, that the liver iron concentration in the iron-loaded rats were also much higher than those found in patients with iron-overload. Human serum ferritin is thought to be secreted from the reticuloendothelial system and to have a low iron content even in ironloaded patients (0.023-0.067 pg Fe/pg of protein, Worwood et al., 1976). The finding of an ironrich serum ferritin (0.21 pg Fe/pg protein) in ironloaded rats suggests that serum ferritin in this case may be released from iron-overloaded, damaged liver cells. This corresponds to results from Ward et al. (1991), who measured an lo-fold increase in total N-acetyl-fl-glucosaminidase activity in the liver homogenate of rats after 6 weeks on TMH-ferrocene-diet, indicating an increase in lysosomal membrane fragility. Using light and electron microscopy, we have recently shown in the same group of iron-loaded rats, that iron storage in the liver starts in hepatocytes (Diillmann et al., 1992). With progression of iron loading iron also accumulated in liver macrophages which finally surpassed the hepatocytes in iron storage. Slight fibrosis was observed above an iron
Lker
iron ,fractions
As expected from earlier studies in moderately iron-loaded rats (Zuyderhoudt et al., 1978a: Longueville et al., 1986, Ward et al., 1991), ferritiniron and haemosiderin increased with the non-haem liver iron concentration in the TMH-ferrocene fed rats. More iron was stored in form of ferritin than in haemosiderin even in heavily iron-loaded rat livers. Even though a considerable variation of the experimental values must be taken into account, the ratio ferritin ironihaemosiderin seemed to be independent from the status of liver siderosis. This is also in agreement with results in hypertransfused rats (Zuyderhoudt et al., 1978a). In human iron overload diseases, controversial results concerning liver iron fractionation are reported. Some authors (Morgan and Walter, 1963; Selden et al., 1980) found the ferritin iron/haemosiderin-ratio gradually falling with increasing iron overload, whereas Zuyderhoudt et al. (1983) described a constant ratio between the two iron storage proteins in patients with primary or secondary iron overload.
.
Y -
-
0
r *
”
0.26
0137
x f
178
.
Fig. 6. The ferritinihaemosiderin-Fe-ratio as a function of liver iron concentration in iron-loaded rats.
Non-transferrin-bound
serum iron in rats
229
NORMAL RAT (Ilver-Fe: 0.4 mg Fe/o w.wt)
terrltln
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*\
36
1
= 2 80s” g 80‘; 2 E g 400”
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20 fractions
IRON-LOADED
RAT
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18 mg Fe/g w.wt)
100 ? L CI 80 B s E 8o E “0 40 ?i C 0
.L,
20
20 fractions -
absorptlon
(280 nm)
-+-
69Fe
m
Fe-AAS
Fig. 7. Size-exclusion-FPLC of rat liver homogenate supernatant of a rat with normal (top) or strongly increased (bottom) iron stores (IO weeks on 0.5% TMH-ferrocene). The liver homogenate was lab&d in uitro with tracer dose s9Fe-citrate. Column, Superose 12; eluant, 30mM Tris-HCI 0.15 M NaCl, (PH 7.5; flow 0.7 ml/min). Peak I, ferritin; Ia, ferritin aggregates in the void volume of the column; peak II, LMW-iron. Law -molecular -weight -iron pool in iron -loaded rat Ever cells
Using two ditl‘erent analytical techniques (ultrafiltration and HPLC), a small amount of low-molecular-weight-iron (LMW-iron) was found in liver homogenates from both normal and iron-loaded rats. Whereas the protein bound iron (mainly ferritin) was strongly increased in iron-loaded rats, absolute values of LMW-iron were only slightly higher (up to 2.5 FM) than in rats with normal iron stores (OS PM) (Table 2). This is in agreement with results from Mulligan et al. (1986), who demonstrated the existence of a LMW-iron fraction in various rat tissue independent from body iron stores. A number of investigators have previously reported the presence of
LMW-iron-components in a variety of cells and tissues. However, the chemical structure (iron complex with citrate, Morley and Bezkorovainy, 1983; phosphate, Pollack et al., 1985; amino acids, Bakkeren et al., 1985, or ATP, Weaver and Pollack, 1989) as well as the function (intracellular iron transport, regulation of the biosynthesis of transferrin receptors and ferritin, Fontecave and Pierre, 1991) remains unclear. Using an in vitro assay, Britton et al. (1990) observed an increased prooxidant action of hepatic cytosolic LMW-iron in carbonyl iron overloaded rats. That would support the hypothesis of iron toxicity being the consequence of increased hepatic pool of LMW-iron which is catalytically active in stimulating the lipid peroxidation. In the present
49.8 rt 13.0
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overloaded rats
Protein fraction’ LMW-iron2 pg iron/g liver (% of inject)
AAS’-iron
in normal and TMH-ferrocene-iron
WFe-activity (in vim labelling) and iron content (measured by atomic adsorption spectroscopy, AAS) of HPLC-fractions and ultrafiltrates from rat liver cytosol fraction (control, normal rats; group A-C, TMH-ferrocene fed rats, A, liver-Fe, 8.43 I 1.34 mgfp; 8, 16.9 + 2.25 mg/g; C, 30.7 _+0.8 mg Fe/g liver wet wt). Size-exclusion HPLC (column, Supcrose 12; eluant, Tris buffer pH 7.5, I mM EDTA). For ultrafiltration experiments, liver homogenates were preincubated with EDTA (+ EDTA) or without EDTA (- EDTA) (all values, mean f SD). ‘Protein-~und~iron fraction (~,~5~,~ Da, corresponds to peak I in Fig. 7). ‘Low-molecular-weight-iron fraction (< 3000 Da, corresponds to peak II in Fig. 7). ‘AAS, atomic absorption spectroscopy. ‘Supernatant of rat liver homogenate, centrifuged 30 min at 12,OOOg. For details see Materials and Methods.
160.9 + 27.6
17.7 * 9.7
4
Control
42.1 +_ 13.8
n
Protein fraction’ LMW-iron’ (% of inject)
HPLC of rat liver cytosol rPFe in vitro label __~~_.____.
Group
Iron concentration liver cytosol fraction’ (mg/t)
Table 2. Low-mol~ular-weight-iron
Non-transferrin-bound
study, we failed to demonstrate a distinctly increased LMW-iron pool in TMH-ferrocene-iron loaded rats, although these rats had a much higher parenchymal liver siderosis than the carbonyl-iron fed rats used by Britton et al. (1990). It can be speculated that the turnover of catalytically active LMW-iron may be increased during iron-loading more than the absolute amount of this special iron fraction. In patients with idiopathic hemochromatosis, the onset of clinical relevant liver damage (liver fibrosis, liver cirrhosis) is known to occur above a liver iron concentration of 4-5 mg Fe/g wet wt (Bassett et al., 1986). Regarding the severe liver siderosis in the iron-loaded rats of the present study (Fig. 1) on one hand and the absence of severe liver damage on the other hand, it is obvious that iron-loaded rats can tolerate much more iron before developing a severe liver fibrosis than humans. It may well be that the alteration of the LMW-iron pool during ironloading is quite different in patients with idiopathic haemochromatosis. Further in vitro and in viuo studies in experimental iron overload and in human iron overload disease are needed to understand the role of the LMW-iron pools in lipid
pet-oxidation
and
the
development
of liver
fibrosis. A~X-notr,ledgement-We techmcal assistance.
thank
Maria
Fiirtjes
for excellent
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