The biochemistry and physiology of iron

by International Conference on Proteins of Iron Storage and Transport

(5th 1981 San Diego)
1982, Elsevier Biomedical (New York, Oxford) (scientific books) 

No abstract available 
 
original article: page 479‐483 
 
On the Role of Rat Liver Lysosomes in the Catabolism of Intravenously
Injected Rat Liver Ferritin

F.M.J. Zuyderhoudt, H.G. Uiterdijk, and

G.G.A. Jörning
 

 
Published 1982 by Elsevier North Holland. Inc.

Paul Salt man and Jack Hegenauer. eds.

Thc Biochemistry and Physiology of Iron

On the Role of Rat Liver Lysosomes in the

Catabolism of Intravenously Injected Rat
Li ver F erri tin

F.M.J. Zuyderhoudt, H .G. Uiterdijk, and

G.G.A. Jörning
Laboratory of Experimental Medicine, University of Amsterdam, Wilhelmina Gasthuis,
Amsterdam, The Netherlands

Summary
During the first 4 hr af ter an intravenous injection of rat liver . ferritin into
anemic riHs, ferritin protein and iron accumulated in liver Iysosomes. Then,
within 24 hr, both iron and ferritin protein disappeared from this fraction. No
change in Iysosomal sedimentation nor Iysosomal disruption occurred. The
ferritin concentration of the bi1e was not increased at 4 hr af ter injection of
ferritin. These results indicate that ferritin can be catabolized in rat liver
Iysosomes.

Introduction
The intracellular degradation of liver ferritin has not yet been studied exten­
sively. Electron microscopic studies indicate that Iysosomes might be active in
this process (Hernandez-Yago et al., 1980). But it has been suggested that
intralysosomal degradation of ferritin results in retention of the iron in these
organelles (Wixom et al., 1980). Previous studies showed that ferritin can be
eliminated from the blood by the liver and is metabolized rapidly (Unger and
Hershko, 1974; Zuyderhoudt et al., 1978).
We used the intravenous injection of liver ferritin as a model to study the
role of liver Iysosomes in the degradation of this ferritin. The rats were made
anemic because catabolism of intravenous injected ferritin is activated in such
animals (Hershko et al., 1973), and because this facilitates the measurement of
the distribution of injected ferritin over the liver eell fractions, since the
endogenous liver ferritin concentration becomes very low.
480

Materials and Methods

Rat liver ferritin and antibodies to this ferritin were obtained as has been
described (Zuyderhoudt et al., 1978). Ferritin protein was measured with an
ELISA (Zuyderhoudt et al., 1978). Total iron was measured using a modifica­
tion of an existing method (Unger and Hershko, 1974). Differential centrifuga­
tion of rat livers, homogenized in 0.3 mol/L sucrose was performed essentially
as has been described by De Duve et al. (1955). Particulate fractions were
washed carefully to remove adhering ferritin. Enzyme activities and tota!
protein concentrations were measured with standard biochemica! methods.
Rats were made anemic by orbita punctions and iron-restricted food (white
bread and water) during three weeks. Liver ferritin (120 p.g of protein contain­
ing 40 p.g iron) was injected intravenously in the dorsal penis vein during ether
anesthesia. The relative specific concentrations of ferritin protein and total iron
were calculated in the same manner as the relative specific activities of the
marker enzymes (De Duve et al., 1955).

Results
Anemia caused the ferritin protein content of rat Jiver (4 mg) to decrease with
>97%.
The distribution of the marker enzymes over the isolated fractions did not
differ during all experimental conditions. Figure 1 shows such a distribution.
The mean recovery of the enzyme activities was between 85% and 100%.
Plasma ferritin concentrations at 2 minutes after an intravenous dose of Jiver
ferritin was 13750 p.g/L plasma (SD::::: 1208 p.g/L; n::::: 4). The mean plasma
half-life of this ferritin was 37 minutes.
Figures 2 and 3 show the relative specific concentrations of ferritin protein
and total iron as measured in the isolated fractions, during 24 hr aftel'
intravenous injection of Jiver ferritin. The only obvious change occurs in the
Iysosomal fractions at 1, 2, and 4 hr af ter injection of ferritin. At 24 hr after
injection, the distributions of ferritin and iron over the fractions are almost the
same as those at 0 hr.
Figure 4 shows the mean amounts of ferritin protein and iron in lysosomal
fractions. Always, the amount of iron is much higher than can be expected on
the basis of the measured amount of ferritiIl protein. This suggests rupie!
degradation of the protein shelI.

Discussion
During Jess than four hours af ter intravenous injection of rat Jiver ferritin, an
accumulation of ferritin protein and iron was measured in the Iysosomal
fraction. The increased amount of iron is in the same range as the amount of
injected ferritin iron (Figure 4). At 4 hr and especially at 24 hrafter the dose of
 481

MARKER ENZYMES
relative
speciflc
acti . . . ity

21 1 lJJ
iI-L~~~N~ --,JM:::::iL!::;===S===:l~5!
___ (plasma
Nucl eotml'mbrane)
ida se

,
- 50·'. 100·'.
5
4

2·1--- --1 Glutamate dehydrogenase

(mitochondria)

100·'.

Acid phosphatase

( Iysosomes)

,
50·'. 100·'.
4 Glucose -6- phosphalase
3 (microsoml'S)

AUto, ("ntrllug~tion

JO', 100000 , 9

50·'. 100·'.
r - - - - -1 pyruvale Iransaminase (ALAT)
1 I (cyloSOI)

__ _ Altf"r c~ntrtfug~'lon

JO: 100000, 9

M L 50·'. 5 100"1. percentage of prote1n In

N
the fractions
MAIN COMPONENTS
N nuclear fractlon L: Iysosomal fraction
M: mltochondrlal trac tlon S :posl·lysosomal SUpNnatant

Figure 1. Marker enzymes identifying the main components of the fractions N, M, L,

and S. Principal details of the centrifugation procedure: N : 10', 1000 g; M: 10',6000 g;
L: 10',21000 g.

ferritin, most of the accumulated ferritin protein and iron had disappeared
from these fractions. Af ter accumulation of ferritin in the lysosomes, no shift
of ferritin protein and iron towards the heavier mitochondrial fraction was
observed (Figure 3). No indication of Iysosomal disruption was obtained as
there was no increase of acid phosphatase . activity in the postlysosomal
supernatant. At4 hr af ter the do~e of ferritin , no increase of ferritin in the bile
was measured.
Thus, our results indicate that the ferritin is taken up by lysosomes, is
degraded, and the iron is mobilized from these organelles.
482

relatil/e speelt ie FERRITIN PROTEIN

concentrations

::i
6

MAIN COMPONEN1S

N: nuclear fractlon

M: mitochondrlal tractlon
L : Iysosomal tractlon

o .L
--'--,-,=*,oL......L..:,-L J1-_ _

-''--",--..l-....L.-,-!.. --'_"'----'--'---'-S : post-I ys osomal

n-=Lt1
superna tanl

NMLS NML NMLS NMLS

t 2 4 24
hours after 1.1/ dosIs ot ferrltln

5 TOTAL IRON

N M L S N M L S N M L S N M L S L S N M
o 2 4 24
hours af ter ivo dosis of terrltm

Figure 2. Relative specific concentrations of ferritin protein and tota! iron in the rat
liver fractions af ter an intravenous dose (120 /-tg) of rat liver ferritin_ Histograms at 0 hr
af ter injection: mean va!ues with the range, n = 3. Histograms at 1-24 hr af ter
injection: duplo experiments (-) and (---), respectively.

Figure 3. Relative specific concentrations of ferritin protein and total iron lil thc
Iysosomal and mitochondria! fnictions (mean values : for details see Figure 2).

FERRiTlN PROTEIN TOTAL IRON

reldtlve spE'Cltic
concentratlons
"LYSOSOMAL" " MITOCHONDRI AL" "LYSOSOMAl" " MITOCHONDRIAL"
10 FRACTION FRACTION FRAC TION FRACT ION
9 ,­

6
5
4
- -

o 2 4 24 o 1 4 24
----> hours af ter ÎV. dosis of territln ~ hours after IV dOSIS ot terrltln
 483

jJg!tiver FERRITIN PROTEIN TOTAL IRON

20

16

r- ­
12 - f-­

I, !­
- -

0 I I

0 2 4 24 0 2 4 24
:Jo hours after I.v. dosis of fl?rritin

Figure 4. Amount of ferritin protein and tolal iron in the lysosomal fractions (mean
values: for details, see Figure 2).

It remains to be studied whether this mechanism of degradation of ferritins

and release of its iron applies to the metabolism of endogenous liver ferritin as
weil. Our results are compatible with such a mechanism.

References
De Duve, c., Pressman, B.C., Giaoetto, R., Wattiaux, R., and Appelmaos, F. 1955. Intracellular
distribution patterns of enzymes in rat-liver tissue. Biochemis/ry 60:604-617.
Hcrnandez-Yago, J., Knecht, E., Martinez-Ramon, A., and Grisolia, S. 1980. Autophagy of ferritin
incorporated into tbe cytosol of HeLa cells by liposomes. Cell Tissue Res. 205:303-309.
Hershko, c., Cook, J.D., and Finch, CA 1973. Storage iron kinetics. III. J. Lab. Clin. Med.
81 :876-886.
Ungcr, A. and Hershko, C. 1974. Hepatocellular uptake of ferritin in the rat. Br. J. Hema/ol.
28:169-179.
Wixom, R.L., Prutkin, L., and Munro, H .N. 1980. Hemosiderin: nature, formation, and signifi­
cance. lil/em. Rev. Exptl. Pa/hol. 22: 193-225. .
Zuyderhoudt, F.M.J., Boers, W., Linthorst, c., Jörning, G.GA, and Hengeveld, P. 1978. An
enzyme-linked immunoassay for human serum and rat plasma and tbe influence of tbe iron in
serum ferritin on serum iron measurement, during acute hepatitis. Clill. ehim. Ac/a 88 :37-44.
Zuyderhoudt, F.M.J., Jörning. G.GA, and Van Gooi. J. 1978. On the nonferritin depot iron
fraction in the rat liver. Biochim. Biophys . Ac/a 543:53-62.