Clinical Science and Molecular Medicine (1976)50, 75-18.
SHORT COMMUNICATION
Acid hydrolase activities and lysosomal integrity
in liver biopsies from patients with iron overload
T. J. P E T E R S
AND
CAROL A. SEYMOUR
Department of Medicine, Royal Postgraduate Medical School, London
(Received 20 September 1975)
Since lysosomal disruption leads to the release of
powerful hydrolytic enzymes into the cytoplasm,
initiating cell damage (de Duve & Wattiaux, 1966),
this may be a mechanism of tissue damage in iron
overload.
1. Iron, acid phosphatase and N-acetyl-8-glucosaminidase were assayed in liver biopsies from control
subjects and patients with primary and secondary
haemochromatosis.
2. The activities of the lysosomal enzymes were
sisnificantly higher in liver biopsies from patients
with iron overload than in those from other patient
groups.
3. Lysosomesfrom the livers of patients with iron
overload were strikingly more fragile than those of
control subjects as demonstrated by assays of latent
and sedimentableN-acetyl-gglucosaminidase.
4. Lysosomal integrity was essentially normal in
biopsies from patients with a wide variety of chronic
liver diseases.
5. It is suggested that iron accumulation damages
the lysosomal membrane, releasing acid hydrolases
into the cytoplasm and thus initiating cell damage.
Methods
Patients
Specimensof liver were obtained with a Menghini
needle from patientswith iron overload, from patients
with idiopathic cirrhosis and from patients without
liver damage. Six patients with untreated primary
haemochromatosis were studied. The eight patients
with secondary haemochromatosis all had thalassaemia major and were treated with a high blood
transfusion regime (Necheles, Chang, Sabbah &
Whitten, 1974). Each patient had been given between
100 and 200 units (approximately 40-80 1) of blood
but had also received chelationtherapy (Barry,Hynn,
Letsky & Risdon, 1974). The group of eight patients
with idiopathic, alcoholic or post-hepatitic cirrhosis
showed no histological evidence of iron overload.
Control specimenswere obtained from patients with
normal serum liver-functiontests and normal hepatic
histologywho had liver biopsies during the investigation of pyrexia of unknown origin or who had open
needle biopsies during surgery for duodenal ulcer.
Specimenswere collected into ice-cold SVE medium,
pH 7.4, which contained sucrose (250 mmol/l), disodium EDTA (1 mmol/l) and ethanol (20 mmol/l).
Representativeportions were taken for routine histological examination. Informed consent for the liver
biopsies was obtained from each subject and the
project was approved by the local ethical committee.
Key words : acid hydrolase, haemochromatosis,
haemosiderosis,iron overload, liver, lysosomes.
Introduction
It is well recognizedthat iron overload causes damage
to the liver and to other organs, The mechanism of
iron toxicity is ill-understood and in this paper evidence is provided which indicates that selective
damage to lysosomes occurs in both primary or
idiopathic haemocbromatosis and in secondary
haemochromatosis due to multiple transfusions.
Correspondence:Dr T. J. Peters, Department of Medicine,
Royal Postgraduate Medical School, Du Cane Road, London
w12 OHS.
P
75
T. J. Peters and Carol A . Seymour
76
TABLE
1. Iron, N-acetyl-8-glucosarninidase,acid phosphatase and latent and sedimentable N-acetyl-B-ghcosaminidase in
liver biopsy specimens
Patient groups
(no.)
Control (8)
Cirrhosis (8)
Primary
haemochromatosis (6)
Secondary
haemochromatosis (8)
Total N-acetyl-8Total iron glucosaminidase
(munits/mg
(nmollmg
of protein)
of protein)
Total acid
phosphatase
(munits/mg
of protein)
Latent N-acetyl- Sedimentable N8-glucosaminidase
acetyl-8(%I
glucosaminidase
(%I
36.0f9.0
21.5f 1.0
P>0.1
750k 120
P < 0.001
4305 110
P<0001
2.1k0.2
3.0+ 06
P>01
6.9& 2.4
P < 0.001
5*8+ 1.2
P < 0.005
Analytical methods
The biopsy was homogenized in 3 ml of SVE
medium with fifteen strokes of a loose-fitting Dounce
homogenizer. Aliquots were taken for assays of iron
(Barry, 1968), acid phosphatase (EC 3.1.3.2) and
N-acetyl-8-glucosaminidase (EC 3.2.1.30) (Peters,
Miiller & de Duve, 1972) and for protein estimation
(Lowry, Rosebrough, Farr & Randall, 1951). The
remainder of the homogenate was centrifuged at 600
g for 10 min and the latent and sedimentableN-acetyl8-glucosaminidase was determined as described by
Peters, Heath, Wansbrough-Jones &Doe (1975).
Results
Table 1 shows the concentration of iron, acid phosphatase and N-acetyl-8-glucosaminidase and the
latent and sedimentable N-acetyl-8-glucosaminidase
in the biopsies from the various patient groups. There
are no significant differences between the results in
the control and the cirrhotic groups. The two groups
of patients with haemochromatosis show marked increases in acid hydrolase activities and decreases in
latent and sedimentable N-acetyl-B-glucosaminidase
activity. The changes tend to be more marked in the
primary haemochromatosis group but apart from the
iron content the differences from secondary haemochromatosis are not statistically significant.
Discussion
Results presented in this paper demonstrate striking
abnormalities in the biochemical properties of the
lysosomes in liver biopsies from patients with iron
overload. There changes appear to be unique to
11-Of1.4
12*1+24
P>O.l
275+ 3.7
P < 0001
24.8k 2.5
P<0001
66.5f 3-9
59.7+ 6 0
P>O*l
29.0f 2.9
P < 0.001
32.1 f2.6
P < 0.001
61-8+4.4
64.4k4.8
P > 0.1
32-5k2.5
P < 0~001
33.0& 3.3
P<O.001
haemochromatosis. The lysosomal properties in biopsies from the patients with other types of cirrhosis
are normal, indicating that the lysosomal changes are
not due to any fibrous tissue in the biopsy affecting
the homogenization procedure. Similar studies in
biopsies from patients with different forms ofchronic
hepatitides including alcoholic liver disease show no
lysosomal abnormalities (Seymour, Neale & Peters,
1975, and unpublished results). The increased acid
hydrolase activities suggest an accumulation of
undegradable material within the lysosomes. Thus
in certain congenital lysosomal storage diseasesthere
is an accumulation of the substrate of the missing
enzyme with increased activities of the other lysosoma1 enzymes(van Hoof & Hers, 1968).
It is probable that iron compounds are accumulating within the lysosome. Dense deposits of ferritin
and haemosiderin in structures tentatively identified
as lysosomes have been noted by several workers in
liver biopsies from patients with haemochromatosis
(Bessis & Caroli, 1959; Essner Kt Novikoff, 1960;
Scheuer, Williams & Muir, 1962). Moreover Trump
and his colleagues have made detailed morphological studies of iron-overloaded rats and have
clearly demonstrated ferritin and haemosiderin within liver cell lysosomes (Bradford, EIchlepp, Arstila,
Trump & KiMey, 1969; Arstila, Bradford, Kinney &
Trump, 1970; Trump, Valigorsky, Arstila, Mergner
& KiMey, 1973). Chronic iron administration to
experimental animals is associated with elevated
liver acid hydrolase activities (Goldberg, Martin &
Batchelor, 1960) and subcellular fractionation
studies have shown a striking increase in the density
of the lysosomes (Arborgh, Ericsson & Glaumann,
1973; Seymour, Budillon & Peters, 1974). It is prob-
Lysosomal integrity and iron ouerload
able that the iron compounds enter the lysosomes by
autophagy, a process in which discrete cytoplasmic
structures become surrounded by unit membranes
(autophagic vacuole). These fuse with primary lysosomes, forming secondary lysosomes, permitting the
acid hydrolases to degrade the contents of the autophagicvacuole(de Duve & Wattiaux, 1966).
The mechanism of increased lysosomal fragility in
haemochromatosis is uncertain. Distension of the
lysosomes with poorly degradable ferritin (Coffey &
de Duve, 1968) may disrupt their membranes, thereby
releasing the hydrolases into the cytoplasm. Precipitation of highly insoluble dense deposits of
haemosiderin, perhaps derived from incompleteproteolysis of ferritin by the lysosomal cathepsins, may
damage the membrane in a similar manner to that
described for silica (Allison, Harrington & Birbeck,
1966) and for urate crystals (Shirahama & Cohen,
1974). Alternatively the iron may catalysefree radical
formation (Bors, Saran, Lengfelder, Spottl &
Michel, 1974), which readily damage lysosomes and
other subcellular organelles by lipid peroxidation
(Chio, Reiss, Fletcher & Tappel, 1969; Dillard &
Tappel, 1971;Fong, McCay, Pover, Keele & Misra,
1973).
It is not suggested that the lysosomal changes in
hamnochromatosis represent a primary lesion. Similar changes are demonstrable in both primary and
secondary haemochromatosis and preliminary
studies have shown a reversion to normal of the
lysosomal abnormalities after removal of the excess
of iron from the liver (C.A. Seymour and T. J.
Peters, unpublished work). Nevertheless haemochromatosis is an example of a secondary lysosomal storage diseaseand lysosomotropicchelatingand stabilizing agents(de Duve, de Barsy, Poole, Trouet, Tulkens
8z van Hoof, 1974) may prove a more useful form of
therapy, particularly in secondaryhaemosiderosis.
Acknowledgments
We thank Professor K. Weinbren for reviewing the
liver histology and Professor G. Neale, Dr B. Model1
and Professor J. White for allowing us to study their
patients. This work is supported by The Wellcome
Trust and the Medical Research Council.
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