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Michigan State
University

 

 

 

 

 

 

 

 

 

 

 

 

r".

This is to certify that the
dissertation entitled

LYSOSOMES IN THE IRON OVERLOAD GUINEA PIG MODEL:

A BIOCHEMICAL AND MORPHOLOGIC EVALUATION

presented by

E . TERENCE ADAMS

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Environmental Toxicology/
Pathology

 

 

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DATE DUE DATE DUE DATE DUE
Mg“, n ‘ _

 

 

 

 

 

 

MSU In An Affirmative Action/Equal Opportunity Institution

 

LYSOSOMES IN THE IRON OVERLOAD GUINEA PIG MODEL: A BIOCHEMICAL AND
MORPHOLOGIC EVALUATION

BY

E. TERENCE ADAMS

A DISSERTATION

Submitted to Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1990

 

(00501430?

ABSTRACT

LYSOSOMES IN THE IRON OVERLOAD GUINEA PIG MODEL: A BIOCHEMICAL AND
MORPHOLOGIC EVALUATION

BY

E. Terence Adams

Lysosomal abnormalities have been associated with hepatic
disease in iron overload of both humans and experimental animals.
To investigate the prospect that cardiac lysosomes are altered in
iron overload cardiomyopathy, we determined the free activity of
selected myocardial and hepatic lysosomal enzymes in iron dextran
treated guinea pigs (0.25, 0.5, 1.0 and 2.0 g Fe/kg BW). In
addition, a serologic profile composed of serum glucosaminidase,
serum iron, total iron binding capacity, and transferrin
saturation was determined. Light and electron microscopic, as
well as lipid peroxidation studies were included.

The free activity of hepatic glucosaminidase (p<0.01) and
glucuronidase (p<0.05) and serum glucosaminidase activity (p<0.01)
were significantly elevated at all levels of iron loading; free
hepatic acid phosphatase (p<0.01) was increased at all but the
lowest iron dose level. The free activity of myocardial
glucosaminidase (p<0.05) was significantly increased at all iron
dose levels when compared to pooled controls; free myocardial acid
phosphatase was elevated only at the highest iron dose level of

2.0 g/kg (p<0.05).

 

Further studies demonstrated enhanced lipid peroxidation (as
determined by the generation of malondialdehyde) in whole
homogenates of liver and heart (p<0.01) at the iron dose level of
1.5 g Pe/kg.

Serologically, both serum iron and total iron binding
capacity were significantly elevated at iron dose levels of 0.5
(p<0.001) and 1.0 g/kg (p<0.05 and p<0.01, respectively).
Transferrin saturation was increased at 0.25 (p<0.01) and 1.0 g
Fe/kg (p<0.05).

Horphologic studies indicated that intraperitoneal injection
of iron dextran resulted in the initial deposition of iron

particles within reticuloendothelial cells of the liver with

‘subsequent hepatocellular iron deposition at higher iron loads.
Myocardial iron particles were primarily located in connective
tissue histiocytes and reticuloendothelial cells with rare
evidence of deposition in cardiac myofibers. Ultrastructurally,
these particles were aggregated in membrane-bound organelles
consistent with lysosomes.

Additional preliminary studies indicated that iron loading
(1.0 g Fe/kg) resulted in severe testicular atrophy and

degeneration in male guinea pigs.

 

To Robert W. Leader, for unyielding support and

encouragement through arduous times

iv

ACKNOWLEDGEMENTS

I wish to express my genuine gratitude to the outstanding members
of my graduate committee, Drs. Kenneth A. Schwartz, Robert W. Bull,
Willimn W. Wells, Park Willis and Behzad Yamini. Their guidance and
assistance were crucial to the success of my graduate training.

In addition, I would like to recognize many individuals who have
sacrificed their time and talent to assist me through this difficult
process. My sincere appreciation goes to John Davis, John Gauger,
Dianne Schwartz, Jonelle Golding, Mark Lewis, Pat Lowrie, Becky McMahon,
Karen Klomparens, Jeff Wilson, Reginald Johnson and John Kaneene.

A special thanks goes to Marty Muth and Ann W. Braman, for without
their vast skills and infinite patience none of this would have been

accomplished.

 

TABLE OF CONTENTS

List of Tables......................................................ix
List of Figures..................................................... x
List of Abbreviations.............................. ..... ...........xii
Introduction.................................................. ...... .1
Review of Literature............ ..... . ..... ..... ...... ... ...... . ..... 5
Primary Iron Overload............................................5
Hereditary Hemochromatosis..................... ...... ........5

Linkage to HLA...............................................7
Population Association.......................... ..... ......7

Antigen Association.... ......... ...... ..... ................8

Secondary Iron Overload....................... ..... ..............9
Iron Overload Secondary to Anemias ....... ....................9

Iron Overload Secondary to Liver Disease....................10

Iron Overload Secondary to Excessive Dietary Ingestion. ..... 11
Manifestations of Iron Overload in Specific Organ Systems.......12

Heart................... ..... ...............................12
Liver............................................. ..... .....15
Skin................................................ ........ 16

Gonads......................................................16
Diagnosis.......................................................17
Serum Ferritin..............................................17
Transferrin Saturation......................................19
Serum Iron Concentration....................................19
chelation Test - Desferrioxamine Excretion........... ....... l9
Hepatic Iron Stores.........................................20
Bone Marrow Iron............................................21
Iron and Free Radical Formation.................................22
Nonenzymic Lipid Peroxidation...................................24
Free Radicals, Lipid Peroxidation and the Pathogenesis of
Iron Overload Disease.........................................25
Iron and In Vivo Lipid Peroxidation ...... .......................27
Iron and Myocardial Cells.......................................29
Iron and Infection..............................................32
siderophores................................................33
The Iron-Withholding System.....................................35
Transferrins................................................35
Iron and Cellular Immunity......................................38
NR Cells....................................................38
Polymorphonuclear Granulocytes..............................39
Peripheral Blood Monocytes..................................40
Lymphocytes.................................................41
References.................................. ..... ...............42

Chapter I: Gram Negative Sepsis in Iron Loaded Guinea Pigs

Abstract........................................................65
Introduction....................................................66

vi

 

Methods.........................................................66
Results.........................................................67
Discussion......................................................7l
References......................................................74

Chapter II: Iron Induced Myocardial and Hepatic Lysosomal
Abnormalities in the Guinea Pig...................................77
Introduction....................................................77
Materials and Methods................................ ........... 78
Animal Treatment....................................... ..... 78
Sample Preparation..........................................79
Serum Iron, TIBC and If Saturation..........................79
Lysosomal Enzyme Assays.....................................80
Malondialdehyde Determination...............................80
statistical Analysis........................................81
Results.........................................................81
Serum Iron, Serum TIBC and Transferrin Saturation...........81
Hepatic Lysosomal Fragility.................................81
Serum Glucosaminidase.......................................86
Myocardial Lysosomal Fragility..............................86

Generation of Malondialdehyde in whole Homogenates
of Heart and Liver........................................86
Discussion......................................................86
References......................................................94

Chapter III: Histologic and Ultrastructural studies in the

Iron Overload Guinea Pig Model....................................99

Introduction....................................................99
Materials and Methods...........................................100
Animal Treatment............................................100
x-ray Microanalysis.........................................101
Results.........................................................101
Light Microscopy............................................101
Liver.....................................................101
Heart.....................................................104
Electron Microscopy.........................................104
Discussion......................................................104
Refsrences......................................................118

Chapter IV: Hypogonadism in Iron Loaded Male Guinea Pigs:
A Preliminary Study.............................................120
Brief Communication.............................................120
References......................................................124

 

APPENDIX

Appendix A: Specific Activity of Hepatic and Myocardial B-N-

acetylglucosaminidase (NAG) in Dextran Control and

Iron Treated Guinea Pigs...............................125
Appendix B: Specific Activity of Hepatic and Myocardial Acid

Phosphatase in Dextran Control and Iron Treated

Guinea Pigs............................................126
Appendix C: Specific Activity of B-glucuronidase in Hepatic

Tissue of Dextran Control (Con) and Iron Treated (Fe)

Guinea Pigs...................................... ...... 127
Appendix D: Serum Iron (SI), Total Iron Binding Capacity (TIBC)

and Transferrin Saturation (Tf Sat) Values in Control

Guinea Pigs...... ..... ........................ ......... 128
Appendix E: Serum Iron (SI), Total Iron Binding Capacity (TIBC)

and Transferrin Saturation (TF Sat) Values in Iron

Dextran Treated Guinea Pigs. ..... ......................129
Appendix F: Serum B-N-acetylglucosaminidase (NAG) activity

(expressed as nmoles p-nitrophenol/min/ug protein

{xlo- }) in Dextran Control and Iron Treated Guinea

Pigs...................................................130
Appendix G: Gross Photographs of Liver from Iron Treated

Septic Guinea Pigs ....................................131
Appendix H: Electron Micrographs of Cardiac Interstitial Cells

from Iron Dextran Treated Guinea Pigs (1.5 g/kg).

Tissues were fixed with 2% glutaraldehyde/2%

paraformaldehyde and postfixed with 2% 0304; thin

sections were stained with saturated aqueous uranyl

acetate and lead citrate or left unstained.............l33

vita ....IOCOOOCI...0.II.IOIIII.IOOOIIIIIIOOIIOQCIOOOCIOOICOIOOI0135

viii

 

1.
2.

LIST OF TABLES

Culture Results................................................68
Comparison of serum iron, total iron binding capacity

(TIBC) and transferrin saturation values in dextran

control (CON) vs iron dextran treated (Fe) guinea pigs.........82

ix

 

10.

11.

LIST OF FIGURES

Focal region of hepatic necrosis with pyknotic nuclei

and extracellular iron particles bordered by non-

degenerate cells. Hematoxylin and eosin; Line = lOOym.........70
Numerous rod shaped bacterial organism surrounded

by heterophils and macrophages in the liver of a

septic guinea pig. Hematoxylin and eosin; Line = 20pm..........70
Comparison of free hepatic B-N-acetylglucosaminidase

(NAG) activity ..... ................................ ..... . ...... 83
Comparison of free hepatic B-glucuronidase in dextran

control and iron treated guinea pigs...........................84
Comparison of free hepatic acid phosphatase in dextran

control and iron dextran treated guinea pigs ..... ...... ........ 85
Comparison of serum B-N-acetylglucosaminidase activity

in dextran control and iron dextran treated guinea pigs........87
Comparison of free myocardial B-N-acetylglucosaminidase

in pooled dextran controls vs. iron treated guinea pigs........88
Comparison of free myocardial acid phosphatase in pooled

dextran controls vs. iron treated guinea pigs..................89
Comparison of malondialdehyde (MDA) levels in whole

homgenates of heart and liver in dextran control and iron

dextran treated guinea pigs....................................90
Hepatic Siderosis:

A) Iron dextran, liver, 0.5 g Fe/Kg, guinea pig. Iron particles
confined to sinusoidal lining cells (mainly Kupffer cells)
surrounding a central vein. Prussian Blue; Line = 100 ym;.....103
3) Iron dextran; liver 1.5 g Fe/Kg, guinea pig. Positive
staining iron particles are located within hepatocytes and

Kupffer cells. Prussian Blue; Line = 50pm............ ......... 103
Cardiac Siderosis:

A) Iron dextran, atrium, 0.5 g Fe/kg, guinea pig. Iron particles
lining epicardial surface. Prussian Blue; Line = lOOpm........106
3) Iron dextran; right ventricle, 0.5 g Fe/kg guinea pig.

Iron particle lining the endocardium. Prussian blue; Line =
50pm...........................................................106
C) Iron dextran, subepicardial region, 1.5 g Fe/kg guinea

pig. Numerous intracellular iron aggregate within connective
tissue macrophages surrounding blood vessels and intermixed

with collagen. Prussian Blue, Line = lOOym....................108
D) Iron dextran; right ventricular myocardium, 1.5 g Fe/kg,

guinea pig. Several aggregates of iron particles in

interstitium of ventricular myocardium between myocytes.

Prussian Blue, Line = 100pm....................................108

 

E)

12.

13.

14.

15.

Iron dextran; atrial myocardium, 0.5 g Fe/kg, guinea pig.

Iron aggregates in atrial interstitial tissue. Prussian

Blue, Line = 50ym..............................................110
F) Iron dextran; right ventricular myocardium, 1.5 g Fe/kg,

guinea pig. Iron particles in cardiac myofibers. Prussian

Blue, Line = 100pm.............................................110
Interstitial cell with numerous perinuclear iron-filled

lysosomes, i.e., sideromes. Stained; Line = 500pm.............111
A) Energy spectrum obtained from interstitial cell lysosome
confirms the presence of iron. Copper peaks are due to copper
mesh grids; osmium peaks are the result of postfixation with
0504........................................... ...... ........113
3) Electron micrograph of cardiac interstitial cell with

iron laden lysosome-evaluated using STEM analysis. Unstained;
Line = lOOOpm..................................................114
Photomicrograph of testicle from iron dextran treated male

guinea pig with atrophy of seminiferous tubules. Note lack of
normal maturation and mitotic activity of spermatogenic
epithelium; marked vacuolation is notable. Hematoxylin

and eosin; Line = 100pm............................. ........... 123
Photomicrograph of testicle from dextran treated control male
guinea pig. Maturation of spermatogenic cells from

spermatogonia to spermatids is normal. Hematoxylin and eosin,
Line = lOOym............. ...... ........... ..... ... ...... .. ..... 123

xi

 

LIST OF ABBREVIATIONS

APO4ase ......................
Df ...........................

2+

Fe .................... .....
Fe3+ ........ ...... ...........
H202 ...... ....... ............
IHC ..........................
IP ................... ........
L' ...........................
LH ...........................
LOz' .........................
LOOH .........................
MDA ..........................
NAG ..........................
02 ..........................
OH' ..........................
SI ...........................
STEM .........................
Tf ...........................

TIBC .........................

acid phosphatase
desferrioxamine

ferrous iron

ferric iron

hydrogen peroxide
idiopathic hemochromatosis
iron particles

lipid alkyl radical
unsaturated fatty acid
lipid peroxy radical
lipid hydroperoxide
malondialdehyde
B-N-acetylglucosaminidase
superoxide radical
hydroxyl radical

serum iron

scanning transmission electron microscopy
transferrin

total iron binding capacity

 

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INTRODUCTION

Primary and secondary iron overload disorders are characterized by
variable degrees of cytosiderosis with the accumulation of iron in
lysosomes of both reticuloendothelial and parenchymal cells of the
liver, spleen, heart, pancreas and other tissues. As a consequence of
severe siderosis a variety of clinical manifestations may evolve
including hepatic cirrhosis, cardiomyopathy, endocrine pancreatic
dysfunction, bronze skin pigmentation, arthropathy and hypogonadism.

The pathogenesis of iron-induced organ injury is poorly understood
and is the subject of much debate. Previous research suggests that
iron-mediated lysosomal disruption may play a role in initiating cell
injury. Numerous reports demonstrate that hepatic lysosomes from iron
overload patients are particularly fragile when compared to control
subjects (1-4). Similar findings are documented in rats fed carbonyl
iron (5,6), injected with a iron-sorbitol—citric acid complex (7) or
iron nitrilotriacetate (8). Favored, mutually compatible, proposals for
the mechanism of lysosomal membrane injury in iron overload include (a)
accumulation of excess hemosiderin leading to physical disruption of the
lysosome and (b) iron-catalyzed lipid peroxidation mediating the loss of
lysosomal membrane integrity. Following lysosomal membrane injury, it
is hypothesized that leakage of acid hydrolases results in destruction

of intracellular constituents eventuating in cell damage (9)

 

Severe cardiomyopathy is a common clinical manifestation of
hereditary and transfusion-induced iron overload. Chronically
transfused thalassemic children with iron cardiomyopathy may develop
severe clinical problems or even die during puberty. Investigation of
this problem has been hampered by lack of an easily reproducible animal
model that mirrors the human condition. Although iron overload has been
reproduced in rats by injection of iron-sorbitol—citric acid complex or
feeding carbonyl iron, these models are expensive and require 3 weeks -
12 months before the animals are iron loaded and studies can begin
(5,10,11) .

We have developed a previously unexplored animal model of iron
overload. Guinea pigs intraperitoneally injected with iron dextran
develop hemosiderosis of both their livers and hearts. This model was
used to investigate the pathophysiology of iron-induced cardiac and
hepatic toxicity.

In this study alterations in the stability of both myocardial and
hepatic lysosomal membranes were examined. We determined the free
activity of hepatic glucosaminidase, glucuronidase, and acid phosphatase
as a measure of hepatic lysosomal fragility. Free myocardial acid
phosphatase and glucosaminidase activity were evaluated as indicators of
cardiac lysosomal fragility. In addition, the activity of serum
glucosaminidase was determined in an effort to correlate the serum
activity of this enzyme with tissue lysosomal enzyme changes.

In addition, we attempted to develop a serologic profile
reflective of the iron status in the iron loaded guinea pig. We

measured serum iron, total iron binding capacity and transferrin

saturation levels and subsequently compared values in this model with
other mammalian species.

Morphologic studies were conducted to determine the distribution
and location of iron in the heart and liver. Tissues were stained with
hematoxylin/eosin and Prussian Blue for histologic evaluation.
Ultrastructural studies were performed on both stained and unstained
sections of left ventricular myocardium; selected sections were assessed
by STEM analysis. A brief preliminary study was conducted to evaluate

iron-induced testicular alterations in male guinea pigs.

References

10.

11.

Seymour CA, Budillon G and Peters TJ. Lysosomal changes in human
and experimental iron overload. Gut 15:838, 1974.

Peters TJ and Seymour CA. Acid hydrolase activities and lysosomal
integrity in liver biopsies from patients with iron overload.
Clin.Sci.:Mol.Med. 50:75-78, 1976.

Selden C, Owen M, Hopkins JMP and Peters TJ. Studies on the
concentration and intra-cellular localization of iron proteins in
liver biopsy specimens from patients with iron overload with
special reference to their role in lysosomal description.
Br.J.Haematol. 44:593-603, 1980.

Seymour CA and Peters TJ. Organelle pathology in primary and
secondary haemochromatosis with special reference to lysosomal
changes. Br.J.Haematol. 40:239-253, 1978.

LeSage GD, Kost LJ, Barham JS and LaRusso NF. Biliary excretion of
iron from hepatocyte lysosomes in the rat. J.Clin.Invest. 77:90-
97, 1986.

Iancu TC, Ward RJ and Peters TJ. Ultrastructural observations in
the carbonyl iron-fed rat, an animal model for hemochromatosis.
Virchows Arch.B. 53:208-217, 1987.

Hultcrantz R, Ahlber J and Glaumann H. Isolation of two lysosomal
populations from iron-overloaded rat liver with different iron
concentration and proteolytic activity. Virchows Arch (Cell
Pathol.) 47:55-65, 1984.

Peters TJ, O'Connell MJ and Ward RJ. Role of free radical mediated
lipid peroxidation in the pathogenesis of hepatic damage by
lysosomal disruption. In: Free radicals in liver injury, edited by
Poli G, Cheeseman KH, Dianzani MV and Slater TF. Oxford: ITP Press
Limited, 1986, p. 107-115.

Peters TJ, Selden C and Seymour CA. Lysosomal disruption in the
pathogenesis of hepatic damage in primary and secondary
haemochromatosis. Iron Metabolism:Ciba Foundation Symposium
51:317-325, 1977.

Hultcrantz R and Arborgh 8. Studies on the rat liver following
iron overload. Fine structural appearance. Acta
Path.Microbiol.Scand.Sect.A 86:143-155, 1978.

Park CH, Bacon BR, Brittenham GM and Tavill AS. Pathology of
dietary carbonyl iron overload in rats. Lab.Invest. 57:555-563,
1987.

 

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REVIEW OF THE LITERATURE

Iron storage diseases in humans may be classified as either
primary or secondary. Primary iron overload specifically refers to
hereditary (idiopathic) hemochromatosis (IHC), the most common cause of
significant iron overload. In this disease, increased intestinal
absorption of iron on a diet with normal iron content, leads to iron
overload with eventual tissue damage. Secondary iron overload defines a
group of disorders (e.g., the thalassemia syndromes, transfusional
hemosiderosis, African Siderosis) in which the iron overload is
attributable to some abnormality other than a primary increase of
intestinal iron absorption. Regardless of the cause, both primary and
secondary iron overload disorders eventuate in a clinical syndrome of
diabetes mellitus and pigmentary cirrhosis, often accompanied by cardiac

dysfunction, gonadal failure and hyperpigmentation of the skin.

I. PRIMARY IRON OVERLOAD

Hereditary (idinnathir) " L L in (Iggy

The clinical syndrome of portal cirrhosis, diabetes mellitus, and
bronze skin pigmentation was first described in 1865 by Trousseau (1).
In 1871, Troisier (2) referred to a similar entity as "pigmentary
cirrhosis in sugar diabetes" and "bronze diabetes." This was first
referred to as hemochromatosis by von Recklinghausen in 1889 (3). The
first major review and organization of the clinical data was published
in 1935 by Sheldon (4), who recounted several instances of familial
hemochromatosis and postulated that the disease was due to an inborn

error of metabolism. MacDonald (5,6) later proposed the opposite

5

 

viewpoint, stating that most, if not all cases of idiopathic
hemochromatosis were acquired. According to this theory, a toxic
process, usually due to alcoholism, damages the liver and other organs
and in some way predisposes this damaged tissue to accumulate excessive
quantities of iron. Most recent investigations have supported the
hypothesis that hereditary hemochromatosis is inherited, and it has been
shown that the gene is linked to the major histocompatibility complex on
the sixth human chromosome (7,8). Nevertheless, it is generally
recognized that variable expressivity, as well as certain exogenous
factors (e.g., alcohol intake), may influence the course and severity of
the disease.

In IHC, increased amounts of iron are absorbed from the diet and
deposited mainly in hepatocytes and in parenchymal cells of other
organs. Increased iron deposition in reticuloendothelial cells, an
important site of iron storage in normal individuals, usually does not
occur in this disease until iron overload is far advanced (9).

The adult form of hereditary hemochromatosis is a disease which
most often becomes clinically manifest during the fifth and sixth decade
of life and is quite unusual before the age of 20 (Juvenile IHC appears
clinically during the second and third decades of life). It is more
common in men than in women. Persons who manifest clinically
significant hemochromatosis undoubtedly have had progressive tissue iron
overload for many years. The development of signs and symptoms of
hemochromatosis is associated with cellular and tissue damage, thought
to be due to toxic levels of iron. Involvement of the liver, heart,
joints, pancreas, and other endocrine organs culminates in overt

manifestations of the disease. The clinical course of the disease in

younger individuals seems to be more fulminant, (10,11) and cardiac

dysfunction more common (12-14).

L e to
A. Population Association

In 1975, Simon et al. (15) reported a relationship between
hemochromatosis and the major histocompatibility (HLA) locus, noting an
association with the HLA antigen A3. This discovery led eventually to
the elucidation of the mode of inheritance. Walters et al. (16) and
Shewan et al. (17) confirmed the findings of Simon and co-workers, and
in addition noted an association with HLA-B7 among patients in Great
Britain. However, the patients studied by Simon et al. (15,18) in
Brittany, France more often possessed the combination of HLA-A3 with -
814. Simon also found that among unrelated patients with hereditary
hemochromatosis the incidence of HLA-A3 was 78.4‘, in contrast to an
incidence of 27% among a group of apparently healthy adults. This
apparent association is surprising because most diseases associated with
a particular HLA antigen involve disorders of the immune system.
Another unusual feature of this observation is that whereas most
associations between HLA and disease involve B-series antigens,
hemochromatosis appears to be more commonly associated with a A-series
antigen. Class II antigens have also been implicated; a significant
increase in DR2 was reported by Doran et al. (19), however the
prevalence of several other Class II antigens (i. e., CW, Bf, DRw6, and

GIo) has not been demonstrated to be significantly altered (20).

 

B. Antigen Association: True significance

Current evidence indicates that hemochromatosis is associated with
HLA-A3 more frequently than with B7 or 814. Doran et al. (19) reported
an increased prevalence of A3 in B7-negative patients. In contrast to
this evidence Valberg (21) suggested that HLA-87 was more prevalent than
A3 in hemochromatosis. Ritter et al. (22) reported a higher relative
risk for 814 than A3. The conflicting evidence implies that A3, as well
as 87 and 814 are all independent markers of the hemochromatosis allele.
However, data on haplotype associations clearly indicate that haplotypes
carrying 87 or 814 without A3 are not more frequent in hemochromatosis
than in controls. However, haplotypes carrying A3 without 87 or A3
without 814, or also A3 without either 87 or 814 are significantly more
frequent in hemochromatosis than in controls. Therefore, the
significance of B7 and 814 as a marker of the hemochromatosis allele
dissolves in the absence of A3; A3 remains the only independent marker.
The presence of HLA-B7 and/or 814 alone is of no significance.
Simon et al. (18) was the first to suggest the possibility that the
particular HLA antigens involved might not be critical and that a
specific gene for hemochromatosis might be linked with the major
histocompatibility complex on chromosome 6. The gene for this disease
has now been mapped on chromosome 6, although its precise location
remains a mystery. Numerous linkage studies (7,19,20,23-26) indicate
the genetic distance between the HLA locus and hemochromatosis locus
ranges from O to 2.5 centimorgans. Lalouel et al. (27) reported a

distance of 1.0 centimorgan in a study involving 147 families.

 

 

II. SECONDARY IRON OVERLOAD
Secondary iron overload may result as a consequence of : a) anemia
and ineffective erythropoiesis (due to long term red cell transfusions);

b) liver disease, and c) excessively high intake of dietary iron.

A. Iron Overload Secondary to Anemias

Patients with iron loading anemias fall into two groups (a) those
with decreased red cell production secondary to marrow failure (e. g.,
anemia secondary to marrow aplasia) whose major source of excess iron is
blood transfusion, and (b) patients with hyperplastic bone marrow but
ineffective erythropoiesis (e.g. thalassemia major). In the latter
patients, the excess iron results not only from transfusion but also
from increased iron absorption secondary to the ineffective
erythropoiesis, thus the incidence of hepatic cirrhosis and cardiac
involvement is much higher in this group. Patients with thalassemia
major can be kept in good health for the first decade of life with
regular blood transfusions. Inevitably, however, iron overload results
with deposition of iron in many organs including the heart, liver and
endocrine glands. The gross deposition of iron in the myocardium and
conducting fibers causes arrhythmias and refractory cardiac failure,
usually resulting in death between the ages of 16 and 22 years of age
(28).

In thalassemia major, hepatic parenchymal iron deposition occurs
even before transfusion therapy has begun; reticuloendothelial iron
loading becomes prominent only after transfusions have been initiated
(29). Even nonthalassemic patients chronically maintained on

transfusion therapy for treatment of other anemic states may eventually

 

 

10

develop parenchymal cell iron overload and progress to a syndrome

similar to hemochromatosis.

8. Iron Overload Secondary to Liver Disease

For the past two decades there has been confusion between IHC and
alcoholic cirrhosis with increased stainable iron in the liver. It is
now known that some stainable iron is quite common in normal subjects
and in patients with cirrhosis.

Patients with alcoholic cirrhosis and stainable liver iron can be

divided into two groups: (1) those patients who have a mild-to-moderate
increase in stainable iron but relatively normal body iron stores, and
(2) those patients with gross iron deposition and increased total body
iron stores of the magnitude seen in IHC (15-50 9 iron stores).
The majority of the patients in the first group have alcoholic liver
disease (usually cirrhosis) with some increase in stainable hepatic
iron, but little increase in total body iron; these patients do not have
IHC. Simon et al. (30) have concluded from their study, which included
determination of HLA antigens, that such subjects are neither homozygous
nor heterozygous for the disease. Liver iron concentration in such
patients is usually less than twice the upper limit of normal (30,31).
The reason for the increased stainable iron is unknown. It may be
related in part to cell necrosis and the uptake of released iron by
adjacent Kupffer and parenchymal cells.

The second group of alcoholic subjects who have cirrhosis with
gross iron overload probably have IHC. A study of the hepatic pathology
of alcoholic patients with a family history of IHC has revealed that in

approximately 75$ of them, the histologic changes are indistinguishable

1
f

 

11

from those in nonalcoholic subjects with IHC (32). The remaining 25%
show very similar changes, but with features of alcoholic liver disease

superimposed.

C. Iron Overload Secondary to Excessive Dietary Ingestion

Dietary iron overload, the prototype of which is African
siderosis, has resulted in varying degrees of hemosiderosis.

African siderosis occurs in South African blacks and is one of the
most thoroughly described examples of acquired iron overload. Initially,
the excess iron deposits in reticuloendothelial cells (usually termed
hemosiderosis). If the level of iron accumulation is sufficiently
great, this syndrome may progress to a clinical picture resembling
hemochromatosis, with iron deposition in hepatic parenchymal cells.
Strachan first called attention to this entity in 1929 (33). Among 1100
autopsies performed in Johannesburg between 1924 and 1928, he found 33
instances of iron deposition comparable to that seen in hereditary
hemochromatosis. Many of these individuals apparently had bronze
diabetes mellitus and cirrhotic livers with large deposits of
hemosiderin. A number of theories were proposed, but the etiology was
not discovered until Walker and Arvidson (34) found that the prepared
diet of these Africans contained approximately ten times more iron than
could be measured in the uncooked food. Subsequently, it was
demonstrated that iron derived from iron cooking pots was responsible
for the excess iron in the diet. The traditional beer prepared by the
Africans was brewed in these iron pots, and the amount of iron dissolved
in the beer (approx. 4 mg Fe/dl) was enhanced by its lower pH. The

distribution pattern of the resulting dietary iron overload differed

 

 

12

from IHC. In African siderosis, more hemosiderin was found in the
spleen than in the liver. In contrast to IHC, hemosiderin was identified
in the pancreas in only the severe cases (4). Hemosiderin deposits were
unusually heavy in the bone marrow and in the duodenal and jejunal
villi. Subsequently, distinct differences were recognized in the liver
iron distribution; in African siderosis the hemosiderin was most
prominent in the Kupffer cells. Only in the advanced stages was
hemosiderin conspicuous in the hepatic parenchymal cells (35,36). In
contrast, hemosiderin was prominent in the hepatic parenchymal cells in
IHC, even in early stages of the disease. Thus, despite similar
endstage disease manifestations, the clinical picture and the initial
histologic distribution of the excess iron differs in these two
conditions, suggesting different etiologic mechanisms. Clinically, the
African siderotic deviates from the patient with IHC in that (1) the
progress of the cirrhosis in the African is more rapid and is the
principal cause of death, (2) there is a high incidence of porphyria
cutanea tarda among African siderotics, and (3) cardiac complications

are rare. (37).

III. NANIFESTATIONS OF IRON OVERLOAD IN SPECIFIC ORGAN SYSTEMS
The physical findings in patients with iron overload are usually

related to involvement of the heart, liver, skin or testicle.

A. Heart
Cardiac failure is the most critical life-limiting complication of
both primary and secondary iron overload. Cardiac dysfunction is

diagnosed in 35% of patients with hereditary hemochromatosis;

 

 

13

approximately one third of untreated hemochromatotic patients die of
cardiac failure or arrhythmia. Cardiac disease is especially common in
younger patients, who often have a more rapidly progressive disease
process.

The cardiac disease in iron overload is most likely due to iron
deposition in heart muscle fibers. In an extensive study by Buja and
Roberts (38) nineteen of 135 patients (four with idiopathic
hemochromatosis and 131 with chronic anemia) had cardiac iron deposits
(CID). The ventricular CID were grossly visible in nine patients and
microscopically visible only in ten patients. Atrial CID were extensive
in six patients with extensive ventricular CID, but in the other
thirteen patients atrial CID were insignificant. Iron deposits in
cardiac conduction tissue were minimal and always less than working
myocardium. Of the nineteen patients with CID, three had idiopathic
hemochromatosis; 16 had chronic anemia. Each anemic patient who
received more than 100 units of blood had extensive CID unless chronic
bleeding diatheses coexisted.

This study Concluded that (1) grossly visible CID were always
associated with cardiac dysfunction and usually chronic cardiac failure;
(2) CID, usually extensive, occur in patients with idiopathic
hemochromatosis; (3) extensive CID occur in patients who receive more
than 100 units of blood unless bleeding diatheses coexist; (4) CID
initially occur in ventricular myocardium, and were usually more
extensive in ventricular than atrial myocardium; (5) CID were always
more extensive in working than in conducting myocardium; (6)

supraventricular arrhythmias correlate with the extent of CID in atrial

14

myocardium. These conclusions refuted the findings of a previous study
(39).

Sanyal et al. (40) investigated both the histochemical and
ultrastructural aspects of the heart in an adolescent with fatal
congestive heart failure resulting from exogenous hemochromatosis.
Histologic sections of myocardial tissue showed extensive iron deposits
in all four chambers, papillary muscles and the conduction system; these
were greatest within the outer third of the left and right ventricular
myocardium. Iron deposits in the middle and inner third of the left
ventricle were chiefly focal, in contrast to a more diffuse pattern in
the outer third. There were no differences present between the other
portions of the ventricular myocardium, however the interventricular
septum contained heavy iron deposits. Degenerative changes of the
ventricular myocardium were minimal and nonspecific, but occurred more
frequently in the left ventricle.

Ultrastructural findings demonstrated that intracytoplasmic iron
deposition followed one of three patterns: (1) paranuclear - a small
collection of iron adjacent to the myofiber nucleus; (2) perinuclear - a
more extensive deposition of iron around the nucleus; (3) diffuse -
maximum quantities of iron present in a diffuse cytoplasmic pattern. It
was noted that regardless of the pattern of deposition, some iron was
consistently present within the nucleus and mitochondria. Another
ultrastructural alteration of the myofibers was an increase in
mitochondria associated with a decrease in myofibrils.

This study concluded that iron deposition in the ventricular
myocardium follows a differential zonal pattern, with the greatest

amount in the subepicardial region, an intermediate ,amount in the

 

 

15

subendocardial region and papillary muscles, and the least amount in the

middle third of the ventricular myocardium. This zonal concept of iron
distribution in ventricular muscles was originally suggested by Cappel
et a1. (41) and further supported by Buja and Roberts (38), as well as

Olson et al. (42).

8. Liver

Manifestations of liver involvement are among the most constant
features of iron overload disease (43); hepatomegaly is diagnosed in
more than 90% of hemochromatotic patients. The enlarged liver usually
is smooth rather than nodular, however hepatomegaly diminishes in many
cases after phlebotomy therapy. Cirrhosis, when present, is of the
micronodular type. If the disease remains untreated, cirrhosis will
slowly progress to hepatic failure with all the associated
complications. Treatment by venesection can reverse many or all of the
abnormal histologic findings and architectural changes of cirrhosis (44-
46). This observation supports the hypothesis that the cirrhosis is due
to the toxic effects of excess iron. Recent reports indicate that the
life expectancy of cirrhotic patients is markedly reduced compared to
noncirrhotics, which had a life expectancy nearly identical to that of
an age-matched normal population (47).

In the past, hepatoma occurred in about 14: (7 to 20:) of patients
with hereditary hemochromatosis (12,39,48,49). Current evidence
indicates that primary hepatocellular carcinoma and bile duct carcinoma

are the leading causes of death in hemochromatotic patients (47).

 

16

C. Skin

Abnormal pigmentation is present in 908 of patients with fully
expressed hemochromatosis. The color is variable, ranging from the
classic bronze pigmentation to a slate gray appearance. The pigmentation
may be patchy with dry, friable skin. The hyperpigmentation is due to
the presence of melanin in the epidermis, however iron deposits may be
found in the dermis, particularly around the sweat glands (50). This
association with the sweat glands is not surprising, since iron is
commonly deposited in other glandular organs of the body. The cause of

the melanoderma is not known.

D. Gonads

Hypogonadism, impotence and loss of libido are prominent clinical
features in men with IHC (51-56). Hypogonadism usually results from
iron-induced damage of the pituitary gonadotrophs or the GnRH
(gonadotropin releasing hormone) cells of the hypothalamus (57,58). The
resulting FSH/LH (follicle stimulating hormone/luteinizing hormone) or
GnRH deficiency leads to variable atrophy of seminiferous tubules and
absence of spermatozoa and spermatids. Typically, the testes contain no
excess iron or may have minimal deposits limited to blood vessel walls,
thus sparing the germinal epithelium and interstitial cells (56).
Bergeron and Kovacs (59) using a combination of immunocytochemical and
Prussian Blue staining technique demonstrated that iron was
preferentially localized in the gonadotropic cells of IHC patients. A
minority of thyrotropic, corticotropic or somatotropic cells contained
iron. Thus, panhypopituitarism with hypothyroidism and adrenal cortical

insufficiency is a rare event in IHC (54). Previously, this form of

17

hypogonadism was considered irreversible and male patients were treated
with testosterone supplementation (57). Recent case reports have
indicated a restoration of normal pituitary and testicular function
following aggressive phlebotomy therapy in hemochromatotic patients with
hypogonadism (55,56)

It is notable that hypogonadism is particularly severe in both

juvenile IHC and young well-transfused thalassemic patients.

IV. DIAGNOSIS

There is no single clinical sign which is pathognomonic of
hemochromatosis, nor is there a single noninvasive test or combination
of tests adequate for its diagnosis. In patients with various types of
acquired iron overload, the underlying cause for the excess iron
accumulation often is obvious, while in hereditary hemochromatosis there
is no apparent inciting event. It is important to exclude causes of
secondary iron overload in evaluating patients with suspected

hemochromatosis.

A. Serum Ferritin

The most valuable test for the detection of tissue iron overload,
regardless of location (parenchymal or reticuloendothelial), is
measurement of serum ferritin. In healthy adults the concentration of
ferritin in serum is directly related to the available storage iron in
the body. The mean concentration of serum ferritin is three times
higher in males than in females, with a range between 12 and 250 ug/
liter (60,61). In patients with iron deficiency anemia concentrations
are below 12 pg/liter, and in patients with iron overload the

concentration may be as high as 10,000 pg/liter (60,62-64). A

18

retrospective study by Milder et al. (51) reported a mean serum ferritin
level of 3,735 pg/liter (range 1,132-7,059 pg/liter) in a group of 34
hemochromatotic patients.

The measurement of mobilizable iron stores by quantitative
phlebotomy in normal subjects show a good correlation with initial serum
ferritin concentration (65) and a similar relation is seen in idiopathic
hemochromatosis (66). In patients with transfusional siderosis the serum
ferritin concentration is related to the amount of blood given (60,64),
and there is a good correlation between serum ferritin concentration
and the chemically determined iron concentration in liver biopsy tissue
(64). Comparisons between serum ferritin concentration and the
subjective semiquantitative visual assessment of stainable iron in bone
marrow smears (63,67) show a crude relation between these two indexes of
iron stores.

In most situations the serum ferritin concentration appears to
reflect reticuloendothelial storage iron fairly accurately. Changes in
reticuloendothelial iron are followed rapidly by changes in the serum
concentration of ferritin. In normal subjects undergoing venesection
the serum ferritin concentrations fall rapidly as iron stores are
mobilized, and in both hemochromatosis and secondary iron overload,
serum ferritin can be used to monitor therapeutic removal of excess
storage iron. In addition, it has been used to evaluate the effect of
chelation therapy in thalassemic patients with iron overload.

A limitation of serum ferritin concentration as a measure of iron
stores is that it is not specific for iron overload; certain disease
states unrelated to iron overload may also result in elevated serum

ferritin levels (68). These include hepatocellular necrosis, malignant

19

neoplasia, leukemia and related disorders of the monocyte-macrophage

system, and acute inflammation (68,69).

8. Transferrin Saturation

Transferrin saturation is the single most reliable screening test
for hemochromatosis (70). Dadone et al. (26) demonstrated a transferrin
saturation level above 62% to be the best indicator of homozygosity for
hemochromatosis (92% correlation). However, other researchers have
shown a cutoff of 55% to be the optimal discriminator between
homozygotes and nonhomozygotes (70,71). Despite this the combination of
serum transferrin and serum ferritin is still considered more sensitive

than either test used alone.

C. Serum Iron Concentration

In primary hemochromatosis the serum iron concentration is
generally increased; a mean value of 211.3 pg/dl was documented by
Milder et al. (51). Serum iron has the disadvantage of being a
nonspecific indicator of total body iron stores and may be markedly
decreased during inflammatory processes (i.e., hypoferremia of

inflammation).

D. Chelation Tests - Desferrioxamine Excretion

Parenchymal as opposed to reticuloendothelial iron overload may be
further identified by chelation tests i.e., after intramuscular
injection of the iron chelating agent desferrioxamine (500 mg), patients
with hemochromatosis or other iron overload states (that maintain
regular renal function and are ascorbic acid replete) usually will

excrete substantially more iron in a 24 hour urine sample (> 2 mg) than

20

will normal subjects (72,73). The estimation of serum ferritin
concentration, however, has now virtually replaced this test as an
indication of excessive iron stores in the diagnosis of uncomplicated
idiopathic hemochromatosis.

Another chelating agent employed in the assessment of iron
overload is diethylenetriamine pentaacetic acid [DTPA] (74,75). The
principles and techniques employed in the diagnostic use of
desferrioxamine may be applied to DTPA, except the latter is given by

i.v. infusion.

E. Hepatic Iron Stores

A definitive diagnosis of iron overload requires a liver biopsy.
A percutaneous needle biopsy provides not only an estimation of tissue
iron deposition, but also enables one to directly assess the
distribution of iron among Kupffer cells and hepatocytes, as well as
document associated changes such as fibrosis or cirrhosis. The extent
of hepatic iron accumulation may be estimated semiquantitatively by
histochemical grading. In a commonly used system (76), a grade of 0
signifies absence of iron, while grade 1, 2, 3, and 4 represent
increasing amounts of stainable iron by Perl's Prussian Blue. Iron
grades of 3 and 4 are cannon in untreated hemochromatosis. The iron
deposition in hereditary hemochromatosis characteristically is greatest
in the periphery of the lobules (32). Reticuloendothelial (Kupffer
cell) iron may be inconspicuous despite significant hepatocellular
involvement, although late in the disease process both types of cells
may be involved. In secondary iron overload, iron accumulation usually

is less, in the range of grade 1 and 2 (77). Reticuloendothelial iron

21

in a periportal distribution is prominent. Forms of severe secondary
iron overload (i.e., advanced transfusional siderosis) result in
markedly higher iron grades, thus may be histologically
indistinguishable from advanced hereditary hemochromastosis.

Powell and Kerr (32) have further defined the criteria for
histochemical grading. In this system, grade 4 is assigned to biopsies
with stainable iron is virtually 100% of hepatocytes, grade 3 to
biopsies with iron in 75‘, and grades 2, 1, and 0 to correspondingly
lesser degrees of hepatocyte involvement. Since stainable iron may be
visualized even when tissue iron concentration is normal or only
slightly’ increased, direct. determinations. of ‘tissue iron. content of
liver biopsy specimens may be useful. Both histochemical and chemical
iron determinations may be obtained conveniently using liver tissue from

a single percutaneous needle biopsy (78,79).

F. Bone Marrow Iron

In normal individuals and in patients with secondary iron
overload, iron stores may be judged by Prussian blue staining of bone
marrow specimens and estimation of the ferritin and hemosiderin content
of bone marrow macrophages; these estimates generally are well-
correlated with RE iron elsewhere in the body (80) . In hereditary
hemochromatosis bone marrow iron often is inconspicuous despite iron
overload in other organs (11,81-83). Thus, a comparison of marrow to
liver iron content may be useful in differentiating hereditary

hemochromatosis from secondary iron overload.

22

V. IRON AND FREE RADICAL FORMATION

Iron is essential for a. wide variety of metabolic processes
functioning primarily as an important biological catalyst (similar to
many transition metals). Despite its physiologically beneficial role as
a catalyst, iron can also have deleterious effects due to its ability to
undergo changes in oxidation states involving one electron. The ability

to access two oxidation states, ferrous iron (Fe2+) and ferric iron

(Fe3+

), allows it to coordinate electron donors and to participate in
redox processes. However, the characteristics that made iron an
excellent catalyst also renders it potentially hazardous since redox
reactions favor a reductive pathway which can lead to the formation of
unstable intermediates with unpaired electrons, i.e., to ”free radical"
formation. The term "free radical" has been broadly defined by
Halliwell and Gutteridge (84) as any species capable of independent
existence that contains one or more unpaired electrons; an unpaired
electron is one which occupies one atomic molecular orbital by itself.
Acceptance of a single electron by the oxygen molecule, 02 in its
ground state (the most stable configuration of the 02 molecule) results
in the production of the superoxide radical, 02'. Addition of a second
electron to the superoxide radical gives the peroxide ion, 022', which
has no unpaired electrons; thus it is not a radical. At physiological
pH the peroxide ion is immediately protonated to produce hydrogen
peroxide, H202 and 02; the reaction can be written:

202'

+ 28+ ———> H202 +02 (1)
It should be noted that both the superoxide radical and hydrogen

peroxide can be detected during normal metabolism in many biological

23

systems and there is sufficient evidence that they exist in vivo (85-
92).

Due to the relatively weak. O-O bond in H202, it is readily
converted into two hydroxyl radicals (OH'); this reaction can be
achieved by heat or by ionizing radiation. Additionally, a mixture of

2+

H202 and Fe (ferrous iron) readily forms the OH' radical. This

reaction is known as the zentgn geggtiog:

Fe2+ + 3202 _—> 03' + Fe3+ + on“ (2)
Also, traces of Fe3+ (ferric iron) can react with 8202 to produce the
Fe2+ required in reaction (2):
3+ 2+ -
Fe + 8202 ———> Fe + 02 + 23+ (3)

Ferric iron also reacts with 02' to generate H202 and Fe2+. This may

result in the generation of the hydroxyl radical through a series of

three reactions collectively known as the Haber-Weisg reaction:

02' + Fe3+ ———> 02 + 1'92" (4)
02' + 02‘ ——> 02 + H202 (1)
3202 + 292+ —> 03' + on' + 1793+ (2)

(The last step is W)-

Of the numerous reduction products derived from oxygen in the presence
of iron, it is the hydroxyl radical, 08', that is by far the most toxic.
This free radical reacts with extremely high rate constants with most
organic molecules found in cells. In particular, it attacks and
destroys cell membranes, as well as DNA. In light of the damaging
effects of the OH’ radical, it is not surprising that aerobic organisms
have evolved two enzymes to remove the two reactants, H202 and 02-, as
rapidly as they appear to minimize the production of the OH’ radical.

The enzyme superoxide dismutase scavenges the 02- and glutathione

24

peroxidase (selenium enzyme) and catalase deal with H202. These enzymes
are present in relatively large amounts in all aerobic organisms and
their role, as well as the role of other molecules, in protecting
biological systems against oxygen radicals is adequately documented
(84). Since the toxicity of the 02' and H202 involves their conversion
to the OH' radical a reaction which in turn will depend on the amount of
catalytic iron available, it can be seen that it is also important that
iron be chelated in a controlled way in vivo so that uncontrolled free
radical reactions are avoided.

In vertebrates most iron is tightly bound to proteins (i.e.,
transferrin, ferritin, lactoferrin) and would be unavailable for the
Fenton reaction. The small pool of non-protein bound iron present
could, however, conceivably provide iron for this reaction;
physiological chelates, such as ferric citrate, have been shown to react
with H202 in vitro. Normally this ”free pool" is kept extremely small
but "free iron" has been detected in individuals with iron-overload

disease (93).

VI. NONENZYNIC LIPID PEROXIDATION

A free radical that has sufficient energy to abstract a hydrogen
atom from a methylene carbon of a unsaturated fatty acid (LH) can
initiate a chain reaction in bulk lipid. The resulting carbon-centered
radical (L') reacts rapidly with molecular oxygen to form a peroxy
radical (L02°), which itself can abstract a hydrogen atom from an
unsaturated fatty acid, leaving a carbon-centered radical and a lipid

hydroperox ide (LOOH) .

25

LH + R' ———> L' + RH Initiation [1]
L . + 02 —> L02 .

LH + .Loz' ———> LOOH + L' Propagation [2]
L' + L’ ——-> LL

L02‘ + L02° ———> LOOL + 02 Termination [3]

L02 ' + L ' —> LOOL

The free-radical chain reaction propagates (reaction 2) until two
free radicals destroy each other to terminate the chain (reaction 3).
In nonenzymic lipid peroxidation, the peroxy radicals last long enough
to be able to move to new fatty acid molecules since they can readily be
intercepted and scavenged by a variety of different antioxidants.
Peroxidic products of the chain reaction (LOOH) are a complex mixture of
isomers.
VII. FREE RADICALS, LIPID PEROXIDATION AND THE PATEOGENESIS OF IRON

ONERLOAD DISEASE

In both primary and secondary iron overload conditions, ferritin,
hemosiderin and transferrin become heavily loaded with iron. As
previously mentioned, "free", non-protein bound iron in the form of low
molecular weight complexes, has been found in the serum of some patients
suffering from idiopathic hemochromatosis and transfusional siderosis
(93,94). Although, iron accumulates in most organs of the body, severe
clinical manifestations result from cytosiderosis of the liver and
heart. The heart is particularly sensitive; in the past as many as one-

third of untreated hemochromatotic patients died as a result of cardiac

26

failure or arrhythmia (12). In addition, many chronically-transfused
thalassemics and juvenile hemochromatotics die during their second and
third decades of life from congestive heart failure and cardiac
arrhythmias (95). The ultrastructural appearance of iron-loaded cells
from hemochromatotic individuals is characterized by an accumulation of
electron-dense ferritin cores, mainly in the cytoplasm and. by the
presence of electron dense deposits of iron within the lysosomes
(96,97). It has been hypothesized that these iron deposits are
responsible for damaging lysosomal membranes, releasing hydrolytic
enzymes into the cytoplasm, thus initiating cell damage (98,99). Work
of Allison and Young (100) provided evidence that hydrolases released in
vivo in cells can cause severe damage to cellular structure. Thus, the
susceptibility of the lysosomal membrane to lipid peroxidation is of
apparent importance to cellular pathology.

In a study of six patients with primary hemochromatosis and eight
patients with secondary iron overload (all with thalassemia major),
Peters and Seymour (98) demonstrated that lysosomes from liver biopsies
of patients with iron overload were strikingly more fragile than control
subjects. Also noted was that the lysosomal integrity of liver biopsies
from patients with other types of cirrhosis were normal, thus indicating
that the lysosomal changes were not due to a cirrhotic process affecting
the homogenization procedure. Similar studies in biopsies from patients
with different forms of chronic hepatitides including alcoholic liver
disease have not demonstrated lysosomal abnormalities (101). Selden et
al. (99) demonstrated a close positive correlation between enhanced
lysosomal fragility and hemosiderin content of liver biopsies from

patients with primary' or secondary hemochromatosis. Thus, it was

27

suggested that the hemosiderin was responsible for the lysosomal
disruption and subsequent tissue damage.

The mechanism of increased lysosomal fragility in hemochromatosis
is uncertain. Early researchers proposed that distension of the
lysosomes with poorly degradable ferritin and hemosiderin lead to
rupture of their membranes (102). However, subsequent data show that
lysosomal membrane rupture is not due ‘to simple distension. of the
lysosomes (104) nor to an in vitro mechanical effect (103). Iron-
generated free radicals have been implicated in lysosomal disruption by
their ability to initiate a chain reaction of lipid peroxidation with

lysosomal disruption in vitro (104—108).

VII. IRON AND IN VIVO LIPID PEROIIDATION

Numerous studies have documented evidence of enhanced lipid
peroxidation in both naturally-occurring and experimental iron overload.
Many of these studies demonstrated a protective role for various
antioxidants, thus providing additional indirect evidence that lipid
peroxidation is associated with iron overload in vivo.

Dougherty et al. (109) measuring exhaled ethane, a volatile
autoxidation product of omega-B-unsaturated fatty acids demonstrated
that vitamin E, the most important in vivo biological free radical
scavenger, and selenium (a critical component of glutathione peroxidase,
an enzyme which. metabolizes hydrogen peroxide and lipid peroxides)
protected rats against lipid peroxidation induced by acutely toxic doses
of ferrous chloride and iron dextran. In a similar study, Dillard et
al. (110) measured pentane (a volatile decomposition product of omega-6-

unsaturated fatty acids) as an index of in vivo lipid peroxidation.

28

This study demonstrated that both dl-alpha-tocopherol (vitamin E) and L-
ascorbic acid (vitamin C) effectively suppressed lipid peroxidation in
rats injected intraperitoneally with iron dextran. Results suggested
that vitamin E was much more effective than vitamin C as an antioxidant
and N,N'-diphenyl-p-phenylenediamine was most effective among a group of
synthetic antioxidants tested. These results confirmed previous
findings (111,112). Bacon et al. (113) provided evidence of in viva
hepatic mitochondrial and microsomal lipid peroxidation as measured by
conjugated diene formation in two models of experimental chronic iron
overload in rats (i.e., parenteral ferric nitrilotriacetate and dietary
supplementation, with carbonyl iron). Results indicated that
mitochondrial lipid peroxidation occurred at several hepatic iron
concentrations in both models of iron overload; whereas, microsomal
lipid peroxidation was detected only at the higher liver iron
concentration achieved by dietary carbonyl iron supplementation. Thus,
in light of these findings sufficient evidence is available to indicate
that lipid peroxidation is associated with experimental iron overload in
viva.

Lipid peroxidation has also been demonstrated in naturally-
occurring iron overload disease. Rachmilewitz et al. (114) showed that
the erythrocytes of thalassemia major patients with transfusional iron
overload underwent excess lipid peroxidation; this was associated with
decreased levels of vitamin E. Heys and Dormandy (115) demonstrated
that iron overloaded spleens from thalassemic patients were more
susceptible to free radical peroxidation in vitro. In addition, it was
found that tocopherol levels in these spleens correlated inversely with

iron overload and homogenates with a low tocopherol content showed the

29

greater tendency to free radical oxidation, thus iron content was the
main but not the only variable governing susceptibility to peroxidation.
This study also investigated the effects of ascorbate on peroxidation
and concluded that added ascorbate had a dose-dependent action either as
an antioxidant or as a pro-oxidant, the direction of its effect

depending mainly on the degree of iron overload.

II. IRON AND MYOCARDIAL CELLS

Most of the studies of iron overload have been focused of the
effect of excess iron on hepatocytes. However, iron cardiotoxicity
remains the major life-limiting complication of thalassemia and other
chronic anemias requiring continued transfusional therapy. Therefore,
understanding the mechanism of myocardial iron toxicity is critical.
Within the last decade, studies utilizing cultured neonatal rat
myocardial cells have greatly enhanced the investigation of iron
cardiotoxicity.

Early studies by Cox et al. (116) demonstrated that ferric
ammonium citrate was readily taken up by cultured neonatal rat
myocardial cells and that approximately 50% of the iron was sequestered
in ferritin. Ultrastructural evaluation of these cells showed that
endocytic vesicles and lysosomes contained iron-filled ferritin
molecules. The number of lysosomes increased with time in both the
control and experimental cultures; however the control cultures
contained considerably fewer molecules of ferritin per lysosome than
iron-treated cultures. Also, the number of ferritin molecules apparent
in lysosomes increased with time and with increasing concentrations of

iron in the medium.

30

Subsequent studies by Link et al. (117,118) correlated structural
and biochemical alterations with functional derangement of myocyte
contractility in cultured myocardial cells. Results of these studies
indicated that roughly one-third of cellular radiolabelled iron was in
ferritin and the rest in an insoluble lysosomal fraction (i. e.,
hemosiderin). However, iron uptake was almost completely inhibited by
reducing the incubation temperature from 37°C to 10°C. Additionally,
studies of iron-induced lipid peroxidation demonstrated that
intracellular concentrations of malondialdehyde (MDA) were double after
15 minutes of iron loading and reached maximal concentrations at 3
hours; cellular MDA concentrations were normalized by iron mobilization
using desferroxamine at concentrations ranging from 0.025 mmol/L to 0.3
mmol/L. Normalization of cellular MDA concentrations was in direct
proportion to the amounts of iron removed. Functional studies
determined the amplitude and rate of cell contractions using a Model 633
Video Analyser; results showed that exposure of myocardial cells to 20
ug/ml of iron gradually reduced the contractility and increased the
rates and irregularity of the heart cell beats. In conclusion, these
findings indicated that cultured myocardial cells were able to
assimilate large amounts of nontransferrin iron and that iron uptake and
mobilization were associated with striking changes in lipid peroxidation
as manifested by the respective increase and decrease in cellular MDA
concentrations. In addition, functional alterations were demonstrable
at high concentrations of iron.

A recent study by Hershko et al. (119) investigated the ability of
ascorbic acid, alpha-tocopherol and hypoxia to modify' iron uptake,

chelation, and toxicity as manifested. by the» generation. of MDA in

31

myocardial cell cultures. Results indicated that ascorbic acid and
tocopherol had opposing effects on iron uptake and cellular MDA
production. Ascorbate inhibited iron uptake 73%, whereas tocopherol
increased iron uptake by 19%. Contrastingly, the addition of ascorbate
greatly enhanced the production of cellular MDA (86% relative to
controls), however tocopherol reduced cellular MDA levels by 75%.
Chelation studies (using desferroxamine) demonstrated significant
reductions in cellular iron content (53%) and MDA production (40%).
Neither simultaneous desferroxamine and ascorbate nor tocopherol
treatment affected iron mobilization, however ascorbic acid entirely
prevented the reduction of MDA concentration by desferroxamine in iron
loaded cells. In contrast, tocopherol potentiated the antioxidant
effect of desferroxamine.

The findings of Hershko's study indicating a marked increase in
myocardial lipid peroxidation after ascorbate therapy underline the
controversy regarding vitamin C supplementation in thalassemic patients
(120). Ascorbate deficiency is a common complication of iron overload
caused by the accelerated conversion of ascorbate to oxalic acid in the
presence of excess tissue iron (121). Furthermore, ascorbate
supplementation potentiates the in viva effect of desferroxamine
probably by enhancing storage iron mobilization and by increasing the
chelatable iron pool (122). However, there have been reports of severe
deterioration of cardiac function in primary and secondary iron overload
patients receiving supplemental ascorbic acid to enhance responsiveness
to desferroxamine therapy (123). Thus, experimental and clinical

evidence indicates that ascorbic acid supplementation in iron overload

32

patients should be carried out with extreme caution, and in many cases
may be entirely contraindicated.

Hershko's study was the first report to directly demonstrate a
protective effect of alpha-tocopherol against lipid peroxidation in
myocardial cells; however, the therapeutic use of alpha-tocopherol for
protecting membranes against lipid peroxidation has been advocated in
the past with evidence of limited success (124). In addition, this
study as well as a previously mentioned study (118) indicated that
desferroxamine was capable of reducing myocardial MDA concentrations in
direct proportion to the amounts of iron removed. Thus, at least two
methods of protecting myocardial cells from increased lipid peroxidation
are available: removal of excess iron by a metal chelator or preventing
the propagation of lipid peroxidation by the chain-breaking antioxidant

alpha-tocopherol.

X. IRON AND INFECTION

Iron is essential for the growth of virtually all bacterial
organisms (125-127). Weinberg (125) demonstrated that the concentration
of iron necessary to promote bacterial growth ranged from 0.4 to 4.0 uM;
this quantity of iron is present in many environments, including
mammalian tissues. However, nearly all of the iron in mammals is
complexed to intracellular proteins (i.e., ferritin, hemosiderin, heme)
or tightly bound extracellularly to high affinity iron-binding
glycoproteins, such as transferrin in serum and lymph; as well as a
related protein, lactoferrin in external secretions and milk (128-130).
Although there is a wealth of iron in mammalian tissues and fluids, the

amount of free iron in equilibrium with iron-binding proteins was

33

calculated by Bullens et al. (131) to be approximately 10-18M, which is
virtually zero. This result was later confirmed by other researchers
(129,130). Thus, under normal conditions the» amount of free iron

available to sustain bacterial growth is much too small (131,132).

A. Siderophores

Bacteria are known to possess two iron transport systems, a low
affinity system that functions when iron is freely available and a high
affinity system which operates during periods of iron restriction. A
critical component of the high affinity system is the synthesis and
release of low molecular weight iron chelators, known as siderophores
(126,133). Siderophores bind and solubilize ferric iron, re-enter the
bacterial cell (via special outer membrane proteins in many cases) where
the iron is utilized to promote microbial growth. These iron chelators
are categorized into two broad chemical classes, hydroxymates and
catecholamides; at least one of which has been identified in almost
every' aerobic or facultatively anaerobic bacterial species examined
(126).

The hydroxymate class of siderophores is typified by
desferrioxamine 8, which possesses an iron ligand formation constant of
1031 M'1 (134). Desferrioxamine 8 is synthesized by Streptomyces
pilosus and is used clinically as a metal chelator to treat iron and in
some cases aluminum overload (94,135,136). Numerous reports have
documented the promotion of Yersinia enterocolitica septicemia by
desferrioxamine in patients with both acute and chronic iron overload
(137-140). Similar case reports demonstrated an increased incidence of

disseminated mucormycosis in desferrioxamine treated patients with

34

aluminum overload resulting from long-term dialysis (141-144). Y.
snterocolitica does not produce siderophores but has receptors for the
iron-siderophore complex (145). It is known that desferrioxamine acts
as a growth factor for Y. enterocolitica in vitro and dramatically
enhances the virulence of this organism in experimental infections by
providing iron that can be utilized for microbial growth (138,146-148).
A similar mechanism is thought to operate with respect to the
development of mucormycosis (142,144).

Alternatively, other reports have suggested that treatment with
desferrioxamine does not favor the development of septicemia or
bacterial infection independently of iron overload and that the iron
load of the host is the most important predisposing factor leading to
infection (149).

The catecholamides, represented by enterochelin (also called
enterobactin) , generally form tighter iron complexes than hydroxymate
siderophores. Enterochelin is synthesized by several genera of bacteria
(e.g., Klebsiella, Escherichia, Salmonella, Shigella) and has the

highest formation constant for ferric iron ever recorded among

chelators, being near 1 M at neutral pH (134,150-155). Thus,
organisms synthesizing enterochelin are capable of competing for
complexed iron (e.g., transferrin - Rf approx. 1028 M'l) due to the
large difference in formation constants.

Pseudomonas species are known to produce several siderophores:
pyochelin, pyoverdin, pseudobactin, ferribactin and ferrioxamines (156-
161). However, only pyochelin and pyoverdin are of significant interest

with regards to infection. Pyoverdin has been demonstrated to promote

the growth of Pseudomonas in the presence of transferrin (162), despite

35

having a binding constant for iron of only 1032 (163). Cox (164)
demonstrated that pyochelin has dramatic effects on the virulence of Pa.
aeruginosa and promotes experimental infections in mice. Additionally,
work by Ankenbauer et al. (165) suggest that the production of pyoverdin
is critical for the serologic growth of Pa. aeruginosa; apparently

pyochelin is poorly synthesized by the organism in serum and offers

minimal benefit .

II. was IRON-WITNEOLDINO SYSTEN

The iron withholding system in mamalian species is composed of
both storage (e.g., ferritin) and transport (e.g., transferrin,
lactoferrin) proteins. Of the several iron containing proteins it is

known that the transferrins have well-defined bacteriostatic properties.

A. Transferrins

Schade and Caroline (166) observed that raw egg white was
inhibitory to bacterial growth and that such inhibition could be
abolished by the addition of iron. The protein responsible for this
phenomenon was identified as conalbumin (later referred to as
ovotransferrin by Alderton et al.) (167) and they noted that it
prevented microbial growth by withholding iron from bacterial and fungal
invaders; it was proposed that a related protein was present in the
plasma.

In 1946, Schade and Caroline (168) identified an iron-binding
plasma protein and applied the name siderophilin. One year later,
Holmberg and Laurell (169) changed the name to transferrin because they
discovered that the protein had a second function of transporting iron

among the various tissues of the body.

36

The various members of the transferrins are glycoproteins (each
consist of a single chain of approx. 680 amino acids) with molecular
weights in the range of 75,000-80,000. They contain two metal binding
sites (each composed of one aspartyl, one histidinyl, and two tyrosyl
residues) in the polypeptide and are capable of binding up to two atoms
of ferric iron at neutral pH values. This binding of iron is
accompanied by the binding of a carbonate ion on a 1:1 molar basis
(170) .

The primary function of plasma transferrin is the transport of
iron to and among cells of erythropoietic bone marrow, spleen, liver,
small intestine, muscle, as well as the reticuloendothelial system
(171). However, considerable evidence suggest an addition
microbiostatic function. Kochan et al (172) clearly showed that the
amount of available iron (1. e., the ratio between iron saturated and
iron-free transferrin) determined the ability of tubercle bacilli to
grow in serum samples. A low value for the ratio (e. 9., human serum,
0.4), was associated with tuberculostasis, whereas a high value (e. 9.,
guinea pig serum, 5.6) was affiliated with support of bacillary
multiplication. Additionally, the degree of bacillary growth correlated
with the level of transferrin saturation in various mamalian species.
Human and bovine sera with a transferrin saturation level of
approximately 30% were tuberculostatic, whereas guinea pig serum (Tf
saturation :- 84.4‘) sustained bacillary growth. Limited bacillary
multiplication was observed in rabbit and mouse sera (Tf saturation
approximately 60%).

Lactoferrin was discovered in milk in 1939 but- not definitively

identified until 1960 (173). In human milk it comprises 20% of the

37

protein content (129) , however this figure is reduced to only 2% in
bovine milk. In addition, lactoferrin is an important component of
other exocrine fluids such as bronchial mucous, nasal exudate, saliva,
gastrointestinal fluid, bile, seminal fluid and tears.

Bezkorovainy (174) suggested that the relatively large quantity of
lactoferrin in human milk is an important contributor to the much lower
incidence of infection in breast-fed infants as compared with the
incidence in infants receiving milk formula or bovine milk. In addition
to its ability to retain iron from potential infant intestinal pathogens
such as Escherichia, Salmonella, and Clostridium, the lactoferrin in
human milk may function to retard the absorption of intestinal iron
(derived from both bile and diet) in the human nursling (175). This
inhibition of iron absorption is significant because as previously
stated the level of saturation of Tf is believed to be critical in the
development of infection. Values (of Tf saturation) less than roughly
30% are helpful in preventing infection (176).

Lactoferrin differs from transferrin and ovotransferrin in that it
retains its binding avidity for ferric iron far below neutrality (pH <
4.0). This ability makes it an effective scavenger of iron in septic
areas in which the pH has been decreased by organic acids derived from
microbial invaders, as well as from infiltrating inflammatory cells,
namely polymorphonuclear leukocytes (PMNs) of which lactoferrin is a
major component (177). Upon migration of PMNs to a site of sepsis
degranulation of specific granules occurs causing the release of
lactoferrin. In the septic area the unsaturated lactoferrin combines
with the increased iron pool that is derived from dying microorganisms

and injured tissue cells. The binding of free iron by lactoferrin

38

serves to retain iron from utilization by microbial pathogens. In
addition, the saturated lactoferrin is ingested by macrophages which
provides a signal to suppress continued metabolic activity. This signal
aids in the down-regulation of the inflammatory process that occurs at
the conclusion of the successful response to insult (178). It should be
noted that humans with PMNs deficient in specific granules containing
lactoferrin are at increased risk of gram-positive and gram-negative

bacterial infections (179).

III. IRON AND CELLULAR IMMUNITY
A. NR Cells

Numerous studies investigating the correlations between iron
status and immune function in clinical situations have been conducted in
thalassemic patients. Akbar et al. (180) noted a reversible,
transfusion-related decrease in natural killer (NR) cell function in 8-
thalassemia major patients who were iron overloaded as a result of
chronic transfusion therapy. The NR cell function was significantly
increased when effector cells were preincubated with an iron chelator
(desferrioxamine or 2,3-dihydroxybenzoic acid), thus indicating that the
decrease was related to iron overload. (Note: Similar results were not
observed when target cells were incubated with chelators) . It was
suggested that iron may influence NR activity by causing changes in the
expression of transferrin receptors. Transferrin receptors (TfR) have
been proposed to be a target structure for NR cells (181,182) and that
TfR on both effector and target cells are important in the recognition
event of cell killing (183). Mattia et al. (184) suggested that the

expression of this receptor can be manipulated by agents (such as

39

desferrioxamine) that alter the iron status of cells. Thus, it was
proposed that the decreased NR cell activity seen in thalassemia
patients is the result of decreased TfR expression due to iron overload.
In addition, the observation that preincubation of thalassemic effector
cells with iron-chelating agents can increase NR function may be related
to an increase in the expression of TfR. due to removal of iron.
Alternatively, work by Shau et al. (185) proposes that the TfR has an
important role in the induction of NR-like activity in long-term culture
with IL-2 and argued against its role as a target recognition
determinant. They suggest that the importance of the TfR is likely
related to its direct interaction with Tf during the induction phase of
NR-like function rather than in its unspecified role during effector

phase cytotoxicity.

8. Polymorphonuclear granulocytes (PMN)

There have been several reports relating the influence of iron on
the phagocytic function of polymorphonuclear granulocytes (PMN) . In
vitro the phagocytic ability of PMN were demonstrated to be reduced
after incubation with ferrous, as well as ferric iron (186-190).
Similarly, PMN from patients with iron overload have also exhibited
impaired phagocytic function (191-193). This impairment is thought to
mediated by non-transferrin plasma iron found in primary and secondary
iron overload patients (93,94,194,195).

waterlot et al. (192) demonstrated altered neutrophil phagocytosis
(of Staphylococcus aureus) and myeloperoxidase activity in a group of
polytransfused patients receiving maintenance hemodialysis. Results

indicated that the index of phagocytosis was inversely correlated with

40

serum ferritin concentrations and following 6 to 18 weeks of
desferrioxamine therapy phagocytic function and myeloperoxidase activity
returned to normal in most patients. These results confirmed in vitro
studies (196) in which neutrophil phagocytosis was altered in an iron
enriched medium and corrected by the addition of desferrioxamine.

Cantinieaux et al. (193) documented both altered PMN phagocytosis
and killing of Y. enterocolitica in a similar group of hemodialysis
patients. Results indicated that the reduced phagocytosis may be the
consequence of a cellular defect since opsonization with normal serum
did not improve phagocytic function. A previous study in thalassemia
major patients (197) revealed a similar defect in PMN phagocytosis of E.
coli. Prussian Blue positive staining of blood PMN indicated a cellular
iron intoxication to» explain. phagocytic dysfunction. Excessive
production of toxic oxygen species under the influence of an increase in
cellular iron is thought to be involved in the pathogenesis of impaired
phagocytosis. These radicals have been reported to be toxic for
cellular membranes (198,199).

Reduced phagocytosis and killing are not the sole alterations in
PMN function associated with iron overload. Khan et al. (200)
demonstrated defective chemotactic and random migration patterns in a
group of 11 thalassemia major patients. It was not determined if this
reflected a primary defect of PMN or was a secondary manifestation of

associated liver disease and/or diabetes mellitus.

C. Peripheral Blood Monocytes (PBMo)
Ballart et al. (201) demonstrated that PBMo from thalassemia major

patients exhibited markedly diminished lytic activity against C.

41

pseudotropicalis, however phagocytic activity did not vary significantly
from controls. In addition, an inverse correlation was documented
between PBMo lytic activity and serum ferritin levels. The results from
this study suggested that PBMo from patients with thalassemia major have
an intracellular defect in their microbicidal mechanisms associated with

the degree of iron overload.

D. Lymphocytes

Studies examining the relationship of iron overload, lymphoid
subsets and immune function have focused on patients with thalassemia
intermedia (202-204) and major (180,205,206). Lymphocytes from a group
of thalassemia intermedia patients exhibited diminished mitogen
responses to stimulation with FHA and Con A but not PWM (203). In
polytransfused thalassemia major patients, Grady et al. (205) observed
increasing preportions of circulation CDB+ cells that correlated
significantly with the number of transfusions.

In IHC patients two conditions appear to persist: (1) unusually
high numbers of thermostable E-rosette forming cells (207), and (2)
Bjorn-Rasmussen et al (208) demonstrated a high percentage of PBMo

expressing receptors for transferrin in many patients.

42

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Hilgartner MW and Grady RW. Decreased natural killer activity in
thalassemia major: A possible consequence of iron overload.
J.Immunol. 136:1635-1640, 1986.

Vodinelich L, Sutherland R, Schneider C, Newman R and Greaves M.
Receptor for transferrin may be a target structure for natural
killer cells. Proc.Natl.Acad.Sci.USA 80:835, 1983.

Alarcon B and Fresno M. Specific effect of anti-transferrin
antibodies on natural killer cells directed against tumor cells:
Evidence for the transferrin receoptor being one of the target
structures recognized by NE cells. J.Immunol. 134:1286, 1985.
Baines MG, Lafleur FL and Holbein BE. Involvement of transferrin
and transferrin receptors in human natural killer effector: Target
interaction. Immunol.Lett. 7:51, 1983.

Mattia E, Rao K, Shapiro DS, Sussman HH and Klausner RD.
Biosynthetic regulation of the human transferrin receptor by

desferrioxamine in K562 cells. J.Biol.Chem. 259:2689, 1984.

185.

186.

187.

188.

189.

190.

191.

192.

62

Shau H, Shen D and Golub SH. The role of transferrin in natural
killer cell and IL-2-induced cytotoxic cell function. Cell.
Immunol. 97:121-130, 1986.

Ward PA, Goldschmidt P and Greens MD. Suppressive effects of metal
salts on leucocyte and fibroblast function. J.Reticuloendothel.
Soc. 18:313-321, 1975.

Gladstone GD and Walton E. The effect of iron and haematin on the
killing of staphylocci by rabbit polymorphs. J.Exp.Pathol. 52:452-
464, 1971.

van Asbeck BS, Verbrugh HA, van Oost BA, Marx JJM, Imhof HW and
verhoef J. Listeria monocytogenes meningitis and decreased
phagocytosis associated with iron overload. Br.Med.J. 284:542-544,
1982.

van Asbeck BS, Marx JJM, Struyvenberg A, van Rats JH and Verhoef
J. Effect of iron (III) in the presence of various ligands on the
phagocytic capacity and metabolic activity of human
polymorphonuclear leukocytes. J.Immunol. 132:851-856, 1974.
Hoepelman IM, Jaarsma EY, Verhoef J and Marx JJM. Polynuclear iron
complexes impair the function of polymorphonuclear leukocytes.
Br.J.Haematol. 68:385-389, 1988.

van Asbeck BS, Marx JJM, Struyvenberg A and Verhoef J. Functional
defects in phagocytic cells from patient with iron overload.
J.Infsc.Dis. 8:232-240, 1984.

Waterlot Y, Cantinieaux B, Hariga-Muller C, DeMaertelaere-Laurent
E, Vanherweghem JL and Fondu P. Impaired phagocytic activity of
neutrophils in patients receiving haemodialysis: the critical role

of iron overload. Br.Med.J. 291:501-504, 1985.

193.

194.

195.

196.

197.

198.

199.

200.

201.

63

Cantinieaux B, Boelaert J, Hariga C and Fondu P. Impaired
neutrophil defense against Yersinia enterocolitica in patients
with iron overload who are undergoing dialysis. J.Lab.Clin.Med.
111:524-527, 1988.

Batey RG, Fong PLC, Shamir S and Sherlock DBE. A non-transferrin
bound serum iron in idiopathic haemochromatosis. Digestive
Diseases and Sciences 25:340-346, 1980.

Hershko C and Peto TEA. Non-transferrin plasma iron. Br.J.
Haematol. 66:149-151, 1987.

van Asbeck BS, Marex JJ, Struyvenberg A, van Kats JH and Verhoef
J. Desferrioxamine enhances phagocytic function of human
polymorphonuclear leukocytes. Blood 63:714-720, 1984.

Cantinieaux B, Hariga C, Ferster A, DeMaertelaere E, Toppet M and
Fondu P. Neutrophil dysfunctions in thalassaemia major: The role
of cell iron overload. Eur.J.Haematol. 39:28-34, 1987.

Lian CJ and Pia CH. Inhibition of human neutrophil
chemiluminescence by plasmid-mediated outer membrane proteins of
Yersinia enterocolitica. Infect.Immunol. 49:145-151, 1985.

Fondu P and Cantinieaux B. Infections and iron overload. Acta
Clin.Belg. 41:1-9, 1986.

Khan AJ, Lee CK, Wolff JA, Chang H, Khan P and Evans HE. Defects
of neutrophil chemotaxis and random migration in thalassemia
major. Pediatrics 60:349-351, 1977.

Ballart IJ, Estevez ME, Sen L, Diez RA, Giuntoli J, deMiani SA and
Penalver J. Progressive dysfunction of monocytes associated with
iron overload and age in patients with thalassemia major. Blood

67:105-109, 1986.

202.

203.

204.

205.

206.

207.

208.

64

Kapadia A, DeSousa M, Markensen A, Miller J, Good RA and Gupta s.
Lymphocyte sets and immunoglobulins in patients with thalassemia
intermedia. Br.J.Haematol. 45:405-416, 1980.

Munn CG, Markensen AJ, Kapadia A and DeSousa M. Impaired T cell
mitogen responses in some patients with thalassemia intermedia.
Thymus 3:119-128, 1981.

Guglielmo P, Cunsolo F, Lombardo T, Sortino G, Giustolisi R and
Cacciola E. T subset abnormalities in thalassemia intermedia:.
Possible evidence for a thymus functional deficiency. Acta
Haematol. 72:361-367, 1984.

Grady RW, Akbar AN, Giardina PJ, Hilgartner MW and DeSousa M.
Disproportionate lymphoid cell subsets in thalassemia major: The
relative contribution of transfusion and splenectomy.
Br.J.Haematol. 59:713-724, 1985.

Akbar AN, Giardina PJ, Hilgartner MW and Grady RW. Immunological
abnormalities in thalassemia major: I. A transfusion related
increase in circulating cytoplasmic immunoglobulin-positive cells.
Clin.Exp.Immunol. 62:397-404, 1985.

Bryan CF, Leech SH, Ducos R, Edwards CO, Kushner JP, Skolnick MH,
Bozelka B, Linn JC and Gaumer R. Thermostable erythrocyte rosette-
forming lymphocyte in hereditary hemochromatosis: I.
Identification in peripheral blood. J.Clin.Immunol. 4:134-142,
1984.

Bjorn-Rasmussen E, Hageman J, van den Dugen P, Prowit-Ksiazek A
and Biberfield P. Transferrin receptors on circulating monocytes

on hereditary hemochromatosis. Scand.J.Haematol. 34:308-311, 1985.

CHAPTER I

GRAN NEGATIVE SEPSIS IN IRON LOADED GUINEA PIGS

Abstract

Susceptibility to infection is a well recognized manifestation of
human iron overload disorders. Existing animal models of iron overload
are time consuming, expensive, and have not been developed for the study
of severe spontaneous gram negative infections. Experiments with the
guinea pig model of iron overload demonstrated that animals receiving
relatively large intraperitoneal injections of iron dextran (200 mg/kg
every other day for five injections) have a high incidence of
spontaneous gram negative infections. Pseudomonas was isolated from the
liver, spleen or blood of 71% of the iron treated guinea pigs (n27). At
necropsy the affected animals had markedly icteric mucous membranes,
roughened hair coats, and numerous small multifocal regions of hepatic
necrosis (6/7). Histologically, these randomly distributed areas of
coagulative necrosis were variably-sized with few inflammatory cells.
Control animals (n24) appeared clinically normal and without gross or
histologic lesions. We conclude that guinea pigs receiving relatively
large injections of iron dextran have a high incidence of gram negative
infections and will provide a useful model for further investigation of

iron induced sepsis.

65

66

I. INTRODUCTION

Acute and chronic iron overload is associated with an increased
susceptibility to infection (1-5). Treatment of both iron and aluminum
overload patients with desferrioxamine (Df), a metal chelator, mobilizes
metal stores and enhances the probability of life-threatening infection
(2-6).

Several mechanisms may be involved in the development of enhanced
susceptibility to infection in iron overload patients: [1] saturation of
the host iron withholding system resulting in diminished bacteriostatic
effectiveness of iron-binding glycoproteins, transferrin and lactoferrin
(7,8); [2] increased virulence of bacterial pathogens in the presence
of freely available iron (9); [3] altered cellular immune responses
involving several types of leukocytes (10-16).

We report an increased incidence of spontaneous gram negative
infections using guinea pigs treated for less than two weeks with

relatively large doses of intraperitoneal iron dextran.

II. MODS

Seven female Hartley guinea pigs (Michigan Dept. of Public Health)
weighing 200-250 grams received 5 intraperitoneal injections of 200 mg
Fe/kg of iron dextran (Sigma Chemical Co., St. Louis, Mo) every other
day. Control animals (n84) were injected with equivalent volumes of
dextran. Animals were humanely sacrificed and tissues collected for
microbiologic and histopathologic evaluation. Samples of liver, spleen
and blood were cultured from iron treated animals. Specimens were

cultured separately on both blood agar and McConkey's agar. Tissues

67

were fixed in 10% buffered formalin and stained with hematoxylin and

eosin for light microscopy; gram staining was performed in select cases.

III. RESULES

Clinically, all iron treated guinea pigs were markedly jaundiced
with evidence of reduced weight gain and roughened hair coats; the
control group appeared healthy and only liver samples were submitted for
evaluation.

Pseudomonas aeruginosa. was cultured from the blood, liver or
spleen of iron dextran treated (5/7: 71.4%) guinea pigs (Table 1).
Pseudomonas was also cultured from the liver of a single control guinea
pig (1/4; 25%), however this was believed to be a subclinical infection
as there were no clinical signs nor pathologic lesions in this animal.

Macroscopically, the liver from 6 of 7 of the iron treated animals
was discolored (brownish-yellow) and contained numerous, multifocal
(<0.5 mm) regions of necrosis distributed throughout the parenchyma. In
addition, moderately-sized (1-2 cm) subcutaneous abscesses were present
at 4 of 7 sites of injection; similar lesions were not observed in the
control group. Histologically, randomly dispersed variably-sized,
multifocal regions of hepatic coagulative necrosis were observed in iron
loaded guinea pigs (Figure l); inflammation was minimal in these areas.
Severe fibrinous peritonitis associated with numerous gram-negative
bacterial organisms developed in one of seven iron treated guinea pigs
(Figure 2). Other histologic lesions in this animal included chronic,
active fibrosing septal panniculitis and myositis in subcutaneous

tissues. These lesions were morphologically consistent with resolving

68

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Figure 1” Focal region of hepatic necrosis with pyknotic
nuclei (arrows) and extracellular iron particles bordered by

nondegenerate cells. H & E; Line = 100pm

Figure 2. Numerous rod-shaped bacterial organisms (arrows)
surrounded by heterophils and macrophages in the liver of a

septic guinea pig. H & E; Line = 20 pm.

70

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71

abscesses or cellulitis. A single sample of this tissue cultured

positive for both, Pseudomonas aeruginosa and Serratia odorifera.

IV. DISCUSSION

This report documents an increased occurrence (71%) of Pseudomonas
infection in guinea pigs given high levels of intraperitoneal iron
dextran. These findings support prior studies in humans and animals
relating iron to infection in both clinical, as well as experimental
studies using mice, rats and guinea pigs (17-19). The median lethal
dose of intraperitoneally administered Yersinia enterocolitica was
reduced 10 to loo-fold in mice injected with iron dextran; parallel
results were demonstrated using guinea pigs in a keratoconjunctivitis
test (17,18). In addition, iron has been demonstrated to enhance the
virulence of Pseudomonas aeruginosa in a chronic pulmonary infection
model in rats (20) and enhanced the growth of Staphylococcus aureus and
Yersinia enterocolitica in human serum (19). The virulence of Listeria
monocytogenes, Klebsiella pneumoniae and Escherichia coli have also been
shown to be accentuated by the injection of iron (9).

Numerous reports relate iron to infection in a variety of clinical
circumstances. Parenteral administration of iron dextran to iron
deficient Polynesian infants was temporally related to an outbreak of
E. coli neonatal sepsis (1). In addition, an unusual cluster of
Acinetobacter pneumonia was reported in foundry workers inhaling high
concentrations of iron particles (21) . Several studies document an
enhanced incidence of Yersinia enterocolitica septicemia in both acute
and chronic iron overload patients, particularly those treated with

desferrioxamine (2-4). The role of desferrioxamine is debated, however

72

it should be noted that recent studies describe an increased occurrence
of mucormycosis in desferrioxamine-treated renal dialysis patients with
aluminum overload (5,6).

The development of enhanced bacterial virulence observed with iron
overload may involve inhibition of transferrin and lactoferrin, whose
bacteriostatic capability is known to be inversely related to their
level of saturation (7). The level of Tf saturation in this report was
not determined. However, prior experiments from this laboratory
demonstrated complete saturation of transferrin (>100%) in guinea pigs
receiving 25% (250 mg/kg) of the amount of injected iron dextran in this
report. Normal guinea pig Tf saturation is 85% thus, the amount of
injected iron required to thoroughly saturate iron-binding proteins was
relatively small.

Iron overload alters other imune defense systems. Studies in
iron overload patients have shown depressed cellular immunity as
evidenced by decreased natural killer (NK) cell activity (10),
suppression of polymorphonuclear granulocytes (PMN) phagocytic function
(13-16), reduced lytic activity in peripheral blood monocytes and
diminished mitogen responses in lymphocytes (22-24). It remains unknown
as to how these and possibly other iron induced defects interact to
produce sepsis.

Results in this report provide additional evidence that iron is
capable of enhancing the development of sepsis in the mammalian host.
We propose that the iron dextran injected guinea pig mimics iron-induced
septicemia observed in human patients and will provide a model to
investigate the relationship between iron overload states, host immunity

and bacterial virulence. In addition, pertinent related questions

73

involving the efficacy of prophylactic or therapeutic antibiotics, as

well as chelation treatment could be examined using this model.

74

REFERENCES

1.

Barry DMJ and Reeve AW. Increased incidence of gram negative
neonatal sepsis with intramuscular iron administration. Pediatrics
60:908-912, 1977.

Melby K, Slordahl S, Gutteberg TJ and Nordbo SA. Septicaemia due
to Yesinia enterocolitica after oral overdoses of iron. Br.Med.J.
285:467-468, 1982.

Mofenson HC, Caraccio TR and Sharieff N. Iron sepsis: Yersinia
enterocolitica septicemia possibly caused by an overdose of iron.
N.Eng.J.Med. 316:1092-1093, 1987.

Gallant T, Vellend H and Francombe WH. Yersinia sepsis in patients
with iron overload treated with deferoxamine. N.Eng.J.Med.
314:1643, 1986.

Goodill JJ and Abuelo JG. Mucormycosis - A new risk of
deferoxamine therapy in dialysis patients with aluminum or iron
overload? N.Eng.J.Med. 317:54, 1987.

Calesciabetta CC, Eribaum AI and Coburn JN. Fatal disseminated
phycomycosis (mucormycosis) in patients on desferoxamine for
aluminum toxicity. Am.J.Kidney Dis. 8:A20, 1986.(Abstract).

Kochan I, Golden CA and Bukovic JA. Mechanism of tuberculostasis
in mammalian serum. II. Induction of serum tuberculostasis in
guinea pigs. J.Bacteriol. 100:64-70, 1969.

Bezkorovainy A. Human milk and colostrum proteins: A review.
J.Dairy Sci. 60:1023-1037, 1977.

Bullen JJ. The significance of iron in infection. Rev.Infect.Dis.

331127-1137, 1981.

10

11.

12.

13.

14.

15.

16.

75

Akbar AN, Fitzgerald-Bocarsly PA, DeSousa M, Giardina PJ,
Hilgartner MW and Grady RW. Decreased natural killer activity in
thalassemia major: A possible consequence of iron overload.
J.Immunol. 136:1635-1640, 1986.

Ballart IJ, Estevez ME, Sen L, Diez RA, Giuntoli J, deMiani SA and
Penalver J. Progressive dysfunction of monocytes associated with
iron overload and age in patients with thalassemia major. Blood
67:105-109, 1986.

van Asbeck BS, Verbrugh HA, van Cost BA, Marx JJM, Imhof HW and
Verhoef J. Listeria monocytogenes meningitis and decreased
phagocytosis associated with iron overload. Br.Med.J. 284:542-544,
1982.

Hoepelman IM, Jaarsma EY, Verhoef J and Marx JJM. Polynuclear iron
complexes impair the function of polymorphonuclear leukocytes.
Br.J.Haematol. 68:385-389, 1988.

Waterlot Y, Cantinieaux B, Hariga-Muller C, DeMaertelaere-Laurent
E, Vanherweghem JL and Fondu P. Impaired phagocytic activity of
neutrophils in patients receiving haemodialysis: the critical role
of iron overload. Br.Med.J. 291:501-504, 1985.

Cantinieaux B, Hariga C, Ferster A, DeMaertelaere E, Toppet M and
Fondu P. Neutrophil dysfunctions in thalassaemia major: The role
of cell iron overload. Eur.J.Haematol. 39:28-34, 1987.

Cantinieaux B, Boelaert J, Hariga C and Fondu P. Impaired
neutrophil defense against ‘Yersinia. enterocolitica. in jpatients
with iron overload who are undergoing dialysis. J.Lab.Clin.Med.

111:524-527, 1988.

17.

18.

19.

20.

21.

22.

23.

24.

76

Robins-Browne RM and Prpic JK. Desferrioxamine and systemic
yersiniosis. Lancet 1:1372, 1983.

Robins-Browne RM and Prpic JK. Effects of iron and desferrioxamine
on infections with Yersinia enterocolitica. Infect.Immunol.
47:774-779, 1985.

Brock JH and Ng J. The effect of desferrioxamine on the growth of
Staphylococcus aureus, Yesinia enterocolitica and Streptococcus
faecalis in human serum: Uptake of desferrioxamine-bound iron.
FEMS Microbiol.Lett. 20:439-442, 1983.

Sokol PA and Woods DE. Relationship of iron and extracellular
virulence factors to Pseudomonas aeruginosa lung infections.
J.Med.Microbiol. 18:125-133, 1984.

Cordes LG, Brink EW, Checko PJ, Lentner A, Lyons RW, Hayes PS, Wu
TC, Thar DC and Fraser DW. A cluster of Acinetobacter pneumonia in
foundry workers. Ann.Intern.Med. 95:688-693, 1981.

Kapadia A, DeSousa M, Markensen A, Miller J, Good RA and Gupta S.
Lymphocyte sets and immunoglobulins in patients with thalassemia
intermedia. Br.J.Haematol. 45:405-416, 1980.

Munn CG, Markensen AJ, Kapadia A and DeSousa M. Impaired T cell
mitogen responses in some patients with thalassemia intermedia.
Thymus 3:119-128, 1981.

Guglielmo P, Cunsolo F, Lombardo T, Sortino G, Giustolisi R and
Cacciola E. T subset abnormalities in thalassemia intermedia:
Possible evidence for a thymus functional deficiency. Acta

Haematol. 72:361-367, 1984.

CHAPTER I I

IRON INDUCED MYOCARDIAL AND HEPATIC LYSOSOMAL ABNORMALITIES

IN THE GUINEA PIG.

I. INTRODUCTION

Primary and secondary iron overload disorders are characterized by
the development of cytosiderosis with accumulation of iron in lysosomes
of both reticuloendothelial and parenchymal cells of the liver, spleen,
heart, pancreas and other tissues. As a consequence of severe siderosis
a variety of clinical disorders may evolve including hepatic cirrhosis,
cardiomyopathy, endocrine pancreatic dysfunction, bronze skin
pigmentation and hypogonadism.

The mechanisms of tissue damage in iron overload diseases are not
clearly defined, however previous research suggests that iron-mediated
lysosomal disruption may play a role in initiating cell injury.
Numerous reports demonstrate that hepatic lysosomes from iron overload
patients are particularly fragile when compared to control subjects (1-
4). Similar findings are documented in rats fed carbonyl iron (5,6),
injected with an iron-sorbitol-citric acid complex (7) or iron
nitrilotriacetate (8). Favored, mutually compatible, proposals for the
mechanism of lysosomal membrane injury in iron overload include (a)
accumulation of excess hemosiderin leading to physical disruption of the
lysosome and (b) iron-catalyzed lipid peroxidation mediating the loss of

lysosomal membrane integrity. Following the lysosomal membrane injury,
77

78

it is hypothesized that leakage of acid hydrolases results in
destruction of intracellular constituents eventuating in cell damage
(9) .

Severe cardiomyopathy is a common clinical manifestation of both
hereditary' and transfusion-induced iron overload. Chronically
transfused thalassemic children with iron cardiomyopathy may develop
severe clinical problems or even die during puberty. Investigation of
this problem has been hampered by lack of an easily reproducible animal
model that mirrors the human condition. Although iron overload has been
reproduced in rats by injection of iron-sorbitol-citric acid complex or
feeding carbonyl iron, these models are expensive and require 3 weeks-12
months before the animals are iron loaded and studies can begin
(5,10,11).

We have developed a previously unexplored animal model of iron
overload. Guinea pigs intraperitoneally injected with iron dextran
develop hemosiderosis of both their livers and hearts. Iron loaded
animals demonstrate increased serum iron, total iron binding capacity,
and percent transferrin saturation. This model was used to investigate
the pathophysiology of iron induced toxicity; abnormalities involving
membrane peroxidation and enhanced liver and myocardial lysosomal enzyme

release are presented.

II. MATERIALS AND METHODS
Aaimal.Irsatment

Female Hartley guinea pigs (Michigan Department of Public Health)
weighing 200-250 grams were housed (4-6/group) in stainless steel cages

on wire grates and fed a commercial diet with vitamin C (Purina Guinea

79

Pig Chow # 5025, St. Louis, MO). Iron dextran (Sigma Co., St. Louis,
MO) [83 mg Fe/kg/injection (pH 7.4)] was injected intraperitoneally on
alternate days to achieve iron loading levels of 0.25, 0.5, 1.0 and 2.0
g Fe/kg BW for lysosomal studies and 0.5 and 1.5 g Fe/kg BW for the
malondialdehyde study. Controls received an equivalent volume of
dextran. All animals were sacrificed 48 hours following the final

injection.

W

Blood was collected via aortic puncture from guinea pigs
anesthetized with pentobarbital in combination with ether. Blood was
flushed from liver vasculature with 50 ml of ice-cold .25 M sucrose with
1 mM disodium EDTA and 10mM Hepes (pH 7.4). The right lobe of the liver
was immediately excised and placed in the perfusion solution. Excised
hearts from the same guinea pigs were placed in an ice-cold .25 M KCl
and 1 mM disodium EDTA with 10 mM Hepes (pH 7.4). Tissues were blotted,
weighed and minced with scissors; livers placed in 10% (w/v) sucrose and
hearts in isotonic KCL. All subsequent steps were conducted at 0-4°C.
Livers were homogenized for 45 seconds (s) with a Potter-Elvehejm
homogenizer with a Teflon pestle. Hearts were homogenized using a
Polytron Kinematica homogenizer (Brinkmann Instruments) for 5s at a low
speed followed by a 30s homogenization with a Potter-Elvehejm
homogenizer
s d aturation

Serum iron and total iron binding capacity were determined
colorimetrically using ferrous ammonium sulfate as a standard (Stanbio

Laboratory, Inc., San Antonio, TX). This method was a modification of

80

that reported by Persijin et al., (12) employing the chromogenic

compound, ferrozine, as described by Stookey (13).

W

Fractions

Following centrifugation of the whole homogenates at 800 x g for
10 minutes the enzyme activity of the supernatant fraction in the
presence of 0.25 (w/v) Triton x-1oo was termed the total enzyme
activity. Free activity indicates that measured in the 22,000 x g (30
minutes) supernatant fraction in the presence of 0.2% (w/v) Triton x-
100. The free activity is expressed as a percentage of the total
activity without regard to quantity of protein.

Reactions were performed at three enzyme concentrations to verify
linearity; all assays were demonstrated to be linear with time. Acid
phosphatase (EC 3.1.3.2) was measured by a modification of the method of
Vaes and Jacques (14); the pH was 5.0 as described by Schroeder et al.
(15). Serum and tissue N-acetyl-B,D-glucosaminidase (EC 3.2.1.30) was
assayed according to Sellinger et al. (16) using p-nitrophenyl-N-acetyl-
B-D-glucosaminide as substrate. N-acetyl-B,D-glucuronidase (EC
3.2.1.31) was assayed according to Schroeder et. al (15). with the
modification that both free and total activities were incubated for 60
minutes. Serum protein was estimated according to Lowry et al. (17)

with bovine serum albumin as the standard.

M nd de Determinat on
Malondialdehyde in whole homogenates of liver and heart was

estimated using thiobarbituric acid as described by Ohkawa et al. (18).

81

The protocol were slightly modified by centrifuging tubes at 175 x g for

20 min. to form the organic layer (pink chromogen).

fit:tiatisal.bnalx§ia
All data was analyzed using an unpaired t-test. Percentage values

were normalized by arcsin square root percentage transformation (19).

II. RESULTS
§arum_Ir9ni_§srum_IIaQ_aad_1ran§ferrin_§aiuratien

Significant elevations in serum iron values occurred at total iron
doses of 0.5 (p<0.001) and 1.0 g/kg (p<0.05); serum TIBC values were
also increased at these dose levels (p<0.001 and 0.01, respectively).
Transferrin saturation was elevated at both 0.25 and 1.0 g Fe/kg BW

(p<0.01) (Table 2).

Hanati£_Lx§stmal_£ragilitx

Iron loaded animals at all dose levels had increased lysosomal
fragility as demonstrated by a significant elevation in the free
activity of hepatic glucosaminidase (p<0.01) and B-glucuronidase
(p<0.05) (Figures 3 and 4). Elevation of free glucosaminidase and
glucuronidase occurred at the lowest iron dose level (0.25 g/kg) and did
not differ significantly between groups of iron treated animals,
indicating an initial maximal or near maximal release of these
hydrolases with no sequential increase at higher iron loads. Free
hepatic acid phosphatase activity was significantly elevated at 0.5, 1.0

and 2.0 g Fe/kg BW (p<0.01) (Figure 5).

82

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Levels of serum glucosaminidase activity were measured in an
effort to relate iron toxicity to changes in serum enzyme activity.
Similar to the findings with hepatic glucosaminidase there was a
significant elevation of serum glucosaminidase activity (Figure 6) at
all levels of iron loading (p<0.01); at 0.25, 0.5, and 2.0 g Fe/kg the
increase was approximately 2-fold over control values and did not vary

significantly between iron treated groups.

W

The pattern of lysosomal enzyme abnormalities differed from that
observed in the liver. Free myocardial glucosamindase (Figure 7) was
significantly elevated at all iron dose levels (p<0.05). However, the
free activity of myocardial acid phosphatase (Figure 8) was increased

only at the highest iron dose of 2.0 g Fe/kg (p<0.05).

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only at the high dose (1. 5 g Fe/kg BW) of iron loading in whole

homogenates of both heart and liver (p<0.01).

IV. DISCUSSION

Investigation of the pathophysiology of excess iron toxicity
utilizes the guinea pig model which is less expensive and requires a
shortened iron loading time than previously developed animal models
(5,10,11). Like humans with iron overload, guinea pigs injected with
iron have increased serum iron and percent transferrin saturation.

Although previous studies have investigated the fragility of hepatic

87

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lysosomes in human iron overload patients (1-4) and experimental animal
models (5-8), myocardial lysosomes have largely been ignored. This study
extends these previous reports by demonstrating similar lysosomal
alterations in the myocardium.

In general, liver lysosomes demonstrated increased fragility at
lower iron loads than myocardial lysosomes. Increases in free hepatic
B-N-acetylglucosaminidase and B-glucuronidase followed a similar pattern
and were suggestive of an initial maximal or near maximal release of
enzyme at even the lowest iron dose level. A comparable response was
shown with myocardial glucosaminidase, yet free myocardial acid
phosphatase was demonstrated to be significantly elevated at only the
highest level of iron loading, 2.0 g/kg. Variation in lysosomal enzyme
analysis between the liver and heart may be partially explained by
different cell types composing these tissues, and by the heterogeneity
of lysosomal organelles and their response to different stimuli (20).

Demonstration of iron-enhanced lipid peroxidation in this report
was consistent with numerous studies (7 ,21-24). Findings of a previous
in vitro investigation indicated that incubation of isolated hepatic
lysosomes in the presence of iron rapidly produced maximal levels of
lysosomal lipid peroxidation with subsequent increase in lysosomal
fragility (25,26). Similar results were demonstrated using mouse
peritoneal macrophages (27). These studies established that lysosomal
membranes were susceptible to peroxidation by both ferrous and ferric
forms of iron, leading to the liberation of acid hydrolases. In
addition, studies of cultured rat myocardial cells have demonstrated
that iron loading results in (l) the rapid assimilation of large amounts

of non-transferrin iron with an associated increase in lipid

92

peroxidation (28); (2) the accumulation of iron-filled ferritin
molecules within endocytic vesicles and lysosomes (29); and (3) altered
cellular contractions (30). The effects of iron loading were
ameliorated by in vitro chelation with desferrioxamine; thus, directly
implicating iron as the causative factor. These observations showed
that cultured myocardial cells and most likely the lysosomes of these
cells were susceptible to the toxic effects of iron. Our results in the
iron loaded guinea pig model demonstrating increased membrane
peroxidation and lysosomal enzyme release are comparable to these prior
studies. The mechanism of iron-initiated peroxidation remain complex
and largely unknown. However, it has been demonstrated that iron
overload disorders result in ”decompartmentalization" of stored iron
leading to elevated levels of non-transferrin bound plasma iron (i.e.,
”free iron”); thus, potentially increasing the possibility of initiating
iron-mediated membrane injury (31,32).

Determination of serum B-N-acetylglucosaminidase levels reflected
tissue enzyme alterations. A previous study (33) demonstrated a linear
correlation between serum NAG concentrations and iron stores (as
determined by serum ferritin levels) in B-thalassemia intermedia
patients. This correlation seemed to support the hypothesis that, in
iron overloaded patients, lysosomal membranes are damaged resulting in
increased permeability, release of lysosomal acid hydrolases and
subsequent tissue injury. Interestingly, our results indicated that
elevations in serum NAG appeared to follow increases in the free
activity of hepatic NAG. Thus, this serologic assay may be useful as an

indirect determinant of lysosomal fragility.

93

In summary, experiments with the iron loaded guinea pig model
demonstrate: 1) serum iron and percent transferrin saturation are
comparable with changes in humans with excess iron; 2) iron loading
increases the fragility of both hepatic and myocardial lysosomes
suggesting that lysosomes may perform an influential role in the
pathogenesis of cardiomyopathy and hepatic disease commonly observed in
iron overload disorders: 3) enhanced lysosomal fragility is likely the
result of iron-catalyzed membrane peroxidation; 4) increases in serum

glucosaminidase may be reflective of lysosomal damage.

94

REFERENCES

1.

Seymour CA, Budillon G and Peters TJ. Lysosomal changes in human
and experimental iron overload. Gut 15:838, 1974.

Peters TJ and Seymour CA. Acid hydrolase activities and lysosomal
integrity in liver biopsies from patients with iron overload.
Clin.Sci.Mol.Med. 50:75-78, 1976.

Selden C, Owen M, Hopkins JMP and Peters TJ. Studies on the
concentration and intracellular localization of iron proteins in
liver’ biopsy specimens from jpatients with iron overload with
special reference to their role in lysosomal disruption.
Br.J.Haematol. 44:593-603, 1980.

Seymour CA and Peters TJ. Organelle pathology in primary and
secondary' haemochromatosis *with. special reference to lysosomal
changes. Br.J.Haematol. 40:239-253, 1978.

LeSage GD, Kost LJ, Barham JS and LaRusso NF. Biliary excretion of
iron from hepatocyte lysosomes in the rat. J.Clin.Invest. 77:90-
97, 1986.

Iancu Tc, Ward RJ and Peters TJ. Ultrastructural observations in
the carbonyl iron-fed rat, an animal model for hemochromatosis.
Virchows Arch.B. 53:208-217, 1987.

Hultcrantz R, Ahlber J and Glaumann H. Isolation of two lysosomal
populations from iron-overloaded rat liver with different iron
concentration and proteolytic activity. Virchows Arch. (Cell

Pathol.) 47:55-65, 1984.

10.

11.

12.

13.

14.

15.

95

Peters TJ, O'Connell MJ and Ward RJ. Role of free radical mediated
lipid peroxidation in’ the pathogenesis of hepatic damage by
lysosomal disruption. In: Free radicals in liver injury, edited by
Poli G, Cheeseman KB, Dianzani MV and Slater TF. Oxford: ITP Press
Limited, 1986, p. 107-115.

Peters TJ, Selden C and Seymour CA. Lysosomal disruption in the
pathogenesis of hepatic damage in primary and secondary
haemochromatosis. Iron Metabolism:Ciba Foundation Symposium
51:317-325, 1977.

Hultcrantz R and Arborgh 8. Studies on the rat liver following
iron overload. Fine structural appearance. Acta
Path.Microbiol.Scand.Sect.A 86:143-155, 1978.

Park CH, Bacon BR, Brittenham GM and Tavill AS. Pathology of
dietary carbonyl iron overload in rats. Lab.Invest. 57:555-563,
1987.

Persijn JP, van der Slik W and Riethorst A. Determination of serum
iron and latent iron-binding capacity (LIBC). Clin.Chem.Acta
35:91-98, 1971.

Stookey LL. Ferrozine - a new spectrophotometric reagent for iron.
Anal.Chem. 42:779-781, 1970.

Vaes G and Jacques P. Studies on bone enzymes: The assay of acid
hydrolases and other enzymes in bone tissue. Biochem.J. 97:380-
387, 1965.

Schroeder HR, Gauger JA and Wells WW. Lysosomal fragility and
induction of liver hexose-monophosphate dehydrogenases in the

fasted-refed rat. Arch.Biochem.Biophys. 172:206-214, 1976.

16.

17.

18.

19.

20.

21.

22.

23.

24.

96

Sellinger oz, Beufau H, Jacques P, Doyen. A. and. DeDuve C.
Intracellular distribution and properties of B-N-
acetylglucosaminidase and B-galactosidase in rat liver. Biochem.J.
74:450-456, 1960.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ. Protein
measurement with the Folin phenol reagent. J.Biol.Chem. 193:265-
275, 1951.
Ohkawa H, Ohishi N and Yagi K. Assay for lipid peroxides in animal
tissues by thiobarbituric acid reaction. Anal.Biochem. 95:351-358,
1979.
Steel RGD and Torrie JN. Principles and procedures of statistics:
A biometrical approach. McGraw-Hill Book Co., 1989. Ed. 2

Welman E and Peters TJ. Properties of lysosomes in guinea pig
heart: Subcellular distribution and in vitro stability.
J.Mol.Cell.Cardiol. 8:443-463, 1976.

Bacon BR, Tavill AS, Brittenham GM, Park CH and Recknagel RD.
Hepatic lipid peroxidation in vivo in rats with chronic iron
overload. J.Clin.Invest. 71:429-439, 1983.

Dillard CJ, Downey JE and Tappel AL. Effect of antioxidants in
lipid peroxidation in iron-loaded rats. Lipids 19:127-133, 1984.

Dougherty JJ, Croft WA and Hoekstra WG. Effects of ferrous
chloride and iron-dextran on lipid peroxidation in vivo in vitamin
E and selenium adequate and deficient rats. J.Nutr. 111:1784-1796,
1981.

Heys AD and Dormandy TL. Lipid peroxidation in iron-overloaded

spleens. Clin.Sci. 60:295-301, 1981.

25.

26.

27.

28.

29.

30.

31.

32.

97

Mak IT, Misra HP and Weglicki WB. Temporal relationship of free
radical induced lipid peroxidation and loss of latent enzyme
activity in highly enriched hepatic lysosomes. J.Biol.Chem.
258:13733-13737, 1983.

Mak IT and Weglicki WB. Characterization of iron-mediated
peroxidative injury in isolated hepatic lysosomes. J.Clin.Invest.
75:58-63, 1985.

Abok K, Hirth T, Ericsson JLE and Brunk U. Effect of iron on the
stability of macrophage lysosomes. Virchows Arch. (Cell Pathol.)
43:85-101, 1983.
Link G, Pinson A and Hershko C. Heart cells in culture: A model of
myocardial iron overload and chelation. J.Lab.Clin.Med. 106:147-
153, 1985.(Abstract).
Cox PG, Harvey NE, Sciortino C and Byers BR. Electron-microscopic
and radio iron studies of iron uptake in newborn rat myocardial
cells. Am.J.Pathol. 102:151-159, 1981.

Link G, Urbach J, Hasin Y, Penson A and Hershko C. Beating rat
heart cell cultures: An in 'vitrol model of iron ‘toxicity and
chelating therapy. Blood (suppl.l,9) 62:38a, 1983.(Abstract).
Hershko C, Graham G, Bates GW and Rachmilewitz EA. Non-specific
serum iron in thalassaemia: An abnormal serum iron fraction of
potential toxicity. Br.J.Haematol. 40:255-264, 1978.

Gutteridge JMC. Rowley DA, Griffths E and Halliwell B. Low-
molecular weight iron complexes and oxygen-radical reaction in

idiopathic hemochromatosis. Clin.Sci. 68:463-467, 1985.

33.

98

Frigerio R, Mela Q, Passiv G, Cacace E, LaNasa G, Perpignano G and
Carcassi VEF. Iron overload and lysosomal stability in B-
thalassaemia intermedia and trait: Correlation between serum
ferritin and serum N-acetyl-B-D-glucosaminidase levels.

Scand.J.Haematol. 33:252-255, 1984.

CHAPTER III

HISTOLOGIC AND ULTRASTRUCTURAL STUDIES

IN THE IRON OVERLOAD GUINEA PIG MODEL

I. INIRODUCTION

Iron overload disorders, both acquired and inherited are
associated with functional impairment of the liver, heart, and endocrine
organs. Study of the pathophysiology of iron excess has been hampered
by the lack of an easily reproducible animal model.

Previously developed animal models have been cumbersome, requiring
up to 12 months of iron loading before experimentation can begin (1-4).
Histological and ultrastructural descriptions of siderosis involving the
liver of rats, as well as cultured neonatal rat myocardial cells have
been published. However, morphological analysis of myocardial siderosis
in an in vivo model has not been described.

Our prior studies using myocardial and hepatic tissue
demonstrated abnormal lysosomal membrane stability and lipid
peroxidation in guinea pigs receiving intraperitoneal injections of iron
dextran. To correlate morphologic findings with these concurrent
biochemical and functional studies we examined the histologic and
ultrastructural features of hepatic and. myocardial tissues in iron

dextran treated guinea pigs.

99

100

11. MATERIALS AND METEODS
531W

Female Hartley guinea pigs (Michigan Department of Public Health)
weighing 200-250 grams were housed (3 per group) in stainless steel
cages on wire grates and fed a commercial diet with vitamin C (Purina
Guinea Pig Chow #5025, St. Louis, MO). Iron dextran (Sigma Co., St.
Louis, MO) [83 mg Fe/kg/injection (pH 7.4)] was injected
intraperitoneally on alternate days to achieve iron loading levels of
0.5, 1.0 and 1.5 g Fe/kg for histopathologic and ultrastructural
evaluation. Controls received an equivalent quantity of dextran. All
animals were sacrificed 48 hours following the final injection. Tissues
(heart and liver) were perfused and fixed in 10% buffered formalin for
histopathologic evaluation. Multiple sections of liver and heart were
paraffin-embedded and stained with both hematoxylin/eosin and Prussian
Blue. Portions of left ventricle were prepared for ultrastructural
evaluation. Samples (lmmz) were placed in an ice-cold fixative of 2%
glutaraldehyde/2% paraformaldehyde in 0.1M NaPO4 buffer (pH 7.4) for 3-4
hours. Tissues were washed several times in sodium phosphate buffer for
30 minutes to an hour and subsequently post-fixed with 2% 0904 in 0.1M
NaPO4, then dehydrated through a graded alcohol series. Following
numerous washings, specimens were embedded in Spurr's low viscosity
epoxy resin. Thin sections were cut on a LXB ultramicrotome-3 with
glass knives, mounted on 300 mesh copper grids, left unstained or
stained with saturated aqueous uranyl acetate and lead citrate, and
viewed on a Philips 201 electron microscope operating at an acceleration

of 60 kV.

101

- oa

For x-ray microanalysis, glutaraldehyde/paraformaldehyde fixed
(postfixation with 2% 0s04) left ventricular myocardial tissue was
carbon-coated and left unstained. Analytical electron microscopy was
performed using a JEOL 100 cx II with STEM attachment and a Link Systems
AN 10000. The tissue was examined in the scanning transmission mode at

100 kv with a 30° tilt for 300 seconds.

III. RESULES
Light Microscopy

No histologic changes except the presence of iron particles (This
term is used throughout since the histologic and ultrastructural
features of hemosiderin and ferritin are not well defined) were observed
in sections of H & E stained myocardial and hepatic tissues. Sections

of liver and heart from dextran treated controls were normal.

Liza:

Prussian Blue staining demonstrated iron particles (IP) in
sinusoidal lining cells (Kupffer cells, endothelial lining cells) in all
areas of liver sections: neither preferential periportal nor
centrilobular distribution of iron particles was observed. In guinea
pigs administered 0.5 g Fe/kg, iron staining was observed exclusively
within sinusoidal cells and not hepatocytes (Figure 10A). However, as
iron loads increased IP within hepatocytes became clearly discernible,

especially at the highest iron dose (Figure 103).

102

Figure 10. Hepatic Siderosis

10A. Iron Dextran, liver, 0.5 g Fe/kg, guinea pig. Iron
particles (arrows) confined to sinusoidal lining cells
(mainly Kupffer cells) surrounding a central vein. Prussian

Blue; Line = 100pm

108. Iron Dextran; liver, 1.5 g Fe/kg, guinea pig.
Positive staining iron particles are located within
hepatocytes (arrows) and Kupffer cells (open arrows).

Prussian Blue: Line = 50pm

103

 

104

323::

Iron was demonstrable in all areas of the heart, particularly
lining the epicardium (Figure 11A), endocardium (Figure 118), and within
macrophages of the subepicardial connective tissue (Figure 11C).
Slightly lesser amounts of iron, located primarily in interstitial cells
was observed in the myocardium (Figure 11D). Atrial myocardium also
contained IP and the distribution was similar to that described for
ventricular myocardium (Figure 11E). In addition, IP were found in
sections of chordae tendineae and there was infrequent evidence of IP
within cardiac myofibers (Figure 11F). Total quantity of iron increased

moderately with increments of iron load.

Wm

Ultrastructural evaluation was performed only on myocardial tissue
from guinea pigs of the high dose group. Aggregates of electron dense,
membrane-bound granules were occasionally observed within mesenchymal
cells. These granules were primarily located perinuclearly (Figure 12)
in lysosomal-like organelles (i.e., siderosomes) and were
morphologically consistent with ferritin particles. In heavily loaded
cells particles were abundantly free within the cytosol and not confined
to membrane-delimited organelles. x-ray microanalysis of lysosomal

structures within these cells confirmed that electron dense particles

were iron (Figure 13A and 138).

IV DISCUSSION

This study documents histologic and ultrastructural findings in

hepatic and myocardial tissue of guinea pigs injected intraperitoneally

105

Figure 11A. Cardiac Siderosis

11A. Iron Dextran; atrium, 0.5 g Fe/kg, guinea pig. Iron
particles lining epicardial surface (arrows). Prussian

Blue, Line = 100pm

11B. Iron Dextran; Right Ventricle, 0.5 g Fe/kg, guinea
pig. Iron particles lining the endocardium (arrows).

Prussian Blue, Line = 50pm.

106

Figures 11 A

 

Figure 11 B

 

107

11C. Iron Dextran; subepicardial region, 1.5 g Fe/kg,
'guinea pig. Numerous intracellular iron aggregates within
connective tissue macrophages (arrows) surrounding blood
vessels (bv) and intermixed with collagen (open arrows).

Prussian Blue, Line = 100nm.

11D . Iron Dextran; right ventricular myocardium, l . 5 g
Fe/kg, guinea pig. Several aggregates of iron particles in
interstitium of ventricular myocardium (arrows) between

myocytes (*). Prussian Blue, Line = 100pm.

Figures 11 c

 

109

11E. Iron Dextran; atrial myocardium, 0.5 g Fe/kg, guinea
pig. Iron aggregates in atrial interstitial tissue

(arrows). Prussian Blue, Line = 50pm.

11F. Iron Dextran; right ventricular myocardium, “1.5 g
Fe/kg, guinea pig. Iron particles in cardiac myofibers

(arrows). Prussian Blue, Line a lOOpm.

Figures 11 E

 

 

Figure 12. Interstitial cell with numerous perinuclear
iron-filled lysosomes, i.e., siderosomes (arrows). Stained;

Line = 500nm.

112

Figure 13A. Energy spectrum «obtained from interstitial cell
lysosomes (see electron micrograph Line = 1000nm; arrow) confirms
the presence of iron (Fe). Copper (Cu) peaks are due to copper
mesh grids; osmium (Os) peaks are the result of postfixation with

0804 e

113

Figure 13A

 

< A “““‘ 3.140 .1 kOV
FS = 511
mam: GP HEART, FE-CONTAINING Lvsosoms

   

 

 

 

 

114

 

Figure 138. Electron micrograph of cardiac interstitial
cell with iron laden lysosome (arrow) - evaluated using STEM
analysis (see energy spectrum in previous figure) Unstained;

Line - lOOOnm.

115

with iron dextran. The morphologic presence of iron complements the
concurrent studies showing altered lysosomes and membrane peroxidation,
thus helps to validate the guinea pig model for investigation of iron
toxicity.

Previous attempts to create an animal model for iron overload have
used various forms of iron, each differing in deposition sites.
Injected iron-nitrilotriacetate (Fe-NTA) and dietary carbonyl iron
preferentially accumulates in hepatocytes with minimal deposition within
Kupffer cells, whereas iron dextran primarily deposits in Kupffer cells
and to a lesser extent hepatocytes (3,5,6). In contrast, parenteral
administration of Jectofer (iron-sorbitol-citric acid complex) leads to
aggregation of iron particles in both parenchymal and Kupffer cells
(1,7). Furthermore, these prior studies have demonstrated a periportal,
as opposed to centrilobular distribution for carbonyl iron and Jectofer,
similar to human hemochromatosis (1,5). Histologic findings in our
study did not indicate a selective hepatic iron deposition pattern
which is consistent with a previous report (6). Iron was initially
confined to Kupffer cells and gradually extended to hepatocytes, a
pattern morphologically more consistent with secondary, rather than
primary forms of iron overload. These findings suggest that in this
model, as in acquired forms of iron overload, parenchymal iron
deposition does not occur until the iron retention capacity of the
reticuloendothelial system is saturated.

Extensive studies on myocardial siderosis in an animal model have
not been reported, however Iancu et al. (5) briefly’ described the
presence of sparse ferritin particles within myocytes and endothelial

cells in a carbonyl iron—fed rat model. Findings in this study show

116

that injection of iron dextran results in aggregation of iron particles
in the heart of this model. The demonstration of interstitial cell
siderosis (probably fibroblasts and connective tissue macrophages)
suggests that in vivo there is an operative defense mechanism resulting
in segregation of iron from myocytes. Nevertheless, results of parallel
studies using this model show significant elevation in myocardial
lysosomal membrane fragility with release of acid hydrolases, in
addition to increased lipid peroxidation. It is important to note that
iron-related release of lysosomal enzymes is thought to play a crucial
role in the pathogenesis of tissue damage in hemochromatotic patients
(8). Collectively, these related studies suggest that liberation of
hydrolytic enzymes from the interstitial cells or possibly myocardial
cells of the heart may initiate the cardiac abnormalities observed in
this model. These findings are supported by prior reports demonstrating
that in neonatal rat cardiac myocytes cultured in the presence of ferric
ammonium citrate iron uptake is associated with sequestration of iron-
filled ferritin into endocytic vesicles and lysosomes (9), increased
intracellular concentrations of malondialdehyde, altered cellular
contractility, and ultrastructural damage as evidenced by mitochondrial
abnormalities and excessive autophagocytosis (5,10,11). Thus, in both
studies iron loading is related to biochemical, morphologic and
functional alterations.

Numerous clinical reports of myocardial hemosiderosis have
described various histologic and ultrastructural changes in both
primary, as well as acquired forms of iron overload. Consistent
findings among these studies include (1) the presence of regional

anatomic differences in ventricular iron deposition with the

117

subepicardial region containing the heaviest iron deposits; intermediate
amounts subendocardially and lesser quantities in the midmyocardium (12-
15); and (2) distinct patterns of intracytoplasmic iron distribution.
Sanyal et al. (13) documented three patterns (1) paranuclear - a small
collection of iron adjacent to the myofiber nucleus; (2) perinuclear - a
more extensive deposition of iron around the nucleus; (3) diffuse -
maximum quantities of iron present in a diffuse cytoplasmic pattern.
These findings supported a previous study (12). Morphologic evaluation
of hearts in this study reveal that iron particles are particularly
prominent in epicardial and endocardial regions with slightly lesser
amounts in the ventricular myocardium; similar findings are noted in
atria. However, ultrastructural assessment clearly demonstrate that
iron particles accumulate within membrane-bound structures around nuclei
of mesenchymal cells.

In conclusion, this model mirrors many of the biochemical and
morphologic findings documented in previous reports of experimental iron
overload. Iron loading is consistently associated with the presence of
iron particles in membrane-bound structures (siderosomes), enhanced
lipid peroxidation, and fragile lysosomal membranes. The findings of
this, as well as concurrent studies in our laboratory suggest the
hypothesis that in the iron-loaded guinea pig, siderosis of myocardial
interstitial cells may be the initial lesion leading to further

biochemical and functional abnormalities.

118

References

1.

Hultcrantz R and Arborgh B. Studies on the rat liver following
iron overload. Fine structural appearance. Acta Path.
Microbiol.Scand.Sect.A 86:143-155, 1978.

Hultcrantz R, Arborgh B, Wroblewski R and Ericsson JLE. Studies on
the rat liver following iron overload. Electron Probe x-ray
microanalysis of acid phosphatase and iron. Am.J.Pathol. 96:625-
640, 1978.

Park CH, Bacon BR, Brittenham GM and Tavill AS. Pathology of
dietary carbonyl iron overload in rats. Lab.Invest. 57:555-563,
1987.

LeSage GD, Kost LJ, Barham JS and LaRusso NF. Biliary excretion of
iron from hepatocyte lysosomes in the rat. J.Clin.Invest. 77:90-
97, 1986.

Iancu TC, Ward RJ and Peters TJ. Ultrastructural observations in
the carbonyl iron-fed rat, an animal model for hemochromatosis.
Virchows Arch.B. 53:208-217, 1987.

Parmley RT, May ME, Spicer SS, Buse MG and Alvarez CJ.
Ultrastructural distribution of inorganic iron in normal and iron-
loaded hepatic cells. Lab.Invest. 44:475-485, 1981.

Hultcrantz R, Arborgh B, Wroblewski R and Ericsson JLE. Acta
Path.Microbiol.Scand.Sect.A 88:341-353, 1980.

Peters TJ, Selden C and Seymour CA. Lysosomal disruption in the
pathogenesis of hepatic damage in primary and secondary
haemochromatosis. Iron Metabolism:Ciba Foundation Symposium

51:317-325, 1977.

10.

11.

12.

13.

14.

15.

119

Cox PG, Harvey NE, Sciortino C and Byers BR. Electron-microscopic
and radio iron studies of iron uptake in newborn rat myocardial
cells. Am.J.Pathol. 102:151-159, 1981.

Link G, Pinson A and Hershko C. Heart cells in culture: A model of
myocardial iron overload and chelation. J.Lab.Clin.Med. 106:147-
153, 1985.(Abstract).

Hershko C, Link G and Pinson A. Modification of iron uptake and
lipid peroxidation by hypoxia, ascorbic acid, and alpha-tocopherol
in iron-loaded rat myocardial cell cultures. J.Lab.Clin.Med.
110:355-361, 1987.

Buja LM and Roberts WC. Iron in the heart: Etiology and clinical
significance. Am.J.Med. 51:209-221, 1971.

Sanyal SK, Johnson W, Jayalakshmamma B and Green AA. Fatal "Iron
Heart” in an adolescent: Biochemical and ultrastructural aspects
of the heart. Pediatrics 55:336-341, 1975.

Cappel DF, Hutchinson HE and Jowett M. Transfusional siderosis:
The effects of excessive iron deposits in the tissues. J.
Path.Bact. 74:245, 1957.

Olson LJ, Edwards ND, McCall JT, Ilstrup DM and Gersh BJ. Cardiac
iron deposition in idiopathic hemochromatosis: histologic and
analytic assessment of 14 hearts from autopsy.

J.Amer.Coll.Cardiol. 10:1239-1243, 1987.

CHAPTER IV

BRIEF COMMUNICATION

Hypogonadism in Iron Loaded Male Guinea Pigs: A Preliminary Study

Hypogonadotropic hypogonadism, accompanied by loss of libido is a
frequent endocrine dysfunction in men ( 1) with idiopathic
hemochromatosis (IHC). The hypogonadism results from the selective
deposition of iron in gonadotropic cells of the pituitary leading to
decreased levels of plasma FSH/LH and testosterone (2). In a
preliminary study, histologic evaluation of testicular tissue from iron
dextran injected male guinea pigs (n82) [83 mg Fe/kg/injection times 5
injection (pH 7.4) on alternate days from a total dose of 1.0 g Fe/kg]
demonstrated moderate to severe atrophy of seminiferous tubules with
decreased numbers of spermatogenic cells (Figure 14) when compared to
dextran treated controls (Figure 15). Occasional intracellular
aggregates of iron particles observed around interstitial (Leydig) cells
of the testes, were absent in sections of pituitary gland. These
results suggest that morphologic changes consistent with testicular
atrophy observed in primary hemochromatosis may be reproduced with this
model. Hypogonadism is particularly severe in young male patients
affected with the juvenile form of IHC (3). Histologic studies from
these patients indicate atrophic seminiferous tubules with scanty
mitoses, absent spermatozoa and spermatids, thickening of the tubular
walls and reduced numbers of Leydig cells (2). Similar findings were

observed in this brief investigation. Future studies with this model

120

121

would be enhanced by using morphometric techniques and assessing
endocrine function, i.e., determining plasma levels of FSH, LH and

testosterone. In addition, analysis of response to hypothalamic

stimulation would be of value.

122

Figure 14

Photomicrograph of testicle from iron dextran treated male
guinea pig with atrophy of seminiferous tubules. Note lack
of normal maturation and mitotic activity of spermatogenic
epithelium; marked vacuolation is notable. Hematoxylin 8:

eosin; Line = 100pm.

Figure 15

Photomicrograph of testicle from dextran treated control
male guinea pig. Maturation of spermatogenic cells from
spermatogonia (arrows) to spermatids (open arrows) is

normal. Hematoxylin and eosin; Line - 100nm.

123

Figure 14

 

Figure 15

 

124

References

Siemons LJ and Mahler CH. Hypogonadotropic hypogonadism in
hemochromatosis: Recovery of reproductive function after iron
depletion. J. Clin. Endocrinol. Metab. 65:585-587, 1987.

Stremmel W, Niederau C, Berger M, Kley HK, Rruskemper HL and
Strohmeyer G. Abnormalities in estrogen, androgen, and insulin
metabolism in idiopathic hemochromatosis. In Hemochromatosis;
Proceedings of the First International Conference. Ann. N.Y.
Acad. Sci. 526:209-223, 1988.

Cazzola M, Ascari E, Barosi G, Claudiani G, Dacco' M, Kaltwasser
JP, Paraiotopoulos N, Schalk KP and Werner EE. Juvenile
idiopathic haemochromatosis : A l ifs-threatening disorder
presenting as hypogonadotropic hypogonadism. Hum. Genet. 65:149-

154, 1983 .

APPENDICES

125

APPENDIXA: Specific Activity of Hepatic and Myocardial
B-N-acetylglucosaminidase (NAG) in Dextran
Control and Iron Treated Guinea Pigs

IRON

 

 

 

 

 

 

 

 

 

 

HEPATIC MYOCARDIAL
DOSAGE NAG NAG
Con 11.48 x 1.859 7.0195 : 0.2782
0'25 Fe 12.41 i 1.91 7.929 r 1.094 .
Con 14.87 i 1.088 8.525 : 0.7998
0'5 Fe 12.82 t 1.575 ' 9.889 : 0.7128 j
Con 125 i 2.885 8.502 t 1.117 ‘
1'0 Fe ”19.84 t 2.38- ' 11.42 1' 0.2258
Con 15.48 4.. 3.87 7.42 t 08871
2'0 Fe 13.95 x 2.082 7.275 t 08898

 

 

 

 

NAG units = nmol p-nitrophenol/ug protein/min (x

Dextran Control a Con

Iron Treated = Fe

' D < 0.05
” D < 0.01

10 '3')

 

 

 

126

APPENDIX 8: Specific Activity of Hepatic and Myocardial
Acid Phosphatase in Dextran Control and
Iron Treated Guinea Pigs

 

 

 

 

 

 

 

 

 

 

 

 

 

HEPATIC MYOCARDIAL
'RON ACID ACID
DOSAGE PHOSPHATASE PHOSPHATASE
-... Con ‘ 8.024 :- 07271 1.301 r 0.08"?
1 “‘3 Fe 4808 : 1.482 1.158 : 01082
i ,- _ Con 4.527 t 04451 1.872 : 033196
“)0 Fe “2.874 :. 0.8877 1.77s : 0.298
1 .. Con 4.27 t 1.188 1.542 t 0.288 .
l U Fe 5.09 _+. 0.9822 1.8282 1‘. 0.38
l . Con 8.429 1' 1.098 1.628 r 0.2084 4',
l 2.0 Fe '12572 i 1.87 "3.248 ~_~ 0.1241
[extran ContrOI = Con ' p < 0.01
:ron Treated = Fe H D < 0004'

Acid Phosphatase expressed 88

(nmol P04 /ug protein/min (x 10 "3)

127

APPENDIX 0: Specific Activity of B-glucuronidase in
Hepatic Tissue of Dextran Control (Don)
and Iron Treated (Fe) Guinea Pigs.

 

 

 

 

 

 

 

 

Heanc
lron Dose B-glucuronidase
Con 2.49 1 0.5748
0.25 _
Fe 2.818 : 0.5781
Con 2.517 : 0.5811
0.5
Fe 2.307 : 0.4662
Con 2.88 .1- 0.4005
1.0
Fe 8558 : 0.6292
Con 2.855 1 0.7351
2.0
Fe 2.489 : 0.3484

 

 

 

 

B-glucuronidase expressed as
(nmol p-nitrophenol/ug protein/min (x 10-
No significant differences noted.

31

128

Appende

Serum Iron (SI), Total Iron Binding Capacity (T IBC) and
Transferrin Saturation (T f Sat) Values in Control Guinea Pigs

 

Iron 0059 Animal SI TIBC TI Sat
(M9) N0- (us/0|) (us/d0 (96)

8—1 304 298 102.01

8-2 228 291 78.35

0.25 8-3 236 286 100.00

(11:4) 8-4 215 274 78.46

245.8:345 274.75127 .72 817021307

41 265 267 99.25
4.2 291 290 96.99
0.5 43 240 233 103.00
(n=6) 4.4 296 310 95.49
45 299 276 104.34
4-6 233 265 97.92
267.16:25.94 2735:2594 97.911592
0-1 259 - .-
0-2 291 303 96.03
1.0 0-3 333 322 10341
(n-s) 0-4 273 279 99.2
0-5 295 295 100.00
0—6 319 354 90.11

295022162 3104:2905 97.552496
m=0 maa

129

AppsndixE

Serum Iron (SI), Total Iron Binding Capacity (T I80)
and Transferrin Saturation (T f Sat) Values in
Iron Dextran Treated Guinea Pigs

 

Iron 0099 Animal SI TIBC Ti Sat
(90(9) NO- (us/d0 (us/d0 (96)
7-1 315 270 116.66
7-2 285 251 1 12.35
0.25 7-3 252 232 108.62
(n=6) 7-4 269 251 107.17
7-5 234 218 107.33
76 218 231 106.92
26652266 2421621866 10984239
3-1 689 745 92.48
3-2 1074 1194 89.94
0.5 3-3 950 1066 69.11
(n-6) 3.4 1141 1200 95.08
3-5 511 588 86.9
3-6 660 930 70.96
8375225332 95383224854 87.41 +8.54
1-1 585 498 117.46
1-3 1313" - —
1.0 1-4 591 492 120.12
15 321 - -
1-6 398 362 109.94

47375213564 4506627684 115842527
(n=4) (n=3) (n=3)

** = outlier

130

Appendix F

Serum B-N-acetylglucosaminidase (NAG) activity
. (expressed as nmoles p-nitrophenol/min/ug protein {x10’3})
in Dextran Control and iron Dextran Treated Guinea Pigs

 

Control F9 Treated
iron Dose Animal Activity Animal Activity
(g/kg) No. No.
8-1 2584 7-1 4.383
8-2 3.802 7-2 6.96
0.25 83 3.277 73 8.195
84 3.015 7-4 6.644
75 8.491
7-6 10.9
3169525093 7.5932217?
4-1 4.059 34 9.802
4-2 3.561 32 9.085
4-3 3.584 3-3 7.035
0.5 44 3.583 3-4 7.655
45 4.225 3-5 6.239
4-6 3.709 3-6 8.344
378622848 802621.318
0-1 3.073 1-1 14.3
0-2 3.01 1 1-2 17.42
1.0 0-3 3.09 143 17.36
04 3.094 1-4 16.16
0-5 3.155 1-6 20.14
0-6 297
306520656 17.0822119
6—1 2717 5-1 1 1.7
6-2 2664 52 7.285
2.0 6-3 2624 54 8.754
6-4 2786 5-5 7.06
65 3.365
28312.3045 8.69922136

131

Appendix G

A. Gross photograph of liver from septic guinea pig. Note
granular, jaundiced appearance of hepatic surface with
numerous, multifocal regions of necrosis.

8. Gross photograph; cross section of liver from septic
guinea pig demonstrates multiple regions of necrosis.

132

 

133

Appendix H

Electron micrographs of cardiac interstitial cells from iron dextran
treated guinea pigs (1.5 g/kg). Tissues were fixed with 2%
glutaraldehyde/2% paraformaldehyde and postfixed with 2% 0304; thin
sections were stained with saturated aqueous uranyl acetate and lead
citrate or left unstained.

A. Siderosomes containing variable amounts of iron (arrows).
Unstained; Magnification x 23,000

8. Interstitial cell containing abundant iron particles within
lysosomes (arrows) and extending beyond lysosomal membrane
boundaries (arrowheads). Unstained; x 20,000.

 

135

Edward Terence Adams was born on October 22, 1958 in New Orleans,
Louisiana, where he was educated in Catholic schools until graduation
from Brother Martin High School in 1976. The author attended college at
the University of Southwestern Louisiana (Lafayette, LA) for 3 semesters
before transferring to Dillard University (New Orleans). In 1979 he
began his veterinary education at Tuskegee University where he received
a Bachelor of Science degree in Animal Science (1982) and a Doctor of
Veterinary Medicine degree (1983). During his tenure at Tuskegee he
served as class president and received the Upjohn Award for Proficiency
in Small Animal Medicine. He pursued post-graduate training in the
Department of Pathology at Michigan State University in 1983 and was
awarded an NIH post-doctoral fellowship in 1984. The author is a member
of the Society of Phi Zeta, American Veterinary Medical Association,
United States and Canadian Academy of Pathology, and Charles Louis Davis

Foundation. He is single and without children.