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PEROXISOMES AND METABOLIC DISEASE

JENNIFER LUCY HUGHES

Thesis submitted for the degree of Doctor of Philosophy

in The University of Adelaide (Faculty of Medicine)

A,^,and."\ ìqq + ii dedication

t dedícate this work to my husband Gregory John Hanisch, and my children Max

(deceased), Nicholas Oscar and Jay Alexander. ¡¡i declaration

This work contains no mater¡al which has been accepted for the award of any other degree or diploma in any university or other tertiary institutlon and, to the best of my knowledge and belief, contains no mater¡al previously published or written by another person, except where due reference has been made ¡n the text.

I give consent to th¡s copy of my thesis, when deposited in the Un¡versity Library, being available for loan and photocopying.

SIGNED: oere:2*:.).:3.:t- iv summary

Peroxisomes are cellular organelles which carry out many metabolic functions including in

particular the breakdown and synthesis of fats. Peroxisomes are limited by a single

membrane, and the integral membrane proteins and the constituent matrix enzymes are

synthesized on free polyribosomes and incorporated post-translationally into pre-existing

organelles. The mechanisms involved in this biosynthetic process are not fully understood.

There is a group of inherited peroxisomal disorders, which are characterised biochemically by

a deficiency of one or more peroxisomal enzymes, and an accumulation of metabolites in the ! ¡,r, patient's tissues and plasma. The affected children most often die before reaching one year of age and suffer severe clinical symptoms including abnormalities of the liver, kidney, brain,

adrenal and b_o_ne. The peroxisomes in the liver of many of these pat¡ents display marked

ultrastructural changes ranging from a complete absence to extremely enlarged, flocculent

organelles.

ln some of the peroxisomal disorders the basic genetic defect is thought to be at the level of

the assembly of the peroxisomes, or the import of the peroxisomal proteins into the

organelles. The aim of this study was to contr¡bute to the understanding of the mechanisms

of peroxisomal assembly in human hepatocytes by investigating the nature of the

ultrastructural changes in peroxisomes in those patients where peroxisomal biogenesis or

function is impaired.

The specific aims of this study were

i.l to determine to what extent the structure of the peroxisomes is affected in the various

phenotypes of the peroxisomal disorders

ii.) to localise peroxisomal matrix and membrane proteins in the liver of peroxisomal disorder pat¡ents in order to find out if they have been normally incorporated into

dysfunctional peroxisomes or are mislocalised within the organelles or elsewhere

within the cells.

¡ii.) to ascertain if the study of peroxisomal morphology and the intracellular localisat¡on of

peroxisomal proteins can contribute to the understanding of the physiopathogenesis

of these metabolic disorders. iv.l to investigate the effect of peroxisomal dysfunction on hepatocyte ultrastructure

The ultrastructural features of the liver from 22 patients with a variety of peroxisomal disorders, and 18 control pat¡ents were investigated using morphological, immunocytochemical and enzyme cytochemical techniques. Morphometric analysis was carried out to quant¡tate the size and number of peroxisomes. The area, diameter, volume density, numerical density and surface density of peroxisomes was recorded from randomly selected electron micrographs. An on-grid immunogold technique was developed to localise and quantitate the peroxisomal matrix proteins catalase, the ß-oxidation enzymes, acyl-CoA

oxidase, bifunctional protein, and 3-ketoacyl-CoA thiolase, and a 68 kDa peroxisomal

integral membrane prote¡n (PMP6B). Liver from 5 control pat¡ents, and patients with disorders affecting peroxisomal assembly [Zellweger syndrome, neonatal

(NALDI, infantile Refsum disease (lRD)1 ; sex-linked

adrenoleukodystrophy (ALD), rhizomelic chondrodysplasia punctata (RCDP), pseudo-

Zellweger syndrome (PZS), and several unclassified peroxisomal disorders (PDl was

investigated.

A range of morphometric values in control pediatric liver was first established. Peroxisomes

which were ultrastructurally abnormal and greatly reduced in size and/or number were found

in several of the Zellweger syndrome pat¡ents, and the NALD and IRD pat¡ents. There was

variation in their numerical density ranging from none at all ¡n four of the Zellweger

syndrome pat¡ents to normal numbers in the IRD patients. ln most pat¡ents there was a

decrease in the immunolabelling of catalase over the peroxisomes. ln the Zellweger

syndrome and NALD pat¡ents, the small, abnormal peroxisomes did not label for any of the

ß-oxidation proteins. The IRD patients and the PD patients however, were heterogeneous

with respect to ß-oxidation labelling.

ln the liver of two RCDP patients, moderately to markedly enlarged, flocculent peroxisomes

were found. ln one pat¡ent they were very heterogeneous with regard to the number per

hepatocyte. lmmunoelectron microscopy was carried out on the liver of one of these

pat¡ents and the peroxisomes had a very low labelling density for catalase. The 3 ß- v table of contents

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 LITERATURE REVIEW

2.1 Morphology of peroxisomes 3

2.2 Distribution and development of peroxisomes 3

2.3 Peroxisomal enzymes 5

2.4 Biogenesis of peroxisomes 6

2.5 Visualisation of peroxisomes 8

2.5.a Enzyme cytochemistry I

2.5.b lmmunolabelling using colloidal gold I

2.5.c lmmunolabelling of peroxisomes 'to

2.5.d Morphometry t5

2.6 lnherited diseases involving peroxisomal defects 18

2.7 Zellweger syndrome 18

2.7.a History t8

2.7.b Heredity t9

2.7.c Clínical features t9

2.7.d Histopathology 20

2.7.e Biochemical findings 22

2.8 Neonatal adrenoleukodystrophy 28

2.8.a History 28

2.8.b Heredity 28

2.8.c Clinical features 28

2.8.d Histopathology 28

2.8.e Biochemical fíndings 29

2.9 lnfantile Refsum's disease 30

2.9.a History 30

2.9.b Heredity 30

2.9.c Clinical features 30 2.9.d HistoPathologY 30

2.9.e Biochemical findings 32

2.1O Sex-linked adrenoleukodystrophy 33

2.1O.a HistorY 33

2.1O.b HereditY 33

2.1O.c Clinical features 33

2.1O.d HistoqathologY 34 2.1O.e Biochemícalfindings 35

2.1O.f AdrenomYeloneuroPathY 36

2.11 Rhizomelicchondrodysplasiapunctata 36

2.12 Pseudo Zellweger and related syndromes 38 43 2.13 Other Peroxisomal disorders

2.14 Peroxisomes and other pathological conditions 44

2.15 ComPlementation analYsis 44

2.16 SummarY and conclusions 45 48 CHAPTER 3 MATERIALS 48 3. 1 Liver bioPsies

3.2 Control liver bioPsies 54

3.3 Animal tissues 54

3.4 Antisera 54 55 3.5 Colloidal gold Probes 57 CHAPTER 4 METHODS

4.1 Electron microscoPY 5l

4.2 Cytochemistry 57

4.3 lmmunocYtochemistrY 58

4.3.a Fixatíon 58

4.3.b Resín embeddíng 58

4.3.c Ultrathin crYosectíons 59 4.3.d Immunolabellingforelectron microscopy 59

4.3.e Quantitatíon of ímmunogold labelling 6't

4.4 Morphometry 61

4.4.a Measurements of peroxisomal size 6t

4.4.b Measurements of peroxisomal number 62

CHAPTER 5 MORPHOMETRY OF HEPATIC PEROXISOMES 64

5.1 Controls 64

5.2 Discussion 66

5.3 Peroxisomal disorder patients 68

5.3.a Generalísed peroxisomal disorders 68

5.3.b Sex-linked adrenoleukodystrophy 69

5.3.c Pseudo-Zellweger sYndrome 69

5.3.d Rhízomelic chondrodysplasía punctata 69

5.3.e Unclassified peroxisomal disorders 70

5.4 Discussion 73

5.4.a Small, abnormal Peroxisomes 73

5.4.b Enlarged peroxisomes 75

5.4.c Giant, flocculent peroxisomes 75

5.5 Conclusion 77

CHAPTER 6 ENZYME CYTOCHEMISTRY 78

6.1 Controls 78

6.2 Peroxisomal disorder patients 78

6.2.a Generalised peroxisomal disorders 78

6.2.b SexJinked adrenoleukodystrophy 78

6.2.c Pseudo-Zellweger sYndrome 78

6.2.d RhÞomelíc chondrodysplasia punctata 78

6.2.e Unclassífied peroxisomal disorders 79

6.3 Discussion 79

CHAPTER 7 IMMUNOCYTOCHEMISTRY OF HEPATIC PEROXISOMES a2 7.1 Controls a2

7.2 Peroxisomal disorder patients 82

7.2.a Generalised peroxisomal dísorders 82

7-2.b Sex-linked adrenoleukodystrophy 83

7.2.c Pseudo-Zellweger syndrome 83

7.2.d Rhízomelicchondrodysplasíapunctata 84

7.2.e Unclassifiedperoxisomaldisorders 84

7.2.f lmmunoelectron microscopy on ultrathin

cryosections 85

7.3 Discussion 88

7.3.a Control líver 88

7.3.b Small, abnormal peroxisomes 89

7.3.c Enlarged or normal sized peroxisomes 9t

7.3.d G iant, flocculent peroxisomes 92

7.3.e Peroxisomal "membrane ghosts " 93

7.3.f Immunoelectron microscopy on ultrathin

cryosections 95

7.3.g Changes in peroxisom)" ou", ,¡-" 97

-7.4 Conclusion 97

CHAPTER 8 MITOCHONDRIA AND LYSOSOMES 103

8.1 Mitochondrial abnormalities 103

8.2 Lysosomal inclusions 103

8.3 Adrenal gland 104

8.4 Discussion 105

CHAPTER 9 CONCLUDING REMARKS 109

CHAPTER 1O REFERENCES 112

APPENDIX A CASE NOTES 168

APPENDIX B PUBLISHED PAPERS AND MANUSCRIPTS 176

APPENDIX C DEVELOPMENT OF METI{ODS 180 v¡ list of tables

Table 1 Major peroxisomal enzymes and their metabolic function

Table 2 lmmunoelectron microscopy of peroxisomal proteins reported in the literature

Table 3 Morphometric values for control liver which appear in the literature

Table 4 Data on pseudo-Zellweger and related syndromes as compiled from the literature

Table 5 Summary of reported clinical, biochemical and histopathological features in peroxisomal disorders

Table 6 Clinical features

Table 7 Biochemical findings

Table 8 Tissues examined, type of fixation and resin used, and the source of the specimen for the patient material in this study.

Table 9 Tissues examined, type of fixation and resin used, and the source of the specimen for the control pat¡ent material in this study.

Table 10 Processing schedule for Lowicryl K1 1M resin

Table 11 Processing schedule for LRWhite resin

Table 12 lmmunolabetling schedule for electron microscopy

Table 13 Morphometry of control pediatric liver

Table 14 Morphometry of peroxisomes in peroxisomal disorder pat¡ents

Table 15 Cellular phenotypes seen in generalised disorders of peroxisomal dysfunction

Table 16 Results of incubation of liver in DAB medium

Table 17 lmmunocytochemistry of peroxisomal proteins in control and patient liver

Table 18 Morphological/immunocytochem¡cal types of peroxisomes identified in generalised peroxisomal disorders

Table 19 Summary of results for each group of peroxisomal disorders

Table 20 Morphological changes in other hepatic organelles v¡¡ list of figures Fig. 1 Control liver

Fig. 2 Frequency histogram of control peroxisomal diameters

Fig. 3 Small, abnormal peroxisomes

Fig. 4 lnfantile Refsum's disease

Fig. 5 Sex-linked adrenoleukodystrophy

Fig. 6 Pseudo Zellweger syndrome

Fig. 7 Rhizomelic chondrodysplas¡a punctata

Fig. 8 PD 1 and PD 2

Fig. 9 PD 3

Fig. 10 Frequency h¡stograms

Fig. 11 DAB sta¡n¡ns

Fig. 12 lmmunocytochemistry of control liver

Fig. 13 lmmunocytochemistry of lRD2

Fig. 14 lmmunocytochemistry of sex-linked ALD

Fig. 15 lmmunocytochemistry of pseudo Zellweger syndrome

Fig. 16 lmmunocytochemistry of RCDP

Fig. 17 lmmunocytochemistry of RCDP

Fig. 18 lmmunocytochemistry of PD1

Fig. 19 lmmunocytochemistry of PD2 and PD3

Fig. 20 lmmunocytochemistry of PD3

Fig. 21 lmmunocytochemistry of control liver using cryosections

Fig. 22 lmmunocytochemistry of NALD using cryosections

Fig. 23 lmmunocytochemistry of IRD using cryosect¡ons

Fig. 24 Mitochondrialabnormalities

Fig. 25 Mitochondrial abnormalities

Fig. 26 Lysosomal inclusions

Fig. 27 Lysosomal inclusions

Fig. 28 Adrenal inclusions Fig. 29 lmmunocytochemistry of rat liver

Fis. 30 Histogram of labelling density in rat liver v¡¡¡ acknowledgements

I wish to acknowledge the support of the Adelaide Children's Hospital (Division of Women's and Children's Hospitatl where this work was carried out. ln particular, the Departments of

Histopathology, Chemical Pathology and Paed¡atr¡cs provided the facilities, equipment, and pat¡ent information to make this study possible.

I would like to thank Dr E Robertson (Adelaide Children's Hospital), Dr CW Chow (Royal

Children's Hospital, Melbournel, Dr G Wise (Prince of Wales Children's Hospital, Sydneyl, Dr

LJ Sheffield (Murdoch lnstitute, Royal Children's Hospital, Melbourne) and Professor D

Sillence ( Unit, Ch¡ldren's Hospital, Camperdown, NSWI for the provision of pat¡ent material and clinical notes. I am also grateful to the many other doctors and clinicians who took the t¡me to carefully prepare and send patient information and tissues for this work. I also acknowledge the expert work of Dr Alf Poulos and co-workers who carried out the biochemical investigations as well as many useful discussions and criticisms. I thank

Dr Barbara Paton for assistance and many discussions in regard to the ant¡body techniques. l.am very grateful to my supervisors, Dr Alf Poulos, Dr Rodney Carter, and Professor Don

Roberton, all of whom encouraged me in this work. I would like to thank Dr Alf Poulos for reading and criticising the thesis.

I am indebted to Helen Thiele, Jenny Harford and Ruth Williams for skilled and willing technical assistance in electron microscopy. I also thank Roland Hermanis for many hours of photographic assistance.

The provision of ant¡bodies from Dr. Denis Crane (Faculty of Science and Technology,

Griffith University, Oueensland, Australia) and Professor T. Hashimoto (Shinsu University,

Japan) is also gratefully acknowledged. ix note regarding publications

Some of the work presented in this thesis has been published during the course of the candidature. The published papers are indicated when cited, and copies of the papers can be found in Appendix B. x abbreviations

ALD: sex-linked adrenoleukodystrophy

AT: 3-amino- 1,2,4-tiazole

BFP: bifunctional protein

D&WB: dilutent and wash buffer

DAB: 3'3'diaminobenzidine

DHAP-AT: dihydroxyacetone-phosphate acyltransf erase

IRD: infantile Refsum's disease

LRW: London Resin White

NALD: neonatal adrenoleukodystrophy

NRS: normal rabbit serum i.e. pre-immune rabbit serum

Nv: numerical density

PBS/BSA: phosphate buffered saline with 1% bovine serum albumin

PBS: phosphate buffered saline

PD: peroxisomal disorder

PLP: paraformaldehyde-lysine-periodate fixative

PMP68: 6BkDa integral membrane protein

PZS: pseudo Zellweger syndrome

RCDP: rhizomelic chondrodysplasia punctata

sv: surface density

VLCFA: very long chain fatty acids

Vv: volume density

ZS: Zellweger syndrome CHAPTER 1

INTRODUCTION Chapter 1 ln tion .t

Peroxisomes are organelles which are found in nearly all mammalian cells and also in cells throughout the plant kingdom. ln early biochemical studies using cell fractionation techniques, peroxisomes co-sedimented with mitochondria and lysosomes. ln electron microscopy studies, the peroxisomes were relatively small, amorphous organelles and were easily overlooked. lt was not until the early 1960's, when advances in both biochemistry and morphology took place that peroxisomes were generally recognised as separate organelles, distinct from mitochondria or lysosomes. ln 1954 Rhodin first described an organelle, which he termed a microbody, in the convoluted tubular cells of mouse kidney.

Almost simultaneously an organelle of similar morphology was described in hepatic parenchymal cells by Roullier and Bernhard (19561. The term microbody describes the morphological appearance of this organelle only and may also be applied to other unrelated structures. The term peroxisome defines an organelle characterised by ¡ts content of catalase and at least one hydrogen peroxide producing oxidase (de Duve 1969). Since then microbodies or peroxisomes have been identified in many tissues and are now believed to be present in varying numbers in all eukaryotic cells (Böck et al. 1980; Tolbert 1981; de Duve

1969,19831.

For many years peroxisomes were not considered significant in cellular metabolic processes.

The discovery that an absence of peroxisomes, as occurs in Zellweger syndrome, causes

severe clinical consequences for the pat¡ent, led to a renewed interest in this organelle.

Since then more than 40 different enzymes, involved predominantly with lipid and

carbohydrate metabolism, have been assoc¡ated with peroxisomes, and several inherited

metabolic disorders have been linked with a defect in the assembly or function of

peroxisomes. Children with genetically inherited disorders now make up approximately 25-

30% of admissions to children's hospitals in the Western world (Milunsky 1986), so the

diagnosis, both pre- and post-natally of inborn errors of metabolism is of great ¡mportance in

the prevention and treatment of these serious diseases. At the same t¡me the study of these

metabolic diseases may increase our knowledge of cell biology, and of the role of

peroxisomes in metabolism. This has been the case with lysosomal storage diseases where

advances in cell biology and molecular genetics led to a group of previously unrelated

1 Chapter I lntroduction

"storage" diseases being recognised as the result of lysosomal enzyme deficiencies. The study of molecular defects and enzyme deficiencies in this group of disorders has greatly increased our understanding of lysosomal enzyme synthesis, modification and transport within the cell. Advances in biochemical and immunochemical procedures, such as enzyme purification, monoclonal and polyclonal antibody production, and immunolabelling procedures for electron microscopy, have now made it possible to study the biosynthesis and post- translational modification and transport of prote¡ns in situ (Galjaard 1986).

Peroxisomes can be identified by both light and electron microscopy using enzyme cytochemistry, but the more recent advances in immunocytochem¡stry will enable the study of many more peroxisomal enzymes and integral membrane proteins, and allow the investigation of peroxisomal biogenesis. The study of inherited disorders involving peroxisomal defects should assist in elucidating this as yet unknown mechanism as well as facilitate the diagnosis and possible treatment of these metabol¡c diseases.

2 CHAPTER 2

LITERATURE REVIEW Chapter 2 Literature review

2.1 MORPHOLOGY OF PEROXISOMES

Peroxisomes are single membrane bounded organelles which vary in diameter from 0.1um to

1.Sum (de Duve and Baudhuin 1966; Tolbert and Essner 1981; Goldfischer and Reddy

19841, The limiting membrane is approximately 6-8 nm in thickness and is thinner than that of lysosomes (Tolbert and Essner 1981) and also of mitochondria which have a double membrane. Furthermore, the peroxisomal membrane differs from that of lysosomes in that it is highly permeable to small molecules such as sucrose, substrates, and inorganic ions

(Masters and Holmes 19771. Peroxisomes have a finely granular matrix and in some species an electron dense core. This core or nucleoid has a para-crystalline structure and can sometimes be seen to be made up of tubules (de Duve and Baudhuin 1966). Urate oxidase tias been localised to this peroxisomal core, which is absent in tissues lacking this enzyme such as human liver and mouse kidney.

The shape of peroxisomes is usually round to ovoid although in some tissues and species they are angular, ellipsoid, elongated or dumbbell shaped (Sternlieb and Ouintana 1982;

Black and Cornacchia 1986; Zaar and Fahimi 1990; Roels 1991). The peroxisomes are usually evenly d¡stributed throughout the cell, although they have been reported as occurring

¡n clusters (Sternlieb and Ouintana 1982; Briére 1986), or in close association w¡th the

endoplasmic reticulum (Novikoff and Novikoff 1982)'

Some peroxisomes have a marginal plate where the peroxisome profile is flattened and more

electron dense (Goldfischer and Reddy 1984; Zaar and Fahimi 1990). The function of this

plate is not known. Marginal plates in bovine kidney have recently been shown to be

associated with the enzyme isozyme B of L-ø-hydroxyacid oxidase (Zaar et al. 1991).

2.2 DISTRIBUTION AND DEVELOPMENT OF PEROXISOMES

Peroxisomes have a wide distribution in mammalian cells (Hrubran et al. 1972), but are most

numerous in the liver and kidney. Peroxisomes have been described in normal human

intestinal ep¡thelium (Black and Cornacchia 1986; Roels et al. 1991a1, in muscle {Riley et al.

1 988), aorta (Hor¡e et al. 19871, -alveolar type ll cells (Fringes et al. 1988) and

interstit¡al cells (Van Meir and Scheuermann 1 9BB), nervous tissue (Holtzman et al. 1973;

3 Chapter 2 Literature review

Arnold and Holtzman 1978; Arnold et al. 1979; Holtzman 1982), and brain (Dabholkar

19881. ln human fetal k¡dney, the mean size of peroxisomes was less than that of adults, and there was little change during the 1Oth to lBth weeks of gestation (Briére 1986). This suggests that a significant increase in the diameter of peroxisomes must take place after the 18th week of gestation or after birfh to reach the level of the adult. There were also variations in the diameter of peroxisomes within the same cell, some being small, electron dense

"microperoxisomes", without cores, and the others being larger and showing less catalase activity.

Similarly in human fetal liver an increase in peroxisomal size takes place during development

(Kerckaert 1990 as quoted in Espeel et al. 1990a and Roels 1991). Espeel et al. (1990a) found an increase in the size of hepatic peroxisomes between the seventh and eighteenth gestational weeks. The number of peroxisomes has been reported to be higher in younger fetuses (8-10 weeks), and then falls abruptly from twelve weeks onwards (Kerckaert 1990, as quoted in Roels 19911. ln rat liver the volume of individual hepatocytes increases with age and variations occur in different organelles (schmucker 1990). The data concerning peroxisomes is very limited and somewhat inconsistent, however it appears that ageing has littte effect on th¡s organelle in rat liver. However, during fetal and postnatal development some changes do occur. ln mouse hepatocytes the volume density of peroxisomes increases 2-3 fold between birth and 10 days of age (Kanamura et al. 1990), and in rat liver hepatocytes peroxisomes increase gradually in size and number during fetal development

(Stefanini 1ggbl. Also in the liver of baby pigs the number of peroxisomes increases 2 fold from 1 to 4 weeks of age (Giesecke et al. 1987).

ln the human brain, peroxisomes in the neurons appear in the basal ganglia, thalamus and

cerebeltum at 27-28 weeks of gestation, and in the frontal cortex at 35 weeks' They

increase in number w¡th gestational age, and gradually decrease with postnatal age. ln the

glial cells, peroxisomes appear in the deep white matter at 31-32 weeks of gestation and

shift to the superficial white matter with increasing age (Houdou et al. 1991).

4 Chapter 2 Literature review

2.3 PEROXISOMAL ENZYMES

More than 40 different enzymes have now been identified as peroxisomal. The enzyme content of peroxisomes and their characteristics have been reviewed by several authors (Tolbert 1971,1981; Masters and Holmes 1977; Böck et al. 1980; Lazarow and Fujiki

1985; van den Bosch et al. 1992). Peroxisomes play an important role in the metabolism of carbohydrate and lipid in the cell. ln 1976 Lazarow and de Duve discovered that rat liver peroxisomes contain a fatty acid oxidation system. Previously it was believed that fatty acid

ß-oxidation in animal cells occurs only in mitochondria. lt is now realised that there are two

ß-oxidation systems, consisting of different sets of enzymes; one in the mitochondria and one in the peroxisomes (Lazarow 1978). Some of the major peroxisomal enzymes and their metabolic functions are summarised in Table 1' and their metabolic function ENZYME FUNCTION

Catalase Degradation oÍ H2O2

Acyl CoA-oxidase ß-oxidation of long chain fatty acids

Bifunctional protein ß-oxidation of long chain fatty acids

3-Keto Acyl-CoA Thiolase ß-oxidation of long chain fatty acids

Di hyd roxyacetone-phosPhate Ether lipid biosynthesis acyltransferase ( DHAP-AT)

Al ky-di hydroxyacetone- Ether lipid biosynthesis phosphate synthetase

3-hydroxy-3-methyl glutaryl synthesis CoA reductase

Phytanate oxidase Degradation of phytanic acid

D-Amino acid oxidase Production of H2O2 Chapter 2 Literature review

2.4 BIOGENESIS OF PEROXISOMES

Early morphological stud¡es showed connections between peroxisomes and the endoplasmic reticulum (de Duve 1969; Reddy and Svoboda 1973¡ Reddy et al. 1974; Böck et al. 1980;

Fringes et al. 1982; Novikoff and Novikoff 1 9821, leading to the theory that peroxisomes are formed by budding from the endoplasmic ret¡culum. However, other investigators failed to find any such continuities even after extensive serial sect¡oning (Gorgas 1982, 19851, and enzyme cytochemistry failed to reveal any reaction product for catalase within the endoplasmic reticulum (Shio and Lazarow 1981; Gorgas 1985). Furthermore, biochemical studies showed that the peroxisomal membrane and the endoplasmic reticulum have different polypeptide and phospholipid composition (Fujiki et al. 19821. Lazarow et al.

(19821 has postulated the existence of clusters of interconnected peroxisomes, termed the

"peroxisomal reticulum", distinct from the endoplasmic reticulum. This "peroxisomal reticulum" has been identified morphologically in regenerat¡ng rat l¡ver by 3-D reconstruction of serial ultrathin sections (Yamamoto and Fahimi 1987a,b). However, this reticulum does not necessarily allow the interchange of matrix proteins between existing peroxisomes (for discussion see Roels 19911.

It is currently understood that peroxisomal proteins are encoded in the nucleus, synthesized on free polyribosomes in the cytosol, and enter pre-existing peroxisomes post-translationally.

New peroxisomes are then formed by growth and fission of pre-existing ones (Goldman and

Blobel 1978; Robbi and Lazarow 1 978; Lazarow et al. 1 982; Fujiki et al. 1 984; Suzuki et al.

1987a and reviewed by Lazarow and Fujiki 1985; Borst 1989). Morphological evidence for this model has been presented as dumb-bell shaped interconnections and clusters within the cell, especially after peroxisome proliferation (Fringes et al. 1982; Lazarow and Fujiki'1985;

Yamamoto and Fahimi 1987a,b). ln a study on regenerating rat liver after partial hepatectomy, Lüers et al. (19901 found that the formation of new peroxisomes is initiated by the synthesis of peroxisomal membrane proteins which is then followed by that of the matrix components. This suggests that the peroxisomal membrane proteins and peroxisomal matrical proteins are independently regulated. Chapter 2 Literature review

ln the liver of rats treated with a drug to induce proliferation of peroxisomes, double membraned loops resembling smooth endoplasmic reticulum were found in proximity to peroxisomes (Baumgart et al. 1989). Serial sectioning showed thatthese loops were always continuous with the peroxisomal membranes, and are part of the peroxisomal membrane system. These membranous loops were catalase negative but labelled immunocytochemically for the 70 kDa peroxisomal membrane protein, suggest¡ng that they may represent a membrane reservoir for the proliferat¡on of peroxisomes.

Most peroxisomal matrix prote¡ns are synthes¡zed at their mature sizes and not

proteolytically processed in the process of import into the peroxisomes. The exception to this is 3-ketoacyl-CoA thiolase, which is synthesized as a large precursor (44 kDa), and then

undergoes proteolytic processing during maturation or import in to the peroxisomes (Miura

et al. 1984; Fujiki et al. 1985). The sterol carrier protein 2 {or non-specific lipid transfer

protein is also synthesized as a larger precursor with a cleavable N-terminal presequence

(Fujiki et al. 19891

The mechanism by which newly synthesized peroxisomal proteins are targeted to, and

translocated into pre-existing peroxisomes is currently under investigation (for recent

reviews see de Hoop and Geert 1992; Subramani 1992; van den Bosch et al. 19921. The

target¡ng information for acyl-CoA oxidase has been shown to reside in two non-overlapping

regions of the carboxy-terminal protein of this enzyme (Small and Lazarow 19871Small et al.

19881. Gould et al. (1988, 19Bg) has now identified a peroxisomal targeting signal

consisting of the tripeptide serine-lysine-leucine (SKL) on the carboxy term¡nus of many

peroxisomal proteins. A second peroxisomal targeting signal at an amino-terminal or ¡nternal

location has also been identífied in peroxisomal thiolases (Swinkels et al. 1991).

The import of acyl-CoA oxidase into peroxisomes requires ATP, but no membrane potent¡al

(lmanaka et al. 19871. This import mechanism may require the presence of membrane

receptors (lmanaka et al. 1987). Kamijo et al. (1990) has cloned the rat-liver cDNA

encoding the 70kDa peroxisomal integral membrane protein, and found that the C-terminal

region of this prote¡n has a strong sequencç similarity to a superfamily of ATP-binding Chapter 2 Literature revíew

proteins. This suggests that this protein may be involved in transport across the peroxisomal membrane.

2.5 VISUALISATION OF PEROXISOMES 2.5.a Enzyme cytochemistry

The cytochemical localisation of peroxisomes is based on the peroxidatic act¡v¡ty of the enzyme catalase on the substrate 3', 3'diaminobenzidine tDABl (Novikoff and Goldfischer

1969; Fahimi 1975: Roels et al. 1975). Controls are carried out by the addition to the medium of a specific inhibitor of catalase, 3-amino-l ,2,4,triazole (Novikoff and Goldfischer

1969). The cytochemical technique is widely employed and has been used to visualize peroxisomes in many different tissues and species (Hruban et al. 1972; Novikoff et al. 1972;

1973). The sensitivity of the DAB technique was improved by Roels and Goldfischer in

1979 by increasing the incubation temperature to 45oC. Using this procedure they were able to identify peroxisomes in human biopsy specimens of liver and kidney. ln tissues obtained at autopsy, renal peroxisomes could be seen but in the liver the sta¡n¡ng reaction was weak or absent. De Craemer et al. (1990b1 have identified peroxisomes by DAB sta¡ning in rat and human autopsy liver up to 48h after death, although some regional sta¡ning loss is seen.

The fixation of the tissues, the section thickness, the composition, pH and temperature of the incubation medium, and postfixation can all influence the staining reaction with DAB

(Fahimi 1975; Roels et al. 19751. An alkaline DAB medium has been found to g¡ve the best result by a number of workers (Novikoff and Goldfischer 1969; Hruban et al. 1972; Novikoff et al. 1972; Fahimi 1975; Roels and Goldfischer 1979). The DAB'cytochemical technique has proved extremely useful in identifyinn O.ro*trornes and microperoxisomes in a wide variety of tissues and species at both the light and electron microscopic levels. Cytoplasmic or extraperoxisomal catalase has also be demonstrated, but only in those species which have a large amount of active enzyme in the cytosol (Holmes and Masters 1972; Roels

1976, 1977¡ Geerts et al. 1980; Geerts and Roels 1981). The DAB technique requires the

presence of an active form of the enzyme catalase and also high levels of activity. ln some

hepatomas with low levels of catalase act¡vity, peroxisomes could not be sta¡ned with DAB Chapter 2 Líterature revíew

(Mochizuki et al. 19711. Also, in some plant tissues, peroxisomes which could be shown biochemically to contain catalase, could not be sta¡ned with DAB (Fahimi 19751.

Nonspecific staining with DAB may occur due to endogenous peroxidases or non-enzymatic reactions with DAB (Goldfischer and Bernstein 1967; Barden 1 969; Holtzman et al. 1973;

Roels et al. 19751. The reaction product of the DAB technique is an electron dense, homogeneous deposit and as such it often obscures the underlying structures making them difficult to identify. The oxidised DAB is also difficult to quantitate especially when extraperoxisomal sta¡n¡ng occurs. Another disadvantage of the DAB method is the poor penetration of reagents into the tissue. This necessitates the use of very small tissue blocks or preferably thin slices of tissue. ln larger blocks the centre of the tissue is often completely unstained and can lead to false interpretation of the results. The use of DAB for the detection of peroxisomes and microperoxisomes presupposes that catalase is the common enzyme in these organelles. More than 40 enzymes have now been identified as peroxisomal, and this has necessitated the means of localizing enzymes other than catalase. 2.5.b lmmunolabelling using colloidal gold

The introduction of colloidal gold probes for immunocytochemistry at the ultrastructural level has proved to be a major advance for ultrastructural immunolabelling. Colloidal gold, as first described by Faulk and Taylor in 1971, is now used in a wide variety of applications for both light and electron microscopic investigations. This field has advanced very rapidly in the

1980's and has been extensively reviewed by several authors (Bendayan and Zollinger 1983;

De Mey 1983; Bendayan 1984; Beesley 1 985, 1 986; Bendayan et al. 1986; Gosselin et al.

1986; Roth 19861. Colloidal gold has several qualities which make it an excellent markerfor immunocytochemical stud¡es. Firstly, because of the high atomic number of gold, it is a very electron dense, spherical part¡cle and is thus readily identified in the electron microscope. The gold particles also have a natural red colour which can be seen in light microscopic sect¡ons, and the ¡ntroduction of silver enhancement (Holgate et al. 1983;

Springall et al. 1984; Scopsi and Larsson 1 985) has allowed it to be even more easily visualised as an intense black sta¡n¡ng. Silver enhancement of colloidal gold has also been utilised in immunoblotting procedures for the characterisation of antibod¡es (Scopsi and Chapter 2 Literature revíew

Larsson 1985). The gold particles can be prepared in homogeneous sizes ranging from 1nm up to 160nm, by using a variety of reducing agents for the reduction of gold chloride (Frens

1973; Slot and Geuze 1981, 1985; De Mey 1983; Baschong et al. 1985; Randal and

Saelinger 1986; van Bergen en Henegouwen and Leunissen 19861. By using two or more different size gold probes more than one antigen can be simultaneously localised on the same section {Geuze et al. 1 9B'l). A variety of macromolecules can be absorbed to colloidal gold (Geoghegan and Ackerman 19771 to produce a stable probe whilst reta¡n¡ng the biological act¡v¡ty of the protein. Staphylococcalprotein A, which binds to the Fc port¡on of immunoglobulin G, is most commonly used, but monoclonal antibodies, lectins, streptavidin, immunoglobulins, glycoproteins, hormones and enzymes have also been used (Hodges et al.

1987). These gold complexes remain stable and bioactive for many months when stored at

4 oC. Another advantage of the use of the post-embedding colloidal gold method is that it is easily quant¡tated, since only antigens exposed at the surface of the section will be labelled

(Bendayan et al. 1980; Griffiths and Hoppeler 19861. A disadvantage of using a post- embedding method however, is that many antigens are sensitive to the fixation, dehydrating and embedding methods which must be used in order to produce sections for the electron microscope. A compromise must be reached between the preservation of ultrastructure and the preservation of antigenic¡ty. The effects of chemical fixation will vary depending on the ant¡gen to be localised, and the optimal conditions must be worked out for each protein,

The use of various fixatives in relation to immunocytochemical studies has been reviewed by

Causton (1984).

Recently, ultrasmall i.e. 1nm diameter, colloidal gold has been used for pre-embedding immunolabelling (DeGraaf et al. 1991; Shimizu et al. 1992). The use of ultrasmall probes allows penetration into the permeablised tissue or cells, and the gold is then visualised by silver enhancement (Danscher 1 981 ). 2.5.c lmmunolabelling of peroxisomes

ln 1974, Yokota and Nagata used a ferritin-conjugated antibody technique to localise

catalase in ultrathin cryosections of mouse liver. Yokota and Fahimi (1980, 1981)then used

monospecific Fab fragments aga¡nst rat liver catalase, conjugated to horseradish peroxidase, Chapter 2 Literature review

to study the intracellular localisation of catalase in rat liver. The resultant electron dense deposit was seen only in the peroxisomal matrix and there was no evidence of cytoplasmic catalase. ln 1982, the prote¡n-A colloidal gold technique was ut¡l¡sed to demonstrate catalase and heat-labile enoyl-CoA hydratase in rat liver (Bendayan and Reddy 1982; Reddy et al. 1982). Both enzymes, as revealed bythe gold label, were confined to the peroxisomal

matr¡x, and no label was seen over the peroxisome core. Other peroxisomal enzymes and

membrane proteins which have been localised using immunoelectron microscopy are

reported in Table 2. TABLE 2: Immunoelectron mícroscopy of peroxísomal proteins reported in the líterature

REFERENCE ANTIBODY LOCATION TISSUE SPECIES

Usuda et al. 1988a; urate ox¡dase px core liver rat,mouse Völk et al, 1988 cow,dog,cat bovine Usuda et al. 1988b urate ox¡dase,catalase px matrix,core kidney fatty acyl-CoA oxidase

gurnea prg Yamamoto et al. 1988 catalase px matrix,cytosol liver nuclear matrix

rat Yamamoto et al. 1988 catalase px matrix liver rat,guinea Yamamoto et al. 1988 ø-hydroxy acid oxidase px matrix liver pis

liver rat Usuda et al, 1991 D-amino acid oxidase px subcomPartment Yokoto 1 990 glycolate oxidase urate oxidase

Litwin et al. 1988a,c catalase, ß-oxidation px matrix kidney human human, rat Litwin et al. 1984,1987,1988b catalase ß-oxidation px matrix liver liver rat Ghesquier et al.1 987 bifunctional protein px matrix liver human Espeel et al. 1990a ß-oxidation px matr¡x fetal px PM liver human Espeel et al. 1 990b catalase,ß-oxidation matrix rat,mouse Hollinshead and Meijer 1988 catalase, soluble ePoxide px matrix liver hydrolase

rat Deguchi et al. 1992 catalase, acyl-CoA px matrix ret¡na oxidase rat Yokota et al. 1985 L-alpha-hydroxyacid periphery of kidney oxidase px matrix o Reddy et al.1 982;Bendayan and catalase,heat labile px matrix liver,kidney rat Reddy 1982;Bendayan et a|.1983 enoyl-CoA hydratase

Yokota and Fahimi 1980, 1981 catalase px matrix liver rat

Yokota and Fahimi 1982 acyl-CoA oxidase px matrix liver,kidney rat

Yokota et al. 1983 enoyl-CoA hydratase specific granules eosinophils rat 3-ketoacyl-CoA thiolase

lozzo et al. 1982 erythrocyte catalase specific granules eosinophils human

Yokota et al. 1984 acyl-CoA oxidase,catalase specific granules intesti nal rat eosinophils

Usuda et al. 1986; Perotti et D-amino acid oxidase central clear kidney,liver rat al. 1987 matrix of px

Beier et al. 1988 catalase, urate oxidase px matrix and core liver rat ø-hydroxy acid oxidase acyl-CoA oxidase,thiolase bifunctional enzyme

Keller et al. 1985, 1986 3- hyd roxy-3-methyl g I utaryl px matrix liver rat coenzyme A reductase

Gutierrez et al. 1988 NADPH cytochrome P-450 px matrix liver rat red uctase

Gutierrez et al. 1988 NADH cytochrome c (b5) px membranes liver rat reductase

Keller et al. 1989; Tsuneoka sterol carrier protein-2 px, mitochondria liver rat et al. 1988 ER, cytosol

Hamilton et al. 1990 apolipiprotein E px matrix, microvilli liver rat endosomes, multi- vesicular bodies

(, Danpure et al. 1989a,b,1990; alanine:glyoxylate px matrix, liver human,baboon Cooper et al. 1988 aminotransferase mitochondria marmoset nonspf lipid transfer cat,rabbit protein

Cuezva et al. 1 990 mitochondrial F1 -ATPase px matr¡x, liver rat ø subunit mitochondria

Yokota et al. 1987 serine:pyruvate px matrix liver human aminotransferase

Usuda et al. 1 991 7OkDa,26kDa,22kDa px membrane liver,kidney membrane proteins small intestine rat

Wiemer et al. 1989 69kDa membrane protein px membrane liver rat catalase fibroblasts human

Baumgart et al. 1989 22,26,7OkDa membrane px membrane liver rat protein

Þ A method has been described for the light microscopic immunocytochemical demonstration of peroxisomal enzymes in Epon sections (Litwin et al. 1984). Tissues that had been

processed for routine electron microscopic studies were used to demonstrate catalase, acyl-

CoA oxidase, enoyl CoA hydratase (bifunctional protein) and 3-ketoacyl-CoA thiolase in

semi-thin sections embedded in epoxy resin. ln subsequent papers Litwin and co-workers

have extended this technique to human liver and kidney embedded in paraffin (Litwin et al.

1988a), to rat and human liver embedded in Epon (Litwin and Beier 1988b) and to human

kidney embedded in Epon (Litwin et al. 1988c). A brief etching in ethanolic sodium

hydroxide is required in order to unmask the antigenic sites in this method.

Ultrastructural immunolabelling of peroxisomal enzymes in human liver biopsies has been

demonstrated by Litwin et al. (1987,1988b) in both Lowicryl sect¡ons and Epon sections

after etching with ethanolic sodium hydroxide. The technique has been applied to human

post mortem liver and human foetal llver for the detection of the peroxisomal ß-oxidation

enzymes (Espeel et al. 1990a; 199Ob). 2.5.d Morphometry of peroxisomes

The measurement of morphometric values of oeroxisomes in the liver has been used

extensively in experimental work on animals, particularly to assess the proliferation of

peroxisomes by various drugs and experimental conditions. However, there are relatively

few publications on ultrastructural morphometric investigations in normal human liver

biopsies. These are documented in Table 3. The methods described by Weibel (1969,

1979) for the estimation of morphometric parameters are the most commonly used.

Beier and Fahimi (1986, 1992) have developed a morphometric technique for the

ultrastructural investigation of peroxisomes using automatic image analysis on rat liver

stained cytochemically for catalase. By using DAB-stained preparations in conjunction with

computer controlled automatic image analysis they showed that the values for volume and

numerical density were higher than in unstained sections, and the average peroxisomal

diameter was smaller. This implies that many of the smaller peroxisomes without nucleoids

are missed in unstained sections. ln a further study, Beier and Fahimi (1992) applied

automatic image analysis to rat liver tissue which had been immunostained for catalase and

15 Chapter 2 Literature review

examined in the light microscope. This has the advantage over DAB-stained tissue of restricting the labelling to the section surface, thus making it independent of section thickness.

The use of morphometry and stereology provides a useful objective view of the changes

which may occur in pathological material'

L6 TABLE 3: Morphometric values for control liver which appear in the literature

AUTHOR SAMPLE MEAN DIAMETER ¡r-* Yv lo/ol Nr, (¡rm-3) Sr, {¡rm-1}

DeCraemer et al. 1991b 6 adult 0.529 1.06 0.096 0.1 08 1 6wk infant o.445 0.70 o.128 0.085 1 4mth infant o.529 1.18 o.1 10 o.131

DeCraemer et al. 1991a I control o.517 1 .02 0.100 0.1 07 (range) (o.445-O.620) (o.71-1.411 (o.o52-o.1 31 ) (0.o83-o.1 49)

Koch et al. 1 978 4 adult 0.97 o.o7

Jezequel et al. 1 983 not stated 0.97 o.o72

Poll-The et al. 1988 1 adult o.534 1 baby o.478

Roels et al. 1988 1 adult 0.534 1 5wk female o.478 1 4mth male 0.481

Roessner et al. 1 978 14 adult 0.99

Rohr et al. 1 976 4 adult 1.2

Sternlieb and 4 0.618 Ouintana 1 977 (12-2Ovrl (0.25-1.34) * d-circle, not corrected for section thickness 2.6 INHERITED DISEASES INVOLVING PEROXISOMAL DEFECTS

Metabolic diseases have been described in which abnormalities of peroxisomal biogenesis, or of peroxisomal enzyme activity may be of pathogenic significance (Borst 1983; Moser and

Goldfischer 1985; Goldfischer 1986; Schutgens et al. 1986; Wanders et al. 1988d; for recent reviews see Moser 1987,1988, 1989; Moser et al. 1991a,b; Schutgens et al. 1989;

Wanders et al. 1990a, 1991; Wilson 1991).

The peroxisomopathies can be divided into three groups, depending on the degree to which peroxisomal functions and structure are affected. The first group includes those disorders where there is evidence of a generalized peroxisomal dysfunction, and the defect here, believed to be in peroxisomal biogenesis, leads to a great reduction or absence of identifiable peroxisomes and at the same time to an accumulation of metabolites in the patient's tissues.

Zellweger syndrome, infantile Refsum's disease, and neonatal adrenoleukodystrophy are examples of this group. ln the second group the defect is confined to an abnormality in the metabolism of a single metabolite or group of metabolites. Patients in this group have peroxisomes in their tissues but show a deficiency in one or more specific peroxisomal enzymes. Sex-linked adrenoleukodystrophy, thiolase deficiency, acyl-CoA oxidase deficiency and bifunctional enzyme deficiency are examples of this group. The third group contains the disorder rhizomelic chondrodysplasia punctata (RCDP) , in which peroxisomes are present in the liver, but more than one peroxisomal enzyme pathway is affected. The "Zellweger-like"

syndrome may also be included in this group as multiple enzyme pathways are affected in this disorder and morphologically normal peroxisomes are present in the liver. The clinical

phenotypes of the various inherited peroxisomal disorders are very diverse and there may be

other as yet unrecognized peroxisomopathies. 2.7 ZELLWEGER SYNDROME 2.7.a History

A syndrome involving multiple congenital defects was first described in two siblings by

Bowen, Lee, Zellweger and Lindenberg in 1 964. Smith and co-workers, in 1965, also

reported two siblings with similar congenital defects. ln 1967, Passarge and McAdams

proposed the descriptive term "cerebro-hepato-renal syndrome", after observing five sisters

1B Chapter 2 Literature review

with a similar disorder. Since the siblings in the first family reported (Bowen et al. 1964) were Professor Zellweger's patients, Opitz et al. 1969 used the term "Zellweger Syndrome" when describing four additional patients. The editor of Birth Defects, in which this article was published, suggested a combination of the two and called the disorder the "cerebro- hepato-renal syndrome of Zellweger". The term Zellweger syndrome is now the most commonly used. 2.7.b Heredity

Zellweger syndrome is believed to be inherited as an autosomal recessive trait (Smith et al.

1965; Opitz et al. 1969; Danks et al. 1975; Govaerts 1984; Schutgens et al. 1986). The syndrome occurs equally in males or females and consanguinity is known in many families, with the parents usually being unaffected. The incidence of Zellweger syndrome is estimated to be between 1:25,000 and 1:100,000 live births (Danks et al. 1975; Zellweger

1 987). 2.7.c Clinical features

The clinical characteristics of Zellweger syndrome have been reviewed by several authors

(Opitz et al. 1969; Danks et al. 1975; Govaerts et al. 1982; Kelley 1983; Govaerts 1984;

Schutgens et al. 1986; Wilson et al. 1986, 1988a; Stephenson 1988; Theil et al. 1992).

The prominent features are a characteristic facial appearance with a high forehead and long face, wide open fontanelles, shallow orbital ridges, external ear deformity, high arched palate, and redundant skin folds of the neck. Severe hypotonia is usually present as well as other neurological manifestations such as abnormal Moro response, hypo-or aref lexia, recurrent seizures and nystagmus. Other abnormalities are cataracts, hepatomegaly, renal cortical cysts, skeletal malformations and chondrodysplasia calcificans. Difficulty with feeding is nearly always encountered due to poor sucking and the patients generally fail to thrive. No effective treatment is known for the Zellweger syndrome. Most Zellweger patients die within the first weeks or months of life, however several patients older than two years have been reported (Govaerts et al. 19821 Bleeker-Wagemakers et al. 1986; Barth et al. 1987).

L9 Chapter 2 Literature review

2.7.d Hístopathology

A detailed review of histopathologic studies in Zellweger syndrome is given by Govaerts

(1984) . She discusses in detail the changes reported in the central nervous system, the liver, the kidneys, skeletal muscle and peripheral nerve, the hemato-lymphatic system, the adrenal glands, the pancreas and the eyes. ln 1972, Volpe and Adams studied a case of

Zellweger syndrome and found a primary disturbance of development of the brain, consisting of an incomplete migration of neuroblasts to form the cerebral cortical plate. Abnormal morphogenesis and delayed maturation of neurones in the brain has also been reported in two cases of Zellweger syndrome by Takashima et al. (1991).

ln 1973, Goldfischer et al. discovered an apparent lack of peroxisomes in hepatocytes and

renal proximal tubular epithelia, in biopsy and autopsy material from two patients with

Zellweger syndrome. Peroxisomes were absent when the tissue was examined

ultrastructurally after using the DAB cytochemical staining technique f or detecting

peroxisomes. They also reported structural abnormalities in the mitochondria of hepatocytes

and cortical astrocytes and concluded that there is an underlying subcellular defect in

Zellweger syndrome, Subsequently, the morphological absence of peroxisomes in the liver

of Zellweger syndrome patients has been reported by many authors (Pfeifer and Sandhage

1g7g; Monnens et al. 1980; Mooi et al. 1983; Auborg et al. 1985; Lazarow et al. 1985;

Roels et al. 1986; Kaplan et al. 1988). Peroxisomes of normal appearance were also absent

from the intestinal epithelium of patients with Zellweger syndrome, however, rare, minute,

DAB positive structures were observed (Black and Cornacchia 1986), This suggests that

peroxisomes in the intestinal epithelium of Zellweger patients are affected, but to a lesser

extent than in the liver. Shimozawa et al. (1988a) were able to detect peroxisomes in the

rectal mucosa of control patients, but peroxisomes were not found in the rectal mucosa of

three Zellweger infants.

Cultured fibroblasts from patients with Zellweger syndrome have also been examined. Arias

et al. (1985) showed the presence of small DAB positive peroxisomes in cells from a

Zellweger patient. The number of these peroxisomes per cell was significantly less than in

the control cells. Santos et al. (1985) also used the DAB cytochemical technique to study

20 Chapter 2 Literature review

cultured cells from a Zellweger patient and concluded that peroxisomes were not detectable.

It appears that the reduction or absence of peroxisomes apparent in the liver of Zellweger patients may not be reflected to the same extent in cultured fibroblasts from the same patient (Beard et al. 1986).

Wanders et al. (1987d, 1987f) has shown that peroxisomes are deficient in cultured myoblasts from Zellweger patients.

Abnormal lysosomal structures have been reported in liver macrophages, in brain astrocytes and in the adrenal cortex of patients with Zellweger syndrome. ln an ultrastructural study of the liver from three patients with Zellweger syndrome, Mooi and co-workers (1983) found macrophages with large angulate or fusiform lysosomes filled with fine double lamellae.

These correspond to the PAS-diastase positive material observed by light microscopy. ln the electron microscope the lamellae appear as pairs of very fine electron-dense lines, slightly less than lOnm thick and usually arranged parallel to one another and to the main axis of the lysosomes. These lysosomes were also seen in small numbers in the hepatocytes of the same patients. These large angulate lysosomes were more abundant in the older patients in the later stage of the disease. Similar structures have also been reported by Pfeifer and

Sandhage (1979) and Roels et al. (1g86) in patients with Zellweger syndrome. Roels et al'

(1986) also noted that these structures were more abundant in an older patient. Goldfischer et al. (1983) describes similar lamellar profiles lying free in adrenocortical parenchymal cells from Zellweger patients. These cytoplasmic inclusions are morphologically identical to those seen in adrenoleukodystrophy (Powers and Schaumburg 1 973) and it has been suggested that they reflect the storage of very long chain fatty acids that accumulate in these patients'

Similar lamellar and trilamellar inclusions have been found in the mesenchymal cells of the liver, in cells of the adrenal glands and in the cytoplasm of cerebral astrocytes in Zellweger syndrome (Auborg et al. 1985; Powers et al. 1989). The inclusions are ultrastructurally similar to those described in infantile Refsum's disease (Scotto et al. 1982; Poulos et al'

1984a; Hughes et al. 1990; for review see Kerckaert et al. 1988 and Roels et al. 1991b), in sex-linked adrenoleukodystrophy (Schaumburg et al. 1975) and in neonatal adrenoleukodystrophy (Partin and Mc Adams 1983). ln a study of mouse heart and liver

2L Chapter 2 Literature review

after treatment of the mice with O.5% chlorpromazine (which inhibits peroxisomal ß- oxidation), lamellar structures resembling those seen in human peroxisomal disorders,

appeared in the liver as well as an increase in very long chain fatty acids in the plasma

(Vamecq et al. 1987).

Goldfischer et al. (1973) described mitochondrial abnormalities in the hepatocytes and

cortical astrocytes of his Zellweger patients. The mitochondria, which were reduced in

number, were very dense with twisted cristae and irregular dilation of their intracristate

space. Oxidative studies on mitochondrial fractions from the brain and liver of these

patients, suggested a defect in mitochondrial respiratory function. Ultrastructural

abnormalities of mitochondria have been reported in two cases of Zellweger syndrome

(Mathis et al. 1980), three cases of Zellweger syndrome (Mooi et al. 1983) and in one

infantile Refsum's disease and one neonatal adrenoleukodystrophy case (Hughes et al.

1g9O). Wanders et al. (1990b) have also reported abnormal mitochondria in one infantile

Refsum's disease patient. Trijbels et al. (1983) studied the oxygen consumption in muscle

and liver mitochondria from a Zellweger patient, Like Goldfischer and coworkers they found

a defect in the electron transport chain before the cytochromes, but attribute this to a

deficiency of peroxisomes resulting in increased levels of peroxides' 2.7.e Biochemical findings

A wide variety of biochemical abnormalities have been reported in Zellweger syndrome

(Patton et al. 1972; Goldfischer et al. 1973; Danks et al. 19751 Hanson et al. 1979;

Monnens et al. 1980; Goldfischer 1982, 1986; Govaerts et al. 1982; Trijbels et al. 1983;

Heymans et al. 1983, 1984; Datta et al. 1984; Schutgens et al. 1984; Stokke et al. 1984;

Wanders et al. 1984, 1987a, c, e; Lazarow et al. 1985; Schrakamp et al. 1985; Tager et al.

1985; Suzuki et al. 1986; Vamecq et al. 1986; Wilson et al. 1986 and reviewed by Kelley

1983; Govaerts'1984; and Kaiser and Kramar 1988).

ß-Oxidatíon Enzymes

It has now been established that peroxisomes as well as mitochondria, are involved in the

metabolism of fatty acids via the ß-oxidation system (Lazarow 1978; Osmundsen et al.

1979). The absence of peroxisomes in Zellweger patients leads to an accumulation of

22 Chapter 2 Literature review

VLCFA (fatty acids with carbon chain lengths above C22l in the tissues and plasma (Brown et al. 1982; Moser et al. 1984b; Aubourg et al. 1985; Hajra et al. 1985; Sharp et al. 1987;

Poulos et al. 1989). The peroxisomal ß-oxidation enzymes, acyl-CoA oxidase, enoyl-CoA hydratase (bifunctional protein),3-hydroxyacyl-CoA dehydrogenase and 3-oxoacyl-CoA thiolase, are involved in the catabolism of VLCFA and are all deficient in Zellweger patients (Tager et al. 1985). This enzyme deficiency is thought to be caused by the rapid degradation of the ß-oxidation enzymes, in the absence of functional peroxisomes, rather than to an impaired synthesis of the enzymes (Suzuki et al. 1986, 1988b). On the other hand, catalase and D-amino acid oxidase and L-a-hydroxy acid oxidase, are stable in the absence of peroxisomes, although they are found in the cytoplasm rather than in the peroxisomal matrix (Wanders et al. 1984; Tager et al. 1985). Cultured skin fibroblasts from

Zellweger patients also show impaired degradation of VLCFA (Moser et al. 1984b; Lazarow et al. 1986; Wanders et al. 1987e).

Hexacosaenoic Acid

The level of hexacosaenoic acid (C26:1) (Moser et al. 1984b; Hajra et al. 1985) and also phytanic acid (3,7,11,15 tetramethylhexadecanoic acid) (Auborg et al. 1985) is also elevated in the plasma and in cultured skin fibroblasts from Zellweger pat¡ents. This abnormally high level of hexacosaenoic acid has been used as the basis of a prenatal test for Zellweger syndrome by assaying the level in cultured chorionic villus samples (Hajra et al. 1985).

Glycerol Ether Lipids

The absence of peroxisomes in Zellweger syndrome also leads to a deficiency of the peroxisomal membrane-bound enzymes involved in plasmalogen biosynthesis, which results in a greatly reduced level of plasmalogens in the tissues (Heymans et al, 1983, 1984; Poulos etal. 1991). Theplasmalogensorglycerol etherlipidscomprisemorethan2Oo/o of thelipid in the human brain and are widely distributed in cell membranes (O'Brien and Sampson

1965). A reduction in plasmalogens has been observed in erythrocytes and fibroblasts of

Zellweger patients (Heymans et al. 1984). The enzyme acyl-CoA: dihydroxyacetone-

phosphate acyltransferase (DHAP-AT), which is involved in the initial step of glycerol ether

lipid (plasmalogen) biosynthesis, has been localised in rat liver peroxisomes (Hajra et al'

23 Chapter 2 Literature review

1979; Hajra and Bishop 1982). This enzyme is significantly reduced in the liver, brain, and cultured skin fibroblasts of Zellweger patients (Schutgens et al. 1984). Datta et al. (1984) also found DHAP-AT levels to be deficient in the fibroblasts from patients with Zellweger syndrome. A second peroxisomal membrane-bound enzyme, alkyl dihydroxyacetone phosphate synthase, which is also involved in ether lipid biosynthesis, has been found to have reduced activity in the liver, kidney, brain, and fibroblasts of Zellweger patients

(Schrakamp et al. 1985).

Bile Acid Metabolism

Abnormalities in bile acid metabolism have been observed in Zellweger syndrome (Hanson et al. 1979; Monnens et al. 198O; Gustafsson et al. 1987) and have been attributed to the absence of peroxisomes (Kelley 1983; Wanders et al. 1987a). Bile acids are synthesized f rom cholesterol in the liver and the accumulation of the bile acid precursors, trihydroxycoprostanic acid and dihydroxycoprostanic acid, in the serum, bile and urine of

Zellweger patients suggests a defect in cholesterol side chain oxidation. This oxidative side chain cleavage of cholesterol is strongly impaired in liver from Zellweger patients (Wanders et al. 1987a; Kase 1989). The accumulation of bile acids in Zellweger syndrome is

attributed to the absence of peroxisomes, since in rat liver, peroxisomes, as well as

mitochondria, endoplasmic reticulum and the cytoplasm are involved in the conversion of

cholesterol into primary bile acids (Hagey and Krisans 1982; Kase et al. 1983; Kase 1989).

Furthermore, there is evidence for a close association between the fatty acid ß-oxidation

system in peroxisomes and the bile acid side chain cleavage system, since both are cofactor

dependent and both systems are enhanced in rats after treatment with peroxisome

proliferators (Wanders et al. 1987a¡ Hayashi and Miwa 1989). There is now evidence that

peroxisomes are involved in cholesterol biosynthesis, as well as cholesterol oxidation (see

Krisans 1992 for review).

Screening for bile acid precursors can be used in the detection of Zellweger syndrome and

related peroxisomal disorders (Van Eldere et al. 1987). Serum bile acids and the percentage

of bile acid precursors are highest in Zellweger syndrome patients who died young. Long-

living Zellweger syndrome patients, neonatal adrenoleukodystrophy and infantile Refsum's

24 Chapter 2 Literature review

patients have a lower level of bile acid precursors, whereas patients with sex-linked ALD, classical Refsum's disease, and rhizomelic chondrodysplasia punctata do not have increased bile acid precursor levels.

Pipecolíc Acíd

Elevated serum and urinary pipecolic acid, an imino acid derived from lysine, has been reported in Zellweger patients (Danks et al. 1975; Trijbels et al. 1979; Arneson et al. 1982;

Govaerts et al. 1982; Mihalik et al. 1989). Evidence that pipecolic acid metabolism takes

place in peroxisomes comes from the ability of normal renal and hepatic peroxisomal

fractions to metabolize pipecolic acid lZaar et al. 1986). A distinct L-pipecolate oxidase is

responsible for the oxidation of L-pipecolate in human liver, and this enzyme has been

located within peroxisomes (Wanders et al. 1989b).

Plasma levels of pipecolic acid are also elevated in patients with chronic liver disease

(Kawasaki et al. 1988) and the levels increase with the severity of liver damage, suggesting

a relationship to the injury of liver peroxisomes which are involved in the degradation of

pipecolic acid.

Other biochemical abnormalities which have been reported in Zellweger patients include

glycogen storage and hypoglycemia, dicarboxylic aciduria and glutaric aciduria, and abnormal

iron metabolism (Kelley 1983; Govaerts 1984).

Peroxísomal Membrane Proteíns

ln 1983 Borst postulated that the absence of peroxisomes in Zellweger syndrome may be

caused by the absence of an essential peroxisomal membrane protein, or of an enzyme

required for the import of proteins into the matrix. Some peroxisomal matrix enzymes, e.g.

catalase and D-amino acid oxidase. are present in Zellweger syndrome, although they are

located in the cell cytosol instead of in functional peroxisomes. However, the peroxisomal

ß-oxidation enzymes, which are membrane bound, are synthesized, but are rapidly degraded

in the absence of peroxisomes. Thus it would seem likely that the basic defect is in the

assembly of the peroxisomal membrane, or in the import of matrix proteins from their point

of synthesis on free polyribosomes into the peroxisomes. The peroxisomal 22-kDa integral

membrane protein l22lMPl has been found in normal amounts in the liver of a Zellweger

25 Chapter 2 Literature review

patient by immunoblotting techniques (Lazarow et al. 1986; Small et al. 1986). This suggests that peroxisomal membranes are present in Zellweger syndrome and that the defect is in the import of the matrix proteins. Further immunoblotting experiments on autopsy liver have shown that three peroxisomal membrane proteins (22, 53 and 68 kDa) were present in normal amounts in the liver of three Zellweger syndrome and one IRD patients (Small et al. 1988a). However, Wiemer et al. (1989) found the 69 kDa membrane protein was greatly reduced in autopsy liver of one Zellweger patient but not in another, and the 22 kDa and 36 kDa membrane proteins were present but in reduced amounts in three

Zellweger patients. Aikawa et al. (1987) also found the 22,26 and 70 kDa integral membrane proteins were deficient in one Zellweger patient. Suzuki et al. (1987b, 1989) also using immunoblot analysis of autopsy liver, found that a 70 kDa peroxisomal membrane polypeptide was deficient in four patients with Zellweger syndrome and that the amount of

22 kDa polypeptide varied from case to case. Pulse chase experiments in cultured fibroblasts revealed that the 70 and 22 kDa polypeptides were synthesized in the patients.

Gartner et al. (1990, 1991) found the 22 kDa integral membrane protein was present but in

variable amounts in seven Zellweger patients, and they recently found normal amounts of

normal sized PMPTO mRNA in fibroblasts from eleven Zellweger probands, suggesting

normal amounts of PMPTO protein (Gartner et al. 1992). Gartner et al. (1992) have cloned a

cDNA for human PMPTO and mapped the gene to chromosome 1. They have found 2

mutant PMPTO alleles in Zellweger syndrome probands from the same complementation

group. This suggests that a mutation in the PMPTO gene may be responsible for a subset of

Zellweger syndrome patients. A human cDNA for the peroxisomal integral membrane

protein PMP35 has also been cloned (Shimozawa et al. 1992), and a mutation in this gene

has been shown to be responsible for a single Japanese Zellweger syndrome proband

belonging to a rare complementation group. The PMP35 membrane protein has been

designated the peroxisome assembly factor-1 (PAF-1) because it restores the assembly of

peroxisomes in one peroxisome-deficient Chinese hamster ovary cell mutant'

26 Chapter 2 Literature review

Peroxisomal "membrane ghosts"

Large empty vesicles which react with antibodies to peroxisomal membrane protein have been described in cultured skin fibroblasts from Zellweger syndrome patients (Santos et al.

1988a; Wiemer et al. 1989). These "membrane ghosts" react with antibodies to peroxisomal integral membrane proteins, but are largely empty and are thought to be aberrant membrane vesicles. They have a density on Nycodenz gradients which is lower than that of normal peroxisomes. Their diameters are generally 2-4 times greater than normal peroxisomes, and they are heterogeneous in size (Santos et al' 1988b).

Balfe et al. (1990) have reported similar peroxisomal "membrane ghosts" in fibroblasts from

Zellweger and RCDP patients, and demonstrated immunoreactivity to thiolase by immunofluoresence. Fibroblasts from these patients contained the unprocessed 44 kDa form of thiolase and this was reported to be associated mainly with the lower density

fractions, although in RCDP cells some immunoreactive protein was also detected in the

peroxisomal fractions. This contrasts with the report of Singh et al. (1991), which showed

that in RCDP f ibroblasts most of the thiolase precursor was present in organelles

sedimenting with the same density as peroxisomes'

Gartner et al. ('1990, 1991)also showed that in Zellweger syndrome fibroblasts the 22 kDa

peroxisomal membrane protein was located in a subcellular membrane fraction with a lower

density than normal peroxisomes or mitochondria, and that the 44 kDa thiolase precursor

protein was located in the same fraction. ln Zellweger fibroblasts unprocessed acyl-CoA

oxidase has also been detected in structures which contain the 69 kDa peroxisomal integral

membrane protein and have a lower density gradient than normal peroxisomes (van

Roermund et al. 1991). The residual DHAPAT activity present in Zellweger cells was also

contained in these structures.

Heikoop et al. (1992b) have carried out double labelling immunoelectronmicroscopy

experiments on Zellweger fibrobtasts using antisera to the 69 kDa peroxisomal membrane

protein and lysosomal hydrolases. Their results indicate that at least 80% of the structures

previously described as peroxisomal "membrane ghosts" contain lysosomal hydrolases, and

21 Chapter 2 Literature review

may represent degradative autophagic vacuoles'

So far the peroxisomal "membrane ghosts" seen in cultured fibroblasts of Zellweger patients have not been described in the liver of these patients (Roels et al. 1991b). 2.A NEONATAL ADRENOLEUKODYSTROPHY

Several types of adrenoleukodystrophy (ALD) are described in the literature, including neonatal ALD, sex-linked ALD, and adrenomyeloneuropathy (Powers 1985). They differ in the age of onset of the disease, the mode of inheritance, and the nature of the underlying cellular defect. 2.8.a History A new form of ALD (neonatal or infantile) was described in 1978 by Ulrich et al. This disorder has a neonatal onset and was thought ¡nitially to be a phenotypic variant of sex- linked ALD (Manz et al. 1980) . However the occurrence of neonatal ALD in a female suggested it to be a variant of sex-linked ALD with the same genetic basis (Davis et al'

1979t Moser et al. 1980b). 2.8.b Heredity

As with Zellweger syndrome this disorder has an autosomal recessive mode of inheritance

(Benke et al. 1981; Moser et al. 198Ob; Jaffe et al' 1982). 2.8.c Clinical features

The clinical features of neonatal ALD have been reviewed by Schutgens et al. (1986). The

onset of neonatal ALD usually occurs at less than one year and the patients show

craniofacial dysmorphism, severe hypotonia, seizures, and psychomotor retardation and

degeneration. Adrenocortical hypofunction is only rarely seen. The patients generally fail to

thrive and die before six years of age (Goldfischer et al. 1985)' 2.8.d Histopathology

Striated lamellar inclusions similar to those observed in sex-linked ALD, have also been

described in the adrenal gland, spleen, thymus, liver and central nervous system of neonatal

ALD patients (Haas et al. 1982). These accumulations of very long chain fatty acids are

more pronounced in neonatal than in sex-linked ALD (Goldfischer and Reddy 1984). The

disorder differs from sex-linked ALD in that in liver biopsies from patients with neonatal

28 Chapter 2 Literature review

ALD, peroxisomes are greatly reduced in number and size (Goldfischer et al. 1983, 1985;

Partin and Mc Adams 1983; Goldfischer and Reddy 1984; Black and Cornacchia 1986;

Vamecq et al. 1986; Hughes et al. 1990). Peroxisomes which are smaller in size, but normal in number have also been reported in the intestinal epithelium of a patient with neonatal ALD (Black and Cornacchia 1986). Roels et al. (1988) described three patients with NALD who had enlarged peroxisomes in the liver. 2.8.e Biochemical findings

Accumulation of VLCFA has been described in the tissues, cultured fibroblasts, leukocytes and plasma in patients with neonatal ALD (Jaffe et al. 1982¡ Goldfischer and Reddy 1984;

Goldfischer et al. 1985; Schutgens et al. 1986; Vameiq et al. 1986; Wanders et al. 1987b).

ln particular, the level of hexacosanoic acid (C26:0) in the liver and adrenal gland was higher than thar found in sex-linked ALD (Jaffe et al. 1982; Goldfischer et al. 1985). Also the

storage in the tissues of these VLCFA was more widespread in neonatal then in sex-linked

ALD (Moser et al. 1 984a). ln contrast to sex-linked ALD, several other biochemical

abnormalities have been f ound in neonatal ALD. These include the accumulation of

trihydroxycoprostanoic acid and pipecolic acid in body fluids (Kelley and Moser 19841

Gotdfischer et al. 1985; Kelley et al. 1986). Wanders et al. (1987b) studied cultured skin

alkyl f ibroblasts f rom patients with neonatal ALD and found that DHAP-AT and

dihydroxyacetone phosphate synthase, the two membrane-bound peroxisomal enzymes

involved in plasmalogen biosynthesis, were both deficient. They also found that de novo

plasmalogen biosynthesis is impaired in the cultured f ibroblasts f rom these patients,

although not to the same extent as in those from Zellweger patients. Wanders et al.

(1g87b) also found that catalase is present in the cultured fibroblasts from neonatal ALD

patients although, as in Zellweger syndrome, it is localised in the cytoplasm, instead of in

peroxisomes. On the basis of these multiple enzyme abnormalities, they conclude that the

primary defect in neonatal ALD is, as in Zellweger syndrome, at the level of the biogenesis

of peroxisomes, although the extent of peroxisomal dysfunction is less severe than in

Zellweger syndrome. This concept of the underlying defect being in peroxisomal assembly

has also been suggested by several others (Goldfischer et al. 1985; Schutgens et al. 1986;

29 Chapter 2 Literature review

Vamecq et al. 1986). 2.9 INFANTILE REFSUM'S DISEASE 2.9.a Hístory ln 1974, Kahlke et al. described a 2 year-old boy with an elevated level of phytanic acid in the blood and liver. However the clinical picture was different to that found in classical

Refsum's disease, and the phytanic acid level, although above normal was lower than that seen in classical Refsum's disease. Since then several authors have reported similar cases

(Boltshauser et al. 1982t Scotto et al. 1982; Poulos et al. 1984a; Poulos and Sharp 1984;

Stokke et al. 1984; Budden et al. 1986; Poll-Thé et al. 1986; Wanders et al. 199Ob). 2.9.b Heredity

The mode of inheritance of infantile Refsum's disease is not known but since the disorder occurs in both sexes and the parents are usually normal, an autosomal recessive mode has been suggested. The parents of one family reported are first cousins (Scotto et al. 1982). 2.9.c Clinical features

The clinical features in infantile Refsum's disease are usually present from birth and include retinitis pigmentosa, deaf ness, mental retardation, dysmorphic f acial features and

hepatomegaly (Budden et al. 1986). Peripheral neuropathy, hypotonia, osteopenia, frequent

seizures, hypocholesterolemia and hypoalphalipoproteinemia have also been reported (Kahlke

et al. 1974; Scotto et al. 1982; Poulos et al. 1984a1Budden et al. 1986). 2.9.d HistopathologY

Liver biopsies have been obtained from most of the patients reported in the literature. Roels

et al. (1986) have described birefringent material in the macrophages, Kupffer cells and

hepatocytes in the livers of infantile Refsum's disease patients. The birefringent material,

observed with polarised light, is resistant to all processing treatments including extraction

with acetone or n-hexane. This accumulated material corresponds to the trilaminar

structures observed ultrastructurally. Electron microscopy of the liver shows the presence

of accumulated material, either membrane-bound within lysosomes, or free in the cytoplasm

within the hepatocytes, Kupffer cells and macrophages. The included material is made up of

lipid droplets of varying density, dark bodies, and trilaminar prof iles. The trilaminar

30 Chapter 2 Literature review

structures are 7-10 nm in width with a clear area between the two dense profiles, and are usually angular shaped (Scotto et al. 1982; Poulos et al. 1984a; Hughes et al. 1990).

Wanders et al. 1990b has reported these trilaminar lamellae in the epithelial cells of eccrine sweat glands as well as the liver in one IRD patient. The frequency of the trilaminar structures varies from patient to patient and may be correlated with the age of the patient, the older patients having greater amounts of stored material. The structures seen in infantile

Refsum's disease are similar to those described in both types of ALD and Zellweger syndrome. lntranuclear glycogen particles and clumps of glycogen within the cytoplasm have also been observed in the hepatocytes of infantile Refsum's disease patients (Scotto et

al. 19821Poulos et al. 1984a). A report by Torvik et al. (1988) describes the post-mortem findings in a 12-year old boy with infantile Refsum' disease, and Chow et al. (1992) have

documented the autopsy findings in two siblings with lRD. Several authors have examined the livers of infantile Refsum's disease pat¡ents for peroxisomes, either morphologically or

using the cytochemical DAB technique. ln most patients none have been detected (Ogier et

al. 1985; Budden et al. 1986; Roels et al. 1986; Poll-The et al. 1987; Wanders et al.

199Ob), suggesting a defect in peroxisomal assembly, however Roels et al. (1986) and

Hughes et al. (1990) found unusual peroxisomes in the liver of some patients. These

organelles were significantly smaller in size and had minimal catalase activ¡ty as measured

cytochemically. Beard et al. (1 986) also found small, slightly DAB reactive, atypical

peroxisomes in the liver of some patients with infantile Refsum's disease. They also

examined cultured skin fibroblasts from six patients using the DAB cytochemical method.

Four of these six cell lines had normal reactive peroxisomes and in the other two,

peroxisomes were rare or absent. This is further evidence that there may be heterogeneity

between cell types f rom the one patient with respect to the extent of peroxisomal

abnormality. Wanders et al. (1986) has shown a deficiency of catalase containing

peroxisomes in fibroblasts from infantile Refsum's disease patients using biochemical

techniques. Poll-The et al. (1987) examined a fetus believed to have infantile Refsum's

disease, and found a complete absence of peroxisomes in the liver and adrenal gland, but

atypical peroxisomes in the kidney. ln contrast, Zellweger syndrome patients are reported to

31 Chapter 2 Literature review

have an absence of peroxisomes in the kidney in postnatal life. 2.9.e Biochemical findíngs

The most characteristic biochemical finding in infantile Refsum's disease is the elevated level of phytanic acid in the plasma (Scotto et al. 1982; Poulos et al. 1984a; Budden et al. 1986).

The level is above normal, but is not as great as in classical Refsum's disease. The ability of cultured skin fibroblasts to metabolize phytanic acid is greatly reduced and this has been attributed to a lack of phytanic acid oxidase, an enzyme involved in the catabolism of phytanic acid (steinberg 1967; Poulos 1981; Poulos et al. 1984b). Other biochemical abnormalities related to peroxisomal functions have been reported in infantile Refsum's disease. An accumulation of very long chain fatty acids in the plasma, and a deficiency of peroxisomal ß-oxidation enzymes in cultured f ibroblasts has been reported in inf antile

Refsum's disease (Poulos et al. 1986; Schram et al. 1986; Wanders et al. 1986). An accumulation of the very long chain fatty acid hexacosanoic acid (C26) has also been found in the plasma of infantile Refsum's disease patients (Poulos and Sharp 1984; Budden et al'

1986). Greatly increased levels of pristanic acid have been found in the plasma of some

patients with infantile Refsum's disease, as well as in some patients with other peroxisomal

disorders (Poulos et al. 1988b). Levels of pipecolic acid are raised in the plasma, urine and

fibroblasts of these patients (Poulos et al. 1984a; Budden et al. 1986; Poll-Thé et al. 1986).

Abnormal bile acid metabolites have been found in the plasma of infantile Refsum's disease

patients (Poulos et al. 1984a; Poulos et al. 1985; Poll-Thé et al. 1986). The activity of the

membrane-bound peroxisomal enzyme acyl CoA:dihydroxyacetone phosphate acyltransferase

is deficient in cultured fibroblasts of infantile Refsum's disease patients (Poll-Thé et al.

1986). An impaired biosynthesis of plasmalogens has also been observed in fibroblasts (poll-Thé et al. 1986). The 22 kDa peroxisomal integral membrane protein studied by

immunoblotting techniques by Small et al. (1986) is also present in normal amounts in the

liver of one infantile Refsum's disease patient, as well as in Zellweger syndrome and

neonatal ALD patients. The spectrum of biochemical abnormalities observed in infantile

Refsum's disease is very similar to those seen in Zellweger syndrome, and to some extent

neonatal ALD, and can all be attributed to a lack of functional peroxisomes. The absence of

32 Chapter 2 Literature review

peroxisomes or reduction in peroxisomal numbers in the liver of these patients also suggests that the primary defect in infantile Refsum's disease may be in the biosynthesis of peroxrsomes.

The treatment of infantile Refsum's disease patients with a low phytanic acid diet produces some clinical improvement as well as a decrease in the plasma levels of phytanic acid and pipecolic acid (Robertson et al. 1988).

2.1O SEX-LINKED ADRENOLEUKODYSTROPHY 2.1O.a History

Classical ALD was first described by Siemerling and Creutzfeldt in 1923 as a syndrome of adrenal disease with accompanying cerebral demyelination. This combination of adrenal dysfunction and cerebral demyelination was considered coincidental and it was not until

1g63 that Fanconi et al. established the condition as a hereditary disease. ln 1972,

Schaumburg et al. discovered specific cytoplasmic inclusions in nine males with Schilder disease. Since then it has been established that most cases of Schilder disease are actually

ALD (Schaumburg 1975). 2.1O.b Heredity

Sex-linked ALD affects young males in the first decade of life, and has been shown to be a

X-linked disorder (Schaumburg et al. 1975; Moser et al. 1980b). The gene for sex-linked

ALD has been mapped to the Xq28 locus. Female carriers of the defect may show clinical

symptoms due to selective favouring of the mutant allele (Migeon et al. 1981; Moser et al,

1980b).

2. 1O.c Clínical features

Sex-linked ALD appears in young boys, usually in the first decade of life, after a period of

normal development. The clinical features have been discussed by Schaumburg et al.

(197b); Moser et al. (198Ob, 1991b) and Jaffe et al. (1982). The disease is characterised

by the progressive demyelination and destruction of both cerebral white matter and adrenal

cortex. The rates of degeneration of the nervous system and the adrenal cortex are

independent of each other (Schaumburg et al. 1975). The clinical symptoms, which reflect

33 Chapter 2 Literature review

the progressive deterioration of motor and intellectual functions include abnormal behaviour, disturbance of gait and loss of vision and hearing. Symptoms of adrenal failure, such as fatigue, hypotension, vomiting and cutaneous pigmentation, are also common (Schaumburg et al. 1975). 2.1O.d Histopathology

The histopathology of sex-linked ALD has been studied by Schaumburg et al. (1975) and reviewed by Kelley et al. (1986). Adrenal biopsy is the most reliable for diagnosis (Powers and Schaumburg 1974; Schaumburg et al. 1975). Characteristic cytoplasmic lipid inclusions have been described in the central nervous system macrophages, adrenal cortical cells, and

Schwann cells (Powers and Schaumburg 1973, 1974; Schaumburg et al. 1975; Johnson et

al. 1976; Ulrich et al. 1978; Powers et al. 1980; Kelley et al. 1986). Ultrastructurally, these

inclusions consist of paired electron-dense leaflets, separated by an electron-lucent space.

They are thought to represent an accumulation of the acetone-insoluble very long chain fatty

acids containing acyl-cholesterol esters which are stored in this disease (Johnson et al.

1976- Powers et al. 198O). ldentical inclusions have been identified in the fetal-zone

adrenocortical and Leydig cells of fetuses with ALD (Powers et al. 1982). Heterozygous

women for sex-linked ALD also have cells containing these inclusions in the adrenal cortex

(Powers et al. 1987). Recently, biopsied rectal mucosa from seven patients with sex-linked

ALD was studied and histiocytes containing the characteristic cytoplasmic lamellar

inclusions were found (Tanaka et al. 1987). These inclusions were also detected in two

asymptomatic younger brothers who had abnormal VLCFA profiles. However, there is no

correlation between VLCFA levels in the plasma and the severity of the neurological deficit

(Moser et al. 1989). The neurological expression of ALD may be influenced by factors in

addition to the VLCFA excess. The onset of the central nervous system demyelination is

accompanied by a perivascular infiltration of mononuclear inflammatory cells, suggesting

that one component of the disease may be immunologically mediated (Griffin et al. 1985).

ln liver biopsies from patients with sex-linked ALD, peroxisomes have been identified

ultrastructurally by cytochemical methods, and are normal in number and size (Goldfischer et

al. 1985).

34 Chapter 2 Literature review

2. lO.e Biochemical findings lncreased concentrations of VLCFA (as first described by lgarshi et al. in 1976) have been reported in the tissues (singh et al. 1981; Goldfischer et al. 1985), blood (Moser et al.

1981; Singh et al. 1984a), cultured skin fibroblasts (Singh et al. 1981; Moser et al. 1980a;

Poulos et al. 1985,1986) and cultured amniotic cells (Singh et al. 1984a) of sex-linked ALD patients. A raised level of VLCFA has also been detected in the plasma and cultured fibroblasts of female heterozygote carriers of the defect (Tönshoff et al. 1982; Moser et al.

1981, 1983). Singh et al. (1984b) studied the white blood cells, cultured fibroblasts and amniocytes in sex-linked ALD patients and found they have a defect in the oxidation of

VLCFA (C24:O and longer) but not for the degradation of fatty acids with a chain length of

18 carbon atoms or less. The oxidation of VLCFA takes place via the peroxisomal ß- oxidation system (Singh et al. 1984b) and the impairment of this metabolic process in sex- linked ALD patients suggests an underlying peroxisomal enzyme deficiency. Chen et al.

(1987) used antibodies to detect peroxisomal ß-oxidation enzymes (acyl-CoA oxidase, the bifunctional protein, and ß-ketothiolase) in liver samples from both sex-linked and neonatal

ALD patients. ln sex-linked ALD, all three enzymes were present, although in neonatal ALD there was an absence of enoyl-CoA hydratase, one of the activities of the bifunctional protein. No abnormalities have been found in plasmalogen levels in the tissues or fibroblasts from sex-linked ALD patients (Schutgens et al. 1986). Pipecolic acid levels and phytanic acid levels are also normal (Moser et al. 1984a) . The abnormalities in bile acid metabolism described in Zellweger syndrome, are not present in sex-linked ALD, lt is now recognised that the accumulation of VLCFA is caused by a deficiency of peroxisomal very long chain fatty acyl-CoA synthetase (Wanders et al. 19879, 1988a). The defect in X-linked ALD is at the level of a deficient ability of peroxisomes to activate VLCFA due to the deficient activity of the peroxisomal very long chain acyl-CoA ligase (Hashmi et al, 1986; Wanders et al.

1988a). A putative ALD gene has recently been identified (Moser et al. 1993) in the distal

part of Xq28. However, this putative gene shows no homology to the VLCFA-CoA

synthetase or any of the other three enzymes involved in peroxisomal ß-oxidation. A

significant sequence identity was found with the TOkDa peroxisomal membrane protein that

35 Chapter 2 Literature review

is believed to be involved in peroxisome biogenesis.

2. I O.f Adrenomyeloneuro7athY

This disorder is a milder form of sex-linked ALD, which progresses more slowly and mainly affects adults (Griffin et al. 1977; Davis et al. 1979; Moser et al. 1980b). The ultrastructural pathology of adrenomyeloneuropathy shows cytoplasmic inclusions similar to

ALD in the adrenocortical cells and brain macrophages (Davis et al. 1979; Powers 1985).

The inf iltration of perivascular lymphocytes is much less prominent in adrenomyeloneuropathy than in sex-linked ALD with childhood onset (Powers 1985), and the nervous system pathology differs from sex-linked ALD in that it mainly involves the long ascending and descending tracts of the spinal cord, and the peripheral nerves. The

biochemical findings in adrenomyeloneuropathy are similar to those described in sex-linked

ALD (Moser et al. 1980a, b). 2.11 RHIZOMELIC CHONDRODYSPLASIA PUNCTATA

This is another autosomal recessive disorder which usually leads to death in infancy. The

clinical characteristics are severe shortening and disturbed ossification of the proximal limbs,

and typical f acial appearance, congenital cataracts, mental retardation, skin changes,

srippting of the epiphysis (Spranger et al. 1971; Gilbert et al. 1976; Wardinsky et al. 1990).

Clinically and biochemically RCDP shows some similarity to Zellweger syndrome (Heymans

et al. 1985), There is a defect in plasmalogen (ether lipid) synthesis, and a reduction of

phytanic acid oxidation. However, unlike Zellweger syndrome, very long chain fatty acids,

pipecolic acid, and bile acid intermediates are normal (Hoefler et al. 1988; Poulos et al.

1988a, 1991). Also, in RCDP the peroxisomal enzyme 3-ketoacyl-CoA thiolase has been

found in an unprocessed form in post-mortem liver (Hoefler et al. 1988), and cultured skin

fibroblasts (Heikoop et al. 1 99O; Singh et al. 1 991 ; Balfe et al. 1 990). Catalase

sedimentation studies in cultured f ibroblasts show that, unlike Zellweger syndrome

peroxisomal structure is not deficient in RCDP (Hoefler et al. 1988).

There are few reports on the ultrastructure of the liver in RCDP patients. One patient has

been described in whom there was an absence of peroxisomes in some hepatocytes, with

other cells having an increased number of exceptionally large and irregularly shaped

36 Chapter 2 Literature review

peroxisomes (Heymans et al. 1986; Wanders et al. 1988d). Very large peroxisomes have been reported in the liver of one case of RCDP by DeCraemer et al. (1991b), and in two patients by Hughes et al. (1992 and see Chapter 5.3.d). ln one of these RCDP patients hepatic peroxisomes were immunoreactive for catalase, the three ß-oxidation enzymes, and a 68kDa integral membrane protein (see Chapter 7.2.d\. ln cultured skin fibroblasts from RCDP patients, normal peroxisomes have been described which react immunocytochemically with antibodies to catalase, and a 69kDa peroxisomal membrane protein. (Heikoop et al. 1 991 ). These peroxisomes did not react with a monoclonal antibody to 3-oxoacyl-CoA thiolase (Heikoop et al. 1991). These results are in conflict with those of Balfe et al. (1990) who found most of the thiolase immunoreactivity in subcellular fractions having a lower density than normal peroxisomes and mitochondria

(peroxisomal membrane ghosts). Some immunoreactive material was also detected in the peroxisomal fractions.

Recently, other syndromes characterised by chondrodysplasia punctata have been investigated for peroxisomal functions (Holmes et al. 1987; Wilson et al. 1988b; Poll-The et al. 1991; Van Maldergem et al. 1991; Emami et al. 1992). The biochemical abnormalities of

RCDP have been found in a patient with a non-rhizomelic type of chondrodysplasia punctata with distinct clinical features (Poll-The et al. 1991), and in a case of the Conradi-Hunerman form of chondrodysplasia punctata (Holmes et al. 1987). Van Maldergem et al. (1991) investigated peroxisomal function in a case of X-linked recessive chondrodysplasia punctata.

They found normal biochemical functions and normal peroxisomes in the liver, indicating a

non-peroxisomal nature in this case. lnvolved skin fibroblasts from one patient with the

CHILD (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) have

been studied and they show a decrease in the number of peroxisomes, and a decrease in the

activities of peroxisomal catalase and DHAP-AT (Emami et al. 1992). Thus, this syndrome,

which shares some clinical features with RCDP, may also be associated with a peroxisomal

defect- Pike et al. (1990) have reported a case of calcific epiphyseal stippling associated

with congenital rubella syndrome. Peroxisomal dysfunction in this infant was indicated by a

greatly increased level of plasma phytanic acid and by a reduced activity of DHAP-AT.

31 Chapter 2 Literature review

Peroxisomes in cultured skin fibroblasts were reduced in size and number, and hepatic peroxisomes were reduced in size. Wanders et al. (1992) have described a patient with all the clinical features of RCDP but in whom plasma phytanic acid levels were normal. The biochemical abnormality in this patient was restricted to a deficiency of DHAP-AT.

2.12 PSEUDO.ZELLWEGER AND RELATED SYNDROMES

Peroxisomes which are enlarged in size have been found in abundance in "pseudo-

Zellweger" patients with some clinical and biochemical similarities to Zellweger syndrome

(Goldfischer et al. 1986; Hughes et al. 199O). The patient described by Goldfischer et al.

(1986), has since been found to have a deficiency of the peroxisomal ß-oxidation enzyme 3- oxoacyl-CoA thiolase (Schram et al. 1987a, 1987b). Three other "pseudo-Zellweger" pat¡ents have been described by Clayton et al. (1988). These sibling patients had abundant catalase positive peroxisomes which were described as normal. Acyl-CoA oxidase, bifunctional protein and thiolase were present in the liver but a functional deficiency of peroxisomal thiolase was suggested.

Enlarged peroxisomes have also been described in 2 siblings with acyl-CoA oxidase deficiency (pseudo-NAlD) (Poll-The et al. 1988). ln one of these patients, the peroxisomes were present in normal numbers but were heterogeneous in size and shape, and in the other patient the peroxisomes were enlarged and were increased in number. Bifunctional protein and thiolase were present in both patients, but acyl-CoA oxidase was deficient. A patient with similar clinical features was reported by Kyllerman et al. (199O) and this patient also had enlarged peroxisomes which were increased in number and showed a marked variability in size. Naidu et al. (1988) found enlarged peroxisomes in a patient with the biochemical features of sex-linked ALD, but with clinical features more similar to neonatal ALD. ln this patient, all three ß-oxidation enzymes were present in an immunoblot of post-mortem liver, and the peroxisomes were catalase positive by enzyme cytochemistry.

Several infants with clinical and biochemical features of Zellweger syndrome have been reported who had morphologically normal peroxisomes in the liver (Paturneau-Jouas et al.

1987; Suzuki et al. 1988a; Mandel et al. 1992). These cases have been termed "Zellweger- like" disorders. Barth et al. (1990) described a patient with an impairment of peroxisomal ß-

38 Chapter 2 Literature review

oxidation resulting in elevated levels of VLCFA in the plasma and fibroblasts. However immunoblot analysis of the peroxisomal ß-oxidation enzymes in the liver of this patient showed normal immunoreactivity to acyl-CoA oxidase, bifunctional protein and th¡olase.

They conclude that this patient had a deficiency of a single peroxisomal ß-oxidation enzyme with retained immunoreactivity. Espeel et al. (1991) has described a newborn with greatly impaired peroxisomal ß-oxidation who had a subpopulation of hepatic peroxisomes, larger than 1¡lm, although the mean diameter, volume density, numerical density and surface density were within normal limits. Catalase, and the three ß-oxidation enzymes were localised normally in these organelles by immunocytochemistry.

The clinical features, peroxisomal morphology, immunoreactivity and biochemical findings in these patients are documented in Table 4.

39 \tc

Data on and related as from the literature ETHER LIPIDS REFERENCE TERMINOLOGY PATIENTS CLINICAL FEATURES PEROXISOMES IMMUNOBLOTTING VLCFA

normal Goldf ischer pseudo- 1F seizures, hypotonia, abundant, acyl-CoA oxidase increased et al. Zellweger areflexia, FTT large, BFP, catalase present ( 1 986) syndrome developmental delay catalase facial diplegia positive thiolase deficient renal cortical cysts (Schram et al. adrenal atrophy 1987a,b) death at 11112

increased def icient Paturneau- Zellweger- 1 typical Zellweger normal acyl-CoA oxidase Jouas like sex not syndrome BFP, thiolase et al. disorder specif ied not detected ( 1 987)

Clayton et pseudo- 3 sibs seizures, hypotonia abundant, acyl-CoA oxidase increased normal al. (1988) Zellweger 1F,2M large fontanelle normal, BFP, thiolase syndrome metopic suture catalase present developmental delay positive BFP functionally adrenal atrophy inactive (Wanders renal cortical cysts et al. 199O) death at 5 I 1 2,9 I 1 2,8.5 I 1 2

Suzuki Zellweger- 1M hypotonia, seizures normal acyl-CoA oxidase increased deficient et al. like aref lexia BFP, thiolase (1 988a) syndrome large fontanelle scanty, but mRNA frontal bossing detected shallow supraorbital ridges, hypertelorism low nasal bridge hepatomegaly

Poll-The pseudo- 2 sibs hypotonia, areflexia enlarged BFP, thiolase increased normal et al. NALD 1F,1M seizures size, present, ( 1 988) acyl-CoA death at 5 years normal no. acyl-CoA oxidase oxidase catalase def icient def iciency positive but uneven s

Naidu new peroxi- 1F hypotonia, seizures enlarged, acyl-CoA oxidase, increased et al, somal delayed development catalase BFP, thiolase (1e88) disorder death at 5/1 2 positive, present in normal amounts

normal Watkins Bif unctional 1M hypotonia, seizures normal, acyl-CoA oxidase, increased et al, enzyme developmental delay catalase thiolase present ( 1 989) def iciency macrocephalic positive BFP deficient large fontanelle mRNA for BFP split metopic suture present small adrenals renal cysts death at 5/1 2 *acyl-CoA Hughes pseudo- 2M FTT, hypotonia enlarged, oxidase, increased normal et al. Zellweger seizures, areflexia abundant BFP,present, (1990) syndrome joint contractures catalase, thiolase prominent midface present but reduced death at 4112,8112

Kyllerman pseudo- 1F hypotonia, seizures enlarged, increased normal et al. NALD areflexia, feeding increased no., ( 1 990) difficulties, poor catalase weight gain, slow pos¡tive but development, facial uneven, variable features anomalous stze cerebellar malformations

Barth ß-oxidation 1F hypotonia, feeding acyl-CoA oxidase, increased normal et al. defect diff iculties BFP, thiolase ( 1 990) deafness, progressive normal loss of psychomotor functions, central demyelination death at 37112 (\ ç

increased normal Espeel ß-oxidation 1M hypotonia, seizures enlarged, acyl-CoA oxidase, et al. defect neuronal migration amorphous BFP, thiolase, (1991) defects nucleoid catalase present death at 3/1 2

increased normal Mandel ß-oxidation 1M,1 F hypotonia, seizures catalase acyl-CoA oxidase, et al. defect (sibs) feeding difficulties positive BFP, thiolase present ( 1 992) no psychomotor progress, facial abnormality death at 3112, 4112 Chapter 2 Literature review

2.13 OTHER PEROXISOMAL DISORDERS

Various other inherited disorders have been linked to a defect in one or more peroxisomal enzymes. These include acatalasemia (Schutgens et al. 1986), Leber disease (Ek et al.

1986; Aikawa et al. 1989), and hyperoxaluria type I (Danpure and Jennings 1986; Latta and

Brodehl 1990). Primary hyperoxaluria type lis associated with a deficiency of the enzyme alanine:glyoxylate aminotransferase (AGT) in the peroxisomes of the liver. The enzyme AGT is normally located entirely in peroxisomes in human liver (Cooper et al. 1988). But in some patients with primary hyperoxaluria type I the AGT appears to be rerouted to the mitochondria (Danpure et al. 1989b). Thus this disease may be caused by a unique trafficking defect. The peroxisomes are of normal ultrastructural appearance in this disorder although somewhat reduced in number and size (lancu and Danpure 1987; De Craemer et al.

1 989).

Hyperpipecolic acidaemia is another disorder with clinical and biochemical features similar to

Zellweger syndrome (Gatfield et al. 1968; Thomas et al. 1975¡ Burton et al. 1981; Arneson et al. 1982; Challa et al. 1983). ln the livers of one patient with hyperpipecolic acidaemia which was studied ultrastructurally, peroxisomes were described as normal (Burton et al.

1981). This is a surprising result given the clinical similarities of this patient to Zellweger syndrome. Arneson et al. (1982) also described the occurrence of hyperpipecolic acidaemia in a patient with clinical and pathological findings consistent with Zellweger syndrome, and it has also been described in neonatal ALD patients (Kelley and Moser 1984).

A five-year-old boy has been described who has all the biochemical evidence of a disorder of peroxisomal biogenesis (MacCollin et al. 199O). However this boy has only very mild clinical symptoms, including hypotonia, areflexia and ataxia. Atypical catalase-positive peroxisomes were identified in the liver and distal tubules of the kidney, however there were no peroxisomes in the renal proximal tubules. This child may be a unique benign variant of peroxisomal dysgenesis.

2.14 PEROXISOMES AND OTHER PATHOLOGICAL CONDITIONS

43 Chapter 2 Literature review

Peroxisomal abnormalities, usually a reduction or increase in average size, increased or decreased numbers or morphological alterations, have been described in patients with other liver diseases including cholestasis, cirrhosis, hepatitis and malignancies (Sternlieb and

Ouintana 1977, 1982; Sternlieb 1979; Roels et al. 1983; De Craemer et al. 1991c, 1992b).

Ultrastructural changes in peroxisomes have also been reported in other metabolic diseases including Wilson's disease, Reye's syndrome, ø-1-antitrypsin deficiency and congenital total lipodystrophy (Goldfischer and Reddy 1984; Lake et al, 1987; Phillips et al. 1987; De

Craemer et al, 1992a).

DeCraemer et al. (1991a) has described changes in shape, size and number of peroxisomes in alcoholic and drug-induced hepatitis in humans, and in viral hepatitis in humans (De

Craemer et al. 1991c) and in mice (DeCraemer et al. 199Oa). Reinke et al. (1988) also

reported peroxisomal changes in patients with chronic hepatitis B. collins et al. (1989) have

described morphologically abnormal peroxisomes (pleomorphic, increased size and matrical

heterogeneity) in a case of abetalipoproteinemia, a disorder of lipid metabolism.

Ultrastructural changes in peroxisomes have been reported in drug and toxic liver injury

(Phillips et al. 1987). These changes are usually an increase in the number of peroxisomes

accompanied by an increase in the size and/or variation in shape and matrical density. 2.15 COMPLEMENTATION ANALYSIS

Complementation analysis experiments have now identif ied at least I diff erent

complementation groups from patients with peroxisomal disorders, implying that at least I

different genes are involved in the assembly of peroxisomes (Brul et al. 1988a; Poll-The et

al. 1989; Roscher et al. 1989; McGuinness et al. 1990; Yajima et al. 1992). ln these

experiments somatic cell fusions are performed with cultured fibroblasts from patients with

peroxisomal disorders. The levels of various peroxisomal functions, plasmalogen synthesis,

phytanic acid oxidation, and oxidation of very long chain fatty acids, and the formation of

peroxisomes (Yajima et al. 1992) are then measured in the resulting multinucleated

heterokaryon. lf the defect observed in one of the original cell lines is corrected in the

heterokaryon, complementation is said to have occurred. The restoration of activity in the

multinucleated cells can only occur if each parental cell line provides the gene product

44 Chapter 2 Literature review

defective in the other. Brul et al. (1988a) identified five complementation groups from patients deficient in peroxisomes. Patients with Zellweger syndrome, infantile Refsum's disease and hyperpipecolic acidemia fell into one group, rhizomelic chondrodysplasia punctata was a separate group, neonatal ALD a separate group, and the other two were individual Zellweger syndrome patients. They concluded that at least 5 genes are required for the assembly of a functional peroxisome. ln a subsequent study (Brul et al. 1988b) they described the kinetics of peroxisome assembly in heterokaryons of complementary cell lines, by measuring the rate of incorporation of catalase into particles. They concluded from this study that the components necessary for the assembly of peroxisomes must have been present in the parental cell lines.

There is no correlation between genotypes and phenotypes, and the currently used classifications of peroxisomal disorders do not represent distinct genotypes (Roscher et al.

1989). Furthermore, studies in cultured skin fibroblasts from patients belonging to the same complementation group, have shown further phenotypic heterogeneity with regard to immunofluorescence microscopy and staining with catalase antibody (Wiemer et al. 1991).

Complementation analyses on 1O patients with RCDP revealed 2 complementation groups, and thus mutations in at least 2 different genes can |ead to the RCDP phenotype (Heikoop et al. 1992a)

2.16 SUMMARY AND CONCLUSIONS

A summary of the reported clinical, histopathological and biochemical findings in the principle ínherited diseases involving peroxisomal dysfunction is presented in Table 5.

45 of biochemical and his features ín disorders FEATURE ZELLWEGER NALD IRD X-LINKED ALD RCDP PZS

Age of onset At birth At birth At birth Childhood At birth Ar birth Mode of inheritance Autosomal Autosomal Autosomal Sex-linked Autosomal Unknown recesstve recessrve recessive recessive recesslve

Clinical features Seizures Seizures Seizures Disturbance Seizures Seizures Hypotonia Hypotonia Hypotonia of gait St¡ppling of Hypotonia Hepatomegaly Psychomotor Peripheral Adrenal epiphyses Failure Renal cysts retardation polyneuropathy dysf unction Skeletal to thrive Skeletal Facial Retinitis Loss of malformation Renal cysts malformation abnormality pigmentosa hearing lmpaired Facial Facial Adrenal atrophy Deafness lmpaired vtslon abnormality abnormality Mental vision Facial Adrenal atrophy lmpaired vision retardation Abnormal abnormality Mental Facial behaviour Mental retardation abnormality retardation Adrenal atrophy Osteopenia

elevated VLCFA elevated elevated elevated elevated normal 'normal thiolase ß-oxidation enzymes deficient def icient def icient normal deficient elevated Pipecolic acid elevated elevated elevated normal normal level normal Phytanic acid elevated elevated elevated normal elevated level partial reduction Phytanic acid def icient def icient def icient normal def icient oxidase(f ibroblasts) impaired normal Plasmalogen impaired impaired impaired normal synthesis (severe) (mild) normal DHAP-AT(f ibroblasts) deficient def icient def icient normal def icient particulate particulate particu late Catalase(f i broblasts) cytoplasmic cytoplasmic cytoplasmic normal impaired Bile acid impaired impaired impaired normal metabolism abundant and Hepatic peroxisomes absent reduced number absent ot rale normal normal, or and size minute increased size larger liver Lamellar inclusions liver,brain spleen,thymus liver brain,adrenal adrenal brain,adrenal rectal mucosa liver

yr O-1yr Usual survival age 0-1yr 0-6yr O-1 1yr 5-1 Syr O-1 Despite the clinical differences there are many pathological and biochemical similarities in

Zellweger syndrome, neonatal ALD and infantile Refsum's disease. These disorders are now referred to as the generalised peroxisomal disorders because they have multiple biochemical defects (Brown et al. 1982; Scotto et al. 1982¡ Poulos et al. 1984a; Stokke et al. 1984;

Goldfischer 1986; Kelley et al. 1986; Poll-Thé et al. 1986, 1988; Schutgens et al. 1986;

Vamecq et al . 1986). Further investigations are required to discover whether or not

Zellweger syndrome, neonatal ALD, infantile Refsum's disease and other disorders involving a deficiency of peroxisomes are genetically distinct and where the primary biochemical defect occurs. The absence or reduction in peroxisomes observed in these diseases appears to vary from one disorder to another but also within each disorder there is variation depending on the age of the patient and which tissue or cells are examined. The relationship between the extent of the clinical features and survival of the patient and the number of peroxisomes and the peroxisomal enzymes synthesized. remains to Oe fully established.

Furthermore the study of these metabolic diseases may lead to a better understanding of peroxisomal biogenesis and the synthesis and import of peroxisomal enzymes. The role of this organelle in cellular functions may be implicated in more inherited diseases than is recognised at present.

4'/ CHAPTER 3

MATERIALS Chapter 3 Materíals

3.1 LIVER BIOPSIES

Liver tissue from patients with a peroxisomal disorder was obtained at needle biopsy or at autopsy. Other tissues including kidney, adrenal, muscle, brain, small intestine and spleen were also obtained where possible. The tissue was taken as quickly as practical depending upon the circumstances, and fixed immediately by immersion fixation. The suspicion of a peroxisomal disorder was determined by the clinician involved, and was largely dependent upon the presence of appropriate clinical symptoms and family history. ln¡tial biochemical tests included measurement of plasma very long chain fatty acid (VLCFA) ratios and phytanic acid levels. The activ¡ty of the enzyme dihydroxyacetone-phosphate acyltransferase (DHAP-AT) was measured in cultured skin fibroblasts. Biochemical tests on the liver obtained at biopsy or autopsy were carried out simultaneously by the Chemical

Pathology Department at the Adelaide Children's Hospital.

Tissue Írom 22 patients was included in this study and the case histories are described in

Appendix A. This included tissue f rom 9 Zellweger syndrome patients; 1 neonatal adrenoleukodystrophy (NALD) patient;2 infantile Refsum's disease (lRD) patients; 3 sex- linked adrenoleukodystrophy (ALD) patients; 2 pseudo-Zellweger syndrome patients |PZSI; 2 rhizomelic chondrodysplasia punctata (RCDP) patients; and 3 unclassified. The cases are arranged according to their provisional clinical diagnoses. The term pseudo-Zellweger syndrome is used here to indicate 2 patients with a peroxisomal disorder which resembles

Zellweger syndrome to some degree but cannot be described as Zellweger syndrome because of the presence of abundant hepatic peroxisomes. lt does not necessarily imply that these patients have a thiolase deficiency as described in the 2 patients of Goldfischer et al. (1986) [see 2.12 for further discussion]. There are three cases which do not fit into any currently recognised category and they are designated peroxisomal disorder (PD). A superscript of a or f denotes examination of autopsy or fetal tissue respectively. ln some cases more than one biopsy was received from the same patient, and for some patients a liver biopsy was obtained and then autopsy tissue was examined when the patient died. ln these cases the number of the biopsy is designated by a postscript b and the number. A summary of the main clinical features of each of the patients is given in Table 6, and the

48 Chapter 3 Materials

biochemical features are in Table 7.

Where the biopsy was performed in another state or country arrangements were made to send fixative to the appropriate hospital by overnight mail or courier, and the tissue was returned the same way. Alternatively, in some cases the technicians in the hospital made up fixative in their laboratory according to my instructions. Unfortunately some tissue was received which had already been fixed, and in some cases embedded, before other arrangements could be made. The different fixatives and embedding resins for all the patients in this study are shown in Table 8, and for the controls in Table 9.

49 Clinical features Condition IUGR FTT Hepatomegaly Dysm.F Ret.Pigm. Deaf Alive/ Stippled Hypotonia Fits Devt. Periph. or Cataract Deceased Epiphysis Delay Neuropathy zs1 ++++ N.R. N.R. died 4112 yr + + + zs2f Fetus zs3a -+++ + died 8112 yr + + + zs4a ++++ N.R N R died 4112 yr N.R. N.R. + zsSa N.R. N.R. + + + N R died < 2112 yr N.R. +++ + + N.R zs6f Fetus lndex case Zellweger syndrome zsTa Clinical suspicion of Zellweger syndrome zsga Clinical suspicion of Zellweger syndrome, dysmorphic, ++ zsga Clinical suspicion of Zellweger syndrome

NALD + +++ + + + + died 3.5 yr + + +

IRDl + +++ + + + + died 2 yr + + + IRD2 + + + + + + alive 11 yr + +

ALDIf Fetus ALD2A died 5.5 yr + + ALD3A died 7 yr late N.R I ate N.R.

PZS 1 Consistent with peroxisomal disorder PZS2 ++ ++ N.R. ++ N.R N.R, died 8112 yr N.R. +++ ++ ++ N.R

RCDPl N.R. ++ + cataract N.R died 3.5 yr +++ +++ N.R. +++ N R RCDP2A N.R. N.R. N.R + cataract N.R died 2 wks ++ N.R. N.R. N.R N R

PD1 prop + + + + N.R N.R. died 5112 yr + + + PD1 Fetus * PD2 + + died 1.5 yr + + PD3 + + alive 7 yr + ++

tlJGR, intra-uterine growth retardation; FTT, failure to thrive; Dysm. F, dysmorphic features; Ret. pigm., Retinitis Pigmentosa; Devt. Delay, developmentat detay; Periph. Neuropathy, peripheral neuropathy; N.R., not recorded accidental death

)'t Biochemical CASE PLASMA CULTURED SKIN FIBROBLASTS

c26t22 c24t22 PHYTANIC c26t22 PHYTANIC DHAP-AT CATALASE ACID ACID OXIDASE %sedimentable zs1 elevated elevated elevated decreased reduced decreased zs2r reduced! zs3a elevated elevated normal elevated reduced zs4a elevated elevated elevated reduced zs5a elevated elevated normal zs6f elevated Pdecreased reduced zsTa elevated elevated normal elevated decreased reduced decreased zsSa elevated elevated normal elevated reduced decreased (sibling ) zsga elevated elevated normal elevated decreased reduced decreased

NALD elevated elevated elevated elevated decreased reduced

IRDl elevated elevated elevated elevated decreased reduced decreased IRD2 elevated elevated elevated decreased reduced

ALDI f elevated ALD2A elevated elevated normal elevated ALD3A elevated elevated normal elevated

PZSl elevated elevated elevated normal PZS2 elevated elevated normal elevated normal normal

RCDPl elevated elevated# elevated normal decreased reduceds ! RCDP2A normal normal normal ' normal decreased normal normal

PD1 P elevated elevated normal elevated * normal PDl f elevated PD2 elevated elevated elevated normal decreased normal decreased PD3 elevated elevated elevated decreased normal ^ ! chorionic villus direct and chorionic villus cultured cells; chorionic villus direct and chorionic villus cultured cells, and cultured amniotic cells; P findings in the propositus; # elevated at 4 months but normal at 2 years; * abnormal in autopsy liver;s alkyl DHAP synthase also reduced; - not measured 8: Tíssues examined, of fixation and resin and the source of the for the material in this PATIENÏ TISSUES FIXATION RESIN SOURCE OF SPECIMEN zs1 liver Kar nov ;4o/o PF + O.25 o/oc Spurrs; Low LT; LRW Royal Childrens Hospital, Vic. zs2f liver, kidney,l u ng,skin Karnovi4o/o PF +O.25%G Spurrs; Low LT Royal Womens Hospital, Vic. zs3a liver 2.5o/oG;2Yo PF +O.5%G Spurrs; Low LT; LRW Royal Hobart Hospital, Tas. zs4a liver,spleen, brai n,adrenal 2.5o/oG Spurrs; Low LT; LRW Princess Margaret Hospital, WA zs5a liver EM Epoxy Wellington Hospital, NZ zs6f liver,kidney Karnov Spurrs Flinders Medical Centre, SA zs7 liver EM Epoxy Oueensland Medical Laboratory zsSa liver EM Spurrs; LRW Perth, WA zsga liver,adrenal, kidney, brai n Karnov Spurrs; Low LT The Children's Hospital, NSW

NALDl b1 liver,skin Karnov Spurrs Adelaide Childrens Hospital, SA NALDl b2 liver Kar nov', 4o/oPF + O.25 o/oG Spurrs; Low LT Adelaide Childrens Hospital, SA A o/oG SA NALDl liver, kidney,adrenal, brai n Kar nov i 4 YoPF + O . 25 Spurrs; Low LT Adelaide Childrens Hospital,

o/oP o/o SA IRDl liver Kar nov | 4 F + O . 25 G Spurrs; Low LT; LRW Adelaide Childrens Hospital, tRD2b1 liver,ski n,appendix,fat Karnov Spurrs Adelaide Childrens Hospital, SA tRD2b2 liver Karnov Spurrs Adelaide Childrens Hospital, SA o/oG SA tRD2b3 liver Kar nov i 4o/oPF + O.25 Spurrs; Low RT Adelaide Childrens Hospital, tRD2b4 liver Karnov; o/oPF +O.25%G Spurrs; LRW Adelaide Childrens Hospital, SA

ALDIf liver,adrenal, brain, kid ney Glutaraldehyde? Spurrs; Low LT Christchurch Womens Hospital, NZ ALD2A liver,adrenal,brain,skin EM fixative? Spurrs Prince of Wales Childrens, NSW ALD3A liver,adrenal EM fixative? Spurrs Newcastle Mater, NSW

PZSl liver 1o/oG Spurrs; LRW Royal Childrens Hospital, Vic PZSl A liver,adrenal 2.5o/oG Spurrs Royal Childrens Hospital, Vic PZS2 liver 2.5o/oG Spurrs Royal Childrens Hospital, Vic

RCDPl liver Karnovi4o/oPF + 0.1 %G Spurrs; LRW Royal Alexandria Hospital, NSW RCDP2A liver 2.5%G Spurrs Royal Childrens Hospital, Vic.

PDl f liver,adrenal Formalin? Spurrs; Low LT; LRW John Hunter Hospital, NSW PD2 liver Karnovi4o/oPF +O.25%G Spurrs; Low LT; LRW Royal Alexandria Hospital, NSW PD3b1 liver Karnovi4o/oPF+0.5%G Spurrs; Low LT: LRW Prince of Wales Childrens, NSW PD3b2 liver EM fixative? Spurrs Prince of Wales Childrens, NSW

Karnov, Karnovsky's fixative (see 4.1); PF,paraformaldehyde; G, glutaraldehyde (see 4'3.a) Low LT, Lowicryl resin at low temperature (see 4.3.b); LRW, London resin white (see 4.3.b) TABLE 9: Ir.ssøes examined, type of fixation and resin used, and the source of the specimen for the control patíent material in this s PATIENT TISSUES FIXATION RESIN SOURCE OF SPECIMEN

c1f liver,kidney Karnov Spurrs Adelaide Childrens Hospital, SA c2f liver,adrenal, brain, kid ney Karnov Spurrs; Low LT Oueen Victoria Hospital, SA c3 liver Karnov Spurrs Adelaide Childrens Hospital, SA c4 liver Karnov; 4o/oPF + O.25 %G Spurrs; Low LT Adelaide Childrens Hospital, SA C5a liver, kid ney,adrenal,brai n Karnov; 4o/oPF +O.25o/oG Spurrs; LRW Adelaide Childrens Hospital, SA c6 liver Karnov Spurrs Adelaide Childrens Hospital, SA c7 liver Karnov Spurrs Adelaide Childrens Hospital, SA CB liver Karnov Spurrs Adelaide Childrens Hospital, SA c9 liver,skin, marrow Karnov; 2o/oPF + 0.5 %G Spurrs; LRW Murdoch lnstitute, Vic c10 liver Karnov Spurrs Adelaide Childrens Hospital, SA c11 liver Karnov Spurrs Adelaide Childrens Hospital, SA c12 liver Karnov Spurrs Adelaide Childrens Hospital, SA c13 liver Karnov Spurrs Adelaide Childrens Hospital, SA c14 liver Karnov Spurrs Adelaide Childrens Hospital, SA c15 liver Karnov Spurrs Adelaide Childrens Hospital, SA c16 liver Karnov Spurrs Adelaide Childrens Hospital, SA c17 liver Karnov Spurrs Adelaide Childrens Hospital, SA c18 liver Karnov Spurrs; LRW Adelaide Childrens Hospital, SA c19 liver Karnov; Ao/oPF +O.25o/oG Spurrs; Low RT Adelaide Childrens Hospital, SA

Karnov, Karnovsky's fixative (see 4.1); PF,paraformaldehyde; G, glutaraldehyde (see 4.3.a) Low LT, Lowicryl resin at low temperature (see +.3.b); LRW, London resin white (see 4.3.b)

(,(¡ Chapter 3 Materials

3.2 CONTROL LIVER BIOPSIES

Control liver biopsies were obtained from 3 pediatric patients without a peroxisomal disorder who were undergoing liver bÌopsy for other investigations, and one autopsy liver and one fetal liver. Liver biopsies which had been processed for routine electron microscopic studies were examined in a retrospective study of peroxisome morphology. Sixteen controls were chosen from biopsies which were classified as normal by histological criteria. The patients ranged in age from 3 months to 18 years and were undergoing investigations for various conditions not related to peroxisomal disorders. Autopsy liver from a 4 month old boy where the post-mortem interval was approximately 8-9 hours was also examined and autopsy liver from a fetus aged 12 weeks was included.

3.3 ANIMAL TISSUES

Rat liver, kidney and brain were taken from rats of varying ages which were being sacrificed for an on-going study involving the isolation and purification of rat liver peroxisomes. The

tissues were removed as quickly as possible after death, sliced into small pieces and fixed

immediately by immersion fixation, The animal tissues were used in this study primarily to

establish the correct fixation, embedding and immunolabelling procedures for the localisation

of each antigen under investigation (see Appendix C). 3.4 ANTISERA

Polyclonal antibodies to catalase, TOkDa bif unctional protein and 68kDa peroxisomal

membrane protein (PMP68) were raised in rabbits against mouse liver catalase or isolated

peroxisomal membranes and kindly donated by Dr. D. l. Crane, School of Science, Griffith

University, Oueensland.

Catalase was purified to homogeneity from mouse liver using the procedure of Price and

coworkers (1962), and antiserum was raised against this protein in rabbits. Affinity

purification of catalase antibody was accomplished using the method described by Lazarow

and de Duve (1973).

Peroxisomal membrane proteins with subunit molecular masses of 68 kDa and 70 kDa were

isolated from peroxisomes purified from mouse liver (Crane et al. 1985). Antisera against

these proteins have been previously characterised and shown to be monospecific for these

54 Chapter 3 Materials

membrane proteins (Chen et al. 1988). The 68 kDa protein has been shown to be completely integral to the peroxisomal membrane (Chen et al. 1988); the 70 kDa protein has been identified as the bifunctional protein of peroxisomes (Crane et al. 1989).

Antisera to rat liver peroxisomal 3-ketoacyl CoA thiolase and acyl-CoA oxidase were generous gifts from Professor T. Hashimoto, Shinsu University, Japan.

3.5 COLLOIDAL GOLD PROBES

Colloidal gold probes were produced by first preparing 12nm gold particles by the boiling citrate method (De Mey and Moeremans 1986). 247.5m1 of clean water was placed in a

SOOml Erlenmeyer flask, and heated to boiling point on a hotplate with a magnetic stirrer.

15ml of freshly prepared, O.2pm millipore filtered, 1% sodium citrate was added and thq mixture was reflux boiled for 5min. The stirring speed was then increased and 2.5m1 of 1o/o gold chloride was rapidly added. This mixture was reflux boiled for 15min and the colour gradually changed from black to purple to an orange-red. The gold sol was cooled to room temperature and millipore filtered using a O.2pm disposable filter. The size of the gold particles was assessed by placing a drop on a celloidon coated grid and examining them in an electron microscope.

The gold sols were then conjugated to Staphylococcal protein A (Pharmacia) which was prepared by dissolving 2mg of protein in 1ml of clean water and dialysing overnight against distilled water to remove salts. This stock solution was stored frozen at -2OoC. The amount of protein required to stab¡lise the gold sol was determined by carrying out a stabilisation test at a pH slightly basic to the isoelectric point of the protein to be conjugated. For protein A this was pH 5.9-6.0. The pH of the gold sol was first adjusted to this pH by the addition of O.1M potassium carbonate. A series of 6 tubes each containing

25Opl of gold sol was set up and increasing amounts of protein A was added from O-6 pl.

After 5 minutes 25Opl of 1Oo/o sodium chloride was added to each tube and the colour change observed. The tubes containing an insufficient amount of protein A to stabilise

25O¡tl of gold sol turned blue and the tubes containing enough protein A remained red. The amount of protein A required to stabilise 3Oml of gold sol was then extrapolated and this amount plus a 2Oo/o excess was rapidly added to the gold sol. After vigorous stirring for

55 Chapter 3 Materials

Smin, 1O% bovine serum albumin (Sigma grade V) was added to a final concentration of

1 o/o.

The resultant gold conjugates were purified by ultra-centrifugation at 60,OOOg (12nm gold) for 45min. The loose pellets were resuspended in phosphate buffered saline (PBS) containing 1% bovine serum albumin (PBS/BSA) and the ultracentrifugation repeated. The final pellets were resuspended in approximately 1ml of PBSiBSA and this solution was combined from all the tubes. This conjugate was serially diluted and the optical density was read in a spectrophotometer at a wavelength of 52Onm. The dilution giving an optical density of 0.14 was used as the working dilution in the incubation of sections. The gold conjugates were stored at 4oC with O.O5% sodium azide. A new batch of gold conjugate was prepared every six months.

The sensitivity of the gold probe was assessed by immunoblotting on nitrocellulose

(Moeremans et al. 1984) using a serial dilution of mouse liver catalase and the catalase antibody, followed by the gold conjugate; or by the "golden-blot" method of Brada and Roth

(1984) (see Appendix C for details).

56 CHAPTER 4

METHODS Chapter 4 Methods

4.1 ELECTRON MICROSCOPY

Liver was obtained at needle biopsy or at autopsy and fixed immediately by immersion in

Karnovsky's fixative consisting of 4o/o formaldehyde and 1% glutaraldehyde in O.1M sodium cacodylate buffer at pH 7.3. After fixation for 2 hours at room temperature, the tissue was

washed twice in 0.1M sodium cacodylate buffer, postfixed in 1o/o osmium tetroxide, washed

in cacodylate buffer again and stained en bloc with 2o/o aqueous uranyl acetate. The tissue

was then dehydrated in a graded series of ethanol and embedded in Spurr's low-viscosity

epoxy resin. Polymerisation was carried out at 7Oo C under vacuum for at least 12 hours.

Silver-gold thin sections were cut on a Reichert Ultracut microtome, stained with uranyl

acetate for 3 min and lead citrate for 3 min, and examined in an Hitachi TOOO electron

mrcroscope.

4.2 CYTOCHEMISTRY

The cytochemical localisation of catalase was carried out as soon as possible after fixation in

Karnovsky's fixative (as described above in 4.1) in order to prevent the d¡ffusion of catalase

out of the peroxisomes (Fahimi 1973). Small pieces of the tissue were incubated in a

medium containing 3',3 diaminobenzidine (DAB) and hydrogen peroxide at pH 9.7 for th at

37oC. Negative controls were performed by adding the catalase inhibitor 3-amino-1 ,2,4-

triazole (AT) to the control incubation medium, and positive controls were carried out by

incubating rat liver in the same incubation medium. The tissue was then washed in sodium

cacodylate buffer, osmicated, dehydrated, embedded, sectioned, stained and examined

similarly as described above.

The DAB medium was made up as follows:

Test medium: l Omg DAB

4.65m1 O.O5M AMPD* buffer pH 9.7

O.1ml 2.5o/o H2O2

Control medium: 10 mg DAB

2.5m1 O.O5M AMPD* buffer pH 9.7

2.25m10.O2M AT

* 2- amino-2-methyl- 1, 3-propaned iol

57 Chapter 4 Methods

4.3 IMMUNOCYTOCHEMISTRY 4.3.a Fixation

Tissue for immunocytochemical investigations was fixed in a mixture of 4o/o freshly prepared paraformaldehyde andO.25o/o glutaraldehyde in 0.1M sodium phosphate buffer at pH 7.4 for

2 hours at room temperature. For transport or storage the tissue was retained in 2% paraformaldehyde in O.1M sodium phosphate buffer at 4oC. 4.3.b Resin embedding

After fixation the tissue was rinsed in PBS containing 50mM ammonium chloride in order to quench free aldehyde groups (Roth et al. 1989), dehydrated in ethanol and embedded in

Lowicryl K1 1 M resin using a modification of the progressive lowering of temperature method described by Carleman (1982). The processing schedule is described in Table 10.

TABLE 1O: Processing schedule for Lowicryl K|1M resin

REAGENT TEMPERATURE PLACE TIME

30% ethanol 40C ln fridge 3Omin

50% ethanol ooc ln freezer 3Omin

70% ethanol -200c lce:Na CL 30min

90% ethanol -250C ln cryostat 3Omin

lOOo/o ethanol -250C ln cryostat 3Omin

lOOo/o ethanol -250C ln cryostat 60min

1 :1 resin:ethanol -250C ln cryostat 6Omin

2:1 resin:ethanol -250C ln cryostat 6Omin

1OO% resin -250C ln cryostat 6Omin

1OO% resin -250C ln cryostat overnight

The samples were then embedded in gelatin capsules using fresh pre-cooled resin. The

capsules were filled to the top to minimize air space over the resin. Polymerisation was

carried out by ultra violet irradiation at -25oC for several days.

Some liver tissue was embedded in LRWhite resin as shown in the table below ladapted

58 Chapter 4 Methods

from Newman et al. (1983)l

TA 11 Processing schedule for LRWhite resin

DEHYDRATE: 70% ethanol 15min

90% ethanol 15min

lOOo/o ethanol 15min

1OO% ethanol 15min

1 :1 resin:ethanol 3Omin

INFILTRATE: 100% resin 6Omin

100% resin 6Omin

1OO% resin overnight

The samples were then embedded in fresh resin in gelatin capsules and polymerised overnight at sOoC.

Silver-gold sections of both Lowicryl-embedded tissues and LRWhite-embedded tissues were cut on a Reichert Ultracut ultramicrotome and mounted on celloidon coated nickel grids. 4.3.c Ultrathin cryosections

After fixation the tissue was rinsed in PBS containing 50mM glycine, and infused with a graded series of sucrose in PBS to a final concentration of 2.3M. 0.8% paraformaldehyde was added to the 2.3M sucrose to prevent reversal of fixation (Tokuyasu 1986). After sucrose infusion for 30 minutes to 2 hours the sample was mounted on a stub, frozen in liquid nitrogen, and cryo-sectioned at -80 to -1OOOC using a Reichert Ultracut ultramicrotome equipped with an FC4D cryo attachment. Ultrathin frozen sections were retrieved in a sucrose loop according to Tokuyasu (1980) , and mounted on celloidon and carbon coated nickel grids. The grids containing the cryo-sections were stored face down on gelatiniagarose plates. 4.3.d lmmunolabelling for electron microscopY lmmunolabelling was carried out using a two-step indirect procedure as described in Table

12.

59 Chapter 4 Methods

schedule for electron

STEP SOLUTION DILUTED TOTAL TIME NO. DROPS

1 PBS/5OmM glycine 15 min 3

2 D&WB 20 min 3

3 Antibody ln D&WB 16 hr at 4oC 1

4 D&WB 30 min 6

5 Protein A gold D&WB to O.D.52g 60 min 1 conjugate of O.14

6 D&WB 30 min 6

7 Clean water 30 sec 3 XSOml

8 Dry the grids 30 min

9 Uranyl acetate 2olo aqueous 30 sec 1

10 Clean water 3O sec 3 XSOml

11 Lead citrate 15 sec 1

12 Clean water 30 sec 3 XSOml

The diluent and wash buffer (D&WB) consisted of PBS at pH 7.3 containing 0.5%

ovalbumin,0.1% gelatin, O.O5% Tween 20 and O.2o/oTeleostean gelatin. All solutions were

millipore filtered (O.22pml before use and the primary antibody and the colloidal gold probes

were spun at 15,OOOg in an Eppendorf bench centrifuge for Smin immediately before use in

order to remove any aggregates. The optimal dilution for each antibody was determined

empirically by a dilution series and was the dilution giving maximum labelling with minimal

background. The protein A-gold conjugate was diluted in D&WB to give an O.D.529 of 0.14

(Roth et al. 1989).

60 Chapter 4 Methods

Negative controls were performed on adjacent sections by omitting the primary antibody and by incubating in normal rabbit serum (NRS) instead of the primary antibody. Positive controls were carried out simultaneously on sections of either normal rat liver or control human liver.

For immunolabelling of ultrathin cryosections, the gelatin/agarose plates containing the sections were melted at 37oC for approximately 3Omin. They were then labelled according to steps 1 to 7 in Table 12 except that the incubation time in the antibodies (step 3) was reduced to 6Omin. After step 7 the grids were stained in uranyl oxalic acid at pH7.4

(Tokuyasu 1986) for lOmin; and embedded in 2Vo methyl cellulose on lce (Griffiths 1984). ln some experiments the sections were embedded in 2o/o aqueous polyvinyl alcohol (Mwt.

1 O,OOO) with O.1 % uranyl acetate (Tokuyasu 1 989). 4.3.e Auantitation of immunogold labelling

Twenty electron micrographs from sections of liver which had been similarly treated were randomly selected. Where possible, sections to be quantitated were incubated at the same time and using the same batch of colloidal gold probe. Five micrographs of each of the control sections were randomly selected to determine the background density. The labelling density (gold particles per ¡rm2) was calculated by measuring the area of the peroxisomes and counting the number of gold part¡cles over each peroxisome. The background density calculated from the controls was automatically subtracted. A Hewlett-Packard 9847A. digitizer linked to a Model 9816 Microcomputer was used for these evaluations. The results were compared using a Students's f test.

4.4 MORPHOMETRY 4.4.a Measurements of peroxisomal size

Twenty electron micrographs of each section of liver were randomly selected at a magnification of xlO,OOO and enlarged photographically to x22,OOO. A carbon grating replica with 2,160 lines per millimeter (Probing and Structure) was photographed and enlarged at the same magnification as each batch of micrographs. The magnification factor was calculated by measuring the length of 5lines on the print of the carbon grating.

6I Chapter 4 Methods

Twenty such measurements were taken f rom each print and the calculated mean magnification factor was used.

The area, perimeter and diameter of the peroxisomes, and the area of the hepatocyte cytoplasm was measured by manual tracing on a digitiser linked to a computer using a Video

Trace software package (Leading Edge Technology, Adelaide, Australia)' ln patients where there was a greatly reduced number of peroxisomes, additional micrographs which contained

peroxisomes were taken at x2O,OOO and enlarged to x5O,OOO and the area and diameter of the peroxisomes was calculated. 4.4.b Measurements of peroxisomal number

The volume density, numerical density, and surface density were calculated using the

methods of Weibel (1979). The volume density Vu is a measure of the total volume of the

peroxisomes as related to the total hepatocyte volume (including the hepatocyte cytoplasm

and nucleus). This was calculated by dividing the sum of the area of all the peroxisomal profiles by the sum of the area of the hepatocyte cytoplasm and nucleus. This was

expressed as the relative volume density (%).

The numerical density Nu is the number of peroxisomes per unit cell volume and was

calculated using the formula

Nrr:1'(N¡)3/2'kv ß (Vu)"'

where k is the size distribution coefficient and ß is the shape related coeff icient as

determined according to Weibel (1979). N4 is the ratio between the number of peroxisomes

and the total area of the hepatocytes'

The surface density (Su) is the total membrane area of the peroxisomes expressed per

cellular volume and was calculated from the formula

S.,=48¡v- tf

where 84 is the ratio between the sum of the peroxisome perimeters and the total area of

the hepatocytes.

The results were not corrected for section thickness, since the sections used were all of

6/ Chapter 4 Methods

similar thickness according to the interference colours on the ultramicrotome during sectioning

63 CHAPTER 5

MORPHOM RY OF HEPATIC PE OXISOMES Chapter 5 Morphometry of hepatic peroxisomes

5.1 CONTROLS

Peroxisomes were readily identified in pediatric liver biopsies as single membrane bounded organelles with a homogeneous moderately electron-dense matrix (Fig. 1a). Electron dense or crystalline cores were not normally present. Section profiles of the peroxisomes were

roughly circular and they ranged in diameter from O.11 to 1 .11 pm. A frequency histogram

of the diameter of peroxisomes in 16 control pediatric liver biopsies is shown in Fig. 2. The

volume density of peroxisomes in the 16 pediatric controls ranged from 0.7 to 3.2 % with a

mean of 1.67o/o. The numerical density of peroxisomes ranged from O.O57 to 0.188 with a

mean of O.125, and the surface density of peroxisomes ranged from O.O73 to O.262 with a

mean of O.1 61 .

Regression analysis showed that there was no correlation between the age of the patient

o/ol and the mean area of peroxisomes (R2 =O.1 .

ln the fetal specimen the peroxisomes could easily be recognised and were slightly smaller in

mean area than in biopsy livers (Fig. 1b and Table 13). ln the autopsy liver the peroxisomes

were larger than in the biopsy and fetal livers and had a flocculent, extracted appearance

(Fig. 1c) . Profiles of smooth endoplasmic reticulum were often flattened and condensed

along the peroxisomal membrane in autopsy tissue (Fig. 1c) .

The mean area of peroxisomes, and the mean diameter (d-circle), the volume, numerical and

surface densities in 18 control liver specimens are shown in Table 1 3.

64 TABLE 13: Morphometry of peroxisomes in control pediatric liver

tÊ Case Age n Mean area# SD Area range Mean d-circle o/o Yy Nv sv

c1 3 mths 67 o.22 o.09 o.o7-0.52 0.56 1.7 o.128 0.165 c2 4 mths 77 o.21 o.12 o.o5-0.64 0.56 2.O o.152 0.1 96 c3 6 mths 93 o.29 o.17 o.oo8-0.77 0.63 3.2 0.1 54 o.262 c4 7 mths 54 o.17 o.08 o.oo2-0.37 0.51 1.1 o.122 o.125 c5 9 mths 31 0.1 9 o.08 o.o8-0.40 0.53 o.7 o.064 o.073 c6 9 mths 58 0.1 I o.o8 o.03-0.34 0.53 1.1 o.112 o.122 C7 21 mths 80 o.21 o.14 0.001-0.56 0.55 2.1 0.163 0.1 94 c8 30 mths 27 o.22 o.09 o.006-0.36 0.59 0.8 0.057 0.076 c9 42 mths 54 o.21 0.11 o.oo3-0.44 0.56 1.3 0.100 o.126 c10 5 yrs 56 o.23 o.09 o.o6-0.44 0,58 1.4 0.098 0.1 35 c11 I yrs 80 o.24 o.12 o.oo8-o.51 o.58 2.3 o.1 48 o.212 c12 9 yrs 't04 o.22 o.09 o.06-0.47 o.58 2.6 0.1 88 o.252 c13 11yrs 86 o.21 o.08 o.oo6-o.46 0.53 1.9 0.1 60 0.1 96 c14 13 yrs 58 o.24 0.1 5 o.oo4-0.96 o.62 1.6 o.1 03 o.1 49 c15 13 yrs 55 o.20 o.09 o.o7-0.46 0.54 1.4 o.117 0.1 38 c16 18 yis 71 o.20 o.09 o.o2-0.43 o.54 1.5 o.1 33 o.1 58

MEAN 60 o.22 o.oo1-o.96 o.56 1.67 o.125 0.1 61

c17f 12 wks 51 0.11 o o4 o.o3-o.23 o.42 o.63 o.128 o.088 c18 a 4 mths 39 o.37 o 14 o.14-O.77 0.75 1.6 o.o56 o.126

f fetal a autopsy n number of peroxisomal profiles measured { r"un area'in pm2 mean diameter (d-circle) in pm

(¡o) FIG. 1 Peroxisomes (P) in control liver a control pediatric liver biopsy X40,00O b control fetal liver X40,O00 c control autopsy liver X40,00O -J.'¡*rlh. ql

. it1t.

l. all ù"t \, tr t

\aar ..f

¿J

'î'ìöii ..,.a tt ,,1 rr "¿ ì

1: t: ,, tt

t o. .l"\ l.' t

a

j

,:¿ ,fr t í, *

á FIG. 2 Frequency histogram of peroxisomal diameter (¡'rm) in control pediatric liver biopsies. Frequency Histogram CONTROL I 200

F 150 r o q

U 100 B n c v 50

o 3g ,05 93 1,2 Poroxisomal diametsr um F1g2 Chapter 5 Morphometry of hepatic peroxisomes

5.2 orscusstoN

The diameter of peroxisomes in the control liver biopsies in this study are similar to those which already appear in the literature (Table 3; see 2.5.d). ln particular there appears to be little difference in the mean diameter of peroxisomes in liver from adult patients and the liver of pediatric patients. Roels et al. (1988) found peroxisomes with a mean diameter slightly less than the adults in two infants aged 5 weeks and 4 months. De Craemer et al. (1991a,

1991b) also reported slightly smaller peroxisomes in one of two infant biopsies. This patient

(and one of Roels et al. [1988]) was only 5 weeks old. There were no patients of this age in my control group.

The relative Vy, Nu, and Su reported in this study are somewhat higher than the data reported in the literature. This may be because the control tissues are from infants and children, compared with adult liver which has been reported in most studies. I have examined more control patients than others reported in the literature and this is another factor which may account for the increased values. lt is also $ossible that some of the control patients in this study had been treated w!!h drugs, or suffered other conditions which may have caused an increase in the number of peroxisomes. All the patients in the control group had a liver biopsy which was classified as normal by histological criteria.

Unfortunately it is not possible to obtain liver biopsies from genuinely "normal" infants.

However it is important to establish a range of morphometric values from these pediatric patients with which to compare liver biopsies from peroxisomal disorder patients.

The identification of peroxisomes in this study was based on purely morphological features.

A proportion of the smaller peroxisomes may be missed by using morphology alone to identify peroxisomes (Beier and Fahimi 1 986), rather than tissue which has been cytochemically stained for catalase to identify peroxisomes (Novikoff and Goldfischer 1969;

Fahimi 1975; Roels et al. 1975). However it would not be possible to use retrospective liver biopsies if this was the case, as all the control liver biopsies had been processed using standard electron microscopic techniques, without cytochemical staining. Also in biopsies from patients w¡th peroxisomal disorders abnormal peroxisomes need to be identified. Often

66 Chapter 5 Morphometry of hepatic peroxisomes

these peroxisomes have low levels of catalase (Roels et al. 1986; Hughes et al. 1992) and the enzyme cytochemical technique may not detect them. Beier and Fahimi (1992) have recently reported the use of immunocytochemical techniques in conjunction with automatic image analysis for identifying peroxisomes in rat liver. This technique is also unsuitable for human liver biopsies as special fixation and processing is required. The figures obtained in this study where peroxisome identification relied on morphology only are actually greater than most of those previously reported. lt therefore seems likely that the use of retrospective t¡ssue which has been routinely processed for electron microscopy has ¡gr had 1 a significant effect on the values of Vy, Nu and Su.

The mean peroxisomal diameter in fetal liver was smaller than that found in pediatric liver.

The volume, numerical and surface densities of peroxisomes in the fetal specimen in this study were at the lower end of the range for biopsy liver. ln human fetal kidney, the mean size of peroxisomes was less than that of adults, and there was little change during the l Oth to 18th weeks of gestation (Briére 1986). This suggests that a significant increase in the diameter of peroxisomes must take place after the 18th week of gestation or after birth to reach the level of the adult. Similarly in human fetal liver an increase in peroxisomal size takes place during development (Kerckaert, 1990, as quoted in Espeel et al. 1990a and

Roels 1991). Espeel et al. 1990a found an increase in the size of hepatic peroxisomes between the seventh and eighteenth gestational weeks. The number of peroxisomes has been reported to be higher in younger fetuses (8-10 weeks), and then falls abruptly from 12 weeks onwards (Kerckaert, 199O, as quoted in Roels 1991). ln rat liver the volume of individual hepatocytes increases with age and variations occur in different organelles (Schmucker 1990). The data concerning peroxisomes is very limited and somewhat inconsistent, however it appears that ageing has little effect on this organelle in rat liver. However, during fetal and postnatal development some changes do occur. ln mouse hepatocytes the volume densíty of peroxisomes increases 2-3 fold between birth and

10 days of age (Kanamura et al. 1990), and in rat hepatocytes the size and number of peroxisomes increases gradually during fetal development (Stefanini 1985). Also in the liver

61 Chapter 5 Morphometry of hepatic peroxisomes

of baby pigs the number of peroxisomes increases 2 fold from 1 to 4 weeks of age

(Giesecke et al. 1987). Our data show there is no correlation between the size or number of hepatic peroxisomes and the age of the patient. However, since our youngest pat¡ent was 3 months old, it is possible that changes may occur from birth to 3 months.

The mean diameter of the peroxisomes in the autopsy tissue was considerably enlarged compared to biopsy liver. This difference is most likely due to post-mortem swelling of the peroxisomes. The peroxisomes have a flocculent empty looking matrix as though swelling and extraction of the contents has taken place. Caution must be exercised in using autopsy tissue for morphometric analysis of hepatic peroxisomes.

5.3 PEROXISOMAL DISORDER PATIENTS 5.3.a Generalised peroxisomal disorders ln four of the Zellweger syndrome patients peroxisomes could not be identified at all. ln the other generalised peroxisomal disorders including five of the Zellweger syndrome patients and the NALD patient small, morphologically abnormal structures were identified in biopsy, autopsy and fetal liver (Fig.3). These organelles were surrounded by a single membrane and contained electron dense centres which often filled the entire organelle except for a clear area just inside the membrane. The area and diameter of these structures was greatly reduced compared to normal peroxisomes, as was their volume, numerical and surface densities (see Table 14 and Fig. 1Oa and 1Ob). ln the two patients to whom a clinical diagnosis of IRD had been assigned, small, abnormal organelles with a similar ultrastructural appearance were seen (Fig.4a). These structures were also reduced in size from normal peroxisomes, however their numerical densities were similar to those of normal peroxisomes in control patients. The volume and surface densities were reduced. ln the lRD2 patient, a few organelles which approximated the size of normal peroxisomes were also present in some cells (Fig.4b) . These peroxisomes also had an electron dense centre along with some finely granular matrix of normal appearance. ln this patient the number of peroxisomes per cell was very heterogeneous with some hepatocytes containing near normal numbers and other having none.

68 FIG. 3 Peroxisomes (P) in the liver of patients with generalised peroxisomal disorders are small compared with those from control liver, and contain electron dense centres. a control pediatric liver X30,000 b a liver biopsy from a Zellweger patient X3O,000 c autopsy liver from a Zellweger patient X3O,000 d liver from a 12 week old fetus affected with Zellweger syndrome x30,ooo e a liver biopsy from a NALD patient X3O,0O0 f autopsy liver from a NALD patient X30,000 I ; at' zs t;. '|l .+ Ç' ìtf*

Þr t ü .'.1 ¡" *üi

¡

3b .:1

ZSt¡ izs ;ft \ú. af ú ,Ìl f s P j1 *

,t t $ t $a ,ù¿ ;ar 3c 3d -1t

a t t I i * l.' o NAtDcr NAt.,i'l - Þ 'àìüt ç ¡: Ç^ + t 4l- ! Þ a t' t i t:i + .:; I l a ?. ..1 P a ¡ pà

r.t dÐ

¡ .ra r't 1 j i' t a , - *.1 "i (t , -*1. . ae _a -.'âû# 'a 3f FIG. 4 Peroxisomes (arrows) in a liver biopsy from an infantile Refsum's patient (lRD2). The peroxisomes vary in size but contain electron dense centres.

a small morphologically abnormal peroxisomes which are similar in size and appearance to those seen in patients with Zellweger syndrome or NALD X42,00O b larger organelles which are closer in size to normal peroxisomes but which still contain an electron dense centre X42,OOO 'ir rl

...1 å' lÇ.

,A d" I t t' , ì ¡..

4 *-r Chapter 5 Morphometry of hepatic peroxisomes

5.3.b Sex-linked adrenoleukodystrophy

The 17week-old fetal liver (ALD1f ), from a fetus affected with ALD, contained morphologically normal peroxisomes with a mean area of O.18+O.06 which is comparable to the mean area found in an age-matched control (O.19+O.06) (FiS. 5a). The values for the volume, numerical and surface densities were within the range of values for control pediatric biopsy liver.

The other two ALD specimens were autopsy liver (Fig. 5b), and the mean areas of peroxisomes in these two samples (O.33+O.12 and O.53+O.26) were higher than control liver biopsies. They were comparable however to the mean area of peroxisomes in one control autopsy liver (0.37+0.14) . The other morphometric values in these two samples were slightly less than the lower end of the control biopsy range. 5.3.c Pseudo-Zellweger syndrome ln the PZS patients (siblings), peroxisomes which were enlarged in size but had a normal volume and surface density were identified (Table 14 and Fig. 6a and 6b). The numerical density in one case (PZS1) was slightly reduced. These peroxisomes had a finely granular homogeneous matrix similar to that observed in control human peroxisomes. The size and shape of the peroxisomes was extremely variable (see Fig. 1Oc) . ln the autopsy material from the first patient (PZS1a) enlarged peroxisomes with a normal volume density were identified. However, the numerical and surface densities were somewhat reduced (Table 14). 5.3.d Rhizomelic chondrodysplasia punctata ln the liver biopsy from patient RCDPl extremely enlarged peroxisomes were present in the cytoplasm of the hepatocytes (Fig.7). These organelles, which were generally round or oval, were up to five times the size of normal peroxisomes (Fig 1Od and Table 14) , The peroxisomes were bounded by a single membrane and the matrix was flocculent with a very heterogeneous electron density resulting in a prominent mosaic pattern. Membranous material was often found enclosed within the peroxisome (Fig.7c) . These membranous inclusions were found in approximately 3O% of peroxisomes and only in those organelles

69 FIG. 5 a a cluster of peroxisomes from a 17 week old fetus affected with ALD. These peroxisomes are normal in size, number and appearance X50,00O b peroxisomes (arrows) in autopsy liver from a patient with ALD, showing some post-mortem change X50,000 ai -È-'- Ur ?l ,Y

.¿ebù

s+.i r Ft ¡ "s

r.. .}. 'Þr' tt* o il .t

(i,-

'¡11 I

!:

t rb FIG. 6 Peroxisomes (P) in pseudo-Zellweger syndrome are enlarged in size and irregular in shape a PZSI X50,000 b PZS2 X50,000 ( \ FIG. 7

Electron micrographs of human liver showing peroxisomes (P) and mitochondria (M).

a normal peroxisomes from a control patient X3O,OOO b liver biopsy from patient RCDPl showing an extremely enlarged peroxisome which is much bigger than the mitochondria X25,2OO c liver biopsy from patient RCDPl showing an extremely enlarged and flocculent peroxisome (P) with membranous material enclosed within the matrix X25,2OO d autopsy liver from patient RCDP2 showing enlarged peroxisomes x30,oo0 ^¡tq Chapter 5 Morphometry of hepatic peroxisomes

which were grossly enlarged. The peroxisomes were frequently partially surrounded by cisternae of smooth endoplasmic reticulum. The numerical and surface densities were reduced and the occurrence of these peroxisomes was very heterogeneous from hepatocyte to hepatocyte, with many cells containing none, and others one or several. Due to the large size of the peroxisomes, the volume density was almost within normal range despite the low numbers. The peroxisomes were heterogeneous in regard to size, ranging from O. 14 ¡tm2 to

5.2O pm2 in area. Normal peroxisomes were not observed in this specimen.

The peroxisomes in the post-mortem liver in patient RCDP2 were also enlarged, although not as severely as RCDPI (Fig.7d and Table 14). Some of the mitochondria showed a slight swelling, which may be due to post-mortem change, however, the rest of the tissue was well preserved. As in RCDPl the peroxisomal matrix was flocculent and non-homogeneous, and cisternae of smooth endoplasmic reticulum often partially surround the peroxisomes

(Fig. 7d). ln this case the enlarged peroxisomes were present in larger numbers (numerical density 0.049), and the volume and surface densities were within normal range.

Membranous inclusions were not observed in this case. 5.3.e Unclassified peroxisomal disorders

The unclassified peroxisomal disorder patient PD1 had peroxisomes which were normal in size, volume, numerical and surface densities. The peroxisomes in this patient did vary from normal in that they had electron dense centres (Fig. 8a). ln the biopsy from the PD2 patient, small peroxisomes with electron dense centres were seen. These peroxisomes resembled those seen in the generalised peroxisomal disorders

(Fig. 8b and 8c). The size of the peroxisomes was greatly reduced in this patient and their volume and surface densities were reduced. However in this case, as in the IRD patients, the numerical density was within normal limits (see Table 14 and Fig. 8c). ln the first biopsy of the third PD patient (PD3b1) similar small peroxisomes with electron dense centres were observed (Fig. 9a) These peroxisomes were reduced in size, and volume and surface density, however the numerical density was normal. This is similar to the picture in the IRD patients. ln the second biopsy from this patient, taken 2 years later,

10 Chapter 5 Morphometry of hepatic peroxisomes

enlarged peroxisomes were observed (Fig.9b and Table 14). These peroxisomes did not have electron dense centres but consisted of a homogeneous matrix bounded by a singte membrane. The numerical and surface densities were normal and the volume density was at the upper end of the control range.

7a FIG. 8 Peroxisomes (P) in two of the unclassified peroxisomal disorder patients a ln patient PD1 normal sized peroxisomes are present but have electron dense cores X50,000 b ln patient PD2 small peroxisomes are present which have electron dense centres X50,000 c These morphologically abnormal peroxisomes have a normal numerical density X9,625 .l \.-.r 1.

lr 4 .,1'

.: ì1, l,,l t.\ FIG. 9 Liver from two biopsies from patient PD3 taken two years apart. M : mitochondria a ln the first biopsy from this patient small, morphologically abnormal

peroxisomes (arrows) with electron dense centres were present in normal numbers X25,0O0 b ln the second biopsy taken two years later, enlarged peroxisomes (P) were present X25,O00 > o i.; a Ç,iì*", 'a tz þ .V ' ¡l ^ . .r .,¿ ?t; 'l 'rl. \ j

*¡i. *r_- rj J)l .a-a t

,1r

,i

ì

I FtG. 10 Frequency histograms of peroxisomal diameter (/¡m) in control pediatric liver and various disorders a control pediatric liver b Zellweger syndrome patients c pseudo Zellweger syndrome patients d RCDP patients FiglO

Frequency Histogram CONTROL Frequency Histogram zeuweoeR I 50 50

F F 37.5 s7.5 I r I I q q U U e e 25 n n c v Y 12.5 % 12.5 %

0 o 1.2 9 Peroxsomal diameter um o b" Peroxisohal diamerer um

Frcqucncy Histogram PSELIDozELLweceR I 50 Frcquency Histogram ncop I 50

F 37.5 F r ( s7.5 e B q q U U e e n n c v v t2.5 % 12.5 %

0 o 't.2 C Peroxisomal

PATTENT Ace AREA DlauEreR Vv NV sv

Controlf 17 wks 0.19 a 0.06 o.49 0.38 o.o35 0.o40 Controla 4 mths 0.37 a 0.14 0.69 1.6 o.o56 o.126 * Control - 0.22 + O.11 0.56 1.67 o.125 0.161 Range (3mth-18yr) (0.17-0.29) (0.51-O.63) lo.7-3.2t (0.057-0.188) (0.o73-0.262)

zs1 2 wks O.O3 r 0.008 o.20 o.o1 0,014 0.003 zs2f 12 wks o.o2 + 0.008 0.18 o.o2 o.021 0.004 ZS3A 8 mths no perox no perox no perox no perox no perox zs4a 3 mths no perox no perox no perox no perox no perox ZS5A 2 mths no perox no perox no perox no perox no perox ZS6f 12 wks O.O2 + 0.007 0.1 8 0.04 o.o47 o.o10 zs7 3 mths 0.06 + O.O2 o.28 o.o1 o.o21 o.oo3 ZSSA no perox no perox no perox no perox no perox ZSgA 3 wks O,O5 + 0,05 o.25 o.27 o.025 o.006

NALDIbl 1 Y( o,03 + 0.009 0 1 6 o.03 o.057 o.oo9 S + NALDl Ab2 2 Y( O.O3 O.o2 0 20 o.o3 o.051 0.o12 NALDl 3 Yt S O,O3 r O.O3 0 20 o.oo6 0.o09 o.oo4

IRDl 5 mths o.o3 + o 008 0,20 o.o5 0.065 o.o'13 tRD2b1 18 mths O.O3 + O o1 0.20 0.04 0.002 o.oo9 tRD2b2 3yr o.o2 + o 006 o.16 o.o1 o.031 o.oo4 tRD2b3 5yr o.o3 + o 005 o.20 o.o5 0.o30 o.014 tRD2b4 6 yrs O.O4 + O o3 o.26 o.13 o.122 o.029 f ALDI 17 wks 0.18 + 0.06 0.48 1.1 0.111 o.122 ALD2A 6yr 0.33 + 0.12 0.65 0.6 o.025 0.o55 ALD3A 7vr o.53 + O.26 o.82 o.7 o.o14 o.045

PZSl 7 mths o.43 + O.27 o.74 1.3 o.o35 0.089 PZS2 2 mths o.3o + o.14 o.62 2.1 o.097 o.1 85 PZSlA 8 mths 1.OO + O.7O 1.13 1.3 0.010 o.o64

RCDPl 8 mths O.77 + O.43 1.11 0,66 o.oo6 0.033 RCDP2 1 3 days 0.61 + O.28 0.88 3.1 0.049 0.1 84

PD1 f 17 wks o.17 + O.12 o.47 0.8 0.086 0.086 PD2 1 1 mths o.o3 + o.o1 o.20 o.09 0.065 o.020 PD3b1 2 yrs 0.06 + o.03 o.28 o.2 0.078 o.028 PD3b2 4 yrs 0.43 + O.23 o.74 3.3 o.089 o.221 I autopsy tissue ' fetal tissue b biopsy perox peroxisome mean of 16 control pediatric liver biopsies (see section 5.1 and Table 13)

72 Chapter 5 Morphometry of hepatic peroxisomes

5.4 DISCUSSION

Three morphological types of peroxisomes have been identified in the peroxisomal disorders included in this study, and these correlate well with the current classification of peroxisomal disorders. These are the very small, morphologically abnormal peroxisomes seen in varying numbers in the generalised disorders of peroxisomal dysfunction (or Group 1 disorders); normal sized or enlarged peroxisomes with a normal appearance which occur in Group 2 disorders, or disorders where there is a defect in a single peroxisomal enzyme; and giant peroxisomes with a flocculent matrix, seen in RCDP patients (Group 3 disorders) where more than one peroxisomal enzyme pathway is affected. 5.4.a Small, abnormal perox¡somes

The small peroxisomes with electron dense centres are associated w¡th the generalised peroxisomal disorders i.e. Zellweger syndrome, NALD and lRD, They have been previously reported in the liver of NALD and IRD patients (Beard et al. 1 986; Goldfischer et al. 1985;

Hughes et al. 1990; Roels et al. 1986; Vamecq et al. 1986). We have found these abnormal peroxisomes also in five of our Zellweger syndrome patients, including one 12 week old fetus which was diagnosed as Zellweger syndrome by biochemical analysis of chorionic villus biopsy. lt should be noted, however, that these peroxisomes are not found in all Zellweger syndrome cases. The difference between an absence of peroxisomes, as observed in some Zellweger syndrome patients, and peroxisomes with a Vu of only O.O1 or

O.O2o/o is not great, and it may be that in those cases described as having no peroxisomes the peroxisomes are so small and scarce that they are not detected by normal morphometric and ultrastructural means.

The Vu of peroxisomes in these generalised peroxisomal disorders is also greatly reduced.

The Nu, however is within normal limits for both the IRD patients, but due to the small size of the peroxisomes, the Vu is still reduced. This suggests that in these cases at least the liver is still able to assemble small peroxisomes in roughly the right number, but these peroxisomes are structurally abnormal and unable to carry out all the biochemical functions.

Five different cellular phenotypes based on morphology of the peroxisomes have been identified in the generalised peroxisomal disorders investigated in this study. These are

73 Chapter 5 Morphometry of hepatic peroxisomes

outlined in Table 15. ln the patients which are the most severely affected clinically, i.e.

Zellweger syndrome, the size, structure and number of peroxisomes vary the most from normal. And in the patients which are the least severe clinically i.e. lRD, particularly lRD2 who is still alive at age 11 years, there are some peroxisomes with an almost normal size and appearance, and the peroxisomes have a normal numerical density.

74 Chapter 5 Morphometry of hepatic peroxisomes

TABLE 15: Cellular phenotypes seer? in generalised disorders of peroxisomal dysfunction

Normal numbers of normal síze peroxísomes with electron dense centres (PD|)

Heterogeneous populations of large and small peroxisomes, some with electron dense

centres and normal numerical densíty (lRD2, PD?)

Small peroxisomes with electron dense centres and normal or near normal numerical

density (lRDl, PD2, NALD)

Small peroxisomes with electron dense centres and greatly reduced numerical density

(Zellweger syndrome)

No peroxisomes detected (Zellweger syndrome)

5.4.b Enlarged peroxisomes

The two sibling PZS cases had enlarged peroxisomes with a normal appearing matrix. The peroxisomal Vu of these cases was normal, however, the Nu was slightly reduced. These cases have been described previously as having abundant peroxisomes (Hughes et al. 199O), based on subjective observation without morphometric analysis. lt is probable that since the peroxisomes are enlarged and the Vv is normal, the actual number of peroxisomes was overestimated. lt has been suggested that an increase in the diameter of peroxisomes leads to decreased surface arealvolume ratio and decreased metabolic capacity (Roels et al. 1988;

Roels and Cornelis 1989) (see Section 6.3 for further discussion). 5.4.c Giant, flocculent peroxisomes

A third morphological type of peroxisomes was identified in the RCDP patients. These peroxisomes were extremely enlarged with a decreased Nu. However the Vv was almost within normal limits due to the large size of small numbers of peroxisomes. The Su of the peroxisomes in this case was reduced indicating a reduction in metabolic capacity (see De

Craemer et al. 1991b; Hughes et al. 1992).

15 Chapter 5 Morphometry of hepatic peroxisomes

The ultrastructural appearance of the peroxisomes in the 2 cases of RCDP varies considerably from the previously reported appearance of peroxisomes in other peroxisomal disorders. The group of genetic diseases known as the peroxisomal disorders can be divided into three groups depending upon the extent of the loss of peroxisomal function (Moser,

1989; Schutgens et al. 1989; Wanders 1990). ln the Group lor generalised disorders (i.e.

Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum's disease), there is a generalised loss of peroxisomal function, and an absence or marked reduction of peroxisomes. The basic defect in the Group I disorders is thought to be at the level of biogenesis or maintenance of peroxisomes, or in the import of peroxisomal proteins from their site of synthesis in free polyribosomes into the peroxisomes. ln the Group ll peroxisomal disorders there is a loss of a single peroxisomal function due to a single enzyme defect and where ultrastructural studies of the liver have been carried out on these patients, peroxisomes were described as either normal or somewhat enlarged and abundant. RCDP is not usually included in the Group I disorders, since very long chain fatty acids, bile acids and pipecolic acids are metabolized normally, and peroxisome structure is not deficient according to catalase sedimentation studies (Wanders et al. 1988d). Nor is it included in the Group ll disorders, since more than one enzyme is deficient. Based on peroxisomal morphology my results support the inclusion of RCDP into a group separate from Group I and ll disorders,

However RCDP should also be described as a disorder of peroxisomal assembly since the structure of the peroxisomes appears to be defective. The extremely large flocculent peroxisomes, displaying heterogeneity in number per hepatocyte, which were observed in

RCDP1, are consistent with the description given by Heymans et al. (1986) in one case of

RCDP. However, since there were no published micrographs of this case and immunocytochemistry was not performed, it is difficult to know the extent of the similarity. ln RCDP2, the peroxisomes are also enlarged and flocculent, although not to the same degree as RCDP1. lt may be argued that some of the enlargement in RCDP2 may be due to post-mortem change. However, since the liver tissue was collected within 2 hours of death and other cellular components, such as mitochondria and glycogen, are well preserved, this is unlikely. Peroxisomes can be readily identified in autopsy material if the post-mortem

'76 Chapter 5 Morphometry of hepatic peroxisomes

interval is minimized. De Craemer et al. (1990b) have shown that peroxisomes can be evaluated in human autopsy liver up to 24 hours after death, and in liver obtained 8.3 hours post-mortem there was very little post-mortem alteration.

Enlarged peroxisomes of similar appearance, size, Nu, Su and Vu as those described here were found in one case of RCDP by De Craemer et al. (1991b). These peroxisomes also had a lower amount of catalase as determined by enzyme cytochemical staining for catalase (see

Chapter 6.2.d).

Espeel et al. (1992) has also described an RCDP patient with extremely enlarged, flocculent organelles which labelled only weakly for catalase, but had normal immunocytochemical labelling for ß-oxidation enzymes.

5.5 CONCLUSTON

The measurement of morphometric values of peroxisomes provides a more accurate description of the types of peroxisomes in peroxisomal disorders. However, qualitative as well as quantitative descriptions are required in order to avoid misinterpretation. This is evidenced by the occurrence of electron dense cores in some peroxisomes which are of normal size and present in normal numbers. We have identified three types of peroxisomes, based primarily on size and ultrastructural appearance, but there is further heterogeneity within each group on the basis of Vv, Nv and Su. These three morphological types of peroxisomes correlate well with the three groups of peroxisomal disorders based upon clinical and biochemical features.

The morphometric analysis of peroxisomes in the liver of patients with a suspected peroxisomal disorder can confirm the clinical diagnosis and in some cases point to atypical features which may be indicative of an unsuspected condition or a new variant of a known disorder.

1'7 CHAPTER 6

ENZYME CYTOCHEMISTRY Chapter 6 Enzyme cytochemistry

6.1 CONTROLS

After incubation of control liver tissue in the DAB medium the peroxisomes became markedly electron dense and were more easily identified (Fig. 1 1a).

6.2 PEROXISOMAL DISORDER PATIENTS 6.2.a Generalised peroxisomal disorders

The small, abnormal peroxisomes observed in some of the Zellweger syndrome patients and in the NALD patient, did not stain cytochemically for catalase. ln routinely processed tissue these peroxisomes had electron-dense centres, but the density was not enhanced by incubation in the DAB medium. The IRD patients, however, were heterogeneous with regard to cytochemical staining. ln the first patient, lRD1, the small abnormal peroxisomes observed did not show any increase in electron density after incubation in the DAB medium.

However in the second patient lRD2, some larger peroxisomes were observed which did show a reaction to DAB (Fig. 11b). The "normal" appearing matrix of these organelles stained for catalase, and the electron dense centre remained the same. 6.2.b Sex-linked Adrenoleukodystrophy

Cytochemical staining for catalase was only carried out on the fetal ALD specimen (ALD1f). ln this specimen the peroxisomes reacted with the DAB medium and were stained with an electron-dense deposit (Fig. 1 1c). 6.2.c PseudoZellweger Syndrome

The enlarged peroxisomes observed in the PZSl patient showed only a very weak reaction for catalase by cytochemical staining (Fig. 11d). Cytochemistry was not carried out on the liver biopsy of the sibling of this patient (PZS2\. 6.2.d Rhizomelic chondrodysplasia punctata lncubation of the liver tissue in both RCDP cases did not reveal any normal catalase-positive peroxisomes. The electron density of the enlarged, f locculent peroxisomes was not increased in DAB incubated tissue as compared to that observed in routinely processed tissue.

78 FtG. 11

Liver tissue after cytochemical staining with DAB

a Control pediatric liver biopsy. The peroxisomes are stained to an intense black density X32,500 b A peroxisome in a liver biospy from an IRD patient. There is some staining of the matrix, and the electron dense core remains x32,500 c Peroxisomes in a 17 week old fetus affected with ALD react with DAB in a manner similar to control tissue X32,500 d Enlarged peroxisomes in a PZS patient show very little reaction with DAB X32,500 ,Í .lr

.lþ ¡_

.4 '.'1. sb .û; '. û

!;rù.

I t I )

'/t.1

fJ ,'{ --+ i l' llc a Chapter 6 Enzyme cytochemistry

6.2.e Unclassified Peroxisomal Dísorders

PD1: This patient had normal numbers of normal sized peroxisomes which had electron dense centres. The peroxisomes were stained cytochemically for catalase.

PD2: This patient had normal numbers of very small abnormal peroxisomes with electron dense centres. These peroxisomes did not show any increase in their electron density after incubation in the DAB medium.

PD3: ln the first biopsy from this patient, small abnormal peroxisomes were identified.

These peroxisomes were stained cytochemically for catalase (see Fig.2b and c in Hughes et al. 199O) . ln the second biopsy from this patient, where enlarged peroxisomes were present cytochemical staining was not carried out.

6.3 DISCUSSION

A summary of the cytochemical results is shown in Table 16.

The incubation of liver tissue in DAB medium to increase the electron density of peroxisomes was useful in cases where normal peroxisomes were present. However, in most of the peroxisomal disorder patients in this study the peroxisomes were abnormal in some way.

Most of the very small peroxisomes with electron dense centres, which were observed in many of tn" g"n"r"tised disorders, did not show any reaction with DAB indicating that active catalase was not present in these organelles.

Even in the disorders with enlarged or giant, flocculent peroxisomes, any reaction with the

DAB medium was only very weak, suggesting that these peroxisomes contain only small amounts of active catalase. One explanation of the low-catalase nature of these peroxisomes is that the structure of the peroxisomes is affected, such that most of the catalase has leaked out. Alternatively, the mechanism for the import of catalase into the peroxisomes may be impaired. Another explanation for the low levels of catalase may be associated with the large size of these organelles. Roels and Cornelius (1989) found heterogeneity of catalase staining in human liver peroxisomes that was related to size, with larger peroxisomes having less catalase activity. They speculate that the diminution of peroxisomal membranes associated with large organelles (i.e. lower surface arealvolume

'79 Chapter 6 Enzyme cytochemistry

ratio) will lead to decreased metabolic capacity, assuming that receptor-mediated import is occurring. Roels et al. (1988) have also described the occurrence of enlarged peroxisomes in several patients with childhood adrenoleukodystrophy and other peroxisomal disorders.

They noted that the occurrence of enlarged peroxisomes, with reduced catalase staining, is associated with the clinically more severe syndromes, suggesting that increased size is associated with lowered metabolic capacity.

80 Chapter 6 Enzyme cytochemistry

TABL Results of incubation of liver in DAB medium PATIENT PEROXISOME TYPE DAB TEST

c1f normal Px stained c2f normal Px stained c4 normal Px stained c5a post-mortem change Px not stained c19 normal Px stained

zs1 small, abnormal Px not stained zs2Í small, abnormal Px not stained zs3a none detected zs4a none detected zsSa none detected not done zs6f small, abnormal Px not stained t,t:'. ' | : zs7 small, abnormal not done zsga none detected zsga small, abnormal Px not stained

NALDl b1 small, abnormal Px not stained NALDl b2 small, abnormal Px not stained NALDl A small, abnormal Px not stained

IRDl small, abnormal Px not stained tRD2b1 small, abnormal not done tRD2b2 small, abnormal Px stained tRD2b3 small, abnormal Px not stained tRD2b4 small, abnormal Px stained

ALDIf normal Px stained ALD2A normal not done ALD3A normal not done

PZSl enlarged Px weakly stained PZSl A enlarged Px not stained PZS2 enlarged not done

RCDPl giant, flocculent Px not stained RCDP2A giant, flocculent Px not stained

PD1 f abnormal Px stained PD2 small, abnormal Px not stained PD3b1 small, abnormal Px stained;heterog PD3b2 enlarged not done

B1 CH ER7

IMMUN ocYToc HEMISTRY OF HEPATI C PEROXI MES Chapter 7 lmmunocytochemístry of hepatic peroxisomes

7.1 CONTROLS ln control liver the labelling for catalase was evenly distributed over the peroxisomal matrix with a mean labelling density of 204.42 gold particles per ¡r^2 çig. 12a and Table 17).

There was very little background labelling as evidenced by the scarcity of gold particles over any other organelles, or the ex-tracellular matrix. ln the fetal control tissue the labelling for catalase was much less than this (62.68 gold particle, p"l. ¡rt2¡. ln control liver the label for all three ß-oxidation enzymes was also distributed over the peroxisomal matrix with a mean labelling density of 89.69 gold particlesl¡rmz for acyl-CoA oxidase, gg.22 gold particles//m2 fo, the bifunctional protein, and 134.54 gold particles//m2 for thiolase (Table 17). As with catalase, the labelling density for the ß- oxidation enzymes was much less in the control fetal tissue (Table 17).

The label for PMP68 in control liver was found over the membrane of the peroxisomes with an average number of 2.11 gold particles per ¡lm of peroxisomal membrane (Fig. 12b and

Table 1 7).

One of the control livers was autopsy tissue and the labelling density for all proteins was similar to the values in biopsy tissue even though the peroxisomes were larger than normal due to post mortem change (see Table 17). 7.2 PEROXISOMAL DISORDER PATIENTS 7.2.a Generalised peroxisomal disorders

ln all of the generalised peroxisomal disorder patients in this study the labelling for catalase

was considerably reduced or absent (see Table 17). ln the Zellweger syndrome and NALD

labelling for catalase over the small, morphologically abnor."' patients, there was no , , peroxisomes. However in the IRD patients some labelling was present although greatly

reduced in density.

The labelling density for acyl-CoA oxidase was not detectable for Zellweger syndrome,

NALD and lRDl patients, but normal for the lRD2 patient. A similar result was observed for

the labelling of bifunctional protein, although a very small amount of labelling for this

enzyme was also present in the lRDl patient. The labelling density for thiolase was zero for

the Zellweger syndrome and NALD patients. ln the IRD patients a very small amount of label

82 FtG. 12 lmmunoelectron microscopy of control pediatric liver

a Liver after incubation with catalase antibody and protein-A-gold showing labelling

for catalase over the matrix of peroxisomes (Px). M = mitochondria X8O,OOO

b Liver after incubation with PMP68 antibody and protein-A-gold showing labelling

for the membrane protein located specifically in the peroxisomal membrane

x80,ooo .riï' li

CONTROL Chapter 7 Immunocytochemistry of hepatic peroxisomes

was present in one patient (lRD1) but in the other patient (lRD2) the labelling density for thiolase was increased approximately two fold (Table 17). ln this case the labelling for

thiolase was concentrated in the thin "halo" just inside the membrane of the small

peroxisomes (Fig. 13a). Labelling was not present over the electron dense centres of these

organelles. ln this patient some peroxisomes were almost normal in size, but still had an

electron dense centre. ln these larger peroxisomes there was little label over the electron

dense centre even though there was labelling of the matrix (Fig. 13b). This phenomenon

was also observed in the PD1 patient (see 7.2.e1 , where peroxisomes of normal size and

number had electron dense centres which did not label with any of the antibodies used' The

rest of the matrix in these peroxisomes had a normal appearance and did label for the matrix

proteins.

ln the Zellweger syndrome and NALD cases, there was no labelling for PMP68 over the

small, abnormal peroxisomes. However in lRD, a very small amount (0.31 particles/¡rm) was

observed in the first patient (lRD1), and the second patient (lRD2) had normal labelling. A

summary of the morphometric and immunocytochemical results for each peroxisomal

disorder group is given in Table 18.

7.2.b Sex-linked adrenoleukodystrophy

lmmunocytochemistry was only carried out on the 17-week old fetal ALD liver (Fig. 1a)'

Labelling for catalase, acyl-CoA oxidase and thiolase was not significantly different from the

control fetal liver of the same age (C2fl. Labelling for bifunctional protein and PMP68 was

greater than in the age-matched control.

7.2.c PseudoZellweger sYndrome

ln the pZSl patient, the labelling density of catalase was significantly decreased compared

with control liver (Fig. 15 and Table 17). The labelling densities for acyl-CoA oxidase,

bifunctional protein and PMP68 were normal, however, the labelling density for thiolase was

significantly decreased.

83 FtG. 13 lmmunolabelling of thiolase in patient lRD2 a Small morphologically abnormal peroxisomes in this IRD patient label for thiolase over the matrix but excluding an electron dense centre. The labelling density of thiolase is increased and is concentrated in the outer rim of matrix just below the membrane

x94,OOO b ln the larger peroxisomes in the same patient the labelling for thiolase is restricted to the peroxisomal matrix and the electron dense core is relatively free of label X94,O00 ÍHl

t.t-r t

Ê

Q ..

l3rl i,ct IRD2

a r ù

I

ì

Ç rf t3b FtG. 14 lmmunolabelling of the ß-oxidation enzymes in 17 week old fetal ALD liver. Labelling for all three enzymes is restricted to the peroxisomes. a acyl CoA oxidase X84,000 b bifunctional protein X84,000 c thiolase. X84,O00 a:+ --

{..,r t .... ì-:-.:i FtG. 15 lmmunolabelling of the enlarged peroxisomes in the liver of a pseudo Zellweger patient (PZS1) a labelling for catalase is reduced X62,0OO b labelling for acyl-CoA oxidase is normal X62,000 c labelling for thiolase is reduced X62'000 q I

THI

¿ô px

'?s-t t't, 'Í l5c :ß .rffi, PZSI Chapter 7 lmmunocytochemistry of hepatic peroxisomes

7.2.d Rhizomelic chondrodysplasia punctata

The giant, flocculent peroxisomes in patient RCDPl showed only a small amount of immunocytochemical label for catalase (Fig. 16a and Table 17) when compared with control liver. The labelling density for the ß-oxidation enzymes, acyl CoA oxidase (Fig. 16b), bifunctional protein (F¡g. 16c), and 3-ketoacyl thiolase (Fig. 16d) was not significantly different from the controls (see Table 17). The label for all three ß-oxidation enzymes was distributed over the peroxisomal matrix. ln some peroxisomes an eccentric electron dense core was observed, and this did not label for any of the antibodies used (Fig. 16d)'

The PMP68 integral membrane protein was localised predominantly over the peroxisomal membranes (Fig. 17a) . Small membranous loops were sometimes observed adjacent to the large peroxisomes (Fig. 17a) , and occasional double membrane loops were also seen (Fig.

17b). These membranes also labelled for the PMP68 membrane protein.

7.2.e Unclassified peroxisomal disorders

PD1 ln the PD1 patient the labelling density of catalase was increased compared to the age- matched control fetal tissue. Similarly the labelling densities for the three ß-oxidation enzymes, and PMP68 were increased compared with the age-matched control. The electron dense centres observed in the normal sized peroxisomes in this patient did not label for any

of the antibodies tested (Fig. 18a,b,c).

PD2

The small, morphologically abnormal peroxisomes with electron dense centres which were

observed in patient PD2 did not react immunocytochemically for catalase, acyl-CoA oxidase,

bifunctional protein, or thiolase. However, these organelles did label with the PMP68

membrane protein despite a complete absence of labelling for any of the matrix proteins (Fig.

19a and Table 17).

84 FtG. 16

lmmunolabelling of the liver in an RCDP patient a labelling for catalase is very sparse X4O,OOO b labelling for acyl-CoA oxidase is normal X40.0O0 c labelling for bifunctional protein is normal X4O,000 d labelling for thiolase is normal X40,000 tFr

-r d't

ród FrG. 17 lmmunoelectron microscopy of liver from patient RCDPl after incubation with the antibody to the 68 kDa integral membrane protein, and protein-A-gold

a Large peroxisome showing labelling over the membrane. Adjacent small

membranous loops also label for the PMP68 membrane protein (arrows)

x74,100

b A double membrane loop with both the inner and outer loops labelling for PMP68

membrane protein. X74,1OO Ì È RCDPI

,l* f;

l, ¡l ..ó,1 t7b FtG. 18 lmmunoelectron microscopy of unclassified patient PD1 a labelling for bifunctional protein is mainly over the matrix of the peroxisomes and the electron dense core is relatively free of label x75,OO0 b labelling for thiolase is also restricted to the matrix of the peroxisomes and excluding the electron dense core X75,000 c labelling for PMP68 is located in the peroxisomal membrane x75,OOO lt -! -.í1 . " _$:. )i!, t'¡. j ¡',-.'¡' -) ,:-...:.iü

;.t :. ..lt \.- -,

!- .¡ , tû\ b ú 'irr ß, û. -t- i¡ I ì' tr ? ..i çr I lfi ,/1 f+t- a

f .' !¡.

, ïr +å r -.1

¡ lr..¡- -¡ f:- ? ¿. l".r¡, . ¡ l. I ¡ t-'), t ,: r:. ¡ :Èb FtG. 19 lmmunolabelling of PMP68 in PD2 and PD3 patients a The membranes of the small, morphologically abnormal peroxisomes in patient PD2 label for PMP68 X48,000 b The membranes of the small, morphologically abnormal peroxisomes in patient PD3 label for PMP68 X48,000 c The membranes of the normal sized peroxisomes in patient PD3 also label for PMP68 X48,OO0 lt ,,' PMPó8 I'i

l9cr

PMPó8

#.

,?-

'i'' . ' PD3sm ft- if'.,

:;',,{Í,

PD3lg Chapter 7 lmmunocytochemistry of hepatic peroxisomes

PD3 ln the PD3 patient two populations of peroxisomes were identified in the tissue examined for immunocytochemistry. Small peroxisomes with electron dense centres were found which had a very reduced labelling density for catalase and no labelling for bifunctional protein.

These small peroxisomes had a normal labelling density for acyl-CoA oxidase and a greatly increased labelling density for thiolase. As in the lRD2 patient the labell¡ng for thiolase and acyl-CoA oxidase was concentrated in the narrow halo just inside the peroxisomal membrane

(Fig. 2Oa).

Larger peroxisomes with a normal ultrastructural appearance were also seen in this PD3 patient. This population of organelles had a reduced labelling density for catalase, acyl-CoA oxidase and thiolase but the labelling density for bifunctional protein was normal (see Table

17 and Fig. 2Ob). Both the large and small peroxisomes identified in this biopsy had a normal labelling density for PMP68 (Fig. 19b and 19c).

7.2.f lmmunoelectron microscopY on ultrathin cryosections ln liver f rom a control patient (C4) most of the catalase was localised within the peroxisomes. Some labelling was also apparent in the hepatocyte cytosol and a small amount in the nucleus and over the mitochondria and the lysosomes (Fig. 21 and Table 20) .

This.labelling was not observed in the negative control sections'

NALDl ln the liver from the patient NALD1, only small, abnormal and very rare peroxisomes could be detected by electron microscopy. However, using ultrathin cryosections and immunocytochemistry, labelling for catalase was observed in the cytoplasm of the hepatocytes. This label was free in the cytosol and not associated with any identifiable organelles (Fig.22al . A small amount of label, similar to the control liver was detected over the mitochondria, the nucleus and the lysosomes. The control sections were negative (Fig.

22b:l. The antibodies to the three ß-oxidation enzymes and the PMP68 antibody were not available when the immunolabelling of the ultrathin cryosections of this patients was carried out.

85 FIG. 20 lmmunolabelling for thiolase in a dual population of peroxisomes in patient

PD3 a ln the small peroxisomes the label is concentrated in the thin rim between the electron dense centre and the peroxisomal membrane x50,o00 b ln the larger peroxisomes observed in the same biopsy the label for thiolase is distributed over the peroxisomal matrix X50,000 THI

.i+

{

2Oc¡ PD3sm

I

,fl* j

2o/b FrG. 21

Ultrathin cryosection of control pediatric liver labelled with catalase and protein-A gold probe. Most of the label is concentrated in the peroxisomes (PER), although a small amount is also seen over the nuclear matrix (NUC), and free within the cytoplasm (arrows). The mitochondria (MlT) are relatively free of the catalase label. X50,000 ?t"¡t " F. J '¡S' ill' #j 9l-

tü t. 6 ê I I "¡,. € "{' ,Ì çr t ¡& '4 t. .. :Ë ¿: .$.: ',Lit{1 ¡. I à ,,1

lÞ -ir. F I l ,d .i ìi la '{

Þ \'¿-. í -È-l ^ . .i F ( ,.y ,{* ¿ a- ir''¡ c{ I TL FtG.22 from the NALD patient Ultrathin cryosections of a liver biopsy probe' catalase and protein-A gold a After immunolabelling with mostofthelabelisfreewithinthecytoplasm.Themitochondria X50'00O (M) are relatively free of catalase label'

b Controlultrathincryosectionlabelledwithnormalrabbitserumand protein-Agoldprobe'Themitochondria(M)andcytoplasmhave only a small amount of label X50'000 cAl ,tF v a

22t¡ ü F .P

I

üfr bz¡ å NALDT Chapter 7 lmmunocytochemistry of hepatic peroxisomes

IRD2 ln the liver of this patient (lRD2b4), the morphologically abnormal peroxisomes labelled for catalase. Labelling for catalase was also present free within the cytosol as in the NALDI patient (Fig. 23). The antibodies to the three ß-oxidation enzymes and the PMP68 antibody were not available at the time when the immunolabelling of the ultrathin cryosections was carried out.

ZSf and ZS4a

All of the antibodies used in this study were applied to ultrathin cryosections of these two patients. Peroxisomes were not identified and there was no evidence of the peroxisomal

"membrane ghosts" which have been described in cultured skin fibroblasts of Zellweger syndrome patients (see 7.3.e) . There was some labelling for catalase lying free within the cytosol.

ZS'l and lRDl

All of the ant¡bodies used in this study were applied to ultrathin cryosections of these two patients. Small, morphologically abnormal peroxisomes with electron dense centres were identified in these two patients by conventional electron microscopy. lmmunocytochemical

labelling was not observed over these organelles in the ultrathin cryosections. Some

labelling for catalase was seen free within the cytosol. There was no evidence of labelling

with the PMP68 antibody over the small abnormal peroxisomes nor any other structures.

TABLE 2O: Labelling density (number of gotd particles per pm2) of catalase in hepatocytes

CONTROL LIVER (C4) NALD LIVER (NALD1)

Peroxisomes 243.73 + 188.57 not measured

Mitochondria 1.95 + 5.24 4.18 + 5.61

Lysosomes 10.77 + 9.82 2.83 + 11.84

Nucleus 2.99 + 3.13 5,14 + 4.98

Cytosol 7.58 a 4.98 12.31 + 11.15

86 FIG. 23 Ultrathin cryosection of liver from patient lRD2 labelled with catalase and protein-A gold probe. Labelling is seen over the nuclear matrix (NUC), and free within the cytoplasm. The mitochondria (M) are relatively free of the catalase label. X50,00O -Gr

F v

_-,:. rll í¡H-' ù- 23 - ]R,D2 TABLE 17: Immunocytochemistry of pero4isomal proteins in control and patient líver (ACoAOX), protein (BFP)' and 3- Labetting density (number of gold particles per ¡tm' of peroxisome) for the matrix enzymes catalase, acyt-CoA oxidase bifunctional deviatíon ketoacyl-CoA (THIOLASE); and the integral membrane protein PMP68 (number of gotd partictes per pm of peroxisomal membrane) ¡ standard The mean area ) of the ís also shown PATIENT PX AREA CATALASE A-COAOX BFP THIOLASE PMP68

'f + O.70 + O.b4 c2 0.1 I + 0.06 62.68 + 39.60 46.23 l 23,89 25.99 + 10.99 82.72 58.55 + c4 o.21 r.o12 189.42 + 139.88 106.1 5 + 88.28 49.14 + 87.39 115.75 t 73.19 3.28 2.15 + x. 122.17 92.85 + 51.53 34.53 + 40.10 139.31 t 91.77 1.14 + O.81 c5a 0.37 O.14 180.00 + c18 0.20 a 0.09 220.7-I ! 128.43 72.46 + 49.73 30.74 + 30.05 1 19.36 t 84.39 1.67 1.O4 c19 o.20 + o.o9 222.22 + 148.68 94.43 + 76.29 46.21 + 35.59 141.59 + 94.86 2.33 r 1.68 * * + * + * 0.00 o.oo 0.00 o.oo * zs1 0.03* + 0.007 0.00 * * * * zs2f o.o2 + o.oo8 0.00 o.oo o.oo o.o0 0.00 not found zs3a Px not found Px not found Px not found Px not found Px not found Px Px not found zs4a Px not found Px not found Px not found Px not *found Px not* found * * * + o.oo zsga o.O5 + O.o5 0.00 0.oo o.oo o.oo * * * * * o oo o.oo o o0 o.00 NALDl b2 o o3 * ro ooI o oo * * * + t NALDl A o o3 +O o3 o oo o oo o.oo o oo 0.00 * * * * * + 0.31 + o.71 IRDl 0.03 +0 oo8 2.90 + 5.30 o.oo 3.24 + 10.31 2.71* 7.81 * * + 1.65 + 1.28 tRD2b3 o.03 +O oo5 31.52 + 5O.52 92.11 + 88.56 34.44 + 41.97 21O.g2# 181.43 * + 1.99 + 1,39 tRD2b4 o.04 ro o3 84.63 + 72.77 57.79 r 1 18.57 29.66 + 41.31 19o.o3# x. 127.3g # + 2.59 + 1.14 ALDl o.1 I + 0.06 87.83 + 64.57 51 .32 t 41 .11 83.23 + 46.50 84.51 47.41 * + 1.1O PZSl O.4g# + O.27 37 .42 + 45.1 6 84.03 + 38.53 14.73 + 13.54 43.73 I 51.99 1.19 * + 1.69 RCDPl O.77# + O.43 b.Bg + 6.66 41.42 + 30.98 21.26 r 18.51 106.25 + 107.61 1.68 2.56# + 1.65 PD1 f o.17 + O.12 143.69# r 60.64 e3.66Í + 44.77 114.13*# + 40.1 135.9o# t lg.ls o.o0 o.oo o.oo 3.76 + 2.83 PD2 0,03 + O.O1 o.00 + * + 62.48 o.oo 430.75,' + 805.44 2.78 x. 1.29 PD3small 0.06 + O.O3 2.93 * ! 7.22 52.62 * * + 31 .10 1.52 + 0.40 PD3large 0.19 + 0.06 72.27 t 62.77 43.93 + 23.37 27.tt + 12.73 48.27 *. significantly decreased at p

7.3 DISCUSSION 7.3.a Control liver

Of the four control liver biopsies in the immunocytochemical study, three were fixed in 4% paraformaldehyde and O.25o/o glutaraldehyde (C4, CSa and C19), and the other (C18) was fixed in Karnovsky's fixative as described in section 4.1 (see Table 9). The labelling density for C18 was not significantly different from the other three controls for any of the antibodies used. This agrees with the results found in the experiments on rat liver (Appendix C,

Experiment 1). Similarly, C4 and C19 were embedded in Lowicryl resin at low temperature,

and C5a and C18 were embedded in LRW resin (see Table 9). There was no significant

difference in labelling density for catalase, acyl-CoA oxidase and thiolase between the

controls embedded in these two different resins.

ln rat liver, the labelling density of bifunctional protein and PMP68 was enhanced by the use

of Lowicryl resin at low temperature as compared to LRW resin (see Appendix C, Experiment

21. This result is reflected in the labelling densities for these two antibodíes in the control

livers, where C5a and C18 (LRW resin) have a slightly lower labelling density for bifunctional

protein and PMP68 than C4 and C19 (Lowicryl resin). However, for the purpose of

comparing the patient material to the control material, the mean labelling densities of the

four controls was used.

ln rat liver, the labelling density of PMP68 was considerably lower in resin embedded

material than in ultrathin cryosections (Appendix C, Experiment 2 and Fig' 29 and 30). The

labelling efficiency of membrane proteins in resin-embedded tissues is usually only 1-10%

(Griffiths and Hoppeler 1986). This is probably due to the small amount of membrane area

exposed at the cut surface of the sections. ln the ultrathin cryosections some penetration of

the antibody or gold probes may occur. Alternatively, the enhancement of the labelling for

PMP68 in cryosections may be related to the avoidance of dehydration in organic solvents in

the preparation of the tissue as opposed to resin embedding. Although in the experiments

with rat liver, the labelling for PMP68 was greatly increased in ultrathin cryosections

compared to resin embedded material, the use of cryosections for human liver biopsies was

88 Chapter 7 lmmunocytochemistry of hepatic peroxisomes

found to be impractical on a routine basis. Some patient material was examined using this technique (see section 7.2.'fl , however, the use of resin embedded tissue allowed the labelling experiments to be repeated and allowed sections to be incubated in batches which were to be compared quantitatively' 7.3.b Small abnormal perox¡somes

The immunocytochemical results show there is no labelling for catalase or the ß-oxidation enzymes over any of the small morphologically abnormal peroxisomes observed in Zellweger syndrome or NALD. ln one IRD patient (lRD1)there is a very small amount of labelling, and

in the other (lRD2), there is normal labelling for acyl-CoA oxidase and bifunctional protein,

and an increased labelling density for thiolase. The peroxisomes in this case have a reduced

amount of label for catalase. The increased labelling for thiolase can be explained by the

concentration of this enzyme in the narrow "halo" of normal matrix just inside the

peroxisomal membrane. The electron dense centre observed in these peroxisomes does not

label for any of the matrix enzymes. This electron dense centre may represent a storage

product as a result of impaired ß-oxidation, or alternatively may represent the breakdown of

the internal structure of the peroxisomes. lt is also possible that this electron dense material

represents an aggregate of a protein or proteins which have been imported but are unable to

be properly localised within the organelle. ln NALD and Zellweger syndrome and some IRD

patients the small peroxisomes consist almost entirely of this abnormal electron dense

material, and there is no "normal" matrix to support the matrix enzymes. This is one

explanation for the severe deficiency of peroxisomal metabolic functions leading to the rap¡d

demise of these patients.

ln our lRD2 patient and the PD3 patient the labelling for tþiolase and acyl-CoA oxidase was

concentrated in a thin rim between the peroxisomal membrane and the electron dense

centre. This suggests that these peroxisomes are still able to import these enzymes but that

they are misplaced within the organelles. These patients both have a biochemical defect in

ß-oxidation, despite the presence of immunologically reactive ß-oxidation enzymes in these

abnormal peroxisomes. However, these two patients are still living at ages 11 years and 7

89 Chapter 7 Immunocytochemistry of hepatic peroxisomes

years respectively, whereas the other patients with generalised peroxisomal disorders all died at an early age. This suggests that the presence in the liver of even a small number of morphologically normal peroxisomes may be related to a better clinical outcome.

Of the small peroxisomes observed in the generalised peroxisomal disorders, .onl'i those in the IRD parients labelled for PMP68. ln one IRD patient (lRD1)the labelling for peroxisomal membrane protein was reduced, but in the other (lRD2) it was normal. This IRD patient was

an older patient who had some larger peroxisomes present in the liver, as well as the small

peroxisomes. These data are in conflict with that found by immunoblotting of autopsy liver

where three peroxisomal membrane proteins (22, 53 and 68 kDa) were present in normal

amounts in the liver of three Zellweger syndrome and one IRD patients (Small et al. 1988a)'

However, Wiemer et al. (1989) found the 69 kDa membrane protein was greatly reduced in

autopsy liver of one Zellweger patient but not in another. Aikawa et al. (1987) also found

the 22, 26 and 70 kDa integral membrane proteins were deficient in one Zellweger patient;

and Suzuki et al. (1987b) found deficient 70 kDa protein in four Zellweger syndrome

patients. However, Gartner et al, (1992) recently found normal amounts of normal sized

PMPTO mRNA in fibroblasts from eleven Zellweger probands, suggesting normal amounts of

PMPTO protein.

Labelling for PMP68 was found in the 3 PD patients in this study. ln the PD2 patient, the

labelling density for PMP68 was increased over the small, abnormal peroxisomes, despite a

lack of labelling for any of the matrix proteins. ln the PD3 patient both the large and small

populations of peroxisomes, labelled for PMP68. This confirms the peroxisomal nature of

these abnormal organelles.

At least six different types of peroxisomal abnormalities based on morphological and

immunocytochemical criteria, were detected in the patients with generalised disorders of

peroxisomal dysfunction. The synthesis of even abnormal peroxisomes appears to take

place in all patients, except perhaps a subset of Zellweger patients,

TABLE 18 Morphological/immunocytochemical types of peroxisomes

90 Chapter 7 lmmunocytochemistry of hepatic peroxisomes identified in disorders

Normal size and morphology peroxisomes, with moderately reduced labelling of matrix

proteins (PD3-largeJ

Normal size peroxisomes with electron dense centres and normal labelling of peroxisomal

matrix and membrane proteins (PDIJ

Smatt peroxisomes with electron dense centres and reduced labelling of some matrix

proteíns (i.e. catalase, lRD2; catalase and bifunctional protein, PDS small)

Smalt peroxisomes with electron dense centres and markedly reduced labellíng of all

matrix proteins, and reduced labelling of PMP68 (lRDl)

Smatl peroxísomes with electron dense centres with undetectable labelling for matrix

proteíns but normal tabetling for membrane proteins (PD2)

Smatl peroxísomes with electron dense centres with undetectable labellíng for matrix and

membrane proteins (Zellweger syndrome, NALD)

7.3.c Enlarged or normal sized peroxisomes

ln the fetal liver from patient PD1 peroxisomes of normal size were found, however the

labelling densities for all the antibodies were increased compared with the age-matched

control fetal liver. The peroxisomes in the PD1 pat¡ent did vary from normal in that most of them contained an electron dense centre, and this did not label for any of the antibodies.

Since the total area of the peroxisome was used to calculate the labelling density, the

presence of the electron dense centre could explain the increase in labelling density for the

matrix enzymes i.e. the same amount of label is present but is localised to a reduced area

thus leading to a higher labelling density.

ln the PZS patient where significantly enlarged peroxisomes were found, there was a

decreased amount of immunocytochemical labelling for catalase and thiolase. Roels and

Cornelius (1989) found heterogeneity of catalase staining in human liver peroxisomes that

91 Chapter 7 Immunocytochemistry of hepatic peroxisomes

was related to size, with larger peroxisomes having less catalase activity. They speculate that the diminution of peroxisomal membranes associated with large organelles (i.e. lower surface area/volume ratio) will lead to decreased metabolic capacity, assuming that receptor- mediated import is occurring.

The decrease in labelling for thiolase in this patient may indicate a defect in this enzyme. ln the pseudo-Zellweger patients of Goldfischer et al. (1986), a deficiency of thiolase was found by immunoblotting of liver (Schram et al. 1987a, 1987b)' 7.3.d Giant, flocculent peroxisomes

The immunoelectron-microscopy results confirm the peroxisomal nature of the enlarged, flocculent organelles observed in RCDP1. The PMP68 integral membrane protein is specific

and is localised only to the membrane of peroxisomes. ln some peroxisomes there is

evidence of extra membranous material (Fig. 17a and 17b) and this also reacts with the

PMP68 membrane antibody. Catalase negative peroxisomal membrane loops have been

described in rat liver after treatment with the hypocholesterolemic drug BM 1 5766 (Baumgart et al. 1989). This drug induces proliferation of peroxisomes many of which

display alterations in their membranes such as invaginations into the matrix, and loop-like

extensions. Double-membraned loops which react with a peroxisomal integral membrane

protein antibody, but are negative for catalase, were also observed. These membranous

structures are thought to be part of the peroxisomal membrane system, and have been

described in experimental conditions associated with peroxisomal proliferation (Baumgart et

al. 1989). My results show a similar type of membrane alteration i.e. membranous material within the peroxisome matrix, membrane loops or extensions adjacent to the large

peroxisomes, and double-membraned peroxisomal loops. lf these are part of the normal

peroxisomal membrane structure one can speculate that the¡r increase in this RCDP patient

may be an attempt to increase the transport of peroxisomal matrix proteins from the cytosol

into the peroxisomes. Alternatively, the membranous alterations may be a secondary effect

due to the impaired activlties of two enzymes (dihydroxyacetone phosphate acyltransferase

and alkyl-dihydroxyacetone phosphate synthase) which are involved in the biosynthesis of

92 Chapter 7 lmmunocytochemistry of hepatic peroxisomes

plasmalogens. The peroxisomal ß-oxidation enzymes are normally localised in these

organelles, and this supports the biochemical findings of normal ß-oxidation in this patient.

The biochemical manifestations in these 2 cases i.e. impaired plasmalogen synthesis and

elevated plasma phytanic acid, indicates that although organelles of a peroxisomal nature are

present, and contain immunoreactive ß-oxidation enzyme proteins, they are unable to carry

out all the necessary functions of normal peroxisomes. Given the enormous enlargement,

and flocculent ultrastructural appearance of these organelles, and the severe deficiency of

catalase, the most likely explanation is that the internal structure of the peroxisomes is

altered in such a way that some biochemical functions cannot be maintained' 7.3.e Peroxisomal "membrane ghosts"

'/. \ Large emptyl vesicles which react with antibodies to peroxisomal membrane protein have

been described in cultured skin fibroblasts from Zellweger syndrome and RCDP patients

(Balfe et al. 199O; Santos et al. 1988a, 1988b; Wiemer et al. 1989). They have a density

on Nycodenz gradients which is lower than that of normal peroxisomes. Their diameters are

generally 2-4 times greater than normal peroxisomes, and they are heterogeneous in size

(Santos et al. 1988b). Balfe et al. (1990) have demonstrated immunoreactivity to thiolase

by immunofluoresence in these peroxisomal membrane ghosts. Fibroblasts from these

patients contained the unprocessed 44 kDa form and this was reported to be associated

mainly w¡th the lower density fractions, although in RCDP cells some immunoreactive

protein was also detected in the peroxisomal fractions. This contrasts with the report of

Singh et al. (1991), which showed that most of the thiolase precursor was present in

organelles sedimenting with the same density as peroxisomes.

We did not find any evidence of these peroxisomal "membrane ghosts" in the liver biopsies

from our generalised peroxisomal disorder patients. Certainly some of the structurally

abnormal peroxisomes described in these patients displayed labelling for PMP68, with or

without concurrent labelling for peroxisomal matrix enzymes. However, there was no

evidence of the very large peroxisomal "membrane ghosts" structures described in cultured

93 Chapter 7 lmmunocytochemistry of hepatic peroxisomes

skin fibroblasts. Even in the several Zellweger syndrome and one IRD patients in which immunolabelling with ultrathin cryosections was carried out using the PMP68 antibody there was no indication of these aberrant organelles. Labelling with the PMP68 antibody was enhanced in rat liver sections when ultrathin cryosections where used in preference to

Lowicryl sections (see Appendix C, Expt. 2)'

It is possible that the large low-catalase peroxisomal organelles described in the liver of the

RCDpI patient may be the equivalent of the "membrane ghosts" reported in cultured

Zellweger and RCDP fibroblasts. However, even though the organelles observed here are

much larger than normal peroxisomes, it is not possible to predict where they would

sediment on a density gradient, especially since much of the matrix is empty-looking. My

results show that immunoreactive acyl-CoA oxidase and bifunctional protein, as well as

thiolase are also detected in these organelles. This is in contrast to the findings in RCDP

fibroblasts, where acyl-CoA oxidase and bifunctional protein were detected predominantly in

a normal peroxisomal fraction (Balfe et al. 1990) . This anomaly may be attributed to the

vastly different size, number and possibly function of peroxisomes from cultured skin

fibroblasts as compared to those occurring in the liver.

ln cultured skin fibroblasts, catalase activity is unaffected in RCDP (Hoefler et al. 1988; Balfe

et al. 1990; Heikoop et al. 1990; ), and yet my results indicate that in the liver of RCDP

patients catalase is not present at normal levels within peroxisomes. This suggests that the

results seen in cultured fibroblasts are not necessarily indicative of what is occurring in the

liver.- The cultured skin fibroblasts may be affected by culturing conditions. Also, in

cultured skin fibroblasts peroxisomes are extremely small, sparse organelles compared to the liver where peroxisomes are normally much larger and abundant. Alternatively the

differences between liver and fibroblast organelles could simply reflect the ultrastructural,

and therefore, presumably, metabolic differences between the organelles in individual

tissues. A difference in the presence of cytochemically recognisable peroxisomes between

cell types has been reported in infantile Refsum's disease (Beard et al. 1986), where

94 Chapter 7 lmmunocytochemistry of hepatic peroxisomes

peroxisomes were markedly affected in the liver of two patients, yet in cultured skin fibroblasts from the same patients normal peroxisomes were identified.

The peroxisomal "membrane ghosts" have recently been reported to co-label with lysosomal enzymes (Heikoop et al. 1992b), and they may represent the degradation of peroxisomes'

This may be a phenomenon peculiar to cultured skin fibroblasts. 7.3.f Immunoelectron microscopy of ultrathin cryosections

The immunoelectron microscopy results show that in human liver catalase is located not only within the peroxisomes but also free in the cytosol. The technique used here avoids the disruption to peroxisomes which may occur during subcellular fractionation procedures, thus preventing the leakage of catalase out of the peroxisomal matrix, or the redistribution of enzymes within the cell. The tissue has undergone only a light fixation, followed by cryo- protection and cryosectioning before the application of immunoreagents. lt therefore seems likely that the cytoplasmic localisation of catalase is not due to leakage from peroxisomes as may be the case in subcellular fractíonation studies. My results are in agreement with those of Hemsley et al. (1988), who showed a cystolic localisation of catalase in rat, mouse and guinea pig cultured hepatocytes using a digitonin permeabilization technique. A variation in the relative proportion of cytoplasmic/peroxisomal catalase between the three species was investigated by these authors. Rat and mouse hepatocytes have only a small percentage of cytoplasmic catalase (28 and 12o/o lespectively), compared with guinea pig hepatocytes

(51%1. This may explain why cytoplasmic catalase can be detected in guinea pig liver by

DAB cytochemistry (Geerts et al. 198O; Roels 1977|r, but not in rat liver (Roels 19771. The relative proportion of cystoliciorganelle-bound catalase in humans may well lie somewhere between the two species. Using the more sensitive techniques of immunocytochemistry, cytoplasmic catalase has only been previously reported in guinea pig liver (Yamamoto et al.

1g88) whereas in rat liver catalase is described as being localised exclusively in the peroxisomes. Hollingshead and Meijer (1988), have used similar techniques to mine (i'e' immunoelectron microscopy on ultrathin cryosections) and also found catalase to be located exclusively within the peroxisomes with no cytoplasmic labelling in rat and mouse liver.

95 Chapter 7 lmmunocytochemistry of hepatic peroxísomes

They conclude that the catalase recovered from the supernatant after cell fractionation is due to leakage from the peroxisomes. However, as discussed by Hemsley et al. (1988), dense immunocytochemical staining within the peroxisomes does not necessarily exclude any existence in the cytoplasm, since the concentration of catalase activity within the peroxisomes is much greater than that of the cytoplasm. ln human liver, catalase has been localised by immunoelectron microscopy within the peroxisomes only, and not within the cytoplasm, or the nuclear matrix (Litwin et al. 1987; Litwin and Beier 1988a). However, since the techniques used by these authors are different to mine there are several reasons to explain this discrepancy. Firstly, the use of a high concentration of glutaraldehyde in the fixative, followed by post-fixation in osmium tetroxide may result in loss of antigenicity' The

many washing and dehydration steps involved in this type of processing may cause

considerable loss of the soluble cytoplasmic catalase. The embedding of the tissue in a

nonpolar epoxy resin which requires dehydration of the tissue in solvents and the application

of high temperature during polymerization may also result in conformational changes in

proteins. The use of resin-embedded sections also means that only those antigenic sites

exposed on the surface of the sections are available to the immunoreagents (Bendayan and

Zollinger 1983), since the rest of the section is cross-linked by the resin. So, even though

Litwin et al. 1987, are able to demonstrate peroxisomal catalase in human liver biopsies, the

use of the hydrophobic Epon resin, or even Lowicryl K4M resin, may interfere with the

detection of cytoplasmic catalase. Once again, the catalase present within the peroxisomes

is much more concentrated than that within the cytoplasm, since the cytoplasm occupies a

much larger area of the cell than do peroxisomes. So, peroxisomal catalase can be

visualised in human liver even after strong fixation, osmication and embedding in Epon resin,

but the less concentrated cytoplasmic catalase may be less easily detected.

The observation of catalase in the NALD patient is further confirmation of the existence of

cytoplasmic catalase in human liver. My results support the hypothesis that the basic defect

in these patients may be in the biogenesis of peroxisomes, or in the incorporation of the

peroxisomal enzymes into the peroxisomes. The immunoelectron microscopy indicates that

96 Chapter 7 Immunocytochemistry of hepatic peroxísomes

the peroxisomal enzyme catalase is still being synthesized by the hepatocytes in NALD patients, but is unable to be properly incorporated into the residual peroxisomes.

7.3.g Changes ín peroxísomes over time ln two of the patients in this study, PD3 and lRD2, more than one liver biopsy was received for assessment of peroxisomal structure. ln lRD2 the major change over a period of several years was an increase in the numerical density of peroxisomes, and the appearance in the last two biopsies of some peroxisomes of almost normal size. ln pD3 there was a dual population of peroxisomes in the first biopsy, but in the second biopsy taken two years later only enlarged peroxisomes with an increased numerical density were visualised. Unfortunately none of this biopsy was processed for immunocytochemical studies due to the small amount of tissue available.

The increases in number and size of the peroxisomes in these two patients over time may

represent the natural progression of the disease or may be age related. These two patients

(now aged 7 and 11 years) are the only ones in this study who are still surviving'

Alternatively, the changes in the nature of the peroxisomes may be a response to treatment

with a low phytanic acid diet or other treatment. Both these boys were treated with a diet

low in phytanic acid and this resulted in a lowering of their plasma phytanic acid levels to

near normal values (see section 8'4 for further discussion)'

Ultrastructural changes in peroxisomes, usually an increase in number accompanied by an

increase in size have been reported in drug and toxic liver injury (Phillips et al. 1987)'

Another explanation is that the changes are related to sampling error in using liver biopsies.

In both patients the number of peroxisomes was very heterogeneous from cell to cell, with

some hepatocytes having near-normal numbers and adjacent cells having no peroxisomes.

This variability ¡n peroxisomal numbers from cell to cell is not usually observed in control

tissues. Given the very small area of tissue examined by electron microscopy, the changes

between biopsies may relate merely to sampling of a different area of the hepatic lobule'

7.4 CONCLUSION

The different types of peroxisomes identified in each of the peroxisomal disorder groups in

91 Chapter 7 lmmunocytochemistry of hepatic peroxisomes

this study are summarised in Table 19.

Complementation analysis experiments have identified at least 8 different complementation groups from patients deficient in peroxisomes (Brul et al. 1988a; Poll-The et al. 1989;

Roscher et al. 1989; Yajima et al. 1992). This suggests that at least I genes may be involved in the assembly of functional peroxisomes, and a mutation in any one of these genes can lead to the biochemical and clinical phenotype of a generalised peroxisomal disorder. Furthermore, studies in cultured skin fibroblasts from patients belonging to the same complementation group, have shown further phenotypic variation with regard to

immunofluorescence microscopy and staining with catalase antibody (Wiemer et al' 1991).

My studies of the morphological characteristics of peroxisomes in the liver of peroxisomal

disorders patients clearly support the complementation analyses where there is genetic

heterogeneity between and within the major groups of peroxisomal disorders. I have found

heterogeneity with regard to number of peroxisomes within the Zellweger syndrome

patients, and heterogeneity between the IRD patients with regard to the size and number of

peroxisomes and the labelling for peroxisomal proteins. lhave also identified 3 patients who

do not fit any current classification of peroxisomal disorders, who display heterogeneity in

the size, number, and immunocytochemical labelling of hepatic peroxisomes.

98 peroxisomes to the control TABLE 19: Summary of results for each gloup of peroxisoryl disorders. The change in size of the relative group ß in¿rcated as well as the tabetting density of each of the antibodies.

PMP68 GROUP PX SIZE CATALASE A-CoAOx BFP THIOLASE

zs reduced/absent none none none none none none NALD reduced none none none none

decreased/i ncreased decreased/normal IRD reduced decreased decreased/normal decreased/normal increased PD2 reduced none none none none

decreased normal PD3large normal decreased decreased normal increased normal PD3small reduced decreased normal none

decreased normal PZS enlarged decreased normal normal

normal normal RCDP enlarged decreased normal normal

normal normal ALDf normal normal normal increased

f increased increased PD1 normal increased increased increased

(o (o CHAPTER8 MITOCH ONDRIA AND LY SOMES +

Chapter I Mitochondria and lysosomes

8.1 MITOCHONDRIAL ABNORMALITIES

Mitochondria with an abnormal orientation of cristae were observed in many of the patients with a generalised peroxisomal disorder (see Table 2O and Figs' 24 and 25a)' These mitochondria were usually enlarged in size and the cristae had a characteristic "whorled" appearance. The majority of the mitochondria in these patients were of normal appearance with only a small number showing the ultrastructural abnormality' ln several patients, giant mitochondria containing paracrystalline inclusions were also seen (Fig. 25b) . An extreme variability in the size of the mitochondria was also apparent in several patients.

The characteristic "whorled" appearance of the mitochondrial cristae was not observed in the ALD, PZS or RCDP patients in this study. A.2 LYSOSOMAL INCLUSIONS

Lamellar-lipid inclusions were visualised in the hepatocytes and the Kupffer cells and

macrophages of many of the patients (Table 2Ol ' These inclusions were varied in

appearance (see Figs. 26 and 271 but were usually spindle shaped, and contained trilaminar

profiles 7-1Onm in width and electron dense granules and lipid. The inclusions were most

notable in the IRD patients, but were also seen in several Zellweger syndrome, NALD, and

the PD3 patient.

The amount of included material varied with age in patient lRD2. The first two biopsies

(taken at ages 1.5yr and 3.5yr) contained large amounts of tr¡laminar material, whereas the

subsequent biopsies (taken at ages 5yr and 6yr), after treatment with a low phytanic acid

diet showed very few of these inclusions. Similarly, in patient PD3, the first biopsy taken at yr age 2yr did contain a few trilaminar inclusions, whereas in the second biopsy, at age 4

and after two years on a low phytanic acid diet, trilaminar inclusions were not seen.

There was no evidence of immunocytochemical labelling with peroxisomal antibodies in

these lysosomal inclusions'

103 FtG. 24

Enlarged mitochondria have a typical abnormal alignment of the cristae in some peroxisomal disorders a mitochondria with "whorled" cristae in a liver biopsy from the

NALD patient XSO,OOO b a giant mitochondrion with "whorled" cristae in a liver biopsy from the PD2 patient X50,000 t

,ù.f, FrG. 25 a a giant mitochondrion in the liver of the lRD2 patient showing "whorled" cristae X50,O0O b a mitochondrion from the same biopsy showing para-crystalline inclusions X50,00O :'.1 ie r' rq { I Ï 4 f' 5r :a

a

_{',t :r{Þ

it it t

rli i . d v i.l. Í ü,:

¡

:iÌ -î

ï ,*t\ -$ *'!,r .( |:

'4., i: FrG. 26 cytoplasm as well as Lysosomal inclusions can be seen within the hepatocyte patients within macrophages in the liver of many peroxisomal disorder a typical spindle shaped, angular inclusions containing trilaminar material within a macrophage from the lRD2 patient x65,000 from a b spindle shaped inclusion within the hepatocyte cytoplasm patient with an unclassified disorder (PD2)' This inclusion also contains electron dense granules X65'O00 >i .-' J \a. ìr { ¡ .¿ ? Y

\

¡ I FtG. 27

a an atypical lysosomal inclusion within an hepatocyte from a Zellweger patient (ZSSa) X24,3OO b an inclusion in autopsy liver from a Zellweger patient (ZS7al which contains trilaminar material and lipid X50,00O *+! Ð ¡

üj'

t I

t't" t ,1..'l ,.:

a T" ,rìr *' 27çÉ.t', '.t- Chapter I Mitochondria and lysosomes

T in other Patient Age Mitochondria Lysosomal lnclusions

zs1 2 wks whorled cristae, enlarged, not seen variable size zs2'f 12 wks normal not seen ZS3a 8 mths whorled cristae Kupffer, hepatocytes ZS4a 3 mths whorled cristae, enlarged Kupffer ZSSa 2 mths PM change not typical zs6f 12 wks whorled cristae, not seen dense matrix zs7 3 mths dense matrix Kupffer, hepatocytes ZSSa PM change not seen ZS9a 3 wks normal not seen

NALDl b1 1Yr whorled cristae Kupffer, hepatocytes NALDl b2 2 yrs whorled cristae Kupffer, hepatocytes NALDI a 3 yrs whorled cristae Kupffer, hepatocytes

IRDl 5 mths variable size Kupffer, hepatocytes tRD2b1 18 mths normal Kupffer, hepatocytes lRD2b2 3 yrs variable size Kupffer, hepatocytes tRD2b3 5 yrs normal hepatocytes tRD2b4 6 yrs whorled cristae, Kupffer, hepatocytes crystalline inclusions

ALDIf 17 wks normal not seen ALD2a 6 yrs PM change,crystalline not seen inclusions ALD3a 7 yrs PM change not seen

PZSl 7 mths normal not seen PZSl a I mths PM change not seen PZS2 2 mths normal not seen

RCDPl 8 mths normal not seen RCDP2a 1 3 days normal,PM swelling not seen

PDl f 17 wks normal adrenal only PD2 11 mths whorled cristae, not seen enlarged, variable size PD3b1 2 yrs normal yes, rare PD3b2 4 yrs normal not seen

8.3 ADRENAL GLAND

lntracytoplasmic inclusions were detected in the adrenal gland of some of the patients in this

study. ln the rhree sex-linked ALD patients, ALD1f, ALD2a and ALD3a, slightly curved

electron lucent clefts or spicules were distributed in the cytoplasm of the adrenocortical cells

(Fig. 2Bb). These inclusions often formed spindle-shaped bundles and sometimes trilaminar

material was also present. All three of these patients had morphologically normal hepatic

104 FtG. 28

Spindle shaped inclusions which contain laminar material can be seen in adrenal tissue as early as 17 weeks a adrenocortical cell with cytoplasmic inclusions from a 17 week old fetus affected with an unclassified peroxisomal disorder (PD1) x13,000

b adrenocortical cell with cytoplasmic inclusions f rom a fetus affected with ALD (ALD1) X13,OO0

Chapter I Mitochondria and lysosomes

peroxisomes, and trilaminar lysosomal inclusions were not visualised in the liver.

Adrenal tissue was examined in only two of the nine Zellweger syndrome patients in this study. However in both of these (ZS4a and ZSga) a large proportion of the adrenocortical cells contained intracytoplasmic angulate clefts and spicules similar in appearance to those observed in the sex-linked ALD patients. ln one of these (ZS4a) a small amount of angulate, trilaminar material was also observed in the liver. Adrenal tissue was also examined at autopsy from the NALDIa patient, and found to contain similar intracytoplasmic inclusions.

Lysosomal trilaminar inclusions were observed within the hepatic cytoplasm and within macrophages in the liver of this patient at biopsy and autopsy. Trilaminar inclusions were also seen in the brain, kidney and spleen of this patient at autopsy.

Similarly in the adrenal gland of the PDlf patient, a large proportion of the cells contained intracytoplasmic, angulate clefts and spicules (Fig.28a) . lnclusions were not present in the were liver of this 17 wk f etus, however peroxisomes were present although they

morphologically abnormal in that they contained an electron dense centre.

Adrenal tissue was not available for examination in the other patients in this study so it is

not possible to indicate how widespread the occurrence of these inclusions is in peroxisomal

disorder patients. 8.4 DISCUSSION

The presence of mitochondrial cristae with a "whorled" appearance would seem to be

characteristic of a generalised peroxisomal disorder. Although many ultrastructural

abnormalities of mitochondria have been described in the literature, there are no reports of

this particular phenomenon except in peroxisomal disorder patients. Of the patients in this study only those with a generalised peroxisomal disorder and including PD2 had

mitochondria with "whorled" cristae. These patients all had small morphologically abnormal

peroxisomes with electron dense centres, except for two of the Zellweger patients where

none were detected. lt ¡s not known if the mitochondrial abnormality is due to a

mitochondrial defect or if the observed morphological changes are a secondary effect due to

an accumulation of metabolites in the hepatocytes with reduced peroxisomal function. An

impairment of the mitochondrial electron transport chain was described by Goldfischer et al.

105 Chapter I Mitochondria and lysosomes

(1g73) in Zellweger patients, and perhaps indicates that there may also be a mitochondrial defect operating in at least some of the generalised peroxisomal disorder patients.

The presence of abnormal mitochondria did not necessarily correlate with the presence of lysosomal lipid inclusions'

Mitochondrial abnormalities were first described by Goldfischer et al. (1973) in the hepatocytes of Zellweger syndrome patients. Mathis et al. (1980) also found mitochondria with a dense matrix and irregular and twisted cristae in two cases of Zellweger syndrome, and Monnens et al. (1980) reported mitochondria with curled cristae in one Zellweger syndrome patient. They suggest that a generalised mitochondrial defect may be responsible for the abnormal bile acids found in Zellweger syndrome infants.

The ultrastructural abnormality of "whorled" cristae described here could be interpreted as

an attempt to increase the surface area of the mitochondrial cristae, possibly in response to

an increased metabolic demand. This seems quite likely since the abnormal mitochondria are

only observed in patients with a greatly reduced volume density of peroxisomes'

The intramitochondrial crystalline inclusions observed in two patients (lRD2b4 and ALD2) are

most probably a nonspecific feature since crystalline structures have been reported in the

mitochondria of normal liver cells (Wills 1965) and, more frequently, in diseased liver

(Sternlieb and Berger 1969). The significance of the inclusions is unknown but they are

believed to be degenerative and associated with cells in a phase of increased metabolic

activity.

The lysosomal lamellar-lipid inclusions observed in the liver and adrenal gland of many of the

patients with a generalised peroxisomal disorder are believed to represent storage of very

long chain fatty acids (Powers et al. 1980) . These structures have been reported in the

liver, brain and adrenal of patients with Zellweger syndrome (Pfeifer and Sandhage 1979;

Mooi et al. 1983; Auborg et al. 1985; Roels et al. 1986; Kerckaert et al. 1988), and are

morphologically similar to those seen in sex-linked ALD (schaumburg et al' 1975), NALD

(partin and McAdams 1983) and IRD (Scotto et al. 1982; Budden et al. 1986; Robertson et

al. 1988; Torvik et al. 1988; Wanders et al. 1990a).

106 Chapter I Mitochondria and lysosomes

A change in the number of lysosomal lamellar-lipid inclusions with age was noticed in patient lRD2. ln the first biopsy at age 1.5 yr the inclusions were very striking. This biopsy contained only very small numbers of small, abnormal peroxisomes with electron dense centres. The introduction of dietary management i.e. a low phytanic acid diet, resulted in a decrease of plasma phytanic acid level to near normal values. Plasma pipecolic acid level also decreased. ln contrast, the plasma long chain fatty acid ratios appeared to increase at first and then gradually diminished (see Robertson et al. 1988 for further details). ln the first post-diet biopsy from this patient (3.5yr, lRD2b2), an'increase in the trilaminar inclusions was observed. However in the liver biopsies taken at age 5yr (lRD2b3) and 6yr (lRD2b4)

(i.e. after 4.b and 5.5 years of dietary management) the number of trilaminar inclusions had decreased remarkably. ln Zellweger syndrome patients the frequency of trilaminar structures increases with the age of the patient (Mooi et al. 1983; Kerckaert et al. 1988) as

presumably more material is stored. ln the lRD2 patient the opposite seems to have

occurred and may be attr¡buted to the dietary treatment. The reasons for the initial increase

remain obscure. An improvement in some clinical parameters was also found after dietary

management. ln the most recent biopsy (6yr) an increase in the number of peroxisomes in

the liver was also apparent and some of these peroxisomes were larger in size than those

seen in previous biopsies, and had some normal appearing matrix as well as electron dense

centres

ln patient PD3, who has a disorder with some similarities to IRD but with normal DHAP-AT

activity, a difference in the frequency of the trilaminar structures was also noted with age.

ln the first biopsy a few of these inclusions were seen, but in the second biopsy (after 2 yrs

of dietary treatment) none were evident. This patient also showed a simultaneous variation

in the nature of peroxisomes between the two biopsies (see section 7.3'g for further

discussion). As in lRD2, the adherence to a diet low in phytanic acid resulted in a decrease

of this boy's plasma phytanic acid levels to normal values. However, his long chain fatty

acid ratios remained abnormal and there was no obvious clinical improvement, nor

deterioration,

It is diff icult to speculate on the nature of the material stored in these inclusions.

l_07 Chapter I Mitochondria and lysosomes

Morphologically there is great variation in their ultrastructural appearance and their frequency varies not only from patient to patient within each group of peroxisomal disorders, but also from biopsy to biopsy from each patient. There is also variation in the presence of these inclusions between different organs. Given the small size of many of the liver biopsies from these infant patients it is not unlikely that sampling error may contribute to this variation since the majority of the material is stored in the non-parenchymal cells' The material is generally assumed to be stored long chain fatty acids and certainly the generalised disorders all have this abnormality and trilaminar inclusions have been reported in varying frequency in

Zellvrieger syndrome, NALD and IRD patients. Further evidence comes from animal studies where Vamecq et al. (1987) examined mouse heart and liver after administration of a drug which inhibits peroxisomal ß-oxidation. They found lamellar structures which resemble those seen in human peroxisomal disorders and an increase in plasma very long chain fatty

acids. However, sex-linked ALD patients also have abnormal long chain fatty acids, and are boys, usually older patients, but trilaminar inclusions are onlrT rgfq[y seen in the liver of these Í I ' i"(' t'' {' although are usually present in the adrenal gland'

Roels et al. (1991b) have recently reported the occurrence of these inclusions in the liver of

an RCDp patlent and long chain fatty acid ratios are usually normal in these patients.

Another possibility is that the inclusions are related to storage of phytanic acid and the data

presented here particularly in relation to the lRD2 and PD3 patients would support this'

However patients with adult Refsum's disease who have very elevated plasma phytanic acid

levels do not display hepatic inclusions (Roels et al. 1991b). The nature of these inclusions

remains unknown but their presence in the liver and other organs of patients with a

deficiency of peroxisomes or with severe peroxisomal dysfunction suggests that they may

represent the storage of material which would normally be metabolized within peroxisomes'

1-0B CHAPTER9 CONCLUDING REMARKS

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1,66 APPENDIX A

CASE NOTES Appendix A Case Notes

A summary of the clinical features of the patients in this study are in Table 6 (see section

3.1) and the deta¡ls of the biochemical findings are recorded in Table 7 (see section 3.1).

The full clinical and biochemical details of several of the patients in this study have been published previously (lRD2 see Hughes et al. 1990 and Robertson et al. 1988; NALD see patient 3 in Hughes et al. 1990; PD3 see patient 2 in Hughes et al. 1990; RCDPI see case 6 in Poulos et al. 1991 and case 1 in Hughes et al. 1992; RCDP2 see patient 1 in Poulos et al.

1g88a, case 5 in Poulos et al. 1991, and case 2 in Hughes et al. 1992). The clinical criteria employed for the diagnoses of Zellweger syndrome, infantile Refsum's disease and neonatal adrenoleukodystrophy have been reported earlier (Poulos et al. 1988a)' The full clinical details of the nine Zellweger syndrome patients, and the three sex-linked ALD patients have not been included as these phenotypes are well described in the literature (see Chapter 2). PATIENT NALDl

This patient was a female born by emergency caesarean at 36 weeks, and was noted to be

mildly dysmorphic and small for gestational age (1.a9 kg). A brief seizure was noted in the

first month but was controlled with phenobarbitone. No further seizures occurred and drug

treatment was discontinued at 11 months. Progress was poor with marked failure to thrive,

gross developmental delay, and hepatomegaly. The patient's weight never exceeded 5 kg

despite an adequate calorie intake. A retinal pallor was observed in the neonatal period and

progressed to severe retinitis pigmentosa. Hearing was normal but deteriorated in the last

three months of life. Gradual loss of motor and developmental milestones occurred just

before death at three years. The diagnosis of neonatal ALD was based on the clinical,

ultrastructural, post-mortem and biochemical findings (Kelley et al. 1986)'

Biochemical findings:

Reduced DHAP-AT and phytanic acid oxidase activities were found in fibroblasts, The levels

of plasma pipecolic acid, phytanic acid, pristanic acid and trihydroxycholestan-2G-oic acid

were increased. PlasmaC26:C22 fatty acid ratios were also elevated. Light microscopy:

Clusters of cells within the lobules, possibly Kupffer cells, and in portal triads contained

pAS-positive, diastase resistant, spiculate material which was non-biref ringent in H&E

168 Appendix A Case Notes

sections. Autopsy findings:

The liver was enlarged and showed moderate diffuse fatty change. The adrenals weighed

O.5g and O.4g and histology showed a thickened and fibrotic capsule. The spinal cord

showed widespread degeneration of long myelinated tracts. The tissue affected showed

numerous foamy macrophages, gemistocytic astrocytes and areas of cystic degeneration.

The cerebral hemispheres and the cerebellum showed extensive demyelination. There was

no significant ocular or renal pathology and there were no renal cortical cysts. The lungs

showed severe and extensive bronchopneumonia with features indicative of a recent onset'

1,69 Appendix A Case Notes

PATIENT IRDl

This patient presented at 4 months with developmental delay, hepatomegaly and dysmorphic features. At 1O months he was noted to be severely hypotonic with hyperreflexia, a sensory neural hearing loss and a retinopathy with optic atrophy. The provisional clinical diagnosis was infantile Refsum's disease. He died at age 2 years and autopsy was refused. Biochemical findings:

Plasma phytanic acid, pristanic acid and C26:O/C22:O fatty acid ratios were elevated. ln cultured fibroblasts the phytanic acid oxidase and DHAP-AT activities were reduced.

PATIENT IRD2

The patient is a male who presented at 19 months of age with pendular nystagmus, hepatomegaly, retinitis pigmentosa, deafness, hypotonia and ataxia. Motor and sensory nerve conduction, and sensory nerve action potential amplitudes were normal. He was treated with a low phytanic acid diet and there was a rapid response biochemically with a reduction in phytanic acid in the plasma and liver. Growth and motor skills improved, and the hepatomegaly was not so pronounced. However vision and fundoscopy changes have remained the same. The patient is now eleven years old. Biochemical findings:

The biochemical findings include reduced levels of DHAP-AT and phytanic acid oxidase in skin fibroblasts; increased plasma phytanic acid, pristanic acid, trihydroxycholestan-2G-oic

acid and pipecolic acid; and elevated plasma C26: C22 and C24: C22 fatty acid ratios. Light microscopy:

The normal hepatic architecture is disturbed with loss of central vein: portal tract relat¡onship

due to moderate fibrosis. The cytoplasm of portal histiocytes of Kupffer cells contains PAS

positive, diastase resistant, spiculate material which is biref ringent in H&E sections.

lntranuclear glycogen is observed in isolated hepatocytes.

PATIENT PZSZ

The parents are first cousin Lebanese. Their first child had a diaphragmatic hernia and is

developmentally delayed. High voltage electrophoresis of urine was normal. Their second

170 Appendix A Case Notes

child (PZS2) was born at forty two weeks gestation, had respiratory distress from birth due

to mild pulmonary hypoplasia and required mechanical ventilation for ten days. He had a

number of dysmorphic features including a prominent nose and premaxilla and contractures

of the large joints, generalized muscle weakness and marked hypotonia. He also had sparse,

abnormal looking hair which was microscopically normal. Seizures developed on day one

which were treated with phenobarbitone and phenytoin. His eyes were normal. His physical

and developmental progress was poor and he remained hypotonic and inactive. He died at

four months of respiratory failure. Post-mortem examination was refused.

lnvestigations undertaken at the age of two and a half months showed a mild anemia (98g/l)

with target cells on blood smear and normal blood clotting studies. Gamma-glutamyl

transferase levels in serum were increased (276 units/l-normal range O-37)' Urinary high

voltage electrophoresis demonstrated raised levels of threonine, glycine and serine. Urinary

organic acids measured by gas liquid chromatography were normal. A skeletal survey

showed no punctate changes or calcification of epiphyses. A short Synacthen test was

normal. Biochemical findings:

plasma and fibroblast C26:C22 and C24:C22 fally acid ratios were increased but pipecolic

acid and phytanic acid levels were normal. DHAP-AT and phytanic acid oxidase levels were

normal. The diagnosis of pseudo-Zellweger syndrome was based on clinical, biochemical

and ultrastructural findings.

PATIENT PZSl

This child is the brother of patient PZS2. The parents received extensive counselling and

requested prenatal diagnosis by amniocentesis with their next pregnancy. The test predicted

that the fetus would be affected but the parents elected to continue with the pregnancy. He

was born at term by lower segment caesarean section because of fetal distress. Apgar

scores were 6 and 9 at 1 and 5 minutes respectively. Birth weight was 2880 grams and he

was noted to be floppy at birth. He developed seizures within the first few days of life.

These were characterised by multiple episodes of generalised and focal attacks which were

l.7L Appendix A Case Notes

partially responsive to phenobarbitone and clonazepam. There was no major facial dysmorphism. He required frequent oralpharangeal suction and nasogastric gavage feeds.

With age it became obvious that he was severely developmentally retarded. He was not responsive to most stimuli, and had only occasional weak spontaneous movements. He was admitted to hospital at 7 months of age because of uncontrolled epilepsy and it was noted at that time that he was not fixing or following and that he had horizontal nystagmus. His marked hypotonia had persisted. There was very little spontaneous movement and his cry was weak. Hepatomegaly was not present. At 8 months of age he developed gastroenteritis and died during an apnoeic episode. A limited post mortem was allowed.

Bíochemical findings:

Plasma and skin taken in the newborn period confirmed the elevated levels of very long chain fatty acids seen at amniocentesis. Autopsy findings:

Autopsy was confined to examination of the abdominal organs, as requested by the parents.

The kidneys were normal with no cortical cysts. The liver showed mild haemosiderosis of the periportal hepatocytes. The adrenals weighed O.4g and O.89 and histology showed extensive atrophy with focal residual clusters of cortical cells and moderate numbers of macrophages with cytoplasm containing linear clefts. The spinal cord showed moderate vacuolation of the white matter. There was no conspicuous gliosis, macrophage infiltration or decrease in myelination. The corticospinal tracts were relatively poorly myelinated, but probably normal in view of the age. Ultrastructure showed membrane-bound organelles,

probably lysosomal, with trilaminar profiles in many macrophages and some cortical cells in the adrenal, occasional Kupffer cells and hepatocytes, and a few interstitial cells in the testis. ln the spinal cord, only a very rare axon showed degeneration associated with

macrophages containing similar lysosomes.

PATIENT RCDPl

The patient was a male who was noted shortly after birth to have short limbs, hypotonia,

cataracts, flexion contractures of the proximal upper and lower limbs, and ulnar deviation at

1,72 Appendix A Case Notes

rhe metacarpo-phalangeal joints. Skeletal x-rays showed typical changes of RCDP although facial features were not typical of RCDP. The plasma 26:0122:0 fatty acid ratio and plasma

phytanic acid, both measured when the patient was 4 months old, were elevated. However,

26:0122:0 fatty acid ratio in cultured skin fibroblasts were normal. At 2 years plasma

26:0122:0 fatty acid ratio were normal but phytanic acid level remained elevated. Skin

f ibroblast phytanic acid oxidase, dihydroxyacetone phosphate acyltranferase and alkyl

dihydroxyacetone phosphate-synthase activities were reduced. Glyceryl ether lipid

(plasmalogens) levels in the liver were also greatly reduced (Poulos et al. 1991). Liver

biopsy was performed at I months, and tissue was collected for electron microscopy,

catalase cytochemistry, and immunocytochemistry'

PATIENT RCDP2

This male patient was noted at birth to have short limbs, bilateral cataracts and dysmorphic

features. Radiological examination showed changes typical of RCDP. The patient died at 13

days. The full clinical and biochemical findings of this patient have been published

previously (patient 1 in Poulos et al. 1988, case 5 in Poulos et al. 1991, and case 2 in

Hughes et al. 1992). Plasma 26:0122:0 fatty acid ratio, pipecolic acid, and PhYtanic acid were normal. Skin fibroblast phytanic acid oxidase, dihydroxyacetone phosphate

acyltransferase and alkyl dihydroxyacetone phosphate-synthase activities were reduced.

Glyceryl ether lipid levels in liver and brain were also greatly reduced (Poulos et al. 1991)'

PATIENT PD 1

Fetal tissue was received after prenatal assessment on chorionic villus biopsy and cultured

amniotic cells showed an affected child. The propositus, a male, was born small for

gestational age and had some mild signs of asphyxia at birth. He started to fit from the first

day of life and, apart from occasional breaks, continued to fit in the first few months of life.

At 3 months he was developmentally delayed, his liver was enlarged, but his optic discs and

maculae were normal. He died aged 6 months. There were fatty changes in the liver and

his adrenals were found to be small on post-mortem examination.

L'73 Appendix A Case Notes

Biochemical findings: ln the propositus plasma and post-mortem liver C26:C22 and C24:C22 fatty acid ratios were elevated. Plasma and liver phytanic acid was normal and plasma pipecolic acid was slightly elevated. ln cultured skin fibroblasts the C26:C22 fatty acid ratios were elevated and

DHAP-AT activ¡ty was normal. The mother's VLCFA values were normal.

PATIENT PD2

This patient presented at 7 weeks with a suspected heart murmur. On examination she was found to have hepatomegaly and a slightly enlarged heart with a small to moderate ventricular septal defect. At 7 months she was developmentally delayed, hypotonic, and showed evidence of a retinal dystrophy and sensineural deafness. She was dysmorphic with a high forehead, a large anterior fontanelle and small mouth. She died accidentally at age 16 months.

Biochemical findings:

The VLCFA ratios in plasma and the plasma phytanic acid levels were elevated. However, in cultured fibroblasts the VLCFA ratios were normal, and DHAP-AT and alkyl DHAP-synthase activities were normal. Phytanic acid oxidase activity was reduced and the percentage of particulate catalase was reduced to l OTo (64-93Vo normal)'

PATIENT PD3

The patient is a male who presented at age 18 months with global developmental delay,

hypotonia, arreflexia and hearing loss. Plasma phytanic acid and C26: C22 fatty acid ratios

were elevated and he commenced a low phytanic acid diet which led to a dramatic decrease

in plasma phytanic acid. He has a cousin, also male, who has peripheral neuropathy and is

thought to have a variant form of Refsum disease (Case number 2 in Poulos et al. 1984b) .

The cousin has normal plasma phytanic acid and elevated C26: C22 fatty acid ratio but a

reduced activity of phytanic acid oxidase (Poulos et al' 1986). Biochemical findings:

plasma C26:C22 fatty acid ratios and phytanic acid are elevated. Fibroblast phytanic acid

l.14 Appendix A Case Notes

oxidase activity is decreased by approximately 50% and DHAP-AT activity is normal

L75 APPENDIX B

PUBLISHED PAPERS AND MANUSCRIPTS Appendix B Published papers

Some of the material presented in this thesis has been published, or submitted for

publication during the term of the candidature. The papers and manuscripts are detailed

below with acknowledgements to the co-authors, and copies of the papers and manuscripts

are included in the remainder of this appendix. Reference to the appropriate chapters in the

thesis is made. Where no reference to a thesis chapter is made, the material in the paper or

manuscript is not reproduced in the body of the thesis'

1. Hughes JL, Poulos A, Robertson E. Chow CW, Sheffield LJ, Christodoulou J, Garter

RF (1990) Pathology of hepatic peroxisomes and mitochondria in patients with

peroxisomal disorders. virchows Archiv A Pathol Anat 4162 255-264

The candidate carried out the electron microscopy, cytochemistry, and morphometry

on the five patients described, and wrote the manuscript. Dr Alf Poulos carried out

the biochemical analysis on plasma and cultured skin fibroblasts. Dr Evelyn Robertson

provided patient material and clinical notes for two patients. Dr CW Chow, Dr LJ

Sheffield and Dr John Christodoulou provided patient material and clinical notes on

two patients. Dr CW Chow also provided the autopsy report on one patient and read

the manuscript. Dr RF Carter reviewed the manuscript and the pathology of two

patients in his capacity as Director of Histopathology, in which department this work

was carried out. (Chapters 5, 6 and 8)

2 Hughes JL, Poulos A, Grane Dl, Chow CW, Sheffield LJ, Sillence D (19921

Ultrastructure and immunocytochemistry of hepatic peroxisomes in rhizomelic

chondrodysplaia punctata. Eur J Pediatrics 151: 829-836

The candidate performed the electron microscopy, cytochemistry, morphometry and

immunoelectron microscopy in this study, and wrote the manuscript. Dr Alf Poulos

carried out the biochemical analysis of the patients. Dr Denis Crane supplied the

antibodies to catalase, bifunctional protein and PMP68. Dr CW Chow and Dr LJ

Sheffield provided patient material and clinical notes for one patient. Professor D

L76 Appendix B Publíshed papers

Sillence provided patient material and clinical notes for the other patient. (Chapters 5,

6 and 7)

3 Klucis E, Crane Dl, Hughes JL, Poulos A, Masters CJ (1991) ldentification of a

catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrate.

Biochimica et Biophysica Acta 1O74:294-301

E. Klucis carried out the biochemical investigations under the supervision of Dl Crane

and CJ Masters. A. Poulos was co-investigator on the grant from the National Health

and Medical Research Council of Australia which funded this research. The candidate

carried out the immunoelectron microscopy on the mouse liver fractions as described.

4. Hughes JL, Bourne AJ, Poulos A (1993) Establishment of a normal range of

morphometric values for peroxisomes in pediatric liver. Virchows Archiv A Pathol

Anat /n press.

The candidate carried out the ultrastructural and morphometric examination of the

tissues in this study and wrote the manuscript. Dr. AJ Bourne performed the

statistical analysis of the data and reviewed the manuscript in his capacity as

Director of Histopathology in which department this work was carried out. Dr. A

Poulos reviewed the manuscript in his capacity as supervisor of the candidate.

(Chapter 5)

5 Hughes JL, Crane Dl, Robertson E, Poulos A (1993) Morphometry of peroxisomes

and immunolocalisation of peroxisomal proteins in the liver of patients with

generalised peroxisomal disorders. Virchows Archiv A ln P¡ess.

The candidate carried out the electron microscopy, the morphometry and the

immunocytochemistry on the patients in this study, analysed the data and wrote the

177 Appendix B Published papers

manuscript. Dr Denis Crane supplied the antibodies to catalase, bifunctional protein

and PMP68. Dr Evelyn Robertson supplied the clinical information on the patients

and Dr Alf Poulos carried out the biochemical analysis of the patients, and reviewed

the manuscript. (Chapters 5 and 7)

6 Hughes JL, Poulos A, Carter RF, Crane Dl, Masters CJ (1988) Cytochemical and

immunocytochemical localization of catalase in peroxisomes of rat liver and human

cultured skin fibroblasts. (Abstractl Aust Paediatr J 24:94

The candidate carried out the immunocytochemical and cytochemical investigations,

wrote the abstract, and presented the work. Dr A Poulos and Dr RF Carter supervised

the work and reviewed the abstract. Dr Dl Crane and Prof CJ Masters provided the

antibody to catalase.

7 Hughes J and Poulos A (1992) Ultrastructural examination of t¡ssues from patients

with peroxisomal disorders. (Abstract) Electron Microscopy 3: 521-522

The candidate performed the ultrastructural examination of pat¡ent tissues, wrote the

abstract and presented the work. Dr A Poulos was the supervisor. (Chapters 5 and

8)

CONFERENCE PRESENTATIONS RELAT¡NG TO THIS THESIS

1. JL Hughes, A Poulos, RF Carter, Dl Crane, CJ Masters (1987) Cytochemical and

immunocytochemical localization of catalase in peroxisomes of rat liver and human

cultured fibroblasts.

Combined meeting of the New Zealand Society for Electron Microscopy and the

Australían and New Zealand Society for Cell Biology, Auckland, May 1987; and the

Human Genetics Society of Australia, Rotorua Conference, May 1987. 2. RF Carter, JL Hughes, A Poulos, Dl Crane, CJ Masters (1987)

lmmunocytochemistry of peroxisomal disorders. RCPA 32nd Meeting, Adelaíde,

October 1987

3. JL Hughes, A Poulos, Dl Crane, CJ Masters, RF Carter (1988) Localization of

L78 Appendix B Published papers

peroxisomal proteins in human tissues and cells. lOth Australian Conference on

Electron Mícroscopy, Adelaide, February 1988

4. H Thiele and JL Hughes (1988) Effects of fixation, embedding and gold probe on the

immunocytochemical labelling of catalase. lOth Australian Conference on Electron

Microscopy, Adelaide, February 1 988

5 JL Hughes, Dl Crane, S Whatley, B Paton, H Thiele, A Poulos (1990) The

distribution of catalase, ß-oxidation enzymes, D-aminoacid oxidase and integral

membrane proteins within rat liver and kidney peroxisomes. llth Australian

Conference on Electron Mícroscopy, Melbourne, February 199O.

6 JC Fanning, JF White, JL Hughes, PS Smith, M Henderson, JS Kumaratilake, R

Polewski, EG Cleary (199O) Preservation, destruction or restoration of antigenic

sites? Choosing the "right" method for immunocytochemistry. llth Australian

Conference on Electron Microscopy, Melbourne, February 199O.

7 JL Hughes, Dl Crane, A Poulos (1992) lmmunolocalisation of peroxisomal proteins

in patients with peroxisomal disorders. 9th International Congress of Histochemistry

and Cytochemístry, Maastrícht, The Netherlands, August 1992

I Hughes J and Poulos A (1992) Ultrastructural examination of tissues from patients

with peroxisomal disorders. EUREM 92, Granada, Spain, September '1992

I R Jamieson, J Christodoulou, M Wilson, J Hughes, A Poulos (1993) Zellweger

syndrome in a premature infant with prolonged conjugated hyperbilirubinemia. Aust

Soc lnborn Errors Metabolism, September 1993

L79 Hughes, J. L., Poulos, A., Robertson, E., Chow, C. W., Sheffield, L. J., Christodoulou, J. & Carter, R. F. (1990). Pathology of hepatic peroxisomes and mitochondria in patients with peroxisomal disorders. Virchows Archiv A, 416(3), 255-264.

NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at: http://dx.doi.org/10.1007/bf01678985 Hughes, J. L., Poulos, A., Crane, D. I., Chow, C. W., Sheffield, L. J. & Sillence, D. (1992). Ultrastructure and immunocytochemistry of hepatic peroxisomes in rhizomelic chondrodysplasia punctata. European Journal of Pediatrics, 151(11), 829-836.

NOTE: This publication is included in the print copy of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at: http://dx.doi.org/10.1007/bf01957935 294 Bioctdmica et Biophysica .4era, 1074 (1991) 294-301 © 1991 Elsevier Science Publishers B.V. 0304-4t65/91/$03.50 ADONIS 030441659100191J

BBAGEN 23550

Identification of a catalase-negative sub-population of peroxisomes induced in mouse liver by clofibrat¢

Elmar Klucis 1, Denis I. Crane 1, Jennifer L. Hughes 2 All Poulos 2 and Colin J. Masters I Division of Science and Technology, Griflfith University, Brisbrme (Australitz) and 2 Departments of Hisiopathology and Chemical Pathology, Adelaide Children's Hospital, North Adelaide (Australia)

(Received 17 October 1990) (Revised manuscript received 21 Match 1991)

Key words: Peroximme proliferation; Clofibrate; (Mouse liver)

The peroxisomal compartment in mouse liver was investigated using rate sedimentation of liver subfractions on sucrose density gradients. Treatment of mice with eloflbrate, a hylmlipidemle agent and ~roxisome proliferator, resulted in the formation of small particles which were devoid of catalase and urate n~Adase, hut wllieh were identified as peroxisomal on the basis of content of the deflbrate-lndaced perosisomal p-oxtdation euz;Inmcs (fatty acyI-CoA oxidase, hydratase/dehydrogenase bitunctienal protein, and thiolas¢) and the 68 kDa INro~somal integral membrane protein, lmmunoelectron microscopy confirmed the membrane-bound orgalllar nature and enzyme compesitien of these particles, These particles were absent in normal mice, and were increased to a maximal level within 2 days of doflbrate treatment. These data have been taken as indicative nf a role of these particles in the mechanism of drog-induced peroxisome proliferation. lmroduction laced from the polydisperse population of peroxisomes may yield information about the detailed biochemical The nature of the peroxisomal compartment in ro- structure of the peroTdsomal retieulum has prompted dent liver has been extensively investigated in recent us 1o undertake an examination of this polydispersity. years. Biochemical data have indicated a reticulum We have employed rate sedimentation of liver subfrac- system for this organelle [1] and morphological exami- tions on sucrose gradients to identify a sub-~pulation nations have detailed the organization of this structure of peroxisomcs which is induced in livers of mice with its appendant loops and interconnections [2-4]. treated with clofibrate. Homogenization of liver is believed to disrupt these peroxisomal structures and yield a sedimentable poly- Materials and Mclhods disperse population of particles [2,5], with this suscepti- bility to disruption presumably being related to the fragile and permeable nature of peroxisomal mem- Matetiab branes in vitro [6,7]. Administration to mice of peroxi- Mature female mice (Quackenbush strain) ~ez~ ob- some proliferators has been reported to result in iso- tained from the Central Animal Breeding House, Meg. lated peroxisomes of increased fragility [6,8]. gill, Brisbane. Clofibratc (p-chiorophenoxyisobutyri~ The possibility that sub-populations of particles iso- acid, ethyl ester; Atromid-S) was p gift from I.C.I. Australia. Diehlorofluorescein was purchased from Eastman Kodak, Rochester, NY, U.S.A. Protein molecular mass markers for SDS.polyaerylamide gel Abbreviatiom: PNS, post-nuclear supernatant; LMF, light milochon- electrophoresis were obtained from Pharmacia. S- drial fraction; PMP6S, 68 kDa peroxisomal integral membrane pro- tein. palmtoyl-CoA was from Boehringer Mannbeim. Io- dine-125 for iodination, and autoradiography film (Hy- Correspondence: D,I. Crane, Division of Science and Technology, perfilm-MP) were from Amersham. 3-Amino-l,2,4-tri- Griffith University, Nathan, QLD 411 I, Australia, azole was from K& K Laboratories, NY, U.S.A. Other 295 biochemical reagents were obtained from Sigma Chem- NADPHcytochre: ~" c reductase, U (/.Lmol cyto- ical, St. Louis, MO. All other materials were of analyti- chrome c redueed/min); thiolase, U (#mol ace- cal grade. toacetyI-CoA/min). Immunoblottiag and SDS-POly- acrylamide electrophoresis were performed as previ- Methods ously [13]. Homogenization efficiency was calculated Treatment of animals. Mature female mice weighing from the partitioning of eatalase between the ~st- about 40 g had access to normal laboratory chow nuclear supernatant and the nuclear pellet, and ',his pellets and water ad libitum. For administration of was used to determine the weight of liver which yielded clofibrate, chuw pellets containing 0.35% (w/w) doff- the sub-cc!!uI,~ eomb'o~zen:~. V~!ues ~h~.'- :,~, figures brate were substituted for normal pellets for the re- are expressc a. in terms of activity per g liver homoge- quired period. Animals were killed by cervlca[ disIoca~ nized. tion. hnmunoelectron microscopy. Relevant gradient frac- Literfraetionation. Livers were excised and immedi- tions were ~oled, diluted with 0.25 M sucrose/0.1% ately placed into cold 0.25 M sucrose containing 0.1% ethanol/5 mM Tris-HCI (pH 7.5) and pelleted by ethanol. The sucrose solution used for liver homoge- ccnlrifugation at 100000 ×g for 60 rain. The pellets nization also included 5 mM Tris (tris(hydroxy- were fixed in a mixture of 4% paraformaldehyde and methyi)aminomethane)-HCI (pH 7.5). The livers were 0.25% glutaraldehyde in 0.I M sodium phosphate minced using a Mellwain tissue chopper and homoge- buffer (pH 7.4.) for l har 4"C, and rinsed in sodium nized in a sucrose solution by one stroke of a loose phosphate buffer containing 0.8% paraformaldeh~,de Potter-Eivehjem homogenizer (0.6 mm diameter clear- for 24 h. Small pieces of ;he pellets were infused with ance) with the pestle rotating at 1000 rpm. 2.3 M sucrose, frozen in liquid ~.!,rogen, and e~osec- Differential fractions were prepared from the ho- tinned at -1U0°C in a Reichert Ultracut ;:!'~amicro- mogenate by centrifugation in a Sorvall SS34 rotor at tome with a FC4D cryoattaehment. The ultrathm 4"C, according to the following scheme: A nuclear c~o-cctions were stored on gelatin/agarose plates. pellet was separated from the homogenate by ¢¢ntrii- tmmunolabelling was carried out using a number of ugation at 800 ×g for 10 rain. The resultant post- incubations/washes as follows: phosphate buffered- nuclear supernatant (PNS) was centrifuged at 2800 × g saline containing 0.5% ovalbumin, 0.1% gelatin, 0.05% for 15 rain to yield a heavy mitochondrial fraction and Tween 20 and 0.2% Teleostean gelatin (buffer A) for su~rnatant. This supcmatant was then centrifuged at 20 rain; primary antibody (diluted in buffer A) for 60 27000×g for 10 rain to generate a light mitochondrial min; six washes in buffer A over 30 rain; protein A-gold fraction (LMF) pellet and a post-LMF supcrnatant. complex diluted in buffer A (to As~0 of 0.14) for 60 Rato sedimentation of humogenate differential frac- rain; six washes in buffer A cater 30 min; six washes in tions was performed in sucrose solutions using a Beck- distilled water over 30 rain. The cryosections were man SW28 rotor in an L8-55 ultracentrifuge. PNS stained in 2% neutral uranyl acetate for 10 rain, then fractions were layered on a 20-45.5% (w/w) gradient in 2% acidic uranyl acetate for 10 rain, and then on top of a 58% (w/w) cushion and centrifuged at embedded in 2% pol~inylalcohol (M, 10000) with 14000×g~ to an integrated ,.3t value of 2.8' 10 ~. 0.1% uranyl acetate [14]. Post-LMF supernatant fractions were centrifuged at Antisera. Aatiscra to the ~roxisomal bifunc~i0nal 10001]0 xg~ on a 13-45.5% (w/w) gradient on a 58% protein and the 68 kDa peroxisomal integral mem- (w/w) cushion to an integrated 102t of 3.8.10 ~°. Is0py- brane protein (PMP68) of mr,use liver ha~ been char- chic sedimentation of fractions from rate gradients was actcrized previously [13,15]. Catalase was purified to ee.,,'r/ed out at 100C00 x g~, on a 25-58% (w/w)gradi- homogeneity from mouse liver using the o~cedm~e of ent on a 60% (w/w) cushion, to an integratc,l ~, of Price and co-workers [16], and antibody to this protein 4.9' 10 n. was raised in rabbits, and affinity-purified as descn'bed Analytical methods. Enzymatic activities were deter- by Lazarow and de Duve [17]. Antisera to rat liver mined on aliqunts of samples diluted in a solution ~roxisomal thiolase and acyl.CoA oxidas¢ [18] were containing l mM sodium carbonate, 1 mM EDTA, generous gifts from Professor T. Hashimotn, Shinsu 0.1% ethanol, 0.1% Triton X-RI0, (pH 7.6). Catalase University, Japan. and NADPH-eytochrome c reductas¢ were assayed as previously described [9]. Thiolase was determined ac- Resulls cording to the procedure of Lazarow [10], fatty aeyl- CoA oxidase as described by Small et al. [11], and Preliminary experiments indicated that subceilular protein by the method of Sedmak and Grossberg [12]. particle aggregation was minimized when the pH of the Enzyme activities are expressed as follows: catalase, solution used to homogenize liver was 7.5. Subsequent LU. (t~mol H2OJmin); urate oxidase, U (nmol uric rate s~dimentation ext~rim0nts of liver post*nuclear acid/rain); fatty acyI-CoA oxidase, aAso2/min; supematants at this pH yielded broad and symmetrical 296 distributions of peroxisomes on gradients, as deter- Exploratory immunoblotting for the peroxisomal mined by the activities of the marker enzymes, eatalase membrane protein, PMP68, of PNS fractions rate sedi- and urate oxidase (data not shown). mented in sucrose gradients, indicated the presence of

I A [.15

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I I I l | l I I i I | I I 10 2O ~O Gradient volume (ml) Fig 1. (A) Main particle zone from tale sedimentalion o[ -~ liver pos1-LMF supernatant haclion from a mou~c tz~ak:d with ~[Lb[at¢ for 10 days. o, ¢atalase; III, urate ox~dase;e, fatty a,.yl-CoA oxidase; 'e, thiolas¢; z~, NADPH-wlochmme c reduclase. (B) ]mmunoblol ~ai~sis of fractions in (A} for the peroxisomal bifunctional proteim (PBPlo) and the 68 kDa peroxJsoma]inlegzal membrane protein (PMP~). 297 peroxisomal membranes in a~d near the starting zone located in the less dense (presumably non-particulate) as well as in the gradient. To sediment the smaller of fractions of the gradient. Results of immunoblotting these particles further into the gradient it was neces- experiments of these gradient fractions further indi- sary to work with homogenates from which both the cated that this particulate material is also coincident heavy and light mitochondr[al fractions had been re- with a peak of the peroxisoma[ bifunctional protein moved. (hydratase/dehydrogenase) of the t~-oxidation path- A post-LMF supernatant from livers of clofibrale- way, as well as peroxisomal membrane, as assessed by treated mice was subjected to rate sedimentation at the content of PMP68, an integral membrane protein 100000 ×g on a 13-45.5% (w/w) sucrose gradient. of these organelles (Fig. IB). Quantitative data relating The gradient was subsequently fractionated and anal- to the (li~tribut_i._~n of fatty acyl-CoA oxidase activity ysed for activities of catalase and urate oxidase, as well following liver subfractionalion are described in Table as for the peroxisomal /]-oxidation enzymes, fatty acyl- i. The specific activity of this enzyme in the peak CoA oxidase and thiolase. From Fig. 1A, a zone of fraction of the eatalase-negative particle zone is twice particulate material is identifiable at the centre of the that found in the post-nuclear superaatant, and repre- gradient which contains fatty acyI-CoA oxidase and se~.~ a 6-fold enrichment over the post.LMF super- thiolase, but which lacks both urate oxidase and cata- natant starting fraction. The content of these catalase- lase activity, with the bulk of this latter enzyme being negative particles (pooled gradienl fractions) is equiva-

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CmdIAnl vuhm18 - fnomt(~p [ml) IQr~nl vll~. ~ lop lml)

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Fig. 2. Rat-" sedinlcnlafion o[ liver pos[-LMF SUl~Zlzatants fTom a normal mouse arid mice tTeated with ¢lofibxate for the pcn~:ls indicated: A, normal mouse:, B-D, mice txeazcd ".:~.:~ clofih~:¢ ~ 2. days (B), 4. d~Ts (C), 7 days (D). o, cataLase, e, fatty acyl-CoA o'xi.dase; ~,, NADPH-c~ochromec rechtcta~e. 298

TABLE l profile of the microsomal marker, NADPH-eytoehrome Specific acticity of.fatty acyl-CoA o.,:kJase in subcellular fractions from c reductase (Fig. 1A). These particles were not found Iicers of clofibrute.treated mice when post-LMF supernatants from livers of normal Data represent means+_S.D, for three mice. untreated mice were similarly fractionated by rate sedi- mentation, even though microsomal membranes still Fraclion Specific aedvily sedimented to this density region (Fig. 2A). This ab- (AAso2/min/mg protein) sene~ of the particles was confirmed by immune- Homogenate 0.91 ± 0.08 blotting for the bifunetional protein and the membrane Pt~xl-nuclear supernatant 123 ~ 0.02 protein, PMP68, and by immunoeleetronmicroscopy of Light mi~ochondria; fraction l,:~0 ~ 0.l l PogI-LM'F supernatant o (I.38 5- 0.08 relevant gradient fractions (data not shown, but see Peak gradient fraction b 2.40 + 0.53 below). The content of these particles in liver as a function of time on dietary clofibrate was assessed by a LMF= light mitochondrial fraction. b Peak fraction of fatty Bcyl-CoA oxidase a,:tMty in the eatalase- fatty aeyl-CoA oxidase levels in gradient fractions from negalive particle zone. Fatty acyI-CoA oxidase activity in the post-LMF supernations gractionated, as before, by rate post-LMF supcrnatant, and in the pooled catalase-negativ¢ parti- sedimentation. The data obtained (Fig 2B-D) indi- cle zone from the gradient, aCCOunted for 18.1 +4.2% and 7.9"k cated that the particles were induced within 2 days of 2.4%, respectively, of that in the postauclear supernataat fraction. clofibrate treatment, and that their liver content was ReeoveTy of activity in the gradient fractions as a percemage of that in the pOSt-LMF superaatant fractinn loaded on the gradient unchanged by longer treatment times. was 84.5 ± 4.9%. Immunoeleetron microscopy of pooled gradient fractions confirmed the particulate nature of this mate- rial, with the particles being identified as small (ap- lent to approximafely 8% of the fatty acyl-CoA oxidase proximately 0,2 /~ in diameter), anucleoid, and mem- activity present in the post-nuclear supcrnatant. brane-bound (Fig. 3A). The particles also labelled posi- Mierosomat membranes also appeared to cosedi- tively m the matrix for the peroxisomal 0-oxidation ment with these particles, as indicated by the activity proteins, viz. the bifunetional protein, fatty acyI-CoA

D

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Fig, 3, ]mmuno~tochemical toea|izatiun of pcro~somal proteins [n uRrathin c~Ceaeclions of cataLase-ncgaliv¢ pasti¢]c'~. Fractions from a rate sedimentation gradient containing peak activities for ratty acyl-CoA oxidas¢ (see Fig. IA) were pooled and the particles therein sedimented at hig[: spee2 and processed for immunoelectron microscopy, lmraunogold labelling for: A, PMPfig; B, bifunelional protein; C, htty ~eyl-CoA oxidase; D, thiolas¢, Magnification, × 110000. 299 oxidase and thiolase (Fig 3B, C and D, respectively) were negative in each ca~. The secondary particulate but not for catatase (data nol showa). Ccmtro[ immuno- material (peak at 32-34 ml in Fig. IA) contained labelling experiments (i.e., pre-immune rabbit serum: particles which more closely reser0bled the type of absence of primary antibody) were carried out on ~dja- peroxisome usually seen in normal liver, in being larger cent sections for each of the antibodies tc~tcd, and and contair, ing catalase and cores. Furthermore, analy- o[ A 1,24,

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Fig. 4. (A) lso~¢nic sedimentation o[ catalase-negaliv¢ particles. Fractions from a rate-sedimentation gradient comaiu[ng peak aclivities for fatty a~l-CoA o.~ddnse (see Fig. IA) were pooled end centrifuged din.'ctly in a 25-58% (w/w) sucrose gradient. Enzyme acti'virie.~are expressed as the percentage of total gradient activity in each fraction (~lat[w: aclivity), o, fatty acyI-CoA oxidase; r,, NADPH ¢'ytochrome c rcductase; C], protein; •, density. (B) SDS-polyac.,3,1amRl¢gel ¢lectfophoresis of fractions it, ',,'.,~. l';~.:ci~ .,=, ~iaiilcd wJlh Coomassi¢ brilliant blue R. 300 sis of the corresponding gradient volume for the cata- The data of Flatmark et al. [5] are also of interest in lase-negative particles from rate sedimentation gradi- this regard. These authors described the polydispersity ents of post-LMF supernatants from normal mice indi- of rat liver peroxisomcs after clofibrate treatment and cated an absence o~ these types of particles, whereas identified an induced particle of sedimentation velocity again catalase- and core-containing normal type perox- of approx. 870S. Microsomes display S-values of this isomes were identifiable and frequent in the 32-34 ml order [19], hence the cosedimentation with microsomes gradient volume zone (data not shown). of the catalasc-negative particles identified in the pre- Rehomogenization of liver light mitochondrial frac- sent studies would argue for a similar S-value, and tions from elofibrate-treated mice did not result in the therefore size, for these particles [5]. A possible simi- generation of further particles of the nature described larity to the 'micropera~isomes' induced in rat liver by (data not shown). thyroid hormones, is also indicated [20]. To examine more clos,=Lythe apparent cosedimenta- An indication of the origin of the particles was tion of the catalase-negative particles with microsomal reflected in the fact that rehomogenization of LMF membrane, a pooled fraction from a rate sedimenta- yielded no further particles of this kind. Such data tion gradient containing the peak peroxisemal and indicate an initial shearing event at singular sites of mierosomal particles was subjected to isopycnie centrif- mechanical weakness in the extended peroxisomes, and ugation in a 25-58% (w/w) sucrose gradient. The further confirm the general fragility of clofibrate-in- pcroxisomal particles were substantially separated from duced structures. Constrictions and loops are the prob. the microsomal membranes by this procedure, to band- able sites of weakness to shear [2], ing densities of 1.195 amd 1.150 g/ml, respectively The enzyme content of the isolated particles also (Fig. 4A). Analysis of these separated particles by suggests their origin from loops and connections of the SDS-palyacrylam[de gel eleetrophoresis showed pro- peroxisomal redeulum. Notable is the absence of mea- files mare typical of peroxisomal and microsomal pro- sureable urate oxidase and eatalase - a property also teins, respectively [13], although some cross-contamina- noted by Flatmark et al. [5] for their 870S particle tion was still evident (Fig. 4B). population. The fact that these partides are devoid of catalase may be construed as reflecting a complete loss Discussion of this enzyme during the fraetionation procedures. Arguing against this possibility is the evidence that The present studies have identified a subpopulation peroxisomes in fractions prepared from livers of elofi- of peroxisomes in liver homogenates which is induced brate-treated mice, although lealq, with respect to a in mice by treatment with clofibrate, a hypolipidemie substantial portion of ¢atalase, do not relinquish com. drag and peroxisome proliferator. pletely this enzyme, and even after subjection to vigor- To place this finding in perspective, we have found ous treatments like sanitation and extraction in 0.2% it useful to correlate our data with the informative Triton X-100, retain 30-50% of the eatahse in particu- findings by Gorgas [2] on the ultrastructure and organi- late form [8]. The low thiofas¢ levels in the catalase- zation of mouse hepatic peroxisomes. In her analyses, negative regions of the gradient (Fig. 1) are similar to Gorges has described three classes of peroxisomes ex- those in predisassembly control particle~ found by isting in periportal hepatocytes of normal mice: (i) Alexson et al. {21]. Hence some portion, if not all, of large nueleoid-containing organdies, (it) small anu- the thiolase initially present has been retained and the cleoid particles and (iii) tail-like extensions, devoid of absence of catalase would indicate its initial absence - both catalase activity and a crystalline core. From the considering the mild homogenization condition used data presented in this report, it would appear that on here and the similar tendonces of catalase and thiolase the basis of the biochemical findings, the peroxisomal to leak from peroxisomes [21]. Morphometric analyses particles induced by elofibrate relate to the tail-like showing the presence of thiolase in membrane loops extensions identified by Gorges [2], although the size of has not been reported. these peroxisomes analysed morphologically would im- The particulate catalase and urate oxidase that is ply more of a relationship to the second class of found on the gradients (Fig. 1) is not in the vicinity of particles - it should be noted however that the actual the sub-population described, but consists instead of a sizes of the peroxisomal vesicles generated through the concentration at or near the usually quoted [22] iaopyc- disruptien and dispersion of this reticulum by liver hie density of peroxisomes, followed by a diminishing homogenization are unknown. The effect of peroxi- residue in the less dense regions of the gradient. These some proliferation on these three classes has also not particles are believed to result from loose upper layers yet been elucidated, although it is the structures re- of the LMF pellet which are disturbed during the sembling tail-like extensions or membraoed loops which removal of the supetnatant. have been suggested to represent the morphological The fatty a~l-CoA oxidase content of the middle equivalents of peroxisome biogenesis [2,4]. zones of the gradients (Fig. 2B-D, Table I) represents 301 approx. 8% of that assayed in the corresponding PNS References fractions, with a similar level retained at the starting zone of the gradient and 5% ~n the eatalase-positive 1 l..azarow. P.B. and Fuiiki, Y. (1985) Annu. Rev. Cell Biol, 1, particles which have sedimented to the bottom of the 489-530. gradients. The calculated recovc~ of fatty acyI-CoA 2 Gorgas. K (t9851 Anal_ EmbrsoL 172_, 21-32. oxidase in these experiments was approx. 85%. The 3 Yamamoto, K- and Fahimi, H.D. (19~7) J. C¢11 Biol. 105, 713-722. specific activity of fatty acyI-CoA oxidase in the peak ,t Bat~mg:~rt. E.. Vok[ A., Hashimom, 1". and Fahiml. H.D. (198'9) J. Celt Biol. )98, 22.21-~,~31. fraction of the catalase-negative particles indicated a 5 Fla~mark, T., Christian~n, E.N. and KWh. H. (1981) Ear. J. Ceil 6*fold enrichment over the post-LMF superuatant. Biol, 24. 62-69. These particles could be further enriched by separating 6 Klucis. E.. Oane, D. and Masters, C. (1985)Mol. Cell. Bioehem. them from contaminating mictosomal membranes b~' bS, 73-82. isooyenic gradient centrifugation, These data are con- 7 Crane, D.I. and Masters, C3+ (19861Biocllim. Biophy~ Ac*a 876, ~6-2fi3. sistent with an enrichment of a particle type generated 8 Crane, DJ. Z,.amatlia, .I. and Masters, C.J. 11990) Mol. Cell. by the subfractionation procedures, rather than a non- Biochem. %. 153-161. specific aggregation phenomenon. 9 Cr~ne, DL, Hemslcy, A.C. and Masters, C3. (1985) Anal. Our overall assessment of these data, taken in rela- B[ochem 148, 436--445. tion to previous data in this regard, is that the particles t0 Lazarow, P.B. (1978)I. Biol. Chem. ~3, 1522-1528. II Small, (3.M., Burdetl, K. and Connc~l~, Md'. (1985) Biochem. L identified in these studies represent structures of the 227. 205-210. peroxisomal retieulum which are induced by peroxi- 12 Sedmak, J J, and Grossbetg, S.E. (1977) Anal Biochem. 79, some proliferators. These particles are either authentic 544-552. separate peroxisomal particles, or, more likely, parti- 13 Chea. N., Crane, D.L and Mas|c~rs, C.J. (198S)Bioehim, Bioph~, cles which have arisen as a result of shearing from a Aeta t~45, 135-144. 14 Toku':/asu. K.T. (1989) Hisloehcln. J. 21, 163-1"/'5'. larger structure, and which have resealed. In particu- 15 Crane. D.I., Chert, N. and Masters. CJ. (1989) Biochem. Bioph:r~. lar, the presence in these particles of peroxisomal Res. Commnn. 160, 503-508. proteins which are induced by dofibrate, viz. the ~- 16 Price, V.E, Striding, W.R., Tarantola, V.A., Hartley, R.W., Jr. oxidation enzymes and the membrane protein PMP6g and Rech¢inl, M., Jr. (1962) J. BioL Chem. 23?, 3468-3475. [2,3], and the apparent absence of those proteins which 17 Lazarow, P.B. and dn Duve, C. (1973) .I. Cell Biol. 59, 507-524. 18 Miura, S., Mort. M., Takiguehi, M., Tal~ana. M,, Fumta, S.. are not induced or depleted by such treatment (viz. Mi~azawa, S- and H~himato, 1". (1984) J. Biol. Chem. 259. eatalase and urate oxidase, respectively) indicates the 63q7-6402. selective formation of peroxisomal structures enriched 19 Anderson, N.G., Harris, W.W., Barber, A.A., Rankia, C.T. and in the enzymes involved in the fatty acid ,8-oxidation Candler. E.L. (19156) Natl. Cancer Inst. Monograph 21.25:L269. pathway. The availability of new drugs such as BM 20 Just, W.W., HurlS, F.-U. and Schimassek, H. (198z) Fur. J. Cell Biol. 26, 249-254. 15766 which induce peroxisome proliferation without 21 Aleph, S.E.H., Fujiki, Y., Shi~, H, and Lazamw, P.B. (1985) J. concomitant induction of /3-oxidation enzymes [24] Cell Biol. 101, 294-305. should further clarify this mechanism of peroxisomal 22 Crane, D., Holmes, R. and Mas|e~, CJ. (1982) Bib:him. [lio- induction. ph~'s. Acla 712, 57-64, 23 Crane, D.I,. Ches, N. and Mastnt's. CA. (1988) Mol. Cell. Biochem. 81, 29-36. Acknowle:lgement 24 Baur~an, E., Vok), h., Pill, J. and Fahinli, i:l.D. (1990) His- Iochem. I. 21, 163-177. The receipt of financial support from the National Health and Medical Research Cotmcil of Australia is gratefully acknowledged. Virchows Archiv A Cell Plant (1993) Wrchows Archiv A Pathological Anatomy and Histopathology O Springer-Vertag 1993

Establishment of a normal range of morphometric values for peroxisomes in paediatric liver

Jennifer L. Hughesl, Änthony J. Bournel, Alf Poulos2 I Department of Histopathology, Adelaide Children's Hospital, Division ol Women's and Children's Hospital, King William Road, North Adelaide, South Australia 5006, Australia 2 Department of Chemical Pathology, Adelaide Children's Hospital, Division of Women's and Childrens' Hospital, King William Road, North Adelaide, South Australia 5006, Australia

Received May 14, 1993 / Recæived after revision September 14, 1993 / Accepted September 20, 1993

Abstract. The size and number of hepatic peroxisomes Roels 1991). Morphometric data on peroxisomes in var- was investigated in l6 control paediatric liver biopsies ious peroxisomal disorders are now appearing in the ranging in age from 3 months to l8 years$ one fetal liver literature [Roels et al. 1988, l99l ; Hughes et al. 1990, specimen and one paediatric autopsy liver. The area, 1992, 1993 (data to be published); De Craemer et al. diameter, volume density (V"), numerical density (N,) l99la, b]. Some data on control patients have been and surface density (S") of the peroxisomes was recorded published (Table 1). However much of these data re- using randomly selected electron micrographs. The mean late to adult patients and there is a need to compare liver diameter of peroxisomes in control paediatric liver was from peroxisomal disorder patients who are usually aged 0.56 pm, the mean V" was 1.67%, the mean N" was 0. 125 from several weeks or months to a few years, with aged 3 per pm - and the mean S" was 0. I 61 per pm. No correla- matched controls. tion was found between the size and number of hepatic The purpose of this study was to establish a range of peroxisomes and the age or sex of the patient. normal morphometric values for peroxisomes in paedi- Peroxisomes in the fetal liver were smaller than those in atric liver with which to compare liver tissues from pa- biopsy tissue and had a mean diameter of 0.42 pm. tients with peroxisomal disorders and also other disease Peroxisomes were identified in autopsy tissue and were states which may have some peroxisomal involvement. enlarged with a mean diameter of 0.75 pm, most prob- ably due to post-mortem swelling. A range of mor- phometric values in paediatric liver has now been estab- Materials and methods lished. Liver biopsies which had been processed for routine electron mi- Key words: Peroxisomes - Morphometry - Liver croscopic studies were examined in a retrospective study of peroxisome morphology. Sixteen controls were chosen lrom biop- sies which were described as normal by histological criteria. The patients ranged in age from 3 months to I 8 years and were undergo- ing investigations for various conditions not related to peroxisomal Introduction disorders. We also examined autopsy liver from a 4-month-old boy where the postmortem interval was approximately 8-9 h. Autopsy Peroxisomes are important organelles which are most liver from a letus aged 12 weeks was also included. numerous in mammalian liver and are involved in many Liver was obtained by needle biopsy or at autopsy and fixed metabolic functions (Hruban et al. 1972). Their impor- immediately by immersion in a mixture of 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.3. After tance in cellular metabolism has been emphasised by the fixation lor 2 h at room temperature the tissue was washed twice discovery of inherited diseases such as Zellweger syn- in 0.1 M sodium cacodylate buffer, postfixed in l% osmium te- drome, neonatal adrenoleukodystrophy and infantile troxide, washed in cacodylate buffer again and stained en bloc with Refsum's disease, where many peroxisomal functions are 2% aqueous uranyl acetate. The tissue was then dehydrated in a deficient, leading to severe clinical consequences with graded series of ethanol and embedded in Spurr's low-viscosity most patients not surviving the first year (Schutgens et epoxy resin. Polymerisation was car¡ied out at 70o C under vacuum lor at least l2 h. Silver-gold thin sections were cut on a Reichert al. 1986 ; Moser et al. I 991). Hepatic peroxlsomes in these Ultracut microtome, stained with uranyl acetate for 3 min and lead fea- patients show alterations in their morphological citrate for 3 min, and examined in an Hitachi 7000 electron micro- tures: usually a reduction or increase in their size and scope. number (Roels et al. 1988, 1991; Hughes et al. 1990; Twenty electron micrographs of each section of liver were ran- domly selected in relation to the grid bars at a magnification of Correspondence to: J. Hughes x 10000 and enlarged photographically to x 22000. No distinction

Ms. No Author Ms PagesT/-

S prin ger-Verlag, Heidelberg Sttirø AG, Würzburg Provisorische Seitenzahlen / Provisional page numbers 1. Korr nur" 2.7,./4.llt L Table l. Morphometric values lor control liver which appear in the literature

Author Sample Mean diameter Volume density Numerical density Surface density (pm)* (range) (%) (per ¡rm3) (per ¡rm)

DeCraemer et al. l99lb Six adults 0.529 1.06 0.096 0.108 One 6-week-old infant 0.Ms 0.70 0.128 0.08s One 4-month-old inlant 0.529 1.18 0.1 l0 0.131 DeCraemer et al. l99la Eight controls 0.5r7 1.02 0.100 0.107

(0A4s4.620) (0.71-1.41) (0.052-{. 1 3 1) (0.083-{.149) Koch et al. 1978 Four adults 0.97 0.07 Jezequel et al. 1983 Not stated 0.97 0.072 Poll-The et al. 1988 One adult 0.534 One baby 0.478 Roels et al. 1988 One adult 0.534 One 5-week-old lemale 0.478 One 4-month-old male 0.48 r Roessner et al. 1978 Fourteen adults 0.99 Rohr et aI.1976 Four adults 1.2 Sternlieb and Four (12-20 years old) 0.618 Quintana 1977 (0.2r1.34) This study Sixteen paediatric 0.56 1.67 0.125 0.161 (3 months-I8 years) (0.51-o.63) (0.7-3.2) (0.057-{. 188) (0.0734.262)

* d-circle, not corrected for section thickness

was made between periportal and centrilobular hepatocytes. Results A carbon grating replica with 2160 lines per millimetre (Probing and Structure) was photographed and enlarged at the same magnifica- Peroxisomes were readily identifred in liver biopsies as tion as each baich of micrographs and the magnification factor calculated. The area, perimeter and diameter of the peroxisomal single membrane bounded organelles with a homoge- profiles, and the area of the hepatocyte cytoplasm were measured neous moderately electron-dense matrix (Fig. 1a). Elec- by manual tracing on a digitiser tinked to a computer using a Video tron-dense or crystalline cores were not normally Trace soltware package (Leading Edge Technology, Adelaide, Aus- preserlt. Section profiles of the peroxisomes were roughly tralia). The diameter was recorded as the d-circle wtrich is the circular and they ranged in diameter from 0.11 to 1.1I pm. diameter ol the circle having same the area as the measured or- A frequency histogram of the diameter of peroxisomes ganelle. The volume density (V"), numerical densíty (N"), and sur- shown peroxisomes 16 lace density (S,) were calculated using the methods of Weibel is in Fig. 2. The V" of in the (1979). The V, is a measure of the total volume ol the peroxisomes paediatric controls ranged ftom 0.7o/o to 3.2% with a as related to the total hepatocyte volume (including the hepatocyte mean of 1.67'/o. The N" of peroxisomes ranged from cytoplasm and nucleus). This was calculated by dividing the sum of 0.057 to 0. I 88 per pm3 with a mean of 0. 125, per pm3 and the area of all the peroxisomal profrles by the sum of the area of the the surface density of peroxisomes ranged from 0.073 to hepatocyte cytoplasm and nucleus and was expressed as the relative 0.262 per pm with a mean of 0.161 per pm. Y (o/o). " Regression analysis showed there was no correla- The N" is the number of peroxisomes per unit cell volume and that was calculated using the formula of Weibel (1919): tion between the age of the patient and the mean area of peroxisomes (R2 : l.(Nn)./r_,- 0.1"/,). rìv-p_ N peroxisomes easily be ^, (Vv)li2 In the fetal specimen the could recognised and were slightly smaller in mean area than where k is the size distribution coeflficient, B is the shape related in biopsy livers (Fig. lb, Table 2). In the autopsy liver the coefftcient as determined according to Weibel (1979), and N¡ is the peroxisomes were larger than in the biopsy and fetal ratio between the number of peroxisomes and the total area of the hepatocytes. livers and had a flocculent, extracted appearance (Fig. lc). The S" is the total membrane area of the peroxisomes expressed Profiles of smooth endoplasmic reticulum were often per cellular volume and was calculated lrom the formula: flattened and condensed along the peroxisomal mem- 4Bn brane in autopsy tissue (Fig. lc). s, -- peroxisomes, mean dia- 1l The mean area of and the meter, the volume, numerical and surface densities in l8 where B^ is the ratio between the sum of the peroxisome perimeters control liver specimens are shown in Table 2. and the total a¡ea of the hepatocytes. The results were not corrected for section thickness, since the sections used were all of similar thickness according to the inter- Discussion ference colours on the ultramicrotome during sectioning. The diameter of peroxisomes in the control liver biopsies in this study is similar to those which already appear in -.'f J ,qtL ç 7 , -.4f :'+ F ).

K

,'' ';

¿ 1,1 1,

l:*?

Fig. 1a-<. Peroxisomes (P) in (a) { control paediatric liver biopsy, (b) .'.| -r.- letal liver and (c) autopsy liver. :tÊ crË x 40000

Frequency Histogram CONTROL I the literature (Table 1). In particular there appears to be 200 little difference in the mean diameter of peroxisomes in liver from adult patients and the liver of paediatric pa- tients. In two infants aged 5 weeks and 4 months, Roels 150 et al. (1988) found peroxisomes with a mean diameter slightly less than that of the adults. De Craemer et al. (l99la, 1991b) also reported slightly smaller peroxisomes in one of two infant biopsies. This patient rco [and one of Roels et al. (1988)l was only 5 weeks old. We did not have any patients this young in our control group. The relative V", N" and S" reported in this study are 50 somewhat higher than the data reported in the literature. This may be because our control tissues are from infants and children, compared with adult liver which has been

o reported in most studies. It is also possible that some of the control patients in this study had been treated with Psroxisomal diam€t€r um drugs, or suffered other conditions, which may have Fig. 2. Frequency histogram of peroxisomal diamete¡ in control caused an increase in the number of peroxisomes. Unfor- paediatric liver biopsies tunately it is not possible to obtain liver biopsies from (

Table 2. Morphometry of peroxisomes in control paediatric liver

Case Age n Mean area SD Area range Mean diamete¡ Volume density Numer density Surlace density (pm') (rm') (d-circle) (pm) (%) (per pm3) (per ¡rm)

I 3 months 67 0.22 0.09 0.07-o.52 0.56 I .'7 0.128 0.165 2 4 months 77 0.21 0.t2 0.0H.64 0.56 2.0 0.t52 0.196 3 ó months 93 0.29 0.17 0-008-{.77 0.63 3-Z 0.1 54 0.262 4 7 months 54 0.17 0.08 0.002-{.37 0.51 l.l 0.t22 0.125 5 9 months 3l 0. l9 0.08 0.08-{.40 0.53 (,. / 0.064 0.073 6 9 months 58 0.18 0.08 0.03-o.34 0.53 1.1 0.1 l2 0.122 '7 2l months 80 0.21 0. 14 0.001-o.56 0.55 2.1 0.163 0.194 8 30 months 27 0.22 0.09 0.00H.36 0.59 0.8 0.057 0.076 9 42 months 54 o.2t 0.11 0.003-{.,{4 0.56 1.3 0.100 0.126 l0 5 years 56 0.23 0.09 0.oil.44 0.58 t.4 0.098 0.135 ll 8 years 80 0.24 0.12 0.008-{.51 0.58 2.3 0.148 0.212 t2 9 years 104 0.22 0.09 0.0ñ.47 0.58 2.6 0.188 0.252 13 I I years 86 0.21 0.08 0.00H.46 0.53 1.9 0.160 0.196 l4 13 years 58 0.24 0.15 0.004-4.96 0.62 1.6 0.103 0.149 l5 13 years 55 0.20 0.09 0.07-{.46 0.54 1.4 0.117 0.138 l6 18 years 7t 0.20 0.09 0.02-{.43 0.54 1.5 0.133 0.1 58 Mean 60 0.22 0.001-{.96 0.56 r.67 0.125 0.t61

12 weeks 51 0.1 I 0.04 0.03-{.23 0.42 0.63 0.1 28 0.088 8¡, 4 months 39 0.37 0.14 0.144.77 0.75 1.6 0.056 0.1 26 " Fetal b Autopsy

genuinely "normal" infants. However, it is important to at the lower end of the range for biopsy liver. In human establish a ràÍLge of morphometric values from these fetal kidney, the mean size of peroxisomes was less than paediatric patients with which to compare liver biopsies that of adults, and there was little change during the from peroxisomal disorder patients. lOth-18th weeks of gestation (Briére 1986). This suggests The identification of peroxisomes in this study was that a significant increase in the diameter of peroxisomes based on purely morphological features. We considered must take place after the 18th week of gestation or after using tissue which had been cytochemically stained for birth to reach the level of the adult. Similarly in human catalase to identify peroxisomes (Novikoff and Gold- fetal liver an increase in peroxisomal size takes place fischer 1969; Fahimi 19751' Roels et al. 1975), as a during development (Kerckaert 1990, as quoted in Espeel proportion of the smaller peroxisomes may be missed by et al. 1990 and Roels l99l). Espeel et al. 1990 found an using morphology alone (Beier and Fahimi 1986). How- increase in the size of hepatic peroxisomes between the ever, it would not be possible to use retrospective liver 7lh and l8th gestational weeks. The number of biopsies if this was the case, as all our liver biopsies have peroxisomes has been reported to be higher in younger been processed using standard electron microscopic fetuses (8-10 weeks) and then falls abruptly from 12 techniques. Also in biopsies from patients with peroxiso- weeks onwards (Kerckaert 1990, as quoted in Roels mal disorders abnormal peroxisomes need to be iden- reel). tified. Often these peroxisomes have low levels of catalase In rat liver the volume of individual hepatocytes in- [Roels et al. 1986; Hughes et al. 1992, 1993 (data to be creases with age and variations occur in different or- published)l and the enzyme cytochemical technique may ganelles (Schmucker 1990). The data concerning not detect them. Beier and Fahimi (1992) have recently peroxisomes are very limited and somewhat inconsistent. reported the use of immunocytochemical techniques in However, it appears thai ageing has little effect on this conjunction with automatic image analysis for identify- organelle in rat liver, but during fetal and postnatal ing peroxisomes in rat liver. This technique is also unsuit- development some changes do occur. In mouse hepa- able for human liver biopsies as special fixation and tocytes the V" of peroxisomes increases 2- to 3-fold be- processing is required. The figures obtained in this study, tween birth and 10 days of age (Kanamura et al. 1990) and where peroxisome identifrcation relied on morphology in rat hepatocytes the size and number of peroxisomes only, are actually greater than most of those previously increases gradually during fetal development (Stefanini reported. We therefore feel that the use of retrospective 1985). Also in the liver of baby pigs the number of tissue which has been routinely processed for electron peroxisomes doubles from I to 4 weeks of age (Giesecke microscopy has not had a significant effect on the values et al. 1987). Our data show there is no correlation be- of V", N" and S,. tween the size or number of hepatic peroxisomes and the The mean peroxisomal diameter in fetal liver was age of the patient. However, since our youngest patient smaller than that found in biopsy liver. The V", N" and was 3 months old, it is possible that changes may occur S" of peroxisomes in the fetal specimen in this study were from birth to 3 months. f The mean diameter of the peroxisomes in the autopsy Jezequel AM, Librari L, Mosca PG, Novelli G (1983) The human tissue was considerably enlarged compared to biopsy liver in extrahepatic cholestasis: ültrastructural morphometric liver. This difference is most likely due to postmortem data. Liver 3:303-314 Kanamura S, Kaai K, Watanabe J (1990) Fine structure swelling of the peroxisomes. The peroxisomes have a and func- tion of hepatocytes during development. J Electron flocculent empty-looking matrix (Fig. as rhough Microsc lb) Tech 14:92 105 swelling and extraction of the contents has taken place. Koch MM, Freddara U, Lorenzini I, Giampieri MP, Jezequel AM, Although peroxisomes can readily be identified in au- Orlandi P (1978) A stereological and biochemical study ol topsy tissue, their morphometric analysis can produce the human liver in uncornplicated cholelithiasis. Digestion unreliable results due to postmortem swelling of thc t8.,162-177 organelles with a subsequent increase in diameter. Moser HW, Bergin A, Cornblath D (1991) Peroxisomal disorders. Biochem Cell Biol 69:4634'74 Novikoff AB, Goldfischer S (1969) Visualization of peroxisomes Acknowledgements. 'We are grateful Helen to Thiele and Ruth (microbodies) and mitochondria with diaminobenzidine. J His- Williams for technical assistance in electron microscopy. tochem Cytochem l'7 :67 5480 Poll-Thé BF, Roels F, Ogier H, Scotto J, Vamecq J, Schutgens RBH, Wanders RJA, Van Roermund CWT, Van Wijland MJA, References Schram A'W, Tager JM, Saudubray jM (1988) A new peroxiso- mal disorder with enlarged peroxisomes and a specific deficiency Beier K, Fahimi HD (1986) Ap image analy- ol acyl-CoA oxidase (pseudo-neonatal adrenoleukodystrophy). sis for morphometric studie ed cytochem- Am J Hum Genet 42:422434 ically for catalase. L Elec lication. Cell Roels F (199 1) Peroxisomes. A personal account. VUB Press, Tissue Res 246:635-640 Brussels Beier K, Fahimi HD (1992) Application of auromatic image analy- Roels F, Wisse E, dePrest B, Meulen J van der (1975) Cytochemical sis lor quantitative morphological studies of peroxisomes in rat discrimination between catalases and peroxidases using diami- liver in conjunction with cytochemical staining with 3-3'-diami- nobenzidine. Histochemistry 4l :281-312 nobenzidine and immunocytochemistry. Microsc Res Tech Roels F, Cornelis A, Poll-Thé BT, Aubourg P, Ogier H, Scorto J, 2l:27 l-282 Saudubray JM (1986) Hepatic peroxisomes are deficient in in- B¡iére (1986) N Peroxisomes in human loetal kidney: variations in fantile Refsum disease: a cytochemical study ol 4 cases. Am J size and number during development. Anat Embryol (Berl) Med Genet 25:257-271 174:235-242 Roels F, Paumels M, Poll-Thé BT, Scotto J, Ogier H, Auborg P, De Craemer D, Kerckaert I, Roels F (l99la) Hepatocellular Saudubray JM (1988) Hepatic peroxisomes in adrenoleukodys- peroxisornes in human alcoholic and drug-induced hepatitis: trophy and related syndromes: cytochemical and morphometric a quantitative study. Hepatology l4: 8 I l-817 data. Virchows Arch [A] 413:275-285 De Craemer D, Zweens MJ, Lyonnet S, Wanders RJA, Poll-Thé Roels F, Espeel M, De Craemer D (1991) Liver pathology and BT, Schutgens RBH, Waelkens JJJ, Saudubray JM, Roels F immunocytochemistry in congenital peroxisomal diseases: (l99lb) Very large peroxisomes in distinct peroxisomal disor- a review. J Inherited Metab Dis 14:853 875 ders (rhizomelic chondrodysplasia punctata and acyl-CoA ox- Roessner A, Kolde G, Stâhl K, Blane G, Husen N van, The- idase deficiency): novel data. Virchows Arch [A] 419 523-525 mann H (1978) Ultrastructural morphometric investigations E,speel M, Jauniaux E, Hashimoto T, Roels F (1990) Immunocyto- on normal human liver biopsies Acta Hepato gastroenterol chemical localization of peroxisomal B-oxidation enzymes in 25: l19-123 human letal liver. Prenat Diagn l0:349 357 Rohr HP, Luthy J, Gudat F, Obe¡holzer M, Gysin C, Bianchi L Fahimi HD (1975) Fine structural cytochemical localization ol (1976) Stereology of liver biopsies lrom healthy volunteers. peroxidatic activity ol catalase. In: Glick D, Rosenbaum RM Virchows Arch [A] 371:251 263 (eds) Techniques ol biochemical and biophysical morphology, Schmucker DL (1990) Hepatocyte fine structure during maturation voI 2 J Wiley New York, pp 19'7-245 and senescence. J Electron Microsc Tech l4: 10Ç125 postnatal Giesecke D, Pospischil A, Laging C, Heiss G (1987) Schutgens RBH, Heymans HSA. Wanders RJA, Bosch H van den, development ol hepatic uricase and peroxisomes in baby pigs Tager JM (1986) Peroxisomal disorders: a newly recognised Comp Biochem Physiol 88:999-1003 group ol genetic diseases. Eur J Pediatr 144:430440 Hruban Z, VigìI EL, Slesers A, Hopkins E (1972) Microbodies. Stefanini S ( 1985) Differentiation ol liver peroxisomes in the loetal Constituent organelles of animal cells. Lab Invest 27: l8+l9l and newborn rat. Cytochemistry of catalase and o-aminoacid Hughes JL, Poulos A, Robertson E, Chow CW, ShefÍield LJ, Chris- oxidase. J Embryol Exp Morphol 88:151-163 todoulou J, Carter RF ( I 990) Pathology of hepatic peroxisomes Sternlieb I, Quintana N (1977) The peroxisomes of human hepa- and mitochondria rn patients with peroxisomal disorders. Vir- tocytes. Lab Invest 36:140-149 chorvs Arch l{l 416:255-264 Weibel ER (1979) Stereological methods, vol l. Practical methods Hughes JL, Poulos A, Crane DI, Chow CW, Sheffield LJ, Sillence lor biological morphometry. Academic Press, London D (1992) Ultrastructure and immunocytochemistry ol hepatic peroxisomes in rhizomelic chondrodysplasia punctata. Eur J Pediatr 151:829 836 Virchows Archiv A (1993) Virchows Archìv A Pathological Anatomy and Histopathology O Springer-Verlag1993

Morphometry of peroxisomes and immunolocalisation of peroxisomal proteins in the liver of patients with generalised peroxisomal disorders

Jennifer L. Hughesl, Denis I. Crane3, Evelyn Robertson2, Alf Poulos2 1 Department of Histopathology, Adelaide Children's Hospital, Division of Women's and Children's Hospital, North Adelaide, South Australia 2 Department of Chemical Pathology, Adelaide Children's Hospital, Division of Women's and Children's Hospital, North Adelaide, South Australia 3 Faculty of Science and Technology, Griffrth University, Queensland, Australia

Received May 14, 1993 / Accepted August 19, 1993

Abstract. Hepatic peroxisomes were studied by mor- Introduction phometric and immunocytochemical techniques in con- trol patients and in four Zellweger syndrome patients, Peroxisomal disorders are a group of iqherited diseases two infantile Refsum's (IRD) patients, one neonatal affecting children which result in severe clinical conse- adrenoleukodystrophy (NALD) patient, and three pa- quences including abnormalities of the liver, kidney, tients with peroxisomal disorders (PD) which do not fit brain, adrenal and bone. The disorders are characterised any currently recognised clássification, but have disor- biochemically by a defrciency of one or more peroxisomal ders involving a defect in peroxisomal biogenesis. enzymes, and an accumulation of substrates in the pa- Peroxisomes which were ultrastructurally abnormal and tient's tissues and plasma (recent reviews see Moser et greatly reduced in size and/or number were found in two al. l99l; Wanders et al. 1991; Wilson 1991). The of the Zellweger syndrome,patients, and the NALD and peroxisomes in the liver of these patients show alterations IRD patients. There was variation in their numerical in their size, number and ultrastructural appearance density ranging from none at all in two of the Zellweger ranging from a complete absence of hepatic peroxisomes syndrome patients to normal numbers in the IRD pa- in some Zellweger patients (Goldfischer et al. 1973) to tients. In most patients there was a decrease in the im- extremely enlarged, flocculent organelles in some munolabelling of catalase over the peroxisomes. In the rhizomelic chondrodysplasia punctata patients (De Zellweger syndrome and NALD patients, the small, ab- Craemer et al. l99l; Hughes et al. 1992). Small, abnor- normal peroxisomes did not label for any of the p-oxida- mal peroxisomes have been found in small numbers in tion proteins. The IRD patients and the PD patients the liver of some patients with generalised peroxisomal however, were heterogeneous with respect to p-oxidation disorders i.e. Zellweger syndrome, neonatal adreno- labelling. The ultrastructural heterogenity of peroxi- leukodystrophy (NALD) and infantile Refsum's disease somes in these peroxisomal disorders patients indicates (IRD) Goldfischer et al. 1985; Roels et al. 1986; Vamecq there may be genotypic differences between the major et al. 1986; Hughes et al. 1990). These patients have a groups and also within each group. The common factor deficiency of multiple enzyme functions. in all the patients in this study where peroxisomes were Ultrastructural immunolabelling of peroxisomal present was the presence in the hepatic peroxisomes of enzymes in liver biopsies has been demonstrated by Lit- an electron dense centre which did not label im- win et al. (1987, 1988) in both Lowicryl sections and munocytochemically for catalase or the B-oxidation en- Epon sections after etching with ethanolic sodium hy- zymes. This electron dense centre may indicate a struc- droxide. The technique has been applied to human post- tural abnormality in the peroxisomes in these patients. mortem liver and human fetal liver for the detection of the peroxisomal p-oxidation enzymes (Espeel et al. Key words: Peroxisomal disorders - Immunocytochemis- 1990a, b). try - Zellweger syndrome - Neonatal adrenoleukodys- In the present study we have used a similar on-grid trophy - Infantile Refsum's disease immunogold technique to localise the peroxisomal ma- trix protein catalase; the p-oxidation enzymes; acyl-CoA oxidase, bifunctional protein, and 3-ketoacyl-CoA thiol- ase; and a 68 kDa peroxisomal integral membrane pro- tein (PMP68). Liver from five control pationts, and pa- three Correspondence to: J.L. Hughes, Department of Histopathology, tients with Zellweger syndrome, NALD, IRD, and Adelaide Children's Hospital, Division of Women's and Children's unclassified peroxisomal disorders (PD) was inves- Hospital, King William Rd., North Adelaide, South Australia 5006 tigated. The results indicate that the peroxisomal mor-

Ms. No. 4% Author Hq6 þ/Er Ms. 4-jcl pages n- 40 Springer-Verlag, Heidelberg I H. Srürrz AG, Würzburg Provisorische Seitenzahlen / Provisional page numbers 1. Korr nur" .1.4: /!,?3 J,

T¡ble l. Clinical leatures Condition IUGR FTT HePato- Dysm. F Ret. Pigm. Deaf Alive/ Stippled Hypo- Fits Devt. Periph. megaly or Cataract Deceased Epiphysis tonla Delay Neuro- pathy

zsl + + N.R. N.R. Died4ll2yr + + + zsz l l (fetus TOP) + + + + Died 8/12 yr + + + zs3 + zs4 + + + + N.R. N.R. Died 4ll2 yr N.R. N.R NALD + + ++ + + + + Died 3.5 yr + + + IRDI + + ++ + + + + Died 2 yr + + + + + + + + + Alive I I yr + + IRD2 + PDI prop + N.R. N.R Died 5/12 yr + + PDl l I l (fetus TOP) + PD2 + Died 1.5 yr' 1 + ++ PD3 + + Alive 7 yr l Adrenoleukodystro- IUGR, Intra-uterine growth retardation; FTT, failure to thrive; ZS, Zellweger syndrome ; NALD, Neonatal PD, Peroxisomal Disorder, Dysm. F, dysmorphic features; RerPigm., Retinitis Pigmentosa; phy; IRD, Infantile Refsum's Disease; Dêvt. Delay, deveiopmental delay; Periph. Neuropathy, peripheral not otherwise classiûed neuropathy; N.R., not recorded " accidental death

Table 2. Biochemical ûndings

Case Plasma Cultured skin fibroblasts c26122 c24122 Phytanic c26122 Phytanic DHAP_AT Catalase acid acid oxidase %sedimentable

zsl Elevated Elevated Elevated Decreased Reduced Decreased ZS2" Reducedb zs3 Elevated Eleváted Norrnal Elevated Reduced zs4 Elevated Elevated Elevated Reduced NALD Elevated Elevated Elevated Decreased Reduced IRDI Elevated Elevated Elevated Elevated Decreased Reduced Decreased IRD2 Elevated Elevated Elevated Decreased Reduced PDld Elevated Elevated Norrral Elevated Normal PDI. Elevated" Decreased PD2 Elevated Elevated Elevated Normal Decreased Normal PD3 Elevated Elevated Elevated Decreased Normal

, Fetal cultured am¡iotic cells d b Chorionic villus direct and chorionic villus cultured cells Findings in the propositus and Not measured " Chorionic villus direct and chorionic villus cultured cells, -,

phology and subcellular localisation of peroxisomal pro- syndrome, IRD and NALD have been reported earlier ieins iiheterogeneous within the generalised peroxisomal (Poulos et al. 1988). prenatal A disorders. PD- fetal tissue was received after assessment Control tissue was obtained from three patients un- of a plegancy at risk. Chorionic vilius biopsy and cul- dergoing biopsy for conditions not related to peroxiso- tured amniotic cells showed an affeÆted child. Clinical the maa disorders. Autopsy tissue was obtained from one features of the index case are shown in Table 1, and patient without a peroxisomal disorder where the post- biochemical information relating to the index case and was mortem interval was approximately 8-9 h. Control fetal to the fetus are in Table 2. Tissue from the index case tissue was obtained from a 17-18 week old fetus. not available for ultrastructural examination. The clinical features of the in this study are documented in Table Materials and methods details of three of 1n y have been published elsewhere (IRD2 see Hughes et al. 1 990 and Eleclron mic at needle bioPsY or at Robertson et al. 1988; NALD see patient 3 in Hughes et autopsy and ion in a mixture of 4% al. 1990; PD3 see patient 2 in Hughes et al. 1990). The formãlâehyd I MsodiumcacodYlate the clinical criteria employed for the diagnoses of Zellweger buffer at pH room temPerature, oc.J- +Ke d.+ct^\* 4 {hc-- \¡¡eq\sc"'-t cd q- cr

Resin embedding. After fixation the tissue was rinsed in phosphate buffer containing 50mM ammonium chloride, dehydrated in etha- and embedded in Lowicryl Kl lM resin at C. Polymerisa- nol -25o small, morphologically abnormal structures were iden- tion was carried out by UV irradiation at C lor several days. -25" liver (Fig. 1b, c). These Silver-gold sections were mounted on celloidon coated nickel grids. tified in biopsy, autopsy and fetal organelles were surrounded by a single membrane and Antisera. Antisera to catalase, bifunctional protein and PMP68 contained electron dense centres which often filled the were raised and purified as previously described (Hughes et al. entire organelle except for a clear area just inside the 1992). The PMP68 lrom mouse liver is homologous to PMP70 from membrane. The area and diameter of these structures rat liver and human liver. Chen and Crane (1992) compared a was greatly reduced compared to normal peroxisomes, as partial cDNA for mouse PMP68 with rat PMP70 (Kamijo et al. surfa*ce densities (see 1990) and found 95% nucleotide homology and 98% amino acid was their volume, numerical and homology. Table 3). In the two patients to whom a clinical diagnosis Antisera to ¡at liver peroxisomal 3-ketoacyl CoA thiolase and of IRD had been assigned, small, abnormal organelles acyl-CoA oxidase weÍe generous gifts from Professor T. Hashimo- with a similar ultrastructural appearance were seen. to, Shinsu University, Japan. These structures were also reduced in size from normal peroxisomes, however their numerical densities were sim- The grids containing the sections were washed in Immunolabelling. patients. three washes of phosphate buffered saline with 50mM glycine lor ilar to those of noünal peroxisomes in control 15 min followed by three washes of D&WB for 20 min. D&V/B The volume and surface densities were reduced. In the consisted ofphosphate-buffered-saline pH 7.3 containing 0.5% ov- IRD2 patient, a few organelles which approximated the gela- albumin, 0. 1 % gelatin, 0.05 % Tween 20 and 0.2% Teleostean size of noÍnal peroxisomes were also present in some primary diluted in tin. They were then incubated in the antibody cells. These peroxisomes also had an electron dense cen- D&WB for 16 h zt4o C. The grids were washed in six washes of tre along with some finely granular matrix of normal D&WB lor a total time of 30 min and then incubated in a protein A gold conjugate (11 nm) lor 60 min at room temperature. The appearance. protein A gotd conjugate was diluted in D&WB to an optical ln the biopsies from the PD2 and PD3 patients, sim- density at 520 nm of 0.14. Following this the grids were washed in ilar small, peroxisomes with electron dense centres were D&WB for a further 30 min, washed in clean water and then seen. The size of the peroxisomes was reduced in these counterstained with uranyl acetate lor 30 s and lead citrate for l5 s' two patients (0.03 pm2 and 0.06 pm2 respectively) and out on adjacent sections by omitting the Controls we¡e carried surface densities were reduced. How- primary antibody, and by incubating in normal rabbit serum in- their volume and patients, the numerical stead of the primary antibody. ever in these cases, as in the IRD When quantitation was to be carried out, grids containing sec- density was within normal limits (see Table 3 and Fig. 2). tions ol each liver sample were immunolabelled at the same time in The other unclassified peroxisomal disorder patient order to reduce variations due to different batches of antibody or PDl had peroxisomes which were nonnal in size, vol- gold probe. ume, numerical and surface densities. The peroxisomes in this patient did vary from normal in that they also had Quantitalion of immunogold labelling. Twenty electron micrographs lrom sections of liver which had been similarly treated were ran- electron dense centres (Fig. ld). domly selected. Five micrographs ol each of the control sections were selected to determine the background density. The labelling Immunocytochemistry.In control liver the labelling for density (gold particles per ¡rm2) was calculated by measuring the catalase was evenly distributed over the peroxisomal particles area of the peroxisomes and counting the number ol gold matrix with an average labelling density of 203.1 gold peroxisome. For quantitation of the peroxisomal mem- over each particles per pm2. In the fetal control tissue the labelling brane protein, the number ol gold particles p€r pm of membrane gold particles was measured. The background density calculated from the con- lor catalase was much less than this (62.68 trols was automatically subtracted. A Hewlett-Packard 9847 A sorder Patients in this digitizer linked to a Model 9816 microcomputer was used for these considerablY reduced evaluations. The results were compared using a Student's / test. he PDI Patient where age-matched control Measuremenls of peroxisomal size and number. Morphometry was Zellweger NALD andPD2 carried out as described earlier (Hughes et al. I 993). In brief, twenty fetal tissue. In the syndrome, electron micrographs oleach section of liver were randomly selected patients, there was no labelling for catalase over the tl

I

liver from the neonatal adrenoleukodYstroPhY (NALD) Patient' c. Small' àbnormat peîoxisome (P) tn the liver ol a fetus with Zellweger sYndrome. d. Peroxisome þ) in the liver olPDl Patient' These peroxisomes are normal in size and number but have an electron dense centre. x 40,000

(d-circle in (Ém2) + standard deviation, the mean diameter age.of, the patient, the mean area Table 3. Morphometry of peroxisomes' The (Pm-l of hepatic density (¡rm-3) and the surface densitY ) Peroxisomes pm), the retative volume density (%), the numerrcal sv Diameter Nv Patient Age Area 0.035 0.040 0.06 0.49 0.38 17 wks 0.19 + 0.056 0.126 Controlb 0.69 1.6 Control" 4 mths 0.37 + 0.14 r.67 0.125 0. l6l 0.22 + 0.1 I 0.56 (0.0?3-{.262) Control" (0.'7 3.2) (0.057-o.188) (0.17-o.29) (0.51-{.63) Range (3 mth-18 Yr) 0.003 0.01 0.014 wks 0.03 + 0.008 0.20 0.0M ZSI 2 0.02 0.021 0.03 + 0.009 0.20 peroxtsomes ZSzh 12 wks No peroxtsomes No peroxisomes No No peroxtsomes No peroxisomes peroxisomes zs3" 8 mths peroxrsomes No peroxisomes No No peroxisomes No peroxtsomes No ZS4" 3 mths 0.012 0.03 0.051 0.03 + 0.02 0.20 NALD 2 yrs 0.013 0.0s 0.065 5 mths 0.03 + 0.008 0.20 0.029 IRDI 0.26 0.13 0.122 IRD2 5 yrs 0.04 + 0.03 0.8 0.08ó O.OEó wks 0.17 + 0.12 0.47 0.020 PDIb l7 0.09 0.065 0.05 + 0.04 0.25 PD2 l l mths 0.078 0.02E + 0.03 0.28 0.2 PD3 2 yrs 0.06 et al. 1993) of l6 control pediatric liver biopsies (see Hughes b letal tissue " mean " autopsy tlssue I

Fig. 2. Hepatocyte from patient PD2 showing normal numbers of small perolisomes with election dense centres. x 8,750

integral membrane protein Table 4. Immunocytochemistry ol peroxisomal enzymes in control CoA thiolase (Thiolase); and the partictes per of peroxisomal mem- and patient livers. Labelling density (number of gold particles per PMP68 (number of gold Pm brane) + standard deviation ¡rm2 of peroxisome) for the matrix enzymes catalase, acyl-CoA bxidase (nConO*¡, bifunctional protein (BFP) and 3-ketoacyl- Thiolase PMP68 Case Catalase A-CoAOx BFP

58.55 0.70 + 0.54 c2b 62.68 + 39.60 46.23 +23.89 25.99 L10.99 82.'.|2 + 1 73.19 3.28 +2.15 C4 189.42 + 139.88 106.15 +88.28 49.14 +87.39 15.75 + 1.14 +0.81 C5' 180.00 + 122.17 92.85 + 51.53 34.53 +40.10 r 39.31 + 91.77 84.39 1.67 +1.04 cl8 220.',17 +128.43 72.46 +49.73 30.74 + 30.05 t 19.36 + 2.33 + 1.68 ct9 222.22 +148.68 94.43 +76.29 46.21 +35.59 141.59 + 94.86 0.00. ZSI 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" ZSzb px not found zs3^ px not lound px not found px not found px not lound px not lound zs4" px not found px not found px not found px not found 0.00" NALD 0.00" 0.00" 0.00" 0.00" 1" 7.8 I 0.31" + 0.71 IRDI 2.90" + 5-30 0.00'+ 0.00 3.24" +10.31 2.7 +. 181.43 1.65 + 1.28 IRD2 31.52" + 50.52 92.11 +88.56 34.44 +41.9'l 210.32d+ 2.56 +1.65 PDIb 143.69d+ 60.64 93.66d +44.77 l 14. l 3d + 40.1 135.96d+ 19.15 3.76d+2.83 PD2 0.00. 0.00" 0.00" 0.00" 2.'18 +1.29 PD3 2.93. + 7.22 52.62 +62.48 0.00" 430.75d+ 805.44 1.52 + 0.40 PD3. 72.27 + 62.'77 43.93 +23.37 27.'1't +12.73 48.27 + 31.10 p<0.001 " Population of large Peroxisomes " Autopsy tissue " Signiûcantly decreased at b Fetal tissue d Signifrcantly increased at p<0.001

syndrome, abnormal peroxisomes. However in the IRD and idase was virtually undetectable for Zellweger small, for the IRD2 PD3 patients some labelling was found although greatly NALD and IRD1 patients, but normal acyl-CoA ox- in densitY. patient. In the PD patients, labelling for reduced normal In control liver the label for all three p-oxidation idur. *ut zero for PD2, in"t"used for PDl, and or decreased for PD3 (see next paragraph). A similar result was observed for the labelling of bifunctional pro- tein, although a very small amouni of labelling for this enzyme waialso present in the IRDI patient' The label- ling density for thiolase was zero for the Zellweger syn- patients a very p-oxidation enzymes was much less in the control fetal drõme and NAID patients. In the IRD present one patient (IRDI) tissue (Table 4). The labelling density for acyl-CoA ox- small amount of labèl was in 6

IR,D2 ln f'

i. -q*' Fig. 3. Immunolabelling of thiolase in peroxisomes from a f¡i'a'' patient with IRD. ¡. A small peroxisome with an electron

I dense centre which is relatively free ol label. The labelling Þ. density of thiolase is increased and labelling is concentrated t. in the outer rim ol matrix. b. Larger peroxisome from the samc patient with label for :s'.of" thiolase over the matrix but excluding the electron dense 3o thiolose 3b ..^'th centre. x 96,000

:t:RÞ3 ¡.d' PD3

Fig. 4. Immunolabelling of thiolase in patient PD3. a. A small peroxisome with an electron dense centre which is relatively free of label. The labelling for thiolase is concentrated in the outer rim tr' of matrix just inside the membrane. b. A large peroxisome of normal morpholo gical appearance which also labels for thiolase although the labelling density h¡ is decreased. x 64,000 but in the other patient (IRD2) the labelling density for In the PD3 patient two populations of peroxisomes thiolase was increased approximately two fold (Table 4 were identified in the tissue examined for im- and Fig. 3a). In this case the labelling for thiolase was munocytochemistry. Small peroxisomes with electron concentrated in the thin "halo" just inside the membrane dense centres were found which showed very reduced of the small peroxisomes. Labelling was not present over labelling density for catalase and no labelling for bifunc- the electron dense centres of these organelles. In this tional protein. These small peroxisomes however had a patient some peroxisomes were almost normal in size, normal labelling density for acyl-CoA oxidase and a but still had an electron dense centre. In these larger greatly increased labelling density for thiolase. As in the peroxisomes there was littte label over the electron dense IRD2 patient the labelling for thiolase änd acyl-CoA centre even though there was labelling of the matrix oxidase was concentrated in the narrow halo just inside (Fig. 3b). This phenomenon was also observed in the the peroxisomal membrane (Fig. 4a). PDI patient, where peroxisomes of normal size and num- Larger peroxisomes with a normal ultrastructure were ber had electron dense centres which did not label with also seen in this PD3 patient. This population of or- any of the antibodies used. The rest of the matrix in these ganelles had a reduced labelling density for catalase, peroxisomes had a normal appearance and did label for acyl-CoA oxidase and thiolase but the labelling density the matrix proteins. for bifunctional protein was normal (see Table 4 and +

.':i PD2,

Fie. 5. Immunolabelling of PúP68. a. In control liver the label is concentrated in the oeroxisomal membrane. t. A small peroxisome with an electron dense centre in an unclassified Peroxisomal disorder (PD2). These oeroxisomes did not labcl for ätulut. or any of the p-oxidation enzymes. x 87,000

the control group is indicated as well as the and structure ol the peroxisomes relative to Tabte 5. SummarY of results' The change in size grouP of peroxisomal disorders labelling densitY of each of the antibodies for each PMP68 A-CoAOx BFP Thiolase Group PX size PX structure Catalase Not detectable Not detectable Not detectable Not detectable Not detectable ZS Reduced" Abnormal Not detectable Not detectable Not detectable Not detectable Not detectable NALD Reduced Abnormal Reduced/increased Reduced/normal Reduced Reduced/normal Reduced/normal IRD Reduced Abhormal Increased Increased Increased Increased Increased PDI Normal Abnormal Not detectable Increased Not detectable Not detectable Not detectable PD2 Reduced Abnormal Increased Normal Reduced Normal Not detectable PD3 Reduced Abnormal Reduced Normal Reduced Normal PD3L Normal Normal Reduced

were detected " In patients where peroxisomes

Discussion Fig. Peroxisomes identified inih ng density for PMP68' nature. thus- - I tt tttt control livers the label for PMP68 was found over the membrane of the p€roxisomes with an average gold particles per pm of p€roxisomal ;;b"r oi z.tt -5a). (Fig' the Zè[weger syndrome and r*-Liu". -there in ÑÀlO cases, was no labelling over the small' peioxisomes. However in IRD, a very small "U"ãã"fã-o""i (0.31 particles/pm) was observed in the first patient (IRD2) h?9 pãti"nt tfÞOf l, and the second -1oI: Iiui fuUèffi"g. Ín the PD2 patient, the label for PMP68 *"r i"..*t"ä, despite a complete absence of labelling for ---b""any of the matrix proteins (Fig. 5b)' of úvers was auiopsy tissue and the label- ling density"ontrol for all proteins was ¡imilar to the values in Uio-pty tissue even though the peroxisomes were larger itrui-t íotrnul due to post mortem change (see Tables 3 and 4). A summary of the morphometric and im- for each peroxisomal disorder -u"oóytoctremical résults group is given in Table 5. means. ç,

ll- I

T¡ble 6. Morphological/immunocytochemical types ol peroxisomes identifred in generalised peroxisomal disorders

Normal size and morphology peroxisomes, with moderately reduced labelling of matrix proteins (PD3-large) Normal size peroxisomes with electron dense centres and normal labelling olperoxisomal matrix and membrane proteins (PDl) Small peroxisomes with electron dense centres and reduced labelting of some matrix proteins (i.e. catalase, IRD2; catalase and bilunctional protein, PD3 small) Small peroxisomes with electron dense centres and markedly reduced labelling of alI matrix proteins, and reduced labelling of PMP68 (IRDI) Small peroxisomes with electron dense centres with undetectable labelling for matrix proteins but normal tabelling îor membrane proteins (PD2) Small peroxisomes with electron dense centre with undetectable labelling for matrix and membrane proteins (Zellweger syndrome, NALD)

Table 7. Cellular phenotypes seen in generalised disorders of peroxisomal dysfunction

Normal numbers of normal size peroxisomes with electron dense centres (PDl) Heterogeneous populations of large and small peroxisomes, some with electron dense centres and normal numerical density (IRD2, PD3) Small peroxisomes with electron den;g-centres and normal or near normal numerical density (IRDl, PD2, NALD) Small peroxisomes with electron dense centres and greatly reduced numerical density (Zellweger syndrome) No peroxisomes detected (Zellweger syndrome)

Conclusion. The different morphological types of per- Balfe A, Hoefler G, Chen WW, Watkins PA (1990) Aberrant sub- oxisomes that we have identified in these patients with cellular localization of peroxisomal 3-ketoacylCoA thiolase in chondrodysplasia pun- generalised peroxisomal disorders are summarised in the Zellweger syndrome and rhizomelic ctata. Pediatr Res 27:30¿l-310 Table 6 and the various cellular phenotypes in Table 7. Beard ME, Moser AB, Sapirstein V, Holtznan E (1986) The measurement of morphometric values of per- Peroxisomes in infantile phytanic acid storage disease: a cyto- oxisomes provides a more accurate description of the chemical study in skin ûbroblasts. J Inherited Metab Dis types of peroxisomes in peroxisomal disorders. However, 9:321-334 qualitative as well as quantitative descriptions are re- B¡ul S, Westerveld A, StrÜland A, Wanders RJA, Schram AV/' RBH, Van Den Bosch H, Tager JM quired order avoid misinterpretation. This is em- Heymans HSA, Schutgens in to (1988) Genetic heterogeneity in the cerebrohepatorenal (Zell- phasised by the occurrence of electron dense centres weger) syndrome and other inherited disorders with a which are immunocytochemically negative, in some generalized impairment olperoxisomal functions. A study using peroxisomes of normal size (e.g. patient PDI). All the complementation analysis. J Clin Invest 8l: l710-1715 patients in this study had peroxisomes in the liver which Chen N, Crane DI (1992) Induction of the major integral membrane proliferators. were ultrastructurally abnormal, in some respect. There protein of mouse liver peroxisomes by peroxisome Biochem J 283:601{10 was heterogeneity \ /ith regard to size and number, but all De Craemer D, Zweens MJ, Lyonnet S, Wanders RJA, Poll-The patients had peroxisomes with electron dense centres. At BT, Schutg least six different types of peroxisomal abnormalities (1991) Very based on morphological and immunocytochemical (rhizomelic criteria, were detected in patients with generalised disor- deflciency): F (1990a) Immuno- ders of peroxisomal dysfunction. We conclude that syn- Espeel M, Hashimoto T, De Craemer D, Roels cytochernical detection of peroxisomal p-oxidation enzymes in thesis of even abnonnal peroxisomes must take place in ciyostat and parafün sections ol human post-mortem liver. all patients. Histochem J 22:5742 Espeel M, Jauniaux E, Hashimoto T, Roels F (1990b) Immuno- Acknowledgemenls.We are gratelul to all the doctors and clinicians cytochemical localization of peroxisomal p-oxidation enzymes who provided patient material and clinical information. We wish to in human fetal liver. Prenat Diagn 10:349-357 thank Professor T. Hashimoto for providing the antibodies to Gartner J, Moser HW, Valle D (1992) Mutations in the 70K thiolase and acyl-CoA oxidase and Helen Thiele and Ruth Williams peroxisomal membrane protein gene in Zellweger syndrome- for technical assistance in electron microscopy. This work was Nature genetics l:lÇ23 supported in part by a grant lrom the Channel 7 Children's Re- Goldfischer S, Moore CL, Johnson AB, Spiro AJ, Valsamis MP, search Foundation of South Australia. Wisniewski HK, Ritch RH, Norton WT, Rapin I, Gartner LM (1973) Peroxisomal and mitochondrial defects in the cerebro- hepato-renal syndrome. Science 182 : 6244 References Goldfischer S, Collins J, Rapin I, Coltofl-Schiller B, Chang C' Nigro M, Black V, Javitt N, Moser H, Lzzarow P (1985) Aikawa J, Ishizawa S, Narisawa K, Tada K, Yokota S, Hashimoto Peroxisomal delects in neonatal-onset and X-linked adreno- T (1987) The abnormality of peroxisomal membrane proteins leukodystroph in Zellweger syndrome. J Inherited Metab Dis l0 [Suppl 2] Heikoop JC, Van A, Weijers PJ, Just W\t/' 211-213 Meijer AJ, Ta er of peroxisomal vesicles 40

ders ol peroxisome biogenesis- a complementation study involv- by autophagic proteolysis in cultured fibro blasts lrom Zellweger ing cell lines lrom 19 patients Pediatr P.es 26:6'1-'12 syndrome patients. Eur J Cell Biol 57: 165- t't 1 Imanaka T, Shio H, Small GM, Lazzrow PB (1988a) Hughes JL, Poulos A, Robertson E, Chow CW, Sheffreld LJ, Chris- Santos MJ, Pc¡oxisomal membrane ghosts in Zellweger syndrome- aberrant todoulou J. Carter RF (1990) Pathology ofhepatic peroxlsomes Science 239: I536 1538 and mitochondria in patients with peroxisornal disorders' Vich- organelle assemblY T, Shio H, Lazarow PB (1988b) Peroxisomal ows Arch 416:255-264 Santos MJ, Imanaka f{l teins in control and Zellweger frbroblasts- Hughes JL, Poulos A, Crane DI, Chow CW, SheffreldLJ,Sillence integralmembran€pro D (1992) Ultrastructure and immunocytochemistrY ol hepatic J Biol Chem 263:10501- 10509 MJ, Imanaka T, Poulos A, Danks DM, Moser peroxisomes in rhizomelic chondrodysplasia punctata' Eur J Small GM, Santos PB (1988) Peroxisomal integral membrane pro- Pediatr 151:829-836 HW, Lazarow livers of with Zellweger syndrome, infantile Hughes JL, Bourne AJ, Poulos A (1993) Establishment of a normal teins in Patients disease and X-linked adrenoleukodystroPhY. J In- range ol liver Refsum's ln Prcss ¡"r¡¡s¿ Metab Dis I I :358-37 N, Orii T' Aikawa J, Tada K, Kuwabara T, o S, Osumi T, Hashimoto'I (leeo) Suzuki Y, Shimozawa (1987) BiosYnthesis ol peroxisomal membrane poly- 70-kDa peroxisomal membrane protein is a member of the Hashimoto with Zellweger syndrome J Inherited Metab Mdr (P-glycopro tein)-related ATP-binding protein superlamily peptides in infants J Biol Chem 265:45344540 Dis l0:297-300 JP, Van Hool F, Misson JP, Evrard P, Vereuen Litwin JA, Beier K (1988) Immunogold localization of peroxisomal Vamecq J, DraYe Elde¡e J, Schutgens RBH, Wanders RJA' enzymes in Epon-embedded liver tissue. HistochemistrY G, Eyssen HJ, Van S (1 986) Multiple peroxisomal enzymatic 88:193-196 Roels F, Goldfrscher A comParatl ve biochemical and mor- Litwin JA, Völkl A, Müller-Höcker J, Hashimoto T, Fahimi HD defrciency disorders. Zellweger cerebrohepatorenal sYndrome and (1e87) Immunocytochemical localization of Peroxisomal en- phologic study of adrenoleukodystroPh y. Am J Pathol 125:524-535 zymes in human liver biopsies. Am J Pathol 128 l4l-150 neonatal Van Roermund CVy'T' Schixgens RBH, Tager JM Moser HW, Bergin A, Cornblath D (1991) Peroxisomal disorders. Wanders RJA, peroxisomal fatty acid P-oxidation. In Biochem Cell Biol 69 463474 (1991) Disorders ol metabolism. Schaub J, Van Hoof F, Vis HL Poll-The, Skjeldal OH Stokke O, Poulos A, Demaugre F, Saudu- Inborn errors ol Workshop Series 24, Vevey/Raüen Press bray J-M (1989) Phytanic acid alpha-oxidation and comPle- (eds) Nestlé Nutrition pp 43-56 mentation analysis ol classical Refsum and peroxisomal disor- New York, Weibel ER (1979) "Stereological methods", vol l. Practical meth- morPhometry Academic Press, London A, Sharp P, Whiting M (1984) Inlantile Refsum's disease ods for biological 'Wiemer V/W, Van Driel R, Brouwer-Kelder E, (phytanic acid storage disease): a variant of Zellweger's syn- EAC, Brul S, Just M, Weijers PJ' Schutgens RBH, Van Den Bosch drome? Clin Genet 26:579-586 Van Den Berg Wanders RJA, Tager JM (1989) Presence ol A, Singh H, Paton B, Sharp P, Derwas N (l 986) Accumula- H, Schram A, Poulos proteins in liver and frbroblasts lrom tion and delective p-oxidation of very long chain fatty acids in peroxisomal memb¡ane the Zellweger syndrome and related disorders Zellwegers syndrome, adrenoleukodystrophY and Refsum's dis- patients with of peroxisomal ghosts. Eur J Cell Biol variants. Clin Genet 29 evidence lor the existence DW (1988) Accumula- 50 407417 Out M, Schelen A, Wanders RJA, Schutgens RBH, tion of pristanic acid (2, 6, 10, I 4 tetramethyl-pentadecanoic Wiemer EAC, Van Den Bosch H, Tager JM (1991) Phenotypic heterogeneity acid) in the plasma of Patients with generalised peroxisomal fibroblasts from with disorders of dysfunction. Eur J Pediatr 147: I 43-147 in cultured skin Patients belonging to the same complementation Robertson EF, Poulos A, SharP P, Manson J, Wise G. Jaunzems peroxlsome biogenesis Biophys Acta 1097 232 23'7 A, Carter R (1988) Treatment of inlantile phytanic acid storage group. Biochem Structure-lunction relationships in the disease : clinical, biochemical and ultrastructural findings in two Wilson GN( l99l) lor human disease Biochem Med children treated lor 2 years. Eur J Pediatr I 47:133-142 peroxrsome imptications 46 288 298 Roels F, Cornelis A, Poll-Thé BT, Aubourg P, Ogier H, Scotto J, Metab Biol S, Suzuki Y, Shimozawa N, Yamaguchi S, Orii T, Fujiki Saudubray JM (1986) Hepatic Peroxisomes are deficient in in- Yajima Hashimoto T, Moser HW (1992) ComPlementa- fantile Refsum disease: a cytochemical study ol 4 cases. Am J Y, Osumi T, peroxisome-deficient disorders by immunofluores- Med Genet 25:257-271 tion study of and characterization of lused cells. Hum Genet Roscher AA, Hoefler S, Hoefler G, Paschke E, Paltauf F, M oser A cence staining 499 Moser H (1989) Genetic and phenotypic heterogeneity in disor- 88:491

Hughes, J. L., Poulos, A., Carter, R. F., Crane, D. & Masters, C. (1988). Cytochemical and immunocytochemical localization of catalase in peroxisomes of rat liver and human cultured fibroblasts. Journal of Paediatrics and Child Health, 24(1), 94.

NOTE:

This publication is included in the print copy

of the thesis held in the University of Adelaide Library.

It is also available online to authorised users at:

http://dx.doi.org/10.1111/j.1440-1754.1988.tb01341.x

Hughes, J. & Poulos, A. (1992). Ultrastructural examination of tissues from patients with peroxisomal disorders. Electron Microscopy, 3, 521– 522.

NOTE:

This publication is included in the print copy of the thesis held in the University of Adelaide Library. APPENDIX C

DEVEL PMENT OF M HODS Appendix C Development of methods

INTRODUCTION

A large number of experiments were carried out on rat and mouse liver and kidney, in order to ascertain the correct methodology for immunolabelling of peroxisomal proteins, and for the morphometric analyses. Some of the more relevant ones are presented here as a series of short experiments. Not all of the antibodies utilised in this study were available at the commencement of the candidature, and also the patient material arrived haphazardly over a period of several years. Therefore, some decisions with regard to fixation and processing of the tissue had to be made early on, before all of the antibodies

had been tested on animal tissues.

180 Appendix C Development of methods

EXPT 1 COMPARISON OF DIFFERENT FIXATIVES

AIM: To determine the best fixative to use in order to give optimal labelling of

peroxisomal proteins

METHODS: Small pieces of rat liver were fixed by immersion in the following freshly

prepared fixatives:

1. Karnovsky's fixative (see chapter 4.1)

2. Periodate-lysine-paraformaldehydefixative (PLP), preparedaccordingtoMclean

and Nakane (1974l,

3. PLP prepared with 2o/o wlv paraformaldehyde, instead of 8o/o paraformaldehyde

4. 1o/o glutaraldehyde in o.1M phosphate buffer alpH7.4 with 8% sucrose

The following paraformaldehyde and glutaraldehyde mixtures were prepared in O.1M

phosphate buffer at pH 7.4

5. 4o/o paraformaldehyde + O.1o/o glutaraldehyde

6. 4Yo paraformaldehyde + O'25o/o glutaraldehyde

7. 4o/o paraformaldehyde + 1o/o glutaraldehyde

8. 2%o paraformaldehyde + O'1o/o glutaraldehyde

9. 2o/o paraformaldehyde + O.25o/o glutaraldehyde

10. 2o/o paraformaldehyde + 1o/o glutaraldehyde

After fixation for 2h at 4oC, the tissue was washed twice in buffer corresponding to the

fixative, for 1 5 min. Processing into Lowicryl resin, immunolabelling and quantitation

were carried out according to Chapters 4.3.b,4.3.d,4.3'e and Tables 1O and 12.

RESULTS: Catalase was detected by the protein A gold probe in all tissue sections, for

fixatives 1-1O. Colloidal gold particles were seen over the peroxisomal matrix, excluding

the crystalline core (Fig. 29). The mean gold labelling density for each specimen is

shown below:

181 FrG. 29 lmmunoelectron microscopy of rat liver a Rat liver embedded in LRWhite resin and immunolabelled for catalase (CAT). The gold particles are located over the peroxisomal matrix, excluding the central core. X50,000 b Ultrathin cryosection of rat liver immunolabelled for catalase (CAT). Labelling is distributed over the peroxisomal matrix. X67,500 c Rat liver embedded in Lowicryl resin at -3OoC and immunolabelled for bifunctional protein (BFP) X67,500 d Ultrathin cryosection of rat liver immunolabelled for bifunctional protein (BFP) X67,50O e Rat liver embedded in Lowicryl resin at -3OoC and immunolabelled for PMP68. The labelling (arrows) is specific for the peroxisomal membrane X67,5O0

f Ultrathin cryosection of rat liver immunolabelled for PMP68. The labelling is over the peroxisomal membrane X67,50O CAT

29b CRYO

BFP

29d CRYO

.: *î'¡7"{.4," lrl PMPó8 PMPóI :É,

29e 2ii CRYO Appendix C Development of methods

FIXATIVE GOLD LABELLING DENSITY

FOR CATALASE + SEM

Karnovsky's 325 * 25.2

2. PLP + 8% PF 368 a 32.8

3. PLP + 2%PF 421 t 28

4. 1% G in phosphate 397 + 26

5. 4o/o PF + 0.1% G 420 + 38.6

6. 40/o PF + O.250/o G 350 t 20.6

7. 4o/o PF + 1% G 353 + 17

L 2% PF + 0.17o G 365 + 23.4

o O.25o/o G 369 t 32.3 2o/o ,PF *

10 2o/o PF * 1o/o G 360 + 27.8

CONCLUSION: Although the use of the PLP fixative resulted in the highest labelling

density for catalase, the tissue was not well preserved and showed poor membrane

definition and a lot of vacuolation. The labelling density for catalase was not signifcantly

affected by the addition of glutaraldehyde above O.1% to the paraformaldehyde fixative.

The use of 4o/o pF + 0.25% glutaraldehyde resulted in a better fixation quality without

affecting the labelling density for catalase significantly. Similarly, the localisation of the

bifunctional protein and PMP6B were only slightly affected by the type of fixation.

NOTE: This experiment was carried out, under the supervision of the candidate, and the

results were presented by Ms. Helen Thiele as part of a project for the Associate

Diploma in Medical Laboratory Science at the South Australian lnstitute of Technology

(now the University of South Australia).

L82 Appendix C Development of methods

EXPT 2 COMPARISON OF DIFFERENT RESINS

AIM: To determine the best resin in which to embed fibsues in order to give optimal

labetling of peroxisomal proteins

METHODS: Small pieces of rat liver were fixed in a mixture of 4o/o freshly prepared

paraformaldehyde and O.25% glutaraldehyde in O.1M sodium phosphate buffer at

pH 7.4 for 2h at room temperature. The tissue was then processed into either

LRWhite resin or Lowicryl resin at -25oC as described in the Methods chapter (see

section 4.3.b) . ln addition some tissue was also processed into Lowicryl resin

according to Table 1O except that the entire processing, embedding and

polymerisation was carried out at room temperature. Ultrathin cryosections were

obtained f rom some of the fixed tissue according to section 4'3'c'

lmmunolabelling for catalase, bifunctional protein and PMP68 membrane protein

was carried out as described in section 4.3.d, and the immunogold labelling was

assessed visually and quantitated according to section 4.3'e'

RESULTS: The results are presented below and in Figs. 29 and 30

The effect of embedding medium on tabelling of catalase, bifunctional protein and pMp6B jn rat liver peroxisomes. Mean gotd tabetting densities (numbe¡ of gold particles pe, pm¿ or peÍ pm) + standard deviation-

Embedding medium Catalase Bifunctional protein PMP68

LR White resin 252+ 78.35 23.94 + 18.37 o.54 + O.61

Lowicryl resin RT 283 + 125.5 26.72+21.27 0.71 +O.58 * * Lowicryl resin -25oC 275 ! 1 59.6 64.77 +39.24 1.91 t 1 .7 * Cryosections 307 r 83.9 165.34 !79.71 35.29 + 14.6 I *

* significant at p

CONCLUSION: There was no significant difference in the labelling density of catalase

183 J

FrG. 30 The effect of the embedding medium on the localisation of three peroxisomal proteins. The labelling density is expressed as the number of gold particles per unit area [for catalase (CAT) and the bifunctional protein (7OK)l and per unit length lfor PMP68 (68K)]. Each histogram represents a different embedding medium: LRWhite resin (LRW); Lowicryl K4M resin embedded and polymerised at room temperature (LOW RT); Lowicryl K4M resin embedded and polymerised at -3OoC (LOW-30); ultrathin cryosections (CRYO). CAT

- CAT o CAT t¡Jz d CAT o 250

r¡¡ d

=z 200 f d, ¡¡¡ CL 70K .t) ¡¡¡ 9 150 d,

CL ô o() 100 o¡¡- 70K d r¡J co = 68K z 70K 70K

68K 68K 68K 0 LRW LOWRT LOW-30 CRYO

Fig30 DeveloPment of methods Appendix C

betweenthethreetypesofresin'Theuseofultrathincryosectionsalsodidnot increasethelabellingdensityofcatalaseovertheperoxisomes'Thelabelling densityofbifunctionalproteinandPMP6EwasenhancedbytheuseofLowicryl cryosections' The use of ultrathin resin at low temperature of the use of ultrathin

cryosectionswasfoundtobeimpractiblewhendealingwithhumantissuesand the use of Lowicryl resin at -25oC' adequate labelling could be acheived by

l.84 Appendix C Development of methods

EXPT 3 SPOT BLOTS ON NITROCELLULOSE gold' AIM: Io assess whether or not the antibodies to be used react with protein A of two METHOD: A one microliter spot of each antibody to be tested was made on each

strips of nitrocellulose (Schleicher and Schuell Membranfilter), and allowed to air

dry for 30 min. The antibodies were used undiluted and normal rabbit serum

was used as a negative control and an antibody to protein A was used as a

positive control. The strips were then covered with the diluent and wash buffer (D&wB) for th. The strips were removed and allowed to air dry, and then gold in covered in a protein A gold solution which consisted of protein A diluted

D&WB to an optical density at 52Onm of 0.14. Different protein A gold

conjugates were tested on each strip. After t h the strips were removed and

rinsed in D&WB for Smin twice, rinsed in clean water for Smin twice, and then

silver enhanced for Tmin in a darkroom. The developer for silver enhancement

was prepared as follows:

1. O.119 of silver lactate was added to 15ml clean water. This solution was

kePt in the dark until readY to use'

2. 2.35g trisodium citrate dihydrate, 2.55g citric acid, and 85ml of clean water were combined. o.85g of hydroquinone was added, shaken vigorously to

dissolve and kePt in the dark.

3. ln the darkroom under safelight, solution 1 and solution 2 were combined and

used immediatelY.

The strips were then rinsed in water for 5min, four times and fixed in llford Rapid

fix diluted 1 + 4 for 3Os. After rinsing in plenty of tap water the strips were

allowed to air dry. The results were assessed visually and recorded using an

increasing series of plusses to indicate an increased reaction.

185 DeveloPment of methods Appendix C

show n below one such eriment are RESULTS: ThE results of PAG New AntibodY OId

PAG

NRS

+ + NRS A2 +++ catalase ++++

+++ ++j TOkDa 1 +++ TOkDa 2 +++ +++ TOkDa 3 +++ +++ 68kDa +++

+ + DaaOx A2B3

+ + DaaOx A2B4

22kDa R184 +++ A-CoAOx +++

A-CoAOx old +++ Thiolase +++ ++++ anti-Protein A +++++

+ protein (all three to catalase' TOkDa bif unctional GONGLUSION: The antibodies thiolase and proteln' acyl CoA oxidase, batches), 68kDa integral membrane a spot blot test strip protein A gold coniugate on protein A all react with the

186 Appendix C Development of methods

This does not necessarily mean that they will react on a section since the

amount of antibody present on a .section will be much less and more diffuse'

The difference between the new and old batch of protein A gold was not

significant.

187 DeveloPment of methods Appendix C

EXPT 4 IMMUNOBLOTTING OF CATALASE antígen whích coutd be detected by AIM: To determine the minímal amount of catalase gold' the catalase antibodY and protein A PBS' to (approximately 4mg/ml) was serially diluted in METHOD: Mouse liver catalase gong/¡rl o.625ng/¡rl' 2¡rl drops of each give a concentration ranging from to dilutionweredotblottedonto2nitrocellulosestripsandairdried'Thestrips

wereplacedinseparateair-tightplasticbagscontainingSmlof3o/onon-fatmilk lOpl of the catalase antibody (raised powder (NFMP)in PBS, for 3Omin at 37oC' inrabbitsagainstmouselivercatalase)wasthenaddedtoonebag,and bag was incubated without antibody as incubated for gomin at 37oc. The other

anegativecontrol'Thestripswereremovedfromthebags,washedinPBSand gold conjugate in 3% NFMP, for 6omin at then incubated in a 12nm protein A

3Toc.TheywerethenwashedextensivelyinPBsandcleanwaterandsilver

enhanced as described in experiment 3'

RESULTS:Thecontrolstripwasnegative.Approximatelylong/¡llofcatalaseantigen

was detected by the protein A gold probe'

1 +++ SOng/pl 2 ++ 40nglpl 3 + 2Ongl¡tl 4 + l Ongi¡rl 5 + 5ng/pl 6 2.5nglul 7 1.2\nglpl I O.625ng/Pl I ++++ undiluted 4mg/ml

coNcLusloN:ThecatalaseantibodyandtheproteinAgoldconjugateareable (1ong&l) which is concentrated on to detect a small amount of catalase antigen anitrocellulosestrip.Howeveronathinsectionthisamountwouldbedifferent and embedding of the tissue' due to the effects of fixation, dehydration

188 DeveloPment of methods Appendix C

EXPT 5 DILUTION OF ANTIBODIES

AIM:Todeterminetheoptimatditutionoftheantibodytocatalaseonth¡nsectíons

(raised in rabbits against mouse liver catalase) METHOD: Purified anti-catalase antibody wasreceivedfromDrDenisCraneattheFacultyofScienceandTechnology, Griffithuniversity,oueensland,Australia.Theproteinconcentrationwas

approximatelyo'7mg/ml.TheantibodywasdilutedinPBscontainingl%bovine Serumalbumintogivedilutionsoflllo,l/50,1/10o,1/500,1/1ooo.The antibodywasappliedtothinsectionsofratliverwhichhadbeenfixedin4%

paraformaldehydeando'25o/oglutaraldehyde,andembeddedinLowicrylresinat to visualise the low temperature. A Protein A gold probe was used

antigen/antibodY comPlex'

result with a large number of gold RESULT: The dilution of 1/5OO gave the best very little background labellling' particles over the peroxisome matrix' There was

Thelowerdilutionsresultedingreaterbackgroundlabel.

CoNCLUSIoN:Theoptimalworkingdilutionofthisbatchofanti-catalaseantibodywas 1/5oo'Similardilutionexperimentswerecarriedoutfortheotherantibodies

utilised in this study'

189 Appendix C Development of methods

EXPT 6 COMPARISON OF DIFFERENT SIZE COLLOIDAL GOLD

AIM: To determine íf better ímmunolabelling of peroxísomat proteíns could be achieved

by using a smaller size gold Probe.

METHOD: Thin sections of rat liver, embedded in Lowicryl resin, were incubated in a

panel of antibodies followed by either a freshly prepared 1 2nm protein A gold gold probe (PAG6). The probe (pAG1 2l; or a freshly prepared 6nm protein A . 6nm gold probe was prepared by using sodium borohydride for the reduction of

tetrachlorauric acid (Tschopp et al' 19821, conjugating to protein A' and

purifying by ultracentrifugation at TO,OOOg for t h, twice'

RESULTS: PAG12 PAG

6 1. Control, no antibodY

2. Control, normal rabbit serum 1/5OO 3. CatalaseantibodYl/5OO ++++ ++ -tOkDa + + 4. antibodY 1 /2O

5. 68kDa antibodY 1/2O + +

6. thiolase antibodY 1/350 + + + +

CONCLUSION: The use of a smaller size colloidal gold probe (6nm) did not increase the

amount of label for peroxisomal proteins. The labelling with the 6nm conjugate

was often too small to be visualised easily against the granular matrix of the

peroxisomes'

r-90 DeveloPment of methods Appendix C

EXPT 7 MORPHOMETRY PILOT STUDY the volume density of peroxisomes AIM: To compare 3 different methods of measuring

in heqatocYtes.

METHoD:Themicroscopemagnificationwasmeasuredbyphotographingacarbon gratingreplicawith2,l60linespermillimeter(ProbingandStructure)atx1o

magnification.Twentymicrographsofasectionofcontrolliverwererecorded randomlybyphotographingwithrespecttoonecornerofthesupportinggrid. Twentymicrographsofasectionfromapatientwithaperoxisomaldeficiency were enlarged disorder (PD3b1) were also recorded' The micrographs photographicallytoamagnificationofx22,ooo,andthevolumedensitywas

assessed using three different methods:

I . Point counting AmultipurposetestSystemMl6s(Weibel1979)wasoverlainonthe micrographsandthenumberofintersectionswhichfellonperoxisomesWaS which fell on the recorded. SimilarlY, the total number of intersections The volume density was hepatocyte cYtoPlasm or nucleus was recorded' peroxisomes divided by the sum of calculated as the sum of the points falling on

the points falling on the hepatocyte cytoplasm or nucleus'

2. Manual tracing. hepatocytes was measured The area of the peroxisomes and the area of the usingadigitiserpadlinkedtoacomputer(seeMethodssection4'4'b\andthe

Vu calculated bY the formula

areas VV = sum of Deroxisomal sum of hePatocYte areas

3. Modificatíon of the method of Peifer (198O) rather than an increase This method was developed for the study of a decrease inthenumberofanorganelleandthusseemedappropriatefortheStudyof

peroxisomaldeficienydisor'ders.Theareaofonecoppergridsquarecompletely

191- Appendix C Development of methods

covered by thin section was measured in um2, and the peroxisomes were then

systematically searched for at a magnification of x2O,OOO'

RESULTS: The results are shown below:

METHOD CoNTROL Vv PD3bl Vv

O.1 1 o/o 1 . Point counting 1Vo

2. Manual tracing 1 .13o/o O.15o/o

3. Peifer 1 grid square O.37o/o O.O2o/o

4. Peifer 4 grid squares O.43o/o o.03%

CONCLUSION: The use of point counting or manual tracing gave comparable results.

These results compare favorably with previously published data (see Table 3 in Chapter

2.5.d). Using the method of Peifer (1980) gave a much reduced volume density and

was considerably more time consuming'

192