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Liver ultrastructure and diagnosis of inborn metabolic disorders
Micron and Microscopica Acre, Vol. 20, No. h pp. 59-62, 1989.
0739-6260/89 $3.01.) + I).00 Maxwell Pergamon Macmillan plc
Printed in Great Britain.
LIVER ULTRASTRUCTURE
AND DIAGNOSIS
OF INBORN METABOLIC
DISORDERS
Franqois Van Hoof (i) & Frank Roels (2) (i) Laboratolre de Chimle Physiologique, Universit~ Catholique de Louvain and International Institute of Cellular and Molecular Pathology, UCL 7539, Avenue Hippocrate 75, B-1200 Bruxelles and (2) Menselijke Anatomic, VriJe Universitelt Brussel, Laarbeeklaan 103, B-IO90 Brussel
In man, over 200 inborn metabolic disorders are known to involve the liver, but only a minority among them are characterized by specific ultrastructural alterations. This group mostly concerns the disorders affecting the function of one type of intracellular organelle. The best known is the group of inborn lysosomal diseases (Figs. 1-3), which involves over 40 different genetic disorders. Among them, about ten mucopolysaccharldoses, as much mucollpidoses and lipidoses, Other lysosomal storage diseases involve the accumulation of glycogen, cerold, lipofuscin, cystin etc .... Inborn lysosomal diseases result usually from the deficiency of an acid hydrolase and sometimes from the lack of a specific carrier. Such carriers allow the egress from lysosomes of some of the products of the action of acid hydrolases, such as cholesterol, slalic acid, cystln or vitamin BI2. These diseases can readily be diagnosed by the accumulation of undigestible substrates in the lysosomes. The accumulated substances are sometimes easily recognized on micrographs, as it is the case for glycogen and
Lysosomal
diseases
:
Fig. i. Intralysosomal accumulation of glucosylcerebroside, displaying a tubular form is typical of Gaucher disease which corresponds to the lack of a specific lysosomal beta-glucosldase. Extralysosomal accumulation of glucosylcerebroslde is also found as there exists no cytosolic enzyme to hydrolyze this molecule. (x 16,500) Fig. 2. In Hurler disease (mucopolysaccharldosls type I, deficiency of alpha-L-iduronidase) the volume of lysosomes can be more than thousand-fold increased. (x 5,300) Fig. 3. Overloaded lysosomes can also be found in acquired disorders, here after acute valproate intoxication. (x 14,300)
59
60
F. Van H o o f and F. Roels
some glycolipids (i). In most of these diseases, several substrates accumulate together because of the lack of a single acid hydrolase (2). This results from the fact that acid hydrolases are specific of one type of chemical linkage, whatever the complex molecule in which it is present. Recognition of peroxisomal disorders also owes very much to the ultrastructural study of the liver. No peroxisome can be recognized in Zellweger's cerebrohepatorenal syndrome, and also in most patients with infantile Refsum disease; no diaminobenzidine-positive structures evoking peroxisomes are detected in hepatocytes from these patients (3). In neonatal adrenoleukodystrophy, an autosomal recessive disorder, peroxisomes are usually scarce and of very small size (4). In the patients with a specific defect of one of the 3 peroxisomal beta-oxidation enzymes (acyl-CoA oxidase, thiolase and probably the bifunctional protein displaying enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity), peroxlsomes are enlarged, sometimes more numerous or tubular shaped (5,6). Clinically, however, these children closely resemble patients with Zellweger disease or neonatal adrenoleukodystrophy. Stacks of regularly spaced, membranous trilaminar structures, believed to reflect accumulation of very long chain fatty acids, are always observed in generalized peroxisomal disorders (7), but only in some of the patients who lack a single beta-oxidation enzyme (Figs. 4-7). In the X-linked form of adrenoleukodystrophy, the only conspicuous abnormality in hepatocytes consists in the accumulation of cholesteryl esters (Fig. 8). It is most likely that our knowledge of the peroxisomal disorders will benefit from the developments in immunocytochemistry.
Peroxisomal
disorders
:
Fig. 4. Short trilaminar inclusions in dense bodies of a macrophage in a baby (age 2 m.) with Zellweger's CHRS. In older patients inclusions form very large stacks (see ref. 7). Numerous iron particles are also typical of this syndrome. (x 66,000) Fig. 5. °'Pseudo-Zellweger", thiolase deficient girl reported by Goldfischer et al., showing trilaminar structures fn dense bodies of macrophage; Such inclusions were not seen in isolated acyl-CoA oxidase deficiency. Iron particles are present. (x 66,000); Marker = 0,I uM. Fig. 6. Rare and short trilaminar inclusions in a hepatocellular lysosome in a patient (19 year) with X-linked adrenoleukodystrophy. (x 66,000) Fig. 7. Hepatocellular lysosome in a girl (8 months) with infantile Refsum's disease containing trilaminar inclusions. (x 66,000) Fig. 8. These cytosolic crystals in a boy with X-linked adrenoleukodystrophy most probably correspond to cholesterol esterified with very long chain fatty acids. (x 27,000)
Inborn Metabolic Disorders
6l
Fig. 9. Deficiency of alpha-l-antitrypsin (Pi ZZ form).(x 8,150) Fig. i0. Dysfibrinogenemia corresponding to the lack of glycan transfer on the fibrlnogen molecule. Absence of the glycan reduces the solubility of the protein which crystallizes in the cisternae of the endoplasmic reticulum. (x 17,500) Fig. ii. In Byler's disease bile thrombi occur i n distended billary capillaries together with an enlargement of the layer of cytoplasm around these minute bile collectors. The picture is, however, not pathognomonlc. (x 23,000) Fig. 12. Giant mltochondrla can occur in severe copper- or riboflavin-deficlency, but apparently also in relatively healthy subjects (here 5 weeks after hepatitis A). (x 8,900) Fig. 13. Type IV glycogenosis is characterized by intracytoplasmic inclusions of poorly branched glycogen. This almost insoluble polysaccharide appears to be responsible for the severe course of the disease, most patients dying from cirrhosis before the age of 5. (x 3,600)
62
F. Van H o o f and F. Roels
Ultrastructural study of the liver also allows recognition of storage diseases involving the endoplasmic reticulum. The pathogeny of this kind of disorder consists in the lack of glycan transfer to secretory proteins, not because of the deficiency of a glycan- or glycosyl transferase - which would affect the synthesis of all glycoproteins, but because a specific aminoacid, usually asparagine, is lacking in a single protein, amino acid which serves as anchoring point for the glycanic structure. To this group of diseases belong the Pi Z-Z form of alpha-l-antitrypsin deficiency (Fig. 9) and some rare patients with dysfibrinogenem a (Fig. i0). The electron microscope may also reveal abnormalities of the biliary pole of hepatocytes. They have been mostly looked for in Byler's disease (Fig. ii), but should not be considered pathognomonic. Most structural abnormalities of mitochondria in hepatocytes are also deprived of diagnostic significance (Fig. 12). An excess of cytosolic glycogen is found in glycogenoses types I, III and VI, but this picture - including the intranuclear localization of the polysaccharide - allows no proof of the disorder and no specific diagnosis. On the contrary, the accumulation of glycogen in lysosomes (glycogenosis type II) or the cytosolic accumulation of a barely soluble amylase-resistent polysaccharide resulting from the defect of glycogen branching enzyme (Fig. 13) in glycogenosis type IV are of great diagnostic value
(7). Current ultrastructural studies of the liver should be completed in patients with inborn metabolic disorders by some quantitative studies (morphometry), and sometimes also by cluster analysis (8). The latter might reveal an additional level of organization of intracellular organelles. References i. Johannessen, J.V., Electron Microscopy in human medicine. Vol. 8 : The Liver : Metabolic disorders, McGraw-Hill, USA, pp. 20-78, 1979. 2. Hers, H.G. & Van Hoof, F. Lysosomes and Storage Diseases. Academic Press, N.Y., 666 pp, 1973. 3. Roels, F., Cornelis, A., Poll-Th~, B.T., Aubourg, P., Ogier, H., Scotto, J. & Saudubray, J.M. Hepatic peroxisomes are deficient in infantile Refsum disease : a cytochemical study of 4 cases. Am. J. Med. Genet. 25:257-271, 1986. 4. Goldfischer, S., Collins, J., Rapin, I., Coltoff-Schiller, B., Chang, Ch., Nigro, M., Black, V.H., Javitt, N.B., Moser, H.W. & Lazarow, P.B. Peroxisomal defects in neonatalonset and X-linked adrenoleukodystrophy. Science 227:67-70, 1985. 5. Poll-Th~, B.T., Roels, F., Ogler, H., Scotto, J., Vamecq, J., Schutgens, R.B.H., Wanders, R.J.A., van Roermund, C.W.T., van Wijland, M.J.A., Schram, A.W., Tager, J.M. & Saudubray, J.M. A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of acyl-CoA oxidase (pseudo-neonatal adrenoleukodystrophy). Am. J. Hum. Genet. 42:422-434, 1988. 6. Roels, F., Pauwels, M., Poll-Th~, B.T., Scotto, J., Ogler, H., Aubourg, P. & Saudubray, J.M. Hepatic peroxisomes in adrenoleukodystrophy and related syndromes. Cytochemical and morphometric data. Virchows Archiv A 413:275-285, 1988. 7. Kerckaert, I., Dingemans, K.P., Heymans, H.S.A., Vamecq, J., Roels, F. Polarizing inclusions in some organs of children with congenital peroxisomal diseases (Zellweger's Refsum's, Chondrodysplasia Punctata (Rhizomelic Form), X-llnked Adrenoleukodystrophy). J. Inher. Metab. Dis. 11:372-386, 1988. 8. Hers, H.G., Van Hoof, F. & de Barsy, Th. Glycogen Storage Diseases. In : The metabolic basis of inherited diseases. 6th edn, C.R. Scriver, A.L. Beaudet, W.S. Sly & D.L. Valle eds, Mc Graw Hill, USA, 1989, in press. 9. Baudhuin, P., Leroy-Houyet, M.A., Quintart, J. & Berthet, P. Application of cluster analysis for characterization of spatial distribution of particles by stereologieal methods. J. Microscopy 115:1-17, 1979.
0739-6260/89 $3.01.) + I).00 Maxwell Pergamon Macmillan plc
Printed in Great Britain.
LIVER ULTRASTRUCTURE
AND DIAGNOSIS
OF INBORN METABOLIC
DISORDERS
Franqois Van Hoof (i) & Frank Roels (2) (i) Laboratolre de Chimle Physiologique, Universit~ Catholique de Louvain and International Institute of Cellular and Molecular Pathology, UCL 7539, Avenue Hippocrate 75, B-1200 Bruxelles and (2) Menselijke Anatomic, VriJe Universitelt Brussel, Laarbeeklaan 103, B-IO90 Brussel
In man, over 200 inborn metabolic disorders are known to involve the liver, but only a minority among them are characterized by specific ultrastructural alterations. This group mostly concerns the disorders affecting the function of one type of intracellular organelle. The best known is the group of inborn lysosomal diseases (Figs. 1-3), which involves over 40 different genetic disorders. Among them, about ten mucopolysaccharldoses, as much mucollpidoses and lipidoses, Other lysosomal storage diseases involve the accumulation of glycogen, cerold, lipofuscin, cystin etc .... Inborn lysosomal diseases result usually from the deficiency of an acid hydrolase and sometimes from the lack of a specific carrier. Such carriers allow the egress from lysosomes of some of the products of the action of acid hydrolases, such as cholesterol, slalic acid, cystln or vitamin BI2. These diseases can readily be diagnosed by the accumulation of undigestible substrates in the lysosomes. The accumulated substances are sometimes easily recognized on micrographs, as it is the case for glycogen and
Lysosomal
diseases
:
Fig. i. Intralysosomal accumulation of glucosylcerebroside, displaying a tubular form is typical of Gaucher disease which corresponds to the lack of a specific lysosomal beta-glucosldase. Extralysosomal accumulation of glucosylcerebroslde is also found as there exists no cytosolic enzyme to hydrolyze this molecule. (x 16,500) Fig. 2. In Hurler disease (mucopolysaccharldosls type I, deficiency of alpha-L-iduronidase) the volume of lysosomes can be more than thousand-fold increased. (x 5,300) Fig. 3. Overloaded lysosomes can also be found in acquired disorders, here after acute valproate intoxication. (x 14,300)
59
60
F. Van H o o f and F. Roels
some glycolipids (i). In most of these diseases, several substrates accumulate together because of the lack of a single acid hydrolase (2). This results from the fact that acid hydrolases are specific of one type of chemical linkage, whatever the complex molecule in which it is present. Recognition of peroxisomal disorders also owes very much to the ultrastructural study of the liver. No peroxisome can be recognized in Zellweger's cerebrohepatorenal syndrome, and also in most patients with infantile Refsum disease; no diaminobenzidine-positive structures evoking peroxisomes are detected in hepatocytes from these patients (3). In neonatal adrenoleukodystrophy, an autosomal recessive disorder, peroxisomes are usually scarce and of very small size (4). In the patients with a specific defect of one of the 3 peroxisomal beta-oxidation enzymes (acyl-CoA oxidase, thiolase and probably the bifunctional protein displaying enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity), peroxlsomes are enlarged, sometimes more numerous or tubular shaped (5,6). Clinically, however, these children closely resemble patients with Zellweger disease or neonatal adrenoleukodystrophy. Stacks of regularly spaced, membranous trilaminar structures, believed to reflect accumulation of very long chain fatty acids, are always observed in generalized peroxisomal disorders (7), but only in some of the patients who lack a single beta-oxidation enzyme (Figs. 4-7). In the X-linked form of adrenoleukodystrophy, the only conspicuous abnormality in hepatocytes consists in the accumulation of cholesteryl esters (Fig. 8). It is most likely that our knowledge of the peroxisomal disorders will benefit from the developments in immunocytochemistry.
Peroxisomal
disorders
:
Fig. 4. Short trilaminar inclusions in dense bodies of a macrophage in a baby (age 2 m.) with Zellweger's CHRS. In older patients inclusions form very large stacks (see ref. 7). Numerous iron particles are also typical of this syndrome. (x 66,000) Fig. 5. °'Pseudo-Zellweger", thiolase deficient girl reported by Goldfischer et al., showing trilaminar structures fn dense bodies of macrophage; Such inclusions were not seen in isolated acyl-CoA oxidase deficiency. Iron particles are present. (x 66,000); Marker = 0,I uM. Fig. 6. Rare and short trilaminar inclusions in a hepatocellular lysosome in a patient (19 year) with X-linked adrenoleukodystrophy. (x 66,000) Fig. 7. Hepatocellular lysosome in a girl (8 months) with infantile Refsum's disease containing trilaminar inclusions. (x 66,000) Fig. 8. These cytosolic crystals in a boy with X-linked adrenoleukodystrophy most probably correspond to cholesterol esterified with very long chain fatty acids. (x 27,000)
Inborn Metabolic Disorders
6l
Fig. 9. Deficiency of alpha-l-antitrypsin (Pi ZZ form).(x 8,150) Fig. i0. Dysfibrinogenemia corresponding to the lack of glycan transfer on the fibrlnogen molecule. Absence of the glycan reduces the solubility of the protein which crystallizes in the cisternae of the endoplasmic reticulum. (x 17,500) Fig. ii. In Byler's disease bile thrombi occur i n distended billary capillaries together with an enlargement of the layer of cytoplasm around these minute bile collectors. The picture is, however, not pathognomonlc. (x 23,000) Fig. 12. Giant mltochondrla can occur in severe copper- or riboflavin-deficlency, but apparently also in relatively healthy subjects (here 5 weeks after hepatitis A). (x 8,900) Fig. 13. Type IV glycogenosis is characterized by intracytoplasmic inclusions of poorly branched glycogen. This almost insoluble polysaccharide appears to be responsible for the severe course of the disease, most patients dying from cirrhosis before the age of 5. (x 3,600)
62
F. Van H o o f and F. Roels
Ultrastructural study of the liver also allows recognition of storage diseases involving the endoplasmic reticulum. The pathogeny of this kind of disorder consists in the lack of glycan transfer to secretory proteins, not because of the deficiency of a glycan- or glycosyl transferase - which would affect the synthesis of all glycoproteins, but because a specific aminoacid, usually asparagine, is lacking in a single protein, amino acid which serves as anchoring point for the glycanic structure. To this group of diseases belong the Pi Z-Z form of alpha-l-antitrypsin deficiency (Fig. 9) and some rare patients with dysfibrinogenem a (Fig. i0). The electron microscope may also reveal abnormalities of the biliary pole of hepatocytes. They have been mostly looked for in Byler's disease (Fig. ii), but should not be considered pathognomonic. Most structural abnormalities of mitochondria in hepatocytes are also deprived of diagnostic significance (Fig. 12). An excess of cytosolic glycogen is found in glycogenoses types I, III and VI, but this picture - including the intranuclear localization of the polysaccharide - allows no proof of the disorder and no specific diagnosis. On the contrary, the accumulation of glycogen in lysosomes (glycogenosis type II) or the cytosolic accumulation of a barely soluble amylase-resistent polysaccharide resulting from the defect of glycogen branching enzyme (Fig. 13) in glycogenosis type IV are of great diagnostic value
(7). Current ultrastructural studies of the liver should be completed in patients with inborn metabolic disorders by some quantitative studies (morphometry), and sometimes also by cluster analysis (8). The latter might reveal an additional level of organization of intracellular organelles. References i. Johannessen, J.V., Electron Microscopy in human medicine. Vol. 8 : The Liver : Metabolic disorders, McGraw-Hill, USA, pp. 20-78, 1979. 2. Hers, H.G. & Van Hoof, F. Lysosomes and Storage Diseases. Academic Press, N.Y., 666 pp, 1973. 3. Roels, F., Cornelis, A., Poll-Th~, B.T., Aubourg, P., Ogier, H., Scotto, J. & Saudubray, J.M. Hepatic peroxisomes are deficient in infantile Refsum disease : a cytochemical study of 4 cases. Am. J. Med. Genet. 25:257-271, 1986. 4. Goldfischer, S., Collins, J., Rapin, I., Coltoff-Schiller, B., Chang, Ch., Nigro, M., Black, V.H., Javitt, N.B., Moser, H.W. & Lazarow, P.B. Peroxisomal defects in neonatalonset and X-linked adrenoleukodystrophy. Science 227:67-70, 1985. 5. Poll-Th~, B.T., Roels, F., Ogler, H., Scotto, J., Vamecq, J., Schutgens, R.B.H., Wanders, R.J.A., van Roermund, C.W.T., van Wijland, M.J.A., Schram, A.W., Tager, J.M. & Saudubray, J.M. A new peroxisomal disorder with enlarged peroxisomes and a specific deficiency of acyl-CoA oxidase (pseudo-neonatal adrenoleukodystrophy). Am. J. Hum. Genet. 42:422-434, 1988. 6. Roels, F., Pauwels, M., Poll-Th~, B.T., Scotto, J., Ogler, H., Aubourg, P. & Saudubray, J.M. Hepatic peroxisomes in adrenoleukodystrophy and related syndromes. Cytochemical and morphometric data. Virchows Archiv A 413:275-285, 1988. 7. Kerckaert, I., Dingemans, K.P., Heymans, H.S.A., Vamecq, J., Roels, F. Polarizing inclusions in some organs of children with congenital peroxisomal diseases (Zellweger's Refsum's, Chondrodysplasia Punctata (Rhizomelic Form), X-llnked Adrenoleukodystrophy). J. Inher. Metab. Dis. 11:372-386, 1988. 8. Hers, H.G., Van Hoof, F. & de Barsy, Th. Glycogen Storage Diseases. In : The metabolic basis of inherited diseases. 6th edn, C.R. Scriver, A.L. Beaudet, W.S. Sly & D.L. Valle eds, Mc Graw Hill, USA, 1989, in press. 9. Baudhuin, P., Leroy-Houyet, M.A., Quintart, J. & Berthet, P. Application of cluster analysis for characterization of spatial distribution of particles by stereologieal methods. J. Microscopy 115:1-17, 1979.