George Constantopoulos

Determination of molecular weight distribution of acid mucopolysaccharides by sephadex gel filtration.

Abstract A method has been developed for the determination of molecular weight distribution of AMPS. Chondroitin sulfate A was fractionated by gel filtration on Sephadex G-200 into bands of narrow distribution of molecular weights. The molecular weights of the AMPS from bands widely separated were determined by a combined diffusion-sedimentation coefficient method. Aliquots from the same AMPS preparations were also subjected to gel filtration on a 1.06 (i.d.) 86.0 cm Sephadex G-200 column. It was found that a linear relationship exists between elution volume and log of molecular weight of the respective AMPS. Chondroitin sulfate B, chondroitin sulfate C, and heparitin sulfate follow the same relationship. Elution diagrams of the urinary AMPS of the three main variants of the Hurler syndrome are characteristically distinct from each other and they are also different from the elution diagrams of AMPS obtained from normal subjects. Heparitin sulfate and a portion of chondroitin sulfate B from Hurler patients are markedly degraded.

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN LIPIDS AND LYSOSOMAL ENZYMES IN PATIENTS WITH FOUR TYPES OF MUCOPOLYSACCHARIDOSIS AND IN NORMAL CONTROLS

Abstract— Lipids and certain lysosomal enzymes were measured in the cerebral gray and white matter and in the liver of unaffected controls and six patients with mucopolysaccharidosis (MPS). Three of the patients had MPS Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie). The glycosaminoglycans (GAG) of those tissues have been fully characterized previously (Constantopouloset al., 1976).

Mucopolysaccharidosis Types IH, IS, II and IIIA: Glycosaminoglycans and Lipids of Isolated Brain Cells and Other Fractions from Autopsied Tissues

Abstract: Brain cellular fractions were prepared in bulk from four non‐neurological patients and from five patients with mucopolysaccharidosis (MPS). Glycosaminoglycans and lipids were isolated and chemically analyzed. Results of the present study: in the normal controls glycosaminoglycans as μg per mg protein (mean) were 2.2 in neuronal perikarya, 2.0 in astroglia, 2.1 in oligodendroglia, 3.3 in neuropile from gray matter and 3.2 in a mixed fraction from white matter. In the partially myelinated axons from gray and white matter of an 8‐month‐old infant, the concentration was 6.9 and 2.6 μg per mg protein, compared with 2.8 and 0.8 μg per mg protein, respectively, in the adult patients. It was estimated that chondroitin sulfates constituted more than one‐half of the total glycosaminoglycan. Hyaluronic acid, heparan sulfate and dermatan sulfate were also present in all cell types and fractions. Cholesterol, phospholipids, cerebrosides, sulfatide and gangliosides were present in all cell types and fractions, but differed widely in concentration. There was a four‐ to sixfold increase in the concentration of total glycosaminoglycans in the neuronal perikarya of patients with MPS IH, II and IIIA. The increased glycosaminoglycans were heparan sulfate in MPS IIIA and dermatan sulfate plus heparan sulfate in MPS IH and II. Similar changes were found in the astroglia and in the other brain fractions of those patients. The concentration of the gangliosides Gm2, Gm3, Gd3 and ceramide dihexoside was markedly increased in the neurons and other brain fractions of the same patients. The quantities of Gm3, Gm2 and Gd3 together amounted to 65% of the total gangliosides of the neurons, indicating changes of the same magnitude seen in the gangliosidoses. All these patients exhibited mental retardation. The concentration and composition of glycosaminoglycans, gangliosides and neutral hexosyl ceramides in the neuronal perikarya of the patient with MPS IS was normal. There was only a small increase of dermatan sulfate content in the neuropile, mixed fraction and myelinated axons from the white matter and some increase of ceramide dihexoside content in the myelinated axons. This patient was an adult of normal intelligence.

Mucopolysaccharidosis types I, II, IIIA and V

SummaryHistochemical and electron microscopic studies of the brains inclusive of the leptomeninges containing large blood vessels from 7 patients with mucopolysaccharidosis (MPS) I, II, IIIA and V showed marked increase in mesenchymal elements and the generalized presence of characteristic lesions around cerebral veins and arteries. The periadventitial space was greatly distended and filled with viscous fluid and numerous mononuclear cells containing large cytoplasmic vacuoles; these cells stained positively for glycosaminoglycans (GAG). In contrast, the neurons showed only a slight increase of GAG over the normal controls but contained an excessive amount of glycolipid-like material.The amount of GAG in the leptomeninges, inclusive of the large blood vessels, was 10.8, 6.5, 4.5 and 2.2 times greater in patients with MPS I, II, V and IIIA respectively, than the mean of unaffected controls. Dermatan sulfate (DS) accounted for most of the GAG increase in MPS I, II and V [mixed excretors of DS and heparan sulfate (HS)], and HS for the GAG increase in MPS IIIA (HS excretor).With the exception of the patient with MPS IIIA, whose GAG content and composition were the same in both the neural and mesenchymal elements, in all the other MPS types the mesenchymal elements contained more GAG, with a preponderance of DS.We conclude that the mesenchymal elements contribute substantially to the increased content of GAG in the brain and its coverings, mostly in the form of dermatan sulfate.

Long-Term Neurological Effects of Bone Marrow Transplantation in a Canine Lysosomal Storage Disease

ABSTRACT: A naturally occurring disease in Plott hound dogs, caused by deficiency of the lysosomal enzyme α-L-iduronidase, was used to study the feasibility of bone marrow transplantation therapy in a neurodegenerative storage disease. Three long-term survivors of transplantation with littermate marrow at 5 months of age (before clinical signs) had CNS enzyme activity, glycosaminoglycan storage, and light microscopic and ultrastructural changes evaluated 594, 628, and 740 days after treatment. Iduronidase activity in small amounts (1–3% of donor values) was detectable in brain tissue. Cerebrospinal fluid had higher iduronidase activity after transplantation (7–15% of donor values). Enzyme activity within the CNS resulted in significant reductions in stored glycosaminoglycans and resolution, to a large extent, of light microscopic and ultrastructural lesions observed in affected, untreated littermate control dogs.

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS IN NORMALS AND FOUR TYPES OF MUCOPOLYSACCHARIDOSES

Glycosaminoglycan content, composition and molecular weight distribution were determined in cerebral gray and white matter, liver and spleen from normals and 7 patients with mucopolysaccharidosis; 4 were of Type I (Hurler), one Type II (Hunter), one Type IIIA (Sanfilippo A) and one Type V (Scheie).

Occurrence of multiple dentigerous cysts in a patient with the Maroteaux-Lamy syndrome (mucopolysaccharidosis, type VI)

The mucopolysaccharidoses (MPS) are a group of genetic lysosomal storage diseases. These diseases result from a defect in specific lysosomal enzymes required for the degradation of specific mucopolysaccharides. These incompletely degraded saccharides accumulate in tissues and are excreted in the urine. A general characteristic of these diseases is dysostosis multiplex. Dental complications can be severe and include unerupted dentition, dentigerous cystlike follicles, malocclusions, condylar defects, and gingival hyperplasia. This report examines multiple dentigerous cysts in a patient with a deficiency in N-acetylgalactosamine-4-sulfatase, Maroteaux-Lamy syndrome (MPS VI). The inability to hydrolyze the sulfate group from N-acetylgalactosamine-4-sulfate residue of dermatan sulfate due to a deficiency in this enzyme results in the accumulation of dermatan sulfate in tissues and its excretion in the urine. Examination of dentigerous cyst fluid revealed glycosaminoglycan content of 397 microgram per milliliter. Compositional analyses revealed 60% hyaluronic acid, 30% chondroitin 4- and -6-sulfate, and only 10% dermatan sulfate. This was consistent with dentigerous cyst fluid derived from persons without mucopolysaccharide-storage disorders but distinctly different from glycosaminoglycans assayed from other body fluids of this patient.

Organomegaly and histopathology in an animal model of mucopolysaccharidosis induced by suramin

SummaryThe trypanocidal drug suramin causes glycosaminoglycan and sphingolipid accumulation in the rat, thus simulating a mucopolysaccharidosis (Constantopoulos et al. 1980). In this paper we report on the extent and nature of the morphological changes that occur in the liver, kidneys, spleen, heart, lung and brain as a result of short or long term suramin administration.The first group of rats received a single intravenous injection of suramin (500 mg/kg) and was sacrificed 3–9 days after the injection. The second group received low doses of suramin (50–90 mg/kg) at 2–3 weekly intervals over 3 months. Samples of the above mentioned organs were processed for light and electronmicroscopy and the remainder of the tissue weighed and assayed for total protein, DNA and RNA content.In both groups of rats, suramin caused an abnormal enlargement of the spleen, kidney, lung and liver, splenomegaly being the most pronounced. The total protein, and DNA content did not alter in the treated rats, however, the RNA content of the spleen increased 100%, 9 days after injection and there was a small but consistent increase in RNA content of the liver, kidney and lung. Significant pathological changes were observed in these organs and also in the brain and heart. The changes were similar in many respects to the pathology seen in the lysosomal storage disorder, mucopolysaccharidosis and further support the proposition that the suramin treated rat might be a useful experimental animal model of the disease. Several mechanisms by which suramin might produce organomegaly in the rat are discussed.

NEUROCHEMISTRY OF THE MUCOPOLYSACCHARIDOSES: BRAIN GLYCOSAMINOGLYCANS, LIPIDS AND LYSOSOMAL ENZYMES IN MUCOPOLYSACCHARIDOSIS TYPE III B (α-N-ACETYLGLUCOSAMINIDASE DEFICIENCY)

Glycosaminoglycans, lipids and lysosomal enzymes were measured in brain, liver and spleen of a patient with mucopolysaccharidosis Type III B (α‐N‐Acetylglucosaminidase deficiency).

Acid mucopolysaccharides in the cerebrospinal fluid of patients with Hunter–Hurler's syndrome

AN IMPORTANT characteristic of the metabolic error in the patients with Hunter-Hurler’s syndrome is excessive storage of acid mucopolysaccharides (AMPS) in many organs and tissues (BRANTE, 1957; DORFMAN, 1966). In addition to hepatosplenomegaly, dwarfism and bony deformities, the affected patients generally exhibit severe neurological abnormalities which eventually culminate in a decerebrate state and death before adult years are reached. Chondroitin sulphate B (CSB) and heparitin sulphate (HS) in varying ratios accumulate in various body organs (MEYER, HOFFMAN, LINKER, GRUMBACH and SAMPSON, 1959) and are also excreted in large amounts in the urine of such patients (DEKABAN, RENNERT and HATHAWAY, 1966). Since the brain in Hunter-Hurler’s syndrome is markedly abnormal, knowledge of the content and composition of AMPS in the cerebrospinal fluid (CSF) in different variants of the diesease may prove valuable. The only pertinent report was published by FRIMAN (1967), who was unable to demonstrate the presence of AMPS in the CSF of patients with Hunter-Hurler’s syndrome. By applying a recent technique (DIFERRANTE. 1967), we have been able to demonstrate a marked increase of the A M P S in the CSF of these patients. Here, we report the values, composition, and certain chemical and physical properties of the AMPS in the CSF of patients with the clinical diagnosis of Hunter-Hurler’s syndrome.