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SUMMARY A case of mucopolysaccharidosis is described, in which the principal glycosaminoglycan stored in the liver and excreted in the urine was chondroitin sulphate. Both isomers were present in equal amounts. The clinical features were similar to those of the Hurler syndrome or mucopolysaccharidoses type I (McKusick 1966). RESUME Une forme de mucopolysaccharidose avec surcharge viscerale et elimination urinaire excessive de chondroitine sulfate Les auteurs decrivent un cas de mucopolysaccharidose dans lequel le principal glycosaminoglycan stocke dans le foie et elimine dans les urines etait la chondroitine sulfate. Les deux isomeres etaient presents en quantiteegaie. Les symptomes cliniques etaient semblables e ceux du syndrome de Hurler autrement dit du type I des mucopolysaccharidoses selon la classification de McKusick en 1966. ZUSAMMENFASSUNG Eine Form von Mukopolysaccharidose, bei der Chondroitinsulfat in Organen gespeichert und in hohem Maβe in Urin ausgeschieden wird Es wird ein Fall von Mukopolysaccharidose beschrieben, bei dem von Glykoaminogly-kanen hauptsachlich das Chondroitinsulfat in der Leber gespeichert und im Urin ausgeschieden wurde. Beide Isomeren waren in gleichem Mase vertreten. Das klinische Bild ahnelte dem beim Hurler Syndrom oder bei der Mukopolysaccharidose Typ I, wie auch von McKusick 1966 berichtet wurde. RESUMEN Una forma de mucopolisacaridosis con almacenamiento visceral y excesiva excrecion urinaria de condroitin sulfato Se describe un caso de mucopolisacaridosis en que el principal acumulo de glicosamino-glican en el higado y excretado en orina era el condroitin sulfato. Ambos isomeros estaban presentes en cantidades iguales. Los caracteres clinicos eran similares a los del sindrome de Hurler o mucopolisacaridosis tipo I descrita por McKusick en 1966.

We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.

A detailed study of the biochemistry of each of the lower lumbar intervertebral discs from 3 spines aged 8, 16, and 44 years has shown progressive changes down the spine in a number of biochemical parameters. These were most apparent in the 44-year-old spine. The chemical composition of proteoglycans of the nucleus pulposus and of its constituent proteoglycans differed from those of the corresponding annulus fibrosus of all three spines. The interaction of proteoglycans with collagen, as assessed by extractability, changed markedly with advancing age, while the molecular size of the proteoglycans from both regions decreased and their keratan sulphate content increased. These changes would be expected to affect the mechanical properties of the disc.

The structure of newly synthesized proteoglycans from explant cultures of cartilage from joints subjected to transection of the anterior cruciate ligament (osteoarthritic) and from normal (non- or sham-operated) joints was examined. The structure of the products of proteoglycan turnover was also examined using explants of normal and osteoarthritic cartilage maintained in culture for a 48 h chase period. The findings were as follows: Newly synthesized (/sup 35/S)-proteoglycans extracted from cartilage explants from osteoarthritic joints whether examined 3 weeks, 3 months, or 6 months after surgery were larger than those from corresponding normal cartilage. This can be explained by the synthesis in osteoarthritic cartilage of abnormally long chondroitin sulfate chains on newly synthesised proteoglycans. The extracts also contained a newly formed small proteoglycan species that was unable to interact with hyaluronic acid. The proportion of this species was higher in osteoarthritic cartilage compared with normal, examined 3 weeks after surgery, but was generally absent from cartilage obtained 3 and 6 months after surgery. Compared with controls, a smaller proportion of the (/sup 35/S)-proteoglycans released into the maintenance medium of explant cultures of osteoarthritic cartilage during a 48 h chase period was able to interact with hyaluronic acid. However, although furnished withmore » longer (/sup 35/S)-glycosaminoglycan chains, these proteoglycans were smaller than those from control explants.« less

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.

Analysis of the knee-joint cartilage of pigs at five ages (namely foetuses from the second half of pregnancy and animals 10 weeks, 25 weeks, 3 years and 5 years old) showed that the composition approached that of adult cartilage by 25 weeks of age, the most marked differences being between foetal and 10 week-old cartilage. Protein–polysaccharides were extracted sequentially, first by brief low-speed homogenization with iso-osmotic sodium acetate, then by two extractions with 2m-CaCl2 for 24h with gentle agitation interspersed with brief low-speed homogenization and agitation for another 24h. About half of the protein–polysaccharides were removed from foetal cartilage by the first extraction and the remainder by the second. The proportion in the first extract declined sharply with the age of the animal, but that in the first CaCl2 extract was similar at all ages other than 10 weeks. The amount left in the residue increased approximately with the collagen content from about one-fifth at 10 weeks of age to one-third in adult and old cartilage. The proportion of medium-sized protein–polysaccharides in the extracts changed little with age after birth, but the glucosamine content increased about fivefold and the protein content almost doubled between 10 weeks and 5 years of age. Other analytical values changed little. These results cannot be explained solely by changes in the proportion of ‘link-glycoprotein’ in the protein–polysaccharides. Since major changes in most parameters had taken place by 25 weeks of age, the first weeks after birth may be a critical period for cartilage development in the pig.

The addition of small amounts of hyaluronic acid (0.01–1.0%) to disaggregated cartilage proteoglyans produced a large increase in hydrodynamic size on gel chromatography. Re-chromatography of the excluded and retarded fractions showed that there was no equilibrium between them. The increase in hydrodynamic size, which was unaffected by the presence of EDTA, was maximal when the proportion of hyaluronic acid was rather less than 1% and decreased with higher proportions up to 50%. There was no such interaction when proteoglycans were mixed with other polyanions in comparable proportions. The interaction was accompanied by a large increase in viscosity which was eliminated in concentrated solutions of guanidine·HCl and at low pH. From the optimal proportions of proteoglycan and hyaluronic acid and estimates of their molecular weights, it was calculated that 10–30 proteoglycans were associated with each hyaluronic acid chain.

The kinetics of incorporation of [35S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ‘pulse–chase’ radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ‘pulse–chase’ experiments over 5h did not demonstrate any precursor–product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ‘pulse–chase’ experiments there was again no evidence for precursor–product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

Proteoglycan extracted from the Kurloff cells of the guinea-pig spleen and its corresponding glycosaminoglycan inhibits the migration of macrophages from capillary tubes in vitro, but the proteoglycan has no effect on lymphocyte transformation in vitro. A proteoglycan, chemically and spectrally similar to Kurloff cell proteoglycan, extracted from human spleen, also inhibits macrophage migration. In contrast, proteoglycan from cartilage and its corresponding glycosaminoglycan had no significant effect. The differences between Kurloff cell proteoglycan and migration inhibitory factor (MIF) are discussed.

1. Protein–polysaccharides of chondroitin 4-sulphate were extracted with neutral calcium chloride from pig laryngeal cartilage that was not completely homogenized. The protein–polysaccharides were purified by precipitation with 9-aminoacridine. On zone electrophoresis in compressed glass fibre at pH7·2 it was separated into two fractions, although two distinct zones were not obtained. These fractions, which had already been shown to differ in their antigenic determinants, also differed considerably in amino acid composition, total protein, hexose and glucosamine contents. 2. The fraction of higher mobility contained approx. 2% of protein and only traces of glucosamine. Serine and glycine accounted for over half the total amino acid residues, but aromatic, basic and sulphur-containing amino acids were not detected. The weight-average molecular weight, determined by sedimentation, was 230000. 3. Assuming that there was the same sequence of neutral sugars at the linkage points as in PP-L fraction (protein–polysaccharide light fraction), the approximate molar ratio of hexose to serine suggested that most of the serine residues were linked to chondroitin sulphate chains. Support for this was derived from the agreement between the weight-average molecular weight of the chondroitin sulphate–peptide after proteolysis, and the chain weight calculated from its serine content. The chain weight based on the serine content of the fraction of higher electrophoretic mobility was approximately similar. 4. In contrast, the fraction of lower electrophoretic mobility resembled PP-L fraction in its amino acid composition, protein and glucosamine contents. The presence of glucosamine, together with the higher hexose content, suggested that this fraction contained some keratan sulphate. 5. The relatively low molecular weight of the fraction of higher mobility enabled it to be extracted without complete disintegration of the cartilage. The unlikelihood of its being produced by autolytic enzymes is discussed.

1. Protein–polysaccharides from pig laryngeal cartilage extracted by two procedures described in the preceding paper (Tsiganos & Muir, 1969) were shown to consist of macromolecules of various sizes as assessed by gel filtration in 4% and 6% agarose. 2. A larger proportion of the smaller molecules was present in the preparation obtained by brief extraction in iso-osmotic sodium acetate (procedure I) than in that obtained by more prolonged extraction in 10% (w/v) calcium chloride (procedure II). 3. Two fractions were separated by gel filtration in 6% agarose and by electrophoresis in compressed glass fibre. These fractions differed in chemical composition and in antigenic determinants. The gel-retarded fraction R and that of higher electrophoretic mobility possessed the same single antigen, whereas the gel-excluded fraction E and the slower electrophoretic fraction contained all the antigens of the starting material including that of fraction R. 4. Five N-terminal amino acid residues were identified in preparation I and fraction E, only two of which were present in fraction R. 5. The relative proportions of gel-excluded and gel-retarded fractions did not change when solutions of high ionic strength, urea or guanidine hydrochloride were used for elution. 6. The differences in chemical and amino acid composition between fractions R and E showed that the latter was not a simple aggregate of the former. Fraction E contained more basic and aromatic amino acids, and some methionine and cystine; the last two were absent from fraction R. Hydroxyproline was not detected in either fraction. 7. The number of glycosidic linkages in both fractions was estimated by alkaline β-elimination. Appreciable amounts of threonine as well as serine were destroyed in both fractions. An average chain length for chondroitin sulphate was calculated from the galactosamine content of both fractions and the amounts of hydroxy amino acid destroyed. Average chain lengths were also calculated from the xylose and galactosamine content of each fraction. Each independent method gave a value of approximately 28 disaccharide units for the chain length in both fractions and hence their difference in size could not be explained by differences in the length of carbohydrate chains. 8. All fractions contained glucosamine, which was attributed to keratan sulphate. Content of both protein and keratan sulphate increased with the size of the macromolecules. 9. It is suggested, from these results, that chondroitin sulphate–protein complexes normally exist as a heterogeneous population of macromolecules in cartilage, and that keratan sulphate is involved in the formation of larger molecules.

A method is described of measuring the tensile stiffness and fracture stress of human femoral condylar cartilage in planes parallel to and at increasing depth below the articular surface. The axis of tension was either parallel or perpendicular to the predominant collagen fibre direction in the superficial zone. Specimens were analysed for their collagen and glycosaminoglycan contents and partial correlation coefficients were determined between the tensile properties and each of the chemical constituents. The correlations between the tensile properties and the collagen content of specimens oriented parallel to the collagen fibre direction was statistically significant in the superficial zone but the significance level decreased with increasing depth. In specimens which were oriented perpendicularly to the collagen fibre direction the correlations between the above variables were less significant. There was no significant correlation between the tensile properties and the glycosaminoglycans in cartilage. Visibly normal specimens from the superficial layer which were situated adjacent to visibly degenerate cartilage were weaker and less stiff than specimens situated on normal joints or remote from visibly degenerate cartilage. Such differences decreased with depth below articular surface and were greater in parallel-oriented specimens.

Explants of cartilage from tibiae of 11–12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.

Pig articular cartilage, from which protein–polysaccharides soluble in iso-osmotic sodium acetate had been removed, was extracted in three further stages with 8m-urea in 2m-sodium acetate and with tris–HCl buffer after bacterial collagenase digestion, followed by the same urea–sodium acetate solution, thus leaving only 2% of the original uronic acid in the tissue. The histological appearance of the cartilage was unaltered until after collagenase digestion. The collagenase used did not affect the viscosity or molecular size of a protein–polysaccharide preparation obtained previously. The protein–polysaccharides in each extract differed in size, amino acid composition and protein content, but protein and keratan sulphate contents were not related to hydrodynamic size, in contrast with protein–polysaccharides extracted previously before collagenase digestion. Hydroxyproline could not be removed from those obtained by the first urea–sodium acetate extraction until degraded by heat. The galactosamine/pentose molar ratio agreed closely with the galactosamine/serine molar ratio that was destroyed on treatment with 0.5m-sodium hydroxide, showing that chondroitin sulphate was attached only to serine residues. From these molar ratios the chondroitin sulphate chains were calculated to be of the same average length in protein–polysaccharides in all three extracts although somewhat shorter than in protein–polysaccharides extracted previously. Some threonine residues were also destroyed on alkali treatment suggesting that keratan sulphate may be attached to threonine. These findings together with previous results show that differences in size, composition and physical state extend to all the protein–polysaccharides in cartilage.

SUMMARY Infusion of a dextran solution of equivalent osmolality to plasma into a patient with the Sanfilippo syndrome caused an increased excretion of uronic acid. This increase was less than that obtained previously, when an equal amount of plasma was infused into the same patient. Moreover, there were no significant changes in the ratio of larger to smaller glycosaminoglycan fragments or in the sulphate/uronic acid ratios, in contrast to the changes noted subsequent to plasma infusion. It is concluded, therefore, that in this case infusion of dextran was not as effective as plasma in inducing a temporary increased mobilisation of pathological glycosaminoglycans. RESUME Effet de perfusion de dextrane sur l'excretion des glucosaminoglycuroniques dans le syndrome de Sanfilippo La perfusion de solution de dextrane isotonique au plasma chez un malade presentant un syndrome de Sanfilippo a provoque une excretion accrue d'acides uroniques. Cet accroissement a ete moindre que celui obtenu anterieurement, apres perfusion d'une quantiteequivalente de plasma chez le meme sujet. De plus, il n'y a pas eu de modifications significa-tives dans le rapport des fragments glucosaminoglycuroniques lourds et legers, ou dans le rapport sulfate-acide uronique, comme cela avait ete note anterieurement lors de la perfusion de plasma. Les auteurs concluent alors que, dans ce cas, la perfusion de dextrane n'a pas ete aussi efficace que celle de plasma pour provoquer un accroissement temporaire de mobilisation des glucosaminoglycuroniques pathologiques. ZUSAM MENFASSUNG Die Wirkung von Dextraninfusionen auf die Glycosaminoglycanausscheidung beim Sanfilippo-Syndrom Die Infusion einer Dextranlosung mit plasmaaquivalenter Osmolalitat steigert bei einem Patienten mit Sanfilippo-Syndrom die Ausscheidung von Harnsaure. Dieser Anstieg war geringer als er bisher durch Infusion gleicher Mengen Plasma bei demselben Patienten erreicht wurde. Daruberhinaus gab es keine signifikante Anderungder Rate vongroszlig;erenzu kleineren Glycosaminoglycanfragmenten oder im Sulfat/Harnsaureverhaltnis wie sie im Anschlus an Plasmainfusionen beobachtet wurden. Die Autoren stellen daher fest, das in diesem Fall die Infusion von Dextran nicht so wie die von Plasma die zeitweise verstarkt auftretende Mobilisation pathologischer Glycosaminoglycane bewirkte. RESUMEN Efecto de perfusiones de dextrano sobre la excrecion de glucosaminogliean en el sindrome de Sanfilippo La perfusion de una solution de dextrano de una osmolaridad equivalente a la del plasma a un paciente con sindrome de Sanfilippo causo una excrecion aumentada de acido uronico. Este aumento fue menor del obtenido previamente, al perfundir una cantidad igual de plasma en el mismo paciente. Ademas no hubo cambios significativos en la relacion entre los fragmentos mas grandes y mas pequenos de glycosaminoglican, o en la relacion sulfato/acido uronico, como los observados tras una perfusion de plasma. Los autores concluyen, por lo tanto, que en este caso la perfusion de dextrano no fue tan efectiva como la de plasma para inducir una movilizacion temporal aumentada de glycosaminoglicanos patologicos.

The glycosaminoglycans in the menisci of beagles 5--7 years old were analyzed at various times after osteoarthritis was induced by sectioning the anterior cruciate ligament of one knee; the unoperated knee served as control. In the first month after induction, there were signs of inflammation in the operated joint. After 1 week, the water content was elevated and the glycosaminoglycan content (per dry weight) was reduced. The content of keratan sulfate decreased more than that of chondroitin sulfate, but the hyaluronic acid content did not change consistently. The relative proportions of chondroitin-4-sulfate, chondroitin-6-sulfate, and dermatan sulfate remained unchanged. After 3--18 months, the glycosaminoglycan levels reverted to normal, and there was some evidence that after 15--18 months, they were elevated above normal. These results, together with results obtained from single examples of mild and severe osteoarthritis in working foxhounds, suggest that, in contrast to articular cartilage, the meniscus is capable of some regeneration in response to injury.

Enzyme Replacement Therapy by Transplantation of HLA-Compatible Fibroblasts in Sanfilippo A Syndrome

The biosynthesis of proteoglycans in the menisci and articular cartilages of the knee (stifle) of mature beagles was studied in the early stages of experimental osteoarthritis. The rate of proteoglycan synthesis, determined by systemic labeling in vivo at 21, 42, and 84 days after sectioning of the anterior cruciate ligament, was generally found to be 1.5-2.5-fold higher than control in articular cartilages and 3-10-fold higher than control in menisci. The medial meniscus was more stimulated than the lateral meniscus, and the medial tibial plateau under the meniscus was more stimulated than the adjacent tibial area. This area-specific stimulation suggests the involvement of mechanical factors in the cellular response. The rate of proteoglycan synthesis determined in vitro at 7, 14, and 21 days after operation was also about 2-fold higher than control in articular cartilages and about 3-fold higher in menisci. This increase in biosynthetic activity in vitro was confirmed by 35S-autoradiography and appeared to be due to general stimulation of existing chondrocytes, particularly in the middle and deep zones of the articular cartilage and throughout the meniscal cartilage. The rate of proteoglycan synthesis determined in vitro in cartilages from 2-week and 3-week sham operated joints was also increased relative to controls, suggesting that humoral as well as mechanical factors are involved in stimulating chondrocyte activity.

ATTEMPTS to treat the mucopolysaccharidoses with infusions of normal plasma or leukocytes have produced mixed results; some workers1–3 reported little or no positive effect, while others4–6 noted both clinical and biochemical changes after treatment. Among the more obvious reasons for these conflicting findings are differences in the responsiveness to treatment of genetically distinct forms of the disorders7, in the volumes of plasma administered and in the activities of the corrective factors in the plasma.

The electrophoresis of proteoglycans and glycosaminoglycans on large-pore polyacrylamide gels which contained agarose as a support medium is described. The average pore diameter of the gels was sufficient to allow penetration of all but the very largest proteoglycans of articular cartilage, making it possible to distinguish “aggregates” from disaggregated molecules. The relative sizes of proteoglycans could be assessed, using only microgram quantities on these gels, as the mobilities were inversely related to hydrodynamic size as determined by gel chromatography. The method was able to separate single from double chains of chondroitin sulfate. On the other hand, the relative mobilities of separate chains of different types of glycosaminoglycans were a function of their charge density. The method has been applied to a pathological condition in which changes in the sizes of proteoglycans were evident.

THE guinea pig lymphocyte has long been known to contain a special inclusion body. Such lymphocytes, called Kurloff cells after their discoverer, increase greatly in number in pregnancy and were shown by Ledingham1 to be produced in both malesand females by administration of œstrogens. Marshall and Swettenham2, by histochemical methods, have shown that the inclusion body is composed of a mucoprotein–sulphated mucopolysaccharide complex. Further work on the chemistry of a mucopolysaccharide fraction by extraction of spleens of œstrogen-treated guinea pigs gave the following results

1. Protein–polysaccharides of chondroitin sulphate were extracted from fresh laryngeal cartilage at pH6·8 by two procedures. Procedure I consisted of brief low-speed homogenization in 0·15m (iso-osmotic) sodium acetate and procedure II consisted of longer homogenization followed by prolonged extraction in 10% calcium chloride solution. 2. The protein–polysaccharides in both extracts were isolated and purified by precipitation with 9-aminoacridine hydrochloride. They were free from serum proteins, collagen and nucleic acids and also of degradative enzymes. The absence of such enzymes was shown by viscosity measurements on solutions of protein–polysaccharides incubated for up to 24hr. at pH4 and 6·8. 3. Mannose, glucose or fucose were not detected by paper chromatography and only traces of sialic acid were present. 4. The yield with procedure II was twice that with procedure I and the products differed in their protein and glucosamine contents. 5. Hyaluronic acid was unlikely to have been precipitated at an acid pH, so the glucosamine was attributed to keratan sulphate, as serum proteins were absent. There was no free keratan sulphate in the preparation. 6. Both preparations were heterogeneous in the ultracentrifuge, showing at least three components.

The excretion and degradation was studied of 35S-labelled 4-chondroitin sulphate injected into guinea pigs in the form of proteoglycan isolated from cartilage and in the form of free chondroitin 4-sulphate prepared from the same proteoglycan by proteolysis. When the proteoglycan was injected there was a delay of about 15–20min before significant amounts or radioactivity were excreted, whereas after injection of chondroitin 4-sulphate a considerable amount of radioactivity was excreted within 10min and a much higher proportion of the radioactive dose was excreted in 1h or 24h compared with the proteoglycan. In both cases, however, a major part of the radioactivity was not excreted even in 24h. Sterile conditions were used to collect the radioactive material directly from the bladder. When chondroitin 4-sulphate was injected, the molecular sizes of injected and excreted materials were similar, as assessed by gel chromatography on Sephadex G-200, whereas when proteoglycan was injected the molecular size of the excreted labelled material was similar to that of the chondroitin 4-sulphate chains in the original proteoglycan. In neither case did the size of the excreted labelled material change with time over 1h, and low-molecular-weight labelled material was virtually absent. In contrast, when urine was collected for 24h without preservative the labelled material in it was extensively degraded after either the proteoglycan or chondroitin 4-sulphate had been given. Chondroitin 4-sulphate became similarly degraded when incubated with non-sterile urine, but not when the urine was passed through a bacterial filter, suggesting that degradation was caused by contaminating micro-organisms in the experiments in which urine was collected for 24 h. It is concluded that chondroitin 4-sulphate chains of about 18000 molecular weight can be excreted readily as such, whereas intact proteoglycans must be degraded to free glycosaminoglycans first, although both are taken up by the tissues more rapidly than they are excreted.

A physical method is described for measuring the concentration of fixed negatively charged groups in articular cartilage. Comparison is made with values of fixed charged density derived from chemical analysis of hexuronic acid and total hexosamine contents. The mean deviation between values derived from the two methods was 10%. This study was carried out on successive tangentially cut slices of human articular cartilage from the femoral condyle. The following observations were made. The fixed charge density increased with depth from the articular surface, as did the uronic acid and total hexosamine contents. In the 25–60 age group neither the hexuronic acid nor the total hexosamine contents showed any correlation with the age of the subject. However, there was considerable local variation and variation between individuals.

In cartilage proteoglycans, chains of chondroitin sulphate are attached through a linkage region of neutral sugars to the protein core. Smith degradation, in which periodate oxidation is employed, cleaves the linkage at the terminal xylose residue, removing the entire chain without modifying the amino acids involved in the linkage. Rabbits were immunised with Smith degraded proteoglycan from bovine nasal cartilage, as well as with the intact proteoglycan and with proteoglycan from pig laryngeal cartilage. Cross reaction between all of the antigens and antisera showed that there was an antigenic determinant common to all three proteoglycans. Results obtained with the Smith degraded proteoglycan and its antiserum showed that the sugars of the linkage region and the chondroitin sulphate chains did not contribute to the expression of the species-common determinant, which must be due solely to amino acid sequence. Immunodiffusion and tanned-cell haemagglutination experiments showed that this species-common determinant was localised in that region of the core protein which in intact proteoglycans is resistant to trypsin and chymotrypsin.

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm−1. Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein–polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline β-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein–polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein–polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein–polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein–polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm−1. Although active incorporation of [35S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.

Protein–polysaccharides of femoral articular cartilage from pigs of ages 9 months and 5 weeks were compared after extraction at pH6·8 with iso-osmotic sodium acetate followed by 0·63m-calcium acetate. The cartilage from the younger animals had a higher moisture content and contained considerably larger amounts of protein–polysaccharide, but less than half as much collagen/g. dry weight, than cartilage from the older pigs. There was notably less keratan sulphate in the fractions from the less mature animals. After gel filtration on 6% agarose, elution profiles of the calcium acetate extracts were similar to those of the sodium acetate extracts of the same tissue. Chemical analyses, however, showed that in both age-groups the extraction procedure had achieved a sequential solubilization of protein–polysaccharides in that the initial extracts contained a higher proportion of keratan sulphate than those that were extracted subsequently. Both extracts from the older animals contained up to 25% of a relatively small protein–polysaccharide that was retarded on 6% agarose and that had a lower protein content and less keratan sulphate than the larger protein–polysaccharides. In contrast, in extracts from the less mature cartilage only about 5% of the protein–polysaccharides were small enough to be retarded by 6% agarose, suggesting that the small components may not be precursors of the larger. The average length of chondroitin sulphate chains, as calculated from the analytical data, was the same in the smaller protein–polysaccharides as in the larger.

Intensive rearing, and restricted activity, induce rapid growth in pigs, but they often become lame. Groups of normal and lame pigs reared intensively were killed when 10 or 25 weeks old. Although there were no differences in the overall composition of the knee-joint cartilage of lame and sound animals, the proteoglycans in the cartilage of the lame pigs were extracted more easily by a standardized sequential procedure and contained a higher proportion of molecules of smaller size as assessed by gel chromatography on 6% agarose and Sepharose 4B. These increased at the expense of both the larger and mediumsized molecules. Differences were most evident at 10 weeks of age, when there was twice as much of the smaller proteoglycans in the cartilage of lame pigs. Despite these size-differences, the compositions of the proteoglycans in corresponding sequential extracts of cartilage of lame and normal groups were the same, as were the changes in chemical composition that accompany development. Proteoglycans from lame animals may have undergone limited proteolysis, thus decreasing their size without changing their composition detectably. As the differences between normal and lame groups were greater at 10 weeks than at 25 weeks of age, the first weeks after birth (when the greatest changes occur in the proteoglycans and in the cartilage) may be a critical period in the maturation of articular cartilage in this species. At this time, rapid gain in weight produced by intensive rearing may be too great for the immature cartilage to bear.

Oligosaccharides derived from hyaluronic acid were shown to inhibit proteoglycan–hyaluronic acid interaction, as measured in a viscometer. The relative inhibition increased with the size of the oligosaccharide and the results suggested that decasaccharides were the smallest fragments able to bind strongly to the proteoglycan.

The structures of the sulfated mucopolysaccharides are outlined and their common and particular features pointed out. All are normally attached to specific proteins by covalent linkages to form large complex molecules. The biosynthesis of the sulfated mucopolysaccharides is considered in relation to the possible genetic defects of the mucopolysaccharidoses. The formation of the core protein is a prerequisite for the subsequent synthesis of the carbohydrate chains in both normal and abnormal cells. A linkage region consisting of atypical neutral sugars that joins the carbohydrate chain to protein is common to all the sulfated mucopolysaccharides apart from keratan sulfate. The formation of this region is strictly controlled by the specificities of the glycosyl transferases that form it. The synthesis of the main carbohydrate chain is dependent on prior formation of the linkage region so that a defect in the formation of the linkage region or in glycosylation of the core protein cannot explain the abnormalities of the mucopolysaccharide diseases. Other possible explanations are considered, including defects in protein-polysaccharide structures that might produce inbalance in synthesis and degradation. Faulty intracellular degradation of mucopolysaccharides has been shown in cultures of fibroblasts from patients with Hurler's and Hunter's syndromes, implying a deficiency of degradative enzymes. Deficiencies of certain lysosomal exo-glycosidases found in some tissues of patients with mucopolysaccharidoses is unlikely to be the primary defect in view of the specificities of these enzymes and the structures of the mucopolysaccharidoses. Some of the clinical features of the mucopolysaccharidoses may be explained by possible secondary effects of the accumulation of abnormal mucopolysaccharides within the body.

Glycosaminoglycans were isolated from the urine of three patients with Hurler's, Hunter's and Morquio's syndromes and also from the liver and spleen of the case of Hurler's syndrome by a procedure avoiding further degradation. A method of determining the proportions of dermatan sulphate, heparan sulphate and chondroitin sulphate in each preparation is described. The relative proportions of these glycosaminoglycans in the urine and organs of the case of Hurler's syndrome were very similar. Glycosaminoglycans from the organs were of much lower molecular weight than normal, consisting of single chains of molecular weight about 5000 together with multiples of up to four such chains attached to peptide moieties. The linkage region normally attaching glycosaminoglycan chains to protein in whole protein–polysaccharides of connective tissue was degraded progressively towards serine. The total output and relative proportions of abnormal glycosaminoglycans in the urine were compared in two brothers with Hunter's syndrome examined on two occasions 4 years apart. At comparable ages they excreted about the same amount, and the relative proportions of each glycosaminoglycan remained essentially constant. The composition and chromatographic behaviour of the glycosaminoglycan in the urine from the case of Morquio's syndrome indicated that it consisted of material containing about one-third keratan sulphate and two-thirds chondroitin sulphate as part of the same molecule, as in proteoglycans of cartilage. The total output of glycosaminoglycans, although higher than normal, was considerably less than in other types of Mucopolysaccharidoses.

1. Serial slices of articular cartilage obtained at necropsy from apparently normal femoral condyles of individuals between the ages of twenty-six and sixty were examined chemically, by electron microscopy and for permeability. 2. The most superficial layer was shown by chemical analysis and electron microscopy to have the highest collagen content, which fell sharply with distance from the articular surface. On the other hand the glycosaminoglycan content was very low in the superficial layers but increased with depth. This variation was found in all specimens tested but the absolute levels of collagen and of glycosaminoglycans were widely different. There was no correlation of chemical composition with age. 3. Collagen fibrils in the superficial layer were of much smaller diameter than in the deeper zones. 4. Hydraulic permeability was shown to depend more on glycosaminoglycan than on collagen content, although it varied inversely with both these factors. 5. The results obtained demonstrate clearly the close relation between the physical properties of cartilage and its chemical composition.

The metabolism of newly-synthesised and total (“resident”) proteoglycans was examined in control and osteoarthritic cartilage explants obtained from an experimental model (Pond and Nuki, 1973) of canine osteoarthritis. The following findings were obtained: (i) Non-labelled proteoglycans extracted from normal cartilage with 4 M guanidine HCl showed two bands visualised by staining with toluidine blue. The electrophoretic mobilities of proteoglycans from osteoarthritic cartilage were unchanged but the relative abundance of the slower migrating band increased with time after surgery. (ii) There were qualitative differences in the proteoglycan breakdown products released into the medium of explant cultures of osteoarthritic compared with control cartilage. This was apparent for both labelled and total unlabelled proteoglycans. (iii) There were similarities in the electrophoretic mobilities of the major labelled and nonlabelled proteoglycan breakdown products suggesting that total (“resident”) proteoglycans and newly-formed proteoglycans were degraded by similar mechanisms. There were however some differences in the labelled and non-labelled proteoglycans, suggesting that the mechanisms of breakdown were not identical. (iv) Immunoblotting techniques showed differences in the distribution of various glycosaminoglycans in proteoglycan breakdown products from control compared with osteoarthritic cartilage explant cultures. (v) Monoclonal antibodies 7-D-4 and 3-B-3 (which recognise unusual native chondroitin sulphate epitopes) showed greatly increased expression on proteoglycans from osteoarthritic cartilage compared with controls.

Purified proteoglycans extracted from pig laryngeal cartilage in 0.15 M-NaCl and 4 M-guanidinium chloride were analysed and their amino acid compositions determined. Selective modification of amino acid residues on the protein core confirmed that binding to hyaluronate was a function of the protein core, and was dependent on disulphide bridges, intact arginine and tryptophan residues, and epsilon-amino groups of lysine. Fluorescence measurement suggested that tryptophan was not involved in direct subsite interactions with the hyaluronate. The polydispersity in size and heterogeneity in composition of the aggregating proteoglycan was compatible with a structure based on a protein core containing a globular hyaluronate-binding region and an extended region of variable length also containing a variable degree of substitution with chondroitin sulphate chains. The non-aggregated proteoglycan extracted preferentially in 0.15 M-NaCl, which was unable to bind to hyaluronate, contained less cysteine and tryptophan than did other aggregating proteoglycans and may be deficient in the hyaluronate-binding region. Its small average size and low protein and keratan sulphate contents suggest that it may be a fragment of the chondroitin sulphate-bearing region of aggregating proteoglycan produced by proteolytic cleavage of newly synthesized molecules before their secretion from the cell.