Helen Muir

A modified uronic acid carbazole reaction

Abstract A modification of Disches carbazole reaction for uronic acid in the presence of borate is described. The advantages of the procedure are: 1. (1) There is an approximately twofold increase of sensitivity. The OD is a linear function of concentration between 4 and 40 μg/ml. 2. (2) Maximum color develops immediately. 3. (3) The color is stable for at least 16 hr. 4. (4) There is greater reproducibility and reduction of interference by chloride ion and oxidants. It has been found possible to distinguish between heparin, heparin derivatives, and other polyuronides of connective tissue by comparing the effect of chlorides on the color yield in both procedures.

THE DISTRIBUTION OF COLLAGEN IN HUMAN ARTICULAR CARTILAGE WITH SOME OF ITS PHYSIOLOGICAL IMPLICATIONS

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.

Gel electrophoresis of proteoglycans and glycosaminoglycans on large-pore composite polyacrylamide-agarose gels

Abstract 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.

Electrophoresis of 35S-labeled proteoglycans on polyacrylamide-agarose composite gels and their visualization by fluorography

We have developed techniques for the electrophoresis of 35S-labeled proteoglycans on polyacrylamide-agarose gel slabs and subsequent fixation, impregnation, and fluorography of such electrophoretograms. The procedure permits the examination of newly synthesized proteoglycan subspecies using a rapid technique, previously unavailable for these labeled molecules.

Mucopolysaccharides of whole human spleens in generalized amyloidosis.

Amyloidosis is a morphological concept (2) the diagnosis of which has been based on histological critera (3). Primary and secondary amyloidosis and the forms of amyloidosis seen in conjunction with familial Mediterranean fever (FMF) and myeloma are all grouped under one name because of morphological similarities, although it is not certain that they represent a single disease. Electron microscopy has, however, revealed characteristic fibrils in all forms of amyloidosis (4-6). Some of the stains that are taken up by amyloid material suggest that it may contain mucopolysaccharides (MPS) (7). Nevertheless, chemical investigations have not previously shown any marked increase in extractable metachromatic material (8) nor in uronic acid (9) or sulfate-containing compounds (10), although heparan sulfate has been isolated from a few amyloid-bearing organs (11, 12). The purpose of the present study was a systematic chemical investigation of the MPS in an extended series of cases and controls that has not hitherto been carried out.

THE DIAGNOSTIC VALUE OF ISOLATED URINARY MUCOPOLYSACCHARIDES AND OF LYMPHOCYTE INCLUSIONS IN GARGOYLISM.

Gargoylism or Hurlers syndrome (Hunter, 1917; Hurler, 1919) is a rare genetically determined disease (McKusick, 1960) appearing in autosomal recessive and sex-linked forms. Two of the six known sulphated mucopolysaccharides are produced in excess, namely chondroitin sulphate B and heparitin sulphate. They accumulate in most organs of the body, especially in the liver and the spleen (Meyer, Grumbach, Linker and Hoffman, 1958; Meyer, Hoffman, Linker, Grumbach and Sampson, 1959). They appear as metachromatic deposits which, however, are very soluble in routine fixatives so that liver biopsies might show vacuoles unless they are fixed by special techniques (Haust and Landing, 1961). Both mucopolysaccharides are excreted in large amounts in the urine (Dorfman and Lorincz, 1957; Lorincz, 1958) and being resistant to hyaluronidase, are distinguishable from the mucopolysaccharides of normal urine, which are excieted only in small amounts (Di Ferrante and Rich, 1956). It has been found that a proportion of the lymphocytes of such patients contains abnormal cytoplasmic inclusions (Mittwoch, 1959, 1961) which can be seen in preparations stained with May-GrunwaldGiemsa. They also stain metachromatically with toluidine blue. These staining reactions agree with the assumption that the inclusions consist of acid mucopolysaccharides. Up to the present the lymphocyte inclusions have been found in a total of 22 patients, in whom a diagnosis of gargoylism had been suggested. In the present investigation these inclusions have been correlated with the presence of abnormal amounts of hyaluronidase-resistant mucopolysaccharides in the urine. Some of the clinical findings on the patients investigated by both methods are summarized in Table 1. Table 2 summarizes the clinical findings of the other patients in whom lymphocyte inclusions were found, without biochemical investigation. Reports on some of the cases have already been published: five cases in Table 2 and Case 2 of Tables 1 and 3 (Mittwoch, 1959); Cases 1, 3, 6 and 7 (Mittwoch, 1961).

Estimation of pentoses and methylpentoses in biopolymers, in particular of fucose and xylose.

Abstract 1. (1) Conditions under which pentoses and methylpentoses react with anthrone in sulfuric acid have been reexamined. 2. (2) The number of steps involved has been reduced and good reproducibility obtained. 3. (3) Xylose was the most reactive pentose; other pentoses and methylpentoses required longer times of heating at 40°C. These differences in reaction rates and absorption spectra were utilized to estimate fucose and xylose in a mixture. 4. (4) Interference due to hexoses, hexuronic acids, hexosamine, sialic acid, and deoxyribose was virtually eliminated and no interference by tryptophan and protein was observed. 5. (5) Bound fucose and xylose reacted as easily as the free sugars. Although ribose in RNA did not, nevertheless the method could be used to estimate RNA in the presence of DNA provided an RNA standard is used.

An automated version of the periodate-thiobarbituric acid assay for analysis of Δ-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acid and chondroitin sulfates☆

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μm) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.

A CONTRIBUTION TO THE DIFFERENTIAL DIAGNOSIS OF HURLER'S DISEASE AND FORMS OF MORQUIO'S SYNDROME

1. Mucopolysaccharides were analysed in the urine of thirteen patients with Morquios syndrome aged between three and fifty-nine years and of fourteen controls of comparable ages.nn2. There were no significant qualitative or quantitative differences between patients and controls.nn3. The clinical and radiological findings suggested that these patients did not have the Morquio-Ullrich form of the disease, which appears on retrospective assessment of case reports to be more uniform and less diffuse than the Morquio-Brailsford form which may include a number of possibly unrelated diseases.nn4. Keratosulphate has so far been demonstrated in the urine only of patients with the Morquio-Ullrich form of the disease, although the mucopolysaccharide excretion has been investigated in only a few patients with the Morquio-Brailsford form. The normal mucopolysaccharide excretion of the present series of patients suggests that a normal mucopolysaccharide excretion distinguishes the Morquio-Brailsford from the Morquio-Ullrich form, the latter having a number of features overlapping with Hurlers disease, where large amounts of mucopolysaccharide other than keratosulphate are excreted.nn5. Both qualitative and quantitative analysis of urinary mucopolysaccharide are thus necessary to distinguish between Hurlers disease, the Morquio-Ullrich form and the Morquio-Brailsfords form of Morquios syndrome.