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Biomedical subjects

B Engfeldt

Publications and source records attributed to B Engfeldt.

At least 37 records · Page 2Linked to original sources

Analysis of dentine glycosaminoglycans using high-performance liquid chromatography.

Puppy dentine was prepared using ultracentrifugation of tooth powder in organic density gradients. The glycosaminoglycans of the obtained tissue fraction were prepared after papain digestion and beta-elimination, using preparative chromatography on DEAE-cellulose and CPC-cellulose. These polysaccharide fractions were analyzed using highly sensitive HPLC procedures. One such HPLC procedure allowed hyaluronic acid to be determined in less than microgram amounts. The glycosaminoglycans thus prepared consisted only of chondroitin-4-sulfate, chondroitin-6-sulfate, and small amounts of highly hybridized dermatan sulfate, while the experiments failed to demonstrate even trace amounts of keratan sulfate, hyaluronic acid or heparan sulfate.

Animals↗

Stereological studies on matrix vesicle distribution in the epiphyseal growth plate during healing of low phosphate, vitamin D deficiency rickets.

A model of the healing phase of low phosphate, vitamin D deficiency was used to investigate the initial stages of mineralization. The matrix vesicle distribution between the zones of the growth plate was found to be bimodal with high volume densities in the resting and hypertrophic zones and low volume densities in the proliferative and calcifying zones. Healing of the rachitic lesion was associated with a decrease in matrix vesicle volume density in the calcifying zone, compared with the lower hypertrophic zone in florid rickets. The volume density differences were due to differences in the number of vesicles, as the variation in mean caliper diameter was rather small. The findings are compatible with the dynamic cell debris theory for matrix vesicle origin and distribution presented earlier, which favours the view that a major part of matrix vesicles are formed from cell debris. A role of matrix vesicles in the mineralization process is indicated by the finding of an association between mineralization and matrix vesicle degradation.

Animals↗

Dyggve-Melchior-Clausen dysplasia. Morphological and biochemical findings in cartilage growth zones.

The results of light and electron microscopic examination and of biochemical proteoglycan studies of costochondral and iliac crest biopsies from a recently diagnosed case of Dyggve-Melchior-Clausen dysplasia are reported. At light microscopy of resting cartilage large lacunae containing clusters of five or more chondrocytes were seen in some areas. In the hyaline cartilage there were scattered fibrous foci but no mineralized areas. Electron microscopy revealed chondrocytes containing widened cisternae of rough endoplasmic reticulum and vesicles coated with a smooth single-layered membrane. The content of the cisternae and of the vesicles was amorphous. Throughout the cartilage a considerable proportion of the chondrocytes displayed more or less pronounced necrobiotic changes. The biochemical analysis showed an increased amount of glucosaminoglycans in the cartilage and indicated that the ability of proteoglycan monomers to reaggregate to hyaluronic acid chains was decreased. Our findings support the suggestion that Dyggve-Melchior-Clausen dysplasia is due to a disturbance in proteoglycan metabolism.

Cartilage↗

Morphological and biochemical analysis of biopsy specimens in disorders of skeletal development.

A programme for combined morphological and biochemical analysis of cartilage-bone biopsy material is described. The structural analysis includes light and electron microscopy, and the biochemistry is focused on polysaccharides. A procedure for isolation and characterization of proteoglycans and glycosaminoglycans from dry cartilage in amounts down to 1 mg is described. The procedure is exemplified by analyses of material from patients with osteogenesis imperfecta, mucopolysaccharidosis IV-Morquio and metaphyseal chondrodysplasia type McKusick. In osteogenesis imperfecta characteristic morphological alterations were demonstrated. Apart from increased chondroitin sulphate molecular weights, the biochemical analyses showed values within the assumed normal range. In Morquio's disease extensive intracellular deposits in single membrane vacuoles were observed in both chondrocytes and osteocytes. There were indications that these deposits contained keratan sulphate, which also occurred in increased amounts in the total tissue preparation. Furthermore, the tendency to formation of proteoglycan aggregates was decreased. In the tissue from the patients with the McKusick syndrome insignificant ultrastructural changes, mainly affecting the mitochondria, were found. The biochemical results were within the assumed normal range. The significance of these findings and the possibilities of further methodological development are discussed.

Bone and Bones↗

Bone mineral content in women with vertebral fractures.

Postmenopausal women, classified as osteoporotics on the basis of clinical and microradiographic findings, were found to have a low bone mineral content as compared with age-matched controls. The bone mineral content in 12 patients on a dietary supplement of calcium and vitamin D did not decrease during a two-year follow-up period.

Aged↗

The occurrence of chondroitin-6-sulphate in adult human compact bone tissue.

Compact bone tissue chondroitin sulphate, previously considered to be pure chondroitin-4-sulphate, was isolated from three adult human femoral diaphyses and digested with chondroitinase ABC. Assaying these digests by means of high performance liquid chromatography we could demonstrate that 12 to 14% of the disaccharide residues were 6-sulphated. The 4/6-ratios were also studied in chondroitin sulphate fractions of different molecular size. Slightly increasing amounts of 6-sulphated disaccharides were found with increasing size of the chondroitin sulphates.

Aged↗

Determination of sulphated disaccharides from chondroitin sulphates by high-performance liquid chromatography.

A sensitive method for the determination of chondroitin 4- and 6-sulphate is presented. After chondroitinase digestion of the chondroitin sulphate preparations, the obtained disaccharides are separated on a weak anion-exchange resin in a high-performance liquid chromatography system. The method was used to study 4-sulphate to 6-sulphate ratios in chondroitin sulphates prepared from bovine nasal cartilage and human nucleus pulposus. The results show clearly that these two preparations contain considerable amounts of both isomers.

Animals↗

Modified procedures for the determination of hexuronic acids, hexoses and proteins using the Auto Analyzer II system.

Previous automated procedures for the determination of hexuronic acids, hexoses and proteins have been modified to suit the Technicon Auto Analyzer II system. The present methods were highly reproducable and the detection limits showed to be in the order of 10 micrograms/l (hexuronic acids and hexoses) and 60 micrograms/l (protein) when 0.13--0.34 ml of the sample was used.

Autoanalysis↗

Proteoglycans of dentine and predentine.

A density gradient system is presented by which dentine and predentine are separated, leaving an intermediate fraction, which contains material from the mineralization front. From the fractions thus obtained the proteoglycans were extracted with 4 M guanidinium chloride and further purified in urea on a DEAE column. The glycosaminoglycans extracted from dentine appeared to be protein-bound, as judged from papain digestion experiments. The polydispersity of dentine proteoglycans seemed to depend, at least partly, upon the polydispersity of its glycosaminoglycans. The materials extracted from the three tissue fractions were ellted in a similar way from Sepharose 6 B, and the amino acid composition of the preparation was determined. The differing proteoglycan patterns of the three tissue fractions indicate a metabolism related to the mineralization front.

Amino Acids↗

Glycosaminoglycans and proteoglycans of human bone tissue at different stages of mineralization.

Using ultracentrifugation, compact bone from a growing 15 years old man was separated into three fractions with different degrees of mineralization. The finely powdered material was fractionated in a density gradient of acetone and bromoform in varying proportions. The glycosaminoglycans were isolated after papain digestion of the tissue fractions. The glycosaminoglycan elution profiles from CPC-cellulose microcolumns indicated the presence of chondroitin-4-sulphate and, possibly, minor traces of hyaluronic acid. The degree of sulphatation was the same in all three fractions, while the total amount and molecular size of the glycosaminoglycans decreased somewhat with increasing degree of mineralization. The glycosaminoglycans of the tissue fractions were also extracted with 4 M guanidinium chloride and then purified chromatographically on DEAE. Gel chromatography of the isolated material showed three different peaks, two probably representing low molecular weight proteoglycans. The proportion of intermediate molecular size material increased with increasing degree of mineralization. The results are discussed briefly in relation to the mineralization process.

Adolescent↗

Glycosaminoglycans of cartilage and bone tissue in two cases of osteogenesis imperfecta congenita.

Epiphyseal cartilage and bone tissue from two cases of osteogenesis imperfecta congenita and one control case with similar skeletal age were examined regarding their glycosaminoglycan content. The diseased bone tissue showed a 3-fold increase in glycosaminoglycans, and 20-25 per cent of the chondroitin sulphate disaccharides seemed to be disulphated. The diseased cartilages showed only traces of material with characteristics of disulphates disaccharides. No disulphates disaccharides were isolated from the control materials. No difference between diseased and control material was indicated regarding molecular size, chondroitin-4-sulphate/chondroitin-6-sulphate ratio or contents of keratan sulphate and hyaluronic acid. The disease is thus associated with fundamental changes of the glycosaminoglycan structure, and these changes may be of functional importance to the mineralization process as well as to the organisation of collagen.

Bone and Bones↗

Morphological effects of rapeseed oil in rats. I. Short-term studies.

Light microscopy of paraffin embedded and frozen sections, supplemented with electron microscopy, was performed on the heart muscle of young rats fed rapeseed oil in short-term experiments. It was confirmed that high levels of rapeseed oil, which contains erucic acid, produce severe lipoidosis of the heart muscle fibres within 10 days. An attempt was made to find out the lowest level of erucic acid in the rat diet to give rise to pathological fatty accumulation. Several frozen sections from each heart or serial sections in combination with electron microscopy were used for this evaluation. The level found to give rise to pathological fatty accumulation was about 2% by weight (w/w), while rats fed 1% erucic acid showed normal myocardium. No direct proof that erucic acid is of importance in human pathophysiology has hitherto been presented. It is concluded, however, that the similarity in reaction among the many species of experimental animals tested by different workers, as well as the basic metabolic disturbances demonstrated, are in strong favour of a similar effect in man.

Animals↗

Morphological effects of rapeseed oil in rats. II. Long-term studies.

In long-term studies covering up to 160 days young Sprague-Dawley rats were fed diets containing 40 cal% of fat. The fat component consisted of either conventional rapeseed oil, or Canadian rapeseed oil low in erucic acid, or arachis oil. Myocardial fatty accumulation was demonstrated in light microscopic studies throughout the experiments in rats fed conventional rapeseed oil, but the number of fat droplets decreased with time. The controls fed arachis oil showed no fatty accumulation. In the rats fed conventional rapeseed oil focal myocardial lesions appeared after 40 days on the diet. These consisted of histiocytic infiltration, occurrence of macrophages, myolysis, proliferation of fibroblasts and finally scarring. Such foci were found widely spread in the myocardium of these rats. In the experimental groups given Canadian rapeseed oil from the cultivar Oro no histiocytic foci or scarring were observed. Small myocardial lesions were occasionally found in the control rats. These latter findings were observed on serial sections. It was concluded that this type of lesion is a "normal" finding. The number and size of the foci observed in animals fed conventional rapeseed oil (10% and 2% (w/w) erucic acid in the diet) indicate, however, that they have to be considered pathological under such circumstances. The pathogenesis of the myocardial alteration is discussed and it is concluded that the long-chain fatty acids are responsible. No direct proof has been presented that the described events are of importance in human pathophysiology, However, several circumstances pointing in this direction are discussed. It is concluded that on the basis of our present knowledge a pathological effect of erucic acid and its homologues in man cannot be excluded.

Animals↗

Morphological effects of rapeseed oil in rats. III. Studies in germ-free rats.

The morphological effects on the myocardium of feeding rapeseed oil were compared in conventional and germ-free rats in short-term experiments (10 days). It was concluded that the fatty accumulation in the heart muscle cells occurring in rats fed rapeseed oil was not influenced by the presence or absence of a normal intestinal flora. In long-term experiments (80 days) under similar conditions, the myocardial effects of feeding germ-free rats with conventional rapeseed oil, rapeseed oil from the Canadian cultivar Oro very low in erucic acid, or arachis oil were studied in serial sections. Severe myocardial lesions developed in the group of rats fed conventional rapeseed oil, while in the other two groups the myocardium was completely normal. These results give no support to the theory that other factors than C22:1 acids in rapeseed oil are responsible for the myocardial lesions.

Animals↗