Moira Breen

Microanalysis and characterization of acidic glycosaminoglycans in human tissues.

Abstract The acid glycosaminoglycans (AG) were isolated from small quantities of human tissue (1.0 gm wet weight or less) and were separated, identified, and measured by zone electrophoresis on cellulose acetate. The major AG components in human skin, sclera, and cornea were identified and measured. The amount of Alcian Blue (dye used to stain the AG) bound to each AG polymer on the cellulose acetate strip was dependent on the number of moles of disaccharide repeating units (DRU) times the number of dissociated carboxylic and sulfate groups per DRU. The degree of dissociation of these groups depended on their corresponding pKs and the pH of the Alcian Blue solution. The glucosamine/galactosamine fraction agreed closely with the hyaluronic acid/sulfated AG (dermatan sulfate + chondroitin 4 6 - sulfate ) fraction obtained by electrophoresis. The mobility of each AG polymer was affected more by the number of negative charges per DRU than by the molecular weight. The identity and number of charged groups per DRU could be deduced from the electrophoretic mobilities and the dye binding data. The combined use of chemical analyses and zone electrophoresis permitted the major AG polymers to be identified and measured in tissues without the use of ion-exchange columns.

Intraocular pressure and glycosaminoglycan distribution in the rabbit eye: Effect of age and dexamethasone

Abstract The effect of dexamethasone on intraocular pressure was studied in young and old rabbits and was related to the concentration and distribution of the glycosaminoglycans obtained from the proteoglycan in the anterior segment of the eye (central cornea, peripheral cornea, sclera and iris). Topical application of dexamethasone resulted in an increase of intraocular pressure of young rabbits but had no effect on the intraocular pressure of old rabbits. The changes in the relative distribution of the glycosaminoglycans components were observed as a function of age and/or dexamethasone treatment. The ratio of non-uronic acid containing glycosaminoglycans (keratan sulfate) to the uronic acid containing glycosaminoglycans decreased with age and was reversed by dexamethasone treatment of the aged animal. Thus, the distribution of the glycosaminoglycans induced by dexamethasone treatment may modulate intraocular pressure.

Acidic glycosaminoglycans in human skin during fetal development and adult life.

Abstract 1. 1. There is little information on the acidic glycosaminoglycans in human skin during fetal development. The present study was undertaken to compare the concentration and composition of skin acidic glycosaminoglycans in the developing human fetus with the adult. 2. 2. Hyaluronic acid, dermatan sulfate and chondroitin 4 (6)-sulfate were found in adult skin and in fetal skin from 5.5 months to term. Skin from a 3-month-old fetus contained only hyaluronic acid and chondroitin 4(6)-sulfate. 3. 3. The concentration of the acidic glycosaminoglycans in fetal skin at 3, 5.5 and 9 months gestation was twenty times, five times, and twice the adult levels respectively; the negative correlation was highly significant. The decrease in skin acidic glycosaminoglycans during this developmental period was due to primarily to the fall in hyaluronic acid. 4. 4. There was a highly significant positive correlation of the galactosamine: hexosamine mole ratio of skin acidic glycosaminoglycans with fetal age, indicating a relative increase in the sulfated skin acidic glycosaminoglycans as the fetus developed. 5. 5. Back and abdominal skin from the same fetal subject had the same acidic glycosaminoglycans concentration and composition; no significant difference was observed between adult abdominal and adult collar-line (exposed part of the back of neck) skin. 6. 6. The changes in the composition and concentration of skin acidic glycosaminoglycans occur primarily during fetal development and childhood growth. Very little change occurs during adult life.

The Acidic Glycosaminoglycans in Human Fetal Development and Adult Life: Cornea, Sclera and Skin

A comparison was made of the concentration and composition of the acidic glycosaminoglycans in human cornea, sclera and skin during five to nine months fetal development and in the adult. In skin, the concentration of the acidic glycosaminoglycans decreases gradually duringfetal development; the adult value is still less than the newborn; hyaluronic acid and dermatan sulfate are always present. In sclera, during the same period of development the concentration of the acidic glycosaminoglycans varied little; dermatan sulfate is the main component in both fetus and adult but the latter also contains some hyaluronic acid and chondroitin 4(6)-sulfate. The concentration of the acidic glycosaminoglycans in cornea increased by 33% at seven months gestation and remained constant; fetal cornea has chondroitin 4(6)-sulfate, chondroitin and a small amount of keratan sulfate (18 to 27% of the acidic glycosaminoglycans); in contrast, the adult cornea is rich in keratan sulfate (64 % of the acidic glycosaminoglycans) a...

The use of high centrifugal force in separating plasma calcium into its protein-bound and protein-free fractions

Abstract The method of obtaining protein-free calcium in plasma using the Spinco/ Beckman Model L Preparative Ultracentrifuge was investigated. It proved to be a simple procedure of high precision requiring the minimum of manipulation. The pH and temperature were easily and effectively controlled throughout the procedure. Equilibration with CO 2 was eliminated. The protein-free calcium in 14 normal subjects was 53.5% of the total plasma calcium as compared with 60% in 3 cases of hyperparathyroidism.

Microanalysis of glycosaminoglycans

Abstract A sensitive and versatile method for the qualitative and quantitative determination of glycosaminoglycans (GAGs) is described. An enriched GAG fraction was subjected to nuclease enzyme treatment and to an appropriate sequence of GAG degrading enzymes—Streptomyces hyaluronidase, chondroitinase AC and ABC, and endo-β- d -galactosidase—and nitrous acid treatment. To determine the result of each degradative procedure, the remaining GAG polymers were subjected to cellulose acetate electrophoresis. The combination of sequential degradation and the monitoring of each step by electrophoresis and densitometry permitted the identification and quantitation of all the GAGs on a microscale basis.

Keratan sulfate-like glycosaminoglycan in the cerebral cortex of the brain and its variation with age.

A keratan sulfate-like substance was found kn the cerebral cortex proteoglycans of rats. This substance tripled during the rapid growth phase from 1 to 3 months of age and then decreased steadily to a negligible quantity in the senescent rat, aged 25 months. In contrast, the uronic acid-containing glycosaminoglycans remained constant during the life span studied. The marked decrease observed in the proportion of keratan sulfate-like moiety to the other glycosaminoglycans in the cerebral cortex of the senescent rat (aged 25 months) compared with the young adult rat (aged 3 months) may explain the reduced extracellular volume observed by others in the senescent brain.

Acidic glycosaminoglycans in developing sterno-costal cartilage of the hydrocephalic (ch+/ch+) mouse

Abstract The sterno-costal cartilage of the hydrocephalic mouse carrying the autosomal recessive gene ( ch +/ ch +) has 40 ± 3% of the acidic glycosaminoglycan concentration of the normal control containing the satin marker (+ sa /+ sa ). The acidic glycosaminoglycan concentration of the sterno-costal cartilage in the heterozygous mouse ( ch +/+ sa ) is significantly higher (114 ± 8%) than the normal control. The distribution of the acidic glycosaminoglycans in the sterno-costal cartilage is similar in the normal, heterozygous and homozygous mice at all stages of development studied, (prenatal, newborn and postnatal) being 78 ± 4% chondroitin 4(6)-sulfate and 22% hyaluronic acid and/or keratan sulfate. The concentration of acidic glycosaminoglycans in the sterno-costal cartilage decreases as development progresses in all three gene types of mice. The reduced level of acidic glycosaminoglycans in the sterno-costal cartilage of the autosomal recessive mouse, ch +/ ch +, is associated with a defect in the formation of the sternum. The higher than normal acidic glycosaminoglycan concentration in the sterno-costal cartilage of the heterozygous mouse ch +/+ sa is associated with delayed calcification of the sternum. This study characterizes the molecular locus of a defect in the extra-cellular matrix of a mouse carrying a lethal gene and may help in understanding proteoglycan disorders (mucopolysaccharidosis) in the human.

Age-related changes in the cerebrospinal fluid outflow glycosaminoglycans ☆

The glycosaminoglycan distribution patterns of the cerebrospinal fluid (CSF) outflow pathway, dura mater and cerebral cortex of young New Zealand red rabbits and 1-, 3- and 12-week-old C-57 mice were identified by analyses of the glycosaminoglycan moieties and by the use of zone electrophoresis. The glycosaminoglycans were identified by specific degradation procedures, i.e., hyaluronate lyase, chondroitin ABC lyase, endo-beta-D-galactosidase and nitrous acid treatment. The CSF outflow pathway and dura mater glycosaminoglycan components were primarily hyaluronic acid and chondroitin sulfate-dermatan sulfate, whereas the cerebral cortex glycosaminoglycan components were hyaluronic acid, chondroitin sulfate-dermatan sulfate, keratan sulfate and heparan sulfate. The glycosaminoglycan components of the dura mater and cerebral cortex decreased and those of the CSF outflow pathway increased as a function of age. These results demonstrate the feasibility of analyses of the CSF outflow pathway glycosaminoglycan components and suggest that topographical changes in the glycosaminoglycan distribution profiles may contribute to the pattern of cerebrospinal fluid outflow.

Cerebral cortex glycosaminoglycans as a function of age in Fischer-344 and King (Sprague-Dawley) cesarean-derived, barrier-raised rats.

The glycosaminoglycan (GAG) concentrations in cerebral cortices of two strains of cesarean-derived, barrier-raised rats were compared as functions of growth and senescence.The qualitative distribution of the GAG was similar in both strains of rats at all ages. The GAG present were hyaluronic acid, chondroitin 4(6)-sulfate, heparan sulfate and a non-uronic acid containing glycoconjugate which was partially degraded by endo-B-D-galactosidase (keratanase). The chondroitin 4(6)-sulfate was remarkably constant throughout the lifespan studied and similar in both strains of rats. A non-uronic acid containing component in the GAG fraction increased significantly to a peak at 4 months of age in the Fischer rat and at 6 months of age in the King rat, but subsequently declined to the pre-peak baseline level in senescence. A parallel rise and fall of sulfate and sialic acid in the GAG fraction was observed in both strains of rats. Our data suggest that the non-uronic acid containing component is a sulfated glycoprotein-like glycoconjugate similar to keratan sulfate. The glycoconjugate peak in the cerebral cortex may be associated with the rapid proliferation of glyco-protein-rich glial cells. The gradual decline in keratan sulfate with age may represent the gradual alteration of glial glycoconjugates.