Torvard C. Laurent

A theory of gel filtration and its exeperimental verification

Abstract The separation by gel filtration of molecules varying in size is explained as a steric exclusion of solutes from the gel phase. The volume available for a solute in the gel phase can be determined from the elution volume, the void volume and the total volume of the gel column. It has been calculated for a number of proteins and dextran fractions and for various dextran gels from data given in the literature as well as from some new data. The values were used to test the hypothesis that the exclusion takes place from a three-dimensional random network of straight polymer fibers distributed in the gel. The experimental data were found to verify the hypothesis. The experimentally determined available volumes in the gel phase for three proteins are approximately the same as the available volumes in dextran solutions, having the same polymer concentrations as the gels. Therefore there seems to be no essential difference between the exclusion phenomenon in a polymer gel and in a polymer solution.

Endothelial cells are a site of uptake and degradation of hyaluronic acid in the liver

Abstract In a recent communication it was shown that intravenously injected radioactively labelled hyaluronic acid was preferentially taken up by the liver and degraded. We now report that uptake occurs in the liver endothelial cells and that these cells degrade the polysaccharide in vitro into low-molecular weight (LMW) products.

Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll)

Abstract A new gradient medium (Percoll) for density gradient centrifugation of cells and subcellular particles is described. It consists of colloidal silica particles which have been firmly coated with a layer of polyvinylpyrrolidone. The particle population is polydisperse, and the average diameter is approximately 17 nm. Both the electrophoretic mobility of the particles and the conductivity of the solution are low, indicating a low surface charge. The colloid has a high solubility and forms clear solutions. It can be used in concentrations which give solution densities between 1.00 and 1.20 g/ml. The solutions have a low osmolality and can be mixed with electrolytes of physiological pH and ionic strength. The sedimentation properties of the particles are described. Density gradients can be formed by highspeed centrifugation. The colloid has been shown to be nontoxic for a number of cells and cell organelles.

On the interaction between polysaccharides and other macromolecules: II. The transport of globular particles through hyaluronic acid solutions☆

Abstract A study of the diffusion behaviour of albumin, α-crystallin, fribrinogen, and turnip yellow mosaic virus in hyaluronic acid media has shown that the diffusion rates of these substances are markedly decreased by the presence of the polysaccharide. The relative decrease in the same as the corresponding effect observed on the sedimentation rates of these substances. Investigation of eleven globular particles with diameters of 47–3650 A established that the relative decrease of the sedimentation rate of a particle in the presence of hyaluronic acid was essentially a function of the diameter of the particle and the concentration of the polysaccharide and that the effect could be expressed by a simple exponential relationship. The results observed have been interpreted as a macromolecular-sieving effect of the polysaccharide.

Concentration of sodium hyaluronate in serum

A radioassay for sodium hyaluronate using high-affinity binding protein from bovine cartilage has been modified for serum analysis. The accuracy of the method was checked by isotope dilution experiments and by recovery studies with exogenous hyaluronate. The between-assay standard deviation in the determination is 15-20%. The concentration of sodium hyaluronate in healthy adults (blood donors) is in the range of 10 to 100 micrograms/l with a mean value in the order of 30 to 40 micrograms/l. This is a lower concentration than previously reported. The same level was found in young people. Higher hyaluronate concentrations were noted in persons above 50 years of age. Analysis of plasma showed a slightly higher average hyaluronate level (5%) than in serum from the same persons. There were no notable sex differences. Analysis of serum and plasma from adult animals (rat, rabbit, dog, pig, goat, sheep, cow and horse) gave hyaluronate concentrations of the same order or higher than in human serum.

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM).

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [125I]asialoceruloplasmin (parenchymal cells) or heat-denatured [125I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.

Biochemistry of Hyaluronan

Hyaluronan (hyaluronic acid) is a linear polysaccharide formed from disaccharide units containing N-acetylglucosamine and glucuronic acid. It is ubiquitously distributed in the organism but is found in the highest concentrations in soft connective tissues. The molecular weight of hyaluronan is usually in the order of 10(6) to 10(7). Due to hydrogen bonding, the chain is rather stiff and the molecule behaves in solution as an extended, randomly kinked coil. Molecules of hyaluronan start to entangle already at concentrations of less than 1 g/l and form a continuous polymer network. Some of the functions of the polysaccharide have been connected with the unique physical chemical characteristics of the network such as its rheological properties, flow resistance, osmotic pressure, exclusion properties and filter effect. Hyaluronan is synthesized in the cell membrane by adding monosaccharides to the reducing end of the chain. The precursors are UDP-glucuronic acid and UDP-N-acetylglucosamine. The polysaccharide grows out from the cell surface and it can be shown that fibroblasts, for example, surround themselves with a coat of hyaluronan. The rate of biosynthesis is regulated by various factors, such as growth factors, hormones, inflammatory mediators, etc. The responsible enzyme, hyaluronan synthase, is a phosphoprotein and the regulation of the synthetic rate is apparently via phosphorylation. The hyaluronan is at least partly carried by lymph flow from the tissues. Part of the material is taken up and degraded in the lymph nodes. Another part is carried to the general circulation and taken up in the endothelial cells in the liver sinusoids. These cells have specific receptors for hyaluronan, which also recognize chondroitin sulphate. The uptake in the liver of high-molecular weight hyaluronan is very efficient and its normal half-life in serum is only in the order of 2 to 5 min. The polysaccharide is rapidly degraded in the lysosomes to low-molecular weight products, lactate and acetate. The total turnover of hyaluronan in serum is in the order of 10-100 mg/24 h. The normal concentration of hyaluronan in serum is less than 100 micrograms/l with a mean of 30-40 micrograms/l. High serum levels have been noted in liver cirrhosis (impaired uptake in the liver) and rheumatoid arthritis (increased synthesis in the tissues). Hyaluronan has been shown to interact specifically with certain proteins and cell surfaces. It binds to proteoglycans in cartilage and other tissues and fills an important structural role in the organization of the extra-cellular matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies on fractionated heparin.

Abstract An investigation of heparin fractionated by cetylpyridinium chloride precipitation has shown that variations in anticoagulant activity are related to molecular weight. Chemical analysis did not reveal any difference in the chemical composition of the individual fractions, and titration indicated that the number of ionizable acid groups per disaccharide unit is constant. The fractions were found to be electrophoretically homogeneous and had the same mobilities. Molecular weights ranged from 7600 to 11,800; the relationship between limiting viscosity number and molecular weight was calculated to be [η] = 1.58 × 10 −3 M . Chromatography on ECTEOLA columns revealed that the fractions are eluted at different ionic strengths.

Uptake of circulating hyaluronic acid by the rat liver

SummaryThe uptake of [3H]acetyl-labelled hyaluronic acid (HA)Abbreviations used in this paper: HA hyaluronic acid; i.v. intravenous was examined in the liver, spleen and kidney of the rat after i.v. injection. 3H-activity was located by light- and electron-microscopic autoradiography after measurement by scintillation counting of tissue digests. In the liver, approximately 90% of the radioactivity was located in the sinusoidal endothelial cells, with autoradiographic grains distributed throughout the cytoplasm; 50% of the grains overlay vacuoles 0.3 to 1.2 μm in diameter. A few grains (4%) were located in Disses space or nearby in the cytoplasm of hepatocytes. No grains were found in Kupffer cells. The remainder were randomly scattered across the sections in a pattern indicating nonspecific background activity. These observations are in accordance with the selective uptake of HA exhibited by dissociated liver cells in vitro. HA concentrations in the spleen and kidney were too low for detection by autoradiography. Splenic concentrations were much lower than in rabbits or mice; in this respect the uptake of circulating HA in the rat resembles that reported for chondroitin 4-sulphate.

Determination of the structure of agarose gels by gel chromatography

Abstract Well-characterized fractions of the synthetic polysaccharide Ficoll have been chromatographed on columns of pearl-condensed agarose gels of concentrations between 2 and 8%. The results agree with the theory that the Ficoll is sterically excluded from the gel, which is made up of a random three-dimensional network of long fibers. The agarose fibers, which were estimated to be approx. 50 A in diameter, appear to contain 35–50 % water.