Ulla B.G. Laurent

Hyaluronan: its nature, distribution, functions and turnover

Fraser JRE, Laurent TC, Laurent UBG (Monash University, Clayton, Victoria, Australia; and University of Uppsala, Uppsala, Sweden). Hyaluronan: its nature, distribution, functions and turnover (Minisymposium: Hyaluronan). J Intern Med 1997; 242: 27–33.

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 molecular weight of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle eyes

The molecular weight distribution of hyaluronate in the aqueous humour and vitreous body of rabbit and cattle has been determined by gel chromatography. The eluate from the column was monitored by a radioassay, whereby the molecular weight distribution of 15-20 microgram samples could be analysed. Control experiments were carried out with radioactively-labelled hyaluronate added to bovine material to estimate the degradation of the polymer during handling of the ocular fluids. It was shown that in vitro degradation does not appreciably affect the results. The analyses show a considerable polydispersity of the hyaluronate preparations. There is also a marked variation in the degree of polymerization between the two species. Hyaluronate from rabbit vitreous has a weight-average molecular weight (MW) of 2-3 X 10(6) while adult bovine vitreous displays a value of about 5-8 X 10(5). The hyaluronate of bovine vitreous varies with age. In newborn calf, a value of 3 X 10(6) was registered. This value dropped to about 5 X 10(5) in old cattle. The hyaluronate in the aqueous humour of rabbit showed a considerably higher molecular weight than that of the vitreous indicating that part of the hyaluronate in the anterior segment originates elsewhere than the vitreous. The differences between hyaluronate from the aqueous humour of adult cattle and that from the vitreous were more complex. As with the rabbit, a relatively large proportion of hyaluronate in the aqueous humour was of high-molecular weight, but, in contrast, the aqueous humour also contained material which had a lower degree of polymerization than the hyaluronate in the vitreous. The proportion of high-molecular weight material in bovine aqueous humour seemed to be lower in the summer than in the winter.

Catabolism of hyaluronan in the knee joint of the rabbit.

Catabolism of hyaluronan was studied by injecting hyaluronan labelled with [125I]-tyramine cellobiose ([125I]-TC) into knee joints of rabbits. After endocytosis [125I]-TC remains intracellularly allowing localization of the site of catabolism. At 6 hours after injection 63% could be recovered in and around the joint, while at 48 hours 32% remained locally. Chromatography showed that 12% of the injected tracer was degraded in joint tissues at 6 hours, increasing to 33% at 24 hours. There was no apparent degradation within the joint fluid. No tracer was found in the regional lymph glands, but 16% of the injected tracer was detected in the liver at 24 hours. This investigation demonstrates that hyaluronan in the joint can be degraded both locally and in the liver.

Turnover of hyaluronan in the tissues

Abstract Hyaluronan, a negatively charged, high-molecular weight polysaccharide is found in all tissues and body fluids of higher animals and most abundantly in soft connective tissues. The total amount in an adult human has been estimated to be 11–17 g. Its turnover has earlier been studied by following the fate of endogenously labelled polysaccharide or injected highly concentrated unlabelled polymer. More recently, hyaluronan biosynthetically labelled with 14C or 3H has been injected into various compartments or tissues and its disappearance followed. These experiments have demonstrated a fairly rapid turnover in many localities ( t 1 2 in the order of one day). This is partly due to lymphatic removal of hyaluronan from the tissues and subsequent degradation in lymph nodes and liver. Recent work with [125I]tyramine cellobiose-labelled hyaluronan has furthermore shown that the polysaccharide can also be endocytosed and degraded locally in the tissues. Thus the catabolism of hyaluronan takes place both by local degradation and drainage via the lymphatic system.

Urinary excretion of hyaluronan in man

A specific assay for hyaluronan (hyaluronic acid) has been applied to the determination of the polysaccharide in urine. The excretion in 22 healthy subjects was 330 micrograms/24 h (SD 77). The excretion was correlated with body weight and was therefore somewhat higher in males than in females. The molecular weight of the main fraction of urinary hyaluronan was in the range of 4000 to 12,000 in accordance with the hypothesis that it originates from blood and arises by glomerular filtration. A small fraction was of higher molecular weight and could have been produced in the urinary tract. Hyaluronan in male and female urine displayed the same molecular weight distributions. Patients with rheumatoid arthritis and primary biliary cirrhosis showed a two-fold and three-fold increase, respectively, of hyaluronan in urine with concurrently high levels of the polysaccharide in serum. A patient with Werners syndrome displayed a ten-fold increase of the polysaccharide in both serum and urine.

Turnover of hyaluronate in the aqueous humour and vitreous body of the rabbit.

Sodium hyaluronates with molecular weights of 18000, 500000 and 4 x 10(6), labelled with either 3H or 14C, were injected into the anterior chamber or into the centre of the vitreous body of rabbits and their rates of disappearance were followed. The out-flow from the anterior chamber in anaesthetized animals was virtually independent of the molecular weight indicating that the disappearance of the polysaccharide is controlled by bulk flow. The rate constant for the disappearance of sodium hyaluronate was 0.0094/min and after treatment with indomethacin 0.0061/min. These figures are in general agreement with published flow-rates of aqueous humour in the rabbit. The disappearance from the vitreous body was strongly molecular weight dependent indicating a diffusion controlled transport. The rate constant for hyaluronate with mean molecular weight of 18000 was 0.16/day and 500000, 0.024/day. The rate constant for endogenous hyaluronate was estimated to be about 0.01/day. A calculation using these rate constants shows that the turnover of sodium hyaluronate in the rabbit anterior chamber is about 3 micrograms per 24 hr while the turnover in the vitreous body is only 15% of that. This confirms an earlier conclusion (Laurent and Granath, 1983) that the preponderant part of the hyaluronate in aqueous humour is not a general degradation product from the vitreous body.

The Catabolic Fate of Hyaluronic Acid

Part of the hyaluronic acid (HA) synthesized in peripheral tissues enters the blood circulation through the lymph. It is rapidly taken up by the endothelial cells in the liver (half-life in blood is 2.5-5.5 minutes) and degraded. Pure primary cultures of liver endothelial cells were obtained by a newly developed technique and used to follow the metabolism of the polysaccharide on the cell surface. At 37 degrees C the HA is effectively endocytosed and degraded to acetate and lactate. A radioassay specific for HA and sensitive in the nanogram range has been developed to follow the concentration of HA in serum. The normal level in man is 10 to 100 micrograms/l. Elevated serum levels of HA are seen in liver cirrhosis, rheumatoid arthritis and scleroderma indicating that both an impaired catabolism in the liver and an increased synthesis in the peripheral tissues can modify the HA level.

Hyaluronan in inflammatory joint disease.

Hyaluronan (HYA) (Laurent et al. 1992) is a linear extracellular polysaccharide found in all tissues and body fluids but in highest concentrations in loose connective tissue. It is present in synovial fluid, where it is responsible for the unique rheological properties, in cartilage, where it forms the backbone of the proteoglycan aggregates and in the synovial tissue and joint capsule. HYA is also present in muscular tissues between the muscle bundles.

The diurnal variation of serum hyaluronan in health and disease

The variation of the serum concentration of hyaluronan during the day and between days has been investigated. In a group of healthy volunteers, the mean hyaluronan level was very stable over time except for a moderate but significant elevation after rising from bed in the morning. Patients with rheumatoid arthritis showed markedly increased hyaluronan concentrations 0.5-2 h after leaving bed. Patients with primary biliary cirrhosis exhibited high and rather constant levels during the day. A reference group of hospitalized patients with other diseases did not show any diurnal variation. The best reproducibility in hyaluronan determinations is obtained if specimens are taken before the subjects rise from bed or a few hours later, i.e. after the morning elevation of serum hyaluronan has subsided. In rheumatoid arthritis valuable information can be obtained by repeated sampling during the morning hours.