Hyaluronate in human aqueous humor.

Samples of aqueous humor obtained at cataract operations were analyzed for sodium hyaluronate by a recently developed radioassay. The mean value +/- SD of 47 samples was 1.14 +/- 0.46 micrograms/g. The samples from patients with diabetes and/or glaucoma did not differ substantially from the general distribution.

Turnover of hyaluronan in the rabbit pleural space.

Hyaluronan influences lung fluid balance. The clearance of lung hyaluronan by way of the pulmonary lymphatics and the pleural space is increased when fluid flux into the interstitium is increased. The purpose of this study was to determine the rate at which hyaluronan is removed from the pleural space. We injected hyaluronan, labeled with tritium in the acetyl group, into the pleural space of six rabbits. The appearance of [3H]H2O in serum was measured over time to calculate the turnover rate of hyaluronan. We found that the half-lives ranged between 8 and 15 h and were positively related to the amount of hyaluronan injected. At the end of the experiment, the contralateral pleural space was irrigated to determine the amount of pleural space hyaluronan, which was 0.3 micrograms/kg body wt.

Concentration and turnover of intraperitoneal hyaluronan during inflammation.

Aseptic peritonitis was induced in rabbits by intraperitoneal injection of irritating agents, mainly starch suspensions. The inflammatory response was followed in the peritoneal lavage fluid by cell counts (average increase about 800-fold the first day) and hyaluronan concentration (average increase about 200-fold on the second and third days). The turnover rate of hyaluronan was studied by injecting tritium-labeled hyaluronan intraperitoneally and by following the appearance of tritiated water in serum. In control animals given trace amounts of hyaluronan, half-lives of 1-14 h were recorded. When the labeled polysaccharide had been mixed with 10 mg/ml of unlabeled hyaluronan, the half-life was approximately one day. Rabbits with ongoing peritonitis exhibited half-lives between 1 and 16 h. It was concluded that there was a large individual variation in uptake kinetics, that the removal process could be receptor mediated, and that the increase in intraperitoneal hyaluronan in peritonitis mainly was due to an increased production of the polysaccharide rather than a decreased rate of removal.

Reduction of the hyaluronate concentration in rabbit aqueous humour by topical prednisolone.

Rabbits were treated topically with prednisolone for 5 or 10 days and aqueous humour removed immediately after treatment as well as 10 and 20 days later. The hyaluronate concentration was significantly lower in the treated eyes compared to control eyes. It returned to the normal level after withdrawal of the drug. Repeated sampling of aqueous humour at intervals of 10 days did not influence the hyaluronate concentration in the control group.

Turnover of hyaluronan in the microcirculation.

Hyaluronan in skin, lung, and intestine turns over within a few days and catabolism takes place locally in the tissues, in local lymph nodes, and in the liver. Hyaluronan will affect microcirculatory exchange through its influence on interstitial volume exclusion, hydraulic conductivity, and diffusivity of macromolecules. Prolonged increase in interstitial fluid flux in intestine has been shown to reduce the hyaluronan content, which in turn increases hydraulic conductivity and diffusivity of macromolecules.

Disappearance of concentrated hyaluronan from the anterior chamber of monkey eyes.

The removal of hyaluronan (HYA) from the primate eye has received attention due to the use of the polymer in ophthalmic surgery. The turnover of concentrated HYA injected into the anterior chamber of 12 cynomolgus monkeys was therefore studied using a technique earlier described for rabbits. The technique is based on the observation that when HYA labelled with tritium in the acetyl group leaves the eye and enters the circulation it is rapidly taken up by the liver and degraded to tritiated water. A mixture of tritium-labelled HYA and high concentration, high molecular weight HYA (Healon) was injected in amounts of 50 microliters (or 75 microliters, for three monkeys) into the anterior chamber. The concentration of tritiated water in blood plasma was followed for up to 3 weeks. An initial rise of radioactivity in blood was followed by an exponential decrease, representing the turnover of water in the body. The initial rise of tritium could be corrected for water losses and thereafter corresponded to the disappearance of HYA from the eye, plus a 45-min time lag due to the catabolism in the liver. Using this technique it was found that half of the material had left the anterior chamber after 20 hr when 50 microliters (or 75 microliters) were injected. Four animals were treated with pilocarpine topically during the first day and the corresponding time was then 8.5 hr. Maximal IOP occurred about 6 and 3 hr after the injection for non-treated and pilocarpine treated animals, respectively.

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 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.

An experimental technique to study the turnover of concentrated hyaluronan in the anterior chamber of the rabbit.

An experimental technique to study the turnover of hyaluronan in the anterior chamber in single animals has been developed. It is based on the fact that circulating hyaluronan labelled with tritium in the acetyl group is rapidly taken up by the liver and degraded to tritiated water. 3H-labelled hyaluronan was mixed with high concentration, high molecular weight hyaluronan (Healon) and injected into the anterior chamber of rabbits. The concentration of tritiated water in blood plasma was followed for up to 13 days. An initial rise of radioactivity in the blood was followed by an exponential decrease. From the exponential disappearance it was possible to calculate the rate of turnover of water in the body. This was used to correct the initial rise of tritium for water losses. After this correction the appearance of radioactive water in blood corresponded to the disappearance of hyaluronan from the eye plus a 45 min time-lag due to the catabolism in the liver. Using this technique it was found that trace amounts of radioactive hyaluronan and 0.055 ml and 0.2 ml of 1% hyaluronan exhibited half-lives in the anterior chamber of 1.5, 7 and 13 hr respectively. When the same technique was used for subcutaneously and intramuscularly injected hyaluronan half-lives of 50 and 30 hr, respectively, were recorded.

The structure and function of hyaluronan: An overview.

Hyaluronan is a major component of synovial tissue and fluid as well as other soft connective tissues. It is a high-Mr polysaccharide which forms entangled networks already at dilute concentrations (< 1 mg/mL) and endows its solutions with unique rheological properties. Physiological functions of hyaluronan (lubrication, water homeostasis, macromolecular filtering, exclusion, etc.) have been ascribed to the properties of these networks. Recently a number of specific interactions between hyaluronan and a group of proteins named hyaladherins have also pointed towards a role of hyaluronan in recognition and the regulation of cellular activities. Many more or less well documented hypotheses have been proposed for the function of hyaluronan in joints, for example, that it should lubricate, protect cartilage surfaces, scavenge free radicals and debris, keep the joint cavities open, form flow barriers in the synovium and prevent capillary growth.

Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver.

The catabolism of hyaluronan has been studied by injecting hyaluronan, labelled with 125I-tyramine cellobiose (125I-TC), subcutaneously into the hindpaw of rabbits. Following endocytosis, 125I-TC remains in the cells at the site of uptake, allowing localization of the site of catabolism. At 6 h after subcutaneous injection, 65% of the injected radioactivity was recovered. The skin at the injection site contained 47%, the popliteal gland at the side of injection 10%, and the liver 8% of the injected dose. At 48 h the three organs contained 40% of the injected dose with 17% in the skin, 10% in the lymph node and 13% in the liver. The decline in recovery could be accounted for by urinary excretion of the tracer, implying that some tracer had been released from the cells after endocytosis. Chromatography revealed that over 85% of 125I-TC-hyaluronan in the lymph nodes and liver was of low molecular mass throughout the experiment. In skin, 4% of the injected tracer was recovered with low molecular mass at 6 h, increasing to 12% of injected dose at 24 and 48 h. Thus, a minimum of 12% of the injected tracer was catabolized per 24 h at the skin injection site. If cells in skin are responsible for the subsequent release of tracer, as seen from the decrease in recovery of the injected dose, another 10-15% of the tracer could have been catabolized locally in the skin per day. The major part of the hyaluronan injected in the skin was, however, catabolized by lymphatic removal and subsequent degradation in local lymph nodes and liver.

Turnover of hyaluronan in synovial joints: elimination of labelled hyaluronan from the knee joint of the rabbit.

After the injection of [3H]acetyl-labelled hyaluronan into normal rabbit knee joints, about 90% of its isotope content was ultimately accounted for as 3H2O. The rate of elimination of hyaluronan from synovial fluid was therefore estimated from changes in the level of 3H2O in plasma. The half-life of plasma 3H2O was 6.2 days (S.D. 0.7). As estimated from its metabolism to 3H2O, the mean intrasynovial half-life of [3H]hyaluronan of high molecular weight (modal relative molecular mass (Mr) greater than 6.0 x 10(6) was 13.2 h (range 11-15.5 h; n = 4); an otherwise identical preparation of low molecular weight (modal Mr 0.09 x 10(6] exhibited a mean half-life of 10.2 h (range 7.8-13.5 h; n = 4). The difference between the two groups was significant (P = 0.029). Both estimates were nevertheless close to those determined by others in the same species for injected proteoglycans (Mr 2.5 x 10(6), t1/2 = 12 h) and for endogenous hyaluronan calculated from changes in concentration during intravenous infusion of fluid under anaesthesia (t1/2 = 16 h). The similarity suggests that hyaluronan and proteoglycan are removed from synovial fluid by a common pathway with limited dependence on their molecular dimensions and concentrations.

Hyaluronan: its nature, distribution, functions and turnover.

Hyaluronan is a polysaccharide found in all tissues and body fluids of vertebrates as well as in some bacteria. It is a linear polymer of exceptional molecular weight, especially abundant in loose connective tissue. Hyaluronan is synthesized in the cellular plasma membrane. It exists as a pool associated with the cell surface, another bound to other matrix components, and a largely mobile pool. A number of proteins, the hyaladherins, specifically recognize the hyaluronan structure. Interactions of this kind bind hyaluronan with proteoglycans to stabilize the structure of the matrix, and with cell surfaces to modify cell behaviour. Because of the striking physicochemical properties of hyaluronan solutions, various physiological functions have been assigned to it, including lubrication, water homeostasis, filtering effects and regulation of plasma protein distribution. In animals and man, the half-life of hyaluronan in tissues ranges from less than 1 to several days. It is catabolized by receptor-mediated endocytosis and lysosomal degradation either locally or after transport by lymph to lymph nodes which degrade much of it. The remainder enters the general circulation and is removed from blood, with a half-life of 2-5 min, mainly by the endothelial cells of the liver sinuoids.

Serum hyaluronan as a disease marker.

Hyaluronan is a connective tissue polysaccharide which has also been found in blood serum in concentrations < 100 micrograms/L (average 30-40 micrograms/L in middle-aged persons). The serum level is regulated by the influx of the polysaccharide from the tissues via lymph and its receptor-mediated clearance by liver endothelial cells. Markedly high serum levels are noted in certain liver diseases, especially in patients with cirrhosis, when the clearance is impaired. In these cases serum hyaluronan can be used to follow the development of the disease. Serum hyaluronan is also a sensitive marker for impending rejection of liver transplants. Patients with rheumatoid arthritis constitute another major group with increased serum hyaluronan, but in this case the level varies markedly during the day corresponding to physical activity. There are good indications that in these subjects the excess hyaluronan comes from the joints. Under stringent sampling conditions of serum it should be possible to extract interesting information on the inflammatory joint process. Increased hyaluronan levels are also seen in other inflammatory diseases and it is of special interest that high hyaluronan levels in patients with septic conditions is a sign of poor prognosis. Certain tumours, notably Wilms' tumour and mesothelioma, produce factors which activate synthesis of hyaluronan and increase its serum level. Rare hereditary diseases with disturbances of hyaluronan metabolism and elevated blood levels have also been discovered, e.g. Werner's syndrome and cutaneous hyaluronanosis. Information accumulated during the last decade regarding the metabolism of hyaluronan has made this polysaccharide an interesting clinical marker for a number of pathological conditions.

Hyaluronan injected in the anterior chamber of the eye is catabolized in the liver.

Catabolism of hyaluronan was studied by injecting hyaluronan (M(r) 200,000) labelled with [125I]-tyramine cellobiose, ([125I]-TC), into the anterior chamber of the rabbit eye. After endocytosis [125I]-TC accumulates in cells, allowing localization of the site of catabolism. When 0.1 ml of the aqueous humour was replaced with an equal volume containing polymer in tracer quantities, 24% was recovered from the eye at 2 hr and 6.5% at 6 hr after injection. With replacement of 0.2 ml of aqueous humour 38% and 19%, respectively, were found within the eye at the same time points. Most of the radioactivity was found in the liver. If the tracer was mixed with Healon the removal from the anterior chamber was delayed. Chromatography on Sephacryl S-300 showed that there was no significant degradation of the labelled polymer in the aqueous humour nor in the remaining parts of the enucleated eye, while most of the tracer extracted from the liver was of low molecular weight. Thus the labelled hyaluronan was being washed out of the eye and subsequently taken up and catabolized by the liver.

The concentration of hyaluronate in amniotic fluid.

The concentration of sodium hyaluronate has been determined in amniotic fluid by a specific radioassay. It shows a large individual variation. The mean concentration is approximately 20 micrograms/ml at Weeks 16-20 of the gestational period. It drops to approximately 1 micrograms/ml at Week 30 and is then constant until the end of the pregnancy.

Removal rate of [3H]hyaluronan injected subcutaneously in rabbits.

Hyaluronan is an important constituent of the extracellular matrix in skin, and recent studies suggest that there is a pool of easily removable ("free") hyaluronan drained by lymph. The removal rate of free hyaluronan in skin was measured from the elimination of [3H]hyaluronan, injected subcutaneously in 13 rabbits. The removal of radioactivity was determined from appearance of 3H in plasma. During the first 24 h after injection, 10-87% of the tracer entered blood, less in injectates with high concentrations of hyaluronan. The removal was monoexponential with a half-life of 0.5-1 day when concentration of hyaluronan was 5 mg/ml or less. When hyaluronan concentration was 10 mg/ml or higher, the removal was slow for about 24 h and then became similar to that in experiments with low hyaluronan concentration. Free hyaluronan at physiological concentrations is thus turned over with the same rate as serum albumin, supporting the concept that hyaluronan is removed essentially by lymph flow to be degraded in lymph nodes and liver.

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.

Effect of increased interstitial fluid flux on fractional catabolic rate of high molecular weight [3H]hyaluronan injected in rabbit skin.

The removal rate of high molecular weight (M(r) 3 x 10(6) [3H]hyaluronan has been measured in rabbit paw skin in control and during increased local venous pressure induced by ligation of the femoral vein. The increased venous pressure resulted in a 20% increase of interstitial volume at 4 h after ligation, a value which relates to at least a doubling of lymph flow, i.e. also to doubling of fluid flux through the tissues. The fractional catabolic rate of hyaluronan was 0.0918 +/- 0.0170 h-1 (n = 10) and 0.0775 +/- 0.0206 h-1 (n = 11) in control and following increased venous pressure respectively (P > 0.05). Since the fractional catabolic rate of hyaluronan is not affected by increased venous pressure, the removal of hyaluronan via lymphatic drainage in rabbit paw skin must be small compared to the local turnover of the polysaccharide.