H. Greiling

Intra-articular sodium hyaluronate in osteoarthritis of the knee: a multicenter, double-blind study

The efficacy and the safety of intra-articular injections of sodium hyaluronate were studied in patients with osteoarthritis of the knee in a randomized multicenter double-blind study. Two hundred and nine patients received five injections of either 25 mg hyaluronate/2.5 ml (verum, N = 102) or 0.25 mg hyaluronate/2.5 ml (control, N = 107) at weekly intervals. Seven patients in each group were excluded from the protocol-correct efficacy analysis. The Lequesne Index, the first main criterion, showed a significant superiority of the verum-treated patients after the third injection up to the final follow-up examination 9 weeks after the last injection (MANOVA, P < 0.025). The consumption of paracetamol was defined as a complementary main criterion that did not reveal significant differences between the treatment groups. Most of the individual secondary endpoints demonstrated a much better response to the active treatment without reaching the significance level in the intergroup comparisons for the single time-points. Side-effects were confined to local reactions of minor severity and short duration in four patients (six events) of the verum group and in five patients of the control group. Clinical chemistry and hematology remained essentially unchanged.

Assay of synovial fluid parameters: hyaluronan concentration as a potential marker for joint diseases.

Synovial fluids from the knees of patients with degenerative joint disease (n = 29), osteoarthritis (n = 16), diabetic arthropathy (n = 12), gout (n = 7) and acute inflammatory joint disease (n = 7) were investigated by high-performance size-exclusion chromatography combined with multiangle laser light scattering detection and differential refractometry. These data were compared with the viscosities of the same samples measured by rotation viscometry with one low shear rate, as well as with C reactive protein. The median value of the weight-average molecular weight of hyaluronan in synovial fluids, which differed less than the viscosity of these groups, varied between 1.09 x 10(6) g/mol (range 0.849-1.63 x 10(6) g/mol) (acute-inflammatory joint disease) and 1.91 x 10(6) g/mol (range 1.06-3.48 x 10(6) g/mol) (degenerative joint disease). The correlation between viscosity and hyaluronan concentration was much better than between viscosity and weight-average molecular weight. Changes in C reactive protein concentration were correlated with the disease activity. The concentration of hyaluronan was significantly higher in the cases of degenerative joint disease and diabetic arthropathy. These results suggest that synovial fluid concentration of hyaluronan is appropriate as a prognostic value in the evaluation of different kinds of joint diseases.

Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.

A Comparative Study of the Concentrations of Hypoxanthine, Xanthine, Uric Acid and Allantoin in the Peripheral Blood of Normals and Patients with Acute Myocardial Infarction and Other Ischaemic Diseases

The aim of this study was the elucidation of the role of the xanthine oxidoreductase in the purine metabolism in ischaemic diseases of man. The serum concentrations of hypoxanthine, xanthine, uric acid and allantoin were determined in peripheral blood samples from patients with angina pectoris, cerebral insult and myocardial infarction with thrombolytic therapy and were compared with the concentrations obtained for healthy males and females. No significant differences were observed for the serum hypoxanthine concentrations, xanthine concentrations, the sum (hypoxanthine+xanthine) and the ratio (xanthine/hypoxanthine) between the healthy males, healthy females, the patients suffering from angina pectoris and the patients suffering from cerebral insult. An increase of the serum xanthine concentration in patients with myocardial infarction indicates a significant metabolic involvement of xanthine oxidoreductase in this disease and therefore a possible role in the development of tissue damage in the postischaemic phase due to oxygen radicals generated by the oxidase activity of this enzyme. The serum concentrations of uric acid and allantoin showed no differences between any of the studied groups. Study of the non-enzymatic oxidation of uric acid to allantoin by oxygen radicals, a relevant radical-scavenging mechanism in other diseases, provided no indication of an increased concentration of oxygen radicals due to the xanthine oxidoreductase reaction or other radical-producing mechanisms.

Effects of oxygen-derived free radicals on the molecular weight and the polydispersity of hyaluronan solutions

Abstract Depolymerization of high-molecular-weight sodium hyaluronan by superoxide anions and hydroxyl free radicals was studied. The changes in number-average and weight-average molecular weight, molecular-weight distribution, polydispersity, and concentration were measured over time by high-performance liquid chromatography on a TSK 6000 PW size-exclusion column coupled to multiangle laser light scattering and refractive index detection. There was no significant change in the elution profile and in the peak height of the refractive index chromatogram for either of the radical species at different reaction times. Furthermore, the weight-average molecular weight of hyaluronan decreased from 1.3 × 10 6 to 3.0 × 10 5 g/mol; however, the polydispersity did not significantly increase. These experimental results indicate that superoxide anions and hydroxyl free radicals do not have different modes of action on hyaluronan. Also, both radical species seem to attack hyaluronan randomly from the end of the molecule. Depolymerization studies on sodium hyaluronate by superoxide anions and hydroxyl free radicals indicate similar modes of action for both species.

The phosphorylation of liver ribosomal protein S6 during the development of acute hepatic cell injury induced by D-galactosamine

The administration of D-galactosamine-HCl (GalN) to rats initiates a sequence of metabolic events in the liver leading to a morphological and biochemical hepatitis-like liver injury [l] . The formation of UDPgalactosamine metabolites plays a pathogenetic key role since they reduce strongly the hepatocellular pool of uridine phosphates and UDP-sugars by a trap mechanism [2]. As a consequence, macromolecular synthesis, e.g., of RNA [3] and protein [4-91, is inhibited. It is presently unknown whether the impairment of protein biosynthesis is due to a direct effect on translation or to a secondary response to inhibition of RNA synthesis [2]. A direct influence on the translation machinery is suggested by experiments which indicate that the inhibition of protein synthesis by GalN can be reversed by uridine in the presence of actinomycin D [9]. However from present knowledge on the molecular mechanism of polypeptide synthesis, in particular on the role of uridine nucleotides, a specific action of UTP deficiency on translation is hardly to explain. Therefore it is conceivable that the effect of GalN on cellular protein synthesis is a direct one but mediated by a common, nonspecific pathway. Postsynthetic modifications of ribosomal proteins have been suggested as a regulatory mechanism of protein biosynthesis [lo] . Recently presented evidence

Characterization of biotin-labeled proteoglycans by electrophoretic separation on minigels and blotting onto nylon membranes prior and after enzymatic digestion.

Biotinylated proteoglycans were separated by sodium dodecyl sulfate electrophoresis prior and after enzymatic digestion by glycan-specific enzymes using polyacrylamide minigels. The biotin-labeled compounds were blotted onto nylon membranes either by electrophoresis or by diffusion and detected by avidin-enzyme conjugates. The method allows the nonisotopic detection of native proteoglycans and core proteins. Proteoglycans can be visualized at protein amounts as low as 0.7 ng per lane. In comparison with sensitive protein stains, compounds of enzyme preparations do not interfere with bands corresponding to core proteins. Electrophoresis, blotting, and staining of up to 12 samples per gel are accomplished in less than 3 h.

The synthesis of total and specific glycosaminoglycans during development of experimental liver cirrhosis

During hepatic fibrogenesis induced by long-term administration of thioacetamide, the synthsis of chondroitin 4,6-sulfate and hyaluronic acid was strongly enhanced; the formation of heparan sulfate comprising at least 70% of total liver GAG synthesis and of a keratan-sulfate-like fraction was stimulated 1.7fold. Formation of dermatan-sulfate in liver could not be detected.

The state of ribosomal protein phosphorylation during thioacetamide-induced liver injury

Abstract The development of thioacetamide (TAA)-induced liver injury is associated with a strong stimulation of phosphorylation of liver ribosomes. The phosphorylation of the 40 S ribosomal subunit was enhanced five- and ninefold 3 and 6 hr after the onset of injury, respectively. By two-dimensional gel electrophoresis and autoradiography it was shown that the increase was due to an eightfold-stimulated phosphorylation of protein S6 of the small ribosomal subunit. No significant change of the specific radioactivity of the 60 S subunit was detected. Two days after injection of TAA a tendency toward regeneration of normal (low phosphorylated) protein S6 was observed.

Decreased glomerular basement membrane heparan sulfate proteoglycan in essential hypertension

Abstract Heparan sulfate proteoglycans are major components of the glomerular basement membrane and play a key role in the molecular organization and function of the basement membrane. Moreover, their presence is essential for maintenance of the selective permeability of the glomerular basement membrane. Recently, we isolated and characterized a novel small basement membrane–associated heparan sulfate proteoglycan from human aorta and kidney. Partial amino acid sequence data clearly show that this heparan sulfate proteoglycan is distinct from the large basement membrane–associated heparan sulfate proteoglycan (perlecan). Using specific monoclonal antibodies, we have shown that the novel heparan sulfate proteoglycan is located predominantly in the glomerular basement membrane and, to a lesser extent, in the basement membrane of tubuli. Turnover or, in the course of kidney diseases, degradation of heparan sulfate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulfate proteoglycan, which can be measured by a sensitive enzyme immunoassay. The aim of the present study was to analyze whether changes in the structure and function of glomerular basement membranes can be directly detected by measurement of the excretion of a component of this basement membrane, eg, heparan sulfate proteoglycan into urine. The excretion of this small heparan sulfate proteoglycan was compared after physical exercise in normotensive and hypertensive subjects. Normotensive subjects and treated, essential hypertensive patients underwent a standardized workload on a bicycle ergometer. Biochemical characterization of the urinary proteins and heparan sulfate proteoglycan was performed before and 15 and 45 minutes after exercise. In both groups, physical exercise induced a significant increase in the excretion of urinary α1 microglobulin and albumin. However, a 10-fold increase in the urinary excretion rate of heparan sulfate proteoglycan was seen in normotensive subjects under exercise. In hypertensive patients, the relative increase in heparan sulfate proteoglycan excretion was significantly diminished ( P <.05). These data, supported by immunohistochemistry, indicate changes in the glomerular basement membrane of the kidney in hypertension. Therefore, determination of urinary excretion of this novel small heparan sulfate proteoglycan after exercise may be a sensitive marker for the detection of basement membrane alterations in hypertension.