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H Greiling

Publications and source records attributed to H Greiling.

At least 37 records · Page 2Linked to original sources
PubMed1993

A radiochemical high-performance liquid chromatographic method for the analysis of 2-fluoro-2-deoxy-D-glucose-derived metabolites in human chondrocytes.

A high-performance liquid chromatography method with on-line radioactivity monitoring was developed for the measurement of 2-fluoro-2-deoxy-D-[U-14C]-glucose-derived metabolites in a cell culture system of human chondrocytes embedded in soft agarose. To optimize the chromatographic procedure, glucose-analogous substrates derived from 2-fluoro-2-deoxy-D-glucose by enzymatic synthesis in vitro were used. The synthesized metabolites could be separated by anion-exchange chromatography on a Partisil 10 SAX cartridge with a LiChrosorb RP 18-5 guard column eluted with a 35-min ion-strength/pH gradient performed from 15 mM NH4H2PO4, pH 3.8, to 0.75 M NH4H2PO4, pH 4.8, at a flow rate of 2 ml/min. Only by using an on-line radioactivity monitor instead of an off-line counting procedure was the resolution obtained sufficient for the determination of these intermediates. This method was applied to studying the metabolic pathway of 2-fluoro-2-deoxy-D-glucose in human chondrocytes. Due to the resistance of the chondrocytes embedded in soft agarose, the usual cell-lysing methods could not be used; therefore, an extraction procedure for acid-stable glucose metabolites, which may also be applied to other resistant cell lines or critical cell culture systems, was developed. With the procedure presented here, the existence of metabolites of 2-fluoro-2-deoxy-D-glucose resulting from enzymatic reactions following the hexokinase reaction could be proven.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

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.

Adjuvants, Immunologic↗
PubMed1993

A high-performance liquid chromatographic method for the determination of hypoxanthine, xanthine, uric acid and allantoin in serum.

A method was developed for the simultaneous determination of hypoxanthine, xanthine, uric acid and allantoin based on isocratic reversed-phase chromatography. This HPLC-method additionally allows the direct determination with UV-detection of inosine-5'-phosphate, uridine, thymine, orotic acid, allopurinol and oxipurinol, besides hypoxanthine, xanthine and uric acid in the same chromatographic run. Allantoin elutes in this system near the void volume and a fraction is collected covering the retention time range for this substance. After hydrolysis allantoin is converted to glyoxylate-2,4-dinitrophenylhydrazone, rechromatographed and detected at 360 nm. The coefficient of variation for this method does not exceed 5.0% for a serum concentration of 0.3 mumol/l hypoxanthine and is not greater than 5.3% for a xanthine concentration of 0.3 mumol/l serum. Recoveries were 90-110% for both hypoxanthine and xanthine. The determination of uric acid had an imprecision and inaccuracy not exceeding 1.45% in the concentration range of 103-568 mumol/l. Due to the more complex procedure required for the determination of allantoin, the coefficient of variation between days was 13.6% for a sample containing 0.8 mumol/l allantoin and the recoveries for this analyte were in the range of 86-93%. Reference ranges (mean +/- SD) determined on 171 serum samples from healthy adults were 12.7 +/- 6.6 mumol/l for hypoxanthine, 3.3 +/- 1.4 mumol/l for xanthine, and 15.7 +/- 7.9 mumol/l for allantoin. No significant age or sex dependence was observed. Uric acid concentrations were 320 +/- 55 mumol/l serum for men and 206 +/- 55 mumol/l for women.

Adolescent↗
PubMed1991

Purification and N-terminal amino acid sequence of a chondroitin sulphate/dermatan sulphate proteoglycan isolated from intima/media preparations of human aorta.

A proteoglycan (PG) was purified to homogeneity from intima/media preparations of human aorta specimens by the following chromatographic steps: Sepharose Q anion exchange, Sepharose CL-4B size exclusion, hydroxyapatite, MonoQ anion exchange and TSK G 4000 SW size exclusion. The purity of the preparation was established by SDS/PAGE using direct staining by silver or Dimethylmethylene Blue, as well as by Western blots of biotin-labelled samples. The electrophoretic mobility of the native PG was less than that of a 200,000-Mr standard protein. After treatment with chondroitin sulphate lyase ABC, a core protein of Mr 15,000 was revealed. The Mr of the glycosaminoglycan (GAG) peptides was less than 24,000, by comparison with a keratan sulphate peptide. The composition of the GAG chains was determined by differential digestion of the PG by chondroitin sulphate lyases AC/ABC or chondroitin sulphate lyase AC alone followed by anion-exchange chromatography of the resulting disaccharides. The GAG chains are composed of approximately one-third of dermatan sulphate and two-thirds chondroitin sulphate disaccharide units. The sequence of the 20 N-terminal amino acids is identical with the sequence previously reported for PG I isolated from human developing bone [Fisher, Termine & Young (1989) J. Biol. Chem. 264, 4571-4576]. The assignment of glycosylation sites to the serine residues in positions 5 and 10 was confirmed. The findings indicate that the chondroitin sulphate/dermatan sulphate PG is a major PG in intima/media preparations of human aorta and represents a biglycan-type PG.

Amino Acid Sequence↗
PubMed1991

Composition of proteoglycan fragments from hyaline cartilage produced by granulocytes in a model of frustrated phagocytosis.

An in vitro model of frustrated phagocytosis was developed in which granulocytes interact with well-defined slices of hyaline cartilage. The composition of the purified proteoglycan fragments released from the cartilage slices by N-formyl-methionyl-leucyl-phenylalanine-stimulated granulocytes was studied after 30, 60 and 90 min incubation time. It was shown that the proteoglycan fragments do not change their composition during incubation. The only change observed during incubation was an increase in the quantity of the fragments. The protein content of the proteoglycan fragments is 7.0-8.6%, corresponding to a peptide chain of 24-28 amino acids, and the relative molecular mass of the total fragment is Mr = 37,600-39,200. On average, each proteoglycan fragment contains two chondroitin sulphate chains (Mr = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (Mr = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide, whereas no N-glycosidic oligosaccharide could be detected. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin 4-sulphate, 12.9-19.4% chondroitin 6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the proteoglycan fragments resemble those of native proteoglycans, the conclusion may be drawn that the degeneration of the proteoglycans occurs by proteases that attack preferably the chondroitin sulphate-rich region of the core protein. This is the first inflammation model of joint destruction, which demonstrates the elution of soluble specific proteoglycan degradation products of defined size.

Amino Acids↗
PubMed1990

Interleukin-1 beta induces synthesis and secretion of interleukin-6 in human chondrocytes.

Increased concentrations of interleukin-6 (IL-6) have been found in the synovial fluid of patients with osteoarthritis, rheumatoid arthritis and crystal-related joint diseases. It is therefore of great interest to identify the cells responsible for the production of IL-6, and to investigate whether IL-6 plays a role in the pathogenesis of degenerative or inflammatory joint diseases. Here we show that human interleukin-1 beta (IL-1 beta) induces IL-6 synthesis and secretion in differentiated human chondrocytes. In organ cultures resembling closely the in vivo system 10(6) chondrocytes incubated with 100 units of interleukin-1 beta per ml of medium led to the release of 6 X 10(3) units of IL-6 within 24 h. Chondrocytes cultured in agarose or as monolayers similarly incubated with IL-1 beta produced even higher amounts of IL-6: 70 X 10(3) units per 10(6) cells within 24 h. The induction of IL-6 synthesis by IL-1 beta was also shown at the mRNA level. IL-6 secreted by stimulated chondrocytes showed heterogeneity upon Western blot analysis.

Adult↗
PubMed1990

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.

Alginates↗
PubMed1990

Analysis of the proteolytic degradation products of hyaline cartilage proteoglycans.

The proteolytic degradation products of nasal hyaline cartilage proteoglycans produced by polymorphonuclear leukocyte lysosomal enzymes were investigated. The protein content of the degradation products is 7.0-8.6% corresponding to a peptide chain of 24-28 amino acids and the relative molecular mass of the total fragment is M(r) = 37,600-39,200. On an average, each proteoglycan fragment contains two chondroitin-sulphate chains (M(r) = 22,000-22,400), every fourth fragment contains a keratan sulphate chain (M(r) = 7000-7200) and every seventh to eighth contains an O-glycosidic oligosaccharide. The results of the disaccharide analysis show that the galactosaminoglycan chains contain 76.2-83.6% chondroitin-4-sulphate, 12.9-19.4% chondroitin-6-sulphate, 3.5-3.8% chondroitin and no dermatan sulphate. Since composition and relative molecular mass of the chondroitin sulphate and keratan sulphate chains from the degradation products resemble those from native proteoglycans, it is suggested that the degradation of the proteoglycans occurs by proteinases that attack preferably the chondroitin sulphate region of the core protein.

Amino Acids↗
PubMed1989

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.

Amino Acids↗
PubMed1989

An enzymic assay for uric acid in serum and urine compared with HPLC.

We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively.

Chemical Phenomena↗
PubMed1989

A candidate reference method for the determination of uric acid in serum based on high performance liquid chromatography, compared with an isotope dilution-gas chromatography-mass spectrometer method.

A method based on isocratic high performance liquid chromatography (HPLC) with UV detection at 292 nm is proposed as a candidate reference method for the determination of uric acid. Data obtained by this method are compared with those from an isotope dilution-gas chromatography-mass spectrometric method (ID-GC-MS), using [1,3-15N2]uric acid as internal standard and selected mass detection at m/z = 456 and m/z = 458. The inaccuracy of the ID-GC-MS method is maximally 0.4% for NBS-SRM-909 control sera with a concentration of 483 mumol/l. The coefficient of variation between days is 0.26%-0.80% and 0.37-0.90% for 14 control sera from other suppliers. The maximum bias of the HPLC method is 0.6%, and the coefficient of variation between days is 0.31%-0.65% for NBS-SRM-909 control sera. The coefficient of variation between days for the other 14 control sera tested is 0.35%-0.66%. Comparison of the HPLC method with the reference ID-GC-MS method resulted in a coefficient of correlation of r = 0.9998 (n = 14). The concentration of uric acid in the tested control sera ranged from 160 to 624 mumol/l.

Chromatography, High Pressure Liquid↗
PubMed1988

Effect of diacetylrhein on the phagocytosis of polymorphonuclear leucocytes and its influence on the biosynthesis of hyaluronate in synovial cells.

1. The influence of diacetylrhein on the luminol-induced chemiluminescence of zymosan-activated polymorphonuclear leucocytes (PMNL) was investigated. At a concentration of 4 x 10(-5) mol/l diacetylrhein an inhibition of about 40% was found. 2. A model for the degradation of hyaline cartilage by frustrated phagocytosis was developed, in which human polymorphonuclear leucocytes cause a release of glycosaminoglycan peptides from hyaline cartilage slices (bovine nasal septum). We observed a 20% inhibition of this release at a concentration of 10(-4) mol/l diacetylrhein. 3. Human synovial fibroblasts synthesize the glycosaminoglycan hyaluronate. As a parameter of the rate of hyaluronate synthesis we measured the incorporation of 14C-glucosamine into hyaluronate. At a concentration of 2 x 10(-4) mol/l diacetylrhein a 4-fold increase of 14C-glucosamine incorporation in the membrane fraction of the synovial cells (tryptic fraction) and a 1.6-fold elevation of glucosamine release into the medium was measured. The synovial fibroblasts show a higher (1.5-fold) glucose consumption and lactate production in the presence of diacetylrhein (2 x 10(-4) mol/l).

Adult↗
PubMed1987

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.

Animals↗
PubMed1987

Proteoglycans from human umbilical vein endothelial cells.

Human umbilical vein endothelial cells were incubated with [35S]sulphate and investigated for their proteoglycan production. By gel chromatography, ion-exchange chromatography and CsCl density-gradient centrifugation we obtained preparative amounts of the endothelial proteoheparan sulphate HSI and of proteochondroitin sulphate from the conditioned medium of mass-cultured human umbilical vein endothelial cells. Approximately 90% of the 35S-labeled material in the endothelial cell conditioned medium was proteochondroitin sulphate. This molecule, with a molecular mass of 180-200 kDa, contains four side-chains of 35-40 kDa and a core protein of 35-40 kDa. Two proteoheparan sulphate forms (HSI and HSII) from the conditioned medium were distinguished by molecular mass and transport kinetics from the cell layer to the medium in pulse-chase experiments. One major form (HSI), with an approximate molecular mass of 160-200 kDa a core protein of 55-60 kDa and three to four polysaccharide side-chains of 35 kDa each, was found enriched in the cellular membrane pellet. Another proteoheparan sulphate (HSII), with polysaccharide moieties of 20 kDa, is enriched in the subendothelial matrix (substratum).

Amino Acids↗