ABSTRACT. Objective. To investigate the effects of doxycycline on disease activity and joint dest... more ABSTRACT. Objective. To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). Methods. A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. Results. The changes of clinical or laboratory disease activity measures, pyridinoline excret...
Journal of Chromatography B Biomedical Sciences and Applications, 1997
Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (G... more Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.
This study aimed to examine whether biochemical characteristics of juvenile articular cartilage a... more This study aimed to examine whether biochemical characteristics of juvenile articular cartilage are changing during the first year post partum and whether they can be influenced by exercise at young age. Water, glycosaminoglycan (GAG), DNA, total collagen, hydroxylysine and hydroxylysylpyridinoline (HP) content were measured in articular cartilage of 43 foals that were subdivided into 3 groups (n = 15, 14 and 14) which were subjected to different exercise regimens from one week after birth to age 5 months. At the age of 5 months all foals were weaned and 8 foals were selected randomly from each exercise group and subjected to euthanasia. The remaining foals (n = 19) were grouped and subjected to a similar exercise regimen for an additional 6 months. Differences were tested by student's t test (P<0.01). No effect of exercise on the water or DNA content was found. GAG content increased with increasing exercise in the 5 months group. These differences had disappeared after 6 months of similar exercise. No influence of exercise could be demonstrated on any of the collagen parameters. When comparing 5 months with 11 months group, all parameters except hydroxylysine changed significantly during these 6 months. Water, DNA and GAG content decreased during maturation. Collagen and HP content increased. It is hypothesised that juvenile equine articular cartilage may be seen as a dynamic, continuously remodelling tissue that is gradually taking on the biochemical characteristics it will have during the rest of the life of the animal. Moderate exercise does not influence the collagen component of the extracellular matrix. It has a beneficial, but reversible, effect on the glycosaminoglycan component.
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (G... more Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pento... more A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.
To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-posi... more To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium. Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry. Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front. The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to de...
In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been sugg... more In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been suggested to be involved in the development of atherosclerosis. alpha-Dicarbonyl compounds were identified as intermediates in the non-enzymatic glycation and increased levels were reported in patients with diabetes. We studied the effect of the alpha-dicarbonyl compound methylglyoxal (MG) on the physicochemical and biological properties of LDL. MG dose-dependently modifies LDL, as indicated by the formation of fluorescent products and the increase of a net negative charge. MG (10 mmol/l) induced major modifications of arginine residues (up to 85%) and minor lysine modifications (less than 6%). MG-LDL preparations generated small amounts of superoxide anion radicals as measured by the reduction of cytochrome c, but this was not accompanied by peroxidation of the polyunsaturated fatty acids of MG-LDL. MG-LDL showed diminished recognition and uptake by the human LDL receptor in cultured cells ...
Dysfunction of the vascular endothelium is considered an early step in the development of diabeti... more Dysfunction of the vascular endothelium is considered an early step in the development of diabetic angiopathy. Hyperglycaemia results in endothelial dysfunction, both through direct effects of glucose and through formation of advanced glycosylation end-products (AGEs). We hypothesized that the effects of glucose and AGEs on endothelial function in insulin-dependent diabetes mellitus (IDDM) are distinct and are reflected by distinct plasma markers of endothelial function. We therefore measured plasma levels of von Willebrand factor (vWF), soluble (s) E-selectin and vascular cell adhesion molecule-1 (sVCAM-1), and evaluated the relationship with HbA1c and urinary excretion of pentosidine, an AGE product, in 56 patients with IDDM. Urinary pentosidine excretion was higher in the diabetic than in a control group (n = 60) of similar age (P < 0.0001) and showed a steeper increase with age (P < 0.02 vs controls). In the diabetic group, sE-selectin was correlated to HbA1c (r = 0.52, P ...
A biomechanical failure of the collagen network is postulated in many hypotheses of the developme... more A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (>20 years), whereas in young cartilage (<10 years) the opposite was seen; highest levels ...
We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arth... more We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS-polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplif...
ABSTRACT. Objective. To investigate the effects of doxycycline on disease activity and joint dest... more ABSTRACT. Objective. To investigate the effects of doxycycline on disease activity and joint destruction in patients with rheumatoid arthritis (RA). Methods. A 36 week double blind, placebo controlled crossover trial was conducted. Patients (n = 66) received 50 mg doxycycline or placebo twice a day during 12, 24, or 36 weeks. Patient assessments were performed before the treatment was administered, at 6, 12, 24 and 36 weeks of treatment, and finally at 4 weeks after cessation of treatment. Patient assessments, swollen and tender joint counts, duration of morning stiffness, erythrocyte sedimentation rate, and Modified Disease Activity Score were used as measures of disease activity. Effects on joint destruction were assessed by urinary excretion of the pyridinolines hydroxylysylpyridinoline and lysylpyridinoline and by scoring radiographic damage of hands and feet before and after treatment. Results. The changes of clinical or laboratory disease activity measures, pyridinoline excret...
Journal of Chromatography B Biomedical Sciences and Applications, 1997
Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (G... more Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.
This study aimed to examine whether biochemical characteristics of juvenile articular cartilage a... more This study aimed to examine whether biochemical characteristics of juvenile articular cartilage are changing during the first year post partum and whether they can be influenced by exercise at young age. Water, glycosaminoglycan (GAG), DNA, total collagen, hydroxylysine and hydroxylysylpyridinoline (HP) content were measured in articular cartilage of 43 foals that were subdivided into 3 groups (n = 15, 14 and 14) which were subjected to different exercise regimens from one week after birth to age 5 months. At the age of 5 months all foals were weaned and 8 foals were selected randomly from each exercise group and subjected to euthanasia. The remaining foals (n = 19) were grouped and subjected to a similar exercise regimen for an additional 6 months. Differences were tested by student&amp;amp;amp;amp;#39;s t test (P&amp;amp;amp;amp;lt;0.01). No effect of exercise on the water or DNA content was found. GAG content increased with increasing exercise in the 5 months group. These differences had disappeared after 6 months of similar exercise. No influence of exercise could be demonstrated on any of the collagen parameters. When comparing 5 months with 11 months group, all parameters except hydroxylysine changed significantly during these 6 months. Water, DNA and GAG content decreased during maturation. Collagen and HP content increased. It is hypothesised that juvenile equine articular cartilage may be seen as a dynamic, continuously remodelling tissue that is gradually taking on the biochemical characteristics it will have during the rest of the life of the animal. Moderate exercise does not influence the collagen component of the extracellular matrix. It has a beneficial, but reversible, effect on the glycosaminoglycan component.
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (G... more Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic method with fluorimetric detection has been developed and employed to determine the glycosylated hydroxylysine residues in alkaline collagen hydrolysates. After hydrolysis, amino acids were derivatised with 9-fluorenylmethyl chloroformate and separated on a Micropak ODS-80TM reversed-phase column (150x4.6 mm). With a multistep gradient system all amino acids were separated in less than 30 min, including the collagen-specific hydroxylysine, hydroxyproline and the glycosylated hydroxylysines. The method was used to evaluate the glycosylation levels of human articular cartilage derived from femoral head, femoral condyle, tibial plateau and ankle. GGH was highest in cartilage from femoral head and ankle; GH showed no differences between the different sources of cartilage.
Journal of Chromatography B: Biomedical Sciences and Applications, 1997
A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pento... more A high-performance liquid chromatographic assay was developed for pyridinium crosslinks and pentosidine in mature collagen of a wide variety of connective tissue hydrolysates by a simple two-step isocratic assay using a reversed-phase column. The crosslinks (including the internal standard pyridoxine) were optimally detected by their native fluorescence by switching wavelengths of the detector during the assay. The method resulted in highly sensitive and accurate measurements, without need for precleaning of the samples: crosslink levels in 200 microm thin slices of the various zones of articular cartilage were easily quantified. The detection limit was as low as 0.4 pmol for the pyridinolines and 0.05 pmol for pentosidine. The intra-assay and inter-assay coefficients of variation were as low as 2% (pyridinolines) and 5% (pentosidine); calibration curves for all compounds were linear over a concentration range larger than two orders of magnitude. With our chromatographic system, the diglycosylated form of hydroxylysylpyridinoline in unhydrolyzed urine was separated as well.
To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-posi... more To investigate the cartilage-degrading capacity of granzyme B and the presence of granzyme B-positive cells at sites of erosion in the rheumatoid synovium. Granzyme B was added to [(3)H]proline/[(35)S]sulphate-labelled cartilage matrices and to cartilage explants. Proteoglycan degradation was assessed by the release of (35)S and glycosaminoglycans into the medium and collagen degradation was assessed by the release of (3)H and hydroxyproline and by measuring the fraction of denatured collagen. Granzyme B expression was studied at the invasive front of the synovium by immunohistochemistry. Granzyme B induced loss of both newly synthesized, radiolabelled proteoglycans in cartilage matrices and resident proteoglycans of the cartilage explants. No effect on collagen degradation was found. Granzyme B-positive cells were present throughout the synovium and at the invasive front. The presence of granzyme B-positive cells at the invasive front of the synovium together with its ability to de...
In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been sugg... more In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been suggested to be involved in the development of atherosclerosis. alpha-Dicarbonyl compounds were identified as intermediates in the non-enzymatic glycation and increased levels were reported in patients with diabetes. We studied the effect of the alpha-dicarbonyl compound methylglyoxal (MG) on the physicochemical and biological properties of LDL. MG dose-dependently modifies LDL, as indicated by the formation of fluorescent products and the increase of a net negative charge. MG (10 mmol/l) induced major modifications of arginine residues (up to 85%) and minor lysine modifications (less than 6%). MG-LDL preparations generated small amounts of superoxide anion radicals as measured by the reduction of cytochrome c, but this was not accompanied by peroxidation of the polyunsaturated fatty acids of MG-LDL. MG-LDL showed diminished recognition and uptake by the human LDL receptor in cultured cells ...
Dysfunction of the vascular endothelium is considered an early step in the development of diabeti... more Dysfunction of the vascular endothelium is considered an early step in the development of diabetic angiopathy. Hyperglycaemia results in endothelial dysfunction, both through direct effects of glucose and through formation of advanced glycosylation end-products (AGEs). We hypothesized that the effects of glucose and AGEs on endothelial function in insulin-dependent diabetes mellitus (IDDM) are distinct and are reflected by distinct plasma markers of endothelial function. We therefore measured plasma levels of von Willebrand factor (vWF), soluble (s) E-selectin and vascular cell adhesion molecule-1 (sVCAM-1), and evaluated the relationship with HbA1c and urinary excretion of pentosidine, an AGE product, in 56 patients with IDDM. Urinary pentosidine excretion was higher in the diabetic than in a control group (n = 60) of similar age (P < 0.0001) and showed a steeper increase with age (P < 0.02 vs controls). In the diabetic group, sE-selectin was correlated to HbA1c (r = 0.52, P ...
A biomechanical failure of the collagen network is postulated in many hypotheses of the developme... more A biomechanical failure of the collagen network is postulated in many hypotheses of the development of osteoarthritis with advancing age. Here we investigate the accumulation of non-enzymatic glycation (NEG) products in healthy human articular cartilage, its relation to tissue remodelling and its role in tissue stiffening. Pentosidine levels were low up to age 20 years, and increased linearly after this age. This indicates extensive tissue remodelling at young age, and slow turnover of collagen after maturity has been reached. The slow remodelling is supported by the finding that enzymatic modifications of collagen (hydroxylysine, hydroxylysylpyridinoline, and lysylpyridinoline) were not related to age. The high remodelling is supported by levels of the crosslink lysylpyridinoline (LP) as a function of distance from the articular surface. LP was highest at the surface in mature cartilage (>20 years), whereas in young cartilage (<10 years) the opposite was seen; highest levels ...
We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arth... more We investigated whether plasma and synovial fluid (SF) samples from patients with rheumatoid arthritis (RA) contained extracellular mitochondrial DNA (mtDNA) or the oxidatively damaged DNA adduct 8-hydroxy-2'-deoxyguanosine (8-oxodG). Moreover, we correlated the laboratory findings of the patients with RA with their levels of mtDNA and 8-oxodG. SF and plasma samples from 54 patients with RA, SF from 30 non-arthritic control subjects, and plasma from 22 healthy volunteers were collected. The samples were subjected to polymerase chain reaction (PCR) using mitochondrial genomic primers, and the products were analyzed by SDS-polyacrylamide-gel electrophoresis. The intensities of the PCR-amplified bands were quantified and normalized to a reference sample. Furthermore, the SF samples were assayed by enzyme-linked immunosorbent assay for 8-oxodG. Extracellular PCR-amplifiable mtDNA was detected in the SF of 38 of 54 (70%) patients with RA, but not in any of the SF controls. PCR-amplif...
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Papers by Johan Koppele