Department of Rheumatology, University Hospital Vrije Universiteit, Amsterdam,
1 Department of Rheumatology, University Hospital Vrije Universiteit, Jan van Breemen Instituut, Slotervaart Ziekenhuis, Amsterdam and
2 Jan van Breemen Instituut, Amsterdam, The Netherlands
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Abstract |
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Patients and methods. The urinary levels of bone resorption markers (pyridinoline, deoxypyridinoline, N-terminal telopeptide and C-terminal telopeptide) were measured in 184 patients with inactive RA, as defined by the preliminary criteria of clinical remission of the American College of Rheumatology, and in 118 healthy individuals.
Results. After adjusting for age, concentrations of all four bone resorption markers were found to be significantly higher in patients with inactive RA than in healthy controls.
Conclusion. The urinary excretion of bone resorption markers is increased in patients classified as having inactive RA. These results suggest that the inflammatory process is not completely absent.
KEY WORDS: Bone resorption markers, Bone metabolism, Disease activity, Remission, Rheumatoid arthritis.
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Introduction |
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Collagen degradation products are known to be increased in RA patients with active disease [7] and correlated with disease activity [8, 9]. Pyridinium cross-links [pyridinoline (Pyr) and deoxypyridinoline (D-pyr)], N-terminal telopeptide (NTx) and ß-C-terminal telopeptide (ß-CTx) are collagen degradation products and play a role in the resorption of collagen in bone, cartilage and synovium. These products are released into the circulation during the breakdown of collagen and are subsequently excreted in the urine.
To answer the question whether inactive disease in RA indeed corresponds to the absence of inflammation, we measured the urinary excretion of bone resorption markers in RA patients with clinically inactive disease, as defined by the American College of Rheumatology (ACR) criteria for clinical remission, and in healthy controls.
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Patients and methods |
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Inactive RA was present for at least 6 months prior to inclusion in the study. At the time of inclusion, the treatment of the patients consisted of non-steroidal anti-inflammatory drugs (NSAIDs) and/or slow-acting anti-rheumatic drugs (SAARDs). Patients treated with corticosteroids were excluded from the study. For all RA patients, a composite index of disease activity, the DAS (disease activity score), was computed [11].
Urine samples from 77 young healthy controls (HCs), aged <60 yr, and 41 elderly HCs, aged >60 yr, were also studied.
Age and sex were recorded for all subjects; menopausal status was also recorded for all female patients and HCs.
All RA patients fulfilled the 1987 American Rheumatism Association criteria for RA [12].
Urine samples
Second-morning void urine samples were collected and stored at 20°C without additives. Samples were centrifuged before use. The creatinine concentration (mmol/litre) was determined in duplicate using the Jaffe rate technique with alkaline picrate.
Measurement of cross-links by high-performance liquid chromatography
Total pyridinoline (Pyr) and deoxypyridinoline (D-pyr) were determined as described previously [13, 14], with some modifications. After acid hydrolysis (in duplicate), interfering fluorophores were removed on CF1 cellulose columns (Whatman). Pyr and D-pyr in the eluates were quantified by ion-pair chromatography on a C18 column (Alltima C18 5u, Alltech, Deerfield, IL, USA) with fluorescence detection using Waters high-performance liquid chromatography (HPLC) equipment (excitation 297 nm, emission 400 nm). A Metra Biosystems (Mountain View, CA, USA) HPLC calibrator was used to calibrate the assay. The intra-assay coefficients of variation were <3% (n = 10) and the inter-assay coefficients of variation were <10% (n = 40).
Measurement of the cross-links by ELISA
Two enzyme-linked immunosorbent assays were performed in duplicate according to the manufacturer's instructions. The Osteomark kit (Ostex International, Seattle, WA, USA) measures the pyridinoline cross-link in the NTx of type I collagen. The inter-assay coefficients of variation were <6% (n = 10). The CrossLaps kit (Osteometer, Hevlev, Denmark) measures the cross-link in the ß-CTx of type I collagen. The inter-assay coefficients of variation were <9% (n = 10).
Statistical analysis
Differences in the demographic data between patients and controls were tested using the MannWhitney U-test for age and the 2 test for sex and menopausal status.
Differences in laboratory data on the bone resorption markers were tested using multiple regression analysis. Logarithmically transformed values for the bone markers were used as dependent variables. Age was used as independent variable. A separate regression analysis was performed for each bone resorption marker.
Spearman's correlation coefficients between markers of bone resorption and parameters of disease activity were calculated. P < 0.05 was considered significant.
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Results |
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A significant correlation was found between the concentration of each bone marker and the number of swollen joints (Pyr, 0.17; D-pyr, 0.18; NTx, 0.29; CTx, 0.25; P < 0.05). The concentrations of Pyr and D-pyr correlated significantly with ESR (Spearman's = 0.23 and 0.16 respectively; P < 0.05). Pyr and D-pyr concentrations also correlated significantly with DAS (Spearman's
= 0.18 and 0.18 respectively; P < 0.05). The concentrations of bone resorption markers did not correlate significantly with the number of tender joints or the Ritchie index.
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Discussion |
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Elevated concentrations of bone resorption markers in the urine can reflect several other pathological processes which occur in RA, including activity of the inflammatory process or a high rate of bone turnover, as occurs in osteoporosis, osteoarthritis and erosive joint destruction, or a combination of these processes [15, 16].
The present study on bone resorption markers in RA patients with inactive disease was prompted by reports on associations between disease activity and the excretion of collagen cross-links in RA patients. In patients with (active) RA, increased urinary excretion of Pyr and D-pyr has been described [7, 15]. In addition, it has been shown that urinary excretion of bone markers correlates with parameters of RA disease activity [8, 9].
Although the extent to which each of the above-mentioned factors contributes to the increased urinary levels of bone resorption markers in patients with inactive RA remains unclear, the results of the present study suggest that an active inflammatory process is still involved in such patients.
Whether the results of this study have clinical significance for all patients with inactive RA is unknown. However, such clinical significance is not excluded in some patients with inactive RA. The increased concentrations found in this study suggest that some patients may have had active disease although they were classified as inactive disease.
Prospective studies are needed to investigate whether increased urinary concentrations of bone resorption markers are associated with the radiological progression of erosive disease in patients with inactive RA. Such studies may also give more information on the contributions of the inflammatory process and of radiological abnormalities to the increased urinary excretion of bone resorption markers. Such studies are currently being carried out.
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Notes |
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References |
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