Levels of markers of bone resorption are moderately increased in patients with inactive rheumatoid arthritis

E. T. H. Molenaar, W. F. Lems1, B. A. C. Dijkmans1, M. H. M. T. de Koning2, R. J. van de Stadt2 and A. E. Voskuyl

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


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objective. Clinical remission occurs in 10–20% of patients with rheumatoid arthritis (RA). However, it is questionable whether clinical remission corresponds to the complete absence of the inflammatory process. To answer this question we measured collagen degradation products (which are known to be increased in active disease) in patients with inactive RA and in healthy controls.

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.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The inflammatory process and functional disability in rheumatoid arthritis (RA) varies considerably among patients. From observational studies it is estimated that functional disability is progressive in 40–70% of patients and that disease activity is characterized by exacerbations and remissions in 20–40% of patients [1, 2]. Long-standing clinical remission has been reported in only 10–20% of RA patients [35]. Since this state is the final goal of therapy, preliminary criteria of clinical remission have been proposed, which are defined as the absence of joint pain and swelling, a normal erythrocyte sedimentation rate, and the absence of fatigue and of morning stiffness [6]. It is unknown whether other parameters of the inflammatory process correspond with this condition.

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.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Subjects
Urine samples were obtained from 184 patients with inactive RA, as defined by the ACR criteria of clinical remission [6]. Clinical remission was considered to be present if at least four out of the following five criteria were satisfied: (1) duration of morning stiffness not exceeding 15 min; (2) no joint pain; (3) no joint tenderness or pain on motion; (4) no soft-tissue swelling in joints or tendon sheets; (5) erythrocyte sedimentation rate (ESR) <30 mm/h for a female or <20 mm/h for a male. The criteria for joint pain and joint tenderness on motion were modified by allowing mild arthralgia if local synovitis was absent and anatomical changes in the joint could be demonstrated radiologically, as described previously [10]. Joint pain and swelling were assessed by the ACR 66 swollen joint count, the 68 tender joint count and the Ritchie index.

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 Mann–Whitney U-test for age and the {chi}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.


    Results
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The demographic characteristics of patients and controls are shown in Table 1Go. The RA patients had a significantly higher median age than the controls (58 vs 53 yrs). The frequencies of female patients and post-menopausal status were similar in the two groups. A large majority of the RA patients had elevated serum rheumatoid factor levels and erosive disease, and were currently being treated with SAARDs. As expected, disease activity was low, as shown by a low median ESR and a low median DAS.


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TABLE 1. Characteristics of patients with inactive RA and healthy controls

 
In patients with inactive RA, the urinary concentrations of Pyr, D-pyr, NTx and ß-CTx were significantly higher than those in HCs (Table 2Go).


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TABLE 2. Urinary concentrations of bone resorption markers in patients with inactive RA and healthy controls

 
When comparing data for patients treated with and without SAARDs, no significant difference in concentrations of bone markers were found between the groups. Also, there was no significant difference in concentrations of bone markers between patients treated with and without NSAIDs.

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 {rho} = 0.23 and 0.16 respectively; P < 0.05). Pyr and D-pyr concentrations also correlated significantly with DAS (Spearman's {rho} = 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.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In this study, excretion of bone resorption markers was higher in patients with inactive disease than in HCs.

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.


    Notes
 
Correspondence to: E. T. H. Molenaar, Department of Rheumatology, University Hospital Vrije Universiteit, P. O. Box 7057, 1007 MB Amsterdam, The Netherlands. Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

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Submitted 24 June 1999; revised version accepted 10 January 2000.