Hypovitaminosis D in recent onset rheumatoid arthritis is predictive of reduced response to treatment and increased disease activity: a 12 month follow-up study

Study population

This is a longitudinal observational retrospective study with a 12 months follow-up. Data from seventy-six consecutive patients referring to the Early Arthritis Clinic, Rheumatology Unit, Sapienza University of Rome, for recently onset RA between 2010 and 2012 were initially obtained. Among them, we enrolled subjects which fulfilled the following inclusion criteria: age between 30 and 65 years, subjects meeting the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for RA diagnosis [22], mean disease duration ≤ 24 weeks, BMI 16–25 kg/m², conscious acceptance of written informed consent. The exclusion criteria were: treatment with vitamin D and/or calcium supplementation or drugs affecting the bone and mineral metabolism, multivitamin supplementation, oral contraceptives, parathyroid hormone, L-tyroxine, diuretics, DMARDs, anti-epilepsies and steroids; intestinal malabsorption; diseases associated with hypercalcaemia (lymphoma, sarcoidosis, tuberculosis infection, primary hyperparathyroidism); acute/chronic kidney failure; acute/chronic hepatic failure; type 1 and type 2 diabetes mellitus; metabolic syndrome; history of drugs and/or alcohol abuse. Among the initial seventy-six patients, thirty-seven subjects were considered eligible for the study: fifteen patients were excluded for treatment with drugs affecting the bone and mineral metabolism, eight for multivitamin supplementation, one for oral contraceptives, three for treatment with steroids, one for intestinal malabsorption, two for chronic kidney failure, five for diabetes mellitus, three for metabolic syndrome and one refused to participate.

Study population underwent 12 months follow-up (mean ± SD: 54 ± 3 weeks) during which they were treated with low doses of corticosteroids (Prednisone < 10 mg/day) and methotrexate (7.5-15 mg/week) and attended to quarterly recall visits for clinical evaluation.

Laboratory tests

For each patient, we collected and analyzed data at baseline (disease onset) and after 12-months follow-up on medical history, physical examination and blood immune-inflammatory parameters, such as: erythrocyte sedimentation rate (ESR) (normal values: ≤15 mm/h in men and ≤20 mm/h in female), C-reactive protein (CRP) level (normal values: <3 mg/L), using standard laboratory methods and to detect IgM-Rhematoid Factor (RF) by nephelometric method (N latex FR, Bering, CV < 4%) and anti-citrullinated protein antibodies (ACPA) by immune-enzymatic method (ImmunoCap, Phadia, <5%). A titre of IgM-RF >15 IU/ml was considered positive, the cut-off point for ACPA positivity was > 12 IU/ml, according to the manufacturer’s instructions.

In order to evaluate vitamin D status in our population at the diagnosis of RA, serum levels of 25(OH) vitamin D (ng/ml), the most stable circulating form of this molecule were also measured [23,24]. In order to minimize the impact of seasonality on circulating vitamin D concentrations, all participants underwent fasting blood sampling for serum 25(OH) vitamin D measurement during autumn-winter period. Vitamin D was measured by immunochemiluminescent method (LIAISON, DiaSorin, CV < 5%) on sera frozen immediately after separation and stored at −25°C for few days.

Clinical assessment

Clinimetric assessment, routinely used in clinical practice and Clinical Trials to evaluate disease activity and response to treatment, was performed by a rheumatologist experienced in clinimetric evaluation and blinded to laboratory data. At baseline and 12 months of follow up we collected data on: the number of tender and swollen joints on the basis of the 28-joint count and 44-joint count, Ritchie index (a graded assessment of 26 joint regions), visual analogue scale (VAS 0–100 mm) for pain and patient’s disease activity, examinator’s global disease activity assessment (EGA), patient global health assessment (GH) and 28-joints Disease Activity Score (DAS28) [25].

At 12 months follow-up, EULAR response was calculated from the DAS28 scores. Study population was divided in three EULAR response groups: no response, moderate response or good response. To be a good responder, a patient had to show an improvement of at least 1.2 units and achieve an absolute score of < 3.2. Non-responders had to show an improvement of < 0.6, or > 0.6 and ≤ 1.2, and have a final DAS28 score of > 5.1. Moderate responses fell in-between these criteria [26].

Musculoskeletal ultrasonography

US evaluation was performed both at baseline and after a 12-months follow-up. A systematic multiplanar grey-scale and powerDoppler (pD) examination of 17 joints [wrist, metacarpophalangeal (MCP) joints, proximal interphalangeal (PIP) joints, knee and metatarsophalangeal (MTP) joints bilaterally] was performed according to standard EULAR guidelines, by MyLab70 XVision Gold (Esaote, Italy) machine equipped with a multifrequency linear probe (6–18 MHz); powerDoppler PFR (peak frequency ratio) was 750 Hz, Doppler frequency was 7.5 MHz. Synovial effusion (SE), synovial hypertrophy (SH) and bone erosions were assessed according to the Outcome Measures in Rheumatoid Arthritis Clinical Trial (OMERACT) definitions [27]. US-detected elementary lesions (SE, SH and pD signal) were scored according to a semiquantitative scale based on the severity of the US modifications (0 = normal; 1 = mild; 2 = moderate; 3 = severe). The total synovitis score was obtained from the sum of the all US modifications identified in all the joints. Bone erosions were assessed with a semiquantitative score as follows: grade 0 = no erosion, grade 1 = 1 erosion, grade 2 = 2 erosions, grade 3 ≥ 3 erosions.

Statistics

SPSS version 17 statistical package was used to perform the analyses. Non-parametric Mann–Whitney test for mean comparison between two independent groups and Wilcoxon test for mean comparison between two related groups were used. The χ-squared test was used to compare the rate of responders to treatment at 12 months follow-up (moderate + good response vs non response) among patients with and without basal hypovitaminosis D. Bivariate correlation analyses were performed by Spearman’s coefficient calculation. Study population was divided in two subgroups according to vitamin D status considering serum 25(OH) vitamin D < 20 ng/ml as a cut-off for insufficient vitamin D levels [28,29]. At the best of our knowledge, clinical remission and response to treatment by DAS28 after a 12 months follow-up in relation to vitamin D status at RA onset have never been studied before. Therefore, in order to confirm the statistical power of this study, we performed a post-hoc sample size calculation considering a prevalence of remission in the subgroup with normal vitamin D status of 68% and of 16% in patients with hypovitaminosis D; thus, we obtained that eleven patients per subgroup were enough to reach the statistical significance with a power of 80% (α error = 0.05, β error = 0.20). For all the above, a p-value < 0.05 was considered statistically significant. Study protocol was reviewed and approved by the Ethics Committee of Policlinico Umberto I, Sapienza University of Rome and conducted in conformance with the Helsinki Declaration. Written consent was obtained from all patients before the study.