Hormone, metabolic peptide, and nutrient levels in the earliest phases of rheumatoid arthritis—contribution of free fatty acids to an increased cardiovascular risk during very early disease

Study subjects

Twenty-two patients with active RA, 45 individuals at risk for developing RA, and 16 healthy controls were included in the first part of this study. The individuals at risk for RA had arthralgia and/or a positive family history for RA, without any evidence of arthritis upon thorough physical examination. They were positive for IgM rheumatoid factor (IgM-RF) and/or anticitrullinated protein antibodies (ACPAs). These individuals at risk were characterized by the presence of systemic autoimmunity associated with RA (phase c according to [14]) with or without environmental risk factors (phase b + c according to [14]), with or without symptoms but without clinical arthritis (phase d according to [14]) and with or without genetic risk factors (phase a + b + c according to [14]). IgM-RF was measured using IgM-RF ELISA from Hycor Biomedical, Indianapolis, IN (ULN 49 IU/ml). ACPA was measured using anti-CCP2 Elisa CCPlus (Eurodiagnostica, Nijmegen, the Netherlands (ULN 25 kAU/L)).

RA patients fulfilled the 2010 ACR/EULAR classification criteria [15, 16] and had active disease defined as DAS28 >2.6 and at least one swollen joint. All RA patients were treated with non-steroidal anti-inflammatory drugs (NSAIDs) and/or disease-modifying antirheumatic drugs (DMARDs) and were biological naive. RA patients and the individuals at risk for RA were recruited via the outpatient clinic of the department of Clinical Immunology and Rheumatology of the Academic Medical Center, Amsterdam, the Netherlands. We included healthy controls, matched for gender and age, without joint complaints, history of clinical evident arthritis, and who were not known with other systemic inflammatory disease for which immunosuppressive therapy was needed; they were negative for IgM-RF and ACPA. The controls were also recruited in the Academic Medical Center, Amsterdam, the Netherlands, via advertisements.

We used another cohort to validate our findings in the test cohort in order to limit the chance on significant results by coincidence. We included an independent validation cohort, consisting of 32 individuals at risk for RA (arthralgia and elevated IgM-RF and/or ACPA serum levels without clinically manifest arthritis), 20 early arthritis patients with disease duration <6 months (n = 16 RA and n = 4 unclassified arthritis (UA)), and 20 age- and gender-matched healthy controls. Patients were classified as UA in the absence of fulfillment of disease-specific criteria, but they had elevated serum levels of RA-specific autoantibodies (IgM-RF and/or ACPA). The RA and UA patients were biologically naive.

All subjects were included at the outpatient clinic of the department of Clinical Immunology and Rheumatology of the Academic Medical Center, Amsterdam, the Netherlands. The study was performed according to the principles of the Declaration of Helsinki, approved by the institutional review board of the Academic Medical Center (2010_327#B2011135), registered in The Netherlands National Trial Register/Nederlands Trial Register (NTR2833), and all study subjects gave written informed consent.

Study procedures

After enrollment, the individuals at risk for developing RA were followed annually for arthritis development, which was the endpoint of the study. Patients with established RA and healthy controls had one study visit. Overnight fasting blood samples were collected between 9 and 10 a.m. at baseline and again when arthritis developed in the at-risk population. Samples were directly stored on ice, centrifuged, aliquotted, and frozen at −80° within 30 min after bleeding of the subject. Catecholamines were determined in tubes containing EGTA/glutathione and glucagon using tubes containing aprotinin.

The hormones and peptides tested are described in Supplementary Table 1. Lipid levels (total cholesterol, high density lipoprotein (HDL)-cholesterol, low density lipoprotein (LDL)-cholesterol, and TG) were measured using standard laboratory techniques. All hormone measurements, except for adrenocorticotropic hormone (ACTH), cortisol, and insulin, were performed in duplicate. The mean of these duplicate means were used for the analysis. The validation cohort was used to confirm the results obtained for lipid levels.

The following clinical and disease activity parameters were obtained during the study visits: age, sex, body mass index (BMI), alcohol use, smoking status, IgM-RF and ACPA status, disease duration, use of NSAIDs, and/or DMARDs. Patient visual analog scale for global disease activity (VAS GDA) of 0–100 mm, tender joint count of 28 joints (TJC28), swollen joint count of 28 joints (SJC28), erythrocyte sedimentation rate (ESR; in mm/h), C-reactive protein (CRP; mg/L), and disease activity score evaluated in 28 joints (DAS28) were also measured. In individuals who developed arthritis, an additional visit was performed and all clinical and disease activity parameters mentioned above were assessed, including a 68 joint score to confirm the presence of arthritis.

Statistical analysis

Data are presented as median (IQR) unless otherwise indicated. Differences between study groups were analyzed using Kruskal-Wallis or Mann-Whitney U test where appropriate. We considered a p value of <0.05 as significant in this exploratory study. The differences found in this exploratory study should be evaluated in future studies. Categorical data were analyzed using chi-square test or, if more appropriate, Fisher’s exact test. Correlations between variables were analyzed using Spearman’s rank correlation coefficient. Statistical analysis was performed using IBM SPSS Statistics 18 (SPSS Inc., Chicago, IL).

Most hormone levels are comparable in at-risk individuals, patients with RA and healthy controls

Figure 1 and Table 2 show the hormone levels and peptide levels in at-risk individuals, patients with RA, and healthy controls. There were significantly higher norepinephrine levels in at-risk individuals (3.02 (2.24–4.15) nmol/L) compared to RA patients (2.45 (1.51–3.26) nmol/L) and healthy controls (2.15 (1.49–2.71) nmol/L; P = 0.017). The norepinephrine levels did not correlate with clinical and laboratory disease parameters in either RA patients or in at-risk individuals. The epinephrine levels were not different between the three groups. The levels of ACTH, cortisol, and cortisol/IL-6 ratio were also on average comparable between the three groups. IL-6 levels were higher in both individuals at risk (25.1 (3.8–54.5) pg/ml) and RA patients (27.7 (9.3–80.9) pg/ml) compared to healthy controls (7.5 (0.9–66.7) pg/ml; P = 0.23). The sex hormones estradiol, follicle stimulating hormone (FSH), and luteinizing hormone (LH) were comparable between the three groups. PRL levels were also similar in the at-risk individuals and RA patients compared with healthy controls. Similarly, the neuroendocrine hormones growth hormone (GH) and thyroid stimulating hormone (TSH) were comparable between the groups.

FFAs are elevated in at-risk individuals and correlated with disease activity parameters

In the test cohort, we found higher TG levels in at-risk individuals and significantly higher TG levels in RA patients compared to healthy controls (0.94 (0.72–1.15) mmol/L, 1.03 (0.75–1.29) mmol/L, and 0.70 (0.59–1.02) mmol/L, respectively; P = 0.11). We could confirm these findings in the independent validation cohort and found significantly increased TG levels in 20 early arthritis patients (consisting of RA and UA patients) compared to 20 healthy controls matched for age and gender (1.05 (0.65–1.54) mmol/L and 0.80 (0.48–0.90), respectively; P = 0.036 (Fig. 2). Moreover, in this validation cohort of 32 at-risk individuals, we observed significantly higher TG levels in baseline serum of 10 individuals who developed arthritis during follow up (1.47 (1.12–1.91) mmol/L) compared to 22 individuals who did not develop arthritis (1.01 (0.68–1.31) mmol/L) (P = 0.030).

In line with this, we found a trend toward increased FFA levels in the at-risk individuals (0.53 (0.40–0.59) mmol/L) and significantly higher levels in RA patients (0.59 (0.47–0.65) mmol/L) compared to healthy controls (0.40 (0.35–0.50) mmol/L; P = 0.024), independent of gender. The association of the FFA levels in the three different groups was not confounded by age, BMI, current smoking, or other variables measured (using regression analysis). Using the reference upper limit of 0.44 mmol/L as cutoff, 30 (67 %) at-risk individuals had elevated FFA levels compared to 18 (82 %) RA patients and 5 (31 %) healthy controls (P = 0.005). Interestingly, the FFA levels correlated in RA patients with TJC28 (r = 0.48 and P = 0.022), CRP (r = 0.47 and P = 0.029), and DAS28 (r = 0.53 and P = 0.012) and a trend toward a positive correlation with VAS GDA (r = 0.39 and P = 0.07) and SJC28 (r = 0.41 and P = 0.06) was seen (Fig. 3). In the at-risk individuals without arthritis, we did not observe a correlation between FFA levels and clinical and laboratory disease parameters. The PP levels were significantly higher both in at-risk individuals (31 (21–45) pmol/L) and RA patients (34 (23–58) pmol/L) compared to healthy controls (10 (6–27) pmol/L); P = 0.003. The PP levels did not correlate with clinical and laboratory disease parameters in either RA patients or in at-risk individuals.

Because of the small number of the at-risk individuals developing arthritis in this study, no conclusions can be drawn on the role of these hormones and peptides in the risk of developing arthritis. The values for these individuals are described in Table 3.

Competing Interests

All authors do not have financial or other relationships that could give conflicts of interest. Dr. Gerlag and Professor Tak have become employees of GSK after completion of this study; GSK has not been involved in this study.

Funding

The study was supported by the European Union Seventh Framework Programme (project EuroTEAM; FP7-Health2–2012; grant number 305549).

Authors’ Contributions

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version of the article to be published; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. M.W.T contributed to research design, acquisition of data, and analyzed and interpreted data; F.A.K was involved in research design and acquisition of data; J.P.M.V. contributed to acquisition of data; M.J.H. was involved in analyzing and interpreting data; D.M.G designed the research and analyzed and interpreted data; P.P.T. designed the research and analyzed and interpreted data.