**2. Measured adrenomedullin (ADM) in patients with RA**

#### **2.1 Patient background**

For measurement of plasma ADM, the study population consisted of 26 patients with RA aged 58–73 years (mean ± SD 62 ± 4 yrs), 10 with osteoarthritis (OA) aged 59–76 years

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 175

It has been reported that some collagenous disorders show increased levels of plasma ADM (Yudoh et al., 1999). We measured and compared plasma ADM concentrations in patients with RA and healthy controls, finding that patients with RA exhibited a 1.7-fold increase in

**Tolal ADM (fmol / ml) Mature ADM (fmol / ml)** 

**2.3 Result of ADM in plasma, joint fluid and joint tissues in patients with RA** 

**Healthy controls** 11.64±2.8 1.34±0.9 **RA patients** 18.35±6.9 1.80±1.4 **OA patients** 12.88±1.9 1.42±0.8

significantly higher than that of OA patients (7.2 ± 1.8 fmol/ml) (Fig. 2).

Fig. 1. Correlation between plasma AM and C-reactive protein

Table 1. Blood adrenomedullin (ADM) levels in RA and OA patients and healthy controls

All values are expressed as means ± SD.※p < 0.01. Patients with RA demonstrated high plasma concentration of ADM (18.35 ± 6.9 fmol/ml) compared to healthy controls (11.64 ±

Moreover, plasma ADM levels in patients with RA were also found to be significantly correlated to CRP levels (Fig. 1). In RA, CRP correlates with disease activity and response to therapy. Our patient data did not include RA disease activity, excluding RA functional class classification. Hamada et al. (2010) stated that no autologous antibody such as rheumatoid factor or anti-CCP antibody showed significant correlation with plasma ADM level. In addition, there was no correlation with disease activity scores such as DAS-28. DAS-28 is not only dependent on the inflammatory level, so the plasma ADM level may escalate before escalation of the activity in synovitis or the amount of actual synovial inflammation (Hamada et al., 2010). Therefore, our results suggest that ADM levels are increased in patients with RA and investigated that they might be correlated with disease activity. Moreover, ADM concentration in the joint fluid of RA patients (10.8 ± 4.3 fmol/ml) was

A significant positive correlation was observed between AM and CRP (correlation coefficient = 0.685, p < 0.01). Plasma ADM and plasma CRP levels were found to be well

correlated. The correlation coefficient between CRP and AM was 0.685, p < 0.01.

**Discussion of joint ADM level in patients with RA** 

2.8 fmol/ml) and OA patients (12.88 ± 1.9 fmol/ml)

plasma ADM levels (Table 1).

(68 ±8), and 10 healthy volunteer controls aged 50–66 years (57 ± 5). All subjects were female.

To clarify further the reason for the increase in plasma ADM, joint fluid, synovial tissue, and cartilage were measured by radioimmunoassay (RIA). These were acquired from surgical subjects during total knee arthroplasty in patients with RA (n = 6) and OA (n = 6). All patients with RA were classified as stage 4, functional class 2, according to the criteria of the American Rheumatism Association (ARA) (Clegg & Ward, 1987); they were medicated with only DMARDs and NSAIDs without steroid. All patients with OA were classified as stage 4 to 5, according to the Kellgren-Lawrence radiographic staging system (Kellgren & Lawrence, 1958). Since plasma ADM concentration has been reported to be elevated in patients with hypertension, renal failure, systemic infections, myocardial infarction, and heart failure (Ishimitsu et al., 1994; Nishikimi et al., 1995; Kobayashi et al., 1996), patients and controls with these conditions were excluded.

## **2.2 Methods of measuring ADM levels in plasma, joint fluid and joint tissues 2.2.1 Methods measuring the plasma and joint fluid ADM level**

Blood and joint fluid samples were transferred into tubes containing 1 mg/ml EDTA-2Na and 500 kallikrein inhibitory units/ml of aprotinin for measurement of ADM. The plasma was kept at –30°C until assayed. Levels of plasma ADM and joint fluid ADM were measured by IRMA using specific kits (AM RIA Shionogi) developed by Shionogi Pharmaceutical Co. Ltd., Osaka, Japan. The limit of detection of human AM is 0.5 pmol/l for these kits.
