ORIGINAL ARTICLE

The synovial fluid adiponectin-leptin ratio predicts painwith knee osteoarthritis

Rajiv Gandhi & Mark Takahashi & Holly Smith &

Randy Rizek & Nizar N. Mahomed

Received: 22 February 2010 /Revised: 27 February 2010 /Accepted: 10 March 2010 /Published online: 28 March 2010# Clinical Rheumatology 2010

Abstract The relationship between adipokines, such asleptin and adiponectin, and cartilage degeneration is beingincreasingly recognized. We asked what the relationship isbetween these hormones and patient-reported knee osteo-arthritis (OA) pain. We collected demographic data, ShortForm McGill Pain scores, Western Ontario and McMasterUniversities Arthritis Index (WOMAC) pain scores, andsynovial fluid (SF) samples from 60 consecutive patientswith severe knee OA at the time of joint replacement

surgery. SF samples were analyzed for leptin and adiponectinusing specific ELISA. Non-parametric correlations andlinear regression modeling were used to identify therelationship between the adipokines and pain levels. Thecorrelations between the individual adipokines and the painscales were low to moderate and consistently less than thatfor the corresponding adiponectin/leptin (A/L) ratio. Linearregression modeling showed that the A/L ratio was asignificant predictor of a greater level of pain on the MPQ-SF (p=0.03) but not the WOMAC pain scale (p=0.77). Agreater A/L ratio was associated with less pain with severeknee OA and this metabolic pathway may represent a targetfor novel therapeutics.

Keywords Leptin . Obesity . Osteoarthritis . Pain

Introduction

Obesity is well known to be a risk factor for the incidenceand progression of prevalent osteoarthritis (OA) [1–3].There is increasing evidence that visceral and sub-cutaneous truncal white adipose tissue is an activeendocrine organ that secretes cytokines and adipokines,such as interleukin 1 (IL-1), leptin and adiponectin, into thesystemic circulation [4, 5]. These inflammatory mediatorshave been shown to have independent relationships withcartilage degeneration [5–7]. This hypothesis supports theepidemiologic relationship between obesity and OA of non-weight-bearing joints such as the hand [2, 8, 9].

Leptin is a hormone that acts centrally at the level of thehypothalamus to regulate appetite and energy expenditure.Serum leptin levels are known to correlate with body massindex (BMI) [5]. Greater circulating levels have beenshown to predict an increased risk of chronic diseases such

R. Gandhi :H. Smith :R. Rizek :N. N. MahomedDivision of Orthopaedic Surgery, University Health Network,399 Bathurst Street, East Wing 1-439,Toronto, ON M5T-2S8, Canada

R. Gandhi :H. Smith :R. RizekDepartment of Surgery, University of Toronto,Toronto, Canda

M. TakahashiMaRS Discovery Institute, University of Toronto,Toronto, Canada

N. N. MahomedDivision of Health Care and Outcomes Research and ArthritisCommunity Research and Evaluation Unit,Toronto Western Research Institute,Toronto, Canada

N. N. MahomedDepartment of Health Policy,Management and Evaluation and Department of Surgery,University of Toronto,Toronto, Canada

R. Gandhi (*)Toronto Western Hospital, University Health Network,399 Bathurst Street, East Wing 1-439,Toronto, ON M5T-2S8, Canadae-mail: rajiv.Gandhi@uhn.on.ca

Clin Rheumatol (2010) 29:1223–1228DOI 10.1007/s10067-010-1429-z

as myocardial infarction [10, 11] and stroke [11, 12].Moreover, laboratory studies have also demonstrated anassociation between synovial fluid (SF) leptin and kneecartilage degeneration [5, 6]. Leptin is believed to induce aproinflammatory and damaging effect on cartilage cellsthrough the production of interleukin 1ß and metallopro-teinases 9 and 13 (MMP9 and MMP13) [6, 13]. Theseinflammatory mediators potentiate the further release oflocal type II nitric oxide synthase (NOS2) leading to furtherchondrocyte death [7]. These proinflammatory cytokinesare also mediators of knee joint pain in OA.

Adiponectin has been shown to decrease inflammation invascular endothelium and therefore may represent an anti-atherosclerotic factor [14]. The adiponectin receptors(AdipoR1 and AdipoR2) have been identified on thesurface of human chondrocytes suggesting a potential rolein joint inflammatory diseases [15]. The anti-inflammatoryeffects of adiponectin are believed to be related to down-regulation of IL 1ß and MMP 13 levels [16], and alsothrough stimulation of the release of anti-inflammatoryfactors such as IL-10 and IL-1 receptor antagonist (IL-1RA)[17]. However, others have shown that adiponectin mayhave a pro-inflammatory effect through increased produc-tion of NOS2, MMP 3, and MMP 9 [15, 18]. Thisdifference between an anti-inflammatory effect at thevascular level and a pro-inflammatory adiponectin effectin the synovial joint may be related to the concentration ofthe protein present [15].

The serum adiponectin/leptin (A/L) ratio has been shownby others to be an important predictor of inflammatorydiseases suggesting that it represents the balance betweenthe proinflammatory effect of leptin and the anti-inflammatory role of adiponectin. The ratio has been shownto predict the risk of cardiovascular in-stent restenosis [19],intima media thickness of the common carotid artery [16],and it may also serve as an atherogenic index in non-insulin-dependent diabetics, above the individual hormonelevels alone [20].

We asked what the relationship is between adiponectin,leptin, and the A/L ratio and patient-reported pain in anend-stage knee OA joint population. We hypothesized thatthe A/L ratio would predict OA pain greater than theadipokine levels alone.

Material and methods

We recruited 60 consecutive patients with a diagnosis ofend stage knee OA from the Toronto Western Hospital,University of Toronto during the year 2008. The diagnosisof OAwas based on the American College of Rheumatologycriteria [21]. By this definition, patients with knee pain andradiographic osteophytes and at least one of the following

three items meet the criteria for knee OA; age>50 years,morning stiffness≤30 min in duration, or crepitus on motion.All patients were on non-steroidal anti-inflammatory medi-cation (NSAID) or narcotic medication for pain control at thetime of study enrolment. All NSAIDs were discontinued10 days prior to surgery. Patients were consented toparticipate by an independent study nurse not involved inthe medical care of the patients. The study protocol wasapproved by the Human Tissue Review Committee.

Demographic data of age, gender, and BMI werecollected by patient self-report. BMI was defined as bodyweight in kilograms (kg) divided by the square of height inmeters. Medical comorbidity was collected by the 14categories of chronic illness from the Cumulative IllnessRating Scale [22, 23]. This scale encompasses the domainsof (1) cardiac, (2) vascular, (3) hematological, (4) respira-tory, (5) otorhinolaryngological and ophthalmological, (6)upper gastrointestinal, (7) lower gastrointestinal, (8) hepaticand pancreatic, (9) renal, (10) genitourinary, (11) musculo-skeletal and tegumental, (12) neurological, (13) endocrine,metabolic and breast, and (14) psychiatric systems.

Outcomes assessment

The Western Ontario and McMaster Universities ArthritisIndex (WOMAC) pain scale consists of five questions thatassess a patient’s level of pain as it relates to functionalactivities. These activities include walking on a flat surface,going up or down stairs, at night while in bed, sitting or lying,and standing upright. Responses are recorded on a five-pointLikert scale and a greater score represents a greater level ofpain. This scale is very widely used and has been shown to bevalid and reliable in an OA population [24].

The Short Form McGill Pain Questionnaire (MPQ-SF)consists of 15 descriptive terms that fall in the categories ofsensory (e.g., sharp, stabbing), affective (e.g., punishing-cruel), and the evaluative aspects of the pain experience(e.g., tiring-exhausting) [25]. The patient rates their painexperience as it relates to these terms on a four-point Likertscale ranging from none to severe. Patients also rate theirpain on a visual analog scale and also the overall intensityof the pain on a five-point scale ranging from no pain toexcruciating pain [25]. A greater score on the MPQ-SFrepresents more pain.

These two pain measures were chosen as they appear tomeasure different aspects of the pain experience. One groupcompared these two pain scales in 68 knee OA patients andconcluded only a moderate correlation of −0.38 betweenthe WOMAC pain scale and the full length MPQ [26]. TheWOMAC pain scale asks about pain with functionalactivities while the MPQ-SF focuses on the experientialfeatures of the pain. Similarly, in our own work presentlysubmitted for publication, we found a moderate correlation

1224 Clin Rheumatol (2010) 29:1223–1228

(r=−0.36) between the WOMAC pain scale and the MPQ-SF for knee OA. In our practice, we now collect data onboth pain measures as together they capture a morecomplete picture of the OA pain experience.

Venous blood samples and knee joint SF samples werecollected under sterile conditions at the time of joint replace-ment surgery. To ensure consistency, all patients were fastingfor 12-16 h prior to sample collection as it has been shown thatfasting will decrease serum leptin levels and levels are highestafter meals [27]. Similarly, serum leptin levels are known tobe greatest between midnight and early morning which isrelated to its effect on suppressing appetite during sleep [28].All samples in our study were consistently collected during afour hour window between 1,000 and 1,400 h.

Samples were kept out of the light and stored in sterilecontainers on ice or at −800C until analyzed to minimizeinterassay variation. Samples were then centrifuged at3,000×g for 30 min. All samples were analyzed within3 weeks of collection. Samples were analyzed for leptin andadiponectin levels using specific enzyme-linked immuno-sorbent assays (ELISA) and commercially available kits(Linco Research, St. Charles, MO, USA). Our ELISA foradiponectin recognizes all multimeric forms; however, notthe monomeric forms or the globular domain. Leptin andadiponectin concentrations were determined in triplicate foreach sample to ensure accuracy.

The coefficient of variation (CV) for leptin and adiponectinwere calculated as a measure of performance characteristicsfor the immunoassays. For the adiponectin assays, we foundthe following: intra-assay CV serum=10.1%, synovial fluid=9.9%; inter-assay CV serum=2.4%, and synovial fluid=2%.For the leptin assays, we found the following: intra-assay CVserum=5.8%, synovial fluid=14.6%; inter-assay CV serum=2.0%, and synovial fluid=2.2%.

Statistical analysis

Continuous data are reported with means and standarddeviations, while categorical data are reported withfrequencies.

Correlations between leptin, adiponectin, and the A/Lratio with the two pain measures were calculated usingSpearman’s coefficients. The correlation was defined as lowif the coefficient was less than 0.3, moderate if it wasbetween 0.3 and 0.5, and strong if it was greater than 0.5[29]. Considering the correlation matrix, Table 2 representssix statistical comparison; we correct our alpha level to0.01. Similar correlations were calculated between theserum and SF adipokine levels and A/L ratios.

Linear regression modeling was used to evaluate therelationship between our predictor of interest, the A/L ratioand the two pain measures, WOMAC pain and MPQ-SF.The independent variables entered into the models wereage, gender, BMI, and medical comorbidity. BMI was takenas a continuous variable, rather than categorized across thetraditional cut points, as it provides the most informationfor each patient. All variables were retained in the modelswhether reaching statistical significance or not to maintainface validity of the models. We intended to include theindividual adipokine levels in the model in addition to theratio; however, we found substantial collinearity betweenthese variables with tolerance statistics.

Sixty patients was the sample size chosen as weanticipated five independent variables in the regressionmodel, thus allowing for greater than ten outcomes perpredictor variable [30].

All statistical analysis was performed with SPSS Version13.0(Chicago, IL, USA). All reported p values are two-tailed with an alpha of 0.05. Unstandardized beta coef-ficients and 95% confidence intervals are reported for theregression models.

Males (n=24; SD) Females(n=36; SD) p value

Mean age 68.1 (11.6) 68.9 (9.6) 0.82

Mean BMI kg/m2 30.2 (5.8) 32.1 (8.5) 0.44

Serum leptin (ng/ml) 11.7 (6.1) 30.5 (20.5) 0.03

Synovial fluid Leptin(ng/ml) 8.5 (4.6) 22.4 (16.6) 0.03

Serum adiponectin (ng/ml) 18006.7 (14756.1) 25068.1 (18754.1) 0.21

Synovial fluid adiponectin (ng/ml) 4773.9 (2991.1) 5555.9 (3921.5) 0.59

Serum A/L ratio 2617.0 (4239.8) 1491.4 (1545.9) 0.17

SF A/L ratio 758.6 (834.4) 457.6 (610.8) 0.16

Table 1 Demographic data,serum, and synovial fluidadipokine levels comparedacross gender

Table 2 Correlation matrix for pain scores, and synovial fluid (SF)adipokine levels, and adiponectin/leptin (A/L) ratio

MPQ-SF WOMAC pain

SF leptin 0.24 0.20

SF adipon −0.38 −0.33A/L ratio −0.46a −0.38

a Statistically significant correlation (p<0.01)

Clin Rheumatol (2010) 29:1223–1228 1225

Results

In our dataset of 60 patients, we had 24 males (40%) and 36females (60%). The mean age of our cohort was 69.0 years(range 48-86 years, SD 10.1) and the mean BMI was31.4 kg/m2(range 18.6-61.6 kg/m2, SD 6.7; Table 1)

The correlations between the individual adipokines andthe pain scales were consistently less than that for thecorresponding adipokine ratio. The A/L ratio correlatedmoderately with the MPQ-SF, (r(58)=−0.46, p<0.01), andthe WOMAC pain score (r(58)=−0.38, p>0.01; Table 2).The serum-SF leptin and adiponectin correlations were0.98, p<0.01, and 0.55, p<0.01, respectively. The correla-tion between the serum-SF A/L ratio was 0.67, p<0.01.

Linear regression modeling demonstrated that the A/Lratio was a significant predictor of a greater level of pain onthe MPQ-SF (p=0.03, Table 3) but not the WOMAC painscale (p=0.77, Table 4). Models were adjusted for age,gender, BMI, and medical comorbidity.

Discussion

Identification of both the leptin and adiponectin receptorson the surface of human chondrocytes has strengthened therelationship between the metabolic effects of obesity andcartilage degeneration [13, 31]. Early research has sug-gested that leptin may have a proinflammatory role whileadiponectin an anti-inflammatory role in synovial jointdiseases. Our study shows that the A/L ratio predictspatient-reported knee OA pain for the MPQ-SF, but not forthe WOMAC pain scale. This effect is above that for theindividual adipokine levels alone.

The A/L ratio has been evaluated by others and it hasbeen found to better predict the risk of cardiovascular

diseases, such as atherosclerosis, as compared to the individ-ual leptin and adiponectin levels alone [16, 20, 29, 32]. All ofthese studies have examined the serum A/L ratio and itsrelation to chronic disease, while our study is the first toexamine the impact of the corresponding SF ratio on OA.The serum and SF levels of both adipokines have beenshown to correlate with measures of body habitus, such asBMI; however, there is presently no evidence that thesehormones cross the joint capsule to enter the joint [5, 33].

In vitro studies have shown that leptin stimulates IL-1and Interferon-γ production of NO in human chondrocytes[34, 35]. NO regulates many chondrocyte functionsincluding apoptosis and extracellular matrix degradation[36]. The proinflammatory role of leptin in OA has beenfurther supported by the findings that it stimulates releaseof IL-8 from synovial fibroblasts [37] and MMP 13 [38],PGE2, and IL-6 [39] from human chondrocytes. In contrast,adiponectin stimulates the systemic release of anti-inflammatory factors such as IL-10 and IL-1RA [17].Moreover, adiponectin has been shown to increase produc-tion of tissue inhibitor of metalloproteinase-2 and to down-regulate IL-1β-induced MMP-13 levels [40]. The literaturehowever, has also suggested that adiponectin may have apro-inflammatory effect in synovial joints through increasedproduction of NOS2, MMP3, and MMP9 [15, 18]. Thisdifference in effect may be dependent upon the concentrationof the hormone present in the joint [15]. Further work isrequired to clearly elucidate these pathways.

No study to our knowledge, has previously examined therelationship between adipokines and OA pain. One grouphas shown a relationship between an elevated systemicinflammatory state as measured by a greater high sensitiveC-reactive protein (hsCRP) and hip and knee OA pain [41].Further to this, it was shown that a greater systemic hsCRPcorrelates with a greater knee joint level of inflammation

Beta coefficient (95% CI) for predicting MPQ-SF p value

Age 0.2 (−0.2, 0.5) 0.34

Gender 3.7 (−4.1, 11.5) 0.34

Comorbidity 1.0 (−0.9, 2.9) 0.30

BMI −0.2 (−0.6, 0.3) 0.48

Adiponectin/leptin ratio −0.006 (−0.01, −0.001) 0.03

Table 3 Linear regressionmodeling predicting MPQ-SFby age, gender, comorbidity,body mass index, andadiponectin/leptin ratio

Beta coefficient (95% CI) for predicting WOMAC pain scores p value

Age −0.06 (−0.2, 0.2) 0.48

Gender 0.3 (−3.8, 4.4) 0.88

Comorbidity −0.2 (−1.2,0.8) 0.70

BMI −0.06 (−0.6, 0.3) 0.48

Adiponectin/leptin ratio −0.0003 (−0.003, 0.002) 0.77

Table 4 Linear regressionmodeling predicting WOMACpain scores by age, gender,comorbidity, body mass index,and adiponectin/leptin ratio

1226 Clin Rheumatol (2010) 29:1223–1228

(elevated interleukin-6) [42]. Our finding of a greater A/Lratio predicting less pain on the MPQ-SF scale, but not theWOMAC pain scale, suggests that these scales aremeasuring different aspects of the pain experience. Focusgroups have shown that patients report distinctly differenttypes of pain in OA; a dull, nagging, throbbing pain that isconstant in nature and also a sharp, episodic, moreemotionally debilitating pain [43]. The later pain having agreater impact on patient quality of life [43]. Some havesuggested that these differences may be related to distinctnociceptive and neuropathic mechanisms of OA pain [43–45]. Perhaps our two pain scales are sensitive to a differenttype of pain and the A/L ratio is associated with one painpattern more so than another.

Our study has several strengths. First, we have adjustedfor BMI and gender as potential confounders of therelationship between the A/L ratio and patient-reportedpain as both have been shown to have independentrelationships to SF hormone levels [5, 33]. This suggeststhat the relationship we report between the A/L ratio andpatient-reported pain is consistent across males and femalesof all levels of BMI. Second, our study enrolled patientsacross a spectrum of BMI, as a narrow spectrum wouldsubstantially limit the generalizability of our findings.Third, we have used validated patient-reported pain scales.Fourth, we have enrolled an adequate patient number forour regression model.

Our study has potential limitations. First, although webelieve our regression model has face validity, there alwaysexists the possibility of unmeasured confounders of therelationship between the A/L ratio and the OA pain scores.Second, we have not employed a control group with earlyOA that would help strengthen the association betweenthese hormones and cartilage degeneration. Third, we havenot used protease inhibitors in the fluid samples so thepossibility of proteolytic cleavage occurring is possible.However, since all samples were immediately frozenand then analyzed immediately after thawing, webelieve this degradation would have been minimal.Fourth, we have used self-report BMI; however, othershave suggested that it correlates highly with objectivemeasurements [46, 47].

In conclusion, a greater A/L ratio predicted lower kneeOA pain as measured by the MPQ-SF, but not on theWOMAC pain scale. This finding was above that of theindividual adipokine levels alone. Some authors havesuggested that leptin may have a proinflammatory rolewhile adiponectin an anti-inflammatory role in synovialjoint diseases. Further work to elucidate these pathwaysmay present a target for novel therapeutics in knee OA.

Disclosures None.

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