A novel visceral adiposity index predicting bone loss in female early rheumatoid arthritis patients: A 1-year HR-pQCT follow-up study

White adipose tissue is considered as an important endocrine organ and regulates metabolism. We assessed the relationship between Chinese visceral adiposity index (CVAI) and the bone microstructure in early rheumatoid arthritis (ERA) patients. The relationship may be existed between bone loss and visceral dysfunction of fat metabolism in female early rheumatoid arthritis patients. Methods 104 female ERA and 100 age-, gender- and BMI-matched healthy controls were recruited for the comparison of CVAI. All ERA patients were prospectively followed for 1 year. HR-pQCT scan of the distal radius, tibia and second metacarpal head were performed at baseline and after one-year. ERA patients were divided into two sub-groups according to the median CVAI value.

activation, articular in ammation, invasion of synovium to adjacent bone and cartilage resulting in bone erosion (1). Excessive production of pro-in ammatory cytokines trigger bone resorption by stimulating osteoclasts directly and by inhibiting osteoblast function, resulting in systemic bone loss manifested as osteoporosis diagnosed by a low bone mineral density (BMD) measured using dual-energy x-ray absorptiometry (DXA) (2). Using high-resolution peripheral quantitative computed tomography (HR-pQCT), our previous studies showed that intra-articular bone loss at the metacarpal head and periarticular bone loss at the distal radius and tibia have also been demonstrated in these patients (3,4).
Obesity has been traditionally considered to be protective against osteoporosis and fracture. Adipose tissue is no longer thought of as solely a triglyceride storage compartment and neighboring tissue insulator, but rather as an active endocrine organ with multiple functions (5). White adipose tissue (WAT) is recognized as an important endocrine organ and regulates metabolism by affecting other organs, such as bone and immune system (6). Visceral adipose tissue (VAT) acts as an active as well as metabolic organ and secretes in ammatory matters (7) and adipokines (8), and plays a critical role in metabolizing steroid hormones (9) that affect skeletal metabolism. VAT has been related to a low BMD and been a signi cant risk factor for some types of fractures (10). Computed tomography (CT) and magnetic resonance imaging (MRI) are applied for quantitatively measuring the VAT, but these imaging techniques are expensive and not routinely assessed (10). Recently, a clinical visceral adiposity index (VAI) calculated from waist circumference (WC), body mass index (BMI), serum triglycerides (TG), and highdensity lipoprotein (HDL) levels, was performed to quantitatively assess the visceral adiposity. VAI was signi cantly associated with a number of metabolic diseases, such as cardiovascular and cerebrovascular disease, metabolic fatty liver disease (MAFLD)/non-alcoholic steatohepatitis (NASH), polycystic ovary syndrome in western countries. However, striking differences in terms of body fat allocation exist between different ethnicities. Asians are featured by a comparatively higher body fat content at lower BMI variables comparing to western population and are more prone to visceral fat accumulation (11). Chinese visceral adiposity index (CVAI) is a clinical con rmed index for the assessment of visceral fat dysfunction. In a study involving 6495 Chinese subjects, CVAI was related to visceral obesity and insulin resistance; and was able to discriminate patients with and without metabolic syndrome, hypertension, diabetes and prediabetes better than BMI and WC (12).
In the general population, a recent HR-pQCT study demonstrated that higher VAT was associated with greater volumetric BMD (vBMD) and better microstructure of the peripheral skeleton despite observation on deleterious changes in the cortical compartment of the non-weight bearing radius (10). Associations were no longer signi cant after adjustment for BMI or weight, suggesting that VAT might not have a substantial effect on the skeleton metabolism independent of BMI or mechanical loading (10). Nonetheless, in patients with RA, moderate in ammatory activity was associated with greater visceral adipose tissue (13). Whether visceral adiposity measured using CVAI in patients with RA may predict accelerated intra-articular and periarticular bone loss in patients remains for our exploration.

Participants
This was one of a group of studies, open-labelled, randomized controlled trials (RCT) in Hong Kong applying for exploring the role of two intense control treatment approaches on vascular stiffness in early RA patients (clinicaltrial.gov no: NCT01768923) (14). One hundred and four female ERA patients in this current study were chosen from this RCT. This study mainly focused on the association between CVAI and bone microstructure detected by HR-pQCT. In other words, 104 consecutive ambulatory Chinese female ERA patients with symptom duration below 24 months were enrolled from the department of rheumatology in the Prince of Wales Hospital and other hospitals in Hong Kong. Participants were recruited once they ful lled the American College of Rheumatology (ACR)/ EULAR revised criteria for RA (2010 version) (15). Participants were excluded if they: 1) had a lower creatinine clearance (<30 ml/min) or cancer treated by chemistry or radiology; 2) were on antiosteoporosis medication, including oral bisphosphonates, teriparatide, double acting bone preparation, hormone replacement therapy, or RANKL inhibitor or bDMARDs; 3) had critical deformities at the metacarpophalangeal joints which may prevent a credible HR-pQCT scan leading to the motion artefacts with inaccurate scan; 4) were pregnant or lactation. All patients were given medical strategies based on a standard method with the purpose of the remission for 1 year (16). One hundred age-, BMI-and gender-matched apparently healthy female volunteers without rheumatic diseases and bone diseases were enrolled in the ward of mouth recommendation department at the Prince of Wales hospital. The study was launched following the

HR-pQCT imaging and image evaluation
The methodology information has been reported before (17,18). In other words, distal radius and tibia in the non-dominant side of the patients, and the second metacarpal head (MC2) of the non-dominant hand of the patients were explored by the rst-generation HR-pQCT (XtremeCT I, Scanco Medical AG, Bruttisellen, Switzerland) at baseline and 12 months (18). HR-pQCT was operated by an experienced practitioner (VH) who was not aware of any clinical data of the patients. All the images were assessed by one professional performer who evaluated motion artifact followed by a mature system published by Pialat and his colleagues, which graded images based on different situation named grade 1 to grade 5 (19). Images were assessed as grade 1 or 2 if there was no or minor motion artifact. If there was severe or extreme motion artifact in these participants, they would be excluded from further study and be considered to unsuccessful scan.
The whole interested volume was divided into cortical and trabecular components by applying a completely automated strategy named cortical compartment segmentation methodology with good short-term and long-term reproducibility (20,21).

Clinical interview, anthropometrical and serum laboratory measurements
Clinical parameters assessed included the extra-articular manifestations, tender and swollen joint count, damaged joints' number, and the modi ed health assessment questionnaire of those patients (HAQ). Previous medical strategies were inquired and recorded when patient was interviewed (22). Standardized medical data collection documents was applied when these patients were interviewed and explored.
Body height and weight were evaluated without shoes and outer clothing. BMI was explored following the formula: weight in kilograms divided by square of height in meters. WC was assessed by using a soft tape at midway between the lowest rib and iliac crest in standardized standing situation.
All the blood samples were taken at fasting status for at least 10 hours. Rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (ACCP) were evaluated at baseline. Other tests including complete blood count, plasma fasting blood glucose, serum total cholesterol, HDL-c, TG, low-density lipoproteincholesterol (LDL-c), erythrocyte sedimentation rate (ESR), C-response protein (CRP), liver and renal function was assessed every 3 months.

Statistical analysis
All statistical analyses were conducted by applying SPSS Statistics Version 22 (SPSS, Chicago, IL). The data were expressed as mean ± Standard Deviation (SD), except for skewed variables, which were shown by median and interquartile range (25-75%) given in parentheses. For continuous parameters, the differences between both groups were calculated by either Mann-Whitney U test or Student's t test, whereas the Chi-squared test was used for comparisons in terms of categorical parameters between two groups. The relationships between levels of CVAI and variables of cortical/trabecular bone density, microstructure, and parameters associated with disease activity/severity were explored by applying Spearman correlation. Linear or logistic regression analysis was conducted to evaluate the relationship between CVAI at baseline and bone microstructure (at distal radius, distal tibia and MC2) after adjusting for other confounding variables at month 12 as well as the change over a period of 12 months. Potential explanatory variables included in the regression analyses were shown in Tables 1& 2. Parameters with a P value of <0.1 in the univariate analysis were recruited in the multivariate analysis. For all the analyses, a two tailed p ≤0.05 was recognized as indicating statistical signi cance.

Results
Characteristics of participants in this study Table 1 demonstrated the characteristics of the patients (at baseline) and controls. ERA patients and healthy controls were comparable in terms of age, BMI, fasting blood glucose (FBG), TG, and HDL-c levels, VAI and the prevalence of overweight/obesity and visceral obesity. Nevertheless, WC, CVAI, TC, LDL-c, ESR and CRP levels were signi cantly higher in ERA patients compared to healthy controls (p<0.05, Table 1).
The association between CVAI, metabolic pro le and clinical parameters of RA patients at baseline HR-pQCT parameters in the two CVAI subgroups ERA patients were then divided into two subgroups according to the median of CVAI (65.73): low CVAI group (<65.73) and high CVAI group (≥65.73). With regards to the clinical features, some markers of disease severity (including the number of damaged joints, ESR level, RF titer and HAQ) were higher in the high CVAI group than the low CVAI group (all p<0.05, Table 2). CRP, disease duration, ACCP titer, tender joint count, swollen joint count, DAS28-CRP, SDAI score and patient VAS global assessment were similar between the two groups (p>0.05, Table 2).
At baseline, cortical porosity diameter at the MCP2 was larger while the cortical vBMD at the tibia and distal radius were signi cantly lower in the high CVAI group compared to the low CVAI group (all p<0.05, Supplementary Tables 2-4). No signi cant differences existed between the two groups in terms of trabecular bone density, trabecular thickness (Tb. Th), cortical thickness (C.Th) and trabecular bone volume fraction (all p>0.05, Supplementary Tables 2-4).
The changes in cortical vBMD at the distal radius and trabecular vBMD at the tibia after 12 Table 5).
In terms of changes in vBMD and microstructure over the period of 12 months, CVAI at baseline was also an independent predictor of the change in trabecular vBMD (tibia: B=0.444, p=0.001, 95% CI: 0.018~0.063; distal radius: B=0.356, p=0.008, 95% CI: 0.403~0.063) ( Table 4). There was no signi cant association between baseline CVAI and HR-pQCT parameters at MCP 2 (at month 12 or changes over the period of 12 months) (data not shown).

Discussions
To the best of our knowledge, this study is a prospective study to explore the associations between visceral dysfunction of fat metabolism (CVAI) and bone microstructure assessed using HR-pQCT in early RA patients. We demonstrated that CVAI was signi cantly higher in female ERA patients compared with age-and BMI-matched healthy controls, which was associated with several disease severity parameters, indicating that adipose tissue dysfunction may be related to in ammation in RA patients. This nding concurs with previous report suggesting that obesity is associated with higher CRP levels and ESR in women with RA. This association is related to fat mass and not RA disease activity (25).
Several studies have demonstrated that VAT rather than subcutaneous adipose tissue plays a crucial role in metabolic/in ammation diseases (26,27). Although the clinical index VAI was signi cantly related to metabolic diseases (12), our data did not show any statistical differences in VAI between the ERA group and healthy controls (p>0.05), which suggests that this parameter may not be able to re ect visceral fat dysfunction in these patients. The underlying reason may be related to ethnic variations in body fat distribution (28), as Asians are characterized by relatively higher body adipose tissue content at a lower BMI comparing with Caucasians, and are more likely to accumulate visceral fat (11).
In contrast, CVAI was signi cantly higher in ERA patients comparing to age-and BMI-matched controls.
Observational studies have identi ed obesity as a risk factor and a poor prognostic marker of RA, and adipose tissue as a possible source of in ammation (29). These ndings are in line with our results showing that higher in ammatory burden, i.e. re ected by the higher ESR level, RF titer and damaged joint count, were observed in the high CVAI group compared to the low CVAI group, indicating that fat dysfunction may contribute towards the pathophysiology of in ammation in RA. Adipose tissue has been implicated in the development of a low-grade in ammatory state in in ammatory conditions including RA by the production of pro-in ammatory adipocytokines (29). Dysregulation of immune-endocrine circuits involved in the progression of chronic metabolic disorders (such as obesity) also plays a critical role in chronic in ammatory diseases such as RA (30). Cytokines secreted by an excess of adipose tissue could have an effect on the whole body, resulting in the progression of a named low-level in ammation, which can be found in obese individuals (31). Chronic in ammation caused by the adipocytokines may contributes towards irreversible damage of cartilage and bone (32). Our data provided preliminary data supporting the relationship between visceral adipose tissue dysfunction and deterioration in bone microstructure, including its index vBMD. It is reported that serum and synovial uid levels of the adipokine fatty acid-binding protein (FABP4) are much higher in patients with RA compared with osteoarthritis (OA) (33). Levels of FABP4 were positively associated with BMI in patients with RA but did not correlate with disease activity and, until now, there is no evidence that FABP4 affects bone remodeling in RA (32). Whether FABP4 may be involved in the association between adipose tissue and bone loss in ERA still needs further investigation.
In our study, there was a correlation between CVAI and damaged joint count, increased cortical porosity diameter at the MCP2, as well as a lower cortical vBMD (at the distal tibia at baseline, and at the distal radius/tibia at month 12), suggesting that adipose tissue may adversely affect the bone microstructure. Indeed, preadipocyte differentiation is a complicated procedure depending on hormonal and nutrient adequacy, which involves activation of various transcription factors (34). Interestingly, adipocytes share the same stem cell precursors with osteoblasts and chondrocytes (35,36) and under certain conditions their precursors can differentiate to osteoblasts and macrophages (35,37). Studies show that adipocytes secrete adipokines, cytokines, chemokines and complement factors, which may be involved in the bone metabolism of RA. Adiponectin, a well-studied adipokine, can increase the capacity of osteoblasts to mineralize bone and increase the expression of osteoblast-related genes during osteogenic differentiation, via phosphorylation of AMP-activated protein kinase (AMPK) pathway (38), suggesting a pro-osteogenic effect of this adipokine during bone remodeling. Leptin, another adipokine, may induce higher expression of vascular cell adhesion protein 1 (VCAM-1) in primary human chondrocytes mediated by Janus kinase 2 (JAK2) and phosphatidylinositol 3-kinase (PI3K) signaling (39). These central signaling pathways contribute to cellular activation and in ammatory processes. ADAMTS4, ADAMTS5 and ADAMTS9 expression could be induced by leptin in human chondrocytes via the activation of proin ammatory signaling pathways involving mitogen-activated protein kinase (MAPK) and NFκB (40). Nonetheless, the relationship between CVAI, adipocytokine (adiponectin and leptin et al) and bone microstructure would need further studies in ERA patients.
Our study has several limitations. First, although our study sample size was su cient to meet the end points by using G-power analysis, this cohort study was still relatively small, consisting of convenience sample of female, and thus the results could have been in uenced by selection bias. Large sample size and male should be enrolled in the future and con rm our results. Second, we did not measure the adipocytokine in our study, which refers to all factors produced by adipocytes, including adipokines, cytokines, chemokines and complement factors. Therefore, we did not know the relationship between adipocytokine and bone microstructure, and we may explore its association in the future study. Third, due to the study, we may not indicate the cause and effect of CVAI on bone microstructure among all the ERA patients. Fourth, because all the patients in this study were Southern Chinese, our results may not be extrapolated in other ethnicities. Last but not the least, we did not measure the sexual hormones in female ERA patients, and not sure whether they may also be an important bio-factor contributing to the association between CVAI and bone microstructure.

Conclusion
The current clinical follow-up study suggests that CVAI is an independent predictor of trabecular bone deterioration in female ERA patients.