Serum levels of sclerostin reflect altered bone microarchitecture in patients with hepatic cirrhosis

In patients with hepatic cirrhosis areal bone mineral density (aBMD) is decreased especially at the lumbar spine [1, 2], whereas either decreased [3] or normal values [1, 2, 4] are observed at the hips. Due to technical limitations, aBMD alone can be insufficient to explain increased fracture risk [5] and bone microarchitecture can provide additional information. We recently investigated bone microarchitecture via high resolution peripheral quantitative computed tomography (HR-pQCT) [3]. As HR-pQCT is technically intensive, expensive and rarely available, serum bone turnover markers (BTMs) should be tested for predictive value. As standard serum markers of bone turnover do not reflect bone microarchitecture in hepatic cirrhosis [3], new markers need to be examined. Sclerostin, a glycoprotein secreted mainly by osteocytes, is a product of the SOST gene. It negatively regulates bone mass via binding to low-density lipoprotein receptor-related protein 5 and/or 6 (LRP5/6) and inhibits the Wnt/beta-catenin pathway, thereby decreasing bone formation [6]. To date, the only study investigating serum sclerostin in hepatic cirrhosis reported increased levels [7]. In patients at increased fracture risk, the results are divergent: Whereas some studies reported higher levels in postmenopausal osteoporotic women with fractures [8], diabetics [9] and alcoholics [10], others observed lower levels in geriatric patients with hip fractures [11]. These differences may result from methodological differences (fragments biasing measurements, different antibodies and therefore epitopes recognized, different assay constructions) [12].

A novel sclerostin ELISA was recently developed and launched (BI-20472, Biomedica, Vienna, Austria), which measures bioactive sclerostin by using a monoclonal antibody directed at the LRP5/6 binding region, thereby capturing all circulating sclerostin forms containing the free-receptor binding site. To ensure the reliability of this ELISA, it was validated in depth according to Food and Drug Administration quality standards. Therefore, the aforementioned limitations could be reduced, and the measured analyte specified.

This study shows validation data of the used ELISA and evaluated sclerostin in patients with hepatic cirrhosis, compared to matched healthy controls. The secondary objectives were first to test for correlations of sclerostin with trabecular and cortical bone microarchitecture and second, to examine relationships with serum markers of bone turnover.

In this study 32 patients (including 12 women) and 32 matched healthy controls (including 12 women) were included. None of the subjects had received a specific treatment for osteoporosis or chronic liver disease. The patients and controls were of similar age (median 62 and 60 years, respectively), 16 patients had alcoholic liver disease (ALD), 8 viral hepatitis, 5 non-alcoholic fatty liver disease, 2 hemochromatosis and 1 autoimmune hepatitis. Age and body mass index did not differ between the patients and controls but alcohol intake was higher in the patients (4 units/day vs. 1 unit/day). None of the patients with ALD were abstinent.

Patients with ALD (63 years) and other etiologies (62 years) were of similar age. The percentages of males were similar for patients with ALD (69%) and controls (63%) but slightly lower for patients with other etiologies (56%). The Child-Pugh score was similar among the patients with ALD (5) and other etiologies (6).

This study assessed the quality of sclerostin to explain bone microarchitecture in hepatic cirrhosis. We observed slightly lower sclerostin levels compared to controls. To date, a single previous study [7] has observed increased levels; however, neither BTMs, aBMD nor microarchitecture were assessed. Therefore, in our patients, aBMD could be lower due to advanced age. Moreover, we investigated Caucasians, whereas Rhee et al. [7] examined Koreans. Furthermore, most of their patients had viral hepatitis, whereas more than 50% of our patients had ALD. In a study on patients with primary biliary cholangitis (PBC), local expression of sclerostin in the bile ducts was reported, especially in early stages of the disease but declined in advanced disease stages [13]. Therefore, we assume that in our patients only a minor fraction of serum sclerostin is derived from hepatic production. Finally, within the characterization and validation of the ELISA used in our study we could show that it uniquely detects sclerostin molecules containing the LRP5/6 interaction site and therefore assumed to have bioactive function. As it is not completely understood which sclerostin molecules circulate (monomers vs. dimers [14], intact vs. fragments [15]), it is even more important to further characterize binding sites of utilized antibodies and therefore specify the detected analyte.

Within the technical validation of specificity, accuracy, intra-assay precision, limit of detection, lower limit of quantification and sample parallelism, we could show that the novel ELISA completely meets all quality standards comparable with other sclerostin assays on the market [16] but in contrast to those assays, the novel ELISA used in this study has clearly defined binding sites.

Sclerostin correlated with the MELD score, but neither the Child-Pugh nor the MELD scores improved the predictive value of sclerostin (data not shown). Albumin improved the R² value of sclerostin for microarchitecture. These findings emphasize the presence of a liver-bone axis in terms of a strong relation between liver function and bone health. Especially albumin (synthetic function) together with sclerostin explained deranged bone structure in our patients. Similarly, in a large population-based outpatient study, lower albumin was independently associated with osteoporosis [17]. Rhee et al. [7] found higher sclerostin levels in advanced disease states and suggested lower hepatic clearance and altered concentrations of sex hormones to explain these findings. Similarly, patients with advanced ALD had higher sclerostin levels than in less pronounced states [10]. In contrast, those of our patients with ALD had significantly lower sclerostin levels, probably as alcohol promotes osteocyte apoptosis [18]. Moreover, maybe more sclerostin fragments are circulating and detected with their assay but not with our ELISA. Adjusted calcium improved R² especially for trabecular microarchitecture, probably due to the correction for low albumin. Moreover, adjusted calcium was decreased in patients in whom we also observed deranged microarchitecture [3]; however, albumin and adjusted calcium did not correlate with sclerostin. Rhee et al. [7] did not test for relations with BTMs. Our patients’ sclerostin levels correlated with aBMD and microarchitecture. Similarly, in older men serum sclerostin [19] is related to microarchitecture and higher fracture risk. Lower serum sclerostin [11] and lower femoral sclerostin expression correlated with impaired trabecular microarchitecture in patients with hip fractures [20].

Sclerostin is produced predominantly by osteocytes. As low sclerostin expression is related to low bone volume and number of osteocytes per volume [21], lower trabecular bone volume and/or increased osteocyte apoptosis could explain the lower sclerostin levels in our patients. Alcoholism promotes osteocyte apoptosis, as demonstrated in a rat model [22]. In addition, in bone samples of patients with hepatic cirrhosis of various etiologies, a decreased number of osteocytes was reported [23]; however, bone histomorphometry was not available in our patients and therefore, further studies are required to confirm our assumption. In contrast, sclerostin levels could be decreased due to a rescue mechanism. A study on male idiopathic osteoporosis reported that lower sclerostin expression could reflect an autoregulatory promotion of bone formation [24]. As osteoblast activity is reduced in hepatic cirrhosis [25], this mechanism could act as an attempt to preserve osteogenesis. The relatively small sample size is a limitation of our study. The possibility exists that in a higher number of subjects some differences in serum sclerostin concentrations could become significant. In conclusion, serum sclerostin levels reflect deterioration of bone microarchitecture and osteocyte function in patients with hepatic cirrhosis. Patients with ALD had significantly lower sclerostin levels compared to other etiologies.