Uncemented hip arthroplasty and denosumab: increased postoperative dipeptide concentrations and identification of potential new bone turnover biomarkers

Abstract

Denosumab is a potent antagonist of RANKL and is widely used to treat severe postmenopausal osteoporosis. Using high-resolution mass spectrometry (HRMS), we aimed to identify molecular mediators associated with the rapid reactivation of osteoclasts following discontinuation of denosumab. In a previously reported randomized controlled trial, 64 patients undergoing uncemented total hip arthroplasty were randomized to receive 2 doses of 60 mg denosumab or placebo, administered 1-3 d and 6 mo postoperatively. Serum samples were analyzed using untargeted HRMS coupled with liquid chromatography, and bone turnover markers were assessed. Data were evaluated using linear mixed-effects models and machine learning techniques. After surgery, 83 metabolite features showed significant concentration changes (p < .0001). Denosumab-treated patients exhibited increased levels of the dipeptides di-L-phenylalanine, phenylalanylleucine, and alpha-Asp-Phe, and decreased levels of fibrinopeptide A and related peptides 24 mo after surgery. The oxidized peptide AP(Ox)GDRGEP(Ox)GPP(Ox)GP, derived from the collagen type I alpha 1 chain (COL1A1) and referred to as COL1A1-OxP, showed a strong correlation with the bone formation marker procollagen type 1 amino-terminal propeptide (P1NP) (p = 4.4E^-83). Similarly, the tripeptide DL-alpha-aspartyl-DL-valyl-DL-proline (DVP) correlated highly with the bone resorption marker carboxy-terminal telopeptide of type 1 collagen (CTX) (p = 1.1E^-222). P1NP and CTX levels were suppressed at 3, 6, and 12 mo postoperatively but exceeded baseline levels by 24 mo. Global metabolic shifts were observed postoperatively, with distinct profiles between treatment groups. The observed increase in specific dipeptides may reflect mechanisms contributing to rebound bone loss following denosumab withdrawal. Fibrinopeptide A and its analogs may play a protective role, while COL1A1-OxP and DVP represent potential new markers of bone turnover. These findings suggest metabolomics-based biomarkers could aid clinical decision-making by allowing earlier detection of rebound effects and guiding individualized treatment strategies after denosumab therapy. Clinical trial registration number: ClinicalTrials.gov, NCT01630941 (URL: https://clinicaltrials.gov/); European Union Clinical Trials Register (EU CTR), EudraCT No. 2011-001481-18 (https://www.clinicaltrialsregister.eu/).

Keywords: bone turnover biomarkers; denosumab; dipeptides; high-resolution mass spectrometry; metabolomics; osteoclast activity; periprosthetic bone loss; rebound bone loss; total hip arthroplasty.

Graphical Abstract

Figure 1

PCA plot based on 1552 metabolites and all time points except T2. Left: PCA plot based on 1552 metabolites (excluding T2) illustrates the distribution of samples over time, with colors indicating time points and shapes representing treatment groups (denosumab or placebo). Right: subplots (A–D) show selected PCs (PC1, PC2, PC4, and PC6) that revealed significant differences between time points in each treatment group relative to baseline (T1). Asterisks denote statistically significant differences (p < 0.05), and error bars represent standard errors. Time points: T1 (baseline), T3 (3 mo), T4 (6 mo), T5 (12 mo), and T6 (24 mo). The second dose of denosumab was administered at 6 mo.

Figure 2

Hierarchical clustering of the significantly affected metabolites. Heatmap displaying log₂ fold changes of 83 significantly affected metabolites, stratified by time and treatment comparisons. Clustering highlights metabolite dynamics within placebo and denosumab groups over time and between-group comparisons (denosumab vs placebo), with and without baseline correction. Asterisks indicate statistical significance (p < 0.05). Metabolite IDs correspond to Table 1 for compound identity and class. Time points: T1 (baseline), T3 (3 mo), T4 (6 mo), T5 (12 mo), and T6 (24 mo). The second dose of denosumab was administered at 6 mo.

Figure 3

Temporal changes in serum dipeptide levels following surgery. Boxplots showing serum concentrations of 4 dipeptides: (A, B) Di-L-phenylalanine (including sodium adduct), (C) phenylalanylleucine, and (D) alpha-Asp-Phe. In denosumab-treated patients, all dipeptides increased significantly over time, starting at 6 mo postoperatively. Asterisks mark statistically significant differences (p < 0.05); exact p-values are indicated when p < 0.1. Time points: T1 (baseline), T3 (3 mo), T4 (6 mo), T5 (12 mo), and T6 (24 mo). The second dose of denosumab was administered at 6 mo.

Figure 4

Trends in bone formation marker P1NP and COL1A1-OxP peptide. Boxplots depicting serum levels of P1NP and the oxidized peptide AP(ox)GDRGEP(ox)GPP(ox)GP (COL1A1-OxP), derived from the collagen type I alpha 1 chain. Both markers showed sustained suppression in denosumab-treated patients up to 12 mo, with a trend toward rebound by 24 mo. Asterisks denote p < 0.05; p < 0.1 values are shown explicitly. Time points: T1 (baseline), T3 (3 mo), T4 (6 mo), T5 (12 mo), and T6 (24 mo). The second dose of denosumab was administered at 6 mo.

Figure 5

Trends in bone resorption marker CTX and tripeptide DVP. Boxplots illustrating serum levels of CTX (a marker of bone resorption) and DL-alpha-aspartyl-DL-valyl-DL-proline (DVP), a newly identified tripeptide. Both markers remained suppressed in the denosumab group until 12 mo, followed by a notable increase at 24 mo. Asterisks represent statistically significant changes (p < 0.05), and exact p-values are noted for trends (p < 0.1). Time points: T1 (baseline), T3 (3 mo), T4 (6 mo), T5 (12 mo), and T6 (24 mo). The second dose of denosumab was administered at 6 mo.