Mass Spectrometry Reveals Molecular Effects of Citrulline Supplementation during Bone Fracture Healing in a Rat Model

Abstract

Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (μCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. μCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with μCT.

Figure 1

Schematic overview of citrulline–arginine–nitric oxide metabolism. The important molecules in fracture healing are marked orange. Abbreviations: Arg1 = arginase 1; ASS = argininosuccinate synthetase; ASL = argininosuccinate lyase; NOS = nitric oxide synthase; ODC = ornithine decarboxylase; OTC = ornithine transcarbamylase.

Figure 2

Overview of the analytical workflow combining the analysis of lipid distributions using MALDI-MSI and proteins using LC-MS/MS. Small pieces of bone were used for lipid distribution analysis. These pieces were embedded and sectioned during sample preparation. Matrix was sprayed onto the section before MALDI-MSI analysis. Crushed bone tissue was used for the protein analysis. Proteins were extracted from the crushed bone tissue and subsequently digested using enzymes. The resulting peptides were analyzed with LC-MS/MS.

Figure 3

Comparison of (a) the fracture callus volume and (b) the fracture callus volume fraction for the citrulline supplementation (Citr) and control (Cont) group for the different time points (3, 7, 14, and 28 DPO). Abbreviations: MVI = mean voxel intensity.

Figure 4

First discriminant function (DF1) for the lipid comparison of the citrulline supplementation (Citr) and control (Cont) group per time point for bone representing the results of the PCA-LDA analyses. DF1 plots are shown for (a) 3, (b) 7, (c) 14, and (d) 28 DPO representing 2.20, 1.47, 1.94, and 1.61% of the variance in the data set, respectively. The DF plots display how well the Citr and Cont groups can be separated based on the lipid profiles acquired from bone, showing the effect of citrulline supplementation on the lipid profiles.

Figure 5

First discriminant function (DF1) for the lipid comparison of the citrulline supplementation (Citr) and control (Cont) group per time point for bone marrow representing the results of the PCA-LDA analyses. DF1 plots are shown for (a) 3, (b) 7, (c) 14, and (d) 28 DPO representing 2.69, 3.46, 3.53, and 2.75% of the variance in the data set, respectively. The DF plots display how well the Citr and Cont groups can be separated based on the lipid profiles acquired from bone marrow, showing the effect of citrulline supplementation on the lipid profiles.

Figure 6

Selected identified pathways related to bone fracture healing for the citrulline supplementation (Citr) and control (Cont) group for 3, 7, 14, and 28 DPO with the related p-value in parentheses. Abbreviations: BMP = bone morphogenetic protein; ECM = extracellular matrix; eNOS = endothelial nitric oxide synthase = NOS3; ERBB4= erb-b2 receptor tyrosine kinase 4; IGF = insulin-like growth factor; IL = interleukin; MMP = matrix metalloproteinase; NFE2L = nuclear factor erythroid 2-related factor; NF-κB = nuclear factor kappa B; NOTCH = neurogenic locus notch homolog; NTRK = neurotrophic receptor tyrosine kinase; ODC = ornithine decarboxylase; PA = glycerophosphates; RAGE = receptor for advanced glycation end-products; RHO GTPases = RAS homolog family member of nucleotide guanosine triphosphate-ases; RUNX = runt-related transcription factor.