The Metabolomics Study Of Osteonecrosis Of The Femoral Head Based On UPLC-MS/MS

Background:Osteonecrosis of the femoral head(ONFH) is a potential is a disease characterized by disabling. The incidence is high in the young, thus, burden the society in economy. Nowadays, the diagnosis of osteonecrosis of the femoral head depends on symptom, physical examination, X-ray, nuclear magnetic resonance image(NMR). The NMR is helpful in early diagnosis, however, the large expense and easily being disturbed refine the wide use of NMR. The early diagnosis is still difficult in ONFH. The study of biomarkers of ONFH is good for the early diagnosis and for the understanding this disease. Metabolomics is a newly-developing technology, the use of this technology is benefit for finding the biomarkers and understanding the mechanism of ONFH. The technology has been used in some common orthopedic disease, such as, rheumatoid arthritis, spondyloarthritis, osteoarthritis, etc. However, the metabolomics technology has not been applied in ONFH, so, we employed the technology to research the metabolism character and figure out the biomarkers in ONFH.Objective:Osteonecrosis of the femoral head(ONFH) frequently affect the young people. It is characterised by difficulty of early diagnosis. Our study aimed to research metabolites characters and identify biomarkers candidates of osteonecrosis of the femoral head using metabonomic technology.Methods:Twenty-eight necrotic bone trabecula and twenty-eight adjacent normal bone trabecula from the ONFH patients and twenty normal femoral heads from femoral neck fracture patients were collected for histopathological examination to confirm the diagnosis of all samples. To research the influence of clinical traits to the metabolism of ONFH, we analyzed the difference of clinical feature and its affection on the metabolite distinction. The reproducibility of the system was assessed by PCA(principal component analysis) of the QC(quality control) group. Then, all the metabolites of the bone trabecula were extracted by solvent. PCA(principal component analysis), PLS-DA(partial least squares-discriminant analysis), OPLS-DA(orthogonal-partial least squares-discriminant analysis) models were employed to confirm the difference between these two groups after UPLC-MS/MS(ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry) analysis. At last, the distinctive varies were assured by multivariate analysis and variate analysis. Sixty-seven metabolites were identified by m/z,retention time and MS/MS aligned in HMDB(human metabolomic database) and Massbank.Results:Obvious necrosis of the bone and bone cells can be detected in the slices of ONFH, while the bone and cells in adjacent normal bone trabecula and fructure of femoral neck were normal. The QCs analysis reflected the good stability of the platform of UPLC-MS/MS. Significant distinction can be seen in PCA, PLS-DA, OPLS-DA models scorinng plots between the ANBT, ONBT and control groups varies. 67 changing metabolites in ONFH bone trabeculas were confirmed by multi-variate statistical analysis and variate statistical analysis. Both osteonecrotic bone trabecula(ONBT) and adjacent ‘‘normal’’ bone trabecula(ANBT) showed higher levels of amino acids, such as proline, arginine, glutamine, dipeptides and lower levels of antioxidants. Most disrupted lipids, such as fatty acid esters, glycerophospholipids, sphingolipids, were found in osteonecrotic zone. The area under the receiver operating characteristic curve of combinational biomarkers(D-arginine, L-proline, L-carnitine, inosine) in ONBT and ANBT was 0.996 and 0.950, respectively.Conclusion:The metabolites of ONFH and control bone trabeculas were successfully extracted by solvent to be analyzed by UPLC-MS/MS. PCA, PLS-DA, OPLS-DA models were built and OPLS-DA model were used to find out the differentiated variables. 67 metabolites were identified by MS/MS and 4 obviously changed metabolites, D-arginine, L-proline, L-carnitine, inosine, were indicated as biomarker candidates. Our work supplies the base of further researching biomarkers of ONFH. However, the sample size is small, so, a experiment with a lager sample size is good for further confirm the disease biomarkers.