A LC-MS/MS method to simultaneously profile 14 free monosaccharides in biofluids.

Monosaccharides are important players in cell metabolism and potential biomarkers. An effective tool to quantify monosaccharides is required in basic research and healthcare. In this study, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that could simultaneously quantify 14 free monosaccharides and evaluated its performance according to clinical guidance. The LC-MS/MS step separated and quantified 14 monosaccharides with 6 min. The coefficient of variation at the lower limit of quantification was less than 20% for each analyte. The R square values from linear regression analyses were all greater than 0.995. The validated assay was employed to profile free monosaccharides in conditioned media from cell culture and patient sera from glucose tolerance test. Both LO2 cells and HEK293 cells consumed D-glucosamine, D-glucose and produced D-glucuronic acid, N-acetyl-D-glucosamine. Additionally, LO2 cells produced D-mannose and L-fucose, whereas HEK293 cells consumed D-mannose. In patient sera from glucose tolerance test, the level of D-glucose increased significantly after glucose intake, but the levels of other monosaccharides didn't. In conclusion, the LC-MS/MS assay we developed for 14-monosaccharide profiling met clinical requirements. The monosaccharide profiling results showed the distinct monosaccharide metabolism between liver and kidney cells, and the ignorable diet effect on 6 serum monosaccharides.

Development and validation of a prediction model for glucocorticoid-associated osteonecrosis of the femoral head by targeted sequencing.

OBJECTIVE: To develop and validate a prediction model based on targeted sequencing for glucocorticoid (GC)-associated osteonecrosis of the femoral head (GA-ONFH) in GC-treated adults. METHODS: This two-centre retrospective study was conducted between July 2015 and April 2019 at Zhongshan Hospital (training set) and the Sixth People's Hospital (test set) in Shanghai, China. All patients had a history of GC therapy, with a dose exceeding 2000 mg equivalent prednisone within 6 weeks. Patients were divided into two groups according to whether they were diagnosed with GA-ONFH within 2 years after GC initiation. Blood or saliva samples were collected for targeted sequencing of 358 single nucleotide polymorphisms and genetic risk score (GRS) calculating for developing GA-ONFH prediction model. Receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA) were performed to evaluate and validate the model. RESULTS: . The training set comprised 117 patients, while the test set comprised 30 patients for external validation. Logistic regression analysis showed that GRS was significantly associated with GA-ONFH (OR 1.87, 95% CI: 1.48, 2.37). The ROC and DCA curves showed that the multivariate model considering GRS, age at GC initial, sex and underlying diseases had a discrimination with area under the ROC curve (AUC) of 0.98 (95% CI: 0.96, 1.00). This model was further externally validated using the test set with an AUC of 0.91 (95% CI: 0.81, 1.00). CONCLUSION: Our prediction model comprising GRS, age, sex and underlying diseases yields valid predictions of GA-ONFH incidence. It may facilitate effective screening and prevention strategies of GA-ONFH.