Measurement of glucose and fructose in clinical samples using gas chromatography/mass spectrometry

Objective:The impact of increased fructose consumption on carbohydrate metabolism is a topic of current interest, but determination of serum level has been hindered due to low concentration and interference from serum glucose. We are reporting a method for the quantification of glucose and fructose in clinical samples using gas chromatography/mass spectrometry (GC/MS). The accuracy and precision of GC/MS and an enzymatic assay were compared.Design and methods:Mass spectrometry fragmentation patterns of methyloxime peracetate derivatized aldose and ketose were determined. Unique fragments for glucose and fructose were used for quantitative analysis using isotope labeled recovery standards.Results:Methyloxime peracetate derivatives of glucose and fructose showed characteristic loss of acetate (M-60) or ketene (M-42) under chemical ionization (CI). Under electron impact (EI) ionization, a unique C1–C2 fragment of glucose was formed, while a C1–C3 fragment was formed from keto-hexoses. These unique fragments were used in the quantitative assay of glucose and fructose in clinical samples. In clinical samples, the GC/MS assay has a lower limit of detection than that of the enzymatic assay. In plasma samples from patients evaluated for diabetes the average serum glucose and fructose were 6.19 ± 2.72 mM and 46 ±  25.22 μM. Fructose concentrations in many of these samples were below the limit of detection of the enzymatic method.Conclusion:Derivatization of aldose and ketose monosaccharides to their respective O-methyloxime acetates for GC/MS analysis is a facile method for determination of serum/plasma glucose and fructose samples.     

The molecular mechanisms of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathways in oral squamous cell carcinoma under endoplasmic reticulum stress

BackgroundProteasome inhibitor Carbobenzoxy-Leu-Leu-leucinal (MG132) induces the unfolded protein response (UPR) in oral squamous cell carcinoma (OSCC). X-box binding protein 1 (XBP1) is a key UPR component that regulates endoplasmic reticulum stress (ER) homeostasis. This study was aimed to investigate the activation of IRE1α-TRAF2-ASK1-JNK pathway by silencing the XBP1 expression in an OSCC cell line.MethodsThe XBP1 specific short hairpin RNA (shRNA) plasmid vector was constructed and then transfected into the Tca-8113 cells. The effect of XBP-1 gene silencing on IRE1α-TRAF2-ASK1-JNK pathway under MG132 induced endoplasmic reticulum stress in Tca-8113 were investigated by real-time RT-PCR or western blot. Cell apoptosis was detected by flow cytometry.ResultsXBP1 expression was reduced in transfected groups and MG132 groups. shRNA-XBP1 induces IRE1α-TRAF2-ASK1 signaling activation to activate pro-apoptotic ASK1-JNK signaling. Moreover, combined shRNA-XBP1 with MG132 further enhanced downregulated XBP1 expression and upregulated activation of ASK1-JNK signaling.ConclusionsSilencing XBP1 expression under MG132 induced ER stress block the XBP1 survival pathway and synergism with MG132 to promote Tca8113 cell apoptosis. These findings provide a therapeutic option in oral squamous cell carcinoma by inhibition of proteasome and XBP1 splicing.