Establishment Of PYY Quantitation Method In Human Plasma

Peptide YY (PYY) is a member of the Neuropeptide Y (NPY) family of regulatorypeptides that includes NPY, PYY and pancreatic polypeptide (PP) with high sequence andstructural homology (PP-fold). PYY is secreted as a36amino acid, straight chain polypeptide,and is found in greatest concentrations in the terminal ileum, colon and rectum. There are twoforms mainly exist in circulation system, i.e. PYY1-36and PYY3-36. The PYY3-36is aproduct derived from dipeptidyl peptidase IV (DPP-IV) cleaves the N-terminalTyrosine-Proline residues from PYY1-36. The current studies of PYY are focus on the effectsof PYY on obesity and diabetes development and treatment. PYY participates in theregulation of appetite and weight balance through hypothalamic-based mechanisms. PYY1-36and PYY3-36cause opposite effects by acting through different Y-receptor sub-types.PYY1-36acts through Y1and Y5receptors in the central nervous system to stimulate appetiteand to promote weight increase, while PYY3-36binds Y2-receptors, which inhibit appetiteand promote weight loss. So it is very important to distinguish these two forms when quantifyPYY levels in biological matrices.In the present study, to establish PYYquantitative analysis method in human plasma, theperformance of PYY1-36calibration curve constructed by standard curve method wasevaluated; digested PYY was prepared and used to improve the method (chromatographicseparation efficiency, ionization efficiency, instrument response, etc.); ultra-high performanceliquid chromatography-tandem mass spectrometry method was established for PYYquantitative analysis in plasma and validated with a set of parameters (linearity, precision,recovery, limit of detection and limit of quantitation). The thesis included the following workand conclusions:1. Construction of calibration curve of PYY1-36Combination of enhanced full-scan and product ion scan mode to establish multiplereaction monitoring (MRM) method transtitions for intact PYY1-36quantitation. Theparameters of liquid chromatography and mass spectrometry were optimized. The extractionprotocols for PYY were compared and optimized, and the comparison result of recovery andreproducibility showed that the SPE HLB cartridge extraction protocol was the best method toextact PYY1-36from human plasma. The standard curve was constructed by standard curve method, and the linear range was250~1000ng/mL with limit of detection and limit ofquantitation were100ng/mL and250ng/mL, respectively, which much higher than theexpected endogenous level of PYY in human plasma.2. Construction of standard curve of PYY1-19The fragment PYY1-19was prepared by digested PYY1-36with trypsin. MRMtransitions for PYY1-19were established by enhanced full-scan and product ion scan mode.The separation efficiency of two liquid chromatography systems, i.e. Ultimate3000highperformance liquid chromatography system (HPLC) and Shimadzu ultra-high performanceliquid chromatography system (UPLC) was compared, and the result showed that the UPLCsystem could improve the separation efficiency greatly (elution gradient reduced from15minto6min) without affecting the response of the instrument to PYY1-19; Mass spectrometerdetection sensitivity for PYY1-19and PYY1-36was analyzed, the result confirm thatapplication of digested small fragments instead of intact PYY can greatly improve ionizationefficiency of the electrospray ion source and increase mass spectrometer sensitivity. Theconditions for better liquid chromatographic separation and mass spectrometry instrumentsensitivity were optimized. The standard curve was constructed by standard curve method.The linear range was0.5~250ng/mL with limit of detection and limit of quantitation were0.1ng/mL and0.5ng/mL, respectively.3. Establishment of calibration curve of PYY1-19in human plasmaThe trypsin digestion condition for completely digestion of PYY in human plasma wasoptimized. The calibration curve of PYY1-19in human plasma was constructed by standardaddition method, the linear range was5~1000ng/mL with limit of detection and limit ofquantitation were1ng/mL and5ng/mL, respectively. Comparison of the calibration curvesprepared in standard solution and complex matrix (human plasma) showed that there was asignificant matrix effect, in consequence, it should use the standard addition method whenquantify the PYY level in human plasma.