ABSTRACT
Pipecolic acid (PA) is an important biochemical marker for the diagnosis of peroxisomal disorders. PA is also a factor responsible for hepatic encephalopathy and a possible biomarker for pyridoxine-dependent seizures. We developed an easy and rapid PA quantification method, by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), requiring no derivatization and applicable to small sample volumes. Plasma (100 µl) is extracted with 500 µl acetonitrile (ACN) containing 2 µmol/l [(2)H5]-phenylalanine as internal standard, vortexed and centrifuged. The supernatant is analyzed by HPLC-MS/MS in positive-ion mode using multiple reaction monitoring scan type. HPLC column is a Luna HILIC (150×3.0mm; 3 µ 200A): Buffer A: ammonium formate 5 mmol/l; Buffer B: ACN/H20 90:10 containing ammonium formate 5 mmol/l. PA retention time is 4.86 min. Recovery was 93.8%, linearity was assessed between 0.05 and 50 µmol/l (R(2)=0.998), lower limit of detection was 0.010 µmol/l and lower limit of quantification was 0.050 µmol/l. Coefficient of variation was 3.2% intra-assay and 3.4% inter-assay, respectively. Clinical validation was obtained by comparing PA plasma values from 5 patients affected by peroxisomal disorders (mean, 23.38 µmol/l; range, 11.20-37.1 µmol/l) to 24 ages related healthy subjects (mean, 1.711 µmol/l; range, 0.517-3.580 µmol/l).
Subject(s)
Chromatography, High Pressure Liquid/methods , Pipecolic Acids/blood , Tandem Mass Spectrometry/methods , Adolescent , Adult , Biomarkers/blood , Calibration , Child , Child, Preschool , Epilepsy/blood , Epilepsy/diagnosis , Female , Humans , Infant , Infant, Newborn , Isonipecotic Acids/isolation & purification , Limit of Detection , Male , Nipecotic Acids/isolation & purification , Peroxisomal Disorders/blood , Peroxisomal Disorders/diagnosis , Pipecolic Acids/isolation & purification , Reference Values , Reproducibility of Results , Young AdultABSTRACT
BACKGROUND: Vascular endothelial growth factor (VEGF165) is stored, transported and released by platelets. Platelet functional abnormalities have been described in patients with hepatocellular carcinoma (HCC). Thus, this study was designed to investigate the behavior of VEGF165 with respect to platelet activation in HCC. METHODS: Plasma and serum VEGF165 and plasma sP-selectin levels were analyzed in patients with HCC (n=70) or cirrhosis (n=45) and control subjects (n=70). Given the thrombocytopenia that characterizes both HCC and cirrhotic patients, plasma VEGF165 and sP-selectin as well as serum VEGF (plt-VEGF165-load) levels were normalized by platelet counts. RESULTS: Median concentrations of plasma VEGF165/platelet (p=0.002) and sP-selectin/platelet (p<0.0001) were higher in HCC or cirrhotic patients compared to controls. Moreover, sP-selectin/platelet was the only independent variable predictive of plasma VEGF165/platelet at multivariate analysis (p<0.0001). Conversely, plt-VEGF165-load correlated with tumor diameter (p<0.05) but not with sP-selectin/platelet and was an independent predictor for 5year overall survival (p=0.012). CONCLUSIONS: The results obtained are suggestive for VEGF165 release by tumor in HCC. It is plt-VEGF165-load, but not plasma VEGF165 or serum VEGF165 that is an independent predictor for overall survival of HCC patients.
