Global metabolic profiling in plasma samples of patients before and after ketogenic diet therapy by monolithic-C18 LC and HILIC/(+)ESI-MS

SOLEDAD CERUTTI; PEGGY R. BORUM; JODIE V. JOHNSON; RICHARD A. YOST; DAVID H. POWELL

Filadelfia, Pensilvania, Estados Unidos

Conferencia; 57th ASMS Conference on Mass Spectrometry; 2009

American Society for Mass Spectrometry

Introduction: A ketogenic diet (KD) is a high-fat, low-carbohydrate/adequate-protein diet designed to increase the body´s dependence on fat rather than glucose for energy and to treat disorders of the brain. The two disorders most commonly treated are epilepsy and certain inborn errors of metabolism involving glucose utilization. Despite its long history of clinical use, it is still not entirely clear what mechanism(s) underlie its epileptic seizure-suppressive action and how the KD affects the brain general metabolism. Mass spectrometry-based metabolomics relies on nonbiased, qualitative, and quantitative analysis of a large number of metabolites, providing insight into the nutritional, metabolic, and general health status during different stages of the life cycle and during pathological conditions. Methods: We are testing the hypothesis that the plasma metabolic profile of healthy female and male adults before and after ketogenic diet therapy will differ. In this work, the putative biomarkers were determined by the analysis of plasma samples via both monolithic C18-chromatography (Phenomenex Onyx monolithic column) and hydrophilic interaction chromatography (Phenomenex Luna HILIC column) coupled to a high resolution time-of-flight mass spectrometer (Agilent 6210 TOF-MS) operated in the positive electrospray ionization [(+)ESI] mode. Performance/contribution of each chromatographic strategy and identification of unique m/z features by processing the raw data with the Agilent MassHunter Workstation and GeneSpring MS software platforms were evaluated. Preliminary Results: Plasma samples from healthy female and male adults before and after ketogenic diet treatment were obtained. After adequate sample preparation and optimization of the chromatographic conditions together with (+)ESI-MS parameters, separation, ionization, and accurate mass spectrometric detection of the ion features/compounds present in the plasma samples were performed. Acidic conditions were used to promote the protonation of some of the substances in the reversed-phase separation strategy. In contrast, ammonium acetate was used to enhance the protonation of metabolites in the hydrophilic interaction chromatographic mode. The use of these two complementary separation methodologies resulted in the separation/identification of unique m/z features for each strategy. Hydrophobic compounds demonstrated excellent retention profiles on the C18 column, while hydrophilic analytes were better retained on the HILIC column. All samples were scanned over a mass range of m/z 50 to 1650 and were analyzed randomly under the same experimental conditions. Our studies involved a large number of samples (biological and technical replicates for each person) that required powerful data processing/analysis capabilities. In this sense, the raw instrument data were processed in several steps by the instrument software. From the obtained chromatograms, features were first extracted, aligned, normalized, filtered (data processing), and then analyzed by different statistical methods, including analysis of variance (ANOVA), principal component analysis (PCA), and Volcano plots. Finally, using the accurate mass criterion of 2 ppm mass error, putative biomarkers responsible of the metabolic differences in patients before and after the KD therapy were identified in the METLIN database (METLIN Personal database software platform.) From a metabolic perspective, the overall analytical procedure that can discriminate metabolic changes from individuals before and after a KD will be discussed and the results presented.