Amniotic Fluid Metabonomics Study Of Congenital Malformation

Metabonomics or metabolomics is a new branch of science under thecondition of development of system biology recently. The metabonomics isdivided into four levels: target metabolites, the metabolic profile (spectrum),metabolomics and metabolic fingerprint analysis. Metabolic finger printingis based on the entire map (fingerprint) for a particular cell or tissuespecific metabolic patterns, not separating single component of specificconstituents. Metabolic finger printing is a rapid, high flux and globalanalysis, as well as relatively simple sample preparation. It is usuallycombined with pattern recognition and discriminant classificationtechnology to recognise some of the characteristics of fingerprint andmetabolic fingerprint. It is the most useful method for different statediscriminant screening, diagnosis and its specific metabolic patterns found.Metabolic finger printing can reflect the overall metabolic fingerprint bymonitoring the metabolic network running status, diagnosis, therapeuticevaluation and forecast development trend of the future health of thehuman body. Therefore, it’s necessary to study metabonomics in prenataldiagnosis. We established two methods (UPLC-MS and GC-MS) to studymetabonomics of congenital malformation in amniotic fluid, details asfollows.1. Amniotic fluid metabonomics study of congenital malformationby ultra-performance liquid chromatography tandem triplequadrupole time-of-flight mass spectrometryCongenital malformation is a leading cause of infant deaths. It couldbe caused by genetic factors, environmental factors and multifactorial inheritance, which is difficult to clarify its pathophysiology byconventional screening methods. Metabonomics is the study of metabolicchanges in biological systems and provides the small molecule fingerprintsrelated to the disease. Here, we presented amniotic fluid metabonomicsstudy of congenital malformation using ultra-performance liquidchromatography tandem triple quadrupole time-of-flight mass spectrometry(UPLC Q-TOF/MS) coupled with pattern recognition methods to determinemetabolite alterations in patients and controls. Total14differentialmetabolites were identified and some of them are well known to relate todisease progress involving several key metabolic pathways, such as leucinemetabolism, glutamic acid, methionine and threonine metabolism, histidinemetabolism, arginine metabolism, phenylalanine metabolism and energymetabolism. These findings might be promising to yield a valuable insightinto the pathophysiology of congenital malformation and to enhance theapproaches of screening in prenatal diagnosis.2. Quantification of amino acids in amniotic fluid of congenitalmalformation by gas chromatography tandem mass spectrometryAmino acids play an important role as intermediates in manymetabolic pathways, such as the biosynthesis of nucleotides, vitamins andproteins. A gas chromatography–mass spectrometry (GC–MS) method wasdeveloped for the quantitative analysis of free amino acids as theirtrimethylsilyl derivatives in amniotic fluid samples. The method wasproved to be sensitive, reproducible, and reliable quantification, and all8amino acids were separated using this method. The temperature and heatingtime of derivatization reaction were optimized. Under the optimumconditions, the calibration curves showed good linearity over theinvestigated concentration range from0.5to10μg/ml with a coefficient ofestimation (R2)>0.99and precision and accuracy examined at three concentration levels ranged from0.84%to9.33%and91.12%to104.41%,respectively. The validated method was successfully applied to thequantification of8amino acids in amniotic fluid of congenitalmalformation. Compared to the controls, there were significant variationsfor the concentration of leucine, isoleucine, serine and threonine in theexperimental patients, which might be a indicator for the development ofcongenital malformation. Therefore, this method is potentially applicablefor amino acid analysis in amniotic fluid, providing a further understandingof congenital malformation.