| Non-Hodgkin’s lymphoma (NHL) occurs in the lymph nodes and (or)extranodal lymphoid tissues. The incidence rate increases steadily yearafter year. It has become the ninth malignant tumors of the incidence inChina. Early diagnosis and treatment will significantly improve thepatient’s survival and increase the survival rate, but there are still fewspecific methods for early diagnosis of NHL.Metabonomics focuses on a systems-based study of all smallmolecule metabolites in the body. One of the major applications ofmetabonomic is disease diagnosis. Since the metabolome changes in thebody with pathological, the analysis of these metabolites can help usunderstand the metabolic pathways of the metabolites and the diseaseprocess, find the biomarker(s) and could be supplementary means inclinical diagnosis.Ultra performance liquid chromatography-quadruple/time-of-flight mass spectrometry (UPLC-QTOFMS) and gas chromatographytime-of-flight mass spectrometry (GC-TOFMS) were used to investigatethe different metabolites between serum or urine samples from NHLpatients and healthy controls. We discriminated the NHL subjects fromthe healthy controls by their serum and urinary metabolic profiles.Combined with supervised pattern recognition and univariate statisticalmethod, it can effectively find out the differential metabolites contributedfor the separation between the NHL patients and the healthy controlsderived from serum or urine sample. A total of41identified potentialbiomarkers were found related to the lymphomagenesis and diseaseprogression. A panel of metabolite markers composed of4metabolites was able to discriminate NHL subjects from their healthy counterparts. Areceiver operating characteristic curve (ROC) analysis of these markersresulted in an area under the receiver operating characteristic curve (AUC)of1. The results may provide an insight into the understanding of thebiochemical network and pathway in NHL.Aminoglycosides are clinically used for their water solubility,stability, broad spectrum, strong antibacterial effect and goodcharacteristics in absorption and excretion. Aminoglycosides are derivedfrom microbial fermentation or produced by semi-synthesis. They containaminosugar substructures. Aminoglycosides lack a suitable chromophore,which is necessary for UV detection. For this reason, except themicrobiological potency assay, pre-column derivatization methods orelectrochemical detection are used in many kinds of pharmacopoeia. Weestablished three rapid, reliable and convenient HPLC-ELSD methods forthree aminoglycosides, with high sensitivity, good specificity forsimultaneous determination of main active ingredient and relatedsubstances in a single run.The key contents and results include:1. A serum metabonomic study using UPLC-QTOFMS combined withmulti-/uni-variate statistical method was carried out to investigate thedifferent metabolic profiles between NHL group and control group.The results showed a clear separation between the patients andhealthy controls in scores plots. By uni-and multivariate statisticalanalysis methods,10most important markers were found, indicatinggreat potential for early diagnosis of NHL. The alteration ofarachidonic acid metabolism, steroid metabolism, bile acidmetabolism, amino acid metabolism, glucose metabolism andporphyrin metabolism may play key roles in the disease. The resultsnot only provided a solid, reliable basis for monitoring disease trends,but also the possible drug targets according to the related pathways.2. A urinary metabonomic study using UPLC-QTOFMS combined withmulti-/uni-variate statistical method was carried out to investigate thedifferent metabolic profiles of NHL patients and healthy controls. Theresults showed that urine metabolic profiles of lymphoma patients and healthy controls significantly differ from each other.26mostimportant markers were found, indicating great potential for earlydiagnosis of NHL. Our findings confirm a distinct urinary metabolicprofile of NHL patients characterized by altered levels of manymetabolites involved in nicotinate and nicotinamide metabolism,histidine metabolism, tryptophan metabolism, phenylalaninemetabolism, purine metabolism, pyrimidine metabolism andglutamate metabolism. A panel of metabolite markers composed of4metabolites (homocysteine,2-(2-Phenylacetoxy)propionylglycine,L-malic acid and L-ascorbic acid) selected by univariate statisticalmethod, was able to discriminate BCL subjects from TCL subjects,with an AUC of0.900.3. The N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA)derivatization, gas chromatography time-of-flight mass spectrometry(GC-TOFMS)-based serum metabonomic analysis method wasestablished to investigate the different metabolic profiles of NHLpatients and healthy controls. The OPLS-DA model indicates a clearseparation between NHL patients and healthy controls. Fiverepresentative differential metabolites with both multivariate andunivariate statistical significance that contributed for the separationbetween the two group were benzenebutanoic acid、 β-hydroxypyruvic acid、D-2-hydroxyoctanoic acid、pyruvic acid andL-serine. Differential metabolites derived from GC-TOFMS analysisare good complements to the result of UPLC-QTOFMS. The changesin glycine, serine and threonine metabolism, straight-chain fatty acidmetabolism, glycolysis, the citric acid cycle, the pentose phosphatepathway, cysteine and methionine metabolism were found in NHLpatients, compared to healthy controls. These biochemical changesprovide a novel molecular diagnostic approach which could behelpful to further understand the pathogenesis and identify thetherapeutic target of NHL.4. Four representative metabolites in serum, two derived fromUPLC-QTOFMS analysis and two from GC-TOFMS analysis:2,3-dinor-6,15-diketo-13,14-dihydro-PGF1a, MG(0:0/20:4(5Z,8Z,11Z,14Z)/0:0), benzenebutanoic acid andβ-hydroxypyruvic acid, were selected as a panel of candidate markersbased on their high VIP values, fold changes, AUC, and low p values.Logistic regression was used to combine the four variables into amultivariable. The panel of metabolite markers was able todiscriminate NHL subjects from their healthy counterparts, the AUCreached1with a sensitivity of100%and specificity of100%. Thesepotential metabolite markers provide a novel and promising moleculardiagnostic approach for the early detection of NHL.5. Three simple, rapid and reliable reversed-phase ion-pairchromatography methods by high-performance liquidchromatography coupled to an evaporative light scattering detector(HPLC-ELSD) have been developed to respectively determinespectinomycin hydrochloride, amikacin sulfate and streptomycinsulfate and their related substances. The optimized methods, whichare validated for good accuracy, precision, specificity and robustness,provide an efficient alternative for the common UV detection. Theresults suggest good applicability in routine assay.These studies in this dissertation covered the metabonomics ondifferent bio-samples and different analysis platforms in a certainmalignant disease of NHL. The results in these studies enriched theunderstanding of metabonomics, and powered the potential earlydiagnositic research and mechanism investigation on lymphoma.Moreover, the HPLC-ELSD methods could be of help in improvement ofthe pharmaceutical standards and quality system. |
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