Preparation, Storage And Experimental Conditions Of Biofluid Samples For NMR Based Metabonomics

The NMR-based metabonomics approach evolved from the pioneering work of Nicholson and co-workers in 1999 has become a novel analytical technique.At present,main targets of NMR-based metabonomics experiments are biofluids,and the experiment results are sensitive to the experimental conditions,sample storage and preparation.Study of these external conditions is important to keep the stability and comparability of biofluid sample data.In this paper,we investigated the effect of experimental conditions of human urine and serum samples,the effect of storage and preparation of human saliva samples.Some parameters are fully researched and optimized.The main contents of this study are as follows:1.Investigations of the effect of experimental conditions to human urine sample NMR spectra.Resonances of acetyl,lycine and histidine in urine sample shift as the temperature changes;signals whose chemical shifts are closed to that of water would decrease their intensity with the saturation power rising.Those results demonstrate that trade-off between the water suppression and signal decrease must be taken into consideration;External method is suitable.2.Investigations of the effect of experimental conditions to human serum sample NMR spectra,intensity of resonance signal of LDL,VLDL,valine and choline would increase obviously in the serum spectra with the experimental temperature increasing.Those evidenccs implied that it is important to acquire the 1H NMR spectra of the samples in the same temperature;internal method is suitablc.3.Investigations of the effect of storage and preparation conditions to human saliva sample NMR spectra.The results indicated that human saliva should be stored at or below -25℃,as no changes in the ~1H NMR fingerprints have been observed during 6 weeks. Formation of phenylalanine and formic acid,probably because of microbe activity,was observed in samples stored at 4℃.Addition of NaF is effective to keep saliva sample at -25℃,whereas the increase of acetate in saliva sample with NaF at the 6th week reflects it is not suitable for long term storage.A study evaluating the effects of phosphate buffer concentration on signal variability led to the conclusion that a higher concentration is adequate for normal saliva.For the comparability of NMR spectral data,making a standard of sample storage and experiment progress is useful and this work needs us to keep on researching in the future.