Metabolic profiling sample preparation

In comparison with other measurement methods, NMR has important strengths. Its great virtue is its noninvasive nature, allowing one to obtain spatially resolved metabolic profiles and to investigate metabolomics in vivo. 10 There is little or no sample preparation. It is nondestructive. It is information-rich with regard to the determination of molecular structures because it can detect different chemical groups of metabolites simultaneously. 

In what many consider to be a landmark publication on metabolomics, Fiehn et al. (2000) state it is crucial to perform unbiased (metabolite) analyses in order to define precisely the biochemical function of plant metabolism. The authors argue that for metabolomics/metabolite profiling to become a robust and sensitive method suited to automation, a mature technology such as gas chromatography-mass spectrometry (GC-MS) is required as an analytical technique. The authors go on to describe a simple sample preparation and analysis regime that allowed for the detection and quantification of more than 300 compounds from a single-leaf sample extract. 

The study was performed employing GC-MS, using bis-trimethylsilyl-trifluor-oacetamide (BSTFA) as derivatization agent during sample preparation. The aim of the study was to compare the metabolic profiles of urine samples from patients with liver cancer (n = 20) and healthy volunteers (n = 20) and, subsequently, to develop a diagnostic model that would include selected metabolites of potential diagnostic significance in HCC [21]. 

In the field of both pharmaceutical and pesticide metabolic research, TLRC can be used in three different ways. First it can be used analytically to determine the pattern of metabolites, and with today s modem detectors the quantitative distribution of metabolites can also be estimated. This can be done using either one- or two-dimensional development and of greater importance, this can be achieved without any purification or preconcentration of the sample. This is important since purification or preconcentration can result in a change of the metabolite profile, which can, for example, be due to volatility or selective binding during cleanup. Therefore, the quantitation of metabolites by TLRC within biological samples can often be achieved without any special sample preparation steps. 

Due to these characteristics, the possibility to build databases of NMR-based metabolic profiles of foodstuffs, in order to assess their quality, is becoming more and more actual [9]. However, when we apply multivariate analysis to our data fields or wish to compare databases obtained by NMR spectroscopy by other researchers, it is very important to consider the source of extrinsic variability originated by the employed experimental conditions, in terms of sample preparation, NMR acquisition parameters, signal-to-noise ratio, NMR spectral pre-treatment, NMR data pre-processing, as well as the strategy for NMR-based metabolomics (for an exhaustive introduction to the reported critical cmicepts see the manual by Axelson [10]). 

The profile of known impurities for imiprimine hydrochloride includes the starting material iminodibenzyl and two of the known metabolic degradation products iminostilbene and N-(dimethylaminopropyl)iminostilbene. A standard solution consisting of these three compounds is prepared and used to calculate the quantity of each present in a sample. 

Comparative metabolism in the rat. Adult male (6) and female (6) Sprague-Dawley rats were housed individually in polycarbonate metabolism cages and were orally administered by gavage an aqueous solution of " C-pirlimycin HCl. Five daily doses of 29 mg/kg/day were administered at 24-hour intervals to each rat. Urine and feces were collected at 24-hour intervals just before dose administration. The animals were sacrificed at 2 to 3 hours post-last-dose and liver, kidneys, and samples of flank muscle and abdominal fat carefully excised and placed into tared bottles. Homogenates of 2 1 water tissue were prepared for combustion/LSC analysis. Metabolite profiles were obtained for liver, urine and feces as described above. 

Selection of the appropriate marker residue may be challenging for some antibiotics, particularly those that typically are prepared from fermentation media and may contain multiple active constituents in variable proportions according to the particular manufacturer, such as gentamicin." For these substances, there is batch-to-batch variability, so the ratios of the components are not necessarily consistent, and therefore standards of individual components are preferable for reliable quantification. The rates at which certain components may be more readily metabolized or eliminated from tissues may differ from those of other components. As a result, the residue profile found can vary according to sample collection time since the last treatment, as well as sample storage time and temperature if particular components are subject to degradation on storage prior to analyses. 

A method has been developed I or measurement of heat production rale on pieces of liver tissue with the aim to avoid eventual alterations of cellular metabolic processes during the cell preparation procedure 1108]. Samples, 5-8 mg, were taken from rats by aspiration needle biopsy. Oxygen consumption was measured at the same time. Sodium fluoride was used for inhibition of the anaerobic pathway. The metabolic aerobic/anaerobic profile showed a good qualitative agreement with the earlier studies on isolated hepatocytes. The results indicated that the technique used, with small liver samples, was suitable for studying overall metabolism of human hepatic tissue in different liver diseases. 

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