Hamedani, Morteza Pirali; (1993) Novel liquid chromatography based assays for s-adenosylmethionine and related metabolites. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

S-Adenosyl-1-methionine (SAM) is an endogenous methyl donor which is actively involved in the methyl and aminopropyl transfer processes of a large number of biologically important molecules. Particularly, SAM is involved in the regulation of nerve cell activity by methylation of phospholipids and biogenic amines and is involved in the biosynthesis of one third of all liver phospholipids via the transmethylation pathway. The major metabolites of SAM are S-adenosyl homocysteine (SAH), decarboxylated SAM (dc-SAM) and methylthioadenosine (MTA). These compounds are also involved in different cellular functions, such as transsulphuration and polyamine biosynthesis. Several HPLC assays have been applied by others to this study but were based on single reversed-phase or cation-exchange chromatography. In reversed-phase HPLC relatively short retention times resulted, causing difficulties in the separation and quantitative determination of SAM from the other polar endogenous compounds. In cation-exchange chromatography, SAM had a much longer retention time, but SAH, MTA and adenosine (ado) showed relatively short retention times. In this study, since the reversed-phase or cation-exchange chromatography methods often resulted in overlapping peaks, a two dimensional HPLC assay was developed, which involved a gradient reversed-phase HPLC separation followed by cation-exchange chromatography. The measured hepatic levels of SAM, SAH, MTA, dc-SAM, adenine (ade) and adenosine (ado) by this method, (nmol g-1 wet weight) were found to be 35.5, 10.2, 0.27. 4.0, 49.3 and 43.3 respectively, from 24 hour starved rats. The absolute values for SAM in rat livers are in good agreement with previous published data obtained from 24 hour starved rats. The hepatic concentrations of SAH and MTA were found to be lower than previously reported. Two dimensional HPLC has proved to be a suitable assay for SAM and its related metabolites in different tissues and cultured cells, and has suggested that assay procedures using single HPLC methods may give erroneous results. Finally, a number of different LC/MS techniques were investigated for these assays. These were performed to try and utilise the greater specificity and sensitivities of the mass spectrometer as an LC detector. The MS techniques investigated included, thermospray, flowing FAB and electrospray. Use of an internal standard S-adenosyl ethionine (SAE) gave good reproducibility within runs and allowed better signal/noise ratios to be achieved. Thermospray was disappointing in sensitivity term for these compounds, but better detection limits were obtained by flowing FAB and electrospray (ESP), with levels of 0.012 nmol, 0.009 nmol and 0.004 nmol for SAM, SAH and MTA respectively being achieved by ESP/MS. These results show that a double HPLC method can serve for routine bioassay in conventional laboratories, and further that LC/ESP MS may form the basis for a very specific and sensitive assay for this important class of compounds, in blood and other body fluids, where the concentrations are less than 0.2 nmol.

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