The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.

Increased concentrations of circulating metal-degradation products derived from the use of Ti orthopaedic implants may have deleterious biological effects over the long term. Therefore, there is an increasing need to establish the basal level of Ti in the serum of the population (exposed and non-exposed) with appropriate highly sensitive techniques and strategies. With this aim, we have developed a quantitative strategy for the determination of total Ti concentration in human serum samples by isotope dilution analysis using a double-focussing inductively coupled plasma mass spectrometer. Minimizing sample handling and therefore contamination issues, we obtained detection limits of about 0.05 μg L(-1) Ti working at medium resolution (m/Δm 4000). Such extremely good sensitivity permitted us to establish the range of Ti concentration in serum of 40 control individuals (mean 0.26 μg L(-1)) and also to compare it with the level in exposed patients with different Ti metal implants. On the other hand, Ti transport "in vivo" studies have been enabled by online coupling of liquid chromatography (anion-exchange) separation and double-focussing inductively coupled plasma mass spectrometry for sensitive detection of Ti. The development of a postcolumn isotope dilution strategy permitted quantitative characterization of the Ti-transporting biomolecules in human serum. The results for unspiked serum revealed that 99.8% of the Ti present in this fluid is bound to the protein transferrin, with column recoveries greater than 95%.

Variations in the distribution of sialoforms of human serum transferrin (Tf) in correlation with pathological states, which are associated with abnormalities in glycosylation, is of great clinical interest. In such studies, the methodologies of analysis are required to be sensitive and selective for observing small variations among isoforms and able to characterize the molecular structure of such forms. Thus, the present work describes, in the first part, the separation of transferrin isoforms, after iron saturation of the protein, by high-performance liquid chromatography (HPLC) and the on-line specific atomic detection of the iron present on each of the separated isoforms by on-line coupling the HPLC system to an inductively coupled plasma mass spectrometer (ICPMS). This allowed low detection levels for the different isoforms (L.D. 0.03 microMTf). After screening of the isoforms containing iron by ICPMS, structural characterization of each isoform can be independently carried out. Thus, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF) and electrospray mass spectrometry (ESI-Q-TOF) are compared in the second part of this study. The different atomic and molecular MS methods revealed the presence of elevated carbohydrate-deficient transferrin (CDT) isoforms in human serum samples from chronic alcohol consumption patients. MALDI-TOF appeared to be sensitive to concentration levels of the analytes, and the observed mass accuracy was highly compromised by the protein heterogeneity (peak width at half-maximum approximately 2000 Da for every fraction). On the other hand, ESI-Q-TOF allowed good mass accuracy (m < or = 0.05%) and peak width of 45 Da in the deconvoluted spectra; while ICPMS detection could be preferable for sensitive protein isoforms determinations, ESI-Q-TOF turns out to be an excellent "fingerprinting" technique for alcoholism diagnosis.

No abstract

No abstract