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The use of 57Fe-isotopically enriched ferritin for the accurate measurement of Fe : ferritin ratios is proposed for metabolic studies. Thus, the synthesis of 57Fe-isotopically enriched ferritin from horse apo-ferritin and isotopically enriched (NH4)257Fe(ii)(SO4)2 (Mohr's salt) is conducted. Size exclusion chromatography on-line with UV-VIS absorption (at 380 nm) is used in order to monitor the loading process of apo-ferritin. These studies revealed that the Fe-incorporation process involves also the formation of protein aggregates (oligomers) showing higher molecular mass than ferritin. A final optimized protocol involving incubation of the synthesized standard with guanidine hydrochloride (pH 3.5) has provided the best conditions for maintaining a stable protein structure without aggregates. Such 57Fe-isotopically enriched ferritin was characterized and contained an average of 2200 atoms of Fe per mole of ferritin. The evaluation of the Fe-core after saturation with 57Fe by Transmission Electron Microscopy (TEM) has revealed the formation of 57Fe nanoparticles with a similar diameter to that of the commercial Fe-containing ferritin, confirming the process of Fe uptake, oxidation and mineralization within the protein cavity. The synthesized 57Fe-ferritin shows great potential as a nanometabolic tracer to study the kinetics of Fe release in the cases of iron metabolic disorders. [ABSTRACT FROM AUTHOR]

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 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) 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%. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass specirometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrngaflol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometiy (MAI.DI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALI)I process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells. [ABSTRACT FROM AUTHOR]