Towards single cell ICP-MS normalized quantitative experiments using certified selenized yeast.

In the search for a normalized procedure to replicate and compare single cell-inductively coupled plasma-mass spectrometry (SC-ICP-MS) experiments, SELM-1, a certified reference material containing selenium enriched yeast cells has been used. Selenium concentrations (both, intra- and extracellular) have been measured using either sequential or simultaneous procedures. Regarding quantitative results, the sequential procedure involving cell washing followed by freeze drying of the washed material and intracellular Se quantification using SC-ICP-MS provided best results. In this case, intracellular Se accounted for 1304 ± 48 mg kg-1 (corresponding to 64% of the certified Se content). The average mass of Se per yeast cell was 41.6 fg Se with a dispersion of 1.6-279 fg Se/cell. In the isolated extracellular Se fraction, the Se concentration accounted for 412 ± 48 mg kg-1 (about 21% of the total Se). Thus, the sequential procedure provided a total Se recovery of about 85% with respect to the certified value. The direct dilution and simultaneous measurement of intra- and extracellular Se by SC-ICP-MS provided results of 1024 ± 42 mg kg-1 for intracellular and 316 ± 30 mg kg-1 for extracellular Se representing a total recovery of about 66%. In both cases, an initial thorough characterization of the cell density per solid weighed material was conducted by flow cytometry and the cell integrity ensured using confocal microscopy. These results clearly demonstrated that with appropriate sample preparation, SC-ICP-MS is a unique tool, which is capable of providing quantitative information about intracellular and extracellular Se. In addition, SELM-1 seems the ideal tool to enable data normalization at the single cell level to replicate, benchmark, and improve new SC-ICP-MS studies by using the same material for data validation.

Addressing the presence of biogenic selenium nanoparticles in yeast cells: analytical strategies based on ICP-TQ-MS.

Several organisms have demonstrated the ability of synthesising biogenic selenium-containing nanoparticles. Such particles from biological sources have attracted great attention due to several proven activities as antioxidants or antimicrobial agents. However, little is known in terms of size (distribution), shapes, chemical composition and number/amount/concentration of these particles. Therefore, in this work, we proposed the use of complementary analytical strategies that enabled the detection and characterization of selenium-containing nanoparticles in selenized yeast (Saccharomyces cerevisiae). The first strategy to address the intracellular presence of Se within yeast cells, involves the use of single cell ICP-TQ-MS (inductively coupled plasma-mass spectrometry). For this aim, selenium and phosphorous (as constitutive element) were measured as oxides (80Se16O+ and 31P16O+, resp.) in the triple-quadrupole mode. Then, a simple and fast cell lysis by mechanical disruption is conducted (approx. 30 min) in order to prove the presence of selenium-containing nanoparticles (SeNPs). The lysate is analysed by single particle ICP-TQ-MS and, complementarily, by liquid chromatography coupled to ICP-TQ-MS to cover a wider range of particle sizes. One of the samples revealed the presence of dispersed SeNPs with sizes between a few nm and up to 250 nm also confirmed by transmission electron microscopy (TEM) in the form of elemental selenium. The analysis of the certified reference material SELM-1 showed the presence of spherical SeNPs of 4 to 7 nm diameter. These biogenic particles, at least partially, were made of elemental selenium as well. The whole study reveals the excellent capabilities of "single" event ICP-MS methodologies in combination with HPLC-based strategies for a complete characterization of nanoparticulated material in biological samples.

Management of elevated intracranial pressure.

The pathophysiology, clinical manifestations, monitoring techniques, and management of elevated intracranial pressure (ICP) are reviewed. The use of barbiturate coma to treat ICP is discussed in detail. Elevated ICP can be associated with severe head injuries and diseases of the central nervous system such as brain tumors and stroke. Symptoms of elevated ICP may be difficult to distinguish from symptoms of other disease states. ICP monitoring techniques such as the intraventricular catheter and the Camino fiber optic system are useful for determination of ICP elevations before any changes in vital signs or neurological status occur. Conventional treatment and control of ICP elevations includes general and physiologic management (cerebrospinal fluid removal, fluid restriction, controlled hyperventilation, sedation, and elevating the patient's head) and pharmacologic management. Osmotic diuretics, (e.g., urea, mannitol, glycerol) and loop diuretics (e.g., furosemide, ethacrynic acid) are first-line pharmacologic agents used to lower elevated ICP. Corticosteroids may be beneficial in some patients. Patients with elevated ICP refractory to conventional treatment may benefit from therapy with high-dose barbiturates. Pentobarbital has been used in the majority of the clinical studies. Pentobarbital serum concentrations should be determined every 24-48 hours when a patient is in a barbiturate coma because pentobarbital clearance increases with continued high-dose therapy. The treatment of elevated ICP requires aggressive therapy and intensive monitoring. In patients whose ICP is refractory to conventional therapies alone, survival rates have been improved by combining high-dose barbiturates with conventional therapies.