Monitoring intracellular melatonin levels in human prostate normal and cancer cells by HPLC

Melatonin (N-acetyl-5-methoxytryptamine) is a potent endogenous antioxidant and free radical scavenger that has attracted much attention as a consequence of its multiple biological functions. In addition to other physiological properties, it has clear antiproliferative activity in several types of cancer cell. The concentration of melatonin necessary to inhibit cell growth is much higher than its blood physiological concentrations in some tumor types. For years its indolic nature has impeded proper monitoring, by molecular or immunological techniques, of its uptake by cancer cells. In this work we developed a simple, rapid, and validated analytical method for detection and quantification of MEL inside normal and cancer cells. For this purpose we performed high-performance liquid chromatographic analysis after liquid–liquid extraction of the indole from biological samples. The method was validated, and the correlation coefficient for amounts from 0.125 to 1.25 μg was higher than 0.999, with a range of recovery near 100%. Precision was evaluated as repeatibility, and for intermediate precision, the relative standard deviation was less than 5%. The method was used to study the stability of the indole in solution and to determine intracellular melatonin concentrations in normal (PNT1A) and several cancer (LNCaP, DU-145, PC-3) prostate cell lines. Intracellular LOQ/LOD were 7.23/2.83, 23.17/9.07, 4.03/1.83, and 6.51/2.53 nmol L⁻¹, or 1.82/4.66, 0.56/1.45, 3.26/8.34, and 2.02/5.17 attogram in each cell in PNT1A, LNCaP, DU145, and PC-3 cells, respectively. Because there was no information about intracellular levels of melatonin inside normal or tumor prostate cells after treatment with the indole, nor a relationship between its antiproliferative activity and its intracellular concentration, this is the first time that, by using an analytical method combined with measurement of cellular volume by flow cytometry, the intracellular concentration of MEL has been estimated. Also, data obtained here explain why the antiproliferative properties of MEL vary in different cell types. This is, moreover, the first time that by increasing the intracellular concentration of melatonin, its antitumor properties have been promoted in prostate cancer cells. This process can be monitored by the method developed here.     

Structural and Magnetic Diversity in Alkali‐Metal Manganate Chemistry: Evaluating Donor and Alkali‐Metal Effects in Co‐complexation Processes

By exploring co‐complexation reactions between the manganese alkyl Mn(CH₂SiMe₃)₂ and the heavier alkali‐metal alkyls M(CH₂SiMe₃) (M=Na, K) in a benzene/hexane solvent mixture and in some cases adding Lewis donors (bidentate TMEDA, 1,4‐dioxane, and 1,4‐diazabicyclo[2,2,2] octane (DABCO)) has produced a new family of alkali‐metal tris(alkyl) manganates. The influences that the alkali metal and the donor solvent impose on the structures and magnetic properties of these ates have been assessed by a combination of X‐ray, SQUID magnetization measurements, and EPR spectroscopy. These studies uncover a diverse structural chemistry ranging from discrete monomers [(TMEDA)₂MMn(CH₂SiMe₃)₃] (M=Na, 3 ; M=K, 4 ) to dimers [{KMn(CH₂SiMe₃)₃?C₆H₆}₂] ( 2 ) and [{NaMn(CH₂SiMe₃)₃}₂(dioxane)₇] ( 5 ); and to more complex supramolecular networks [{NaMn(CH₂SiMe₃)₃}_∞] ( 1 ) and [{Na₂Mn₂(CH₂SiMe₃)₆(DABCO)₂}_∞] ( 7 )). Interestingly, the identity of the alkali metal exerts a significant effect in the reactions of 1 and 2 with 1,4‐dioxane, as 1 produces coordination adduct 5 , while 2 forms heteroleptic [{(dioxane)₆K₂Mn₂(CH₂SiMe₃)₄(O(CH₂)₂OCH=CH₂)₂}_∞] ( 6 ) containing two alkoxide–vinyl anions resulting from α‐metalation and ring opening of dioxane. Compounds 6 and 7 , containing two spin carriers, exhibit antiferromagnetic coupling of their S=5/2 moments with varying intensity depending on the nature of the exchange pathways.     

Introducing Deep Eutectic Solvents to Polar Organometallic Chemistry: Chemoselective Addition of Organolithium and Grignard Reagents to Ketones in Air

Despite their enormous synthetic relevance, the use of polar organolithium and Grignard reagents is greatly limited by their requirements of low temperatures in order to control their reactivity as well as the need of dry organic solvents and inert atmosphere protocols to avoid their fast decomposition. Breaking new ground on the applications of these commodity organometallics in synthesis under more environmentally friendly conditions, this work introduces deep eutetic solvents (DESs) as a green alternative media to carry out chemoselective additions of ketones in air at room temperature. Comparing their reactivities in DES with those observed in pure water suggest that a kinetic activation of the alkylating reagents is taking place, favoring nucleophilic addition over the competitive hydrolysis, which can be rationalized through formation of halide‐rich magnesiate or lithiate species.     

Selenium content of Argentinean infant formulae and baby foods by pseudo-cyclic instrumental neutron activation analysis coupled to Compton suppression

The selenium levels of Argentinean infant formulae and baby food were measured using the 162-keV gamma-ray of ^77mSe (t _½ = 17.4 s) by a pseudo-cyclic instrumental neutron activation analysis (PC-INAA) method in conjunction with Compton suppression spectrometry (CSS). For comparison purposes, 5 selected infant formulae were also analyzed for selenium by a radiochemical neutron activation analysis (RNAA) method. The selenium levels for three samples agreed between ±2.8 and 6.5 % while the other two differed by 12 and 17 % which could perhaps be attributed to sample inhomogeneity. The selenium content of cow milk-based infant formulae varied from 42–146 μg kg^?1 compared to 52–63 μg kg^?1 for soy-based milk formulae. In the case of baby foods, the selenium levels varied from 34 to 74 μg kg^?1. The detection limits for selenium by PC-INAA–CSS for all the samples analyzed in this work were between 8.5 and 65 μg kg^?1 depending on the major elements present in the samples, while it was 20 μg kg^?1 for the RNAA method. The expanded uncertainty (κ = 2) of the PC-INAA–CSS method was 7.0 % at the end of cycle #4 for a sample containing 73.7 μg kg^?1 selenium compared to the RNAA value of 24.2 % for a sample of 67.0 μg kg^?1 selenium content.     

Electronic structure and excited states of rhenium(I) amido and phosphido carbonyl-bipyridine complexes studied by picosecond time-resolved IR spectroscopy and DFT calculations

UV-vis absorption and picosecond time-resolved IR (TRIR) spectra of amido and phosphido complexes fac-[Re(ER2)(CO)3(bpy)] (ER2 = NHPh, NTol2, PPh2, bpy = 2,2'-bipyridine, Tol = 4-methylphenyl) were investigated in conjunction with DFT and TD-DFT calculations in order to understand their ground-state electronic structure, low-lying electronic transitions and excited-state character and dynamics. The HOMO is localized at the amido/phosphido ligand. Amide and phosphide ligands are sigma-bonded to Re, the pi interaction being negligible. Absorption spectra show a weak band at low energies (1.7-2.1 eV) that arises from essentially pure ER(2) --> bpy ligand-to-ligand charge transfer (LLCT). The lowest excited state is the corresponding triplet, (3)LLCT. Low triplet energies and large distortions diminish the excited-state lifetimes to 85 and 270 ps for NHPh and NTol(2), respectively, and to ca. 30 ps for PPh2. nu(CO) vibrations undergo only very small ( bpy MLCT character, is a unique feature of the amido/phoshido complexes, whose lowest excited state can be viewed as containing a highly unusual aminyl/phosphinyl radical-cationic ligand. For comparison, the amino and phosphino complexes fac-[Re(NHPh(2))(CO)3(bpy)]+ and fac-[Re(PPh3)(CO)3(bpy)]+ are shown to have the usual Re --> bpy (3)MLCT lowest excited states, characterized by upshifted nu(CO) bands.