RESUMO
This article describes a novel digital L-band EPR spectrometer. The spectrometer uses direct digital detection with time-locked subsampling (TLSS). The device consists of a microwave bridge equipped with a microwave source based on direct digital synthesis (DDS) and a digital receiver. DDS technology combined with an ultra-low noise 1 GHz master clock allowed the development of a digitally controlled microwave source with exceptionally good phase noise characteristics. The obtained level of phase noise is as low as -140 dBc/Hz at 30.5 kHz from the carrier frequency of 1.15 GHz, which is important when registering the EPR dispersion signal. The receiver is equipped with a high-speed A/D converter that enables direct digitalization of the L-band microwave signal. The obtained discrete data are then buffered and averaged in a programmable logic FPGA device. Data packets from FPGA are transferred to a DSP microcontroller that correlates them with the appropriate reference signals. This detection algorithm requires time locking of the generator and the receiver, which is ensured by clocking both devices from the same reference source. This procedure allows the simultaneous detection of the absorption and dispersion signals at the magnetic field modulation frequency and at any of its harmonics. The software to control the spectrometer was designed in the LabView programming environment. The program also allows further data processing. To the best of our knowledge, the described spectrometer is one of the first full implementation of the direct digital detection technique which could replace conventional analog CW spectrometers that utilize magnetic field modulation. For an 11 µm aqueous TEMPOL solution, the new spectrometer obtained a S/N ratio greater than 160 for an EPR spectrum registered in 69 s.
RESUMO
The mammalian retina contains a high level of polyunsaturated fatty acids, including docosahexaenoic acid (22:6) (DHA), which are highly susceptible to oxidation. It has been shown that one of the products of DHA oxidation-carboxyethylpyrrole (CEP), generated in situ, causes modifications of retinal proteins and induces inflammation response in the outer retina. These contributing factors may play a role in the development of age-related macular degeneration (AMD). It is also possible that some of the lipid oxidation products are photoreactive, and upon irradiation with blue light may generate reactive oxygen species. Therefore, in this work we analysed oxidation-induced changes in photoreactivity of lipids extracted from bovine neural retinas. Lipid composition of bovine neural retinas closely resembles that of human retinas making the bovine tissue a convenient model for studying the photoreactivity and potential phototoxicity of oxidized human retinal lipids. Lipid composition of bovine neural retinas Folch' extracts (BRex) was determined by gas chromatography (GC) and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS) analysis. Liposomes prepared from BRex, equilibrated with air, were oxidized in the dark at 37 °C for up to 400 h. The photoreactivity of BRex at different stages of oxidation was studied by EPR-oximetry and EPR-spin trapping. Photogeneration of singlet oxygen (1O2, 1Δg) by BRex was measured using time-resolved detection of the characteristic phosphorescence at 1270 nm. To establish contribution of lipid components to the analysed photoreactivity of Folch' extract of bovine retinas, a mixture of selected synthetic lipids in percent by weight (w/w %) ratio resembling that of the BRex has been also studied. Folch's extraction of bovine neural retinas was very susceptible to oxidation despite the presence of powerful endogenous antioxidants such as α-tocopherol and zeaxanthin. Non-oxidized and oxidized BRex photogenerated singlet oxygen with moderate quantum yield. Blue-light induced generation of superoxide anion by Folch' extract of bovine neural retinas strongly depended on the oxidation time. The observed photoreactivity of the studied extract gradually increased during its in vitro oxidation.