PubMed · 15534687
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Paal Røiri. 2004-11-04. [INTERHEART--a doubtful study].. https://pubmed.ncbi.nlm.nih.gov/15534687/
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Thiol-dependent peroxidases catalyzing the reductive detoxification of lipid hydroperoxides (LOOHs) are crucial antioxidant components of mammalian cells. There is a growing interest in manipulating expression of such enzymes to better understand their biological roles. A new approach for determining their cellular activity is described, whereby LOOH reduction kinetics are tracked by high performance thin layer chromatography with peroxide-sensitive tetramethyl-p-phenylenediamine detection (HPTLC-TPD). The approach was tested on a tumor cell transfectant clone (7G4) over-expressing selenoperoxidase GP x 4. Timed incubation of Triton-solubilized 7G4 cells with GSH and peroxidized phosphatidylcholine (PCOOH), followed by lipid extraction, HPTLC-TPD and densitometry revealed an exponential decay of PCOOH at a rate approximately 80-times greater than that for GP x 4-deficient controls (VC). A TPD-detectable cholesterol hydroperoxide (7alpha-OOH) was also reduced much faster by 7G4 than VC extracts. Spraying with H(2)SO(4) after TPD revealed both 7alpha-OOH loss and resolved diol product (7alpha-OH) accumulation, the kinetics of which were identical. The approach described is relatively convenient, highly specific, and much more sensitive than conventional assays for cellular LOOH reducing enzymes.
The mechanical characteristics of non-ionic bilayer membranes composed of sorbitan monostearate, cholesterol and poly-24-oxyethylene cholesteryl were studied by measuring the modulus of surface elasticity (mu), a measure of membrane strength, as a function of cholesterol content and temperature. The modulus of surface elasticity increased slowly with increasing cholesterol concentration, with a sharp increase around 40 mol% cholesterol (on average an increment of 0.43x10(6) Nm(-2) per molar percentage), and displayed a maximum of 6.5x10(6) Nm(-2) around 47.5 mol% cholesterol. Further cholesterol resulted in a decrease in mu. Generally the interaction of cholesterol with sorbitan monostearate should increase the rigidity of the membrane. However, the latter effect may be due to the formation of cholesterol clusters at high cholesterol content where excess amounts of cholesterol cannot interact with the sorbitan monostearate, and deposits on the bilayers compromising their uniformity, strength and permeability. This behavior was evident when measurements were carried out above and below 25 degrees C.
X-band EPR spectroscopy has been employed to study the dynamic properties of magnetically aligned phospholipid bilayers (bicelles) utilizing a variety of phosphocholine spin labels (n-PCSL) as a function of cholesterol content. The utilization of both perpendicular and parallel aligned bicelles in EPR spectroscopy provides a more detailed structural and orientational picture of the phospholipid bilayers. The magnetically aligned EPR spectra of the bicelles and the hyperfine splitting values reveal that the addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC bicelles. The corresponding molecular order parameter, Smol, of the DMPC/DHPC bicelles increased upon addition of cholesterol. Cholesterol also decreased the rotational motion and increased the degree of anisotropy in the interior region of the bicelles. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other model membrane systems and that the magnetically aligned bicelles are an excellent model membrane system.