Background: Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins, mainly VLDL, HDL, and chylomicrons. It is involved in the redistribution of lipids in the liver and is implicated in growth and repair of injured neurons in the nervous system. apoE has also been associated with the risk of developing cardiovascular diseases and in familial type III hyperlipoproteinemia. Methods: We developed a new procedure for detecting genetic polymorphisms of apoE in human blood samples. The procedure is based on coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood. The DNA electrochemical sensor incorporated single-stranded oligonucleotides immobilized on graphite screen-printed electrodes (SPEs) by adsorption at controlled potential. The hybridization reaction on the electrode surface was monitored by chronopotentiometric stripping analysis (PSA), using daunomycin as indicator. Results: With use of two different probes, it was possible to investigate both DNA positions in which the apoE polymorphism takes place and thus to distinguish different genotypes. Real samples containing only complementary sequences gave a good increase in the area of the daunomycin peak (∼600 ms) compared with the peak observed with the buffer. Samples containing 50% complementary sequences gave a much lower increase, and samples containing only mismatch sequences gave a decrease in the daunomycin area. The procedure was validated by comparison with a method based on polyacrylamide gel electrophoresis. Conclusion: The coupling of DNA electrochemical sensors with PCR allowed quick discrimination between the different genotypes of apoE.

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In 63 patients affected by Systemic Sclerosis (SSc) (limited subset: 40; diffuse subset: 23; early: 30; advanced: 33) the peroxidation product diene-conjugates (DC) and antibodies against oxidised low density lipoproteins (Ab oxLDL) were tested in serum by a spectrophotometer (absorbance 234 mn) and by a standard ELISA respectively. The data were compared with those obtained by 21 healthy subjects. DC was significantly higher in patients (73.3 +/- 37.2 microM/l; p < 0.0001) than in controls (48.4 +/- 16.7) as well as in the limited (80 +/- 48.8; p < 0.05) than in the diffuse subset (64.5 +/- 36.4); and in early (84.1 +/- 31.4; p < 0.05) than in advanced stage of the disease (67.9 +/- 42.5). The levels of Ab oxLDL were significantly higher in SSc patients (309.5 +/- 367.2 mU/ml; p < 0.0001) in all its subsets (limited: 351.9 +/- 351.1, p < 0.0001; diffuse: 207.7 +/- 316.1, p < 0.05; early: 428.9 +/- 417.1, p < 0.001; advanced: 302.7 +/- 89.9, p < 0.0001) than in controls (89.3 +/- 29.1). These antibodies levels were higher in limited subset than in diffuse (p < 0.05) and in early SSc than in advanced SSc (p < 0.05). The highest values of parameters of oxidative stress are found in the early stages, when the episodes of reperfusion after ischemic episodes (Raynaud's phenomenon) are very frequent. Moreover, the damage is higher in the early stages of SSc, with intact microvessels, than in late stages, when microvessels are very reduced in number, destroyed by the worsening of the disease. These data show that the respiratory burst deduced by the lipoperoxidation is higher in SSc than in controls, and may be an important pathogenetic factors involved in tissue changes in SSc.