Quantitation of oxidative damage to tissue proteins.

Active oxygen species are thought to be involved in many physiological and pathological processes and are known to oxidatively modify DNA, lipids and proteins. One such modification is the addition of carbonyl groups to amino acid residues in proteins. The number of carbonyl groups on proteins can be quantitated spectrophotometrically using 2,4-dinitrophenylhydrazine (DNPH). The DNPH assay described in the literature was found to be unreliable in samples containing high amounts of chromophore (e.g. hemoglobin, myoglobin, retinoids). By using an HCl-acetone wash, hemes from the chromophores could be extracted, enabling the determination of carbonyl content to be made even in highly colored tissue extracts. Residual DNPH, which was also found to interfere with the assay, was removed by additional washes with trichloroacetic acid and ethanol-ethylacetate. These improvements are known to remove lipids, do not lengthen the time required to do the assay, permit quantification of carbonyl content in 1-4 mg protein from a variety of tissue types and provide a sensitive and reliable method for assessing oxidative damage to tissue proteins.

[Collagen metabolism disturbances with experimentally increased pulmonary flow--experimental model of left to right shunt with congenital heart defects in children]. / Zaburzenia metabolizmu kolagenu w doswiadczalnie wywolanym zwiekszonym przeplywie plucnym--model eksperymentalny przeciekowych wad wrodzonych serca u dzieci.

The studies carried out on young piglets were to demonstrate that experimentally increased pulmonary flow resulted in collagen hyperproduction in the pulmonary tissue. In 14 piglets a modified Blalock-Taussig anastomosis was performed, 9 animals constituted the controls. The survivors included 9 experimental and 8 control piglets. In direct lung biopsies the biochemical collagen content was assessed, whereas histopathology confirmed the development of vascular lesions characteristic for pulmonary hypertension. A significant increase of collagen level in the pulmonary tissue was demonstrated in experimental animals. Determinations were also made of serum and urine hydroxyproline values. A significant increase was observed in serum and urine hydroxyproline values in experimental animals in comparison to the controls when determinations were made 7 days to 3 months after the anastomosis had been performed (p < 0.01). The authors showed that an increase of pulmonary flow in piglets results in collagen metabolism disturbances which are seen both in an increased collagen level in the tissue and in increased serum and hydroxyproline levels.

Development of an in vivo rat screen model to predict pharmacokinetic interactions of CYP3A4 substrates.

With the advent of polytherapy, drug interactions have become a common clinical problem. Although in vitro data are routinely used for the prediction of drug interactions, in vitro systems are not dynamic and sometimes fail to predict drug interactions. We sought to use the rat as an in vivo screening model to predict pharmacokinetic interactions with ketoconazole. The pharmacokinetic studies were conducted following an oral dose of CYP3A substrates and an optimized oral regimen of ketoconazole. In vitro reaction phenotyping was conducted using individual human and rat cDNA-expressed CYP enzymes and human or rat liver microsomes in the presence of ketoconazole. The in vitro experiments indicated that the test compounds were largely metabolized by CYP3A in both human and rat. The compounds could be rank-ordered with respect to the increase in C(max) and area under the curve (AUC) values relative to midazolam in the presence of ketoconazole. The degree of pharmacokinetic interaction with ketoconazole was dependent, in part, upon their in vitro metabolism in the presence of rat CYP3A1/3A2 and in rat and human microsomes, co-incubated with ketoconazole, and on their fraction metabolized (f(m)) in the rat relative to other disposition pathways. Based on the rank-order of interaction, the compounds could be prioritized for further preclinical development.