Diagnosing and quantification of acute alcohol intoxication--comparison of dual-energy CT with biochemical analysis: initial experience.

PURPOSE: To quantify the correlation between fat content of an acute alcohol intoxication and the difference of computer tomography attenuation value in dual-energy CT in comparison to biochemical triglyceride analysis and to evaluate qualitatively the value of DECT in the diagnosis of fatty liver caused by ethanol-dosage in rats. MATERIALS AND METHODS: DECT at 140 kV and 80 kV was performed on 20 rats before and two days after the administration of 3 ml of 50% ethanol. The CT attenuation value in the livers at 140 kV, 80 kV and the differences between them in Hounsfield units (ΔH) were collected. Parts of the liver (100 mg) were measured in biochemical triglyceride analysis as the reference standard. A blood sample was also taken to measure specific liver enzymes. RESULTS: Linear correlation between biochemical triglyceride analysis and CT density of ΔH was found (r=0.949). 140 kV attenuation data were between 44 HU and 61.3 HU, 80 kV attenuation data were between 58.4 HU and 64.7 HU, and ΔH data were between 3.4 HU and 14.4 HU (p≤0.037). The biochemical triglyceride analysis data were between 7.1 mg/g and 41.1 mg/g. The hepatic enzymes serum aspartate (ASAT) aminotransferase and alanine aminotransferase (ALAT) were elevated in all rats. ASAT correlated directly with ΔHU (r=-0.86). CONCLUSION: DECT provides a non-invasive method to determine and evaluate hepatic fat content after acute alcohol intoxication. It provides the possibility to detect and quantify the hepatic fat content of liver graft.

Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite. Stopped-flow measurements prove ferryl intermediates.

Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett. 382, 101-104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem. 2, 259-269]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450BM-3 (wild-type and F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 microM of PN and was half-maximal between 100 and 150 microM of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2-3 microM. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Lack of direct connection between arachidonic acid release and prostanoid synthesis upon differentiation of U937 cell.

The changes in AA incorporation and release as well as prostanoid synthesis upon differentiation of human premonocytic cell line, U937, induced by three functionally diverse agents--phorbol ester (TPA), dimethyl sulfoxide (DMSO), and retinoic acid (RA) have been investigated. The rate of AA incorporation into the cells remained unchanged whereas a 3- to 6-fold increase in AA release upon stimulation with Ca(2+)-ionophore A23187 as compared to undifferentiated cells was observed. While undifferentiated cells were incapable to metabolise AA via the cyclooxygenase pathway all three types of differentiated U937 cells produced TxB2 and PGE2. Only TPA-differentiated cells responded with a 6-fold increase of prostanoid synthesis after A23187 stimulation, whereas in DMSO-differentiated cells prostanoid synthesis was slightly stimulated by A23187 and in RA-differentiated cells it was not stimulated at all. Thus, agonist-induced prostanoid synthesis in differentiated cells is dependent on the nature of differentiating agent and does not correlate with AA liberation.

Prostaglandin H synthase of mouse macrophages: inhibiting and activating action of ibuprofen.

The effect of low (10(-10)-10(-14 M) ibuprofen concentrations on the release of labeled arachidonic acid metabolites by mouse peritoneal macrophages containing a constitutive isoform of prostaglandin H synthase was investigated. It was found that during the activation the cells metabolized AA through the cyclooxygenase pathway, synthesizing PGE2 (110 +/- 10 ng per 10(6) cells), PGF(2 alpha) (120 +/- 15 ng per 10(6) cells), and TxB2 (48 +/- 5 ng per 10(6) cells). Incubation of the macrophages with 10(-12) M ibuprofen leads to a sharp increase of PGf2 and PGF(2 alpha) synthesis (315 +/- 84 and 320 +/- 20 ng per 10(6) cells, respectively). A microsomal preparation of macrophage PGH-synthase was compared with PGH-synthase of ram seminal vesicles and analogy of their properties was shown (Km = 5.3.10(-5) M, IC50, ibuprofen = 15 microM). Some particularities of the PGH-synthase consecutive isoform (PGH-synthase-1) cellular regulation are discussed.