Acceleration of lipid peroxidation in alpha-tocopherol transfer protein-knockout mice following the consumption of drinking water containing a radical initiator.

To assess the antioxidative role of vitamin E (VE) in a mouse model of severe VE deficiency by using biomarkers, alpha-tocopherol transfer protein (alpha-TTP(-/-))-knockout mice were maintained on a VE-deficient diet for 28 weeks [KO group, n = 6]. Wild-type C57BL/6 mice were maintained on a diet containing 0.002% alpha-tocopherol [WT group, n = 6]. The animals were housed individually in a metabolic cage from the age of 9 weeks (Week 0) to 27 weeks. Urine was collected every week, and the levels of total hydroxyoctadecadienoic acid (tHODE), 7-hydroxycholesterol (t7-OHCh), and 8-iso-prostaglandin F(2alpha)(t8-isoPGF(2alpha)), which are biomarkers for lipid peroxidation, were measured by gas chromatography (GC)-mass spectrometry. From the age of 21 weeks (Week 12), three mice in each group were provided drinking water containing the water-soluble radical initiator 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH) until the end of the study (Week 19). Blood and tissue samples were collected, and the levels of the abovementioned biomarkers therein were assessed. AIPH consumption clearly elevated the plasma and erythrocyte levels of tHODE and t8-isoPGF(2alpha) in both the WT and KO groups except for the erythrocyte level of tHODE in the WT group. Furthermore, this elevation was more prominent in the KO group than in the WT group. Interestingly, AIPH consumption reduced the stereoisomer ratio of HODE (ZE/EE), which is reflective of the efficacy of a compound as an antioxidant in vivo; this suggests that free radical-mediated oxidation reduces the antioxidant capacity in vivo. The urine levels of tHODE, t7-OHCh, and t8-isoPGF(2alpha) tended to increase with AIPH consumption, but these individual levels fluctuated. It was clearly demonstrated by the proposed biomarkers that maintaining alpha-TTP(-/-) mice on a VE-deficient diet results in a severe VE deficiency and promotes lipid peroxidation.

Effect of high-dose statin treatment on plasma concentrations of endogenous nitric oxide synthase inhibitors.

Asymmetric dimethylarginine (ADMA) and monomethylarginine (LMMA) are endogenous inhibitors of nitric oxide synthase. A high level of ADMA in plasma has shown to be a significant risk factor for acute coronary syndromes and elevated plasma ADMA levels are prevalent in patients with hypercholesterolemia. It was therefore hypothesized that lowering plasma cholesterol levels with statin treatment would also lower ADMA concentrations. This double-blind study addressed the effect of high-dose statin treatment on plasma levels of ADMA and LMMA. Forty-eight subjects with mild hypercholesterolemia were randomly assigned to receive simvastatin 80 mg/d, atorvastatin 40 mg/d, or placebo for 8 weeks. Both statins decreased low-density lipoprotein cholesterol effectively (simvastatin 54% and atorvastatin 49%). However, concentrations of arginine derivatives remained unchanged during statin treatment and did not correlate with cholesterol levels. This study indicates that statin treatment has no clear influence on plasma ADMA or LMMA concentrations.

Plasma pharmacokinetics of a liver-selective nitric oxide-donating diazeniumdiolate in the male C57BL/6 mouse.

1. The single-dose plasma pharmacokinetics of O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) following intravenous (i.v.) and intraperitoneal (i.p.) bolus administration to the male C57BL/6 mouse was studied in an effort to characterize the disposition of the agent and to serve as a basis for the design of in vivo efficacy studies. 2. Plasma V-PYRRO/NO concentrations declined rapidly in a bi-exponential manner after i.v. administration of 5 mg kg(-1) body weight to mouse. The terminal half-life was 9.4 min and the mean residence time was 3.4 min. 3. V-PYRRO/NO was absorbed rapidly following i.p. administration, with peak plasma concentrations being observed 3 min after injection. Levels then declined with a terminal half-life of 11.7 min. The bioavailable fraction from the i.p. compartment was 19%, indicating a high first-pass effect. 4. The results provide additional evidence for a liver-selective metabolism of this nitric oxide-donating prodrug.

O/W lipid emulsions for parenteral drug delivery. II. Effect of composition on pharmacokinetics of incorporated drug.

The potential usefulness of O/W lipid emulsions as injectable drug delivery systems for lipophilic drugs was examined using a model lipophilic drug, sudan II (clogP = 5.4) in the normal rats. The standard lipid emulsion composed of soybean oil and egg yolk phosphatides increased the blood concentration of sudan II after i.v. injection when compared with its solubilized solution by plasma. However, it was still lower than that of the oil particles, and the distribution of sudan II to liver, lungs, adipose tissue, heart, and muscle was not altered, and only that to brain and kidneys was decreased. Herein, the effect of extensive alterations in the lipid emulsion composition on the blood concentration and organ distribution of sudan II was examined in comparison with the standard formulation. Addition of cholesterol, use of pure egg yolk phosphatidylcholine, use of phospholipids with saturated alkyl chain, use of saturated long chain triglycerides, and use of saturated medium chain triglycerides were tested. The oil particles of all tested lipid emulsions were still located in plasma space, and use of saturated medium chain triglycerides was the most effective way to increase blood concentration of sudan II, resulting in higher distribution to liver, lungs, spleen, and brain. This was caused by the increase of the steady-state partition of sudan II to the oil particles, and not by alteration of their organ distribution clearance.

In vitro oxidation of alpha-tocopherol (vitamin E) in human platelets upon incubation with unsaturated fatty acids, diamide and superoxide.

Incubation of human blood platelets in vitro in Tyrode solution with unsaturated fatty acids, diamide or superoxide (generated in situ) resulted in the oxidation of tocopherol in the platelets. Arachidonate concentrations of (3-5).10(-4) M caused a 50% decrease in platelet alpha-tocopherol. The addition of saturated fatty acids or platelet-active substances such as ADP, dibutyryl cyclic AMP, and some prostaglandins, or peroxidizing agents such as hydrogen peroxide and tert-butylhydroperoxide to the incubation medium did not cause any change in platelet tocopherol content. During incubations of platelets with arachidonate, malonaldehyde as well as alpha-tocopherolquinone were produced. The latter was also produced during incubations with diamide or superoxide. The oxidation of tocopherol induced by unsaturated fatty acids may be one factor responsible for the well-known increase in dietary vitamin E requirements induced by polyunsaturated fatty acids. The oxidative consumption of tocopherol in the membranes could be expected to take place during localized release of oxidants such as superoxide and polyunsaturated fatty acids during normal biological function (e.g., phagocytosis) or pathological processes (e.g., ischemia). Tocopherol utilization is kept low probably by the regeneration of the compound by vitamin C and/or the preferential utilization of the other biological antioxidants.

The effect of multiple exchange transfusions on bilirubin binding.

Three bilirubin binding tests (hydroxybenzene-azobenzoic acid dye binding method, the estimation of unbound bilirubin by horseradish peroxidase assay and the saturation of albumin by the salicylate saturation index) were performed on pre-exchange samples of blood and repeated 24 hours after the procedure. No significant improvement in bilirubin binding was found even in infants receiving as many as four exchange transfusions. Based on these bilirubin binding tests, we find no evidence that the criteria for subsequent exchange transfusions should be different from the first exchange transfusion.

Differential serum protein binding of benzidine- and benzidine-congener based dyes and their derivatives.

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3'-dimethylbenzidine, and 3,3'-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3'-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3'-dimethoxybenzidine-based dye), Direct Black 38 (a benzidine-based dye), and Evans Blue (a 3,3'-dimethylbenzidine-based dye) all bound to albumin, alpha 1-lipoprotein, beta-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and alpha 1-antichymotrypsin, and degraded C3 globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3'-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of alpha 1-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3'-dimethylbenzidine to both alpha 1- and beta-lipoproteins.

The effect of ethanol on the passive Ca permeability of human red cell ghosts measured by means of arsenazo III.

Ethanol in the range of 0.76-2.40 M caused an immediate increase in the Ca permeability of the plasma membrane of resealed human red blood cell ghosts in which intracellular free Ca could be continuously monitored by means of the Ca chromophore arsenazo III. At a given concentration of ethanol, the Ca permeability increased markedly a few minutes following the mixing of the ghosts and the ethanol, and continued to increase over at least the next 30 min. Preincubating the ghosts in ethanol for 15, 60 and 120 min before measuring the rate of free Ca accumulation, progressively increased the effect of a given concentration of ethanol. These results indicate that the effect of a given concentration of ethanol is a complex function of concentration and exposure time. The effects of ethanol in this concentration range were completely reversible. The resealed ghosts used in these experiments were depleted of ATP to avoid interference from the Ca pump and all experiments were carried out with 150 mM KCl on both sides of the membrane to minimize changes in either the volume or membrane potential associated with activation of the Ca-dependent K channel.

Characterization of microenvironments of 2-(4'-hydroxyphenylazo)benzoic acid bound to bovine serum albumin by studying the solvent effects.

The binding and thermodynamic parameters of the two binding forms of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin were estimated. The number of the binding sites of the azo form was 3.2 and that of the hydrazone form was 0.7. The binding of the hydrazone form was affected only by enthalpy changes, in contrast to that of the azo form which was affected by both enthalpy and entropy changes. To estimate the complex microenvironments of 2-(4'-hydroxyphenylazo)benzoic acid molecules on bovine serum albumin, the solvent effects were studied. The 2-carboxyl group of 2-(4'-hydroxyphenylazo)benzoic acid participates in the azo-hydrazone tautomerism . The 2-carboxylate ion forms an ion pair with triethylamine in ethylene dichloride, chloroform, and benzene, resulting in the appearance of the hydrazone form. The hydrazone formation in the system of 2-(4'-hydroxyphenylazo)benzoic acid-triethylamine (1:1) in chloroform was affected only by enthalpy changes, in the same manner as in the system of 2-(4'-hydroxyphenylazo)benzoic acid-bovine serum albumin. We speculate the presence of the two kinds of ion pairs on the basis of the changes of the azo-hydrazone tautomerism in chloroform, and that the azo form takes the contact ion pair and the hydrazone form takes the solvent-separated ion pair. A new possible model for the interaction of the azo and hydrazone forms and bovine serum albumin is proposed.

Drug binding defect of uraemic plasma: unlikely involvement of carbamoylated albumin.

Carbamoylation of bovine and human albumin in vitro decreased the binding of methyl red and salicylic acid. Charcoal extraction of the carbamoylated albumin under acid conditions produced some decrease in the degree of carbamoylation, but did not substantially improve the binding of methyl red and salicylate. Albumin from rats with glycerol-induced renal failure showed no significant degree of carbamoylation compared to controls. Carbamoylation is not responsible for the binding defect of uraemic rat plasma, nor is likely to be involved in the case of human uraemic plasma.

[A modified dye dilution method to estimate leakage during regional isolated perfusion of the extremity (author's transl)]. / Eine modifizierte Farbstoffverdünnungsmethode zur Uberprüfung der Kreislaufisolierung der Extremität während regionaler perfusion.

In experiments on dogs (n=13) we tested a procedure for estimating leakage during regional perfusion of the extremity by means of a dye dilution method. After systematic application of 0.5% Evans blue solution (0.1 ml/kg b.w.) we measured the dye concentration in plasma by means of a spectral photometer. The plasma volume (5.5 ml/100 gb.w.) and the disappearance rate of the dye (10%/h) were calculated. In a second procedure the supplying artery and vein of the extremity were proximally clamped and distally connected to an extracorporeal circulation unit consisting of oxygenator, pump, and heat exchanger, and the isolation of the extremity was tested. By simulated of leakage it was possible to detect a little amount of shunt of about 1% escaping from isolated region into the systemic circulation. There were no hints to toxicity when the same dye concentration. There were no hints to toxicity when the same dye concentration was applied. The standardized method was using during 132 cytostatic hyperthermic perfusion in man. In 20 patients we determined shunts of less than 5%, in 104 patients shunts between 5 and 10%, and in eight patients shunts of 10-20% of the extracorporeal circulation. The benefits of the described method are simplicity to carry out and missing of toxicity or radiation.

A one year feeding study with carmoisine in rats.

Rat fed carmoisine (C.I. (1956) No. 14720) at 0.35, 0.8 or 2.0% in the diet for 52 weeks showed no adverse effects of the dye when compared to control animals. Mortality, weight gain, hematological values and relative organ weights were the same in control and treated animals. There was no increase in tumor incidence due to carmoisine treatment. Male rats fed 2.0% carmoisine showed an increased incidence of several mild subclinical conditions (minimal bronchitis and tracheal inflammation). Because of this occurrence, the maximum no-effect level after 1 year exposure appears to be 0.8%, equivalent to a daily intake of approx. 400 mg/kg.