Giacinto Abele Donato Miggiano

Prooxidant action of desferrioxamine: enhancement of alkaline phosphatase inactivation by interaction with ascorbate system

Desferrioxamine (DFO) nearly doubles alkaline phosphatase oxidative inactivation by the ascorbate system. The effect is dependent on ascorbate and desferrioxamine concentrations, exhibiting in both cases a saturation mechanism. Conversion of desferrioxamine to ferrioxamine abolishes the prooxidant action. Desferrioxamine also increases ascorbate-dependent oxygen consumption and nitroblue tetrazolium reduction. Superoxide dismutase, which blocks the desferrioxamine enhancing effect on enzyme inactivation, markedly slows down nitroblue tetrazolium reduction as well as oxygen consumption by ascorbate plus desferrioxamine, while it fails to protect against the ascorbate system alone. Therefore, in the presence of desferrioxamine, the metal-catalyzed ascorbate autooxidation becomes superoxide-dependent and thus inhibitable by superoxide dismutase. Catalase, peroxidase, and ascorbate oxidase protect alkaline phosphatase from inactivation by both ascorbate and ascorbate-desferrioxamine systems. Hemin shields the enzyme from ascorbate plus DFO attack but not from ascorbate alone. In air-saturated solution, desferrioxamine seems to mediate one electron transfer from ascorbate to oxygen, generating superoxide anions, which can either trigger a Fenton reaction or produce desferal nitroxide radicals. In the absence of oxygen, ascorbate alone is ineffective, but the ascorbate plus desferrioxamine system still inactivates the enzyme; catalase, peroxidase, and ascorbate oxidase, but not superoxide dismutase, afford protection.

Digestive and nutritional consequences of pancreatic resections. The classical vs the pylorus-sparing procedure.

SummaryDigestive and nutritional alterations are a common occurrence after pancreatic resections. The authors report the results of a multiparametric evaluation performed in a group of 26 patients submitted to total or cephalic pancreatectomy. Patients were divided into two groups according to the surgical procedure; group A(n=13) included gastroresected patients and group B (n=13) included those submitted to pylorus-sparing pancreatic resection. Subclinical digestive and absorptive impairment has been found in 61.5% of group A patients; the nutritional status was clinically poor in four cases from the same group. Digestive alterations have also been found in 69.2% of group B cases, but nutritional status was always satisfactory in the whole group. The more positive results obtained with the pylorus-sparing technique encourage wider adoption of this procedure.

Antioxidant effect of coenzyme Q on hydrogen peroxide-activated myoglobin

SummaryIn recent years increased attention has been focused on the reduced forms of coenzyme Q as antioxidant compounds inhibiting lipid peroxidation in model systems and in biological membranes, but in spite of extensive experimental evidences the molecular mechanisms responsible for the antioxidant activity of ubiquinones are still debated. Ferrylmyoglobin and/or its free radical form are regarded as powerful oxidizing agents capable of promoting oxidation of essential cellular constituents, particularly cell membranes. Therefore, we investigated the effects of ubiquinol on the formation and survival of ferryl species of myoglobin and on metmyoglobin itself. The addition of a threefold molar excess of hydrogen peroxide to a solution of metmyoglobin induces the rapid formation of a compound with the spectral characteristics of ferrylmyoglobin. The reaction is complete within 4 min, producing up to 76% of ferrylmyoglobin, which remains stable for at least 30 min. The addition of ubiquinol-1 to the same solution provokes a rapid and progressive reduction of ferrylmyoglobin to metmyoglobin and oxymyoglobin. Ubiquinol-1, furthermore, is also capable of protecting metmyoglobin against oxidation when added in the solution before hydrogen peroxide. Ubiquinol-1, indeed, is effective at both limiting the maximal ferrylmyoglobin level attained (59% inhibition) and accomplishing complete removal of the ferryl form (in about 15 min). The results demonstrate that ubiquinol is capable of reducing both ferrylmyoglobin and metmyoglobin to oxymyoglobin, providing a novel antioxidant mechanism for coenzyme Q.

Dietary intake of macronutrients and fiber in Mediterranean patients on chronic hemodialysis.

BACKGROUND We aimed to measure the dietary intake of calories, proteins, carbohydrates, lipids and fiber in patients on chronic hemodialysis (HD) at 3 centers in 1 metropolitan and 2 urban areas of Italy, and to evaluate whether it met the dietary guidelines for cardiovascular risk reduction. METHODS Daily dietary intake was assessed through a 3-day diet diary in 128 HD patients at the hemodialysis units of the Catholic University of Rome, Hospital A. Murri of Jesi and Hospital Principe di Piemonte of Senigallia, Italy. RESULTS Mean dietary calorie and protein intakes were 22.9 ± 9.1 kcal/kg per day and 0.95 ± 0.76 g protein/kg per day, respectively. Daily carbohydrate and lipid intakes as a percentage of total calorie intake were 51.8% ± 8.9% and 32.1% ± 7.1%. Mean daily dietary cholesterol intake was 206.6 ± 173.6 mg. Mean daily dietary intakes of omega-3 and omega-6 fatty acids were 0.49 ± 0.28 g and 5.1 ± 2.5 g, respectively, while the mean ratio of omega-6 to omega-3 intake was 11.5 ± 4.8. Forty-eighty percent of patients had an omega-6 to omega-3 ratio =10. Mean daily dietary intakes of saturated fatty acids (SFAs), monounsaturated fatty acids and polyunsaturated fatty acids were 5.5 ± 3.3 g, 28.9 ± 9.1 g and 3.1 ± 1.7 g, respectively. Ninety-six percent of HD patients had an SFA intake <10% of total calories. Most unsaturated fatty acids intakes were under the value of =30%. Mean daily dietary fiber intake was 11.8 ± 6.1 g. CONCLUSION In HD patients from a Mediterranean country (Italy), daily intakes of calories, proteins and fiber were lower than the recommended values, whereas the intake of lipids was closer to being adequate.

Characterization of alkaline phosphatase inactivation by ascorbic acid

Ascorbic acid, isoascorbic acid and dehydroascorbic acid inhibit bovine kidney alkaline phosphatase activity. Ascorbic acid free radicals seem not to be involved. Dialysis does not make the inactivation reversible. A competitive mechanism can be inferred from experiments with phosphate and substrates, which block the activity decay. The influence of temperature, pH, other inhibitors and tertiary structure modifications on the inactivation process is also investigated.

Mixed function oxidation and enzymes: kinetic and structural properties of an oxidatively modified alkaline phosphatase.

No major structural alteration of alkaline phosphatase can be observed in the early stages of enzyme oxidative inactivation by the ascorbate model system. Fluorescence changes of protein-bound 8-anilino-1-naphthalenesulfonic acid suggest, however, that localized modifications take place. Oxidized alkaline phosphatase displays less catalytic efficiency (decrease of Vmax), while retaining the other kinetic properties, including the same affinity for substrates and inhibitors and the same activation energy of the native enzyme. Typical features of the modified protein are a decreased thermal stability and a biphasic heat inactivation profile, which make the oxidized form quite similar to aged enzymes. The lower response to Mg2+ activation indicates that the magnesium binding sites of alkaline phosphatase are probably the targets of the ascorbate system oxidative modifications.

Free radical production by activated haem proteins: protective effect of coenzyme Q.

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (MbIV and/or HbIV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (MbIV and/or HbIV) back to met and oxy forms (Mb and/or HbIIO2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

Nutrition and IBD: Malnutrition and/or Sarcopenia? A Practical Guide

Malnutrition is a major complication of inflammatory bowel disease (IBD). This mini review is focusing on main determinants of malnutrition in IBD, the most important components of malnutrition, including lean mass loss and sarcopenia, as an emerging problem. Each one of these components needs to be well considered in a correct nutritional evaluation of an IBD patient in order to build a correct multidisciplinary approach. The review is then focusing on possible instrumental and clinical armamentarium for the nutritional evaluation.

Fluorescence studies of ascorbate―melittin interaction

Abstract Tryptophanyl fluorescence quenching by ascorbate has been studied in monomeric and tetrameric forms of melittin. The strong efficiency of ascorbate quenching is inconsistent with a collisional type of mechanism. Moreover, from absorption spectra alteration, temperature dependence of the quenching, lack of diffusion control and the unchanged fluorescence lifetime, it is demonstrated that the mechanism of the process is static (formation of a complex) and not dynamic. Thermodynamic parameters suggest that polar interactions mostly contribute to the formation of the complex (negative ΔH value) but that some hydrophobic interactions also occur in the stabilization of the complex. The bell-shaped relationship of the association constants vs. pH indicates that monoprotonated melittin is quenched more easily and that the active species is the reduced ascorbate monoanion. It can be inferred from the pK values (about 6.8 and 8.0) that the involved ionizable residues are probably Lys-21 and Lys-23. Polarization experiments reveal that ascorbate induces a large phosphate-like effect on melittin self-association, increasing the degree of structural organization of the peptide.

Biochemical properties of alkaline phosphatase from endometrial cancer cells

The occurrence of alkaline phosphatase (AP) activity was examined in several human endometrial adenocarcinomas. Catalytic activities were detectable only in 10 out of 15 tumors, with no apparent correlation between elevated AP and histological type. The apparent molecular weight of the enzyme after partial purification was about 140,000 daltons. Kinetic activity, thermodynamic properties and the pH dependence of the activity were in the ranges reported for other subforms. Several other physicochemical properties were also investigated and compared with those displayed by enzymes obtained from normal human tissues. The inhibition studies show that the enzyme shares several properties with the placental form, particularly in resistance to zinc chloride and EDTA action. On the other hand, in sensitivity to uncompetitive inhibitors and to urea and ascorbic acid, it is closer to other non-Regan heat-sensitive forms. The results support the view that a polymorphism in the expression of AP in neoplastic tissues can occur. A wider spectrum of physicochemical properties is clearly needed to define better the characteristics of oncodevelopmental enzymes.