Isolation, characterization, and evaluation of metallothionein in renal transplant patients.

The plasma metallothionein concentration was studied in renal transplant patients. These patients are submitted to an attack of free radicals catalyzed by metals such as copper, zinc, cadmium, and others. The function of metallothionein is to bind toxic metals inhibiting the attack of free radicals and oxidative stress that patients receiving renal transplants are submitted to. This is the reason for studying this protein in this work. The metalloprotein was separated from the plasma by thermoprecipitation and molecular exclusion chromatography with Sephadex G-75 followed by anionic-ionic exchange chromatographic purification with a CINa gradient. Identification was done by SDS electrophoresis in acrylamide gel with markers and commercial protein. Finally, determinations were made by atomic absorption, silver saturation method. In this work, determinations were made in the plasma of 11 patients before and 48 h and 1 and 2 weeks after renal transplantation. The same study was carried out in parallel in a control group of 11 blood donors. The results obtained show the existence in the plasma of metallothionein, with lower concentrations in patients than in controls (19 +/- 1.2 mg vs. 12 +/- 1.2 mg). The levels were lowest in the patient group analyzed 48 h after having received the transplant (6.34 mg) and had recovered slightly one and two weeks later.

Study of the activity of glutathione-peroxidase, glutathione-transferase, and glutathione-reductase in renal transplants.

The aim of this work was to study the temporare variation of oxidative stress in the plasma and erythrocytes (CR) of renal transplant patients We determined total glutathione (GST), as well as oxidized (GSSG) and reduced (GSH) fractions and the activity of glutathione peroxidase (G-px), glutathione reductase (G-red) and glutathione transferase (GSt). Determinations were performed 48 hours before transplant as well as 1 and 2 weeks after the renal transplant. The results showed a high "oxidative stress" rate, resulting from the equilibrium between the production of free radicals and the activity of antioxidants, the former being higher proportionally. Immediately after the transplant, there was an increase in oxidative stress, which resulted in an increased G-red, a marked decrease in plasma and in erythrocyte G-px (CR9 and an abrupt drop both in GST levels in plasma and in GSG (as well as in the [GSH]/[GSSG] relationship). Thereafter there was a significant improvement in the activity of antioxidant enzymes, but without normalization; the total glutathione levels and the activity of various enzymes approached the average values of the control group.

Antioxidant enzyme determination and a study of lipid peroxidation in renal transplantation.

The aim of this study was to evaluate the antioxidant enzymes, catalase (CAT) and superoxide dismutase (SOD), in renal transplant patients and at the same time to report on the degree of lipid peroxidation observed in these patients. In order to do so we measured erythrocyte malondialdehyde (MDA), a product of lipid peroxidation. These measurements were made at different times: before the transplant, 48 h after the transplant, a week after the transplant and two weeks after the transplant. The values reported were compared with a control group. The results showed that there was a higher MDA level in the transplant group than in the control group one week after the transplant. In addition, two weeks after the transplant, the activities of CAT and SOD were higher in the transplant group than in the control group.

Oxidative stress in chronic renal failure.

This study focuses on the oxidative stress in patients with severe chronic renal failure who are not undergoing dialysis treatment. The erythocyte levels, creatinine clearing and plasma- and cell activities of the following enzymes were determined: glutathione peroxidase (Gpx), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), and glutathione transferase (GT). The concentrations of non-enzyme molecules such as total glutathione in both oxidized and reduced forms, and malonyldialdehyde (MDA) were also measured. The obtained values were compared with those in healthy blood donors of comparable age and social status. The results indicate that chronic renal patients have lower glutathione levels and reduced activities of glutathione peroxidase and of glutathione reductase, while exhibiting elevated levels of malonyldialdehyde and activities of superoxide dismutase, glutathione transferase, and catalase. Finally, creatinine clearing was found to be correlated (p < 0.001) to total (oxidized and reduced) glutathione, Gpx and MDA. These observations may serve to establish a simple protocol for evaluation of renal function.

Oxidative stress and enzyme activity in ambulatory renal patients undergoing continuous peritoneal dialysis.

Some parameters indicating the possibility of oxidative stress in chronic renal failure patients (CRF) undergoing continuous ambulatory peritoneal dialysis (CAPD), are presented in this study. Oxidative stress appears to be lower than in hemodialysis patients. In a group of CRF patients and in a control group malondialdehyde (MDA) was determined as an indicator of red-blood-cell membrane lipid peroxidation. Also, the three glutathione forms, total (GST), oxidated (GSSG) and reduced (GSH) and the levels of glutathione peroxidase (GPX), the levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) have been measured. Significantly higher MDA values (p < .05) have been found in CRF patients when compared to controls. The three forms of glutathione and GPX are also significantly decreased in the CRF group (p < .05 and p < .001, respectively). The levels of SOD and CAT are increased with respect to the control group (p < .001).

Catalase activity in erythrocytes from colon and gastric cancer patients. Influence of nickel, lead, mercury, and cadmium.

Catalase (CAT) is an enzyme that is involved in antioxidant defense, cell growth, and is possibly associated with tumoral processes. In this paper, the results of experiments designed to determine the influence of metallic carcinogens such as nickel (Ni), lead (Pb), mercury (Hg), and cadmium (Cd), on CAT activity are reported. CAT activity was measured in erythrocytes from three groups: a group of colon cancer patients, a group of gastric cancer patients before clinical treatment, and a control group of healthy blood donors. Concentrations of this enzyme are significantly higher than controls in the colon cancer group, but lower in gastric neoplasia. By generating highly reactive oxygenated species, Ni, Pb, Hg, and Cd alter catalase activity. Solutions of Ni, Cd, and Pb at 0.2 mM concentrations inhibit CAT activity in colon cancer, but increase it in gastric neoplasia. Hg activates CAT in colon cancer, and causes a slightly increased activity in gastric cancer. No complete deactivation of the enzyme was observed.

Assay for erythrocyte superoxide dismutase activity in patients with lung cancer and effects on pollution and smoke trace elements.

The antioxidative effect of CuZnSOD, which catalyzes the dismutation of superoxide anion (O2-), provides a defense against the oxygen toxicity. The object of the study is to evaluate the erythrocytes superoxide dismutase (SOD) activity in two groups of persons (Group I, healthy blood donors; Group II, lung cancer patients), using the spectrophotometric assay of NADH oxidation and the indirect method. The effect of trace elements, such as Al3-, Cr3+, Fe3+, Hg2+, NI2+, and Pb2+ (producing free radicals oxygen and present in pollution and smoke) is also evaluated. The results show the decrease of SOD activity in lung cancer patients with respect to healthy individuals. Likewise, in those patients the enzymatic activity is bigger in early stage (I,II) with respect to advanced one (III) (p < 0.05). The lesser activity when the samples are incubated with Ni or Pb point out that these metals play a role in neoplasm development. In short, the oxidant-antioxidant balance is altered in lung cancer patients.

Induction and characterization of metallothionein in different organs of Ostrea edulis L.

In this study, the induction of metallothionein (MT) in one species of oyster Ostrea edulis exposed to copper (Cu) and zinc (Zn) was examined. The quantity of total protein (mg protein/mg dry wt) in each sample and the measurement of MT were determined by the Lowry and silver (Ag)-saturation methods. Our results show that the gonads are the organs producing the greatest quantity of MT in the controls and in the groups induced by Cu and Zn, followed by the mantle, gut, and by muscle and plasma. Competition by Zn with respect to Cu for the production of MT has been established.

Purification and analysis of plasmatic components of middle molecular weight in patients with uremic syndrome.

Many hormonal, metabolic and enzymatic alterations have been described in patients with chronic renal failure (CRF) after prolonged hemodialysis treatment. This toxicity has been associated with the accumulation of middle molecular weight components, called 'middle molecules', in their blood. To investigate the chemical nature of these molecules, 20 CRF patients were studied and compared with 10 control subjects. Sera from these individuals were treated with molecular exclusion chromatography, the components of high molecular weight were eliminated from the elution peaks, and the remainder was then concentrated by lyophilization. The zones of greatest concentration were fractioned by ionic exchange chromatography and by HPLC in reverse phase. Infrared spectroscopy was also performed on the most relevant zones, as well as SDS/PAGE. Finally, the amino acid sequence of a peak presenting the best conditions was analyzed. The results indicate that the majority of the compounds isolated are peptides, amino acids or amino alcohols, and that many of these 'middle molecules' are joined to trace metals.

Inhibitory effect of cysteine and methionine on free radicals induced by mercury in red blood cells of patients undergoing haemodialysis.

The inhibitory effects of cysteine and methionine on mercury-induced free radicals were studied in the red blood cells (RBC) of haemodialysed (HD) patients, using polyacrylonitrile membranes. RBC were taken from a control group of 10 healthy subjects and a group of 30 HD patients. The following were determined before and after HD: malonyldialdehyde (MDA), total glutathione (GST), reduced (GSH) and oxidized (GSSG) glutathione, and percentage haemolysis (before HD). RBC were incubated for 6 hr with mercury (Hg(2+); 10(-5)m), mercury and cysteine (0.001 m) and mercury and methionine (0.005 m). The percentage of mercury induced haemolysis differed between the control group and HD patients. The addition of cysteine and methionine to RBC has a markedly inhibitory effect on the mercury-induced haemolysis (a reduction from about 80 to 5% for cysteine and to 15% for methionine). MDA was increased in HD patients and increased further after HD. GST, GSH and GSSG were lower than normal in HD patients, both before and after HD. There was very little change in MDA concentration when the RBC were incubated with mercury for 6 hr.

Erythrocyte catalase activity in chronic renal failure patients and donors in the presence of metal ions.

The effect of hemodialysis on erythrocyte catalase (CAT) activity was studied in chronic renal failure (CRF) patients. This enzyme was analyzed in vitro, and its activity throughout the incubation period was found to be 34% lower than in healthy donors. The influence of Al3+, Cu2+, and Ni2+ on CAT activity in donor's blood (in vitro) is also studied, for short incubation periods at trace levels of 2.5, 0.25, and 0.196 mM, respectively. With Al3+ and Cu2+, there is a decrease in the enzyme activity. With Ni2+, there is at first a similar decrease, followed by a recovery in activity up to control values.

Study of ceruloplasmin in renal insufficiency.

Ceruloplasmin, a copper carrier protein in mammal serum, was isolated by a variation of the Neo-Zgirski method consisting of a chromatography column in DEAE Sephadex A-50 gel, using different buffers for its selective elution. This protein was then precipitated with ammonium sulfate and an ethanol chloroform mixture. Finally, a dialysis against sodium acetate was performed and the samples were purified. In addition, the protein concentration and its characterization were determined. Enzyme kinetics were calculated using paraphenylenediamine as an artificial substrate, determined by the Michaelis constant. Furthermore, an SDS electrophoresis in polyacrylamide gel was performed, and the samples of porcine ceruloplasmin were compared with commercial bovine ceruloplasmin. Finally, a comparative study was carried out between the ceruloplasmin levels in serum from patients with chronic renal insufficiency (CRI) undergoing hemodialysis treatment in artificial kidneys with a cuprophan membrane, with a control group of donors. A similar study was performed with a group of patients suffering from the same illness, but undergoing dialysis treatment with polyacrylonitrile membrane. These results were expressed in mg% in relation to the ceruloplasmin isolated previously.

Comparative analysis of certain metals and tumor markers in bronchopulmonary cancer and colorectal cancers. Metals and tumor markers in the neoplastic process.

Carcinogenic metal levels in serum and tissue samples were measured in patients with bronchopulmonary or colorectal cancer. The cadmium and nickel tissue levels in the patients with lung cancer were significantly higher than in the controls. A statistical correlation was found between chromium and cadmium, as well as between cadmium and nickel in patients with colorectal cancer. In addition, prior to the operation, the tumor markers alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen (Ca 19-9), polypeptide histidio antigen (TPA) and ferritin were analyzed. Their average concentrations were correlated with the existing concentrations of the metals. This was done for both types of cancer. Tumor marker detection showed an increase of CEA and TPA in patients with colorectal cancer. A statistical correlation was observed between AFP and zinc tumor tissue.

Isolation and characterization of chicken muscle ferritin.

Two ferritins (fast and slow) have been found to exist in the chicken muscle. Ferritin was isolated from the muscle by means of a method based on pH changes and saline fractioning, followed by purification in Ultrogel AcA-3A and ultracentrifugation at 100,000 g. Identification of the two ferritins shown in the chromatogram was carried out by electrophoresis in polyacrylamide gel, the typical Prussian Blue band with ferrocyamide appearing in both cases. Ferritin characterization was carried out by means of molecular weight determination, amino acid analysis, number of Fe atoms linked by ferritin molecule and other parameters.

Influence of metallic carcinogenesis in lung and colorectal neoplasia. Metals in neoplastic processes.

We have found changes in the metabolism of trace elements undergoing cancerogenic processes. Using chromatography we have been able to prove that both their distribution among the different plasma proteins and their total concentration in serum and neoplastic tissue are altered. Some differences were found in the values of ceruloplasmin against controls by measuring its oxidizing activity as opposed to O-phenylenediamine. At the same time, using electrophoretic techniques, we have studied the global modifications in the different groups of plasma proteins in neoplastic serum. This report is especially interested in the metabolism of copper, chrome and zinc in serum and tissue in about 20 patients suffering from lung and colorectal neoplasia, in order to compare the parameters which were studied.

[Determination of trace elements, ceruloplasmin and blood proteins in pulmonary neoplasms]. / Determinación de elementos traza, ceruloplasmina y proteínas plasmática en las neoplasias pulmonares.

In cancer process there have been found metabolic changes in several elements, especially in respect to copper. It has been proved that there is an alteration in the serum levels of some metals, and in their distribution in the proteins binding them. On the other hand, there exist, also, ceruloplasmin differences between patients serum and the control. Our work has been carried out to study the trace element metabolism in lung cancer, as well as chromium and zinc contents. Electrophoretic techniques have been applied to study the protein levels.

Comparative study of ferritins from dove Columba oena and chicken Gallus domesticus livers.

A comparative study of ferritin from the liver of doves and chicken has been carried out. In both cases, the molecular weight, the number of iron atoms bounded to the protein and composition of amino acids were determined. The ferritins were isolated by the Luxton method. The identification of both proteins was carried out by acrylamide gel electrophoresis. Ferritin from the dove showed two bands while that from the chicken only one.

Isolation, purification and characterization of spleen ferritin of Gallus domesticus L.

The ferritin from the spleen of the chickens has been isolated by a method of salt fractionation and by a pH change followed by purification in sephadex G-200. 2. The identification of the protein was carried out by acrylamide gel electrophoresis showing a single band. 3. The characterization of ferritin has been made by determination of molecular weight, amino acids analysis and the number of iron atoms (4520) which bound the ferritin. 4. The ferritin from the spleen of chicken is compared with the ferritin from the liver of pigeon.

Serum copper, ceruloplasmin, lactic-dehydrogenase and alpha 2-globulin in lung cancer.

Twenty-six patients with primary lung cancer were studied in whose blood we found a significant increase in the levels of copper, ceruloplasmin, lactic-dehydrogenase and alpha 2-globulins. The role of copper in pulmonary cancerogenesis is discussed.