Robert A. DiSilvestro

Comparison of four indirect methods for fluid superoxide dismutase activities

Relatively small sample dilutions could render fluid extracellular (EC) superoxide dismutase (SOD) activity assays more subject to interfering compounds than tissue SOD assays. Highly variable relative SOD activities were obtained when comparing four indirect assays for several fluid samples (human plasma, human synovial fluid, and plasma from healthy or inflamed rats). Analysis of rat plasma fractionated with Sephadex G-150 showed that each assay (three xanthine oxidase based assays plus a modified pyrogallol assay) detected apparent SOD activity almost entirely at the same molecular weight as rat lung EC SOD. However, unfractionated fluid samples caused interferences with the xanthine oxidase based SOD assays, though not with the pyrogallol method. Example of interference were stimulation of xanthine oxidase activity, color formation without xanthine oxidase, color formation despite excess Cu-Zn SOD addition, and absorbance changes with cyanide inhibition of EC SOD that were above or below blank values. In summary, relative fluid SOD values depended on the assay used, and a modified pyrogallol assay was not subject to several interferences found for three xanthine oxidase based assays of fluid SOD activity.

Influence of dietary copper, copper injections and inflammation on rat serum ceruloplasmin activity levels

Abstract Inflammation hormonally elevates activity levels of the serum copper protein ceruloplasmin (Cp). Marginal copper status could limit this rise to the extent that potential antiinflammatory actions are limited, but not to the extent that Cp activity levels reliably assess copper status during inflammation. This study demonstrated such behavior using rats fed graded amounts of copper (0.3, 3, 6, or 15 ppm Cu). Inflammation produced Cp activity levels inversely proportional to copper intake. However, inflammation in rats fed 3 or 6 ppm copper raised initially low Cp activities to values equal or exceeding, respectively, those of rats fed 15 ppm copper without inflammation. Injection of CuSO 4 (0.5 mg Cu/kg, ip) raised Cp activities after 20 h in inflamed and noninflamed rats from each dietary treatment except the 15 ppm copper. The percent increases tended to be inversely proportional to copper intake. Serum copper responses to copper injections resembled those of Cp activities, but actual percent increases for the serum copper were sometimes different from those for Cp. Possibly, copper status in people with inflammatory diseases could be assessed by Cp activity response to bolus copper administration.

Copper activation of superoxide dismutase in rat erythrocytes

Several lines of evidence suggested that copper can activate a preexisting pool of superoxide dismutase (SOD) apoprotein in erythrocytes from copper-deficient rats. First, feeding adequate copper to copper-deficient rats raised initially low erythrocyte SOD activities to normal values in under one-third the time needed to replace the entire red cell population. Moreover, copper injection (1 mg Cu/kg, sc) doubled erythrocyte SOD activity levels in 16 h. Since protein synthesis is restricted in mature erythrocytes, these results imply that copper activated apoSOD in vivo. Furthermore, injected copper raised SOD activity contents of both young and old erythrocytes. Neither dietary copper status nor copper injection influenced red cell SOD immunoreactive protein levels. In contrast, copper injection increased the amount of copper associated with the SOD activity peak region resulting from gel filtration of hemoglobin-free erythrocyte proteins on Sephadex G-75. Copper ions (3 microM) elevated SOD activity levels in vitro by 63% in 4 h in intact red cells from copper-deficient rats. No activation took place in lysed red cells from the same rats or in intact cells from copper-adequate rats. These results all suggest that copper can activate SOD apoprotein in erythrocytes by a specific, saturable process.

Plasma Levels of Immunoreactive Ceruloplasmin and Other Acute Phase Proteins during Lactation

Abstract Lactation elevates plasma copper as well as oxidase activity levels of the copper-containing, acute phase protein ceruloplasmin (Cp). The present study provides an initial inquiry into the mechanisms behind these changes. Plasma obtained from 12 lactating women, 1 month postpartum, displayed a greater percentage increase in immunoreactive Cp levels (mean increase = 89%) than in plasma copper (mean = 66%) or Cp oxidase activity (mean = 42%). Lactation did not increase plasma content of C-reactive protein or α1-antitrypsin but significantly elevated haptoglobin concentrations. Plasma α2-macroglobulin contents correlated with immunoreactive Cp levels in lactating women but not in controls. These results strengthen the hypothesis that plasma content of individual acute phase proteins is regulated by both overlapping and individualized processes. In addition, the present findings raise the possibility that lactation increases both Cp synthesis and plasma turnover time of Cp-bound copper.

Effects of Inflammation on Copper Antioxidant Enzyme Levels

Inflammation increases plasma levels of ceruloplasmin, a copper protein with possible antioxidant function. This paper describes modulation of these increases by copper intake, and describes combined effects of inflammation and copper intake on Cu-Zn and extracellular (EC) superoxide dismutase (SOD) activities. Turpentine injections in rats fed 1 of 4 copper levels increased ceruloplasmin activities, but values were sensitively limited by copper intake. Cu-Zn SOD activities in the liver, but not in erythrocytes or lungs, were reduced by inflammation in each dietary copper group. Inflammation in rats fed a standard mixed feed diet reduced plasma EC superoxide dismutase activities measured by inhibition of pyrogallol autoxidation. Different results were obtained with 3 xanthine oxidase based SOD assays which were each subject to assay interference. Studies in humans found a group of rheumatoid arthritis patients to possess relatively low erythrocyte SOD and relatively high ceruloplasmin activities. Activity levels of SOD, but not of ceruloplasmin, increased after 4 weeks of copper supplementation (2 mg/day). The fate of cellular Cu-Zn SOD activity contents in inflamed tissues is largely uninvestigated. However, interleukin-1, a hormone released at inflammation sites, elevated Cu-Zn SOD activities in cultured fibroblasts.

An enzyme-linked immunoadsorbent assay for rat ceruloplasmin

A noncompetitive, colorimetric enzyme-linked immunoadsorbent assay (ELISA)† was developed for measuring rat ceruloplasmin in serum and in medium from culture hepatocytes. The assay utilized polystyrene immobilized antibody which bound ceruloplasmin which then bound biotinylated antibody. The biotinylated antibody-antigen complex was detected with strepavidin-alkaline phosphatase conjugate. Standard curves for rat ceruloplasmin were constructed in the range between 10 and 50 ng/mL. Increases of 10 ng produced an increase inA403 of more than 0.2. With this immunoassay, serum ceruloplasmin levels were found to average 35 mg Cp/dL in control rats and 87 mg/dL in rats with experimental inflammation. Liver parenchymal cells secreted 1.6μg Cp/5×105 cells/24h. This ELISA assay for ceruloplasmin will facilitate studies on the regulation of ceruloplasmin synthesis and secretion in both intact rats and isolated hepatocytes.

Enzyme immunoassay for ceruloplasmin: application to cancer patient serum

Ceruloplasmin (Cp), a glycoprotein of molecular weight 132 000 contains most of the copper found in human serum [I]. Cp has been proposed to act as a copper transport agent and to carry out enzymatic functions [2]. Serum or plasma copper concentrations rise during many disease states such as cancer [3], arthritis [4] and infection [3,5] as well as during nonpathological stresses such as pregnancy [5], lactation [7] and chronic exercise [8]. Generally, increases in serum or plasma copper have been found to correspond to elevated levels of Cp oxidase activity toward one of several substrates [3-81. In the case of cancer, Cp oxidase reaches highest levels during the most severe stage of the disease [3]. On the other hand, one study found that successful treatment of several types of cancer was accompanied by a return of Cp oxidase activity concentrations toward normal [9]. Despite these various studies, no functional or diagnostic significance has been firmly established for stress-induced elevations in serum or plasma copper and Cp oxidase activity. It is very possible that the mechanisms regulating activity levels as well as the functional consequences of changes may vary for different stress conditions. Further characterization of the different stress-induced increases in serum copper and Cp oxidase activity levels requires comparison of these parameters with immunoreactive Cp levels. Recently, work in this laboratory [lo] showed that lactation raised immunoreactive Cp concentrations to a greater extent than serum copper or Cp oxidase activity. Immunoreactive Cp levels were measured using commercially available radial immunodiffusion (RID) plates (Calbiochem-Behring Diagnostics). The present paper describes an enzyme-linked immunoadsorbant assay (ELISA) for ceruloplasmin. The ELISA is cheaper and faster than the RID approach and utilizes commercially available antibodies. No conjugation synthesis is required of the user. The ELISA was applied to serum from cancer patients and controls but should be sensitive enough for application to appropriate cell cultures.

Interleukin 1 slowly increases lung fibroblast Cu-Zn superoxide dismutase activity levels.

Abstract Certain pulmonary stress raises rat lung Cu-Zn superoxide dismutase (SOD) activity levels, but cytokines released during stress are reported to exert no regulatory effects on Cu-Zn SOD levels in cultured cells. In contrast, our study found that interleukin 1 (IL-1) can increase Cu-Zn SOD activities in human WI38 lung fibroblasts. The difference in results could be explained by differences in experimental conditions. The increases seen here did not occur during the first 24 hr, but Cu-Zn SOD activities more than doubled by 3 days. In addition, little increase occurred unless the medium was changed at 24-hr intervals. On the other hand, some other potential experimental variables showed little or no effects on IL-1-induced increases in Cu-Zn SOD activities. These variables included IL-1 isoform (α, β, or both), IL-1 concentration (0.5, 2, 5, or 7 units IL-1α/ml medium), and the presence or absence of exogenously added copper as CuO or ceruloplasmin. In addition, combining IL-1 with dexamethasone, a synthetic glucocorticoid that enhances some IL-1 actions, produced only additive, not synergistic, increases in Cu-Zn SOD activities. In conclusion, IL-1, in several different experimental protocols, raised lung fibroblast Cu-Zn SOD activity levels, but only after a 1 day lag time. Stress-induced increases in Cu-Zn SOD activity levels in vivo also tend to occur only after lag times.

Effects of ascorbic acid and inflammation on ceruloplasmin activity levels in guinea pigs

Abstract The possibility that ascorbic acid exerts a postabsorptive effect on copper metabolism in guinea pigs was examined. A pair of ascorbic acid injections (20 mg/kg, ip, 18 h apart) caused a small but statistically significant increase in oxidase activity levels of ceruloplasmin, a serum copper protein. In contrast, dietary ascorbic acid deprivation for 14 days did not prevent increases in ceruloplasmin activity levels caused by turpentine-induced inflammation. The ascorbic acid deprivation did reduce weight gain and adrenal ascorbic acid contents but serum ascorbic acid concentrations were not significantly lowered. In summary, ascorbic acid can produce postabsorptive changes in copper metabolism but adequate intake of the vitamin is not necessary for inflammation-induced elevations of ceruloplasmin activity levels.

Influence of Diet and Inflammation on Serum Copper Enzymes

Copper is believed to function primarily as a catalyst in copper metalloenzymes. A number of these enzymes perform functions which in principle should protect against tissue injury caused by stress such as inflammation. Three types of observations support the notion that copper enzymes work against stress induced tissue damage. First, dietary copper restriction reduces activity levels of several copper metalloenzymes while increasing susceptibility to injury (1). For instance, copper deficient rats show especially high sensitivity to CC1, hepatoxicity (2) and adjuvant arthritis stimulated footpaw swelling (3). A second set of observations implying a protective role for copper enzymes is the linking of many stress states with elevated serum levels of the copper protein ceruloplasmin (4). These elevations have been hypothesized to represent an attempt to minimize stress-induced tissue damage.