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Nutrition Research Division, Bureau of Nutritional Sciences, Food Directorate, Health Canada, Ottawa, ON, Canada K1A 0L2
2To whom correspondence should be addressed. E-mail: kevin_cockell{at}hc-sc.gc.ca.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • protein oxidation • carbonyl • Western blotting • copper deficiency • plasma proteins • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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).3
Loss of antioxidant enzyme activity can contribute to oxidative stress. For example, aorta tissue from copper-deficient rats has decreased Cu,Zn-SOD activity and increased levels of lipid peroxidation, measured as thiobarbituric acid-reactive substances (TBARS) (2
). Plasma lipoproteins and tissue homogenates from copper-deficient rats are more susceptible to in vitro oxidation, compared with controls (3
). The higher levels of ethane expired by copper-deficient than by copper-adequate rats support the hypothesis that increased lipid peroxidation occurs in vivo in copper-deficient rats (4
).
Plasma is easily obtainable from animals and humans and contains both lipid and protein components that may be susceptible to oxidation, making it appropriate to investigate the suitability of plasma oxidation variables as biomarkers of in vivo oxidative stress. Oxidation of plasma proteins may be discernible as an increased content of carbonyl (aldehyde or ketone) adducts on the proteins. Protein oxidation, measured as an increase in dinitrophenylhydrazine (DNP)-reactive carbonyl groups, has been shown to be an early event in oxidative stress in vitro (5
). The increase in protein carbonyls occurs rapidly (within minutes) in cultured endothelial cells exposed to oxidative stress, preceding loss of cellular ATP and eventual cell death (6
). A substantial accumulation of protein carbonyl groups occurred at a stage in oxidative damage in which no change in malondialdehyde levels was apparent in endothelial cells in vitro (6
). Measurement of protein carbonyls has also been used to show accumulation of oxidative damage to proteins over the longer term, for example, in studies of aging (7
). Measurement of protein oxidation offers several advantages over the monitoring of lipid peroxidation, including the early formation and relative stability of oxidized proteins (8
). Oxidative modification of protein has been suggested to be not only a marker for oxidative damage but also a causal factor in oxidative injury (6
). Oxidative damage to proteins may be a more critical pathological event than damage to lipids because enzyme inactivation can have rapid and suprastoichiometric effects, by nature of the catalytic functions of enzymes (9
).
The protein carbonyl content of 
- and ß-spectrin from RBC membranes has been shown to be higher in copper-deficient rats than in controls (10
). Also, the carbonyl contents of unspecified proteins with molecular weights of 90 and 100 kDa were greater in mitochondria of copper-deficient HL-60 cells than in copper-supplemented controls (11
).
The present work was undertaken to test the hypothesis that copper deficiency, through loss of antioxidant enzyme capacity such as plasma Cu,Zn-SOD, would result in oxidative modification of circulating plasma proteins. Previous work in our laboratory had indicated a small but significant decrease in plasma total protein carbonyl content in copper-deficient compared with control rats, measured spectrophotometrically (12
). However, this was opposite to the hypothesized increase in carbonyl content, and it has been suggested that the spectrophotometric method may be less reliable for analysis of protein carbonyls in tissues than in isolated proteins in vitro (13
). Analysis of total protein carbonyl content could not reveal whether there might have been increases in the carbonyl content of some proteins and decreases in others. This has been shown under other circumstances because streptozotocin-induced diabetes in rats was associated with increased plasma protein oxidation evident in some protein bands, but decreased in other bands (14
). We therefore undertook the investigations reported here, using a more sensitive Western-blotting technique (15
) to examine the carbonyl content of electrophoretically separated plasma proteins from copper-deficient and copper-adequate rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Weanling male Long-Evans rats were obtained from Charles River Canada (St. Constant, Canada). Upon arrival, rats were randomly assigned to two groups of 18 rats each, fed sucrose-based modified4
AIN-93G diets (16
) containing normal or low copper levels [-Cu: 0.43 ± 0.03 mg Cu/kg dry diet; +Cu: 6.19 ± 1.24 mg/kg dry diet (mean ± SD), n = 6] for 4 wk. Sucrose-based diets were used to take advantage of the known interaction between dietary carbohydrate source and copper deficiency in male rats, which exacerbates the signs of copper deficiency and results in higher susceptibility of liver and heart tissues to in vitro oxidation (17
). Food consumption by copper-deficient rats was measured daily and control rats were pair-fed at the mean food intake of the deficient group. Body weights were measured weekly and at killing. The experiment protocol was approved by the institutional Animal Care Committee of the Health Products and Food Branch of Health Canada.
Tissue sampling.
Rats were killed at 0800 or 1300 h by exsanguination while under isoflurane anesthesia. Blood was withdrawn from the abdominal aorta and immediately transferred into tubes containing EDTA (Vacutainer, Becton Dickinson, Franklin Lakes, NJ), on ice. Free-flowing blood was collected into heparinized capillary tubes for determination of Cu,Zn-SOD activities in erythrocytes and plasma and selenium-dependent glutathione peroxidase (Se-GSH-Px) activity in plasma (see below). Hearts were removed, cut in half, blotted free of blood and weighed accurately. Livers were removed, rinsed in 0.27 mol/L sucrose (a concentration providing a physiologic level of osmolarity analogous to physiologic saline), blotted dry and divided into seven anatomically distinct portions as described elsewhere (18
). A portion of the EDTA-treated blood was used for hematology (erythrocyte count, hemoglobin concentration and hematocrit) measured on a Coulter S-Plus IV (Coulter Electronics of Canada, Burlington, Canada). The remaining EDTA-treated blood was centrifuged at 1500 x g for 5 min to separate plasma from cellular components. Plasma was stored at -80°C until analysis of plasma protein oxidation (see below). EDTA was used as anticoagulant and the headspace of plasma samples was flushed with nitrogen before storage at -80°C; both of these measures should have minimized artifactual postsampling oxidation of the plasma. Cell pellets were used to prepare washed erythrocytes for 13C-nuclear magnetic resonance (NMR) assays to investigate copper deficiency effects on carbohydrate metabolism (unpublished data). It was necessary to pool blood from two rats to provide enough erythrocytes for NMR spectroscopy. Samples used for evaluation of plasma protein oxidation in this report therefore represent pooled plasma from two rats from the same dietary group.
Assessment of copper status.
Diet and liver copper levels (18
), heart weight:body weight ratios and activity of Cu,Zn-SOD in plasma and erythrocytes (19
) were measured to ascertain the copper status of the rats. Cu,Zn-SOD activity was measured as the inhibition by Cu,Zn-SOD in the plasma or erythrocyte samples of the reduction of cytochrome c by superoxide radical generated using the xanthine/xanthine oxidase system. One unit of Cu,Zn-SOD activity is defined as the activity that inhibits the rate of cytochrome c reduction by 50% under the assay conditions used (19
).
Assessment of plasma protein oxidation.
Protein concentration in plasma samples was assayed using the Abbott QuickStart Total Protein kit (Abbott Laboratories, North Chicago, IL) on the Abbott VP SuperSystem autoanalyzer. For the assessment of protein carbonyl content, plasma proteins were derivatized with DNP before SDS-polyacrylamide gel electrophoresis on 4–12% gradient gels, followed by Western transfer to nitrocellulose filters (15
). Protein equivalent to 0.12 µL of plasma was loaded onto each lane for electrophoresis. DNP-derivatized plasma protein carbonyl groups were sequentially reacted with rabbit anti-DNP and goat anti-rabbit immunoglobulin G (IgG) antibodies (OxyBlot Oxidized Protein Detection Kit, Oncor, Gaithersburg, MD, now supplied by Intergen Company, Purchase, NY) followed by chemiluminescence detection with ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech, Baie d’Urfé, Canada). Total protein staining with amido black was done on duplicate nitrocellulose filters prepared at the same time from the same DNP-derivatized samples. Quantitation of densitometry scans from chemiluminescence detection and of total protein staining was done using a Howtek Scanmaster 3 scanning densitometer and Quantity One software for analysis of 1D gels (PDI, Huntington Station, NY).
Additional analyses.
Samples of diets or livers were dry ashed in a programmable furnace (Model 497, Fisher Scientific, Nepean, Canada) at 450°C using concentrated nitric acid as an oxidizing agent. The ash was dissolved in 2.9 mol/L hydrochloric acid and analyzed for copper by flame atomic absorption spectrophotometry (20
) (Perkin Elmer 5100PC, Perkin-Elmer, Norwalk, CT). Analytical standards were prepared from certified single-element stock solutions (SPEX Chemical, Metuchen, NJ). These analytical methods have been verified in multilaboratory quality control studies (21
) and analysis of NBS Bovine Liver (1577a, National Institute of Standards and Technology, Gaithersburg, MD) gave results within 5% of certified values. Plasma TBARS were assayed by a spectrometric method based on that of Ohkawa et al. (22
). Thiobarbituric acid (TBA) reagent was prepared in 0.1 mol/L NaOH instead of water, which facilitated dissolution and stability of the TBA reagent without adversely affecting the analytical response of the assay. To assess whether copper deficiency affected plasma antioxidant enzymes beyond known cuproenzymes, plasma Se-GSH-Px activity was determined by the automated method of L’Abbé et al. (23
) on an Abbott VP SuperSystem with a 340/380 nm filter (Abbott Laboratories, Mississauga, Canada), using 0.3 nmol/L t-butylhydroperoxide as substrate.
Statistical analyses.
Most results were analyzed using t test for independent samples (P < 0.05) and are presented as means ± SD. Because of unequal variances, hematology results were analyzed using the nonparametric Mann-Whitney U test. All statistical analyses were performed using STATISTICA for Windows, version 5.1 (StatSoft, 1997, Tulsa, OK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Body weight was not affected, indicating the success of the pair-feeding protocol. Hematocrit and hemoglobin concentration were significantly lower in the rats fed the copper-deficient diet, whereas erythrocyte counts did not differ from controls (Table 1)
, indicating a mild copper-deficiency anemia in these rats. Plasma lipid peroxidation, as indicated by plasma TBARS, was not affected by copper deficiency (2.3 ± 0.6 vs. 2.5 ± 0.4 µmol/L plasma, n = 9). Plasma Se-GSH-Px activity was also not affected by copper deficiency (69 ± 10 vs. 73 ± 12 U/g protein, n = 9).
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). Densitometric analysis of several highly stained protein bands supported the conclusion of equal protein loading between lanes (data not shown), although careful inspection of the amido black–stained filters revealed that the intensity of one minor plasma protein band (
145 kDa) was lower in copper-deficient rat plasma (Fig. 1
A).
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B). The most visible of these on the chemiluminescence films had molecular weights (based on comigration of protein standards in electrophoresis) of 145, 125, 80, 55 and 50 kDa. These generally did not correspond to the major bands indicated by amido black staining for total protein (Fig. 1
A). The carbonyl content of four of these plasma proteins, immunodetected after DNP derivatization, was significantly lower in plasma from copper-deficient rats; the carbonyl content of the remaining one also tended to be lower (P = 0.08) (Table 2
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Measurable basal levels of protein carbonyls have been reported in a number of tissues including plasma from control animals in a variety of experiments (25
–27
).
Carbonyl groups can be introduced into proteins by several mechanisms including oxidative cleavage of proteins, direct oxidation of lysine, arginine, proline and threonine residues, reaction with aldehydic products of lipid peroxidation (e.g., hydroxynonenal, malondialdehyde) or through glycation or glycoxidation reactions (7
). Protein oxidation has been noted as a general feature of aging and of many diseases that have been related to oxidative stress (7
). Oxidation of plasma proteins has been demonstrated in experimental models of ischemia-reperfusion injury of rat small bowel (28
), and has been exploited in monitoring ischemia-reperfusion injury after human surgical procedures (8
). Accumulation of plasma protein carbonyls, which has been termed "carbonyl stress," is a complication of uremia (29
). Carbonyl stress has also been implicated in the development of diabetic complications (30
). Thus, monitoring of changes in protein oxidation has been shown to have practical application in a variety of medical circumstances.
In the present study, copper deficiency decreased the levels of carbonyl groups in a number of electrophoretically separated plasma proteins. This is consistent with our previous observations using spectrophotometric detection of DNP-derivatized total plasma protein carbonyls under conditions in which Cu,Zn-SOD activity had declined to a similar extent and plasma ceruloplasmin activity was essentially abolished (12
); this also refutes our original hypothesis that plasma protein carbonyls, as a potential biomarker of in vivo oxidation, would be increased after a significant decline in plasma copper-dependent antioxidant enzyme activities. The observed decrease in plasma protein carbonyl groups could have resulted from decreased production of protein carbonyl or from increased removal of oxidized plasma proteins.
Dietary copper deficiency has been shown to increase protein carbonyl content in - and ß-spectrin of erythrocyte membranes of rats under experimental conditions that yielded a similar level of copper-deficiency anemia, based on measurement of hemoglobin and hematocrit (10
). Also, the carbonyl content of unspecified proteins with molecular weights of 90 and 100 kDa was higher in mitochondria of copper-deficient HL-60 cells than in copper-supplemented controls (11
). However, it has been noted that there may be a greater likelihood of oxidation occurring in the intracellular microenvironment compared with free in the plasma (31
). This has been demonstrated for indices of protein oxidation including 3-nitrotyrosine and dityrosine (32
). Proteins in plasma may be relatively resistant to oxidative change in vivo as a result of copper deficiency if total antioxidant capacity is not compromised, despite significant loss of ceruloplasmin and plasma SOD activities specifically. This is because of the diverse and abundant antioxidant capacity of plasma, including nonenzymatic components such as vitamins C and E and ubiquinol, among others, and enzymatic components such as glutathione peroxidase. In the present work, plasma Se-GSH-Px activity was not affected in copper-deficient rats. Plasma TBARS also were not increased, and others have shown accumulation of protein carbonyl in the absence of evidence of lipid peroxidation (6
); this fact therefore does not rule out the possibility of plasma protein oxidation in these copper-deficient rats.
It has been noted that the level of oxidized protein in a cell reflects the balance between the rate of protein oxidation and the rate of oxidized protein degradation (7
). Enhanced rates of proteolytic degradation have been noted for a variety of experimentally oxidized proteins (5
). In an extracellular compartment such as plasma, the balance between possible release of oxidized proteins from intracellular compartments and removal or clearance from the plasma compartment must also be considered. Free radical–damaged bovine serum albumin (BSA) has been shown to be endocytosed and degraded more rapidly than native BSA by cultured murine peritoneal macrophages, although a portion of the endocytosed free radical–damaged BSA was resistant to degradation and consequently accumulated intracellularly (33
). Accumulation of oxidized protein has been suggested to play a role in atherosclerosis (9
), thus making it relevant to investigate further the possibility that protein oxidation products may accumulate in the blood vessel walls in copper deficiency. It is of interest to note that our band 1, which showed decreased intensity on Western blots for carbonyl content, is the same apparent size (145 kDa) as a protein band that diminished in total-protein staining intensity with amido black in copper-deficient rats, thus suggesting increased clearance of this protein from plasma. However, other protein bands did not have diminished amido black staining intensity. Additional study will be required to show whether the decrease in plasma protein carbonyls is due to decreased synthesis or increased removal.
Plasma total protein carbonyl content measured by HPLC was increased after 1 wk of feeding a very high protein (60% casein) diet to rats, although this increase was not evident after 14 wk (34
). Thus, it is possible that measurement of plasma protein carbonyls after 4 wk of feeding a copper-deficient diet may have missed an earlier transient increase.
The results reported herein reinforce our earlier observations of decreased plasma protein carbonyl content in copper-deficient rats (12
), extending those observations to specific electrophoretically separated plasma protein bands. Thus, although dietary copper deficiency may increase susceptibility to oxidative stress (3
), it does not lead to an accumulation of oxidized plasma proteins in this model system.
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; +Cu, -Cu, sucrose-based modified AIN-93G diets with and without copper in the mineral premix; Cu,Zn-SOD, copper-zinc superoxide dismutase (EC 1.15.1.1); DNP, dinitrophenylhydrazine; IgG, immunoglobulin G; NMR, nuclear magnetic resonance; Se-GSH-Px, selenium-dependent glutathione peroxidase (EC 1.11.1.9); TBA, thiobarbituric acid; TBARS, thiobarbituric acid-reactive substances.
4 Composition in g/kg diet: casein, vitamin free 200.0; cornstarch 132.0; sucrose 497.5; nonnutritive fiber 50.0; vitamin mix AIN-93-VX 10.0; modified mineral mix AIN-93G-MX, without Cu 35.0; L-cystine 3.0; choline bitartrate 2.5; soybean oil (containing t-butylhydroquinone 0.2 g/kg oil) 70.0. For the copper-adequate controls, AIN-93G-MX was used; cupric carbonate was added at the expense of sucrose.
Manuscript received 12 April 2002. Initial review completed 10 May 2002. Revision accepted 3 June 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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