27.4: Erythrocyte superoxide dismutase (24b.3)
- Page ID
- 117274
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Approximately 60% of the copper found in erythrocytes is present in the cytosol as superoxide dismutase (Cu,Zn-SOD, EC 1.15.1.1). This enzyme, which contains both divalent copper and divalent zinc, has a molecular weight of approximately 32,500. It is important as a scavenger of the free radical \(\mathrm{O}_2^{-}\) that causes damage to membranes and biological structures. It catalyzes the dismutation reaction, converting the highly reactive superoxide anion \(\mathrm{O}_2^{-}\), to less reactive hydrogen peroxide and oxygen:
\[\mathrm{O}_2^{-}+\mathrm{O}_2^{-}+2 \mathrm{H}^{+} \rightarrow \mathrm{SOD} \rightarrow \mathrm{H}_2 \mathrm{O}_2+\mathrm{O}_2\nonumber\]
| Measure | Time | Study (n=8) |
Control (n=9) |
|---|---|---|---|
| Plasma copper (µmol/L) |
Before After |
15.4±10.5 26.0±5.4 |
22.4±4.1 |
| Ceruloplasmin (mg/L) |
Before After |
330±260 500±140 |
540±120 |
| SOD (U/g Hb) |
Before After |
1073±312 1371±207 |
1461±451 |
Studies with both animal models and humans have shown that the activity of erythrocyte Cu,Zn-SOD is depressed in copper deficiency and that the activity is often, but not always, restored during copper repletion (Bettger et al., 1979; Okahata et al., 1980; Uauy et al., 1985; Milne et al., 1990; Milne and Nielsen, 1996). Table 24b.7 presents data on erythrocyte Cu,Zn-SOD activity in infants recovering from malnutrition and receiving marginal copper intakes: levels returned to normal after copper supplementation (Uauy et al., 1985) and there was a significant positive correlation between erythrocyte Cu,ZnSOD and plasma copper concentrations in these malnourished infants (Figure 24b.4). Such a correlation was not reported by Fischer et al., (1990). in their study of healthy adults, probably because the individuals studied had a copper status within the normal range.
Positive correlations between erythrocyte Cu,Zn-SOD activity and other measures of copper status (e.g., liver cytochrome c oxidase) have also been observed in some animal studies (Bettger et al., 1979; Andrewartha and Caple, 1980).
Erythrocyte Cu,Zn-SOD activity appears to be a more sensitive index of copper depletion than serum copper or ceruloplasmin. In several experimental copper depletion-repletion studies in humans (Reiser et al., 1985; Milne and Nielsen, 1996), erythrocyte Cu,ZnSOD activity declined significantly during the depletion phase, despite no detectable decrease in serum copper or ceruloplasmin. This trend is shown in Figure 24b.2.
Nevertheless, the response of erythrocyte Cu,Zn-SOD activity to copper repletion in these experimental studies has not been consistent. Several investigators have failed to show a response by erythrocyte Cu,Zn-SOD to copper supplementation (Turnlund et al., 1990; Medeiros et al., 1991). Even in a later study by Kehoe et al. (2000) involving two different copper compounds and two levels of copper supplementation, the activity of Cu,Zn-SOD in erythrocytes (and leukocytes) did not respond to the copper supplements.
It appears that the study duration, amount of copper fed, and probably rates of cell turnover all influence the response of erythrocyte Cu,Zn-SOD activity to copper repletion (Klevay et al., 1984; Milne et al., 1988; Turnlund et al., 1990; Medeiros et al., 1991; Milne and Nielsen, 1996; Kehoe et al., 2000). In Figure 24b.2 the erythrocyte Cu,Zn-SOD activity remained at a plateau during copper repletion, perhaps associated with the rate at which new erythrocytes were being synthesized. In contrast, in the study of malnourished copper-deficient infants (Table 24b.7), erythrocyte Cu,Zn-SOD activity increased to normal levels after supplementation with copper (80µg/kg/d) for 120d. Furthermore, Cu,Zn-SOD levels increase in a variety of diseases, including pancreatitis (Ściskalska et al. 2020). In some conditions that produce oxidative stress (Lukaski et al., 1990), including alcoholism and Down's syndrome (Del Villano et al., 1980; Porstmann et al., 1990), erythrocyte Cu,Zn-SOD activity may be elevated. In rheumatoid arthritis patients, lower levels than those in controls have been observed (DiSilvestro et al., 1992). Activity levels, but not total concentration, appears to be reduced in hyperglycemic, but not normal, diabetic patients possibly by glycosylation of the active site of the enzyme (Kotake et al., 1998).
Very few investigators have used erythrocyte Cu,Zn-SOD activity as an index of copper status in community-based studies. Fischer et al. (Fischer et al., 1990) measured erythrocyte Cu,Zn-SOD activity (and serum copper and ceruloplasmin) in 384 free-living adults and examined the relationships with age, sex, estrogen use, smoking, alcohol use, and exercise, as noted earlier. Table 24b.8 presents the mean ±SEM for erythrocyte Cu,Zn-SOD activity for males and females by age. Unlike serum copper levels and ceruloplasmin activity, erythrocyte Cu,Zn-SOD activity appears to be unaffected by age, gender (Table 24b.8), hormone use, or the acute phase status (Solomons, 1985; Fischer et al., 1990; Milne and Johnson, 1993).
| Age group (years) |
Men (Mean±SEM) |
Women (Mean±SEM) |
|---|---|---|
| 25–34 | 82.1±6.3 | 74.7±2.9 |
| 35–44 | 76.7±1.4 | 75.3±2.7 |
| 45–54 | 76.2±2.1 | 79.0±5.7 |
| 55–64 | 77.8±1.8 | 75.5±3.9 |
| 65+ | 77.4±2.9 | 68.9±3.9 |
| Women Pre- menopausal |
Non-users of oral contr. 75.1±2.1 |
Users of oral contr. 77.2±6.1 |
| Women Post- menopausal |
Non-users of estrogens 76.7±3.5 |
Users of estrogens 73.8±7.9 |
• Interpretive criteria
Interpretive criteria for erythrocyte Cu,ZnSOD activity are limited. Laboratories should establish their own reference range for erythrocyte Cu,Zn-SOD activity. Milne and Johnson (1993) have compiled ranges for healthy adults based on the assay of Winterbourne et al. (1975). However, there were relatively small numbers of subjects in each age group. Activity levels are unaffected by age, gender, and hormone use. The U.K. National Diet and Nutrition Survey of young people aged 4–18y measured erythrocyte Cu,Zn-SOD activity; levels decreased for both boys and girls with increasing age. Mean, median, and the lower and upper 2.5 percentile values by age and sex are presented (Gregory et al., 2000). However, work is still required to confirm the utility of erythrocyte Cu,Zn-SOD activity in studies of the copper status of the general population.
• Measuring erythrocyte Cu,Zn-SOD
There is no standard assay for the measurement of erythrocyte Cu,Zn-SOD activity. Several methods are available; some are very time consuming, require large samples, necessitate the removal of hemoglobin immediately after sample collection, and are subject to interferences.
The assay generally involves the inhibition of oxidation-reduction reactions which are catalyzed by the superoxide anion. The latter may be generated enzymatically by, for example, xanthine plus xanthine oxidase, which, in turn, reduces cytochrome c. The reduction of cytochrome c is followed spectrophotometrically (Marklund and Marklund, 1974).
Several of these procedures are now automated. L'Abbé and Fischer (1986) developed a method for determining Cu,Zn-SOD activity that can be used with small samples. Commercial kits based on either an enzyme-linked immunosorbent assay or on a spectrophotometric assay are now available for measuring Cu,Zn-SOD activity in human serum and urine.
The analytical coefficient of variation for the erythrocyte superoxide dismutase assay when determined using an automated method ranges from 1.9% to 11.9%. The within subject variation for erythrocyte Cu,Zn-SOD activity is generally high (Gallagher et al., 1989).