Full text of DOI:10.1385/BTER:73:3:269

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Cytochrome c Oxidase,
Cu,Zn-Superoxide Dismutase,
and Ceruloplasmin Activities
in Copper-Deficient Bovines
S. I. CERONE,* A. S. SANSINANEA, S. A. STREITENBERGER,
M. C. GARCIA, AND N. J. AUZA
Departamento de Fisiopatología, Facultad de Ciencias
Veterinarias, Universidad Nacional del Centro de la Provincia
de Buenos Aires, Buenos Aires, Argentina
Received April 10, 1999; Accepted May 11, 1999
ABSTRACT
The activity of several cuproenzymes in relation to the immune
system was examined in serum and blood cells from bovines with
molybdenum-induced copper deficiency. Five female cattle were given
molybdenum (30 ppm) and sulfate (225 ppm) to induce experimental
secondary copper deficiency. Ceruloplasmin activity was determined
in serum. The Cu,Zn-superoxide dismutase and cytochrome c oxidase
activities were measured in peripheral blood lymphocytes, neutro-
phils, and monocyte-derived macrophages. Copper deficiency was
confirmed from decreased serum copper levels and the animals with
values less than 5.6 µmol/L were considered deficient. The content of
intracellular copper decreased between 40% and 70% in deficient cells
compared with the controls. In copper-deficient animals, the serum
ceruloplasmin activity decreased to half of the control value. Both of
them, the Cu,Zn-superoxide dismutase and the cytochrome c oxidase
activities, undergo a significant reduction in leukocytes, showing dif-
ferences among diverse cell populations. We concluded that the cop-
per deficiency alters the activity of several enzymes, which mediate
antioxidant defenses and ATP formation. These effects may impair the
cell immune functionality, affecting the bactericidal capacity and mak-
ing the animals more susceptible to infection.
Index Entries: Bovine; ceruloplasmin; cytochrome c oxidase;
copper; Cu,Zn-superoxide dismutase; leukocytes.
*Author to whom all correspondence and reprint requests should be addressed.
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INTRODUCTION
Copper is an essential trace element that has an important role in
many physiological functions in nervous, hematological, cardiovascular,
reproduction and immune systems (1). Copper plays a significant role,
being associated with specific proteins. The majority of the biological
functions of copper are believed to be associated with copper’s role as a
ligand in the active site of metalloenzymes. Among the principal
enzymes, it can be mentioned that the ceruloplasmin, a plasma glyco-
protein, may function as a copper transport and as an antioxidant. The
amine oxidases, isolated from plasma and organs, are responsible of oxi-
dative deamination. Dopamine-β-monooxygenase located in noradrener-
gic neurons and involved in conversion of dopamine to norepinephrine.
Cytochrome c oxidase (CcO) is the terminal mitochondrial electron carrier.
Lysyl oxidase is reponsible for oxidative deamination of peptidyllysine.
Cu,Zn-Superoxide dismutase is a cytosolic protein that speeds up the dis-
mutation of superoxide at neutral pH. Tyrosinase, located in melanocytes,
is involved in the conversion of tyrosine into melanin.
Some of these enzymes such as superoxide dismutase and cyto-
chrome c oxidase are ubiquitous enzymes underscoring the importance
of Cu in all animal cells (1). In this article, we fix our attention to copper
enzymes related with the immune response either humoral or cellular
and the impact of copper deficiency on this system.
Cytochrome c oxidase (EC 1.9.3.1) is the terminal member of the res-
piratory chain, catalyzing the reaction 4H+ + 4 e− + O2 → 2H2O and cou-
pling this exergonic reaction to the translocation of a proton across a
membrane barrier, generating a proton electrochemical gradient that is
then used to synthesize ATP. Eukaryote CcO contain two heme prosthetic
groups and three copper atoms (2).
Cu,Zn-superoxide dismutase (Cu,Zn-SOD, EC 1.15.1.1) (3) is a cupro-
enzyme that catalyzes the dismutation of O−2 to H2O2. Cu,Zn-SOD is
located in the cytosol of most tissues and is an integral part of the body’s
defense mechanism against the consequences of reactive oxygen inter-
mediates, which are deleterious to cell metabolism.
Ceruloplasmin (EC 1.16.3.1) is a single chain α2-glycoprotein that
binds up to 95% of serum copper. Its most important functions are the
transport of copper from liver to peripheral, nonhepatic tissues, catalysis of
the oxidation of ferrous ions, and action as an extracellular antioxidant (4).
Immunoglobulins, synthesized by lymphocytes, plays an important
role in host immunity because the F(abЈ)2 portion of the Ig mediates anti-
gen binding and is reponsible for the specific immune response, whereas
the Fc portion of it determines the effector function of the antibody as Fc
receptor binding.
Neutrophils and macrophages defend against bacterial infection by
various effector mechanisms, including the production of a group of
powerful oxidizing species (H2O2, ClO−, and others) that derive from a
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Enzyme Activities in Copper-Deficient Bovines
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single, relatively less reactive precursor: the superoxide radical (O−2 (5).
Following activation, these O−2 radicals are generated in large quantities
by the membrane-bound NADPH oxidase systems. Although production
of O−2 and the metabolites such as H2O2 and HO− are key steps in the
oxidative damage of the invading pathogens, overproduction of these
radicals can damage the host tissues (6). In order to protect the host cells
from any unwarranted oxidative destruction, the cells are provided with
antioxidant enzymes such as superoxide dismutase, catalase, and glu-
tathione peroxidase. The Cu,Zn-SOD catalyses the dismutation of O−2 to
H2O2 and catalase dismutates hydrogen peroxide to water and oxygen.
Both soluble and particulate agents stimulate neutrophils and macro-
phages to produce a high flux of O2− through the reduction of molecular
oxygen, known as the respiratory burst (7).
In this study, we examined the effect of molybdenum-induced Cu
deficiency of several cuproenzyme activities in serum, lymphocytes, neu-
trophils, and macrophages.
MATERIALS AND METHODS
Animals
Eight female Aberdeen Angus calves were separated into control
(n = 3, group 1) or Cu-deficient (n = 5, group 2) groups. All animals
were fed with basal diet containing alfalfa hay and concentrate ration
with 6.8 and 3.6 mg of Cu and Mo per gram of dry matter, respectively,
in a small pen at the experimental farm of our university. The animals
had free access to water. Additionally, the heifers in the Cu-deficient
group were supplemented orally with 30 ppm of Mo as ammonium
molybdate and 225 ppm sulfate as sodium sulfate, 5 days a week. This
supplementation was provided to induce a low Cu status (8), for 120 d,
controling the copper status every 30 d. The decrease of serum copper
level was used as indicator of body copper status.
Blood samples were taken from the jugular vein. When the animals
had a low copper status, peripheral blood leukocyte cells were isolated
as described in the following.
Copper Levels
In both serum and blood cells, copper levels were determined by
atomic absorption spectrophotometry (AAS) with a Perkin-Elmer spec-
trophotometer (Norwalk, CT). In serum, the copper level was determined
following 1 :1 dilution with trichloroacetic acid (TCA) (200 g/L). Cells
were washed three times in saline solution (9 g NaCl/L), resuspended in
deionized water at 6 × 107 cells/mL, lysed by freezing-thawing, and
diluted 1 :1 with TCA.
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Ceruloplasmin
Ceruloplasmin (Cp) activity was assayed by the method of Ravin (9)
using 100 µL of serum, 2 mL of acetate buffer, pH 6.4, and 1 mL of 0.5%
p-phenylenediamine in 42 mM sodium hydroxide as substrate. The reac-
tion was stopped by addition of 1 mL 0.11% sodium azide solution. The
absorbance was measured at 530 nm and results are expressed as in units
of IU/mL.
Cell Isolations
Blood from each cow was drawn into acid citrate dextrose anticoag-
ulant at a ratio of 10 :1. The anticoagulated blood was centrifuged. The
plasma and top one-third of the red blood cell pellet were discarded.
Neutrophils were recovered from the packed erythrocytes after erythro-
cyte lysing. The cells recovered were rinsed and suspended at 2 × 106
cells/mL in Hanks balanced salt solution (HBSS). The mononuclear cell
layer was removed and layered over Ficoll–Hypaque 1.083 (10). The mono-
nuclear cell layer was harvested and placed into a tissue-culture Petri
dish with RPMI 1640 media supplemented with 10% autologous serum.
After incubation at 37°C with 5% CO2 for 60 min, nonadherent cells were
recovered (lymphocytes) and fresh media was added to the flasks. Mono-
cytes were incubated for an additional 36 h in order to obtain a more uni-
form and pure population of cells. Then, the adherent cells were gently
scraped from the bottom of the flasks (11). The scraped cells were
washed in phosphate-buffer solution–calcium magnesium-free (PBS-CMF)
and resuspended to 5 × 107 cells/mL in serum-free RPMI 1640 medium.
Recovered cells were identified as macrophages based on morphological
features, adherence to plastic, and nonspecific esterase staining; 85–90%
were viable as determined by exclusion of 0.2% Trypan Blue.
Determination of Cu,Zn-SOD Activity
Cells isolated or harvested were washed twice in saline solution and
lysed by sonication. Following centrifugation of the lysate, the super-
natant fraction was used to measure the SOD activity, by a modification
of the method of Beauchamp and Fridovich (12). The assay involves the
ability of SOD to inhibit the reduction of nitroblue tetrazolium (NBT) by
superoxide, which is generated by the reaction of photoreduced
riboflavin and oxygen. For each sample, six tubes were set up containing
0.1 mol/L EDTA buffer, 0.15 mol/L phosphate buffer, 1.5 mmol/L NBT,
0.12 mM riboflavin, and various quantities of cell extract. The tubes were
placed in a light box containing an 18-W fluorescent tube and received
uniform illumination for 12 min. Optical densities were measured at
560 nm. Mn-SOD activity was measured by the addition of 5 mM NaCN
to the reaction mixture and Cu,Zn-SOD was calculated by subtraction of
the Mn-SOD value from the total. A unit of SOD enzyme activity is defined
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Enzyme Activities in Copper-Deficient Bovines
273
as the amount of enzyme that inhibits the reaction by 50%. Cellular SOD
activity was expressed as U/107 cells.
Determination of Cytochrome c Oxidase
Activity
In the supernatant, the fraction of lysate was assayed for CcO activ-
ity by the method of Cooperstein and Lazarow (13). Briefly, 3 mL of
reduced cytochrome c solution and 80 µL of the lysate were added into
the spectrophotometer cuvet. Readings were taken every 30 s at 550 nm
for 3 min, until a few grains of potassium ferricyanide were added (to
oxidize the cytochrome c completely) and the extinction redetermined.
Results are expressed as U/mg cellular protein.
Statistical Analysis
All values are expressed as means SD. The results were analyzed
using the Student’s t-test. A p-value of <0.05 was taken to indicate sta-
tistical significance.
RESULTS
Bovines supplemented with molybdate and sulfate had significantly
lower levels of Cu in serum than those not supplemented, as deter-
mined by atomic absorption spectrophotometry, decreasing from 11.8 to
5.6
0.7 µmol/L at 120 d after starting the trial, whereas the control
group maintained the initial values (11.8 0.5 µmol/L). When the ani-
mals from group 2 reached those values, the blood samples were
processed and the cells cultivated, as mentioned earlier (Table 1).
The levels of intracellular copper decreased notably in neutrophils
from deficient bovines in relation to the controls, reaching values of
0.032 µg Cu/6 × 107 cells for deficient bovines and 0.116 µg Cu/6 × 107
cells for the control group; this is a decrease of 72% of intracellular metal
element content. The quantity of intracellular copper was 35–40% less in
monocyte-derived macrophages from deficient bovines than that in control
cells, being 0.028 0.005 µg/6 × 107 cell and 0.045 0.004 µg/6 × 107
cell, respectively. Data are shown in Table 1.
Before the treatment started, the ceruloplasmin activity in the serum of
control animals was 46 6 × 10−2 IU/mL. Copper deficiency decreased the
enzyme activity by approximately 50%, attaining values of 22 5 × 10−2
IU/mL (Table 1).
Results of the intracellular Cu,Zn-SOD activity measurement are
shown in Fig. 1. The addition of cyanide to the reaction mixture abol-
ished the Cu,Z,-SOD activity and allowed the determination of its activ-
ity by subtraction from the total value. Cellular Cu,Zn-SOD activity was
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Table 1
Copper Content in Serum and White Blood Cells,
and Ceruloplasmin Activity
Note: Values represent mean SD. Significant differences (p < 0,05) are indi-
cated by footnotes a–d.
Fig. 1. Cu,Zn-SOD activity in neutrophils and macrophages, expressed as
percentage/107 cells (control group [striped bars, n = 3] and deficient group
[open bars, n = 5]). Values represent mean SD. Significant diferences (p < 0.05)
between groups are indicated by an asterisk.
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Enzyme Activities in Copper-Deficient Bovines
275
Fig. 2. CcO activity in lymphocytes, neutrophils, and macrophages,
expressed as percentage/mg protein cell (control group [striped bars, n = 3] and
deficient group [open bars, n = 5]). Values represent mean
SD. Significant
diferences (p < 0.05) between groups are indicated by an asterisk.
significantly reduced, to 31% of the value in control neutrophils, whereas
this reduction reached 39% in macrophages, expressed as U/107 cells.
Results of the mitochondrial CcO activity measurements are shown
in Fig. 2. It can be seen that the enzyme activity was more intense in con-
trol lymphocytes than in control neutrophils and macrophages. Depletion
of Cu in animals from group 2 caused a significant reduction of CcO
activity in all cell populations. This decrease was more evident in lympho-
cytes than in polymorphonuclear neutrophils and macrophages, as the
same quantity of cellular protein from deficient group was able to trans-
form only 50% of reduced cytochrome c to oxidized cytochrome c com-
pared to the control group, whereas in neutrophils and macrophages, this
reduction reached 32% and 27% of the control values, respectively.
DISCUSSION
The concept that nutritional status influences the susceptibility of a
host to infectious disease is well established. Copper is an essential trace
element that has an important role in the immune response; nonetheless,
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Cerone et al.
the precise mechanism by which Cu deficiency alters host inmunity is
still uncertain.
Active oxygen species, including the O−2, H2O2 and the OH−, are pro-
duced through a number of reaction mechanisms as part of physiologi-
cal enzyme functions. The species that escape being scavenged can
induce oxidative stress in vivo. Antioxidant enzymes are thought to play
an important role in providing protection from the deleterious effects of
active oxygen species, both by decreasing their levels and by preventing
the destructive propagation of radical chain reactions (14).
It is assumed that SOD protects the cells from uncontrolled and
damaging reactions of O−2 through catalyzing its dismutation to molecu-
lar oxygen and hydrogen peroxide. The dismutation of the superoxide
anion to hydrogen peroxide and molecular oxygen by SOD is often
called the primary defense (15), as it prevents radicals derived from O2−
such as hydroxyl, peroxinitrite, and the perferyl ion complex, which are
more oxidant and able to initiate lipid peroxidation by extraction of
hydrogen from unsaturated fatty acid, thereby destroying the membrane
integrity (16). Inadequate copper levels have adverse effects on the syn-
thesis and activity of cuproenzymes. It is probable that the less SOD
activity determined in Cu-deficient neutrophils and macrophages com-
pared with control group impaired not only the primary defense but also
induce the lipid peroxidation.
The lipid peroxidation in a localizated membrane segment may per-
turb the microenvironment of membrane-bound proteins, altering their
function (17). Oxidative damage to lipid components of leukocyte mem-
branes initiate the process of prostaglandin and leukotriene synthesis
leads to a loss of fluidity as well as alteration of the receptor function (18).
These alterations in receptor structure because of liperoxidation can be the
responsible for the lower activity in the burst respiratory in copper defi-
cient neutrophils (19) and macrophages when they were stimulated.
Copper, through its cofactor role in CcO, is directly involved in
oxidative phosphorylation. The observed reduction in CcO levels in cop-
per-deficient lymphocytes may affect intracellular ATP levels. It is known
that the synthesis of macromolecules formation of antibody molecules
requires energy. When the CcO activity falls enough to reduce the elec-
tron flux to molecular oxygen, the mitochondrial energy metabolism is
impaired and this can be the cause of lower antibody synthesis, as was
demonstrated previously (20). The morphological appearance of abnor-
mal mitochondria in lymphoid tissue of copper-deficient mice (21) adds
further credence to this hypothesis.
In addition, bovine IgG through Fc induces superoxide production
in bovine neutrophils and is one of the mayor components of serum
stimulatory factor, playing an important role in the antimicrobial func-
tion of them in tissue (22). If the IgG production is less than normal, it is
probably an impaired stimulatory effect on polymorphonuclear mono-
cytes with diminished activity.
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Enzyme Activities in Copper-Deficient Bovines
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Ceruloplasmin is among those enzymes involved in the acute-phase
reaction of inflammation and in the scavenging of oxygen radicals to pro-
tect cells against oxidative damage. Copper deficiency, associated with a
low level of plasma Cp, leads to a decrease of the antimicrobial activity
of phagocytes (23). This enzyme may be needed in inflammation because
it functions as a copper-transport protein, delivering copper to Cu-
dependent enzymes, such as lysyl oxidase, which is involved in the
repair of damaged tissues and Cu,Zn-SOD. The copper-transporting
function of Cp is important for the maintenance of the activity of leuko-
cyte enzymes involved in the respiratory burst.
We concluded that the limitation of a single nutrient, copper, can
adversely affect immunity and host resistance. The immunocompetent
cells could suffer a functional defect and can, therefore, neither synthe-
size enough antibody nor undergo an adequate respiratory burst. As a
consequence, the animals suffer severe bacterial infections, because the
decreased function of phagocytic cells compromises the nonspecific
immune defense system of Cu-deficient animals and contribute to their
greater susceptibility to infections.
ACKNOWLEDGMENTS
This study was supported by a grant from Secretaría de Ciencia y
Tecnología of Universidad Nacional del Centro de la Provincia de Buenos
Aires and Comisión de Investigaciones Científicas de la Provincia de
Buenos Aires.
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