Clinical Science (1991) 81, 413-417
413
Plasma ascorbate levels and inhibition of the antioxidant
activity of caeruloplasmin
JOHN M. C. GUTTERIDGE
Oxygen Chemistry Laboratory, Department of Anaesthesia and Intensive Care, Royal Brompton Hospital and National Heart and
Lung Institute, London
(Received 11February/25 March 1991; accepted 11April 1991)
SUMMARY
1. T h e copper-containing protein caeruloplasmin has
several oxidase activities.
2. Its ability to catalyse the oxidation of ferrous ions to
the ferric state (ferroxidase activity) makes it an important
antioxidant in vivo.
3. Recent reports have suggested that oral supplementation with vitamin C can inhibit the oxidase activities
of caeruloplasmin.
4. As expected, damage to DNA and membrane lipids
was stimulated by mixtures of iron salt and ascorbate, and
this damage could be inhibited by caeruloplasmin
provided the molar ratio of ascorbate to caeruloplasmin
was kept sufficiently low.
5. When the molar ratio of ascorbate to caeruloplasmin was greater than 200 substantial loss of ferroxidase
antioxidant activity occurred.
6. It is unlikely, however, that oral supplementation
with vitamin C can raise plasma levels sufficiently to
inhibit caeruloplasmin activity in vivo.
Key words: antioxidant, bleomycin, caeruloplasmin, lipid
peroxidation, oxygen radicals, vitamin C supplementation.
INTRODUCTION
The copper-containing protein caeruloplasmin, at a concentration of 300 mg/l, accounts for almost all the detectable copper in normal human plasma. Caeruloplasmin has
oxidase activities towards polyamines, polyphenols and
inorganic ferrous ions (for a review, see [l]). However,
only ferrous ions have been considered a biological substrate for caeruloplasmin, which can load the resulting
ferric ions on to the iron-transport protein transferrin.
The catalytic oxidation by caeruloplasmin of ferrous
ions, o r of various ferrous complexes, to the ferric state is
known as plasma ferroxidase I activity, since stored or
mishandled human plasma also contain a ferroxidase I1
activity [2]. This second ferroxidase activity arises after
the proteolytic cleavage of copper-containing fragments
from caeruloplasmin and their association with lipoproteins to produce peroxidized lipid-protein-copper
complexes [3]. Ferroxidase I activity is inhibitable by
azide, whereas ferroxidase I1 activity is not.
A n important biological role has been proposed for
caeruloplasmin as an extracellular antioxidant [ 1, 4-61.
The protective activity of caeruloplasmin has been
ascribed mainly to its ferroxidase activity, which prevents
the stimulation by ferrous ions of lipid peroxidation and
Fenton chemistry [l, 4-61. By catalysing the oxidation of
ferrous ions at physiological pH values, without the
release of reactive oxygen intermediates into extracellular
fluids, caeruloplasmin prevents the formation of the
reactive and damaging hydroxyl radical [7].
Ascorbic acid is present in freshly taken normal human
plasma at concentrations ranging from 30 to 100 pmol/l,
and concentrations some tenfold higher than these are
present in normal human cerebrospinal fluids [S].
Recent studies have suggested that vitamin C (ascorbic
acid), a widely taken nutritional supplement in Western
countries, can decrease the oxidase activity of plasma
caeruloplasmin [9, 101. Since the protein plays an important biological role as an extracellular antioxidant [ 1 , 4 4 1
the following experiments were undertaken to assess in
vitro the effect of different levels of ascorbate on the
ferroxidase activity of caeruloplasmin.
MATERIALS AND METHODS
Materials
Correspondence: Dr J. M. C. Gutteridge, Oxygen Chemistry
Laboratory, Department of Anaesthesia and Intensive Care,
Royal Brompton Hospital and National Heart and Lung Institute, Dovehouse Street, London SW3 6LY.
Caeruloplasmin (human), bleomycin sulphate, DNA
(calf thymus) and ovotransferrin (conalbumin) were from
Sigma Chemical Co. Ltd (Poole, Dorset, U.K.). Ascorbate
414
J. M. C. Gutteridge
oxidase (Calbiochem, U.K.; 1 unit oxidizes 1.0 pmol of
ascorbate min-' I-' at pH 5.6,25"C). All other chemicals
were of the highest purity available from BDH Chemicals
Ltd (Poole, Dorset, U.K.).
Ferroxidase activity of caeruloplasmin
The ability of caeruloplasmin to load iron on to the
iron-binding protein ovotransferrin in the presence of
ascorbate was determined by the method of Johnson et al.
[ 111, but using ovotransferrin instead of transferrin.
Antioxidant activity towards iron-ascorbate-stimulated
lipid peroxidation
Liposomes were prepared from bovine brain phospholipids (5 mg/ml) as previously described [ 121. These were
peroxidized by incubating the following reactants in a
volume of 1.2 ml for 30 min at 37°C with 0.8 mg of
phospholipid, 0.025 mol/l sodium phosphate buffer, pH
7.4, and 1.7 pmol/l ferric chloride. The reaction was
started by the addition of ascorbate at the concentrations
shown in Table 1. The inhibitory effect of caeruloplasmin
(0.1 ml) was assessed by adding the protein before the
ascorbate. After incubation the tube contents were mixed
with 0.5 ml of 1% (w/v) thiobarbituric acid in 50 mmol/l
NaOH and 0.5 ml of 25% (v/v) HCI and heated at 100°C
for 15 min. The resulting pink chromogen was extracted
into 1.5 ml of butan-1-01, and the absorbance of the
organic phase was measured at 532 nm against appropriate blanks.
DNA degradation by iron-bleomycin
The bleomycin assay for chelatable iron [ 131 was used
to assess the inhibition by caeruloplasmin of DNA
degradation at different ascorbate concentrations. The
reaction mixture in a volume of 1.2 ml contained: 0.1 mg
of DNA, 0.025 unit of bleomycin, 1.7 pmol/l ferric
chloride and 0.125 mol/l Tris buffer, pH 7.4. The reaction
was started by the addition of ascorbate at the concentrations shown in Table 2. Caeruloplasmin was added before
the ascorbate, and the reaction mixture was incubated at
37°C for 1 h. After incubation the tube contents were
treated in the same way as the lipid peroxidation samples
and the resulting thiobarbituric acid-reactivity was
measured at 532 nm. The reproducibilities of the
methods have been described previously [l l-141 and the
results shown here are the mean of three highly reproducible experiments. Molar ratios for caeruloplasmin
Table 1. Inhibition by caeruloplasmin of lipid peroxidation stimulated by iron-ascorbate
Final reaction concentrations are shown. The peroxidation blank value (without ascorbate) of
0.005 was subtracted from the AS3*value shown. Abbreviation: TBA, thiobarbituric acid.
Ascorbate concn.
( WW)
Lipid peroxidation as
TBA-reactivity ( A , , ? )
320
160
80
40
20
10
5
2.5
1.25
0.762
0.671
0.644
0.522
0.379
0.22 1
0.093
0.05 1
0.012
Inhibition of peroxidation
by caeruloplasmin
(0.03 mg/rnl)
7
30
53
63
94
98
98
100
100
(YO)
Axorbate/
caeruloplasmin molar
ratio
1391
6 96
348
174
87
44
22
10.9
5.4
Table 2. Inhibition by caeruloplasmin of DNA degradation with iron-bleomycin-ascorbate
Final reaction concentrations are shown. The bleomycin-iron value in the absence of ascorbate
was 0.002 and this was deducted from the A,3zvalues shown. Abbreviation: TBA, thiobarbituric
acid.
Ascorbate concn.
(FOl/l)
DNA dcgradation as
TBA-reactivity
('4532)
320
160
80
40
20
10
5
1.78
1.45
0.62
0.29
0.12
0.06
0.03
Inhibition of DNA
degradation (YO)
by
caeruloplasmin
(0.3 mg/ml)
Axorbate/
caeruloplasmin molar
ratio
68
71
82
99
100
100
100
139
70
35
17
9
4
2
415
Ascorbate and plasma antioxidants
were based on a relative molecular mass of 130000
(reviewed in [l]).
1
Measurement of low-molecular-mass copper
Caeruloplasmin (19 pmol/l) was incubated in 0.25
mol/l Tris buffer, pH 7.4, with different ascorbate concentrations ranging from 320 to 1.25 pmolll, for 1 h at
37°C. After incubation, the ascorbate remaining in the
reaction was removed by adding 50 units of ascorbate
oxidase and incubating for a further hour at 30°C. The
release of low-molecular-mass copper from caeruloplasmin was measured by the phenanthroline assay [ 141 with
the inclusion of appropriate controls for copper release
from ascorbate oxidase and the uniform removal of
ascorbate from the reaction.
10
30
40
50
60
Time (s)
RESULTS
The ferroxidase activity of caeruloplasmin can be
measured as the ability of the protein to catalyse the
oxidation of ferrous ions to the ferric state and to bind the
ferric ions to ovotransferrin forming a pink-coloured
complex. As can be seen from Fig. 1, ferrous ions in the
absence of caeruloplasmin only slowly oxidize at pH 6.0
in acetate buffer to form ferric ions. Ascorbate added
over the concentration range 10-160 pmol/l did not
appear to inhibit the ferroxidase activity of caeruloplasmin. As expected, the addition of azide did inhibit the
ferroxidase I activity of caeruloplasmin.
Caeruloplasmin at a physiological concentration of 300
mg/l inhibited the iron-ascorbate-stimulated peroxidation of phospholipid membranes by 67% at a molar ratio
of ascorbate (320 pmol/l) to caeruloplasmin of 139, by
93% at a molar ratio of 70 (ascorbate 160 pmolll), and by
99% at a molar ratio of 35 (ascorbate 80 pmol/l) (data not
shown). With a decreased concentration of caeruloplasmin (30 mg/l), molar ratios of ascorbate to caeruloplasmin ranging from 5.4 to 1391 could be achieved for use
with phospholipid peroxidation (Table 1). Using these
ratios it can be shown that ascorbate inhibits the ferroxidase activity of caeruloplasmin by up to 50% when
present at a ratio of 348 and by 93% at a ratio of 1391
(Table 1).
Degradation of DNA by iron-bleomycin-ascorbate
can, like the peroxidation of lipid, be inhibited by caeruloplasmin provided the molar ratio of ascorbate to caeruloplasmin is kept sufficiently low (Table 2). When the
concentration of bleomycin in the reaction was increased
fivefold over the value described in the Materials and
methods section, the loss of inhibitory activity of caeruloplasmin (at a molar ratio of ascorbate to caeruloplasmin
139) was some 66% greater than that shown in Table 2
(data not shown).
When caeruloplasmin was incubated with ascorbate
concentrations in the range 320-1.25 pmol/l there was
no detectable difference in the amount of low-molecularmass copper released in the reaction (data not shown).
20
Fig. 1. Ferrous ions do not readily bind to ovotransferrin
but when oxidized to the ferric state they are bound with
high affinity to produce a pink-coloured complex
absorbing at 460 nm. The ability of caeruloplasmin to
catalyse the oxidation of ferrous ions (ferroxidase activity)
to ferric ions which bind. to ovotransferrin has been
determined here (final concentrations are shown). 1,
Ascorbate (160 pmol/l) plus caeruloplasmin (0.3 mg/ml);
2, ascorbate (80 prnol/l) plus caeruloplasmin (0.3 mg/ml);
3, ascorbate (40 pmolll) plus caeruloplasmin (0.3 mg/ml);
4, ascorbate (10 pmol/l) plus caeruloplasmin (0.3 mg/ml);
5, caeruloplasmin (0.3 mg/ml) without ascorbate present;
6, caeruloplasmin (0.3 mg/ml) with azide (1 mmol/l); 7,
ascorbate ( 160 pmol/l) without caeruloplasmin.
Most of the copper detected by the phenanthroline assay
appeared to come from the ascorbate oxidase.
DISCUSSION
The normal plasma molar ratio of caeruloplasmin to
ascorbate is probably between 1:13 and 1:65 (assuming a
normal caeruloplasmin concentration of 300 mg/l and
ascorbate concentrations of 30-150 pmol/l). A caeruloplasmin concentration of 30 mg/l was used in our experiments for the technical reasons described; however,
values as low as this can occur in the plasma of full-term 8
newborn infants and of patients with Wilson’s disease
(reviewed in [l]).It has recently been suggested that
nutritional supplementation with vitamin C can decrease
the oxidase activity of plasma caeruloplasmin in vivo [9,
101. Ascorbate has been ascribed both pro-oxidant and
antioxidant functions, and these appear to be concentration-dependent. Ascorbate can redox-cycle with iron and
copper salts to generate hydroxyl radicals (OH’) and can
also scavenge these when the concentration of ascorbate
is sufficiently high. When human plasma is tested for its
ability to scavenge water-soluble peroxyl radicals, ascorbate stands out as a particularly effective antioxidant [15].
416
J. M. C. Gutteridge
However, situations can rise whereby ascorbate promotes
the release of iron and copper ions from metal-storage
and -transport proteins [16], and it has been proposed
that nutritional supplementation with ascorbate may
cause the release of copper from caeruloplasmin [lo],
although this could not be substantiated in the present
study. Assessing the effect of a high ascorbic acid intake
(605 mg/day), Jacob et af. [lo] reported a 21% decrease
in plasma caeruloplasmin oxidase activity when plasma
concentrations of ascorbate of around 80 pmol/l were
achieved. However, when they added 600 pmol/l ascorbate to serum in vitro, giving a molar ratio of ascorbate to
caeruloplasmin of about 260, no significant loss of
caeruloplasmin oxidase activity was observed [lo]. From
our studies here it is clear that the ratio of ascorbate to
caeruloplasmin is important in determining inhibition of
its ferroxidase activity. This concentration-dependent
inhibitory property of ascorbate has been used previously
to inhibit the ferroxidase activity of caeruloplasmin in
phospholipid antioxidant assays [17] and in the bleomycin
assay for iron [18]. When measuring the protective antioxidant properties of caeruloplasmin towards ferrousion-stimulated lipid peroxidation of liposomal membranes
and ferrous-ion-bleomycin-dependentdamage to DNA,
ferroxidase activity begins to be lost when ratios around
100-200 are approached, with greater inhibition occurring above these values.
It is therefore extremely unlikely that sufficiently high
levels of ascorbate to inhibit the ferroxidase antioxidant
functions of caeruloplasmin can be obtained in plasma by
oral intake of ascorbate. Several known functions of
caeruloplasmin contribute to its antioxidant activities and
these include: catalytic ferroxidase activity [ 1, 4-61,
stoichiometric scavenging of superoxide [ 191 and hydrogen peroxide [20], and weak ascorbate oxidase activity
[21] when ascorbate is functioning as a pro-oxidant.
The inhibition of the ferroxidase activity of caeruloplasmin by ascorbate in the DNA-bleomycin-iron reaction was higher than that seen with phospholipids, and
this could be due to factors other than ascorbate concentration. Damage to DNA by bleomycin-iron releases
numerous reactive products, such as malondialdehyde,
which are able to cross-link proteins and thereby possibly
damage caeruloplasmin. It is also likely that bleomycin
itself becomes a competing ferroxidase [22].
At physiologically achievable plasma levels of
ascorbate no impairment of the ability of caeruloplasmin
to oxidize ferrous ions to the ferric state and to load these
on to ovotransferrin was observed. It is concluded that in
normal subjects the plasma levels of ascorbate reached
during oral supplementation with ascorbic acid are insufficiently high to inhibit the important antioxidant
activities of caeruloplasmin. However, in other body
fluids, such as cerebrospinal fluid, the low levels of caeruloplasmin and the high levels of ascorbate normally present mean that ratios of 25 000 are likely to occur, which
would inhibit the ferroxidase activity of caeruloplasmin
and keep any low-molecular-mass iron present [23] in the
reduced ferrous state (J. M. C. Gutteridge, unpublished
work).
ACKNOWLEDGMENTS
J.M.C.G. holds the first British Lung Foundation/British
Oxygen Company Senior Research Fellowship in
Respiratory Critical Care, and gratefully thanks the
British Lung Foundation and British Oxygen plc for their
generous support.
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