315
Clinical Science (1992) 82, 3 15-320 (Printed in G r e a t Britain)
Ferrous ions detected in cerebrospinal fluid by using
bleomycin and DNA damage
John M. C. GUTTERIDGE
Oxygen Chemistry Laboratory, Department of Anaesthesia and Intensive Care,
Royal Brompton Hospital and National Heart and Lung Institute, London, U.K.
(Received
2 M a y l 2 l O c t o b e r 1991; accepted 24 O c t o b e r 1991)
1. During pathological states of iron-overload or oxidant
stress, low-molecular-mass iron can become available
within extracellular fluids.
2. This iron would be converted to the ferrous state were
it not for the protective anti-oxidant protein caeruloplasmin.
3. The ferrous-ion-oxidizing activity of caeruloplasmin
rapidly converts ferrous ions back to the less reactive
ferric state so that they can bind to available binding sites
on transferrin.
4. Cerebrospinal fluids, however, often appear to contain
low-molecular-massiron, high levels of ascorbate and low
levels of ferroxidase activity with little or no iron-binding
capacity.
5. When iron ions are present in cerebrospinal fluid they
are therefore likely to be in the ferrous state.
6. The development and application of an assay to
speciate and measure ferrous ions in simple aqueous
solution and their redox cycling activity in biological
fluids is described.
INTRODUCTION
Iron ions and certain low-molecular-mass iron
complexes have the potential in a biological system to
react with molecular oxygen and its reduction intermediates to generate highly reactive and damaging species
such as the hydroxyl radical (.OH). The chemistry leading
to *OH radical formation is complex, involving
iron-oxygen intermediates such as ferry1 and perferryl
ions which may also contribute to any damage observed
(for a review, see [l]).
Since iron ions are themselves free
radicals that can readily enter into reactions with oxygen,
the body takes greater care to safely sequester them to
high-affinity iron-binding and -storage proteins such as
transferrin and ferritin. Transferrin is normally only onethird loaded with iron and retains a considerable ironbinding capacity which makes the level of plasma iron
ions undetectable [2]. The anti-oxidant property this
confers on transferrin is considered an important part of
safe iron retention and recycling within the body [l].It
may also allow reduced intermediates of oxygen, such as
superoxide and hydrogen peroxide to survive long enough
to perform useful signal and messenger functions in the
extracellular environment [3,4].
Inside the cell, where oxygen is metabolized, highmolecular-mass specific proteins such as superoxide dismutase, catalase and glutathione peroxidase (selenium
enzyme) remove superoxide and hydrogen peroxide. This
allows a small pool of low-molecular-mass iron [ 5 ] to
safely exist within cells for the synthesis of iron-containing
proteins. The reductive environment inside the cell would
keep this iron in a soluble ferrous state.
During pathological conditions of iron-overload or
severe oxidant stress, low-molecular-mass chelatable iron
can be detected in human plasma [6-91. When simple
ferric complexes are incubated with molecules normally
present in human plasma capable of reducing iron, such
as ascorbate, urate and a-tocopherol, the iron is
converted to the ferrous form [lo], suggesting that iron
ions not bound to transferrin would also exist in plasma in
the ferrous state. However, plasma contains the ferrousion oxidizing (ferroxidase) protein caeruloplasmin [ 11,
121, which rapidly converts ferrous ion and many of their
simple complexes back to the less reactive ferric state.
The purpose of this study was to develop and apply a
method both sensitive and specific enough to speciate and
detect ferrous ions in cerebrospinal fluid (CSF). As
expected, ferrous ions were not detected in normal freshly
obtained human sera, even when caeruloplasmin was
inhibited with azide, but could be detected in normal
human CSF.
MATERIALS AND METHODS
Materials
DNA (herring testes), caeruloplasmin (human plasma),
ascorbate oxidase (1unit oxidizes 1.O pmol of axorbate/
min at pH 5.6 and 25"C), bleomycin sulphate and conalbumin were from the Sigma Chemical Company, Poole,
Dorset, U.K. All other chemicals were of the highest
Key words: anti-oxidants, ascorbate, bleomycin-iron, caeruloplasmin, cerebrospinal fluid, ferrous ions, low-molecular-mass iron.
Abbreviations: CSF, cerebrospinal fluid; RFI, relative fluorescence intensity; TBA, thiobarbituric acid.
Correspondence: Professor 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, U.K.
316
J. M. C. Gutteridge
purity available from BDH Chemicals, Poole, Dorset,
U.K.
Collection of samples
CSF samples were obtained for authentic medical
purposes. All those analysed in this study were classified
as 'normal' by the diagnostic service laboratory in respect
of cell counts and protein and glucose contents. Before
storage at - 20°C, the fluids were centrifuged at 2000 g to
remove cells from the retained supernatant. Serum was
obtained from six healthy male subjects and was assayed
within the shortest possible time.
Measurement of ferrous ions
Into new clean plastic tubes (known to be free of iron
and other trace metals) were placed the following reagents
in the order indicated. (1)0.4 ml of DNA solution (1 mg/
ml), stored over 1/20th its volume of Chelex-100 resin;
the resin was retained for further use and was replenished
with DNA solution. (2) 0.1 ml of bleomycin sulphate (1.5
mg units/ml) stored over a 5% (w/v) solution of conalbumin retained in sealed dialysis tubing [13]; where
appropriate, 10 pl of 120 mmol/l sodium azide was
added to inhibit the ferroxidase activity of caeruloplasmin. (3)0.2 ml of sample or ferrous salt standard (for
CSF this was reduced to 0.1 ml of sample and the appropriate corrections were introduced). (4) 0.4 ml of 0.5
mol/l sodium phosphate buffer, pH 7.0, treated with 5%
(w/v) conalbumin retained in a sealed dialysis tube. A
blank was prepared for each biological sample by
omitting bleomycin. The reaction mixtures were
incubated at 37°C for 30 min to bring about ferrous iondependent degradation of DNA.
Preparation of ferrous salt standards
Ammonium ferrous sulphate (1 mmol/l) was dissolved
in Chelex-resin-treated distilled water containing 1
mmol/l HCI. The solution was de-oxygenated by sparging
with nitrogen. Working ferrous salt standards, 1-10
pmol/l, were prepared from the stock iron solution by
diluting, just before use, with Chelex-resin-treated
distilled water.
Measurement of DNA damage as thiobarbituric acid
(TBA)-reactivity
To the tube contents, after incubation, was added 0.5
ml of TBA [1.0"/0(w/v)in 50 mmol/l NaOH] and 0.5 ml of
25"/0 (v/v) HCl. The tubes (plastic or contents transferred
to glass) were heated at 100°C for 5 min in a dry-block
heater. When cool, the tube contents were extracted with
1.5 ml of butan-1-01 and were centrifuged at 2000 gfor 8
min to separate the phases. The clear upper organic layer
containing low levels of TBA-reactive material was
measured spectrofluorimetrically. Fluorescence emission
was measured at 553 nm with excitation at 532 nm.
Relative fluorescence intensity (RFI)units were expressed
relative to a standard of rhodamine B ( 3 pmol/l) set to
100 units. Appropriate blank values were subtracted from
the test samples for calculation of ferrous ion content.
RESULTS
Ferrous ion assay
The measurement of ferrous ions in aqueous solution,
using the bleomycin reaction with fluorescence detection
(Fig. I), was linear from 1 to 10 pmol/l (Fig. 2). Optimal
conditions for the detection of ferrous ions were found to
be a pH value of 7.0, using a phosphate buffer with a
reaction molarity of 0.18 mol/l, and 0.135 mg units of
bleomycin incubated at 37°C for 30 min. Ferric ions will
also bind to bleomycin under these conditions but in the
absence of a suitable reductant will not damage DNA
with the release of TBA-reactive material (Table 1).
Copper ions also bind to bleomycin and could conceivably interfere with the binding or redox cycling of ferrous ions. Concentrations of cupric salt from 1 to 20
pmol/l did not, however, inhibit the reactivity of 10
pmol/l ferrous ions when degrading DNA (Table 1).The
sensitivity of detection for ferrous ions was around 0.5
pmol/l and the coefficient of variation at 5.0 pmolll
ferrous ions was 4.4% and that at 10 pmol/l was 2.3%.
The extracellular copper-containing protein caeruloplasmin has a ferroxidase activity and is able to catalyse
the oxidation of ferrous ions and simple ferrous complexes. When caeruloplasmin (diluted in Chelex-resintreated distilled water) was added to the DNA-degrading
reaction of ferrous-bleomycin it showed a concentrationdependent ability to inhibit DNA damage (measured as
TBA-reactivity) (Table 1). Caeruloplasmin was inhibitory
whether added before the reaction was started by the
addition of ferrous salt or just after the addition of ferrous
salt (Table 1).Azide at a final reaction concentration of
1 mmol/l prevented the inhibitory activity of caeruloplasmin on DNA degradation (Table 1). A variety of
scavengers of -OH, such as mannitol, formate and thiourea, and the hydrogen peroxide-removing enzyme catalase, had no effect on the detection and measurement of
ferrous ions with the bleomycin reaction (data not shown).
Application of the ferrous ion assay to biological samples
Freshly obtained human serum from six normal healthy
subjects had no detectable levels of ferrous ions, and
addition of azide to the reaction did not cause such iron to
appear in the assay (data not shown). Each sample had a
paired blank which did not contain bleomycin and this
was included to control for TBA-reactive material present
in plasma not derived from DNA degradation by ferrous
ions.
CSF samples obtained for authentic medical purposes
and retrospectively classified as 'normal' by cell count and
protein and glucose contents were assayed for ferrous
ions. When compared with paired controls, to which
bleomycin had not been added, degradation of DNA
occurred and the levels could be measured spectrofluorimetrically. When the damage occurring to DNA was
Assay for ferrous ions in cerebrospinal fluid
+
317
Bleomycin ---+
bound iron (Ill) 3
and a high ferroxidase
activity
+ 0,
inhibits
ferroxidase
activity
‘Active oxyzen’
.- species
.
degrades DNA with release
of base-propenals
-1
4.
MDA
TBA
(TBA),-MDA (A,,, “A
(TBA),-MDA (excitation, 532 nm,
emission, 553 nmj
$
+
B-in-+$
Biological sample containing loosely bound
iron which is converted to iron (11) by a
reductant, such as ascorbate, and maintained
by a low or inhibited ferroxidase activity
T
As above
Fig. I . Diagrammatic representation of the two bleomycin assays used to detect low-molecular-mass iron in the
ferrous state. (a) Bleomycin assay for total loosely bound iron. (b) Bleomycin assay for ferrous ions and their redox cycling
activity. Abbreviation: MDA, malondialdehyde
calculated in ferrous ion equivalents, it suggested a range
of 3.6-55.6 ,umol/l (Table 2). However, when total loosely
bound iron was measured by the standard bleomycin
assay [I41 (Fig. I), to which high concentrations of
ascorbate (7.5 mmol/l) were added to both samples and
standards to inhibit caeruloplasmin and make the reaction
limiting to sample iron content (ferrous and ferric), values
for total loosely bound CSF iron were only within the
range 0.05-1.00 pmol/l (mean+ SD 0.55 k 0.27) (Table 2),
in agreement with previously published results [I].Under
these conditions the degradation of DNA is extensive and
measurement of TBA-reactivity is performed spectrophotometrically (Fig. 1).
The large discrepancy between the assay used for
ferrous ions to which an iron reductant essential for DNA
damage is not added, and the total loosely bound iron
assay, suggests that iron bound to bleomycin in the
ferrous ion assay is undergoing several redox cycles by a
reductant present in CSF. It is well established that CSF
contains high levels of ascorbate (up to ten times the level
in plasma), and incubation of CSF with ascorbate oxidase
(10 units/ml) abolished the ability of the assay to detect
ferrous ions in CSF but not total loosely bound iron
(Table 2). Intrinsic and variable ascorbate levels in CSF
therefore appear to determine the ability of low-molecular-mass iron to undergo redox cycling and to damage
DNA. This was further investigated by adding a range of
ascorbate concentrations (4-800 pmol/l) to the ferrous
ion assay in simple aqueous solution using the trace
amounts of adventitious iron present in the reagents
(0.5-1.5 pmolll) as the source of iron in the reaction. Fig.
3 shows ascorbate values from 0 to 30 ,umol/l (the
relationship was linear up to 800 pmol/l). The mean CSF
value for DNA degradation expressed as ascorbatedriven redox cycling ferrous ions, stored for an average of
28 days, would be 23.8 k 16.0 pmol/l (Table 2); however,
this represents an ‘iron-ascorbate activity’ and not an
absolute amount of ferrous ions.
DISCUSSION
Iron, in the ferric state, is transported and stored in the
body by specific proteins such as transferrin and ferritin
[15]. Without attachment to a ligand ferric ions rapidly
become insoluble at physiological pH values, forming
ferric polynuclear hydroxide complexes. Unlike ferrous
ions, which are soluble at physiological pH values, ferric
ions are poorly reactive with oxygen and its reduction
intermediates. Ferrous ions, by their solubility, reactivity
and inability to bind to transferrin, therefore pose a
potential hazard to biological systems. Intracellularly,
where low-molecular-mass iron is required for the
synthesis of iron-containing proteins, problems of iron
reactivity are limited by the specific removal of reduction
intermediates of oxygen [3]. Extracellularly, in contrast
however, iron is kept tightly sequestered in non or poorly
reactive forms by specific iron-binding proteins such as
transferrin, lactoferrin, haemopexin and haptoglobins (for
a review, see [16]) making the detectable levels of lowmolecular-mass iron in plasma effectively nil [2].
J. M. C. Gutteridge
318
In pathological states of iron-overload, such as
idiopathic
haemochromatosis,
thalassaemia
and
leukaemic patients on chemotherapy, chelatable redox
active iron can be detected in plasma [7,8, 171, suggesting
that the transferrin is fully saturated with iron o r that
some of the iron is not correctly loaded on to the protein
[18].Iron ions in the presence of normal concentrations of
plasma reductants, such as ascorbate, urate and atocopherol, are converted to the reactive ferrous state
2
4
6
8
10
12
14
[Ferrous ions] ( p o l i l )
Fig. 2. Dose-response curve for ferrous ions detected as TBAreactive material released from DNA in the presence of
bleomycin
[lo]. The copper-containing plasma protein caeruloplasmin, however, performs an important anti-oxidant
role in vivo by catalysing the oxidation of ferrous ions to
the less reactive ferric state, and in the process transfers
electrons to oxygen to form water (for a review, see [19]).
The introduction, several years ago, of the bleomycin
assay for total chelatable redox active loosely bound iron
[2] allows the simple detection and measurement in
biological fluids of iron likely to take part in oxygenradical chemistry (for a review, see [ 11).In the assay, iron
ions, both ferrous and ferric, are chelated to bleomycin
bound to DNA. When high concentrations of ascorbate
are added to both samples and standards, to inhibit the
ferroxidase activity of caeruloplasmin and make the
reaction limiting on iron content, DNA is substantially
degraded with the release of base-propenals [20] that
break down to give malondialdehyde [21], which can be
detected spectrophotometrically as a thiobarbiturate
adduct. The damage done to DNA by bleomycin and iron
is site-specific, making most biological anti-oxidants and
scavengers ineffective at preventing iron-dependent
damage to DNA. All laboratory reagents and chemicals
contain trace amounts of adventitious iron [22], and by
reducing this with Chelex resin and conalbumin the sensitivity of the bleomycin assay has been increased. The
modified bleomycin assay for ferrous ions described here
does not have ascorbate or other reductants added to
samples or standards, and damage to DNA is small,
necessitating the use of spectrofluorimetry. The assay can
be used to speciate and measure micromolar concentrations of ferrous ions in simple aqueous solution not
Table I, Oxidation by caeruloplasmin of ferrous ions measured with the bleomycin assay. Concentrations for caeruloplasmin and azide are the final reaction concentrations; those for iron and copper salts are addition concentrations. The blank
value for the bleomycin reaction in the absence of added ferrous ions o r reductant was 5 RFI units. This was subtracted t o
calculate the values shown.
TBA fluorescence
at 553 nm
(RFI) units
I. Ammonium ferrous sulphate standard (10 pmolll)
2. Ferric chloride (10 pmolll)
57
0
Reaction I plus caeruloplasmin 0.30 mglrnl
0. I5 mg/ml
0. I 2 mglml
0.08 mgiml
0.04 mglml
0.02 mglml
0.01 mglml
0.005 mgiml
Caeruloplasmin (0. I 2 mg/ml), heat-denatured
Caeruloplasmin (0. I 2 mglml) plus azide I mmolll
Reaction I plus cupric chloride I pmolil
5 pmolil
10 pmolll
20 pmolil
Addition of caeruloplasmin (0.1 2 mgiml) t o reaction I before the iron salt
Addition of caeruloplasmin (0.1 2 mglml) t o reaction I after the iron salt
Loss of ferrous
ions detected
in the assay (%)
I00
I00
95
88
78
56
40
16
0
0
0
0
0
0
96
95
Assay for ferrous ions in cerebrospinal fluid
319
Table 2. Bleomycin-detectable iron (total) and ferrous ion activity in CSF
CSF sample
No. of days stored at
- 20°C
I
2
3
21
9
7
4
5
6
15
20
37
7
44
8
9
32
10
II
12
13
14
before analysis
31
5
44
36
44
44
Pooled CSF (six samples)
Pooled CSF (six samples) t ascorbate
oxidase (10 unitslml)
Bleomycin-detectable iron
(total) (,umol/l)
Redox cycling iron and ascorbate
measured as ferrous ion activity
and expressed as [Fe'+] (pmolll)
0.09
0.40
I .oo
0.30
0.05
0.6I
0.35
5.4
48.0
39.0
33.0
10.5
28.0
16.0
3.6
55.6
0.70
0.70
0.87
0.6 I
31.2
28.8
0.61
0.78
0.6 I
I8.4
I0.8
3.6
0.40
16.4
-
containing an iron reductant, but in biological fluids the
assay is complicated by the presence of iron reductants
which limit it to detecting ferrous ions and their activity.
Blank values in the ferrous ion assay are virtually zero,
since adventitious ferrous ions are unstable in aerobic
solution at a pH value of 7.0. When the assay is applied to
normal human serum it does not detect ferrous ions,
either in the presence o r absence of a caeruloplasmin
inhibitor, as would be expected from previous studies
using bleomycin for total loosely bound iron [ 2 ] .
When normal human CSF is assayed for ferrous ion
content, using the method described here, levels some 40
times greater than total loosely bound iron are found.
This anomaly arises because of the high ascorbate and
high iron-saturation of transferrin in CSF. Low levels of
transferrin with high iron levels [23] mean that CSF
transferrin is probably fully saturated with iron and has no
further iron-binding capacity. Low levels of caeruloplasmin and high levels of ascorbate are likely to produce
a molar ratio of ascorbate to caeruloplasmin of around
25 000, which would be inhibitory to the ferroxidase
activity of caeruloplasmin [24]. When ferrous ions in
simple aqueous solution are measured with the spectrofluorimetric bleomycin assay, the reaction appears to be
quantitative for ferrous ions. This is not so, however,
when applied to a fluid such as CSF containing high levels
of ascorbate. Unlike the bleomycin assay for total loosely
bound iron, to which high levels of ascorbate are added to
both samples and standards to inhibit ferroxidase activity
and make the reaction independent of small variations in
intrinsic sample ascorbate concentration, the ferrous ion
assay does not have ascorbate or other iron reductants
added to it, and the redox activity of iron becomes
dependent on sample ascorbate levels. Iron will be
0
2.
2.
-.z
"7
=
1.6
"?
ga
u-
1.2
0.8
0.4
[Ascorbate] (pmoVl)
Fig. 3. Relationship between ascorbate concentration added to
the bleomycin reaction, in the presence of trace amounts of
adventitious ferric salts, and the damage done to DNA by
ferrous ions calculated from the standard curve shown in Fig. I
present in a ferric state in trace amounts in all assay
reagents and will be reduced to the ferrous state by sample ascorbate, although this will not occur in reagent
blanks. For these reasons it is at present, not possible to
320
J. M. C. Gutteridge
directly quantify ferrous irons in CSF by using the
bleomycin-ferrous ion assay. However, its application to
biological fluids does allow the detection of ferrous ions,
and it may be reasonable to assume that the total
bleomycin-iron value is closely related to the total ferrous
iron content.
ACKNOWLEDGMENTS
I am grateful to Mr P. Lamport, Department of Microbiology, Whittington Hospital, London, for the gift of CSF
samples. 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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