Vol.1, No.2, 104-110 (2009) Health
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
Ceruloplasmin levels in human sera from various
diseases and their correlation with patient’s age and
Viorica Lopez-Avila1*, William H. Robinson2, Kirk Lokits3
1Agilent Technologies, Santa Clara, CA, USA; Corresponding author: viorica_lopez-avila@agilent.com
2Stanford University, Stanford, CA, USA
3University of Cincinnati, Cincinnati, OH, USA; Present address: Midwest Research Institute, 425 Volker Blvd, Kansas City, MO, USA
Received 7 July 2009; revised 5 August 2009; accepted 7 August 2009.
Ceruloplasmin (Cp), a copper metalloprotein in
human serum has been a valuable diagnostic
marker in Wilson’s disease where Cp levels tend
to be low while high levels in serum were asso-
ciated with myocardial infarction, neoplastic
and inflammatory conditions. There is no stan-
dardized reference method for Cp and current
immunologic and bichromatic assays have a
number of drawbacks. The method described
here uses immunoaffinity chromatography to
remove six of the most abundant proteins from
a serum sample and high-pressure liquid chro-
matography (HPLC) with a size-exclusion col-
umn to separate Cp from other serum proteins
and any free Cu prior to analysis of 63Cu and
65Cu by inductively-coupled plasma mass spec-
trometry (ICPMS). Identification of Cp is based
on retention time match of the unknown protein
in the serum sample with the Cp external stan-
dard and the presence of 63Cu and 65Cu at a ratio
of 2.2 ± 0.1. The method accuracy, as estab-
lished independently by two of the authors with
a reference serum certified for Cp, is 98 to 101%
and the coefficient of variation is 6.4% and 5.4%,
respectively. The assay was used to analyze a
total of 167 human sera for Cp from patients
with myocardial infarction (MI), pulmonary em-
bolism (PE), rheumatoid arthritis (RA), systemic
lupus erythematosus (SLE), other forms of ar-
thritis, and a set of healthy patients as normal
controls (NC). Our data show that Cp concen-
trations tend to be higher in MI, RA, and SLE
patients, higher in female as compared to male
patients, and we did not observe a correlation
between Cp concentration and patient’s age for
the set of 70 patients for which we had gender
and age information.
Keywords: Ceruloplasmin; HPLC-ICPMS;
Immunoaffinity Chromatography
Ceruloplasmin (Cp) is a blue alpha-2 glycoprotein with a
molecular weight of 132,000 u [1]. It binds 90-95% of
blood plasma copper (Cu), has 6-7 Cu ions per molecule
(1) and exhibits ferroxidase activity [1,2], amine oxidase
activity [1], superoxidase activity [1] as well as it is in-
volved in Cu transport and homeostasis [1]. Hellman and
Gitlin, however, reported that Cp plays no essential role
in the transport and metabolism of Cu [2] and in a sepa-
rate study [3] reported that analysis of Cu incorporation
into apoceruloplasmin (apoCp) in vitro showed that fail-
ure is intrinsic to mutant proteins. Linder et al. [4] claim
that newly absorbed dietary Cu is transported by plasma
protein cariers (i.e., albumin, transcuprein, and Cp) from
intestine to liver and kidney, and that Cp is involved
primarily in transport of Cu from liver to other organs.
Prohanska and Gybina [5] provide details on the trans-
port process in which Cu, imported by plasma mem-
brane protein Ctr1, binds to Cu chaperone proteins like
Atox1, which then docks with ATP7B and delivers Cu to
plasma Cp.
Current analytical procedures for the determination of
Cp include immunoturbidimetry and nephelometry [6],
in which Cp is reacted with anti-Cp antibodies to give
insoluble aggregates whose absorbance is proportional to
the concentration of Cp in the sample [6], radial immu-
nodifussion (RID) test [7], and bichromatic assay [8].
When comparing RID with immunonephelometry a sig-
nificant bias was found that was in part attributed to the
variation in the antisera sources used in the two methods
[7]. In the case of the bichromatic method, the proce-
dures are based on the oxidase activity of Cp on dia-
mines such as benzidine. The bichromatic method re-
quires special precautions (i.e., benzidine is a known
V. Lopez-Avila et al. / HEALTH 1 (2009) 104-110
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
carcinogen) and purification of substrates [9], detects
only Cp and not the apoCp [10], and it is not very effec-
tive since Cp does not have its own substrate [11]. The
immunologic methods also have drawbacks because
antisera cross-react with apoCp thus giving higher con-
centrations for Cp [9]. Evidence suggests that patients
with Wilson’s disease may have exhibited normal serum
concentrations of Cp because the immunologic assay
could not distinguish between the apoCp and Cp [10]. In
general, a normal person has 0.2 to 0.5 mg/mL of Cp in
serum [11].
Although low serum concentration of Cp has been an
important diagnostic indicator of Wilson’s disease [10],
high Cp concentrations were reported in patients with
macular degenerations as compared with controls (i.e.,
Cp concentration 0.691 ± 0.153 mg/mL vs 0.312 ± 0.064
mg/mL) by Newsome et al. [12], in patients with MI by
Reunanen et al. [13], and in a variety of neoplastic and
inflammatory conditions like carcinomas, leukemia,
Hodgkin disease, primary biliary cirrhosis, systemic
lupus erythematosus, and rheumatoid arthritis [14].
This paper describes the peer verification of a new
method for the determination of Cp in human serum at
biologically relevant concentrations > 0.01 mg/mL using
a reference serum certified for Cp and a set of 167 hu-
man sera from several diseases. This method, which was
published recently [15], uses HPLC to separate Cp from
other proteins including transcuprein (molecular weight
270,000 u) and from inorganic ions, and ICPMS to de-
tect Cu isotopes at mass-to-charge (m/z) ratios of 63 and
65, and to identify Cp from the HPLC retention time and
the signal ratios of Cu isotopes 63Cu and 65Cu measured
with ICPMS. To eliminate possible interference from
highly abundant proteins, some of which may bind Cu to
form protein-Cu complexes, the serum sample is first
depleted of albumin, IgG, IgA, transferrin, haptoglobin,
and anti-trypsin using immunoaffinity chromatography
prior to HPLC. Quantitation of Cp in the depleted serum
is performed by external standard calibration with a Cp
Materials: the standard of Cp purified from human
plasma was from EMD Biosciences/Calbiochem (La
Jolla, CA) in lyophilized form from 133 µL of 50 mM
potassium phosphate, pH 6.8, 100 mM potassium chlo-
ride, 200 mM ε-amino-n-caproic acid and 5mM EDTA,
with a purity of >95%. The 167 serum samples were as
follows: 37 patients with MI, 50 with RA, 24 with SLE,
8 with PE, 16 NC, and 32 sera (identified as “other” in
this paper) were from patients with different forms of
arthritis: osteoarthritis, juvenile rheumatoid arthritis,
reactive arthritis, inflammatory arthritis; myositis and
dermatomyositis, fibromyalgia, anthralgia, ankylosing
spondilitis, spinal stenosis, Sjogren, Reiter’s syndrome,
connective tissue disease, scleroderma, polymyalgia
rheumatica and palindromic rheumatism gout and
CREST syndrome.
ERM DA470 is a human serum certified for 15 pro-
teins including Cp [16,17] and was purchased from RTC
(Laramie, WY).
Serum preparation: all human samples were col-
lected and utilized under Institutional Review Board
approved protocols and with informed consent. To
summarize, blood samples were withdrawn using sterile
conditions and allowed to clot at room temperature for a
minimum of 10 min. Serum was separated by centrifu-
gation for 10 min at 4000 rpm, divided among several
vials to minimize freeze-thawing, and kept at -80°C until
Immunoaffinity chromatography: high-abundant
protein removal from human serum was performed on a
4.6 x 100 mm immunodepletion column (Agilent Tech-
nologies) with a capacity of 40 μL of non-diluted human
serum (capacity is defined as the amount of original se-
rum that can be loaded onto the column such that 99% of
the targeted high-abundant proteins are removed for at
least 200 injections on a particular column). After a 5-
fold dilution of serum sample with buffer A and filtration
through a 0.22 μm spin filter, 150 μL of the diluted sam-
ple was injected onto the column in 100% Buffer A at a
flow rate of 0.5 mL/min for 10.0 min. After collection of
the flow-through fraction (2 mL), the column was
washed and the bound proteins were eluted with 100%
Buffer B at a flow rate of 1.0 mL/min (volume of bound
protein fraction 3 mL). The immunoaffinity column was
then regenerated by equilibrating it with Buffer A for 13
min bringing the total run cycle to 30.0 min. Fraction
collection of flow-through proteins was time-controlled
and corresponded to the UV 280 nm absorbance of the
eluting proteins. The flow-through fraction was collected
and kept at 4º C using the thermostatted fraction collec-
tor, was reduced to a final volume of 30 µL using spin
concentrators and analyzed by HPLC-ICPMS. Bound
proteins (i.e., albumin, IgG, IgA, transferin, haptoglobin
and anti-trypsin) were eluted from the immunodepletion
column and selected samples were analyzed by ICPMS
(data not included here). Buffer A is a phosphate buffer
(pH 7.4) and buffer B is a concentrated urea buffer in
water (pH 2.25).
Instrumentation: An Agilent 1100 LC system
equipped with a binary pump, degasser, autosampler
(300 µL loop) with thermostat, diode array detector with
6 mm flow cell, and a thermostated fraction collector
was used for the immunodepletion work. Protein separa-
tion was achieved on a silica TSKGel column SW3000
(30 cm x 4.6 mm id x 4 μm particles x 250 nm pore size)
V. Lopez-Avila et al. / HEALTH 1 (2009) 104-110
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
from Tosoh Bioscience (Montgomerryville, PA). All
HPLC analyses were performed on an Agilent Tech-
nologies 1100 Series High Performance Liquid Chro-
matography system equipped with a binary pump, de-
gasser, autosampler (100 μL loop) and diode array de-
tector (215 nm and 280 nm). 0.1 M Tris (pH 7) was used
as mobile phase at a flow rate of 0.3 mL /min. The liquid
flow from the HPLC column was converted into aerosol
droplets by a Micromist nebulizer with a dual pass spray
chamber. 63Cu and 65Cu scan was performed on an
Agilent 7500ce ICPMS system with a quadrupole mass
analyzer and an Octapole Reaction System (ORS) for
matrix-based interference removal. High levels of Na in
the sample can cause the formation of 40Ar 23Na polya-
tomic species that overlap with 63Cu. Similarly, 31P based
molecular species (31P 16O 16O and 31P 18O 16O) can
overlap with the 63Cu and 65Cu isotopes. The ORS with
He (99.999 % purity) as collision gas at 3.5 mL/min was
used to eliminate these interfering species and to im-
prove signal to noise. ICPMS conditions: outer gas (Ar)
flowrate 15 L/min; carrier gas (Ar) flowrate 0.8 L/min;
makeup gas (Ar) flowrate 0.15 L/min; RF power 1.55kW,
sampling depth 8 mm.
3.1. Method Performance
The performance of this method (see Table 1) was es-
tablished independently by two of the authors (in sepa-
rate laboratories) with a reference human serum ERM
DA470 that is certified for Cp at 0.205 mg/mL using
identical instrumentation. This serum was reconstituted
with high purity water and analyzed in triplicate in
Laboratory 1 and in seven replicates over a period of two
months in Laboratory 2. The results are summarized in
Table 1. The agreement between the concentration of Cp
in the certified serum and the reconstituted serum ana-
lyzed by this method is excellent (method accuracy is
101% in Laboratory 1 and 98.0% in Laboratory 2). The
coefficient of variation (CV) for the three replicate
measurements of the freshly reconstituted serum in
Laboratory 1 is 5.4 %. The CV of the seven replicates
performed over a period of two months in Laboratory 2
is 6.4%.
Method performance data are included in Table 2.
The method detection limit was established from the
instrument detection limit and applies only to sample
injection volumes of 5 µL; larger injection volumes
would allow a lower method detection limit but such
experiments were not pursued here. The method dy-
namic range is given as 0.01 to 5 mg/mL since this is the
range of concentrations that were tested here. Although
the instrument dynamic range is 9 orders of magnitude,
that would involve adjustments in ICPMS operating pa-
rameters to accommodate such a wide range. Expected
Table 1. Concentration of Cp in the ERM DA 470 reference
Conc meas-
ured in this
ERM DA 470
Reference Serum
(Laboratory 1)
(0.011)a 0.208 (5.4 %)b 2.1 (3.6%)b
ERM DA 470
Reference Serum
(Laboratory 2)
(0.011)a 0.201 (6.4%)c 2.2 (7.3%)c
aUncertainty (mg/mL) – defined as half-width of the 95% confidence
interval of the mean value(K factors were chosen according to the
t-distribution depending on the number of labs) [12, 13].
bAverage of 3 determinations; value given in parentheses is the coeffi-
cient of variation.
cAverage of 7 determinations; value given in parentheses is the coeffi-
cient of variation.
Table 2. Cp determination by HPLC-ICPMS-method perfor-
Method indicatorValue
Detection Limit 0.01 mg/mL
Dynamic Range 0.01 – 5.0 mg/mL (tested only until 5
CV for immunodepletion : 0.07% to 2.2 %
CV for injection into HPLC: 5.3%
(Cp standard at 1 mg/mL)
Overall CV: <10%
Accuracy using
101 % (Laboratory 1);
98.0% (Laboratory 2)
Cp Identification
from retention time match of the unknown
peak in the sample to the Cp standard and
the presence of 63Cu and 65Cu at a ratio of
2.2 ± 0.1
aThis method takes approximately 95 min/sample from start to finish
(15 min dilution and filtration, 30 min immunoaffinity chromatography,
20-30 min concentration, and 20 min HPLC-ICPMS analysis).
concentrations of Cp in human sera are in the 0.1-2
mg/mL range, therefore a 30 µL volume of the original
serum is sufficient to detect Cp at 0.1 mg/mL if the final
volume of the depleted serum is 30 µL. The overall CV
for method reproducibility is <10 % and it is shown in
Table 2 for various steps in the analysis. The identifica-
tion of Cp is based on retention time match of the un-
known peak in the sample to the Cp standard and the
ratio of 63Cu/65Cu. The average HPLC retention time for
8 consecutive injections of the Cp standard is 8.389 min
with a CV of 0.059%. The relative abundance of the
63Cu is 69.17% and 65Cu is 30.83%, thus the theoretical
ratio for 63Cu /65Cu is 2.24; based on our experimental
data we set the acceptance limits for 63Cu /65Cu to 2.2 ±
0.1 [15].
In addition, we validated the Cp measurements by
performing a total Cu analysis on a set of 23 depleted
sera and compared those measurements with the Cp
concentrations measured by HPLC-ICPMS (see Figure
1). Regression analysis gave a correlation coefficient of
V. Lopez-Avila et al. / HEALTH 1 (2009) 104-110
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
0.9, which confirms literature reports that most Cu in
depleted serum is bound by Cp [1]. Furthermore, we
verified the number of Cu atoms bound by Cp in the
ERM DA470 reference serum by determining the total
Cu in the depleted sample. At a Cp concentration of 0.2
Total Cu conc vs Cp conc (n=23 samples)
y = 1073x + 641.98
= 0.805
Cp conc (mg/mL)
Total Cu conc (ng/mL)
Figure 1. Total Cu vs Cp concentration for 23 serum samples
(7 PE, 1 NC, 14 MI and ERM DA470).
mg/mL (measured in this study for the ERM DA470
serum), 6 Cu atoms per Cp molecule would correspond
to a total Cu concentration of 596 ng/mL and 7 Cu atoms
per Cp molecule would correspond to 695 ng/mL. Be-
cause the total Cu measured in the depleted reference
serum was in the range of 618-661 ng/mL, we concluded
that Cp must contains between 6 and 7 atoms per mole-
cule, consistent with the published data for Cp [1] Also
as part of method validation, a Cp standard, the ER-
MDA470 certified serum and one of the depleted MI
sera were fractionated by HPLC and the corresponding
fractions containing Cp were collected manually, and
were then subjected to one gel electrophoresis followed
by Cp band excision, in-gel digestion, and electrospray
MS of the digest to confirm the presence of Cp [15].
Figure 2 shows HPLC-UV and ICPMS chroma
tograms for a Cp standard and a depleted serum sample;
the HPLC chromatograms show the complexity of the
Figure 2. HPLC-ICPMS chromatograms for Cp standard and depleted serum sample.
V. Lopez-Avila et al. / HEALTH 1 (2009) 104-110
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
serum sample even after its depletion of the high abun-
dant proteins whereas the ICPMS chromatograms show
only the 63Cu and 65Cu signals at a retention time that
matches that of the Cp standard and are in a ratio corre-
sponding to 6-7 Cu atoms per Cp molecule.
3.2. Ceruloplasmin Levels in Sera from
Different Diseases
Figure 3 shows the distribution of Cp concentration
across several diseases, including MI, PE, RA, SLE,
other forms of arthritis (i.e., osteoarthritis, juvenile
rheumatoid arthritis, reactive arthritis, inflammatory
arthritis) and NC sera (167 serum samples in all). Sam-
ples derived from patients experiencing MI (37 in our
study) had an average Cp concentration of 0.402 ± 0.377
mg/mL and exhibited Cp concentrations as high as 1.64
mg/mL, while a subset of 50 RA patients and 24 SLE
patients had average concentrations of 0.447 ± 0.215
mg/mL and 0.426 ± 0.264 mg/mL and exhibited elevated
Cp concentrations as high as 1.23 mg/mL and 1.24
mg/mL, respectively (Figure 3). Normal Cp concentra-
tions are in the 0.2-0.5 mg/mL range [11] and the aver-
age Cp concentration in the set of NC sera in our study
(16 patients) was 0.316 ± 0.120 mg/mL. When compar-
ing Cp concentrations for our MI, RA, SLE, and
“other”sera with our set of NC sera, only the RA and
SLE data were statistically different from the NC in a t
test (i.e., p values were 0.0037 and 0.0837 for RA and
SLE sera, respectively). The MI data reported here show
a much higher variation than our NC data, and this
variation is statistically significant (F value is 9.92, F crit
is 2.22, and p<0.001). Reunanen et al. [13], using serum
from 104 patients with MI or stroke and 104 matched
controls, concluded that high Cp concentrations in serum
were significantly associated with higher incidents of MI
but not of stroke.
Hantzschel et al. [18] reported for RA and polymyal-
gia rheumatica an average Cp concentration for 23 RA
patients (22 females) of 0.7 ± 0.4 mg/mL and for 16 po-
lymyalgia rheumatica patients (all females) 0.5 ± 0.1
mg/mL. The authors suggested that clinical data, includ-
ing a history of hip and shoulder muscle tenderness and
lack of positive rheumatoid factor, and a normal Cp level
could distinguish polymyalgia rheumatica from rheuma-
toid arthritis. We observed a similar trend for RA pa-
tients as compared with patients with “other” forms of
arthritis. The average Cp concentrations for RA of 0.447
± 0.215 mg/mL were significantly different from the
average Cp concentrations for “other” diseases, which
had an average Cp concentration of 0.376 ± 0.145
mg/mL, only when doing a one-tail test (p value was
0.041). Perhaps Cp concentrations above 0.5 mg/mL
would be indicative of disease severity, however charac-
terization of larger sample sets will be necessary to sub-
stantiate this observation.
Figures 4 and 5 show Cp concentrations as a function
of patient’s gender and age, respectively. Although this is
a very limited sample set (70 sera from 33 RA, 5 SLE
and 32 “other”arthritis patients with 49 females and 21
males) it is interesting to note that female patients exhib-
ited slightly higher Cp concentrations (ave ± SD of
0.392 ± 0.153 mg/mL) than male patients (0.319 ± 0.123
mg/mL) that were statistically significant at 5% signifi-
cance level (48 degrees of freedom, tstat. 2.104, tcrit 2.011,
probability for a two-tail test was 0.0410). However,
when we averaged only the Cp concentrations for the 33
RA patients by gender (24 females and 9 males) we
found a larger difference between the female patients
and male patients (0.417 ± 0.158 mg/mL vs 0.278 ±
0.096 mg/mL, respectively) that was statistically sig-
nificant at 5% significance level (24 degrees of freedom,
tstat. 3.059 , tcrit 2.064, probability for a two-tail test was
0.005 ). Data reported by Lyngbye and Kroll [19] for a
normal population (280 patients, 149 males and 111 fe-
males) also indicate significantly higher concentrations
of Cp in female patients which are known to be caused
by use of oral contraceptives [20].
There does not seem to be a correlation between the
Cp concentration and patient’s age across these 70 pa-
tients with RA and arthritis (Figure 5). The average Cp
concentration for RA patients was 0.370 ± 0.149 mg/mL
and 0.384 ± 0.163 mg/mL for <50 y.o. and > 50 y.o.,
respectively ( tstat. of 0.247 is less than t crit 2.059, p for a
two-tail test was 0.807, indicating that the results were
not statistically different). The average Cp concentra-
tions for “other” arthritis patients were 0.387 ± 0.162
mg/mL and 0.356 ± 0.110 mg/mL for <50 y.o. and > 50
y.o., respectively (tstat. 0.645 is less than tcrit 2.048, p for
a two-tail test was 0.524), indicating again that the
167 serum samples consist of 37 MI (myocardial infarction), 16 NC
(normal controls), 8 PE (pulmonary embolism), 50 RA (rheumatoid
arthritis), 24 SLE (systemic lupus erythematosus) and 32 other diseases
(osteoarthitis, gout, dermatomyositis, ankylosing spondylitis, myositis,
juvenile rheumatoid arthritis, etc)
Figure 3. Cp concentration for various diseases and normal
V. Lopez-Avila et al. / HEALTH 1 (2009) 104-110
SciRes Copyright © 2009 Openly accessible at http://www.scirp.org/journal/HEALTH/
70 serum samples from 33 RA patients, 5 SLE, and 32 other diseases
M – male (21 patients)
F – female (49 patients)
Figure 4. Cp concentration as a function of patient’s gender.
70 serum samples from 33 RA patients, 5 SLE, and 32 other diseases
Figure 5. Cp concentration as a function of patient’s age.
results were not statistically significant). Results for a
normal population indicated no age variation in adults
[19], however in another study Revnic [21] reported
differences (p<0.05) between Cp concentrations in RA
patients < 50 y.o. and >70 y.o. We have looked at Cp
concentration for 22 RA patients < 50 y.o. (12 patients)
and > 66.6 y.o (10 patients) and found no significant
differences at p<0.05. Age related changes in human Cp
concentrations were attributed to oxidative modifications,
which can likely cause conformational changes around
the Cu sites [22].
The method described here uses immunoaffinity chro-
matography and HPLC to separate Cp from the serum
proteins prior to analysis by ICPMS. By removing the
six most abundant proteins from serum with immunoaf-
finity chromatography and by using HPLC to separate
Cu bound by Cp from any free Cu in the serum sample,
we demonstrated that we can measure Cp in the ERM
DA470 reference serum with an accuracy of 98 to 101%.
The HPLC-ICPMS method was used to analyze 167
serum samples from several diseases and a set of NC for
Cp. Our data for the 167 human sera show that Cp con-
centrations tend to be higher in MI, RA, and SLE pa-
tients. Cp concentrations were higher in female as com-
pared to male patients, and this trend was most promi-
nent in patients with RA. We did not observe a correla-
tion between Cp concentration and patient’s age for the
limited set of 70 patients for which we had gender and
age information. Thus, measurement of Cp levels by
ICPMS represents a biomarker that when combined with
conventional clinical and laboratory data may provide
increased diagnostic value.
The authors thank Toshiaki Matsuda of Agilent Tokyo Analytical Divi-
sion, Tokyo, Japan, for making available an ICPMS system for per-
forming this research, Alex Apffel of Agilent Labs, Santa Clara, CA for
making available an HPLC system with fraction collection and for
assistance with the immunodepletion process.
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List of Abbreviations:
Cp - ceruloplasmin
HPLC - high-pressure liquid chromatography
ICPMS - inductively-coupled plasma mass spectrometry
MI - myocardial infarction
NC - normal control
PE - pulmonary embolism
RA - rheumatoid arthritis
SLE - systemic lupus erythematosus
CREST- form of systemic sclerosis
ERM - European Reference Materials