3,4-Dihydroxy-l-phenylalanine as a biomarker of oxidative damage in proteins: Improved detection using cloud-point extraction and HPLC

Article abstract of DOI:10.1016/j.bbrc.2014.08.080

Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker for oxidized proteins is protein-bound 3,4-dihydrox

Full text of DOI:10.1016/j.bbrc.2014.08.080

Biochemical and Biophysical Research Communications 452 (2014) 376–381
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Biochemical and Biophysical Research Communications
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3,4-Dihydroxy-L-phenylalanine as a biomarker of oxidative damage
in proteins: Improved detection using cloud-point extraction and HPLC
⇑
Peter A.C. McPherson , Bryn T. Türemen
School of Applied Science, Belfast Metropolitan College, Belfast, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 August 2014
Available online 23 August 2014
Oxidized protein adducts are formed under conditions of oxidative stress and may represent a valuable
biomarker for a variety of diseases which share this common aetiology. A suitable candidate biomarker
for oxidized proteins is protein-bound 3,4-dihydroxyl-
L
-phenylalanine (
L
-DOPA), which is formed on
0
3
-hydroxylation of tyrosine residues by hydroxyl radicals. Existing methodologies to measure protein-
Keywords:
Protein oxidation
Biomarker
bound
L
-DOPA employ lengthy acid hydrolysis steps (ca. 16 h) which may cause artifactual protein
-DOPA. We report a
-DOPA which involves rapid hydrolysis followed
oxidation, followed by HPLC with detection based on the intrinsic fluorescence of
novel method for the measurement of protein-bound
L
L
Protein hydrolysis
by pre-column concentration of 6-aminoquinolyl-derivatives using cloud-point extraction. The deriva-
tized material is resolved by reversed-phase HPLC in less than 30 min and has derivatization chemistry
compatible with both UV and fluorescent detection, providing detection down to the femtomole level.
The method provides identical results to those found with highly specific ELISA-based techniques and
requires only basic instrumentation. The stability of the 6-aminoquinolyl-derivatives together with the
fast and sensitive nature of the assay will be appealing to those who require large sample throughput.
Ó 2014 Elsevier Inc. All rights reserved.
6
-Aminoquinolyl-N-hydroxyl-succinimidyl
carbamate
1
. Introduction
oxidation are redox active and mediate further damage to the pro-
tein [9], measurement of these latter species may provide a more
Oxidative modification of proteins is believed to play a major
role in the pathogenesis of a variety of conditions, including cancer
1], Alzheimer’s disease [2] and atherosclerosis [3]. Although virtu-
representative measure of protein oxidation in vivo. The aromatic
amino acids (viz. phenylalanine, tyrosine, tryptophan and
histidine) are particularly susceptible to oxidation and satisfy
this criterion [10]. The oxidation of tyrosine mainly produces 3,4-
[
ally all classes of biological molecule undergo oxidation, the pre-
eminence of proteins in vivo makes these a likely first target for
oxidants such as hydroxyl radicals (HO ) [4], superoxide (HOO)
dihydroxy-L-phenylalanine (L-DOPA) and a small quantity of its
2,4-isomer, ostensibly through a disproportionation reaction in
anaerobic conditions [11], or by elimination of hydroperoxyl radi-
Å
[
5] and reactive nitrogen species (RNS) [6]. The reaction of proteins
with oxidants may bring about damage in a number of ways,
including formation of protein hydroperoxides which mediate fur-
ther oxidation [7], and formation of imines which lead to fragmen-
tation of the polypeptide backbone [8]. Overall, the conformation
and activity of the protein can be substantially altered, leading to
disease progression. It therefore follows that the products of pro-
tein oxidation may serve as a useful metric of disease progression.
Although all twenty amino acids undergo oxidation modifica-
tion, in order for the products of such reactions to be suitable bio-
markers, they must be relatively stable and present at levels
amenable to detection. As many of the products of protein
cal in the presence of oxygen [12]. L-DOPA is prone to further oxi-
dation to produce a variety of o-benzoquinones which have a
tendency to undergo Michael additions with thiols to produce
2-,5- and 6-cysteinyl-DOPA adducts which are involved in protein
cross-linking [13,14]. L-DOPA itself is redox active and has suffi-
o
cient reactivity (E = 0.745 V) to bring about the reduction of cop-
per(II) to copper(I) which may be associated with the
concomitant production of superoxide anion which is a powerful
oxidizing species [15,16]. The production of superoxide in this
manner has been implicated in the formation of 8-oxo-7,8-dihy-
0
droxy-2 -deoxyguanosine, a key biomarker of oxidative DNA dam-
age [17,18].
In considering the chemical and spectroscopic properties of our
⇑
Corresponding author. Address: School of Applied Science (Chemistry), Belfast
Metropolitan College, Belfast BT3 9DT, UK. Fax: +44 (00)2890635001.
target analytes, we see that free tyrosine is marginally polar
3
E-mail address: pmcpherson@belfastmet.ac.uk (P.A.C. McPherson).
and has modest UV absorption (kmax 278 nm;
e
max 1.10 ꢀ 10
http://dx.doi.org/10.1016/j.bbrc.2014.08.080
0
006-291X/Ó 2014 Elsevier Inc. All rights reserved.

P.A.C. McPherson, B.T. Türemen / Biochemical and Biophysical Research Communications 452 (2014) 376–381
377
ꢁ
1
ꢁ1
M
cm ) with conversion to
L
-DOPA being accompanied by a
2. Materials and methods
3
ꢁ1
slight bathochromic shift (kmax 280 nm;
emax 2.71 ꢀ 10 M
ꢁ
1
cm ); however, this is virtually indistinguishable under normal
conditions. Accordingly, UV absorption alone cannot be utilized
for measurement of these compounds and several alternative
methods have been proposed. An early attempt by Arnow [19]
was an adaptation of the xanthoproteic test for proteins, in which
an alkaline nitrite/molybdate reagent reacts specifically with
2.1. Preparation of reagents
All chemicals were of at least analytical grade and purchased
from Sigma–Aldrich (Dorset, UK) unless otherwise stated. Aqueous
solutions were prepared using Milli-Q double-deionized water
2
(resistance > 18 m
X
/cm ) (Millipore, Bedford, MA, USA) stored
L
-DOPA in the presence of tyrosine to produce an orange
chromophore (kmax 510 nm) and is sensitive down to ca. 2.5 nmol
per sample. Later, Waite [20] reported measurement of -DOPA
over Chelex-100 resin to eliminate adventitial transition metal
ions. Protein (fatty acid free bovine serum albumin, BSA) was used
as supplied or oxidized (0.06 mg) by AAPH (10 mg/mL) at 50 °C for
3 h in sodium phosphate buffer (10 mmol/L, pH 7.4). The oxidized
proteins were stored under helium at ꢁ20 °C until required.
L
by UV difference spectroscopy of (catechelo)borate complexes.
This latter assay is less sensitive than the colorimetric procedure
(
ca. 10 nmol per sample) and is subject to interference from both
tyrosine and tryptophan. Improved detection of -DOPA can be
obtained by employing HPLC with fluorescent detection (E
80 nm; E 320 nm) [21]. However, as this method relies on the
intrinsic fluorescence of -DOPA, results are subject to the usual
variations associated with small changes in temperature and pH,
as well as self-absorption at high concentrations of -DOPA. Fur-
L
2.2. Protein purification and hydrolysis
m
2
x
Fasting peripheral venous blood samples were collected from
healthy volunteers by standard venipuncture of a prominent vein
in the antecubital fossa. Collected blood was transferred into
L
L
K
3
-EDTA tubes (Vacuette) containing 100
recovered by low-speed centrifugation. Plasma (700
bined with 100 L trichloroacetic acid (50%) and 50
l
mol/L BHT and plasma
L) was com-
L sodium
thermore, the level of background fluorescence in serum-derived
samples would be expected to be high without substantial sample
extraction.
l
l
l
deoxycholate (0.3%) and the protein precipitate recovered by cen-
One potential solution to the issues of sensitivity and selectivity
is to employ any of the amino acid derivatization agents, such as
trifugation at 6000 rpm for 10 min. The precipitate was washed
three times (alternately) with 800
ether and dried under vacuum.
lL of acetone and 800 lL diethyl
that reported by Waite, who derivatized peptidyl-
Edman’s reagent [22]. However, in seeking to develop an assay
for routine measurement of -DOPA, we were keen to employ a
L-DOPA using
Proteins (oxidized BSA and purified human plasma proteins)
were hydrolyzed according to the method of Tsugita et al. with
L
derivatization procedure which would produce relatively stable
derivatives which could be stored until analysis. A review of the
common amino acid derivatization agents commercially available
revealed 6-aminoquinolyl-N-hydroxyl-succinimidyl carbamate
minor modification [27]. Briefly, aliquots (500 lL) of protein
(0.1 mg/mL) were transferred to gas-tight MininertÒ vials (Supelco,
PA, USA) and combined with concentrated hydrochloric acid/triflu-
oroacetic acid (2:1 v/v) containing phenol (1%) and mercaptoacetic
acid (5%). The tube was sealed, evacuated of air and the solution
deoxygenated by bubbling with helium for 30 s; this process was
repeated a further three times. Samples were heated at 160 °C
for 25 min, dried and reconstituted immediately before use in
(
AccQ) as an obvious choice. AccQ reacts with primary and second-
ary amines to yield 6-aminoquinolyl-derivatives and N-hydroxy-
succinimide (Scheme 1). This reaction is rapid and produces
derivatives which are stable and respond by fluorescence (E
48 nm; E 395 nm), UV absorption (kmax 254 nm) or electrochem-
ical detection [23,24].
In this work, we report an improved HPLC-based method for
the determination of -DOPA in both purified proteins and human
m
3
x
2
1 mL ddH O.
2.3. Derivatization and concentration of hydrolysates
L
serum. By employing a rapid, anaerobic acid hydrolysis step, we
have minimized artifactual oxidation of amino acid residues and
have used AccQ reagent to produce 6-aminoquinolyl-derivatives
which are resolved by reverse-phase HPLC. The sensitivity of the
assay has been significantly increased by incorporating a concen-
tration step utilizing cloud-point extraction. In this procedure,
hydrophobic compounds (in this case, mainly the 6-aminoquinolyl
derivatives) are partitioned in a small, surfactant-rich layer,
which on addition of concentrated salt solution becomes turbid
Hydrolyzed samples (1 mL) were combined with 70 lL borate
buffer (0.2 mol/L, pH 8.8), 40 lL AccQ reagent (Waters, Milford,
MA, USA) which was used neat (for fluorescence detection) and also
at a 1/10 dilution (for UV detection). Samples were vortexed for 30 s
and heated at 70 °C for 30 min. As preliminary results for UV detec-
tion were poor (see Results), a pre-column concentration step was
added in the form of cloud-point extraction. To derivatized samples,
200 lL of Triton X-114 and 300 lL of ammonium sulfate (3 mol/L)
were added. The mixture was gently inverted to allow the cloud
point to form and complete separation was achieved by centrifuga-
tion at 6000 rpm for 10 min. The upper aqueous phase was dis-
(
the ‘cloud-point’), permitting separation from underivatized
components by centrifugation [25,26]. This rapid, cost-effective
method is ideal for large-scale studies where high sample
throughput is a desired and is sufficiently versatile to permit
carded and the volume of the lower phase (ca. 100 lL) adjusted to
200
l
L with ddH O, vortexing briefly to ensure a homogeneous
2
measurement on basic HPLC systems without
decrease in sensitivity.
a
significant
solution.
L
-DOPA standards (0.005–100 nmol/L) and a set of twenty
amino acids standards were also prepared.
1 2 1 2
Scheme 1. Derivitization of L-DOPA. Tyr R = 1; R = OH; L-DOPA R = R = OH.

3
78
P.A.C. McPherson, B.T. Türemen / Biochemical and Biophysical Research Communications 452 (2014) 376–381
2
.4. HPLC of derivatized amino acids
(non-polar) amino acids. Results (Fig. 1) are in keeping with this
prediction, with 6-aminoquinolyl-tyrosine and - -DOPA eluting
mid-profile with average retention times (R ) of 7.8 ± 0.02 and
L
Derivatized amino acids (20
lL sample injection) were resolved
t
on a Phenomenex C18 column (3.9 ꢀ 300 mm; 5
l
m) using a Per-
7.5 ± 0.09 min, respectively (n = 12). These retention times
matched those obtained for a simple sample containing only
kin-Elmer Series 200 HPLC system. A gradient of solvent A (Waters
AccQ.Tag™ Eluent A) in solvent B (60% acetonitrile; Ridel-de Haen,
Steinheim, Germany) was used at a flow rate of 1 mL/min. The
gradient was programmed as follows: 0–5 min, 0–6% B;
L-DOPA and L-tyrosine (Fig. 1, inset). The resolution of the amino
acid derivatives was virtually identical for both detection modes
(UV, Method A and fluorescence, Method B), as expected. The large
5
3
1
–6.5 min, 6–10% B; 6.5–11 min, 10–33% B; 11–13 min, isocratic
3% B. A wash-out sequence of 13–14 min, 33–100% B; 14–
5 min, isocratic 100% B; 15–16 min, 100–0% B completed a sam-
initial peak (R
t
ꢂ 2 min) was the co-product of the derivatization
reaction, N-hydroxysuccinimide, though it appeared that neither
the presence of this additional peak nor those of any of the twenty
common amino acids interfered with the identification of the tar-
get analyte.
ple run. The eluent was monitored by either diode array detection
at kmax 254 nm (Method A) or fluorescence (E 395 nm; E 348 nm)
Method B). For comparison, HPLC employing detection by intrinsic
fluorescence of tyrosine/ -DOPA (E 280 nm; E 320 nm) (Method
C) was also employed as previously reported [16].
x
m
(
L
x
m
3.3. Analytical performance
The measurement of
over the calibration range employed for both Method A and
Method (0.005–100 nmol/L; correlation coefficient of
r = 0.9998) with an identical relative standard deviation (RSD) of
.7% for both methods. This made the routine determination of
-DOPA straightforward as a non-weighted linear calibration func-
tion (peak response vs. concentration) could be used. The limits of
detection (LOD) and limits of quantification (LOQ) were calculated
from the standard deviation of blank samples and the gradient of
the calibration line, assuming a signal-to-noise ratio of 3 and 10,
respectively (Table 1). The repeatability and reproducibility of
the assay, taken as intra-day and inter-day RSD, was similar for
both methods (Table 1). Overall, the performance characteristics
for Methods A and B were superior to those found for the assay
employing intrinsic fluorescence (Method C), particularly with
regard to the LOQ and inter-day CV.
In establishing our assay, we selected detection by either UV or
fluorescence as at least one of these detection modes will be avail-
able on even the most basic HPLC instrument and is therefore
accessible to a wide-range of laboratories. We did not investigate
the use of electrochemical detection as this is generally compara-
ble to fluorescence detection in terms of sensitivity, as shown by
Pappa-Louisi et al. [32] and would therefore offer no analytical
advantage. We found that the anticipated detection method had
an impact on the derivatization procedure employed, as prelimin-
ary experiments utilizing neat AccQ reagent and UV detection
demonstrated poorer resolution of peaks, due to the dominance
of the N-hydroxysuccinimide peak. It was found that when using
UV detection, a 1/10 dilution of AccQ reagent could be used with
no observable effect on the derivatization reaction, yet substan-
tially improving the resolution of peaks. The neat reagent was
required for optimum detection by fluorescence down to the
pmol/sample level.
L-DOPA was found to be completely linear
2
.5. Miscellaneous methods
B
Independent measures of protein oxidation were used at vari-
2
L
ous stages in the development of the analytical procedure. Total
protein carbonyls were determined spectrophotometrically as
their 2,4-dinitrophenylhydrazone derivatives [28]. The concentra-
tion of
L-tyrosine and L-DOPA were determined by commercially
available enzyme-linked immunosorbent assays (tyrosine:
Immundiagnostik AG, K7015;
MBS162177).
L-DOPA: BioSource ELISA kit,
2.6. Method validation and statistical analysis
Routine descriptive statistics were generated from analysis of
data using Microsoft Excel (2007) and results expressed as
mean ± standard deviation. Non-parametrically distributed results
were analyzed by the Mann Whitney U-test. A comparison of the
methods was achieved by two-way analysis of variance (ANOVA).
Both statistical tests were performed using the Statistics Package
for Social Sciences (SPSS) Version 17.0 for Windows. P < 0.05 was
considered as statistically significant.
3
. Results and discussion
3.1. Preparation of BSA sample material
To provide readily available source of sample material for the
development and characterization of the assay, BSA was oxidized
by alkylperoxyl radicals generated by the thermal decomposition
of the azo-initiator AAPH [29]. The levels of protein carbonyls
were assessed to verify adequate oxidation of the protein, and
provided results in keeping with those available in the literature
(
2
4.9 ± 0.2 nmol/mg BSA) [30]. The levels of
L-tyrosine (30.9 ±
3.4. Impact of cloud-point extraction
.3 nmol/nmol BSA) and -DOPA (39.6 ± 2.3 pmol/nmol BSA) were
L
determined by ELISA and provided an estimate of the expected
concentrations of these analytes.
When dealing with samples of a more complex nature (BSA or
human plasma), it was found that detection of tyrosine and L-DOPA
was limited to the submicromolar range for UV detection and the
nanomolar range for fluorescent detection, and thus did not repre-
sent a particularly significant improvement. However, when sam-
ples were pre-concentrated using cloud-point extraction, the
limit of detection was shifted by an order of magnitude for both
3
.2. Reversed-phase HPLC of derivatized amino acids and
L-DOPA
A set of twenty standard amino acids plus -DOPA (2.5 mol/L)
L
were derivatized and subjected to HPLC initially using conditions
recommended by the manufacturer [31]. The modification of these
conditions reported in the Experimental section afforded good res-
olution of the amino acids peaks, with peak heights in keeping with
the derivatization chemistry. The position of the L-DOPA peak was
predicted to be in advance of tyrosine, based on the presence of an
additional –OH group which increases the polarity of the molecule,
detection methods and enabled detection of
BSA using UV detection (Table 2).
L-DOPA in unoxidised
The effect of cloud-point extraction is twofold. Firstly, as Triton-
X114 is amphiphilic, micelles are formed in aqueous solution with
a highly hydrophobic core. The non-polar components of the reac-
tion mixture (mainly AccQ-derivatives) migrate from the aqueous
buffer solution to the hydrophobic core, separating them from
resulting in L-DOPA eluting before tyrosine and the remaining

P.A.C. McPherson, B.T. Türemen / Biochemical and Biophysical Research Communications 452 (2014) 376–381
379
Fig. 1. Representative chromatogram for separation of 6-aminoquinoyl amino acid derivatives. L-DOPA eluted between serine and tyrosine, which is consistent with the
polarity of the molecule under the solvent conditions employed.
the incorporation of this additional step is worthwhile, especially
if the UV detection mode is used.
Table 1
Performance characteristics for measurement of L-DOPA.
Method Aa
Method Bb
Method Cc
LOD (20
injection)
LOQ (20
injection)
Recovery
l
L
0.42 ± 0.01 pmol 2.10 ± 0.11 fmol 0.15 ± 0.03 nmol
1.41 ± 0.05 pmol 7.06 ± 0.16 fmol 1.08 ± 0.07 nmol
3
.5. Effect of protein hydrolysis on
L-DOPA
lL
Owing to the labile nature of tyrosine, it is possible that a por-
98.4 ± 1.2%
1.1%
97.8 ± 1.9%
1.8%
119.2 ± 1.5%
3.9%
tion of the tyrosine residues in a given sample may undergo oxida-
tion during the acid hydrolysis step. Such artifactual alterations in
Intra-day CV
Inter-day CV
2.3%
3.4%
10.8%
L-DOPA expression were assessed by determining the amount of
a
b
c
tyrosine present in a BSA sample by ELISA and then subjecting
the sample to our hydrolysis procedure and re-assessing the levels
of tyrosine present by ELISA and HPLC. The effect of acid hydrolysis
Method A, UV absorption (kmax 254 nm).
Method B, fluorescent detection (E 395 nm; E
Method C, HPLC employing intrinsic fluorescence (included for comparison).
x
m
348 nm).
on the levels of protein-bound L-DOPA was assessed in a similar
fashion. Results (Table 3) demonstrated no significant decrease in
the amount of tyrosine detected, implying that virtually no tyro-
Table 2
sine residues had undergone conversion to
quence of acid hydrolysis. There was a slightly larger decrease in
the levels of -DOPA following hydrolysis, most likely due to fur-
ther oxidation of -DOPA to a variety of o-benzoquinone interme-
diates. However, this decrease was not statistically significant
(P > 0.05, n = 12) and should therefore not present any major
L-DOPA as a conse-
Effect of cloud-point extraction on measurement of tyrosine and L-DOPA.
L
Method A
Method B
L
Without cloud-point extraction
a
Tyrosine (nmol/nmol BSA)
47.12 ± 1.43
–
41.49 ± 3.22
2.67 ± 1.38
L
-DOPA (pmol/nmol BSA)b
underestimation of L-DOPA.
With cloud-point extraction
Taking these findings into consideration, the hydrolysis proce-
dure presented in this work seems preferable to other methods,
not only because it requires a much shorter incubation time
Tyrosine (nmol/nmol BSA)
34.13 ± 2.92
33.80 ± 2.16
1.57 ± 0.19
L
-DOPA (pmol/nmol BSA)
1
.59 ± 0.16
a
Mean tyrosine concentration (by ELISA) for same samples = 30.9 ± 2.3 nmol/
nmol BSA.
Mean L-DOPA concentration (by ELISA) for same samples = 1.62 ± 0.3 pmol/
nmol BSA.
(
25 min vs. 16 h), but also because it is performed in an inert atmo-
b
sphere in the presence of mild reducing agents. These precautions
were worthwhile, as BSA hydrolyzed by a traditional method (6 M
Table 3
potential hydrophilic interferences which may have UV or fluores-
cent properties. Secondly, as the AccQ-derivatives are transferred
from a larger volume (the aqueous phase) to a smaller volume in
an equilibrium-dependent manner, the desired components are
concentrated by a factor easily determined from experimental data
Levels of tyrosine and L-DOPA before and after protein hydrolysis.
Tyrosinea
b
L-DOPA
Before
After
Before
After
Method A
Method B
ELISA
32.14 ± 1.9
33.23 ± 0.6
33.98 ± 3.7
31.07 ± 1.3
32.44 ± 2.1
34.63 ± 6.1
1.64 ± 0.11
1.71 ± 0.31
1.75 ± 1.99
1.49 ± 0.23
1.68 ± 0.13
1.73 ± 2.93
[
33]. In addition to improving the sensitivity of the assay,
cloud-point extraction substantially reduced the peak due to
N-hydroxysuccinimide and completely eliminated a peak believed
to be due to free phenol (used in the hydrolysis step). Therefore,
a
nmol tyrosine/nmol BSA.
pmol L-DOPA/nmol BSA.
b

3
80
P.A.C. McPherson, B.T. Türemen / Biochemical and Biophysical Research Communications 452 (2014) 376–381
HCl for 16 h at 110 °C) showed substantial over-recovery of
142.0 ± 2.3%; P < 0.05; n = 3).
L
-DOPA
of intrinsic fluorescence in terms of sensitivity and precision, and
on the basis of comparison to ELISA data, exhibits notable specific-
ity. This method will be particularly appealing to those laboratories
equipped with minimal or limited instrumentation, with the
method employing UV detection still offering adequate sensitivity
for routine measurements.
(
3.6. Application to human plasma samples
To validate our method for a more complex sample matrix,
plasma was obtained from twenty volunteers and subjected to
our analytical method as well as independent ELISA-based meth-
ods. Results (Fig. 2) show a strong correlation between the two
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L
-DOPA concentration of
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