Role of plasma protein and low-molecular weight substances in the change of hydroxyl radical scavenging activity in hemodialysis patients.

Abstract

While it is well known that hemodialysis (HD) patients with end stage renal failure are exposed to high oxidative stress, there is not a general opinion regarding whether antioxidant activity is high or low in these patients. We evaluated the variation of plasma hydroxyl radical scavenging activity (p-HRSA) by a single-session of HD in 69 patients by using a new system, reactive flow-injection electron spin resonance. And then comparing p-HRSA with their biochemical parameters, we tried to elucidate what components affected p-HRSA in the HD patients. The average of p-HRSA significantly increased after HD and the variation of p-HRSA by HD was correlated with that of plasma total protein (TP). In 5 patients however, their p-HRSA decreased after HD, in spite of increasing TP. In pre-HD, the p-HRSA values and hydroxyl radical scavenging activity of low-molecular weight fraction of plasma were significantly higher in these 5 patients than in patients whose p-HRSA increased after HD. These 5 patients were in an inflammatory state. These findings suggest that p-HRSA is mainly affected by TP, but caution should be exercised in patients who have high p-HRSA before HD and whose p-HRSA does not increase after HD.

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Original Article
J. Clin. Biochem. Nutr., 42, 111–117, March 2008
JCBN Journal of Clinical Biochemistry and Nutrition0912-00091880-5086 the Society for Free Radical Research JapanKyoto, Japanjcbn200801610.3164/jcbn.2008016Original Article
Role of Plasma Protein and Low-Molecular Weight Substances
in the Change of Hydroxyl Radical Scavenging Activity
in Hemodialysis Patients
Akiko Hirakawa1, Kazumasa Aoyagi2,*, Motoo Nakajima2, Syuichi Kikuchi3, Syoji Ohba3,
and Kunihiro Yamagata4
1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
2Center of Integrative Medicine, Tsukuba University of Technology, Tsukuba, Ibaraki 305-0821, Japan
3Ohba Renal Clinic, Mito, Ibaraki 310-0841, Japan
4Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-0006, Japan
*To whom correspondence should be addressed.
Tel: +81-29-858-9537 Fax: +81-29-858-9591
E-mail: aoyagi@k.tsukuba-tech.ac.jp
3
Received 13 August, 2007; Accepted 31 August, 2007
Copyright © 2008 JCBN
SummaryWhile it is well known that hemodialysis (HD) patients with end stage renal failure
are exposed to high oxidative stress, there is not a general opinion regarding whether
antioxidant activity is high or low in these patients. We evaluated the variation of plasma
hydroxyl radical scavenging activity (p-HRSA) by a single-session of HD in 69 patients by
using a new system, reactive flow-injection electron spin resonance. And then comparing
p-HRSA with their biochemical parameters, we tried to elucidate what components affected
p-HRSA in the HD patients. The average of p-HRSA significantly increased after HD and the
variation of p-HRSA by HD was correlated with that of plasma total protein (TP). In 5
patients however, their p-HRSA decreased after HD, in spite of increasing TP. In pre-HD, the
p-HRSA values and hydroxyl radical scavenging activity of low-molecular weight fraction of
plasma were significantly higher in these 5 patients than in patients whose p-HRSA increased
after HD. These 5 patients were in an inflammatory state. These findings suggest that p-HRSA
is mainly affected by TP, but caution should be exercised in patients who have high p-HRSA
before HD and whose p-HRSA does not increase after HD.
Key Words:hemodialysis, plasma hydroxyl radical scavenging activity, flow-injection electron
spin resonance, total protein, low-molecular weight substance
Introduction
The high incidence of early cardiovascular disease in
patients with chronic renal failure, either on dialysis or not,
is well documented [1–3]. This phenomenon is regarded as
being caused by increasing oxidative damage due to reactive
oxygen species (ROS). The over production of ROS in these
patients is evidenced by several oxidative stress markers
[3–7]. In addition, hemodialysis (HD) patients are exposed
to more oxidative damage because neutrophils and the
complement pathways may be activated during HD with a
subsequent increase in the neutrophil free radical production
[2, 6–8]. On the other hand, Soejima et al. reported that HD
procedure reduced oxidative albumin [9]. Given that HD
removes uremic toxins, a hemodialysis session does not
always induce malignancies.
To date, there are several reports about antioxidants and
antioxidant capacity in HD patients. Some reports have
said that antioxidant capacity was higher in HD patients

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J. Clin. Biochem. Nutr.
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compared with healthy subjects [1, 2, 10, 11], but others
have stated that antioxidant status was not different [12, 13].
It is difficult to attribute whether antioxidant activity is
stronger or not because their methods had different
approaches against ROS. And it was unclear what kinds of
ROS were scavenged in each report. Thus, in HD patients
there is an increase of oxidative stress and the impairment of
antioxidant defense. The imbalance between overproduction
of oxidants and antioxidant defense mechanism is well
established in HD patients [6–8].
Electron spin resonance (ESR) is the only direct method
for both detection and identification of free radicals. In
previous reports, we have already demonstrated by ESR
measurement that hydroxyl radical scavenging activity
(HRSA) of plasma in HD patients significantly decreased
compared with controls and recovered after HD [14]. Shirai
et al. also reported that, by using ESR in HD patients, HRSA
was higher than that in healthy subjects but it never changed
by HD session [15]. These differences may contribute to
defects in the conventional ESR method. In addition, the
existing X-band ESR method has such difficulties as low-
sensitivity, a longer time requirment, and variable of sen-
sitivity. Therefore, we developed a reactive flow-injection
ESR system (FI-ESR), using three high-pressure liquid
chromatography (HPLC) pumps and ultraviolet (UV) lamp.
The problems were solved by holding flow-cell in the cavity,
mixing solutions short of cell, and measuring of fixing
magnetic field. When hydrogen peroxide is exposed to UV
in the cell, hydroxyl radical is generated and is transiently
trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). This
system makes it possible to measure HRSA for various
antioxidants. In this study, we investigated the variation of
plasma HRSA (p-HRSA) by a single-session of HD by using
this new system, FI-ESR. Moreover, by comparing it with
their biochemical parameters, we tried to elucidate what
components affected p-HRSA in HD patients.
Subjects and Methods
Patients
All studies were performed after 48 hours passed since
the last HD. Sixty-nine hemodialysis patients (39 male, 30
female) were studied. Sixty-eight patients underwent
hemodialysis three times a week and one patient did so
twice. The age was 63 ± 10 (mean ± SD, range 36–81) years
and the duration of HD treatment was 96 ± 79 (5–330)
months. Origins of the end stage renal disease (ESRD) in
the group were: chronic glomerulonephritis (n = 24),
diabetic nephropathy (n = 19), polycystic kidney (n = 3),
autoimmune disease (n = 2), chronic pyelonephritis (n = 1),
ureter tumor (n = 1), reflux nephropathy (n = 1), pregnancy-
induced hypertension syndrome (n = 1), and unknown
(n = 17). Clinical characteristics are shown in Table 1.
Measurement of hydroxyl radical scavenging activity
(HRSA)
HRSA was observed by reactive flow-injection electron
spin resonance (FI-ESR) system. It consisted of three inert
HPLC pumps (PU-2080i; JASCO, Tokyo, Japan), automatic
injector (AS-2057i; JASCO), ultraviolet lamp (RUVF-203S;
Radical Research Inc., Tokyo, Japan), and ESR spectrometer
(RRX-1X; Radical Research Inc.) (Fig. 1). Ultrapure water,
10 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin
trap agent, and 20 mM hydrogen peroxide, were flown into
a capillary quartz cell at each flow rate of 0.4 ml/min. The
cell was exposed to ultraviolet light and generated hydroxyl
radical reacted DMPO. The formed DMPO-OH adduct was
detected by ESR and we obtained its spectra. Then, we fixed
a magnetic field at the second peak. Samples were then
injected into an ultrapure water line. When antioxidant was
injected, reduced signal peak was observed (Fig. 2). The
HRSA was the negative peak of the FI-ESR signal, and the
HRSA value was calculated as relative intensity against
0.5 mmol dimethyl sulfoxide.
Samples
Before measurement all plasma was stored at −24°C.
In plasma HRSA (p-HRSA) determination, plasma was
undiluted and used directly. Low-molecular weight fraction
Table 1. Clinical characteristics of hemodialysis patients before
(pre-HD) and after (post-HD)
pre-HDpost-HD
Total Protein (g/dl)6.58 ± 0.41 7.71 ± 0.66
Albumin (g/dl)3.84 ± 0.314.46 ± 0.45
Glucose (mg/dl)124.4 ± 45.1 97.5 ± 43.9
Uric Acid (mg/dl) 8.2 ± 1.42.5 ± 0.6
Creatinine (mg/dl) 11.9 ± 2.54.9 ± 1.3
Plasma Urea Nitrogen (mg/dl)70.3 ± 14.11 24.5 ± 6.7
CRP (mg/dl)0.29 ± 0.5—
Hemoglobin (g/dl) 10.3 ± 1.0—
Hematocrit (%)32.6 ± 3.0—
Fig. 1.Schematic flow diagrams of reactive flow-injection ESR
system. P1, P2, and P3 pumps sent ultrapure water,
10 mM DMPO, and 20 mM H2O2, respectively. Each
pump of flow rate was 0.4 mL/min.

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113
of plasma was collected by ultrafilteration (Ultrafree-MC
filter, Millipore Corporation, Billerica, MA; cut off M.W.
10,000).
Statistical analysis
Data were expressed as mean ± SD. Correlation coefficient
was obtained using simple regression analysis (Excel
software). Statistical significance was determined using
paired or un-paired t-test. The differences were considered
statistically significant when the calculated p value was less
than 5%.
Results
p-HRSA value of pre- and post-HD
Using this new system, FI-ESR, the significant increase in
the average of p-HRSA values was observed after HD (from
0.44 ± 0.06 to 0.49 ± 0,06, n = 69). The p-HRSA value
increased over 5% after HD in 46 patients (increased group),
and in 18 patients, their p-HRSA values showed little
variation (invariant group; within 5% variation). Interestingly
enough, in the remaining 5 patients, this value significantly
decreased over 5% (reduced group) (Table 2). In a
comparison of the p-HRSA values between each group,
p-HRSA of pre-HD in the invariant group and the reduced
group were stronger than in the increased group (p = 0.002,
and p<0.01, respectively). Furthermore, in post-HD, the
value of the reduced group was lower than that of the
increased group (p<0.05).
Relationship between p-HRSA and plasma total protein
Protein is a major component in plasma and an easy target
of hydroxyl radicals. In addition, plasma total protein
becomes thicker by HD procedure because of hemo-
concentration. We investigated the relationship between p-
HRSA and the concentration of plasma total protein (TP).
Fig. 3 shows the correlation of the before-after HD ratio
between the p-HRSA and the concentration of TP. Variation
of p-HRSA was correlated with that of TP (r = 0.69,
p<0.0001). Both pre- and pro-HD p-HRSA values were
also correlated to plasma TP levels significantly (r = 0.41,
p = 0.0005; r = 0.69, p<0.0001, respectively) (Fig. 4).
The ratio of p-HRSA/TP of the increased, invariant, and
decreased group
To eliminate the influence of HRSA of plasma protein, we
calculated p-HRSA value/TP for each group (Fig. 5). In the
Fig. 2. FI-ESR chart of plasma and 0.5 mmol DMSO. When
UV lamp was turned on, hydroxyl radical generated
and upper baseline was shown, and when it was turned
off, hydroxyl radical stopped generating and the detected
point went down. Hydroxyl scavenging activity
represented these downward peaks. These peak areas
were calculated and p-HRSA values were elucidated by
plasma/DMSO ratio.
Table 2.p-HRSA value of hemodialysis patients
Statistical significance: *p = 0.001, **p<0.001 vs pre-HD,
+p = 0.002, ++p<0.01, +++p<0.05 vs increased group.
The grouping was done in over 5% increase, within 5% invariant,
and over 5% decrease after HD.
p-HRSA Value
pre-HDpost-HD
All patient (n = 69)0.44 ± 0.06 0.49 ± 0.06**
Increased group (n = 46)0.42 ± 0.010.51 ± 0.01**
Invariant group (n = 18)0.47 ± 0.01+
0.48 ± 0.01
Reduced group (n = 5)0.50 ± 0.02++
0.44 ± 0.02*+++
Fig. 3.Correlation between the variation of total protein and p-
HRSA by single session of HD (n = 69, r = 0.69,
p<0.0001)

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increased group, means of the p-HRSA value/TP were the
same as pre- and post-HD (0.064 ± 0.008 and 0.064 ± 0.006,
respectively). In the invariant and reduced group, these
values were higher in pre-HD than in post-HD (0.071 ±
0.006 vs 0.065 ± 0.006, p<0.001; 0.079 ± 0.01 vs 0.063 ±
0.007, p = 0.001). Calculation of p-HRSA value/TP in pre-
HD revealed a significant difference in each group (invariant
group; p = 0.002, reduced group; p<0.001 vs increased
group, respectively).
Correlation between the p-HRSA value and the plasma
concentration of albumin
Plasma protein includes many kinds of proteins but
albumin is known to be the most abundant protein. We also
investigated the possibility that there is correlation between
p-HRSA value and plasma albumin. Before HD they were
not correlated (r = 0.02, p = 0.88), whereas after HD, p-
HRSA values were correlated with plasma albumin levels
(r = 0.45, p<0.0001) (Fig. 6).
Comparison of the HRSA of low-molecular weight fraction
(lmf-HRSA) of plasma for the increased group and reduced
group
In an attempt to compare the lmf-HRSA between the
increased and reduced group, we randomly selected eight
patients in the increased group. The lmf-HRSA of the
reduced group was stronger than the increased group in
pre-HD (0.18 ± 0.03, n = 5 vs 0.13 ± 0.03, n = 8, p = 0.02)
(Fig. 7). However, levels of the conceivable low molecular
weight substances that expressed hydroxyl radical scavenging
activity (i.e., glucose, uric acid, and creatinine) were not
different (data not shown).
Relationship between p-HRSA and C-reactive protein
In the grouping described above, quite a few patients of
Fig. 4. Correlation between plasma concentration of total
protein and p-HRSA value in pre-HD and post-HD. Pre-
HD are shown by closed circles (r = 0.41, p<0.0005),
post-HD are open circles (r = 0.68, p<0.0001).
Fig. 5. Ratio of p-HRSA/TP of the increased, invariant, and
decreased group. Increased group are shown by circles,
invariant group are shown by diamonds, and reduced
group are shown by squares. Each plot displayed
graphically by pre-HD are closed and post-HD are
opened.
Fig. 6.Correlation between plasma concentration of albumin
and p-HRSA value in pre-HD and post-HD. Pre-HD are
shown by closed circles (r = 0.02, p = 0.88), post-HD are
open circles (r = 0.45, p<0.0001)
Fig. 7. HRSA value of low-molecular weight fraction of plasma
in increased group and reduced group.

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the invariant and reduced groups had high C-reactive protein
(CRP) level (≥0.3 mg/dL), leukocyte count, or inflammatory
diseases (invariant group; 7 of 18 subjects, reduced group; 5
of 5 subjects). We then divided patients into normal
(<0.3 mg/dL) and high (≥0.3 mg/dL) CRP group, and
investigated pre- and pro-HD p-HRSA values of each group.
In normal CRP patients, both pre- and pro-HD p-HRSA
values were correlated to plasma TP levels significantly
(r = 0.55, p<0.0001; r = 0.69, p<0.0001, respectively). But
in high CRP patients there was not a significant correlation
for pre-HD (r = 0.25, p = 0.33) (Fig. 8). In pre-HD, the ratio
of p-HRSA/TP in high CRP patients was significantly larger
than that in the normal CRP patients (p = 0.01) (Table 3).
Discussion
In conventional ESR measurement, radical sources, a
spin-trapping reagent, and samples are first mixed and they
reacted for a set time in a test tube. Then, the mixture is
transferred into a cell and inserted into the ESR spectro-
meter. These operations were not only troublesome but also
caused measurement error because observed ·OH was easily
affected by the measurement time. Our new method, the
reactive FI-ESR system, gives hydroxyl radical generation
first in the cell and is able to measure scavenging activity of
samples in real time. In addition, holding the cell avoids
measurement error by moving over in the cavity and mixing
reaction solutions before the cell insures shorter and more
accurate reaction time. Measurement of fixing magnetic
field at the point of DMPO-OH adduct peak specifically
does not need sweeping time, therefore, the measuring time
can be shortened. This reactive FI-ESR solves several
problems and is a not only practical but also more powerful
system for measuring hydroxyl radical.
In the present study, we showed that the average value of
p-HRSA of post-HD was significantly higher than that of
pre-HD. This result corresponded with previous data [14].
However, comparing patients individually, in 33% of the
patients the p-HRSA did not increase after dialysis. This
result indicates that a single-session of HD did not always
make HRSA stronger.
The variation of p-HRSA by a single-session of HD had a
positive correlation to that of plasma TP level. It suggests
that the variation of p-HRSA is affected by TP concentra-
tion. But in albumin, which accounts for 60% of TP, p-
HRSA of pre-HD was not correlated with the albumin level.
Human serum albumin is composed of mercaptalbumin
(reduced form) and nonmercaptalbumin (oxidized form),
that is, a protein redox couple. Soejima et al. have recently
shown that the percentage of the reduced form of human
Fig. 8. Correlation between p-HRSA and total protein in (a)
normal or (b) high CRP group. Pre-HD are shown by
closed circles (a; n = 51, r = 0.55, p<0.0001, b; n = 18,
r = 0.25, p = 0.33), post-HD are open circles (a; r = 0.69,
p<0.0001, b; r = 0.71, p = 0.001)
Table 3.Comparison between normal and high CRP patient
Statistical significant: *p = 0.01. **p<0.01 vs normal CRP group
normal CRP group (n = 51)high CRP group (n = 18)
pre-HDpost-HDpre-HD post-HD
CRP (mg/dL)0.093 ± 0.066— 0.85 ± 0.79—
Total Protein (g/dL) 6.57 ± 0.377.75 ± 0.636.62 ± 0.517.59 ± 0.73
albumin (g/dL)3.91 ± 0.27 4.57 ± 0.373.66 ± 0.36**4.14 ± 0.52
p-HRSA value0.43 ± 0.060.49 ± 0.060.47 ± 0.05*0.50 ± 0.07
p-HRSA/TP 0.065 ± 0.009 0.063 ± 0.006 0.071 ± 0.008**0.066 ± 0.006