Life Sciences 78 (2006) 1754 – 1759
www.elsevier.com/locate/lifescie
Interdependency of the oxidizability of lipoproteins and peroxidase
activity with base excess, HCO3, pH and magnesium in human
venous and capillary blood
Sabine Wurzinger a,b, Mirela Bratu c, Willibald Wonisch d, Reinhold Wintersteiger b,
Gabriele Halwachs-Baumann d, Sepp Porta a,e,*
a
Institute for Applied Stress Research, Bad Radkersburg, Austria
Institute of Pharmaceutical Sciences, Karl-Franzens University, Graz, Austria
c
Ovidius University, Constanta, Romania
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria
e
Institute of Pathophysiology, Medical University of Graz, Austria
b
d
Received 27 April 2005; accepted 10 August 2005
Abstract
As continuous production of free radicals and reactive oxygen species is a normal metabolic process, increased metabolism during exercise/
workload should increase free radical generation and oxidative stress. Oxidative stress intensity should then depend on the intensity of
metabolic stress effects. Intensity of stress is usually reflected in norepinephrine (NE) levels, which correlate linearly and significantly with
changes in blood gases, blood buffer systems, blood electrolytes, blood glucose and lactate [Porta, S., Leitner, G., Heidinger, D., Lang, T.,
Weiss, U., Smolle, K.H., Hasiba, K., 1997. Magnesium während der Alpinausbildung bringt um 30% bessere Energieverwertung. MagnesiumBulletin 19(2), 59 – 61].
Those parameters were used in an open study design to screen 64 subjects for metabolic stress effects along with their antioxidative capacity
using both venous and capillary blood. To compare venous and capillary blood, we took venous blood samples from 12 healthy volunteers and
capillary blood from 52 other healthy subjects.
To show whether free radical changes indeed go along with metabolic stress effects, we tried to quantify relations between metabolic stress
effects and oxidative stress by linear correlations.
In conclusion, both venous and capillary blood are suitable for determining at least those parameters of the oxidative state that we used. All
significant correlations of peroxidase activity and oxidation lag time (OLT) with pH, bicarbonate (HCO3), base excess (BE) and magnesium (Mg)
indicate that free radical production increases with metabolism.
Those relationships could help to evaluate the oxidative state more precisely.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Venous blood; Capillary blood; Peroxidase activity; Oxidation lag time measurement
.
Introduction
1. Free radicals and reactive oxygen species are produced
continuously during normal metabolism (Cao and Prior,
2000). As increased metabolism during exercise/workload
* Corresponding author. Institute of Pathophysiology, Heinrichstrasse 31,
8010 Graz, Austria. Tel.: +43 316 380 4291; fax: +43 316 380 9640.
E-mail address: stresscenter@netway.at (S. Porta).
0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.lfs.2005.08.010
also increases free radical generation and oxidative stress
(Ji, 1995; Cooper et al., 2002; Schmidt et al., 2002; Urso
and Clarkson, 2003; Banerjee et al., 2003), oxidative stress
intensity seems to depend largely (Wolf et al., 2005) upon
metabolic intensity.
2. As intensity of stress is usually characterized by changes in
NE levels, which in turn correlate linearly and significantly
with changes in blood gases, blood buffer systems, blood
electrolytes, blood glucose and lactate, the effects of NE
upon metabolism can be estimated from changes in those
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S. Wurzinger et al. / Life Sciences 78 (2006) 1754 – 1759
Material and methods
Subjects
In the course of a routine stress assessment, 64 subjects (35
males and 29 females, aged between 19 and 82 years) were
screened for metabolic stress effects and their antioxidative
capacity in an open study design.
Collection of blood samples and preparation
To compare venous blood and capillary blood, we divided
the subjects into two groups, i.e. we took venous blood from
the median cubital vein (using a Monovette Z 2.7 mL, Sarstedt)
of 12 healthy volunteers (11 males and 1 female), and capillary
blood from 52 subjects (24 males and 28 females) by pricking a
hyperemized fingertip with a lancet (Accu-Chek\Softclix\,
Roche Diagnostics, Basel, Switzerland) and collecting the
blood in a 200 AL Microvette (Sarstedt). The first drop of
capillary blood was discarded.
After collection, the samples coagulated (30 min) at room
temperature and afterwards were centrifugated at 3000 rpm for
10 min (Eppendorf). All serum samples were stored at 20 -C
until use, but not longer than 2 weeks.
Diagnostic parameters
Clinical stress assessment (CSA)
All the following parameters (pH, partial pressure of carbon
dioxide ( pCO2), BE, HCO3, calcium (Ca), Mg, glucose, lactate
and potassium) were measured according to the Clinical Stress
Assessment (CSA) method (Porta et al., 1993) with a ‘‘Critical
Care X-Press (CCX)’’ Nova analyzer.
The applied test method (CSA) is based on the assumption
that changes in the parameters mentioned above follow or even
are instigated by changes in NE and correlate linearly with
them since both free radical production and increased
metabolism during stress have increased oxidative phosphorylation as their common denominator (Kang and Hamasaki,
2003).
Linear correlations with p < 0.01 between pH, HCO3, BE,
pO2 and NE have been found in an experiment using 21 human
probants. NE has been determined by high pressure liquid
chromatography (HPLC, Beckman Gold, electrochemical
detector) and the other parameters by ion selective electrodes
(Porta et al., 1997).
Lipid soluble and endogenous antioxidants
Determination of the oxidizability of lipoproteins in human
sera. The lag time of ex vivo degradation of the
fluorophore 1-palmitoyl-2-((2-(4-(6-phenyl-trans-1,3,5-hexatrienyl)phenyl)ethyl)-carbonyl)-sn-glycero-3-phosphocholine
(DPHPC) by reactive oxygen species (ROS) in serum was
determined with a fluorescent diagnostic assay (Lipid-Ox,
Tatzber KEG, Austria) according to Hofer (Hofer et al.,
1995), with modifications to the oxidation part. Briefly, 100
AL serum was incubated with 2 nM DPHPC and kept under
argon at 37 -C for 12 h. Oxidation was started via a
peroxide – peroxidase reaction and the time-dependent decrease in fluorescence intensity at 430 nm (excitation at 360
nm) was monitored on a FluoStar fluorometer (BMG LabTechnologies; D-77656 Offenburg, Germany). Results were
expressed as lag time in minutes.
Endogenous antioxidants. Serum total peroxidase activity
was determined by a rapid enzymatic in vitro diagnostic
assay (ARS\, Tatzber KEG, Klosterneuburg, Austria) as
previously described (Tatzber et al., 2003). The test system
was based on the reaction of endogenous peroxidases with
hydrogen peroxide, using 3,5,3V, 5V- tetramethylbenzidine
(TMB) as chromogenic substrate. Oxidation of TMB by
horseradish peroxidase (HRP)/H2O2 was reported by Josephy
(Josephy et al., 1982), with the advantage of very stable
TMB oxidation products. Standards and samples were
incubated with the reaction mixture (buffer, hydrogen
peroxide and TMB) in microtiter plates. The first absorbance
reading was done at a wavelength of 450 nm in a plate
reader. After 15 min (T 5 min) of incubation the reaction was
stopped through the addition of the stop solution, and a
second absorbance reading was done at 450 nm. Results
were calculated from the linear horseradish-peroxidase
standard curve by subtracting the first absorbance reading
from the second. Peroxidase activity was expressed as
‘‘milliunits per millilitre’’ (mU/mL) according to the linear
horseradish-peroxidase standard curve.
Statistical analysis
All data were collected and processed in a software
immanent pattern recognition system, including semi-automatOLT/BE
BE (mmol/L)
parameters, using a few microlitres of capillary blood
(Clinical stress assessment, CSA method—see Material
and methods).
3. Since the actual volume of antecubital venous blood required
to determine oxidative stress is not much greater than the
volume of capillary blood required to determine metabolic
stress effects, we investigated the feasibility of using capillary
blood samples to examine both oxidative stress and metabolic
stress effects. This sampling technique would have the
advantage of being more comfortable for patients.
8
7
6
5
4
3
2
1
0
-1
20
-2
30
40
50
60
70
80
OLT (min)
Fig. 1. Capillary blood. Abscissa: OLT (Oxidation lag time) in minutes;
Ordinate: BE (Base Excess) in mmol/L.
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S. Wurzinger et al. / Life Sciences 78 (2006) 1754 – 1759
OLT/HCO3
OLT/HCO3
40
HCO3 (mmol/L)
HCO3 (mmol/L)
40
35
30
25
35
30
25
20
20
20
30
40
50
60
70
20
80
30
40
Fig. 2. Capillary blood. Abscissa: OLT (Oxidation lag time) in minutes;
Ordinate: HCO3 (Bicarbonate) in mmol/L.
ic calculations of means, standard errors of mean, significance
tests and correlations (CSA software, Austria).
Results
Comparison between venous and capillary fingertip blood
(Table 1)
pH and blood – gas levels
As expected, there were differences between capillary and
venous blood in the form of increased pH (Matthiesen et al.,
2002) (7.486 T 0.003 to 7.42 T 0.011) and lower levels of pCO2,
BE and HCO3 in capillary blood.
Calcium
Calcium levels in venous blood (1.227 T 0.007 mmol/L)
were significantly higher than in capillary blood (1.068 T 0.009
mmol/L).
Magnesium
Venous ionized Mg (0.564 T 0.013 mmol/L) was significantly higher than capillary ionized Mg (0.497 T 0.007 mmol/L),
see also Matthiesen et al. (2002).
Blood glucose
Blood glucose levels (99.387T 3.094 mg/dL) were significantly
higher in capillary than in venous blood (78.308T 2.491 mg/dL).
60
70
80
Fig. 4. Venous blood. Abscissa: OLT (Oxidation lag time) in minutes; Ordinate:
HCO3 (Bicarbonate) in mmol/L.
Potassium
There were no significant differences between potassium
levels in capillary (4.313 T 0.034 mmol/L) and venous blood
(4.3 T 0.063 mmol/L).
Peroxidase activity and oxidizability of lipoproteins (OLT = oxidation lag time)
Peroxidase activity was significantly higher in capillary
blood (10.447 T 0.854 mU/mL) than in venous blood
( 5 . 8 1 8 T 0 . 5 3 6 m U / m L ) . O LT i n c a p i l l a r y b l o o d
(47.696 T 1.834 min) was not significantly higher than in
venous blood (42.833 T 2.011 min).
Since it is not unconceivable that free radical changes go
along with physical stress (Pincemail et al., 2000; Schippinger
et al., 2002), we tried to quantify possible relationships
between metabolic stress effects and oxidative stress by
looking for linear correlations between them.
The group averages of all measured metabolic parameters–
except potassium –were significantly different in capillary and
venous blood.
Correlations in both capillary and venous blood
Capillary blood
OLT and BE ( p < 0.01).
The higher BE, the longer the OLT (Fig. 1).
OLT and HCO3 ( p < 0.05).
The higher HCO3, the longer the OLT (Fig. 2).
Venous blood
OLT and BE ( p < 0.05).
High BE levels go along with high OLT (Fig. 3).
OLT/BE
OLT/pH
7.54
7.52
7.5
pH
BE (mmol/L)
Lactate
We saw a significantly higher lactate level in venous blood
(1.918 T 0.132 mmol/L) than in capillary blood (1.018 T 0.062
mmol/L).
8
7
6
5
4
3
2
1
0
-1
20
-2
50
OLT (min)
OLT (min)
7.48
7.46
7.44
7.42
30
40
50
60
70
80
OLT (min)
Fig. 3. Venous blood. Abscissa: OLT (Oxidation lag time) in minutes; Ordinate:
BE (Base Excess) in mmol/L.
20
30
40
50
60
70
80
OLT (min)
Fig. 5. Capillary blood. Abscissa: OLT (Oxidation lag time) in minutes;
Ordinate: pH.
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S. Wurzinger et al. / Life Sciences 78 (2006) 1754 – 1759
OLT/Mg
Mg (mmol/L)
0.7
0.6
0.5
0.4
0.3
20
30
40
50
60
70
80
OLT (min)
Fig. 6. Capillary blood. Abscissa: OLT (Oxidation lag time) in minutes;
Ordinate: ionized Mg (Magnesium) in mmol/L.
Peroxidase/Mg
Mg (mmol/L)
0.7
0.6
0.5
0.4
0.3
0
5
10
15
20
25
30
Peroxidase (mU/mL)
Fig. 7. Capillary blood. Abscissa: Peroxidase (Peroxidase activity) in mU/mL;
Ordinate: ionized Mg (Magnesium) in mmol/L.
OLT and HCO3 ( p < 0.05).
The higher HCO3, the longer the OLT (Fig. 4).
Correlations typical for capillary blood
Capillary blood
OLT and pH showed a significantly positive correlation: the
higher the pH values, the longer the OLT ( p <0.01) (Fig. 5).
OLT and Mg, peroxidase activity and Mg.
There were significant correlations between Mg and OLT
( p < 0.001) peroxidase activity ( p < 0.05). The higher the Mg
levels in capillary blood, the longer the OLT (Fig. 6). The
higher the Mg levels, the lower the peroxidase activity (Fig. 7).
Discussion
As the metabolic states of the members of an experimental
group may vary considerably for no obvious reason, there
may be considerable statistical variance in the group’s
oxidative parameters. In fact, high statistical variation of
group averages due to the different momentary metabolic
states of the group members may importantly prevent the
evaluation of differently treated groups from reaching significance level. If, however, the metabolic state of a given
individual could be precisely determined, statistical group
deviations would decrease considerably. A suitable tool for
describing metabolic states seems to be simultaneous determination of parameters of blood buffer systems, of carbohydrate turnover and electrolyte status with pH, pCO2, BE,
HCO3, Ca, Mg, potassium, blood glucose and lactate. Most of
these values have been seen to correlate linearly and highly
significantly with changes in NE (Porta et al., 1997).
To increase sample numbers, we also tried using capillary
blood to determine oxidative parameters.
The advantage of capillary sampling is that as there is only
one sampling site for both oxidative stress and metabolic stress
effects, the procedure is simpler and patient discomfort is
minimal.
The disadvantage (which can be quickly overcome with
some experience) is an increased inclination to haemolysis due
to the small sample size.
Free radicals are involved in lipid peroxidation (Esterbauer
et al., 1992; Esterbauer et al., 1993; Lindschinger et al., 2004),
DNA damage and protein degradation. In vitro, the interaction
between free radicals and lipids involves three processes:
initiation, propagation and termination. Oxidative damage to
membranes increases membrane fluidity and compromises
integrity. Put briefly, since radicals interact with lipids, DNA
and proteins (Clarkson and Thompson, 2000), oxidative stress
is an important modulator of vascular cell functions (Kunsch
and Medford, 1999) and leads to initiation and progression of
numerous diseases as coronary artery disease, stroke, rheumatoid arthritis and cancer (Prior, 2003, 2004).
A considerable number of papers deal with the fact that
increased radical production goes along with increased
metabolism (Hruszkewycz and Bergtold, 1988; Ji, 1995;
Clarkson and Thompson, 2000; Cooper et al., 2002; Schippinger et al., 2002; Schmidt et al., 2002; Urso and Clarkson, 2003;
Banerjee et al., 2003).
Since the use of venous blood is recommended with the
commercially available kits we used to determine oxidation lag
time of lipids and peroxidase activity, we also determined the
metabolic parameters from those samples. It turned out that
oxidation lag time was positively and significantly correlated
with BE and HCO3. This means that increased metabolism,
characterized by lower blood buffer capacities, goes along with
increased free radical activity in a mathematically predictable
manner.
Table 1
Comparison of pH, pCO2, BE, HCO3, Ca, Mg, Blood Glucose, Lactate, Potassium, Lag-time and ARS in venous and capillary blood (mean values and standard
error of means (SEM))
Venous blood
Capillary blood
Mean
SEM
Mean
SEM
t-test, p =
pH
pCO2
(mm Hg)
BE
(mmol/L)
HCO3
(mmol/L)
Ca
(mmol/L)
Mg
(mmol/L)
BG
(mg/dL)
Lactate
(mmol/L)
Potassium
(mmol/L)
Lag-time
(min)
ARS
(mU/mL)
7.420
0.011
7.486
0.003
<0.001
47.573
1.921
32.192
0.368
<0.001
4.180
0.521
2.375
0.192
<0.01
30.390
0.755
24.310
0.262
<0.001
1.227
0.007
1.068
0.009
<0.001
0.564
0.013
0.497
0.007
<0.001
78.308
2.491
99.387
3.094
<0.001
1.918
0.132
1.018
0.062
<0.001
4.30
0.063
4.313
0.034
n.s.
42.833
2.011
47.696
1.834
n.s.
5.818
0.536
10.447
0.854
<0.01
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S. Wurzinger et al. / Life Sciences 78 (2006) 1754 – 1759
Since determination of all the metabolic parameters mentioned
above can be done with 200 AL capillary blood, we tried to
adapt OLT and peroxidase activity determination to this simpler
and smoother sampling method.
Another advantage of this much simpler sampling procedure
lies in the larger numbers of samples that can be tested within a
given time. This is the main reason why our 12 subjects with
venous sampling could be compared with 52 subjects with
capillary sampling. The most important drawback of capillary
blood sampling for the determination of oxidative state is that it
is more difficult to avoid haemolysis. We had to establish that
e.g. potassium is within the normal range in both venous and
capillary samples. In our subjects, the mean potassium
concentration was 4.313 T 0.034 mmol/L in capillary blood
and 4.3 T 0.063 mmol/L in venous blood; the respective normal
values for adults are 3.6 –4.8 mmol/L (Thomas, 2000) and
3.6 –5.4 mmol/L (Hildebrandt, 1998).
On the other hand, venous and capillary blood shows
significant differences in all other metabolic parameters. Those
differences are shown in Table 1 and are in accord with well
established observations (Thomas, 2000; Matthiesen et al.,
2002).
Peroxidase activity is significantly higher in capillary blood,
but OLT values are not. Generally, free radical concentration in
venous blood is higher than in capillary blood (Shi et al.,
2001). As far as peroxidase activity is concerned, there may be
a higher radical scavenging capacity in capillary blood.
Preliminary experiments with increased antioxidant capacity
by application of radical scavengers suggest a shifting of the
correlation curves to the right due to extended OLTs.
Nevertheless, the positive correlations between OLT and
BE, HCO3 are also significant in capillary blood. This means
that either venous or capillary blood can be used to identify
differences in OLT due to differences in metabolic states.
There are, however, correlations between oxidative and
metabolic parameters that are typical for capillary blood and
are not seen in venous blood. A positive correlation between
OLT and pH points in the same direction as the correlations
between OLT and BE or OLT and HCO3. A positive
correlation between OLT and ionized Mg shows that low
Mg values go along with short OLT, whereby peroxidase
activity increases with lower concentrations of ionized Mg.
Since low Mg – except in hyperproteinaemia – nearly always
accompanies (chronically) elevated metabolism, there is
obviously a logical connection between all the correlations
seen in capillary blood.
Conclusion
1. Both venous and capillary blood are suitable for determining at least those parameters of the oxidative state that we
used in our study.
2. About 400 AL of capillary blood suffices to determine
oxidative parameters and the metabolic stress effects.
3. All significant correlations of peroxidase activity and OLT
with pH, HCO3, BE, and Mg point toward increased free
radical production with a higher metabolic rate.
4. There is a mathematical predictability (within the range of
our results) of the proportional changes of parameters of
oxidative stress and metabolic stress effects. Such proportional changes can be used to develop standard curves that
fit oxidative values (e.g. OLT) to the proper metabolic
parameter (e.g. BE). Thus a more precise evaluation of the
momentary oxidative state dependent upon the metabolic
state is possible.
Acknowledgements
The authors gratefully acknowledge the valuable help of
Mrs. B. Poncza, Mrs. G. Porta and members of the Austrian
Military Academy.
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