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 1755 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. 1756 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. 1757 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 1758 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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