Luminescence 2005; 20: 321–325 Comparison of photosensitized and thermo-initiated CL Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bio.856
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Photochemiluminescent detection of antiradical activity. VII. Comparison with a modified method of thermo-initiated free radical generation with chemiluminescent detection I. Popov* and G. Lewin Research Institute for Antioxidant Therapy GmbH, Berlin, Germany
ABSTRACT: The method of photosensitized chemiluminescence (PCL) allows the quantification of water- and lipid-soluble antioxidants and activity of superoxide dismutase (SOD) in the same measuring system. However, it needs a special device, which we have described in a previous paper in this series. Another method suitable for the assay of water- and lipid-soluble antioxidants is the thermo-initiated decay of azo-compounds combined with the measurement of O2 consumption (Niki, 1985; Wayner et al., 1985). Its long duration and the complicated measuring procedure is not acceptable for routine medical applications. We show that a modification using CL detection of free radicals with luminol, has results comparable with PCL for the determination of nonenzymic water- and lipid-soluble antioxidants, SOD activity and oxidative modification of proteins. In contrast to PCL, it is possible to use any luminometer with a heatable measuring cell and to investigate coloured samples. While the new method has an overall higher sensitivity and is scalable to microtitre plates, PCL measurements can be made at different pH. The advantages and analytical information content of certain components of the integral antioxidative capacity of blood plasma are discussed in comparison with other methods. Copyright © 2005 John Wiley & Sons, Ltd. KEYWORDS: chemiluminescence; antioxidant status; antioxidative homeostasis; antiradical ability of proteins; diabetes
INTRODUCTION Free radicals, inducing damage to lipids, proteins, carbohydrates and DNA, are involved in the aetiopathogenesis of ‘civilization’ diseases such as atherosclerosis, cancer and diabetes. In physiological processes they are normally in a steady state with antioxidants. This antioxidative homeostasis is maintained by the antioxidative system of the organism, regulating absorption, synthesis, activation, release and excretion of exogenous and endogenous antioxidants (1). Its disturbance, e.g. due to exhaustion of antioxidants, is a precursor of the above processes. The analysis of the antiradical capacity of watersoluble substances (ACW) of blood plasma by means of the method of photosensitized chemiluminescence (PCL) (2, 3) showed that, under normal conditions, the main water-soluble components are urate (UA) and ascorbate (ASC). The rest of the total antiradical capacity was assumed to be the sum of minor antioxidants, including many low molecular weight substances and serum albumin. Investigation of the effects of UV- and hypochloriteinduced oxidative modifications of proteins and amino *Correspondence to: I. Popov, Central laboratory Oxidative Stress (ZOS) in IFLB (Institute for Laboratory Medicine Berlin), Windscheidstr. 18, 10627 Berlin-Charlottenburg, Germany. Email:
[email protected] Copyright © 2005 John Wiley & Sons, Ltd.
acids on their antiradical properties showed that the antiradical capacity of blood plasma proteins is not an a priori characteristic of antioxidant defence, but rather a feature of preceding free radical processes (4). More precisely, the antiradical ability of proteins (ARAP) reflects the degree of oxidative stress and can be utilized for an otherwise difficult detection of an undersupply of antioxidants by their direct measurement. Similar arguments apply to nucleotides (5). Here, we compare PCL with the method of thermoinitiated chemiluminescence (TIC), showing that similar results can be obtained, but that the latter is more suitable for the characterization of antioxidative homeostasis in medical diagnostics (6, 7).
METHODS The principle of PCL is based on an UV-A light (365 nm)-induced photochemical reaction, consisting of two steps: 1. Absorption of light, substrate excitation: S + hν S* 2. Generation of free radicals and/or singlet oxygen: S* + O2 → S+• + O2• S* + O2 → S + 1O2 Luminescence 2005;20:321–325
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If luminol (3-aminophthalhydrazide, L) is used as a photosensitizer, excited state aminophthalate anion will be produced, followed by chemiluminescence: LH− + hν1 → L• + O2• → N2 + AP2− + hν2 All substances that are able to react with luminol or oxygen radicals affect the light output, hν2. It should be mentioned that coloured substances may modify the intensity of hν1 and, as such, the amount of generated radicals. The photosensitized chemiluminescence was measured with the device Photochem® (now manufactured by Analytik Jena AG, Germany) (2). TIC measurements were made using a Minilum® chemiluminometer and ARAP and ARAP-Plus kits for antioxidant analysis of blood plasma (both from ABCD GmbH, Germany). Thermally initiated decay of waterand fat-soluble azo-compounds is used in this equipment as the source of free radicals, according to the following equations:
Figure 1. Original curves registered with the device Minilum®: a, blank; b, 0.5 µL blood plasma.
37°C → 2R• + N2↑ R–N = N–R
R• + O2 → ROO• Subsequently, the free radicals generated are detected via a chemiluminescent reaction with luminol (LH2):
According to above equations, one of the intermediates of the reactions is the luminol radical. Since it also participates in the SOD-dependent PCL reaction, we have tested and observed inhibition of TIC by SOD (unpublished data).
RESULTS AND DISCUSSION A blank sample with no antioxidants causes immediate light output in TIC and PCL systems (Fig. 1, curve a). In a plasma sample, the antioxidant species react with free radicals and delay the generation of photons until antioxidants are being consumed (curve b). The evaluation parameter Lag is the time difference of the chemiluminescence inhibition due to plasma sample in comparison to the blank: Lag = L1 − L0 Lag is directly proportional to the amount of antioxidant species in the sample. Fig. 2 shows examples of Copyright © 2005 John Wiley & Sons, Ltd.
Figure 2. Comparison of calibrations with ascorbic acid in PCL (upper diagram) and TIC (lower diagram) systems.
calibrations prepared with ascorbic acid as the standard substance in both systems. The main goal of the elaboration of the TIC method was to deliver a simple and reliable apparatus for Luminescence 2005;20:321–325
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Figure 3. Main components of the integral antioxidant capacity of blood plasma and an example of TIC analysis of a sample from a healthy volunteer. Ascorbic acid was used as the calibrating substance.
routine medical applications. For characterization of the degree of oxidative stress in the human organism as a precursor and/or as an accompanying phenomenon of ‘civilization’ diseases and subsequent therapeutic decisions, one needs to analyse the antioxidants and the degree of oxidative damage to the blood plasma components. It is insufficient to use only the quantities of selected substances (i.e. profiles of 10 or more single antioxidative effective components), to measure the sum of antioxidant capacities. In our previous investigations we showed that analysis of the integral antiradical capacity of blood plasma for its main components yields more information (see Fig. 3), especially for ascorbic acid (ASC) and uric acid (UA), which are subject to homeostatic control. With both methods comparable results can be obtained, but the contribution of ASC in ACW measured with PCL is lower than that measured with TIC, because of weaker sensitivity of PCL for ascorbate in comparison with TIC. The phenomenon of different degrees of effectiveness of UA and ASC to react with free radicals in various test systems, despite their stoichiometric equality, is well known. It depends not only on the kind of free radicals generated, but also on the physicochemical properties of the medium. This could be easily demonstrated in a PCL system after replacing some of the water in the measuring cell with methanol. UA has a very low level of antiradical activity under such conditions in comparison with ASC. It is on this basis that the ASC assay in blood plasma in PCL system works (8). As an example of practical application of the TIC method, Fig. 4 shows the results of measurements of the Copyright © 2005 John Wiley & Sons, Ltd.
urate-independent component of ACW in blood plasma in comparison with oxidative damage to plasma proteins in the same samples. Despite the small number of measurements, the results are significant and consistent with earlier findings in cancer patients (9, 10) and the hypothesis of the existence of an antioxidative system in the body (1). We have postulated that ACW is the main homeostatically regulated parameter. In effect, we showed that ACW is species-specific and individually constant. Nevertheless, it reacts very quickly to physical or emotional stress by elevation of ASC and UA levels in blood, which correlate negatively with each other: the lower the ASC, the higher the UA (6). Unfortunately it was discovered that ACW values, measured directly or calculated after analysis of single
Figure 4. Correlation of ACU and ARAP in blood plasma of healthy persons. Luminescence 2005;20:321–325
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components with the help of different methods, sometimes show considerable differences. Although many methodologies are used to measure antioxidants, for the above reasons all attempts to find the optimal system for determination of the hypothetical ‘total antioxidant status’ will fail. Similarly, it is impossible to compare the results of the determination of the integral antioxidative capacity of the blood plasma (IAC) determined by different methods simply by finding a coefficient, e.g. IAC = α ·ACW (3) = ß·TAS (11) = ·ORAC [12] = δ ·TRAP (13). Therefore, the clinical relevance of a technology can be decided independently of the measuring method and absolute values and distribution of the antiradical capacities of blood plasma components. On the basis of existing correlations of the individual parameters and the clinical picture, a useful formula for the IAC computation on the basis of its individual components can be found. Thus, our earlier results showed the significantly negative correlation of ASC and UA under the conditions of oxidative stress (6), as well as the correlation of the antioxidative parameters with the severity of illness (9) and therapy effectiveness (14). For substances which are foreign to the body and for which the blood level is not homeostatically regulated, the determination of high antioxidant activity in vitro is not evidence for the situation in vivo. It pertains in particular to vegetable components of currently very popular nutritional supplements, which are captured in the AOW share of ACW. A further problem in this regard is the significance of the antiradical properties of proteins as a part of the IAC/ACW. The common opinion is that ARAP is an inherent property of proteins and an important part of antioxidant defence. Our findings show that ARAP is a measure of the degree of oxidative stress. The measurement of the effects of UV irradiation of histidine on its antiradical efficacy with the TIC method using the ARAP-kit (Fig. 5) shows complete consistency with, and better sensitivity than, earlier PCL results (data from Fig. 4) (4). A higher sensitivity of the TIC method was confirmed also in investigations of oxidative modification of HSA under UV irradiation (Fig. 6). Our findings on the dependency of antioxidant properties on the measuring conditions (nature of free radicals in PCL and TIC systems, medium composition) show that in comparing different methods to characterize antioxidative homeostasis in the human body, and in the selection of a suitable method for clinical practice, the ARAP as a parameter of antioxidant defence must be excluded from IAC and considered separately. TIC measurements of blood samples from diabetes patients (Fig. 7) revealed, as expected, a higher disturbance of antioxidant homeostasis (a higher level of Copyright © 2005 John Wiley & Sons, Ltd.
I. Popov and G. Lewin
Figure 5. TIC and PCL assay of ACW of histidine immediately after and on the 5th day after UV irradiation at 254 nm.
Figure 6. Antiradical ability of HSA (60 g/L) after UV irradiation at 254 nm measured in PCL and TIC systems.
Figure 7. Comparison of blood parameters in patients with diabetes type 1 (n = 10; grey columns) and type 2 (n = 10, black columns). Luminescence 2005;20:321–325
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oxidative damage to proteins and a lower level of antioxidant defence) in patients suffering from type 2 diabetes in contrast to patients with type 1 diabetes. The results indirectly confirm an elevated relative risk in these patients, of 18% for type 2 diabetes, of oxidative stress-dependent cardiovascular complications and mortality, proportional to the 1% increase in HbA1c level (with normal values of 6.0–6.5%) (15).
CONCLUSION Detection of the degree of oxidative stress and determination of antioxidant defence in patients is not yet a common medical test. The biggest drawback of available methods is that they do not reflect the interplay of exogenous and endogenous antioxidants, with respect to their antiradical effectiveness and not to their quantity, by measuring all parameters in the same system using the same calibrating substance. Both PCL (2) and TIC (16) are free from this disadvantage and allow characterization of the antioxidant homeostasis in the human body, and therefore monitoring of the efficiency of conventional as well as antioxidant therapies. The main purpose of their application is in prepreclinical diagnostics—the recognition of disturbances of the state of health in a reversible stage, not manifested by specific signs of tissue damage. Here, we have compared both methods with the following results: (a) comparable analysis of antiradical properties of blood plasma; (b) better overall sensitivity of TIC, especially for ASC and ARAP; (c) simpler instrumentation of TIC; and (d) TIC allows an adaptation to microtitre plate applications.
REFERENCES 1. Lewin G, Popov I. The antioxidative system of the organism. Theoretical basis and practical consequences. Med. Hypoth. 1994; 42: 269–275. 2. Popov I, Hörnig J, v. Baehr R. Die Photochemolumineszenzmethode zur Bestimmung der Superoxiddismutaseaktivität. Z. Med. Labor. Diagn. 1985; 26: 417–421.
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3. Popov I, Lewin G. Photochemiluminescent detection of antiradical activity. II. Testing of nonenzymic water-soluble antioxidants. Free Rad. Biol. Med. 1994; 17: 267–271. 4. Popov I, Lewin G. Photochemiluminescent detection of antiradical activity. VI. Antioxidant characteristics of human blood plasma, low density lipoprotein, serum albumin and aminoacids during in vitro oxidation. Luminescence 1999; 14: 169–174. 5. Popov M, Popov I, Lewin G. Anti-radical properties of oxidatively modified nucleotides and DNA measured in a chemiluminescence detection system with free radical azo-initiator. In Summer Meeting of the Society for Free Radical Research, European Section, Ioannina, Greece, 26–29 June 2003; Book of Abstracts; 109 pp. 6. Popov I, Lewin G. Antioxidative homeostasis: characterization by means of chemiluminescent technique. In Methods in Enzymology, Packer L, Glazer AN (eds). Academic Press: New York, 1999; 300: 437–456. 7. Popov I, Lewin G. Photosensitized chemiluminescence, its medical and industrial applications for anti-oxidizability tests. In Chemiluminescence in Analytical Chemistry, Garcia-Campana AM, Baeyens W (eds). Marcel Dekker: New York, 2000; 497–527. 8. Lewin G, Popov I. Photochemiluminescent detection of antiradical activity. III. A simple assay of ascorbate in blood plasma. J. Biochem. Biophys. Methods 1994; 28: 277–282. 9. Völker H, Lewin G, Winzer K-J, Popov I. Die antioxidative Kapazität des Blutplasmas bei Patientinnen mit MammaTumoren. Zeitschr. Onkol. 1997; 29: 40–43. 10. Popov I, Völker H, Lewin G. Photochemiluminescent detection of antiradical activity. V: Application in combination with the hydrogen peroxide-initiated chemiluminescence of blood plasma proteins to evaluate the antioxidant homeostasis in humans. Redox Rep. 2001; 6: 43–48. 11. Miller NJ, Rice-Evans C, Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin. Sci. 1993; 84: 407–412. 12. Cao G, Alessio HM, Cutler R. Oxygen-radical absorbance capacity assay for antioxidants. Free Rad. Biol. Med. 1993; 14: 303– 311. 13. Wayner DDM, Burton GW, Ingold KU, Locke S. Quantitative measurement of the total, peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. FEBS Lett. 1985; 187: 33–37. 14. Popov I, Lewin G, Scherf H-P, Meffert H. Zum Verhalten der antioxidativen Kapazität des Plasmas nach UV-Blutbestrahlung. II. Untersuchungen an Patienten mit Psoriasis und arterieller Verschlusskrankheit. Z. Klin. Med. 1989; 44: 1857–1860. 15. Selvin E, Marinopoulos S, Berkenblit G et al. Meta-analysis: glycosylated hemoglobin and cardiovascular disease in diabetes mellutus. Ann. Intern. Med. 2004; 141: 421–431. 16. Popov I, Popov M, Lewin G. Assessing the risk of diseases by means of oxidative stress parameters of blood plasma. In Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications. Meeting of the Society for Free Radical Research, European Section. Ioannina, Greece, 26–29 June 2003. International Proceedings, Galaris D (ed.). Medimond: Bologna, 2003; 219–223.
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