Chemiluminescence in Analytical Chemistry

1. ANTIOXIDIZABILITY AND ITS QUANTIFICATION

Antioxidizability and its control are relevant for various areas in medicine and industry. Atherosclerosis, cardiac infarction, malignant growth, and aging are consequences of uncontrolled oxidation. Currently, oxidizability and antioxidants are also actual problems for alternative and complementary therapies like phyto-, helio-, and aero-ion therapy.

Among others, in the development of suntan lotions or in the production of beer, the oxidation of ingredients during storage is a known cause of reduced product quality.

A principal characteristic of living organisms is their capability to actively protect themselves against uncontrolled oxidation. Although all organisms are subject to the permanent influence of oxygen and other oxidatively active causes (UV sun irradiation, atmospheric noxae, natural and artificial radiation, etc.), they maintain their integrity due to the effect of a special antioxidative system that developed in the course of phylogenesis [1, 2]. This is in contrast to avital compounds the oxidizability of which depends only on their chemical composition. Thus, antioxidizability is a characteristic of living organisms.

Antioxidative protection mechanisms can be classified into at least four categories [2], i.e., compartmentation, detoxification, repair, and utilization; the first two have a direct relationship to antioxidizability.

Compartmentation means both spatial separation of potentially harmful but essential compounds (e.g., storage of iron in ferritin) and celland tissue-specific distribution of antioxidative compounds, and it serves to prevent uncontrolled oxidation.

Detoxification of pro-oxidatively active molecules (radicals, peroxides) is ensured by enzymatic and nonenzymatic compounds.

In addition to the antioxidative detoxificating enzymes, superoxide dismutase, catalase, and glutathione peroxidase with primarily intracellular occurrence that protect the cells from the destructive side effects of the physiological metabolism by prevention of initiation and branching of free-radical chain reactions, nonenzymatic antioxidants are of essential relevance. In contrast to enzymes, they are more or less equally distributed in all compartments of an organism. Exogenous stimuli, like X-rays, UV and ionizing radiation, lesions, inflammations, etc., can cause oxidative stress anywhere in the organism. Therefore, easily oxidizable structures are protected by a permanent homeostatically controlled antioxidant influx. The maintenance of antioxidative homeostasis is ensured by the antioxidative system with all its mechanisms such as release from depots, new synthesis, and control of excretion. In ex vivo studies, these mechanisms cannot be detected; the same applies to most industrial applications.

The antioxidative state of the organism can be defined by the antioxidizability and the oxidation state of blood components. The investigation includes mea-

surement of the antioxidative capacity of the blood plasma and the degree of existing oxidative damage to lipids, proteins, and nucleic acids.

A large number of nonenzymatic compounds, including tocopherols, carotinoids, vitamins C and D, steroids, ubiquinones, thiols, uric acid, bilirubin, inosine, taurine, pyruvate, CRP, and so on, demonstrate qualitative antioxidant properties under experimental conditions. However, the quantitative relevance of most findings remains unclear.

Owing to phenomena such as synergism, antagonism, competition, potentiation, mimicry, sparing, pseudoactivity, etc., it does not seem relevant to perform an assessment of the antioxidative state of the organism merely by selective determination of the physiologically most relevant antioxidants in blood plasma, vitamins C and E, carotinoids, and the compounds uric acid and bilirubin synthetizable in the organism.

Measurement of the total antioxidative overall efficacy in an oxidant-gener- ating test system is considered a physiologically relevant alternative to selective determination of individual components.

A system for determination of antioxidizability consists of two compo-

nents:

1.A generator of pro-oxidatively acting species, e.g., free radicals

2.Their detector allowing quantification of the generated species and indicating changes in the measured signal as a response to the presence of antioxidative compounds

In the case of free radicals, all generation systems can be classified into physical (radiolysis, photolysis, electrolysis, etc.), physicochemical (thermic decomposition of nitrogen compounds, photosensitized generation), chemical (Fe2 /H2O2 system, KO2 decomposition), and biochemical systems of varying complexity including individual enzymes (e.g., xanthine oxidase), subcellular fractions (NADPH-consuming microsomes), and tissue homogenates (e.g., brain homogenate).

Detection of free radicals can be performed using light absorption, luminescence, oxygen consumption, electrical conductivity, and enzyme activity measurements. A number of examples of relatively common systems are given in Table 1.

Despite the diversity of analytical procedures, no valid selection of solely ‘‘right methods’’ exists. Depending on the problem, the nature of the free radicals themselves or the physicochemical properties of the test system such as composition and hydrophobicity of the medium, oxygen partial pressure, pH value, etc., are important as well [15–17]. An adequate test system is characterized by the fact that with regard to size, lipophilicity, and reactivity the biological target substrate reacts with the most relevant radicals.

As the superoxide radical is a precursor of the other reactive oxygen species and interacts with blood plasma components under physiological and pathological conditions as well, systems related to its generation are biologically relevant. It should be noted, however, that with respect to the initiation of lipid peroxidation as one of the main causes of oxidative cell damage, its own reactivity is very weak and that only in protonized form is its toxicity comparable to that of lipid peroxyl radicals [18].

Systems generating only hydroxyl radicals are of minor relevance because of the high reactivity of these radicals resulting in oxidation of all types of molecules at the site of origin. This approach makes it possible to declare almost every compound an ‘‘antioxidant,’’ although this thesis will not stand in vivo examination. According to Halliwell and Gutteridge [19], who proposed a practicable definition, an antioxidant is ‘‘any substance that, when present at low concentrations compared to those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate.’’

According to this principle, all antioxidant detection methods can be categorized in two groups, i.e., undefined and defined. The first group includes those with free radicals of unknown characteristics, e.g., the oxidation of a suitable object (unsaturated fatty acids, tissue homogenate of a lab animal, egg yolk) in vitro by treatment with air or pure oxygen. The time when the antioxidants are used up (‘‘lag phase’’) is recognizable and measurable by the accelerated generation of oxidation products and increased oxygen consumption. The antioxidative effect of a test substance can be estimated after repeated oxidation of the sample in the presence of this substance followed by calculation of the difference between the first and second lag phase. In addition to shortcomings of chemical nature, particularly in the use of egg yolk or tissue homogenate, the major disadvantage of the method is its long duration. It takes several hours to perform one single test. Another shortcoming is due to the fact that the nature of the free radicals involved remains insufficiently defined. The methods based on systems, in which the nature of the generated radicals is known, for example thermic decomposition of nitrogen compounds, photosensitized generation, KO2 decomposition, etc., are free of this disadvantage.

Below several useful methods are discussed in greater detail.

1.1 Autoxidation of Unsaturated Fatty Acids

Approximately 10 mL of oil are fumigated with oxygen in a closed system, so that the gas stream from the reactor cell is directed into the measuring cell containing distilled water. After the antioxidants are used up, the lipid peroxidation is initiated, and the volatile reaction products get into the measuring cell improving its electrical conductivity. This is recorded graphically as the lag phase. According to this principle, the antioxidizability of oils is investigated with the Ran-

cimat device (manufacturer: Metrohm, Switzerland) [20]. A single measurement with this instrument takes several minutes to hours, and it is suitable only for investigation of fat-soluble compounds.

1.2 Enzymatic Radical Generation

The reaction of peroxidase (metmyoglobin) with hydrogen peroxide leads to the generation of a green-blue radical from a colorless compound 2,2′-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS). It is slowed down in the presence of an antioxidant, an effect that is used for its quantitation in the Total Antioxidant Status Kit (manufacturer: Randox, UK) [10]. Problems associated with this method are due to potential interference of the reaction compound H2O2 with components of the sample to be investigated. No investigation of fat-soluble compounds is possible.

1.3Thermally Induced Decomposition of Nitrogen Compounds

A number of waterand fat-soluble nitrogen compounds, e.g., 2,2′-azo-bis(2- amidinopropane) dihydrochloride (ABAP), 2,2′-azo-bis(2,4-dimethylvaleroni- trile) (AMVN), and 2,2′-azo-bis(2-cyanopropane) (ABCP), form free radicals during decomposition that in the sample to be investigated initiate lipid peroxidation [16]:

RENCNER → 2R• N2 R• O2 → ROO•

ROO• LEH → ROOH L•

As in the first example, the time when the antioxidants are used up can be determined by measurement of the lag phase of oxygen consumption or of the generation of oxidation products. Although the measuring time is shorter than it is with the other variants mentioned, it still takes many minutes to obtain a result, obviously too long a period for routine tests.

Pronounced improvement of quantity and quality in the determination of antioxidizability is obtained by the photosensibilized chemiluminescence (PCL) method, realized in the Photochem device (manufacturer: Analytik Jena AG, Germany).

2. PHYSICOCHEMICAL BASIS OF PCL

The main feature of the PCL measuring method is combination of the simple and reliable photochemical generation of free radicals with their very sensitive

chemiluminometric detection. Compared to standard conditions, the oxidative reaction is accelerated by a factor of 1000. This results in a reduction in measuring time by a factor of 10–1000 compared to other methods.

2.1 Photosensitized Generation of Free Radicals

This is obtained by optical excitation of the photosensitizer (S) and followed by oxygen reduction.

There are two possible initial steps of photosensitized reactions, leading to the formation of superoxide radical:

I. S hν → S* R(RH) → R • S

'

R• •SH

where S* is the photosensitizer in triplet state, and R is the reducing substance. Various dyes can be used as photosensitizers, including methylene blue, riboflavine, and hematoporphyrin derivative. The selection of the photosensitizer should be in favor of a compound that exclusively leads to Reaction (b), so that

a clear interpretation of the results is possible.

2.2 Chemiluminometric Detection of Free Radicals

The dismutation (disproportioning) of two free radicals is accompanied by release of a portion of reaction energy as a light quantum. As the quantum yield of such a process is extremely low, the detection of this type of chemiluminescence is technically complicated. Several compounds like lucigenin and luminol have a high quantum yield after reaction with peroxide radicals. Therefore, they are widely used for the detection of these radicals, particularly in the examination of phagocyting cells.

The design of a device that would unify the photochemical method, i.e., the generation of free radicals, and chemiluminometric detection conflicts with controversial requirements—irradiation of the photosensitizer-containing solution with high-intensity light and the need to completely darken the environment during registration of the chemiluminescence signal.

The solution of this problem was to design a device with circular sample transfer. Irradiation and chemiluminescence measurement are spatially separated;

Figure 2 Chemical mechanisms of superoxide-dependent PCL with riboflavin as photosensitizer. (From Ref. 22.)

1LH hν → 3LH → 3LH O2 → 1LH• O2

↓ H

1L2 hν → 3L2 → 3L2 O2 → 1L O2

Thereafter the free radicals can react with one another and with the initial molecules. The reaction LH• O2 → O2 LH is not accompanied by chemiluminescence [23].

The reaction L O2 → LHOO leads, after some intermediate stages, to generation of electronically excited compound AP* (aminophthalate anion) [23, 24]. In these reactions, let k1 31k2, but at pH 9.2 both proceed equally fast.

Independent of the precise mechanism of the chemiluminescence-produc- ing reaction, the process of luminol-dependent PCL can be subdivided into two stages:

I. L hν1 → L O2 (generation of free radicals)

II. L O2 → N2 AP2 hν2 (chemiluminescence).

The reaction partners for antiradical substances are products of the first reaction. The SOD reacts selectively with O2 ; the nonenzymic antioxidants can react with both superoxide and luminol radicals. Theoretically, the carbonate radicals can also be involved in the PCL [25, 26].