Chemiluminescence in Analytical Chemistry

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3. THE ‘‘PHOTOCHEM’’ DEVICE FOR PCL MEASUREMENT

The measuring principle of photosensitized chemiluminescence is applied in the Photochem. The principal functional units of the instrument are shown in Figure 3.

Irradiation of the photosensitizer solution takes place in a vessel (1) by means of a lamp (2). During measurements, a continuously irradiated solution is transferred with a peristaltic pump (5) from the vessel (1) into the measuring cell (6) of a chemiluminometer (7). Control of the unit, signal recording, and data processing are performed by a computer (8). The tube (4) serves for injection of various components during measurement.

Data acquisition software (Fig. 4) makes possible:

Recording and plotting of PCL signals

Evaluation of curves with and without calibration

Archiving and printing of curves and tables of results

3.1 Testing of Water-Soluble Compounds

A typical time course of PCL with luminol as the photosensitizer is shown in Figure 5, as ‘‘blank.’’ The presence of a water-soluble antioxidant leads to dosedependent temporary inhibition of PCL. ‘‘ACW’’ (antioxidant capacity of watersoluble compounds) represents the effect of human blood plasma (2 L) on PCL; all tested antioxidants, such as ascorbic acid, uric acid, Trolox, taurine, bilirubin, ceruloplasmin, etc., produced the same effects.

An evaluation parameter of the graphs is the duration of the lag phase L of the PCL (L L1 L0, where L1 is the lag phase of a sample with an antioxidant and L0 is the same of a blank sample). All compounds tested demonstrate

Figure 3 Scheme of the apparatus for measurement of PCL. 1, Vessel for irradiation of a photosensitizer-containing solution; 2, UV lamp; 3, shutter; 4, injection tube; 5, peristaltic pump; 6, measuring cell; 7, photomultiplier; 8, computer. (From Ref. 45.)

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Figure 4 Poplab software.

a linear dependence of the lag phase on the substance quantity. Figure 6 shows the effects of increasing concentrations of Trolox on ACW.

‘‘SOD’’ in Figure 5 demonstrates the effect of superoxide dismutase on PCL. Its effect is characterized by an inhibition of the chemiluminescence intensity without having an impact on the lag phase. According to McCord and Fridovich [27] (50% test signal inhibition), 1 activity unit in this system refers to approximately 100 ng of the enzyme preparation of the Sigma Co.

3.2 Testing of Lipid-Soluble Compounds

A modification of the measuring system concerns the composition of the assay mixture, in which a part of the water (90%) is replaced by methanol to provide for solubility of hydrophobous substances.

Figure 7 shows an example of the measurements of the antioxidant capacity of lipid-soluble compounds (ACL). In this figure, recording 1 corresponds to ‘‘blank,’’ while recordings 2–5 demonstrate the effect of adding lipid extracts from equivalently 20, 40, 60, and 80 L of blood plasma from a healthy volunteer

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Figure 5 Time course of PCL of luminol. Blank, without additions; ACW, effect of human blood plasma (2 L); SOD, effect of superoxide dismutase (150 ng). (From Ref. 34.)

to the assay mixture. The evaluation parameter of measurement is inhibition of PCL (I), which can be calculated as: I 1 S1/S0. Here S0 is the integral of PCL intensity recorded during 2 min, S1 is the same parameter in the presence of an antioxidant.

A comparison of the PCL method with HPLC gave the results shown in Figure 8. The measurements were performed with the both methods on the same

Figure 6 ACW calibration curve for Trolox.

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Figure 7 PCL recordings in ACL system. 1, blank; 2–5, effects of lipid extracts from, respectively, 20, 40, 60, and 80 µL of blood plasma from a healthy blood donor. (From Ref. 28.)

Figure 8 Effect of LDL supplementation with α-tocopherol in vitro determined by HPLC and PCL techniques. The surplus in comparison to the initial value is represented for each parameter. (From Ref. 28.)

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lipid extracts from LDL, supplemented in vitro with α-tocopherol [28]. Supplementation of LDL was made before isolation. Plasma was enriched with increasing concentrations of α-tocopherol in dimethylsulfoxide (10 µL DMSO/1 mL plasma). The LDL was isolated after an incubation time of 3 h at 37°C using the microultracentrifuge HITACHI CS 120 as described in Ref. 28, covered with nitrogen, and stored in the refrigerator at 4°C until the lipid extraction. A good quantitative coincidence of both methods was established with r 0.998, p 0.001.

3.3 Specialized Analytical Opportunities

In the context of the theoretically existing problems, a need exists to investigate the antiradical efficacy of compounds independent of pH value or polarity of the solvent, respectively. These problems are particularly relevant in the design of new pharmaceutical antioxidant preparations for specialized therapeutic applications.

It is well known that under pathological conditions the pH value in tissues may significantly differ from the physiological pH value of 7.4. No direct measurement with the Photochem is possible at various pH values, including particularly the acidic range. The reason is the pronounced decrease in chemiluminescence intensity caused by spontaneous dismutation of the superoxide radicals during transfer of the irradiated solution into the measuring cell, according to the following equation:

2O2 2H → H2O2 O2

Already at a pH value of 8.0, the signal amplitude is so low that no sufficient accuracy will be obtained during measurement. In the physiological pH value range, the signal equals zero. In such cases, a special pH jump method is a valuable tool. Analogously to measurement of the optical spectrum of chemiluminescence using cutoff filters, the total graph expected in PCL is separated into sections. Each measurement starts with the preferred pH value, while the measuring curve tends to the zero line. At a predefined time, the pH value is changed in a substantial step by the injection of buffer solution, and starting from this time, a signal can be detected. It has been demonstrated by suitable experiments that the change in pH value has no impact on photochemical radical generation.

4.MEDICAL APPLICATIONS OF PCL: CHARACTERIZATION OF ANTIOXIDATIVE HOMEOSTASIS

4.1 Parameters Assayed in Blood Plasma

A large number of reports on the biological activity of antioxidants in blood plasma and cell membranes verify that vitamin E (VE) is the most significant

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antioxidant in the lipid phase and vitamin C in the aqueous phase. Vitamin C causes vitamin E to change from the oxidized into the reduced state (vitamin E changes into the oxidized state while performing its main function, namely inhibition of free-radical-mediated lipid peroxidation).

Aside from these two vitamins, uric acid has also been assigned an important role as antioxidant [29]. It has the ability to protect vitamin C from oxidizing [30]. Under strong oxidative stress, bilirubin also seems to be important [31, 32].

4.1.1ACW—Integral Antiradical Capacity of Water-Soluble Compounds

During determination of the integral antiradical capacity of blood plasma (ACW) by the PCL method, the above-mentioned substances, among others, will be detected primarily.

Basic procedure (ACW kit): Mix 1500 µL of ACW reagent 1 (diluter) with 1000 µL of ACW reagent 2 (buffer) and 25 µL of photosensitizer reagent (luminol based). Start measurement after brief vortexing. Assayed solution (or control) is added before addition of photosensitizer reagent. Volume of ACW reagent 1 is reduced by the volume of assayed plasma sample. Standard substance: ascorbic acid. Duration of measurement: 2–3 min. Measured parameter: effective lag phase lag-phase sample lag-phase blank. Assayed amount of human blood plasma: 2 µL.

Tests performed on humans and animals under normal and pathological conditions showed that under normal conditions the ACW is a stable parameter specific to species and age. On the contrary it is very sensitive to any stress factors (bacterial and sterile inflammations, surgical and psychoemotional stress) and to the application of β-agonists [33].

These and other findings relating to the importance of ACW justified the concept of antioxidant homeostasis in organisms and postulate the ACW as a central and evidently regulated parameter (comparable with pH or body temperature) [2].

4.1.2ACL—Integral Antiradical Capacity of Lipid-Soluble Compounds

Basic procedure (ACL kit): Mix 2400 µL of ACL reagent 1 (diluter) with 100 µL of ACL reagent 2 (buffer) and 25 µL of photosensitizer reagent (luminol based). Start measurement after brief vortexing. Assayed solution (lipid extract) is added before addition of photosensitizer reagent. Volume of ACL reagent 1 is reduced by the volume of assayed solution. Standard substance: α-tocopherol or Trolox. Duration of measurement: 1 min. Measured parameter: integral (area under the kinetic curve of PCL).

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Lipid extraction: 200 µL of plasma sample is mixed by brief vortexing with 200 µL of ethanol followed 1 min of vortexing with 800 µL hexane. After centrifugation for 5 min at 5000 rpm, 400 µL of upper (hexane) phase is transferred to a glass tube with screwed cap, dried under nitrogen, and kept at 60°C until PCL analysis. For PCL analysis the sample is dissolved in 400 µL of MeOH by 30 s of vortexing, and 100 µL aliquots are taken for ACL assay.

Similar to ACW corresponding to water-soluble compounds, ACL is a sum parameter including various fat-soluble compounds, the majority of which belong to the vitamin E group. The scatterplot diagram in Figure 9 shows the results of PCL (ACL) and HPLC (VE) investigations of antioxidants in 142 plasma samples from healthy donors. It illustrates a good linear correlation between VE as a sum of α- and γ-tocopherols and ACL: r 0.811, p 0.001. In the average belongs to the VE (23.54 8.78 µmol/L) a share of about 84% in the ACL (28.03 8.02 µmol/L).

Figure 10 presents the results of assay of VE and ACL in blood plasma of rabbits treated with probucol and two other synthetic antioxidants (S-1, S-2) for 4 weeks. In addition to an improvement in antioxidative blood plasma protection, in the case of compound S-2 a statistically significant (p 0.01) decrease in vitamin E content was detected, a finding considered physiologically unfavorable.

Figure 9 Antiradical capacity in the lipid phase of blood plasma (ACL) determined with the PCL method versus vitamin E (VE) as a sum of α- and γ-tocopherols determined with HPLC. (From Ref. 28.)

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Figure 10 Lipid-soluble antioxidants in blood plasma in rabbits fed 4 weeks with probucol and two newly synthesized compounds. All products were administered in equal amounts. The VE portion is the dark gray portion of the columns. Contr., control group; Prob., group fed with probucol; S-1 and S-2, groups fed with new antioxidants.

4.1.3ACU—Urate-Independent Component of ACW

Using suitable oxidases it is possible to analyze the proportion of individual ACW compounds.

Modified procedure (ACU kit): The plasma sample is preincubated with uricase (urate oxidase) and then quantified in the ACW assay. The method requires 10 L of plasma. It is also possible to determine the uric acid (UA) portion in ACW: UA ACW ACU.

The main value of ACU measurement is that it makes it possible to give a fast estimate of vitamin C deficiency because in healthy subjects ascorbic acid is about 90% of total ACU [33]. A strong deficit of ACU, possibly due to vitamin C deficiency, was found in patients with breast tumors (Fig. 11).

These results do not have any diagnostic meaning. The dynamics of the ACU values after an operative intervention could be taken into account regarding the existence of metastases. As demonstrated in Figure 12, prognostic relevance can be ascribed to ACU. The rise in ACU value is caused in this case by increase of the bilirubin level under the conditions of the life-threatening inflammatory processes [1].

4.1.4ASC—Vitamin C

Modified procedure (ASC kit): Ascorbic acid is isolated from plasma proteins and uric acid in a single-step liquid gel chromatography procedure and its amount

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Figure 11 Blood plasma ACU in patients with mammary tumors. N, healthy women (n 24); Be., benign mastopathies (n 40); C, carcinomas in stages I (n 14), II (n 13), III (n 9), and IV (n 7). Compared to healthy subjects, p 0.001 for all carcinomas.

Figure 12 ACU course in the blood plasma of two intensive-care patients with peritoni-

tis. Lethal outcome in patient 1. Normal ACU value, determined in 144 healthy blood donors: 70 27 mol/L ASC.

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(based on its antiradical capacity) is measured in a modified ACW assay. The method requires 500 L of plasma; its sensitivity is 5 M [34].

An example of the prognostic meaning of vitamin C measurements is given in Figure 13 [33]. In this study on neurosurgical patients drastic reduction of the vitamin C concentration in blood during the operation was associated with postoperative brain edema.

4.1.5ACP—Antiradical Capacity of Proteins

Modified procedure (ACP kit): Total plasma protein is isolated from low-molecu- lar-weight plasma antioxidants in a single-step liquid gel chromatography procedure and its antiradical capacity ACP is measured in the ACW assay.

The antiradical capacity of proteins is thought to be an important component of the total antioxidant capacity of blood plasma. Analysis of the ACW of blood plasma showed, that under normal conditions, its main components are the UA and ASC. The rest of the total antiradical capacity (ACR), which can be

Figure 13 Dynamics of ASC in the blood plasma of neurosurgical patients. Conditions of blood taking referring to the sample number: 0, 1 day before operation; 1, before the first cut; 2 and 3, during the operation, in 30–40-min intervals; 4, at the end of the operation; 5, the next day; 6, at discharge from the hospital. Mean values from n 14 in the group without complications (circles) and n 6 in the group with brain edema (squares). p 0.05 for 1st and 3rd samples, and p 0.01 for 2nd sample. (From Ref. 33.)