Chemiluminescence in Analytical Chemistry

calculated as: ACR ACW (UA ASC), was assumed to be the sum of minor antioxidants, including serum albumin, tocopherols, carotenoids, vitamin D, steroids, ubiquinones, thiols, bilirubin, inosine, taurine, pyruvate, C-reactive protein, etc. According to the literature, these substances have antioxidant properties. The practical quantitative relevance of these findings for ACR, however, remains unknown.

In investigations of healthy persons under normal conditions and after UV irradiation of the whole body we have found that ASC correlates significantly (p 0.001) negatively with ACR [35]. A plausible explanation was that some of the above antioxidants can replace ASC in case of its increased consumption during an oxidative stress.

Investigations of the effects of UVand hypochlorite-induced oxidative modification of 20 amino acids and human serum albumin (HSA) on their antiradical properties showed unexpected results [36]. Seven amino acids (cystine, histidine, methionine, phenylalanine, serine, tryptophan, and tyrosine) and HSA developed ACW following oxidation (see examples in Fig. 14). The fresh (produced in 1998) HSA from Serva had no antiradical capacity, but it acquired this quality during irradiation. The out-of-date HSA sample (Dessau, GDR, 1987, expiration date 7/1/1992) showed a remarkable ACW even in an unirradiated state.

Figure 15 presents a qualitative comparison of different oxidation types on histidine ACW. Hence, the ACP cannot be seen to be characteristic for antioxidant defense, but more likely it is a feature of prehistory connected with the freeradical processes, reflecting the degree of oxidative stress.

A check of the results of the parameter ACP of human blood plasma after removal of low-molecular-weight antioxidants by means of gel filtration was positive and showed a clear difference between the results in healthy donors and cancer patients, as can be seen in Figure 16 [35].

Figure 14 Time course of ACW of HSA (60 g/L) and cystine (2 mmol/L) during (filled circles) and 24 h after UV irradiation (open circles). (From Ref. 36.)

by environmental pollutants or professional noxious factors, and diagnosis of the seriousness as well as control of the therapy efficacy of illnesses like atherosclerosis and heart attack, cancer, diabetes, asthma, allergies, and others.

Analysis of antioxidants (antioxidant status) for characterization of the antioxidative homeostasis in organisms by selective measurement of the ACP can be very meaningful for efficient supervision of antioxidant therapy as well.

4.2 Parameters Assayed for Plasma LDL

Oxidized low-density lipoprotein (LDL) may play a key role in the initiation and progression of atherosclerosis. Risk factors for elevated levels of oxidized LDL are not well established and may be important in identifying individuals who may benefit from antioxidant supplementation or interventions to reduce oxidant stress.

4.2.1ACW, ACL, and ACL0 for Isolated LDL

Basic procedure: LDL is isolated from fresh EDTA(Na2) plasma by sequential ultracentrifugation in KBr step gradient at 120,000 rpm (microultracentrifuge HITACHI CS 120) and a temperature of 10°C for 2 2 h. The LDL was covered with nitrogen in a screw-cap tube and stored in the refrigerator at 4°C until further use.

Prior to application, the LDL proceeded through the gel filtration desalting procedure and its cholesterol content was adjusted to 0.4 mM.

LDL isolation is used for measurement of its oxidizability under the influence of various factors in a model system using CuCl2 (final concentration 3.3 µM) as the initiator of lipid peroxidation. Oxidation of LDL was followed by changes in optical density at 234 nm (conjugated dienes formation assay) [37].

ACL0 is a simplified variation of the ACL assay with direct LDL investigation in the ACL system without prior lipid extraction. The ACL0 value correlates with the ACL value but is lower by approximately 25–30%.

In Figure 17 the changes in ACW and ACL0 during Cu2 -initiated LDL oxidation are shown. It should be noted that the native LDL has negligible ACW and that the ACL0 kinetic course is composed of two distinct phases. The same processes such as UV-induced oxidation of proteins obviously take place during the relatively slow Cu2 -initiated LDL oxidation. It was demonstrated that approximately 80% of the antioxidant capacity of LDL can be accounted for by α- tocopherol [38, 39]. It was assumed that after all antioxidant capacity is exhausted, the oxidative stress imposed by copper ions or by the peroxidase-cata- lyzed reaction results in lipid peroxidation [40, 41]. The formation of conjugated dienes is conditioned by exhaustion of endogenous antioxidants, but the increase

Figure 17 Time course of the antiradical parameters ACL0 and ACW of LDL measured by the PCL method; its α-tocopherol content (AT) measured by the HPLC technique and conjugated dienes (LDL-abs. at 234 nm) during Cu2 -initiated oxidation in vitro. (From Ref. 36.)

of the ACW and ACL0 is obviously the consequence of acquisition of antiradical activity by proteins, possibly apolipoprotein B [42].

In contrast to lipid peroxidation the products of which typically appear after a lag phase, protein damage by reactive oxygen species takes place directly and immediately [43, 44]. In contrast to single amino acids and HSA, the ACW of LDL was not elevated until all lipid-soluble antioxidants were exhausted. Hence the first points of the ACL0 curve in Figure 17 represent the sum of antioxidants without a contribution from protein-dependent ACW. The late was detected at the same time as the formation of conjugated dienes showing the possible protective effect of endogenous antioxidants in LDL on free-radical-induced modification of apolipoprotein B.

4.2.2ACW, ACL, and ACL0 for Nonisolated LDL

As isolation of LDL for determination of its antiradical properties is a tedious task, it was alternatively selectively removed from the blood plasma.

Modified procedure: HDL plasma—plasma depleted with apo B–con- taining lipoproteins by pretreatment with dextran sulfate/Mg2 HDL reagent (Sigma, procedure 352-3). Plasma LDL-bound antiradical parameters can be calculated as the difference between ACW/ACL/ACL0 for whole plasma and for HDL plasma as well.

An example is shown in Figure 21 (see below). Further investigations are in progress by our group.

5. INDUSTRIAL APPLICATIONS OF PCL

In many industrial areas, antioxidants play a significant role in the context of stability improvement of easily oxidizable compounds. There is a considerable interest in antioxidants as bioactive components of food and as nutritional agents with a role in the maintenance of health and in disease prevention.

5.1 Detection of Consequences of Food Irradiation

The objective of irradiation of food with γ-rays is elimination of parasitizing insects, fungi, and bacteria to prevent premature spoiling of the food and the outbreak of diseases. In addition, retardation of aging and ripening of fruits and vegetables can be achieved. In the Federal Republic of Germany, irradiation of food with the exception of spices is not permissible.

The examination was performed with the apple variety Jonagold. Apple pieces weighting 0.3–0.5 g without skin were homogenized in a Potter-type glass homogenizer and then centrifuged. Before examination, the supernatant was diluted 1:10 with double distilled water. PCL measurements were performed with 2 µL of the diluted supernatant. Apple juice contains mainly vitamin C and carotinoids as principal ACW components.

A typical change in ACW as measured in various apples 3 days after irradiation is shown in Figure 18. The results are in agreement with the expectation that ACW is reduced due to an interaction of the free-radical scavengers with the free radicals originating during irradiation.

5.2 Antiradical Properties of Wines

People in France eat a lot of fatty foods but suffer less from fatal heart strokes than people in the northern regions of Europe or in North America, where wine is not consumed on a regular basis (‘‘French paradox’’). There is an increased favorable effect from red wine. The unique cardioprotective properties of red wine are due to the action of flavonoids, which are minimal in white wine. The best-researched flavonoids are resveratrol and quercetin, which confer antioxidant properties more potent than α-tocopherol.

Some structural changes of the native flavonoids occur during wine conservation, and one of the most studied of those changes concerns red wine color evolution, called ‘‘wine aging.’’ It has been demonstrated that as a wine ages, the initially present grape pigments slowly turn into new, more stable red pig-

Figure 18 Dosage-dependent decrease in ACW detected in apple juice after γ-irradia- tion of apples.

ments. That phenomenon continues for many weeks and years. The main questions to be answered in PCL investigations of wines are:

Can antiradical properties of wines be quantified by means of PCL? Which subpopulations of blood plasma lipoproteins bind the wine-derived

antioxidants?

Do the results of oxidizability tests of LDL in the Cu2 system correlate with the results of PCL examinations after treatment with wine?

Can technological processes of wine production be followed using the PCL method?

Figure 19 presents results of PCL examination of wines, grape skins, and grape pips. The very low antioxidant capacity of white wine is obviously related to the technology of its manufacturing: it is prepared from pure juce without grape skins and pips, in contrast to red wine. The dependency of ACW on the storage time of red wine is depicted in Figure 20. White wine is stable in this sense.

Three different types of white wines and four red wines were compared after PCL testing in an ex vivo model system of the influence of oxidation resistance on LDL.

Results of PCL measurements and LDL investigations are depicted in Table 2 and Figure 21. Dr. V. Ivanov (Linus Pauling Institute, Oregon State University), a recipient of a grant from the German Federal Ministry for Research and Technology (AiF FKP 0034904B7B), was involved in this part of the study.

Figure 19 Antioxidative qualities of wines (white rectangles) in comparison to watery extracts from solid grape constituents: skins (gray rectangles) and pips (black rectangles) in equivalent concentrations of ascorbic acid (mmol/L, mmol/g).

The results of the LDL protection determined with both methods correlate very well with each other. The linear regression between LDL oxidation assay by conjugated dienes formation and PCL (ACWLDL) for all wines was: y 0.2505x 16.563, R2 0.9348.

Figure 21 shows the distribution of wine-derived antioxidants between apo B–containing lipoproteins and other high-molecular-weight components of

Figure 20 Comparison of ACW values of human blood plasma (A) with different wine types: Riesling Spa¨tlese 1989 (B), Riesling Kabinett 1996 (C), Spa¨tburgunder 1996 (D), Spa¨tburgunder 1994 (E), Spa¨tburgunder 1990 (F). B and C, white wines; D, E, F, red wines.

Table 2 Antiradical Activity of Red and White Wines (ACWw) and Their Antioxidative Protective Effectiveness in the Test System of the Cu2 -Initiated LDL Oxidation

Parameters of protection: ∆(Lag), change of the lag phase of conjugated dienes formation, ∆(ACWLDL), change of the ACW of LDL after its preincubation with wine, both expressed in percentage of the initial value.

plasma, mainly HDL and serum albumin. After preincubation of plasma with wine in vitro the apo B–containing lipoproteins were removed by pretreatment with dextran sulfate/Mg2 HDL Sigma reagent.

The plasma LDL/VLDL-bound antiradical parameter was calculated as the difference between ACP for whole plasma and for HDL plasma as well.

Figure 21 Ex vivo effects of wines on ACP of plasma, HDL plasma, and LDL-VLDL fraction.

5.3 Antiradical Properties of Different Tea Types

Tea also contains phenolic substances and carotenoids. The long-living Caucasian population regularly consume wine and tea. The aim of the studies was to compare the antiradical properties of different tea types. The results of measurements of ACW and ACL0 for 16 various types are shown in Figure 23.

In the investigated teas the lowest content of antioxidants is in cumin tea, the highest in sage tea, followed by green and black tea.

6. CONCLUSIONS

The method of photosensitized chemiluminescence is suitable for antioxidizability testing of biological fluids. In addition to measurement of the integral antiradical capacity of blood plasma [45], the PCL method allows selective determination of uric acid and of vitamin C [34] and the degree of oxidative modification of proteins as well, therefore offering the possibility of screening tests of new artificial, herbal, and gene technologically obtained compounds in vitro and in vivo.

The significant benefits of the PCL method include speed, precision, and the possibility to investigate waterand lipid-soluble compounds in a single system and to alter the type of radicals originated by selection of the respective photosensitizer. In contrast to other methods currently in use, the method discussed here is not restricted to a limited pH or temperature range.

Potential PCL applications in medicine (these investigations are in progress) include characterization of disturbances in antioxidative homeostasis, in the following areas:

Surgery (diagnosis and prevention of ischemic damage to tissues) Internal medicine (characterization of the oxidation state of LDL relevant

for atherosclerosis and cardiac infarction)

Anesthesiology and intensive care medicine (control of blood damage in a heart-lung machine)

Obstetrics and gynecology (diagnosis of pregnancy pathology) Oncology (assessment of antioxidative homeostasis in tumor patients) Transfusion (quality control of stored blood)

Sports medicine (antioxidative vitamins and cell damage in competitive sportsmen)

Medical genetics (diagnostics of Down’s syndrome and familial hypercholesterinemia)

Naturopathy (objectivization of the efficacy of helio-, phyto-, oxygen, and other kinds of unconventional therapy)

Public health (studies on antioxidative homeostasis in various segments of the population the meaning of which could potentially unravel the rele-