Background/Aim:L-Ascorbic acid (AA) is the predominant circulating form of vitamin C found in human blood. It has been hypothesized that surgical stress increases the vitamin C metabolite dehydroascorbic acid (DHAA). Vitamin C is mainly excreted through the kidneys. In this study, the ratio of AA to DHAA excreted in urine was determined in patients who had undergone total hip joint endoprosthesis surgery (n = 12), and the results were compared with data obtained from healthy controls (n = 12). Methods: All subjects received 1,000 mg sodium ascorbate intravenously three times a day (every 8 h) for 8 days, starting 2 days prior to surgery. Total urine was collected daily while subsequent determinations of AA and DHAA were performed photometrically. Results: Administration of vitamin C led to average daily excretions of the combined products AA + DHAA of 2,343 ± 438 mg/day (mean value ± confidence intervals). The initial average ratio DHAA/AA of all 24 probands was 0.064 (6% DHAA; 153 ± 76 mg/day). One day after surgery, an increase in the DHAA/AA ratio to 0.165 (15% DHAA; 332 ± 107 mg/day) was measured in the patients. The ratio decreased 2 days after surgery and returned to normal within 5 days. Conclusion: Our data indicate that surgery increases the oxidation of AA and urinary excretion of DHAA, as a result of the enhanced formation of free radicals.

Alternative methods for the decontamination of foods and surfaces in the food processing industry are under research. We introduce a new approach: the use of visible light plus photosensitizers, the photodynamic effect. First, we established an experimental model to examine the photosensitivity of immobilized micro-organisms. We tested subsequently several Gram-positive bacteria and yeasts for their photosensitivity after incubation with haematoporphyrin (HP) and sodium chlorophyllin (CHL). The survival levels of the bacteria fixed on solid media ranged from 0.0006% to 0.08% (3–5 log10 inactivations), and those of the yeasts from <0.1% to 50% (0.3–3 log10 inactivations) under the experimental conditions chosen.

Photodynamic therapy involves the application of a photosensitizer activated by visible light to generate cytotoxic reactive oxygen. In addition to clinical investigations, in vitro studies concerning photodynamic potency of sensitizers as well as quantification of illumination procedures are necessary. In our investigation, the objective was to evaluate not only the effects of photosensitizer and light on Gram-positive Staphylococcus aureus, but also to investigate possible synergistic or antagonistic effects of these sensitizers. Therefore, we used hypericin, Photofrin II, porfimer sodium and meso-tetrahydroxyphenylchlorin (mTHPC) alone, as well as in combination. Log-phase cells of S. aureus exhibited a marked sensitivity to white thermal light irradiation in the presence of Photofrin II and mTHPC. However, hypericin caused a rather stimulated growth expressed in increased optical density (OD) and increase of total cell count (TCC) of the culture. Combination sensitization of S. aureus by Photofrin II and mTHPC with hypericin likewise caused a stimulation of bacterial growth. No synergistic effects were obtained by combination of Photofrin II and mTHPC; photoresponse of S. aureus was rather decreased by using combined porphyrins. In comparison, TCC and colony-forming units (CFU) were suppressed in the presence of mTHPC after an illumination procedure as well as in dark reactions. These effects were also obtained in the combination photosensitization by mTHPC and Photofrin II. In the presence the of hypericin, photodynamic effects of mTHPC and Photofrin II were inhibited. It was finally concluded that hypericin in our model is not a proper sensitizer for combination photo-sensitization due to antagonistic effects on photodynamic activity of mTHPC and Photofrin II.

Thiol oxidation by plant peroxidases is an oxygen consuming process independent of hydrogen peroxide addition. The in vitro system consists of two interdependent reactions, i.e. hydrogen peroxide mediated oxidation of thiols via the enzymatic intermediates compound I and II yielding thiyl radicals, and further the non-enzymatic reaction of thiyl radicals to the corresponding disulphides or reaction with molecular oxygen yielding superoxide radicals and hydrogen peroxide. The thiyl radical generation per time (regulated by enzyme, thiol, and phenol concentration) determines the amount of hydrogen peroxide generated and as a consequence the amount of phenol which is oxidized by this plant enzyme. In the presence of phenols and with increasing pH oxygen consumption, thiol oxidation, and as a consequence the amount of hydrogen peroxide generated, increases. Considering phenol oxidation the system apparently becomes biphasic with a slow phenol oxidation phase A followed by a rapid phenol oxidation phase B. During phase A oxygen consumption occurs and thiol is oxidized completely. We present reaction schemes and discuss the biological relevance with regard to a possible biosynthetic route of hydrogen peroxide in plants.

In order to measure inotropic influences of physiologically occurring substances and drugs we used a newly developed guinea pig papillary muscle (GPPM) bioassay. GPPM were suspended in air and surface coated with buffer (Krebs-Henseleit solution). The muscles were stimulated (pulsating direct current, 1.5 V; 0.5 Hz, 20 ms duration) which led to contraction. This method enables measurements of inotropic effects up to 5 days, contrary to previous studies (1 day), in which immersions of GPPM in buffer were performed. In order to investigate the comparability of the new method we measured the effect of metabolites (citric acid cycle), lactic acid, lactate, and extracellular pH on muscle contractility. The H(+)-dependent decrease of the contractile force of the GPPM can be compensated by an increased Ca(2+)-concentration. Further, the influence of catecholamines (isoproterenol) on the contractility was investigated. As a result, isoproterenol caused arrhythmias and extrasystoles as it was observed in clinical studies. Several pharmaceutical substances were tested to show the reproducibility and repeatability of the bioassay.

Photodynamic eradication of tumour cells in vivo depends on the presence of a photosensitizer, light delivery to the cells, and an oxygen supply. Hypericin, a polycyclic quinone with absorption maxima in the ultraviolet and visible ranges, was prepared for clinical use as a photosensitizer. Due to antitumoral and antineoplastic activities as well as the generation of singlet oxygen after photoexcitation, hypericin was applied in clinical oncology and photodynamic therapy. Hypericin was administered subcutaneously (20 micrograms hypericin in 200 microliters Nacl/pyridine solution) into the ante brachium (forearm) of two volunteers. After the diffusion and equilibration of 120 min phototesting was carried out using outdoor light exposure, halogen lamp, laser 514 nm (argon), laser 632 nm (argon dye) and laser 670 nm (diode laser), from 60 to 120 J cm-2. Positive phototests to outdoor light exposure, halogen lamp and laser 514 nm were characterized by rubescence, oozing, vesiculation and darting pain. Phototests with laser 632 nm and 670 nm showed no effects after irradiation. When hypericin was administered topically on skin, erythema and flaring could not be induced by any irradiation. These results suggest that hypericin is a potent photosensitizer only within the UV and green light ranges. This characteristic photoresponse could also be obtained in guinea pig papillary muscle (GPPM) bioassay, which may be established as a model for photosensitizer testing. Irradiation of hypericin-incubated GPPM with 514 nm (20 J cm-2) led to a decrease of the contractile force of about 31%. However, excitation with 632 nm and 670 nm did not cause inotropic effects on GPPM. In addition, hypericin and Photosan 3 were shown to be capable of sensitizing the photo-oxidation of sodium linoleate. This assay should be established for testing interactions between photosensitizers and light sources in vitro.

Chlorophyll and some of its synthetically produced derivatives are important sensitizers in photodynamic cancer therapy. Other natural products from plants with light dependent activity include quinones like hypericin and fagopyrin. These compounds have extended π-electron systems which upon photoexcitation with visible light are responsible for singlet oxygen production.

For the quantitative determination of singlet oxygen the formation of dimethyl-(2,5)-methoxy-5-hydroperoxydihydrofuran (2,5-DMFPO) as a consequence of porphyrin photosensitization was used. Acid hydrolysis of this peroxide with 0.05 M sulfuric acid stochiometrically generates hydrogen peroxide. This can be determined by the horseradish-peroxidase catalysed dimerisation of homovanillic acid. Since DMFPO is a crystalline compound with sufficient stability it can be used as a primary standard for calibration. The described method allows the detection of singlet oxygen in methanol down to concentrations as low as 0.1 μM.

We describe the first local use of hypericin as photosensitizer for photodynamic therapy in a patient with recurrent malignant mesothelioma. Hypericin is a polycyclic quinone, which has been shown to possess in vivo and in vitro antiretroviral and photosensitizing activity; moreover, it is used in depressive disorders. The semiquinone radical, singlet oxygen, and superoxide anion radical are reported to be the toxic agents in hypericin phototherapy. Our first experience with locally applied hypericin in a superficial tumor-plate was performed 8 weeks after the systemic administration of hematoporphyrin derivatives. For tumor light illumination we used an argon pumped dye laser tuned to 632 nm. Owing to satisfactory results, we repeated the same therapy 4 weeks later — and no therapeutic effect was noted. Following this, we proved the interstitial application of HPD and the combination of interstitial HDP and superficially applied hypericin. The subsequent light illumination 6 hours later had no efficacy in the HDP-photosensitized area but there was tumor destruction in the field with both administered photosensitizers. Our first experience suggests a potentiation of two photosensitizers: hematoporphyrin derivatives and hypericin.

The described staining techniques for glucose oxidase activity on polyacrylamide gels are based on the fact that glucose oxidase reacts with one-electron scavengers in a reaction dependent on pH and pO2. Glucose oxidase separated in PAGE shows reduction of ferricyanide, cuprisulfate, ferricytochrome c and nitrotetrazolium blue reduction leading to sharp coloured bands under aerobic conditions. In contrast to the conventional peroxidase/o-dianisidine-assay, the staining procedures only need about 30 min and are specific for glucose oxidase using glucose as electron donor. Enzyme activity can be detected down to 0.2 units.

In order to develop an accurate and quick method for the determination of Photosan III in human plasma, we used statistically planned experiments with an aim to identify the factors that can influence the analysis. Through a series of 20 experiments based on acid extraction of the porphyrin from the plasma and subsequent fluorescence analysis a calibration was obtained between 0 and 3.3 X 10(-5) M. The deviation of the parameters around the regression line is 3.02%, the coefficient of variation 3.55%.

*Ludwig Bottzmann Institut für Onkologie und Photodynamische Therapie A-1130WIEN, Wolkersbergenstrasse 1 **Department of Chemistry University of Bodenkultur, A - 1190WIEN. Peter-Jordanstrasse 70. ***Department of Cardiothoracic-Anaesthesia and Intensive Care. University of Vienna Austria A - 1090 WIEN Wähnnger Gürtel AKH

*Department of Chemistry, University of Bodenkultur Vienna, Austria. **Department of Cardiothoracic-Anaesthesia and Intensive Care, University of Vienna, Austria

There is a lot of botanical, historical and linguistic evidence that the use of turnip-type Brassica rapa is very old in Europe. It was probably domesticated directly from its wild progenitor, the wild- type B. rapa. The use of European oilseed-type Brassica crops is relatively new. This paper gives a short overview on the history of B. rapa and presents botanical, historical and linguistic facts for a better understanding wild-, turnip- and oilseed-type B. rapa which will be useful for the interpretation of systematic and genetic work and future breeding.

In the presence of Fe(II)-ions the glucosinolates from the leaves of Brassicaceae are degraded by myrosinase to nitriles and sulphur. On polyacrylamide the myrosinase was separated by electrophoresis, followed by Fe(II)-S-detection. Thus myrosinase of wild growing Brassicaceae, mainly from pannonic climate area of Austria, was made visible.

The influence of externally applied gaseous nitric oxide (NO) on spruce needles in relation to cGMP formation has been examined. Exposure of spruce needles (Picea abies) to gaseous nitric oxide leads to a strong and rapid increase of the cGMP concentration. Depending on the base level of cGMP in the untreated needles, the concentration of cGMP in the NO exposed needles increased up to four degrees of magnitude. The content of adenosine-3′,5′-cyclic monophosphate (cAMP) remained below the detection limit of the HPLC-method used (10− 7 moll− 1). Therefore, it was not subject to further investigation.

A singlet oxygen determination method based on the formation of dimethyl-(2,5)-2-methoxy-5-hydroperoxydihydrofuran (DMFO2) followed by acid hydrolysis with 0.1 N H2SO4 is described. Hydrogen peroxide is formed thereby as a product of hydrolysis which is then determined by the horseradish peroxidase-catalyzed formation of the homovanillic acid dimer. Since DMFO2 is a crystalline compound with sufficient stability it can be used as a primary standard for the determination. The peroxidase-catalyzed hydrogen peroxide determination allows detection of singlet oxygen concentrations as low as 10(-7) M.