Redox reaction-modulated fluorescence biosensor for ascorbic acid oxidase assay by using MoS2 quantum dots as fluorescence probe.

Herein, a sensitive fluorescence (FL) biosensor for the assay of ascorbic acid oxidase (AAO) was established based on the fluorescence resonance energy transfer (FRET) between MoS2 quantum dots (MQDs) and CoOOH nanoflakes. CoOOH nanoflakes as effective FL quencher could quench the FL signal of MQDs on the basis of FRET. When ascorbic acid (AA) was added to the MQDs/CoOOH nanoflakes system, the FL signal was restored due to the redox reaction between CoOOH nanoflakes and AA, in which CoOOH nanoflakes were reduced to Co2+ by AA. In the presence of AAO, the recovered FL signal of MQDs was quenched again because of the enzymatic catalytically reaction between AAO and AA, in which AA was oxidized to dehydroascorbic acid (DHA) and then prevented the decomposition of CoOOH nanoflakes. Under the optimal experimental conditions, this developed fluorescence method exhibited good linear ranges from 2 to 10 mU mL-1 and 10-40 mU mL-1 with a low detection of limit of 0.8 mU mL-1 for AAO detection. And the limit of quantification (LOQ) of 2.6 mU mL-1 was obtained. The proposed biosensor showed high sensitivity and selectivity, and was successfully applied for AAO determination in human serum samples.

A fluorescence "off-on-off" sensing platform based on bimetallic gold/silver nanoclusters for ascorbate oxidase activity monitoring.

Herein, papain-protected bimetallic gold/silver nanoclusters (Au/Ag NCs) were successfully synthesized and applied for the detection of ascorbate oxidase (AAO). The doping of papain-protected Au nanoclusters with Ag enhanced the fluorescence intensity with an intense red fluorescence peak at 617 nm, and the red-emitting Au/Ag nanoclusters were further used to monitor the AAO activity. The fluorescence of Au/Ag NCs could be quenched by hydrogen peroxide (H2O2) due to the generation of hydroxyl radicals (˙OH) from the reaction of Ag/Au nanoclusters and H2O2. However, the addition of ascorbic acid (AA) effectively reacted with the free radicals and caused the fluorescence recovery of the Au/Ag NCs. Furthermore, AAO could catalyze the oxidation of AA to form dehydro-ascorbate (DHA). As a result, there was not enough AA to consume the hydroxyl radicals, which resulted in a decrease in the fluorescence of the papain-capped Au/Ag NCs. Therefore, the AAO activity can be monitored by measuring the fluorescence intensity of the red-emitting Au/Ag NCs. Moreover, the developed method for AAO detection displayed a good linear relationship from 5 to 80 mU mL-1 and the detection limit was 1.72 mU mL-1. Thus, a simple and selective method for the determination of the AAO activity was constructed and satisfactory results were obtained in real sample detection.

A carbon dot-based ratiometric fluorometric and colorimetric method for determination of ascorbic acid and of the activity of ascorbic acid oxidase.

A dual-mode method was developed for the determination of ascorbic acid (AA) and of ascorbic acid oxidase (AA-Ox) activity. It combines the advantages of ratiometric fluorometry and colorimetry. The assay is based on the oxidation of o-phenylenediamine (OPDA) by permanganate (KMnO4). A yellow substance (referred to as oxOPDA) with an absorption peak at 425 nm is rapidly produced in the presence of the oxidant. oxOPDA reduces the blue fluorescence of carbon dots (C-dots) peaking at 450 nm (upon 380-nm excitation), and a new emission peak is found at 565 nm. If AA is pesent, it consumes a certain fraction of KMnO4, so that less OPDA will be oxidized. This is accompanied by a decrease in the intensity of the fluorescence at 565 nm and an increase in the intensity at 450 nm. In parallel, the color of the solution changes from yellow to colorless. The determination of the activity of ascorbic acid oxidase (AA-Ox) is performed as follows: AA is oxidized by AA-Ox. This causes the fluorescence and colors to change in the opposite directions compared with AA detection. The ratio of fluorescences (I565/I450) becomes larger if the color the solution turns from colorless to yellow. Thus, the fluorescence intensity ratio (I565/I450) and colorimetric "bare-eye" readout can be used for determination of both the concentration of AA and the activity of AA-Ox. The fluorometric assay for AA has a linear range that extends from 0.6 to 40 µM, and the colorimetric assay from 0.2 to 70 µM. The respective data for AA-Ox activity are 0.04 ~ 5 mU·mL-1 and 0.04 ~ 8 mU·mL-1, respectively. The limits of detection for AA are 9 and 40 nM, and the LODs for AA-Ox activity are 0.017 and 0.012 mU·mL-1. Graphical abstract Schematic presentation of the assay. Permanganate (KMnO4) rapidly oxidizes ortho-phenylenediamine oxide to form a product (oxOPDA) having a yellow fluorescence peaking at 565 nm. The yellow color of oxOPDA can be detected visually. It also reduces the intensity of the blue fluorescence of carbon dots (C-dots) peaking at 450 nm. Ascorbic acid (AA) can consume permanganate, and this results less oxidation of OPDA. Ascorbic acid oxidase (AA-Ox) catalyzes the oxidization of AA by oxygen, and this - in turn - causes the changes in absorbance and fluorescence to change in the opposite directions.

The effects of cilostazol on exercise-induced ischaemia-reperfusion injury in patients with peripheral arterial disease.

OBJECTIVES: Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemia-reperfusion injury in such patients. METHODS: PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the inflammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. Inflammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, alpha-tocopherol, beta-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B(2) (TXB(2)), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albumin-creatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks. RESULTS: One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more significant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, p=0.026 and 161.7% vs. 79.0% at 24 weeks, p=0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: -11.8% vs. +5.8%, p=0.003 and post-exercise: -12.3% vs. +13.9%, p=0.007 and 24 weeks: pre-exercise -15.5% vs. +12.0%, p=0.025 and post-exercise: -9.2% vs. +1.9%, p=0.028). beta-Carotene levels were significantly increased in the cilostazol group, compared to placebo, before exercise at 6 and 24 weeks (6 weeks: 34.5% vs. -7.4%, p=0.028; 24 weeks: 34.3% vs. 17.7%, p=0.048). Cilostazol also significantly reduced P-selectin, I-CAM and V-CAM levels at 24 weeks as compared to baseline (p<0.05). There was no difference between treatment groups for ascorbate, alpha-tocopherol, interleukin-6 and -10, hsCRP and p75TNF receptor levels. CONCLUSIONS: Cilostazol significantly improves ACD, in addition to attenuating exercise-induced ischaemia-reperfusion injury, in PAD patients.

Extracellular reduction of the ascorbate free radical by human erythrocytes.

We investigated the possibility that human erythrocytes can reduce extracellular ascorbate free radical (AFR). When the AFR was generated from ascorbate by ascorbate oxidase, intact cells slowed the loss of extracellular ascorbate, an effect that could not be explained by changes in enzyme activity or by release of ascorbate from the cells. If cells preserve extracellular ascorbate by regenerating it from the AFR, then they should decrease the steady-state concentration of the AFR. This was confirmed directly by electron paramagnetic resonance spectroscopy, in which the steady-state extracellular AFR signal varied inversely with the cell concentration and was a saturable function of the absolute AFR concentration. Treatment of cells N-ethylmaleimide (2 mM) impaired their ability both to preserve extracellular ascorbate, and to decrease the extracellular AFR concentration. These results suggest that erythrocytes spare extracellular ascorbate by enhancing recycling of the AFR, which could help to maintain extracellular concentrations of the vitamin.

[The status of ascorbate oxidation-reduction system of blood in patients with chronic pancreatitis throughout parenteral nutrition]. / Sostoianie askorbatnoi okislitel'no-vosstanovitel'noi sistemy krovi u bol'nykh khronicheskim pankreatitom v usloviiakh primeneniia parenteral'nogo pitaniia.

There was conducted operative treatment of patients with complicated chronic pancreatitis. Intravenous infusion of aminoacid cocktail with enhanced content of glutamine, methionine with the selenium addition was applied in the complex of therapy. Under the influence of modified cocktail during 3 days there was observed more complete restoration of the ascorbic acid (AA) level and lowering of her oxidated forms content. This effect became more potent in addition of AA (0.75 per one infusion).

Effects of increasing doses of alpha-tocopherol in providing protection of low-density lipoprotein from oxidation.

In this study, we tested whether 1,200 IU/day of alpha-tocopherol was more potent than 400 and 800 IU of alpha-tocopherol in decreasing low-density lipoprotein (LDL) oxidative susceptibility in a 2-month study. The decrease in LDL oxidation was significantly greater with 1,200 IU/day than 400 IU/day.

Ascorbate radical and ascorbic acid level in human serum and age.

Free radical in human serum was observed by an electron spin resonance (ESR) technique and was assigned to ascorbate radical. Quantitative estimation revealed that the ESR intensity could be used as an expedient mean. The ESR intensity of ascorbate radical, the concentrations of total ascorbic acid and oxidized form of ascorbic acid were determined on sera of 200 healthy individuals whose ages ranged from 12 to 96 years. The ESR intensity as well as the concentrations of total and oxidized form of ascorbic acid declined with age. There were significant correlations between the ESR intensity and the concentrations of total and oxidized form of ascorbic acid. From these results, the clinical significance of the concentrations of total ascorbic acid, ascorbate radical and the oxidized form was discussed in relation to age.

Ascorbate oxidase and its possible involvement in cancer.

The electron spin resonance (ESR) spectrum of erythrocytes of a health male volunteer exhibited 2 min after intravenous administration of 1g of ascorbic acid a considerable increase in spin concentration and an new signal at about g-2.005 which we previously had found to correlate to the semidehydroascorbate (SDA) radical and which is not identical with the O2 radical. Moreover, the vitamin C concentration in erythrocytes and plasma was considerably higher than in comparable samples of other volunteers treated identically. In the latter cases, the ESR spectrum of the erythrocytes was not modified at all. These findings suggest that there must be a substance which reacts with ascorbic acid specifically. It can be assumed that the enzyme ascorbate oxidase plays this decisive role in the ascorbic acid metabolism. For this reason, different amounts of ascorbate oxidase have been added to healthy erythrocytes treated in vitro with ascorbic acid and to tissue samples of lung cancer. As expected, the vitamin C effect as expressed by the appearance of the SDA signal and the increase in spin concentrations could be reversed. It is suggested, therefore, that in special types of cancer, such as acute lymphatic leukemia and lung cancer, the concentration of ascorbate oxidase or of an enzyme acting like it is, primarily, diminished, while in other types it might be enlarged.