On-line antioxidant activity determination: comparison of hydrophilic and lipophilic antioxidant activity using the ABTS*+ assay

The ABTS/H(2)O(2)/HRP decoloration method is capable of determining both hydrophilic (in buffered media) and lipophilic (in organic media) antioxidant properties in complex samples. Now, we have adapted this method for on-line chromatographic determination. The easy, rapid and controlled generation of the ABTS radical and its great stability in buffered and organic media were important characteristics in the measurement of antioxidant activities. The HPLC-ABTS method used two pumps (one for isocratic eluting-phase and the other for preformed ABTS radical) and an UV-VIS diode array detector. The dual analysis of samples -- conventional (with UV-VIS detection) and ABTS-scavenging (at 600 nm) -- provided valuable on-line information about the correspondence between the presence of a determined compound and its possible antioxidant activity, and was applicable to both hydrophilic and lipophilic antioxidants (HAA and LAA). A comparison between HAA and LAA determined by the end-point method and by the on-line HPLC method is presented. The application to juices showed that both methods are suitable, sensitive and selective, gave similar values, and the HPLC-ABTS method contributed additional information about the antioxidant activity profile.     

A newly developed assay for the quantitative determination of antimicrobial (anticyanobacterial) activity of both hydrophilic and lipophilic test compounds without any restriction

This paper reports about a newly developed assay which enables the user to quantify and compare the antimicrobial (anticyanobacterial) activity of both hydrophilic and lipophilic test compounds without any restriction. The assay is characterised by the application of a test compound solution on a porous matrix (silica gel) in well-defined concentrations per unit area, and by the coating of this matrix by a concentrated suspension of the tested living cyanobacterium (using a spraying or a dipping technique). The organism was only applied after the complete evaporation of the solvent used for preparation of the test compound solution to avert potential influences of this solvent on micro-organisms' viability. Toxic concentrations of a test compound caused a clearly contoured regional damage in the blue-green coloured uniform layer of the cyanobacterial test organism. The resulting regional decolourisation was readily identifiable within a species-dependent short time (e.g. for the species Arthrospira laxissima after 24h). This newly developed assay is applied for a patent in Germany.     

A fluorometric assay for angiotensin-converting enzyme activity

A simple and sensitive assay for angiotensin-converting enzyme (ACE; EC 3.4.15.1) activity has been developed which employs fluorescently labeled tripeptides. ACE hydrolyzes dansylphenylalanyl-arginyl-tryptophan or dansyl-phenylalanyl-arginyl-phenylalanine, liberating dansyl-phenylalanine and a dipeptide. Dansyl-phenylalanine partitions quantitatively into chloroform, whereas the substrates are virtually insoluble in chloroform. This allows rapid measurement of ACE activity with high signal-to-noise ratios even when microliter aliquots of human serum are assayed. Inhibition studies of the dansyl-tripeptide cleaving activity of human serum and rat lung, the identity of the products of enzyme action, and the regional distribution of enzyme activity among rat tissues demonstrate that only ACE cleaves these substrates under the conditions employed here. This assay may be useful for the clinical measurement of human serum ACE activity and for research investigations of ACE from a variety of tissues.     

A fluorometric assay for cholesterol reductase activity.

A fluorometric method for the assay of cholesterol reductase activity from pea leaves (Pisum sativum) is presented. This method is based on the decrease in relative fluorescence occurring as a result of the oxidation of NADH when cholesterol is reduced catalytically to coprostanol by cholesterol reductase. The reaction mixture consisted of micellar cholesterol, NADH, and cytosol of pea leaves in a phosphate buffer. After incubation for 1 h, the reaction mixture were diluted with 2-(N-cyclohexylamino)ethanesulfonic acid buffer (50 mM, pH 10.0) to an appropriate concentration for NADH quantification. The relative fluorescence was measured at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. This fluorometric method is relatively rapid, simple, and inexpensive. The results obtained show close correlation (R = 0.997) with those obtained by the more time-consuming and expensive radiometric method for assay of cholesterol reductase activity. Results suggest that the fluorometric method is useful for the accurate determination of cholesterol reductase activity in biological specimens.     

A novel fluorometric assay for ADP-ribose pyrophosphatase activity

ADP-ribose pyrophosphatase (ADPRase) hydrolyzes ADP-ribose to ribose-5-phosphate and AMP. The ADPRase activity have been assessed by coupling the reaction to alkaline phosphatase and colorimetrically measuring the amount of inorganic phosphate released from AMP that is one of the products of ADPRase. Another but less sensitive colorimetric method has been employed: the reaction mixture was treated with charcoal to adsorb the adenine-containing compounds such as AMP and ADPR and subsequently remaining ribose-5-phosphate was measured colorimetrically. However, the measurement of inorganic phosphate cannot be feasible to assay ADPRase in phosphate-containing samples and the determination of ribose-5-phosphate also is less sensitive. Here we develop a fluorescent assay for ADPRase that utilizes 1, N(6)-etheno ADP-ribose, a fluorescent analogue of ADP-ribose. This method measures fluorescent 1, N(6)-etheno adenosine that is produced by coupling the hydrolysis of 1, N(6)-etheno ADP-ribose to dephosphorylation with alkaline phosphatase. The fluorometric assay is comparable in sensitivity and useful for ADPRase assay in phosphate-containing samples.     

Biomarkers of antioxidant capacity in the hydrophilic and lipophilic compartments of human plasma

Antioxidants located in both the hydrophilic and lipophilic compartments of plasma are actively involved as a defense system against reactive oxygen species (ROS), which are continuously generated in the body due to both normal metabolism and disease. However, when the production of ROS is not controlled, it leads to cellular lipid, protein, and DNA damage in biological systems. Several assays to measure 'total' antioxidant capacity of plasma have been developed to study the involvement of oxidative stress in pathological conditions and to evaluate the functional bioavailability of dietary antioxidants. Conventional assays to determine antioxidant capacity primarily measure the antioxidant capacity in the aqueous compartment of plasma. Consequently, water-soluble antioxidants such as ascorbic acid, uric acid and protein thiols mainly influence these assays, whereas fat-soluble antioxidants such as tocopherols and carotenoids play only a minor role. However, there are active interactions among antioxidants located in the hydrophilic and lipophilic compartments of plasma. Therefore, new approaches to define the 'true' total antioxidant capacity of plasma should reflect the antioxidant network between water- and fat-soluble antioxidants in plasma. Revelation of the mechanism of action of antioxidants and their true antioxidant potential will help us to optimize the antioxidant defenses in the body.     

A gel filtration assay to determine glycogen synthase activity

We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.     

A method to measure antioxidant activity in organic media: application to lipophilic vitamins

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS(.+)) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10(-3) (-deltaabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as alpha-tocopherol or beta-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for alpha-tocopherol and beta-carotene. The relative antioxidant activity values obtained by different antioxidants showed that alpha-tocopherol has a similar antioxidant potential to Trolox and that beta-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.     

Synthesis and antioxidant activity of new lipophilic dihydropyridines

Dihydropyridines (DHPs) obtained from Hantzsch multicomponent reactions are an important pharmaceutical class of compounds marketed as antihypertensive (e.g., nifedipine, nitrendipine, and amlodipine) drugs. This study synthesized new symmetrical and unsymmetrical long-chain fatty DHPs using multicomponent reactions under metal-free conditions with sulfamic acid as a catalyst. The DHPs were tested for antioxidant activity using three different methods. The insertion of a long chain into the DHP core contributed to antioxidant potential, and compounds derived from nitro aldehydes have better antioxidant potential than the antihypertensive drug nifedipine. In addition, fatty analogs to nifedipine derived from palmitic and oleic chains showed similar antioxidant activity to the common standards butylated hydroxytoluene and vitamin E. These results showed that our new synthesized products may find novel applications as antioxidant additives or for tools for use in drug discovery.     

A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.     

A fluorometric assay for HIV-protease activity using high-performance liquid chromatography

A rapid sensitive method for the quantification of in vitro HIV-protease activity has been developed on the basis of the endoproteolytic conversion of N-Dns-SQ-NYPIV to N-Dns-SQNY. The use of the N-dansyl group as a fluorescence label was shown to not significantly alter the apparent kinetic parameters for the peptide-enzyme interaction. Using fluorescence detection, the dansylated product and unconverted substrate are detected in a single rapid (3 min) isocratic reverse-phase HPLC separation in quantities as low as 0.2 pmol. The method is highly reproducible and suited to a variety of applications including the analysis of large sample numbers and rigorous enzymological studies.     

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present, the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as postsegregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. Thus, it appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. This article describes a simple and convenient continuous assay for the assessment of MazF activity. The assay uses an oligonucleotide with a fluorophore on the 5' end and a quencher on the 3' end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay, we have for the first time determined K(M) and V(max) values for this enzyme and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE/MazF.     

Modified enzyme activity assay to determine biofilm biomass

An assay of potential exoproteolytic enzyme activity was modified to quantitatively measure the biomass of attached biofilm. The assay utilized the nonfluorescent compound L-leucine-beta-naphthylamide (LLbetaN) that becomes fluorescent when bacterial exoenzymes break the peptide bond, releasing the fluorochrome beta-naphthylamine. Fluorescence development was measured by pumping the liquid phase of a biofilm sample through a fluorescence detector and recording the detector output using a personal computer. A significant linear relationship was shown to exist between the rate of fluorescence development and the biofilm's biomass as carbon, determined using total direct cell counts, measured cell volumes and an existing relationship between cell volume and cell carbon. The technique was used to measure biofilm biomass for experiments where iron oxides were the substratum. Biofilm biomass measurements made using heterotrophic plate counts (HPCs) on R2A medium were shown to correlate well to biomass measurements made using the modified enzyme assay. The technique was shown to be sufficiently sensitive to measure biomass on samples containing little biofilm biomass, such as those exposed to free chlorine. While granular and porous media were used for the experiments presented, small biofilm coupons could easily be used to measure biofilm biomass, expanding the number of possible applications for the enzyme assay technique.     

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity.

A fluorometric, high-performance liquid chromatographic assay for transglutaminase activity is described. The method uses the small synthetic peptide benzyloxycarbonyl-L-glutaminylglycine and the fluorescent amine monodansylcadaverine as substrates. Very small amounts of substrates and enzyme are required for this assay. The reaction product is separated from substrates on a reversed-phase, C-18 column, using an isocratic elution solvent consisting of 50% methanol in water, and is detected fluorometrically with didansylcadaverine as standard. A detection limit of 31 pmol of product per injection was measured. An apparent Km of 34.7 +/- 2.4 mM was determined for the peptide substrate with purified guinea pig liver enzyme. Using this assay, a series of alkyl aldehydes was shown to inhibit transglutaminase. Modification of this assay using either gradient or isocratic elution with various proportions of acetonitrile (0.1% trifluoroacetic acid)/water (0.1% trifluoroacetic acid) afforded assays for a series of glutamine-containing peptides including substance P, alpha-endorphin, and two small, synthetic peptides. The assay is suitable for measurement of transglutaminase activity with purified enzyme or with crude preparations. This method provides a sensitive, quantitative assay for the determination of substrate and inhibitor properties of small peptides toward transglutaminases.     

A fluorometric assay for peptidyl alpha-amidation activity using high-performance liquid chromatography

A rapid and sensitive method for the determination of peptidyl alpha-amidation activity has been developed and is based on reverse-phase high-performance liquid chromatographic separation and fluorometric detection. A dansylated tripeptide, N-dansyl-Tyr-Val-Gly-OH, is used as the substrate in the assay and the amount of alpha-amidation activity is determined by quantitating the extent of its conversion to product, N-dansyl-Tyr-Val-NH2. Both product and substrate can be detected in a single assay in quantities as low as 5 fmol by isocratic elution using C-18 reverse-phase columns. The method yields highly reproducible results and requires less than 3 min per sample for separation and quantitation. The assay procedure is applicable to the screening of a large number of samples under different pH conditions and is readily adaptable for use in a variety of studies. For example, the procedure is ideal for detecting alpha-amidation activity in various tissues, monitoring activity at the different stages during purification of a particular alpha-amidation enzyme, determining kinetic parameters of the purified enzyme, and identifying both competitive and noncompetitive inhibitors.     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.