Chemiluminescent determination of acetylcholine,and continuous detection of its release from torpedo electric organ synapses and synaptosomes

A chemiluminescent procedure to determine acetylcholine is described. The enzyme choline oxidase recently purified, oxidises choline to betaine, the H₂O₂ generated is continuously measured with the luminol-peroxidase chemiluminescent reaction for H₂O₂. Other chemi or bioluminescent detectors for H₂O₂ would probably work as well. The chemiluminescent step provides great sensitivity to the method which is slightly less sensitive than the leech bio-assay but much more sensitive than the frog rectus preparation. The specificity of the chemiluminescent method depends on the fact that choline oxidase receives its substrate only when acetylcholine is hydrolysed by acetylcholinesterase. The acetylcholine content of tissue extracts was determined with the chemiluminescent method, and with the frog rectus assay, the values found were very comparable. The chemiluminescent procedure was used to follow the release of acetylcholine from tissues. When a slice of electric organ is incubated with choline oxidase, luminol and peroxidase, KCl depolarization or electrical stimulation in critical experimental conditions triggered an important light emission, which was blocked in high Mg²⁺. The venom of Glycera convoluta, known to induce a substantial transmitter release, was also found to trigger the light emission from tissue slices. Suspensions of synaptosomes release relatively large amounts of acetylcholine following Glycera venom action; this was confirmed with the chemiluminescent reaction. The demonstration that the light emission reflects the release of acetylcholine is supported by several observations. First, when the tissue is omitted no light emission is triggered after KCl or venom addition to the reagents. Second, the time course of the light emission record is very similar to the time course previously found for ACh release with radioactive methods. Third, if choline oxidase is omitted, or if acetylcholinesterase is inhibited by phospholine, the light emission is blocked, showing that the substance released has to be hydrolyzed by acetylcholinesterase and oxidised by choline oxidase to generate chemiluminescence.The procedure described has important potential applications since other transmitters can similarly be measured upon changing the oxidase.     

Application to Mammalian Tissues of the Chemiluminescent Method for Detecting Acetylcholine

Abstract: It is now possible to extend to mammalian tissues the chemilumi-nescent acetylcholine assay. Mammalian tissue extracts must be treated with oxidants (which is not necessary for electric organ extracts). The assay can then be performed as previously described (acetylcholinesterase hydrolyses acetylcholine; choline oxidase converts choline to betaine and H₂O₂, which gives off light in the presence of luminol and peroxidase). It is also shown that release experiments can be performed on mammalian tissue slices (mouse caudate nucleus) after the slice is washed in oxygenated saline solutions.     

Chemiluminescent assays for choline and phospholipase-D using a flow injection system

A flow injection chemiluminescent method is described for the determination of choline. The method is based on the production of hydrogen peroxide from choline using on-line covalently bound immobilized choline oxidase column. The product is mixed downstream and detected via the cobalt catalyzed chemiluminescent oxidation of luminol. The detection limit is 1×10⁻⁷ mol/L, with rsd 1.8 to 2.8% in the range 2–10×10⁻⁵ mol/L. The sample throughput is 30 per hour. The method was applied to the determination of choline produced off-line from phosphatidylcholine using phospholipase-D isolated from cabbage. © 1997 John Wiley & Sons, Ltd.     

Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proehancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picornole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D -glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7- phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.     

The photodynamic effect: the comparison of chemiexcitation by luminol and phthalhydrazide

The presence of light, oxygen and photosensitizer (organic dye) is required for the photodynamic effect. Light and photosensitizer are harmless by themselves, but when combined with oxygen, reactive oxygen species (ROS) can be produced. This photodynamic effect is used in photodynamic therapy (PDT); the production of ROS as lethal cytotoxic agents can inactivate tumor cells. However, during PDT, there are many difficulties, so it is not possible to excite the photosensitizer using a laser, a source of light at the wavelengths specific to the photosensitizer (in visible region of the spectrum). Chemiluminescence is the light emission as a result of a chemical reaction. It is possible to use a chemiluminescent mixture to excite the photosensitizer even if the light emission does not conform to the absorption maximum of the photosensitizer. Luciferin and luminol have been used as chemiluminescent compounds (energizers) for the excitation of the photosensitizers. The aim of this work was to compare the chemiexcitation of some selected photosensitizers (e.g. fluorescein, eosin, methylene blue, hypericin and phthalocyanines) by chemiluminescent mixtures containing luminol (high chemiluminescent quantum yield) or phthalhydrazide (low chemiluminescent quantum yield) on some Gram‐positive (Enterococcus faecalis, Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa, E. coli) bacteria and some cell lines (NIH3T3 and MCF7). The efficiency of the chemiexcitation was dependent on the kind of the photosensitizer and on the type of the bacterial strain or cell line and was independent of the energizers. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase

Chemiluminescent assay for menadione-catalyzed H₂O₂ production by mammalian cells was modified by luminol chemiluminescence with microperoxidase instead of peroxyoxalate chemiluminescence with carcinogenic fluorescent materials. Luminol can be used as a common chemiluminescent reagent for the determination of viable mammalian cells and bacteria.     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

Chemiluminescent assay for detection of viable microorganisms

The redox reaction between quinone and viable microorganisms produces active oxygen species. In this study, the production rates of active oxygen species were determined by a luminol chemiluminescent assay, and the luminescence intensity was found to be proportional to the viable cell number. The high sensitivity of the luminol chemiluminescent assay was achieved with Mo-ethylenediaminetetraacetate complex and menadione or coenzyme Q1. The detectable cell densities of bacteria and yeasts were found to be approximately several thousand colony-forming units (CFU/ml) when assays were performed with a 96-well microplate luminometer. The chemiluminescent assay requires 10 min for incubation of quinone and microorganisms and 2s for photon counting. Single Escherichia coli was detected after 4h of cultivation and centrifugation (5 min x 2). This simple chemiluminescent assay is expected to be useful for the rapid detection of viable bacteria and yeast.     

Determination of cis,cis-methylene interrupted polyunsaturated fatty acids in aqueous solutions by lipoxygenase chemiluminescence

The chemiluminescent reaction of luminol during lipoxygenase-catalyzed oxygenations was studied with the purpose of developing a specific luminometric assay for cis,cis-1,4-pentadiene fatty acids directly in aqueous solutions. The addition of picomole levels of either linoleic or arachidonic acids to reaction systems containing 0.04 mM luminol and 40 micrograms/ml of purified soybean lipoxygenase-1 gave light emission curves with a single sharp maximum. Under these conditions the peak heights were linearly dependent on the fatty acid concentration and the detection limit for both of the fatty acids was 2 pmol with a signal to noise ratio of 2. For maximum reproducibility of the assays a procedure for the proper quantitation of the enzyme was developed. The fact that the assay proved to be relatively interference-free was ascribed to the high molar enzyme/substrate ratio (above 1).     

Comparison of seven bio- and chemiluminescent reagents for in situ detection of antigens and nucleic acids

Bio- and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio-or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, aikaline phosphatase, β-galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission. An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears. Among the enzyme-luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.     

Determination of phenol using an enhanced chemiluminescent assay

Enhanced chemiluminescence (ECL) describes the phenomenon of increased light output in the luminol oxidation reaction catalysed by horseradish peroxidase (HRP) in the presence of certain compounds, such as para‐iodophenol. In this work, the effects of phenol on the para‐iodophenol‐enhanced HRP‐catalysed chemiluninescent reaction intensity in an aqueous buffer (Tris–HCl buffer, pH 8.5) and in a surfactant–water–octane mixture were compared. Preincubation of HRP at low phenol concentrations stimulated the chemiluminescent intensity in the assay performed in an aqueous buffer, but did not have significant effect in the sodium bis(2‐ethylhexyl)sulphosuccinate) (Aerosol OT, AOT) applied system. It was also observed that HRP preincubation with phenol concentration higher than 0.003 mg/mL produced an inhibitory effect on the enzyme activity for both assay systems. Only an inhibitory effect of phenol on the chemiluminescent intensity in the surfactant system in octane (as organic solvent) was observed. Three assays were developed to determine phenol concentration in water and in an organic solvent mixture. The detection limits were 0.006, 0.003 and 0.0005 mg/mL, respectively, for the buffer‐containing system, the AOT‐applied system with phenol standard solutions in water and for the AOT‐applied system with phenol standard solutions in octane. Copyright © 2002 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

Determination of sulphite using an immobilized enzyme with flow injection chemiluminescence detection

A ?ow injection method is reported for the determination of sulphite‐based on chemiluminescent detection. Hydro‐gen peroxide is produced from sulphite using on‐line covalently bound immobilized sulphite oxidase packed in a mini‐column, which was mixed downstream and detected via cobalt(II)‐catalysed chemiluminescent oxidation of luminol. The limit of detection (2 × standard deviation of the blank) was 1 × 10^?3 mmol/L with sample throughput 60 h^?1. The calibration data was linear over the range of 0.2–1.0 mmol/L with relative standard deviation (n = 4) in the range 0.9–2.0%. Copyright © 2004 John Wiley & Sons, Ltd.     

Flow-injection chemiluminescent determination of glycerol-3-phosphate and glycerophosphorylcholine using immobilized enzymes

Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5×10⁻⁷ M and 1×10⁻⁶ M respectively with r.s.d. <2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months. © 1997 John Wiley & Sons, Ltd.     

Determination of picomole quantities of acetylcholine and choline in physiologic salt solutions

An assay capable of detecting tens-of-picomole quantities of choline and acetylcholine in milliliter volumes of a physiological salt solution has been developed. Silica column chromatography was used to bind and separate 10–3000 pmol [¹⁴C]choline and [¹⁴C]acetylcholine standards made up in 3 ml of a bicarbonate-buffered Krebs-Ringer solution. The silica columns bound 95–98% of both choline and acetylcholine. Of the bound choline 84–87% was eluted in 1.5 ml of 0.075 n HCl, whereas 95–98% of the bound acetylcholine was eluted in a subsequent wash with 1.5 ml of 0.030 n HCl in 10% 2-butanone. Vacuum centrifugation of the eluants yielded small white pellets with losses of choline and acetylcholine of only 1%. Dried pellets of unlabeled choline and acetylcholine standards were assayed radioenzymatically using [γ-³²P]ATP, choline kinase, and acetylcholinesterase. The net disintegrations per minute of choline[³²P]phosphate product was proportional to both the acetylcholine (10–3000 pmol) and choline (30–3000 pmol) standards. The “limit sensitivity” was 8.5 pmol for acetylcholine and 11.4 pmol for choline. Cross-contamination of the choline assay by acetylcholine averaged 1.3%, whereas contamination of the acetylcholine assay by choline averaged 3.1%.     

Possible Effect of Activation of α7-Nicotinic Acetylcholine Receptors in the Mitochondrial Membrane on the Development of Apoptosis

Using flow cytometry and sandwich-immunoenzyme assay, we showed that nicotinic acetylcholine receptors with a subunit α7 (nAChRs α7) expressed in the outer mitochondrial membrane are involved in the control of mitochondria-dependent apoptosis. Pre-incubation of the mitochondria with an nAChRs α7 agonist, choline, decreased dissipation of the membrane potential of these organelles induced by the action of 0.5 mM hydrogen peroxide (H₂O₂) but did not influence the analogous effect of a high Ca²⁺ concentration (90 μM). Agonists of nAChRs α7 (choline, acetylcholine, and PNU 282987), or an inhibitor of voltage-dependent anion channels, DIDS, prevented the release of cytochrome c from the intermembrane mitochondrial space under the action of H₂O₂. In contrast, an antagonist of nAChRs α7, methyllycaconitine, promoted the release of cytochrome c and prevented the effects of agonists. The obtained data confirm the active involvement of nAChRs α7 and voltage-dependent anion channels in the process of formation of mitochondrial pores. In this case, agonists of mitochondrial nAChRs α7 subunits exert an antiapoptotic effect, while antagonists of mitochondrial nAChRs α7 subunits manifest a proapoptotic action.     

Assay of cholinesterase using a proenhancer of the luminol–H2O2–horseradish peroxidase reaction

2-Naphthyl acetate acts as a pro-enhancer of the luminol–H₂O₂–horseradish peroxidase reaction. Cholinesterase hydrolyses the bound acetyl group and produces 2-naphthol, and this compound is an enhancer of the chemiluminescent reaction. We studied the kinetics of chemiluminescent emission and the influence of 2-naphthyl acetate and cholinesterase enzyme concentration. The cholinesterase concentration versus chemiluminescence intensity maximum was linear for cholinesterase between 0 and 181 μU/mL, with a detection limit of 8 μU/mL and a relative standard deviation of 9.5% (n = 3), for a sample containing 90.67 μU/mL of cholinesterase.     

Menadione-catalyzed luminol chemiluminescent assay for the viability of Escherichia coli ATCC 25922.

Escherichia coli ATCC 25922 produced O2- in the presence of menadione, and O2- -dependent luminol chemiluminescence intensity was proportional to colony-forming unit (CFU) in the exponential phase. CFU was determined by using a 96-well plate at a range of 3 X 10(3) to 8 x 10(7) CFU /well (0.1 ml) after a 10-min incubation with menadione, followed by chemiluminescent assay for 5 s. After a 4-hr incubation of E. coli (10(5) CFU/0.1 ml) with menadione and an antimicrobial agent inhibiting the synthesis of peptidoglycan, protein, and DNA, the inhibitory concentration (IC) of the antimicrobial agent determined by menadione-catalyzed luminol chemiluminescent assay was in good agreement with minimal inhibitory concentration (MIC) of the NCCLS (National Committee for Clinical Laboratory Standard) method requiring 18 hr. Menadione-catalyzed luminol chemiluminescent assay is expected to be useful for the rapid determination of cell viability under the conditions of various cell growths and stresses.