Methodological problems of direct bioluminescent ATP assay in platelets and erythrocytes

Direct bioluminescent ATP determination in platelets and erythrocytes involves the study of different parameters which are discussed here. Some parameters are linked to the bioluminescent reaction and to the analyte (ATP); others have regard to the biological matrix. The composition of bioluminescent reagents and the preparation and conservation of the ATP standard, also in the presence of excipients, are among the first given. Matrix problems involve cell characteristics related to age and form, lysis resistance and the possible formation of aggregates (platelets) that may inhibit the complete release of ATP. For these reasons we used the most efficient ATP release agent with the lowest inhibitory effect on luciferase. The data obtained correlate well with a bioluminescent method requiring extraction with ethanol/EDTA, and therefore more time, for ATP determination in platelets and erythrocytes.     

A bioluminescent assay for the determination of phosphoenolpyruvate carboxykinase activity in nanogram-sized tissue samples

A highly specific and sensitive assay for the determination of phosphoenolpyruvate carboxykinase (PEPCK) in nanogram-sized tissue samples is described. This test system is based on the stoichiometric transformation of phosphoenolpyruvate into ATP. In a subsequent step ATP is quantified by bioluminescent techniques. The applicability of this assay system is shown by measurements in liver samples with normal and high PEPCK activity levels.     

A rapid sensitive method for the measurement of guanine ribonucleotides in bacterial and environmental extracts.

A method was devised for the quantitative determination of guanine ribonucleotides (GTP, GDP, and GMP) in extracts of biological materlals. The technique is based upon selective enzymatic hydrolysis of UTP and ATP contained within the cell extracts, followed by a quantitative determination of GTP. GTP is measured using a nucleoside diphosphate kinase-firefly luciferase coupled bioluminescent reaction, during which the GTP is enzymatically coupled to ATP production, resulting in ATP-dependent light emission. The methods are simple and reproducible and extremely sensitive (≤ 10^?9m GTP), and require no preparatory chromatographic separation procedures. Methods are also presented for the enzymatic conversions of GDP and GMP to GTP in addition to the determination of GTP.     

Methodological problems of direct bioluminescent ADP assay in platelets and erythrocytes

We have developed a method for ADP bioluminescent measurement in platelets and erythrocytes which complements our previous method for ATP assay. When the different parameters of the system under investigation are taken into account, a linea range between 10(-9) and 10(-7) g/ml can be obtained without incubation or troublesome extraction. This makes the method easy and useful for identifying any disease-induced alterations in ATP and/or ADP levels in these blood cells. The data obtained correlate well with those of a bioluminescent method requiring extraction with ethanol/EDTA and incubation, giving the reference intervals of 3.5-5.5 mumol/10(11) PLT for ATP determination and 1.9-3.7 mumol/10(11) PLT for ADP determination in platelets, and 3.2-3.8 mumol/g Hgb for ATP determination and 0.56-0.73 mumol/g Hgb for ADP in erythrocytes. This assay was applied to quality control on blood bags in transfusion centers and proved to be a rapid and reliable method for testing the viability of stored blood cells.     

Chemiluminescent assay of co-factors

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperox-idase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 × 10^?14 to 5 × 10^?12mol/assay. The detection limit of NADH was 30fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.     

Measurement of the adenosine triphosphate content of cysts of Globodera rostochiensis as a method for assessing the efficacy of fumigant nematicides

The measurement of the adenosine triphosphate (ATP) content of samples of 50 cysts of Globodera rostochiensis collected from 40 fields in the Netherlands has been tested as a method for determining the mortality of the nematodes achieved by fumigation of these field soils. The technique used is based on bioluminescent photometry and it has been fully described previously. The ATP assessment has been compared with results obtained by the advisory service in the Netherlands using hatching tests based on samples of 100 cysts from the same field soils. The ratio of the values before and after soil treatment showed that the methods usually provided similar estimates of mortality. The relationship between ATP and hatched juveniles was also similar from pre-treatment and post-treatment samples. The amount of ATP, calculated from these results, which is equivalent to the maximum post-treatment hatch allowed for a successful treatment in the Netherlands was used in conjunction with the mortality estimates to assess the success of the fumigation. On this basis the two methods disagreed in the overall assessment of fumigation in only five of the 40 fields. This frequency of disagreement is not significantly greater than expected from the variability of either method. The ATP technique clearly offers a reliable and relatively inexpensive method for estimating the efficacy of fumigant nematicides and is much more rapid than the alternative hatching tests or egg counts. Therefore, it could replace these in situations where the grower wanted a rapid assessment on the success, or otherwise, of his fumigation.     

Application of intracellular ATP determination in lymphocytes for HLA-typing

The amount of adenosine triphosphate (ATP) in human lymphocytes was determined using a technique based on light emission from a bioluminescent reaction with luciferin-luciferase. The amount of ATP changed when cells were incubated in the presence of specific HLA antisera and complement. For determination of intracellular ATP a modified method was applied, which was based on reduction of extracellular ATP by the addition of ATPase. The results of titration of an anti-human lymphocyte serum using the bioluminescence assay were in agreement with the results of fluorescence vitality staining. Bioluminescent HLA-determination in 57 cell samples each tested with 5 different antisera also gave good agreement (95.8%) with the conventional method. From these experimental data the calculated ATP content per lymphocyte was 0.135 ± 0.058 pg ATP.     

An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA)

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. This resulted in constant luminescence once the stable phase had been reached. Background luminescence of the reagent was reduced with adenosine phosphate deaminase by degrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10(-17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were subjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amount of ATP as an index.     

Simple and rapid bioluminescent detection of two verotoxin genes using allele‐specific PCR of E. coli O157: H7

Allele‐specific PCR for E. coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2). VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide. We developed a simple, rapid bioluminescent detection method for E. coli O157. The method is based on the determination of pyrophosphoric acid (PPi) released during allele‐specific PCR. Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction. As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method. This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly. Additionally, this methodology can be used as a high‐throughput approach for measuring PCR products. Copyright © 2003 John Wiley & Sons, Ltd.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

The continuous bioluminescent assay of ATP has been adapted to the study of Mg²⁺-dependent ATPases, including the (Na⁺,K⁺) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg²⁺-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

Screening for resistance to potato cyst nematodes using bioluminescent photometry

The measurement of the adenosine triphosphate (ATP) content of cysts by bioluminescent photometry has been used as a screen for assessing the resistance of five potato clones to Globodera rostochiensis Rol and G. pallida Pa3. The multiplication rates (P_f/P_i) of the nematodes are based on the ratio of ATP levels for the cyst inoculum and their progeny. These have been compared to the corresponding ratios based on conventional visual counts of eggs and juveniles. The use of recoverable standard volumes of cysts to inoculate pots has allowed an assessment of the unhatched proportion (C_p) of juveniles from the parental cysts by both methods and an estimate of the corrected multiplication rate (P/P^l-Cp)). This is an accurate assessment of resistance for a clone and the use of standard inocula and the ATP method will enable the criterion adopted for establishing resistance to be measured rapidly and precisely.     

Adenosine triphosphate assay of <Emphasis Type="Italic">Bactrocera dorsalis</Emphasis> (Diptera: Tephritidae) eggs treated with low or high temperatures

A bioluminescent adenosine triphosphate (ATP) assay using a luciferin-luciferase reagent was conducted as a viability test for Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) eggs treated with low or high temperatures to evaluate the potential into use for quarantine operations. ATP concentrations in B. dorsalis eggs treated with different temperatures, including freezing, cold treatment, and rapid and slow heating, simulating a hot water immersion treatment and vapor heat treatment, respectively, were measured. Mean ATP concentrations in untreated eggs decreased as egg age increased. For freezing and rapid heating, the mean ATP concentrations in treated eggs significantly decreased 24 h after the treatments, and the maximum ATP concentrations were lower than the minimum ones for untreated eggs. Most ATP concentrations in the cold treatment group exceeded the minimum ones in untreated eggs. Mean ATP concentrations in eggs treated with slow heating decreased less than those in eggs treated with rapid heating. There is potential to use ATP assays in plant quarantine operations for the rapid determination of the viability of fruit fly eggs treated with hot water immersion, although more validation research is first required. Verification tests should be performed by applying ATP assays during quarantine, by using flies and host fruit subjected to different temperature treatments.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Lysostaphin lysis procedure for detection of Staphylococcus aureus by the firefly bioluminescent ATP method.

The objective of this study was to examine the use of lysostaphin as an ATP-extracting agent for the estimation of Staphylococcus aureus cell number by a rapid bioluminescent ATP method. The results of the study showed that lysostaphin (22 U/ml) was able to lyse most of the S. aureus cells (greater than 99.9%) at room temperature in 1 min; ATP of S. aureus cells extracted by the lysostaphin lysis procedure was stable for 24 h in the presence of EDTA; there was a linear relationship between the ATP content and the number of S. aureus cells (ranging from 10(4) to 10(6) CFU/ml); and the lysis of S. aureus cells by lysostaphin allowed estimation of the number of S. aureus cells in mixed cultures and in meat samples.     

Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria

Background

Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.

Methodology/Principal Findings

The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically) and phosphorylation (determined using the glucose - hexokinase - glucose-6-phosphate dehydrogenase - NADP⁺ enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K_mVox) and phosphorylation (K_mVp) rates. We also demonstrate that determination of K_mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.

Conclusions/Significance

Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.     

Bioluminescent determination of free fatty acids

A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes.     

Bioluminescent assay of total bacterial contamination of drinking water.

A bioluminescent assay of total bacterial contamination (TBC) of drinking water (DW) with a detection limit of approximately 1 CFU/mL and duration of less than 7 h has been developed. The protocol of the TBC assay comprises: incubation of water sample in nutrition broth supplemented with salts mixture, up to 6 h; filtration of bacterial suspension obtained through membrane filter (pore size 0.45 microm); release of bacterial ATP by dimethyl sulphoxide; determination of bacterial ATP concentration using highly sensitive ATP reagent based on recombinant Luciola mingrelica luciferase. To simplify the assay, special luminometer microcuvette Filtravette (New Horizons Diagnostics Corp., USA) are used. A good correlation (R=0.98) between ATP concentration measured after 6 h incubation and initial bacterial titre in DW was observed. Semi-quantitative TBC assay of DW is also available. The TBC value in DW is assessed by the fixation of incubation time required to detect a measurable bioluminescent signal: 3, 4 and 6 h corresponds to 100-1000, 10-100 and 1-10 CFU/mL, respectively.     

Microanalysis of the metabolic intermediates of lactose synthesis in human milk and plasma using bioluminescent methods

Sensitive bioluminescent methods were developed to measure the metabolites glucose, glucose 6-phosphate (G6P), glucose 1-phosphate (G1P), UDP-glucose, and UDP-galactose in human milk and lactose and galactose in human plasma. The bioluminescent methods measured NADH produced by coupled enzymatic assays derived from equivalent spectrophotometric methods. We found that the long chain fatty acids in human milk (C10-C16) inhibited the bioluminescent reactions. This inhibition was overcome by adding defatted bovine serum albumin to the reaction mixture containing the bioluminescent enzymes. It also was necessary to modify methods of deproteinizing milk and blood plasma to accommodate small sample volumes. In the development of these assays emphasis was given to simplicity of reagent preparation, minimizing cost, and ease of use. The detection limit for the bioluminescent method for NADH was 0.28 nM for a 20-microliters sample. For the assays of the metabolites, recoveries ranged from 91 to 107%. For sample sizes of 2 to 5 microliters of protein free sample, the detection limits for milk were G1P, 0.09 microM; G6P, 0.05 microM; UDPhexose, 0.07 microM; UDP-Glc, 0.03 microM; glucose, 9 microM; and for plasma, lactose, 0.76 microM, galactose, 0.31 microM. The bioluminescent methods gave equivalent results to spectrophotometric methods for the measurement of blood lactose and milk glucose.     

Double-receptor sandwich supramolecule sensing method for the determination of ATP based on uranyl-salophen complex and aptamer

In this study, we report a double-receptor sandwich supramolecule sensing method for the determination of adenosine triphosphate (ATP). One receptor is a uranyl-salophen complex which can bind the triphosphate group in ATP selectively, and another is an anti-adenosine aptamer which is a single-stranded oligonucleotide and can recognize the adenosine group in ATP specifically. The uranyl-salophen complex was immobilized on the surface of amino-silica gel particles and used as the solid phase receptor of ATP. The anti-adenosine aptamer was labeled with a fluorescent group and used as the labeled receptor of ATP. In the procedure of ATP detection, ATP was first combined with the solid phase receptor and then conjugated with the labeled receptor to form a sandwich-type supramolecule. The conditions of fabricating solid phase receptor and the influence of manifold variables on the determination were studied. The experimental results demonstrate that the method has a number of advantages such as high selectivity, high sensitivity, good stability and low cost. Under optimal conditions, the linear range for detection of ATP is 0.2-5.0 nmol/mL with a detection limit of 0.037 nmol/mL. The proposed method was successfully applied for the determination of ATP in real samples with the recoveries of 96.8-103.3%.